Detection and species identification of Old World Leishmania in clinical samples using a PCR-based method.
The aim of this study was to develop a simple, low-cost method for the detection and species differentiation of Leishmania directly from clinical samples, for routine use in a parasitology laboratory. A total of 87 samples was used, including 60 peripheral blood, seven bone marrow and 17 skin lesion material samples, derived from Greek patients with visceral or cutaneous leishmaniasis, and three reference strains. PCR was performed using primers designed to amplify the internal transcribed spacer 1 (ITS1) region of the rRNA gene. Identification of the Leishmania species studied was achieved by digestion with a single restriction endonuclease (RFLP), single-strand conformational polymorphism (SSCP) and DNA sequencing of the PCR-generated fragments. Typing identified all visceral and one cutaneous leishmaniasis strains as L. infantum, twelve of the cutaneous leishmaniasis strains as L. tropica and four as L. major. The described PCR method proved efficient for the detection of pathogenic Leishmania species in various clinical samples, most importantly in peripheral blood samples. Furthermore, PCR followed by a simple RFLP using a single restriction endonuclease was capable of identifying all Leishmania species commonly encountered in Greece.
Department of Parasitology, Entomology and Tropical Diseases, National School of Public Health, 196 Alexandras Ave, 11521 Athens, Greece. grspan@yahoo.com
Gregory
G
Spanakos
Evangelia-Theophano
ET
Piperaki
Panagiotis G
PG
Menounos
Nicholas
N
Tegos
Alexander
A
Flemetakis
Nicholas C
NC
Vakalis
D000818:Animals; D016054:DNA, Protozoan / Q000032:analysis* / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000032:analysis* / Q000235:genetics; D018503:Genome, Protozoan / Q000235:genetics; D006115:Greece / Q000453:epidemiology; D006801:Humans; D007891:Leishmania / Q000145:classification / Q000302:isolation & purification*; D007896:Leishmaniasis / Q000175:diagnosis* / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D016054:DNA, Protozoan; D021903:DNA, Ribosomal Spacer