Serum HBV Research Articles

Does Aflatoxin B1 Play a Role in the Etiology of Hepatocellular Carcinoma in the United States?

Previous research showed that risk factors associated with hepatocellular carcinoma (HCC) include infection with hepatitis B (HBV) and hepatitis C (HCV) viruses, exposure to aflatoxin B1 (AFB1), and liver cirrhosis, due primarily to alcohol consumption. To determine whether AFB1 may play a role in HCC in the United States, a search for AFB1 adducts and p53 alterations, potentially induced by AFB1, was conducted in the United States in 23 HCC patients with available tissue samples. The presence of AFB12tumor-DNA and -serum lysine adducts and mutant p53 product was determined by immunoassays and codon 249 p53 mutation by restriction enzyme analysis. HBV and HCV serology and serum HBV-DNA were also determined. Thirteen patients were positive for HBV by HBs antigen or anti-HBc antigen or by polymerase chain reaction for HBV-DNA sequences. Nine patients were free of HBV and HCV markers; 5 of 22 sera tested were anti-HCV positive. p53 Protein expression, determined by immunohistochemical staining, was present in 5 of the 23 tumor tissues, whereas p53 codon 249 mutations were not observed in the 5 cases in which tissue was available for study. AFB1 tumor-DNA adducts were present in 3 of 19 tumor tissues, and in 1 of these 3 samples p53 protein was also detected. Sera from only 5 of the patients were tested for AFB1-lysine adducts, and all were positive. In these five patients, neither p53 protein nor a mutation on codon 249 was detected. The demonstration that AFB1-DNA and -lysine adducts are present in HCC patients in the United States is intriguing but requires further substantiation because of the small number of subjects in this pilot study. To elucidate the pathogenetic significance of these findings, further investigation, including studies in larger patient cohorts and properly selected controls, is warranted.

HBV serum marker detection and relative factor analysis of 2925 new students

HBV serum marker detection and relative factor analysis of 2925 new students

HBV serum marker detection and relative factor analysis of 2925 new students

The HBV serum marker of 2925 new students was detected by enzyme linked immunosorbent assay. This produced results with a high degree of precision in a large study sample. The detection results showed that the HBsAg positive rate of new students is closely related to their family location, being either from urban districts or rural areas, and also the educational levels of their parents.

Hepatocellular carcinoma in Brazil: report of a national survey (Florianópolis, SC, 1995).

In order to investigate epidemiological aspects of hepatocellular carcinoma (HCC) in Brazil, basic informations about cases diagnosed from January 1992 to December 1994 were requested to several medical centers of different Brazilian States. A simple questionnaire included age, sex, alcohol abuse (over 80g/day), associated liver cirrhosis, persistent HBV infection (HBsAg), HCV infection (anti-HCV) and serum levels of alpha fetoprotein. 287 cases, over 16 years old, from 19 medical centers of 8 States (Pará, Bahia, Minas Gerais, Espirito Santo, Rio de Janeiro, São Paulo, Paraná and Rio Grande do Sul) were analysed. The results showed: (a) Mean age was 56.3 +/- 14.4 for men and 54.7 +/- 16.8 yr for women and the male/female ratio was 3.4:1. (b) 69.6% were caucasians, 21.8% mullatoes, 4.8% orientals and 3.7% blacks. (c) HBsAg (+) in 77/236 cases (41.6%) without differences between males and females. (d) Anti-HCV (+) in 52/193 cases (26.9%). (e) 7/180 cases were positive both for HBsAg and anti-HCV (3.8%). (f) There was chronic alcoholism in 88/235 cases (37%). (g) HCC was found in cirrhotic livers in 71.2% of 202 cases in which the presence or absence of cirrhosis was reported. (h) Alpha-fetoprotein above 20 ng/ml was found in 124/172 cases (72%) and above 500 ng/ml only in 40 cases (23.2%). These results showed that the HCC in Brazil has an intermediate epidemiological pattern as compared to those from areas of low and high incidence of the tumor. In spite of the high frequency of the association of HCC with the HBV and/or HCV infections, 42% of 180 cases were negative both for HBsAg and anti-HCV, indicating the possible role of other etiological factors. The comparison of data from different States showed some regional differences: higher frequency of associated HBsAg in Pará, Bahia, Minas Gerais and Espírito Santo, higher frequency of associated HCV infection in Rio de Janeiro, São Paulo and States of the Southern region and low frequency of associated liver cirrhosis in Salvador and Rio de Janeiro (55.5 and 50% respectively). Further investigation will be necessary to study the presence of other possible etiological factors as aflatoxins, suggested by the favourable climatic conditions for food contamination by fungi in the majority Brazilian regions.

