{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T20:51:10Z","timestamp":1793393470079,"version":"4.5.0"},"reference-count":50,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2020,7,25]],"date-time":"2020-07-25T00:00:00Z","timestamp":1595635200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2020,7,25]],"date-time":"2020-07-25T00:00:00Z","timestamp":1595635200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Malar J"],"published-print":{"date-parts":[[2020,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      High rates of dihydroartemisinin\u2013piperaquine (DHA\u2013PPQ) treatment failures have been documented for uncomplicated\n                      <jats:italic>Plasmodium falciparum<\/jats:italic>\n                      in Cambodia. The genetic markers plasmepsin 2 (\n                      <jats:italic>pfpm2<\/jats:italic>\n                      ), exonuclease (\n                      <jats:italic>pfexo<\/jats:italic>\n                      ) and chloroquine resistance transporter (\n                      <jats:italic>pfcrt<\/jats:italic>\n                      ) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      isolates from northern Cambodia, and the presence of E415G-Exo and\n                      <jats:italic>pfcrt<\/jats:italic>\n                      mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as\n                      <jats:italic>pfpm2<\/jats:italic>\n                      copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      Cambodian isolates was also determined.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The characterization of culture-adapted isolates revealed that the presence of novel\n                      <jats:italic>pfcrt<\/jats:italic>\n                      mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of\n                      <jats:italic>pfpm2<\/jats:italic>\n                      amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose\u2013response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone\u2013proguanil combinations revealed synergistic antimalarial activity.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>\n                      Surveillance for PPQ resistance in regions relying on DHA\u2013PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance.\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      harbouring novel\n                      <jats:italic>pfcrt<\/jats:italic>\n                      mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of\n                      <jats:italic>pfpm2<\/jats:italic>\n                      amplification was not required to render parasites PPQ resistant suggesting that the increase in\n                      <jats:italic>pfpm2<\/jats:italic>\n                      copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against\n                      <jats:italic>P. falciparum<\/jats:italic>\n                      strains and when down-selecting novel drug candidates.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1186\/s12936-020-03339-w","type":"journal-article","created":{"date-parts":[[2020,7,25]],"date-time":"2020-07-25T07:02:48Z","timestamp":1595660568000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":37,"title":["Piperaquine resistant Cambodian Plasmodium falciparum clinical isolates: in vitro genotypic and phenotypic characterization"],"prefix":"10.1186","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9058-0840","authenticated-orcid":false,"given":"Nonlawat","family":"Boonyalai","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brian A.","family":"Vesely","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chatchadaporn","family":"Thamnurak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chantida","family":"Praditpol","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Watcharintorn","family":"Fagnark","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kirakarn","family":"Kirativanich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Piyaporn","family":"Saingam","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chaiyaporn","family":"Chaisatit","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paphavee","family":"Lertsethtakarn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Panita","family":"Gosi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Worachet","family":"Kuntawunginn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pattaraporn","family":"Vanachayangkul","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michele