AnBrasDermatol.2020;95(4):459---468
Anais
Brasileiros
de
Dermatologia
www.anaisdedermatologia.org.brINVESTIGATION
Identification
of
Leishmania
species
by
high-resolution
DNA
dissociation
in
cases
of
American
cutaneous
leishmaniasis
夽,夽夽
John
Verrinder
Veasey
a,∗,
Ricardo
Andrade
Zampieri
b,
Rute
Facchini
Lellis
c,
Thaís
Helena
Proenc
¸a
de
Freitas
a,
Lucile
Maria
Floeter
Winter
baDermatologyClinic,FaculdadedeCiênciasMédicas,HospitaldaSantaCasadeMisericórdiadeSãoPaulo,SantaCasade
MisericórdiadeSãoPaulo,SãoPaulo,SP,Brazil
bDepartmentofPhysiology,InstitutodeBiociências,UniversidadedeSãoPaulo,SãoPaulo,SP,Brazil
cDepartmentofPathologicalAnatomy,HospitaldaSantaCasadeMisericórdiadeSãoPaulo,SãoPaulo,SP,Brazil
Received8September2019;accepted4February2020 Availableonline15May2020
KEYWORDS Diagnosis; Histology; Leishmania braziliensis; Leishmania guyanensis; Leishmaniainfantum; Leishmaniasis; Leishmaniasis, cutaneous; Leishmaniasis, mucocutaneous; Polymerasechain reaction Abstract
Background: Americancutaneousleishmaniasisisaninfectiousdermatosiscausedbyprotozoa ofthegenusLeishmania,whichcomprisesabroadspectrumofclinicalmanifestationsdepending ontheparasitespeciesinvolvedintheinfectionsandtheimmunogeneticresponseofthehost. TheuseoftechniquesforamplificationoftheparasitesDNAbasedonpolymerasechainreaction polymerasechainreactionandtherecentapplicationofcombinedtechniques,suchas high-resolutionDNAdissociation,havebeendescribedasaviablealternativeforthedetectionand identificationofLeishmaniaspp.inbiologicalsamples.
Objectives: To identify the Leishmania species using the polymerase chain reaction high-resolutionDNAdissociationtechniqueinskinbiopsiesofhospital-treatedpatients,andcompare withresultsobtainedbyothermolecularidentificationtechniques.
Methods: AretrospectivestudyassessingpatientswithsuspectedAmericancutaneous leishma-niasisseenatahospitalinSãoPaulo/Brazilwasconducted.Theparaffinblocksof22patients were analyzedbypolymerasechainreactionhigh-resolution DNAdissociationtoconfirmthe diagnosisandidentifythespecies.
Results: Ofthe22patientswithsuspectedAmericancutaneousleishmaniasis,theparasitewas identifiedin14,comprisingfivecases(35.6%)ofinfectionbyL.amazonensis,four(28.5%)byL. braziliensis,two(14.4%)byL.amazonensis+L.infantumchagasi,two(14.4%)byL.guyanensis,
andone(7.1%)byLeishmaniainfantumchagasi.Inoneofthesamples,inwhichthepresence
夽 Howtocitethisarticle:VeaseyJV,ZampieriRA,LellisRF,FreitasTHP,WinterLMF.IdentificationofLeishmaniaspeciesbyhigh-resolution
DNAdissociationincasesofAmericancutaneousleishmaniasis.AnBrasDermatol.2020;95:459---68.
夽夽StudyconductedattheDermatologyClinic,HospitaldaSantaCasadeMisericórdiadeSãoPaulo,SãoPaulo,SP,Brazil. ∗Correspondingauthor.
E-mail:johnveasey@uol.com.br(J.V.Veasey).
https://doi.org/10.1016/j.abd.2020.02.003
0365-0596/©2020SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCC BYlicense(http://creativecommons.org/licenses/by/4.0/).
ofamastigoteswasconfirmedonhistopathologicalexamination,thepolymerasechainreaction high-resolutionDNAdissociationtechniquefailedtodetecttheDNAoftheparasite.
Studylimitations: Theretrospectivenatureofthestudyandsmallnumberofpatients.
Conclusions: ThemethoddetectedandidentifiedLeishmaniaspeciesinparaffin-embeddedskin biopsieswithasensitivityof96.4%andcouldberoutinelyusedinthepublichealthsystem. ©2020SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).