Detection of HIV, Hepatitis B and Hepatitis C markers in discarded syringes and bloodstains

Puncture injuries from discarded syringes continue to increase and testing these syringes for viral markers of HIV, HBV and HCV infection is now frequently requested. Workers in forensic medicine also regularly deal with potentially infected material. A simulation study was carried out. Syringes (with attached needles) and cotton gauze were contaminated with HIV, HBV and HCV infected blood or serum and tested at intervals over five weeks. Sensitive ELISA techniques, giving a result within three hours, were used to detect viral antibodies (HIV, HCV) or antigens (HBV). Blood residues inside the syringes and on the gauze gave positive results for viral markers throughout the period of study; the needle-tips gave intermittently positive reactions. It was concluded that standard ELISAs for detecting markers of HIV, HBV and HCV could provide valuable guidance on potential infection hazards of material being examined in a forensic situation.

Prevalence of serologic markers of hepatitis B and D viruses in children of the Caiabi and Txucarramãe tribes from the Indian Reservation of Xingu, central Brazil

The medical literature has shown that the inhabitants of the Amazon region are highly affected by hepatitis B and delta viruses infection, but this has never been studied in Indian children by age group. A study of the prevalence of serological markers of hepatitis B and delta viruses in Indian children aged 0 to 14 years living in the PIX was carried out. This Park is located in Central Brazil, a region which is in the transition between the savannab to the south and the Amazon jungle to the north. To determine the prevalence of HBV and HDV markers in Indian children and to characterize the route of HBV transmission in this region. Out of the 17 tribes living in the PIX, two--the Caiabi and the Txucarramãe--were chosen because both live in the North part of the Park, but have quite different ways of life. The overall prevalence of HBV serum markers was: HBsAg, 4.5%; anti-HBs, 39.6%; anti-HBc, 44.1%; any marker of HBV, 47.3%; and anti-HDV, 0.0%. However, a striking difference in the prevalence of hepatitis B markers was observed between the two tribes: younger Caiabi children were much less affected than the Txucarramãe ones. The prevalence of HBsAg in fertile women was 12%, being anti-HBe positive. Our data suggest that HBV infection is highly prevalent among Indian children living in this Indigenous Park and vertical infection is not an important route of transmission in either tribes.

Evaluation of the potent anti-hepatitis B virus agent (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in a novel in vivo model.

A murine model was developed to investigate the in vivo activity of anti-hepatitis B virus (HBV) agents. Mice with subcutaneous tumors of HBV-producing 2.2.15 cells showed reductions in levels of HBV in serum and in intracellular levels of HBV when the mice were orally dosed with (-) cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (FTC). No effects on tumor size or alpha-fetoprotein levels were observed. FTC can selectively inhibit HBV replication at nontoxic doses.