D.","family":"Spring","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark M.","family":"Fukuda","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chanthap","family":"Lon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Philip L.","family":"Smith","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Norman C.","family":"Waters","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David L.","family":"Saunders","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariusz","family":"Wojnarski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,7,25]]},"reference":[{"key":"3339_CR1","volume-title":"World malaria report","author":"WHO","year":"2019","unstructured":"WHO. World malaria report. Geneva: World Health Organization; 2019."},{"key":"3339_CR2","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1016\/S1473-3099(17)30048-8","volume":"17","author":"M Imwong","year":"2017","unstructured":"Imwong M, Suwannasin K, Kunasol C, Sutawong K, Mayxay M, Rekol H, et al. The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion: a molecular epidemiology observational study. Lancet Infect Dis. 2017;17:491\u20137.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR3","volume-title":"Guidelines fro the treatment of malaria","author":"WHO","year":"2015","unstructured":"WHO. Guidelines fro the treatment of malaria. 3rd ed. Geneva: World Health Organization Press; 2015.","edition":"3"},{"key":"3339_CR4","doi-asserted-by":"crossref","first-page":"484","DOI":"10.1056\/NEJMc1403007","volume":"371","author":"DL Saunders","year":"2014","unstructured":"Saunders DL, Vanachayangkul P, Lon C, Program USAMMR, National Center for Parasitology, Entomology, Malaria Control, Royal Cambodian Armed Forces. Dihydroartemisinin\u2013piperaquine failure in Cambodia. N Engl J Med. 2014;371:484\u20135.","journal-title":"N Engl J Med."},{"key":"3339_CR5","doi-asserted-by":"crossref","first-page":"4719","DOI":"10.1128\/AAC.00835-15","volume":"59","author":"R Leang","year":"2015","unstructured":"Leang R, Taylor WR, Bouth DM, Song L, Tarning J, Char MC, et al. Evidence of Plasmodium falciparum malaria multidrug resistance to artemisinin and piperaquine in Western Cambodia: dihydroartemisinin-piperaquine open-label multicenter clinical assessment. Antimicrob Agents Chemother. 2015;59:4719\u201326.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR6","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1016\/S1473-3099(15)70049-6","volume":"15","author":"MD Spring","year":"2015","unstructured":"Spring MD, Lin JT, Manning JE, Vanachayangkul P, Somethy S, Bun R, et al. Dihydroartemisinin-piperaquine failure associated with a triple mutant including kelch13 C580Y in Cambodia: an observational cohort study. Lancet Infect Dis. 2015;15:683\u201391.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR7","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1016\/S1473-3099(15)00487-9","volume":"16","author":"C Amaratunga","year":"2016","unstructured":"Amaratunga C, Lim P, Suon S, Sreng S, Mao S, Sopha C, et al. Dihydroartemisinin-piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study. Lancet Infect Dis. 2016;16:357\u201365.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR8","doi-asserted-by":"crossref","first-page":"715","DOI":"10.3201\/eid2304.161872","volume":"23","author":"BQ Phuc","year":"2017","unstructured":"Phuc BQ, Rasmussen C, Duong TT, Dong LT, Loi MA, Menard D, et al. Treatment failure of dihydroartemisinin\/piperaquine for Plasmodium falciparum malaria, Vietnam. Emerg Infect Dis. 2017;23:715\u20137.","journal-title":"Emerg Infect Dis."},{"key":"3339_CR9","doi-asserted-by":"crossref","first-page":"952","DOI":"10.1016\/S1473-3099(19)30391-3","volume":"19","author":"RW van der Pluijm","year":"2019","unstructured":"van der Pluijm RW, Imwong M, Chau NH, Hoa NT, Thuy-Nhien NT, Thanh NV, et al. Determinants of dihydroartemisinin-piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study. Lancet Infect Dis. 2019;19:952\u201361.