Molecular-based assay for genotyping Leishmania spp. from clinically suspected cutaneous leishmaniasis lesions in the Garmian area, Kurdistan Region of Iraq

Abstract

Cutaneous leishmaniasis (CL) is highly prevalent in southern Iraq and neighboring countries, but is non-endemic to the Kurdistan Region of Iraq, particularly in the Garmian area. This study aimed to investigate the causative agent of CL at the molecular level by amplifying the small subunit (18S) rRNA and internal transcribed spacer 1 (ITS1) region. The present study was conducted from December 2019 to December 2020 at Kalar General Hospital, Kalar, Kurdistan Region, Iraq. Eighty-five clinical specimens were collected selectively from patients with suspected CL lesions via fine needle aspiration. After parasitic genomic DNA was extracted from the removed fluid, PCR and DNA sequencing targeting the 18S rRNA and ITS1 region were performed for molecular detection and species identification. Additionally, for 14 samples, the target bands of amplified DNA fragments for both 18S rRNA and ITS1 were extracted and sequenced via Sanger method using both the directional primers employed in the PCR. Seventy-one (83.53%) of the 85 suspected patients had CL, based on amplification of 18S rRNA and ITS1 via PCR. The sequence analysis revealed that all samples were Leishmania major. Phylogenetic analysis based on ITS1 was also performed. Our study revealed that our molecular method was an efficient technique for detecting CL and a valuable method for identifying Leishmania species in clinical samples. Sequence analysis indicated that the causative agent of CL in the Garmian area was L. major and the disease was rural in origin.