乙型肝炎病毒基因型与病毒复制的关系

检测慢性乙型肝炎患者血清乙型肝炎病毒(HBV)基因型和病毒载量(HBV-DNA定量),探讨它们之间的关系。用微板核酸杂交-ELISA方法对HBV进行基因分型,用PCR荧光定量检测血清HBV-DNA水平,共318例。检测到HBV B型111例(35％);C型128(40％);混合型(B+C,C+D,B+C+D)45例(14％);D型2例;F型2例;未分型30例(9.4％);没有发现A、E型。结果发现,C型和混合型HBV的血清DNA水平高于B型(1.14×107vs2.2×107vsl.60×106拷贝/ml,P<0.05);而混合型HBV的血清DNA水平与C型无差别(P=0.127)。研究提示,我国HBV以B,C两基因型为主,HBV基因型可能是影响HBV复制的重要因素之一。     

乙型肝炎病毒前S1蛋白在血清学诊断乙肝病毒复制中的研究

目的：探讨前S1蛋白（Pre—S1）在血清学诊断乙肝病毒复制中的意义。方法：选择慢性乙型肝炎患者血清250例。分别测定每份患者血清中HBV标志物、前S1蛋白和HBV DNA。结果：HBeAg（＋）组中前S1蛋白阳性率97．2％（70／72）,HBV DNA阳性率100％（72／72）。HBeAg（-）组中前S1蛋白阳性率60．1％（107／178）,HBV DNA阳性率63．5％（113／178）。前S1蛋白和HBV DNA的总符合率为89．6%（224／250）,2者差异无统计学意义（P〉0．05）。HBeAg和HBV DNA的总符合率为54．8％（137／250）,2者差异有统计学意义（P〈0．01）。HBsAg（-）中检出前S1蛋白阳性2例。结论：前S1蛋白比HBeAg更敏感地反映HBV复制情况,且可弥补由于HBsAg基因编码区突变造成的漏诊。     

肝功能与病毒性肝炎免疫及病毒复制的关系

研究肝功能与病毒性肝炎免疫状态、病毒复制的关系.淋巴细胞亚群用碱性磷酸酶法检测.聚合人血清白蛋白受体（PHSA-R）用固相放射免疫法检测.ALT与CD20呈正相关关系;TBil与CD20呈正相关关系,与CD3/CD20呈负相关关系;AST/ALT与CD8呈负相关关系;TBil/ALT与CD8、CD3呈负相关关系.AST/ALT比值与PHSA-R滴度呈正相关.AST/ALT比值升高提示细胞免疫底下及病毒复制活跃,反之提示细胞免疫增高及病毒复制受抑制;ALT体现着免疫对肝细胞膜的损伤,AST体现了病毒复制对肝细胞的破坏.酶胆比值的增高与细胞免疫的相对降低及体液免疫的过度亢进有关.因此,临床研究中应同时参照ALT、AST的值及它们的相对值.     

Clinical and serological events accompanying changes in hepatitis B viral replication: case reports

We measured serum markers of hepatitis B virus replication in two HBsAg-, HBeAg-positive hepatitis B carriers with chronic active hepatitis and cirrhosis. The first of these patients was HBsAg-, HBeAg-, HBV DNA- and HBV DNA polymerase-positive initially and spontaneously lost HBV DNA polymerase and HBV DNA. During the HBeAg-positive, DNA polymerase-negative "window phase", an increase in viral replication, characterized by the reappearance of HBV DNA and HBV DNA polymerase occurred, together with an aggravation of the underlying chronic hepatitis. In the second HBsAg-, HBeAg-positive carrier, spontaneous fluctuations in HBV replication were associated with clinical deterioration. Delta agent and hepatitis A virus superinfection were excluded. These observations suggest that spontaneous low-grade fluctuations of HBV replication accompanied by an increase in the biochemical activity of the underlying chronic hepatitis can be observed in certain HBV carriers.     

