Tracking the Spread of a Naturally Occurring Leishmania infantumMutant: 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 tools 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. Author SummaryLeishmania infantum is the parasite responsible for visceral leishmaniasis in the Americas. Recent studies have shown that two distinct genotypes--those carrying a specific gene deletion (DEL) and those without it (NonDEL)--coexist and circulate in the region. Understanding how these genotypes are distributed and whether they affect disease transmission or treatment is critical, especially because Miltefosine, a drug used to treat dogs, may not work equally well against both genotypes. In this study, we developed a simple and accurate molecular test to detect and quantify these genotypes in both cultured parasites and clinical samples from different animal hosts, including humans. We found that DEL genotypes are common and widely distributed, and that some animals carry both types of parasites simultaneously. Our method also distinguishes L. infantum from other related species, making it useful for diagnosis and surveillance. This tool can help researchers and health authorities monitor parasite populations more effectively and better understand how different genotypes might impact disease outcomes and control strategies.