Mixed cryoglobulinemia and hepatitis C virus

background: Mixed cryoglobulinemia (MC),is frequently associated with clinical and biological evidence of liver disease and has recently been reported in cases of hepatitis C virus (HCV) infection. The aim of this study was to assess prospectively in a large series of MC patients: (1) the prevalence of HCV markers (anti-HCV antibodies and HCV RNA in serum and cryoprecipitate); (2) the main clinical, biologic and liver histologic features in patients with or without HCV infection. patients: One hundred fifteen consecutive unselected MC patients were studied: 45% had well-defined underlying diseases (“nonessential” MC). Fifty-five percent with no cause of MC were considered to have “essential” MC and were subjected to indepth examination. methods: Patients were considered to have MC if two successive determinations of their serum cryoglobulin level were above 0.05 g/L. Anti-HCV antibodies (Ab) were detected in all patients by second-generation tests (ELISA, RIBA). We also looked for HCV RNA sequences amplified by polymerase chain reaction (PCR) in the sera and cryoprecipitates of 39 patients; HBs antigen, anti-HBs Ab and anti-HBc Ab in all patients; and HBV DNA in 20 sera and 17 cryoprecipitates. Quantitative HCV Ab and RNA studies were performed on whole serum, cryoprecipitates, and supernatants. Clinical features were recorded retrospectively for each patient. Liver biopsies from 23 anti-HCV Ab-positive and 7 anti-HCV Ab-negative patients were examined histologically, with qualitative and quantitative analysis. results: Anti-HCV Ab were found in 47/ 115 (41%) patients by ELISA and RIBA: 33/63 (52%) essential MC and 14/52 (27%) nonessential MC. Among the 63 essential MC patients, the 33 anti-HCV Ab-positive (Group 1) were compared to the 30 anti-HCV Ab-negative patients (Group 2). Group 1 patients had more cutaneous involvement (Raynaud's phenomenon, purpura, livedo, distal ulcers, or gangrenous changes) (17 versus 5: p = 0.004), higher alanine aminotransferase levels (110 ± 22 versus 41 ± 10 IU; p < 0.005), higher serum cryoglobulin levels (0.35 ± 0.07 versus 0.12 ± 0.04 g/L; p = 0.01), lower CH50 (28 ± 3 versus 44 ± 2 CH50/mL; p = 0.0001) and lower C4 levels (0.20 ± 0.02 versus 0.29 ± 0.03 g/L; p < 0.04). The prevalence of HBV serum markers was low in both groups, and HBV DNA was never detected in any of the sera and cryoprecipitates tested. HCV RNA sequences were detected in 10/16 (63%) sera and 12/16 (75%) cryoprecipitates from Group 1 patients, whereas they were not in the sera or cryoprecipitates from 23 Group 2 patients. Using quantitative PCR, HCV RNA in cryoprecipitates was concentrated 20 to 100 times despite the absence of significant anti-HCV Ab concentration in these samples. Histologic examination of liver biopsies revealed a spectrum of lesions ranging from chronic active hepatitis to cirrhosis, but Knodell's score did not differ between the groups. conclusion: (1) About 50% of the essential MC patients had anti-HCV Ab, and these patients had more severe cryoglobulinemia-associated clinical and biological signs; (2) HCV RNA sequences were found in the large majority of sera and cryoprecipitates from patients with essential MC and anti-HCV Ab and were more concentrated in cryoprecipitates than in supernatants. These results suggest a role for HCV in the pathogenesis of MC and indicate that many cases of essential MC may be secondary to HCV infection and thus nonessential.

Suppression of the inhibitory effect of denatured albumin on the polymerase chain reaction by sodium octanoate: Application to routine clinical detection of hepatitis B virus at its infectivity threshold in serum

Ten to fifteen percent of posttransfusion viral hepatitis cases are still caused by HBV despite mandatory third generation screening procedures for HBsAg. There is thus an urgent need for a simple, time-cost-effective, but very sensitive test for routine HBV DNA detection in serum. Nested-primed PCR has been shown to detect purified HBV DNA at its infectivity threshold in serum. Since this is too labor-intensive for routine testing, we assessed the efficiency of a Fast PCR procedure, of three pairs of primers, and of thirty-five simple serum pretreatments with the aim to achieve the same sensitivity level. Using ten-fold dilution in phosphate buffered saline as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at the 2 × 10 3/ml level in serum. Using either NaOH denaturation or sodium octanoate thermoprotection as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at its infectivity threshold in serum, while the classical phenol/chloroform/ isoamylic alcohol/isopropanol/ethanol DNA purification procedure enabled us to reach the 10 virus particles ml level. These results suggest that denatured albumin is responsible for the well known inhibitory effect of serum proteins on Taq polymerase. Because of its simplicity and its lower risk of sample-to-sample crosscontamination, the sodium octanoate thermoprotection method was chosen for routine clinical detection of HBV in serum. The clinical usefulness of this approach is demonstrated by the results obtained with HBsAg-negative acute hepatitis B incubation sera and with anti HBe-positive chronic hepatitis B sera.

