长沙市122例输入性恶性疟原虫pfpm2基因拷贝数变异的分析

Analysis of copy number variation in pfpm2 gene of Plasmodium falciparum in 122 imported cases in Changsha from 2016 to 2019

目的了解长沙市近4年来输入性恶性疟原虫(Plasmodium falciparum,P.f)的血浆蛋白酶基因2(plasmodium falciparum plasmepsin 2,pfpm2)基因拷贝数变异(copy number variations,CNVs)情况,用以预测输入性抗药性虫株出现风险。方法收集长沙市输入性疟疾定点医疗机构在2016-2019年收治的单一感染P.f患者外周静脉血制作的干血斑样本122份,采用荧光实时定量PCR检测P.f的pfpm2基因CNVs,以P.f的pfpm2基因CNVs增加参数来分析抗药虫株在不同输入年份间和不同输出国间产生的可能性。结果来自25个输入国122例P.f患者的临床虫株pfpm2基因CNVs值为1.222±0.422,显著高于标准虫株3D7 CNVs值(1.007±0.023,P<0.001)。在122个临床虫株中,有23个pfpm2基因的CNVs值>1.5,分别来源于尼日利亚(6/15)、刚果(5/18)、科特迪瓦(2/5)、塞拉利昂(2/8)等12个国家的恶性疟病例。在2016-2019年输入的病例数中,pfpm2基因CNVs值>1.5的临床虫株数在各年度输入总数中所占比率分别为13.2%(5/38)、16.0%(4/25)、22.9%(8/35)和25.0%(6/24),各年度总虫株数的CNVs值分别为1.061±0.323、1.184±0.247、1.292±0.571和1.414±0.360。经t检验,除2017与2018年、2018与2019年之间的差异无统计学意义外,其余年度间比较差异均有统计学意义(P<0.05)。来源于尼日利亚、科特迪瓦、坦桑尼亚和布隆迪4个国家的临床虫株CNVs均值显著高于总体(122个病例来源)均值(P<0.05)。结论长沙市输入性P.f的pfpm2基因CNVs值逐年提高,提示抗药性虫株有发展趋势;某些来源于西非国家的临床虫株可能产生了抗药性,提示我国疟疾防控机构要注意现行抗疟药对某些输入性恶性疟患者的治疗效果可能不佳。

Objective To analyze copy number variations(CNVs)of pfpm2 gene in 122 imported cases of Palciparum falciparum(P.f)infection in Changsha from 2016 to 2019 for predicting the risk of imported drug-resistant P.f strains in Changsha.Methods We collected dried blood spot samples of anticoagulated peripheral venous blood from 122 patients with a single infection of P.f,who were treated in designated medical facilities for malaria in Changsha in the years of 2016-2019.For each specimen,the CNVs of pfpm2 gene were detected in parallel with the standard strain 3 D7 using real-time PCR.The CNVs values of the samples were compared across the years and also among different source countries of the cases.Results We analyzed a total of 122 P.f strains isolated from imported cases from 25 countries.The mean value of CNVs of pfpm2 gene in the 122 strains was significantly higher than that of the standard strain 3 D7(1.222±0.422 vs 1.007±0.023,P<0.001).There were 23 Strains Whose CNVs Values of pfpm2 gene were higher than 1.5,which were imported from 12 countries including Nigeria(6/15),Congo(5/18),Cote d’Ivoire(2/5)and Sierra Leone(2/8).The percentage of strains with pfpm2 CNVs>1.5 was 13.2%(5/38)in 2016,16.0%(4/25)in 2017,22.9%(8/35)in 2018 and 25.0%(6/24)in 2019 respectively.The mean value of pfpm2 gene CNVs was 1.061±0.323 in 2016,1.184±0.247 in 2017,1.292±0.571 in 2018 and 1.414±0.360 in 2019,showing significant differences across the years except the years of 2017 and 2018 or 2018 and 2019(P<0.05,P<0.001).The mean values of pfpm2 gene CNVs of the strains from Nigeria,Cote d’Ivoire,Tanzania and Burundi were significantly higher than the overall mean of the 122 strains(P<0.05).Conclusion The CNVs of pfpm2 gene of P.f imported from African countries to Changsha are increased year by year,suggesting an increase in the risk of drug-resistant P.f strains in Changsha.Some clinical P.f strains from West Africa may have developed drug resistance,and thus may show poor response to current antimalarial drugs.