Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 18 3 2023 09 22 331 341 EN Mahyar Khorram Department of New Sciences and Technologies, South Tehran Branch, Islamic Azad University, Tehran, Iran Heidar Masjedi Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran Fatemeh Tabrizi Department of Parasitology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran Mitra Sadat Rezaee 1. Genomic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2. Clinical Tuberculosis and Epidemiology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran Payam Tabarsi Clinical Tuberculosis and Epidemiology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran Majid Marjani Clinical Tuberculosis and Epidemiology Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran Mihan Pourabdoullah Toutkaboni Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran Fatemeh Sheikholeslami 1. Pediatric Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran 2. Department of Molecular Biology, Dr. Khosroshahi’s Pathobiology Laboratory, Tehran, Iran 2022 10 24 2023 08 12 Background: We aimed to evaluate the accuracy of genotyping of Leishmania species by the spliced leader mini-exon gene. Methods: Suspected leishmaniasis patients, referred to Masieh Daneshvary Hospital, Tehran, Iran were included from May 2017 to September 2021. The Leishmania species were genotyped by PCR-RFLP based on the SL mini-exon gene and the ITS1 region of SSU-rRNA gene and compared with the sequencing results. The expressed metabolites of metacyclic promastigotes were evaluated by Proton nuclear magnetic resonance (1H-NMR). Results: Out of 66 suspected cases, 36 (54.4%) were positive for Leishmania species based on the PCR assays. In 21 (31.8%) cases, promastigotes grew on culture tubes. Based on the RFLP of SL RNA profile, 13 (19.7%) L. tropica, 9 (13.6%) L. major, 3 (4.5%) L. infantum, and 8 (12.1%) C. fasciculata isolates, isolated from culture media, were identified; however, 3 (4.5%) cases were unidentifiable due to the low number of parasites. Seventeen metabolites were expressed by the metacyclic forms of L. major, L. tropica and C. fasciculata isolates. The top differential metabolites expressed more in C. fasciculata were FAD, p-Methoxybenzyl alcohol and S-b-G-5, 5-G-b-S (A = CH2) (P<0.005) whereas Veratryl glycerols and D-(+)-Mannose were significantly increased in L. major and Betulin, L-Tyrosine in L. tropica (P<0.01). Conclusion: The invaluable techniques such as sequencing and 1H-NMR confirmed the results of genotyping of Leishmania species based on the SL mini-exon gene.  SL mini exon gene can be used as a diagnostic tool to differentiate various Leishmania genotypes and detect contamination of culture media with C. fasciculata.  https://ijpa.tums.ac.ir/index.php/ijpa/article/view/3765 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/3765/1306