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

Malaria Journal

Research

Pietro Alano¹  Giulia Siciliano¹  David Modiano²  Pamela Avellino²  Federica Santolamazza²  Fabrizio Lombardo²  Valentina Dianora Mangano³  Franck Adama Yao⁴  Jean Bosco Ouédraogo⁵ 

[1] Dipartimento di Malattie Infettive, Istituto Superiore di Sanità, Roma, Italy;Dipartimento di Sanità Pubblica e Malattie Infettive, Sapienza Università di Roma, Roma, Italy;Dipartimento di Sanità Pubblica e Malattie Infettive, Sapienza Università di Roma, Roma, Italy;Dipartimento di Ricerca Traslazionale e Nuove Tecnologie in Medicina e Chirurgia, Università di Pisa, Pisa, Italy;Institut de Recherche en Sciences de la Santé, Bobo Dioulasso, Burkina Faso;Institut de Recherche en Sciences de la Santé, Bobo Dioulasso, Burkina Faso;Centre Muraz, Bobo Dioulasso, Burkina Faso;

关键词: Malaria transmission;    Plasmodium falciparum;    Gametocytes;    Sex-ratio;    Real Time qPCR;   

DOI  :  10.1186/s12936-017-2118-z

 received in 2017-07-15, accepted in 2017-11-11,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundThe 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 objectivesThe 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.MethodsRTqPCR 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.ResultsSYBR 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.ConclusionNovel, sensitive, cheap and robust molecular assays were developed for the detection and quantification of female and male P. falciparum gametocytes. In particular, the RTqPCR assays based on the female-specific pfs25 and the newly described male gametocyte-specific pf13 transcripts, including normalization by the human 18S, reliably assess presence and abundance of female and male gametocytes and enable to determine their sex-ratio in human subjects in endemic areas.

【 授权许可】

CC BY   
© The Author(s) 2017

【 图 表 】

12936_2017_2118_Article_IEq1.gif