Combination of Cpb-Hsp70 typing methods reveals genetic divergence between Leishmania infantum strains causing human tegumentary leishmaniasis in northern Italy and central Spain: a retrospective study

Background: Tegumentary leishmaniasis (TL) caused by Leishmania infantum is an overlooked yet re-emerging disease endemic in Mediterranean Europe. Currently, no standardized molecular surveillance of circulating Leishmania strains is performed in European endemic areas, despite the potential public health implications of parasite biodiversity. This study aims to characterize parasite population haplogroups causing TL in two active endemic areas in southern Europe, i.e. Bologna (northern Italy) and Fuenlabrada (central Spain). Methods: In this retrospective study, we typed 87 L. infantum samples from TL cases in the areas of Bologna and Fuenlabrada; these areas hosted the main European foci of human TL occurring in the last 15 years. Two Leishmania genomic typing targets were used: the heat shock protein 70 (Hsp70) and the cysteine peptidase b (Cpb). Simpson’s index was used to calculate the discriminatory power of the used typing methods. Results: Typing results depicted the presence of a heterogeneous parasite population circulating in Bologna with two main haplogroups, i.e. Hsp70(A)_Cpb(F) (n = 7, 30.4%) and Hsp70(G)_Cpb(E/F) (n = 7, 30.4%), differing from the reference L. infantum strain JPCM5 haplogroup and partially overlapping with L. donovani lineages. Among the samples from Fuenlabrada, n = 19 samples were typed by both targets, revealing a homogeneous population expressing Hsp70(A) and Cpb(E), matching the JPCM5 reference strain haplogroup. Overall, the Cpb typing method exhibited higher discrimination power as compared to the Hsp70 method (Simpson’s index of diversity, P-value < 0.05). Conclusions: Our findings show differences among L. infantum populations causing TL in two southern European epidemiological foci of human leishmaniasis and support the recent discovery of L. infantum/L.donovani hybrid strains circulating in northern Italy. These results underscore the critical need to identify the circulating Leishmania strains in endemic areas and assess their potential public health implications in active foci.