A hepatitis B virus variant found in the sera of immunised children induces a conformational change in the HBsAg "a" determinant.

The emergence of variants in the outer envelope proteins of hepatitis B virus (HBV) are found among individuals vaccinated against HBV and asymptomatic carriers of the infection. For example, children in The Gambia vaccinated against hepatitis B may show serological evidence of breakthrough infections, particularly if anti-HBs antibodies induced by the vaccine are low in titre. A single-point mutation at nucleotide 421 of the S gene is associated with such breakthrough infections. In the present study, the antigenicity of variant HBV S protein expressed as HBsAg particles in a vaccinia virus expression system has been characterised using a panel of monoclonal antibodies directed against linear and conformational determinations of the S protein. A cellular ELISA procedure using expressed antigen in Vero cells revealed differences in reactivity using four of the six antibodies that had been raised against the adw subtype of HBV and recognise conformational epitopes in the a determinant. In two instances, an enhanced reactivity for the variant antigen was found, confirming that point mutations in the a determinant of the S protein between residues 139 and 147 may result in significant changes in conformation. These findings also demonstrate that there are distinct antigenic differences between the vaccine strains of HBsAg/ adw subtype and the predominant HBsAg subtype circulating in West Africa. The implications of this work are that serodiagnosis of HBV infections may be unreliable in populations where there is a possibility of variant HBV infections emerging in the face of increasing herd immunity to HBV as a result of vaccination, particularly using monoclonal antibody-based diagnostic tests. Such variants may play a role in the maintenance of HBV infections in endemic regions.     

A low rate of hepatitis B virus vaccine breakthrough infections in Mongolia

A nation-wide hepatitis B virus (HBV) immunization program of all newborn babies was launched in Mongolia in 1991. However, the continuation of clinical icteric viral hepatitis infections in children led to the investigation to determine whether HBV breakthrough infections were occurring and if any were due to hepatitis B surface antigen (HBsAg) mutants. Hepatitis A virus (HAV) infections accounted for most of these cases with 3% of the jaundiced children shown to have acute hepatitis B. Hepatitis B vaccine protection was 93% against HBV infection and 97% against HBV carriage. A G145A "escape mutant" was found in one HBV carrier child only. Anti-HBs levels, however, were low with 85% having titers less than 100 IU/L, 46% of whom had levels less than 10 IU/L. The results from this study demonstrate that the HBV immunization program in Mongolia provides an effective level of protection. However, continued surveillance of breakthrough infections and close monitoring of "vaccine escape" mutants is required.     

Analysis of hepatitis B surface antigen mutations in Mongolia: Molecular epidemiology and implications for mass vaccination

Summary. Although the potential significance of hepatitis B surface antigen (HBsAg) mutants for failure of immunization has been studied in some endemic countries, whether the “a” determinant variants are responsible for vaccine failure in Mongolia remains unknown. Fifty-nine HBsAg-positive children (age: 8.8 ± 0.9 years) who had been observed during the nationwide survey of vaccinated cohorts conducted in 2004 were subjected to molecular analyses of hepatitis B virus (HBV). Partial S gene sequences encoding amino acids (aa) 40–171 of HBsAg were determined in 57 children (96.6%) who had detectable HBV DNA. Phylogenetic analysis of the S gene sequences revealed that genotype D accounted for 93.0% and genotype A for 5.3%. Only one child (1.7%) had HBVs of genotypes A and D. HBsAg mutations were found in 17 (29.8%) children ranging from 1 to 4 aa per subject (mean ± SD, 1.6 ± 0.9 aa). Pro127Thr and Thr118Ala were the most common substitutions, which occurred in 6 (10.5%) and 3 (5.3%) subjects, respectively; none had Gly145Arg. There were no significant associations in the prevalence of HBsAg mutations with age, sex, residential area, or vaccination status against hepatitis B. Analysis of the deduced amino acid sequence of the entire preS1/preS2/S gene revealed that eight genotype D isolates and one genotype A isolate were quite similar to previously-reported wild-type isolates, suggesting that they are essentially wild-type, but not vaccine-induced mutants. In conclusion, the results demonstrate that hepatitis B surface gene mutants do not play a significant role in vaccination failure in Mongolia.     

