HBV genotypes and response to tenofovir disoproxil fumarate in HIV/HBV-coinfected persons.

BACKGROUND: Hepatitis B virus (HBV) genotypes can influence treatment outcome in HBV-monoinfected and human immunodeficiency virus (HIV)/HBV-coinfected patients. Tenofovir disoproxil fumarate (TDF) plays a pivotal role in antiretroviral therapy (ART) of HIV/HBV-coinfected patients. The influence of HBV genotypes on the response to antiviral drugs, particularly TDF, is poorly understood. METHODS: HIV/HBV-co-infected participants with detectable HBV DNA prior to TDF therapy were selected from the Swiss HIV Cohort Study. HBV genotypes were identified and resistance testing was performed prior to antiviral therapy, and in patients with delayed treatment response (>6 months). The efficacy of TDF to suppress HBV (HBV DNA <20 IU/mL) and the influence of HBV genotypes were determined. RESULTS: 143 HIV/HBV-coinfected participants with detectable HBV DNA were identified. The predominant HBV genotypes were A (82 patients, 57 %); and D (35 patients, 24 %); 20 patients (14 %) were infected with multiple genotypes (3 % A + D and 11 % A + G); and genotypes B, C and E were each present in two patients (1 %). TDF completely suppressed HBV DNA in 131 patients (92 %) within 6 months; and in 12 patients (8 %), HBV DNA suppression was delayed. No HBV resistance mutations to TDF were found in patients with delayed response, but all were infected with HBV genotype A (among these, 5 patients with genotype A + G), and all had previously been exposed to lamivudine. CONCLUSION: In HIV/HBV-coinfected patients, infection with multiple HBV genotypes was more frequent than previously reported. The large majority of patients had an undetectable HBV viral load at six months of TDF-containing ART. In patients without viral suppression, no TDF-related resistance mutations were found. The role of specific genotypes and prior lamivudine treatment in the delayed response to TDF warrant further investigation.

Absence of hepatitis B virus DNA in patients with hepatitis C and non-A-E hepatitis in the State of São Paulo, Brazil

Occult hepatitis B virus (HBV) infection has been reported as cases in which HBV DNA was detected despite the absence of any HBV serological markers or in cases in which anti-HBc antibody was the sole marker. The aim of the present study was to determine, using the polymerase chain reaction (PCR), whether HBV infection occurs in hepatitis C and non-A-E hepatitis patients without serological evidence of hepatitis B infection in São Paulo State. Two different populations were analyzed: 1) non-A-E hepatitis patients, including 12 patients with acute and 50 patients with chronic hepatic disorders without serological evidence of infection with known hepatitis viruses; 2) 43 patients previously diagnosed as hepatitis C with positive results for anti-HCV and HCV RNA. Among hepatitis C patients, anti-HBc was detected in 18.6% of the subjects. Three different sets of primers were employed for HBV DNA detection by nested PCR, covering different HBV genes: C, S and X. HBV-DNA was not detected in any sample, whereas the positive controls did produce signals. The lack of HBV DNA detection with these pairs of primers could be due to a very low viral load or to the presence of mutations in their annealing sites. The latter is unlikely as these primers were screened against an extensive dataset of HBV sequences. The development of more sensitive methods, such as real time PCR, to detect circular covalent closed DNA is necessary in order to evaluate this question since previous studies have shown that cryptic hepatitis B might occur.

