A Molecular Assay to Quantify Male and Female Plasmodium falciparum Gametocytes: Results From 2 Randomized Controlled Trials Using Primaquine for Gametocyte Clearance

Abstract

Background: Single low-dose primaquine (PQ) reduces Plasmodium falciparum infectivity before it impacts gametocyte density. Here, we examined the effect of PQ on gametocyte sex ratio as a possible explanation for this early sterilizing effect.

Methods: Quantitative reverse-transcription polymerase chain reaction assays were developed to quantify female gametocytes (targeting Pfs25 messenger RNA [mRNA]) and male gametocytes (targeting Pf3D7_1469900 mRNA) in 2 randomized trials in Kenya and Mali, comparing dihydroartemisinin-piperaquine (DP) alone to DP with PQ. Gametocyte sex ratio was examined in relation to time since treatment and infectivity to mosquitoes.

Results: In Kenya, the median proportion of male gametocytes was 0.33 at baseline. Seven days after treatment, gametocyte density was significantly reduced in the DP-PQ arm relative to the DP arm (females: 0.05% [interquartile range {IQR}, 0.0-0.7%] of baseline; males: 3.4% [IQR, 0.4%-32.9%] of baseline; P < .001). Twenty-four hours after treatment, gametocyte sex ratio became male-biased and was not significantly different between the DP and DP-PQ groups. In Mali, there was no significant difference in sex ratio between the DP and DP-PQ groups (>0.125 mg/kg) 48 hours after treatment, and gametocyte sex ratio was not associated with mosquito infection rates.

Conclusions: The early sterilizing effects of PQ may not be explained by the preferential clearance of male gametocytes and may be due to an effect on gametocyte fitness.

Keywords: artemisinin; dihydroartemisinin; piperaquine; primaquine; sex ratio.

Figure 1.

Screening and enrollment criteria. Abbreviations: ♀, female; ♂, male; DP, dihydroartemisinin-piperaquine; Hb, hemoglobin; PQ, primaquine (0.25 mg/kg).

Figure 2.

Gametocyte prevalence and density. A, Gametocyte density determined by microscopy at all sampling points, presented as the median, interquartile range, and 10th–90th percentiles of gametocytes/µL for gametocyte-positive individuals only. For clarity, 4 data points were not included in the graph: 3 in the dihydroartemisinin-piperaquine (DP)–only arm at day 0 (576, 608, and 686/µL), and 1 in the primaquine (PQ) arm at day 0 (1072/µL). B, Gametocyte prevalence determined by microscopy or Pfs25-based quantitative nucleic acid–based sequence amplification (QT-NASBA), presented as percentage positive, with 95% confidence interval.

Figure 3.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)–based male and female gametocyte density and proportion male in the Kenyan study. A, Proportion of total gametocyte density that is male (males per µL / [males per µL plus females per µL]), presented as individual data points, with the median and interquartile range (IQR). Proportion male was only presented or included in analyses if total gametocyte density was estimated to be ≥16 gametocytes per sample (1 gametocyte/500 µL = 0.032 gametocytes/µL). B, Gametocyte density determined by qRT-PCR at baseline (day 0), and 24 (day 3) or 120 (day 7) hours after dihydroartemisinin-piperaquine (DP) or DP plus primaquine (PQ). Female gametocytes were quantified by extrapolating Pfs25 messenger RNA abundance from standard curves of known quantities of female gametocytes, and vice versa. Density is presented as median, IQR, and 10th–90th percentiles of gametocytes/µL for gametocyte-positive individuals only.

Figure 4.

Infectiousness to mosquitoes, quantitative reverse-transcription polymerase chain reaction (qRT-PCR)–based male and female gametocyte density, and proportion male in the Malian study. A, Prevalence of infectiousness to mosquitoes among the study population in the direct membrane-feeding assay at days 0, 2, and 7. Asterisks (*) indicate significantly different infectiousness in logistic regressions relative to control. Mosquito infection was determined as the presence of any number of oocysts in the mosquito mid-gut 7 days after feeding. B, Proportion of total gametocyte density that is male (males per µL / [males per µL plus females per µL]), presented as individual data points, with the median and interquartile range (IQR). Proportion male was only presented or included in analyses if total gametocyte density was estimated to be ≥16 gametocytes per sample (1 gametocyte/50 µL = 0.32 gametocytes/µL). Data from each dose arm and time point are not included in the graph if proportion male was calculable for ≤4 individuals per arm. Asterisks (*) indicate significantly different proportion male in logistic regressions relative to control, adjusted for baseline density. C, Gametocyte density determined by qRT-PCR at baseline (day 0), and 24 (day 3) or 120 (day 7) hours after dihydroartemisinin-piperaquine (DP) or DP plus primaquine (PQ). Female gametocytes were quantified by extrapolating Pfs25 messenger RNA abundance from standard curves of known quantities of female gametocytes, and vice versa. Density is presented as median, IQR, and 10th–90th percentiles of gametocytes/µL for gametocyte-positive individuals only. Median female gametocyte density was calculated from <5 individuals in some dose arms at day 7 (0.5 mg/kg, n = 4) and day 14 (0.5 mg/kg, n = 2). Median male gametocyte density was calculated from <5 individuals in some dose arms at day 14 (0.25 mg/kg, n = 3; 0.5 mg/kg, n = 4).

Figure 5.

Proportion of male gametocytes in infectious (I) and noninfectious (UI) samples before and after treatment in the Malian study. A, Proportions of gametocytes that were male in samples taken at baseline from individuals whose whole blood was infectious or noninfectious to mosquitoes in the direct membrane feeding assay. Mosquito infection was determined as the presence of any number of oocysts in the mosquito mid-gut 7 days after feeding. B, Proportions of gametocytes that were male in samples taken at day 2 (48 hours after first dose of dihydroartemisinin-piperaquine [DP] in both the DP and DP plus primaquine [PQ] arms, and only dose of PQ in DP-PQ arms only) from individuals whose whole blood was infectious or noninfectious to mosquitoes in the direct membrane-feeding assay. “n” indicates number of individuals for which proportion male was calculable, and for which mosquito feeding assays were conducted.