Hepatitis B virus precore mutations and HBeAg negative reactivation of chronic hepatitis B after interferon therapy

The objective of this study was to look for HBV precore mutations in three patients with chronic active hepatitis B who developed HBV-DNA-positive/HBeAg-negative reactivation after HBe seroconversion induced by interferon therapy. Direct sequencing of polymerase chain reaction products was performed on serum collected before and after HBe seroconversion. In two patients precore sequence showed only wild-type HBV before and after interferon therapy. In one patient, precore sequence showed only wild-type HBV before interferon therapy and a mixed infection by wild-type HBV and precore mutant viruses (1858 and 1896 nucleotide mutations) after treatment. The presence of HBeAg/anti-HBe immune complexes was found after HBe seroconversion in all cases. Our results suggest that: 1) precore mutations are not always found in patients with chronic hepatitis B who develop HBV DNA-positive/HBeAg-negative reactivation; and 2) HBeAg negativity, despite the presence of wild-type HBV, might be due to HBeAg/anti-HBe immune complexes. We speculate that the production of these immune complexes may be favored by interferon therapy.     

Hepatitis flares and hepatitis B e antigen seroconversion: implication in anti-hepatitis B virus therapy

Hepatitis flares or acute exacerbations, defined as an abrupt elevation of serum alanine aminotransferase (ALT) over fivefold the upper limit of normal (ULN), of chronic hepatitis B virus (HBV) infection are the results of HLA-I restricted, cytotoxic T lymphocyte (CTL)-mediated immune response against HBV and its downstream mechanisms. Higher ALT levels reflect a more vigorous immune response and a more extensive hepatolysis that, in the extreme situation, may lead to decompensation and failure. In contrast, higher ALT also reflects a more robust immune clearance of HBV and, therefore, a higher chance of HBV-DNA loss and hepatitis B e antigen (HBeAg) seroconversion, both in the setting of natural course and drug therapy. Alanine aminotransferase of fivefold the ULN appears to be a significant cut-off level to categorize the patients in terms of endogenous immune response against HBV. Patients with ALT levels less than fivefold the ULN or those with a less vigorous immune response require immunomodulation to induce robust immune response to enhance HBV clearance. In contrast, those with a more vigorous immune response or those with ALT flare over fivefold the ULN should be monitored closely for spontaneous HBV clearance/HBeAg seroconversion or to start direct antiviral therapy in time to prevent the occurrence or deterioration of hepatic decompensation. In conclusion, a better understanding of the pathogenetic mechanisms and natural course of hepatitis flares, wiser selection of patients and the timing of drug therapy are crucial to achieve better treatment results.     

Hepatitis B genotypes and precore/basal core promoter mutants in HBeAg-negative chronic hepatitis B

Background. Mutations in the precore stop codon (G1896A) and the basal core promoter (A1762T and G1764A) are frequently found in hepatitis B envelope antigen (HBeAg)-negative chronic hepatitis B. However, the clinical significance of these mutations remains controversial. We therefore investigated the influence of hepatitis B virus (HBV) genotypes, as well as precore/basal core promoter mutants, on the clinical and virological features of patients with HBeAg-negative chronic hepatitis B. Methods. Serum samples from 37 patients with HBeAg-negative chronic hepatitis B were collected for serological and molecular assays. The precore and basal core promoter regions were amplified by polymerase chain reaction and the amplicons were directly sequenced and analyzed. HBV geno-type was determined by polymerase chain reaction-restriction fragment length polymorphism. Results. Most of the patients had detectable serum HBV DNA, and genotypes B and C were the predominant strains. The overall prevalence of the precore stop codon mutant and basal core promoter mutant was 67% and 60%, respectively. The baseline clinical and virological features of patients with genotype B and genotype C infection were comparable. However, in the patients with precore/basal core promoter dual mutations there was a significantly lower proportion of individuals with a high detectable serum HBV DNA level (>100 pg/ml) than in the patients with either the precore stop codon mutation alone or the basal core promoter mutation alone (P = 0.04 by the logistic regression test for the trend). Conclusions. Our data suggest a high prevalence of precore stop codon and basal core promoter mutation in Taiwanese patients with HBeAg-negative chronic hepatitis B, and the influence of the basal core promoter mutation on HBV replication is modulated by the emergence of the precore stop codon mutation. Received: May 14, 2001 / Accepted: September 14, 2001     

