Molecular identification and antifungal susceptibility profiles of clinical strains of Fonsecaea spp

15 Pages·2017·1.96 MB·English

RESEARCHARTICLE
Molecular identification and antifungal
susceptibility profiles of clinical strains of
Fonsecaea spp. isolated from patients with
chromoblastomycosis in Rio de Janeiro, Brazil
RowenaAlvesCoelho1,Fa´bioBrito-Santos1,MariaHelenaGaldinoFigueiredo-Carvalho1,
JulianaVitoriadosSantosSilva1,MariaClaraGutierrez-Galhardo2,AntonioCarlos
FrancesconidoValle2,RoselyMariaZancope´-Oliveira1,LucianaTrilles1,WielandMeyer3,
a1111111111 DayvisonFrancisSaraivaFreitas2,RodrigoAlmeida-Paes1*
a1111111111
1 LaboratoryofMycology,EvandroChagasNationalInstituteofInfectiousDiseases,OswaldoCruz
a1111111111
Foundation(Fiocruz),RiodeJaneiro,RJ,Brazil,2 LaboratoryofClinicalResearchinInfectiousDermatology,
a1111111111
EvandroChagasNationalInstituteofInfectiousDiseases,OswaldoCruzFoundation(Fiocruz),Riode
a1111111111
Janeiro,RJ,Brazil,3 MolecularMycologyResearchLaboratory,CentreforInfectiousDiseasesand
Microbiology,WestmeadClinicalSchool-SydneyMedicalSchool,MarieBashirInstituteforInfectious
DiseasesandBiosecurity,UniversityofSydney,WestmeadInstituteforMedicalResearch,Sydney,Australia
*[email protected]
OPENACCESS
Citation:CoelhoRA,Brito-SantosF,Figueiredo- Abstract
CarvalhoMHG,SilvaJVdS,Gutierrez-GalhardoMC,
doValleACF,etal.(2018)Molecularidentification
andantifungalsusceptibilityprofilesofclinical
strainsofFonsecaeaspp.isolatedfrompatients Background
withchromoblastomycosisinRiodeJaneiro,
Brazil.PLoSNeglTropDis12(7):e0006675. Chromoblastomycosis(CBM)isadifficult-to-treatchronicsubcutaneousmycosis.InBrazil,
https://doi.org/10.1371/journal.pntd.0006675 themainagentofthisdiseaseisFonsecaeapedrosoi,whichisphenotypicallyverysimilarto
Editor:ToddB.Reynolds,UniversityofTennessee, otherFonsecaeaspecies,differingonlygenetically.Thecorrectspeciesidentificationisrel-
UNITEDSTATES evantsincedifferentspeciesmaydifferintheirepidemiologicaspects,clinicalpresentation,
Received:May22,2018 andtreatmentresponse.
Accepted:July9,2018
Methodology/Principalfindings
Published:July26,2018
Copyright:©2018Coelhoetal.Thisisanopen Partialsequencingoftheinternaltranscribedspacer(ITS)wasusedtoidentifytwentyclini-
accessarticledistributedunderthetermsofthe calisolatesofFonsecaeaspp.Theirinvitroantifungalsusceptibilitywasdeterminedusing
CreativeCommonsAttributionLicense,which
thebrothmicrodilutionmethod,accordingtotheM38-A2protocol.AmphotericinB(AMB),
permitsunrestricteduse,distribution,and
flucytosine(5FC),terbinafine(TRB),fluconazole(FLC),itraconazole(ITC),ketoconazole
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. (KTC),posaconazole(POS),voriconazole(VRC),ravuconazole(RVC),caspofungin
(CAS),andmicafungin(MFG)weretested.TheassociationbetweenITC/TRB,AMB/5FC,
DataAvailabilityStatement:Theauthorsconfirm
thatalldataunderlyingthefindingsarefully andITC/CASwasstudiedbythecheckerboardmethodtochecksynergism.Theavailable
availablewithoutrestriction.Thesequences patients’datawerecorrelatedwiththeobtainedlaboratoryresults.Fonsecaeamonophora
obtainedinthisstudyhavebeendepositedin
(n=10),F.pedrosoi(n=5),andF.nubica(n=5)wereidentifiedasCBM’agentsinthe
GenBankundertheaccessionnumbersMF616485
study.TRBandVRCwerethedrugswiththebestinvitroactivitywithminimalinhibitorycon-
–MF616504.Allotherrelevantdataarewithinthe
paperanditsSupportingInformationfiles. centrations(MIC)lowerthan0.25mg/L.Ontheotherhand,FLC,5FC,AMB,andMFG
showedhighMICs.TheAMB/5FCcombinationwassynergisticforthreeF.monophora
Funding:Thisworkwassupportedby“Conselho
NacionaldeDesenvolvimentoCient´ıficoe strainswhiletheotherswereindifferent.PatientshadmoderateorsevereCBM,andITC
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Fonsecaeaspp.identificationandsusceptibility
Tecnolo´gico”[grantnumber449184/2014-5to therapywasnotsufficientforcompletecureinmostofthecases,requiringadjuvantsurgical
RA-P]and“Fundac¸a˜odeAmparoa`Pesquisado approaches.