Multiple hepatitis virus infections in chronic HBsAg carriers in Naples

Summary.  In order to determine the prevalence of multiple infections with hepatitis viruses in chronic HBsAg carriers in Naples, to assess the interaction between HBV, HDV and HCV infections and to evaluate the influence of multiple virus hepatitis infections on the clinical presentation, we studied 198 HBsAg chronic carriers observed consecutively from 1971 to 1988 at our Liver Unit. Of the 198 HBsAg chronic carriers, 171 had undergone percutaneous liver biopsy. The presence of HBcAg or HDAg in the liver biopsy was considered a marker of HBV or HDV replication, respectively; the presence of anti-HCV was considered a marker of HCV infection. Anti-HCV was observed in 13.6% of the 22 subjects with normal liver, in 27.7% of the 47 patients with minimal chronic hepatitis, in 40% of the 50 with mild chronic hepatitis, in 70.6% of the 17 with moderate hepatitis, in 66.7% of the 3 with severe chronic hepatitis and in 65.6% of the 32 with active cirrhosis. Anti-HCV positive cases were antiHD positive more frequently than the anti-HCV negative (59.2% vs. 43%, p=0.05). HDV infection exerted a clear inhibition on the HBV genome. Among the 171 HBsAg chronic carriers, the finding of an active chronic hepatitis (moderate chronic hepatitis+severe chronic hepatitis+active cirrhosis) is less frequent in subjects with HBV replication alone than in those with HDV replication or HCV infection. Patients with both HBV replication and HCV infection and those with both HDV replication and HCV infection showed a very high prevelance of active chronic hepatitis.     

Seroprevalence of hepatitis B virus infection in children in Taipei, 1989: five years after a mass hepatitis B vaccination program

A nationwide hepatitis B vaccination program was launched in Taiwan in 1984. To study the impact of this ongoing program on hepatitis B virus (HBV) infection, a follow-up seroepidemiologic study was carried out in 1989 in a Taipei district where pre-vaccination seroepidemiology had been studied. HBV markers were studied in 1134 apparently healthy children (619 boys and 515 girls) under 13 years of age between March and July 1989. The prevalence of hepatitis B surface antigen (HBsAg) in children under 5 years of age decreased from 9.3% in 1984 to approximately 2% in 1989. A significant decrease in HBsAg prevalence and hepatitis B core antibody in 5- to 8-year-old children who were not immunized against HBV showed that horizontal infection among the older children had also decreased. Thus, this program not only protected vaccinated subjects; the reduction in numbers of highly infectious young HBV carriers also contributed to a lower prevalence of hepatitis B infection and carrier rates in some older children. This study demonstrates that hepatitis B vaccination is effective in protecting the majority of children in hyperendemic areas from HBV infection and from becoming chronic carriers.     

Prevalence of precore mutants in anti-HBe-positive hepatitis B virus carriers in Germany.

Hepatitis B virus (HBV) precore mutants are associated often with highly productive infection in hepatitis B surface antigen (HBsAg) carriers lacking hepatitis B e antigen (HBeAg) but positive for anti-HBe, rendering serological identification of infectious individuals unreliable. Although considered initially to be limited mostly to the Mediterranean area, more recent studies suggest a significant presence of these mutants in northern European countries. The sequence of the precore region was determined and examined for mutations from HBV isolates of 99 German chronic HBsAg carriers positive for HBV-DNA and either HBeAg (n = 15) or anti-HBe (n = 84). In addition, clinical data of individuals carrying wild-type virus and those with precore mutants were compared. HBV precore mutants were found in more than half (44/84) of all HBeAg-negative, anti-HBe-positive virus carriers. There was no difference between carriers of wild-type and precore mutant HBV in the level of viremia or in the clinical course of chronic infection. In conclusion, HBV precore mutants are common in Germany and can therefore present a diagnostic problem for serological testing. However, precore mutants do not appear to have a detrimental effect on the course of chronic HBV infection.     

