Drug-Resistance Biomarkers in Leishmania infantum through Nanopore-Based Detection of Aneuploidy and Gene Copy Number Variations with LeishGenApp

The emergence of drug-resistant strains of Leishmania infantum, a parasite that infects dogs and humans, poses an increasing threat, underscoring the need for a One Health framework for adequate control. Aneuploidy and gene copy-number variation (CNV) are associated with resistance to first-line drugs. We analyzed aneuploidies, genome plasticity, and 15 CNVs as drug-resistance biomarkers in 35 L. infantum isolates from human and canine host across the Mediterranean by nanopore-only whole-genome sequencing and the automated diagnostic platform LeishGenApp. Results highlighted different levels of aneuploidy turnover among chromosomes and characterized the structural organization of resistance-associated genomic regions, emphasizing the role of genomic repeated sequences in the extended H-locus, METK and SMT locus. At least one genomic resistance biomarker was detected in 80% of the Mediterranean isolates, being the most prevalent to antimonials (50%) and allopurinol (50%) in canine isolates and to antimonials (61%) and paramomycin (38%) in human isolates. Monoresistance (50%) predominates in dogs, while resistance to two drugs is most common in human isolates (38%). Differences in genomic resistance biomarkers prevalence in our cohort underscore the importance of targeted drug policies to mitigate resistance dissemination. Our results provide an effective approach for detecting aneuploidies and CNVs as drug-resistance biomarkers in Leishmania parasites by nanopore-only whole-genome sequencing. This powerful tool is significant for monitoring resistant L. infantum strains, particularly in dogs, the primary reservoir, lessening resistance-related relapses, limiting resistant Leishmania transmission by sand flies, and shaping One Health strategies.