{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T05:12:05Z","timestamp":1772169125947,"version":"3.50.1"},"update-to":[{"DOI":"10.12688\/wellcomeopenres.16564.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T00:00:00Z","timestamp":1648771200000}},{"DOI":"10.12688\/wellcomeopenres.16564.2","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T00:00:00Z","timestamp":1648771200000}}],"reference-count":13,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T00:00:00Z","timestamp":1648771200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["WT101148MA"],"award-info":[{"award-number":["WT101148MA"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000865","name":"Bill and Melinda Gates Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100000865","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011744","name":"National University Health System","doi-asserted-by":"publisher","award":["NUHSRO\/2018\/006\/SU\/01"],"award-info":[{"award-number":["NUHSRO\/2018\/006\/SU\/01"]}],"id":[{"id":"10.13039\/501100011744","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011744","name":"National University Health System","doi-asserted-by":"publisher","award":["NUHSRO\/2018\/094\/T1"],"award-info":[{"award-number":["NUHSRO\/2018\/094\/T1"]}],"id":[{"id":"10.13039\/501100011744","id-type":"DOI","asserted-by":"publisher"}]},{"name":"New Zealand HRC eASIA","award":["17\/678"],"award-info":[{"award-number":["17\/678"]}]},{"name":"The Singapore Immunology Network, A*STAR core fund"}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns3:p>\n                    <ns3:bold>Background:<\/ns3:bold>\n                    Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Methods:<\/ns3:bold>\n                    Here we evaluated a real-time reverse transcription PCR (RT-qPCR) method that reduces the required sample volume compared to uPCR. To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were compared using uPCR and RT-qPCR.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Results:<\/ns3:bold>\n                    <ns3:italic>Plasmodium<\/ns3:italic>\n                    spp. RT-qPCR\u00a0confirmed 18 of 21 uPCR\n                    <ns3:italic>Plasmodium falciparum<\/ns3:italic>\n                    positives, while\n                    <ns3:italic>P. falciparum<\/ns3:italic>\n                    specific\u00a0RT-qPCR confirmed 17 of the 21 uPCR\n                    <ns3:italic>P. falciparum<\/ns3:italic>\n                    positives. Combining both RT-qPCR results increased the sensitivity to 100% and specificity was 95.1%.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Conclusion:<\/ns3:bold>\n                    Malaria detection in areas of low transmission and LDMI can benefit from the increased sensitivity of ribosomal RNA detection by RT-PCR, especially where sample volume is limited. Isolation of high quality RNA also allows for downstream analysis of malaria transcripts.\n                  <\/ns3:p>","DOI":"10.12688\/wellcomeopenres.16564.3","type":"journal-article","created":{"date-parts":[[2022,4,1]],"date-time":"2022-04-01T09:40:20Z","timestamp":1648806020000},"page":"39","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Reverse transcription PCR to detect low density malaria infections"],"prefix":"10.12688","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4690-270X","authenticated-orcid":false,"given":"Peter","family":"Christensen","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zbynek","family":"Bozdech","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wanitda","family":"Watthanaworawit","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mallika","family":"Imwong","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laurent","family":"R\u00e9nia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9658-7528","authenticated-orcid":false,"given":"Beno\u00eet","family":"Malleret","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Clare","family":"Ling","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7951-0745","authenticated-orcid":false,"given":"Fran\u00e7ois","family":"Nosten","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2560","published-online":{"date-parts":[[2022,4,1]]},"reference":[{"key":"ref-1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pmed.1001467","article-title":"Targeting asymptomatic malaria infections: active surveillance in control and elimination.","volume":"10","author":"H Sturrock","year":"2013","journal-title":"PLoS Med."},{"key":"ref-2","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms2241","article-title":"Factors determining the occurrence of submicroscopic malaria infections and their relevance for control.","volume":"3","author":"L Okell","year":"2012","journal-title":"Nat Commun."