Control of HBV and HDV infection in an isolated Pacific Island: 1. Pattern of infection

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) infections are known to be hyperendemic in Nauru. Because of the consequences of chronic HBV infection, the Nauruan Government has commenced a program that aims to reduce and eventually eliminate hepatitis B infection by immunizing susceptible adults and children on the island and every newborn baby. At the outset of this program, a national seroepidemiological survey was undertaken. Eighty-eight percent of the population were tested, of whom 69.1% had markers of HBV infection. Evidence of superinfection with HDV was found in 22.7% of HBV carriers, with the highest prevalence in adolescents and young adults. All seronegative individuals were offered three doses of plasma derived hepatitis B vaccine. A post-vaccination survey of 64% of those vaccinated showed that 98% had developed circulating antibodies.     

High prevalence of antibodies to hepatitis A and E viruses and viremia of hepatitis B, C, and D viruses among apparently healthy populations in Mongolia

The prevalence of infection with hepatitis A virus (HAV), HBV, HCV, HDV, and HEV was evaluated in 249 apparently healthy individuals, including 122 inhabitants in Ulaanbaatar, the capital city of Mongolia, and 127 age- and sex-matched members of nomadic tribes who lived around the capital city. Overall, hepatitis B surface antigen (HBsAg) was detected in 24 subjects (10%), of whom 22 (92%) had detectable HBV DNA. Surprisingly, HDV RNA was detectable in 20 (83%) of the 24 HBsAg-positive subjects. HCV-associated antibodies were detected in 41 (16%) and HCV RNA was detected in 36 (14%) subjects, none of whom was coinfected with HBV, indicating that HBV/HCV carriers account for one-fourth of this population. Antibodies to HAV and HEV were detected in 249 (100%) and 28 (11%) subjects, respectively. Of 22 HBV DNA-positive subjects, genotype D was detected in 21 subjects and genotype F was detected in 1 subject. All 20 HDV isolates recovered from HDV RNA-positive subjects segregated into genotype I, but these differed by 2.1 to 11.4% from each other in the 522- to 526-nucleotide sequence. Of 36 HCV RNA-positive samples, 35 (97%) were genotype 1b and 1 was genotype 2a. Reflecting an extremely high prevalence of hepatitis virus infections, there were no appreciable differences in the prevalence of hepatitis virus markers between the two studied populations with distinct living place and lifestyle. A nationwide epidemiological survey of hepatitis viruses should be conducted in an effort to prevent de novo infection with hepatitis viruses in Mongolia.     

High Prevalence of Antibodies to Hepatitis A and E Viruses and Viremia of Hepatitis B, C, and D Viruses among Apparently Healthy Populations in Mongolia

The prevalence of infection with hepatitis A virus (HAV), HBV, HCV, HDV, and HEV was evaluated in 249 apparently healthy individuals, including 122 inhabitants in Ulaanbaatar, the capital city of Mongolia, and 127 age- and sex-matched members of nomadic tribes who lived around the capital city. Overall, hepatitis B surface antigen (HBsAg) was detected in 24 subjects (10%), of whom 22 (92%) had detectable HBV DNA. Surprisingly, HDV RNA was detectable in 20 (83%) of the 24 HBsAg-positive subjects. HCV-associated antibodies were detected in 41 (16%) and HCV RNA was detected in 36 (14%) subjects, none of whom was coinfected with HBV, indicating that HBV/HCV carriers account for one-fourth of this population. Antibodies to HAV and HEV were detected in 249 (100%) and 28 (11%) subjects, respectively. Of 22 HBV DNA-positive subjects, genotype D was detected in 21 subjects and genotype F was detected in 1 subject. All 20 HDV isolates recovered from HDV RNA-positive subjects segregated into genotype I, but these differed by 2.1 to 11.4% from each other in the 522- to 526-nucleotide sequence. Of 36 HCV RNA-positive samples, 35 (97%) were genotype 1b and 1 was genotype 2a. Reflecting an extremely high prevalence of hepatitis virus infections, there were no appreciable differences in the prevalence of hepatitis virus markers between the two studied populations with distinct living place and lifestyle. A nationwide epidemiological survey of hepatitis viruses should be conducted in an effort to prevent de novo infection with hepatitis viruses in Mongolia.     

