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Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR
BACKGROUND: Evaluating the efficacy of transmission-blocking interventions relies on mosquito-feeding assays, with transmission typically assessed by microscopic identification of oocysts in mosquito midguts; however, microscopy has limited throughput, sensitivity and specificity. Where low prevalen...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034226/ https://www.ncbi.nlm.nih.gov/pubmed/29976199 http://dx.doi.org/10.1186/s12936-018-2382-6 |
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author | Wang, Claire Y. T. McCarthy, James S. Stone, Will J. Bousema, Teun Collins, Katharine A. Bialasiewicz, Seweryn |
author_facet | Wang, Claire Y. T. McCarthy, James S. Stone, Will J. Bousema, Teun Collins, Katharine A. Bialasiewicz, Seweryn |
author_sort | Wang, Claire Y. T. |
collection | PubMed |
description | BACKGROUND: Evaluating the efficacy of transmission-blocking interventions relies on mosquito-feeding assays, with transmission typically assessed by microscopic identification of oocysts in mosquito midguts; however, microscopy has limited throughput, sensitivity and specificity. Where low prevalence and intensity mosquito infections occur, as observed during controlled human malaria infection studies or natural transmission, a reliable method for detection and quantification of low-level midgut infection is required. Here, a semi-automated, Taqman quantitative PCR (qPCR) assay sufficiently sensitive to detect a single-oocyst midgut infection is described. RESULTS: Extraction of genomic DNA from Anopheles stephensi midguts using a semi-automated extraction process was shown to have equivalent extraction efficiency to manual DNA extraction. An 18S Plasmodium falciparum qPCR assay was adapted for quantitative detection of P. falciparum midgut oocyst infection using synthetic DNA standards. The assay was validated for sensitivity and specificity, and the limit of detection was 0.7 genomes/µL (95% CI 0.4–1.6 genomes/µL). All microscopy-confirmed oocyst infected midgut samples were detected by qPCR, including all single-oocyst positive midguts. The genome number per oocyst was assessed 8–9 days after feeding assay using both qPCR and droplet digital PCR and was 3722 (IQR: 2951–5453) and 3490 (IQR: 2720–4182), respectively. CONCLUSIONS: This semi-automated qPCR method enables accurate detection of low-level P. falciparum oocyst infections in mosquito midguts, and may improve the sensitivity, specificity and throughput of assays used to evaluate candidate transmission-blocking interventions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12936-018-2382-6) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6034226 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-60342262018-07-12 Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR Wang, Claire Y. T. McCarthy, James S. Stone, Will J. Bousema, Teun Collins, Katharine A. Bialasiewicz, Seweryn Malar J Research BACKGROUND: Evaluating the efficacy of transmission-blocking interventions relies on mosquito-feeding assays, with transmission typically assessed by microscopic identification of oocysts in mosquito midguts; however, microscopy has limited throughput, sensitivity and specificity. Where low prevalence and intensity mosquito infections occur, as observed during controlled human malaria infection studies or natural transmission, a reliable method for detection and quantification of low-level midgut infection is required. Here, a semi-automated, Taqman quantitative PCR (qPCR) assay sufficiently sensitive to detect a single-oocyst midgut infection is described. RESULTS: Extraction of genomic DNA from Anopheles stephensi midguts using a semi-automated extraction process was shown to have equivalent extraction efficiency to manual DNA extraction. An 18S Plasmodium falciparum qPCR assay was adapted for quantitative detection of P. falciparum midgut oocyst infection using synthetic DNA standards. The assay was validated for sensitivity and specificity, and the limit of detection was 0.7 genomes/µL (95% CI 0.4–1.6 genomes/µL). All microscopy-confirmed oocyst infected midgut samples were detected by qPCR, including all single-oocyst positive midguts. The genome number per oocyst was assessed 8–9 days after feeding assay using both qPCR and droplet digital PCR and was 3722 (IQR: 2951–5453) and 3490 (IQR: 2720–4182), respectively. CONCLUSIONS: This semi-automated qPCR method enables accurate detection of low-level P. falciparum oocyst infections in mosquito midguts, and may improve the sensitivity, specificity and throughput of assays used to evaluate candidate transmission-blocking interventions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12936-018-2382-6) contains supplementary material, which is available to authorized users. BioMed Central 2018-07-05 /pmc/articles/PMC6034226/ /pubmed/29976199 http://dx.doi.org/10.1186/s12936-018-2382-6 Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Wang, Claire Y. T. McCarthy, James S. Stone, Will J. Bousema, Teun Collins, Katharine A. Bialasiewicz, Seweryn Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR |
title | Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR |
title_full | Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR |
title_fullStr | Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR |
title_full_unstemmed | Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR |
title_short | Assessing Plasmodium falciparum transmission in mosquito-feeding assays using quantitative PCR |
title_sort | assessing plasmodium falciparum transmission in mosquito-feeding assays using quantitative pcr |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6034226/ https://www.ncbi.nlm.nih.gov/pubmed/29976199 http://dx.doi.org/10.1186/s12936-018-2382-6 |
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