New genetic marker for piperaquine resistance in Plasmodium falciparum
Authors: Arjen M Dondorp
Journal: The Lancet Infectious Diseases (2016)

In the Greater Mekong subregion, Plasmodium falciparum resistance to artemisinins is increasingly compounded by concurrent resistance to partner drugs in combination therapies, which results in high treatment failure rates.1Carrara VI Lwin KM Phyo AP Ashley E Wiladphaingern J Sriprawat K et al.Malaria burden and artemisinin resistance in the mobile and migrant population on the Thai–Myanmar border, 1999–2011: an observational study.PLoS Med. 2013; 10: e1001398Crossref PubMed Scopus (136) Google Scholar, 2Amaratunga C Lim P Suon S et al.Dihydroartemisinin-piperaquine resistance in Plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study.Lancet Infect Dis. 2016; 16: 357-365Summary Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 3Leang R Barrette A Bouth DM et al.Efficacy of dihydroartemisinin-piperaquine for treatment of uncomplicated Plasmodium falciparum and Plasmodium vivax in Cambodia, 2008 to 2010.Antimicrob Agents Chemother. 2013; 57: 818-826Crossref PubMed Scopus (145) Google Scholar, 4Saunders DL Vanachayangkul P Lon C Dihydroartemisinin–piperaquine failure in Cambodia.N Engl J Med. 2014; 371: 484-485Crossref PubMed Scopus (175) Google Scholar, 5Leang 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: 4719-4726Crossref PubMed Scopus (217) Google Scholar, 6Duru V Khim N Leang R 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: 305Crossref PubMed Scopus (111) Google Scholar Artemisinin combination therapies are first-line drugs for the treatment of falciparum malaria in all endemic countries; new antimalarials will probably not be marketed within this decade.7White NJ Can new treatment developments combat resistance in malaria?.Exp Opin Pharmacother. 2016; 17: 1303-1307Crossref PubMed Scopus (30) Google Scholar Clinical drug efficacy studies are time and resource consuming and availability of molecular markers for drug resistance can facilitate near real-time monitoring, which is needed for guiding policy changes towards locally effective combination therapies in the Greater Mekong subregion. Markers are available for artemisinin resistance (Kelch13 mutations8Ariey F Witkowski B Amaratunga C et al.A molecular marker of artemisinin-resistant Plasmodium falciparum malaria.Nature. 2014; 505: 50-55Crossref PubMed Scopus (1283) Google Scholar) and mefloquine resistance (mdr1 amplification9Price RN Uhlemann AC Brockman A et al.Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.Lancet. 2004; 364: 438-447Summary Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Markers for other artemisinin partner drugs used in the region, including lumefantrine, amodiaquine, and piperaquine, are less well defined. In The Lancet Infectious Diseases, two separate studies by Benoit Witkowski and colleagues10Witkowski B Duru V Khim N et al.A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype–genotype association study.Lancet Infect Dis. 2016; (published online Nov 3.)http://dx.doi.org/10.1016/S1473-3099(16)30415-7PubMed Google Scholar and Roberto Amato and colleagues11Amato R Lim P Miotto O et al.Genetic markers associated with dihydroartemisinin–piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype–phenotype association study.Lancet Infect Dis. 2016; (published online Nov 3.)http://dx.doi.org/10.1016/S1473-3099(16)30409-1Google Scholar both done in Cambodia, derived a much needed molecular marker for piperaquine resistance, a rapidly increasing problem in the Greater Mekong subregion: plasmepsin 2 and plasmepsin 3 gene amplifications on chromosome 14. Amato and colleagues11Amato R Lim P Miotto O et al.Genetic markers associated with dihydroartemisinin–piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype–phenotype association study.Lancet Infect Dis. 2016; (published online Nov 3.)http://dx.doi.org/10.1016/S1473-3099(16)30409-1Google Scholar identified an additional marker, an exonuclease gene polymorphism on chromosome 13 (exo-E415G), in strong linkage disequilibrium with plasmepsin 2–3 amplification. Slightly different approaches were used. Witkowski and colleagues10Witkowski B Duru V Khim N et al.A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype–genotype association study.Lancet Infect Dis. 2016; (published online Nov 3.)http://dx.doi.org/10.1016/S1473-3099(16)30415-7PubMed Google Scholar did a genome-wide association study (GWAS) in a relatively small sample of 31 Cambodian parasite strains, using a previously validated piperaquine survival assay (PSA)6Duru V Khim N Leang R 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: 305Crossref PubMed Scopus (111) Google Scholar to define the resistant in-vitro phenotype. The identified plasmepsin 2, as an amplicon reporter, was then assessed in 134 clinical field isolates, showing a much higher median ex-vivo PSA survival rate with two or more plasmepsin 2 copies compared with single copy (51·7% [IQR 29·7–75·1] vs 0·004% [IQR 0·003–0·39]). Additionally, in 725 Cambodian patients with uncomplicated falciparum malaria treated between 2009 and 2015 with dihydroartemisinin–piperaquine, multicopy plasmepsin 2 was associated with an adjusted hazard ratio for treatment failure at 42 days of 20·4 (95% CI 9·1–45·5, p<0·0001). Amato and colleagues11Amato R Lim P Miotto O et al.Genetic markers associated with dihydroartemisinin–piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype–phenotype association study.Lancet Infect Dis. 2016; (published online Nov 3.)http://dx.doi.org/10.1016/S1473-3099(16)30409-1Google