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 g...
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ftdoajarticles:oai:doaj.org/article:dc36368b9fea482cab39012e6fb17dbd 2023-05-15T15:18:35+02:00 Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards Claire Y. T. Wang Emma Ballard Stacey Llewellyn Louise Marquart Teun Bousema James S. McCarthy Katharine A. Collins 2020-06-01T00:00:00Z https://doi.org/10.1186/s12936-020-03291-9 https://doaj.org/article/dc36368b9fea482cab39012e6fb17dbd EN eng BMC http://link.springer.com/article/10.1186/s12936-020-03291-9 https://doaj.org/toc/1475-2875 doi:10.1186/s12936-020-03291-9 1475-2875 https://doaj.org/article/dc36368b9fea482cab39012e6fb17dbd Malaria Journal, Vol 19, Iss 1, Pp 1-10 (2020) Malaria Gametocytes VIS CHMI QRT-PCR Droplet digital PCR Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 article 2020 ftdoajarticles https://doi.org/10.1186/s12936-020-03291-9 2022-12-31T03:58:08Z 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 < 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 a high-level agreement (ICC = 0.998 for pfs25 and 0.995 for pfMGET). Conclusions This study reports the ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Malaria Journal 19 1 |
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Directory of Open Access Journals: DOAJ Articles |
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English |
topic |
Malaria Gametocytes VIS CHMI QRT-PCR Droplet digital PCR Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
spellingShingle |
Malaria Gametocytes VIS CHMI QRT-PCR Droplet digital PCR Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 Claire Y. T. Wang Emma Ballard Stacey Llewellyn Louise Marquart Teun Bousema James S. McCarthy Katharine A. Collins Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards |
topic_facet |
Malaria Gametocytes VIS CHMI QRT-PCR Droplet digital PCR Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
description |
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 < 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 a high-level agreement (ICC = 0.998 for pfs25 and 0.995 for pfMGET). Conclusions This study reports the ... |
format |
Article in Journal/Newspaper |
author |
Claire Y. T. Wang Emma Ballard Stacey Llewellyn Louise Marquart Teun Bousema James S. McCarthy Katharine A. Collins |
author_facet |
Claire Y. T. Wang Emma Ballard Stacey Llewellyn Louise Marquart Teun Bousema James S. McCarthy Katharine A. Collins |
author_sort |
Claire Y. T. Wang |
title |
Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards |
title_short |
Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards |
title_full |
Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards |
title_fullStr |
Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards |
title_full_unstemmed |
Assays for quantification of male and female gametocytes in human blood by qRT-PCR in the absence of pure sex-specific gametocyte standards |
title_sort |
assays for quantification of male and female gametocytes in human blood by qrt-pcr in the absence of pure sex-specific gametocyte standards |
publisher |
BMC |
publishDate |
2020 |
url |
https://doi.org/10.1186/s12936-020-03291-9 https://doaj.org/article/dc36368b9fea482cab39012e6fb17dbd |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Malaria Journal, Vol 19, Iss 1, Pp 1-10 (2020) |
op_relation |
http://link.springer.com/article/10.1186/s12936-020-03291-9 https://doaj.org/toc/1475-2875 doi:10.1186/s12936-020-03291-9 1475-2875 https://doaj.org/article/dc36368b9fea482cab39012e6fb17dbd |
op_doi |
https://doi.org/10.1186/s12936-020-03291-9 |
container_title |
Malaria Journal |
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19 |
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1 |
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1766348783284649984 |