Research Article ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and...

Research ArticleITS1 PCR-RFLP Diagnosis and Characterization of Leishmaniain Clinical Samples and Strains from Cases of Human CutaneousLeishmaniasis in States of the Mexican Southeast

Amalia Monroy-Ostria1 Abedelmajeed Nasereddin2 Victor M Monteon3

Carmen Guzmaacuten-Bracho4 and Charles L Jaffe2

1 Escuela Nacional de Ciencias Biologicas IPN Carpio y Plan de Ayala 11340 Mexico City Mexico2 School of Medicine Department of Microbiology and Molecular Genetics IMRIC The Hebrew University of Jerusalem9112102 Jerusalem Israel

3 Centro de Investigaciones Biomedicas Universidad Autonoma de Campeche Patricio Trueba 24090 Campeche Mexico4 Instituto de Diagnostico y Referencia Epidemiologicos Francisco P de Miranda Lomas de Plateros 01480 Mexico City Mexico

Correspondence should be addressed to Amalia Monroy-Ostria amaliahmogmailcom

Received 10 January 2014 Accepted 9 June 2014 Published 1 July 2014

Academic Editor Stephane Picot

Copyright copy 2014 Amalia Monroy-Ostria et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

American cutaneous leishmaniasis includes a spectrum of clinical forms localized cutaneous diffuse cutaneous and mucocuta-neous leishmaniasis which can be caused by different strains of Leishmania belonging to the Lmexicana or L braziliensis complexeswhich may coexist in the same endemic area We evaluated the PCR-RFLP assay of the ITS1 genes for direct identification ofLeishmania species in 163 clinical samples and 21 Mexican isolates of Leishmania In relation to the Mexican isolates of Leishmania52 displayed a pattern similar to the L (L) mexicana 5 showed a mixed pattern compatible with L (L) mexicana and L (V)braziliensis eight with L (L) amazonensis and L (L) mexicana and one to L (V) braziliensis Most of the clinical samples 109116(94) gave a pattern similar to that of the L mexicana two clinical samples gave similar patterns to that of Leishmania braziliensisand 5 samples gave patterns that suggest a coinfection of L (L) mexicana and L (V) braziliensis or L (L) mexicana and L (L)amazonensis The ITS1 PCR-RFLP assay is a multipurpose tool for diagnosis of Leishmania from clinical samples and enablesdetermination of the infecting species of NewWorld Leishmania in the field in relatively short time and low cost

1 Introduction

Leishmaniasis is a group of parasitic diseases with world-wide distribution Cutaneous leishmaniasis (CL) is the mostwidespread form of leishmaniasis causing primary localizedskin lesions (LCL) that can self-heal but fromwhich parasitescan disseminate to the nasopharyngeal mucosa and causesecondary lesions typical of mucocutaneous leishmaniasis(MCL) or disseminate to the entire body in the form of nodu-lar lesions in diffuse cutaneous leishmaniasis (DCL) WorldHealth Organization estimates a worldwide prevalence ofapproximately 12 million cases with an annual mortality rateof 60000 The size of the population at risk is approximately350 million [1]

American cutaneous leishmaniasis includes LCL causedby Leishmania (L)mexicana DCL caused by Leishmania (L)amazonensis Leishmania (L) venezuelensis and Leishmania(L) pifanoi andMCLcaused bymembers of theL braziliensiscomplex [2]

In endemic regions multiple species of Leishmania maycoexist Identification of the infecting species based onclinical symptoms is difficult since several species can causeboth LCL and MCL In some villages in Mexico patientswith lesions produced by both L braziliensis and L mexicanacomplex members can be found as well as patients with LCLand patients with DCL in the same village [3] Moreoverreports indicate that the response to therapeutic drugs canvary among different species present in the same area [4]

Hindawi Publishing CorporationInterdisciplinary Perspectives on Infectious DiseasesVolume 2014 Article ID 607287 6 pageshttpdxdoiorg1011552014607287

2 Interdisciplinary Perspectives on Infectious Diseases

Diagnostic confirmation and correct identification of theLeishmania species are important for appropriate species-specific therapeutic as well as epidemiologic studies