Evaluation of viral replication in children with chronic hepatitis B with and without interferon treatment

BACKGROUND: In chronic infection with hepatitis virus B the fact that HBeAg becomes negative does not always mean suppression of viral replication. METHOD: HBV replication was assessed in 74 patients with chronic hepatitis or viral B cirrhosis, in whom diagnosis was made according to clinical, biological, and histological criteria. The patients were divided into two groups: group I (36 patients with interferon- therapy, 3 million U/m 2/ dose, 3 doses/week over a period of 4-6 months) and group II (control group of 38 patients who did not undergo interferon therapy). After a follow up period of 6 years in which patients underwent clinical, biochemical and serologic monitorization, HBV DNA was detected by the hybridization method on solid medium. RESULTS: During evolution the levels of transaminases became normal in both groups. The HBe Ag/Ab seroconversion rate at the end of the interferon therapy was 52.8% and the spontaneous HBe Ag/Ab seroconversion rate was 72.7% in group II after an average evolution of 6 years. HBs Ag/Ab seroconversion was not detected in any patient. Assessment of viral replication by HBV DNA testing at the end of the follow up period showed higher levels as compared to the HBeAg testing (69.4% vs. 25% in group I, 55.2% vs. 7.9% in group II). The absence of viral replication (HBV DNA negative) had similar rates in both groups (30.6% in group I vs. 44.8% in group II, p>0.9) and HBV DNA titers in the two groups were not significantly different at the end of the follow up period. In both groups, HBV DNA titers were significantly higher in patients with positive HBeAg. The concordance between the two viral markers was 100%. CONCLUSION: Because of the fluctuating evolution, long-term follow up and monitorization (including HBV DNA testing) of patients with chronic hepatitis B and of inactive HBsAg carriers are necessary.     

Hepatocyte hepatitis B surface antigen expression in chronic hepatitis B virus carriers in Singapore: Correlation with viral replication and liver pathology

The hepatocyte hepatitis B surface antigen (HBsAg) expression in 149 liver biopsies from 124 chronic hepatitis B virus (HBV) carriers was correlated with serum HBV DNA status and histologic activity. Hepatocyte HBsAg was stained by the peroxidase-antiperoxidase method and serum HBV DNA was determined by dot blot hybridization. Sixty-five biopsies (44%) showed minimal changes (MC), 82 biopsies (55%) showed chronic liver disease (CLD) and 2 biopsies (1%) showed hepatocellular carcinoma. Hepatocyte HBsAg was found in 144 biopsies (97%). It was present in the cytoplasm of 141 specimens (95%) and/or plasma membrane of 48 specimens (32%). Approximately half (45%) of the cytoplasmic HBsAg-positive biopsies showed discrete distribution, while the other half (55%) were grouped. Fifty-five per cent (77 of 141) of cytoplasmic HbsAg-positive biopsies had CLD, while 44% (62 of 141) showed MC. There was no relationship between the presence of cytoplasmic HBsAg or its topographic distribution with disease activity. Membrane HBsAg distribution was similar for both groups of patients (MC vs CLD: 25 of 65 (38%) vs 23 of 82 (28%); P = NS). Serum HBV DNA was detected in 98 patients (66%) and was seen mostly in association with CLD (CLD vs MC: 61% vs 39%, P less than 0.001). It was also detected more often in the sera of patients with membrane HBsAg than in those with cytoplasmic HBsAg staining (41 of 48 (85%) vs 97 of 141 (67%); P less than 0.02). However, discrete distribution of cytoplasmic HBsAg was associated with positive serum HBV DNA when compared with grouped distribution (52 of 63 (83%) vs 43 of 78 vs (55%); P less than 0.005).     

