PP 13 - Molecular confirmation of cutaneous leishmaniasis species in Sri Lanka: Evidence from genetic sequence analysis

Karunarathna TD¹, Dewasurendra R², Pathirana S³, Samaranayake N⁴  , ¹Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, ²The PDRU Laboratory, Department of Parasitology, Faculty of Medicine, University of Colombo,³Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, ⁴The PDRU Laboratory, Department of Parasitology, Faculty of Medicine, University of Colombo

Abstract

Introduction

Leishmania donovani which causes visceral leishmaniasis in other geographical regions has been identified as the causative agent of the cutaneous form of the disease in Sri Lanka. However, recent whole-genome sequencing has identified Leishmania tropica in a few Sri Lankan patients, suggesting greater species diversity with important implications for diagnosis and treatment.

Objectives

To identify and confirm the Leishmania species causing CL in Sri Lanka using molecular and bioinformatics methods.

 Design, setting, and methods

A cross-sectional laboratory-based study was conducted. Twenty-one lesion samples from patients with suspected CL, referred to the Parasitic Diseases Research Unit Laboratory, Department of Parasitology, Faculty of Medicine, University of Colombo, were evaluated. DNA was extracted from clinical samples (lesion aspirate/slit skin tissue in saline) positive by direct microscopy and culture using the QIAamp DNA Mini Kit and quantified by NanoDrop spectrophotometry. Genus-specific PCR was performed (for the ITS1 region using LITSR/L5.8S primers). Optimal conditions for annealing temperature, MgCl₂ concentration, and primer concentration were determined. PCR products of 10 samples underwent bidirectional Sanger sequencing followed by comparison with reference sequences and NCBI BLAST analysis.

 Results

Twelve out of the 21 samples were positive by microscopy and culture. Of these 11 gave positive results by PCR (11/12, 91.6%). Optimal conventional PCR conditions obtained were 53°C for annealing temperature, 1.5mM for MgCl₂ concentration, and 0.5μM primer concentration, yielding amplification at 320 bp. The samples evaluated by sequencing were from patients residing in Western (n=5), Southern (n=4) and North Western (n=1) provinces. All sample sequences (n=10) were 99% identical to the L. donovani sequence of GenBank sequence ID FJ753386.1 and 98% identical to the L. donovani sequence of GenBank sequence ID CL-9 (KT273408.1).

 Conclusions

The discrepancies among the diagnostic methods could be due to DNA degradation, low parasite burden, specimen quality issues, or PCR inhibition. Sequence analysis confirmed all the tested isolates were compatible with Leishmania donovani and adds to existing evidence on local patterns of circulating Leishmania species. An expanded study with larger sample numbers from different geographical locations is needed to comprehensively characterize Leishmania species diversity in the country.