Kelch Mutations in Plasmodium falciparum Protein K13 Do Not Modulate Dormancy after Artemisinin Exposure and Sorbitol Selection In Vitro.

Some Kelch mutations of the Plasmodium falciparum K13 protein confer increased survival to dihydroartemisinin (DHA)-treated ring-stage parasites. Here, we asked if K13 mutations affect a dormancy phenotype allowing parasites to survive DHA exposure and then sorbitol selection. Although recrudescence from dormancy differed between two distinct parasite lines, it was similar for isogenic lines carrying single-site substitutions in K13. Therefore, K13 mutations do not alter the DHA-sorbitol combined dormancy phenotype; rather, traits from other loci likely determine this phenotype.

Fitness of artemisinin-resistant Plasmodium falciparum in vitro.

OBJECTIVES: Drug resistance confers a fitness advantage to parasites exposed to frequent drug pressure, yet these mutations also may incur a fitness cost. We assessed fitness advantages and costs of artemisinin resistance in Plasmodium falciparum in vitro to understand how drug resistance will spread and evolve in a competitive environment. METHODS: Genotyping of SNPs, drug susceptibility assays and copy number determination were used to assess the impact of artemisinin resistance on parasite fitness. An artemisinin-resistant clone (C9) selected in vitro from an isogenic parental clone (D6) was used to conduct competitive growth studies to assess fitness of artemisinin resistance. The resistant and susceptible clones were mixed or grown alone in the presence and absence of drug pressure (dihydroartemisinin or pyrimethamine) to quantify the rate at which artemisinin resistance was gained or lost. RESULTS: We experimentally demonstrate for the first time that artemisinin resistance provides a fitness advantage that is selected for with infrequent exposure to drug, but is lost in the absence of exposure to artemisinin drugs. The best correlations with artemisinin resistance were decreased in vitro drug susceptibility to artemisinin derivatives, increased copy number of Pf3D7_1030100 and an SNP in Pf3D7_0307600. An SNP conferring an E208K mutation in the kelch gene (Pf3D7_1343700) was not associated with resistance. Furthermore, we observed second-cycle ring-stage dormancy induced by pyrimethamine, suggesting that dormancy is a fitness trait that provides an advantage for survival from antimalarial drug stress. CONCLUSIONS: Artemisinin-resistant P. falciparum have a fitness advantage to survive and predominate in the population even in the face of infrequent exposure to artemisinin drugs.

Artemisinin-resistant Plasmodium falciparum parasites exhibit altered patterns of development in infected erythrocytes.

Artemisinin derivatives are used in combination with other antimalarial drugs for treatment of multidrug-resistant malaria worldwide. Clinical resistance to artemisinin recently emerged in southeast Asia, yet in vitro phenotypes for discerning mechanism(s) of resistance remain elusive. Here, we describe novel phenotypic resistance traits expressed by artemisinin-resistant Plasmodium falciparum. The resistant parasites exhibit altered patterns of development that result in reduced exposure to drug at the most susceptible stage of development in erythrocytes (trophozoites) and increased exposure in the most resistant stage (rings). In addition, a novel in vitro delayed clearance assay (DCA) that assesses drug effects on asexual stages was found to correlate with parasite clearance half-life in vivo as well as with mutations in the Kelch domain gene associated with resistance (Pf3D7_1343700). Importantly, all of the resistance phenotypes were stable in cloned parasites for more than 2 years without drug pressure. The results demonstrate artemisinin-resistant P. falciparum has evolved a novel mechanism of phenotypic resistance to artemisinin drugs linked to abnormal cell cycle regulation. These results offer insights into a novel mechanism of drug resistance in P. falciparum and new tools for monitoring the spread of artemisinin resistance.