Artemisinin resistance in Plasmodium falciparum, the agent of severe malaria, is currently a major obstacle to malaria control in Southeast Asia. A gene named "kelch13" has been associated with artemisinin resistance in P. falciparum The orthologue of the kelch gene in P. vivax was identified and a small number of mutations were found in previous studies. The kelch orthologues in the other two human malaria parasites, P. malariae and P. ovale, have not yet been studied. Therefore, in this study, the orthologous kelch genes of P. malariae, P. ovale wallikeri, and P. ovale curtisi were isolated and analyzed for the first time. The homologies of the kelch genes of P. malariae and P. ovale were 84.8% and 82.7%, respectively, compared to the gene in P. falciparum kelch polymorphisms were studied in 13 P. malariae and 5 P. ovale isolates from Thailand. There were 2 nonsynonymous mutations found in these samples. One mutation was P533L, which was found in 1 of 13 P. malariae isolates, and the other was K137R, found in 1 isolate of P. ovale wallikeri (n = 4). This result needs to be considered in the context of widespread artemisinin used within the region; their functional consequences for artemisinin sensitivity in P. malariae and P. ovale will need to be elucidated.

Dihydroartemisinin-piperaquine is an artemisinin-based combination treatment (ACT) recommended by the WHO for uncomplicated Plasmodium falciparum malaria, and it is being used increasingly for resistant vivax malaria where combination with primaquine is required for radical cure. The WHO recently reinforced its recommendations to add a single dose of primaquine to ACTs to reduce P. falciparum transmission in low-transmission settings. The pharmacokinetics of primaquine and dihydroartemisinin-piperaquine were evaluated in 16 healthy Thai adult volunteers in a randomized crossover study. Volunteers were randomized to two groups of three sequential hospital admissions to receive 30 mg (base) primaquine, 3 tablets of dihydroartemisinin-piperaquine (120/960 mg), and the drugs together at the same doses. Blood sampling was performed over 3 days following primaquine and 36 days following dihydroartemisinin-piperaquine dosing. Pharmacokinetic assessment was done with a noncompartmental approach. The drugs were well tolerated. There were no statistically significant differences in dihydroartemisinin and piperaquine pharmacokinetics with or without primaquine. Dihydroartemisinin-piperaquine coadministration significantly increased plasma primaquine levels; geometric mean ratios (90% confidence interval [CI]) of primaquine combined versus primaquine alone for maximum concentration (Cmax), area under the concentration-time curve from 0 h to the end of the study (AUC0-last), and area under the concentration-time curve from 0 h to infinity (AUC0-∞) were 148% (117 to 187%), 129% (103 to 163%), and 128% (102 to 161%), respectively. This interaction is similar to that described recently with chloroquine and may result in an enhanced radical curative effect. (This study has been registered at ClinicalTrials.gov under registration no. NCT01525511.).

Artemisinin-based combination therapies (ACTs) have contributed substantially to the global decline in Plasmodium falciparum morbidity and mortality, but resistance to artemisinins and their partner drugs is increasing in Southeast Asia, threatening malaria control. New antimalarial compounds will not be generally available soon. Combining three existing antimalarials in the form of triple ACTs, including dihydroartemisinin (DHA)-piperaquine + mefloquine, is a potential treatment option for multidrug-resistant Plasmodium falciparum malaria. In a sequential open-label study, healthy Thai volunteers were treated with DHA-piperaquine (120 to 960 mg), mefloquine (500 mg), and DHA-piperaquine + mefloquine (120 to 960 mg + 500 mg), and serial symptom questionnaires, biochemistry, full blood counts, pharmacokinetic profiles, and electrocardiographic measurements were performed. Fifteen healthy subjects were enrolled. There was no difference in the incidence or severity of adverse events between the three treatment arms. The slight prolongation in QTc (QT interval corrected for heart rate) associated with DHA-piperaquine administration did not increase after administration of DHA-piperaquine + mefloquine. The addition of mefloquine had no significant effect on the pharmacokinetic properties of piperaquine. However, coadministration of mefloquine significantly reduced the exposures to dihydroartemisinin for area under the concentration-time curve (-22.6%; 90% confidence interval [CI], -33.1, -10.4; P = 0.0039) and maximum concentration of drug in serum (-29.0%; 90% CI, -40.6, -15.1; P = 0.0079). Mefloquine can be added safely to dihydroartemisinin-piperaquine in malaria treatment. (This study has been registered at ClinicalTrials.gov under identifier NCT02324738.).