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR10","doi-asserted-by":"crossref","first-page":"2619","DOI":"10.1056\/NEJMc0805011","volume":"359","author":"H Noedl","year":"2008","unstructured":"Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Artemisinin Resistance in Cambodia 1 Study C: evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359:2619\u201320.","journal-title":"N Engl J Med"},{"key":"3339_CR11","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1056\/NEJMoa0808859","volume":"361","author":"AM Dondorp","year":"2009","unstructured":"Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009;361:455\u201367.","journal-title":"N Engl J Med"},{"key":"3339_CR12","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1038\/nature12876","volume":"505","author":"F Ariey","year":"2014","unstructured":"Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505:50\u20135.","journal-title":"Nature"},{"key":"3339_CR13","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1097\/QCO.0000000000000199","volume":"28","author":"RM Fairhurst","year":"2015","unstructured":"Fairhurst RM. Understanding artemisinin-resistant malaria: what a difference a year makes. Curr Opin Infect Dis. 2015;28:417\u201325.","journal-title":"Curr Opin Infect Dis."},{"key":"3339_CR14","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1038\/nbt.2925","volume":"32","author":"M Ghorbal","year":"2014","unstructured":"Ghorbal M, Gorman M, Macpherson CR, Martins RM, Scherf A, Lopez-Rubio JJ. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol. 2014;32:819\u201321.","journal-title":"Nat Biotechnol"},{"key":"3339_CR15","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1126\/science.1260867","volume":"347","author":"J Straimer","year":"2015","unstructured":"Straimer J, Gnadig NF, Witkowski B, Amaratunga C, Duru V, Ramadani AP, et al. Drug resistance K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science. 2015;347:428\u201331.","journal-title":"Science."},{"key":"3339_CR16","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1016\/S1473-3099(13)70252-4","volume":"13","author":"B Witkowski","year":"2013","unstructured":"Witkowski B, Amaratunga C, Khim N, Sreng S, Chim P, Kim S, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in vitro and ex vivo drug-response studies. Lancet Infect Dis. 2013;13:1043\u20139.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR17","doi-asserted-by":"crossref","first-page":"4631","DOI":"10.1128\/AAC.00366-15","volume":"59","author":"S Chaorattanakawee","year":"2015","unstructured":"Chaorattanakawee S, Saunders DL, Sea D, Chanarat N, Yingyuen K, Sundrakes S, et al. Ex vivo drug susceptibility testing and molecular profiling of clinical Plasmodium falciparum isolates from Cambodia from 2008 to 2013 suggest emerging piperaquine resistance. Antimicrob Agents Chemother. 2015;59:4631\u201343.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR18","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1186\/s12916-015-0539-5","volume":"13","author":"V Duru","year":"2015","unstructured":"Duru V, Khim N, Leang R, Kim S, Domergue A, Kloeung N, et al. Plasmodium falciparum dihydroartemisinin-piperaquine failures in Cambodia are associated with mutant K13 parasites presenting high survival rates in novel piperaquine in vitro assays: retrospective and prospective investigations. BMC Med. 2015;13:305.","journal-title":"BMC Med."},{"key":"3339_CR19","doi-asserted-by":"crossref","first-page":"3615","DOI":"10.1128\/AAC.06350-11","volume":"56","author":"MI Veiga","year":"2012","unstructured":"Veiga MI, Ferreira PE, Malmberg M, Jornhagen L, Bjorkman A, Nosten F, et al. pfmdr1 amplification is related to increased Plasmodium falciparum in vitro sensitivity to the bisquinoline piperaquine. Antimicrob Agents Chemother. 2012;56:3615\u20139.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR20","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/S1473-3099(16)30409-1","volume":"17","author":"R Amato","year":"2017","unstructured":"Amato R, Lim P, Miotto O, Amaratunga C, Dek D, Pearson RD, et al. Genetic markers associated with dihydroartemisinin-piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype-phenotype association study. Lancet Infect Dis. 2017;17:164\u201373.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR21","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1016\/S1473-3099(16)30415-7","volume":"17","author":"B Witkowski","year":"2017","unstructured":"Witkowski B, Duru V, Khim N, Ross LS, Saintpierre B, Beghain J, et al. A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype-genotype association study. Lancet Infect Dis. 2017;17:174\u201383.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR22","doi-asserted-by":"crossref","first-page":"1769","DOI":"10.1038\/s41467-018-04104-z","volume":"9","author":"S Bopp","year":"2018","unstructured":"Bopp S, Magistrado P, Wong W, Schaffner SF, Mukherjee A, Lim P, et al. Plasmepsin II-III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum. Nat Commun. 