Detection of Hepatitis B Virus Antigens in Paraffin-embedded Liver Specimens from the Amazon Region, Brazil

Hepatic viscerotomy of paraffin-preserved old specimens, collected in the period from 1934 to 1967, were analyzed by immunohistochemical assays to detect hepatitis B, hepatitis D, dengue and yellow fever virus antigens. The material belongs to the Yellow Fever Collection, Department of Pathology, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil and the cases were diagnosed at that time according to clinical aspects and histopathological findings reporting viral hepatitis, yellow fever, focal necrosis and hepatic atrophy. From the 79 specimens, 69 were collected at the Labrea Region and the other 10 in different other localities in the Amazon Region. The five micra thick histological slices were analyzed for the presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) by immunoperoxidase technique. An immunofluorescence assay was applied to the detection of hepatitis D, yellow fever and dengue virus antigens. Nine (11.4%) histological samples were HBsAg reactive and 5 (6.3%) were HBcAg reactive. The oldest reactive sample was from 1934. Viral antigens related to the other pathologies were not detected in this study. Our results confirm that the methodology described may be used to elucidate the aetiology of hepatitis diseases even after a long time of conservation of the specimens.

Clinical, demographic and epidemiological characteristics of patients with hepatitis B followed at a university hospital in southeastern Brazil: predominance of HBeAg negative cases

INTRODUCTION: Hepatitis B is common in Brazil, although there are regional differences regarding the degree of endemicity, the most frequent forms of transmission and the presence of different evolutive stages of chronic disease. The present study aimed to determine the clinical, demographic and epidemiological characteristics of patients chronically infected with hepatitis B virus (HBV) residing in the Ribeirão Preto region, southeastern Brazil. METHODS: A total of 529 medical records of individuals with HBV monoinfection were reviewed. RESULTS: More than 60% of the subjects were males, with a mean age of 38 years-old. The HBeAg-negative serological pattern was verified in 84.4% of the patients, among whom the risk of vertical/intrafamily transmission was 43.2% (p = 0.02). The consumption of alcohol in amounts exceeding 20g a day was observed in 21.3% of the subjects and was more frequent among men (33%) (p < 0.001). Among patients with cirrhosis, 54.1% were alcohol abusers (p = 0.04), all of them males. The presence of cirrhosis was more frequent in the HBeAg-positive group (24.4%) than in the HBeAg-negative group (10.2%) (p < 0.001). CONCLUSIONS: High proportions of HBV-infected subjects with an HBeAg-negative pattern were observed, with a higher risk of vertical/intrafamily transmission. Alcohol abuse was associated with male subjects and with cirrhosis of the liver in this group. A tendency toward an increase in the number of HBeAg-negative cases was observed over time.

Liver enzyme elevation after lamivudine withdrawal in HIV-hepatitis B virus co-infected patients: the Swiss HIV Cohort Study.

Background The principal causes of liver enzyme elevation among HIV-hepatitis B virus (HBV) co-infected patients are the hepatotoxic effects of antiretroviral therapy (ART), alcohol abuse, ART-induced immune reconstitution and the exacerbation of chronic HBV infection. Objectives To investigate the incidence and severity of liver enzyme elevation, liver failure and death following lamivudine (3TC) withdrawal in HIV-HBV co-infected patients. Methods Retrospective analysis of the Swiss HIV Cohort Study database to assess the clinical and biological consequences of the discontinuation of 3TC. Variables considered for analysis included liver enzyme, HIV virological and immunological parameters, and medication prescribed during a 6-month period following 3TC withdrawal. Results 3TC was discontinued in 255 patients on 363 occasions. On 147 occasions (109 patients), a follow-up visit within 6 months following 3TC withdrawal was recorded. Among these patients, liver enzyme elevation occurred on 42 occasions (29%), three of them (2%) with severity grade III and five of them (3.4%) with severity grade IV elevations (as defined by the AIDS Clinical Trials Group). Three patients presented with fulminant hepatitis. One death (0.7%) was recorded. Conclusions HBV reactivation leading to liver dysfunction may be an under-reported consequence of 3TC withdrawal in HIV-HBV co-infected patients. Regular monitoring of HBV markers is warranted if active therapy against HBV is discontinued.

Immune response to hepatitis B vaccination in HIV-positive adults with isolated antibodies to HBV core antigen.

