Detection of P. malariae using a new rapid isothermal amplification lateral flow assay
Abstract Background While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-ca...
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ftdoajarticles:oai:doaj.org/article:fd4c03140d8c4a7e99d53cb6e3e27944 2024-09-09T19:27:58+00:00 Detection of P. malariae using a new rapid isothermal amplification lateral flow assay Ashenafi Assefa Kevin Wamae Christopher M. Hennelly Billy Ngasala Meredith Muller Albert Kalonji Fernandine Phanzu Clark H. Cunningham Jessica T. Lin Jonathan B. Parr 2024-04-01T00:00:00Z https://doi.org/10.1186/s12936-024-04928-9 https://doaj.org/article/fd4c03140d8c4a7e99d53cb6e3e27944 EN eng BMC https://doi.org/10.1186/s12936-024-04928-9 https://doaj.org/toc/1475-2875 doi:10.1186/s12936-024-04928-9 1475-2875 https://doaj.org/article/fd4c03140d8c4a7e99d53cb6e3e27944 Malaria Journal, Vol 23, Iss 1, Pp 1-7 (2024) RPA Recombinase polymerase amplification Lateral flow Point-of-care testing Rapid test Isothermal nucleic acid amplification Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 article 2024 ftdoajarticles https://doi.org/10.1186/s12936-024-04928-9 2024-08-05T17:49:36Z Abstract Background While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. Methods Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. Results Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. Conclusion Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory ... Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic Malaria Journal 23 1 |
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ftdoajarticles |
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RPA Recombinase polymerase amplification Lateral flow Point-of-care testing Rapid test Isothermal nucleic acid amplification Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
spellingShingle |
RPA Recombinase polymerase amplification Lateral flow Point-of-care testing Rapid test Isothermal nucleic acid amplification Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 Ashenafi Assefa Kevin Wamae Christopher M. Hennelly Billy Ngasala Meredith Muller Albert Kalonji Fernandine Phanzu Clark H. Cunningham Jessica T. Lin Jonathan B. Parr Detection of P. malariae using a new rapid isothermal amplification lateral flow assay |
topic_facet |
RPA Recombinase polymerase amplification Lateral flow Point-of-care testing Rapid test Isothermal nucleic acid amplification Arctic medicine. Tropical medicine RC955-962 Infectious and parasitic diseases RC109-216 |
description |
Abstract Background While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection. Methods Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo. Results Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min. Conclusion Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory ... |
format |
Article in Journal/Newspaper |
author |
Ashenafi Assefa Kevin Wamae Christopher M. Hennelly Billy Ngasala Meredith Muller Albert Kalonji Fernandine Phanzu Clark H. Cunningham Jessica T. Lin Jonathan B. Parr |
author_facet |
Ashenafi Assefa Kevin Wamae Christopher M. Hennelly Billy Ngasala Meredith Muller Albert Kalonji Fernandine Phanzu Clark H. Cunningham Jessica T. Lin Jonathan B. Parr |
author_sort |
Ashenafi Assefa |
title |
Detection of P. malariae using a new rapid isothermal amplification lateral flow assay |
title_short |
Detection of P. malariae using a new rapid isothermal amplification lateral flow assay |
title_full |
Detection of P. malariae using a new rapid isothermal amplification lateral flow assay |
title_fullStr |
Detection of P. malariae using a new rapid isothermal amplification lateral flow assay |
title_full_unstemmed |
Detection of P. malariae using a new rapid isothermal amplification lateral flow assay |
title_sort |
detection of p. malariae using a new rapid isothermal amplification lateral flow assay |
publisher |
BMC |
publishDate |
2024 |
url |
https://doi.org/10.1186/s12936-024-04928-9 https://doaj.org/article/fd4c03140d8c4a7e99d53cb6e3e27944 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
Malaria Journal, Vol 23, Iss 1, Pp 1-7 (2024) |
op_relation |
https://doi.org/10.1186/s12936-024-04928-9 https://doaj.org/toc/1475-2875 doi:10.1186/s12936-024-04928-9 1475-2875 https://doaj.org/article/fd4c03140d8c4a7e99d53cb6e3e27944 |
op_doi |
https://doi.org/10.1186/s12936-024-04928-9 |
container_title |
Malaria Journal |
container_volume |
23 |
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1 |
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1809897281365737472 |