肝炎基因诊断芯片检测肝炎患者血清及肝组织中HBVDNA、HCVRNA的临床研究

肝炎基因芯片是根据肝炎病毒的特征基因片段设计探针序列，将探针以微阵列形式排列在经过特殊处理的玻片上，从待测血液、组织标本中抽取微量肝炎病原体DNA（RNA），与探针杂交，用特有的荧光波长扫描芯片，通过计算机软件进行分析诊断。我们用病毒性肝炎基因诊断芯片，分别双盲检测40份乙型肝炎患者和健康人、丙型肝炎患者血清，99份肝炎后肝硬化肝组织蜡块标本，15份慢性乙型肝炎患者肝组织活检标本；同时用荧光微粒子定量法测定血清中乙型肝炎病毒标志物HBcAg，PCR法检测血清中HBVDNA、HCVRNA．用免疫组化法、原位分子杂交法分别检测肝组织HBcAg、HBVDNA。结果报道如下。     

丁型肝炎病人肝组织Fas/FasL和HDAg表达

为研究丁型肝炎病人肝组织Ｆａｓ／ＦａｓＬ和ＨＤＡｇ表达及意义,采用免疫组化单,双标记技术,检测了４８例丁型型肝炎病人肝组织Ｆａｓ,ＦａｓＬ和ＨＤＡｇ表达,且以５４例乙型肝炎作对照,结果发现,Ｆａｓ以及肝细胞浆表达为主,ＨＤＡｇ以肝细胞核和胞浆表达为主,二抗原表达及分布呈一致性。     

HDAg和bcl—2、bax在丁型肝炎病人肝组织中的表达及相关性

目的探讨 bcl- 2、bax在丁型肝炎发病机理中的作用。方法采用免疫组化双标记染色和连续切片技术 ,检测 77例丁型肝炎病人肝组织 HDAg、bcl- 2和 bax表达 ,以 6 7例乙型肝炎作对照。结果 HDAg以肝细胞核表达为主 ,bcl- 2以肝细胞浆及核膜表达为主 ,bax以肝细胞浆表达为主 ,胞核也可呈阳性表达。bax和 HDAg表达及分布有相关性 ,在各临床类型肝炎中的表达强度有显著性差异 (P<0 .0 1)。结论 HDV感染可诱导肝细胞表达 bax,增强 bax促细胞凋亡作用 ,bcl- 2 / bax途径介导的细胞凋亡在丁型肝炎发病机理中可能有一定作用     

肝病患者血清cccDNA与HBV复制及肝组织损伤的关系

细胞外乙型肝炎病毒（HBV）DNA是一种松弛环状的部分双链DNA分子，而在感染的肝细胞核内则是另一种不同形式的病毒DNA，即共价闭合环状DNA（covalently closed circular DNA，cccDNA）。近来研究发现血清中存在cccDNA；另有研究证实外周血淋巴细胞同样存在HBVDNA与cccDNA。因此血清cccDNA的来源可能不只是肝组织，其临床意义尚不十分清楚。我们检测了228例肝病患者血清cccDNA。现将有关资料报告于下。     

慢性乙肝患者肝炎活动度、纤维化程度与肝组织HBcAg关系

目的 探讨慢性乙肝患者肝炎活动度、纤维化程度与肝组织HBcAg表达的相关性.方法 选择慢性乙型肝炎126例,并行肝穿刺取肝组织,用HE染色及免疫组化染色,光镜观察肝炎活动度及纤维化程度与肝组织HBcAg各亚型表达的关系.结果 随慢性乙肝患者肝炎活动度及纤维化程度的增加,各亚型HBcAg阳性率差异有统计学意义(P≤0.05).结论 慢性乙肝患者肝炎活动度及纤维化与肝组织HBcAg免疫组化阳性亚型有关.     

分子杂交法研究肝炎病人血清和肝组织中输血传播病毒

目的 对各型肝炎病人血清和肝组织中输血传播病毒（TTV）核酸检测分析,探讨病毒的致病性。方法 以地高辛为标记物制备TTV DNA探针,斑点杂交法、原位杂交法分别检测血清中及肝组织中TTV DNA。结果 检测103例血清,TTV总阳性率为25．24％（26／103）;甲－戊型肝炎组检出率21．81％（12／55）、非甲－非庚型患者检出率47．37％（9／19）,显著高于正常对照组15％（3／20）。临床可见TTV的单独感染和重叠感染;出现急性、慢性甚至重度肝损伤。12 肝组织可见TTV阳性,阳性颗粒主要见于肝细胞核内。结论 从血清和肝组织证实了TTV的存在,提示这种病毒可能是导致肝脏炎症的一种新病原。     