OBJECTIVE: To investigate the merits of vaccination against hepatitis B virus (HBV) in HIV-positive individuals with isolated antibodies to hepatitis B core antigen (anti-HBc). METHODS: HIV-positive patients with isolated anti-HBc and CD4 counts >200 cells/mm(3) received HBV vaccination. An antibody titre to hepatitis B surface antigen (anti-HBs titres) ≥10 IU/L one month post-vaccination was termed an anamnestic response; a titre <10 IU/L was termed a primary response. Patients with primary responses received a 3-dose vaccine course. Anti-HBs titres in all responders were measured 12 and 24 months post-vaccination. RESULTS: 37 patients were studied: 19 (51%) were co-infected with hepatitis C; median CD4 count was 443 cells/mm(3). 8/37 patients (22%) elicited an anamnestic response. 29/37 patients (78%) elicited a primary response. After a 3-dose vaccine course, 15/25 primary responders (60%) achieved anti-HBs titres ≥10 IU/L. HIV acquisition through injecting drug use was the only independent predictor of an anamnestic response (OR 22.9, CI 1.71-306.74, P=0.018). Median anti-HBs titres for anamnestic and primary responders were 51 IU/L (13-127) and 157 IU/L (25-650) respectively. Of all responders, 12/23 (52%) retained anti-HBs titres ≥10 IU/L at 24 months. Anti-HBs duration was not significantly different between anamnestic and primary responders. CONCLUSIONS: 23/37 HIV-positive patients (62%) with isolated anti-HBc achieved anti-HBs titres ≥10 IU/L after 1-3 vaccine doses. However, duration of this immune response was short-lived (ears) in over half the responders. The implications regarding re-infection risk in this population are yet to be determined.

Liver HLA-G expression is associated with multiple clinical and histopathological forms of chronic hepatitis B virus infection

As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and as the histocompatibility leucocyte antigen (HLA)-G is well described as a tolerogenic molecule, we evaluated HLA-G expression in 74 specimens of HBV liver biopsies and in 10 specimens obtained from previously healthy cadaver liver donors. HBV specimens were reviewed and classified by the METAVIR score, and HLA-G expression was assessed by immunohistochemistry. No HLA-G expression was observed in control hepatocytes. In contrast, 57 (77%) of 74 HBV specimens showed soluble and membrane-bound HLA-G expression in hepatocytes, biliary epithelial cells or both. No associations between the intensity of HLA-G expression and patient age or gender, HBeAg status, severity of liver fibrosis, and grade of histological findings were observed. Although significance was not reached (P = 0.180), patients exhibiting HLA-G expression presented a higher median HBV DNA viral load (105 copies/mL) than those who did not express HLA-G (103.7 copies/mL). These results indicate that HLA-G is expressed in most cases of chronic HBV infection in all stages and may play a role in the persistency of HBV infection.

Seroprevalence of Hepatitis B virus infection among an afro-descendant community in Brazil

Furnas dos Dionísios is an Afro-Brazilian black community whose descendants were mainly fugitive slaves that established themselves in the State of Mato Grosso do Sul (MS), Brazil. The population is comprised mainly of low socioeconomic individuals who are engaged in agricultural activities. The objective of this study was to investigate the prevalence of hepatitis B (HB) and its correlation with epidemiological data obtained from the community. The studied population totaled 260 individuals with ages varying from 1 to 79 years (median 20). One hundred thirty-three (51.2%) were females and 127 (48.8%) were males. A high prevalence for anti-HBc was observed (42.7%), with present infection detected in 9.2% of the subjects who were also HB surface antigens (HBs Ag) positive; 27.3% were anti-HBc and anti-HBs reactive, and 6.2% had anti-HBc as only marker. The prevalence for anti-HBc was proportional to age, reaching its highest peak in age categories greater than 50. No serological marker was detected in children under the age of 2 years, however anti-HBc was present in 12 subjects with ages between 2 and 14 years, of these 8 (7.4%) were HBsAg positive. Among individuals over the age of 15 years, 99 were anti-HBc reactive, of these 16 (10.5%) were also HBsAg positive, thus suggesting an increased prevalence of HBV carriers among children and adolescents. The risk factors observed in this community that were significantly associated with anti-HBc positivity were age (over 20 years) and having an anti-HBc positive mother. Both HBeAg and anti-HBe were detected in 44.4% of the samples tested. HBsAg subtypes found in the studied population were adw2 (77.7%) and ayw2 (23.3%). While intrafamilial transmission was most likely responsible for HBV infection among children, other routes such as sexual contact might be considered for individuals with ages over 15 years.

The promise of advanced imaging techniques for the detection of hepatitis C virus antigens in the infected liver.

Injection drug use before and after liver transplantation: a retrospective multicenter analysis on incidence and outcome. Clin Transplant 2009 DOI: 10.1111/j.1399-0012.2009.01121.x. Background and aims: Injecting drug use (IDU) before and after liver transplantation (LT) is poorly described. The aim of this study was to quantify relapse and survival in this population and to describe the causes of mortality after LT. Methods: Past injection drug users were identified from the LT listing protocols from four centers in Switzerland and France. Data on survival and relapse were collected and used for uni- and multivariate analysis. Results: Between 1988 and 2006, we identified 59 patients with a past history of IDU. The mean age at transplantation was 42.4 yr and the majority of patients were men (84.7%). The indication for LT was for the vast majority viral cirrhosis accounting for 91.5% of cases, while alcoholic cirrhosis was 5.1%. There were 16.9% of patients who had a substitution therapy before and 6.8% who continued after LT. Two patients (3.4%) relapsed into IDU after LT and died at 18 and 41 months. The mean follow-up was 51 months. Overall survival was 84%, 66%, and 61% at 1, 5, and 10 yr after transplantation. Conclusions: Documented IDU was rare in liver transplanted patients. Past IDU was not associated with poorer survival after LT, and relapse after LT occurred in 3.4%.

