Evaluative Assay of Nuclear and Mitochondrial Genes to Diagnose Leishmania Species in Clinical Specimens

BACKGROUND: Leishmaniasis as an emerging and reemerging disease is increasing worldwide with high prevalence and new incidence in recent years. For epidemiological investigation and accurate identification of Leishmania species, three nuclear and mitochondrial genes (ITS-rDNA, Hsp70, and Cyt b) were employed and analyzed from clinical samples in three important Zoonotic Cutaneous Leishmaniasis (ZCL) foci of Iran. METHODS: In this cross-sectional/descriptive study conducted in 2014–15, serous smears of lesions were directly prepared from suspected patients of ZCL in Turkmen in northeast, Abarkouh in center and Shush district in southwest of Iran. They were directly prepared from suspected patients and DNA was extracted. Two nuclear genes of ITS-rDNA, Hsp70 and one mitochondrial gene of Cyt b within Leishmania parasites were amplified. RFLP was performed on PCR-positive samples. PCR products were sequenced, aligned and edited with sequencher 4.1.4 and phylogenic analyses performed using MEGA 5.05 software. RESULTS: Overall, 203 out of 360 clinical samples from suspected patients were Leishmania positive using routine laboratory methods and 231 samples were positive by molecular techniques. L. major L. tropica, and L. turanica were firmly identified by employing different molecular genes and phylogenic analyses. CONCLUSION: By combining different molecular genes, Leishmania parasites were identified accurately. The sensitivity and specificity three genes were evaluated and had more advantages to compare routine laboratory methods. ITS-rDNA gene is more appropriate for firm identification of Leishmania species.