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 (EC5072h) 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 RSA6h and EC5072h 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.

中文翻译：

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中缅边境地区恶性疟原虫对青蒿素的体外敏感性及其与K13突变的相关性


K13 基因 Kelch 结构域 (PF3D7_1343700) 的突变此前被认为与恶性疟原虫中的青蒿素耐药性相关。本研究追踪2007-2016年获得的中缅边境地区恶性疟原虫K13多态性动态及其对青蒿琥酯（AS）和双氢青蒿素（DHA）的体外敏感性。通过传统的 72 小时 SYBR Green I 测定法测量，133 种适应培养的野外分离株对 AS 和 DHA 的 50% 有效浓度 (EC5072h) 值在不同年份的寄生虫之间存在显着差异；均显着高于参考菌株3D7。与2007年至2008年的寄生虫相比，后来获得的寄生虫的环存活率几乎翻了一番。对全长 K13 基因进行测序，发现 85 个（63.9%）寄生虫分离株中存在 11 个点突变。 F446I是主要的（55/133）变体，其频率从2007年的17.6%（3/17）增加到2014-2016年的55.9%（19/34）。 2014年至2016年获得的34个样本中未发现野生型（WT）Kelch结构域序列。2014-2016年样本中出现了新的突变（G533S）并达到44.1%（15/34）。总的来说，具有 Kelch 结构域突变（氨基酸 440 之后）的寄生虫比 WT 寄生虫具有显着更高的环存活率。单独而言，F446I、G533S 和 A676D 表现出比 WT 显着更高的环存活率。尽管 RSA6h 和 EC5072h 测定法测量的药物敏感性表型可能与体内临床疗效数据有内在联系，但这两种测定法测定的值并不显着相关。 这项研究确定了中缅边境地区寄生虫种群中K13突变的趋势，证实了Kelch结构域突变与环期存活率升高的总体相关性，并强调了监测青蒿素减少的寄生虫进化和传播的重要性消除疟疾过程中的敏感性。