Little Polymorphism at the K13 Propeller Locus in Worldwide Plasmodium falciparum Populations Prior to the Introduction of Artemisinin Combination Therapies

Abstract

The emergence and spread of artemisinin-resistant Plasmodium falciparum is of huge concern for the global effort toward malaria control and elimination. Artemisinin resistance, defined as a delayed time to parasite clearance following administration of artemisinin, is associated with mutations in the Pfkelch13 gene of resistant parasites. To date, as many as 60 nonsynonymous mutations have been identified in this gene, but whether these mutations have been selected by artemisinin usage or merely reflect natural polymorphism independent of selection is currently unknown. To clarify this, we sequenced the Pfkelch13 propeller domain in 581 isolates collected before (420 isolates) and after (161 isolates) the implementation of artemisinin combination therapies (ACTs), from various regions of endemicity worldwide. Nonsynonymous mutations were observed in 1% of parasites isolated prior to the introduction of ACTs. Frequencies of mutant isolates, nucleotide diversity, and haplotype diversity were significantly higher in the parasites isolated from populations exposed to artemisinin than in those from populations that had not been exposed to the drug. In the artemisinin-exposed population, a significant excess of dN compared to dS was observed, suggesting the presence of positive selection. In contrast, pairwise comparison of dN and dS and the McDonald and Kreitman test indicate that purifying selection acts on the Pfkelch13 propeller domain in populations not exposed to ACTs. These population genetic analyses reveal a low baseline of Pfkelch13 polymorphism, probably due to purifying selection in the absence of artemisinin selection. In contrast, various Pfkelch13 mutations have been selected under artemisinin pressure.

FIG 1

Polymorphism in the Plasmodium falciparum Kelch13 propeller domain from worldwide parasite populations taken before the first report of artemisinin resistance. Nonsynonymous substitutions and synonymous substitutions observed in 581 samples examined in this study are colored in yellow and blue, respectively. The sequence from strain 3D7 (PlasmoDB, PF3D7_1343700) is used as a reference. Substitutions reported by other investigators are framed by a bold line. *, singleton.

FIG 2

Haplotypes of Pfkelch13 observed in 581 worldwide Plasmodium falciparum isolates. Nucleotide and amino acid positions are numbered according to the strain 3D7 sequence (PF3D7_1343700).