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Dried blood spots (DBS) are a practical sample type for malaria field studies to collect, store, and transport large quantities of blood samples for diagnostic testing. Pooled testing is a common way to reduce reagent costs and labour. This study examined performance of the <jats:italic>Plasmodium<\/jats:italic> 18S rRNA biomarker assay for DBS, improved assay sensitivity for pooled samples, and created graphical user interface (GUI) programmes for facilitating optimal pooling.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>DBS samples of varied parasite densities from clinical specimens, <jats:italic>Plasmodium falciparum <\/jats:italic>in vitro culture, and <jats:italic>P. falciparum<\/jats:italic> Armored RNA\u00ae were tested using the <jats:italic>Plasmodium<\/jats:italic> 18S rRNA quantitative triplex reverse transcription polymerase chain reaction (qRT-PCR) assay and a simplified duplex assay. DBS sample precision, linearity, limit of detection (LoD) and stability at varied storage temperatures were evaluated. Novel GUIs were created to model two-stage hierarchy, square matrix, and three-stage hierarchy pooling strategies with samples of varying positivity rates and estimated test counts. Seventy-eight DBS samples from persons residing in endemic regions with sub-patent infections were tested in pools and deconvoluted to identify positive cases.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>Assay performance showed linearity for DBS from 4\u2009\u00d7\u200910<jats:sup>7<\/jats:sup> to 5\u2009\u00d7\u200910<jats:sup>2<\/jats:sup> parasites\/mL with strong correlation to liquid blood samples (r<jats:sup>2<\/jats:sup>\u2009&gt;\u20090.96). There was a minor quantitative reduction in DBS rRNA copies\/mL compared to liquid blood samples. Analytical sensitivity for DBS was estimated 5.3 log copies 18S rRNA\/mL blood (28 estimated parasites\/mL). Properly preserved DBS demonstrated minimal degradation of 18S rRNA when stored at ambient temperatures for one month. A simplified duplex qRT-PCR assay omitting the human mRNA target showed improved analytical sensitivity, 1 parasite\/mL blood, and was optimized for pooling. Optimal pooling sizes varied depending on prevalence. A pilot DBS study of the two-stage hierarchy pooling scheme corroborated results previously determined by testing individual DBS.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>The <jats:italic>Plasmodium<\/jats:italic> 18S rRNA biomarker assay can be applied to DBS collected in field studies. The simplified <jats:italic>Plasmodium<\/jats:italic> qRT-PCR assay and GUIs have been established to provide efficient means to test large quantities of DBS samples.<\/jats:p>\n              <\/jats:sec>","DOI":"10.1186\/s12936-021-03907-8","type":"journal-article","created":{"date-parts":[[2021,10,7]],"date-time":"2021-10-07T13:22:00Z","timestamp":1633612920000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Application of dried blood spot sample pooling strategies for Plasmodium 18S rRNA biomarker testing to facilitate identification of infected persons in large-scale epidemiological studies"],"prefix":"10.1186","volume":"20","author":[{"given":"Ming","family":"Chang","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Selena","family":"Johnston","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Annette M.","family":"Seilie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dianna","family":"Hergott","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2048-0131","authenticated-orcid":false,"given":"Sean C.","family":"Murphy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,10,7]]},"reference":[{"key":"3907_CR1","volume-title":"World malaria report 2019","author":"WHO","year":"2019","unstructured":"WHO. 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