The Accuracy of Diagnosis and Genotyping of Leishmania  Species Based on Spliced Leader Mini-Exon Gene by Nuclear Magnetic Resonance and Sequencing Assays

Mahyar  Khorram; Heidar  Masjedi; Fatemeh  Tabrizi; Mitra  Rezaei; Payam  Tabarsi; Majid  Marjani; Mihan  Pourabdoullah; Fatemeh-Maryam  Sheikholeslami

doi:10.18502/ijpa.v18i3.13756

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 Be-heshti University of Medical Sciences, Tehran, Iran
Fatemeh Tabrizi Department of Parasitology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Mitra Rezaei Genomic Research Center, 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 Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Be-heshti University of Medical Sciences, Tehran, Iran
Fatemeh-Maryam Sheikholeslami Pediatric Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Be-heshti University of Medical Sciences, Tehran, Iran

DOI: https://doi.org/10.18502/ijpa.v18i3.13756

Keywords: Proton nuclear magnetic resonance; Leishmaniasis; Spliced leader mini-exon gene; Genotyping; C. fasciculata

Abstract

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.