Development of molecular assays on Plasmodium falciparum gametocytes for functional analysis and novel diagnostics on malaria parasite transmission

Transmission of P. falciparum malaria parasite from human host to mosquito is mediated by gametocytes in human peripheral blood. Both male and female gametocytes are required for the development of the parasite inside a female Anopheles mosquito. This work aims first to develop a molecular method for detecting gametocyte sex ratios as a parameter to predict parasite infectiousness to mosquito and then to investigate the effects of antimalarial compounds on gametocytes from natural infections. The results were compared with Direct Membrane Feeding Assay (DMFA) on mosquito data. Two multiplex RTqPCR assays, targeting sex-specific transcripts (pfCCp4 and pfs25 for females, pfMGET for males), were developed for simultaneous assessment of male and female gametocytes. These assays were applied to three laboratory lines to measure their sex ratio by using two analytical approaches: the DCt and the TSEX RATIO. The results were compared with morphological sex ratios obtained by immunofluorescence assays (IFA). A linear relation exists between molecular parameters obtained in RTqPCT and the sex ratios determined by IFA analysis only in the lines NF54 and HB3 sel 4 lines but not in the case of the transgenic line pfDynGFP/P47mCherry. TSEX RATIO parameter represents a novel approach to track the copy number of sex-specific transcripts in a gametocyte population offering indirect insights into how the proportion of male and female gametocytes evolves over time and under various experimental conditions. The above RTqPCR assays were used on gametocytes from infected individuals treated with gametocytocidal compounds to evaluate their effect on sex specific transcripts. The data of these assays were analyzed with ΔΔCt method. Molecular analysis performed after 24 hours of treatment did not align with DMFA outcomes. However, compound effects on transcript abundance became evident after an extended cultivation period of 48 hours following the 24 hours of treatment in NF54 gametocytes