Use of PCR-restriction fragment length polymorphism analysis to identify the main new world Leishmania species and analyze their taxonomic properties and polymorphism by application of the assay to clinical samples.
At least 13 characterized Leishmania species are known to infect humans in South America. Five of these parasites are transmitted in the sylvatic ecotopes of the whole French Guianan territory and responsible for cutaneous leishmaniasis. For the diagnosis of cutaneous leishmaniasis, restriction fragment length polymorphism (RFLP) analyses have shown promising results. Thus, the end of the small subunit and internal transcribed spacer 1 of the rRNA genes were sequenced and targeted by PCR-RFLP analysis in the 10 main New World (NW) Leishmania species from the two subgenera. Then, the procedure was tested on 40 samples from patients with cutaneous leishmaniasis, and its results were compared with those of conventional methods. (i) The results of this simple genus-specific method were in agreement with those of previous isoenzyme analyses. (ii) This method distinguished the most medically relevant Leishmania species with only one enzyme (RsaI). (iii) This method could be performed directly on human biopsy specimens (sensitivity of 85.7%). Performing NW Leishmania species typing rapidly and easily in the field constitutes a very valuable improvement for detection of Leishmania spp. Revealing great diversity with several enzymes, this method could also be useful for taxonomic, ecological, and epidemiological studies in space and time.
Rotureau
Brice
B
Laboratoire Hospitalo-universitaire de Parasitologie et Mycologie Médicale, Equipe EA 3593, UFR de Médecine de l'Université des Antilles et de la Guyane, Campus Saint-Denis, BP 718, 97336 Cayenne, Guyane Française.
Ravel
Christophe
C
Couppié
Pierre
P
Pratlong
Francine
F
Nacher
Mathieu
M
Dedet
Jean-Pierre
JP
Carme
Bernard
B
D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis / Q000302:isolation & purification; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D006801:Humans; D007891:Leishmania / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D016773:Leishmaniasis, Cutaneous / Q000175:diagnosis* / Q000469:parasitology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013045:Species Specificity
D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer