中国血液净化

中国血液净化 ›› 2013, Vol. 12 ›› Issue (08): 425-431.doi: 10.3969/j.issn.1671-4091.2013.08.00

• 临床研究 • 上一篇    下一篇

维持性血液透析患者丙型肝炎病毒感染基因型及同源性分子流行病学研究

陈嘉,栾韶东,何永成   

  1. 深圳市第二人民医院
  • 收稿日期:2013-03-20 修回日期:2013-06-01 出版日期:2013-08-12 发布日期:2013-08-12
  • 通讯作者: 何永成 E-mail:heyongcheng@medmail.com.cn
  • 基金资助:

    广东省科技计划项目立项资助

Genotype Distribution and Homological Analysis of Hepatitis C Virus in Maintenance Hemodialysis Patients by molecular epidemiology

  • Received:2013-03-20 Revised:2013-06-01 Online:2013-08-12 Published:2013-08-12

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摘要： 目 的 研究深圳市第二人民医院血透中心维持性血液透析（MHD）患者丙型肝炎病毒（HCV）感染的基因型及同源性，结合临床流行病学资料，为血液透析中心HCV感染的防治提供依据。方 法 荧光定量聚合酶链反应（PCR）法测定抗-HCV阳性的MHD患者的HCV-RNA定量，对HCV-RNA定量≥1×103 IU/mL的MHD患者，通过巢氏反转录（RT）-PCR对5’UTR及NS5B区进行扩增，扩增产物通过Sequence Scanner v1.0导出峰图，Muscle进行序列比对，MEGA 5.1构建进化树以确定基因分型，bootstrap 1000检验进化树可靠性，Blat进行序列同源性分析。结 果 183例MHD患者中，抗-HCV阳性13例，抗-HCV阳性率为7.1%。HCV-RNA定量≥1×103 IU/mL者9例中，1b型7例，6a型2例。1b型中，PC1与PC2间的遗传距离为0.000，PBC17与PC2间的遗传距离为0.008， PC29与PBC31间的遗传距离为0.035， PC32与PBC34间的遗传距离为0.102；6a型中，PC11与PC20间的遗传距离为0.068。PC1与PC2的同源性为100%，PC2与PBC17的同源性为99.23%，PC29与PBC31同源性为96.63%， PC32与PBC34的同源性为90.64%，PC11与PC20同源性为93.63%。9例患者的平均感染时间为（67.4±26.3）月，平均透析时间为（81.5±38.1）月，4例（PC2、PC11、PC29和PBC31）为输入性HCV感染者，另5例（PC1、PBC17、PC20、PC32和PC34）是在本中心接受MHD后的8～77月间发现HCV感染。在本中心感染的5例患者中，2例（PC32和PC34）有输血史及更换透析单位，3例（PC1、PBC17、PC20）无输血、更换透析单位、肾移植等感染HCV危险因素，根据流行病学调查，具有同源性的病例PC1和PBC17与感染源（输入性感染者）PC2患者有多次同班或邻班、相邻机位、同 一护士操作的血透记录。结 论 深圳市第二人民医院血液透析中心的抗-HCV阳性率为7.1%。1b型为深圳市第二人民医院血液透析中心HCV感染的最常见基因型，其次为6a型。其中3例1b型患者（样本PC1、PC2和PBC17）HCV感染为同一流行株，同源性达99%以上，结合流行病学资料确定1例患者（PC2）为输入性传染源，与之同源感染的PC1和PBC17患者的可能传播途径为，因当时未对HCV阳性和阴性患者分区治疗、同一护士同时操作HCV阳性和阴性患者、护士未规范手卫生、护士操作不同患者未更换手套、共用肝素等不规范操作。近3年，本中心将抗-HCV阴性及HCV感染、HBV感染者实行分房间（分区）、专用透析机治疗、专人管理阳性患者、严格实施卫生部《血液净化标准化操作规范》管理，未再发新的感染患者，表明上述措施可有效防止血液透析中心内的交叉感染。本研究为血液透析中心HCV医源性感染的防治提供了重要的科学依据。

关键词: 丙型肝炎病毒, 基因型, 血液透析, 系统进化树

Abstract: Objective To determine the genotype distribution and homology of hepatitis C virus (HCV) by the way of phylogenetic tree in maintenance hemodialysis (MHD) patients of Shenzhen second people’s hospital. To provide scientific evidence for preventing HCV infection in hemodialysis unit with molecular epidemiology. Methods The anti-HCV positive dialysis patients were screened for HCV RNA in sera.When the viral load levels were 1×103 IU/mL and more, fragment of HCV 5’UTR and NS5B was severally amplified by nest RT—PCR. The cloned sequences were analyzed using Sequence Scanner v1.0. The genotype of each sample was determined by comparing its nucleotide sequence with those of HCV prototype representing different genotype obtained from sequence database. The DNA alignments were performed with Muscle program.The phylogenetic tree was constructed with MEGA 5.1 software using neighbor-joining method and Kimura-two parameter and its reliability was assessed by bootstrap 1000. Homologies of sequences were analyzed by Blat software. Results Of the 183 MHD patients enrolled in this study, anti-HCV positive results presented in 13 patients, and HCV infection rate was 7.1%. There were 9 patients with HCV RAN ≥1×103 IU/mL, including 7 with genotype 1b and 2 with genotype 6a. In genotype 1b samples, genetic distance between PC1 and PC2, between PC2 and PBC17, between PC29 and PBC31, between PC32 and PC34 , was 0.000, 0.008 , 0.035 and 0.102 , respectively. In genotype 6a samples, genetic distance between PC11 and PC20 was 0.068. Homology between PC1 and PC2, between PC2 and PBC 17, between PC29 and PBC31, between PC32 and PC34 ,between PC11 and PC20 was 100%, 99.23%, 96.63%, 90.64% and 93.63%, respectively. The nine patients infected HCV for（67.4±26.3） months and dialysis lasted for （81.5±38.1） months. Four patients (sample PC2, PC11, PC29 and PBC31) were imported, another five patients (sample PC1, PBC17, PC20, PC32 and PC34) were found HCV infection during 8 to 77 months of hemodialysis. In the five new infected patients, two had been blood transfusion and changed hemodialysis unit history. Another three patients had denied risk factors of HCV infection, such as blood transfusion, chang hemodialysis unit and renal transplant, except hemodialysis. According to the results of clinical epidemiological investigation, among the three homologous patients (PC1, PC2 and PBC17), two new infected patients (PC1 and PBC17) had dialyzed in the same or neighboring round and been operated by the same nurse with the imported patients PC2. Conclusions Anti-HCV positive rate is 7.1% in this hemodialysis unit of Shenzhen second people’s hospital. Genotype 1b is the most common in MHD patients with HCV in the second people’s hospital of Shenzhen, followed by Genotype 6a. Three patients (sample PC1, PC2 and PBC17) have the same HCV epidemic strain（homology over 99%）and PC2 is the imported infection source that is testified to epidemiological data. Among the three homologous patients, the underlying transmission route is attributed not to partition space, segregate positive and negative patients with given management , replace gloves between different patients, stress importance hand hygiene of nurse or abide by standard operating procedure. In our hemodialysis unit, there are no new. In recently three years, there are no fresh HCV infections in our hemodialysis unit , which prove that segregate positive (including HCV and HBV )and negative patients with given management and space, use special hemodialysis machine and strict implement blood purification standard operating procedure(SOP) can effectively control cross infection in hospital. This study provides important scientific evidence for preventing nosocomial infection of HCV in hemodialysis unit.

Key words: Hepatitis C virus, Genotype, hemodialysis, Polygenetic tree