湖州地区乙肝疫苗免疫失败儿童乙肝病毒S基因“a"决定簇变异分析

目的 了解湖州地区乙肝疫苗免疫失败儿童病毒S基因型变异情况.方法 应用聚合酶链反应(PCR)方法对29例HBV-DNA阳性乙肝疫苗免疫失败儿童乙肝病毒S基因进行扩增,PCR扩增产物进行序列分析.结果 29例样本中测序结果显示,B基因型26例,C基因型3例,有9例样本在S区“a”决定簇发生变异,变异主要存在“a”决定簇内127、129、131、133、141、142、144等氨基酸位点.结论 湖州地区乙肝疫苗免疫失败儿童在“a”决定簇区确实存在变异,可能是发生免疫失败的原因,但并未形成明显优势变异株.     

Naturally occurring hepatitis B surface gene variants in chronic hepatitis B virus infection: correlation with viral serotypes and clinical stages of liver disease

Virus variants escaping from host immunity may be implicated in the pathogenesis of hepatitis B virus (HBV) infection. In this cross-sectional study, the association was evaluated of the frequency of amino acid variation within the immunogenic epitopes of surface gene with different disease stages of chronic HBV infection. The surface gene of HBV encompassing the a determinant (amino acids 124-148) and the putative HLA class I restricted cytotoxic T lymphocyte (CTL) epitope (amino acids 28- 51) were amplified and directly sequenced in 33 asymptomatic carriers (Group I), 31 patients with chronic hepatitis (Group II), 22 with cirrhosis (Group III), and 36 with hepatocellular carcinoma (Group IV). The amino acid sequences were compared subsequently with the consensus sequences of HBV serotype adw or adr. The frequency of amino acid variation per site per sequence (FEQ) was analyzed by generalized estimating equation with Poisson model after stratification by clinical and virological features. The FEQ was 1.21% overall, and was highest in Group IV patients and in patients above 50 years of age. In contrast, nine Group IV patients aged below 50 years who were infected with serotype adw had an inversely higher FEQ than those above 50; the age effect among hepatocellular carcinoma patients was significantly different from that among non-cancerous patients (P = 0.04). Variation of amino acid clustered within a determinant and CTL epitope for serotype adw but was distributed at random for serotype adr. Mutation hotspots differed between serotypes adw and adr. The FEQ of HBV surface protein is correlated positively with advancing age and severity of liver disease, and certain variants may contribute to the persistence of HBV infection.     

Hepatitis B virus transmission pattern and vaccination efficiency in Uzbekistan

A national program of universal vaccination for the prevention of chronic hepatitis B virus (HBV) infection was launched in Uzbekistan since 1998. To evaluate the 6 years' outcome of the program, 567 children were enrolled in the study. Among those enrolled, 333 had immunized with adw2 type based Engerix-B (Glaxo Smith Kline Beechem, Rixensart, Belgium) and 48 with adr type based Hepavax-Gene (Green Cross Vaccine Corporation, Korea). A cohort of 186 children born before the immunization program, was also included in the study. When 45 vaccinated children were compared to age/sex-matched 45 unvaccinated children, the sero-prevalence of HBsAg was 0 versus 11% (P = 0.56), and of anti-HBc was 0% versus 44% (P < 0.0001), respectively. Loss of anti-HBs was observed in 18.4% after 5 years. Among 13 HBsAg carriers found in this study, genotype HBV/D was found in 69%, HBV/A in 23% (all in unvaccinated group) and HBV/C in 8% (in vaccinated group). No significant differences were observed in this study between groups which received different vaccine formulation. Phylogenetic analysis of the HBV isolates obtained from family members of the HBsAg-positive children, revealed evidence suggesting that transmission in the vaccinated group was exclusively perinatal, whereas in the unvaccinated group horizontal transmission pattern predominated. In conclusion, HBV universal vaccination is efficient in Uzbekistan irrespective of the vaccine formulation used, because the horizontal transmission pattern predominates currently in this endemic region.     

Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen

The integration of hepatitis B virus (HBV) DNA in the liver of chronic HBV carriers has been documented extensively. However, the status of the viral genome during acute infection has not been assessed conclusively. While HBV DNA sequences are detected often in serum, liver, and peripheral blood mononuclear cells (PBMCs) after the clearance of serum the hepatitis B virus surface antigen (HBsAg), the precise status of the viral genome, and in particular the possible persistence of integrated genomes in PBMCs, has not been established. A highly sensitive PCR-derived assay (Alu-PCR) was employed to re-examine liver and PBMC specimens obtained from patients with acute (n = 19) and chronic (n = 22) hepatitis in whom serum HBsAg was present (n = 12) (HBV-related chronic active hepatitis) or absent with anti-HCV (n = 10) (HCV-related chronic active hepatitis). Viral integration was demonstrated in 3 out of 19 liver specimens from patients with acute hepatitis and 12 out of 12 specimens from patients with chronic hepatitis. Viral integration was also observed in 4 out of 7 PBMC samples from HBV-related chronic active hepatitis patients and 2 out of 10 liver and PBMC samples from HCV-related chronic active hepatitis patients. In one liver specimen from an acute hepatitis patient, HBV DNA was found integrated in the intronic sequence of the tumour necrosis factor (TNF)-induced protein gene; viral integration into cellular sequences was also found in the PBMCs of four HBV-related chronic active hepatitis and two HCV-related chronic active hepatitis. The results demonstrate the early integration of HBV genome during acute viral infections and the persistence of the viral genome in an integrated form in PBMCs.     

Cloning and expression of surface antigens from occult chronic hepatitis B virus infections and their recognition by commercial detection assays

Occult hepatitis B virus (HBV) infections show little or no serological markers of viral infection, including the absence of hepatitis B surface antigen (HBsAg) which is the main marker of ongoing HBV infection. Such infections can be important in the context of blood and/or organ donations. To study whether mutations contribute to HBsAg seronegativity, S gene sequences from such patients were amplified and cloned. Sequencing revealed 12 clones from seven different patients which contained potentially important mutations. The sequences were subcloned into an expression vector and mutant HBsAgs were expressed in cell culture. The capacity of three HBsAg detection assays to recognise the mutant HBsAgs was studied. Three categories were found: mutant HBsAgs that are not recognised by the assays, those that are recognised as well as wild-type (WT) antigen and an intermediate category where detection of the mutant HBsAgs is reduced with respect to WT. Most of the isolates fall into the second category. Mutations can therefore contribute to HBsAg seronegativity in occult HBV infections, but in most cases the explanation is probably the low level of viral replication.     

Impact of universal vaccination on intrafamilial transmission of hepatitis B virus