Effects of interferon-alpha therapy on serum and liver HBV DNA in patients with chronic hepatitis B.

The aim of this study was to evaluate the effect of interferon-alpha therapy on serum and liver HBV DNA in 20 patients with chronic hepatitis B and to correlate the presence or absence of HBV DNA with the clinical response. There were 11 responders and all lost HBV DNA from the serum. Ten of the 11 were followed for 36 months following IFN treatment and remained well with absence of HBeAg and HBV DNA from the serum and with normal ALT. Five also lost HBsAg. HBV DNA became undetectable in the liver of nine of 10 of these patients in whom liver tissue was available for study. HBV DNA persisted in the liver of seven of nine nonresponders and was not detected in two in spite of the presence of HBV DNA and HBeAg in the serum of these two patients. We conclude that IFN may induce long remissions in patients with chronic hepatitis B with loss of HBV DNA from the serum and that occasionally HBV DNA may persist in the liver of such patients.

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This investigation presents the results of hepatitis B virus screening among leprosy patients conducted in central Brazil as a preliminary information for a HBV vaccination programme. The main objectives were to assess the seroprevalence of HBV serum markers among lepromatous patients and to analyse institutionalization as risk factor for HBV infection in this population. Two groups of lepromatous patients were studied, 83 outpatients and 171 institutionalized ones. Screening for HBV serum markers included the detection of HBsAg, anti-HBc by radioimmune assay (RIA). The prevalence of carrier state (HBsAg) was 4.8% and 8.8% among outpatients and institutionalized, respectively, (p > 0.05). Seroprevalence of exposure (all markers) was statistically significant different between outpatients (16.9%) and institutionalized ones (50.3%). Institutionalized patients had an almost four fold risk of HBV infection when compared to the outpatients, and the highest risks were among patients with more than 21 years of residence in the colony, after adjusting for age and sex.

Anti-hepatitis C virus in the elderly: a seroepidemiological study in a home for the aged.

A seroepidemiological survey of anti-hepatitis C virus (anti-HCV) was carried out in 315 institutionalized elderly people. HBV serum markers were tested in the same sera. Clinical details were also studied in the anti-HCV-positive subjects. The overall prevalence of anti-HCV was 2.2%, while the prevalence of HBV serum markers was 36.8% (the HBsAg prevalence was 0.6%). In 1 subject anti-HCV was found in association with HBsAg positivity. Serum transaminase levels were found within the normal range in all 315 subjects (either anti-HCV+ve and anti-HCV-ve), except in the subject who was found to be HBsAg-positive and anti-HCV+ve. In conclusion we found in the institutionalized elderly people a similar prevalence of anti-HCV compared to blood donors of the same geographical area; homes for the aged appear to bring together subjects with previously acquired infections.

Pre‐S1 and pre‐S2 gene‐encoded proteins in liver and serum in chronic hepatitis delta infection

