Two quantitative multiplex assays for the detection of P. falciparum asexual rings and male/female gametocyte developmental stages from human tissues

Abstract Background Malaria, caused by Plasmodium parasites, remains a significant global health challenge. Gametocytes are the sexual forms of the parasites which do not cause disease but are responsible for parasite transmission to the mosquito vector, often even after successful treatment of infected individuals. The density and sex ratio of gametocytes play a major role in determining malaria transmission dynamics. Sensitive methods for detecting and quantifying gametocyte developmental stages and sex ratios are critical tools for achieving Plasmodium transmission reduction in the malaria elimination agenda. Methods Two sensitive triplex real-time PCR gametocyte sex-specific (Assay I) and gametocyte developmental stage-specific (Assay II) assays were developed. Assay I was designed for the detection and quantification of male and female gametocytes and of asexual rings, based on the amplification of male (PF3D7 1311100, Pf13), female (PF3D7 0903800, CCp4) gametocyte- and asexual ring- (PF3D7_0501300, SBP1) transcripts. Assay II was designed for the detection and quantification of transcripts marking early (PF3D7_1477700, PHISTa), intermediate (PF3D7_1346700, Pfs48/45) and late (PF3D7_1031000, Pfs25) stage gametocytes. Assays were tested and validated using 10 matched treatment day 0 and 7 venous blood samples from a study evaluating gametocyte immune responses in children (GI study) and 10 matched peripheral blood samples and bone marrow aspirates obtained from a study investigating gametocyte development in semi-immune Ghanaian individuals (BM study). Results Assay I detected transcripts of ring stages (SBP1), male (Pf13) and female (CCp4) gametocytes in 40%, 20% and 70% of the samples in the GI study and in 60%, 30% and 70%, respectively, in samples of the BM study. Assay II detected transcripts of early (PHISTa), intermediate (Pfs48/45) and late stage (Pfs25) gametocytes in 40%, 50% and 40% of the samples from peripheral blood compared to 70%, 20% and 40%, respectively, from bone marrow aspirates of the BM study. Conclusions The one-step multiplex assays described here can be readily used to measure densities of male and female gametocytes for sex ratio determinations and to quantify proportions of gametocyte developmental stages in peripheral blood samples and in bone marrow aspirates of malaria-infected individuals.