Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand

Abstract

Resistance of Plasmodium falciparum to artemisinin combination therapy (ACT) in Southeast Asia can have a devastating impact on chemotherapy and control measures. In this study, the evolution of artemisinin-resistant P. falciparum in Thailand was assessed by exploring mutations in the K13 locus believed to confer drug resistance phenotype. P. falciparum-infected blood samples were obtained from patients in eight provinces of Thailand over two decades (1991-2014; n = 904). Analysis of the K13 gene was performed by either sequencing the complete coding region (n = 259) or mutation-specific PCR-restriction fragment length polymorphism method (n = 645). K13 mutations related to artesunate resistance were detected in isolates from Trat province bordering Cambodia in 1991, about 4 years preceding widespread deployment of ACT in Thailand and increased in frequency over time. Nonsynonymous nucleotide diversity exceeded synonymous nucleotide diversity in the propeller region of the K13 gene, supporting the hypothesis that this diversity was driven by natural selection. No single mutant appeared to be favoured in every population, and propeller-region mutants were rarely observed in linkage with each other in the same haplotype. On the other hand, there was a highly significant association between the occurrence of a propeller mutant and the insertion of two or three asparagines after residue 139 of K13. Whether this insertion plays a compensatory role for deleterious effects of propeller mutants on the function of the K13 protein requires further investigation. However, modification of duration of ACT from 2-day to 3-day regimens in 2008 throughout the country does not halt the increase in frequency of mutants conferring artemisinin resistance phenotype.

Declining Efficacy of Artemisinin Combination Therapy AgainstP. FalciparumMalaria on the Thai–Myanmar Border (2003–2013): The Role of Parasite Genetic Factors

“…In most areas where AL is used, there is no evidence for resistance to either component. However, in Myanmar where AL has recently been introduced, K13 propeller mutants and Pfmdr1 amplification are widespread [28, 35–38]. The therapeutic lifetime of AL in Myanmar and other areas with a high prevalence of K13 mutations may be relatively short.…”

Emergence and Spread of kelch13 Mutations Associated with Artemisinin Resistance in Plasmodium falciparum Parasites in 12 Thai Provinces from 2007 to 2016

“…Results from the 277 samples from this study were combined with data from 1,148 samples from previously published work (12,20,22,23,33,37) and the Worldwide Antimalarial Resistance Network (WWARN; http://www.wwarn.org). Previously published results included Sanger sequencing (20,23,33,38) and next-generation sequencing (NGS) (12,22,37) data. The previously called alleles were combined with this study's data and curated to produce a summary table of kelch13 alleles (Table S1).…”

“…In comparison, another 180 km further south, in Chumphon, C580Y was found in 2007 at a frequency of 16% (2 of 12 samples). In the neighboring province, Ranong, (12,20,22,23,33,37,38), denoted by the "number" symbol (#) above the pie charts. References for each corresponding number (#) are provided in Table S1.…”

Artemisinin-Resistant Plasmodium falciparum Malaria

“…Retrospective data from Cambodia, Thailand, and Myanmar indicate that these mutations arose as early as 2001. Molecular surveillance studies have greatly expanded the map of K13-propeller polymorphism to include additional areas of Cambodia(42)(43)(44), Thailand(45,46), Myanmar(47)(48)(49), China(48,50), Laos (51), Bangladesh(52), and India(53). While some of these mutations have already been associated with slow parasite clearance in patients at other sites, most have not and require validation.…”