Between June 2007 and May 2008, a total of 80 patients were enrolled: 40 in Pailin, of whom 11 (28%) were under 16 years of age, and 40 in Wang Pha, where children were not enrolled ( and , and Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org
). The number of days of fever before enrollment was similar in Pailin (median, 3 days; range, 1.5 to 8) and in Wang Pha (median, 2 days; range, 1 to 6) (P = 0.21).
Enrollment, Randomization, and Follow-up of the Patients in the Two Studies
Baseline Characteristics of the Study Patients, According to Location.*
Parasitologic and Clinical Responses
Parasite clearance was markedly slower in Pailin (), with a median parasite clearance time for both Pailin treatment groups combined of 84 hours (interquartile range, 60 to 96), as compared with 48 hours (interquartile range, 36 to 66) in both Wang Pha groups (P<0.001). After the start of treatment in Pailin, 73% of patients had parasitemia at 48 hours, 55% at 72 hours, and 20% at 96 hours; the corresponding percentages in Wang Pha were 45%, 8%, and 3% (P<0.001). In Pailin, the parasite density at baseline was similar in children under 16 years of age (geometric mean, 50,046 per cubic millimeter; 95% confidence interval [CI], 22,767 to 110,007) and in children at least 16 years of age and adults (geometric mean, 71,366 per cubic millimeter; 95% CI, 49,614 to 102,654) (P = 0.33). In Pailin, the median parasite clearance time among the 11 children was 78 hours (interquartile range, 60 to 96), as compared with 84 hours (interquartile range, 66 to 96) among the 29 adults and children at least 16 years of age (P = 0.67). To assess whether exclusion of children from the Wang Pha, Thailand, study influenced the results, data from the Cambodian children were excluded from the analysis; the difference in parasite clearance times between the two study sites remained significant (P<0.001) (Table S2 in the Supplementary Appendix). We also compared the parasite clearance times as calculated on the basis of daily parasite counts in 3264 patients (1996 adults and children at least 16 years of age, and 1268 children under 16 years of age) in the Thailand study area between 1995 and 2007, as well as the times as calculated on the basis of counts every 6 hours in 1214 patients with hyperparasitemia (449 adults and children at least 16 years of age, and 765 children under 16 years of age) who were treated with artesunate-containing regimens. After adjustment for parasitemia, study location, and year of admission, there was no significant difference for children under 16 years of age as compared with adults and children at least 16 years of age in parasite clearance times (P>0.20 for all comparisons).
Clinical and Parasitologic Responses in the Study Patients, According to Location.*
The difference in parasite clearance times also remained significant when the analysis was stratified according to the log10-normalized parasite density on admission (P = 0.008). Further analysis involving linear regression, adjusted for baseline parasite density, showed that site was a significant independent predictor of the time to parasite clearance, whether children were included in the model (coefficient, 18.1; 95% CI, 8.9 to 27.2; P<0.001) or excluded from the model (coefficient, 18.7; 95% CI, 8.0 to 29.4; P = 0.001). Other measures of parasite reduction that are less dependent on the initial parasite load, including the parasite-reduction ratios after 24 and 48 hours and the slope of the log10-normalized parasite clearance over time, were also markedly different between the study sites ( and ).
In Pailin, there was no evident dose–response relationship for artesunate: the median parasite clearance time was 84 hours (interquartile range, 54 to 96) for the dose of 2 mg per kilogram and 72 hours (interquartile range, 60 to 96) for the dose of 4 mg per kilogram (P = 0.61). In contrast, in Wang Pha, patients treated with artesunate at a dose of 4 mg per kilogram had faster parasite clearance (median, 48 hours; interquartile range, 30 to 54) than those treated with artesunate at a dose of 2 mg per kilogram (median, 54; interquartile range, 42 to 72) (P = 0.02) ().
Fever clearance times did not differ significantly between the two study sites (). Early treatment failure, defined as parasitemia and fever persisting for more than 3 days (≥72 hours) after the start of treatment,2
occurred in 3 of the 40 patients (8%) in Pailin, as compared with none in Wang Pha (P = 0.20). In the artesunate-monotherapy group, late parasitologic failure (defined as PCR-confirmed recrudescence of P. falciparum
infection >7 days after the start of treatment)2
occurred in 6 of the 20 patients (30%) in Pailin after a median of 28 days (range, 21 to 35) and in 2 of the 20 patients (10%) in Wang Pha, after 21 days for both patients. In the artesunate–mefloquine group, there were no occurrences of early treatment failure, and late parasitologic failure occurred in 1 of the 20 patients (5%) at each site, after 49 days in Pailin and 34 days in Wang Pha. Reinfection was more common in Wang Pha than in Pailin (P = 0.001) (). Recurrent infection with P. vivax
was common at both study sites. The median duration of patent gametocytemia per person-weeks of follow-up was 0.06 weeks (95% CI, 0.05 to 0.07) in Wang Pha and 0.07 weeks (95% CI, 0.06 to 0.08) in Pailin (P = 0.13). The duration of gametocyte carriage in patients who had gametocytemia at any time also did not differ significantly between the sites ().