EstadodoRiodeJaneiro”[grantnumberE-26/
200.839/2017].RA-P,RMZ-OandLTare
Conclusions/Significance
partiallysupportedby“ConselhoNacionalde
desenvolvimentoCient´ıficoeTecnolo´gico”[grant F.monophora,thesecondmostfrequentFonsecaeaspeciesinSouthAmerica,predomi-
numbers305487/2015-9,304976/2013-0and
natedinpatientsraisedandborninRiodeJaneiro,Brazil,withoutcerebralinvolvementin
442837-2014-3].Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisionto thesecases.TRB,VRC,andtheAMB/5FCcombinationshouldbefurtherinvestigatedasa
publish,orpreparationofthemanuscript. treatmentoptionforCBM.
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Authorsummary
Chromoblastomycosisisadisfiguringdiseaseusuallyoccurringinruralworkersfrom
poorandremotecommunities.InBrazil,themostfrequentagentsofthisneglecteddis-
easearethespeciesbelongingtothegenusFonsecaea.Thediseaseoccursaftertraumatic
inoculationduringwork.Asthelesionsprogress,itchingbecomessevere,andscratching
mayresultinfurtherinoculationtootherbodysites.Whenpatientsseekmedicalhelp,the
lesionsareusuallyextensiveanddisfiguring.Forthisreason,amoreeffectiveandless
time-consumingtreatmentisimportant.Oralantifungaltherapyisnotveryeffective,
mustbetakenformonthsoryears,itiscostlyformostpatientsandoftenunavailable.
Hence,itisimportanttodeterminetheinvitroantifungalsusceptibilityandcorrelateit
withtheisolatedspecies.Inthisstudy,Fonsecaeamonophorawasthepredominantspecies
and,differentlyfromsomestudies,disseminationtothecentralnervoussystemwasnot
observed.Invitroanalysisshowedthatthemosteffectiveantifungaldrugswereterbina-
fineandvoriconazole,followedbyitraconazole,themostuseddruginthetreatmentof
thisdisease.ThecombinationofamphotericinBandflucytosinemaybesynergistic,
dependingontheinfectivestrain.
Introduction
Chromoblastomycosis(CBM)isachronicfungalinfectionofcutaneousandsubcutaneoustis-
suescausedbytraumaticimplantationofseveralspeciesofdematiaceousfungi[1].In2017,
thismycosiswasrecognizedasaneglectedtropicaldiseasebytheWorldHealthOrganization
[2].
TheetiologicalagentsofCBMbelongmainlytothegeneraCladophialophora,Phialophora,
andFonsecaea[3].Inthelastdecade,newspeciesofthegenusFonsecaeahavebeendescribed,
basedonmolecularcriteria:Fonsecaeamonophora[4],Fonsecaeanubica[5],Fonsecaeamulti-
morphosa[6],andFonsecaeapugnacius[7].Thesespeciescanbefoundinnature,trace
amountsinplantdebris,thorns,andwoodcortex,whichprovidemicrohabitatsforthesefungi
[8].IntheBrazilianStateofMaranhão,ontheborderoftheBrazilianAmazonrainforest,sev-
eralagriculturalcommunitiesworkonharvestingbabassu(Orbignyaphalerata),awildpalma-
ceaspecimenthatwasdescribedasprobableinfectionsourceinthisarea[9].
Intheenvironment,allagentsofCBMpresentintheirmycelialform,whichiscomposed
bydematiaceoushyphaeandconidia,whicharespecificforeachgenus.Infectionusuallyfol-
lowsahumantraumawithacontaminatedorganicmaterialsuchasplantthorns,wood,plant
debris,grass,treecortexamongothers,leadingtotheimplantationofthefungusinthesubcu-
taneoustissues,wherethefunguschangestoitsparasiticformcomposedbymuriformcells.
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Thesecellsareheavilymelanisedandareextremelyresistanttotheharshconditionsimposed
bythehostimmunesystem[10].
CBMcanbecausedbyfourFonsecaeaspecies:F.pedrosoi,F.nubica[4–6],F.monophora
andF.pugnacius.Thelattertwoshowsignificantneurotropism,eventuallyleadingtodissemi-
nationtothebrainandotherorgans[4,7]orcausingprimarybraininfectionwithoutskin
lesions,whichareclassifiedasphaeohyphomycosissincenomuriformcellsareseenintissues
[10,11].CBMmayassumeseveralclinicalformswithdifferentdegreesofseverity[10].
Thereisnotreatmentprotocoltobefollowed,andantifungaltherapyisoftencombined
withphysicalmethodssuchascryosurgeryorsurgicalexcisionforsmalllesions[12].Itracona-
zole(ITC)andterbinafine(TRB)arethemostuseddrugsinthetreatmentofCBM[10,13–15].