Cellular immune responses in hepatitis B virus e antigen negative chronic hepatitis B

The immunopathogenesis of hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) virus (HBV) infection has not been adequately investigated. We studied the cellular immune responses of peripheral lymphocytes using proliferating assays, intracellular cytokine staining (ICS) and ELISPOT interferon-gamma (IFN-gamma) assays after non-specific and specific stimulation with whole HBV proteins and synthetic peptides. Thirty patients with HBeAg negative CHB, eleven HBsAg inactive carriers, nine patients with acute hepatitis B and 22 healthy controls were included in the study. Patients with HBeAg negative CHB demonstrated an increased number of peripheral CD8+ T cells while their peripheral blood mononuclear cells showed increased proliferation after in vitro stimulation with overlapping hepatitis B core derived peptides and an envelope derived epitope (HBs 182-191 aa), similar to those observed in acute hepatitis B. Using ICS, we found an expanded population of IFN-gamma producing T lymphocytes, CD4+ and CD8+, after non-specific stimulation, in HBeAg negative CHB compared to all other groups. HBeAg negative CHB and acute hepatitis B patients had a similarly increased number of core specific T cells measured by the IFN-gamma assays. Inactive HBsAg carriers showed minimal proliferative responses overall while they exhibited an increased number of envelope specific effector T cells (measured by ICS). In conclusion, we showed that overall CD4+ T cell responses from patients with HBeAg negative CHB were comparable to those of acute hepatitis B, while inactive HBsAg carriers despite their limited proliferative capacity the effector activity of their peripheral T cells was maintained.     

Cytoplasmic expression of hepatitis B core antigen in chronic hepatitis B virus infection: role of precore stop mutants

Abstract: Aims/Background: The cytoplasmic expression of HBcAg is a possible target of immune hepatocytolysis and it is important for the pathogenesis of hepatic injury in chronic hepatitis B virus (HBV) infection. Cytoplasmic HBcAg expression has been suggested to be related to the precore sequence of HBV, HBV DNA level or cell cycle of hepatocytes and the aim of this study was to understand its mechanism. Material/Methods: We studied the expression pattern of HBcAg and its relationship to serum HBV DNA levels, cell proliferation activity of hepatocytes and precore mutation using 66 patients' sera and biopsied liver specimens of chronic hepatitis B. Results: The expression patterns of HBcAg were cytoplasmic predominant (CP) in 17 cases, nuclear and cytoplasmic (NC) in 10 cases and nuclear predominant (NP) in 9 cases and negative in 30 cases. CP expression cases showed a higher grade of hepatitis activity than NP expression cases. Serum HBV DNA levels showed a wide range and there was no significant difference according to the expression pattern of HBcAg. Cell proliferation activity of hepatocytes, measured by Ki-67 (MIB-1) labelling index was higher in CP expression cases than in NP expression cases and it was also significantly higher in cases of high grade than in low grade hepatitis activity. The precore region was evaluated by primer extension assay in 51 cases and there were 16 cases of 1896 precore mutant, 23 cases of wild type, 12 cases of mixed infection of 1896 precore mutant type and wild type. CP expression of HBcAg was significantly more frequent in 1896 precore mutant cases (86%) than in wild type cases (26%). Conclusion: CP expression of HBcAg is the major pattern of 1896 precore mutant cases and it might be involved in the severe liver injury of precore mutants. One of the mechanisms regulating CP HBcAg expression is suggested to be precore mutation of HBV as well as cell cycle of hepatocyte.     

