BACKGROUND/AIMS: Entecavir is a potent nucleoside analogue with high efficacy and barrier for resistance. We aimed to investigate the long-term efficacy and viral resistance rate of entecavir and explore the factors associated with virologic response, including quantitative hepatitis B surface antigen (qHBsAg) levels. METHODS: One thousand and nine treatment-naïve chronic hepatitis B (CHB) patients were evaluated for cumulative rates of virologic response, biochemical response, and entecavir mutations. The role of baseline qHBsAg for virologic response was assessed in 271 patients with qHBsAg prior to entecavir treatment. RESULTS: The median duration of entecavir treatment was 26.5 months. The cumulative rate of virologic response at years 1, 3, and 5 were 79.0%, 95.6%, and 99.4%, respectively. The cumulative rate of entecavir resistance was 1.0% and 2.1% in years 3 and 5. Multivariate analysis identified baseline hepatitis B e antigen (HBeAg) negative status (p < 0.001) and lower hepatitis B virus (HBV) DNA (p < 0.001) as predictors of virologic response. Lower qHBsAg was an independent predictor of virologic response in patients with baseline qHBsAg. There were no serious adverse events during treatment. CONCLUSIONS: Long-term entecavir treatment of nucleos(t)ide-naïve CHB patients was associated with an excellent virologic response and a low rate of entecavir-resistant mutations at 5 years. Baseline HBV DNA load, qHBsAg levels, and HBeAg status were predictors of virologic response during entecavir treatment.
DNA ; Hepatitis B e Antigens ; Hepatitis B Surface Antigens* ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis B, Chronic ; Hepatitis* ; Humans ; Multivariate Analysis

Adult ; Female ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B virus ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Ovary ; virology

Occult HBV infection is characterized by the absence of serum HBsAg with persistence of low level of intrahepatic HBV DNA. Several suggested mechanisms for the origin of occult HBV infection include strong suppression of viral replication and gene expression, mutation in the regulatory regions of HBV genome, formation of immunoglobulin-bound HBsAg, viral interference, and blockage of HBsAg secretion from infected hepatocytes. Standardized assays are not yet available, and sensitive HBV DNA amplification assay is necessary for the diagnosis of cryptic infection. Detection rate of HBV DNA is highest in IgG anti-HBc positive population. However, neither anti-HBc nor anti-HBs can be detected in a significant proportion of infected persons. Occult HBV infection occurs in a number of clinical settings and is highly prevalent in HCV-infected patients as well as in patients with cryptogenic chronic liver disease including hepatocellular carcinoma.
DNA, Viral/analysis ; Hepatitis B/*diagnosis/*epidemiology/metabolism ; Hepatitis B Antibodies/blood ; Hepatitis B Core Antigens/immunology ; Hepatitis B Surface Antigens/blood ; Humans

No abstract available.
DNA, Circular/*analysis ; Female ; Hepatitis B/*pathology ; Hepatitis B Surface Antigens/*genetics ; Hepatitis B virus/*metabolism ; Humans ; Male

BACKGROUND/AIMS: Long-term use of nucleos(t)ide analogues (NA) may lead to genotypic and/or phenotypic resistance of the hepatitis B virus (HBV). We investigated the efficacy of tenofovir-based rescue therapy in chronic hepatitis B (CHB) patients with newly developed genotypic resistance to prior NAs or partial virologic response to sequential rescue therapies. METHODS: Fifty-four CHB patients were included retrospectively. The patients were treated with tenofovir alone or combined with lamivudine or entecavir. RESULTS: There were 26 forms of genotypic resistance at enrollment. The median amount of serum HBV-DNA was 18,438 IU/mL and 83% of samples were positive for hepatitis B e antigen (HBeAg). Serum HBV-DNA was undetectable in 50%, 61%, and 76% of the patients at 3, 6, and 12 months, respectively. In multivariate analysis, HBV-DNA < 20,000 IU/mL and negative HBeAg at baseline were independent predictors of negativity for serum HBV-DNA. Interestingly, the rtS202 mutation tended to be associated with an unfavorable response. Other clinical variables and viral resistance genotypes showed non-significant viral response. CONCLUSIONS: Lower serum HBV-DNA, negative HBeAg and lack of rtS202G mutations at baseline may predict a favorable response to tenofovir-based rescue therapies in CHB patients with newly developed genotypic resistance to prior NAs or a partial virologic response to sequential rescue therapies.
Drug Resistance ; Genotype ; Hepatitis B ; Hepatitis B e Antigens ; Hepatitis B virus ; Hepatitis B, Chronic* ; Hepatitis, Chronic* ; Humans ; Lamivudine ; Multivariate Analysis ; Retrospective Studies ; Tenofovir

