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Background & Aims: Occult HBV infection (OBI) is defined by the presence of HBV DNA in the liver and/or serum and negative HBsAg testing. Since the implementation of highly sensitive HBV DNA screening, OBI is also detected in healthy blood donors. The aims of this study were to investigate HBV-specific immune responses and genetic variability in donors with OBI, established by HBV DNA in serum., Methods: HBV-specific T-cell responses to HBV antigens were tested in 34 OBI donors by IFN-γ ELISpot, cytometric bead array, and intracellular cytokine staining. As comparison populations, 36 inactive HBV carriers, 22 donors with spontaneously resolved HBV infection, 24 vaccinated donors, and 25 seronegative donors were also included. Surface, pre-S, and pre-c/core genes from 44 genotype D isolates (24 OBI and 20 HBsAg-positive) were sequenced., Results: The immune response of OBI donors to the 3 HBV antigens was 29-41%, similar to the response in subjects with resolved HBV infection and higher than that in HBsAg-positive subjects. On sequence analysis, OBI donors presented a higher HBsAg mutation rate than HBsAg-positive subjects. Mutations were clustered in the major hydrophilic region of HBsAg, and no stop codons or relevant mutations that could affect antigen formation or detection were observed., Conclusions: Our results suggest that immune response can suppress viral replication to low levels and HBsAg expression to undetectable levels in OBI blood donors. Relevant mutations were not found in the genomic HBsAg coding region. Hence, the fact that HBsAg was not detected in OBI is likely due to low HBsAg production, rather than to a failure of laboratory reagents., (Copyright © 2011 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

De novo hepatitis B virus (HBV) infection after orthotopic liver transplantation (OLT) in patients negative for hepatitis B surface antigen (HBsAg) is between 1.7% and 3.5% in areas with a low prevalence of HBV infection. The importance of this problem and the efficacy of lamivudine treatment has not been defined in areas with a high prevalence of positivity to antibody to hepatitis B core antigen (Anti-HBc). To define the characteristics and the clinical impact of de novo HBV infection in OLT recipients and to evaluate the efficacy of lamivudine treatment in this context, 229 HBsAg (-) donors (145 men, 84 women) were retrospectively evaluated between June 1994 and June 2000. Forty-eight recipients were excluded for various reasons. The final study population included 181 patients that were prospectively followed up for more than 6 months after OLT. When de novo HBV infection was detected, liver allograft biopsy was performed and treatment with lamivudine was indicated if patients were HBV-DNA-positive with elevated ALT levels. Survival time was defined as the interval between diagnosis of HBV infection and death or last follow-up visit. Thirty-one of 229 liver donors (13.5%) were anti-HBc(+). After a mean follow-up of 54.4+/-30 months, 9 of the 181 recipients (5%) developed de novo HBV infection; 8 of 27 recipients (29.6%) of livers from anti-HBc(+) donors as compared with only one of 154 recipients (0.6%) of livers from anti-HBc(-) donors P < 0.005). Liver biopsies performed in 8 of 9 cases showed chronic active hepatitis in 7 patients and acute hepatitis in one patient who cleared HBV spontaneously during the first 3 months. Seven patients were treated with lamivudine for a mean period of 24.5 months; HBV-DNA became negative in 5 of 7 (71.4%), and HBeAg became undetectable in 3 of 6 patients (50%). Patient actuarial survival rates at 1, 3, and 5 years were 100%, 94.7%, and 81.2% for recipients of anti-HBc (+) livers and 95.2%, 83%, and 77.3% for recipients of anti-HBc (-) livers P = ns). In our area, the appearance of de novo HBV infection after OLT is related to grafting livers from anti-HBc (+) donors is associated with a benign outcome, with no liver failure or graft loss, and treatment with lamivudine is highly effective in the control of HBV replication.

