Dataset Information

Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards.

ABSTRACT:

Background

Malaria transmission from humans to Anopheles 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 male and female 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 complimentary RNA standards and in vitro cultured gametocytes. Assays were validated and assay performance was investigated in blood samples of clinical trial participants 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 5.71?×?10⁶ to 5.71 female gametocytes/mL for pfs25, and 1.73?×?10⁷ to 1.73?×?10¹ male gametocytes/mL for pfMGET. The limit of detection was 3.9 (95% CI 2.5-8.2) female gametocytes/mL for pfs25, and 26.9 (95% CI 19.3-51.7) male gametocytes/mL for PfMGET. Both assays showed minimal intra-assay and inter-assay variability with coefficient of variation?ConclusionsThis study reports the validation of qRT-PCR assays that are able to accurately quantify female and male P. falciparum gametocytes at sub-microscopic 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.

SUBMITTER: Wang CYT

PROVIDER: S-EPMC7310411 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Publications

Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards.

Wang Claire Y T CYT Ballard Emma E Llewellyn Stacey S Marquart Louise L Bousema Teun T McCarthy James S JS Collins Katharine A KA

Malaria journal 20200623 1

<h4>Background</h4>Malaria transmission from humans to Anopheles 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 male and female gametocytes with ...[more]

PMID: 32576184

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Dataset Information

Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards.

Background

Methods

Results

Publications

Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards.

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