Virus Precore Mutations Research Articles

Specific determination of hepatitis B e antigen by antibodies targeting precore unique epitope facilitates clinical diagnosis and drug evaluation against hepatitis B virus infection

ABSTRACT Hepatitis B e antigen (HBeAg) is a widely used marker both for chronic hepatitis B (CHB) clinical management and HBV-related basic research. However, due to its high amino acid sequence homology to hepatitis B core antigen (HBcAg), most of available anti-HBe antibodies are cross-reactive with HBcAg resulting in high interference against accurate measurement of the status and level of HBeAg. In the study, we generated several monoclonal antibodies (mAbs) targeting various epitopes on HBeAg and HBcAg. Among these mAbs, a novel mAb 16D9, which recognizes the SKLCLG (aa −10 to −5) motif on the N-terminal residues of HBeAg that is absent on HBcAg, exhibited excellent detection sensitivity and specificity in pairing with another 14A7 mAb targeting the HBeAg C-terminus (STLPETTVVRRRGR, aa141 to 154). Based on these two mAbs, we developed a novel chemiluminescent HBeAg immunoassay (NTR-HBeAg) which could detect HBeAg derived from various HBV genotypes. In contrast to widely used commercial assays, the NTR-HBeAg completely eliminated the cross-reactivity with secreted HBcAg from precore mutant (G1896A) virus in either cell culture or patient sera. The improved specificity of the NTR-HBeAg assay enables its applicability in cccDNA-targeting drug screening in cell culture systems and also provides an accurate tool for clinical HBeAg detection.

Mutations of pre-core and basal core promoter before and after hepatitis B e antigen seroconversion

To investigate the role of pre-core and basal core promoter (BCP) mutations before and after hepatitis B e antigen (HBeAg) seroconversion. The proportion of pre-core (G1896A) and basal core promoter (A1762T and G1764A) mutant viruses and serum levels of hepatitis B virus (HBV) DNA, hepatitis B surface antigen (HBsAg), and HB core-related antigen were analyzed in chronic hepatitis B patients before and after HBeAg seroconversion (n = 25), in those who were persistently HBeAg positive (n = 18), and in those who were persistently anti-HBe positive (n = 43). All patients were infected with HBV genotype C and were followed for a median of 9 years. Although the pre-core mutant became predominant (24% to 65%, P = 0.022) in the HBeAg seroconversion group during follow-up, the proportion of the basal core promoter mutation did not change. Median HBV viral markers were significantly higher in patients without the mutations in an HBeAg positive status (HBV DNA: P = 0.003; HBsAg: P < 0.001; HB core-related antigen: P = 0.001). In contrast, HBV DNA (P = 0.012) and HBsAg (P = 0.041) levels were significantly higher in patients with the pre-core mutation in an anti-HBe positive status. There is an opposite association of the pre-core mutation with viral load before and after HBeAg seroconversion in patients with HBV infection.

Detection of Hepatitis B Virus Pre-core Mutant by Allele Specific Polymerase Chain Reaction

Introduction : Mutation in pre-core region is characterized by negative HBeAg and positive anti-HBe despite active replications of the virus. The mutation has diagnostic and prognostic implications. Therefore, detection of pre-core mutant is important. Standard diagnosis approach for detecting pre-core mutant is through DNA sequencing of hepatitis B virus (HBV) pre-core region. Unfortunately, DNA sequencing is not available in most centers. Hence, a simpler diagnostic approach is necessary. Method : An observational-analytic design study was performed. Detection of pre-core mutant was conducted in individuals with positive HBsAg and HBV DNA that had various patterns of HBeAg and anti HBe. HBsAg, HBeAg and anti-HBe was detected using immunochromatography technique. The HBV DNA was evaluated by using qualitative polymerase chain reaction (PCR) testing. PCR was done by three rounds of amplification with primers derived from wild type pre-core and mutant pre-core. Results : Of 25 sera with HBeAg negative, anti-HBe positive and HBV DNA positive, allele specific (AS) PCR pre-core mutant was detected in 20 (80%) sera. Two sera with HBeAg negative, anti HBe negative and HBV DNA positive were negative for pre-core mutant. Of 8 sera with HBeAg positive, anti HBe negative and HBV DNA positive, pre-core mutant was detected in 2 (25%) sera. Conclusion : Most of individuals with HBV DNA positive, HBeAg negative and anti-HBe positive have harbored pre-core mutant. The finding indicated that all patients with HBsAg positive, HBV DNA positive and HBeAg negative, but anti-HBe positive should be examined for the presence of pre-core mutant. Pre-core mutant is also found in HBeAg positive individual. Keywords : HBV, pre-core mutant, polymerase chain reaction