The polymerase chain reaction (PCR) approach wasdeveloped as an alternative to existing diagnostic proceduressuch as direct detection of parasites by microscopic examina-tion of clinical specimens or by cultivation

Several molecular targets for a diagnostic PCR have beenevaluated in Leishmania including minicircle kinetoplastDNA (kDNA) [3] the miniexon (spliced leader RNA) gene[5] the gp63 PCR-RFLP [6] and the internal transcribedspacer (ITS) [7ndash9] among others

In the present study as described by Cupolillo et al [10]and Schonian et al [11] samples spotted on filter paper andLeishmania isolates from patients with cutaneous ulcers sus-pected of having LC were analyzed by PCR amplification ofthe internal transcribed spacer 1 genes (ITS1) and restrictionfragment length polymorphism (ITS1 PCR-RFLP) for thedirect diagnosis of leishmaniasis and parasite identification

The aim of this study was to look for a diagnostic methodfor leishmaniasis that combines high sensitivity with speciesdifferentiation in the field in short time and low cost

2 Materials and Methods

21 Ethical Considerations Informed consent was obtainedfrom all the adults who participated in the study Consentfor inclusion of young children was obtained from parentsor guardians The protocol of the present study was reviewedand approved by the Ethics Committee of Health Authoritiesof Calakmul Campeche Mexico in agreement with Interna-tional Ethics Guidelines for Biomedical Research involvinghuman subjects (Norma Oficial Mexicana de Salud NOM-003-SSA 2-1993) for bleeding human beings for diagnosisand therapeutics

22 Leishmania Cultures and Clinical Samples This studywas conducted with 21 cultures of Leishmania isolated frompatients with cutaneous ulcer from different states of Mexicokindly donated by Instituto de Diagnostico y ReferenciaSecretaria de Salud Mexico

The clinical samples (163) were kindly donated by Centrode Investigaciones Biomedicas Universidad de Campecheand Los Servicios de Salud del Municipio de CalakmulCampeche Mexico The clinical samples were taken on filterpapers or smears from the cutaneous lesions of patientssuspected of having CL from different endemic areas ofMexico

23 Leishmania Reference Strains Leishmania (V) pana-mensis (MHOMCR87NEL3) Leishmania (V) panamensisMHOMPA72LS94 Leishmania (V) guyanensis (MHOMBR75M4147)L (L)mexi-cana (MHOMMX85SOLIS)L(V) braziliensis (MHOMBR75M2903) and L (L) amazo-nensis (MHOMBR73M2269) reference strains were usedas controls The strains of Leishmania were cultured inRPMI medium supplemented with 10 fetal calf serum at26∘C

24 DNA Extraction Each clinical specimen was cut fromthe filter paper or eluted from the smear and incubated in250 120583L cell lysis buffer for 1 h at 56∘C DNA from Leishmaniacultures was prepared by centrifuging 108 parasites in theexponential phase of growth at 2000 g for 10min at 4∘CThe DNA was extracted from the pellet using the High PurePCR template preparation kit (Roche Diagnostics GmbHMannheim Germany) following the manufacturerrsquos instruc-tions The DNA was stored at minus20∘C until being used

25 PCR Analysis of the Internal Transcribed Spacer 1 (ITS1)The samples were analyzed for ITS1 PCR using 400 nMprimers LITSR 51015840-CTTG GATCATTTTCCGATG-31015840 andL58S 51015840-TGA TAC CAC TTA TCG CAT T-31015840 [12] Thereaction was carried out with the PCR-Ready Suprememix (Syntezza Bioscience Jerusalem Israel) in 25120583L oftotal reaction Amplification conditions were as describedpreviously [12] PCR products (8ndash15 120583L) were digested withHae III enzyme according to themanufacturerrsquos instructionsThe amplicons of about 300ndash350 bp were analyzed on 15agarose gels and the restriction fragments on 4 agarose gelsby electrophoresis at 100V in 1X Tris-acetate-EDTA buffer(004M Tris acetate and 1mM EDTA pH 8) and visualizedby UV light after being stained with ethidium bromide(03 120583gmL) The GeneRuler DNA ladder Mix (FermentasMBI) was used as the DNA molecular marker