The interactions among hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) were studied by measuring HBV-DNA and HCV-RNA levels and by determining the influence of viral genotypes and mutations in HBV basal core promoter (BCP) and precore regions. We included 65 consecutive patients, 25 HBV/HCV, 18 HBV/HDV, and 22 HBV/HCV/HDV. Controls consisted of 55 patients with chronic HBV and 55 with chronic HCV infection. HBV-DNA and HCV-RNA levels were lower in coinfections than in single infections (P <.05). HBV/HCV coinfection was associated with lower HBV viremia (8.2 x 10(4) copies/mL) and lower HCV-RNA levels (7 x 10(5) IU/mL), than the corresponding control group (P <.05), with more marked decrease in HBV replication (P <.05). Moreover, in HBV/HCV coinfection and in triple coinfection we observed an inverse relationship between HBV-DNA and HCV-RNA levels (P <.05). HBV/HDV coinfection was associated with lower HBV viremia (2.5 x 10(4) copies/mL) than that found in HBV infection (P <.05). Patients with triple coinfection showed lower HBV-DNA and HCV-RNA levels than control groups (P <.05). Prevalence of precore mutations was lower in HCV coinfections (P <.05). No significant association was observed between HCV-RNA levels and HBV precore mutations, BCP mutations or HBV genotypes, or between HBV-DNA levels and HCV genotypes (P <.05). In conclusion, HCV exhibited stronger inhibitory action in the reciprocal inhibition seen in HBV/HCV coinfection. HDV was the dominant virus in HBV/HDV coinfection and in triple coinfection, and had a greater unfavorable influence on HCV than on HBV replication. The reciprocal inhibition of viral replication seemed to be little influenced by the inherent genomic factors studied.

Background: Several studies have shown that hepatitis B immunoglobulin (HBIG) imposes a selection pressure on the hepatitis B virus (HBV) S gene, and that the emergence of mutations in this region would make reinfection after orthotopic liver transplantation (OLT) possible., Aims: This study was undertaken to analyze the presence of HBV S-gene mutations in the different stages of HBV infection and the relationship between HBIG therapy and the emergence of mutations in liver transplant recipients., Methods: The frequency and location of mutations in the coding region of the HBV S gene were studied by PCR and direct sequencing in 30 patients (7 with acute self-limited hepatitis B, 16 with chronic hepatitis B and 7 recipients of (OLT) for HBV-related end stage liver disease who became reinfected)., Results: The average number of amino acid changes was higher in patients with a more advanced stage of disease, 0.57 mutations/100 positions in acute hepatitis B and 1.57 in chronic hepatitis B (1.28 in HBeAg-positive and 1.8 in anti-HBe-positive patients). The average number of substitutions in the transplanted patients was 2.7 before OLT and 3 after OLT. No amino acid substitutions were detected in the "a" determinant of HBsAg in acute hepatitis B, however, 8 substitutions were observed in 6 chronic patients. In 3 OLT patients, 4 substitutions were observed in samples before and after OLT. One of these patients, who had protective levels of anti-HBs, showed 3 additional new amino acid substitutions after OLT, suggesting escape mutant selection by the effect of HBIG therapy. No changes were observed between the consensus sequences obtained several years before and after transplantation, indicating consensus sequence stability., Conclusion: These results show that there is an accumulation of HBV S-gene mutations in HBV-related end-stage liver disease. Prophylaxis with HBIG mainly obtained from acute self-limited hepatitis patients who have a highly homogeneous viral population, may be one factor underlying the reinfection after liver transplantation.

The aim of this study was to analyze the variability of the HLA-II system in a series of patients with chronic hepatitis B, chronic hepatitis C and acute hepatitis B to know whether there is any relationship between the polymorphism of the HLA system, the different types of hepatitis and the evolution of the infection. HLA-II antigens were determined by a PCR technique in serum samples of 24 controls, 22 cases of chronic hepatitis C, 38 cases of chronic hepatitis B and 11 with acute hepatitis B. The prevalence of the HLA-DR4 antigen was lower in the cases of chronic hepatitis B (10.5%) and C (13.6%) than in the controls (33.3%), particularly the DRB1*0401 allele (p = NS). The prevalence of HLA-DR6 was similar in chronic hepatitis B (42.1%) and acute hepatitis B (45.5%). Predominance of the DRB1*1301 and DRB1*1302 alleles were, however, observed in acute hepatitis B (36.4%) versus chronic hepatitis B (13%). These data suggest that immunologic factors such as HLA antigens may influence in the susceptibility to infection by HBV and HCV. The use of PCR techniques which discriminate between the different alleles of the HLA antigens may provide better knowledge of the immune response.

