Evidence of Artemisinin-Resistant Malaria in Western Cambodia

Figure 1. Parasite Density, Parasite-Clearance Time, and 50% Inhibitory Concentration (IC50) among Patients Receiving Artesunate, According to Clinical Outcome.

Panel A shows the parasite-reduction curves for the 56 patients who were cured, the 2 patients classified as having artemisinin-resistant infections, and the 2 with drug failures (i.e., patients who had recrudescence but who were not classified as having artemisinin-resistant infection, since the drug level was inadequate). The data points and horizontal I bars denote the means and standard errors. Panel B shows the parasite-clearance times in the artesunate group, as compared with the IC50 for dihydroartemisinin (R=0.31, P=0.03). Orange circles indicate patients whose infection was classified as artemisinin-resistant, and blue squares patients in whom treatment failed but whose infection was not classified as resistant.

To the Editor: Although artemisinins are potent and rapidly acting antimalarial drugs, their widespread use for treating patients with Plasmodium falciparum malaria raises the question of emerging drug resistance.1,2 Artemisinin monotherapy should not be used in areas where malaria is endemic; it requires an extended administration period and may lead to treatment failure, most frequently because of problems with compliance. Recent reports of high failure rates associated with artemisinin-based combination therapy, as well as in vitro drug-susceptibility data, suggest the possibility of clinical artemisinin resistance along the Thai–Cambodian border.3,4 We studied the potential emergence of artemisinin resistance using in vivo, in vitro, molecular, and pharmacokinetic methods specifically designed to address the question of potential artemisinin resistance.

We randomly assigned, in a ratio of 2:1, 94 adults from Battambang Province presenting with uncomplicated P. falciparum malaria (100 to 100,000 parasites per microliter) to receive either high-dose artesunate therapy (4 mg per kilogram of body weight per day, orally, for 7 days) (60 patients) or quinine (30 mg per kilogram per day) plus tetracycline (25 mg per kilogram per day) in a split dose every 8 hours for 7 days (34 patients). The study was approved by ethics review committees in Cambodia and the United States and was conducted from October 2006 through March 2007. Written informed consent was obtained from all study participants.

Patients were admitted for 28 days to rule out reinfection. The clinical outcome was recorded, plasma drug concentrations and in vitro drug susceptibility were measured, and molecular assays were performed to investigate genetic resistance markers and to rule out reinfection. Only patients who met all the following criteria were classified as having artemisinin-resistant infection: persistence of parasites 7 days after the start of treatment or reemergence of parasites within 28 days after the start of treatment; adequate plasma concentrations of dihydroartemisinin, a major artemisinin metabolite; prolonged time to parasite clearance; and reduced in vitro susceptibility to dihydroartemisinin.5

Four of the 60 patients who received artesunate had reemergence of parasitemia between days 21 and 28 after the start of treatment (with a Kaplan–Meier probability estimate for cure at day 28 of 93.6%; 95% confidence interval [CI], 84.7 to 97.7); 2 of these patients (3.3%) were classified as having artemisinin-resistant infection, according to the criteria listed above (Figure 1A). These two patients had parasite-clearance times that were prolonged (133 and 95 hours, as compared with a median of 52.2 hours for patients who were cured), and the plasma drug concentrations after the first dose were classified as adequate (greater than the mean for the cured patients minus 1 SD) (see the Supplementary Appendix, available with the full text of this letter at www.nejm.org). For these subjects, the 50% inhibitory concentrations for dihydroartemisinin were up to 4 times the geometric mean for cured patients and almost 10 times that for the reference clone W2. In the artesunate group, 47.9% (95% CI, 36.1 to 60.0) of the patients still had parasitemia 48 hours after the start of treatment, as did 21.9% (95% CI, 13.1 to 33.1) 72 hours after the start of treatment. Clinical and in vitro data suggest that artemisinin resistance may be more accurately portrayed as the long tail of a single distribution than as the result of a sudden change in sensitivity (Figure 1B). Resistance did not appear to be mediated by the number of copies of the P. falciparum multidrug resistance gene pfmdr1 or selected PfATPase6 polymorphisms tested in this study.

The high overall treatment efficacy seen in patients treated with artesunate indicates that relatively few parasite isolates have crossed the threshold of artemisinin resistance as defined in our study. Artemisinin resistance does not seem to be a widespread epidemiologic phenomenon at this time. The prolonged parasite-clearance times and the two cases meeting our definition of artesunate resistance are nonetheless a concern.

Harald Noedl, M.D., Ph.D.
Medical University of Vienna
A-1090 Vienna, Austria
harald.noedl@meduniwien.ac.at

Youry Se, M.D.
Kurt Schaecher, Ph.D.
Bryan L. Smith, M.D.
Armed Forces Research Institute of Medical Sciences
Bangkok 10400, Thailand

Duong Socheat, M.D.
National Center for Parasitology, Entomology, and Malaria Control
Phnom Penh, Cambodia

Mark M. Fukuda, M.D.
Armed Forces Research Institute of Medical Sciences
Bangkok 10400, Thailand

for the Artemisinin Resistance in Cambodia 1 (ARC1) Study Consortium

Supported by the U.S. Department of Defense Global Emerging Infections System Program. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

This letter (10.1056/NEJMc0805011) was published at www.nejm.org on December 8, 2008. It will appear in the December 11 issue of the Journal.

References

Duffy PE, Sibley CH. Are we losing artemisinin combination therapy already? Lancet 2005;366:1908-1909.

Krishna S, Bustamante L, Haynes RK, Staines HM. Artemisinins: their growing importance in medicine. Trends Pharmacol Sci 2008;29:520-527.

Jambou R, Legrand E, Niang M, et al. Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6. Lancet 2005;366:1960-1963. [CrossRef][Medline]

Vijaykadga S, Rojanawatsirivej C, Cholpol S, Phoungmanee D, Nakavej A, Wongsrichanalai C. In vivo sensitivity monitoring of mefloquine monotherapy and artesunate-mefloquine combinations for the treatment of uncomplicated falciparum malaria in Thailand in 2003. Trop Med Int Health 2006;11:211-219. [ISI][Medline]

Noedl H. Artemisinin resistance: how can we find it? Trends Parasitol 2005;21:404-405.
[CrossRef][ISI][Medline]