丙型肝炎病毒基因分型与肝病程度关系的研究

目的探索丙型肝炎病毒（HCV）基因型在辽宁省大连地区的分布及流行的优势型,并探讨HCV基因型与肝病程度的关系。方法采用型特异性引物逆转录巢式PCR法和限制性片段长度多态性分析（RFLP）法对85例抗-HCV阳性的慢性丙型肝炎、丙肝肝硬化、丙肝相关肝癌病人进行HCV的基因分型。结果85份抗HCV阳性标本中,HCVRNA检出率为61例,占71．76％（61／85）。在85份抗HCV阳性血清中,仅检出了61例1b和2a型感染者,其中1b型32例,占52％（32／61）;2a型29例,占48％（29／61）。结论大连地区基因型为1b型和2a型两种型,符合中国丙肝病毒基因分型,大连地区1b型和2a型基本相等。未发现1b和2a基因型因肝病严重程度而有所不同。     

丙型肝炎病毒窗口期感染核酸阳性血清检测和基因型别分析

目的分析丙型肝炎病毒（HCV）早期感染血液样品核酸（RNA）检测的意义，并对检出的核酸阳性血清进行基因分型。方法对8649份自然供血员及566份HCV可疑感染者血清进行核酸（TMA RNA试剂）检测，检测出的HCV RNA阳性样品利用基因分型芯片进行分型。同时利用HCV核心总抗原（HCVc Ag）和游离抗原（HCV free Ag）试剂以及抗体（Anti—HCV）试剂进行检测。并对检测数据进行比较分析。结果在所有血液样品中发现1份HCV抗体、HCVc Ag、HCV free Ag全部阴性但RNA阳性的样品，基因型别为1b型。在8649份自然供血员血清中筛出1份HCVc Ag、HCV free Ag以及HCVRNA阳性但抗体阴性血清，基因型别为1b型。在566份HCV可疑感染者血清中，筛出1份HCV free Ag和核心抗体同时阳性血清，基因型别为1b型。TMA试剂筛出5份RNA阳性血清但微粒子抗体试剂检测阴性，TMA试剂筛出6份RNA阳性的血清而酶标抗体试剂检测阴性。这6份血清，利用傲拓HCV总抗原试剂检测，其中5份HCV总抗原阳性。在澳大利亚国家血液中心进行复核，其中5份RNA阳性。在566份血清中，TMA试剂检出RNA阳性495份，阳性率87．4％。对RNA阳性血清进行型别分析，以1b、2a型为主，分别为46．5％和27％。结论由于检出了HCV RNA单独阳性的窗口期样品，认为利用HCV抗体试剂对血液进行筛查的同时，使用HCV RNA试剂做必要的补充实验，可以减少因窗口期造成的漏检。型别分析认为，窗口期感染样品基因型别也以1b，2a型为主。     

江苏省宜兴地区丙型肝炎病毒基因分型研究

目的了解江苏省宜兴地区丙型肝炎病毒（HCV）基因型分布特征和病毒变异情况。方法对宜兴市人民医院收治的158例HCV抗体阳性患者血清进行HCV RNA检测，对阳性标本进行Simmonds分型，采用5’非编码区（5’NCR）1、2、3、1b型特异性引物进行PCR扩增，并对1b型和2型各1株的PCR产物测序验证。分析不同性别、临床类型的丙型肝炎患者基因型分布差异。结果158份血清中，有95份为HCV RNA阳性，其中1b型80份（84．2％），2型5份（5．3％），1b／2型的混合感染5份（5．3％），不能分型的5份（5．3％），1b型的序列与GenBank J-4株的同源性为99．2％，2型与GenBank J-6株的同源性为97．7％。男性和女性在基因型总体分布上存在差异（P〈0．05），并且在单一型感染和混合型感染的分布上差异亦有统计学意义，不同临床类型的丙型肝炎中HCV基因型分布未显示差异。结论江苏省宜兴地区HCV以1b型为主，男女患者HCV基因型分布差异有统计学意义，而不同临床类型的HCV基因型分布无显著差异，基本反映了本地区HCV感染的特点。     