Anti-HBe-positive patients with precore mutants may have severe, progressive liver disease. Therapy with interferon has been effective, but relapses are frequent. To evaluate and compare two antiviral treatments, lymphoblastoid interferon (ly-IFN) and adenine arabinoside 5'-monophosphate (ARA-AMP), 20 patients with anti-HBe-positive chronic hepatitis (5 cirrhosis and 15 CAH) and viral replication (HBcAg in the liver and HBV DNA in serum) were treated. Patients were randomized into two groups: 11 patients received ARA-AMP, 5 mg/kg/day during 7 weeks, and 9 received human ly-IFN, 5,000,000 units, three times per week, during 4 months. Baseline clinical, biochemical and histological features were not significantly different between the two groups. At the end of therapy, 8 (89%) patients in the interferon group and 5 (45%) in the ARA-AMP group showed normal ALT levels and no HBV DNA in serum by a liquid hybridization assay (P < 0.05). At 1 year of follow-up, a persistent response was observed in 33% of ly-IFN patients and in 27% of ARA-AMP patients, a transient response in 56% and 18%, and nonresponse in 11% and 55%, respectively. HBV DNA remained detectable by polymerase chain reaction (PCR) in 19 of the 20 patients. Among the responders, an improvement in histological lesion and the disappearance of intrahepatic HBcAg were observed; in the nonresponders, histological lesion remained stable or worsened. In conclusion, the efficacy of interferon and ARA-AMP was similar in treating anti-HBe-positive chronic hepatitis. Although interferon treatment led to initial improvement in a larger number of patients, there was a much higher rate of relapses with this drug.

Background/aims: This study aimed to evaluate the usefulness of quantifying HBV-DNA amplified by polymerase chain reaction in chronic hepatitis B infection., Methods: Serum samples were obtained from 32 asymptomatic HBV carriers and 99 chronic hepatitis B patients (62 positive for anti-HBe and 37 positive for HBeAg). In addition, serial serum samples were analyzed from 15 HBeAg positive patients undergoing antiviral therapy and 17 anti-HBe positive patients with precore mutation. HBV-DNA quantification was carried out using an enzyme immunoassay with an HBV-DNA plasma standard., Results: The digoxigenin-incorporated polymerase chain reaction method detected HBV-DNA in 34.3% of the asymptomatic HBV carriers with a median HBV-DNA concentration of about 0.18 x 10(5) mol/ml (range 0.08-0.4), in 87% of the anti-HBe positive chronic hepatitis cases with a range of 0.2 to > 2 x 10(5) mol/ml and in 100% of the HBeAg positive patients, with a value in all cases over 2 x 10(5) mol/ml. We observed that after treatment, HBV-DNA tested negative in only two of the eight HBeAg positive chronic hepatitis patients who seroconverted to anti-HBe, and was positive in the seven remaining, with a median HBV-DNA value of about 0.2 x 10(5) mol/ml (0.09-0.4). In the precore mutants HBV-DNA values ranged from 0.2 to > 2 x 10(5) mol/ml., Conclusions: Polymerase chain reaction HBV-DNA quantification is a sensitive method for managing chronic hepatitis B patients, especially those with low viremia, and may be a valuable tool for evaluating the efficacy of antiviral therapy.

