Chagas disease remains a major neglected tropical disease, highlighting the need for new anti-Trypanosoma cruzi compounds and improved approaches for their phenotypic evaluation. Here, we extended a previously developed Lab-on-a-Chip (LOC) platform integrating a serial dilution generator and automated label-free motility analysis (TR_KF) to the direct screening of extracellular T. cruzi trypomastigotes. The platform was validated in two steps using benznidazole as the reference drug. First, automated motility quantification in conventional 96-well plates was compared with manual Neubauer counting, yielding comparable concentration–response curves and IC₅₀ values. Second, the complete LOC–TR_KF platform was compared with the conventional assay, producing an IC₅₀ that was not significantly different from the reference method. The validated platform was subsequently applied to screen ten candidate samples, identifying combinatorial libraries 2161-X and 2160-X as the most active against trypomastigotes. Comparison with previously reported intracellular amastigote data revealed marked stage-dependent differences in compound activity, while pre-treatment with both libraries reduced parasite infectivity by approximately 50%. Overall, the integrated LOC–TR_KF platform provides pharmacological responses comparable to conventional assays while reducing assay-solution requirements, manual handling and enabling automated, label-free phenotypic screening of T. cruzi trypomastigotes.

Leishmaniases remain a significant global public health threat, with Leishmania amazonensis and Leishmania infantum representing the etiological agents of the cutaneous and visceral forms in the Americas, respectively. Building on our previous identification of Phospholipase A ₁ (PLA ₁ ) in Leishmania braziliensis , this study provides a comprehensive molecular, immunological, and biochemical characterization of PLA ₁ in L. amazonensis and L. infantum promastigotes. We analyzed PLA ₁ activity and expression, purified the recombinant enzyme from L. amazonensis , and validated protein expression using a specific anti-PLA ₁ serum. The major contribution of this research is the first description of the subcellular localization of a PLA ₁ within the Leishmania genus. Moreover, our results reveal an unprecedented association between PLA ₁ and lipid droplets within the parasites. This discovery is of particular interest as it provides the first evidence linking this enzyme to lipid storage organelles in Leishmania . Given that PLA ₁ is an established virulence factor in other trypanosomatids, these findings suggest a specialized role for the enzyme in parasite lipid metabolism and potentially in its pathogenic mechanisms, opening new perspectives for understanding Leishmania biology.