Hepatitis B virus genotype and basal core promoter/precore mutations are associated with hepatitis B-related acute-on-chronic liver failure without pre-existing liver cirrhosis

The study was undertaken to investigate the features and clinical implications of hepatitis B virus (HBV) genotypes, basal core promoter (BCP) and precore (PC) mutations in hepatitis B-related acute-on-chronic liver failure (HB-ACLF). Samples from 75 patients with HB-ACLF and without pre-existing liver cirrhosis and 328 age-matched patients with chronic hepatitis B (CHB) were analyzed. HBV genotype and BCP/PC mutations were determined by direct sequencing. Mutations at 8 sites of the BCP/PC region were compared between the two groups of patients. A significantly higher ratio of genotype B to C was found in patients with HB-ACLF than in patients with CHB (30.7–69.3%vs16.5–82.6%, P < 0.01). Single mutations including T1753V (C/A/G), A1762T, G1764A, G1896A and G1899A and triple mutations T1753V/A1762T/G1764A and A1762T/G1764A/C1766T (or T1768A) were more frequently detected in patients with HB-ACLF than in patients with CHB. Correspondingly, BCP/PC wild-type sequences were absent in patients with HB-ACLF in contrast to 27.1% in patients with CHB. The BCP/PC mutations were found to be associated with increased HBeAg negativity, higher alanine aminotransferase level and lower viral load. Patients with HB-ACLF infected with the PC mutant virus had a higher mortality. The findings suggest that patients with CHB infected with genotype B with BCP/PC mutations were more likely to develop HB-ACLF than those with genotype C with wild-type BCP/PC regions, and patients with HB-ACLF with the PC mutation had increased risk of a fatal outcome.

Changes in different regions of hepatitis B virus gene in hepatitis B ‘e’ antigen‐negative patients with chronic hepatitis B: the effect of long‐term lamivudine therapy

Lamivudine therapy for chronic hepatitis B has been associated with changes in different regions of the hepatitis B virus nucleotide sequence. To study changes in the sequences of polymerase and precore/core promoter regions of hepatitis B virus, before and during 5 years of therapy with lamivudine. Eighty consecutive samples were taken from 10 chronic hepatitis B 'e' antigen-negative patients. Nine patients carried hepatitis B virus precore mutations during the study. Before therapy, wild type was replaced by A1896 in two (20%) cases. During treatment, A1896 reverted transitory to wild type in five cases (50%) and in one case wild type was replaced by A1896. The continuous detection of precore mutations during therapy was associated with a lower response rate. YMDD mutations were observed in nine cases and both, L180M and M204V/I mutations were simultaneously detected in six cases. About 75% of the patients with M204V mutations were responders and none with M204I or mixed pattern sustained response. Hepatitis B 'e' antigen-negative patients exhibit changes in the precore regions both spontaneously and under lamivudine therapy, the transitory reversion to wild type being most frequently witnessed. Patients carrying M204V mutations are more likely to respond to therapy. If, in further studies, these results are confirmed some patients with YMDD mutations could benefit from prolonging the duration of lamivudine therapy.

