{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T14:03:53Z","timestamp":1787580233367,"version":"build-2736575974"},"reference-count":41,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2022,12,16]],"date-time":"2022-12-16T00:00:00Z","timestamp":1671148800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004156","name":"Mahidol University","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100004156","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100017170","name":"Thailand Science Research and Innovation","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100017170","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Fundamental Fund, Thailand","award":["RTA6280006"],"award-info":[{"award-number":["RTA6280006"]}]},{"name":"Mahidol-University Oxford Tropical Medicine Research Programme"},{"name":"Wellcome Trust of the United Kingdom","award":["106698\/B\/14\/Z"],"award-info":[{"award-number":["106698\/B\/14\/Z"]}]},{"name":"Wellcome OA statement"},{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["220211"],"award-info":[{"award-number":["220211"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:sec id=\"sec001\">\n<jats:title>Background<\/jats:title>\n<jats:p>The amplification of <jats:italic>GTP cyclohydrolase 1<\/jats:italic> (<jats:italic>pfgch1<\/jats:italic>) in <jats:italic>Plasmodium falciparum<\/jats:italic> has been linked to the upregulation of the <jats:italic>pfdhfr<\/jats:italic> and <jats:italic>pfdhps<\/jats:italic> genes associated with resistance to the antimalarial drug sulfadoxine-pyrimethamine. During the 1990s and 2000s, sulfadoxine-pyrimethamine was withdrawn from use as first-line treatment in southeast Asia due to clinical drug resistance. This study assessed the temporal and geographic changes in the prevalence of <jats:italic>pfdhfr<\/jats:italic> and <jats:italic>pfdhps<\/jats:italic> gene mutations and <jats:italic>pfgch1<\/jats:italic> amplification a decade after sulfadoxine-pyrimethamine had no longer been widely used.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec002\">\n<jats:title>Methods<\/jats:title>\n<jats:p>A total of 536 <jats:italic>P<\/jats:italic>. <jats:italic>falciparum<\/jats:italic> isolates collected from clinical trials in Thailand, Cambodia, and Lao PDR between 2008 and 2018 were assayed. Single nucleotide polymorphisms of the <jats:italic>pfdhfr<\/jats:italic> and <jats:italic>pfdhps<\/jats:italic> genes were analyzed using nested PCR and Sanger sequencing. Gene copy number variations of <jats:italic>pfgch<\/jats:italic>1 were investigated using real-time polymerase chain reaction assay.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec003\">\n<jats:title>Results<\/jats:title>\n<jats:p>Sequences of the <jats:italic>pfdhfr<\/jats:italic> and <jats:italic>pfdhps<\/jats:italic> genes were obtained from 96% (517\/536) and 91% (486\/536) of the samples, respectively. There were 59 distinct haplotypes, including single to octuple mutations. The two major haplotypes observed included <jats:bold>IRN<\/jats:bold>I-<jats:bold>AGE<\/jats:bold>AA (25%) and <jats:bold>IRNL<\/jats:bold>-S<jats:bold>G<\/jats:bold>K<jats:bold>G<\/jats:bold>A (19%). The sextuple mutation <jats:bold>IRNL<\/jats:bold>-S<jats:bold>G<\/jats:bold>K<jats:bold>G<\/jats:bold>A increased markedly over time in several study sites, including Pailin, Preah Vihear, Ratanakiri, and Ubon Ratchathani, whereas <jats:bold>IRNI<\/jats:bold>-<jats:bold>AGE<\/jats:bold>AA decreased over time in Preah Vihear, Champasak, and Ubon Ratchathani. Octuple mutations were first observed in west Cambodia in 2011 and subsequently in northeast Cambodia, as well as in southern Laos by 2018. Amplification of the <jats:italic>pfgch1<\/jats:italic> gene increased over time across the region, particularly in northeast Thailand close to the border with Laos and Cambodia.<\/jats:p>\n<\/jats:sec>\n<jats:sec id=\"sec004\">\n<jats:title>Conclusion<\/jats:title>\n<jats:p>Despite the fact that SP therapy was discontinued in Thailand, Cambodia, and Laos decades ago, parasites retained the <jats:italic>pfdhfr<\/jats:italic> and <jats:italic>pfdhps<\/jats:italic> mutations. Numerous haplotypes were found to be prevalent among the parasites. Frequent monitoring of <jats:italic>pfdhfr<\/jats:italic> and <jats:italic>pfdhps<\/jats:italic> in these areas is required due to the relatively rapid evolution of mutation patterns.