HBV vaccination of HCV-infected patients with occult HBV infection and anti-HBc-positive blood donors

Anti-HBc positivity is a frequent cause of donation rejection at blood banks. Hepatitis B virus (HBV) infection may also occur in HBsAg-negative patients, a situation denoted occult infection. Similarly, very low levels of HBV-DNA have also been found in the sera of patients with chronic hepatitis C virus (HCV) infection, even in the absence of serum HBsAg. Initially we searched for HBV-DNA in serum of 100 blood donors and 50 HCV-infected patients who were HBsAg negative/anti-HBc positive by nested-PCR and by an HBV monitor commercial test for HBV-DNA. Anti-HBs seroconversion rates were measured in 100 blood donors and in 22 patients with chronic HCV infection after HBV vaccination to determine if the HBV vaccination could eliminate an occult HBV infection in these individuals. Occult HBV infection was detected in proportionally fewer blood donors (6/100 = 6%) than chronic hepatitis C patients (12/50 = 24%) (P < 0.05). We noted seroconversion in 6/6 (100%) HBV-DNA(+) and in 84/94 (89.4%) HBV-DNA(-) blood donors (P > 0.05). All subjects who were HBV-DNA(+) before the first dose of HBV vaccine (D1), became HBV-DNA(-) after D1, D2, and D3. Among 22 HCV-positive patients, 10 HBV-DNA(+) and 12 HBV-DNA(-), seroconversion was observed in 9/10 (90%) HBV-DNA(+) and in 9/12 (75%) HBV-DNA(-) subjects (P > 0.05). The disappearance of HBV-DNA in the majority of vaccinated patients suggests that residual HBV can be eliminated in patients with occult infection.

Studio immunologico per l'individuazione di pazienti trapiantati per cirrosi HBV relata che potrebbero sospedere la terapia con immunoglobine specifiche (HBIG)

Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT) is associated with poor graft and patient survival. Treatment with HBV-specific immunoglobulins (HBIG) in combination with nucleos(t)ide analogs is effective in preventing HBV reinfection of the graft and improving OLT outcome. However, the combined immunoprophylaxis has several limitations, mainly the high cost and the lack of standard schedules about duration. So far, the identification of markers able to predict the reinfection risk is needed. Although the HBV-specific immune response is believed to play an essential role in disease outcome, HBV-specific cellular immunity in viral containment in OLT recipients is unclear. To test whether or not OLT recipients maintain robust HBV-specific cellular immunity, the cellular immune response against viral nucleocapsid and envelope-protein of HBV was assessed in 15 OLT recipients and 27 individuals with chronic and 24 subjects with self-limited HBV infection, respectively. The data demonstrate that OLT recipients mounted fewer but stronger clusters of differentiation (CD)8 T cell responses than subjects with self-limited HBV infection and showed a preferential targeting of the nucleocapsid antigen. This focused response pattern was similar to responses seen in chronically infected subjects with undetectable viremia, but significantly different from patients who presented with elevated HBV viremia and who mounted mainly immune responses against the envelope protein. In conclusion, virus-specific CD4 T cell–mediated responses were only detected in subjects with self-limited HBV infection. Thus, the profile of the cellular immunity against HBV was in immune suppressed patients similar to subjects with chronic HBV infection with suppressed HBV-DNA.

HBV genotypes and response to tenofovir disoproxil fumarate in HIV/HBV-coinfected persons

BACKGROUND Hepatitis B virus (HBV) genotypes can influence treatment outcome in HBV-monoinfected and human immunodeficiency virus (HIV)/HBV-coinfected patients. Tenofovir disoproxil fumarate (TDF) plays a pivotal role in antiretroviral therapy (ART) of HIV/HBV-coinfected patients. The influence of HBV genotypes on the response to antiviral drugs, particularly TDF, is poorly understood. METHODS HIV/HBV-co-infected participants with detectable HBV DNA prior to TDF therapy were selected from the Swiss HIV Cohort Study. HBV genotypes were identified and resistance testing was performed prior to antiviral therapy, and in patients with delayed treatment response (>6 months). The efficacy of TDF to suppress HBV (HBV DNA <20 IU/mL) and the influence of HBV genotypes were determined. RESULTS 143 HIV/HBV-coinfected participants with detectable HBV DNA were identified. The predominant HBV genotypes were A (82 patients, 57 %); and D (35 patients, 24 %); 20 patients (14 %) were infected with multiple genotypes (3 % A + D and 11 % A + G); and genotypes B, C and E were each present in two patients (1 %). TDF completely suppressed HBV DNA in 131 patients (92 %) within 6 months; and in 12 patients (8 %), HBV DNA suppression was delayed. No HBV resistance mutations to TDF were found in patients with delayed response, but all were infected with HBV genotype A (among these, 5 patients with genotype A + G), and all had previously been exposed to lamivudine. CONCLUSION In HIV/HBV-coinfected patients, infection with multiple HBV genotypes was more frequent than previously reported. The large majority of patients had an undetectable HBV viral load at six months of TDF-containing ART. In patients without viral suppression, no TDF-related resistance mutations were found. The role of specific genotypes and prior lamivudine treatment in the delayed response to TDF warrant further investigation.