基因优化对基因工程丁型肝炎小抗原蛋白表达和纯化的影响

目的 探讨基因优化对基因工程丁型肝炎(丁肝)小抗原蛋白表达和纯化的影响.方法 依据GenBank上的丁肝小抗原原始基因,参照大肠埃希菌优势密码子谱进行优化;再分别将优化前后的两组基因进行常规原核表达,用SDS-PAGE电泳比较目的蛋白表达量及优势表达方式;进一步以柱层析进行纯化,再以Image Lab软件分析SDS-PAGE电泳条带,比较两组目的蛋白的纯度.结果 SDS-PAGE显示,两组表达的目的蛋白均与预期相对分子质量大小一致,优化基因组的蛋白表达量高于原始基因组,且更倾向于可溶性表达;经柱层析纯化后,前者的目的蛋白纯度也明显高于后者,可达96.3％.结论 基因优化可提高基因工程丁肝小抗原蛋白表达量,增加可溶性蛋白的比例;且表达产物更易纯化至较高纯度以供诊断使用.     

中国西南地区丁型肝炎的流行状况与临床特征分析

目的:探讨丁型肝炎患者的流行状况及临床特征。方法:筛查2010年1月1日至2020年12月31日陆军军医大学西南医院感染病研究所HBsAg阳性病例832 144例,进行HDV-Ag和(或)HDV-IgG检测,共13 585例。最终收集157例丁型肝炎患者的资料,年龄22~85(53±13)岁,其中男性122例,女性35...     

Persistent delta antigenaemia in chronic delta hepatitis and its relation with human immunodeficiency virus infection.

The prevalence of persistent hepatitis delta (HD) antigenaemia and associated factors in patients with chronic infection with the hepatitis delta virus (HDV) were investigated. Among 157 consecutive patients known to be carriers of hepatitis B surface antigen (HBsAg), 36 (23%) had one serum marker of HDV infection (anti-HD and/or HDAg). Nine of the patients with an HDV marker were HDAg positive, including three who were anti-HD negative. A follow-up over a mean period of 13 months showed that five of five patients had a persistent HD antigenaemia. This serological profile was associated with the presence of antibody to the human immunodeficiency virus (anti-HIV) (P < 0.01), serum HIV antigen (HIVAg) (P < 0.2), and the female sex (P < 0.05). Persistent HD antigenaemia could be the consequence of the suppression of T cell cytotoxic activity against hepatocytes expressing HDAg, a lower humoral response, and/or hormonal factors.     

Factors associated with viremia and elevated transaminase levels in asymptomatic hepatitis D virus-infected risk groups

To detect hepatitis D virus (HDV) RNA in asymptomatic HDV-infected risk groups, Northern blot hybridization was carried out using a strand-specific riboprobe. Univariate and multivariate analyses were carried out to evaluate factors associated with HDV viremia and elevated transaminase levels in these subjects. Two (15%) of 13 antibody to HDV (anti-HDV) -positive intravenous drug addicts, 15 (33%) of 45 anti-HDV positive prostitutes, and 6 (40%) of 15 anti-HDV positive brothel goers had detectable serum HDV RNA. Older age (>31 years old) was negatively associated with HDV RNA (P < 0.04), while hepatitis B e antigen (HBeAg) was positively associated with it (P < 0.002) in univariate analysis. Only HBeAg was still significant in multivariate analysis (P < .05). Of the 76 asymptomatic anti-HDV positive case, 28 (37%) had mildly elevated serum ALT levels and only 5 (7%) had ALT levels more than twice normal (>80 U/L). HBeAg (P < .05) and HDV RNA (P < .02) were two factors associated with ALT elevation in univariate analysis, and HDV RNA was the only significant factor in multivariate analysis (P < .005). In summary, active replication of HBV seemed to be of help for the assembly of HDV and viremia. However, active replication of HDV was associated mostly with mildly elevated ALT levels in these subjects. These cases may represent a particular group in the disease spectrum of HDV infection. © 1994 Wiiey-Liss, Inc.     

