河南省自赤道几内亚输入的恶性疟原虫抗药性基因多态性分析  

Polymorphism analysis of drug resistance genes in imported Plasmodium falciparum isolates from Equatorial Guinea in Henan Province

作　　者：周瑞敏[1] 纪鹏慧 李素华[1] 杨成运[1] 刘颖[1] 钱丹[1] 邓艳[1] 鲁德领[1] 赵玉玲[1] 赵东阳[1] 张红卫[1] ZHOU Ruimin;JI Penghui;LI Suhua;YANG Chengyun;LIU Ying;QIAN Dan;DENG Yan;LU Deling;ZHAO Yuling;ZHAO Dongyang;ZHANG Hongwei(Henan Provincial Center for Disease Control and Prevention,Henan Provincial Key Laboratory of Pathogenic Microbiology,Henan Provincial Medical Key Laboratory of Parasitic Diseases and Vector,Zhengzhou 450016,China)

机构地区：[1]河南省疾病预防控制中心寄生虫病预防控制所,河南省病原微生物重点实验室,河南省寄生虫病与媒介医学重点实验室,郑州450016

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第5期593-600,608,共9页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：河南省医学科技攻关计划联合共建项目(LHGJ20220157,LHGJ20210134)。

摘　　要：目的分析河南省自赤道几内亚输入性恶性疟原虫抗药性基因多态性,了解恶性疟原虫基因突变情况,评估其流行特征。方法收集河南省2012—2019年自赤道几内亚输入性恶性疟病例信息和外周血样,提取血样中恶性疟原虫基因组DNA,巢式PCR扩增恶性疟原虫Kelch 13螺旋体(PfK13)基因、恶性疟原虫氯喹抗性转运蛋白(Pfcrt)基因、恶性疟原虫多药物抗性基因1(Pfmdr1)、恶性疟原虫二氢叶酸还原酶(Pfdhfr)基因和恶性疟原虫二氢蝶酸合酶(Pfdhps)基因,琼脂糖凝胶电泳后进行双向测序。获得的基因序列通过MEGA7软件与参考基因组进行比较获得突变位点信息,参考基因组来自GenBank的恶性疟原虫3D7野生虫株(GenBank登录号分别为PF3D7_1343700、PF3D7_0709000、PF3D7_0523000、PF3D7_0417200和PF3D7_1324800)。使用SPPS 21.0软件对数据进行统计学分析。结果2012—2019年,河南省共报告输入性疟疾病例1522例,其中自赤道几内亚输入病例117例,包括恶性疟病例97例、卵形疟病例16例、间日疟和三日疟病例各1例、恶性疟原虫/三日疟原虫混合感染和恶性疟原虫/卵形疟原虫混合感染病例各1例。测序获得91份恶性疟原虫样品的PfK13基因序列,基因突变率为8.8%(8/91);突变样品中共检测出7个非同义突变位点和3个同义突变位点,其中非同义突变位点分别为M476I混合型(混)、A481V混、A564E混、P574L混、A578S、V589I和N609I混,同义突变位点分别为G625G、N664N和C469C。测序获得91份恶性疟原虫样品的Pfcrt基因序列,基因突变率为18.7%(17/91);检测出3种Pfcrt基因型,分别为野生型C72V73M74N75K76(81.3%,74/91)、突变型C72V73I74E75T76(12.1%,11/91)和混合型C72V73M/I74N/E75K/T76(6.6%,6/91)。测序获得92份恶性疟原虫样品的Pfmdr1基因序列,基因突变率为77.2%(71/92);检测出3个突变位点,分别为N86Y(41.3%,38/92)、Y184F(75.0%,69/92)和D1246Y(1.1%,1/92),其中N86Y突变率从2012年的68.8%(11/16)下降到Objective To analyze the imported Equatorial Guinean Plasmodium falciparum drug resistance gene polymorphisms in Henan Province,and provide a reference for the treatment of imported P.falciparum infections.Methods The medical records and peripheral blood samples were collected from the imported P.falciparum malaria cases original from Equatorial Guinea in Henan Province from 2012 to 2019.The P.falciparum genomic DNA was ex⁃tracted and the P.falciparum genes,including Kelch 13⁃propeller(PfK13),chloroquine resistance transporter(Pfcrt),multidrug resistance 1(Pfmdr1),dihydrofolate reductase(Pfdhfr)and dihydropteroate synthase(Pfdhps),were amplified by nested PCR.Bidirectional sequencing of the secondary PCR products was performed after agarose gel electrophoresis.The obtained sequences were aligned with the corresponding reference P.falciparum 3D7 strain genomes by MEGA7 software.The reference genomes were obtained from the GenBank(GenBank accession numbers:PF3D7_1343700,PF3D7_0709000,PF3D7_0523000,PF3D7_0417200 and PF3D7_1324800 respectively).The data were analyzed by SPPS 21.0 software.Results A total of 1522 imported malaria cases were reported in Henan Province during 2012 to 2019,including 117 cases imported from Equatorial Guinea.Among the 117 cases,97 cases were infected with P.falciparum,16 cases were infected with P.ovale,1 case was infected with P.vivax,1 case was infected with P.ma⁃lariae,1 case was mixed infected with P.falciparum and P.malariae and 1 case was mixed infected with P.falci⁃parum and P.ovale.The PfK13 gene was successfully amplified from 91 P.falciparum samples and the mutant preva⁃lence was 8.8%(8/91).The non⁃synonymous mutation sites were M476I mixed type(mixed),A481V mixed,A564E mixed,P574L mixed,A578S,V589I and N609I mixed respectively.The synonymous mutation sites were G625G,N664N and C469C respectively.The Pfcrt gene was successfully amplified from 91 P.falciparum samples and the mu⁃tant prevalence was 18.7%(17/91).Three Pfcrt haplotypes were identified,including wild⁃

关 键 词：恶性疟原虫 药物抗性 PfK13 PFCRT Pfmdr1 Pfdhfr Pfdhps 赤道几内亚 

分 类 号：R382.31[医药卫生—医学寄生虫学]