广东地区丙型肝炎患者几种常见HCV基因型的快速检测

目的快速准确检测广东地区广泛分布的主要HCV基因型，为HCV的临床诊断和治疗提供依据。方法收集53例HCV—RNA阳性血清标本，选取丙型肝炎病毒（HCV）基因组保守序列为扩增靶序列，设计通用引物和特异型别探针（1，2，3，6型），通过逆转录将RNA逆转录为cDNA后，利用通用引物对cDNA进行PCR扩增，将扩增产物利用反向点杂交（RDB）技术进行基因分型。结果HCVRNA阳性血清53例，共检出标本52例，4种基因型所占的比例为1b型32例占60．38％，2a型4例占7．55％，3b型8例占15．09％，6a型8例占15．09％。结论所测患者的HCV基因型分布以1b为主，6a和3b次之，2a相对较低；反向点杂交技术能对其进行快速分型，符合率高达98．11％。     

蒙古国人丙型肝炎患者HCV RNA检测及基因型的研究

目的通过检测丙型肝炎病毒基因分型,探讨蒙古国抗-HCV阳性患者血清中HCV基因型流行株特征。方法采用基因芯片法对蒙古国183例抗-HCV阳性患者血清标本进行基因型检测,同时检测相关病毒学及肝功能生化指标,分析基因型分布特点和临床意义。结果 183例抗-HCV阳性患者中HCV RNA阳性134例(73.2%),其中基因1b型68例(50.7%),2a型59例(44.0%),并有少量的1a、3a、1b+2a和其它3例混合型。结论抗-HCV阳性并非HCV感染直接标志,蒙古国感染HCV患者的基因型以1b和2a型为主,比例基本相当;基因型在性别上人群分布差异无统计学意义。     

丙型肝炎患者泪液中丙型肝炎RNA的检测和基因分型

目的：研究泪液是否与丙型肝炎（HCV）传播有关。方法：采用套式－PCR法检测慢性丙型肝炎血液和泪液中的HCV RNA并进行了基因分型。结果：HCV可在泪液中存在；基因分型以基因型1b型检测到的频率最高。结论：加强眼科医疗设备的消毒，预防医源性感染，很有必要。     

广西肝癌高、低发区HCV基因型的比较研究

目的：探讨HCV基因型在广西原发性肝癌高发区与低发区的致癌作用。方法：采用PCR微板核酸杂交结合ELISA检测显示技术，检测广西肝癌高发区的原发性肝癌病人52例及低发区内的原发性肝癌病人120例的HCV RNA及其基因型。结果：肝癌高发区组与低发区组HCV RNA阳性率分别为7．7％（4／52）和5．0％（6／120），经统计学处理无显著性差异（P＞0．05）；肝癌高发区组与低发区组HCV RNA基因型均以Ⅱ型为主，阳性率分别为7．7％（4／52）和3．3％（4／120），两者无显著性差异（P＞0．05）。结论：广西肝癌高发区与低发区肝癌病人感染的HCV均以Ⅱ型为主，且广西肝癌的发生似乎与HCV的感染关系不大。     

皖南地区丙型肝炎病毒基因型研究

目的 探讨安徽南方地区慢性丙型肝炎患者血清ALT水平、HCV RNA定量检测和HCV基因分型的相关性.方法 用速率法和荧光定量PCR法,检测141例安徽南方地区慢性丙型肝炎患者血清ALT水平和HCVRNA定量结果,应用RT-PCR和型特异性引物法对其中50例标本进行基因分型.结果 141例患者的血清ALT与HCV RNA两指标间的相关性采用Spearman等级相关分析显示,rs=0.213,P=0.011<0.05,差异有统计学意义,存在正相关;在50例标本中,HCV基因分型Ⅰ型有45例(95.7%),Ⅱ型2例(4.3%),未分型3例.结论 安徽南方地区丙型肝炎病毒感染以Ⅰ型为主;ALT、HCV RNA和基因型是可靠且重要的治疗反应指标.     