Artemether-lumefantrine antimalarial efficacy in pregnancy could be compromised by reduced drug exposure. Population-based simulations suggested that therapeutic efficacy would be improved if the treatment duration was increased. We assessed the efficacy, tolerability, and pharmacokinetics of an extended 5-day regimen of artemether-lumefantrine compared to the standard 3-day treatment in 48 pregnant women and 48 nonpregnant women with uncomplicated falciparum malaria in an open-label, randomized clinical trial. Babies were assessed at birth and 1, 3, 6, and 12 months. Nonlinear mixed-effects modeling was used to characterize the plasma concentration-time profiles of artemether and lumefantrine and their metabolites. Both regimens were highly efficacious (100% PCR-corrected cure rates) and well tolerated. Babies followed up to 1 year had normal development. Parasite clearance half-lives were longer in pregnant women (median [range], 3.30 h [1.39 to 7.83 h]) than in nonpregnant women (2.43 h [1.05 to 6.00 h]) (P=0.005). Pregnant women had lower exposures to artemether and dihydroartemisinin than nonpregnant women, resulting in 1.2% decreased exposure for each additional week of gestational age. By term, these exposures were reduced by 48% compared to nonpregnant patients. The overall exposure to lumefantrine was improved with the extended regimen, with no significant differences in exposures to lumefantrine or desbutyl-lumefantrine between pregnant and nonpregnant women. The extended artemether-lumefantrine regimen was well tolerated and safe and increased the overall antimalarial drug exposure and so could be a promising treatment option in pregnancy in areas with lower rates of malaria transmission and/or emerging drug resistance. (This study has been registered at ClinicalTrials.gov under identifier NCT01916954.).

When severe malaria is suspected in children, the WHO recommends pretreatment with a single rectal dose of artesunate before referral to an appropriate facility. This was an individually randomized, open-label, 2-arm, crossover clinical trial in 82 Congolese children with severe falciparum malaria to characterize the pharmacokinetics of rectal artesunate. At admission, children received a single dose of rectal artesunate (10 mg/kg of body weight) followed 12 h later by intravenous artesunate (2.4 mg/kg) or the reverse order. All children also received standard doses of intravenous quinine. Artesunate and dihydroartemisinin were measured at 11 fixed intervals, following 0- and 12-h drug administrations. Clinical, laboratory, and parasitological parameters were measured. After rectal artesunate, artesunate and dihydroartemisinin showed large interindividual variability (peak concentrations of dihydroartemisinin ranged from 5.63 to 8,090 nM). The majority of patients, however, reached previously suggested in vivo IC50 and IC90 values (98.7% and 92.5%, respectively) of combined concentrations of artesunate and dihydroartemisinin between 15 and 30 min after drug administration. The median (interquartile range [IQR]) time above IC50 and IC90 was 5.68 h (2.90 to 6.08) and 2.74 h (1.52 to 3.75), respectively. The absolute rectal bioavailability (IQR) was 25.6% (11.7 to 54.5) for artesunate and 19.8% (10.3 to 35.3) for dihydroartemisinin. The initial 12-h parasite reduction ratio was comparable between rectal and intravenous artesunate: median (IQR), 84.3% (50.0 to 95.4) versus 69.2% (45.7 to 93.6), respectively (P = 0.49). Despite large interindividual variability, rectal artesunate can initiate and sustain rapid parasiticidal activity in most children with severe falciparum malaria while they are transferred to a facility where parenteral artesunate is available. (This study has been registered at ClinicalTrials.gov under identifier NCT02492178.).

Primaquine is the only widely available drug for radical cure of Plasmodium vivax malaria. There is uncertainty whether the pharmacokinetic properties of primaquine are altered significantly in childhood or not. Patients with uncomplicated P. vivax malaria and with normal glucose-6-phosphate dehydrogenase were randomized to receive either chloroquine (25 mg base/kg of body weight) or dihydroartemisinin-piperaquine (dihydroartemisinin at 7 mg/kg and piperaquine at 55 mg/kg) plus primaquine, given either as 0.5 mg base/kg/day for 14 days or 1 mg/kg/day for 7 days. Predose day 7 venous plasma concentrations of chloroquine, desethylchloroquine, piperaquine, primaquine, and carboxyprimaquine were measured. Methemoglobin levels were measured at frequent intervals. Day 7 primaquine and carboxyprimaquine concentrations were available for 641 patients. After adjustment for the milligram-per-kilogram primaquine daily dose, day of sampling, partner drug, and fever clearance, there was a significant nonlinear relationship between age and trough primaquine and carboxyprimaquine concentrations and daily methemoglobin levels. Compared to adults 30 years of age, children 5 years of age had trough primaquine concentrations that were 0.53 (95% confidence interval [CI], 0.39 to 0.73)-fold lower, trough carboxyprimaquine concentrations that were 0.45 (95% CI, 0.35 to 0.55)-fold lower, and day 7 methemoglobin levels that were 0.87 (95% CI, 0.58 to 1.27)-fold lower. Increasing plasma concentrations of piperaquine and chloroquine and poor metabolizer CYP 2D6 alleles were associated with higher day 7 primaquine and carboxyprimaquine plasma concentrations. Higher blood methemoglobin concentrations were associated with a lower risk of recurrence. Young children have lower primaquine and carboxyprimaquine exposures and lower levels of methemoglobinemia than adults. Young children may need higher weight-adjusted primaquine doses than adults. (This study has been registered at ClinicalTrials.gov under identifier NCT01640574.).