3 Results

PCR with specific primers for ITS1 resulted in the ampli-fication of the Leishmania reference strains the Mexicancultures and the clinical samples giving 300 to 350 bpamplification bands Restriction of the ITS1 gene amplicons ofL (V) panamensis L (V) guyanensis and L (L) braziliensisreference strains with the endonuclease Hae III generatedpatterns with two bands of 170 and 150 bp L (L) amazonensisgenerated two bands of 220 and 140 bp and L mexicanagenerated three bands of 200 80 and 40 bp (Figure 1)

Most of the Mexican isolates of Leishmania 1121 (52)displayed a restriction pattern of three bands (200 80 and40 bp) similar to that of L (L) mexicana reference strainnine of these were obtained from patients from Campeche121 (5) showed a mixed pattern compatible with L (L)mexicana and L (V) braziliensis (lane 15) and one culturewith L (V) braziliensis (lane 3) eight showed amixed patterncompatible with L (L) amazonensis and L (L) mexicanaIn few samples an incomplete digestion can be appreciated(Figure 2) (Table 1) these results were in agreement witha previous study in PCR TS1-RFLP analysis to identifyLeishmania species in clinical samplesby Rotureau et al [13]and in the study of PCR diagnosis and characterization ofLeishmania in clinical samples by Schonian et al [11]

In relation to the clinical samples 116163 (71) wereamplified 109116 (94) giving a ITS1 PCR-RFLP patternsimilar to the L (L) mexicana reference strain in sevensamples (6) extra bands of 50 and 25 bp were observedsuggesting a coinfection as it was found in the previous studyof Hernandez-Montes et al [3] with kDNA PCR analysis

Interdisciplinary Perspectives on Infectious Diseases 3

100

1 765432200

(a)

100

1 765432

200

(b)

Figure 1 (a) Electrophoresis run at 100V for 30min (b) Electrophoresis run at 199V for 60min PCR-RFLP of the ITS1 of Leishmaniareference strains Lane 1 Leishmania (V) panamensis MHOMCR87NEL3 lane 2 Leishmania (V) panamensis MHOMPA72LS9 lane3 Leishmania (V) guyanensis (MHOMBR75M4147) lane 4 Leishmania (L) mexicana (MHOMMX85SOLIS) lane 5 Leishmania (V)braziliensis (MHOMBR75M2903) lane 6 Leishmania (L) amazonensis (MHOMBR73M2269) lane 7 MWMX174 Hae III

Table 1 ITS1 PCR-RFLP of isolates of Leishmania analyzed in this study

Number Code Origin Pathology ITS1 PCR-RFLP (bp) Leishmania species1 MHOMMX84ISET GS Tabasco DCL (220 200 140 40) L am+L mex2 MHOMMX88HRCMC Tabasco LCL (200 8040) L mexicana3 MHOMMX88HRC JS Tabasco LCL (200 8040) L mexicana4 AMG Tabasco DCL (220 200 140 40) L am+L mex5 HC Tabasco LCL (220 200 140 40) L am+L mex6 L527 Tabasco LCL (220 200 140 40) L am+L mex7 MHOMMX85ISET HF Veracruz DCL (220 200 140 40) L am+L mex8 MHOMMX92INDRE AM Veracruz DCL (220 200 140 40) L am+L mex9 LVER Veracruz DCL (220 200 140 40) L am+L mex10 MHOMMX83UAVY Q Roo LCL (200 8040) L mexicana11 MHMMX06ENCBMIC Campeche LCL (200 8040) L mexicana12 MHMMX06ENCB CDL Campeche LCL (200 8040) L mexicana13 MHMMX06ENCB FDL Campeche LCL (200 8040) L mexicana14 MHMMX07ENCB NDM Campeche LCL (200 8040) L mexicana15 RMA Campeche LCL (200 170 150 80 40) L mex + L bra16 REP Campeche LCL (220 200 140 40) L am+L mex17 FAD Campeche LCL (200 8040) L mexicana18 A MJ Campeche LCL (200 8040) L mexicana20 DON Campeche LCL (200 8040) L mexicana20 L528 Campeche LCL (200 8040) L mexicana21 CR Campeche LCL (170 150) L braziliensisL am+L mex L (L) amazonensis + L (L) mexicanaL mexicana L (L) mexicanaL mex + L bra L (L) mexicana + L (V) braziliensis