不同感染途径丙型肝炎患者HCV基因分型

目的探讨江苏省丙型肝炎病毒(HCV)基因型的分布及其与感染途径、肝功能等因素的关系。方法按照Simmonds分型方法对505例不同感染途径的丙型肝炎患者进行HCV基因型分型检测并分析年龄、性别、肝功能以及感染途径与HCV基因型的关系。结果 505例HCV RNA阳性标本中,1a型8例(1.6%),1b型348例(68.9%),2型24例(4.8%),3型40例(7.9%),1b/2混合型67例(13.3%),1b/3混合型13例(2.6%),2/3混合型4例(0.8%),1a/1b混合型1例(0.2%);不同性别、年龄人群HCV基因型分布差异均无统计学意义(均P>0.05);不同HCV基因型人群丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)水平差异均无统计学意义(均P>0.05);不同感染途径人群HCV基因型分布差异有统计学意义(χ2=73.348,P<0.001)。结论江苏省丙型肝炎患者HCV基因型以1b型为主,1a型HCV在中国大陆地区流行程度有扩大趋势,HCV基因型与其感染途径有关。     

宜兴地区丙型肝炎病毒基因型及变异情况分析

目的了解江苏宜兴地区丙型肝炎病毒（HCV）基因型分布特点和变异情况对疾病的影响。方法对宜兴市人民医院收治的166例HCV抗体阳性患者血清进行HCVRNA检测，采用5′非编码区（5′NCR）1，2，3，1b型特异性引物。对阳性标本进行Simmonds分型。选取未经干扰素治疗的43份慢性丙型肝炎患者的HCVRNA阳性血清，采用RT—PCR扩增HCV包膜区（E2），单链构象多态性聚合酶链反应法（SSCP）获得HCV准种条带数，准种数〉2为复杂准种，比较不同肝病活动度患者的准种复杂情况。结果166份血清中，有102份为HCVRNA阳性。其中1b型86份占84．3％，2型6份占5．9％，1b／2型的混合感染5份占4．9％，不能分型的5份占4．9％。男性和女性在基因型总体分布差异有统计学意义。在单一型感染和混合型感染的分布差异亦有统计学意义，而在不同年龄、不同临床类型的肝炎中分布差异无统计学意义。HCV包膜区准种复杂与否和丙氨酸转氨酶（ALT）的正常与否具有相关性。结论江苏宜兴地区的HCV感染主要为1b型，病毒的变异程度与ALT成正相关。     

HCV genotyping by combination of the Cobas Amplicor HCV 2.0 test and the reverse hybridization Versant HCV Genotype Assay

Genotyping of hepatitis C virus (HCV) is of relevance to scheduling the treatment of patients with chronic hepatitis C (VHC), making their prognosis and monitoring the treatment efficacy. A set of 62 sera testing HCV RNA positive in Cobas Amplicor HCV 2.0 test (CA) were genotyped using Versant HCV Genotype Assay (LiPA) Bayer, i.e. the reverse hybridization method, with the CA amplified product being directly used in the assay. Fifty-six out of 57 samples reactive in reverse hybridization (92%) were genotyped. One sample showed a profile differing from any genotype, five samples were not reactive and one sample was not tested within this study design. Two out of five non-reactive sera and one non-tested serum could be genotyped by nested PCR based reverse hybridization. It can be concluded that the CA product resulting from one-step HCV RNA amplification is suitable for use in genotyping by reverse hybridization. The CA product based genotyping procedure is easier to perform, less time-consuming and less costly. The nested PCR based procedure could be used for typing of sera with lower HCV concentrations nontypeable with the combination of CA and Versant HCV Genotype Assay. Forty-eight selected samples were typed not only by reverse hybridization but also by a serological kit Murex HCV Serotyping 1-6 Assay (Abbot Murex). Thirty-seven (77%) of these sera, including all of three sera negative in reverse hybridization, appeared typeable by this kit. Although less sensitive, serotyping may be of relevance to typing of sera with low HCV levels or not containing detectable viral NA which are nontypeable by reverse hybridization. Thirty-three sera appeared genotypeable by both of the methods tested with the results being in good agreement. In two cases only the serotyping method revealed one more type of virus (mixed genotype) compared to the reverse hybridization.     

