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.     

Hepatitis B virus DNA polymerase activity and hepatitis B e antigen (HBeAg)/anti-HBe status among type B chronic liver diseases

To investigate the relationship between hepatitis B virus (HBV) replication and chronic liver disease, age-specific prevalences of HBeAg, anti-HBe, and HBV DNA polymerase (DNAP) activity were studied in 295 asymptomatic HBV carriers and 183 patients with B-type chronic liver diseases. DNAP activity decreased with age, and there was no difference in the overall prevalence of DNAP activity between the asymptomatic carriers (27.8%) and the chronic liver disease patients (27.3%). The prevalence of DNAP activity in the 40-and-over age group was significantly higher for the chronic liver disease patients (17.5%) than for the asymptomatic carriers (1.7%). The prevalence of HBeAg in the 40-and-over age group was also significantly higher for the chronic liver disease patients (33.8%) than for the asymptomatic carriers (2.6%). These data suggest that viral replication decreased with age and that viral replication occurring in older persons closely related to chronic liver disease.     

Hepatitis B virus DNA polymerase activity and hepatitis B e antigen (HBeAg)/anti-HBe status among type B chronic liver diseases

To investigate the relationship between hepatitis B virus (HBV) replication and chronic liver disease, age-specific prevalences of HBeAg, anti-HBe, and HBV DNA polymerase (DNAP) activity were studied in 295 asymptomatic HBV carriers and 183 patients with B-type chronic liver diseases. DNAP activity decreased with age, and there was no difference in the overall prevalence of DNAP activity between the asymptomatic carriers (27.8%) and the chronic liver disease patients (27.3%). The prevalence of DNAP activity in the 40-and-over age group was significantly higher for the chronic liver disease patients (17.5%) than for the asymptomatic carriers (1.7%). The prevalence of HBeAg in the 40- and-over age group was also significantly higher for the chronic liver disease patients (33.8%) than for the asymptomatic carriers (2.6%). These data suggest that viral replication decreased with age and that viral replication occurring in older persons closely related to chronic liver disease.     

Liver disease activity and hepatitis B virus replication in chronic delta antigen-positive hepatitis B virus carriers

Delta antigen is currently thought to reflect superinfection of the liver with a defective RNA virus (delta agent), requiring helper function from hepatitis B virus for its replication. To assess the influence of delta agent on hepatitis B virus replication in patients persistently infected with both viruses and showing chronic liver disease, we measured serum and liver hepatitis B virus DNA in HBsAg-positive chronic liver disease patients who were either positive or negative for delta antigen in the liver. Hepatitis B virus DNA was assayed in the serum of 21 patients with delta antigen-positive/HBsAg-positive chronic liver disease and in 21 patients with delta antigen-negative/HBsAg-positive chronic liver disease matched for HBeAg/anti-HBe status and underlying liver histology. HBcAg and delta antigen in liver was determined by immunofluorescence or immunoperoxidase staining. In delta antigen-positive/HBsAg-positive chronic liver disease, serum hepatitis B virus DNA was detected transiently in 4 of 21 cases (19%) and was present in these patients at low levels (trace to 2+). In contrast, 9 of 21 (43%) delta antigen-negative/HBsAg-positive chronic liver disease patients were serum hepatitis B virus DNA positive, and five of these had high serum hepatitis B virus DNA levels (3+ to 4+). Serum HBsAg and anti-HBc titers were significantly lower in delta antigen-positive cases and correlated with reduced amount of HBcAg in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatitis B virus DNA in chronic HBsAg carriers: correlation with HBeAg/anti-HBe status, anti-HD and liver histology.

