新疆新源县多房棘球蚴病病例特征及其病原基因多态性分析

Characteristics of alveolar echinococcosis cases and genetic polymorphism of the parasite from Xinyuan County,Xinjiang

目的了解新疆新源县人群多房棘球蚴病(AE)病例特征及其病原基因多态性情况,为制定AE预防控制策略提供参考。方法收集2016—2020年新疆医科大学第一附属医院生物样品库中新源县AE患者的病例信息及其手术切除的肝脏病灶样品,提取肝脏病灶样品基因组DNA,PCR扩增线粒体NADH脱氢酶2(nad2)基因并测序,PCR产物经测序剪接后进行BLAST比对,用MEGA10.0软件采用邻接法构建系统进化树,DnaSPv5软件分析扩增序列的基因型,Network10.0软件制作nad2基因型网络图。结果共收集AE病例28例,其中农区7例,巩乃斯河两岸流域21例;男性17例,女性11例,二者差异无统计学意义(χ^(2)=0.704,P>0.05);民族分布中,汉族12例、哈萨克族11例、维吾尔族3例、蒙古族1例、其他民族1例;职业分布中以农牧民为主,占92.86%(26/28),其他职业仅占7.14%(2/28),不同职业间病例数差异有统计学意义(χ^(2)=3.89,P<0.05);年龄分布中以20~49岁年龄组为主,占78.57%(22/28),其他年龄组占21.43%(6/28),不同年龄组间病例数差异无统计学意义(χ^(2)=0.418,P>0.05)。25份多房棘球蚴肝组织病灶样品PCR扩增出大小约1031 bp的片段,与预期大小一致,另3份未扩增出条带。序列比对结果显示,25条序列分属H1、H2、H3和H4等4个基因型,其中H1为主要基因型(占72.0%,18/25),与哈萨克斯坦(GenBank登录号AB461406)分离株序列一致性为100%。系统进化树结果显示,新源县病例分离株的4个基因型与波兰(GenBank登录号KY205700)和哈萨克斯坦(GenBank登录号AB461406)分离株在同一条分支上,但与法国(GenBank登录号AB461404)、奥地利(GenBank登录号AB461403)、加拿大(GenBank登录号JF751036)、美国阿拉斯加(GenBank登录号MT429275)、美国印第安纳(GenBank登录号AB461409)及中国内蒙古(GenBank登录号AB461411)分离株亲缘关系较远。基因型网络图结果显示,以H1为中心,H2、H3、H4、中国四川,波兰、加拿大及日本分离株呈散射状分布,表明这些分离株均与H1亲缘关系较近,但与法国、奥地利、加拿大,美国阿拉斯加等序列不在同一分支,亲缘关系较远。结论本研究分析的28例AE患者主要为汉族和哈萨克族,农牧民占比较高,AE患者样品共有4个基因型,其中H1属新源县主要基因型。

Objective To understand the characteristics of alveolar echinococcosis(AE)cases and the genetic polymorphism of the parasites in Xinyuan County,Xinjiang,to provide reference information for formulating developing prevention and control strategies for AE.Methods During 2016—2020,information on AE patients and some surgically resected liver lesion samples from Xinyuan County in the biological sample bank of the First Affiliated Hospital of Xinjiang Medical University were collected.Genomic DNA of the liver lesion samples was extracted,and the mitochondrial NADH dehydrogenase 2(nad2)gene was amplified by PCR and sequenced.The PCR products were sequenced and spliced for Blast comparison.Mega10.0 software was used to construct a phylogenetic tree by the neighbor-joining method,DnaSPv5 software was used to analyze the genotype of amplified sequence,and Network10.0software was used to make nad2 genotype network map.Results A total of 28 AE cases were collected,including 7in rural areas and 21 in Kunes River Basin,among which 60.71%(17/28)were male,and 39.29%(11/28)were female(χ^(2)=0.704,P<0.05).Ethnic distribution was mainly Han(42.86%,12/28)and Kazak(39.29%,11/28),and the highest infection rate among Mongolians was 11.17%.There was a significant difference in the infection rate among different ethnic groups(χ^(2)=61.08,P<0.01).Farmers and herdsmen were the dominant occupations,accounting for 92.86%(26/28),while only 7.14%(2/28)were of other occupations.There was no significant difference in infection rate among different age groups(χ^(2)=0.418,P>0.05).A expected,25 samples were amplified by PCR with a fragment size of1031 bp.Sequence alignment results showed that there were four genotypes:H1,H2,H3 and H4,among which H1 was the main genotype and the sequence consistency was 100% with Kazakhstan(GenBank accession no.AB461406).Phylogenetic tree results showed that the four genotypes in Xinyuan county isolates were in the same branch as the Poland(GenBank accession no.KY205700)and Kazakhstan(GenBank accession no.AB461406),but more distantly related to France(GenBank Accession no.AB461404),Austria(GenBank accession no.AB461403),Canada(GenBank accession no.JF751036),Alaska(GenBank accession no MT429275),Indiana,USA(GenBank accession no.AB461409)and Inner Mongolia,China(GenBank accession no.AB461411).According to the results of centering on the H1,the genotype network diagram showed H2,H3,H4,Sichuan of China,Poland,Canada and Japan were scattered distributed which indicated that the isolates with H1 has a very close relationship,but it is not in the same clade with France,Austria,Canada,Alaska,USA isolates,and had a distant relationship.Conclusion The majority of AE patients were Han and Kazakian farmers and herdsmen,and the Mongolians had the highest infection rate.There were 4 genotypes in AE patients,among which H1 was the main genotype in Xinyuan County.