Prevalence and clinical significance of hepatitis D virus co-infection in patients with chronic hepatitis B in Korea

Hepatitis D virus (HDV) infection can cause severe acute and chronic liver disease in patients infected with hepatitis B virus (HBV). Despite the significant decline in the global HDV infection, it remains a major health concern in some countries. This study aimed to investigate the prevalence and clinical features of HDV co-infection in patients with chronic HBV infection in Korea, where HBV infection is endemic. Nine hundred forty patients [median age, 48 (18-94) years; men, 64.5%] infected chronically with HBV were enrolled consecutively. All patients who were positive for hepatitis B surface antigen (HBsAg) for at least 6 months and were tested for anti-HDV. A portion of the HDV delta antigen was amplified, sequenced, and subjected to molecular and phylogenetic analysis using sera from the patients who were anti-HDV positive. Clinical features and virologic markers were evaluated. Inactive HBsAg carriers, chronic hepatitis B, cirrhosis and hepatocellular carcinoma accounted for 29.5%, 44.7%, 17.9%, and 8.0%, respectively. Only three patients were positive for anti-HDV, corresponding to a 0.32% positive rate. All patients who were positive for anti-HDV were inactive HBsAg carriers. HDV RNA could be amplified by PCR from the sera of two patients. Phylogenetic analysis showed that both carried HDV genotype 1. In conclusion, the prevalence of HDV infection is very low (0.32%) in Korea. All HDVs were genotype 1 and detected in inactive HBsAg carriers. Therefore, HDV co-infection may not have a significant clinical impact in Korean patients with chronic HBV infection.     

Differentiation of core gene products of the hepatitis B virus in infected liver tissue using monoclonal antibodies

Hepatitis B virus (HBV) core gene translational products were localised previously in the cytoplasm and/or in the nuclei of infected cells. We investigated in naturally infected human hepatocytes whether this variation in the subcellular expression is due to differences in the presence of assembled core particles and other core gene derived proteins, the expression of HBeAg and the processing of liver tissue. By immunostaining of liver specimens infected with HBeAg-positive and HBeAg-minus variants of HBV, using monoclonal antibodies specific for assembled core particles and for various epitopes on denatured core protein, it was shown that virtually all immunoreactive core gene products are assembled into core particles. The latter are present both in the nuclei and in the cytoplasm of hepatocytes, independent of the infecting virus strain. A marked reduction or absence of immunoreactivity, observed with some monoclonal antibodies, was shown to result from nucleotide sequence variations within or close to the corresponding epitope. These results demonstrate that immunoreactive products, derived from the HBV core gene, in the nuclei and cytoplasm of human hepatocytes represent assembled core particles and that monoclonal antibodies with known recognition sites can reveal region-specific core gene variation of the infecting HBV population. J. Med. Virol. 53:127–138, 1997. © 1997 Wiley-Liss, Inc.     

Loss of hepatitis B surface antigen from the serum of patients with chronic hepatitis treated with lamivudine

Although loss of hepatitis B e antigen (HBeAg) from the serum is sought by treatment with lamivudine, clearance of hepatitis B surface antigen (HBsAg) is the eventual goal of any antiviral therapy. In a single hepatology center in the Metropolitan Tokyo, 486 patients with chronic hepatitis B were followed up for longer than 3 years after they started treatment with lamivudine. HBsAg disappeared from the serum in 17 (3.5%). Age >or=50 years and low HBsAg levels (hemagglutination titer or=50 years at the start of lamivudine was the only factor predicting the loss of HBsAg (hazard ratio: 2.96 [95% confidence interval: 1.14-7.68], P = 0.028). By the method of Kaplan-Meier performed on the 486 patients, the loss of HBsAg was estimated to occur in 3% and 13% of patients, respectively, who had received lamivudine therapy for 5 and 10 years. These results indicate that loss of HBsAg occurs in a minority (3.5%) of patients with chronic hepatitis B who receive lamivudine therapy and more frequently in those with lower HBsAg titers and older ages at the start of treatment.     

Hepatitis D virus antibody in HBsAg-positive and HBsAg-negative substance abusers with chronic liver disease

The hepatitis D virus (HDV; previously called the "delta agent") is a defective organism which can replicate only in the presence of the hepatitis B virus (HBV). We tested the serum of 95 substance abusers, all of whom had sufficient evidence of chronic liver disease to warrant a liver biopsy, for hepatitis D virus antibody (anti-HDV). Anti-HDV was detected in five of eight hepatitis B surface antigen (HBsAg)-positive patients and 12 of 87 (14%) HBsAg-negative patients. Antibody to the hepatitis B core antigen (anti-HBc) was the sole hepatitis B marker in eight of the 12 (67%) anti-HDV-positive, HBsAg-negative patients but in only 14 of 75 (19%) anti-HDV-negative, HBsAg-negative patients (P less than 0.005). None of the anti-HDV-positive, HBsAg-negative patients had detectable IgM anti-HBc in the serum or hepatitis D antigen in liver tissue, and they had similar clinical features and liver biopsy diagnoses to HBsAg-negative patients without anti-HDV. We conclude that anti-HDV in HBsAg-negative substance abusers reflects infection with HDV and HBV in the distant past and does not indicate more severe liver disease than that seen in HBsAg-negative patients without anti-HDV.     

