Identification of Leishmania spp. by Molecular Amplification and DNA Sequencing Analysis of a Fragment of rRNA Internal Transcribed Spacer 2

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...

Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples

“…As a final verification, which could be considered optional, especially in settings where cost is a primary issue, a TaqMan assay specific for the infecting Leishmania species may be used. The inability of any primers to distinguish between L. chagasi and L. infantum is consistent with the current belief that these species seem to be genetically identical (16,42).…”

Sensitivity and Specificity of In Situ Hybridization for Diagnosis of Cutaneous Infection by Leishmania infantum in Dogs

“…Furthermore, parasitological culture may be difficult to perform due to poor adaptation of some isolates to the medium (1,10). The drawbacks of PCR are lack of standardization of the different protocols used among laboratories, possibility of contamination, and the fact that it does not necessarily indicate infection with live Leishmania (1,13,18,30). Serologic assays may yield false-positive results due to cross-reactivity with sera of dogs infected with L. braziliensis, T. cruzi, T. caninum, and Ehrlichia canis (15,(31)(32)(33).…”

Leishmaniasis in travelers: A literature review

“…PCR, especially using kinetoplast minicircle DNA or internal transcribed spacer of the multi-copy nuclear ribosomal genes, appears to be the best method for confirming a diagnosis of CL, because of the low parasite load needed, with a sensitivity of 98e100% and a specificity of 100% [118,121,122]. PCR has also proved superior to conventional methods in the diagnosis of ML, showing better sensitivity (97e100%) than the direct visualization of parasites in biopsies (hematoxylin-eosin staining) (33e57%), immunohistochemical testing of biopsy sections (69%), culture (67%), or serological testing (83%).…”