HBsAg expression of liver correlates with histological activities and viral replication in chronic hepatitis B

Introduction. The intrahepatic hepatitis B surface antigens (HBsAg) expression is related to disease progression of chronic hepatitis B. We examined the features of intrahepatic HBsAg expression.Material and methods. A total of 181 patients with e antigen positive chronic hepatitis B were enrolled. Patterns and semi-quantitative measurement of intrahepatic HBsAg expression were analyzed. The association of intra-hepatic hepatitis HBsAg expression with clinical, viral, and histological characteristics was evaluated.Results. Higher necroinflammation grade and greater fibrosis stage accompanied with lower serum HBV DNA and HBsAg levels were observed in patients with type II ground glass hepatocytes and 2+/3+ scales of intrahepatic HBsAg expression. Basal core promoter T1762/A1764 mutations were strongly associated with the pattern of type II ground glass hepatocytes expression (P < 0.001) and higher level of HBsAg expression (9.3 ± 8.0% vs. 4.3 ± 5.0%, P = 0.008). In multivariate analysis, basal core promoter mutations (Odds ratio: 6.356, 95% confidence interval: 1.204 ~ 33.356, P = 0.029) was associated with 2+/3+ scale of HBsAg expression.Conclusion. Both pattern and levels of intrahepatic HBsAg expression were associated with severity of liver disease in e antigen positive chronic hepatitis B. Strong relationship between intrahepatic HBsAg expression and basal core promoter 1762/A1764 mutations indicated that HBsAg expression may be the histological manifestation of hepatitis B virus genomic evolution under host immune surveillance.     

慢性重型肝炎患者中性粒细胞超微结构的改变

目的观察慢性重型肝炎患者中性粒细胞超微结构的改变及其意义。方法采用透射电镜对１５例慢性重型肝炎患者中性粒细胞超微结构进行观察，并以１５例正常人为对照。结果慢性重型肝炎患者中性粒细胞有明显异常，表现为胞浆稀疏，吞噬空泡，细胞器及细胞颗粒明显减少，线粒体水肿、空化、退变，细胞核浓缩等，病情严重及继发严重感染者尤著。结论观察中性粒细胞的超微结构变化对判断慢性重型肝炎患者防御细菌感染能力以及肝衰竭程度有一定的参考价值。     

Correlation of age with the status of hepatitis B virus replication and histological changes in chronic type B hepatitis

The interrelationships of age, hepatitis B virus (HBV)-DNA, hepatitis B e antigen (HBeAg)/antibody (anti-HBe) and hepatic histological changes were examined in a large series of patients with chronic type B hepatitis. A significant inverse relationship between serum levels of HBV-DNA and the age of patients was demonstrated:a high level of HBV-DNA (greater than 500 pg/ml) was found in 100%, 50%, 34%, 10% and 0% of patients in the second, third, fourth, fifth and sixth decade, respectively (P less than 0.001), whereas serum HBV-DNA was negative in 0%, 6%, 8%, 40%, and 50% of cases in the respective decades (P less than 0.001). The correlation of HBV-DNA with age was more pronounced in the HBeAg-negative cases, of whom 81.3% (13/16) of patients below age 40 were positive for HBV-DNA, in contrast to 33.3% (3/9) of those over 40 years (P less than 0.05). In addition, the mean age increased from 25.3 years for HBeAg-positive patients with chronic persistent hepatitis (CPH) and nonspecific reactive histological changes (NSRH), to 32.7 years for HBeAg-positive and 34.9 years for anti-HBe positive patients with chronic active hepatitis and chronic lobular hepatitis (P less than 0.001), and 40.9 years for anti-HBe positive CPH and NSRH (P less than 0.001). These data suggested that age correlated closely with the status of HBV replication and hepatic changes in chronic type B hepatitis.     

Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/anti-HBe system, viral DNA polymerase and HBV-DNA

The detection of HBV-DNA in serum by molecular hybridization is the most sensitive and specific marker or replication and infectivity of hepatitis B virus and currently is proposed as a routine diagnostic technique in the follow-up of HBV-related diseases. Comparing different techniques already described, we found that direct spotting of serum samples on nitrocellulose membranes under vacuum filtration, followed by denaturing and neutralizing washes is more practical, simple, sensible and reproducible. DNA polymerase assay using phosphonoformic acid as specific viral inhibitor has shown 86.8% of concordance with HBV-DNA detection, and so, it is an useful alternative in the follow-up of hepatitis B chronic patients. We found 19.2% HBeAg positive samples with no other markers of viral replication and no anti-HBe positive sample had detectable HBV-DNA. Discordance between the 2 systems have been extensively described, and we confirm this for the first time in our country. Molecular biological techniques are essential to determine the replication status of chronic hepatitis B patients.     

DNA测序与线性反向探针杂交法检测HBV P基因耐药突变的比较

目的建立和优化扩增慢性乙型肝炎患者血清HBV P基因的PCR方法,比较DNA测序法与线性反向探针杂交法检测HBV基因突变情况,并对耐药相关基因突变及其意义进行分析。方法采用巢式PCR法扩增93例慢性乙型肝炎患者血清HBV P基因反转录酶区,对PCR产物进行DNA测序,采用Bioedit6．0．5、Seqman TMⅡ、EditSeq TM和MegAlign软件分析结果;同时随机抽取40份血清进行线性反向探针杂交,比较两者的检测结果,并对11个已知耐药相关突变位点进行分析。结果在93份标本中DNA序列测定法检测到碱基突变113个,其中与LAM相关突变50份（53．78％）,与ADV相关突变6份（6．46％）,与LdT相关突变3份（3．23％）,与ETV相关突变3份（3．23％）;在40份血清中与线性反向探针杂交检测结果一致的突变为87．5％（35140）,氨基酸位点一致为98．0％（431／440）。此外,还检测到V84I、S85C、A181S、L229W、N238T等与耐药相关的突变位点。结论应用线性反向探针杂交检测虽然比较快捷,但其价格昂贵且只能检测已知常见的变异位点;建立在巢式PCR基础上的DNA序列测定法敏感性与线性反向探针杂交法相当,两者检测结果有较高的符合率,但似可检出更多的耐药相关突变。     

Relationship between histology, aminotransferase levels, and viral replication in chronic hepatitis B

To further clarify whether hepatitis B virus is cytopathic, the degree of hepatic histological activity was assessed and compared with levels of replicating virus in serum and liver of 74 untreated patients with chronic hepatitis B. Male homosexuals (n = 35) had significantly greater levels of DNA polymerase (P less than 0.05) and a trend toward higher hepatitis B virus DNA levels than heterosexuals (n = 39). Significantly greater DNA polymerase and hepatitis B virus DNA levels were observed in homosexuals infected with human immunodeficiency virus than in heterosexuals (P less than 0.005 and P less than 0.01, respectively) or human immunodeficiency virus-negative homosexuals (P less than 0.03 for both). Moreover, a trend was observed for higher grades of hepatitis B core antigen staining in the human immunodeficiency virus-infected population than in the human immunodeficiency virus-negative cohort. Hepatitis B virus DNA and DNA polymerase levels in the 74 patients were inversely related to total histological scores, and the degree of portal infiltrate and periportal necrosis bore an inverse relationship to peripheral markers of viral replication in human immunodeficiency virus-negative homosexuals and heterosexuals. Taken together, the data support the view that hepatitis B virus is not cytopathic because the amount of replicating virus does not directly correlate with the degree of histological activity.     

慢性乙型肝炎病毒感染的疾病进展:病毒持续复制的作用

目前全球大约有3.5亿人感染乙型肝炎病毒(HBV),绝大多数感染者在中国.据估计,大约有1.12亿中国人慢性感染HBV[1],约占全世界感染者的33%.     