<\/jats:p>\n<\/jats:sec>","DOI":"10.1371\/journal.pone.0278928","type":"journal-article","created":{"date-parts":[[2022,12,16]],"date-time":"2022-12-16T18:31:15Z","timestamp":1671215475000},"page":"e0278928","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":9,"title":["Ten-year persistence and evolution of Plasmodium falciparum antifolate and anti-sulfonamide resistance markers pfdhfr and pfdhps in three Asian countries"],"prefix":"10.1371","volume":"17","author":[{"given":"Suttipat","family":"Srisutham","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanassanan","family":"Madmanee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jindarat","family":"Kouhathong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kreepol","family":"Sutawong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3281-9702","authenticated-orcid":true,"given":"Rupam","family":"Tripura","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas J.","family":"Peto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rob W.","family":"van der Pluijm","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3218-2166","authenticated-orcid":true,"given":"James J.","family":"Callery","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lek","family":"Dysoley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mayfong","family":"Mayxay","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul N.","family":"Newton","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tiengkham","family":"Pongvongsa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bouasy","family":"Hongvanthong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas P. J.","family":"Day","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas J.","family":"White","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Arjen M.","family":"Dondorp","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0857-1855","authenticated-orcid":true,"given":"Mallika","family":"Imwong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2022,12,16]]},"reference":[{"key":"pone.0278928.ref001","article-title":"The \"World malaria report 2019\" at a glance","author":"WHO","year":"2019"},{"issue":"1","key":"pone.0278928.ref002","first-page":"41","article-title":"Drug resistant malaria on the Thai-Myanmar and Thai-Cambodian borders","volume":"32","author":"C Wongsrichanalai","year":"2001","journal-title":"Southeast Asian J Trop Med Public Health"},{"key":"pone.0278928.ref003","doi-asserted-by":"crossref","first-page":"840","DOI":"10.4269\/ajtmh.1963.12.840","article-title":"Plasmodium Falciparum Resistant to Chloroquine in Cambodia","volume":"12","author":"DE Eyles","year":"1963","journal-title":"Am J Trop Med Hyg"},{"issue":"1","key":"pone.0278928.ref004","first-page":"111","article-title":"Epidemiology of emergence and spread of drug-resistant falciparum malaria in Southeast Asia","volume":"17","author":"J. Verdrager","year":"1986","journal-title":"Southeast Asian J Trop Med Public Health"},{"key":"pone.0278928.ref005","volume-title":"Report on the informal consultation on monitoring resistance to antimalarial drugs in the Mekong Region","author":"WHO","year":"2000"},{"issue":"5","key":"pone.0278928.ref006","doi-asserted-by":"crossref","first-page":"716","DOI":"10.3201\/eid1405.071601","article-title":"Declining artesunate-mefloquine efficacy against falciparum malaria on the Cambodia-Thailand border","volume":"14","author":"C Wongsrichanalai","year":"2008","journal-title":"Emerg Infect Dis"},{"issue":"6","key":"pone.0278928.ref007","doi-asserted-by":"crossref","first-page":"1221","DOI":"10.4269\/ajtmh.2010.10-0276","article-title":"A phase III, randomized, non-inferiority trial to assess the efficacy and safety of dihydroartemisinin-piperaquine in comparison with artesunate-mefloquine in patients with uncomplicated Plasmodium falciparum malaria in southern Laos","volume":"83","author":"M Mayxay","year":"2010","journal-title":"Am J Trop Med Hyg"},{"issue":"14","key":"pone.0278928.ref008","doi-asserted-by":"crossref","first-page":"3807","DOI":"10.1093\/emboj\/17.14.3807","article-title":"Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria","volume":"17","author":"T Triglia","year":"1998","journal-title":"EMBO J"},{"key":"pone.0278928.ref009","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1016\/j.meegid.2015.04.007","article-title":"High prevalence of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum field isolates from pregnant women in Brazzaville, Republic of Congo","volume":"33","author":"F Koukouikila-Koussounda","year":"2015","journal-title":"Infect Genet Evol"},{"issue":"5","key":"pone.0278928.ref010","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1046\/j.1365-2958.1997.2821646.x","article-title":"Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization","volume":"23","author":"P Wang","year":"1997","journal-title":"Mol Microbiol"},{"issue":"1","key":"pone.0278928.ref011","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/0166-6851(94)00207-4","article-title":"Point mutations in the dihydrofolate reductase-thymidylate synthase gene and pyrimethamine and cycloguanil resistance in Plasmodium falciparum","volume":"69","author":"LK Basco","year":"1995","journal-title":"Mol Biochem Parasitol"},{"issue":"9","key":"pone.0278928.ref012","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1046\/j.1365-3156.2003.01099.x","article-title":"Plasmodium falciparum: correlation of in vivo resistance to chloroquine and antifolates with genetic polymorphisms in