Evaluation of liver tissue by polymerase chain reaction for hepatitis B virus in patients with negative viremia

AIM: To assess the clinical significance of Hepatitis B virus (HBV) DNA localization in the liver tissue of patients with positive HBsAg and negative viremia. METHODS: HBV virological parameters of 33 HBsAg positive chronic hepatitis patients, including seromarkers and HBV DNA amplification in both sera and liver biopsies, were evaluated. RESULTS: Ten patients had negative viremia and positive HBV DNA in their liver biopsies. Most of them had HBeAg-negative/HBeAb-positive chronic hepatitis. Their liver biochemical and histopathological profiles were different from the viremic patients. Their disease pattern was designated as "hepatitis B in situ". CONCLUSION: Hepatitis B in situ is a consequential entity which can be missed in clinical practice. It is a new clinical pattern of chronic HBV infection that considers HBV in liver biopsy and adds a new indication for antiviral therapy.     

Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay.

We compared the sensitivity of the polymerase chain-reaction (PCR) assay to that of slot-blot hybridization for detecting hepatitis B virus (HBV) DNA in the serum of 31 patients with chronic hepatitis. Of 14 chronic hepatitis patients positive for both HBV surface and HBV e antigens, 9 were positive for HBV DNA by slot-blot hybridization and all 14 by PCR. Also, of 9 patients positive for HBV surface antigen and antibody against HBV e antigen, 2 were positive for HBV DNA by slot-blot analysis and 8 by PCR. Finally, in 8 patients positive for HBV DNA by slot-blot hybridization, but 4 were positive by PCR. We find that analysis by the PCR technique provides a greater than 10(4)-fold increase in sensitivity over the slot-blot hybridization assay. This result represents an important breakthrough in sensitivity because it is now possible to detect as few as three HBV DNA genomes per sample of serum.     

Evolution of hepatitis B virus polymerase gene mutations in hepatitis B e antigen-negative patients receiving lamivudine therapy

Lamivudine has been shown to be effective in patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, but its long-term efficacy and the rate of resistant mutations in patients with HBeAg-negative chronic hepatitis B is less clear. Twenty-nine patients with HBeAg-negative chronic hepatitis B, who have received lamivudine for at least 1 year were studied to determine the antiviral response, the rate and pattern of lamivudine-resistant mutations, and the effect of lamivudine-resistant mutations on HBeAg status. The mean duration of treatment was 21 +/- 7 months. Before treatment, core promoter variant was detected in 16 (55%) patients and precore stop codon variant in 18 (62%) patients. Serum hepatitis B virus (HBV) DNA was detected by solution hybridization assay in 62%, 4%, and 24% and by polymerase chain reaction (PCR) assay in 100%, 31%, and 40% at months 0, 6, and 24, respectively. The cumulative rates of detection of lamivudine-resistant mutations after 1 and 2 years of treatment were 10% and 56%, respectively. In addition to the duration of treatment, core promoter mutation was associated with the selection of lamivudine-resistant mutants. Three patients with lamivudine-resistant mutations had reversion of the precore stop codon mutation; in 2 patients this was accompanied by the reappearance of HBeAg. We found that lamivudine-resistant mutants were detected at similar rates in patients with HBeAg-negative as in patients with HBeAg-positive chronic hepatitis B. Additional changes in other parts of the HBV genome may restore the replication fitness of lamivudine-resistant mutants.     

Serum cytokine profile in patients with hepatitis B e antigen-negative chronic active hepatitis B and inactive hepatitis B virus carriers

An insufficient cellular immune response seems to be critical for the immunopathogenesis of chronic hepatitis B virus infection.We have previously demonstrated no differences of T-lymphocyte subsets in blood between inactive hepatitis B s antigen(HBsAg) carriers and patients with HBeAg-negative chronic active hepatitis B.This study investigated the peripheral blood cytokine profile in patients with HBeAg-negative chronic active hepatitis B infection(Group A,n = 21) and inactive HBsAg carriers(Group B,n = 13).Serum cytokines [interferon(IFN)-γ,tumor necrosis factor-α,interleukin(IL)-1b,IL-4,IL-12,IL-10,IL-2,IL-5,IL-8] were analyzed by using flow cytometry.Patients with chronic active disease presented with significantly decreased levels of IFN-γ and IL-10 compared to inac-tive carriers(P = 0.048 and P = 0.008,respectively).In HBeAg-negative chronic active hepatitis B patients,a significant negative correlation of IFN-γ levels with serum hepatitis B viral load was noted(P = 0.021).In conclusion,patients with HBeAg-negative chronic active hepatitis B and HBsAg inactive carriers display a different cytokine profile.Decreased Th1 response observed in patients with chronic active hepatitis B could be implicated in the persistence of virus replication and ongoing progression of liver disease.     