Hepatitis B virus DNA was determined in the sera of 198 chronic hepatitis B surface antigen (HBsAg) carriers by the spot hybridization technique. The results were correlated with hepatitis Be antigen (HBeAg) and antibody (anti-HBe), delta antibody (anti-HD) and liver histology. All subjects had a liver biopsy. The prevalence of HBV DNA was 63% in HBeAg-positive subjects and 8.8% in anti-HBe positives. HBV DNA was not found more frequently in chronic HBsAg carriers who had histological evidence of liver disease than in carriers without such evidence. Anti-HD was detected in 48.5% of subjects, with an increasing trend (p less than 0.001) according to the severity of liver disease. Among patients with more severe liver disease (CAH and cirrhosis), HBV DNA and HBeAg were detected less frequently in anti-HD-positive than in anti-HD-negative subjects (7% vs. 42.3%, p less than 0.001 and 7% vs. 34.4%, p less than 0.005, respectively). These findings indicate that HDV infection jointly affects both HBeAg status and HBV DNA.     

Anti-HBc of IgM-class,HBeAg and anti-HBe in acute and chronic hepatitis B

ABSTRACT— The presence and persistence of IgM antibody against hepatitis B core antigen (anti-HBc IgM) and the correlation with other HBV markers were studied in 42 patients, all of whom had acute HBsAg-positive hepatitis but whose subsequent diseases differed. All patients initially had anti-HBc IgM. In 13 out of 15 patients with uncomplicated acute hepatitis, anti-HBc IgM disappeared within 6 months after onset of the disease. In five out of 12 patients, who in spite of transient HBsAg developed chronic liver disease, the anti-HBc IgM persisted for more than 2 years. Among 15 patients with persistent HBsAg, anti-HBc IgM was present from 7 months to more than 8 years. Seroconversion from HBeAg to anti-HBe was observed in seven patients and in five of these anti-HBc IgM disappeared during the follow-up period. These results indicate that anti-HBc IgM can be used as a serological marker of recent or ongoing HBV infection.     

Delta-infection and suppression of hepatitis B virus replication in chronic HBsAg carriers

The presence of hepatitis B virus DNA and anti-delta was examined in a longitudinal study of 24 patients known to be delta-infected during the course from acute to chronic hepatitis B virus infection. Fifteen patients (63%) were hepatitis B virus DNA positive in the first serum sample. Eleven of 14 patients, who cleared hepatitis B virus DNA, did so following or at the same time as onset of delta-infection. Duration of hepatitis B virus DNA positivity in these 11 patients was shorter than in 11 anti-delta-negative controls matched according to duration of preceding hepatitis B virus DNA positivity, but the difference was not statistically significant. Considering only patients positive for IgM anti-delta in the last serum sample (eight patients), a statistically significant shorter duration of hepatitis B virus DNA positivity was found in delta-infected patients than in the controls (p less than 0.02). The study indicates that the delta-agent may have the capacity to inhibit hepatitis B virus replication and that a chronic delta-infection may lead to a termination of the period of active viral replication.     

Hepatitis B virus precore mutants in HBeAg carriers with chronic hepatitis treated with interferon

Summary. Precore mutants of hepatitis B virus (HBV) were looked for in 18 hepatitis B e antigen (HBeAg) carriers who were treated with recombinant interferon-a (rIFN) and the results were compared with those obtained in 12 untreated carriers who underwent spontaneous HBeAb seroconversion. Molecular analysis of the HBV precore region was carried out by polymerase chain reaction (PCR) amplification and direct sequencing. Precore mutants with a stop codon at codon 28 were detectable at baseline in 19/30 carriers. However, wild-type strains predominated in the baseline sera of both treated ( n = 16) and untreated ( n = 10) patients. Sera from the remaining four patients contained predominantly or exclusively mutant virions. Following IFN treatment, there was a shift from the wild-type pattern to the mutant pattern in all patients, with the precore pattern prevailing in long-term responders (six out of nine) compared with the non-responders (none of nine). The wild-type pattern predominated among the non-responders (eight vs three), suggesting that the long-term response to IFN was associated with take-over of precore mutants. There were no relationships between any pre-treatment precore molecular pattern and disease severity or outcome of treatment. Precore mutants also took over in 10 of the 12 untreated patients (83%), who underwent spontaneous HBeAb seroconversion. Thus, a shift from wild-type to precore mutant pattern occurs in most Italian patients undergoing IFN-induced or spontaneous HBeAb seroconversion.     

