{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T23:23:29Z","timestamp":1774567409610,"version":"3.50.1"},"update-to":[{"DOI":"10.1371\/journal.pntd.0010327","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,4,20]],"date-time":"2022-04-20T00:00:00Z","timestamp":1650412800000}}],"reference-count":72,"publisher":"Public Library of Science (PLoS)","issue":"4","license":[{"start":{"date-parts":[[2022,4,8]],"date-time":"2022-04-08T00:00:00Z","timestamp":1649376000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Gorgas Memorial Institute of Health Studies of Panama"},{"DOI":"10.13039\/100008548","name":"Harvard TH Chan School of Public Health","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100008548","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Ministry of Health of Panama"},{"name":"Sistema Nacional de Investigacion"},{"DOI":"10.13039\/501100007061","name":"Secretar\u00eda Nacional de Ciencia, Tecnolog\u00eda e Innovaci\u00f3n","doi-asserted-by":"publisher","award":["ITE15-004"],"award-info":[{"award-number":["ITE15-004"]}],"id":[{"id":"10.13039\/501100007061","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Royal Society Wolfson Merit"},{"DOI":"10.13039\/100004440","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["104111"],"award-info":[{"award-number":["104111"]}],"id":[{"id":"10.13039\/100004440","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosntds.org"],"crossmark-restriction":false},"short-container-title":["PLoS Negl Trop Dis"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background<\/jats:title>\n<jats:p>As the elimination of malaria in Mesoamerica progresses, detection of <jats:italic>Plasmodium vivax<\/jats:italic> using light microscopy (LM) becomes more difficult. Highly sensitive molecular tools have been developed to help determine the hidden reservoir of malaria transmission in low transmission settings. In this study we compare the performance of <jats:italic>PvLAP5<\/jats:italic> and <jats:italic>Pvs25<\/jats:italic> qRT-PCR assays to LM for the detection of <jats:italic>Plasmodium vivax<\/jats:italic> gametocytes in field samples preserved at ambient temperature from malaria endemic regions of Panama.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methods<\/jats:title>\n<jats:p>For this purpose, we collected a total of 83 malaria field samples during 2017-2020 preserved in RNAprotect (RNAp) of which 63 (76%) were confirmed <jats:italic>P<\/jats:italic>. <jats:italic>vivax<\/jats:italic> by LM and selected for further analysis. Additionally, 16 blood samples from local healthy malaria smear negative volunteers, as well as, from 15 malaria na\u00efve lab-bred <jats:italic>Aotus<\/jats:italic> monkeys were used as controls. To optimize the assays, we first determined the minimum blood volume sufficient for detection of <jats:italic>PvLAP5<\/jats:italic> and <jats:italic>Pv18SrRNA<\/jats:italic> using <jats:italic>P<\/jats:italic>. <jats:italic>vivax<\/jats:italic> infected Aotus blood that was preserved in RNAp and kept either at ambient temperature for up to 8 days before freezing or was snap-frozen at -80\u00b0 Celsius at the time of bleeding. We then compared the mean differences in gametocyte detection rates of both qRT-PCR assays to LM and performed a multivariate correlation analysis of study variables. Finally, we determined the sensitivity (Se) and specificity (Sp) of the assays at detecting gametocytes compared to LM.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Results<\/jats:title>\n<jats:p>Blood volume optimization indicated that a blood volume of at least 60 \u03bcL was sufficient for detection of <jats:italic>PvLAP5<\/jats:italic> and <jats:italic>Pv18SrRNA<\/jats:italic> and no significant differences were found between RNA storage conditions. Both <jats:italic>PvLAP5<\/jats:italic> and <jats:italic>Pvs25<\/jats:italic> qRT-PCR assays showed a 37-39% increase in gametocyte detection rate compared to LM respectively. Strong positive correlations were found between gametocytemia and parasitemia and both <jats:italic>PvLAP5<\/jats:italic> and <jats:italic>Pvs25<\/jats:italic> gametocyte markers. However, no significant differences were detected in the Se and Sp of the <jats:italic>Pvs25<\/jats:italic> and <jats:italic>PvLAP5<\/jats:italic> qRT-PCR assays, even though data from control samples suggested <jats:italic>Pvs25<\/jats:italic> to be more abundant than <jats:italic>PvLAP5<\/jats:italic>.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusions<\/jats:title>\n<jats:p>This study shows that the <jats:italic>PvLAP5<\/jats:italic> qRT-PCR assay is as Se and Sp as the gold standard <jats:italic>Pvs25<\/jats:italic> assay and is at least 37% more sensitive than LM at detecting <jats:italic>P<\/jats:italic>. <jats:italic>vivax<\/jats:italic> gametocytes in field samples preserved in RNAp at ambient temperature from malaria endemic regions of Panama.