Open AccessDetection and Identification of Leishmania Species from Clinical Specimens by Using a Real-Time PCR Assay and Sequencing of the Cytochrome b Gene
Author(s) -
Françoise Foulet,
Françoise Botterel,
Pierre Buffet,
Gloria Morizot,
D. Rivollet,
M. Deniau,
Francine Pratlong,
J. F. da V. Costa,
Stéphane Bretagne
Publication year - 2007
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.02555-06
Subject(s) - biology , leishmania , genbank , gene , leishmaniasis , cytochrome b , dna sequencing , visceral leishmaniasis , polymerase chain reaction , genetics , identification (biology) , computational biology , microbiology and biotechnology , mitochondrial dna , parasite hosting , botany , world wide web , computer science
Visceral and cutaneous leishmaniases are heterogenous entities. TheLeishmania 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 allLeishmania species but which is not specific for a givenLeishmania species. We developed a species identification based on sequencing of the cytochromeb (cytb ) 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 theLeishmania cytb gene sequences available in GenBank. Fifty-three consecutive real-time PCR-positive specimens were studied for species identification. The cytb 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 cytb 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.