Polymorphisms in the K13-propeller gene in artemisinin-susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali

Abstract

Artemisinin-resistant Plasmodium falciparum malaria has been documented in southeast Asia and may already be spreading in that region. Molecular markers are important tools for monitoring the spread of antimalarial drug resistance. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain were shown to be associated with artemisinin resistance in vivo and in vitro. The prevalence and role of K13-propeller mutations are poorly known in sub-Saharan Africa. K13-propeller mutations were genotyped by direct sequencing of nested polymerase chain reaction (PCR) amplicons from dried blood spots of pre-treatment falciparum malaria infections collected before and after the use of artemisinin-based combination therapy (ACT) as first-line therapy in Mali. Although K13-propeller mutations previously associated with delayed parasite clearance in Cambodia were not identified, 26 K13-propeller mutations were identified in both recent samples and pre-ACT infections. Parasite clearance time was comparable between infections with non-synonymous K13-propeller mutations and infections with the reference allele. These findings suggest that K13-propeller mutations are present in artemisinin-sensitive parasites and that they preceded the wide use of ACTs in Mali.

Figure 1.

Haplotypes of K13 propeller observed in Bougoula-Hameau and Bandiagara, Mali. Sequence alignment of P. falciparum 3D7 and 25 haplotypes (blue) showing polymorphisms in the K13-propeller gene. Numbers in the top row are amino acid positions. Amino acids positions highlighted in yellow have been described by Ariey and others. Amino acids in green were identified in this study dataset. *A mutation in Mali at the same position as a mutation described in the work by Ariey and others but with a different amino acid present.

Figure 2.

Prevalence of K13-propeller mutations at two sites before the use of ACTs and one site after the introduction of ACTs. Prevalence of K13 mutations may not add up to 100%, because infections may have more than one K13 mutation present in one sample.

Figure 3.

(A) Dot plot of the median slope half-lives of parasites in Bougoula-Hameau in 2010 and 2011. (B) Dot plot of mean PC99 of parasites in Bougoula-Hameau in 2010 and 2011. A parametric t test was used to compare PC99, whereas a Wilcoxon rank sum test was used to compare parasite clearance slope half-life.