qRT-PCR versus IFA-based Quantification of Male and Female Gametocytes in Low-Density Plasmodium falciparum Infections and Their Relevance for Transmission

Abstract

Background Accurate quantification of female and male gametocytes and sex ratios in asymptomatic low-density malaria infections are important for assessing their transmission potential. Gametocytes often escape detection even by molecular methods, therefore ultralow gametocyte densities were quantified in large blood volumes. Methods Female and male gametocytes were quantified in 161 PCR-positive Plasmodium falciparum infections from a cross-sectional survey in Papua New Guinea. Ten-fold concentrated RNA from 800 µL blood was analyzed using female-specific pfs25 and male-specific pfmget or mssp qRT-PCR. Gametocyte sex ratios from qRT-PCR were compared with those from immunofluorescence assays (IFA). Results Gametocytes were identified in 58% (93/161) P. falciparum-positive individuals. Mean gametocyte densities were frequently below 1 female and 1 male gametocyte/µL by qRT-PCR. The mean proportion of males was 0.39 (95% confidence interval, 0.33–0.44) by pfs25/pfmget qRT-PCR; this correlated well with IFA results (Pearsons r2 = 0.91; P < .001). A Poisson model fitted to our data predicted 16% P. falciparum-positive individuals that are likely to transmit, assuming at least 1 female and 1 male gametocyte per 2.5 µL mosquito bloodmeal. Conclusions Based on model estimates of female and male gametocytes per 2.5 µL blood, P. falciparum-positive individuals detected exclusively by ultrasensitive diagnostics are negligible for human-to-mosquito transmission. Estimating the transmission potential of ultralow-density malaria infections informs interventions. Almost all infections with ≥1 female and male gametocyte per 2.5 µL mosquito bloodmeal, and thus with highest likelihood of contributing to human-to-mosquito transmission, were detectable by standard molecular diagnostics.

Molecular methods for tracking residual Plasmodium falciparum transmission in a close-to-elimination setting in Zanzibar

“…3 Zanzibar Malaria Elimination Programme, Ministry of Health, Zanzibar, United Republic of Tanzania. 4 School of Public Health and Tropical Medicine, Tulane University, New Orleans, USA. 5 Ifakara Health Institute, Dar es Salaam, United Republic of Tanzania.…”

“…A key interest is to understand population prevalence as a proxy for the transmission reservoir and transmission potential in a certain area. A number of publications have presented mathematical models and gametocyte data from endemic communities [2][3][4][5][6], all concurring in that, in addition to symptomatic malaria cases, also individuals with asymptomatic as well as submicroscopic infections contribute to the infectious reservoir. This silent reservoir is currently under investigation in many malaria endemic countries, and malaria elimination programmes aim to uncover and potentially track this source of transmission.…”

Utility of ultra-sensitive qPCR to detect Plasmodium falciparum and Plasmodium vivax infections under different transmission intensities

“…Gametocyte densities were 10-fold lower in infections detected by standard 18S rRNA qPCR compared to infections only detectable by us-qPCR and were below 1 male and 1 female gametocyte/µL [22]. Although the gametocyte sex-ratio suggested a very low likelihood of transmission [22], it cannot be excluded that parasite densities in untreated, chronic malaria infections in the community may uctuate and transiently reach levels that could potentially be infective to mosquitoes [23].…”

“…In this recent study, more than half of the P. falciparumand P. vivaxpositive samples that were only detected by us-qPCR, carried gametocytes, (10 gametocyte carriers in 15 P. falciparum-infections and 11 gametocyte carriers in 19 P. vivax-infections) [12]. Gametocyte densities were 10-fold lower in infections detected by standard 18S rRNA qPCR compared to infections only detectable by us-qPCR and were below 1 male and 1 female gametocyte/µL [22]. Although the gametocyte sex-ratio suggested a very low likelihood of transmission [22], it cannot be excluded that parasite densities in untreated, chronic malaria infections in the community may uctuate and transiently reach levels that could potentially be infective to mosquitoes [23].…”

An assay for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure gametocyte standards

“…The use of both female-and male-specific assays allows evaluation of the gametocyte sex ratio, a parameter of interest in quantifying infectivity to mosquitoes, particularly at lower gametocyte densities (18). The Pfs25-PfMGET combination for sex ratio determination was recently validated by immunofluorescence assays on field samples from gametocyte donors (19).This ratio may be particularly important for assessment of gametocytocidal drug activity, as the less abundant male gametocytes may be more readily sterilised or killed by some antimalarial drugs (14,20,21).…”