The precore-core gene of hepatitis B virus (HBV) was directly sequenced from serum samples of 42 patients with chronic B hepatitis (19 hepatitis B e antigen [HBeAg]+ and 23 anti-HBE+). Viral genotypes were determined by comparison with 11 reference sequences and by restriction analysis. Genotype A was identified in 16 cases, genotype D in 24 cases, and other genotypes in 2 cases. Precore mutations, mainly M1 (stop at codon 28), were differently distributed among the viral genotypes: 3 cases (18.8%) with genotype A and 18 cases (75%) with genotype D. In sequences with precore mutants, the encapsidation signal was more stable (negative stabilization energy) than in sequences without precore mutants. In genotype A, the M1 mutation coexisted with a second mutation (C-->T at position 1858 in codon 15), and both mutations were paired in the secondary structure of the RNA encapsidation signal, which justified the rare presence of precore mutants in this genotype. The analysis showed different distribution of mutations depending on the viral genotype; patients with genotype D were more likely to have persistent HBV infection by selection of precore mutants. Multiple amino acid substitutions were detected in the core region, mainly in two subsequences that have been previously described as epitopes (flanked by codons 11 to 27 and 74 to 83); the presence of these mutations was significantly related to the presence of precore variants which abolished the expression of HBeAg.(ABSTRACT TRUNCATED AT 250 WORDS)

To evaluate the presence of precore mutants, serum samples from 25 patients with chronic hepatitis B, HBV-DNA positive (5 HBeAg and 20 anti-HBe positive) were studied. The complete precore-core region of HBV-DNA was directly sequenced after polymerase chain reaction amplification by a fluorescent linear polymerase chain reaction sequencing method. Precore variants were detected in one HBeAg positive and in all 20 anti-HBe positive patients: in 19 cases, G to A at position 1896, with or without the substitution G to A at position 1899, in two cases C to T substitution at position 1817 which also produces a stop codon (CAA to TAA), one accompanied by the mutation G to A at position 1896. The only mutation observed in the patient who was initially HBeAg positive patient was a G to A substitution at position 1899. Consecutive serum samples from a patient with chronic hepatitis B, initially had the simultaneous presence of wild type and variant strains. Elimination of the wild-type strain was associated with reactivation of liver disease. Analysis of the sequences obtained demonstrated the heterogeneity of the hepatitis B virus genome in the precore-core region. These results indicate that the main cause of non-expression of HBeAg in chronic hepatitis B in our country is the substitution of G to A at nucleotide 1896, alone or accompanied by other variants. Fluorescent linear polymerase chain reaction is a fast and sensitive method to study heterogeneity in the precore-core region.

The aim of antiviral therapy in chronic hepatitis B is the cessation of viral replication, which may be demonstrated by the loss of hepatitis B "e" antigen (HBeAg) and serum hepatitis B virus DNA (HBV-DNA) detected by dot-blot hybridization. With the development of the sensitive polymerase chain reaction (PCR) technique for detecting HBV-DNA, it has become apparent that many HBeAg negative patients may still have small amounts of circulating viral DNA. We assessed 19 of 25 patients with chronic hepatitis B who seroconverted from HBeAg to anti-HBe after adenine arabinoside 5'-monophosphate therapy (5 mg.kg-1.day-1 for 7 weeks) to determine whether serum HBV-DNA became undetectable. Sixteen of the 19 HBeAg negative patients remained hepatitis B surface antigen (HBsAg) positive, and the other three lost HBsAg during follow-up. All of them were HBV-DNA negative by dot-blot hybridization. Using the PCR technique, HBV-DNA became negative in 13 (81.2%) of the 16 patients who seroconverted to anti-HBe without losing HBsAg, and in all the patients who lost HBsAg. These data suggest that the majority of patients who respond to adenine arabinoside 5'-monophosphate show a complete inhibition of hepatitis B virus replication, as demonstrated by the absence of viral DNA by PCR. This inhibition was present in all patients who, at the same time, lost HBsAg.

