In vitro susceptibility of Plasmodium falciparum isolates from the China-Myanmar border area to artemisinins and correlation with K13 mutations

Abstract

Mutations in the Kelch domain of the K13 gene (PF3D7_1343700) were previously associated with artemisinin resistance in Plasmodium falciparum . This study followed the dynamics of the K13 polymorphisms in P. falciparum parasites from the China-Myanmar border area obtained in 2007–2016, and their in vitro sensitivities to artesunate (AS) and dihydroartemisinin (DHA). The 50% effective concentration (EC 50 72h ) values of 133 culture-adapted field isolates to AS and DHA, measured by the conventional 72 h SYBR Green I-based assay, varied significantly among the parasites from different years; all were significantly higher than that of the reference strain 3D7. Compared with parasites from 2007 to 2008, ring survival rates almost doubled in parasites obtained in later years. Sequencing the full-length K13 genes identified 11 point mutations present in 85 (63.9%) parasite isolates. F446I was the predominant (55/133) variant, and its frequency was increased from 17.6% (3/17) in 2007 to 55.9% (19/34) in 2014–2016. No wild-type (WT) Kelch domain sequences were found in the 34 samples obtained from 2014 to 2016. In the 2014–2016 samples, a new mutation (G533S) appeared and reached 44.1% (15/34). Collectively, parasites with the Kelch domain mutations (after amino acid 440) had significantly higher ring survival rates than the WT parasites. Individually, F446I, G533S and A676D showed significantly higher ring survival rates than the WT. Although the drug sensitivity phenotypes measured by the RSA 6h and EC 50 72h assays may be intrinsically linked to the in vivo clinical efficacy data, the values determined by these two assays were not significantly correlated. This study identified the trend of K13 mutations in parasite populations from the China-Myanmar border area, confirmed an overall correlation of Kelch domain mutations with elevated ring-stage survival rates, and emphasized the importance of monitoring the evolution and spread of parasites with reduced artemisinin sensitivity along the malaria elimination course.

Genetic features of P. falciparum parasites collected in 2012-2016 and anti-malaria resistance along China-Myanmar border

“…In this study, F446I isolates did not show significant longer PCTs than those in W group, this meant mutation of F446I along China-Myanmar border was not confirmed as the resistant marker. This view was contrary to other research results [50,[59][60]. Bases on careful comparison, the conflict was attributed to the fact that this study was in vivo test and the internal period that the protozoemia was checked was every 24 hrs in this study, which was different from the in vitro tests and 8 hrs internal period in other studies.…”

Efficacy of artemether-lumefantrine for treating uncomplicated Plasmodium falciparum cases and molecular surveillance of drug resistance genes in Western Myanmar

“…Likewise, within the GMS, P. falciparum populations showed striking disparity in the prevalence and distribution of pfk13 mutations, with the C580Y and F446I being the predominant pfk13 mutations in east and west GMS, respectively [ 27 , 29 – 31 ]. The NN insertion between amino acids 136 and 137 was associated with artemisinin resistance and its prevalence has increased dramatically over the years along the China-Myanmar border [ 20 , 32 ].…”

Susceptibility of Plasmodium falciparum isolates to antimalarial drugs in a highly seasonal malaria endemic village in Mali

“…However, no molecular marker of such resistance has yet been identified [10]. Some of the mutations in the gene kelch (K13) have been associated with resistance to artemisinin in vitro in Asia [11][12][13]. Up-to-date, resistance to artemisinin has not been reported in Africa and ACTs have remained very effective [11,14].…”