Real-time PCR assays for detection and quantification of early P. falciparum gametocyte stages

The use of quantitative qRT-PCR assays for detection and quantification of late gametocyte stages has revealed the high transmission capacity of the human malaria parasite, Plasmodium falciparum . To understand how the parasite adjusts its transmission in response to in-host environmental conditions including antimalarials requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that specifically detect and quantify early-stage P. falciparum gametocytes. The assays are based on expression of known early and late gametocyte genes and were developed using purified stage II and stage V gametocytes and tested in natural and controlled human infections. Genes pfpeg4 and pfg27 are specifically expressed at significant levels in early gametocytes with a limit of quantification of 190 and 390 gametocytes/mL, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection. In natural infections, both early ( pfpeg4 / pfg27 ) and late gametocyte transcripts (pfs25 ) were detected in 71.2% of individuals, only early gametocyte transcripts in 12.6%, and only late gametocyte transcripts in 15.2%. The pfpeg4 / pfg27 qRT-PCR assays are sensitive and specific for quantification of circulating sexually committed ring stages/early gametocytes and can be used to increase our understanding of epidemiological processes that modulate P. falciparum transmission.