应用竞争性荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的:建立竞争性荧光定量聚合酶链反应(CFQ-PCR),并探讨CFQ-PCR在乙型肝炎病毒(HBV)临床检测中的意义.方法:根据HBV病毒adr亚型基因组序列合成一对HBV特异的引物,和一条特异的TaqMan探针;根据上述引物序列,采用分子克隆技术制备内对照DNA;再根据内对照序列合成一条内对照DNA特异的与上述TaqMan探针不同标记的TaqMan探针;将适量的内对照DNA加入到PCR反应体系中,使其与HBV靶序列共扩增.结果:在30μL CFQ-PCR反应体系中,加入约20拷贝内对照DNA能够稳定地获得共扩增曲线;经琼脂糖凝胶电泳分析,加入约100-500拷贝内对照DNA能够有效地获得共扩增产物条带信号;在210个临床HBsAg阳性血清标本的CFQ-PCR扩增中识别出8个未能有效扩增的标本,60份HBsAg阴性血清标本中识别出2个内对照未能有效扩增的标本,后经DNA纯化处理,上述全部标本的内对照均获得阳性扩增结果,其中有7个HBsAg阳性血清标本获得HBV DNA扩增阳性结果.结论:CFQ-PCR能够有效地提示临床标本HBV DNA体外扩增时由于扩增失败导致的假阴性,适合临床推广应用.     

Hepatitis B virus DNA prediction rules for hepatitis B e antigen-negative chronic hepatitis B

After hepatitis B e antigen (HBeAg) seroconversion, hepatitis B may become inactive or progress to HBeAg-negative hepatitis with persistent or intermittent alanine aminotransferase (ALT) elevation. The aim of this study was to prospectively identify factors predictive of the clinical course in HBeAg-negative chronic hepatitis B (CHB). Patients were stratified by ALT and HBeAg status and followed every 3 months for up to 5 years. Kaplan-Meier and Cox regression analysis using the change from normal ALT to elevated ALT as endpoints were performed to determine factors associated with ALT elevation/normalization. Seventy-four HBeAg-negative and 32 HBeAg-positive patients were prospectively evaluated. For HBeAg-negative patients, hepatitis B virus (HBV) DNA was predictive of future ALT. Only 1 patient with normal ALT and an HBV DNA value lower than 10,000 copies/mL developed an elevated ALT within the subsequent year, whereas 67% with an HBV DNA value greater than 100,000 copies/mL had a rise in ALT above normal within 1 year. Patients with a previous history of ALT elevation and longer follow-up at all levels of HBV DNA were more likely to experience ALT elevations. For HBeAg-negative patients with elevated ALT and all HBeAg-positive patients, HBV DNA did not predict future ALT. Other viral and host factors were not predictive of future ALT. CONCLUSION: HBeAg-negative CHB has a fluctuating course. HBV DNA values lower than 10,000 copies/mL predict persistently normal ALT for at least 1 year. Patients with HBV DNA values between 10,000 and 100,000 copies/mL can safely be followed at 6 monthly intervals, whereas HBV DNA values greater than 100,000 copies/mL are highly predictive of future ALT elevation and should prompt regular follow-up.     