},{"key":"ref-3","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.00565-18","article-title":"Operational Performance of a Plasmodium falciparum Ultrasensitive Rapid Diagnostic Test for Detection of Asymptomatic Infections in Eastern Myanmar.","volume":"56","author":"J Landier","year":"2018","journal-title":"J Clin Microbiol."},{"key":"ref-4","doi-asserted-by":"publisher","first-page":"98","DOI":"10.12688\/wellcomeopenres.12741.2","article-title":"Scale up of a Plasmodium falciparum elimination program and surveillance system in Kayin State, Myanmar [version 2; peer review: 2 approved].","volume":"2","author":"D Parker","year":"2017","journal-title":"Wellcome Open Res."},{"key":"ref-5","article-title":"Prevalence of submicroscopic Plasmodium falciparum infections in asymptomatic children in low transmission settings in Bagamoyo, Tanzania.","volume":"7","author":"D Sumari","year":"2016","journal-title":"MWJ."},{"key":"ref-6","doi-asserted-by":"publisher","first-page":"3303-9","DOI":"10.1128\/JCM.01057-14","article-title":"High-throughput ultrasensitive molecular techniques for quantifying low-density malaria parasitemias.","volume":"52","author":"M Imwong","year":"2014","journal-title":"J Clin Microbiol."},{"key":"ref-7","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pmed.1002745","article-title":"The impact of targeted malaria elimination with mass drug administrations on falciparum malaria in Southeast Asia: A cluster randomised trial.","volume":"16","author":"L von Seidlein","year":"2019","journal-title":"PLoS Med."},{"key":"ref-8","doi-asserted-by":"publisher","first-page":"2946-53","DOI":"10.1128\/JCM.00276-11","article-title":"Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of plasmodium by amplifying RNA and DNA of the 18S rRNA genes.","volume":"49","author":"E Kamau","year":"2011","journal-title":"J Clin Microbiol."},{"key":"ref-9","doi-asserted-by":"publisher","first-page":"81","DOI":"10.12688\/wellcomeopenres.12240.1","article-title":"Safety and effectiveness of mass drug administration to accelerate elimination of artemisinin-resistant falciparum malaria: A pilot trial in four villages of Eastern Myanmar [version 1; peer review: 2 approved].","volume":"2","author":"J Landier","year":"2017","journal-title":"Wellcome Open Res."},{"key":"ref-10","doi-asserted-by":"publisher","first-page":"1214-9","DOI":"10.1128\/jcm.42.3.1214-1219.2004","article-title":"Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis.","volume":"42","author":"F Perandin","year":"2004","journal-title":"J Clin Microbiol."},{"key":"ref-11","doi-asserted-by":"publisher","first-page":"118","DOI":"10.1038\/srep00118","article-title":"A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development.","volume":"1","author":"B Malleret","year":"2011","journal-title":"Sci Rep."},{"key":"ref-12","article-title":"Reverse transcription PCR to detect low density malaria infections","author":"P Christensen","year":"2021"},{"key":"ref-13","doi-asserted-by":"publisher","first-page":"363","DOI":"10.1186\/s12936-020-03436-w","article-title":"A comprehensive RNA handling and transcriptomics guide for high-throughput processing of Plasmodium blood-stage samples.","volume":"19","author":"M Kucharski","year":"2020","journal-title":"Malar J."}],"container-title":["Wellcome Open Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v3\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v3\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v3\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,5,9]],"date-time":"2022-05-09T05:30:18Z","timestamp":1652074218000},"score":1,"resource":{"primary":{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v3"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,4,1]]},"references-count":13,"URL":"https:\/\/doi.org\/10.12688\/wellcomeopenres.16564.3","relation":{"has-review":[{"id-type":"doi","id":"10.21956\/wellcomeopenres.19701.r49634","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.19701.r49635","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.19701.r49634","asserted-by":"object"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.19701.r49635","asserted-by":"object"}]},"ISSN":["2398-502X"],"issn-type":[{"value":"2398-502X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,4,1]]},"assertion":[{"value":"Indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v3#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.18253.r42783, Gisely Melo, Tropical Medicine Foundation Dr. Heitor Vieira Dourado, Instituto de Pesquisa Cl\u00ednica Carlos Borborema, Manaus, Brazil, 20 Apr 2021, version 1, 2 approved with reservations, 1 not approved","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v1#referee-response-42783","order":0,"name":"referee-response-42783","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Peter Christensen<\/b>; \n<i>Posted: 22 Apr 2021<\/i>; Thank you for your time reviewing this paper, I appreciate the effort. &nbsp; Hopefully I can address all your comments in the next draft. &nbsp; \n<i>Info on target &amp; sensitivity specifictity data:<\/i> I will update the paper to include more information about the primer targets earlier and will add the previously published Sens and Spec for the Plasmodium spp. primer set used by uPCR and RT-PCR. It has been done in the original paper for both qPCR and qRT-PCR and then again for high volume uPCR. \n<b>Will adding previously published data be satisfactory?