Frozen cryostat sections and sera from 30 patients with chronic delta infection were examined for pre-S1 and pre-S2 gene-encoded proteins, and the results were compared to markers in liver and serum HBV and HDV replication. Pre-S1 and pre-S2 were detected by indirect immunofluorescence (IF) in the liver in all 26 patients with histochemically demonstrable HBsAg. Pre-S peptides were found by double IF to have a predominantly cytoplasmic expression and to be located in the same hepatocytes expressing HBsAg. Liver cells expressing hepatitis delta antigen (HDAg) were frequently negative or very weakly positive for HBsAg and pre-S peptides, but occasional HDAg positive hepatocytes were also strongly positive for HBsAg and for pre-S peptides, particularly pre-S2. Circulating pre-S1 was detected in 24 patients (80%) and pre-S2 in 27 (90%). Detection of pre-S peptides in liver and serum was independent of HBV and HDV replication and of the HBV-DNA integration state. There was no correlation between the amount of circulating pre-S peptides and serum HBV-DNA and HDV-RNA. These results indicate that in chronic HDV infection, formation and secretion of pre-S peptides and of HBsAg occur independently of HBV and HDV replication and secretion. They further indicate that in the acquisition by replicating HDV of an HBV-derived envelope in the liver, both HBsAg and pre-S peptides are concomitantly available but circulating HDV-RNA is not invariably associated with the presence of these peptides in serum.

Correlation of Serum Delta RNA with Clinical Course of Acute Hepatitis Delta Virus Superinfection in Taiwan: A Longitudinal Study

Twenty of 22 hepatitis delta virus (HDV)-superinfected chronic hepatitis B carriers who had detectable HDV RNA at the acute stage progressed to chronicity, while only 6 of 16 patients without HDV RNA did so (P less than .005). The poor outcome of patients with persistently positive or fluctuating HDV RNA has been indicated by the following findings: 24 of 38 patients suffered from prolonged hepatic inflammation complicated by three to eight episodes of exacerbations; among them, 5 developed cirrhosis and 2 died in a follow-up period of 4 years. For most patients, the replication of HBV was suppressed at acute stage; only 3 of the 38 cases had detectable HBV DNA in sera. While reactivation of HBV was found in another 8 patients in the follow-up period, for 5 it was in the presence of serum HDV RNA and 2 developed cirrhosis. Therefore, serial assays of serum HDV RNA and HBV DNA appeared to be of value in monitoring the clinical course and outcome of acute HDV superinfection and in the study of the long-term interactions between these two viruses.

HBV-DNA sequences are rarely detected in the liver of patients with HBsAg-negative chronic active liver disease and with hepatocellular carcinoma in Italy.

Hepatitis B virus sequences were studied by molecular hybridization in liver biopsies from patients with HBsAg-negative chronic liver disease or hepatocellular carcinoma, collected in Italy. Among the 42 patients with chronic liver disease who had no history of drug addiction, alcohol abuse nor evidence of metabolic and autoimmune disorders, only two (5%) had HBV-DNA sequences in the liver, although 23 of them (57%) were positive for antibodies to HBV in serum. HBV-DNA was also demonstrated in integrated form in the tumorous tissue of one out of eight cases with HBsAg-negative hepatocellular carcinoma. These incidences of HBV-DNA positivity in the liver are lower than those reported from other Mediterranean areas and similar to those of North Europe, United States and Japan, suggesting that etiologic factors other than HBV are responsible for the majority of HBsAg-negative chronic liver diseases in our region.

Relation between HBsAg Binding with Polymerized Human Serum Albumin and HBV Replication

The binding between hepatitis B surface antigen (HBsAg) and polymerized human serum albumin (poly-HSA) was studied in HBsAg-negative (25) and HBsAg-positive (92) sera by a sensitive enzyme-linked immunosorbent assay technique, and a correlation of binding activity was made with HBe-markers and hepatitis B virus-specific DNA polymerase. The binding could be detected only in HBsAg-positive sera and was found to be independent of the presence of HBe-markers and DNA polymerase activity. Further, binding was noted in significantly higher proportions of sera samples from the patient group compared with the healthy carrier group (p less than 0.01).