Dually Active HIV/HBV Antiretrovirals Protect Against Incident Hepatitis B Infections: Potential for Prophylaxis.

BACKGROUND  Hepatitis-B virus (HBV) has a detrimental effect on HIV natural course, and HBV vaccination is less effective in the HIV infected. We examine the protective effect of dually active antiretroviral therapy (DAART) for HIV/HBV (Tenofovir/Lamivudine/Emtricitabine) in a large cohort encompassing heterosexuals, men-who-have-sex-with-men (MSM), and intravenous drug users (IDU), who are HIV-infected yet susceptible to HBV, with comprehensive follow-up data about risky behavior and immunological profile. METHODS  We defined an incident HBV infection as the presence of any of HBV serological markers (HBsAg/AntiHBc/HBV-DNA) following a negative baseline AntiHBc test. Patients with positive AntiHBs were excluded. Cox proportional hazard models were utilized, with an incident case of HBV infection as the outcome variable. RESULTS  We analyzed 1,716 eligible patients from the Swiss HIV Cohort Study with 177 incident HBV cases. DAART was negatively associated with incident HBV infection (hazard ratio 0.4, 95%CI 0.2-0.6). This protective association was robust to adjustment (0.3, 0.2-0.5) for condomless sex, √CD4 count, drug use, and patients' demographics. Condomless sex (1.9,1.4-2.6), belonging to MSM (2.7,1.7-4.2) or IDU (3.8,2.4-6.1) were all associated with higher HBV hazard. CONCLUSIONS  Our study suggests that DAART, independently of CD4 count and risky behavior, has a potentially strong public health impact including pre-exposure prophylaxis of HBV co-infection.

Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

Aim: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. Methods: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. Results: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 104/ mL, 6.3 × 102/mL and 1.6 × 103/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 109/mL, 2.08 × 106/mL and 4.40 × 107/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 105/ mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples, Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. Conclusion: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA. © 2006 The WJG Press. All rights reserved.

249 TP53 mutation has high prevalence and is correlated with larger and poorly differentiated HCC in Brazilian patients

Background: Ser-249 TP53 mutation (249(Ser)) is a molecular evidence for aflatoxin-related carcinogenesis in Hepatocellular Carcinoma (HCC) and it is frequent in some African and Asian regions, but it is unusual in Western countries. HBV has been claimed to add a synergic effect on genesis of this particular mutation with aflatoxin. The aim of this study was to investigate the frequency of 249Ser mutation in HCC from patients in Brazil. Methods: We studied 74 HCC formalin fixed paraffin blocks samples of patients whom underwent surgical resection in Brazil. 249Ser mutation was analyzed by RFLP and DNA sequencing. HBV DNA presence was determined by Real-Time PCR. Results: 249Ser mutation was found in 21/74 (28%) samples while HBV DNA was detected in 13/74 (16%). 249Ser mutation was detected in 21/74 samples by RFLP assay, of which 14 were confirmed by 249Ser mutant-specific PCR, and 12 by nucleic acid sequencing. All HCC cases with p53-249ser mutation displayed also wild-type p53 sequences. Poorly differentiated HCC was more likely to have 249Ser mutation (OR = 2.415, 95% CI = 1.001 - 5.824, p = 0.05). The mean size of 249Ser HCC tumor was 9.4 cm versus 5.5 cm on wild type HCC (p = 0.012). HBV DNA detection was not related to 249Ser mutation. Conclusion: Our results indicate that 249Ser mutation is a HCC important factor of carcinogenesis in Brazil and it is associated to large and poorly differentiated tumors.

