Diagnosis and Characterization of Leishmania Species in Giemsa-Stained slides by PCR-RFLP

Abstract: Direct identification of Leishmania species in Giemsa-stained slides without parasite culturing in the areas where multiple species exist, is very helpful. This study was designed to isolate Leishmani spp. from Giemsa-stained smears and to characterize them by PCR technique. A total of 48 Giemsa-Stained slides from confirmed cases of leishmaniasis were examined under a light micro­scope at×1000 and classified based on grading of Leishmania parasites. DNA from each slide was extracted separately and sub­jected to PCR. The ribosomal internal transcribed spacer 1 (ITS1) was amplified with specific primers and the PCR prod­ucts were digested with a restriction enzyme (HaeIII). Of the 48 microscopy-positive slides, 43(89.6%) were positive by PCR-RFLP and Leishmania species were identi­fied. A statistically significant difference was observed between the both methods (P< 0.05) and also a concordance was found between microscopy and PCR-RFLP (k= 0.55). PCR-RFLP seems to be an effective method to identify Leishmania species from Giemsa-stained smears which have been collected from both infected humans and animal reservoir hosts in Iran.