Clonal analysis of intrahepatic T lymphocytes in chronic active hepatitis: Isolation of a T-cell line specific for hepatitis B core antigen from a patient with serological evidence of exposure to HBV

We have evaluated whether peripheral blood and hepatic lymphocytes from a patient with chronic active hepatitis (CAH) and antibodies to HBV in serum were specifically sensitized to HBV envelope antigens (HBsAg and pre-S Ag) or to HBcAg. No proliferation to HBV antigens was demonstrated upon stimulation of peripheral blood mononuclear cells either unfractionated or enriched in CD4+ (helper/inducer) T cells. Of 15 T-cell cloned lines (7 CD8+ and 8 CD4+) obtained by limiting dilution in the presence of PHA and recombinant IL2 from liver-infiltrating lymphocytes, one, designated H2, showed specific sensitization to HBcAg, whereas none demonstrated sensitization to viral envelope antigens. The H2 line displayed the CD8+ phenotype, suppressor activity on polyclonal immunoglobulin production and IL2-dependent, HBcAg-specific proliferation. These results suggest that in patients with CAH and serological evidence of previous exposure to HBV, it is possible to obtain lymphocytes specifically sensitized to HBcAg from liver biopsy.

Lack of anti-HBc IgM in neonates with HBsAg carrier mothers argues against transplacental transmission of hepatitis B virus infection.

Transplacental transmission of hepatitis B virus infection was studied in 51 Senegalese neonates born to mothers who were chronic carriers of HBsAg. 13 mothers were positive for both HBsAg and HBeAg. Mother-to-infant transmission of these two markers was different with 3 children being HBsAg positive and HBeAg negative at birth and 6 being HBsAg negative but HBeAg positive. At birth none of the 51 children had anti-HBc IgM detected by a highly specific enzyme immunoassay, indicating that none had had a primary immune response in utero to HBV infection. These HBV serum markers in newborn infants indicate contamination by maternal blood at delivery rather than active infection in utero. Prophylaxis at birth is advisable in children of mothers who are chronic carriers.

Hepatitis A and hepatitis B virus infection in children and adolescents in north-east Italy.

The sera of 722 children and adolescents without overt liver disease were tested for hepatitis B surface antigen (HBsAg), antiHBs and anti-hepatitis B core anti-HBc; 658 of the sera were also tested for anti-hepatitis A virus anti-HAV. Except for the "passive" antibody peak observed in babies, the anti-HAV age-specific prevalence was negligible until the age of 3; it then increased, reaching 35% by the age of 15. Serological evidence of HBV was present in 16% of the subjects: this prevalence was almost constant at all ages. The HBsAg carrier rate was highest in children under 5 years of age (7.6%) and decreased with age. However, only one HBsAg carrier was under 1 year of age. Anti-HBs age-specific prevalence increased progressively from 2.7% to 11.4%. Anti-HBc alone was present in 4.1% of the subjects. No significant sex differences were found in the prevalence of HBV serum markers or in the HBsAg carrier rate. Neither HAV nor HBV infection was significantly influenced by place of residence or socioeconomic status. It is concluded that in this area both HAV and HBV are endemic, but while HAV is mainly acquired at school, most of the HBV infections occur within the household. The results suggest that not only perinatal transmission, but also intrafamilial horizontal infection, plays a role in HBV spread among infants.

Hepatitis B virus infection in prisons. A seroepidemiological survey in prisoners and attending staff.

Prisoners and attending staff from six houses of detention were screened for serum HBsAg, anti HBs, antiHBc and transaminases. Both prisoners and warders showed an increased prevalence of HBV serum markers with respect to age- and sex-matched general population control groups. The HBsAg carrier rate was 6.7% in prisoners and 6.6% in staff. Sixty-five per cent of the HBsAg-positive subjects were antiHBe-positive. When the blood samples were taken, all the HBsAg-positive subjects were asymptomatic; transaminases were normal in 80% and only slightly elevated in 20%. Illicit drug abuse was found to be a relevant risk factor for HBV infection among prisoners under 35 yearts of age, but not in the older group, whereas no correlation emerged between presence of HBV serum markers and tattoos or admitted homosexuality. A high prevalance of HBV serum markers as also found among young warders who had been in service for a short time: most of them, however, come from areas of Italy with a particularly elevated HBV circulation. Although the results suggest that many of the HBV infections are not recently acquired within the institution, this survey confirms that prisons should be regarded as high risk areas for HBV infection, both for prisoners and warders.