云南省恶性疟原虫青蒿素耐药性相关基因K13 kelch结构域序列多态性的分析

Polymorphism Analysis of Plasmodium falciparum K13 Gene Kelch Domain Associated with Resistance to Artemisinin in Yunnan Province

目的 分析云南省恶性疟现症病例中恶性疟原虫（Plasmodium falciparum）K13基因kelch结构域的序列多态性，为掌握云南省恶性疟青蒿素耐药性提供依据。 方法 2013年1月-2015年12月，从云南省16个州（市）收集恶性疟现症病例的滤纸血样和相关信息，通过流行病学调查判定其感染来源，根据中国疾病预防控制信息系统疫情登记确认病例发现地。使用巢式PCR扩增恶性疟原虫K13基因kelch结构域并测序，与恶性疟原虫标准株3D7（PF3D7_1343700）序列进行比对。使用Mega 5.04分析序列多态，计算序列间变异位点、遗传距离等，计算氨基酸突变位点构成比并进行χ^2检验。 结果 2013-2015年共收集恶性疟现症病例血样202例，190例为输入性病例，12例为云南本地感染病例，各年的病例构成比分别为30.7％（62/202）、34.2％（69/202）和35.1％（71/202），呈逐年增多趋势。192例血样K13基因kelch结构域巢式扩增阳性，190例测序成功，66例存在K13基因错义突变，突变率为34.7%（66/190）。各年的突变病例检出构成比分别为40.9%（27/66）、37.9%（25/66）和21.2%（14/66），呈逐年减少趋势。共检出F446I、A578S、N458Y、P574L、A676D、G449A、C469Y、V566I、E556D和S16L等10种突变型，突变率最高的为F446I，占72.7%（48/66）。F446I突变型在18~56岁、农民、感染地为东南亚等病例中的检出比例分别为58.3%（28/48）、70.8%（34/48）和91.7%（44/48），高于同组其他病例的41.7%（20/48）、29.2%（14/48）和8.3%（4/48）（χ2=4.633、5.556、5.152，P＜0.05）。190例K13基因kelch结构域的同源性片段为248 bp，其中保守位点占94.8％（235/248），变异位点占5.2％（13/248），简约信息位点占2.0％（5/248），单态位点占3.2％（8/248）。190例DNA序列间遗传距离为0.000~0.036，平均为（0.001±0.001）。 结论 云南省恶性疟现症病例的K13基因kelch结构域存在10种突变型，以F446I位点突变最常见。

Objective To investigate the polymorphism of Plasmodium falciparum K13 gene kelch domain region and provide basis for understanding the artemisinin resistance of falciparum malaria in Yunnan Province. Methods The filter blood samples and relative information of falciparum malaria cases were collected in 16 prefectures of Yunnan Province from January 2013 to December 2015. The source of infection was determined by epidemiological investigation and the place of case discovery was confirmed according to the China Information System for Disease Control and Prevention Epidemic Registration. The K13 gene kelch domain region was amplified by nested PCR, sequenced, and blasted against the reference strain 3D7（PF3D7_1343700）. The K13 gene kelch domain region polymorphism was analyzed with Mega 5.04. The variable sites and genetic distance between sequences were analyzed. The constituent ratio of amino acid mutation sites was calculated and analyzed with χ^2 test. Results A total of 202 blood samples were collected from 2013 to 2015, comprising 190 from imported cases, 12 from local cases in Yunnan Province. The constitutent ratio of infection cases were 30.7% （62/202）, 34.2% （69/202） and 35.1% （71/202） respectively, increased year by year. The K13 gene kelch domain was successfully amplified from 192 samples and 190 were successfully sequenced, detecting missense mutation of K13 gene in 66 samples, the mutation rate was 34.7% （66/190）. The detection rate of K13 gene mutation was 40.9% （27/66）, 37.9% （25/66） and 21.2% （14/66） respectively, decreased year by year. In this study, ten types of mutations were detected, which were F446I, A578S, N458Y, P574L, A676D, G449A, C469Y, V494I, E556D and S16L. The highest mutation rate occurred in F446I which was 72.7% （48/66）. The proportion of F446I mutation type was 58.3% （28/48） in an age-range of 18-56 years, 70.8% （34/48） in farmers, and 91.7% （44/48） in patients with infection source in Southeast Asia, all significantly higher than that of other groups with the same characteristics （41.7%, 20/48; 29.2%, 14/48; and 8.3%, 4/48, respectively）（χ2=4.633, 5.556 and 5.152, both P〈0.05）. There was a 248 bp homologous sequence in the 190 sequences, composed of 235 conservative sites （94.8%）, 13 variable sites （5.2%）, 5 parsim-info sites （2.0%）, and 8 singleton sites （3.2%）. The genetic distance among the 190 sequences ranged 0.000-0.036, with an average of 0.001±0.001. Conclusion There are 10 types of mutations in the K13 kelch domain in Yunnan Province, the predominant mutation type was F446I.