中国部分地区丙型肝炎病毒核心区基因分型的研究

〔目的〕了解中国部分地区丙型肝炎病毒的基因分型。〔方法〕采用HCV核心区的型特异性引物PCR检测并以基因序列系统进化树法对中国部分地区人群的HCV进行基因分型。〔结果〕在来自中国福建、山东、台湾等9个省、市的HCV-RNA阳性标本中共发现4种HCV基因型和6种HCV基因亚型,各型名称为:1b、2a、3b、6a、6n和6。其中1b型占29.7%(19/64);2a型占4.7%(3/64);3b型占28.1%(18/64);6a型占34.4%(22/64);6n型占1.6%(1/64);6型占1.6%(1/64)。另从1名巴基斯坦藉入境人员血清标本中检出HCV1a型。〔结论〕HCV核心区的型特异性引物PCR和基因序列系统进化树法可较好地对HCV进行基因分型,中国与相邻的东南亚国家或地区一样存在着HCV基因多样性。     

Hepatitis C virus genotypes in southern Taiwan: prevalence and clinical implications

The role of hepatitis C virus (HCV) genotypes in the development of hepatocellular carcinoma (HCC) is still controversial. To determine the distribution and clinical implications of HCV genotypes in southern Taiwan, we analysed 418 patients with chronic HCV infections. HCV genotypes were determined using an HCV Line Probe Assay. The predominant HCV genotype was 1b (45.5%), followed by 2a/2c (30.9%) and 2b (6.9%). The prevalence of genotype 1b in HCC patients (60.3%) was significantly higher than in those with liver cirrhosis (38.7%) and chronic hepatitis (38.7%) (P=0.003 and P<0.001, respectively). Patients with chronic HCV 2a/2c infection had higher alanine aminotransferase (ALT) levels than those with chronic HCV 1b infection (P<0.001). Univariate analysis revealed that disease severity was significantly correlated with older age, genotype 1b, lower ALT levels and lower viral load. Based on multiple logistic regression analysis, after adjusting for age and serum HCV RNA levels, HCV 1b infection was still a significant risk factor for HCC. In conclusion, the predominant genotypes in southern Taiwan were 1b and 2a/2c, and disease severity was associated with genotype 1b.     

宁波地区丙型肝炎病毒基因分型检测

目的:通过检测丙型肝炎病毒基因分型,探讨宁波地区HCV感染基因型流行株特征。方法:本文共收集231例HCV感染者的血清,采用基因芯片技术进行HCV基因分型。结果:231例丙型肝炎病毒基因分型1b型162例,占70.12%;2a型25例,占10.82%;1b+2a混合型15例,占6.49%;并有少数的3b3、a6、型和其它混合型,均占<5%。结论:宁波地区HCV感染基因型以lb型为主、其次为2a型。基因芯片法检测丙型肝炎病毒基因分型,具有微量、多位点、准确性好、灵敏度高、特异性强等特点。     

