Microscopic and Molecular Detection of Leishmania Species Among Suspected Patients of Cutaneous Leishmaniasis Using ITS-r DNA in Fars Province

Fars province is one of the most important foci of leishmaniasis that includes two types of cutaneous (urban and rural forms) and visceral leishmaniasis in sympatry. To study leishmaniasis among suspected patients of cutaneous leishmaniasis in 5 counties of Shiraz, Firouz abad, GhirـKarzin, Farashband and Larestan, both microscopic and molecular analysis were carried out in the present study. The samples were smeared on the microscopic slides and were also stained by Giemsa. All smears were examined under a light microscope and the positive smears were scored for amastigote frequency. DNA was extracted from stained smears and Leishmania DNA was detected by amplification of ITS1ـ5. 8sـITS2 fragments. Amplicons were analyzed using electrophoresis in 1. 5 % agarose gel containing ethidium bromide. Among 34 studied patients, 29 cases (%85) were positive in microscopic and 32 (%94) in molecular analysis using standard PCR. All examined samples were infected with L. major except one (%3) that was infected with L. tropica. Most lesions due to L. major were located on the feet, whereas ulcer due to L. tropica was on the forehead. Preparing stained smears from active lesions of suspected patients (microscopic analysis) removes the problem of Leishmania preservation and transition to culture media. Also, intergenic variations in amplified fragment of ITS-rDNA cause to produce fragments with different length and based on this difference, we can identify L. major and L. tropica separately. Using microscopic and molecular methods in present study confirmed presence of L. major and L. tropica as the causative agents of CL in studied regions of Fars.