1 2 3 4 5 6 7 8 109 11 141312 15 16 17 18 19

500400300200

100

Figure 2 ITS1 PCR-RFPLC of Mexican Leishmania cultures Lane1 MWMX174Hae III lanes 2 to 19 Mexican cultures of Leishmania

of Mexican Leishmania species where they identified inclinical samples both DNA from L (L) mexicana and L(V) braziliensis In lanes 3-4 and 7-8 the pattern of bands

of 200 170 and 140 bp observed suggests the presence of L(L) mexicana L (V) braziliensis and L (L) amazonensisrespectively (Figure 3)

4 Discussion

Molecular techniques have proved to be sensitive and power-ful tools for detecting Leishmania directly in clinical samplesas well as for parasite characterization using the PCR

Several scientific papers based on ITS analysis havebeen published on the diagnosis of leishmaniasis and theidentification of the Leishmania species Cupolillo et al [10]evaluated the ITS using restriction patterns of Leishmaniaand Viannia rDNA isolates from different hosts and geo-graphical areas found high levels of intra- and interspecific


1 2 3 4 5 6 7 8 9 10 1211

500400300

200

100

Figure 3 ITS1 PCR-RFLP of Clinical samples Lane 1 MWMX174Hae III lanes 2 to 12 clinical samples taken on filter paper or smear

variation and showed that the ITS of these genera is evolvingfast enough to enable the species to be discriminated

Interestingly Schonian et al [11] established a diagnosticITS1 PCR-RFLPmethod using the restriction enzymeHae IIIfor leishmaniasis it combines high sensitivity for detectingLeishmania directly in clinical materials and the ability toidentify all medically relevant species groups On the otherhand Spanakos et al [14] developed an ITS1 PCR-RFLPmethod with the endonuclease Apo I for the detection andspecies differentiation of Leishmania directly from clinicalsamples specific enough to identify all Leishmania speciescommonly encountered in Greece Slami et al [15] studieda CL endemic area of Central Iran by using ITS1 PCR-RFLPanalysis for diagnosis of Leishmania species in clinical sam-ples and found changes in the profile of Leishmania speciesthat could have implications on treatment andor controlstrategies On the other hand El-Beshbishy et al [16] studieswith both ITS1 PCR RFLP and kDNA PCR assays in clinicalsamples from CL patients from western Saudi Arabia foundL major and L tropica and that kDNA PCR had a sensitivityof 907 and ITS1 PCR of 701That facilitated the diagnosisand the species identification using both techniques whereasparasite culture alone detected 392 and smear alone 553of the positive samples Furthermore Kumar et al [17] in aCL endemic area of India using ITS1 PCR-RFLP kDNA PCRand specific antibody detection found similar results and Ltropica as the causative parasite

On the other handAbbasi et al [18] performed a prospec-tive cohort study on the transmission dynamics of VL inblood samples collected from villagers in the Tahtay Adiabodistrict of northern Ethiopia combining quantitative real-time kinetoplast DNAPCR (qRT-kDNA PCR) for detectingsmall quantity of Leishmania parasites (1ndash10mL of blood)and sequencing the ITS1 PCR amplicon in order to identifythe Leishmania species

On the other hand Rotureau et al [13] in diagnosis ofCL andMCLNewWorld Leishmania species using ITS1 PCRRFLP found that only one digestion with Rsa I is requiredto identify parasites in clinical samples to the species leveldigestion but restriction with Hae III was not sufficient todistinguish all species in the Viannia subgenus especiallyL (V) braziliensisL (V) naiffi and L (V) lainsoniL (V)guyanensis

In Mexico Perez-Vega et al [19] in Durango State andOchoa-Diaz et al [20] in Sinaloa State identified Leishmaniamexicana in clinical samples with ITS1 PCR RFLP assay

In the present study following themethodology describedby Schonian et al [11] most of the DCL cases were foundin Tabasco and Veracruz States and were caused by L (L)mexicana whereas most of the LCL cases produced by L (L)mexicana belonged to Campeche State as well as the LCLcases caused by L braziliensis complex members (Table 1)All these states are located very close to each other insoutheastern Mexico (Figure 4) They all have rain forestareas where CL is endemic In Tabasco DCL and LCLcoexist in Campeche it is possible to find LCL caused byL (L) mexicana or L braziliensis complex members andwe were able to detect mixed infections in clinical samplesand cultures [3 21] This method was very useful for theanalysis of the Mexican strains of Leishmania and clinicalsamples because we could perform relatively easy diagnosisand characterization of Leishmania species and its possiblerelationship with the clinical manifestation