Introduction
Leishmaniasis is a chronic infectious-parasitic disease causedby severalspeciesof protozoa ofthe genus Leish-mania. In Brazil, the species involved in infections with cutaneousmanifestationsaremainlyL.(V.)braziliensisand
L.(L.)amazonensis,followedbyL.(V.)guyanensis,L.(V.) lainsoni,L.(V.)shawi,L.(V.)naiffiandL.(V.)linderbergi,
with lesser incidence.1---3 More recently, isolated cases of
Americancutaneous leishmaniasis (ACL) caused by Leish-mania(L.)infantumchagasihavebeenreportedinpatients withorwithoutHIVco-infectioninthecentral-westernand southwesternregionsofthecountry.4,5
Ontheskin,themostcommoninitialclinicalpresentation of ACL is a framed ulcer at the site of parasite inocula-tion.Inthisform,itispossibletofindtheparasitethrough directsearchofthesmearofthebaseofrecentulcers. How-ever, there are other forms of cutaneous manifestations, suchasverrucous,papular,nodular,sarcoid,andinfiltrated andvegetative plaques,inwhichthepresenceof the par-asiteinthetissueisrareandhistopathologyisnonspecific, rangingfrominflammatorylymphoplasmacytic infiltrateto granulomaswithor withoutnecrosis, thus makingthe dif-ferentialdiagnosiswithotherinfectious,inflammatory,and even neoplastic diseases difficult.6---9 Tissue
immunohisto-chemistry with anti-Leishmania antibodies contributes to thediagnosis, butitssensitivityis around66% ofcases.A positiveMontenegro skin test (>5mm) canhelp, asit has ahigh predictivevalue. However,thistest hassome limi-tations:itisnegativeinthefirstmonthsofinfectionandin anergicpatients,andremainspositiveevenafterthedisease iscured.1,6
Therefore, leishmaniasis can be diagnosed through severalmethods,allofwhichpresentadvantagesand limi-tations.However,thereisnogoldstandarddiagnostictest. Microscopyisuseful inendemic areas,butit haslow sen-sitivityanddepends onthe assessment ofan experienced pathologist;furthermore,itdoes not allowthe identifica-tionof theparasite species. Serologicaltestscan provide aquickdiagnosis, areeasytointerpret,andarerelatively inexpensive.However,theirsensitivityandspecificityvary indifferentendemicregions;moreover,itisnotpossibleto identifythespeciesthatcausedthedisease.1,3,6
Different methods with molecular approaches that explorespecificcharacteristics ofparasiteDNAhave been tested for the diagnosis of leishmaniasis. Initially, the analysisofthepatternsof fragmentgenerated by restric-tionenzymes,restrictionfragmentslength polymorphisms (RFLPs),wastheidentificationmethodforLeishmaniaand othertrypanosomatids usingDNAasatarget.10---12 Another
typeof genotype analysis explored is the construction of
probesusedinmolecularhybridizationtests.Thistechnique wasusedbothindifferentiationstudiesofstronglyrelated speciesandforthediagnosisofinfections.13,14 Therandom
amplifiedpolymorphicDNA(RAPD)technique,which ampli-fies fragments with random primers through polymerase chainreaction(PCR),wasalsoatechniqueadoptedto ana-lyze specificpatternscapable ofdifferentiatingorganisms oftheLeishmaniagenus.15,16 Duetonumerousadvantages,
PCR-basedtestsarethemaintoolsusedinthedetectionand identification of Leishmania.This methodology allowsthe useofminuteamountsofbiologicalmaterial,aswellasthe specificdiscriminationoforganismsthroughtheanalysisof targetpolymorphisms.DifferentPCR-basedtechniqueshave beenusedintheidentificationofLeishmania,duetocertain advantages, suchashigh speedand sensitivity/specificity, when compared with conventional techniques based on microscopyandcellculture.17
Several DNA sequences have been used as targets for the specific identification of Leishmania, such as kineto-plastDNA(kDNA),18ribosomalDNA(rDNA),13,14andtheg6pd
gene,amongother targets.19,20 kDNAmakesupabout 15%
of theparasite’s DNAand isformed by anetwork of con-catenatedcircularmolecules(maxicirclesandminicircles). Per organism, between 10,000 and 20,000 minicircles are found,rangingfrom0.5to2.5kbinsize,andapproximately 50maxicirclesfrom20to35kb,whichencodemitochondrial proteinsandribosomalRNAoftheorganelle.21,22Amongthe
targets described in the scientific literature,kDNA is one ofthemost usedin moleculardiagnosis.The highnumber of copies of thetarget per genome ensures a high sensi-tivity, representingthe main advantageof the technique. However,theaccuracyoftheassayscanbeaffectedbythe considerableheterogeneityofkDNAsequences.23
