Detection and Identification ofLeishmaniaSpecies from Clinical Specimens by Using a Real-Time PCR Assay and Sequencing of the CytochromebGene
- 1 July 2007
- journal article
- Published by American Society for Microbiology in Journal of Clinical Microbiology
- Vol. 45 (7), 2110-2115
- https://doi.org/10.1128/jcm.02555-06
Abstract
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 >or=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.Keywords
This publication has 37 references indexed in Scilit:
- Advances in leishmaniasisThe Lancet, 2005
- Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High SensitivityJournal of Clinical Microbiology, 2004
- Miltefosine for New World Cutaneous LeishmaniasisClinical Infectious Diseases, 2004
- Antigen Genes for Molecular Epidemiology of Leishmaniasis: Polymorphism of Cysteine Proteinase B and Surface Metalloprotease Glycoprotein 63 in theLeishmaniadonovaniComplexThe Journal of Infectious Diseases, 2004
- Real-Time PCR Assay for Clinical Management of Human Immunodeficiency Virus-Infected Patients with Visceral LeishmaniasisJournal of Clinical Microbiology, 2003
- Real-Time PCR as a New Tool for Quantifying Leishmania infantum in Liver in Infected MiceClinical and Diagnostic Laboratory Immunology, 2001
- Genetic typing and phylogeny of the Leishmania donovani complex by restriction analysis of PCR amplified gp63 intergenic regionsParasitology, 2001
- Leishmaniasis presenting as a dermatomyositis-like eruption in AIDSJournal of the American Academy of Dermatology, 1996
- Subcellular distribution and characterization of glucosephosphate isomerase in Leishmania mexicana mexicanaMolecular and Biochemical Parasitology, 1994
- Placebo-Controlled Clinical Trial of Sodium Stibogluconate (Pentostam) versus Ketoconazole for Treating Cutaneous Leishmaniasis in GuatemalaThe Journal of Infectious Diseases, 1992