Otherdrugsusedincludeposaconazole(POS),voriconazole(VRC),amphotericinB(AMB)
andflucytosine(5FC)[10,16,17].Inaddition,combinedtherapiesofITCwithTRB[10,18],
5FCwithAMB[17],orITCwith5FChavebeenused[19].
Itisimportanttodeterminetheinvitrosusceptibilityoftheseisolatesbecauseofthediffi-
cultyfoundinthetreatmentofthismycosisandthefrequencyofrefractorycasesandrelapses.
Thepresentstudyaimedtomolecularlyidentifythespecies,toevaluatetheinvitrosusceptibil-
itytoantifungals,andtoidentifypossiblecombinationsofdrugswithsynergismagainst
strainsisolatedfrompatientswithCBMdiagnosedinRiodeJaneirostate,SoutheastBrazil,an
areaoflowoccurrenceofthismycosis.Moreover,aclinicalandlaboratorialdataassociationis
providedforsomepatients.
Materialandmethods
Ethicalstatement
ThisstudywasapprovedbytheEthicsCommitteeBoardoftheEvandroChagasNational
InstituteofInfectiousDiseases(INI),OswaldoCruzFoundation(Fiocruz),underthenumber
CAAE:52247016.0.0000.5262.
Fungalisolates
Twentyisolatesofdematiaceousfungifromskinlesionsof17patientswithCBMwere
includedinthisstudy.Theseisolateswerestoredfrom1999to2015attheINIMycologyLabo-
ratoryandidentifiedphenotypicallyasFonsecaeapedrosoi.Fromthetotalof17patients,12
weretreatedattheINI’sInfectiousDermatologyOutpatientClinic,7ofwhichwerepreviously
studiedbyMouchalouatetal.,[20]andtheremaining5werefollowedupatotherinstitutions
aftermycologicaldiagnosisattheINI(Table1).AllstoredfungiwererecoveredonSabouraud
DextroseAgar(DifcoLaboratories,Sparks,MD,USA)incubatedat25˚Cfor10days.Micro-
scopically,hyphaewereseptate,branched,andbrownstainingwiththepredominanceof
conidiophoreswithshortchainsofsmooth,thin-walleddematiaceousconidia.
Molecularidentification
PutativeFonsecaeaspp.colonieswereassessedonpotatodextroseagar(PDA)(HiMediaLabo-
ratoriesPvt.Ltd.,India)at25˚Cafter7–14daysofinoculation.TheDNAextractionandpoly-
merasechainreaction(PCR)oftheITS1-5.8S-ITS2region,theofficialFungalDNAbarcode,
wereperformedaccordingtoBrito-Santosetal.[21].PCRproductswerepurifiedusingthe
WizardSVGelandPCRClean-UpSystemkit(PromegaCorporation,Madison,USA)and
sequencedatthePlatformforDNASequencingPDTIS/Fiocruz.Sequenceswereeditedusing
Sequencher4.9(GeneCodesCorporation,AnnArbor,MI,USA),alignedandanalyzedwith
MEGA6.06[22],andcomparedbyBLASTwithsequencesavailableattheISHAMITS
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Table1. Demographics,clinicalfeatures,timeofevolutionandfollow-upofCBMcases(20strainsfrom17patients).
N Strainnumber Species Sexa Age Occupation State/regionof Cityof Timeof Severity Outcome
(s) Originb Residence evolution
1 16751–1 F.pedrosoi M Nodataavailable
2 16451 F.nubica M Nodataavailable
3c 19571/19889 F.pedrosoi F 42 Maid PB Tereso´polis 20years Moderate LossofFollow-up
4c 25543 F. M 72 Gardener Portugal RiodeJaneiro 32years Severe Cure
monophora
5c 25811 F. M 65 Farmer RJ BomJardim 2years Severe Transferredtoanother
monophora healthunity
6c 28479 F.nubica F 50 Housewife ES SãoJoãode 8months Moderate Cure
Meriti
7c 32999 F. F 36 Housewife RJ RiodeJaneiro 4months Moderate Cure
monophora
8c 33420 F. M 45 Bricklayer PB RiodeJaneiro 9months Moderate Cure
monophora
9 34113/34242 F.nubica M 83 Gardener MG Itatiaia 10years Moderate LossofFollow-up
10 34904 F. M Nodataavailable
monophora
11 35962/36831 F. M 53 HousePainter PB RiodeJaneiro 3years Severe Cure
monophora
12 36134 F. M 35 Farmer MG RiodeJaneiro Nodata Moderate Cure
monophora available
13 38437 F.pedrosoi M Nodataavailable
14 38714 F.pedrosoi M Nodataavailable
15 38833 F. M 60 Bricklayer RJ RiodeJaneiro 8months Moderate Cure
monophora
16 41080 F. M 58 Bricklayer PB RiodeJaneiro 2months Moderate Cure
monophora
17 48262 F.nubica M 65 Snackbar CE RiodeJaneiro 10years Severe Cure
attendant
aM:Male,F:Female.
bStateinBrazilwherethepatientwasborn,exceptpatient4,whowasfromPortugal.CE:Ceara´,ES:Esp´ıritoSanto,MG:MinasGerais,PB:Para´ıba,RJ:RiodeJaneiro.
cCases3–9werepreviouslyreportedbyMouchalouatetal.2011.