Thecharacterizationofribosomalcistronsfromdifferent trypanosomatidsallowedthecreationofprobescapableof identifyingsomeorganisms.Atfirst.theexistenceof poly-morphism in the spacer sequences of ribosomal genes in
Trypanosomaspp. wasdemonstrated.11 The description of
ribosomalcistronsfromsixspeciesofLeishmaniaevidenced acharacteristicpatternofthesespecies,punctuatedbythe existenceofarestrictionsiteidentifiedbytheenzymePvuII. Thissite,presentinthecodingsequenceofthe18SrRNAor SSU,was exploredin the construction of oligonucleotides capableofidentifyingthegenus.13Basedonpolymorphisms
in the sequence ofthat same gene,probes that couldbe usedtoidentifydifferentgroups ofLeishmaniahavebeen described.14 Ampliconsequencinginthisgenesegmenthas
been used to identify Leishmania. Two mutation points, foundatpositions1714and1721oftheSSUrDNAsequence, areabletodiscriminate(L.)amazonensisandL.(Viannia)
IdentificationofLeishmaniaspbyhigh-resolutionDNAdissociation 461 polymorphic characteristics of theSSU rDNAwere carried
outwithdifferentapproaches,focusingonclinical, epidemi-ological,andecologicalaspects,amongothers.24---30
Among the most used molecular identification tech-niques, the analysis by isoenzyme (zymodemes) elec-trophoresis is noteworthy. Glucose-6-phosphate dehydro-genase (G6PD) is one of the main enzymes used in the identification of Leishmania by zymodeme analysis. The polymorphisminthestructureoftheseproteinsisa reflec-tion of polymorphisms at the gene level, which allowed the use of molecular markers capable of discriminating andquantifyingLeishmaniaby conventionalandreal-time PCR.19,20
Inadditiontothese,otherDNAtargetsdescribedinthe literature, such as the internal transcribed spacers (ITS) presentintherDNAcistron,theheatshockprotein(Hsp70) gene, the cysteine proteinase (cpb) gene, among others, havebeenstudiedinLeishmaniaidentificationtests,based onthepolymorphiccharacteristicsofDNA.17
Thesimultaneoususeofdifferenttargetshasalsobeen usedinconventionalPCRreactions,aimingtodiscriminate thepathogensofleishmaniasis.Thecombinationof oligonu-cleotidesdesignedfromthesequenceofthegeneencoding themini-exonaddedbytrans-splicingtoalltrypanosomatid messengerRNAs(SLRNA)hasbeendescribedascapableof discriminating, in a singlePCR reaction, the species that causethevisceralformofthediseaseinonegroupandthe speciesresponsibleforthecutaneousformofthediseasein twoother groups,which aredistinctbecausetheyinclude speciesofthesubgeneraL.(Leishmania)orL.(Viannia).31,32
Recently, an important polymorphism wasdescribed in thehsp70gene,associatedwiththeanalysisthrough high-resolution melting (HRM) analyses, a method capable of detectingdifferencesinthenucleotidecompositionof spe-cificPCRproductsthatallowsdiscriminatingallspeciesthat circulateinBrazilandthemostimportantspeciesfoundin theMediterranean,India, andNorthAfrica.33 The
applica-tionofthistechniquewasshowntobeusefulinthediagnosis andidentificationofLeishmaniaindifferentsamplesfrom patients(freshbiopsiesandinparaffin-embeddedmaterial) andfromreservoiranimals,suchasdogs,andphlebotomine hosts.
Diagnostic teststhat involve thedetection of parasitic nucleicacids, mainlythosebasedonspecificDNA amplifi-cation such asPCR, aredescribed as highlysensitive and specificoptions,withthepotentialtoquantifyandidentify theinfectiousspecies.8,34---36HRMDNAanalysesperformedat
theendofreal-timePCRreactionsareabletodetect ther-modynamicdifferencesintheamplicondissociationprofile bydeterminingspecific signaturesasa resultof small dif-ferencesinthenucleotidecompositionbetweenthespecies. ThePCR-HRMtechniquehasbeenusedtoidentifynotonly
Leishmaniaspecies,butalsootherinfectiousagents,andhas beendescribedasarelativelycheap,fast,androbusttool forthedetectionanddiscriminationofLeishmaniaspecies inallendemicregionsoftheworld.34,37,38
Methods
Thisretrospectivestudywascarriedoutatthedermatology clinic ofa tertiaryhospitalin SãoPaulo, Brazil,analyzing
thedata of 22 patients diagnosedwith ACL treated from January2005toDecember2018.This study wasapproved bytheethicscommittee(CAAE:48719115.0.0000.5479),and clinical,epidemiological,andcomplementaryexamresults wereobtainedfrommedicalrecordanalysis.