Predictors of HBeAg status and hepatitis B viraemia in HIV-infected patients with chronic hepatitis B in the HAART era in Brazil

Background: HBV-HIV co-infection is associated with an increased liver-related morbidity and mortality. However, little is known about the natural history of chronic hepatitis B in HIV-infected individuals under highly active antiretroviral therapy (HAART) receiving at least one of the two drugs that also affect HBV (TDF and LAM). Information about HBeAg status and HBV viremia in HIV/HBV co-infected patients is scarce. The objective of this study was to search for clinical and virological variables associated with HBeAg status and HBV viremia in patients of an HIV/HBV co-infected cohort. Methods: A retrospective cross-sectional study was performed, of HBsAg-positive HIV-infected patients in treatment between 1994 and 2007 in two AIDS outpatient clinics located in the Sao Paulo metropolitan area, Brazil. The baseline data were age, sex, CD4 T+ cell count, ALT level, HIV and HBV viral load, HBV genotype, and duration of antiretroviral use. The variables associated to HBeAg status and HBV viremia were assessed using logistic regression. Results: A total of 86 HBsAg patients were included in the study. Of these, 48 (56%) were using combination therapy that included lamivudine (LAM) and tenofovir (TDF), 31 (36%) were using LAM monotherapy, and 7 patients had no previous use of either one. Duration of use of TDF and LAM varied from 4 to 21 and 7 to 144 months, respectively. A total of 42 (48. 9%) patients were HBeAg positive and 44 (51. 1%) were HBeAg negative. The multivariate analysis revealed that the use of TDF for longer than 12 months was associated with undetectable HBV DNA viral load (serum HBV DNA level < 60 UI/ml) (p = 0. 047). HBeAg positivity was associated with HBV DNA > 60 UI/ml (p = 0. 001) and ALT levels above normality (p = 0. 038). Conclusion: Prolonged use of TDF containing HAART is associated with undetectable HBV DNA viral load. HBeAg positivity is associated with HBV viremia and increased ALT levels.

Hepatitis B Infection Is Associated with Asymptomatic Malaria in the Brazilian Amazon

Background: Areas that are endemic for malaria are also highly endemic for hepatitis B virus (HBV) infection. Nevertheless, it is unknown whether HBV infection modifies the clinical presentation of malaria. This study aimed to address this question. Methodology and Findings: An observational study of 636 individuals was performed in Rondonia, western Amazon, Brazil between 2006 and 2007. Active and passive case detections identified Plasmodium infection by field microscopy and nested Polymerase Chain Reaction (PCR). HBV infections were identified by serology and confirmed by real-time PCR. Epidemiological information and plasma cytokine profiles were studied. The data were analyzed using adjusted multinomial logistic regression. Plasmodium-infected individuals with active HBV infection were more likely to be asymptomatic (OR: 120.13, P < 0.0001), present with lower levels of parasitemia and demonstrate a decreased inflammatory cytokine profile. Nevertheless, co-infected individuals presented higher HBV viremia. Plasmodium parasitemia inversely correlated with plasma HBV DNA levels (r=-0.6; P=0.0003). Conclusion: HBV infection diminishes the intensity of malaria infection in individuals from this endemic area. This effect seems related to cytokine balance and control of inflammatory responses. These findings add important insights to the understanding of the factors affecting the clinical outcomes of malaria in endemic regions.