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database(http://its.mycologylab.org).Themolecularidentificationwasconsideredvalidwhen
itpresentedmorethan98.5%ofidentity,comparedtothesequencesavailableintheISHA-
M-ITSdatabase[23].TheevolutionaryhistorywasinferredbyusingtheMaximumLikelihood
methodbasedontheTamura-Neimodel[24].
Antifungalsusceptibilitytesting
Invitroantifungalsusceptibilitytestingwasperformedaccordingtotherecommendations
proposedintheClinicalandLaboratoryStandardsInstitute(CLSI)M38-A2protocol[25]
withmodifications.AMB,FLC,ketoconazole(KTC),POS,ITC,VRC,ravuconazole(RVC),
5FC,TRB,caspofungin(CAS)(allfromSigma-AldrichChemicalCorporation,St.Louis,MO,
USA)andmicafungin(MFG)(AstellasPharmaTechCorporation,Takaokacity,Toyama,
Japan)weretested.Theinoculumwaspreparedfromaseven-dayoldPDAculture;thecells
wereharvestedinRPMImediumanddilutedtoapproximately0.4–5×104cells/mL.The
plateswereincubatedat35˚Cforfivedays[14].Theminimalinhibitoryconcentration(MIC)
forAMB,FLC,KTC,POS,ITC,VRC,RVC,5FC,andTRB;andtheminimaleffectiveconcen-
tration(MEC)forCASandMFGweredeterminedaccordingtotheCLSIM38-A2protocol
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[25].ThereferencestrainsAspergillusflavus(ATCC204304),Aspergillusfumigatus(ATCC
204305),Candidakrusei(ATCC6258)andCandidaparapsilosis(ATCC22019)wereusedfor
qualitycontrol.
Antifungalcombination
Thesusceptibilitytestwithantifungalcombinationswasperformedbythecheckerboard
method,wheretwodifferentdrugswereappliedatdifferentconcentrationsinasingle96-well
plate,sothatineachwellthereweredifferentconcentrationsoftheantifungalsincombina-
tion.TheconcentrationsassayedinthecombinationswereITC0.0075–4mg/LwithTRB
0.015–1mg/L;AMB0.0075–4mg/Lwith5FC0.06–4mg/L;CAS0.0075–4mg/LwithITC
0.06–4mg/L.Druginteraction,classifiedaccordingtothefractionalinhibitoryconcentration
index(FICI),whichdefinesthetypeofinteractionbetweentheantifungalagentsincombina-
tion,wasasfollows:synergismifFICI(cid:20)0.5;indifferenceif0.5<FICI(cid:20)4andantagonismif
FICI>4[26,27].TheFICIwasobtainedbythesumofthefractionalinhibitoryconcentrations
(FIC)orbytheformula:FICI═(A/MIC(a))+(B/MIC(b)),where:A=MICofthedrug(a)in
combination;MIC(a)=MICofdrug(a)alone;B=MICofthedrug(b)incombination;MIC
(b)=MICofdrug(b)alone[28].
Statisticalanalyses
ThegeometricmeanofMIC/MECs,MIC/MEC ,MIC/MEC andtheMIC/MECranges
50 90
werecalculatedusingtheStatisticalPackagefortheSocialSciencesv.17.0(SPSSInc,USA).
DataanalysiswasperformedintheGraphPadPrism5software.Kruskal-Wallistestwasused
tocompareMICofeachantifungaldrugbetweenthedifferentspecies.TheWilcoxonmatched
pairstestwasusedtocompareMICsoftwodifferentdrugsandtheFriedmantesttocompare
MICsofthreeormoreantifungaldrugs.Pvalueslowerthan0.05wereconsideredsignificant.
Results
Molecularidentification
TenisolateswereidentifiedasF.monophora(50%),fiveasF.pedrosoi(25%)andfiveasF.
nubica(25%).TheITSsequencingalignmentscoresofthefungalisolateshereinstudiedexhib-
ited99–100%identitycomparedwithcorrespondingITSsequencesdepositedintheISHA-
M-ITSdatabase(Fig1).TheITSsequencesobtainedduringthisstudyweredepositedin
NCBI/GenBankundertheaccessionnumbersMF616485–MF616504.