Leishmania spp. were identified through PCR-HRM, as previouslydescribed by this group,33 froma purifiedDNA
sample from paraffin-embedded tissueobtained fromthe hospital’spathologylaboratory.Samplesweresentinablind testcriterion,evenin caseswheremorethan onesample was obtained from the same patient. Paraffin-embedded biopsies underwent a paraffin removal process with suc-cessive heated xylol baths, followed by ethanol baths, and subsequent hydration of the sample. The DNA from thebiopsy sampleswas then extracted andpurifiedusing theDNeasy Blood& Tissue kit (Qiagen), according tothe protocolsuggestedbythemanufacturer.Afirststage,called pre-amplification, was performed with primers based on theLeishmaniahsp70genesequencethatamplifies,by con-ventionalPCR,asegmentofDNAthatcontainspolymorphic sites thatcan be analyzed byHRM. The pre-amplification products were then subjected toreal-time PCR reaction, inwhichthreedistinctregionsof thegenewereamplified independentlyusingtheMeltDocMasterMixkit(Life Tech-nologies).HRManalyseswereperformedattheendofeach real-timePCRreaction;theDNAdissociationprofileswere generatedbytheHighResolutionMeltingsoftwarev.3.0.1 (Life Technologies) and compared to profiles generated from DNA samples from reference strains of Leishmania
spp. (L. (L.) infantum chagasi (MCER/BR/1981/M6445),
L. (L.) major MHOM/IL/81/Friedlin), L. (L.) amazonensis (MHOM/BR/1973/M2269), L. (L.) mexicana (MNYC/BZ/62/M379), L. (L.) lain-soni (MHOM/BR/81/M6426), L. (V.) braziliensis
(MHOM/BR/1975/M2903), L. (V.) guyanen-sis (MHOM/BR/1975/M4147), L. (V.) naiffi
(MDAS/BR/1979/M5533), and L. (V.) shawi
(MCEB/BR/84/M8408).
The results obtained by PCR-HRM were compared to methods already described in the literature, such as sequencingofasegmentoftheSSUrDNAgene,PCR-kDNA, andrestriction fragment lengthpolymorphisms (RFLPs)of thehsp70gene.
Results
Duringthestudy period,22 patients withsuspected diag-nosisofACLweretreated, ofwhom12weremaleand10 werefemale.Agerangedfrom13to78years,withamean of45.72±18.41yearsandamedianof43.5years.The dura-tionoftheskinlesionrangedfromoneto108months,witha meanof28.31±35.49monthsandamedianoftenmonths. Themostcommonclinicalpresentationwasskinulcers(13 patients), followed by mucosal ulcers (four patients with nasallesionsandtwowithorallesions),papularskinlesions (twopatients), andonenodularskinlesion inonepatient (Figs.1and2).
Theepidemiological,clinical,andhistopathological char-acteristics,aswellastheresultsofthedirectsearchofthe parasiteintheinjuredtissueofthe22patientsincludedin thestudy,arespecifiedintable1.
V
easey
JV
et
al.
Table1 Clinical,epidemiological,andlaboratorycharacteristicsofthecasesstudied.
Samplecharacteristics Lesioncharacteristics Evidenceofagent Complementarytests
Patient Origin (state) Clinical pre-sentation Tissue/location Time-lengthof lesion(months) Direct exam Histopa-thology PCRSSU PCR SSU hsp70 hsp70
IHQ kDNA Seq. RFLP HRM
1 MG Ulcer Skin/limb 4 + NP + + NP L.amazonensis L.
amazonensis
2 PR Ulcer Skin/limb 4 + + + + NP L.amazonensis NP
3 BA Ulcer Nasalmucosa 72 − NP + + NP L.amazonensis L.
amazonensis
4 BA Papule Skin/Nose 24 + − + + L.amazonensis L.amazonensis NP
5 SE Nodules Skin/limb 108 − NP + + L.amazonensis L.amazonensis L.
amazonensis
6 BA Ulcer Skin/Ear 6 + NP + + NP NP L.
braziliensis
7 MG Ulcer Oralmucosa 3 NP + + + L.(Viannia) NP L.
braziliensis 8 MG Papule Skin/Periocular 18 + + + + NP NP L. braziliensis 9 BA Ulcer Skin/limb 12 + NP + NP NP NP L. braziliensis 10 BA Ulcerated plaque Skin/limb 12 NP − + − NP NP L. guyanensis 11 AL Ulcer Skin/limb 96 − NP + − NP NP L. guyanensis
12 BA Ulcer Skin/limb 2 + NP + + L.i.chagasi L.i.chagasi L.i.chagasi
13a RN Ulcerated plaque Skin/limb 7 NP + + + L. amazonensis/ L.i.chagasi L. amazonensis/ L.i.chagasi L. amazo-nensis/L.i. chagasi
14& BA Ulcer Skin/Ear 5 + NP + + L.
