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

Autor: Claire Y. T. Wang, Emma Ballard, Stacey Llewellyn, Louise Marquart, Teun Bousema, James S. McCarthy, Katharine A. Collins
Jazyk: angličtina
Rok vydání: 2020
Předmět:
Zdroj: Malaria Journal, Vol 19, Iss 1, Pp 1-10 (2020)
Druh dokumentu: article
ISSN: 1475-2875
DOI: 10.1186/s12936-020-03291-9
Popis: 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
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