Antifungalsusceptibilitytesting
Table2depictsthesusceptibilityprofileofthestrainsincludedinthisstudy.TRB(MICrange
0.015–0.25mg/L)andVRC(MICrange0.12–0.25mg/L)weretheantifungaldrugsthat
showedthebestinvitroactivityagainsttheFonsecaeaspp.isolates.FLC(MICrange8–32mg/
L),5FC(MICrange2–32mg/L),AMB(MICrange4->16mg/L)andechinocandins(MEC
range1–8mg/L)showedhigherMIC/MECvalues.Overall,FLCwastheazolewiththepoorest
activity(P<0.0001)andamongtheechinocandins,CASwasmoreeffectivethanMFG
(P=0.003).Thesusceptibilityprofilebetweenthedifferentspecieswasverysimilarforthe
drugstested.Thefewdifferencesobservedwereasfollows:F.pedrosoipresentedMEC of1
90
mg/LforMFG,whileF.monophoraandF.nubicapresentedbothMEC of8mg/L
90
(P=0.0009)andF.monophorapresentedMIC andMIC of8mg/LforAMB,whileF.ped-
50 90
rosoiandF.nubicapresentedMIC andMIC of4mg/Lforthesamepolyenedrug(P=
50 90
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Fig1.MolecularphylogeneticanalysisusingMaximumLikelihood.Thepercentageoftreesinwhichtheassociated
taxaclusteredtogetherisshownnexttothebranches.Initialtree(s)fortheheuristicsearchwereobtained
automaticallybyapplyingNeighbor-JoiningandBioNJalgorithmstoamatrixofpairwisedistancesestimatedusing
theMaximumCompositeLikelihood(MCL)approach,andthenselectingthetopologywithsuperiorloglikelihood
value.Thetreeisdrawntoscale,withbranchlengthsmeasuredinthenumberofsubstitutionspersite.Theanalysis
involved72nucleotidesequences.Allpositionscontaininggapsandmissingdatawereeliminated.Evolutionary
analyseswereconductedinMEGA6software[22].Isolatesofthisstudyaremarkedwithafilledcircle.ITSSequences
ofFonsecaeabrasiliensis,Fonsecaeaerecta,andFonsecaeamultimorphosawereusedasoutgroups.
https://doi.org/10.1371/journal.pntd.0006675.g001
0.0447).Althoughnotsignificant(P=0.0871),F.nubicapresentedMIC of4mg/Lfor5FC,
90
whilefortheotherspeciestheMIC wastwo-dilutionshigher,thatis,16mg/L.
90
Antifungalcombination
AccordingtotheFICI,when5FCandAMBweretestedincombination,synergisticinterac-
tion(FICI(cid:20)0.5)wasobservedin3F.monophoraisolates(30%).ForthecombinationsITC/
TRBandITC/CAS,anindifferentinteraction(0.5<FICI(cid:20)4.0)wasobservedforallisolates
tested.S1Tabledepictstheresultsofthethreecombinationsofantifungaldrugsherein
studied.
Clinicalandlaboratorialcorrelation
Itwaspossibletodeterminetheprobablesiteofinfectionfor9outofthe12patientswithdoc-
umenteddata,8oftheminRiodeJaneiroand1intheEsp´ıritoSantostate.Amongthepatients
infectedinRiodeJaneiro,sixwerebyF.monophora(75%)andtwobyF.nubica(25%).
Regardingthegeographiclocation,itisimportanttonotethatthethreepatientsbornand
raisedinRiodeJaneirostatewereinfectedwithF.monophoraandallpatientsinfectedwithF.
pedrosoiwerebornoutsidetheRiodeJaneirostate.Inaddition,twocasesofF.pedrosoiwith-
outclinicaldataavailable(cases13and14,Table1)werediagnosedinpatientsfromtheBra-
zilianAmazonregion.
Table2. Minimalinhibitoryconcentrations(MIC)orminimaleffectiveconcentrations(MEC)aof11antifungaldrugsagainst20clinicalisolatesofFonsecaeaspp
obtainedfrom17differentpatients.