输血后丙型肝炎病人10年前瞻性研究

目的研究26例丙肝病人的演变过程.方法采用前瞻性的研究方法,以26例已跟踪10年以上的丙肝病人作为研究对象,对所采集到的输血前及输血后血清,进行ALT和抗HCV检测及HCV RNA检测.结果对该26例输血后丙肝病人跟踪时间平均128个月,输血后抗HCV最早于第15天被检测到,最晚出现于第80天,平均窗口期为42d,至第11年时其抗HCV阳性率仍维持在约70%.大多数病人在输血后的30d内其ALT即出现异常,随后其ALT的变化大致可分为3种类型:短期异常后转为正常并维持至今;ALT维持较长时间异常;ALT呈波浪式反复异常.笔者所跟踪的26例输血后丙肝病人中,24例病人的ALT或只有一个异常峰,但维持时间较长(平均16个月),或呈现波浪式多次的高异常,其抗HCV在第¨年时仍绝大多数维持阳性,说明丙肝病毒在人体内引起的免疫反应较弱而持久.2例病人在ALT异常5个月后转为正常,随后其抗HCV及HCV RNA亦转为阴性,至今已超过10年未发现肝炎症状,HCV感染人体以后能引起较快、较强的免疫反应,该被感染者又能通过免疫系统将病毒清除掉.结论大多数丙型肝炎病人转为慢性化,少数丙型肝炎病人则存在自愈.     

Changes in the distribution of hepatitis C virus genotypes and their association with shifts in transmission routes in Henan,China

Aim

Changes in the prevalence of hepatitis C virus (HCV) genotypes could have considerable impacts on prognosis and therapy of hepatitis C. We aimed to investigate the current distribution of HCV genotypes in Henan province, China, and to assess factors associated with the sort of genotype and evolution of the distribution pattern of HCV genotypes.

Subject and methods

A total of 362 chronic hepatitis C (CHC) patients were enrolled retrospectively in the study. HCV genotypes were determined by DNA sequencing and phylogenetic analysis of the core and/or NS5B regions. Univariate or multivariate analysis was performed for factors associated with HCV genotypes distribution.

Results

Among the 362 samples, the most prevalent genotype was 1b (58.6%), followed by 2a (15.7%), 6a (13.8%), 3a (7.5%), 3b (2.8%), 1a (0.8%), and 2b (0.8%). Multivariate logistic regression models showed that transmission routes and age were independently associated with HCV genotypes. Transfusion of blood or blood product was independently associated with genotype 1b (OR:2.22; 95%CI 1.22–4.04, P = 0.009), while IDU was independently associated with genotype 6a (OR:3.18; 95%CI 1.34–7.51, P = 0.009). Genotype 1b was more frequent in older age patients (OR:1.02; 95%CI 1.00–1.04, P = 0.023), and genotype 6a was more common in younger age individuals (OR:0.97; 95%CI 0.94–0.99, P = 0.029). Changes in HCV genotype distribution were present depending on the year of infection. The prevalence of the HCV genotype 1b decreased over time, while the prevalence of the HCV genotype 6a increased over time.

Conclusions

The most prevalent genotype was still 1b among CHC patients in the Henan province, China. The prevalence of genotype 1b has decreased overtime, and that of genotype 6a has increased in younger patients. The change is attributed to a shift in HCV transmission routes. This study provided further evidence for the prevention and treatment of hepatitis C in future.

     

Examination of hepatitis c virus antibody seropositive blood donors and their relatives