Profile of hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND: Although chronic hepatitis B occurs in hepatitis B e antigen (HBeAg)-negative patients, its prevalence and clinical significance are not known. AIM: To determine the prevalence and profile of HBeAg-negative chronic hepatitis B virus (HBV) infection. METHODS: A retrospective analysis of 363 consecutive patients (mean age 36 y; 288 men) with chronic HBV infection was performed. All patients were HBsAg-positive. Tests for liver profile, HBeAg and anti-HBe antibody were performed in all patients. Serum HBV DNA was tested using branched DNA assay in 245 patients. The patients were classified into three groups: no cirrhosis with normal ALT levels, no cirrhosis with elevated ALT levels, and clinical or histological evidence of cirrhosis. RESULTS: Of 363 patients, 141 (39%) were HBeAg-positive and 222 (61%) HBeAg-negative. Of HBeAg-negative patients, 120 (54%) had normal ALT, 45 (20%) had elevated ALT and 57 (26%) had evidence of cirrhosis; corresponding figures in the HBeAg-positive patients were 40 (28%), 66 (47%) and 35 (25%). HBV DNA was positive in 53 of 131 (40%) HBeAg-negative patients tested; of these 53 patients, 9 (17%) had normal ALT, 20 (38%) had elevated ALT and 24 (45%) had cirrhosis. Thus, 72% of HBeAg-positive and 46% of HBeAg-negative patients had elevated ALT and/or cirrhosis. Among the latter group, 83% of HBV DNA-positive patients had elevated ALT and/or cirrhosis. Overall, 18% of HBsAg-positive patients had HBeAg-negative, HBV DNA-positive liver disease. CONCLUSION: HBeAg-negative chronic hepatitis B is not an uncommon and benign entity and chronic liver disease develops in a significant proportion of such patients.     

Treatment of hepatitis B e antigen-negative patients

Opinion statement Hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) occurs at the late phase in hepatitis B virus (HBV) infection’s natural history. The disease is characterized by progressive liver damage due to variants with mutations in the precore/core promoter region that reduce or abolish HBeAg expression. Chronic HBeAg-negative disease’s prognosis is poor, with only rare incidences of spontaneous remission. Recent studies in Europe, Asia, and the United States all have reported an increased prevalence of HBeAg-negative and a decreased prevalence of HBeAg-positive chronic hepatitis; this may be related to increased awareness, decrease in new HBV infections, and aging of existing carriers. The end point of therapy for HBeAg-negative CHB patients is difficult to assess. In most studies, HBV DNA suppression and normalization of serum alanine aminotransaminase levels have been used to indicate therapeutic response. Six drugs currently are licensed for the treatment of CHB infection. These are the immunomodulatory agents (conventional interferon-α-2b and pegylated interferon-α-2a) and the nucleoside/nucleotide analogues (lamivudine, adefovir dipivoxil, entecavir, and telbivudine). Sustained treatment response rates generally are poor due to the high probability of relapse, particularly following nucleoside/nucleotide analogue therapy. As not all patients can tolerate or will respond to interferon-based therapy, maintenance therapy with nucleoside/nucleotide therapy is the alternative. However, this latter approach can lead to development of viral resistance and long-term safety concerns.     

Detection of hepatitis B virus infection in hepatitis B surface antigen-negative hemodialysis patients by monoclonal radioimmunoassays

We studied 375 chronic hemodialysis patients for evidence of hepatitis B virus infection using first- and second-generation monoclonal radioimmunoassays. These assays employ high-affinity monoclonal antibodies produced against antigenic determinants that reside on hepatitis B surface antigen. Such assays have a lower limit of detection for hepatitis B surface antigen-associated determinants in serum of approximately 55 and 15 pg/ml, respectively. We found that 14 of 375 chronic hemodialysis patients were positive for hepatitis B surface antigen by both polyclonal and monoclonal radioimmunoassay. However, an additional 17, some of whom had chronic hepatitis and hepatocellular carcinoma, were identified as harboring hepatitis B virus infection only by the monoclonal radioimmunoassays. Thus the monoclonal radioimmunoassays improved the hepatitis B virus detection rate by 120% (3.7% vs. 8.3%). More importantly, 6 of the 17 monoclonal radioimmunoassay-reactive patients had no serologic evidence of recent or past hepatitis B virus exposure as shown by the absence of antibodies to the hepatitis B core and surface antigens in the blood. We conclude that there are hemodialysis patients with hepatitis B virus infection undetectable by conventional polyclonal radioimmunoassays.     