However this study contradicts the observation ofBerzunza-Cruz et al [22] with ITS restriction patterns andthe small subunit rRNA genes of Mexican isolates of Lmexicana finding that all strains showed invariant patternsfor both genes

The PCR-based assays are advantageous over immuno-logical techniques such as enzyme linked immunosorbentassay (ELISA) and immunofluorescence antibody test (IFAT)as host species-specific reagents are not required which isimportant in patients with MCL and the immunocompro-mised ones in which both give negative serological tests[3] In particular in chronic CL patients who constitute thegreater diagnostic challenge due to their low parasite densityPCR assays for the detection of Leishmania DNA presented100 sensitivity Moreover the fact that antibodies remaindetectable for years after successful treatment makes theapplication of PCR a necessity [4] Furthermore persistentinfection has been found in apparently healed scars fromMCL patients [7] the presence of Leishmania braziliensiswasreported in patients previously treated by immunotherapy orpatients being at different stages of treatment and in subjectswhohadnever presented clinicalmanifestations but they hadlived in endemic areas and migrated to no endemic regions[8]

These results raise questions on (i) the identity ofthe Mexican strains that displayed restriction patterns thatwere not compatible with any of the restriction patternsof the reference strains used in this study but suggestingcoinfections (ii) the pathogenicity of these strains and(iii) their geographic distribution [23] In order to answerthese questions and to establish the identity of the MexicanLeishmania strains and their geographical distribution itwould be necessary following themethodology developed byVan der et al [24] to analyze several single-locus markerssequencing of the Mexican Leishmania strains from mostof the endemic areas of Mexico and from patients with allthe clinical manifestations of CL (LCL MCL and DCL)Furthermore the Reverse Line Blot Hybridization Assay forMolecular Diagnosis of Old World cutaneous Leishmaniasis


Ocean Pacific

United States of America

Gulf of Mexico

Veracruz

Tabasco

Campeche

QuintanaRoo

Figure 4 Map of Mexico showing the endemic regions studied in this work Veracruz Tabasco Campeche and Quintana Roo

developed byNasereddin et al [25] will be useful with probesdesigned from ITS1 PCR amplicons of Mexican strains forepidemiological studies where a large number of samplesneed to be screened in order to test potential reservoir hostsand vectors and for epidemiological surveillance

5 Conclusion

The ITS1PCR-RFLP assay analyzed in this work was a valu-able multipurpose tool for diagnosis directly from clinicalsamples without parasite isolation and enables determinationof the infecting species of New World Leishmania in thefield in a relatively short time The ITS1 PCR-RFLP assay isrecommended for the reliable characterization of Leishmaniaspecies mainly in endemic areas where the presence ofmultiple species of Leishmania overlapping clinical picturesdemands simultaneous species identification at a relativelow cost Although in areas where both Leishmania andViannia subgenus species are present ITS1 PCR-RFLP mustbe combined with kDNA PCR in order to improve thesensitivity for diagnosis of CL or MCL

Disclosure

This research received no specific grant from any fundingagency in the public commercial or not-for-profit sectors

Conflict of Interests

No conflict of interests exists for the authors to declare

Acknowledgment

Financial support for this researchwas provided by Secretariade Investigacion y Posgrado IPN Mexico Amalia Monroy-Ostria is a fellow of CONACyT EDI COFAA Mexico

References

[1] R Reithinger J Dujardin H Louzir C Pirmez B Alexanderand S Brooker ldquoCutaneous leishmaniasisrdquo Lancet InfectiousDiseases vol 7 no 9 pp 581ndash596 2007

[2] R Lainson and J J Shaw ldquoEvolution classification a ndgeographical distribution of Leishmaniardquo inThe LeishmaniasisR Killick-Kendrick and W Peters Eds pp 1ndash20 AcademicPress London UK 1987