Genetic Heterogeneity of Hepatitis Viruses and its Clinical Significance

The genetic heterogeneity of hepatitis B virus (HBV) (8 genotypes A-H) has been applied for tracing the route of HBV transmission and the geographical migration of HBV carriers but it also appeared to have clinical implications. The secondary structure of e encapsidation signal could explain why the precore mutant virus prevails in Mediterranean countries, where genotype D is most prevalent, while the wild type virus is frequent in Western countries, where genotype A is most prevalent. There is increasing evidence that patients infected with genotype C have more severe outcome of chronic liver disease than those infected with genotype B. Genotype B was associated with fulminant hepatitis and more severe episodes of acute exacerbation of chronic HBV infection. Patients infected with genotype B appeared to seroconvert earlier than those infected with genotype C. The hepatitis delta virus (HDV) has 3 genotypes (I, II, III) which are associated with different disease patterns. Genotype III is the most distantly related HDV genotype and is associated with the most severe outcome while genotype II with relatively mild liver disease. The most geographically widespread genotype is I and is associated with a broad spectrum of chronic liver disease. The hepatitis C virus (HCV) displays high genetic heterogeneity with six genotypes (1-6), multiple subtypes and quasispecies. This viral diversity has epidemiological and clinical implications and has been associated with the severity of liver disease, prognosis, response to treatment and failure to generate an effective protective vaccine. HCV genotype 1 is the predominant genotype in Western countries and has been associated with a low response rate to interferon-alpha (IFN-alpha) or to the combination of ribavirin and IFN-alpha. Consequently the duration of treatment has been tailored according to HCV genotype.

Precore mutant hepatitis B reactivation after treatment with CHOP???rituximab

A patient with stage IV non-Hodgkin's lymphoma and chronic hepatitis who was treated with chemotherapy and rituximab developed fatal reactivation of hepatitis B. High viral load titers of hepatitis B DNA were demonstrated. DNA sequencing analysis revealed the presence of the precore mutant strain. Hepatitis B reactivation in patients receiving rituxmab-based chemotherapy is reviewed and the role of precore mutant virus strains resulting in fatal complications is discussed.

Genotype prevalence, viral load and outcome of hepatitis B virus precore mutant infection in stable patients and in patients after liver transplantation.

The precore mutant is detectable in most Israeli patients with persistent hepatitis B virus (HBV) infection. The aim of this study was to determine the prevalence of HBV genotypes, viral load and outcome of precore mutant infection in stable patients and in patients after liver transplantation. The prevalence of HBV genotype and viral load were investigated in 81 patients with HBV precore mutant infection. Of these, 50 patients (40 males, 10 females; mean age 43.4 +/- 11.0 yr) underwent liver transplantation and were serum HBV DNA-negative by hybridization at the time of transplantation. Patients received long-term HBV immunoprophylaxis and immunosuppression, and lamivudine in cases of graft HBV recurrence. The remaining 31 patients were stable, with serum anti-HBe-positivity. Genotypes were tested by restriction fragment length polymorphism of an S gene amplicon. Precore mutations were studied with an INNO-LiPA probe assay. Follow-up was 46.6 +/- 37.7 months. Most of the transplanted group was of Middle Eastern origin (53.6%); the remainder were from Eastern Europe (21.4%), Western Europe and the USA (10.8%), Africa (7.1%), and Asia (7.1%). In the transplanted group, the pre-transplant HBV genotype D was the most prevalent (96%), while genotype A was found in only 4%. Eleven patients (22%) developed recurrent HBV infection post-transplantation. There were no differences in genotype distribution between patients with graft reinfection or lamivudine resistance and patients without recurrence. Mean viral load at recurrence was 148.4 x 10(6) +/- 60.4 x 10(6) copies/mL. The stable group had a similar origin and HBV genotype prevalence, but a lower mean viral load of 12.4 x 10(6) +/- 29.4 x 10(6) copies/mL (p = 0.007). The prevalence of mutations at the precore region and codon 28 was similar in both groups. The chronic precore mutant HBV-infected patients were characterized as follows: (i) genotype D was the most frequent genotype, (ii) the HBV genotype distribution was similar in patients with stable infection and after liver transplantation, (iii) viral load at recurrence was significantly higher than in stable infection, and (iv) HBV genotype was unrelated to the development of recurrence or lamivudine resistance in the tested population.

