{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:29:50Z","timestamp":1787027390392,"version":"build-2736575974"},"reference-count":31,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,1,26]],"date-time":"2023-01-26T00:00:00Z","timestamp":1674691200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001409","name":"Department of Science and Technology, Ministry of Science and Technology, India","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001409","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Vet. Sci."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>\n                      The imminent risk of zoonoses of non-human malaria parasites is not far from reality in India, as has been observed in the case of Plasmodium knowlesi (Pk), and so is possible with\n                      <jats:italic>P. cynomolgi<\/jats:italic>\n                      (Pc), already reported from South East Asian countries. Therefore, a novel multiplex qPCR assay was developed and evaluated for detection of non-human malaria parasites- Pk and Pc in populations at risk.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>The qPCR primers were designed in-house with fluorescence labeled probes (HEX for Pk and FAM for Pc). DNA samples of Pk and Pc were used as templates and further the qPCR assay was evaluated in 250 symptomatic and asymptomatic suspected human blood samples from malaria endemic areas of North Eastern states of India.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The qPCR assay successfully amplified the target 18S rRNA gene segment from Pk and Pc and was highly specific for Pk and Pc parasites only, as no cross reactivity was observed with\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      (Pf),\n                      <jats:italic>P. vivax<\/jats:italic>\n                      (Pv),\n                      <jats:italic>P. malariae<\/jats:italic>\n                      (Pm), and\n                      <jats:italic>P. ovale<\/jats:italic>\n                      (Po). Standard curves were generated to estimate the limit of detection (LOD) of Pk and Pc parasites DNA (0.00275 &amp;amp; 0.075 ng\/\u03bcl, respectively). Due to COVID-19 pandemic situation during 2020\u201321, the sample accessibility was difficult, however, we managed to collect 250 samples. The samples were tested for Pf and Pv using conventional PCR- 14 Pf and 11 Pv infections were observed, but no Pk and Pc infections were detected. For Pk infections, previously reported conventional PCR was also performed, but no Pk infection was detected.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>The multiplex qPCR assay was observed to be robust, quick, cost-effective and highly sensitive as compared to the currently available conventional PCR methods. Further validation of the multiplex qPCR assay in field setting is desirable, especially from the high-risk populations. We anticipate that the multiplex qPCR assay would prove to be a useful tool in mass screening and surveillance programs for detection of non-human malaria parasites toward the control and elimination of malaria from India by 2030.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fvets.2023.1127273","type":"journal-article","created":{"date-parts":[[2023,1,26]],"date-time":"2023-01-26T06:47:42Z","timestamp":1674715662000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":3,"title":["A novel multiplex qPCR assay for clinical diagnosis of non-human malaria parasites-Plasmodium knowlesi and Plasmodium cynomolgi"],"prefix":"10.3389","volume":"10","author":[{"given":"Ram","family":"Das","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kapil","family":"Vashisht","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kailash C.","family":"Pandey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2023,1,26]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1016\/j.actatropica.2012.06.011","article-title":"Anopheles (Cellia) maculatus group: its spatial distribution and molecular characterization of member species in north-east India","volume":"124","author":"Singh","year":"2012","journal-title":"Acta Trop"},{"key":"B2","doi-asserted-by":"publisher","first-page":"1081","DOI":"10.1093\/jac\/dks508","article-title":"Discordance in drug resistance-associated mutation patterns in marker genes of Plasmodium falciparum and Plasmodium knowlesi during coinfections","volume":"183","author":"Tyagi","year":"2013","journal-title":"Chemother J Antimicrob."},{"key":"B3","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1186\/1756-3305-7-186","article-title":"Spatial distribution of malaria in Peninsular Malaysia from 2000 to 2009","volume":"7","author":"Alias","year":"2014","journal-title":"Parasites Vectors."},{"key":"B4","doi-asserted-by":"publisher","first-page":"e0006801","DOI":"10.1371\/journal.pntd.0006801","article-title":"Reinvestigating the status of malaria parasite (Plasmodium sp.) in Indian non-human primates","volume":"12","author":"Dixit","year":"2018","journal-title":"PLOS Neglect Trop Dis."},{"key":"B5","doi-asserted-by":"publisher","first-page":"817","DOI":"10.3201\/eid2504.181668","article-title":"Anopheles sundaicus Mosquitoes as Vector for Plasmodium knowlesi, Andaman and Nicobar Islands, India","volume":"25","author":"Vidhya","year":"2019","journal-title":"Emerg Infect Dis."},{"key":"B6","doi-asserted-by":"publisher","first-page":"37","DOI":"10.1186\/s13104-020-4900-1","article-title":"Renal detection of Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi in malaria associated acute kidney injury: a retrospective case\u2013control study","volume":"13","author":"Kaur","year":"2020","journal-title":"BMC Res Notes."},{"key":"B7","doi-asserted-by":"publisher","first-page":"100190","DOI":"10.1016\/j.onehlt.2020.100190","article-title":"Trends of neglected Plasmodium species infection in humans over the past century in India","volume":"186","author":"Chaturvedi","year":"2021","journal-title":"One Health."},{"key":"B8","doi-asserted-by":"publisher","first-page":"2187","DOI":"10.3201\/eid2708.204502","article-title":"Natural Human Infections with Plasmodium cynomolgi, P. inui, and 4 other Simian Malaria Parasites, Malaysia","volume":"216","author":"Yap","year":"2021","journal-title":"Emerg Infect Dis."},{"key":"B9","doi-asserted-by":"publisher","first-page":"e1002015","DOI":"10.1371\/journal.ppat.1002015","article-title":"Plasmodium knowlesi: Reservoir Hosts and Tracking the Emergence in Humans and Macaques","volume":"7","author":"Lee","year":"2011","journal-title":"PLOS Pathogens."},{"key":"B10","doi-asserted-by":"publisher","first-page":"123","DOI":"10.3389\/fpubh.2014.00123","article-title":"Zoonotic malaria-global overview and research and policy needs","volume":"190","author":"Ramasamy","year":"2014","journal-title":"Front Public Health."},{"key":"B11","first-page":"2","article-title":"Guest commentary: Plasmodium knowlesi-need to diagnose in India","volume":"189","author":"Mewara","year":"2017","journal-title":"Trop Parasitol."},{"key":"B12","doi-asserted-by":"publisher","first-page":"673","DOI":"10.4269\/ajtmh.1961.10.673","article-title":"Transmission of the M strain of Plasmodium cynomolgi to man","volume":"199","author":"Coatney","year":"1961","journal-title":"Am J. 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