Abstract
This study analyzed DNA minicircles of Mexican isolates of L. (Leishmania) mexicana to look for genetic differences between strains isolated from patients with diffuse cutaneous (DCL) and localized (LCL) leishmaniasis. The kDNA was analyzed using polymerase chain reaction (PCR), restriction fragment polymorphism analysis of the PCR products (PCR-RFLP) and the PCR products were sequenced. In the PCR with primers specific for the subgenus Leishmania, the Mexican isolates gave higher amplification products than the other L. mexicana complex strains and with specific primers for the L. mexicana complex they were poorly amplified. In the PCR-RFLP analysis with the Eco RV, Hae III, and Mbo I endonucleases, the Mexican isolates displayed similar restriction patterns, but different from the patterns of the other members of the L. mexicana complex. In the phylogenetic tree constructed, the kDNA sequences of the Mexican clones formed two groups including sequences of LCD or LCL clones, apart from the other L. mexicana complex members. These results suggest that the kDNA minicircles of the Mexican isolates are more polymorphic than the kDNA of other members of the L. mexicana complex and have different recognition sites for the restriction enzymes used in this study.
Highlights
Cutaneous leishmaniasis (CL) is the most widespread form of leishmaniasis, causing primary localized skin lesions (LCL) from which parasites can disseminate to the nasopharyngeal mucosa and cause the disfiguring lesions typical of mucocutaneous leishmaniasis (MCL) or disseminated to the entire body as nodular lesions (DCL) [1].American cutaneous leishmaniasis is characterized by a spectrum of clinical presentations
These include LCL caused by L. (Leishmania) mexicana, DCL caused by L. (Leishmania) amazonensis, L. (Leishmania) venezuelensis, and L. (Leishmania) pifanoi, all belonging to the Leishmania mexicana complex, and MCL caused by members of the Leishmania braziliensis complex [2]
Polymerase chain reaction (PCR) specific for the Leishmania subgenus performed with the AJS1 and DeB8 primers resulted in the amplification of the kinetoplast DNA (kDNA) of L. (L.) mexicana BEL21 and L. (L.) mexicana M379, the reference strains, and the Mexican isolates giving 700 to 870 bp amplification bands; L. (L.) amazonensis M2269 and PH8 as well as L. (L.) garnhami HM76 and JAP78 gave bands less than 700 bp (Figure 2)
Summary
American cutaneous leishmaniasis is characterized by a spectrum of clinical presentations. (Leishmania) pifanoi, all belonging to the Leishmania mexicana complex, and MCL caused by members of the Leishmania braziliensis complex [2]. Cutaneous leishmaniasis in Mexico is highly endemic with broad geographical distribution of the different clinical manifestations. LCL or MCL can be caused by members of both L. mexicana and L. braziliensis complexes [3], making more accurate analysis and identification of the Leishmania strains imperative so that opportune and appropriate treatment can be administered. Several PCR molecular targets have been developed, including minicircle kinetoplast DNA (kDNA) [3], the miniexon (spliced leader RNA) gene [4], and the gp PCR-RFLP [5], among others
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