Artemisinin Resistance in Plasmodium falciparum Malaria

Arjen M. Dondorp, M.D., François Nosten, M.D., Poravuth Yi, M.D., Debashish Das, M.D., Aung Phae Phyo, M.D., Joel Tarning, Ph.D., Khin Maung Lwin, M.D., Frederic Ariey, M.D., Warunee Hanpithakpong, Ph.D., Sue J. Lee, Ph.D., Pascal Ringwald, M.D., Kamolrat Silamut, Ph.D., Mallika Imwong, Ph.D., Kesinee Chotivanich, Ph.D., Pharath Lim, M.D., Trent Herdman, Ph.D., Sen Sam An, Shunmay Yeung, Ph.D., Pratap Singhasivanon, M.D., Nicholas P.J. Day, D.M., Niklas Lindegardh, Ph.D., Duong Socheat, M.D., and Nicholas J. White, F.R.S.


ABSTRACT

Background Artemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai–Cambodian border, historically a site of emerging antimalarial-drug resistance.

Methods In two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance.

Results We studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). p="0.31)." style="font-weight: bold;">


Discussion

Our study shows that P. falciparum parasites from patients in Pailin, western Cambodia, had significantly reduced in vivo susceptibility to artesunate, as compared with parasites from northwestern Thailand — or indeed, apparently, anywhere else in the world. Artesunate resistance was characterized by a markedly prolonged time to parasite clearance, with relatively little heterogeneity among patients. These reduced parasitologic responses could not be explained by pharmacokinetic or other host factors.

The principal pharmacodynamic advantage of using artemisinins rather than other antimalarial drugs is that they accelerate parasite clearance1 by clearing young, circulating, ring-stage parasites and preventing the further maturation and sequestration of these parasites.19,20 This effect accounts for the rapidity of the therapeutic response, its lifesaving benefit in patients with severe malaria, and the notable gametocytocidal activity of the drugs. A few cases of reduced efficacy of artemisinin-based combination therapy have been reported previously. Most of these cases can be explained by the counterfeit nature of the drugs tested.21 In 2002–2003, decreased in vitro sensitivity of P. falciparum to artemether was reported in French Guiana (but not in Cambodia) and was associated with the S769N mutation in the PfSERCA gene.22,23 There were no clinical correlates. In our study, there was no consistent pattern of PfSERCA mutations, despite clear evidence of in vivo artesunate resistance.

Monitoring in western Cambodia has shown a decline in the efficacy of artemisinin-based combination therapy in recent years, but the relative contributions of resistance to artemisinin, the partner drug, and other factors have been unclear.7,8,9,10,24 In a recent Cambodian study, increased numbers of copies of the PfMDR1 gene, a well-established cause of mefloquine resistance,18 was associated with high failure rates with artesunate–mefloquine therapy.7,25 In the current study, despite artesunate resistance, the efficacy of artesunate–mefloquine use was excellent in Pailin (63-day cure rate, 95%), presumably because 95% of parasite isolates had a single PfMDR1 copy. PfMDR1 amplification also causes a small decrease in artemisinin susceptibility,18 but neither this nor any other molecular markers of antimalarial-drug resistance that we evaluated could explain the resistance to artesunate in Pailin.

There was an unexpected discrepancy between the slow clinical and parasitologic responses in Pailin and the relatively normal in vitro susceptibility. The discrepancy can be explained by the reduced susceptibility of the young, ring-form parasites as compared with the more mature trophozoites and schizonts. Currently available in vitro susceptibility tests assess drug exposure over the entire parasite cycle; therefore, the results would be relatively unaffected if only part of the cycle were affected by the mechanism underlying the resistance. Previous studies have not indicated significant differences in parasitologic responses within the dose range that we evaluated (2 to 4 mg per kilogram per day).26 A concentration–effect relationship was evident in Wang Pha but not in Pailin, suggesting that a small shift to the right along the dose–response curve may also have occurred at the Thai–Burmese border.27

Chloroquine and sulfadoxine–pyrimethamine resistance in P. falciparum emerged in the late 1950s and 1960s on the Thai–Cambodian border and spread across Asia and then Africa, contributing to millions of deaths from malaria.28,29 Artemisinins have been available as monotherapies in western Cambodia for more than 30 years, in a variety of forms and doses, whereas in most countries (other than China, where they were discovered), they have been a relatively recent introduction.1 Despite the early implementation of an active malaria-control program by the Ministry of Health of Cambodia, including the introduction of artemisinin-based combination therapies in 2001, a recent survey showed that 78% of artemisinin use in western Cambodia consisted of monotherapy provided through the private sector.30 The extended period of often-suboptimal use, and the genetic background of parasites from this region,31 might have contributed to the emergence and subsequent spread of these new artemisinin-resistant parasites in western Cambodia. In contrast, artemisinin derivatives have been used almost exclusively in combination with mefloquine on the Thai–Burmese border, where parasitologic responses to artemisinins remain good, even after 15 years of intensive use.27 Measures for containment are now urgently needed to limit the spread of these parasites from western Cambodia and to prevent a major threat to current plans for eliminating malaria.

Supported by grants from the Wellcome Trust of Great Britain (Major Overseas Programme–Thailand Unit Core Grant), the Li Ka Shing Foundation (B9RMXT0-2), the Global Malaria Program, and the WHO (Bill and Melinda Gates Foundation grant 48821 and Western Pacific Regional Office grant WP/07/66224 CAM).

No potential conflict of interest relevant to this article was reported.

We thank the patients for their participation; all staff members of the Pailin Referral Hospital and the SMRU clinic for their dedicated care for the patients and assistance in the study; Tim Anderson for help with the in vitro sensitivity testing; the Village Malaria Workers in Pailin for their collaboration; and Vasee Moorthy, Golam Kibria, Mark Fukuda, and George Watt for help in executing and organizing the trial in Pailin.