Precore and core promoter mutations of hepatitis B virus and hepatitis B e antigen-negative chronic hepatitis B in Korea

BACKGROUND/AIMS: The aims of this study were to determine the frequency of precore/core promoter mutations and hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (e-CHB) in Korea. METHODS: Patients with chronic hepatitis B virus (HBV) infection were tested for HBeAg, anti-HBe, liver profile and HBV-DNA by a branched DNA (bDNA) assay. Serum HBV-DNA was amplified by a polymerase chain reaction and the precore/core promoter sequence was determined. RESULTS: Among the 413 consecutive HBeAg-negative patients, 19.6% were bDNA-positive. Evidence of liver disease was found in 90.1% of bDNA-positive and 41.7% of bDNA-negative patients. Overall, 17.7% of HBeAg-negative patients had e-CHB. Precore mutation (A1896) was detected in 93.7% of HBeAg-negative bDNA-positive and 93.9% of HBeAg-negative bDNA-negative patients. In 59 HBeAg-positive patients, 78% had wild-type and 22% had a mixture of wild-type and A1896 mutant. Core promoter TA mutation was detected in 89.9% of HBeAg-negative bDNA-positive patients, 89.8% of HBeAg-negative bDNA-negative patients, and 74.6% of HBeAg-positive patients. No correlation was found between the presence of precore/core promoter mutations and HBV-DNA levels or disease severity. CONCLUSIONS: In Korean patients infected with HBV genotype C, precore mutation occurred almost invariably along with HBeAg seroconversion and core promoter TA mutation was frequent irrespective of viral replication levels or disease severity.     

Hepatitis B e antigen-negative chronic hepatitis B in Hong Kong

Hepatitis B e antigen-negative chronic hepatitis B (e-CHB) has been reported in Asia but its prevalence and clinical significance have not been determined. The aims of this study were to determine the prevalence of e-CHB in Hong Kong and the frequency of precore and core promoter mutations in these patients. A cross-sectional study was performed in 350 consecutive Chinese patients (230 men and 120 women; mean age +/-SD, 42 +/- 13 years) with chronic hepatitis B virus infection. A total of 243 (69%) patients were hepatitis B e antigen (HBeAg)-negative of whom 15% had clinical cirrhosis. In the remaining 85% of patients, 63% had normal and 22% had elevated transaminases. Serum hepatitis B virus (HBV) DNA was detectable using branched DNA assay in 46% of HBeAg-negative patients with clinical cirrhosis/elevated transaminases. Forty-five percent of the patients with e-CHB had the precore stop codon mutation, and an additional 41% had core promoter changes. There was no correlation between the presence of precore/core promoter mutations and liver disease or HBV-DNA levels. Overall, 17% of HBeAg-negative patients were viremic and had evidence of chronic liver disease (e-CHB) with mean HBV-DNA levels comparable with that in HBeAg-positive patients. In summary, we found that e-CHB may be present in up to 17% of HBeAg-negative patients seen in a tertiary referral center in Hong Kong. e-CHB may be a heterogeneous condition and is not invariably associated with the precore HBV mutant. Population studies are needed to determine the true prevalence of e-CHB in Asia and to assess its natural course and response to treatment.     

拉米夫定治疗e抗原阴性慢性乙型肝炎的早期预测指标分析

目的 评价拉米夫定(LAM)治疗e抗原阴性慢性乙型肝炎患者治疗前基线ALT、HBsAg、HBV DNA水平以及治疗4周和12周时HBV DNA<1×10~3拷贝/ml对其治疗104周时抗HBV疗效的预测价值. 方法 127例成年e抗原阴性慢性乙型肝炎患者均接受LAM 100 mg/d治疗,且均完成≥104周的治疗.治疗期间定期复查肝功能、HBV标志物(HBsAg、抗-HBs,HBeAg、抗-HBe、抗-HBc)及HBV DNA水平.分别比较和分析不同基线ALT、HBsAg、HBV DNA水平及治疗4周和12周时不同HBV DNA水平与治疗104周时疗效的关系.数据采用x~2检验及多元逐步Logistic回归分析.结果 基线ALT<5×正常值上限(ULN)和ALT≥5×ULN两组患者,治疗104周血清HBV DNA<1×10~3拷贝/ml的比例分别为50.0%和86.8%(P<0.01).基线HBsAg<2000 COI和HBsAg≥2000 COI两组患者,治疗104周时HBsAg<500 COI的比例分别为19.1%和17.5%(P>0.05);HBsAg/抗-HBS血清学转换率分别为2.1%和2.5%(P>0.05),血清HBV DNA<1×10~3拷贝/ml的比例分别为61.7%和67.5%(P>0.05).基线HBV DNA<1×10~6拷贝/ml和HBV DNA≥1×10~6拷贝/ml两组患者,至治疗4周和12周时HBV DNA<1×10~3拷贝/ml的比例差异均有统计学意义(P值均<0.01),但至治疗104周时HBV DNA<1×10~3拷贝/ml的比例分别为62.7%和67.1%,差异无统计学意义(P>0.05).治疗4周时HBVDNA<1×10~3拷贝/ml和HBV DNA≥1×10~3拷贝/ml两组患者,104周时HBV DNA<1×10~3拷贝/ml的比例分别为70.7%和60.9%(P>0.05);治疗12周时HBV DNA<1×10~3拷贝/ml和HBV DNA≥1×10~3拷贝/ml两组患者,104周时HBV DNA<1×10~3拷贝/ml的比例分别为78.8%和38.1%(P<0.01).结论 基线ALT≥5×ULN和治疗12周HBV DNA<1×10~3拷贝/ml的e抗原阴性慢性乙型肝炎患者用LAM继续治疗至104周时可以取得较好的病毒学应答.治疗前不同基线HBsAg水平对治疗104周时HBsAg的水平、HBsAg/抗-HBs血清学转换率和HBV载量的预测价值不大;基线HBV DNA水平对104周时是否获得病毒学应答的预测价值也不大.     

