Novel qRT-PCR assay for the detection and quantification of male and female Plasmodium falciparum gametocytes in human populations

The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito malaria transmission. The detection and quantification of gametocytes as well as the estimation of the sex-ratio are essential to assess the human host ability to infect mosquitoes, and comprehensively evaluate malaria transmission risk. The mature gametocytes, responsible for parasite transmission, often circulate at low densities, therefore sensitive molecular tools are necessary to improve detection of low densities of gametocytes and submicroscopic infections to identify potential transmission reservoir. The aim of our work is to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on the detection and quantification of gametocyte-specific transcripts. RT-qPCR methods based on SYBR Green were developed for target genes expressed by male and female gametocytes. We selected two genes Pfs25 and Pfs230p known to be expressed in females and males respectively, and two new target genes GK (female) and Pf13(male). RNA was extracted from blood samples collected in a village of Burkina Faso. To overcome the inherent variability of RNA and different reverse transcription and PCR efficiencies, the quantification obtained for each target were normalized by human housekeeping gene (18S). Preliminary results obtained for 50 samples showed that, as expected, the assays are more sensitive than microscopic examinations. Pf13 male target gene seems to be more sensitive than Pfs230p. The normalization by 18S quantity, improved the correlation between gametocytes density estimated by microscopy and qPCR (correlation coefficient 0.13 vs 0.42). Ongoing work is aimed at the conversion of transcript copies/µl into gametocytes/µl and at sex-ratio determination.