Antifungaldrug MIC/MECa(mg/L)
Fonsecaeaspp.(n=20) F.monophora(n=10) F.pedrosoi(n=5) F.nubica(n=5)
Range MIC b MIC GMc Range MIC MIC GM Range MIC MIC GM Range MIC MIC GM
50 90 50 90 50 90 50 90
AmphotericinB 4->16 8 8 5.77 4->16 8 8 7.46 4–8 4 4 4.59 4->16 4 4 5.28
Ketoconazole 0.06–0.50 0.25 0.50 0.19 0.12–0.25 0.25 0.25 0.17 0.06–0.50 0.25 0.25 0.19 0.12–0.50 0.25 0.25 0.25
Fluconazole 8–32 16 16 12.55 8–16 16 16 11.31 8–16 16 16 12.13 8–32 16 16 16.00
Itraconazole 0.25–1 0.50 1 0.57 0.25–1 0.50 1 0.57 0.25–1 0.50 0.50 0.44 0.25–1 0.25 0.25 0.76
Posaconazole 0.06–0.50 0.12 0.50 0.16 0.06–0.25 0.12 0.25 0.13 0.06–0.50 0.25 0.25 0.19 0.12–0.50 0.25 0.25 0.21
Ravuconazole 0.25–1 0.50 1 0.64 0.25–1 0.50 1 0.62 0.25–1 0.50 0.50 0.50 0.50–1 1 1 0.87
Voriconazole 0.12–0.25 0.12 0.25 0.14 0.12–0.25 0.12 0.25 0.13 0.12–0.25 0.12 0.12 0.14 0.12–0.25 0.25 0.25 0.19
Flucytosine 2–32 8 16 6.28 2–16 8 16 6.50 4–32 16 16 10.56 2–4 4 4 3.48
Terbinafine 0.015–0.25 0.12 0.25 0.09 0.06–0.25 0.12 0.25 0.09 0.06–0.12 0.12 0.12 0.10 0.015–0.25 0.12 0.12 0.08
Caspofungin 1->8 2 4 1.73 1–4 2 4 1.74 2–2 2 2 2.00 1->8 2 2 2.00
Micafungin 1–8 8 8 5.66 8–8 8 8 8.00 1–8 1 1 2.00 8–8 8 8 8.00
aTheminimaleffectiveconcentrations(MEC)refertocaspofunginandmicafungin.Theminimalinhibitoryconcentrations(MIC)refertotheotherantifungaldrugs.
bTheMIC andMIC valuescorrespondtotheminimalinhibitoryconcentrationoftheantifungalabletoinhibitthegrowthof50and90%ofallfungalisolates,
50 90
respectively.
cGM:Geometricalmean
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Table3. Relationshipsbetweenlaboratorialandclinicaldataofthe12patientsfollowedupatINI/Fiocruz.
Case Strain Species MICa(mg/L) Treatment(months) TotalTime
ITCb FLCc TRBd Initial Changecriterion Subsequent Outcome (months)
3 19571 F.pedrosoi 0.5 8 0.12 ITC200mg/day Slowimprovement ITC400mg/day(5) Slowimprovement 91
(8)
19889 1 16 0.12 ITC400mg/day+FLC200 Slowimprovement
mg/day(60)+cryosurgery
(13sessions)
ITC400mg/day+TRB250 Slowimprovement
mg/day(6)
ITC400mg/day+TRB500 Abandonment
mg/day(12)
4 25543 F. 0.5 16 0.12 ITC400mg/day Improvement ITC200mg/day+FLC200 Cure 22
monophora +FLC200mg/ mg/day(4)
day(18)
5 25811 F. 0.25 8 0.06 ITC400mg/day Strokenotrelatedto ITC200mg/day(1) Transferredto -
monophora (6) chromoblastomycosis otherhealthunit
6 28479 F.nubica 0.25 16 0.12 Surgery - - Cure -
7 32999 F. 0.25 8 0.06 Surgery - - Cure -
monophora
8 33420 F. 1 8 0.06 ITC200mg/day Singlelesionin Surgery Cure 5
monophora (4.5) immunosuppressed
9 34113 F.nubica 1 32 0.12 ITC200mg/day Slowimprovement Cryosurgery(2sessions) Abandonment 10
(6)
34242 1 16 0.25
11 35962 F. 0.5 16 0.25 Surgery Lesionin Cryosurgery(2sessions) Cure 17
36831 monophora 1 16 0.06 immunosuppressed
12 36134 F. 0.5 8 0.12 ITC300mg/day - - Cure 2
monophora (2)
15 38833 F. 1 16 0.12 ITC200mg/day Uncontrolleddiabetes Cryosurgery(18sessions) Cure 13
monophora (3) mellitus +surgery
16 41080 F. 0.5 16 0.12 ITC300mg/day - - Cure 1
monophora (1)
17 48262 F.nubica 1 8 0.015 ITC400mg/day Slowimprovement Cryosurgery+surgery Cure 87
+TRB250mg/
day(87)
aMIC:Minimalinhibitoryconcentration
bITC:itraconazole
cFLC:fluconazole
dTRB:terbinafine
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Ofthe12patientsfollowedupatINI,ninewerecured;threeofthemusedonlyantifungal
drugs,twounderwentsurgicalprocedures,threeusedantifungaldrugsassociatedwithphysi-
calmethods(cryosurgeryand/orsurgery)andoneunderwentsurgeryplustwosessionsof
cryosurgery(Table3).Theextensionoftreatmentconsideringonlythesixpatientswhoused
antifungaldrugs(ITCaloneorincombination)rangedfrom1to87months(median=9
months).