Review of genetic and epigenetic alterations in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is associated with multiple risk factors and is believed to arise from pre-neoplastic lesions, usually in the background of cirrhosis. However, the genetic and epigenetic events of hepatocarcinogenesis are relatively poorly understood. HCC display gross genomic alterations, including chromosomal instability (CIN), CpG island methylation, DNA rearrangements associated with hepatitis B virus (HBV) DNA integration, DNA hypomethylation and, to a lesser degree, microsatellite instability. Various studies have reported CIN at chromosomal regions, 1p, 4q, 5q, 6q, 8p, 10q, 11p, 16p, 16q, 17p and 22q. Frequent promoter hypermethylation and subsequent loss of protein expression has also been demonstrated in HCC at tumor suppressor gene (TSG), p16, p14, p15, SOCS1, RIZ1, E-cadherin and 14-3-3 sigma. An interesting observation emerging from these studies is the presence of a methylator phenotype in hepatocarcinogenesis, although it does not seem advantageous to have high levels of microsatellite instability. Methylation also appears to be an early event, suggesting that this may precede cirrhosis. However, these genes have been studied in isolation and global studies of methylator phenotype are required to assess the significance of epigenetic silencing in hepatocarcinogenesis. Based on previous data there are obvious fundamental differences in the mechanisms of hepatic carcinogenesis, with at least two distinct mechanisms of malignant transformation in the liver, related to CIN and CpG island methylation. The reason for these differences and the relative importance of these mechanisms are not clear but likely relate to the etiopathogenesis of HCC. Defining these broad mechanisms is a necessary prelude to determine the timing of events in malignant transformation of the liver and to investigate the role of known risk factors for HCC.

High Frequency of Lamivudine Resistance Mutations in Brazilian Patients Co-Infected With HIV and Hepatitis B

This study analyzed the genotype distribution and frequency of lamivudine (LAM) and tenofovir (TDF) resistance mutations in a group of patients co-infected with HIV and hepatitis B virus (HBV). A cross-sectional study of 847 patients with HIV was conducted. Patients provided blood samples for HBsAg detection. The load of HBV was determined using an ""in-house"" real-time polymerase chain reaction. HBV genotypes/subgenotypes, antiviral resistance, basal core promoter (BCP), and precore mutations were detected by DNA sequencing. Twenty-eight patients with co-infection were identified. The distribution of HBV genotypes among these patients was A (n = 9; 50%), D (n = 4; 22.2%), G (n = 3; 16.7%), and F (n = 2; 11.1%). Eighteen patients were treated with LAM and six patients were treated with LAM plus TDF. The length of exposure to LAM and TDF varied from 4 to 216 months. LAM resistance substitutions (rtL180M + rtM204V) were detected in 10 (50%) of the 20 patients with viremia. This pattern and an accompanying rtV173L mutation was found in four patients. Three patients with the triple polymerase substitution pattern (rtV173L+ rtL180M + rtM204V) had associated changes in the envelope gene (sE164D + sl195M). Mutations in the BCP region (A1762T, G1764A) and in the precore region (G1896A, G1899A) were also found. No putative TDF resistance substitution was detected. The data suggest that prolonged LAM use is associated with the emergence of particular changes in the HBV genome, including substitutions that may elicit a vaccine escape phenotype. No putative TDF resistance change was detected after prolonged use of TDF. J. Med. Virol. 82:1481-1488, 2010. (C) 2010 Wiley-Liss, Inc.