The role of immune responses in the pathogenesis of hepatitis C virus infection

Summary. Hepatitis C virus (HCV) is notable for the high rate of chronic infection, which occurs in nearly all individuals who become infected. Liver biopsies from individuals with chronic HCV infection are notable for the presence of numerous mononuclear cells, at least some of which are CD4+ and CD8+ T lymphocytes. The immune response to HCV is polyclonal and multispecific, both in terms of antibody and cellular immune responses. Individuals who recover from acute HCV infection appear to have quantitatively more vigorous CD4+ proliferative responses against one or more HCV proteins compared with those individuals who develop chronic disease. CD8+ responses are less well characterized, in part because of the technical difficulties involved in isolating and characterizing these cells. HCV-specific CTL can be readily isolated from the liver and PBMC of chronically infected individuals, and recognize multiple epitopes. Even individuals with the same HLA type do not consistently recognize the same epitope. Thus, there does not appear to be an immunodominant response on the CD8+ level in this infection. CD8+ cells do appear to play some role in limiting viral replication. These responses are insufficient to eradicate virus completely, however, and may cause liver injury once chronic infection is established. Cytokines produced by both CD4+ and CD8+ cells may play an important role in both inhibiting viral replication and causing liver injury. A better understanding of the role of cellular immunity in the pathogenesis of HCV infection may aid in the development of vaccines and immunotherapeutic intervention strategies.     

Association of core promoter/precore mutations and viral load in e antigen-negative chronic hepatitis B patients

Apart from core promoter A1762T/G1764A and precore G1896A mutations, other hepatitis B virus (HBV) mutants are detected in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB). The aim of this study was to determine the effects of those mutants on clinical manifestation and viral loads of genotypes B and C HBV. Seventy-nine HBeAg-negative CHB patients with hepatitis flare were enrolled in this study and their HBV precore/core region were sequenced. Serial biochemical profiles and viral loads were assessed and compared. Fifty-three patients (67%) were infected by genotype B HBV and 26 (33%) were infected by genotype C HBV. The clinical manifestation and HBV viral loads were comparable between the two groups. However, genotype B was significantly associated with precore G1896A mutation (92.5%), and more mutations within nucleotide 1809-1817 were detected in patients infected by genotype B as compared with those infected by genotype C (18.9%vs 3.8%). Most of the cases had mutations at the -2, -3 or -5 position from the precore AUG initiation codon. Triple core promoter mutations T1753C/A1762T/G1764A [corrected] appeared to be linked to genotype C rather than genotype B HBV (19.2%vs 1.9%; P = 0.013). In multivariate analysis, the presence of either triple core promoter 1753/1762/1764 mutation or nucleotide 1809-1817 mutation was the only factor associated with lower HBV viral load (<70 Meq/mL) (odds ratio = 9.01; 95% CI 1.11-71.43; P = 0.04). In conclusion, minor HBV variants with mutations in the core promoter and precore region were detectable in genotypes B and C. Such HBV variants are genotype specific and related to viraemia levels.     

Hepatitis B virus genotypes and clinical manifestation among hepatitis B carriers in Thailand

BACKGROUND: Hepatitis B virus (HBV) genotypes have distinct geographic distributions. The aim of the present study was to evaluate the distribution of HBV genotypes and their clinical relevance in Thailand. METHODS: Hepatitis B virus genotypes among 107 hepatitis B carriers residing in Thailand were evaluated using serologic and genetic methods. They were clinically classified into asymptomatic carriers with normal serum alanine transaminase (ALT) levels and patients with chronic liver disease, such as those with chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). RESULTS: Hepatitis B virus genotype distribution among the 107 patients was 25.2% for genotype B, 72.0% for genotype C and 2.8% for genotype D. The serum ALT levels, HBV-DNA and hepatitis B e antigen positivity were significantly higher in carriers infected with genotype C HBV than in those infected with genotype B (P < 0.05). The proportion of genotype B HBV was higher in asymptomatic carriers than in patients with CH and those who developed liver disease, such as LC and HCC (45.5, 16.9 and 25.0%, respectively; P < 0.05). In contrast, the proportion of genotype C HBV was higher in patients who developed liver disease and CH than in asymptomatic carriers (68.7, 83.0 and 50.0%, respectively; P < 0.05). Phylogenetic analysis based on entire genome sequences revealed three HBV isolates, which were classified into a subgroup of genotype C in isolates from South-East Asian countries. CONCLUSIONS: Genotypes B and C are the predominant types among hepatitis B carriers residing in Thailand and those genotypes influence the clinical manifestation in carriers with chronic hepatitis B infection.     