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.     

Chronic carriers of hepatitis B virus in Bangladesh: a comparative analysis of HBV-DNA,HBeAg/anti-HBe,and liver function tests

Serological markers of hepatitis B virus (HBV), liver function tests and quantitative estimation of HBV-DNA are important in the assessment of the state of infection and prognosis following treatment for hepatitis B. This study aimed to determine whether low-cost assays, eg hepatitis B e antigen (HBeAg) and liver function tests, could be used for the assessment of infectivity as an alternative to HBV-DNA estimation. We tested 125 hepatitis B carriers for HBeAg, antibody to HBeAg (anti-HBe), and serum HBV-DNA; we also carried out a range of standard liver function tests. Seventy-three subjects were positive and 52 were negative for HBeAg. Of the HBeAg positive cases, 3 were also positive for anti-HBe; of the HBeAg negative cases, 5 were also negative for anti-HBe. Of these 8 cases, 7 had no detectable HBV-DNA. Most of the HBeAg positive but anti-HBe negative subjects were positive for HBV-DNA (74.3%; 52/ 70) whereas most of the HBeAg negative and anti-HBe positive subjects (93.6%; 44/47) were also negative for HBV-DNA. Of 56 HBV-DNA positive individuals, alanine transaminase (ALT) was found to be raised in 69.6% (p=0.066) and aspartate transaminase (AST) was raised in 66.1% (p=0.011), while 67.9% had normal alkaline phosphatase (ALP) (p=0.054). HBeAg (p=0.018) and raised ALT (p=0.008) were found to be independent predictors for HBV-DNA positivity among HBV carriers. This study suggests that HBeAg positive and anti-HBe negative hepatitis B carriers with raised ALT and AST are likely to be positive for HBV-DNA; the combination of routine serology and biochemical tests may be considered as an alternative to HBV-DNA in evaluating the state of chronic HBV infection. However, HBV-DNA should be specifically assessed if discordance is observed between seromarkers and transaminases.     

Analysis of hepatitis B virus DNA,liver disease and influence of antibody to hepatitis C virus in anti-HBe chronic carriers

ABSTRACT— Hepatitis B virus DNA (HBV-DNA) in serum was studied in 67 anti-HBe patients using dot-blot hybridization, a modified technique and polymerase chain reaction (PCR). All patients had abnormal aminotransferase (ALT) levels. Serum HBV-DNA was detected in 14/67 cases by dot-blot and in 39/67 (DNA probe) and 45/67 (RNA probe) using the modified technique. The RNA probe was more sensitive than the DNA probe when they were compared, using serial dilutions of HBV-DNA of known concentration (0.1 pg vs 1 pg, respectively). HBV-DNA was found by PCR in 57/67 patients. Ten patients were negative to serum HBV-DNA. The ALT level was significantly higher in patients with serum HBV-DNA by dot-blot as compared to those who had serum HBV-DNA by the modified technique and PCR. With respect to the presence of anti-HCV, 6/67 had anti-HCV confirmed by RIBA test. These 6 patients had serum HBV-DNA. The route of acquisition of HBV infection in anti-HCV positive patients was by parenteral exposure in 67% of the cases and 15% in HCV negative (p<0.05). All patients were negative to nonorganic specific autoantibodies. In summary, most of the anti-HBe patients (85%) had viral replication. HCV superinfection plays a minor role in the activity of liver disease.     

Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China

AIM： TO determine the genotype distribution of hepatitis B virus （HBV） with a newly oligonucleotide chip assay among the HBV carriers in Eastern China. METHODS： An assay using oligonucleotide chip was developed for detection of HBV genotypes in serum samples from HBV DNA-positive patients in Eastern China. This method is based on the principle of reverse hybridization with Cy5-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides. The results of 80 randomly chosen sera were confirmed by direct sequencing. RESULTS： HBV genotype B, C and mixed genotype were detected in 400 serum samples, accounting for 8.3% （n = 33）, 83.2% （n = 333）, and 8.5% （n = 34）, respectively. The evaluation of the oligonucleotide assay showed 100% concordance with the amplicon phylogenetic analysis except 9 mixed genotype infections undetected by sequencing. CONCLUSION： The study indicates that HBV genotype C and B prevail in the Eastern China. It is suggested that the oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotyping.     

