Advances in leishmaniasis: development of molecular diagnostic approaches and identification of new compounds against Leishmania (Leishmania) infantum

Leishmaniases are a group of anthropo-zoonic parasitic diseases caused by a protozoan belonging to Leishmania genus with a worldwide distribution that affects mainly the developing countries. The parasite has different hosts such as humans, dogs, and wild animals. Despite the surveillance programs introduced by the WHO, the disease still causes relevant problems in poor countries. Moreover, the treatments available are few and present several limitations such as toxicity, difficulty in administration, relapse, high production cost, and the appearance of resistant strains. In this view, the aims of the thesis were the development of molecular assays mainly based on qPCR and HRM analysis to differentiate the most common pathogenic species for humans and the discovery of new classes of compounds more effective and safer against L. infantum. We established a diagnostic algorithm involving 3 different SYBR green-based qPCR assays for the effective discrimination among Viannia subgenus, L. mexicana complex and L. (L.) infantum, appliable on clinical samples without the parasite isolation. On the other hand, we tested a library of 3,3’- diindolylmethane (DIM) derivatives for their anti-leishmanial potential, first in terms of efficacy on L. infantum promastigotes and then on intracellular amastigotes. The results allowed us to find new promising classes of compounds never tested before with a good IC50 laying the bases for future studies of optimization in term of pharmacokinetics, pharmacodynamics and bioavailability. Once entered in the host macrophages, Leishmania promastigotes are able to subvert the innate immune response and metabolic pathways of the host. The characterization of the molecular mechanisms underlying Leishmania infection and the pathogen survival in host cells can contribute to identifying new targets for therapeutic approaches. Since dogs represent one of the most important reservoirs of infection for leishmaniasis, in the optic of a "one health" approach, we evaluated the gene expression of some ER stress-related genes and miR-346 by qPCR in a canine macrophage cell line (DH82) infected by Leishmania spp. and we compared the presence of the micro-RNA cfa-miR-346 in plasma of dogs non-infected and naturally infected with L. infantum. The results in DH82 cells showed that cfa-mir-346 was induced post-infection with all Leishmania strain/isolates tested. Moreover, the cfa-miR-346 expression analysis on dogs’ plasma revealed a significant upregulation in infected dogs compared to non-infected dogs identifying the miRNA-346 as an infection marker in dogs affected by leishmaniasis. In summary, this thesis illustrates our attempt to contribute to the improvement of both diagnostics and therapeutics, in the context of a one health approach, to reduce the burden of this complex and heterogeneous disease.