Sullivan D G, Kim S S, Wilson J J, Stehman-Breen C, Gretch D R
Department of Laboratory Medicine, University of Washington Medical Center, Seattle, WA 98104-2499, USA.
J Virol Methods. 2001 Jul;96(1):5-16. doi: 10.1016/s0166-0934(01)00303-2.
Hepatitis C virus (HCV) infection is very common among chronic hemodialysis patients. In the past, blood transfusion appeared to be the primary risk factor; however evidence of nosocomial HCV transmission in the hemodialysis setting has recently been reported. This report describes a molecular investigation of HCV isolates obtained from a population of 670 patients attending six different Seattle-King County based hemodialysis centers in order to identify potential common source infections. 733 serum specimens were collected from hemodialysis patients in 1992 and 1996, and were tested for HCV antibodies and RNA. Overall, 115 of 670 (17%) patients were positive for HCV RNA, and thus were considered actively infected by HCV. HCV genotype was determined in all cases by restriction fragment length polymorphism, and 93 patients were found to be infected by HCV genotype 1. HCV envelope genes were amplified from the 93 patients with genotype 1 infection, and were studied in further detail by heteroduplex tracking analysis (HTA) using genotype 1a and 1b specific probes derived from the envelope 1 (E1) and envelope 2 (E2) genes. Genetic relatedness between pairs of HCV envelope genes was estimated by calculating the degree of gel shift relative to homoduplex controls. Nucleotide sequencing and phylogenetic analysis was used to confirm genetic relatedness detected by HTA. When HTA was performed using the E1 gene probe, 12 apparently related infections were detected; 10 of 12 (83%) of these infections were confirmed as truly related using the gold standard method of nucleotide sequencing plus phylogenetic analysis. Using an E2 gene probe, 24 infections were apparently related, but only six (25%) were confirmed by sequencing. As a control, 41 envelope genes, which were unrelated by HTA, were sequenced; 0 of 41 (0%) were truly related. In summary, HTA provides a rapid and effective molecular technique for screening HCV genetic relatedness in population-based studies, and should prove valuable in future studies of HCV molecular epidemiology.
丙型肝炎病毒（HCV）感染在慢性血液透析患者中非常常见。过去，输血似乎是主要的危险因素；然而，最近有报道称在血液透析环境中存在医院内HCV传播的证据。本报告描述了对从西雅图 - 金县六个不同血液透析中心的670名患者群体中获得的HCV分离株进行的分子研究，以确定潜在的共同来源感染。1992年和1996年从血液透析患者中收集了733份血清标本，并检测了HCV抗体和RNA。总体而言，670名患者中有115名（17%）HCV RNA呈阳性，因此被认为是HCV的活跃感染者。所有病例均通过限制性片段长度多态性确定HCV基因型，发现93名患者感染了HCV 1型。从93名1型感染患者中扩增出HCV包膜基因，并使用源自包膜1（E1）和包膜2（E2）基因的1a和1b型特异性探针通过异源双链追踪分析（HTA）进行更详细的研究。通过计算相对于同源双链对照的凝胶迁移程度来估计成对HCV包膜基因之间的遗传相关性。核苷酸测序和系统发育分析用于确认HTA检测到的遗传相关性。当使用E1基因探针进行HTA时，检测到12例明显相关的感染；其中12例中的10例（83%）使用核苷酸测序加系统发育分析的金标准方法被确认为真正相关。使用E2基因探针时，24例感染明显相关，但只有6例（25%）通过测序得到确认。作为对照，对41个通过HTA不相关的包膜基因进行了测序；41例中的0例（0%）真正相关。总之，HTA为基于人群的研究中筛选HCV遗传相关性提供了一种快速有效的分子技术，并且在未来的HCV分子流行病学研究中应被证明是有价值的。
