Multilocus microsatellite typing (MLMT) reveals high genetic diversity of Leishmania infantum strains causing tegumentary leishmaniasis in northern Italy

Abstract Background Tegumentary leishmaniasis (TL) caused by Leishmania infantum has re-emerged in northern Italy, raising questions about the genetic diversity and population structure of circulating parasites and their potential role in shaping different clinical outcomes. Methodology/Principal findings Multilocus microsatellite typing (MLMT) based on 15 polymorphic loci was applied to 44 L. infantum strains obtained from TL cases diagnosed between 2013 and 2024 in the Emilia-Romagna region. These strains were compared with sympatric isolates from VL cases, dogs and sand flies. MLMT revealed a considerable genetic variation among TL-associated strains, with 43 distinct multilocus genotypes identified. Population structure analyses using Bayesian clustering, multivariate approaches and phylogenetic reconstruction consistently identified three highly differentiated genetic populations (F st >0.25). TL strains were divided into two main populations: one shared with VL-associated strains (PopB; 9/44) and a second population found exclusively among TL cases (PopC; 28/44). In contrast, the canine-associated population (PopA) showed no overlap with TL cases in this region. Populations also displayed divergent heterozygosity patterns, as indicated by positive and negative Fis values. Conclusions/Significance These findings revealed previously unknown diversity within L. infantum in the study area and demonstrated that inclusion of tegumentary strains is essential to uncover hidden components of parasite population structure. The identification of a TL-associated population supports the existence of multiple evolutionary pathways and emphasises the importance of integrated One Health surveillance, which combines data from humans, animal hosts and vectors to improve understanding of the epidemiology of leishmaniasis in Italy. Author summary Leishmania infantum is a parasite transmitted to humans through the bite of infected insect vectors. It can cause different forms of leishmaniasis, ranging from a systemic disease known as visceral leishmaniasis to a less common form that affects the skin and mucous membranes, called tegumentary leishmaniasis. Dogs are the main reservoir of the parasite and play a key role in maintaining its circulation in endemic areas. In recent years, cases of tegumentary leishmaniasis have re-emerged in northern Italy. This unexpected increase has raised questions about how the parasite is spreading and whether genetic differences among the parasites could explain these new patterns. To explore this, we examined parasitic DNA obtained from tegumentary leishmaniasis cases and compared it with DNA from patients with visceral leishmaniasis, from dogs and insect vectors from the same area. By examining multiple genetic markers, we found that parasites causing the tegumentary form of the disease are genetically diverse and belong to different groups. Notably, one parasite group was found only in cases of tegumentary leishmaniasis and not in visceral infections nor in infected dogs, suggesting that some parasite lineages may be more closely associated with skin and mucosal disease. Overall, our findings show that studying parasites from cutaneous and mucosal lesions provides important information that would otherwise remain hidden. By combining data from humans, animals and insect vectors, our study highlights the importance of integrated surveillance systems for improving our understanding of disease spread and supporting effective public health strategies.