amazonensis/ L.i.chagasi L. amazonensis/ L.i.chagasi L. amazo-nensis/L.i. chagasi 15 SP Ulcer Skin/limb 5 NP − − NP NP NP NP
16 BA Ulcer Oralmucosa 12 − NP − NP NP NP NP
17 BA Ulcer Skin/limb 8 − NP − NP NP NP NP
18 BA Ulcer Nasalmucosa 60 − NP − NP NP NP NP
19 MG Ulcer Skin/limb 4 − NP − NP NP NP NP
20 BA Ulcer Nasalmucosa 60 − NP − NP NP NP NP
21 SP Ulcer Skin/limb 5 NP + − NP NP NP NP
22 SP Ulcer Nasalmucosa 96 − NP − NP NP NP NP
NP,notperformed,andmoleculartestsappliedtotwodifferentsamples,oneindicatingthepresenceofL.amazonensisandtheotherofL.infantumchagasi.RFLM,restrictionfragment
lengthpolymorphisms;PCR,polymerasechainreaction;HRM,high-resolutionmelting.
IdentificationofLeishmaniaspbyhigh-resolutionDNAdissociation 463
Figure1 Clinicalexamplesofpatientswithclinicallesionsforwhichpolymerasechainreactionhigh-resolutionmelting(PCR-HRM) assessmentwasabletodeterminethespeciesofLeishmania.A,Patient11(L.infantumchagasi);B,Patient13(L.amazonensis+L. infantumchagasi);C,Patient14(L.amazonensis+L.infantumchagasi);D,Patient7(L.brasiliensis);E,Patient3(L.amazonensis);
F,Patient5(L.amazonensis).(Figurenotvisible).
Figure2 Clinicalimagesoflesionsforwhichpolymerasechainreactionhigh-resolutionmelting(PCR-HRM)assessmentwasunable todeterminethespeciesofLeishmania.A,Patient17,ulcerintheupperlimb;B,Patient21,ulcerinthelowerlimbwhosebiopsy showedthepresenceofamastigotesinthetissue;C,Patient21,ulcerinthelowerlimb.
For diagnosis, smears were performed on the lesions of 17 patients (eight positive) and skin biopsies in all 22 patients (14 positive); all samples were assessed by routinestaining(hematoxylin---eosin),andan immunohisto-chemicalstudyfordetectionofLeishmaniawasconducted in eight patients. In ten patients, more than one biopsy was performed in order to diagnose the disease. In all, 38 blocks were analyzed by histology, PCR-SSU,
PCR-kDNA, SSU-Seq, PCR hsp70-RFLP, and PCR hsp70-HRM (Table1).
Elevenpatientspresentedamastigotesonsmearand/or histologicalexamination,andonlytwopatientswere posi-tiveinbothtests.In11patients,noamastigotesformswere observedineithertest(Table1).
Of22patientswithsuspectedACLtreatedintheperiod, 14 had the parasite species identified by SSU-Seq, PCR
hsp70-RFLP,or PCR hsp70-HRM, withfive cases(35.6%) of infectionsbyL.amazonensis,four(28.5%)byL. brazilien-sis, two(14.4%) by L.amazonensis+L. infantumchagasi,
two(14.4%)byL.guyanensis,andone(7.1%)byLeishmania infantumchagasi.Itisinterestingtonotethatthe sequenc-ingof the SSUfragment does notallow the identification of speciesof the sub-genus L.(Viannia), but therewasa coincidenceoftheidentifiedspecies,eitherwithinthis sub-genus,orincaseswheretherewasadoubleidentification ofL.amazonensis+L.infantumchagasi(Table1).