Changes in virus loads and precore mutations in chronic hepatitis B patients treated with 4 weeks of daily interferon alfa-2a therapy

Interferon (IFN) alfa-2a was administered to 23 patients with chronic hepatitis B daily for 4 weeks and the relation between the efficacy of the treatment and changes in total hepatitis B virus (HBV) DNA and precore mutant levels was investigated. At 6 and 12 months after the completion of IFN therapy, 39.1% (9/23) and 36.8% (7/19) of patients, respectively, showed alanine transaminase (ALT) normalization; 31.3% (5/16) and 50.0% (7/14), respectively, became negative for HBe-antigen (HBeAg); and 42.1% (8/19) and 41.2% (7/17), respectively, became undetectable for HBV DNA. All 18 of the patients who were positive for HBeAg at baseline nevertheless had the precore mutation. The level of precore mutant as a proportion of the total HBV DNA level was constant at baseline, and 3 and 6 months after the completion of therapy. Thus, the investigation showed that in chronic hepatitis B, the precore mutation occurs at a constant proportion beginning in the HBeAg-positive phase, and IFN therapy inhibits the growth of the wild-type and precore mutant viruses equally.

Hepatitis B Exacerbation with a Precore Mutant Virus Following Withdrawal of Lamivudine in a Human Immunodeficiency Virus-infected Patient

Chronic active hepatitis B exacerbations have been reported following development of resistance to or withdrawal of lamivudine in HIV-infected patients. A 38-year-old woman with HIV and chronic HBV infections was hospitalized because of acute hepatitis. The occurrence of cytolysis with replication of HBV 2 months after withdrawing lamivudine suggests that our patient experienced a severe reactivation of HBV infection due to the modification of her treatment. Sequencing of the HBV precore region showed the strain to be a mutant. We conclude that lamivudine should not be stopped in HIV- and HBV-infected patients, but could be continued at the dose of 100mg/day as used in isolated HBV infection.

Rapid detection of genotypes and mutations in the pre-core promoter and the pre-core region of hepatitis B virus genome: correlation with viral persistence and disease severity

Background/Aims: We aimed to clarify the clinical relevance of hepatitis B virus pre-core mutant detection in patients with chronic hepatitis B using a newly developed assay. Methods: Viral genotypes and pre-core mutations were studied in relation to viral persistence and liver disease severity using INNO-LiPA methodology. The study group included 151 patients with chronic hepatitis B, 85 positive for HBeAg (group I) and 66 positive for anti-HBe (group II). Results: The prevalence of viral genotypes in group I was: 64% A, 1% B, 15% C, 19% D, 0% E, 0% F and in group II: 39% A, 0% B, 2% C, 56% D, 2% E, 2% F ( p<0.001). The prevalence of mutations at pre-core codon 28 (M2) was lower in group I (5%) than in group II (64%)( p<0.001). The prevalence of pre-core promoter mutations was also lower in group I (21%) than in group II (61%)( p<0.001). M2 mutations were more frequently detected in genotype D than in genotype A ( p<0.001), while the other mutations were not influenced by viral genotype. Serum HBV DNA levels were significantly lower in group II versus group I ( p<0.001), and in patients with any of the pre-core mutations versus wild-type sequence ( p<0.01). Although cirrhosis was more frequent in group II (37%) versus group I (22%) and in patients with either one of the pre-core mutation (31%) versus wild-type sequence (25%), there was no statistical difference in liver severity assessed by ALT levels and Knodell score. Conclusion: Pre-core mutants, whose molecular pattern is strongly dependent on viral genotypes, are associated with viral persistence in anti-HBe positive patients with ongoing chronic hepatitis B. The availability of this rapid assay should allow a precise monitoring of viral pre-core mutants during the course of chronic hepatitis B.

Enrichment of a precore-minus mutant of duck hepatitis B virus in experimental mixed infections.

