Department of Pathogen Biology and Immunology, Kunming Medical University, Kunming, Yunnan Province, 650500, China.
Department of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Suite 304, Tampa, FL, 33612, USA.
Int J Parasitol Drugs Drug Resist. 2019 Aug;10:20-27. doi: 10.1016/j.ijpddr.2019.04.002. Epub 2019 Apr 10.
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) 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 and EC 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.
先前的研究表明，疟原虫 Kelch 结构域基因（PF3D7_1343700）的突变与青蒿素抗药性有关。本研究对 2007 年至 2016 年期间在中国-缅甸边境地区采集的疟原虫寄生虫中的 K13 多态性进行了动态监测，并对青蒿琥酯（AS）和双氢青蒿素（DHA）的体外敏感性进行了检测。采用传统的 72 小时 SYBR Green I 检测法，测定了 133 株经培养适应的野外分离株对 AS 和 DHA 的 50%有效浓度（EC）值，结果表明不同年份的寄生虫之间存在显著差异；所有 EC 值均明显高于参考株 3D7。与 2007 年至 2008 年的寄生虫相比，2009 年以后采集的寄生虫环期存活率几乎翻了一番。对全长 K13 基因进行测序，发现 85 株（63.9%）寄生虫分离株中存在 11 个点突变。F446I 是主要（55/133）突变，其频率从 2007 年的 17.6%（3/17）增加到 2014-2016 年的 55.9%（19/34）。在 2014 年至 2016 年采集的 34 个样本中未发现野生型（WT）Kelch 结构域序列。在 2014-2016 年的样本中，出现了一个新的突变（G533S），并达到 44.1%（15/34）。总的来说，具有 Kelch 结构域突变（第 440 位氨基酸后）的寄生虫环期存活率明显高于 WT 寄生虫。单独来看，F446I、G533S 和 A676D 的环期存活率明显高于 WT。尽管 RSA 和 EC 测定的药物敏感性表型可能与体内临床疗效数据有内在联系，但这两种测定方法确定的值之间没有显著相关性。本研究确定了中国-缅甸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，并强调了在消除疟疾过程中监测青蒿素敏感性降低的寄生虫的演变和传播的重要性。