[3] O Hernandez-Montes A Monroy-Ostria S McCann and DC Barker ldquoIdentification of Mexican Leishmania species byanalysis of PCR amplified DNArdquo Acta Tropica vol 71 no 2 pp139ndash153 1998

[4] G A S Romero M V De Farias Guerra M G Paes and VDe Oliveira Macedo ldquoComparison of cutaneous leishmaniasisdue to Leishmania (Viannia) braziliensis and L (V) guyanensisin Brazil therapeutic response to meglumine antimoniaterdquoAmerican Journal of Tropical Medicine and Hygiene vol 65 no5 pp 456ndash465 2001

[5] O Fernandes V K Murthy U Kurath W M Degrave and DA Campbell ldquoMini-exon gene variation in human pathogenicLeishmania speciesrdquo Molecular and Biochemical Parasitologyvol 66 no 2 pp 261ndash271 1994

[6] K Victoir S de Doncker L Cabrera et al ldquoDirect identificationof Leishmania species in biopsies from patients with Americantegumentary leishmaniasisrdquo Transactions of the Royal Society ofTropical Medicine and Hygiene vol 97 no 1 pp 80ndash87 2003

[7] A M R Davila and H Momen ldquoInternal-transcribed-spacer(ITS) sequences used to explore phylogenetic relationships


within Leishmaniardquo Annals of Tropical Medicine and Parasitol-ogy vol 94 no 6 pp 651ndash654 2000

[8] E Bensoussan A Nasereddin F Jonas L F Schnur and C LJaffe ldquoComparison of PCR assays for diagnosis of cutaneousleishmaniasisrdquo Journal of Clinical Microbiology vol 44 no 4pp 1435ndash1439 2006

[9] R Reithinger and J C Dujardin ldquoMolecular diagnosis ofleishmaniasis current status and future applicationsrdquo Journal ofClinical Microbiology vol 45 no 1 pp 21ndash25 2007

[10] E Cupolillo G Grimaldi Jr H Momen and S M BeverleyldquoIntergenic region typing (IRT) a rapid molecular approach tothe characterization and evolution of Leishmaniardquo Molecularand Biochemical Parasitology vol 73 no 1-2 pp 145ndash155 1995

[11] G Schonian ANasereddin NDinse et al ldquoPCRdiagnosis andcharacterization of Leishmania in local and imported clinicalsamplesrdquoDiagnosticMicrobiology and InfectiousDisease vol 47no 1 pp 349ndash358 2003

[12] N O El TaiM El Fari I Mauricio et al ldquoLeishmania donovaniintraspecific polymorphisms of Sudanese isolates revealed byPCR-based analyses and DNA sequencingrdquo Experimental Par-asitology vol 97 no 1 pp 35ndash44 2001

[13] B Rotureau C Ravel P Couppie et al ldquoUse of PCR-restrictionfragment length polymorphism analysis to identify the mainnew world Leishmania species and analyze their taxonomicproperties and polymorphism by application of the assay toclinical samplesrdquo Journal of Clinical Microbiology vol 44 no2 pp 459ndash467 2006

[14] G Spanakos E Piperaki P G Menounos N Tegos AFlemetakis andNCVakalis ldquoDetection and species identifica-tion of Old World Leishmania in clinical samples using a PCR-based methodrdquo Transactions of the Royal Society of TropicalMedicine and Hygiene vol 102 no 1 pp 46ndash53 2008

[15] G Slami H Anvari M Ebadi et al ldquoThe changing profile ofcutaneous leishmaniasis agent in a central province of IranrdquoTanzania Journal of Health Research vol 15 no 1 2013

[16] HA El-Beshbishy KHAl-Ali andAA El-Badry ldquoMolecularcharacterization of cutaneous leishmaniasis in Al-Madinah Al-Munawarah province Western Saudi Arabiardquo InternationalJournal of Infectious Diseases vol 17 no 5 pp 334ndash338 2013

[17] R Kumar R A Bumb N A Ansari R D Mehta and PSalotra ldquoCutaneous leishmaniasis caused by Leishmania tropicain Bikaner India parasite identification and characterizationusing molecular and immunologic toolsrdquo American Journal ofTropical Medicine and Hygiene vol 76 no 5 pp 896ndash901 2007