isolates from the south of Lao PDR","volume":"8","author":"N Berens","year":"2003","journal-title":"Trop Med Int Health"},{"issue":"4","key":"pone.0278928.ref013","doi-asserted-by":"crossref","first-page":"702","DOI":"10.1111\/mmi.12162","article-title":"Direct evidence for the adaptive role of copy number variation on antifolate susceptibility in Plasmodium falciparum","volume":"88","author":"A Heinberg","year":"2013","journal-title":"Mol Microbiol"},{"issue":"1","key":"pone.0278928.ref014","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1186\/s12936-020-03176-x","article-title":"Molecular characterization of Plasmodium falciparum antifolate resistance markers in Thailand between 2008 and 2016","volume":"19","author":"R Sugaram","year":"2020","journal-title":"Malar J"},{"issue":"1","key":"pone.0278928.ref015","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1006\/expr.1998.4274","article-title":"Plasmodium falciparum: detection of polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes by PCR and restriction digestion","volume":"89","author":"MT Duraisingh","year":"1998","journal-title":"Exp Parasitol"},{"issue":"3","key":"pone.0278928.ref016","doi-asserted-by":"crossref","first-page":"431","DOI":"10.4269\/ajtmh.2007.77.431","article-title":"Antimalarial drug susceptibility and point mutations associated with drug resistance in 248 Plasmodium falciparum isolates imported from Comoros to Marseille, France in 2004 2006","volume":"77","author":"P Parola","year":"2007","journal-title":"Am J Trop Med Hyg"},{"issue":"10","key":"pone.0278928.ref017","doi-asserted-by":"crossref","first-page":"3523","DOI":"10.1128\/AAC.00234-07","article-title":"Genetic analysis of the dihydrofolate reductase-thymidylate synthase gene from geographically diverse isolates of Plasmodium malariae","volume":"51","author":"N Tanomsing","year":"2007","journal-title":"Antimicrob Agents Chemother"},{"issue":"3","key":"pone.0278928.ref018","doi-asserted-by":"crossref","first-page":"e1000830","DOI":"10.1371\/journal.ppat.1000830","article-title":"Origin and evolution of sulfadoxine resistant Plasmodium falciparum","volume":"6","author":"S Vinayak","year":"2010","journal-title":"PLoS Pathog"},{"issue":"10","key":"pone.0278928.ref019","doi-asserted-by":"crossref","first-page":"e1000243","DOI":"10.1371\/journal.pgen.1000243","article-title":"Adaptive copy number evolution in malaria parasites","volume":"4","author":"S Nair","year":"2008","journal-title":"PLoS Genet"},{"key":"pone.0278928.ref020","first-page":"1987","volume-title":"Molecular evolutionary genetics","author":"N M"},{"issue":"9","key":"pone.0278928.ref021","doi-asserted-by":"crossref","first-page":"3793","DOI":"10.1128\/AAC.00308-09","article-title":"In vitro activity of antifolate and polymorphism in dihydrofolate reductase of Plasmodium falciparum isolates from the Kenyan coast: emergence of parasites with Ile-164-Leu mutation","volume":"53","author":"SM Kiara","year":"2009","journal-title":"Antimicrob Agents Chemother"},{"issue":"1","key":"pone.0278928.ref022","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1128\/AAC.00691-10","article-title":"Tracking origins and spread of sulfadoxine-resistant Plasmodium falciparum dhps alleles in Thailand","volume":"55","author":"MT Alam","year":"2011","journal-title":"Antimicrob Agents Chemother"},{"key":"pone.0278928.ref023","doi-asserted-by":"crossref","first-page":"17412","DOI":"10.1038\/srep17412","article-title":"An outbreak of artemisinin resistant falciparum malaria in Eastern Thailand","volume":"5","author":"M Imwong","year":"2015","journal-title":"Sci Rep"},{"issue":"4","key":"pone.0278928.ref024","doi-asserted-by":"crossref","first-page":"1124","DOI":"10.1073\/pnas.94.4.1124","article-title":"Antifolate-resistant mutants of Plasmodium falciparum dihydrofolate reductase","volume":"94","author":"W Sirawaraporn","year":"1997","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"23","key":"pone.0278928.ref025","doi-asserted-by":"crossref","first-page":"9109","DOI":"10.1073\/pnas.85.23.9109","article-title":"Amino acid changes linked to pyrimethamine resistance in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum","volume":"85","author":"AF Cowman","year":"1988","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"23","key":"pone.0278928.ref026","doi-asserted-by":"crossref","first-page":"9114","DOI":"10.1073\/pnas.85.23.9114","article-title":"Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria","volume":"85","author":"DS Peterson","year":"1988","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"2","key":"pone.0278928.ref027","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/0166-6851(92)90047-N","article-title":"Pyrimethamine resistant mutations in Plasmodium falciparum","volume":"52","author":"S Thaithong","year":"1992","journal-title":"Mol Biochem Parasitol"},{"issue":"5","key":"pone.0278928.ref028","doi-asserted-by":"crossref","first-page":"624","DOI":"10.1111\/j.1365-3156.2004.01239.x","article-title":"Relationship