Patterns of viral load in chronic hepatitis B patients in Brazil and their association with ALT levels and HBeAg status

Serum hepatitis B virus (HBV) DNA [eve[ is a predictor of the development of cirrhosis and hepatocellullar carcinoma in chronic hepatitis B patients. Nevertheless, the distribution of viral load levels in chronic HBV patients in Brazil has yet to be described. This cross-sectional study included 564 participants selected in nine Brazilian cities located in four of the five regions of the country using the database of a medical diagnostics company. Admission criteria included hepatitis B surface antigen seropositivity, availability of HBV viral toad samples and age >= 18 years. Mates comprised 64.5% of the study population. Mean age was 43.7 years. Most individuals (62.1%) were seronegative for the hepatitis B e antigen (HBeAg). Median serum ALT level was 34 U/L. In 58.5% of the patients HBV-DNA levels ranged from 300 to 99,999 copies/mL; however, in 21.6% levels were undetectable. Median HBV-DNA level was 2,351 copies/mL. Over 60% of the patients who tested negative for HBeAg and in whom ALT level was less than 1.5 times the upper limit of the normal range had HBV-DNA levels > 2,000 IU/mL, which has been considered a cut-off point for indicating a liver biopsy and/or treatment. In conclusion, HBV-DNA level identified a significant proportion of Brazilian individuals with chronic hepatitis B at risk of disease progression. Furthermore, this tool. enables those individuals with high HBV-DNA levels who are susceptible to disease progression to be identified among patients with normal or stightly elevated ALT.

Entecavir Treatment for up to 5 Years in Patients with Hepatitis B e Antigen-Positive Chronic Hepatitis B

Sustained virologic suppression is a primary goal of therapy for chronic hepatitis B (CHB). In study entecavir (ETV)-022, 48 weeks of entecavir 0.5 mg was superior to lamivudine for virologic suppression for hepatitis B e antigen (HBeAg)-positive CHB. A total of 183 entecavir-treated patients from ETV-022 subsequently enrolled in study ETV-901. We present the results after up to 5 years (240 weeks) of continuous entecavir therapy. The entecavir long-term cohort consists of patients who received >= 1 year of entecavir 0.5 mg in ETV-022 and then entered ETV-901 with a treatment gap <= 35 days. In ETV-901 the entecavir dose was 1.0 mg daily. For patients with samples available at Year 5, proportions with hepatitis B virus (HBV) DNA <300 copies/mL, normal alanine aminotransferase (ALT) levels, HBeAg loss, and HBeAg seroconversion were determined. In all, 146 patients met criteria for inclusion in the entecavir long-term cohort. At Year 5, 94% (88/94) had HBV DNA <300 copies/mL and 80% (78/98) had normal ALT levels. In addition to patients who achieved serologic responses during study ETV-022, 23% (33/141) achieved HBeAg seroconversion and 1.4% (2/145) lost hepatitis B surface antigen (HBsAg) during study ETV-901. Through 5 years, entecavir resistance emerged in one patient. The safety profile of entecavir was consistent with previous reports. Conclusion: Extended therapy with entecavir through 5 years maintained or increased rates of HBV DNA suppression and ALT normalization. Additional patients also achieved HBeAg loss and seroconversion. Entecavir provides sustained viral suppression with minimal resistance during long-term treatment of HBeAg-positive CHB. (HEPATOLOGY 2010;51:422-430.)

Hepatitis B virus and hepatitis delta virus genotypes in outbreaks of fulminant hepatitis (Labrea black fever) in the western Brazilian Amazon region

The genotypes of hepatitis B (HBV) and delta (HDV) viruses circulating among fulminant hepatitis cases from the western Amazon Basin of Brazil were characterized in this study. HBV and HDV isolates were obtained from liver samples from 14 patients who developed fulminant hepatitis and died during 1978-1989. HBV DNA and HDV RNA were detected in all samples. Phylogenetic analyses of HDV sequences showed that they all clustered with previously characterized sequences of HDV genotype 3 (HDV-3). HBV genotypes F, A and D were found in 50.0, 28.6 and 21.4% of cases, respectively. These results confirm the predominance of HDV-3 in South America and its association with the severe form of hepatitis, and the finding of the co-infection of HDV-3 with different genotypes of HBV suggests that the association between HDV-3 and HBV-F is not necessarily causally related to a more severe clinical course of infection.