Treatment of chronic hepatitis B or C in HIV-infected patients with dual viral hepatitis

Dual chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are recognized in 3%-5% of human immunodeficiency virus (HIV)-infected individuals. More severe liver disease is seen in these patients. Viral interference may account for the fact that replication of one virus generally predominates over replication of the other. The impact that treatment of HBV or HCV infection has on this reciprocal inhibition is not well established. No evidence of reactivation of either HBV or HCV was seen when complete suppression of the other predominant virus was achieved with specific therapy in 21 subjects with HIV infection and dual HBV/HCV infections. This information has important pathogenic implications and may influence therapeutic decisions.     

Effect of glycyrrhizin on viral replication and quasispecies in patients with type C chronic hepatitis

The effects of glycyrrihizin on liver-function, amounts of HCV RNA and viral complexity in patients with chronic hepatitis C were examined. The subjects were 27 type C chronic hepatitis patients who did not respond to previous interferon therapy. They were given 60 ml of Stronger Neo-Minophagen C, a preparation of glycyrrhizin combined with glycine and cysteine, three times a week for 16 weeks. The treatment was effective in 20 patients (74.1%), whose serum levels of aminotransferase fell to one half of that before treatment, but was not effective in seven patients (25.9%). No significant differences were noted at the beginning of the therapy in the backgrounds (age, sex, serum levels of aminotransferase, etc.( of the effective and non-effective groups. Also, no significant differences in HCV RNA quantity and viral complexity of HCV RNA were observed between the two groups. The results suggest that glycyrrhizin improves serum ALT levels without changing the amounts of HCV RNA and viral complexity, suggesting that the effect might depend on host factors such as the immune response.     

Sequential changes of serum ferritin levels and their clinical significance in lamivudine-treated patients with chronic viral hepatitis B

AIM: To study the sequential changes of serum ferritin levels in lamivudine-treated patients with chronic viral hepatitis B and the clinical implications. METHODS: Thirty-eight patients with chronic viral hepatitis B were prospectively studied during their treatment with lamivudine. Each patient received 100 mg oral lamivudine daily for 12 mo, and was observed and tested for blood biochemistry and hepatitis B virus (HBV) DNA levels and serum ferritin levels at baseline and at 3, 6 and 12 mo during the treatment. Serum HBV DNA levels were quantitatively determined using fluorescent quantitative polymerase chain reaction (FQ-PCR), and serum ferritin levels were measured by radioimmunoassay. The sequential changes of serum ferdtin levels and their relationships with virological, serological and biochemical responses in the patients were analyzed. RESULTS: All the patients had a baseline HBV DNA level higher than 1&#215;10^7 copies/L as determined by FQ-PCR and positive HBsAg and HBeAg and abnormal ALT levels. At the end of the 12-mo treatment, 19 of the 38(50.00%) patients had undetectable serum HBV DNA levels by FQ-PCR, and 12(31.58%) became negative for serum HSeAg and 10(26.32%) had seroconversion from HBeAg to HBeAb. Nineteen out of the 38(50.00%) patients had biochemically normal ALT levels after 12-mo lamivudine treatment. Sequential determination showed bhat lamivudine treatment significantly reduced ferritin levels in chronic hepatitis B patients. When the patients were divided into different groups according to their posttreatment virological, serological and biochemical responses for analysis of the sequential changes of ferritin levels, it was found bhat the decrease of ferritin levels in HBV DNA-negative group was significantly more obvious than that in HBV DNApositive group at 6 mo during the treatment (P=-0.013). Consecutive comparisons showed that ferritin levels at 3 mo of treatment were obviously decreased as compared with the baseline levels (P&lt;0.05) in HBeAg-negative group, and the decrease of serum ferritin levels in patients with normalized ALT was more significant than that in patients with abnormal ALT at the end of the 12-mo treatment (P=0.048). CONCLUSION: Lamivudine treatment can reduce the serum ferritin levels in chronic viral hepatitis B patients and decreases of ferdtin levels can be more significant in patients exhibiting virological, serological and biochemical responses, indicating that dynamic observation of serum ferritin levels in patients with chronic viral hepatitis B during lamivudine treatment might be helpful for monitoring and predicting patients‘ responses to the therapy.