2018;9:1769.","journal-title":"Nat Commun."},{"key":"3339_CR23","doi-asserted-by":"crossref","first-page":"1673","DOI":"10.1093\/gbe\/evx126","volume":"9","author":"CM Parobek","year":"2017","unstructured":"Parobek CM, Parr JB, Brazeau NF, Lon C, Chaorattanakawee S, Gosi P, et al. Partner-drug resistance and population substructuring of artemisinin-resistant Plasmodium falciparum in Cambodia. Genome Biol Evol. 2017;9:1673\u201386.","journal-title":"Genome Biol Evol."},{"key":"3339_CR24","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1186\/s12936-017-1680-8","volume":"16","author":"NV Thanh","year":"2017","unstructured":"Thanh NV, Thuy-Nhien N, Tuyen NT, Tong NT, Nha-Ca NT, Dong LT, et al. Rapid decline in the susceptibility of Plasmodium falciparum to dihydroartemisinin-piperaquine in the south of Vietnam. Malar J. 2017;16:27.","journal-title":"Malar J."},{"key":"3339_CR25","doi-asserted-by":"crossref","first-page":"468","DOI":"10.1093\/infdis\/jix334","volume":"216","author":"S Agrawal","year":"2017","unstructured":"Agrawal S, Moser KA, Morton L, Cummings MP, Parihar A, Dwivedi A, et al. Association of a novel mutation in the Plasmodium falciparum chloroquine resistance transporter with decreased piperaquine sensitivity. J Infect Dis. 2017;216:468\u201376.","journal-title":"J Infect Dis"},{"key":"3339_CR26","doi-asserted-by":"crossref","first-page":"3314","DOI":"10.1038\/s41467-018-05652-0","volume":"9","author":"LS Ross","year":"2018","unstructured":"Ross LS, Dhingra SK, Mok S, Yeo T, Wicht KJ, Kumpornsin K, et al. Emerging Southeast Asian PfCRT mutations confer Plasmodium falciparum resistance to the first-line antimalarial piperaquine. Nat Commun. 2018;9:3314.","journal-title":"Nat Commun."},{"key":"3339_CR27","doi-asserted-by":"crossref","first-page":"1168","DOI":"10.1016\/S1473-3099(19)30543-2","volume":"19","author":"SK Dhingra","year":"2019","unstructured":"Dhingra SK, Small-Saunders JL, Menard D, Fidock DA. Plasmodium falciparum resistance to piperaquine driven by PfCRT. Lancet Infect Dis. 2019;19:1168\u20139.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR28","doi-asserted-by":"crossref","first-page":"5277","DOI":"10.1128\/AAC.00687-13","volume":"57","author":"P Lim","year":"2013","unstructured":"Lim P, Dek D, Try V, Eastman RT, Chy S, Sreng S, et al. Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in western, northern, and eastern Cambodia, 2011-2012: association with molecular markers. Antimicrob Agents Chemother. 2013;57:5277\u201383.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR29","doi-asserted-by":"crossref","first-page":"181","DOI":"10.1186\/s12936-020-03249-x","volume":"19","author":"MR Ansbro","year":"2020","unstructured":"Ansbro MR, Jacob CG, Amato R, Kekre M, Amaratunga C, Sreng S, et al. Development of copy number assays for detection and surveillance of piperaquine resistance associated plasmepsin 2\/3 copy number variation in Plasmodium falciparum. Malar J. 2020;19:181.","journal-title":"Malar J."},{"key":"3339_CR30","doi-asserted-by":"crossref","first-page":"e02309","DOI":"10.1128\/AAC.02309-17","volume":"62","author":"A Mukherjee","year":"2018","unstructured":"Mukherjee A, Gagnon D, Wirth DF, Richard D. Inactivation of plasmepsins 2 and 3 sensitizes Plasmodium falciparum to the antimalarial drug piperaquine. Antimicrob Agents Chemother. 2018;62:e02309\u201317.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR31","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.ijpddr.2018.11.004","volume":"9","author":"D Loesbanluechai","year":"2018","unstructured":"Loesbanluechai D, Kotanan N, de Cozar C, Kochakarn T, Ansbro MR, Chotivanich K, et al. Overexpression of plasmepsin II and plasmepsin III does not directly cause reduction in Plasmodium falciparum sensitivity to artesunate, chloroquine and piperaquine. Int J Parasitol Drugs Drug Resist. 2018;9:16\u201322.","journal-title":"Int J Parasitol Drugs Drug Resist."},{"key":"3339_CR32","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/S1473-3099(19)30689-9","volume":"20","author":"M Silva","year":"2020","unstructured":"Silva M, Calcada C, Teixeira M, Veiga MI, Ferreira PE. Multigenic architecture of piperaquine resistance trait in Plasmodium falciparum. Lancet Infect Dis. 2020;20:26\u20137.","journal-title":"Lancet Infect Dis."