Cytotoxic CD4 T cells in viral hepatitis

CD4+ T cells are thought to contribute to antiviral immune responses by secretion of cytokines thereby providing help to CD8+ T and B cells. However, perforin-positive cytotoxic CD4+ T cells have been described in human immunodeficiency virus-positive patients suggesting a role not only of CD8+ but also of CD4+ T cells for killing virus-infected cells. We investigated 76 patients with viral hepatitis [15 hepatitis B virus (HBV), 22 HBV/hepatitis D virus and 17 hepatitis C virus (HCV)] for cytotoxic CD4+ T cells. The frequency of perforin-positive CD4+ T cells in viral hepatitis was highly variable ranging from < 1% to more than 25%. Perforin-positive CD4+ T cells displayed the phenotype of terminally differentiated effector cells (CD28-, CD27-). The highest frequencies of CD4+ cytotoxic T lymphocytes (CTLs) were found in patients with delta hepatitis (P = 0.04 vs HBV and HCV patients), and the presence of CD4+ CTLs was associated with elevated aspartate aminotransferase levels (P = 0.01) and decreased platelet counts (P = 0.03). Perforin-positive CD4+ T cells decreased in two individuals during spontaneous clearance of acute hepatitis C. Significant associations were found between the frequency of perforin-expressing CD4+ cells and age (P = 0.04), perforin-positive CD8+ cells (P < 0.001) and perforin-positive CD4-/CD8- lymphoid cells (P = 0.002). Differentiated CD27- effector CD4+ CTLs can be detected in patients with viral hepatitis. In particular in patients with more advanced liver disease, the accumulation of perforin-positive T cells with age could be one correlate for the more severe course of viral hepatitis in elderly individuals.     

Incidence and clinical significance of hepatitis B virus precore gene translation initiation mutations in e antigen-negative patients

Hepatitis Be antigen (HBeAg)-negative chronic hepatitis B (CHB) is associated with hepatitis B virus (HBV) variants harbouring changes in the precore region. Most commonly, a G to A point mutation at nucleotide 1896 (m1896) creates a novel translation stop codon that prevents HBeAg production. In the Mediterranean region the m1896 mutation prevails in greater than 98% of HBeAg-negative CHB patients. In this study the prevalence of additional mutations in the precore region was investigated among patients with chronic HBV infection. Precore sequences were determined by sequencing serum HBV DNA amplified by polymerase chain reaction (PCR) with primers flanking the precore/core region. Thirty-one HBeAg-negative and five HBeAg-positive individuals were studied. All HBeAg-negative patients (100%) harboured the m1896 mutation and 20 (64.5%) also had a G to A mutation at nucleotide 1899 (m1899). Additional mutations affecting the translation initiation of the precore gene were found in seven (22.5%) patients, all with active liver disease, five of whom had episodes of HBV reactivation. HBeAg-positive patients had no mutations in these positions and neither did any of the five HBeAg-negative patients with normal levels of liver enzymes, representing the healthy carrier state of HBV infection. Serial sample analysis from one patient revealed that the initiation codon mutation developed following HBeAg seroconversion and the appearance of m1896. During periods of high HBV replication, the ratio of mutant to wild-type ATG was found to increase in parallel with HBV DNA levels. These data show that a significant proportion of HBeAg-negative patients who already harbour the 1896 stop codon mutation may subsequently develop precore translation initiation mutations, which appear to be associated with enhanced HBV replication and severe liver disease.     