Regardingtheseverityofthedisease,oftheninepatientsinfectedbyF.monophora,clinical
informationwasavailableforeight,fiveofwhichwerecharacterizedbythemoderateform
andthreewiththesevereform.ModerateandsevereCBMwasalsoobservedinpatients
infectedwithF.nubica,andmoderateCBMwasobservedinthepatientinfectedbyF.pedrosoi
withavailableclinicaldata.TheonlycaseofcutaneousdisseminatedCBMwasobservedina
patientwithF.monophora.Theonlycasewithtumorlesionwasobservedinanotherpatient
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Fonsecaeaspp.identificationandsusceptibility
withF.monophora.OfthepatientsinfectedbyF.monophora,sevenwerecuredandonehad
nooutcomeinformation.Regardingthefivepatientswithnoclinicaldata,threewereinfected
withF.pedrosoi,onewithF.monophoraandonewithF.nubica.
Extracutaneousmanifestationsofthediseasewerenotobservedinanycase,regardlessof
theisolatedspecies.
Discussion
ThisworkrepresentsacaseseriesstudyofCBMforaperiodof16yearsinoneofthemainref-
erencecentersforinfectiousdiseasesinRiodeJaneiro.StudiesonCBMinotherBrazilian
regionshavebeenconductedinthisdecadebyseveralgroups[29–31],butinallthesestudies,
datafromRiodeJaneirowasmissing.Webelievethatthisstudycanbeanimportantclinic-
laboratorialcontributiontotheknowledgeofthediseaseandanupdatetotheactualBrazilian
situationofCBM.
AllfourspeciesofFonsecaeauptonowrelatedtoCBMarefoundinBrazil[5,7,32,33].F.
pedrosoiisthepredominantspeciesinSouthAmerica,followedbyF.monophora[34].Riode
Janeiro,thegeographicregionwherethisstudywasconducted,isanareaoflowoccurrenceof
CBMinBrazil[20],whichexplainsthelimitednumberofcasesduringthestudiedperiod.The
highfrequencyofF.monophorainourstudymayindicateareservoirforthisspeciesinthis
region.ThefactthatallpatientsbornandraisedinRiodeJaneirowereinfectedbyF.mono-
phorasupportsthishypothesis.
ThisisfirststudythatevaluateinvitroantifungalsusceptibilityofCBMisolatesinRiode
Janeiro.WefoundhighMICvaluesforAMBcorroboratingotherauthors[35,36].Treatment
withAMBaloneorcombinedwith5FChasnotbeenusedsincetheintroductionofITCdur-
ingthe1980s.Thefrequentoccurrenceofnephrotoxicity,duetothedrugcharacteristicsand
prolongedtreatment[37,38],togetherwiththereactivationoftheinfectionwiththedrugdis-
continuation[10,34]arefactorsthathindertheuseofAMBforCBMtherapy.
TheFonsecaeastrainsincludedinthisstudypresentedlowMICvaluesforKTC,similarto
otherstudies[14,35,39].Thisdrugwasthefirstsystemicimidazoleavailable,butitisrarely
usedduetoserioushepaticreactions,aswellasseveredruginteractions[40,41].Nowadays,
ITCisconsideredthemostcommonlyuseddrugforCBMtreatment[10].Inthisstudy,the
MICvaluesforthisdrugindicatesusceptibilityoftheisolatestotheantifungalagent[42],and
theschemesusingITCaloneorassociatedwithotherantifungalorsurgicalmodalitieswas
abletolead7patientstocure.
POS,VRCandRVCrepresentthenewgenerationoftriazoleswithabroadspectrumof
activityandafavourablepharmacokineticprofile[43].POSisknowntohaveabetterinvitro
activitythanITCagainstclinicalFonsecaeaisolates[44]inaccordancewiththeresultsofthis
study.VRChasgoodinvitroactivityagainstCBMagents,includingFonsecaeaspp.[45].How-
ever,inadditiontoitshighcost[46],theprescriptionofVRCshouldbedonewithcaution,
sinceitpresentsriskofphototoxicityandcutaneouscarcinomainprolongedperiodsoftreat-
ment[47].Tothebestofourknowledge,thisisthefirststudyonFonsecaeaspp.susceptibility
toRVCusingtheCLSIM38-A2protocol.Gonza´lezetal.[48]reportedtheinvitroactivityof
RVCagainstisolatesofF.pedrosoiwithMICsrangingbetween0.125–0.5mg/LusingM38-A
CLSIprotocol.Despitetheuseofanotherprotocol,thisworkreportsMICvalues(cid:20)1mg/Lfor
thesamedrugagainstclinicalFonsecaeaisolates.However,basedonourresultsthatshowed
POSandVRCwithabetterinvitroprofilethanRVC,thefirsttwoazolesshouldbeconsidered
inthetreatmentofCBM,insteadofRVC.