Hepatitis D virus infection among intravenous drug abusers in Taiwan: analysis of risk factors and liver function tests

To investigate the prevalence of hepatitis D virus (HDV) and hepatitis B virus (HBV) infection among intravenous drug abusers in Taiwan, a total of 761 male prisoners, including 680 intravenous drug abusers, were studied for serological markers of HBV and HDV. Questionnaires were distributed to evaluate the risk factors for HDV infection and also to estimate the strength of association among HDV infection and the risk factors. HBV infection was common, and the positive rates of HBV markers between intravenous drug abusers and non-drug abusers were not statistically different. However, the positive rate of the antibody to HDV was significantly higher among intravenous drug abusers than among non-drug abusers (21.3% vs. 8.6%). Of 131 chronic HBV carriers with intravenous drug abuse, 119 (91%) were anti-HD positive. Using multiple logistic regression models, we found that the most important risk factor for HDV infection was hepatitis B surface antigen (HBsAg) carriage, and intravenous drug addiction the next. A matched case-control study also was conducted to compare liver function tests among both anti-HD- and HBsAg-positive group anti-HD-negative, and HBs-AG-positive group as well as those with neither positive. Statistically significant difference in liver function tests was not found. It is concluded that the HBsAg carriers with intravenous drug abuse in Taiwan are commonly HDV infected with and that the infection does not seem to affect the liver as assessed by liver function tests.     

慢性乙型肝炎患者血清HBsAg水平与肝脏炎症和纤维化程度相关性研究

目的 探讨慢性乙型肝炎患者HBsAg水平与肝脏炎症和纤维化的关系.方法 301例确诊为慢性乙型肝炎的患者纳入研究,同时进行肝脏活检术检查和生物化学、铁蛋白、血清HBsAg、HBV DNA定量检测.Spearman等级相关分析血清HBsAg水平与肝脏炎症分级和纤维化分期间的关系;logistic回归分析法分析相关指标的诊断意义,受试者工作曲线法评价血清HBsAg水平预测肝脏炎症分级和纤维化分期的准确性.结果 体质量指数、年龄、性别、基因型和家族史对CHB患者肝脏炎症和纤维化无明显影响(P＜0.05).AST、ALT随着炎症、纤维化进展而升高,差异具有统计学意义(x2=71.193、96.344、47.847、63.981;P =0.000、0.000、0.000、0.000).纤维化为S4时,HBV DNA明显下降(x2=33.322,P=0.000).HBsAg水平随着炎症和纤维化程度加重而逐步下降(x2=68.173,15.719;P =0.000,0.000).HBsAg水平预测≤G3和≤S3的曲线下面积分别为0.732和0.793,特异度分别为0.778、0.891,灵敏度依次为0.685、0.633.结论 中国CHB患者HBsAg水平随着肝脏炎症分级和纤维化分期程度加重而逐渐下降.血清HBsAg水平对CHB患者肝脏炎症≤G3和纤维化≤S3的预测具有较高的特异性,且对纤维化的预测优于炎症.     

Peroxidase-anti-peroxidase detection of hepatitis B surface and core antigen in liver biopsy specimens from patients with chronic type B hepatitis

Liver biopsy specimens from 58 American patients with chronic type B hepatitis were investigated for the presence and distribution of the hepatitis B core (HBcAg) and surface (HBsAg) antigens by peroxidase-anti-peroxidase techniques. HBsAg was detected in 43 (77%) and HBcAg in 52 (90%) patients. HBcAg was present in 50 of 51 (98%) patients with hepatitis B e antigen (HBeAg) but in only two of seven (29%) of patients with antibody to HBeAg (anti-HBe). There was no correlation between severity of hepatitis or height of aminotransferase activities and the amount of HBsAg or HBcAg in hepatocytes but there was a positive correlation between amount of HBcAg and height of HBV-DNA and DNA polymerase activity in serum. Follow-up liver biopsies, taken 1 to 3 yr later, were available from 39 patients. HBcAg remained detectable in 25 of 26 patients with persistence of HBeAg but disappeared in 12 patients who had lost HBeAg. In nine patients, HBcAg was cytoplasmic as well as nuclear in distribution. Seven of these patients had an intense lobular hepatitis with marked elevations in aminotransferase activities. These findings indicate that the amount of HBcAg in liver correlates with the amount of serum hepatitis B virus as quantified by serum levels of DNA polymerase and HBV-DNA. The amount of nuclear HBcAg does not correlate with the severity of the liver disease, but the presence of cytoplasmic HBcAg usually reflects an active and severe ongoing hepatitis.