Pyronaridine-artesunate is a newly introduced artemisinin-based combination treatment which may be deployed together with primaquine. A single-dose, randomized, three-sequence crossover study was conducted in healthy Thai volunteers to characterize potential pharmacokinetic interactions between these drugs. Seventeen healthy adults received a single oral dose of primaquine alone (30 mg base) and were then randomized to receive pyronaridine-artesunate alone (540-180 mg) or pyronaridine-artesunate plus primaquine in combination, with intervening washout periods between all treatments. The pharmacokinetic properties of primaquine, its metabolite carboxyprimaquine, artesunate, its metabolite dihydroartemisinin, and pyronaridine were assessed in 15 subjects using a noncompartmental approach followed by a bioequivalence evaluation. All drugs were well tolerated. The single oral dose of primaquine did not result in any clinically relevant pharmacokinetic alterations to pyronaridine, artesunate, or dihydroartemisinin exposures. There were significantly higher primaquine maximum plasma drug concentrations (geometric mean ratio, 30%; 90% confidence interval [CI], 17% to 46%) and total exposures (15%; 6.4% to 24%) during coadministration with pyronaridine-artesunate than when primaquine was given alone. Pyronaridine, like chloroquine and piperaquine, increases plasma primaquine concentrations. (This study has been registered at ClinicalTrials.gov under registration no. NCT01552330.).

The spread of artemisinin-resistant Plasmodium falciparum compromises the therapeutic efficacy of artemisinin combination therapies (ACTs) and is considered the greatest threat to current global initiatives to control and eliminate malaria. This is particularly relevant in Vietnam, where dihydroartemisinin-piperaquine (DP) is the recommended ACT for P. falciparum infection. The propeller domain gene of K13, a molecular marker of artemisinin resistance, was successfully sequenced in 1,060 P. falciparum isolates collected at 3 malaria hot spots in Vietnam between 2009 and 2016. Eight K13 propeller mutations (Thr474Ile, Tyr493His, Arg539Thr, Ile543Thr, Pro553Leu, Val568Gly, Pro574Leu, and Cys580Tyr), including several that have been validated to be artemisinin resistance markers, were found. The prevalences of K13 mutations were 29% (222/767), 6% (11/188), and 43% (45/105) in the Binh Phuoc, Ninh Thuan, and Gia Lai Provinces of Vietnam, respectively. Cys580Tyr became the dominant genotype in recent years, with 79.1% (34/43) of isolates in Binh Phuoc Province and 63% (17/27) of isolates in Gia Lai Province carrying this mutation. K13 mutations were associated with reduced ring-stage susceptibility to dihydroartemisinin (DHA) in vitro and prolonged parasite clearance in vivo An analysis of haplotypes flanking K13 suggested the presence of multiple strains with the Cys580Tyr mutation rather than a single strain expanding across the three sites.

Increasing resistance in Plasmodium falciparum to artemisinins and their artemisinin combination therapy (ACT) partner drugs jeopardizes effective antimalarial treatment. Resistance is worst in the Greater Mekong subregion. Monitoring genetic markers of resistance can help to guide antimalarial therapy. Markers of resistance to artemisinins (PfKelch mutations), mefloquine (amplification of P. falciparum multidrug resistance-1 [PfMDR1]), and piperaquine (PfPlasmepsin2/3 amplification and specific P. falciparum chloroquine resistance transporter [PfCRT] mutations) were assessed in 6,722 P. falciparum samples from Vietnam, Lao People's Democratic Republic (PDR), Cambodia, Thailand, and Myanmar between 2007 and 2019. Against a high background prevalence of PfKelch mutations, PfMDR1 and PfPlasmepsin2/3 amplification closely followed regional drug pressures over time. PfPlasmepsin2/3 amplification preceded piperaquine resistance-associated PfCRT mutations in Cambodia and reached a peak prevalence of 23/28 (82%) in 2015. This declined to 57/156 (38%) after first-line treatment was changed from dihydroartemisinin-piperaquine to artesunate-mefloquine (ASMQ) between 2014 and 2017. The frequency of PfMDR1 amplification increased from 0/293 (0%) between 2012 and 2017 to 12/156 (8%) in 2019. Amplification of PfMDR1 and PfPlasmepsin2/3 in the same parasites was extremely rare (4/6,722 [0.06%]) and was dispersed over time. The mechanisms conferring mefloquine and piperaquine resistance may be counterbalancing. This supports the development of ASMQ plus piperaquine as a triple artemisinin combination therapy.