Flow cytometry-based evaluation of hepatic infection by non-fluorescent Plasmodium parasites

The complex life cycle of Plasmodium parasites, involving both liver and blood stages of infection in the mammalian host, presents significant challenges for malaria research. Although advances have been made in malaria vaccination and treatment strategies, important gaps in our understanding of the asymptomatic liver stage of Plasmodium infection remain. While reporter gene-expressing parasites are commonly used for drug screening and parasite biology studies during this phase of the Plasmodium life cycle, tools for assessing and quantifying hepatic infection in the absence of parasite-encoded reporter genes are limited. Here, we present a novel flow cytometry-based method that enables the quantitative assessment of infection of hepatic cells by non-fluorescent Plasmodium parasites. This method uses two parasite proteins, heat shock protein 70 (HSP70), found in the parasite cytoplasm, and upregulated in infectious sporozoites 4 (UIS4), located on the parasitophorous vacuole membrane, as markers for parasite detection and quantification. We demonstrate that the use of these markers facilitates the rapid and cost-effective quantification of hepatic infection and intracellular development of Plasmodium parasites devoid of fluorescent reporter genes. This method addresses critical regulatory and technical challenges to the evaluation of reporter-free whole-sporozoite vaccine candidates and could serve as a versatile tool for broader malaria research.