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec005\">\n<jats:title>Author summary<\/jats:title>\n<jats:p><jats:italic>Plasmodium vivax<\/jats:italic> is one of the five species of malaria (<jats:italic>P<\/jats:italic>. <jats:italic>falciparum<\/jats:italic>, <jats:italic>P<\/jats:italic>. <jats:italic>malariae<\/jats:italic>, <jats:italic>P<\/jats:italic>. <jats:italic>ovale<\/jats:italic> and <jats:italic>P<\/jats:italic>. <jats:italic>knowlesi)<\/jats:italic> that are transmitted to man by the bite of female anopheles mosquitoes. It causes ~14.3 million cases mainly in Southeast Asia, India, the Western Pacific and the Americas annually. In the Americas, malaria remains a major problem in underdeveloped areas and indigenous communities in the Amazon region and eastern Panama, where it is endemic and difficult to eliminate. As malaria elimination progresses, detection of <jats:italic>P<\/jats:italic>. <jats:italic>vivax<\/jats:italic> by light microscopy (LM) becomes more difficult. Therefore, highly sensitive molecular tools have been developed that use genetic markers for the parasite to help determine the hidden reservoir of malaria transmission. This study compares the performance of two molecular assays based on the genetic markers of mature gametocytes <jats:italic>PvLAP5<\/jats:italic> and <jats:italic>Pvs25<\/jats:italic> with LM. The study shows that the <jats:italic>PvLAP5<\/jats:italic> qRT-PCR assay is as sensitive and specific as the gold standard <jats:italic>Pvs25<\/jats:italic> assay and is at least 37% more sensitive than LM at detecting <jats:italic>P<\/jats:italic>. <jats:italic>vivax<\/jats:italic> gametocytes. These data suggest that the <jats:italic>PvLAP5<\/jats:italic> qRT-PCR assay can be a useful tool to help determine the hidden reservoir of transmission in endemic foci approaching elimination.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pntd.0010327","type":"journal-article","created":{"date-parts":[[2022,4,8]],"date-time":"2022-04-08T17:37:36Z","timestamp":1649439456000},"page":"e0010327","update-policy":"https:\/\/doi.org\/10.1371\/journal.pntd.corrections_policy","source":"Crossref","is-referenced-by-count":9,"title":["Comparison of PvLAP5 and Pvs25 qRT-PCR assays for the detection of Plasmodium vivax gametocytes in field samples preserved at ambient temperature from remote malaria endemic regions of Panama"],"prefix":"10.1371","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3711-9449","authenticated-orcid":true,"given":"Nicanor","family":"Obald\u00eda","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Itza","family":"Barahona","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jos\u00e9","family":"Lasso","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mario","family":"Avila","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mario","family":"Quijada","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marlon","family":"Nu\u00f1ez","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthias","family":"Marti","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2022,4,8]]},"reference":[{"key":"pntd.0010327.ref001","volume-title":"World Malaria Report","author":"WHO","year":"2020"},{"issue":"11","key":"pntd.0010327.ref002","doi-asserted-by":"crossref","first-page":"e0004222","DOI":"10.1371\/journal.pntd.0004222","article-title":"Plasmodium vivax Transmission in Africa","volume":"9","author":"RE Howes","year":"2015","journal-title":"PLoS Negl Trop Dis"},{"issue":"10","key":"pntd.0010327.ref003","doi-asserted-by":"crossref","first-page":"e1003799","DOI":"10.1371\/journal.pmed.1003799","article-title":"The global burden of Plasmodium vivax malaria is obscure and insidious","volume":"18","author":"KE Battle","year":"2021","journal-title":"PLoS Med"},{"issue":"7","key":"pntd.0010327.ref004","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1093\/infdis\/jiu575","article-title":"Clonal outbreak of Plasmodium falciparum infection in eastern Panama","volume":"211","author":"N Obaldia","year":"2015","journal-title":"J Infect Dis"},{"issue":"4","key":"pntd.0010327.ref005","first-page":"e27","article-title":"When climate change couples social neglect: malaria dynamics in Panama","volume":"3","author":"LA Hurtado","year":"2014","journal-title":"Emerg Microbes Infect"},{"issue":"12","key":"pntd.0010327.ref006","doi-asserted-by":"crossref","first-page":"e0008962","DOI":"10.1371\/journal.pntd.0008962","article-title":"Population genomics of Plasmodium vivax in Panama to assess the risk of case importation on malaria elimination","volume":"14","author":"LE Buyon","year":"2020","journal-title":"PLoS Negl Trop Dis"},{"issue":"3","key":"pntd.0010327.ref007","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1016\/j.actatropica.2011.06.008","article-title":"Malaria in selected non-Amazonian countries of Latin America","volume":"121","author":"M Arevalo-Herrera","year":"2012","journal-title":"Acta Trop"},{"key":"pntd.0010327.ref008","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1186\/s12936-014-0529-7","article-title":"Determinants of low socio-economic status and risk of Plasmodium vivax malaria infection in Panama (2009-2012): a case-control study","volume":"14","author":"N Obaldia","year":"2015","journal-title":"Malar J"},{"issue":"1","key":"pntd.0010327.ref009","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1038\/nm0111-48","article-title":"Defying malaria: Fathoming severe Plasmodium vivax disease","volume":"17","author":"Q Bassat","year":"2011","journal-title":"Nat Med"},{"key":"pntd.0010327.ref010","first-page":"1","article-title":"A research agenda for malaria eradication: Modeling","volume":"8","author":"The malERA CGoM","year":"2011","journal-title":"PLoS Medicine"},{"issue":"9597","key":"pntd.0010327.ref011","doi-asserted-by":"crossref","first-page":"1459","DOI":"10.1016\/S0140-6736(07)61609-2","article-title":"Is malaria eradication possible?","volume":"370","year":"2007","journal-title":"Lancet"},{"key":"pntd.0010327.ref012","volume-title":"InWHO, editor. 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