Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia

Abstract

The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.

Fig. 1.

Distribution and heritability of parasite clearance phenotypes. (A and B) Distribution of parasite clearance half-life and parasite clearance time at the study sites. (C and D) Distribution of clearance half-life and clearance time in 23 identical clones from Tasanh and Pailin, Cambodia. The heritability of each phenotype (adjusted for confounding factors) is shown in C and D Bottom Right.

Fig. 2.

Population structure by geography. A plot of the first two principal components from a principal components analysis demonstrates evidence of population structure among parasites based on geographic region.

Fig. 3.

SNPs associated with parasite clearance in regression models. Manhattan plots showing –log₁₀ P values for each SNP tested in EMMA models of (A) parasite clearance half-life and (C) parasite clearance time for the dataset including all study samples. Corresponding quantile–quantile plots for models of (B) parasite clearance half-life and (D) clearance time are also shown. The red lines in B and D indicate the distribution of expected P values in the absence of any association or confounding. Early deviation from the expected line suggests the presence of confounding that has not been adequately controlled for in the analysis, whereas deviations at the highest –log₁₀ P-value ranges represent the loci most strongly associated with the phenotype. In all panels, the dashed horizontal lines indicate thresholds for genome-wide significance determined using a phenotype-permutation approach.