Digital PCR-based quantification of Leishmania infantum in Lutzomyia longipalpis: comparison between promastigote and macrophage-derived amastigote infection models.

Visceral leishmaniasis (VL) remains a major public health concern in Latin America, particularly in Brazil, where Leishmania infantum is primarily transmitted by the sand fly Lutzomyia longipalpis. Understanding parasite development within the vector is essential for elucidating transmission dynamics, yet classical microscopic assessments often lack sensitivity for detecting early or low intensity infections. In this study, Lu. longipalpis females were experimentally infected with either promastigote or amastigote forms of Le. infantum and the infection dynamics were evaluated by both optical microscopy and digital PCR (dPCR). Microscopic examination revealed successful midgut colonization in both groups, with higher parasite counts in amastigote-infected sand fly females during the early phase of infection. Quantification by dPCR confirmed this pattern, detecting significantly higher parasite loads at the initial time points in amastigote-fed sand flies. However, parasite loads converged between groups at later stages, indicating comparable infection dynamics regardless of the parasite infective form. Both methods showed consistent patterns of parasite dynamics, whereas dPCR provided greater sensitivity for detecting and quantifying low parasite burdens, particularly during periods of reduced infection intensity. Overall, our findings provide new insights into parasite-vector interactions and validate dPCR as a precise and sensitive tool for assessing Leishmania infections in sand fly vectors, with potential applications for entomological surveillance and transmission studies.