Household survey of hepatitis B vaccine coverage among Brazilian children

We conducted a multi-stage household cluster survey to calculate hepatitis B vaccine coverage among children 18-30 months of age in 27 Brazilian cities. Hepatitis B vaccine is administered at birth, 1 month and 6 months of age by Brazil`s national immunization program. Among 17,749 children surveyed, 40.2% received a birth dose within one day of birth, 94.8% received at least one dose of hepatitis B vaccine, and 86.7% completed the three-dose series by 12 months of age. Increased coverage with the birth dose and administration of hepatitis B in combination with diphtheria-tetanus-pertussis-Haemophilus influenzae type b antigens could improve protection against hepatitis B. (C) 2009 Elsevier Ltd. All rights reserved.

Transgenic lettuce seedlings carrying hepatitis B virus antigen HBsAg

The obtainment of transgenic edible plants carrying recombinant antigens is a desired issue in search for economic alternatives viewing vaccine production. Here we report a strategy for genetic transformation of lettuce plants (Lactuca sativa L.) using the surface antigen HBsAg of hepatitis B virus. Transgenic lettuce seedlings were obtained through the application of a regulated balance of plant growth regulators. Genetic transformation process was acquired by cocultivation of cotyledons with Agrobacterium tumefaciens harboring the recombinant plasmid. It is the first description of a lettuce Brazilian variety Vitória de Verão genetically modified.

Liver HLA-G expression is associated with multiple clinical and histopathological forms of chronic hepatitis B virus infection

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Comparative immunogenicity of a PreS/S hepatitis B vaccine in non- and low responders to conventional vaccine

Conventional hepatitis B vaccines do not elicit adequate antibody production in 5-10% vaccinees. This trial tests the ability of a third-generation vaccine, containing PreS1 and PreS2 antigens in addition to the S antigen, to elicit seroprotective titres in documented non- and low-responders, compared with those to a conventional vaccine. In the primary population of non-responders (<10 IU/l anti-HBs antibodies after > or = 4 previous injections of conventional vaccine) an enhanced antibody response was seen to additional injections of the third-generation vaccine compared with a conventional vaccine (absolute difference 14.9%; P = 0.006). Enhanced antibody responses were also found in a population that included low responders.

DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.

Immunogenicity of transgenic plant-derived hepatitis B surface antigen.

The focus of the Children's Vaccine Initiative is to encourage the discovery of technology that will make vaccines more readily available to developing countries. Our strategy has been to genetically engineer plants so that they can be used as inexpensive alternatives to fermentation systems for production of subunit antigens. In this paper we report on the immunological response elicited in vivo by using recombinant hepatitis B surface antigen (rHBsAg) purified from transgenic tobacco leaves. The anti-hepatitis B response to the tobacco-derived rHBsAg was qualitatively similar to that obtained by immunizing mice with yeast-derived rHBsAg (commercial vaccine). Additionally, T cells obtained from mice primed with the tobacco-derived rHBsAg could be stimulated in vitro by the tobacco-derived rHBsAg, yeast-derived rHBsAg, and by a synthetic peptide that represents part of the a determinant located in the S region (139-147) of HBsAg. Further support for the integrity of the T-cell epitope of the tobacco-derived rHBsAg was obtained by testing the ability of the primed T cells to proliferate in vitro after stimulation with a monoclonal anti-idiotype and an anti-idiotype-derived peptide, both of which mimic the group-specific a determinant of HBsAg. In total, we have conclusively demonstrated that both B- and T-cell epitopes of HBsAg are preserved when the antigen is expressed in a transgenic plant.

Chronic Active Hepatitis B with HBV-Associated Nephropathy: Close Resemblance to Lupus Nephritis

We present a rare case of a 23-year-old male incidentally detected with hepatitis B virus (HBV) infection presenting with features suggestive of HBV-associated nephropathy. A renal biopsy specimen suggested a mesangioproliferative glomerulonephritis with a full-house pattern on immunoflourescence consistent with a diagnosis of diffuse lupus nephritis. Glomerular HbeAg and HbsAg antigens were not detectable by immunofluorescence. Antiviral therapy was instituted to suppress viral replication, thereby leading to clinical and virological remission, including that of the glomerulonephritis, without the need for additional immunosuppressant therapy. This case depicts the uniqueness of the presentation of the two conditions mimicking each other, the strategy adopted to prevent the activation of viral replication and the achievement of clinical remission.