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

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. 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.

Keywords: Gametocytes; Malaria transmission; Plasmodium falciparum; Real Time qPCR; Sex-ratio.

Fig. 1

Gametocyte-specific expression of target genes. Copy number of the indicated transcripts measured in asexual (rings, trophozoites) and sexual (female and male gametocytes) stages of the F12 and PfDynGFP parasite lines, respectively

Fig. 2

Sex-specific expression of target genes. Box and whiskers plot reporting data from assays on different sorted gametocyte populations. Two independent female/male sex-specific sorting by FACS procedure (see “Methods”) were used to test each target gene (the graph reports data from 8, 9, 5 and 12 replicates for pf13, pfs230p, pfGK and pfs25 respectively). Copies number for each target were obtained as described in the text, setting established putative amount of female/male gametocyte as reference. For each target, number of copies calculated in the female sorted population were divided with number of copies calculated in the male sorted population. The fold difference of expression (FD) obtained was converted to logarithmic scale for plotting purposes

Fig. 3

Correlation curves between the ratio of male to female transcript copies and the ratio of male to female gametocytes. For each combination of targets, the figure shows the correlation curve between the ratio of male to female mean transcript copy number (x axis) and the ratio of male to female gametocyte (y axis). The data used to build the curves are shown in Additional file 1: Figure S3. The mean transcript copy number was obtained for each target by averaging the number of copies obtained from replicates of sorting experiments. Gametocytes sex ratios were as follows: 0.01 (2.5M/250F), 0.1 (25M/250F, 2.5M/25F), 1 (2.5M/2. 5F, 25M/25F, 250M/250F), 10 (25M/2.5F, 250M/25F), 100 (250M/2.5F). The following formula was used to extrapolate gametocyte sex ratio from RTqPCR data for mixed cultures and field samples: $\text{Female/male}\text{gametocyte}\text{ratio}={10}^{(\text{slope}\times log(\text{n}\text{male}\text{transcript}\text{copies/n}\text{female}\text{transcript}\text{copies)}+\text{Y}\times \text{intercept)}}$. Slope and intercept for the different target combinations are shown in Additional file 1: Table S4

Fig. 4

Number of samples identified as P. falciparum gametocyte positive by microscopy and by RTqPCR assays. The figure shows the number of samples identified as P. falciparum gametocyte positive by microscopy and by RTqPCR assays. For qPCR assays, the number of positive samples is expressed as a sum of microscopy positive and microscopy negative samples

Fig. 5

Correlation between the number of gametocytes and the number of P. falciparum gametocyte transcript copies, before and after data normalization by human 18S. The Figure shows the linear correlation between the number of gametocytes per μl (log 10 gametocyte density, x axis) and the number of P. falciparum gametocyte transcript copies per μl (log10 copies, y axis), before (left panel: pfs25 and pf13) and after (right panel: pfs25 normalised and pf13 normalised) data normalisation by human 18S

Fig. 6

Sex ratio values determined by different male–female target pairs. The figure shows the value of the male/female gametocyte ratio calculated in field samples using RTqPCR data for the four different combination of male and female markers