Detection of Plasmodium falciparum male and female gametocytes and determination of parasite sex ratio in human endemic populations by novel, cheap and robust RTqPCR assays

• Published in: Malaria Journal
• Main Authors: Federica Santolamazza, Pamela Avellino, Giulia Siciliano, Franck Adama Yao, Fabrizio Lombardo, Jean Bosco Ouédraogo, David Modiano, Pietro Alano, Valentina Dianora Mangano
• Format: Article in Journal/Newspaper
• Language: English
• Published: BMC 2017
• Subjects: Malaria transmission; Plasmodium falciparum; Gametocytes; Sex-ratio; Real Time qPCR; Arctic medicine. Tropical medicine; RC955-962; Infectious and parasitic diseases; RC109-216; Arctic
• Online Access: https://doi.org/10.1186/s12936-017-2118-z; https://doaj.org/article/6a10068912fe464abd223bd2781968c1

Abstract Background The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites. Aim and objectives The aim of this work was to develop sensitive and cheap Real Time qPCR assays for large-scale epidemiological surveys, based on detection and amplification of gametocyte sex specific transcripts selected from the literature: the female-specific pfs25 and pf glycerol kinase (pfGK) and the male-specific pfs230p and pf13 transcripts. Methods RTqPCR assays were used to test the gametocyte- and sex-specific expression of the target genes using asexual stages of the gametocyteless parasite clone F12 and FACS purified male and female gametocytes of the PfDynGFP/P47mCherry line. Assays were performed on 50 blood samples collected during an epidemiological survey in the Soumousso village, Burkina Faso, West-Africa, and amplification of the human housekeeping gene 18S rRNA was employed to normalize RNA sample variability. Results SYBR Green assays were developed that showed higher sensitivity compared to Taqman assays at a reduced cost. RTqPCR results confirmed that expression of pfs25 and pfs230p are female and male-specific, respectively, and introduced two novel markers, the female-specific pfGK and the male-specific pf13. A formula was derived to calculate the ratio of male to female gametocytes based on the ratio of male to female transcript copy number. Use of these assays in the field samples showed, as expected, a higher sensitivity of RTqPCR compared to microscopy. Importantly, similar values of gametocyte sex-ratio were obtained in the field samples based on the four different target combinations. Conclusion Novel, sensitive, cheap and robust molecular assays were developed for the detection and quantification of female and male P. ...