From the total of samples from 11 patients in which amastigotesweredetectedthroughdirectresearchonthe smearofthelesionand/orhistopathologicalexaminationof askinfragment,itwaspossibletoidentifythespeciesofthe parasiteintenofthem,andonlyonewasnegativebythe molecularmethodsemployed.Incontrast,ofthe11 nega-tivesamplesbysmearand/orhistopathologicalexams,the protozoanwasidentifiedinfour(Table2).Itisnoteworthy thatoneofthesefourpatients(patient5)presented nodu-larskinlesionsofdifficultclinicalandpathologicaldiagnosis for108months(Fig.1andTable1).
PCR-SSU was positive for 14 of the patients, and the specieswereidentifiedin all ofthem, either bySSU-Seq, PCR hsp70-RFLP, or PCR hsp70-HRM. Moreover, PCR-kDNA wasperformedforthese14patients,resultingin12positive samples.
Discussion
Eventoday,itisstilldifficulttomakeanaccuratediagnosis inpatientswithsuspectedcutaneousand/ormucous leish-maniasiswhennoparasitesarefoundinthelesions.Several complementaryexamsare requested toattemptto reach adiagnosisandexcludeotherdiseasessuchas mycobacte-riosis,sarcoidosis, lymphomas, and deepmycoses, among others.1,6,8 Thus,itisimportanttohaveaquickdiagnostic
test,withhighspecificityandsensitivity,toavoidexcessive testsandconsultations,aswellastheneedfortherapeutic testswithundesirablesideeffects.Inrecentyears,theuse ofPCRtodeterminetheparasitehasevolvedconsiderably; theuseofthistechniquetogetherwithHRM,targetingthe
Leishmaniahsp70gene,allowsthelaboratoryidentification ofallspecieswithhighspecificityandsensitivity.34,37,38
Inaddition,the presentresults highlight thefactthat, eveninthecaseofapublictertiaryhospitalofreferencein thelargestcityinBrazil,accesstosimplediagnostictests is notalways available. Of the 22 cases studied, for only eightcases(36.3%)wasitpossibletoperforman immuno-histochemicalstudyforLeishmania.Adirectsearchforthe parasiteinlesionsmearstainedwithGiemsawasthemost accessibletest,performedin17patients(77.2%).
Thepresenceoftheparasiteisdemonstratedby detect-ingitsDNAinpotentiallysensitive tests,sincepositivityis the result of exponential target amplification by PCR. In fact,the PCR testsappliedto14 of the22 patientswere positiveintheSSUassessment(63.64%)and12were posi-tiveinthekDNAassessment(54.55%),andthesewerealso positivefor PCR-SSU.However,thesetargets donotallow discriminationoftheparasitespecies.
Therewasagreementintheidentificationofthespecies throughSSU-Seq,hsp70-RFLP,andhsp70-HRM;however,the
firsttwoarelaborioustests,whichrequirethemanipulation of thePCRproduct,in additiontoatrainedtechnicianto performandanalyzetheresults.
Ina patientin whomthe presence of amastigoteswas detected by histopathology, the PCR-SSU and hsp70-HRM tests were negative. A possible failure in detecting the parasite’sDNAcanhavebiologicalorphysicochemical expla-nations.The firstisbased onthefactthat cutaneousand mucocutaneouslesionscausedmainlybyL.braziliensishave asacommoncharacteristicthescarcityorabsenceof para-sites, hinderinglaboratory diagnosis. The second is based on the fact that biological samples that undergofixation for histologicalanalysisaresubjecttoDNAdegradationor polymerase enzyme inhibition, mainlydue toexposure to formalin or reagents used in sample preparation.1,3,6,38 In
the case of the patient withevidence of amastigotes by immunohistochemistry,thesamplewassubjectedtoahost DNA amplification test by conventional PCR, and the tar-getwasamplified.Thistestisroutinelyperformedtoassess thequalityoftheDNAandtheinterferenceofthepossible presenceofpolymeraseinhibitorsinthesamples.Thisresult suggests,then,thattheabsenceorextremescarcityof par-asitesinthissamplecanbeaconvincingexplanationforthe negativityoftheparasitedetectionandidentificationtest. The identification of L. amazonensis in lesions of five patientsfromBahia(two),Sergipe(one),Paraná(one),and MinasGerais(one)initiallysurprisedtheauthors,asinthese statesL.braziliensisisthespeciesmostcommonlyrelated tocasesof muco-cutaneous leishmaniasis.L.amazonensis
isclassicallyconsideredrestrictedtoAmazonianareas;itis transmittedthroughwildvectorsandrodentsarethemain reservoirs.However,changesintheepidemiologicalprofile ofthisspecieshaverecentlybeendescribed.Inthestateof Bahia,where thepresenceofL.braziliensisis more com-mon,thepresenceof L.amazonensiscausing skinlesions, andmorerarely,mucosallesions,wasdemonstratedin1988 throughpanelsofmonoclonalantibodies.39Inotherregions
suchasMatoGrossodoSul,SantaCatarina,Paraná,andRio deJaneiro,isolatedhumancasesofcutaneousleishmaniasis byL.amazonensishavebeen identified.40 This specieshas
alsobeenidentifiedindogsinthestateofSãoPaulo41,42and
morerecently inendemic areasin southeasternBrazil43,44
Thus, the authors believe that an unknown proportion of cases ofcutaneous leishmaniasisoutside theAmazon may be caused by L. amazonensis, and that further epidemi-ological studies are needed since evidenceindicates that thetransmissionprofileofcutaneousleishmaniasismaybe changing.