The effect of indomethacin on hepatitis B virus replication in chronic healthy carriers

BACKGROUND: A chronic HBsAg carrier state, a major cause of viral spread in a community, is one of the consequences of hepatitis B virus (HBV) infection. Although successful immunization programs have been initiated to eliminate the virus, there is still a large number of people with HBV infection worldwide. This study was designed to investigate the effect of indomethacin treatment on HBV markers in humans, in comparison with a control group. METHODS: In total, 65 chronic 'healthy' HBV carriers were involved in the study. Patients were divided randomly into two groups. Group I (n = 42) received oral indomethacin 75 mg daily for 6 months. Group II (n = 23) acted as control. Patients in both groups were followed up for 6 months, during which laboratory tests, including viral parameters, were performed periodically. Liver biopsy was done in 17 patients (11/42 of the indomethacin group and 6/23 of the control group). RESULTS: All liver biopsies showed grade 0-2 and stage 0-1 HBV in both groups (P > 0.05). HBsAg positivity did not change in any patient in either group. Five patients who had positive HBeAg in group I became negative 4 months later, while patients in group II continued to be positive at 6 months (P < 0.001). Similarly, all patients receiving indomethacin exhibited a total anti-HBeAg immunoglobulin response at 6 months, while the control group remained the same during this period (P < 0.05). HBV DNA, as detected by polymerase chain reaction in 20/22 (91%), was negative in group I at the end of 6 months. No change was observed in group II (P = 0.007). CONCLUSIONS: Although no biochemical analyses were performed on prostaglandins in the present study, the results suggest that the prostaglandin pathway may be involved in the pathogenesis of the immune response against HBV, and that the suppression of viral replication is achieved as indicated by the disappearance of HBeAg and HBV DNA in healthy chronic HBV carriers.     

Reactivation of viral replication in anti-HBe positive chronic HBsAg carriers

Reactivation of hepatitis B virus replication was investigated in an unselected group of 44 HBV DNA negative, anti-HBe positive chronic HBsAg carriers. Twenty-five patients (54%) were intravenous drug addicts and 7 (16%) were male homosexuals. Sixteen patients had evidence of delta infection and five of the seven male homosexuals had human immunodeficiency virus infection. The patients were followed for 1 to 180 months (median, 24 months) while HBV DNA negative, anti-HBe positive. Reactivation, defined as reappearance of HBV DNA or HBeAg, or both, was detected in six patients corresponding to an annual reactivation rate of 5%. Reactivation in four patients was detected by reversion to HBV DNA positivity only, whereas HBeAg/anti-HBe status remained unchanged. Two patients became both HBV DNA and HBeAg positive. None of the patients developed hepatitis-like symptoms and transaminase elevation was only observed in two patients. Reactivation in two patients was ascribed to human immunodeficiency virus infection and in one patient to chronic lymphatic leukaemia. It is concluded that HBV DNA seems to be superior to HBeAg in the detection of reactivation of HBV replication and that reactivation associated with clinical symptoms leading to progression in chronic liver disease is a rare event in the population studied.     

Analysis of hepatitis B virus DNA, liver disease and influence of antibody to hepatitis C virus in anti-HBe chronic carriers.