To control hepatitis B virus (HBV) infection, a nationwide vaccination program was launched in 1984 and resulted in a significant reduction in the rate of persistent infection of children. However, the relative contribution of vaccination to the intrafamilial clustering of HBV infection remains unclear. The rate of intrafamilial HBV transmission in vaccinated children was investigated. Eighty-four sera from vaccinated children were enrolled and HBV serum markers were determined. The modes of intrafamilial HBV transmission were investigated by history taking and serological assay, and confirmed by genotyping and phylogenetic analysis. The results showed 66 (78.6%) vaccinated children born to hepatitis B surface antigen (HBsAg)-negative parents were HBsAg-negative. Eighteen vaccinees were born to HBsAg-positive parents; four (21.4%) of the children were HBsAg-positive. According to the parents' HBsAg status, three patterns of HBsAg-positive parents were identified. Serological analysis showed that three of 15 children born to HBsAg-positive mother (pattern I) and one of two children born to HBsAg-positive father became infected (pattern II). The remaining one child was HBsAg negative with both parents positive for HBsAg (pattern III). Genotyping and phyogenetic analysis confirmed the mode of intrafamilial transmissions. Sequence analysis of S and pre-S genes showed that HBV isolates of HBsAg-positive vaccinees were variants; no G145R but G145A and other substitutions were found. In conclusion, this small study showed that both maternal and paternal transmissions are important of the intrafamilial spread of HBV infection. In addition, the introduction of HBV vaccination has resulted in a reduction of intrafamilial transmission, but a study of a large population is needed.     

Asymptomatic hepatitis B carriers who were vaccinated at birth

The long-term persistence of immunity following universal infant immunization against hepatitis B virus (HBV) and the need for a subsequent booster dose in adolescence remain under debate. With data derived from Long'an County, Guangxi, China, we reported previously that the prevalence of hepatitis B surface antigen (HBsAg) among adults born from 1987 to 1993 increases with age, although these individuals had received a first dose of the vaccine within 24 hours of birth. Here, we sought the source of transmission by comparison of genotypes among their family members using phylogenetic analysis of complete HBV S gene sequences. For comparison, we screened 2199 vaccinated individuals aged 5 to 17 in Cang Wu County and 1592 vaccinated individuals aged 3 to 7 in Ling Shan County in Guangxi for HBsAg carriers and investigate their family members. In total, 50 asymptomatic HBsAg carriers who were vaccinated at birth and 152 family members were analyzed. The results showed that 25% (95% CI: 6.0-44.0) of the HBsAg-positive children had not acquired their HBV infection from their mothers. This phenomenon showed a trend that increases with age. Antibody escape mutations were detected in 22.9% (95% CI: 11.0-34.8) of the isolates. In conclusion, a booster dose may be necessary for adolescence who were vaccinated at birth in highly endemic countries.     

Use of PCR in resolving diagnostic difficulties potentially caused by genetic variation of hepatitis B virus.

AIMS: To assess the relevance of genetic variants of hepatitis B virus (HBV) and to demonstrate the usefulness of the polymerase chain reaction (PCR) in cases of HBV diagnostic difficulty. METHODS: Five serum samples from patients that presented diagnostic difficulty in routine laboratories were sent to a research laboratory for PCR, and if appropriate, S gene sequencing, in vitro expression, and antigenic analysis. RESULTS: The demonstration of HBV in serum by PCR allowed a definitive diagnosis of current infection. One serum sample with poor reactivity in a diagnostic assay had a minor hepatitis B surface antigen (HBsAg) variant and another with very poor reactivity had multiple variants of HBsAg. Transient HBsAg reactivity was observed in a recently vaccinated patient. A hepatitis Be antigen (HBeAg) false positive reaction was noted in a patient from a well defined risk group for HBV. One patient who was strongly HBsAg/HBeAg positive, but anti-hepatitis B core antibody negative, was viraemic. CONCLUSIONS: PCR may become the gold standard for the diagnosis of current HBV infection. HBV variants are responsible for a proportion of diagnostically difficult cases. Modification of commercial assays is necessary to increase the sensitivity of detection of such variants.     

