Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia

Abstract

Background: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia.

Methods: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently.

Results: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas.

Conclusions: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

Keywords: Plasmodium falciparum; Southeast Asia; artemisinin resistance; kelch; malaria.

Figure 1.

Distribution of parasite clearance half-life by geography in the previous genome-wide associated study (GWAS) and replication data sets. A, Previous GWAS data set. B, Replication data set. A large proportion of parasites from western Cambodia demonstrate delayed parasite clearance, as do a subset of parasites from Myanmar and Vietnam. Parasites from Laos and Bangladesh demonstrated mostly rapid parasite clearance.

Figure 2.

Population structure by geography in the previous genome-wide associated study (GWAS) and replication data sets. A, Previous GWAS data set. B, Replication data set. In the imputed data set representing the previous GWAS, most of the population structure was accounted for by the second principal component. As most of the samples from this data set were genotyped with the least dense single-nucleotide polymorphism array, the first principal component accounted for most of the missing data. In the replication data set, most of the population structure was accounted for by the first principal component.

Figure 3.

Manhattan and random forest plots for the gene-based genome-wide association study. Meta-analysis of the association between the presence of nonreference alleles in each gene in the genome and parasite clearance half-life. A, −Log₁₀ P values for all genes, with both the K13 protein (PF3D7_1343700) and PF3D7_1442600 achieving genome-wide significance. B, The percentage increase in mean squared error for the top 20 single-nucleotide polymorphisms or covariates in the random forests analysis. The presence of nonreference alleles in the K13 gene was the best predictor of parasite clearance half-life after the second principal component, which accounted for most of the population structure.

Figure 4.

Prevalence of K13 mutations and association with parasite clearance half-life. A, Parasite clearance half-life for parasites with K13 mutations (colors) or wild-type alleles (green). Parasites with wild-type alleles are grouped by country (LAO = Laos, BGD = Bangladesh, MMR = Myanmar, VNM = Vietnam, KHM = Cambodia), and parasites with K13 mutations are designated by symbols (Laos = circles, Bangladesh = squares, Myanmar = triangle, Vietnam = inverted triangle, and Cambodia = diamond). Horizontal lines represent median half-lives and 95% confidence intervals for wild-type and K13 mutants represented more than twice in the data set. B, Prevalence of parasites with K13 mutations, by country.

Figure 5.

Median joining haplotype network of K13 mutations and single-nucleotide polymorphisms within linkage disequilibrium of the K13 gene. Haplotype prevalence is proportional to size of the circle, with circle color corresponding to population of origin: Cambodia (red), Myanmar (purple), or Vietnam (green). Individual mutations are labeled by amino acid position, and wild-type haplotypes are not labeled.