Detection of Leishmania Parasites from Cutaneous Leishmaniasis Patients with Negative Direct Microscopy using NNN and PCR-RFLP

Abstract

Background: Leishmaniasis is a parasitic infectious disease caused by a protozoan parasite from trypanosomatidae family with a wide spectrum in tropical and subtropical areas. The purpose of the study was to characterize various species of Leishmania isolates from Isfahan, Iran with negative direct smears. Methods: This study used culture methods and PCR-RFLP to detect cutaneous leishmaniasis (CL) in patients who referred to the Center for Research in Skin Disease and Leishmaniasis, Isfahan University of Medical Sciences, Isfahan Iran, with negative direct smears. Samples taken from the lesions of patients were cultured on NNN and RPMI1640 mediums. Out of 100 smear negative samples, 50 turned positive for leishmania promastigotes whose DNA was extracted. The amplified ITS1 region of DNA was then analyzed through PCR-RFLP. After digesting the isolates by HaeIII enzyme, the species were determined according to standard strains. Findings: Among 50 isolates from Isfahan region, 46 isolates were identified as L. major and 4 as L. tropica. Conclusion: The present research showed that PCR-RFLP is an accurate method for specifying agent species of CL. It was also found that L. major and L. tropica were the main two species responsible for various kinds of skin involvement in the region.

Keywords

• PCR-RFLP
• Cutaneous leishmaniasis
• Isfahan
• Parasite