A precore-deficient mutant of duck hepatitis B virus (DHBV) produced by site-directed mutagenesis was tested for its ability to compete with wild-type virus in a mixed infection of 3-day-old ducklings. The mutation was shown to produce a cis-acting defect, resulting in a replication rate that was about one-half that of wild-type virus. Accordingly, wild-type virus was rapidly selected during the spread of infection. During the chronic phase of the infection, however, two selection patterns were seen. In 4 of 10 ducks, the wild-type virus slowly replaced the precore mutant. In another four ducks, the precore mutant virus slowly replaced the wild-type virus. In the remaining two ducklings, ratios of wild-type and precore mutant virus fluctuated, with wild-type virus slowly predominating. The replacement of wild-type virus was not due to the emergence of a rapidly replicating variant of the precore mutant, since genomes cloned from the infected ducks retained their original replication defect. Replacement of wild-type virus, however, correlated with elevated anti-core antibody titers, which continued to increase with time. The selection of a precore-negative strain of DHBV may be analogous to the selection for precore mutants of HBV during chronic hepatitis in humans.

EFFICACY AND SAFETY OF LAMIVUDINE (3TC) IN RENAL TRANSPLANT PATIENTS WITH CHRONIC HEPATITIS B.

841 Renal transplantation is associated with long-lasting HBV replication probably due to immunosuppression. HBV-infected kidney recipients are at risk for development of more active hepatitis, cirrhosis and, less frequently, hepatocellular carcinoma. To date, Interferon α may be the best treatment of chronic hepatitis B but has been reported to cause severe adverse events and to induce rejection. Lamivudine (3TC) is a new nucleoside analog with potent antiviral effects against HBV and has already been used with success in immunocompetent HBV infected patients (pts) or to prevent HBV reinfection of the graft in liver transplantation. 12 kidney recipients, including 9 men, with a mean age of 50 years (38 to 61), a mean time post transplantation of 11 years (2 to 22), HBV infected since 18 years (11 to 25 years) received an uninterrupted treatment by 3TC. Three pts were HCV co-infected and 41.6% were infected by precore mutant viruses. Liver biopsy was performed in all cases with a median Knodell score of 6.5 ± 2.5 (3-12). 3TC was given for a median of 9 months (range 3.5-23 months) and adapted to renal function. Rapid disappearance of serum HBV DNA was observed in 10 pts and the viral load was decreased of 98% for the last 2 pts. The disappearance of serum AgHBe was achieved in 66.7% of cases and the 2 pts who were CHILD B before 3TC therapy, converted to CHILD A score. Liver biopsy was performed in 2 pts, showing significal improvment in necroinflammatory activity. We did not observe adverse events, interaction with immunosuppressive drugs, or acute rejection. Unlike previous studies in liver transplantation, we did not observe any viral resistance to treatment. This good result could be explained by a low pretreatment HBV DNA level. In conclusion, Lamivudine is a safe and effective treatment for replicating hepatitis B in kidney recipients, with histologic improvment although therapy may have to be continued indefinitively. Effects of long term treatment and/or combination chemotherapy on viral replication and on fibrosis and hepatocellular carcinoma should be evaluated.

Efficacy of lamivudine in patients with hepatitis B virus precore mutant infection before and after liver transplantation

OBJECTIVE: Hepatitis B virus (HBV) precore mutant infection is associated with a more severe liver disease and a poorer response to interferon. We evaluated the efficacy and tolerance of lamivudine to induce complete and sustained suppression of viral replication in seven patients infected with HBV precore mutant (HBeAg−/HBeAb+/HBV DNA+) (in three patients mutation at codon 1896 was detected by direct sequencing). METHODS: Of the seven patients, five had decompensated HBV cirrhosis in a replicative phase and were liver transplant candidates (Group A) and two patients underwent orthotopic liver transplantation (OLT) for HBV liver cirrhosis and developed recurrent HBV infection in the grafted liver (Group B). Lamivudine 100 mg daily was administered orally for a period of 6–75 wk. RESULTS: After 6–8 wk lamivudine therapy was well tolerated and successfully suppressed HBV replication to an undetectable serum level of HBV DNA by polymerase chain reaction in six patients. In Group A, two patients underwent successful OLT with no evidence of HBV reinfection 2–14 months later. Lamivudine was continued after OLT with no episodes of rejection. Three patients died before a suitable liver could be found (one remained serum HBV DNA+ after 6 wk of lamivudine therapy). In Group B, 9–14 months after lamivudine therapy both patients developed lamivudine resistance (increased liver enzymes, reappearance of serum HBsAg and HBV DNA [by hybridization]). In both patients liver histology had progressed and in both, mutation at codon 552 of the HBV polymerase gene was detected. CONCLUSIONS: Lamivudine is well tolerated in patients with decompensated liver cirrhosis due to HBV precore mutant infection who are liver transplant candidates. In four patients (80%) potent suppression of viral replication was detected, allowing OLT to be performed. However, post-OLT, a resistant mutant developed under lamivudine therapy. Combination therapy with other antiviral agents should be evaluated to discourage the emergence of lamivudine-resistant mutants.

