NEW INSIGHTS INTO LEISHMANIASIS: IDENTIFICATION OF POTENTIAL DRUGS, INFECTION BIOMARKERS AND NON-INVASIVE DIAGNOSTIC APPROACHES

Leishmaniasis is a vector-borne parasitic disease and a major global health concern, caused by protozoa of the Leishmania genus. These parasites exist as promastigotes in insect vectors and amastigotes within host macrophages. In humans, the disease manifests in three primary forms: cutaneous, mucocutaneous and visceral leishmaniasis, the latter being potentially fatal if not diagnosed and adequately treated. In dogs, infection typically presents with skin lesions, organomegaly and fever. Currently, no effective vaccine is available and existing treatments are limited and often toxic, underscoring the urgent need for new therapeutic options. An effective management of this re-emerging disease needs combined strategies including the discovery of safer treatments and an accurate, early and non-invasive diagnosis. In this context, microRNAs (miRNAs), which several studies have been shown to be influenced by Leishmania infection, could serve as potential biomarkers for rapid diagnosis and disease monitoring. This thesis focuses on three major research areas in leishmaniasis: drug discovery, biomarker identification and the development of a non-invasive diagnostic approach. In the drug discovery section, four different compound libraries were screened against Leishmania infantum through phenotypic assays, leading to the identification of nine compounds that significantly reduced in vitro infection rates, two of which appear suitable for in vivo testing. Additionally, Gallium Protoporphyrin (GaPPIX), a heme analog, was evaluated as a repurposed compound. GaPPIX demonstrated potent activity against both L. infantum and L. major promastigotes by inhibiting cytochrome c oxidase (COX) activity. Notably, it showed synergy with miltefosine, a current anti-leishmanial drug, and exhibited low toxicity toward human cells. For biomarker identification, human, canine and murine in vitro infection models were used. In human and canine macrophage-like cells and/or in extracellular vesicles (EVs), miR-1246, miR-451, and miR-1290 were found to be dysregulated during L. infantum infection, suggesting their potential as diagnostic biomarkers. Instead, in L. amazonensis-infected murine bone marrow-derived macrophages miR-686, miR-99 and miR-342 were found dysregulated and their differential regulation was also validated ex vivo in lesions from infected mice. Lastly, a novel diagnostic approach based on a previously published qPCR assay was tested using conjunctival swabs as non-invasive clinical sample. This approach successfully diagnosed a clinical case of human visceral leishmaniasis, demonstrating its potential as a rapid, non-invasive diagnostic tool that could also facilitate in treatment monitoring.