Double point mutation in the core promoter region of hepatitis B virus (HBV) genotype C may be related to liver deterioration in patients with chronic HBV infection

BACKGROUND AND AIM: Hepatitis B virus (HBV) genotype C has a more severe pathogenesis than genotype B in Japan. We retrospectively investigated the relationship between HBV genotype and the core promoter (CP) (nt 1762 and 1764) and precore (PreC) (nt 1896) mutations of the HBV genome. METHODS: A total of 129 Japanese patients (42 genotype B and 87 genotype C) with chronic HBV infection, living in two different geographical areas in Japan, were evaluated (mean follow-up period 10.1 +/- 3.8 years). In 2000, CP and PreC HBV mutations were analyzed by direct sequencing from sera. Hepatitis B e antigen (HBeAg), HBV DNA and serial alanine aminotransferase (ALT) changes were followed and determined using serological methods. RESULTS: Genotype C patients had significantly higher rates of HBeAg (40.2%vs 2.4%), HBV DNA positivity (75.9%vs 7.1%) and ALT abnormality (71.3%vs 11.9%) than genotype B patients (all P < 0.05). Among genotype B patients, CP wild type (92.9%) was predominant and PreC mutation (88.1%) was predominant. However, among genotype C patients, CP mutation (75.9%) was predominant and PreC mutation (66.7%) was predominant. The CP mutation was found significantly more in genotype C than in genotype B (P < 0.05). Of the 67 patients with ALT abnormality, five (7.5%) genotype B and 62 (92.5%) genotype C patients (31 HBeAg positive and 31 negative) were found. Among the 31 genotype C patients who were HBeAg positive, the combination of CP mutation and PreC wild (54.8%) was predominant, while among the remaining 31 genotype C patients who were HBeAg negative, the combination of CP mutation and PreC mutant (71.0%) was predominant. CONCLUSION: Genotype C might be one of the worse prognostic markers in patients with chronic HBV infection, possibly because of mutation in the CP region.     

经树突状细胞递呈的HBcAg特异性细胞毒T淋巴细胞的体外研究

目的　从人的外周血树突状细胞 (DC)的抗原递呈方面研究慢性乙型肝炎的发病机制 ,并诱导出针对HBcAg特异性的细胞毒T淋巴细胞 (CTL)。方法　取健康人DC和患者DC ,比较两组的抗原递呈功能是否存在差异。以HBcAg体外冲击致敏DC ,与自体T淋巴细胞共育 ,诱导出HBcAg特异性CTL ;以HepG2细胞为对照靶细胞 ,转染HBVDNA的HepG2 2 15细胞为靶细胞 ,分别测定CTL在效靶比为 2∶1、6∶1和 2 0∶1时对HepG2细胞的非特异性杀伤率及对HepG2 2 15细胞的特异性杀伤率 ,并比较患者组与正常组特异性杀伤率的差异。结果　患者DC的抗原递呈功能(10 99.2 6 7± 2 39.12 ,1374 .8± 36 4 .15 5 ,2 717.78± 15 89.72 )较健康人 (314 7.933± 72 6 .5 7,384 3.0 0 0±10 6 0 .85 ,5 4 86 .86 7± 1790 .6 4 )弱 ;健康组与患者组CTL对HepG2各效靶比的非特异性杀伤作用差异无显著性。健康组与患者组CTL对HepG2 2 15的特异性杀伤作用差异有显著性 ;患者组CTL的活性 (7.1± 4 .33,15 .6 8± 3.3,2 7.6 6± 4 .5 9)较健康组 (2 0 .76± 6 .0 8,33.97± 8.0 0 ,4 9.6 3± 9.4 8)弱。结论　用HBcAg体外负载患者DC ,能诱导出抗原特异性的CTL ,这些CTL能特异性地杀伤相应靶细胞。     

Frequent integration of precore/core mutants of hepatitis B virus in human hepatocellular carcinoma tissues

