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Journal of Clinical Microbiology, July 2007, p. 2110-2115, Vol. 45, No. 7
0095-1137/07/$08.00+0     doi:10.1128/JCM.02555-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Detection and Identification of Leishmania Species from Clinical Specimens by Using a Real-Time PCR Assay and Sequencing of the Cytochrome b Gene

Laboratoire de Parasitologie-Mycologie, Hôpital Henri Mondor-APHP and UMR BIPAR 956, Créteil, France,¹ Pôle de Recherche Biomédicale and Unité d'Immunologie Moléculaire des Parasites, Institut Pasteur, Paris, France,² Centre National de Référence des Leishmanies, Montpellier, France,³ Laboratoire de Biologie Moléculaire, Hôpital Américain de Paris, Neuilly, France⁴

Received 20 December 2006/ Returned for modification 15 February 2007/ Accepted 25 April 2007

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.

Published ahead of print on 2 May 2007.

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