No abstract

Isoenzyme analysis of cultured parasites is the conventional approach for Leishmania species identification. Molecular approaches have the potential to be more sensitive and rapid. We designed PCR generic primers to amplify a segment of the rRNA internal transcribed spacer 2 (ITS2) from multiple Leishmania species. To validate the selected ITS2 fragment, we tested clinical specimens and compared the species results obtained by the molecular approach (PCR followed by DNA sequencing analysis) with those from the parasitologic approach (in vitro culture followed by isoenzyme analysis). Among the 159 patients with clinical specimens positive by both approaches, a total of eight Leishmania species were identified. The species results were concordant for all but two patients: for one patient, the results were Leishmania (Viannia) guyanensis by the molecular approach versus L. (V.) braziliensis by the parasitologic approach; for the other patient, the results were L. (Leishmania) tropica versus L. (L.) major, respectively. ITS2 PCR, followed by sequencing analysis, can be used to detect and discriminate among Leishmania species. The results confirmed our hypothesis that a region of the ITS2 gene can complement the characterization of Leishmania parasites at the species level. The approach we developed can be used as a diagnostic tool in reference laboratories with adequate infrastructure to perform molecular characterization of pathogens.Leishmaniasis encompasses multiple clinical syndromes, most notably, visceral, cutaneous, and mucosal forms (9,13,14,22,27,30). Leishmaniasis is endemic in focal areas in the tropics, subtropics, and southern Europe and is caused by species in the Leishmania and Viannia subgenera (14). Different species can be associated with diverse clinical manifestations and sequelae. Species identification can facilitate clinical management, such as decisions regarding whether/which treatment is indicated.Isoenzyme analysis of cultured parasites is the conventional diagnostic approach for Leishmania species identification. Molecular approaches have the potential to be more sensitive and rapid; e.g., the results can be available within days versus weeks or months. However, Leishmania PCR-based assays have varied in sensitivity, specificity, and utility for genus-or complexlevel versus species-level characterization (2, 3, 7, 10, 24-26, 28); some approaches have been highly sensitive for genuslevel detection, e.g., PCR methods that target minicircle kinetoplast DNA (kDNA), whereas others have been tailored for particular species/settings. We sought to develop a comprehensive molecular approach for characterizing Leishmania species; we focused on the rRNA internal transcribed spacer (ITS) region, which has been sequenced by other investigators and proposed as a target for molecular typing (12, 25). Here we describe our identification and validation of a diagnostic fragment that was amplified from the ITS2 with a PCR generic primer pair that we designed. Rather than conduct a formal evaluation of the m...