TheMICvaluesfoundfor5FCandFLCwerecompatiblewithotherstudies,showingthat
thesedrugsareineffectiveinvitroagainstFonsecaeaspp.,discouragingtheiruseinCBM
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Fonsecaeaspp.identificationandsusceptibility
treatment[35,39,44].Asfor5FC,itsuseinthemid-1960smarkedthebeginningofchemother-
apyapproachesforCBM[45].However,itwaslaterobservedthatF.pedrosoiisabletodevelop
invitroresistanceto5FC[45,49–53].Althoughnotprohibited,thedrugisnotregisteredinthe
Brazilianregulatoryagency(ANVISA)andisnotcommercializedontheBrazilianmarket
[54],becausethereisnopharmaceuticalindustrythatmanufacturesthisantifungaldrugin
ourcountry,whichleadstoaneedtoitsimportbytertiaryhospitals.
SeveralstudieshavereportedthesusceptibilityofFonsecaeaspp.toTRB,revealingits
potentactionagainstvariousfilamentousfungiandinthetreatmentofCBM,demonstrating
upto80%ofcurerate[14,39,55,56].Ourresultswereconsistentwiththosestudiesshowing
lowMICvaluesforthisdrug.Inaddition,TRBshowsapotentantifibroticeffectinrecent
lesions[57].ThisdrughaslittleaffinityforthecytochromeP450enzymesystem,resultingin
lessinteractionwithothersubstances[58]andinageneralway,iswell-tolerated,indicatingan
effectiveoptionforthetreatmentofCBM.
EchinocandinshavealimitedroleinthetreatmentofCBMduetohighMECvaluesforF.
monophoraandF.nubica,asobservedinotherstudies[44,59].IsolatesofF.pedrosoipresented
abetterinvitroresponsetoMFG.Nevertheless,duetothelownumberofanalysedisolates,it
issuggestedthatfurtherstudieswillassesswhetherthisechinocandinisspecificallyeffective
againstF.pedrosoi.
Accordingtosomestudies,inparticularCBMcases,thebesttherapeuticstrategywouldbe
theassociationoftwoantifungalsbasedontheresultsofprevioussusceptibilitytests[17].Someof
thesuggestedcombinationsareAMBand5FC[45,60]orITCandTRB[61].Ourworkshoweda
synergismbetweenAMBand5FCinthreeF.monophoraisolates.Thiscombinationhastwodis-
tinctmechanismsenhancingtheantifungalaction:AMBbindstoergosterolofthefungusmem-
braneformingporesand5FCactsinhibitingthesynthesisofnucleicacids.Thiscombinationis
widelyusedincasesofcryptococcalmeningitisbecauseithasamoreeffectivepenetrationinto
theSNC[62].Inthepast,thecombinationAMB/5FChadbeenusedforCBMtreatment
[17,63,64],butnowadaysitisnolongerconsideredduetotheadversesideeffects[10].However,
webelievethatthisassociationcouldbebeneficialinseverecasesofCBM,especiallythosewith
braininvolvement.Thesynergismfoundfor100%oftheisolatesofPhialophoraverrucosabyLi
etal.[18]encouragedtheuseofITCandCAScombinationinourstudy.Asinglestudy[65]
foundsynergismforisolatesofF.monophora.AnindifferentinteractionwasobservedinallFon-
secaeaisolatesofthisstudy,whichiscompatiblewithmoststudies[15,17,61].
TherearefewstudiescomparinginvitrosusceptibilityamongclinicalFonsecaeaisolates.
Najafzadehetal.[44]foundnosignificantdifferencesamongspeciesintheactivityofeight
antifungals(AMB,FLC,ITC,VRC,POS,CAS,anidulafunginandisavuconazole)againstF.
pedrosoi,F.monophoraandF.nubica.However,inthisstudy,F.monophorashowedhigher
MICvaluesthanF.nubicaforAMB(P=0.0447),andthespeciesF.monophoraandF.nubica
(P=0.0009)hadhigherMECvaluesforMFGwhencomparedtoF.pedrosoi.
CBMisknownasadifficult-to-treatdisease,thereisnostandarddrugofchoiceand
relapsesarefrequent[10,34,45,57,66].Therearemanyfactorsthatcaninfluencethepatient
outcomethatcanberelatedtothehostorthefungalspecies.Thehostimmuneresponse,local
lymphedema,andfibrosisbecomeabarrierforaproperdrugbioavailabilityatthesiteofinfec-
tion.Inaddition,muriformcellsareheavilypigmentedandrepresentaresistantfungalform
againstantimicrobialcompounds[67].Inthesamevein,thereisnoclearcorrelationbetween
invitrosusceptibilityandclinicalpractice[45].Ingeneral,MICvalues(cid:20)1mg/Lusuallyindi-
cateapotentialsusceptibilityofmostdrugsusedinthetreatmentofinfectionsbydematiac-
eousfungi[14,42],asoccurredinthisstudy.Itisalsopossiblethatthebrothmicrodilutiontest
isnotthebestmethodtoguidetherapeuticmanagementinCBM.Infact,inacorrelation
studybetweendifferentantifungalsusceptibilitymethodologiesandclinicaloutcomeof
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