Molecular and serological evaluation of surface antigen negative hepatitis B virus infection in blood donors from Venezuela

Surface antigen negative hepatitis B virus (HBV) infection was evaluated in Venezuela, by molecular characterization of blood samples positive for antibodies to core antigen (anti-HBc) and negative for surface antigen (HBsAg) in blood donors (residual infections). HBV DNA was found in 11/258 samples (4.3%), and was significantly associated with high levels of anti-HBc antibodies (>25 UI/ml, P < 0.05), while no correlation was found between the presence of HBV DNA and the levels of anti-HBs. Synonymous and non-synonymous mutations were found in the HBV surface region (but not vaccine escape mutants) and in the precore/core region (precore mutants in 2/7 samples and 33-45 bp deletions near the N-terminal core region in 4/19 samples). While HBV genotype F prevails among HBsAg positive samples from blood donors in Venezuela, residual infection isolates were mainly genotypes A and D. Phylogenetic analysis of viral surface and core region revealed discrepancies in genotype designation in 6/9 samples, suggesting the presence of mixed infection or recombination. In conclusion, HBV residual infection in Venezuela does not seem to be frequently observed in HBV genotype F. This type of infection is frequently associated with variants exhibiting mutations in the surface gene that might be affecting the correct recognition by commercial tests, with precore mutants and with core internal deletions. These variants do not seem to cause severe liver disease, and on the contrary, were found circulating at low viremia.     

Diagnostic value of anti-HBc IgM in high HBV prevalence areas

The diagnostic value of an anti-mu-capture immunoassay for the detection of IgM antibody against hepatitis B core antigen (anti-HBc) was evaluated. Strongly positive results were obtained from the acute phase sera of the 25 acute hepatitis B patients who were hepatitis B surface antigen (HBsAg) positive and of the 18 confirmed acute hepatitis B patients who had already cleared HBsAg when symptoms developed. Negative results were obtained in 5 hepatitis A patients, 20 non-A, non-B acute hepatitis patients serologically susceptible to HBV, 22 patients with chronic hepatitis B liver disease, 15 asymptomatic HBsAg carriers, and 10 healthy patients immune from past HBV infection. Fourteen of the acute hepatitis patients remained HBsAg positive for a follow-up period of at least 6 months, and 12 of these were found consistently anti-HBc IgM negative. These were considered as chronic HBsAg carriers with a superimposed form of acute liver injury. These data show that this assay can differentiate between acute from chronic (HBsAg positive) and recent from old (HBsAg negative) hepatitis B virus infection. Thus, it should be very useful in the complex diagnostic situations encountered commonly in areas with high prevalence of HBV infections.     

Analysis of HCV co-infection with occult hepatitis B virus in patients undergoing IFN therapy.

BACKGROUND/AIM: Occult hepatitis B virus (HBV) infection is characterized by the presence of HBV DNA in the absence of hepatitis B surface antigen (HBsAg) in the patient serum. Although such infections have been identified in patients with chronic hepatitis C, the clinical significance of those co-infections is still not understood. Our aim was, therefore, to assess the prevalence and clinical consequences of occult HBV infection in chronic hepatitis C patients undergoing antiviral therapy. METHODS: The study population consisted of 53 HBsAg-negative patients with chronic hepatitis C treated with IFN/ribavirin or IFN/ribavirin/amantadine. Nine patients experienced a viral breakthrough (BT), 30 were non-responders (NR) and 14 were responders (R). HBV-DNA detection by PCR was performed using primers specific for the S region of the HBV genome and HCV-RNA detection by PCR with primers localised in both the 5'NC and core region of HCV genome, before, during and after treatment. Viral genome sequences were also studied. RESULTS: Occult HBV genomes were found in the serum of four of 53 (7.5%) patients, unrelated to anti-HBc status. No significant differences in biochemical, virological, or histological markers, age, duration of infection, were observed in patients with or without HBV DNA. There was an inverse correlation in the evolution of HBV DNA and HCV RNA levels. Direct sequencing showed that S gene of occult HBV presented mutations in the "a" determinant while no specific mutation in the core region of HCV was observed. None of the four patients co-infected with HBV and HCV were responders to anti-HCV therapy. CONCLUSION: In our clinical setting, the prevalence of occult HBV co-infection among patients with chronic hepatitis C was low and independent of the presence of markers of previous HBV infection. Further studies in larger cohort of patients are warranted to determine if occult HBV co-infection may be involved in HCV resistance to combination therapy.