The determination of serum hepatitis B virus DNA by polymerase chain reaction in hepatitis B patients treated with alpha-interferon]

To clarify the status of HBV in serum of chronic hepatitis B (CHB) patients who were treated with alpha-interferon, we determined the serum HBV DNA before and after treatment in 15 CHB patients with polymerase chain reaction (PCR). Before treatment all the 15 patients were HBsAg and HBeAg positive. HBV DNA was also positive with dot-blot hybridization (DB), PCR, ethidium bromide (PCR-EB) and PCR Southern-blot hybridization (PCR-SBH). HBeAb was negative in all the 15 patients. 12 patients has the determination repeated 2 to 39 weeks after treatment, 7 out of the 12 patients became HBeAg negative and HBV DNA also negative with DB and PCR-EB. However, in 5 of the 7 patients HBV DNA was still positive with PCR-SBH. Seroconversion of HBeAb from negative to positive occurred in 4 of the 12, but HBV DNA of the 4 patients remained positive with PCR-SBH. After an interval of one and half year or more following treatment 12 patients repeated the examination, only 5 of the 12 patients became seronegative for HBeAg and HBV DNA with DB and PCR-EB, but in 4 of the 5 HBV DNA was positive with PCR-SBH. Two of the 5 were seropositive for HBeAb and HBV DNA with PCR-SBH. The mechanism of residual viraemia after alpha-interferon treatment in CHB patients is uncertain.     

Relationship of clinical and virological factors with hepatitis activity in hepatitis B e antigen-negative chronic hepatitis B virus-infected patients

To investigate the factors associated with active disease among hepatitis B surface antigen (HBsAg) positive/hepatitis B e antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection we studied chronic HBV infected patients who had undetectable HBeAg at the first visit. HBV DNA was determined by the cross-linking assay (NAXCOR) and polymerase chain reaction (PCR). Mutations in the core promoter and precore regions and viral genotypes were studied. Clinical outcome of these patients were followed and categorized as: (i) relapse (ALT > 200 IU/L or three times the previous levels); (ii) active hepatitis (elevated ALT < 200 IU/L with concomitant detectable HBV DNA); or (iii) remission. A total of eighty-five patients were followed up for 5.5 ± 1.0 years. At first visit, 31 (36.5%) patients had elevated ALT levels, 12 (14.1%) had measurable HBV DNA by the cross-linking assay and 26 (30.6%) by PCR. Sixteen (18.8%) patients had hepatitis relapse, 13 (15.3%) had active hepatitis, and 56 (65.9%) remained in remission. Core promoter and precore stop codon mutants were found in 27 and 12 patients, respectively. Eleven and 20 had genotype B and C HBV, respectively. Initial elevated ALT and detectable HBV DNA were associated with active liver disease. Patient demographics, viral mutants or genotypes failed to predict disease activity. Hence, serum ALT and HBV DNA levels offer the best prediction of natural course of HBeAg-negative chronic HBV infection.