Although the efficacy of hepatitis B vaccines in patients under chronic hemodialysis treatment has been well documented, the persistence of immunity in this population remains largely unknown. In this study we have followed 60 hemodialysis patients up to 3 years after primary hepatitis B vaccination (four doses of recombinant hepatitis B vaccine; Engerix B, 20 mg/dose) to evaluate the persistence of immunity (as indicated by serum levels of antibody to hepatitis B surface antigen-anti-HBs-higher than or equal to 10 mIU/ml). Fourty-four (73%) patients developed anti-HBs levels above 10 mIU/ml after vaccination; the remaining 16 (27%) vaccinees were considered nonresponders and were given a booster dose that again failed to elicit an immunoresponse. After 3 years of follow-up, 18 out of 44 (41%) responders had no detectable anti-HBs levels in the serum (antibody loss occurring within 8 and 12 months in 3 cases, within 1 and 2 years in 13, and within 2 and 3 years in 2 other cases). When compared with the responders that lost their antibodies during the follow-up period, those who remained immunoreactive 3 years after vaccination was initiated were younger and had higher anti-HBs levels at 8 months of follow-up. Two hepatitis B virus infections were detected among nonresponders during the follow-up period. Based on these data, we conclude that patients undergoing chronic hemodialysis therapy not only have lower response rates to hepatitis B vaccination than healthy adults, but also that these are frequently transient.(ABSTRACT TRUNCATED AT 250 WORDS)

The sensitivity and specificity of two non-radioactive spot hybridization assays for hepatitis B virus DNA (HBV-DNA) using biotin and digoxygenin-labelled DNA probes were investigated in parallel in 122 serum samples from patients with chronic hepatitis B and 50 controls. The results were compared with an isotopic technique using a 32P-labelled probe. HBV-DNA was detected in 56 (80%) out of 70 hepatitis B "e" antigen (HBeAg)-positive cases and in 4 (8%) out of 52 antibody to hepatitis B "e" antigen (anti-HBe)-positive cases using the digoxygenin or 32P-labelled probes. No false positives were found with either method. Using the biotin-labelled probe, 16% of sera gave discordant results, which were considered to be false positive. The time required for detection of serum HBV-DNA was 2 hours for the non-radioactive probes and 16 hours for the isotopic probes. This study suggests that the digoxygenin-labelled probe for detection of HBV-DNA is the most rapid and sensitive method for routine diagnosis of viral replication in clinical laboratories.

232 asymptomatic HBsAg carriers were controlled between May/1986 and May/1990. The were detected in our blood bank between 1974 and 1976 or during pregnancy or delivery period after 1981. 23 of 232 (10%) became negative during the follow-up, this happening more frequently in carriers after 5 years of follow-up. Only 2 of 209 carriers with HBsAg were HBeAg positive and 1 of these was also DNA-VHB positive. None of the HBeAg negative carriers showed DNA-VHB; only 5 had increased transaminases. 7 were anti-VHC positive, these individuals experiencing more frequent increases in transaminases. The results of this study suggests that the determination of transaminases, viral activity markers, anti-HD, anti-VHC in HBsAg carriers are enough to identify those with hepatic disease.

One hundred eleven newborn infants born of Spanish hepatitis B surface antigen (HBsAg) carrier mothers were consecutively assigned to one of three treatment groups. Group A was treated with three or four doses of hepatitis B immune globulin (HBIG) in one of three different schedules. Group B received one dose of hepatitis B vaccine (Hevac-B, Pasteur) at birth and at 1, 2 and 12 months. Group C was treated with the same vaccination schedule as group B and in addition received a single dose of HBIG at birth. Comparisons were made in the 85 babies who had strictly completed the immunization schedule and had been followed for at least 12 months. The three immunization protocols were equally effective, since none of the children became a chronic HBsAg carrier or developed acute symptomatic infection. There were five transient and subclinical infections among children who received only HBIG (group A), one transient infection in group B, and one in group C. There seems to be some correlation between anti-HBs levels and degree of protection, since all transient infections in group A occurred in the subgroups who did not maintain protective antibodies during the first 6 months. Although the percentage of responders in the two vaccinated groups did not differ significantly, children who received only vaccine reached higher antibody levels than those who also received HBIG. Our results suggest that any immunization schedule able to maintain anti-HBs levels during the first 6 months of life would be useful to prevent mother-to-infant transmission of the hepatitis B virus in areas where most of the carrier women are expected to be anti-HBe positive and hence relatively less infectious.

