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The ring-stage survival assay was utilized to assess the impact of physiological hyperoxic stress on dihydroartemisinin (DHA) tolerance for a panel of Plasmodium falciparum strains with and without Kelch13 mutations. Strains without naturally acquired Kelch13 mutations or the postulated genetic background associated with delayed parasite clearance time demonstrated reduced proliferation under hyperoxic conditions in the subsequent proliferation cycle. Dihydroartemisinin tolerance in three isolates with naturally acquired Kelch13 mutations but not two genetically manipulated laboratory strains was modulated by in vitro hyperoxic stress exposure of early-ring-stage parasites in the cycle before drug exposure. Reduced parasite tolerance to additional derivatives, including artemisinin, artesunate, and OZ277, was observed within the second proliferation cycle. OZ439 and epoxomicin completely prevented parasite survival under both hyperoxia and normoxic in vitro culture conditions, highlighting the unique relationship between DHA tolerance and Kelch13 mutation-associated genetic background. IMPORTANCE Artemisinin-based combination therapy (ACT) for treating malaria is under intense scrutiny following treatment failures in the Greater Mekong subregion of Asia. This is further compounded by the potential for extensive loss of life if treatment failures extend to the African continent. Although Plasmodium falciparum has become resistant to all antimalarial drugs, artemisinin "resistance" does not present in the same way as resistance to other antimalarial drugs. Instead, a partial resistance or tolerance is demonstrated, associated with the parasite's genetic profile and linked to a molecular marker referred to as K13. It is suggested that parasites may have adapted to drug treatment, as well as the presence of underlying population health issues such as hemoglobinopathies, and/or environmental pressures, resulting in parasite tolerance to ACT. Understanding parasite evolution and control of artemisinin tolerance will provide innovative approaches to mitigate the development of artemisinin tolerance and thereby artemisinin-based drug treatment failure and loss of life globally to malaria infections.

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 ( pfk13 ) gene. Here, we carried out in vitro selection over a 1-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA 0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

Background: Deployment of mefloquine-artesunate (MAS3) on the Thailand-Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial., Methods: Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance., Results: Polymerase chain reaction (PCR)-adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8% (95% confidence interval [CI], 45.4, 68.3) compared with 97.8% (95% CI, 93.3, 99.3) in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%., Conclusions: The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand-Myanmar border., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

BackgroundArtemisinin resistance, linked to polymorphisms in the Kelch gene on chromosome 13 of Plasmodium falciparum (k13), has outpaced containment efforts in South East Asia. For national malaria control programmes in the region, it is important to establish a surveillance system which includes monitoring for k13 polymorphisms associated with the clinical phenotype.MethodsBetween February and December 2013, parasite clearance was assessed in 35 patients with uncomplicated P. falciparum treated with artesunate monotherapy followed by 3-day ACT in an isolated area on the Myanmar-Thai border with relatively low artemisinin drug pressure. Molecular testing for k13 mutations was performed on dry blood spots collected on admission.ResultsThe proportion of k13 mutations in these patients was 41.7%, and only 5 alleles were detected: C580Y, I205T, M476I, R561H, and F446I. Of these, F446I was the most common, and was associated with a longer parasite clearance half-life (median) 4.1 (min-max 2.3-6.7) hours compared to 2.5 (min-max 1.6-8.7) in wildtype (p = 0·01). The prevalence of k13 mutant parasites was much lower than the proportion of k13 mutants detected 200 km south in a much less remote setting where the prevalence of k13 mutants was 84% with 15 distinct alleles in 2013 of which C580Y predominated.ConclusionsThis study provides evidence of artemisinin resistance in a remote part of eastern Myanmar. The prevalence of k13 mutations as well as allele diversity varies considerably across short distances, presumably because of historical patterns of artemisinin use and population movements.