Leishmania (Viannia) braziliensis genotypes identified in lesions of patients with atypical or typical manifestations of tegumentary leishmaniasis: Evaluation by two molecular markers

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Abstract

Analyses of MLEE, RAPD and LSSP-PCR were used to compare the panel of american tegumentary leishmaniasis (ATL) isolates obtained from lesions of patients with rare clinical manifestations of the disease and typical lesions. All of the 34 samples analyzed by MLEE demonstrated similar electromorphic profiles with Leishmania (Viannia) braziliensis reference strain. Through the RAPD analysis, nine genetic profiles (genotypes) were identified. LSSP-PCR corroborates the initial screening and phenetic analysis has grouped the isolates into two major clusters comprising the nine different genotypes. Prevalent genotype defined as LbmtDNAgen1 was detected in the largest number of isolates. There was no association between genotypes and clinical symptoms. However, two different genotypes could be identified in the initial (LbmtDNAGen9) and reactivated lesion (LbmtDNAGen3) of the same patient. Our results support the idea of a less pronounced genotypic diversity among L. (V.) braziliensis circulating in the State of Rio de Janeiro and demonstrate the useful application of these molecular markers in genetics variability studies.

Introduction

American tegumentary leishmaniasis (ATL) is caused by Leishmania species of the subgenera Leishmania (Viannia) and Leishmania (Leishmania), which are transmitted to man and other domestic and wild mammals by the bite of sandflies, and is distributed from the south of the United States to the north of Argentina. In Brazil, ATL is found in all states and has shown a high incidence over the last 20 years. The main clinical forms of ATL are cutaneous leishmaniasis, mucosal or mucocutaneous leishmaniasis, and diffuse cutaneous leishmaniasis (Marzochi, 1992).

In the State of Rio de Janeiro, L. (V.) braziliensis is the most prevalent species that has so far been implicated in the cycle of ATL. However, more recently an autochthonous case of diffuse leishmaniasis due to L (L) amazonensis has been reported (Azeredo-Coutinho et al., 2007). Transmission mainly occurs in periurban areas where the primitive rain forest vegetation is being replaced with disorderly human occupation. In this context, adaptation of the vector Lutzomyia intermedia to the domiciliary and peridomiciliary environment has been observed, as well as the presence of infected humans, dogs and horses (Aguilar et al., 1987; Barbosa-Santos et al., 1994, Marzochi, 1992, Marzochi and Marzochi, 1994). The most frequent clinical manifestation is a recent single or double cutaneous ulcer located in areas exposed to the bites of sandflies, which shows a favorable response to antimonial therapy. Multiple or localized skin lesions in areas normally covered by clothing or lesions of long duration, mucosal lesions, an unfavorable response to antimonial treatment, and co-infection with Mycobacterium are eventually observed (Matos et al., 2005, Schubach et al., 2005).

It is known that the clinical manifestations of the disease (Alexander and Russell, 1992, Berman, 1997, Marzochi and Marzochi, 1994) and the response to treatment are related in part to the Leishmania species involved (Grogl et al., 1992, Navin et al., 1992). However, little is known about the importance of biological variability within the same Leishmania species. Studies conducted in Rio de Janeiro suggest that only one zymodeme (Z 27) of L. (V.) braziliensis is involved in the disease in humans and animals (Cupolillo et al., 2003). Several methods can be applied to molecular epidemiology and to the study of genetic variability. Multilocus enzyme electrophoresis (MLEE) is widely used for the taxonomic characterization of Leishmania species (Cupolillo et al., 1994, Momen et al., 1985, Pacheco et al., 1999) but is limited because the marker is less polymorphic. Some techniques among others, are more frequently applied to the study of genetic diversity in trypanosomatids, including the restriction fragment length polymorphisms of kDNA (RFLP) analysis (Tojal Silva et al., 2006), random amplified polymorphic DNA (RAPD) analysis (Hanafi et al., 2001, Ishikawa et al., 2002, Martinez et al., 2003, Pacheco et al., 2005) and low stringency single specific primer—polymerase chain reaction (LSSP-PCR) analysis (Ferreira et al., 2007).

The objective of the present study was to determine the genetic variability among human ATL isolates in the State of Rio de Janeiro using MLEE as taxonomic tool and, RAPD and LSSP-PCR as polymorfic molecular markers to compare the panel of isolates obtained from lesions of patients with rare clinical manifestations of the disease with that of isolates obtained from patients with manifestations typically found in the State.

Section snippets

Samples and patients

A total of 34 Leishmania isolates obtained from tegumentary lesions of 32 patients living in the State of Rio de Janeiro and diagnosed at Centro de Referência em Leishmanioses, Instituto de Pesquisa Clínica Evandro Chagas, Fundação Oswaldo Cruz (CRLeish/IPEC/FIOCRUZ) were studied.

Group I consisted of 25 Leishmania isolates obtained from 23 patients with the following clinical and epidemiological characteristics of ATL rarely reported in this region: co-infection with Mycobacterium sp.,

Results

All of the 34 human samples analyzed by MLEE demonstrated similar electromorphic profiles which were similar to that of L. (V.) braziliensis reference strain (MHOM/BR/75/M2903). No isoenzymatic variants were observed between parasites isolated from groups I and II, or between isolates obtained from initial and reactivated lesions.

For RAPD analysis, all conditions such as reagents and DNA concentrations of the isolates and thermocycling conditions were the same during all experiments. Three

Discussion

The aim of the present investigation was to evaluate the genetic variability between Leishmania samples studied by RAPD and LSSP-PCR obtained from lesions of patients with typical or atypical clinical characteristics of ATL seen at the IPEC-Fiocruz outpatient clinic, Rio de Janeiro, Brazil.

The most frequent manifestation of ATL is a single cutaneous ulcer located in uncovered areas of the body, which presents a circular shape, elevated and well-defined borders and a granular base (Oliveira-Neto

Acknowledgments

This work received financial support from Instituto Kinder do Brasil (IKB), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ). C.Baptista benefited from funding by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Coordenação de Aperfeiçoamento do Pessoal de Ensino). A. Schubach is investigator of CNPq, Brazil. We also thank the Reference Center of Leishmaniasis—IPEC/Fiocruz

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