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The diagnostic tools available to diagnose malaria, such as microscopy and rapid diagnostic test (RDT), are less sensitive at detecting lower parasite density. Droplet digital polymerase chain reaction (ddPCR), which has been shown to have higher sensitivity at diagnosing malaria, allows direct quantification without the need for a standard curve. The aim of this study is to develop and use a duplex ddPCR assay for the detection of\n                      <jats:italic>P. knowlesi<\/jats:italic>\n                      and\n                      <jats:italic>P. vivax<\/jats:italic>\n                      , and compare this method to nested PCR and qPCR.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The duplex ddPCR assay had higher analytical sensitivity (\n                      <jats:italic>P. vivax<\/jats:italic>\n                      \u2009=\u200910 copies\/\u00b5L and\n                      <jats:italic>P. knowlesi<\/jats:italic>\n                      \u2009=\u20090.01 copies\/\u00b5L) compared to qPCR (\n                      <jats:italic>P. vivax<\/jats:italic>\n                      \u2009=\u2009100 copies\/\u00b5L and\n                      <jats:italic>P. knowlesi<\/jats:italic>\n                      \u2009=\u200910 copies\/\u00b5L). Moreover, the ddPCR assay had acceptable clinical sensitivity (\n                      <jats:italic>P. vivax\u2009<\/jats:italic>\n                      =\u200980% and\n                      <jats:italic>P. knowlesi<\/jats:italic>\n                      \u2009=\u200990%) and clinical specificity (\n                      <jats:italic>P. vivax<\/jats:italic>\n                      \u2009=\u200987.84% and\n                      <jats:italic>P. knowlesi<\/jats:italic>\n                      \u2009=\u200981.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      Overall, the ddPCR assay demonstrated acceptable efficiency in detection of\n                      <jats:italic>P<\/jats:italic>\n                      .\n                      <jats:italic>knowlesi<\/jats:italic>\n                      and\n                      <jats:italic>P<\/jats:italic>\n                      .\u00a0\n                      <jats:italic>vivax<\/jats:italic>\n                      , and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay\u2019s clinical sensitivity and specificity.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12936-020-03314-5","type":"journal-article","created":{"date-parts":[[2020,7,10]],"date-time":"2020-07-10T05:03:44Z","timestamp":1594357424000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":29,"title":["Droplet digital polymerase chain reaction (ddPCR) for the detection of Plasmodium knowlesi and Plasmodium vivax"],"prefix":"10.1186","volume":"19","author":[{"given":"Punitha","family":"Mahendran","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan Wee Kent","family":"Liew","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amirah","family":"Amir","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiao-Teng","family":"Ching","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yee-Ling","family":"Lau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,7,10]]},"reference":[{"key":"3314_CR1","unstructured":"WHO. 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Informed verbal consent from the patient was obtained for use of the samples for diagnosis and research. Written consent was found to be unnecessary as verbal consent would be sufficient for the purpose of this study and patient details were noted down solely for record keeping. This consent procedure was approved by the University of Malaya Medical Centre Ethic Committee.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"This manuscript does not contain any individualized data. The confidentiality of the patients\u2019 records has been observed according to ethical regulations.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare that they have no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"241"}}