Relationship of hepatocellular carcinogenesis with precore mutant virus and serum hepatitis B virus DNA concentration: A longitudinal analysis of patients with cirrhosis

In order to elucidate the influence of hepatitis B virus on the hepatocellular carcinogenesis in cirrhotic liver, quantitative analysis of precore region of hepatitis B virus was sequentially carried out. Among 191 patients who were diagnosed with cirrhosis in our hospital between 1974 and 1992 and had received no anti-viral therapy, 52 patients were selected from the following three patient groups: group A, 17 patients with liver cancer development within 5 years after the diagnosis of cirrhosis; group B, 12 patients with cancer development between 5 and 10 years after the diagnosis; and group C, 23 patients without cancer development during the observation period of 10 years or more. In group A, 16 out of 17 patients steadily showed a large amount of precore mutant virus with a high concentration of 10 4 copy/ml or more throughout the observation period. In group B, precore mutant virus was persistently high in eight of 12 patients. The other four patients showed an increase in mutant virus until the appearance of HCC, from a low concentration of less than 10 4 copy/ml to a high concentration. Of 23 patients in group C, no mutant virus was detected throughout the 10 year observation period in 11 patients and a complete disappearance of the mutant virus was found during the observation period in seven patients. Mutant virus was detected at least once in the clinical courses in the five remaining of group C. The amount of precore mutant virus at the endpoint of observation was significantly associated with HCC development (28/29 vs. 5/23, P<0.000001). To conclude therefore, appearance or persistence of precore mutant HBV suggested an increased risk of HCC development and its sequential analysis could be very useful in prediction and early detection of small HCC.

Hepatitis B virus precore mutants in serum and liver of Southern African Blacks with hepatocellular carcinoma

Background/Aim: The aim of this study was to sequence the precore region of HBV isolated from serum and tumorous and non-tumorous liver tissue from patients with hepatocellular carcinoma to identify mutations that might play a role in malignant transformation. Methods: HBV DNA was extracted from 62 sera, 14 tumorous and 12 non-tumorous liver tissue samples of patients with hepatocellular carcinoma, amplified by the polymerase chain reaction and sequenced directly. Results: Thirty-nine patients were HBeAg-negative and 23 HBeAg-positive. Missense mutations were present predominantly in HBeAg-negative sera. The most common missense mutation, a guanine to thymine transversion, occurred at nucleotide 1862 in the bulge of the encapsidation signal; it was more prevalent in HBeAg-negative ( 10 39 ) than in HBeAg-positive patients ( 1 23 ) ( p=0.03). Mutations known to prevent HBeAg synthesis were detected in seven sera; five with an 1896 stop-codon mutation, one with an 1817 nonsense mutation, and one with a frameshift mutation caused by an insertion between 1838 and 1839. Missense mutations and deletions were present more often in tumorous tissue derived from HBsAg-negative patients. In the tumours missense mutations occurred at position 1862 and 1899, and the deletions affected direct repeat 1 and/or the encapsidation signal and included the x gene stop-codon. Conclusions: The 1862 mutation, and other missense mutations and deletions detected in the precore gene, may disrupt HBV DNA replication and/or signal peptide cleavage leading to HBeAg-negativity. Disruption of viral replication may promote integration of unencapsidated replicative intermediates and hence contribute to hepatocarcinogenesis.