Hepatitis B virus DNA (HBV-DNA) in serum was studied in 67 anti-HBe patients using dot-blot hybridization, a modified technique and polymerase chain reaction (PCR). All patients had abnormal aminotransferase (ALT) levels. Serum HBV-DNA was detected in 14/67 cases by dot-blot and in 39/67 (DNA probe) and 45/67 (RNA probe) using the modified technique. The RNA probe was more sensitive than the DNA probe when they were compared, using serial dilutions of HBV-DNA of known concentration (0.1 pg vs 1 pg, respectively). HBV-DNA was found by PCR in 57/67 patients. Ten patients were negative to serum HBV-DNA. The ALT level was significantly higher in patients with serum HBV-DNA by dot-blot as compared to those who had serum HBV-DNA by the modified technique and PCR. With respect to the presence of anti-HCV, 6/67 had anti-HCV confirmed by RIBA test. These 6 patients had serum HBV-DNA. The route of acquisition of HBV infection in anti-HCV positive patients was by parenteral exposure in 67% of the cases and 15% in HCV negative (p less than 0.05). All patients were negative to nonorganic specific autoantibodies. In summary, most of the anti-HBe patients (85%) had viral replication. HCV superinfection plays a minor role in the activity of liver disease.     

Lamivudine prophylaxis for prevention of chemotherapy-induced hepatitis B virus reactivation in hepatitis B virus carriers with malignancies

Summary.  Although hepatitis B virus (HBV) reactivation in HBV carriers undergoing immunosuppressive therapy is clearly documented, the role of antiviral prophylaxis in such individuals is still controversial. The aim of this study was to determine the efficacy of lamivudine prophylaxis in HBV carriers with haemato/oncological malignancies, who receive chemotherapy. Eighteen HBV carriers with malignancy, who were candidates for chemotherapy, were enrolled. Eight subjects (three with leukaemia, four with lymphoma and one with multiple myeloma) were enrolled for prophylactic lamivudine therapy. The remaining 10 patients (six with leukaemia, three with lymphoma and one with breast cancer) were not treated with lamivudine and were used as a control. Lamivudine was administered beginning on the same day as the chemotherapy and was maintained for a year after chemotherapy was discontinued. No HBV-related mortality was observed in either group. In the lamivudine-treated group, none of the subjects had clinical, biochemical or serological evidence of HBV reactivation during the time they were receiving chemotherapy and after their chemotherapy was discontinued. In contrast, five of the 10 HBV carriers not receiving lamivudine therapy experienced a reactivation of HBV infection. This reactivation of HBV was observed during the chemotherapy in four with one individual experiencing a HBV activation 12 months after chemotherapy was discontinued. No lamivudine-related major adverse effects were observed. Hence prophylactic lamivudine treatment in HBV carriers with haemato/oncological malignancy receiving chemotherapy prevents chemotherapy-induced HBV reactivation.     

Spontaneous reactivation of chronic hepatitis B virus infection

Studies on the natural history of chronic type B hepatitis have shown that loss of hepatitis B e antigen and seroconversion to antibody to hepatitis B e antigen are usually accompanied by remission of disease activity and improvement in serum aminotransferase levels. Twenty-five symptomatic patients with biopsy-documented chronic type B hepatitis were followed for 25 +/- 2 mo (mean +/- SEM) after disappearance of hepatitis B e antigen, hepatitis B virus-deoxyribonucleic acid, and deoxyribonucleic acid polymerase activity from the serum. Twenty-four patients developed the antibody to hepatitis B e antigen. All 25 patients demonstrated a decrease in serum aminotransferase levels, and most became asymptomatic. However, during subsequent follow-up, 8 of the 25 patients (32%) exhibited reactivation of chronic type B hepatitis manifested by abrupt elevation of serum aminotransferase levels and reappearance of serum hepatitis B virus-deoxyribonucleic acid, deoxyribonucleic acid polymerase activity, and, in 7 patients, hepatitis B e antigen. All 8 patients developed symptoms: 3 became icteric, 3 developed ascites, and 2 bled from esophageal varices. One of these patients died. Episodes of reactivation invariably occurred within 1 yr of loss of hepatitis B e antigen and lasted for up to 13 mo. These observations suggest that loss of hepatitis B e antigen and seroconversion to the antibody to hepatitis B e antigen do not necessarily imply permanent remission of chronic type B hepatitis, and subsequent spontaneous reactivation may be an important cause of progression of hepatic injury.