Background Malaria transmission from humans to mosquitoes requires the presence of gametocytes in human peripheral circulation, and the dynamics of transmission are determined largely by the density and sex ratio of the gametocytes. Molecular methods are thus employed to measure gametocyte densities, particularly when assessing transmission epidemiology and the efficacy of transmission-blocking interventions. However accurate quantification of gametocytes with molecular methods requires pure male and female gametocytes as reference standards, which are not widely available.
Methods qRT-PCR assays were used to quantify levels of sex-specific mRNA transcripts in Plasmodium falciparum female and male gametocytes ( pfs25 and pfMGET respectively) using synthetic cRNA standards and in-vitro cultured gametocytes. Assay were validated and assay performance was investigated using blood samples of clinical trial participants (ClinicalTrials.gov reference number NCT02431637 and NCT02431650) using these standards and compared to absolute quantification by droplet digital PCR (ddPCR).
Results The number of transcript copies per gametocyte were determined to be 279.3 (95% CI 253.5 - 307.6) for the female-specific transcript pfs25, and 12.5 (95% CI 10.6 - 14.9) for the male-specific transcript pfMGET . These numbers can be used to convert from transcript copies/mL to gametocyte/mL. The reportable range was determined to be 5.71x10 6 to 5.71 gametocytes/mL for pfs25, and 1.73x10 7 to 5.59 for pfMGET. The limit of detection was 3.9 (95% CI 2.5-8.2) gametocytes/mL for pfs25, and 26.9 (95% CI 19.3 - 51.7) gametocytes/mL for PfMGET . Both assays showed minimal intra-assay and inter-assay variability with coefficient of variation < 3%. No cross-reactivity was observed in both assays in uninfected human blood samples. Comparison of results from ddPCR to qRT-PCR assays on clinical blood samples indicated high level agreement (ICC=0.998 for pfs25 and 0.995 for pfMGET ).
Conclusions We developed and validated qRT-PCR assays that are able to accurately quantify levels of female and male P. falciparum gametocytes at submicroscopic densities. The assays showed excellent reproducibility, sensitivity, precision, specificity and accuracy. The methodology will enable the estimation of gametocyte density in the absence of pure female and male gametocyte standards, and will facilitate clinical trials and epidemiological studies.