},{"key":"3339_CR33","doi-asserted-by":"crossref","first-page":"11672","DOI":"10.1073\/pnas.1507142112","volume":"112","author":"S Pelleau","year":"2015","unstructured":"Pelleau S, Moss EL, Dhingra SK, Volney B, Casteras J, Gabryszewski SJ, et al. Adaptive evolution of malaria parasites in French Guiana: reversal of chloroquine resistance by acquisition of a mutation in pfcrt. Proc Natl Acad Sci USA. 2015;112:11672\u20137.","journal-title":"Proc Natl Acad Sci USA"},{"key":"3339_CR34","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1093\/ofid\/ofz314","volume":"6","author":"M Wojnarski","year":"2019","unstructured":"Wojnarski M, Lon C, Vanachayangkul P, Gosi P, Sok S, Rachmat A, et al. Atovaquone-proguanil in combination with artesunate to treat multidrug-resistant P. falciparum malaria in Cambodia: an open-label randomized trial. Open Forum Infect Dis. 2019;6:1\u201314.","journal-title":"Open Forum Infect Dis."},{"key":"3339_CR35","doi-asserted-by":"crossref","first-page":"e1004789","DOI":"10.1371\/journal.ppat.1004789","volume":"11","author":"E Talundzic","year":"2015","unstructured":"Talundzic E, Okoth SA, Congpuong K, Plucinski MM, Morton L, Goldman IF, et al. Selection and spread of artemisinin-resistant alleles in Thailand prior to the global artemisinin resistance containment campaign. PLoS Pathog. 2015;11:e1004789.","journal-title":"PLoS Pathog"},{"key":"3339_CR36","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1016\/S0140-6736(04)16767-6","volume":"364","author":"RN Price","year":"2004","unstructured":"Price RN, Uhlemann AC, Brockman A, McGready R, Ashley E, Phaipun L, et al. Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number. Lancet. 2004;364:438\u201347.","journal-title":"Lancet"},{"key":"3339_CR37","unstructured":"Molecular Module. Copy number estimation of P. falciparum pfmdr1. v1.1. WWARN Procedure. 2011."},{"key":"3339_CR38","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1186\/1475-2875-12-239","volume":"12","author":"S Chaorattanakawee","year":"2013","unstructured":"Chaorattanakawee S, Tyner SD, Lon C, Yingyuen K, Ruttvisutinunt W, Sundrakes S, et al. Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia. Malar J. 2013;12:239.","journal-title":"Malar J."},{"key":"3339_CR39","doi-asserted-by":"crossref","first-page":"519","DOI":"10.1186\/s12936-016-1569-y","volume":"15","author":"S Chaorattanakawee","year":"2016","unstructured":"Chaorattanakawee S, Lon C, Jongsakul K, Gawee J, Sok S, Sundrakes S, et al. Ex vivo piperaquine resistance developed rapidly in Plasmodium falciparum isolates in northern Cambodia compared to Thailand. Malar J. 2016;15:519.","journal-title":"Malar J."},{"key":"3339_CR40","doi-asserted-by":"crossref","first-page":"e0157873","DOI":"10.1371\/journal.pone.0157873","volume":"11","author":"JA Thomas","year":"2016","unstructured":"Thomas JA, Collins CR, Das S, Hackett F, Graindorge A, Bell D, et al. Development and application of a simple plaque assay for the human malaria parasite Plasmodium falciparum. PLoS ONE. 2016;11:e0157873.","journal-title":"PLoS ONE"},{"key":"3339_CR41","doi-asserted-by":"crossref","first-page":"4097","DOI":"10.1128\/AAC.48.11.4097-4102.2004","volume":"48","author":"QL Fivelman","year":"2004","unstructured":"Fivelman QL, Adagu IS, Warhurst DC. Modified fixed-ratio isobologram method for studying in vitro interactions between atovaquone and proguanil or dihydroartemisinin against drug-resistant strains of Plasmodium falciparum. Antimicrob Agents Chemother. 2004;48:4097\u2013102.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR42","doi-asserted-by":"crossref","first-page":"2557","DOI":"10.1128\/AAC.01370-08","volume":"53","author":"EM Co","year":"2009","unstructured":"Co EM, Dennull RA, Reinbold DD, Waters NC, Johnson JD. Assessment of malaria in vitro drug combination screening and mixed-strain infections using the malaria Sybr green I-based fluorescence assay. Antimicrob Agents Chemother. 2009;53:2557\u201363.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR43","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1093\/infdis\/137.2.122","volume":"137","author":"MC Berenbaum","year":"1978","unstructured":"Berenbaum MC. A method for testing for synergy with any number of agents. J Infect Dis. 1978;137:122\u201330.","journal-title":"J Infect Dis"},{"key":"3339_CR44","doi-asserted-by":"crossref","first-page":"1362","DOI":"10.1128\/AAC.01656-08","volume":"53","author":"S Muangnoicharoen","year":"2009","unstructured":"Muangnoicharoen S, Johnson DJ, Looareesuwan S, Krudsood S, Ward SA. Role of known molecular markers of resistance in the antimalarial potency of piperaquine and dihydroartemisinin in vitro. Antimicrob Agents Chemother. 