INTRODUCTION: 239 anti-HCV seropositive blood donors (132 male, 107 female, age: 19-61, mean: 40.59 y.) and 174 family members of them (74 male, 100 female, age: 4-65, mean: 23.67 y.) were studied for chronic hepatitis C virus infection and chronic liver disease. Detailed virus serology, ultrasonography, and 6 months follow-up and--in patients with HCV RNA--liver biopsy were made. RESULTS: HCV RNA was determined in 165 patients. 70% of them were HCV RNA positive. The ALT level was normal in 95 cases (57%), and lower, than twice of the normal was in 34 cases (20%) among them. Liver biopsy was made in 79 patients; chronic C hepatitis was proven in 75 cases (steatosis in 3 cases, alcoholic liver disease in 1 case was observed). Inflammatory activity was minimal (HAI < 3) in 17, mild (HAI: 3-6) in 41, moderate (HAI: 7-9) in 7, and severe (HAI > 9) in 10 cases. There was no correlation between the serum ALT levels and the severity of the histological activity of chronic C hepatitis. Authors stress the importance of the fact, that 2 patients had normal ALT and 5 patients ALT levels were lower, than the twice of the normal of the 17 patients with significant inflammatory activity (HAI < 6). Chronic C hepatitis need for antiviral therapy was occurred in 45% of patients who known themselves previously healthy. CONCLUSIONS: The necessity of the systematic examination of anti-HCV seropositive patients and of the importance of the liver biopsy in patients with HCV RNA positivity is stressed. 3 anti-HCV seropositive cases of 174 family members of the blood donors were observed, but none of them was HCV RNA positive. It seems to be, family members of the HCV infected patients have no increased risk for HCV infection.     

丙型肝炎病毒逆向点杂交基因分型方法的建立及初步应用

目的 利用逆向点杂交技术建立丙型肝炎病毒(HCV)基因分型新方法。方法在HCV5’端非编码区(5’UTR)设计引物与分型探针，将活性氨基标记的分型探针依次固定在尼龙膜上，制成HCV基因分型逆向点杂交检测膜条。分离纯化血清中的HCV RNA，经逆转录反应和生物素标记引物聚合酶链反应(PCR)巢式扩增后，将扩增产物与检测膜条杂交，过氧化物酶和四甲基联苯胺显色，判断基因分型结果。最后，通过与基因测序结果比较确定新方法的有效性。从佛山地区丙型肝炎患者中随机抽取60份经荧光定量PCR方法对HCV RNA进行过定量分析的血清，用新建方法进行HCV基因分型检测。结果新建HCV逆向点杂交基因分型方法可对所有60份HCV RNA拷贝数在10。～10。／ml之间的抽检血清进行基因分型，发现1b型50例，占83．3％；1a型2例，占3．3％；2a型2例，占3．3％；1a、1b混合型5例，占8．0％；1b、2a混合型1例，占1．7％；未发现2b、3a和3b型。新方法基因分型结果与测序分型结果一致。结论PCR一逆向点杂交技术可以准确有效和简便经济地进行HCV基因分型．适用于临床检测与流行病学研究。在佛山地区人群中感染的HCV以1b型为主。     

肝病患者肝炎病毒感染状况的血清学调查

应用第二代抗ＨＣＶ试剂对我国不同地区肝病患者血清抗ＨＣＶ进行了检测，同时调查了ＨＢＶ和 ＨＡＶ感染状况。 ２０１６例肝病患者血清中抗 ＨＣＶ检出率为 １２． ２５％，ＨＢＶｍ 检出率为 ７４． ５５％，抗 ＨＡＶ－ＩｇＭ检出率为２９．３１％。各型肝病患者抗ＨＣＶ检出情况分别是：急性肝炎６２／７５２（８．２４％）；慢迁肝４８／５４１（８．８７％）；慢活肝９３／５２３（１７．７８％），慢性重症肝炎５／３２（１５．６３％）；肝硬化３４／１４３（２３．７８％）；肝癌５／２５（２０． ００％）。对不同年龄组肝病患者抗 ＨＣＶ检出率的比较表明，抗 ＨＣＶ检出率随患者年龄增加而增高。受血史及其他经血液暴露史与 ＨＣＶ感染密切相关，但仍有近半数肝病患者 ＨＣＶ感染途径不明。各型肝病患者中仅４７／２０１６（２．１３％）为单纯ＨＣＶ感染，其他ＨＣＶ感染者则为重叠感染。不同地区肝病患者血清抗ＨＣＶ检出率间差异较大（２．１５％～３０．９５％），北方地区较高，对其影响因素进行了探讨。