between age, molecular markers, and response to sulphadoxine-pyrimethamine treatment in Kampala, Uganda","volume":"9","author":"SG Staedke","year":"2004","journal-title":"Trop Med Int Health"},{"issue":"3","key":"pone.0278928.ref029","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1086\/338566","article-title":"Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria","volume":"185","author":"JG Kublin","year":"2002","journal-title":"J Infect Dis"},{"issue":"8","key":"pone.0278928.ref030","doi-asserted-by":"crossref","first-page":"3018","DOI":"10.1073\/pnas.87.8.3018","article-title":"Molecular basis of differential resistance to cycloguanil and pyrimethamine in Plasmodium falciparum malaria","volume":"87","author":"DS Peterson","year":"1990","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"4","key":"pone.0278928.ref031","doi-asserted-by":"crossref","first-page":"861","DOI":"10.1016\/S1097-2765(05)00077-8","article-title":"Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance","volume":"6","author":"DA Fidock","year":"2000","journal-title":"Mol Cell"},{"issue":"7","key":"pone.0278928.ref032","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1016\/j.ijpara.2020.12.011","article-title":"Geographical spread and structural basis of sulfadoxine-pyrimethamine drug-resistant malaria parasites","volume":"51","author":"R Chaturvedi","year":"2021","journal-title":"Int J Parasitol"},{"issue":"9287","key":"pone.0278928.ref033","doi-asserted-by":"crossref","first-page":"1066","DOI":"10.1016\/S0140-6736(01)06201-8","article-title":"Plasmodium falciparum cross-resistance between trimethoprim and pyrimethamine","volume":"358","author":"JK Iyer","year":"2001","journal-title":"Lancet"},{"issue":"9448","key":"pone.0278928.ref034","doi-asserted-by":"crossref","first-page":"1865","DOI":"10.1016\/S0140-6736(04)17442-4","article-title":"Co-trimoxazole as prophylaxis against opportunistic infections in HIV-infected Zambian children (CHAP): a double-blind randomised placebo-controlled trial","volume":"364","author":"C Chintu","year":"2004","journal-title":"Lancet"},{"issue":"9722","key":"pone.0278928.ref035","doi-asserted-by":"crossref","first-page":"1278","DOI":"10.1016\/S0140-6736(10)60057-8","article-title":"Daily co-trimoxazole prophylaxis in severely immunosuppressed HIV-infected adults in Africa started on combination antiretroviral therapy: an observational analysis of the DART cohort","volume":"375","author":"AS Walker","year":"2010","journal-title":"Lancet"},{"issue":"4","key":"pone.0278928.ref036","doi-asserted-by":"crossref","first-page":"338","DOI":"10.1016\/j.jemermed.2007.08.068","article-title":"Trimethoprim\/sulfamethoxazole resistance in urinary tract infections","volume":"36","author":"O Guneysel","year":"2009","journal-title":"J Emerg Med"},{"issue":"10","key":"pone.0278928.ref037","doi-asserted-by":"crossref","first-page":"4193","DOI":"10.1128\/AAC.01301-08","article-title":"Dosing regimens of cotrimoxazole (trimethoprim-sulfamethoxazole) for melioidosis","volume":"53","author":"AC Cheng","year":"2009","journal-title":"Antimicrob Agents Chemother"},{"issue":"3","key":"pone.0278928.ref038","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1016\/j.actatropica.2005.04.010","article-title":"The origins and spread of antimalarial drug resistance: lessons for policy makers","volume":"94","author":"TJ Anderson","year":"2005","journal-title":"Acta Trop"},{"issue":"9","key":"pone.0278928.ref039","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1016\/j.pt.2010.05.001","article-title":"Following the path of most resistance: dhps K540E dispersal in African Plasmodium falciparum","volume":"26","author":"I Naidoo","year":"2010","journal-title":"Trends Parasitol"},{"issue":"2","key":"pone.0278928.ref040","doi-asserted-by":"crossref","first-page":"103","DOI":"10.3201\/eid0602.000202","article-title":"Malaria on the move: human population movement and malaria transmission","volume":"6","author":"P Martens","year":"2000","journal-title":"Emerg Infect Dis"},{"issue":"6","key":"pone.0278928.ref041","doi-asserted-by":"crossref","first-page":"e57","DOI":"10.1371\/journal.ppat.0020057","article-title":"A systematic map of genetic variation in Plasmodium falciparum","volume":"2","author":"C Kidgell","year":"2006","journal-title":"PLoS Pathog"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0278928","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,16]],"date-time":"2022-12-16T18:31:54Z","timestamp":1671215514000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0278928"}},"subtitle":[],"editor":[{"given":"David J.","family":"Sullivan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2022,12,16]]},"references-count":41,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2022,12,16]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0278928","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,16]]}}}