[18] I Abbasi S Aramin A Hailu et al ldquoEvaluation of PCRprocedures for detecting and quantifying Leishmania donovaniDNA in large numbers of dried human blood samples froma visceral leishmaniasis focus in Northern Ethiopiardquo BMCInfectious Diseases vol 13 no 1 article 153 2013

[19] J H Perez-Vega C Y Lopez-Moreno J A Lopez-Valenzuela etal ldquoLeishmaniasis cutanea causada por Leishmania mexicanaen Durango Mexico Informe del primer caso clınicordquo GacetaMedica de Mexico vol 145 pp 433ndash435 2009

[20] Y O Ochoa-Diaz C Y Lopez-Moreno J G Rendon-Maldonado and H S Lopez-Moreno ldquoMolecular diagnosisof Leishmania mexicana in a cutaneous Leishmaniasis case insinaloa Mexicordquo Vector-Borne and Zoonotic Diseases vol 12no 1 pp 78ndash80 2012

[21] F Andrade-Narvaez D E Simmonds and A S Rico ldquoInci-dence of localized cuta neous leishmaniasis ( chiclerorsquos ulcer) inMexicordquo Transactions of the Royal Society of Tropical Medicineand Hygiene vol 84 pp 219ndash220 1990

[22] M Berzunza-Cruz N Cabrera M Crippa-Rossi T SosaCabrera R Perez-Montfort and I Becker ldquoPolymorphismanalysis of the internal transcribed spacer and small subunitof ribosomal RNA genes of Leishmania mexicanardquo ParasitologyResearch vol 88 no 10 pp 918ndash925 2002

[23] J Shaw ldquoThe leishmaniasis survival and expansion in a chang-ing worldrdquo Memorias do Instituto Oswaldo Cruz vol 102 pp541ndash547 2007

[24] G Van der C Auwera J Verweij et al ldquoEvaluation of foursingle-locus markers for Leishmania species discrimination bysequencingrdquo Journal of Clinical Microbiology vol 52 pp 1098ndash1104 2014

[25] A Nasereddin E Bensoussan-Hermano G Schonian GBaneth and C L Jaffe ldquoMolecular diagnosis of old worldcutaneous leishmaniasis and species identification by use ofa reverse line blot hybridization assayrdquo Journal of ClinicalMicrobiology vol 46 no 9 pp 2848ndash2855 2008

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Diagnostic confirmation and correct identification of theLeishmania species are important for appropriate species-specific therapeutic as well as epidemiologic studies

The polymerase chain reaction (PCR) approach wasdeveloped as an alternative to existing diagnostic proceduressuch as direct detection of parasites by microscopic examina-tion of clinical specimens or by cultivation

Several molecular targets for a diagnostic PCR have beenevaluated in Leishmania including minicircle kinetoplastDNA (kDNA) [3] the miniexon (spliced leader RNA) gene[5] the gp63 PCR-RFLP [6] and the internal transcribedspacer (ITS) [7ndash9] among others

In the present study as described by Cupolillo et al [10]and Schonian et al [11] samples spotted on filter paper andLeishmania isolates from patients with cutaneous ulcers sus-pected of having LC were analyzed by PCR amplification ofthe internal transcribed spacer 1 genes (ITS1) and restrictionfragment length polymorphism (ITS1 PCR-RFLP) for thedirect diagnosis of leishmaniasis and parasite identification

The aim of this study was to look for a diagnostic methodfor leishmaniasis that combines high sensitivity with speciesdifferentiation in the field in short time and low cost

2 Materials and Methods

21 Ethical Considerations Informed consent was obtainedfrom all the adults who participated in the study Consentfor inclusion of young children was obtained from parentsor guardians The protocol of the present study was reviewedand approved by the Ethics Committee of Health Authoritiesof Calakmul Campeche Mexico in agreement with Interna-tional Ethics Guidelines for Biomedical Research involvinghuman subjects (Norma Oficial Mexicana de Salud NOM-003-SSA 2-1993) for bleeding human beings for diagnosisand therapeutics

22 Leishmania Cultures and Clinical Samples This studywas conducted with 21 cultures of Leishmania isolated frompatients with cutaneous ulcer from different states of Mexicokindly donated by Instituto de Diagnostico y ReferenciaSecretaria de Salud Mexico