The development of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) frequently follows persistent HBV infection and may arise in individuals who are hepatitis B e antigen (HBeAg) negative, indicating the possible presence of precore/core mutants. It is unclear whether precore/core mutants are associated with tumour development or are selected for after chromosomal integration of the wild-type viral DNA. We studied the status and sequence variation of the precore/core region of HBV in 56 patients with HBV-associated HCC and in various corresponding non-tumour tissues by Southern blot analysis, polymerase chain reaction and direct sequencing. Southern blot showed that integrated HBV DNA existed in 43 of 56 HCC tissues. Sequence analysis revealed mutations in 65% of the HCC (26/40) and 45% (14/31) of the corresponding non-tumour tissues. The mutation at nucleotide (nt) 1896, known to prevent HBeAg synthesis, was detected in 40% (16/40) of the tumours and in 35.4% (11/31) of the non-tumour tissues. Other mutations were found at nt 1899 (eight of 40 in HCC; three of 31 in non-tumour tissues), nt 1898 (seven of 40 in HCC; two of 31 in non-tumour tissues), nt 1912 (seven of 40 in HCC; none of 31 in non-tumour tissues) and nt 1886 (three of 40 in HCC; none of 31 in non-tumour tissues). To determine whether this finding merely reflected the prevalence of such mutants in this geographical region, HBV DNA from the sera of patients (also in this region) with acute and chronic hepatitis were sequenced. The nt 1896 mutant was found in 5.6% (one of 18) of patients with acute hepatitis B and in 22.8% (nine of 35) of patients with chronic hepatitis B. However, the nt 1898 mutation was not found in any of these sera. The precore/core mutant was observed with increasing frequency from acute hepatitis to chronic hepatitis, non-tumour and HCC, and this difference in frequency was significant between HCC and acute hepatitis B groups (P < 0.01), suggesting that the precore/core mutant or hepatocytes harbouring this mutant may be under immune selection and that such mutations may facilitate integration and subsequent tumour development.     

外周血DNT细胞和T细胞亚群检测在乙型肝炎病毒感染者诊断中的意义

目的：探讨乙型肝炎病毒感染者外周血CD3＋CD4-CD8-T细胞（DNT）和T细胞亚群的变化及意义。方法使用流式细胞仪检测136例乙型肝炎病毒感染者,包括33例无症状携带者、28例急性乙型肝炎患者、28例轻度慢性乙型肝炎患者、25例中度慢性乙型肝炎患者、22例重度慢性乙型肝炎患者和39例健康人外周血DNT细胞及T细胞亚群。结果健康人群和急性乙型肝炎患者外周血DNT细胞比例分别为（4.82±3.43）%和（4.75±2.71）%,显著低于无症状携带者[（5.43±3.31）%,P〈0.05]和慢性乙型肝炎患者（P〈0.05）;轻度慢性乙型肝炎患者DNT细胞比例为（7.97±4.12）%,显著低于重度慢性乙型肝炎患者[（11.36±5.01）%,P〈0.05];中度慢性乙型肝炎患者DNT细胞比例为（8.41±4.93）%,也显著低于重度慢性乙型肝炎患者（P〈0.05）;健康人、急性乙型肝炎患者和无症状携带者之间 T 淋巴细胞亚群分布无明显差异,但随着慢性乙型肝炎患者病情加重,外周血 CD3＋、CD3＋CD4＋CD8-细胞比例降低（P〈0.05）,CD3＋CD4-CD8＋细胞比例升高（P〈0.05）。结论外周血DNT细胞比例的升高与乙型肝炎病毒感染者慢性化及慢性乙型肝炎患者的疾病进程有关。     

Variations of hepatitis B virus precore/core gene sequence in acute and fulminant hepatitis B

Variations of the hepatitis B virus (HBV) precore/core sequence has been shown to play a role in the development of active liver disease in chronic hepatitis B. Whether this is also an important viral factor in the pathogenesis of acute and fulminant hepatitis B is unknown. To determine the precore/core gene sequence in patients with acute and fulminant hepatitis B, 11 patients with fulminant hepatitis B and seven patients with acute hepatitis B were studied. The sequences of precore/core gene were determined by direct sequencing of the polymerase chain reaction amplicons generated from the HBV isolated from patients' serum. For the 11 patients with fulminant hepatitis B, the precore/core regions were successfully amplified in 10 patients. Eight patients exhibited precore stop codon mutations. In addition, nine of the 10 fulminant hepatitis B patients had frequent nucleotide substitutions with corresponding changes in the predicted amino acid sequences in the mid-core and the 5 terminus region of the core gene. In contrast, precore stop codon mutants were not detected, and variations of the HBV core gene were minimal in patients with acute hepatitis B. The association of HBV precore mutants and HBV core gene variations with fulminant hepatitis B and not acute hepatitis B suggested that these variations may be important in modulating the clinical course of HBV infection.     