The number of patients reporting an adverse event did not differ significantly between the two treatment groups (Table S3 in the Supplementary Appendix). Dizziness, joint pain, muscle pain, and sleepiness were more common in Wang Pha, whereas nausea, abdominal pain, and fatigue were reported more frequently in Pailin. No serious adverse events were observed.
Body-weight–normalized doses of the study drugs were similar at the two study sites. Plasma-concentration profiles of artesunate and dihydroartemisinin, and the pharmacokinetic variables derived from them, were generally similar at the two sites, although the areas under the concentration–time curves were slightly greater in Pailin (). There was no significant correlation between the maximum concentration of artesunate or dihydroartemisinin, or the area under the concentration–time curve, and measures of parasite clearance (parasite clearance time and parasite-reduction ratios after 24 and 48 hours) or the risk of treatment failure (Fig. S1 in the Supplementary Appendix). Exclusion of children from the analysis did not affect these findings.
Results of Noncompartmental Analysis of First-Dose Pharmacokinetics, According to Study Drug.*
In Vitro Sensitivity Testing
Reliable fitting of the hypoxanthine-uptake curves for artesunate and dihydroartemisinin was possible for 18 of the 40 patients for both drugs in Pailin and 32 of 40 patients and 30 of 40 patients, respectively, in Wang Pha. The median 50% inhibitory concentration of artesunate in Pailin was 1.9 nM (interquartile range, 1.3 to 3.4), as compared with 3.2 nM (interquartile range, 1.7 to 4.1) in Wang Pha (P = 0.07) and 2.7 nM for the reference strain 3D7. The median 50% inhibitory concentration of artesunate in Pailin was 1.9 nM (interquartile range, 1.3 to 3.4), as compared with 3.2 nM (interquartile range, 1.7 to 4.1) in Wang Pha (P = 0.07) and 2.7 nM for the reference strain 3D7. The median 50% inhibitory concentration of dihydroartemisinin in Pailin was 2.3 nM (interquartile range, 1.1 to 3.2), as compared with 1.5 nM (interquartile range, 0.7 to 2.2) in Wang Pha (P = 0.04) and 2.3 nM for the reference 3D7 (). There were no significant correlations between the 50% inhibitory concentration of artesunate or dihydroartemisinin and the parasite clearance times in Pailin (artesunate: r = 0.07, P = 0.70; dihydroartemisinin: r=0.19, P = 0.33) or Wang Pha (artesunate: r = 0.20, P = 0.44; dihydroartemisinin: r = 0.28, P = 0.23). The 50% inhibitory concentrations of mefloquine and chloroquine did not differ significantly between the two study sites ().
The 50% Inhibitory Concentration (IC50) for Plasmodium falciparum, According to Antimalarial Drug
Molecular Markers of Resistance
In Pailin, 38 of 40 patients (95%) had parasites that contained single copies of PfMDR1, and the 2 remaining patients (5%) had two copies, both associated with 50% inhibitory concentrations of mefloquine exceeding 150 nM. In Wang Pha, 18 of the 40 patients (45%) had isolates that contained multiple copies of PfMDR1 (median, 2; range, 1 to 3; P<0.001). We did not detect any point mutations in the PfMDR1 gene at positions N86Y, S1034C, N1042D, and D1246Y, but the mutant codon Y184F was found in isolates from 32 of the 40 patients (80%) in Pailin and 12 of the 40 patients (30%) in Wang Pha. All parasites had the K76T mutation in the PfCRT gene. Full-length sequencing of the PfSERCA gene revealed sporadic point mutations noted as I89T (in isolates from 5 of 50 patients), N465S (in isolates from 7 of 44 patients), and E847K (in isolates from 1 of 45 patients), with similar frequencies at the two study sites. The number of insertion–deletion mutations of T-A repeats located in the intron region of the gene (nt3621-3671) varied among parasites (range, 10 to 19) but did not differ between the two sites. The PfSERCA L263E and S769 N polymorphisms, proposed to confer artemisinin resistance, were not detected.