The clinical samples (163) were kindly donated by Centrode Investigaciones Biomedicas Universidad de Campecheand Los Servicios de Salud del Municipio de CalakmulCampeche Mexico The clinical samples were taken on filterpapers or smears from the cutaneous lesions of patientssuspected of having CL from different endemic areas ofMexico

23 Leishmania Reference Strains Leishmania (V) pana-mensis (MHOMCR87NEL3) Leishmania (V) panamensisMHOMPA72LS94 Leishmania (V) guyanensis (MHOMBR75M4147)L (L)mexi-cana (MHOMMX85SOLIS)L(V) braziliensis (MHOMBR75M2903) and L (L) amazo-nensis (MHOMBR73M2269) reference strains were usedas controls The strains of Leishmania were cultured inRPMI medium supplemented with 10 fetal calf serum at26∘C

24 DNA Extraction Each clinical specimen was cut fromthe filter paper or eluted from the smear and incubated in250 120583L cell lysis buffer for 1 h at 56∘C DNA from Leishmaniacultures was prepared by centrifuging 108 parasites in theexponential phase of growth at 2000 g for 10min at 4∘CThe DNA was extracted from the pellet using the High PurePCR template preparation kit (Roche Diagnostics GmbHMannheim Germany) following the manufacturerrsquos instruc-tions The DNA was stored at minus20∘C until being used

25 PCR Analysis of the Internal Transcribed Spacer 1 (ITS1)The samples were analyzed for ITS1 PCR using 400 nMprimers LITSR 51015840-CTTG GATCATTTTCCGATG-31015840 andL58S 51015840-TGA TAC CAC TTA TCG CAT T-31015840 [12] Thereaction was carried out with the PCR-Ready Suprememix (Syntezza Bioscience Jerusalem Israel) in 25120583L oftotal reaction Amplification conditions were as describedpreviously [12] PCR products (8ndash15 120583L) were digested withHae III enzyme according to themanufacturerrsquos instructionsThe amplicons of about 300ndash350 bp were analyzed on 15agarose gels and the restriction fragments on 4 agarose gelsby electrophoresis at 100V in 1X Tris-acetate-EDTA buffer(004M Tris acetate and 1mM EDTA pH 8) and visualizedby UV light after being stained with ethidium bromide(03 120583gmL) The GeneRuler DNA ladder Mix (FermentasMBI) was used as the DNA molecular marker

3 Results

PCR with specific primers for ITS1 resulted in the ampli-fication of the Leishmania reference strains the Mexicancultures and the clinical samples giving 300 to 350 bpamplification bands Restriction of the ITS1 gene amplicons ofL (V) panamensis L (V) guyanensis and L (L) braziliensisreference strains with the endonuclease Hae III generatedpatterns with two bands of 170 and 150 bp L (L) amazonensisgenerated two bands of 220 and 140 bp and L mexicanagenerated three bands of 200 80 and 40 bp (Figure 1)

Most of the Mexican isolates of Leishmania 1121 (52)displayed a restriction pattern of three bands (200 80 and40 bp) similar to that of L (L) mexicana reference strainnine of these were obtained from patients from Campeche121 (5) showed a mixed pattern compatible with L (L)mexicana and L (V) braziliensis (lane 15) and one culturewith L (V) braziliensis (lane 3) eight showed amixed patterncompatible with L (L) amazonensis and L (L) mexicanaIn few samples an incomplete digestion can be appreciated(Figure 2) (Table 1) these results were in agreement witha previous study in PCR TS1-RFLP analysis to identifyLeishmania species in clinical samplesby Rotureau et al [13]and in the study of PCR diagnosis and characterization ofLeishmania in clinical samples by Schonian et al [11]

In relation to the clinical samples 116163 (71) wereamplified 109116 (94) giving a ITS1 PCR-RFLP patternsimilar to the L (L) mexicana reference strain in sevensamples (6) extra bands of 50 and 25 bp were observedsuggesting a coinfection as it was found in the previous studyof Hernandez-Montes et al [3] with kDNA PCR analysis


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Research Article ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and...

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Transcript of Research Article ITS1 PCR-RFLP Diagnosis and Characterization of Leishmania in Clinical Samples and...