ThefindingofamucosallesioncausedbyL. amazonen-sis was also unexpected. Rarely have human cases been describedintheliterature,despitehavingbeenreproduced experimentallyin rats.45 The identification ofL.
guyanen-sis in two patients from Northeastern Brazil was another unexpected finding, since this species has a transmission cyclethatisapparentlylimitedtotheNorthernregion.46,47
However,itisnoteworthythatthegeographicalorigin men-tionedinthemedicalrecordsdoesnotindicatewherethe patientswereinfected,sincetheymayhavemovedacross the different regions of the country, and more accurate information is often scarce in retrospective studies. Con-versely, the occurrence of these species outside endemic areas warns to the possibility of a new epidemiology of
IdentificationofLeishmaniaspbyhigh-resolutionDNAdissociation 465
Table2 Resultsofevidenceoftheparasitebydirectexaminationonlesionsmearand/orhistopathologicalexaminationand identificationofLeishmaniaspp.throughPCR-HRM,correlatedwiththeclinicalpresentation.
Muco-cutaneouslesion Parasitepresence(DE/HE) Absenceofparasite(DE/HE) Total PositivePCR-HRM NegativePCR-HRM PositivePCR-HRM NegativePCR-HRM
Skin Skinulcer 7 1 2 3 13
Papules 2 0 0 0 2
Nodules 0 0 1 0 1
Mucosa Oralulcer 1 0 0 1 2
Nasalulcer 0 0 1 3 4
Total 10 1 4 7 22
DE,directexamination;HE,histopathologicalexamination;PCR,polymerasechainreaction;HRM,high-resolutionmelting.
thedisease.Itisunknownwhethertheirgeographic distri-butionhas expanded or whetherthese specieshave been distributedinthenationalterritoryforsometime,and envi-ronmentalchangesareincreasingtheexposureof animals (suchasdogs)andhumanstoparasitesinareaswherethey hadnot been described. Newmethods for identifying the speciesofparasitesallowtheirgeographicaldistributionto bereviewed,contributingtotheunderstandingofthe epi-demiologyofthedisease.
The identificationofacase withL.infantumchagasiis considered a rarity in ACL in Brazil, since this species is generallyrelatedtovisceralpresentations.4,5Isolatedcases
have been described in association withHIV infection,in young patients, and evenone in an urban areaof Rio de Janeiro,withoutanyassociatedcomorbidity,5aswasoneof
thepresentcases.Albeitrare,mixedinfectionscausedby more thanone speciesof Leishmania,or even trypanoso-matids of other genera parasitizing the same host, have beendescribedintheliterature.48 In2002,Martinezetal.
describedthefirstcaseofdetectionofL.amazonensisand
L.infantumchagasiinthesamelesioninaBolivianpatient, asituationsimilartothatidentifiedinoneof thepresent cases.49 Thesetwospecieswerefoundinanotherpatient,
but in different samples, which does not allow inferring whethertheyweretwodifferenttemporalinfections or a doubleinitialinfection.
Thisstudyhaslimitationsduetoitsretrospectivenature and smallsample size; however, by expanding the obser-vations to more cases, it may point to new paradigms in the identification of Leishmania species relatedto the different geographical areas and the forms of the dis-ease. DNA samples from patients with a negative final diagnosisforleishmaniasisshouldalsobeconsidered infor-mative. There was an agreement between the negative results and the absence of amastigotes in 19 paraffin-embedded tissue blocks from eight patients. In practical terms,areliablenegativeresultcanpreventthecollection of more samples, due to failure of diagnosis, in addition to preventing the patient from undergoing unnecessary chemotherapy.