Long-term follow-up of patients with hepatitis B e antigen negative chronic hepatitis B

Background and Aim: After hepatitis B virus (HBV) e antigen (HBeAg) seroconversion, HBV‐DNA continues to replicate, and HBeAg‐negative patients still face the risk of liver disease progression. We investigated the predictive factors for alanine aminotransferase (ALT) elevation, antiviral drug use, and hepatocellular carcinoma (HCC) occurrence in HBeAg‐negative patients. Methods: Age, sex, ALT, platelet counts, HBV‐DNA levels, genotype, antidiabetic drug use, body mass index, smoking, and alcohol consumption were analyzed for a total of 244 HBV carriers who were HBeAg‐negative. Results: Of 244 HBeAg‐negative patients, 158 (64.8%) showed normal ALT levels at baseline. Multivariate Cox hazard regression analysis identified high HBV‐DNA levels and high ALT at baseline as independent risk factors for ALT elevation in the patients with normal ALT at baseline. The threshold ALT and HBV‐DNA levels were determined to be 31 IU/L and 5.3 logcopies/mL, respectively. Seventeen (7.0%) patients used antiviral drugs. Multivariate Cox hazard regression analysis identified high HBV‐DNA levels (threshold, 5.7 log copies/mL), the use of antidiabetic drugs, and daily alcohol consumption at baseline as an independent risk factor for the use of antiviral drugs in HBeAg‐negative patients. In 10 patients (4.1%), HCC was detected, and a low platelet count (threshold, 10.0 × 10⁴/mm³) was associated with the occurrence of HCC. Conclusion: This study identified predictors of future active liver disease in HBeAg‐negative patients, i.e. ALT elevation, unavoidable use of antiviral drugs, and occurrence of HCC.     

Detection of hepatitis B virus precore stop codon mutants by selective amplification method: Frequent detection of precore mutants in hepatitis B e antigen positive healthy carriers

The precore region of hepatitis B virus (HBV) is indispensable for secretion of e antigen protein. Therefore, the precore stop codon mutants may play an important role in the process of e antigen seroconversion. However, the presence of the mutants in hepatitis B e antigen positive carriers has not been fully studied because of difficulties in detecting the mutants in the presence of large amounts of wild-type viruses. To overcome this, a sensitive method has been developed to detect the presence of G to A stop codon mutants at codon 28 of precore region. Primers for polymerase chain reaction (PCR) were devised to introduce restriction enzyme site Sty I for wild-type viruses and Dde I for the mutants. The amplification products with these primers were digested with Sty I to exclude the products from wild-type viruses. The remaining amplicon from precore mutants were re-amplified, and the presence of precore mutant was confirmed with Dde I digestion. The presence of precore mutants was examined in 61 HBV carriers by the method combining PCR and restriction enzyme digestion. Approximately 0.1% of precore mutant DNA among 10⁶ copies of wild-type virus DNA was detectable by this method. The presence of the precore mutants was detected in seven of 10 (70%) e antigen positive asymptomatic carriers, and in 29 of 36 (81%) e antigen positive patients with chronic liver diseases, and in all 15 (100%) anti-e antibody positive patients with chronic liver diseases. This study revealed that a small amount of the precore mutants was present in the majority of HBV carriers.     