Scholar did GWAS on a larger sample of 297 Cambodian strains, allowing better correction for confounders such as parasite population structure; the piperaquine resistance phenotype was defined by the 50% inhibitory concentration (IC50) of conventional in-vitro drug sensitivity testing. Samples were collected between 2010 and 2013 from three different provinces with marked different levels of artemisinin resistance and dihydroartemisinin–piperaquine failure. Both the exo-E415G and plasmepsin 2–3 markers were strongly associated with piperaquine IC50. In 133 patients treated with dihydroartemisinin–piperaquine and who had 63-day clinical efficacy data available, adjusted treatment success rate with or without plasmepsin 2–3 amplification was 0·41 (95% CI 0·28–0·53) and 0·95 (0·87–0·98), respectively. Results for the exo-E415G marker were similar. The converging results of these studies make plasmepsin 2–3 amplification a robust marker for piperaquine resistance in the region. There is a suggestion of a causal relationship, since both plasmepsins and 4-aminoquinolines (like piperaquine) affect haemoglobin degradation and haem detoxification processes. P falciparum transfected with multicopy plasmepsin 2–3 will be an invaluable tool to explore underlying resistance mechanisms. The studies contain a wealth of additional information. There was a consistent absence of mdr1 amplification in plasmepsin 2–3 amplified parasite strains. Although interrupted mefloquine drug pressure could also have contributed, this finding could imply that these markers represent counteracting resistance mechanisms. This possibility would make alternating mefloquine and piperaquine as partner drug, or combining them in triple combinations (currently under trial), attractive treatment modalities. The geotemporal trends in molecular markers show that the sequence of events has been first arrival of artemisinin resistance followed by a sharp increase in piperaquine resistance. Resistance to short half-life artemisinins after 3-day artemisinin combination therapy regimens leads to a greater than 100 times larger residual parasite biomass exposed to the partner drug,12Dondorp AM Fairhurst RM Slutsker L et al.The threat of artemisinin-resistant malaria.N Engl J Med. 2011; 365: 1073-1075Crossref PubMed Scopus (210) Google Scholar which selects for partner drug resistance (as observed with mefloquine on the Thai–Myanmar border).1Carrara VI Lwin KM Phyo AP Ashley E Wiladphaingern J Sriprawat K et al.Malaria burden and artemisinin resistance in the mobile and migrant population on the Thai–Myanmar border, 1999–2011: an observational study.PLoS Med. 2013; 10: e1001398Crossref PubMed Scopus (136) Google Scholar The pharmacokinetics of piperaquine shows an initial relatively rapid plasma clearance followed by a long terminal half-life (2–4 weeks),13Nguyen DV Nguyen QP Nguyen ND et al.Pharmacokinetics and ex vivo pharmacodynamic antimalarial activity of dihydroartemisinin–piperaquine in patients with uncomplicated falciparum malaria in Vietnam.Antimicrob Agents Chemother. 2009; 53: 3534-3537Crossref PubMed Scopus (37) Google Scholar so that small increases in minimal parasitocidal concentrations will result in a much shorter period of time parasites are exposed to parasitocidal concentrations, further contributing to selection. Once resistance to both artemisinin combination therapy components appear, selection of multidrug resistant parasites will strongly amplify because of high treatment failure rates and higher gametocytaemia in recrudescent infections,14Bousema T Drakeley C Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination.Clin Microbiol Rev. 2011; 24: 377-410Crossref PubMed Scopus (482) Google Scholar driving transmission to the Anopheles spp vector. Antimalarial drug resistance threatens to offset the impressive gains made over the past decade in reducing falciparum malaria transmission in the Greater Mekong subregion to the current historically low numbers. We have to eliminate falciparum malaria from the region now before it becomes close to untreatable. AMD declares that Olivo Miotto (an author on the Article by Amato and colleagues) is affiliated with his research unit, and he is co-author with Didier Ménard (an author on the Article by Witkowski and colleagues) of a jointly submitted paper, assessing the plasmepsin molecular marker in the Greater Mekong subregion. Genetic markers associated with dihydroartemisinin–piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype–phenotype association studyThe exo-E415G SNP and plasmepsin 2–3 amplification are markers of piperaquine resistance and dihydroartemisinin–piperaquine failures in Cambodia, and can help monitor the spread of these phenotypes into other countries of the Greater Mekong subregion, and elucidate the mechanism of piperaquine resistance. Since plasmepsins are involved in the parasite's haemoglobin-to-haemozoin conversion pathway, targeted by related antimalarials, plasmepsin 2–3 amplification probably mediates piperaquine resistance. Full-Text PDF Open AccessA surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype–genotype association studyPiperaquine resistance in Cambodia is strongly associated with amplification of plasmepsin 2–3, encoding haemoglobin-digesting proteases, regardless of the location. Multicopy plasmepsin 2 constitutes a surrogate molecular marker to track piperaquine resistance. A molecular toolkit combining plasmepsin 2 with K13 and mdr1 monitoring should provide timely information for antimalarial treatment and containment policies. Full-Text PDF Open Access