<\/b> &nbsp; \n<i>Why only falciparum<\/i>: We used Pf only because we can produce very young rings \n<i>invitro <\/i>that are then FACS sorted for a more consistent standard\n<i>, <\/i>and because clinical vivax samples often contain late stages (which may contain more than one nucleus per infected red cell and also contain more ribosomal material). Furthermore, although there is a growing realisation of vivax malaria\u2019s importance; the funder and clinicians at the field site still consider the sensitive and timely diagnosis of Pf a priority. Nonetheless I wish to assure the reviewer that we also would have preferred to have a Pv data set (esp. considering Pv is the most prevalent in the region); however due to the large proportion of sample per reaction (4ul of DNA template per rxn &amp; only 20ul DNA eluted) this means we can only do two PCRs in duplicate. This is a limitation of this type of high blood volume per reaction assay. I can discuss this limitation. &nbsp; \n<i>Clarify targets<\/i>: Only 2 new RT-PCR results were produced in this study, both target 18S RNA. One was Plasmodium genus specific, the other was \n<i>P. falciparum<\/i> specific. The original Pf classification given by uPCR used another Pf primer set from Perandin et al. JCM 2004. In hindsight I should have used this same Pf primer set for my RT-PCR assays. The reason I used primers from Sumari et al. is because they had been used in RT-PCR format and we originally included gametocyte data using PfS25 primers from that paper as well. We didn\u2019t have enough sample to complete the data set, so it was removed from the paper. &nbsp; \n<i>RDT and Microscopy<\/i>: Microscopy would be better as it is the gold standard. But the LOD is so low it would not be able to detect the majority of samples in this data set (13 of 21 are submicroscopic (&lt;50,000 p\/mL)). Unfortunately RDT data from the malaria elimination program, was found to have some important QA issues (the specific batch of PfHRP-2 RDTs provided by Global Fund)). &nbsp; \n<i>Statistics:<\/i> I thought correlation was appropriate here as well, but our statistician sent me this paper (https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC4470095\/). So I used bland-altman for assessing agreement (both similarities and differences) between test measurements. Originally I wanted this assay to be yes\/no and semi-quantitative and expected the analysis to \n<b><u>not<\/u><\/b> show agreement between tests. The reason thought this is because Kamau \n<i>et al<\/i> have already shown this primer set is less accurate at low parasitaemia when used as qRT-PCR.  \n<i>Number of LDMI<\/i>: I will add the number of samples that were low density (less than 50 parasites\/uL) by uPCR result: 13 of 21 were LDMI. &nbsp;  Again we wish to thank the reviewer for their time and expertise, Regards, Peter C","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v3#referee-comment-4592","order":1,"name":"referee-comment-4592","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.19701.r49634, Muneaki Hashimoto, Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Hayashi-cho, Takamatsu, 761-0395, Japan, 09 May 2022, version 3, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v3#referee-response-49634","order":2,"name":"referee-response-49634","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.19701.r49635, Julian Tanner, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, 08 Apr 2022, version 3, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-39\/v3#referee-response-49635","order":3,"name":"referee-response-49635","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This study was supported by the Wellcome Trust through a strategic award \u201cEliminating malaria to counter artemisinin resistance\u201d [101148,  <ahref=https:\/\/doi.org\/10.35802\/101148> https:\/\/doi.org\/10.35802\/101148<\/a>].\n\nFunding was also obtained from the following sources; the Bill and Melinda Gates Foundation; The Singapore Immunology Network, A*STAR core fund; the NUHS start-up funding [NUHSRO\/2018\/006\/SU\/01]; NUHS seed fund [NUHSRO\/2018\/094\/T1]; the Wellcome Trust Mahidol University Oxford Tropical Medicine Research Programme and the New Zealand HRC eASIA [17\/678] project grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}