Interferon-α2a for chronic hepatitis B with e antigen or antibody: comparable antiviral effects on wild-type virus and precore mutant

Summary. Recombinant interferon-α2a (IFN-α2a) in a total dose of 702 MU was given to 31 patients: nine with wild-type hepatitis B virus (HBV) and hepatitis B e antigen (HBeAg) (A); four with HBeAg and a mixed infection with wild-type HBV and precore mutants (B); 11 with antibody to HBeAg (HBeAb) and a mixed infection (C); and seven with HBeAb and precore mutants alone (D). HBV DNA was not cleared in any patient in groups A and B. By contrast, in patients with HBeAb, HBV DNA was ultimately lost in four patients in group C, as well as in 10 patients in group D. Thus, patients with HBeAb and infected with precore mutants alone (D) lost serum HBV DNA more often than those with HBeAg and wild-type HBV (A). Patients with low pre-treatment levels of HBV DNA cleared virus more frequently, and the response of precore mutant to IFN was comparable with that of wild-type HBV in patients who had a mixed infection. Based on these results, precore mutants do respond to IFN, and therefore, IFN is indicated in patients with HBeAb, especially those with low serum HBV DNA levels.     

Analysis of clinical,biochemical and viral factors associated with early relapse after lamivudine treatment for hepatitis B e antigen-negative chronic hepatitis B patients in Taiwan

Summary.  The efficacy of lamivudine for HBeAg-negative chronic hepatitis B (CHB) Chinese patients has not been fully investigated. The role of the Hepatitis B virus (HBV) genotype on the treatment effect of lamivudine is controversial. Thirty-two consecutive patients with HBeAg-negative CHB were enrolled. All patients were treated with lamivudine 100 mg once daily of 7–12 months duration. The mean total period of follow-up since entry for all patients was 24 ± 3.5 months. HBV genotypes were classified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and verified by sequencing. Precore (G1896A) and basic core promoter (BCP, A1762T & G1764A) mutations were determined by PCR and direct sequencing. Twenty-one (65.6%) patients were infected by genotype B and, 11 (34.4%) by genotype C. G1896A was predominant in genotype B infected patients (95.2% vs 63.6%, P  = 0.037). At the end of treatment, 31 (96.8%) and 14 (43.8%) patients achieved biochemical and virological responses, respectively. The biochemical and virological response rates were 40.6 and 0% at 12 months after treatment. Eighteen (56.3%) patients had biochemical relapse within 12 months after withdrawal of lamivudine. By multivariate analysis, the pretreatment serum level of HBV DNA ≥12 Meq/mL was the only factor associated with early biochemical relapse (Odds ratio = 9.333, 95% CI = 1.497 ∼ 58.197, P  = 0.017). In conclusion, the virological effect of lamivudine for HBeAg-negative CHB is transient. Most patients had biochemical relapse within 12 months after lamivudine treatment regardless of HBV genotype. A high pretreatment viral load is the determinant for early biochemical relapse.     

Basal core-promoter mutant of hepatitis B virus and progression of liver disease in hepatitis B e antigen-negative chronic hepatitis B.

BACKGROUND/AIMS: The long-term outcomes in hepatitis B e antigen (HBeAg)-negative chronic hepatitis B are distinct from those in HBeAg-positive chronic hepatitis. However, the molecular virological factors that contribute to the progression of liver disease in this special clinical setting remain largely unknown. We thus investigated the association of hepatitis B virus (HBV) genotypes as well as precore/basal core-promoter mutations with the clinical and virological characteristics of patients with HBeAg-negative chronic hepatitis B in Taiwan. METHODS: HBV genotypes and sequences of precore and basal core-promoter regions of the HBV genome were determined in 174 HBeAg-negative chronic HBV infection patients including 62 inactive carriers and 112 with different stages of liver disease. RESULTS: HBV carriers with older age (> 50 years) (odds ratio, 9.09; 95% confidence interval (CI), 3.22-25, P < 0.001) and basal core-promoter mutant of HBV (odds ratio, 4.12; 95% CI, 1.41-12.03, P = 0.01) were associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). The gender-related risk factors associated with the development of liver cirrhosis and HCC were further analyzed, and basal core-promoter mutant was only associated with the development of liver cirrhosis and HCC in male carriers (odds ratio, 4.35; 95% CI, 1.30-14.52, P = 0.02). CONCLUSIONS: The risk of development of liver cirrhosis and HCC is significantly increased in patients with advanced age as well as with basal core-promoter mutant of HBV. In addition, basal core-promoter mutant might contribute to the gender difference of the progression of liver disease in HBeAg-negative chronic hepatitis B in Taiwan.