Efficacy of lamivudine in patients with hepatitis B virus pre-core mutant infection pre- and post- liver transplantation

Efficacy of lamivudine in patients with hepatitis B virus pre-core mutant infection pre- and post- liver transplantation

Distribution of the predominant hepatitis B virus precore variants in hepatitis B e antigen-positive children and their effect on treatment response.

Little is known about the distribution of precore hepatitis B virus mutants and their influence on the outcome of interferon therapy in children with chronic hepatitis B. In this study, serum samples were analyzed from 60 children with chronic hepatitis B e antigen+ (HBeAg+) hepatitis. Fifty-two of these children underwent different interferon trials, and a second serum sample was taken from 25 of them at the end of therapy. Fifty-six of the original 60 children were simultaneously infected by wild-type and precore mutant hepatitis B virus variants. The remaining four children were infected by the wild type alone. In 50/56 of children with a mixed viral population, the wild-type variant comprised more than 50% of the total viremia. With respect to the influence of precore variants on the outcome of interferon treatment, the prevalence of mixed viral population was similar in responders and nonresponders (96 versus 88%, respectively). However, precore mutant variants were prevalent (> 50% of the viral population) in 21% of the nonresponders, but in none of the responder children (p < 0.05). Viremia levels were significantly higher in nonresponder than in responder children (p < 0.05). No substantial changes in the prevalence of mutants were observed throughout the interferon therapy. In conclusion, mixed viral infection is found in a high percentage of children with chronic B HBeAg+ hepatitis. Response to interferon therapy does not seem to be related to the presence of hepatitis B virus precore mutants, but rather to the levels of viremia.

Epidemiology of hepatitis B virus infection in India.

The average estimated carrier rate of hepatitis B virus (HBV) in India is 4%, with a total pool of approximately 36 million carriers. Wide variations in social, economic, and health factors in different regions may explain variations in carrier rates from one part of the country to another. Professional blood donors constitute the major high risk group for HBV infection in India, with a hepatitis B surface antigen positivity rate of 14%. Blood transfusions represent the most important route of HBV transmission among adults. However, most of India's carrier pool is established in early childhood, predominantly by horizontal spread due to crowded living conditions and poor hygiene. Acute and subacute liver failure are common complications of viral hepatitis in India and HBV is reckoned to be the aetiological agent in 42% and 45% of adult cases, respectively. HBV is reported to be responsible for 70% of cases of chronic hepatitis and 80% of cases of cirrhosis of the liver. About 60% of patients with hepatocellular carcinoma are HBV marker positive. Small numbers of patients have been reported to be infected with the pre-core mutant virus but none with the S mutant. Coinfection with hepatitis C virus or hepatitis delta virus is comparatively uncommon. In conclusion, hepatitis B is a major public health problem in India and will continue to be until appropriate nationwide vaccination programmes and other control measures are established.

Hepatitis B virus infection in Singapore.

Although Singapore is in an endemic region for hepatitis B infection, the hepatitis B carriage rate of 5-6% is relatively low. The highest positivity rates for hepatitis B surface antigen (HBsAg) are found in the paediatric age group, with another peak in 40-49 year olds. Studies suggest that, although perinatal transmission is an important route of infection, most children acquire the virus through horizontal transmission between family members. Viral replication continues at a high rate in young carriers and tends to slow down with increasing age. Up to 50% of hepatitis B carriers in Singapore have chronic hepatitis, shown by raised serum ALT values and liver histology, and about 10% are infected with the precore mutant virus. About 20% of carriers have cirrhosis. Among patients with HCC, up to 75% are HBsAg positive, of whom 45% are still viraemic. Mass vaccination against hepatitis B was introduced into Singapore on a voluntary basis in 1983, with compulsory vaccination of babies born to HBeAg positive mothers since 1985. The number of cases of acute hepatitis B has fallen by 60% between 1989 and 1995 although the problems of the longterm complications of chronic hepatitis B still need to be tackled.