Genetic diversity, phylogeny, and population structure of Leishmania isolates from cutaneous leishmaniasis patients in Ethiopia

Abstract Cutaneous leishmaniasis (CL) in Ethiopia has historically been attributed almost exclusively to Leishmania aethiopica . However, recent molecular evidence indicated greater species complexity. This study assessed genetic diversity, geographic association and evolutionary patterns of CL isolates using ITS1 sequencing of 96 Slit skin smear samples from parasitologically confirmed cases. Of the 96 successfully typed isolates, (88.5%, 85/96) clustered with L. aethiopica , while 11.5% belonged to the L. donovani species complex or L . tropica , with uneven geographic distribution. Leishmania aethiopica displayed high haplotype diversity (Hd = 0.9929) and low nucleotide diversity (π = 0.0144), a pattern consistent with recent demographic expansion, and weak geographic differentiation (F ST ≈ 0.031). ITS1 sequencing of L. aethiopica samples revealed 96 high-confidence SNPs, including 11 fully fixed (frequency = 1) and 5 near-fixed (≥ 0.97) variants across all sites. Our findings revealed high genetic heterogeneity among L. aethiopica isolates and underscore a complex, multi-species aetiology driving the CL burden in Ethiopia. The fixed and near-fixed ITS1 SNPs identified in L. aethiopica may serve as targets for ecotype identification. Thus, enhanced molecular surveillance and genome-wide sequencing are recommended to elucidate transmission dynamics and guide clinical management of CL in Ethiopia.