Ideally,PCRexamsforthedetectionandidentificationof
Leishmaniashouldbeaccessibletomosthealthcareservices in Brazil.Prior tothisstudy and accesstospecies identi-ficationbyhsp70-HRM inparaffin-embeddedtissueblocks, patients were treated at this service based on clinical (presence of mucocutaneous lesions)and epidemiological data, as well as complementary exams, when available.
In thepresent cases, it wasdemonstrated that access to complementaryexamsisnotroutine,andthatinthose with-out amastigotes, diagnosis confirmation is difficult; thus, therapeutictestingwithpossiblesideeffectsisusually indi-cated,which oftencausesdiscomfortandsuffering tothe patient.
The identification of Leishmania species is impor-tant for the choice of treatment.1,3,47 L. braziliensis can
lead to mucosal involvement, even in patients who had a single ulcer,3,6 and there is a possibility of visceral
involvement in patients with skin lesions caused by L. infantumchagasi.4,5,50Thus,inthesecases,systemic
treat-ment should be preferred over topic treatments such as intralesionalinfiltrationswithmeglumineantimoniate, rec-ommendedforthetreatmentofsingleulceratedlesionson theskin.3
Conclusions
Thediagnosisofleishmaniasisisonethegreatestchallenges inthefightagainstthedisease.Thecorrectidentificationof theLeishmaniaspeciesinparaffinizedsamplesofskinand mucosais an important source of data for ecologicaland epidemiologicalstudies,aswellasforstudiesofthebiology oftheparasiteanditsinteractionwiththehost,andmay beindicativeofthedesignofthetherapeuticprotocol.
Of the three tests used to identify the species, two are very laborious and require a trained technician to interpretresults.Thefactthatthehsp70-HRMresultis pro-vided by a machine not only allows for automation, but alsoeliminatespotentialcontaminationresultingfromthe manipulationsnecessaryfortheanalysisofresultsobtained byconventionalPCR,asthistechniqueisbasedonanalysis ofthe PCR productin sealedtubes,which donotrequire handling.
This study was ableto identifythe parasite speciesin clinicalsamplesin10/11patientswithpositiveamastigotes. Furthermore,itdemonstratedthepresenceofLeishmaniain 4/11patientsinwhomamastigoteformshadnotbeenfound inthestudiedsamplesanddefinitelyruledoutthediagnosis ofleishmaniasisin7/11patientswithnoparasitesondirect exams.
In additiontoL.braziliensis,L. guyanensis,L. amazo-nensis,andL.infantumchagasi,anassociationofthelatter twospecieswasdetected.Inpractice,theseresultsindicate
thattheoccurrenceofrarecasesmaybemorecommonthan previouslydescribed.
The authorsbelieve thatprospective studiesshouldbe conductedwith largerpopulations; the use of the hsp70-HRMtechniquewithmaterialobtainedfromsmearsis also suggested,asitwouldallowstudiesinremoteregionsofthe country.
Financial
support
Nonedeclared.
Authors’
contributions
JohnVerrinderVeasey:Statisticalanalysis;approvalofthe final version of the manuscript; conception and planning of the study; elaboration and writing of the manuscript; obtaining, analyzing, and interpreting the data; effective participationinresearchorientation;intellectual participa-tioninpropaedeuticand/ortherapeuticconductofstudied cases;criticalreviewoftheliterature;criticalreviewofthe manuscript.
RicardoAndradeZampieri:Approvalofthefinalversion of themanuscript; conception and planningof the study; elaborationandwritingofthemanuscript;obtaining, ana-lyzing,andinterpretingthedata;effectiveparticipationin researchorientation;criticalreviewoftheliterature; criti-calreviewofthemanuscript.
Rute Facchini Lellis: Obtaining, analyzing, and inter-pretingthedata;intellectualparticipationinpropaedeutic and/ortherapeuticconductofstudiedcases.
Thaís Helena Proenc¸a de Freitas: Statistical analysis; approvalofthefinalversionofthemanuscript;conception andplanning of thestudy; elaboration andwriting of the manuscript;obtaining,analyzing,andinterpretingthedata; effectiveparticipationinresearchorientation;intellectual participationin propaedeuticand/or therapeutic conduct ofstudied cases; criticalreview of theliterature; critical reviewofthemanuscript.
LucileMariaFloeterWinter:Statisticalanalysis;approval ofthefinalversionofthemanuscript;conceptionand plan-ningofthestudy;elaborationandwritingofthemanuscript; obtaining, analyzing, and interpreting the data; effective participationinresearchorientation;criticalreviewofthe literature;criticalreviewofthemanuscript.
Conflicts
of
interest
Nonedeclared.
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