2009;53:1362\u20136.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR45","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1038\/nature14412","volume":"520","author":"A Mbengue","year":"2015","unstructured":"Mbengue A, Bhattacharjee S, Pandharkar T, Liu H, Estiu G, Stahelin RV, et al. A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria. Nature. 2015;520:683\u20137.","journal-title":"Nature"},{"key":"3339_CR46","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1186\/s12936-017-1845-5","volume":"16","author":"A Mukherjee","year":"2017","unstructured":"Mukherjee A, Bopp S, Magistrado P, Wong W, Daniels R, Demas A, et al. Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia. Malar J. 2017;16:195.","journal-title":"Malar J."},{"key":"3339_CR47","doi-asserted-by":"crossref","first-page":"12799","DOI":"10.1073\/pnas.1812317115","volume":"115","author":"AR Demas","year":"2018","unstructured":"Demas AR, Sharma AI, Wong W, Early AM, Redmond S, Bopp S, et al. Mutations in Plasmodium falciparum actin-binding protein coronin confer reduced artemisinin susceptibility. Proc Natl Acad Sci USA. 2018;115:12799\u2013804.","journal-title":"Proc Natl Acad Sci USA"},{"key":"3339_CR48","doi-asserted-by":"crossref","first-page":"e00374-18","DOI":"10.1128\/AAC.00374-18","volume":"62","author":"MG Robert","year":"2018","unstructured":"Robert MG, Foguim Tsombeng F, Gendrot M, Mosnier J, Amalvict R, Benoit N, et al. Absence of a high level of duplication of the plasmepsin II gene in Africa. Antimicrob Agents Chemother. 2018;62:e00374-18.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR49","doi-asserted-by":"crossref","first-page":"e02445-18","DOI":"10.1128\/AAC.02445-18","volume":"63","author":"MG Robert","year":"2019","unstructured":"Robert MG, Tsombeng FF, Gendrot M, Diawara S, Madamet M, Kounta MB, et al. Baseline ex vivo and molecular responses of Plasmodium faciparum isolates to piperaquine before implementation of dihydroartemisinin-piperaquine in Senegal. Antimicrob Agents Chemother. 2019;63:e02445-18.","journal-title":"Antimicrob Agents Chemother"},{"key":"3339_CR50","doi-asserted-by":"crossref","first-page":"14552","DOI":"10.1038\/srep14552","volume":"5","author":"S Pulcini","year":"2015","unstructured":"Pulcini S, Staines HM, Lee AH, Shafik SH, Bouyer G, Moore CM, et al. Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite\u2019s food vacuole and alter drug sensitivities. Sci Rep. 2015;5:14552.","journal-title":"Sci Rep."}],"container-title":["Malaria Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12936-020-03339-w.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1186\/s12936-020-03339-w\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/s12936-020-03339-w.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,7,24]],"date-time":"2021-07-24T19:41:12Z","timestamp":1627155672000},"score":1,"resource":{"primary":{"URL":"https:\/\/malariajournal.biomedcentral.com\/articles\/10.1186\/s12936-020-03339-w"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,7,25]]},"references-count":50,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2020,12]]}},"alternative-id":["3339"],"URL":"https:\/\/doi.org\/10.1186\/s12936-020-03339-w","relation":{"has-preprint":[{"id-type":"doi","id":"10.21203\/rs.3.rs-22444\/v1","asserted-by":"object"},{"id-type":"doi","id":"10.21203\/rs.3.rs-22444\/v2","asserted-by":"object"},{"id-type":"doi","id":"10.21203\/rs.3.rs-22444\/v3","asserted-by":"object"}]},"ISSN":["1475-2875"],"issn-type":[{"value":"1475-2875","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,7,25]]},"assertion":[{"value":"10 April 2020","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"14 July 2020","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"25 July 2020","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"All participants or guardians provided written consent and samples were collected under approval from the Cambodian National Ethics Committee for Health Research and Walter Reed Army Institute of Research Institutional Review board.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"Not applicable.","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":"269"}}