HBeAg and Ab are investigated by simple radial immunodiffusion in dextrane following Magnius. 14 patients (28%) were HBeAg positive and 2 patients HBeAb positive (4%). HBsAg subtyping was ad in 6 cases and ay in further 5. No relation was found between both determinants and HBeAg. Six of the 10 patients with HBeAg in their sera developed chronic liver disease o fulminant hepatitis, while only 3 of the 21 HBeAg negative controls did so (p less than 0.05). HBeAg is a marker of the evolution of virus B hepatitis.

Eight patients with chronic (mean 52 months) type B hepatitis (chronic active hepatitis and active cirrhosis) and markers of active viral replication (presence of HBeAg and HBV-DNA in serum) were treated with Ara-AMP for 7-12 weeks. The mean follow-up time was 18.3 months. All but one patient responded to treatment. In seven patients, HBeAg and HBV-DNA became negative and developed anti-HBe. One patient lost HBsAg as well as HBeAg and HBV-DNA. HBcAg in liver tissue became undetectable or greatly reduced in the seven patients in whom it was strongly positive. Aminotransferase and immunoglobulin levels reverted to normal in the seven responders and remained normal through the entire follow-up period. A second liver biopsy, performed after completion of therapy, showed improvement in six of eight cases, with disappearance of lobular activity. Two patients treated for 12 weeks developed a severe polyneuropathy lasting for 6 months. A 7-week course of Ara-AMP seems to induce long-lasting inhibition of viral replication, along with an improvement in liver function tests and liver histology, without significant side effects. On the other hand, a 12-week course was associated with the development of a severe and prolonged polyneuropathy.

To evaluate the effect of hepatitis delta virus on the level of replication of hepatitis B virus and to assess the clinical significance that such an effect might have on the final outcome of the infection, the serological profile of hepatitis B virus DNA was investigated in 153 patients with acute or chronic hepatitis B virus infection with or without associated delta infection. Serum hepatitis B virus DNA was detected in 57% of patients with acute hepatitis B, 67% of those with acute hepatitis B virus-hepatitis delta virus coinfection and 25% of HBsAg carriers with hepatitis delta virus superinfection during the first week after the onset of symptoms. Patients with acute hepatitis B and those with acute hepatitis B virus-hepatitis delta virus coinfection did not differ significantly with respect to the serological profile of hepatitis B virus DNA and final clinical outcome. Within the group of HBsAg carriers with hepatitis delta virus superinfection, all patients who were initially negative for hepatitis B virus DNA developed chronic hepatitis delta virus infection, whereas 3 of the 4 patients with active hepatitis B virus infection at the time of superinfection showed transient inhibition of hepatitis B virus replication followed by termination of hepatitis delta virus infection in two patients. Therefore, although delta virus may inhibit the replication of hepatitis B virus among chronic HBsAg carriers, this effect is not readily apparent among patients with hepatitis B virus-hepatitis delta virus coinfection.

Association of primary liver carcinoma with virus of type B hepatitis has been demonstrated around the world, especially in African and Asian countries where high titles for surface antigen of B hepatitis (HBsAg) have been found. Presence of viral markers of B hepatitis has been studied in a group of 34 patients with primary liver carcinoma, in 139 cirrhotic patients, and in 100 normal individuals. Positive titles of HBsAg or of antibodies against "core" antigen (anti-HBc) without evidence of antibodies against HBsAg (anti-HBs) were considered as positive markers for virus B infection. Percentages of positive markers in the series studied were as follows: 52% in the cases of primary liver carcinoma, 38% in the cirrhotic cases, and 5% in the control subjects. There is a significant difference in relation to the incidence of viral markers between patients with liver carcinoma and normal controls. A possible role of the virus of B hepatitis in the pathogenesis of primary liver carcinoma could be suggested on the basis of these results.