Visceral and cutaneous leishmaniases are heterogenous entities. The Leishmania species that a given patient harbors usually cannot be determined clinically, and this identification is essential to prescribe the best species-specific therapeutic regimen. Our diagnosis procedure includes a real-time PCR assay targeted at the 18S rRNA gene, which detects all Leishmania species but which is not specific for a given Leishmania species. We developed a species identification based on sequencing of the cytochrome b (cyt b) gene directly from the DNA extracted from the clinical specimen. The sequences were analyzed using the Sequence Analysis/Seqscape v2.1 software (Applied Biosystems). This software is designed to automatically identify the closest sequences from a reference library after analysis of all known or unknown polymorphic positions. The library was built with the Leishmania cyt b gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cyt b gene was amplified in the 53 specimens. Sequencing resulted in the identification of six different species with >99% identity with the reference sequences over 872 nucleotides. The identification was obtained in two working days and was in accordance with the multilocus enzyme electrophoresis identification when available. Real-time PCR followed by sequencing of the cyt b gene confirmed the diagnosis of leishmaniasis and rapidly determined the infecting species directly from the clinical specimen without the need for the isolation of parasites. This technique has the potential to significantly accelerate species-adapted therapeutic decisions regarding treatment of leishmaniasis.The leishmaniases are a group of parasitic diseases of major and growing public health importance (12,22). Leishmaniasis is endemic in many countries that are destinations for millions of travelers or migrant workers from Northern countries, including patients with immunodepression, each year (9). About 21 Leishmania species have been reported to cause human infection (12). Some species causing cutaneous leishmaniasis, mainly Leishmania braziliensis and to a lesser extent L. panamensis and L. guyanensis, are associated with the risk of delayed mucosal leishmaniasis, and the response to antileishmanial agents is influenced by the species (16, 23). Although the clinical presentation of cutaneous leishmaniasis is influenced by the infecting species (13, 22), on an individual basis, the clinical presentation is not specific enough to allow a reliable species determination (1, 7, 13, 32). Identification of the species can also help predict the risk of dissemination in immunocompromised patients (10, 11). Thus, species identification is important to determine the clinical prognosis and to select the most appropriate therapeutic regimen to be administered to each individual.The reference positive diagnosis methods for leishmaniasis, i.e., direct smear examination and culture, have important limitations (8). Direct examinatio...

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