Tracking the Spread of a Naturally Occurring Leishmania infantum Mutant: A qPCR-Based Investigation in Strains and Clinical Samples

The circulation of Leishmania infantum strains carrying a 12 Kb genomic deletion (DEL) alongside wild-type (NonDEL) parasites in the Americas raises critical epidemiological questions. To support molecular surveillance efforts, we developed and validated a qPCR-based genotyping tool capable of detecting and quantifying DEL, NonDEL, and coinfection (MIX) profiles. The strategy targets a constitutive region present in all strains and a specific region exclusive to NonDEL parasites. It was validated using DNA from cultured parasites and applied to a panel of diverse clinical samples from dogs, humans, and other hosts across South and Central America. DEL genotypes were found to be more frequent and widespread, both in clinical samples and cultured parasites, suggesting their significant role in the transmission cycle. The tool’s application revealed coinfections in paired samples and enabled relative quantification of genotypes. It also showed good correlation with parasite load estimations and high discriminatory power when integrated with the Genotype function of real-time PCR software. This protocol provides a robust, scalable approach for monitoring L. infantum genotypes, with direct implications for understanding infection dynamics, treatment outcomes and public health surveillance.