BACKGROUND: The emergence of YIDD or YVDD mutant hepatitis B virus (HBV), with point mutation in the YMDD motif of DNA polymerase gene, has been reported in patients with lamivudine treatment group. The aims of this study was to investigate the emergence of mutant HBV during long-term lamivudine therapy using nested polymerase chain reaction (PCR) method and direct DNA sequencing. METHODS: Twenty-one chronic hepatitis B patients with HBeAg and HBV DNA positive were evaluated. During lamivudine therapy, there were reported breakthroughs of HBV DNA (over 50 pg/mL) when investigated the emergence of YMDD mutants by nested PCR method using restriction fragment length polymorphism (RFLP) in all patients. Direct DNA sequencing of HBV DNA polymerase gene including YMDD motif was also performed. RESULTS: There were 13 patients (61.9%) with YIDD mutant and 8 patients (38.1%) with YVDD mutant. The results of direct DNA sequencing were consistent with those of nested PCR data based on RFLP. The breakthrough was occurred at 15 to 106 weeks (57.9+/-23.6). At the point of breakthrough, the level of ALT was 74.8+/-117.7 (14-546) IU/L, and it was lower than the level of ALT before the therapy. CONCLUSION: In the long-term therapy of lamivudine, the emergence of YMDD motif mutant HBV was related to the breakthrough of HBV DNA and YIDD mutant was frequent. The nested PCR method using RFLP may be simple and sensitive to detect the YMDD motif mutant HBV.
DNA ; Hepatitis B e Antigens ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis B, Chronic ; Hepatitis* ; Humans ; Lamivudine* ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA

<b>OBJECTIVEb>To establish an in vitro model of hepatitis B virus (HBV) replication and expression in primary rat hepatocytes (PRH) transfected with circular viral DNA for further study on the interaction of HBV with hepatocytes.

<b>METHODSb>Circular viral DNA containing complete HBV genome were transfected into PRH by electroporation (transfected group, about 4mug of circular viral DNA/1 10(7)cells). From day 1 to day 10 after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH were measured with IMX system. HBcAg was assayed with western blotting, immunol dot blotting and immunocytochemistry. Meanwhile, HBV S-mRNA and X-mRNA were tested with RT-PCR, and replicative intermediates of HBV DNA were analyzed by southern blotting and dot blotting. Moreover, Transmission electron microscopy was used if viral particles were produced in transfected rat hepatocytes. PRH electroporated only was used as control group.

<b>RESULTSb>(1) Viral antigen production in transfected rat hepatocytes: HBsAg in cell lysates was positive. P/N values ranged from 4.83 to 85.69, and could be maintained for 10 days after transfection. The average P/N values was 18.239 27.459. Whereas, HBsAg was negative in the supernatants of transfected group (P/N values, negative<2.1). HBeAg in the supernatants and lysates of transfected hepatocytes all was negative (P/N values<2.1) during 10 days following transfection. HBcAg was only found positive in transfected hepatocytes by immunol dot blotting. (2) Detection of viral transcripts: transcription of HBV DNA was investigated by preparing total RNA from rat hepatocytes 2 days after transfection and looking for S-mRNA and X-mRNA by RT-PCR. Results showed S-mRNA positive, X-mRNA negative. (3) HBV DNA replication analysis: intracellular total DNA was extracted 2 days after transfection and analysed by southern blotting. All replicative DNA intermediates, including relaxed circular (rcDNA), covalently closed circular (cccDNA), and single-stranded (ssDNA) linear HBV DNA forms, were indicated. Dot blotting showed intracellular HBV DNA positive in transfected group during 10 days after transfection. However, viral particles were not found in transfected hepatocytes during 3 days after transfection.

<b>CONCLUSIONSb>Circular HBV DNA transfected into primary adult rat hepatocytes could obtain continuous replication and stable expression of HBV surface antigen. This in vitro model has high reproducibility and stability, and is useful for directly studying the interaction of HBV with hepatocytes.

Animals ; DNA Replication ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Gene Expression ; Hepatitis B Core Antigens ; analysis ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; Hepatocytes ; virology ; Male ; Rats ; Rats, Wistar ; Transfection ; Virus Replication

Background: Cytokines play an important role in occurrence and recovery of hepatitis B virus (HBV) infection. The aim of this study was to investigate the changes of cytokines concentration and its correlation to alanine aminotransferase (ALT), HBV deoxyribonucleic acid (HBV-DNA), hepatitis B envelope antigen (HBeAg), and HBV surface antigen (HBsAg) in the development of chronic hepatitis B (CHB). Methods: Thirteen healthy individuals (HI), 30 chronic HBV-infected patients in immune tolerant (IT) phase, and 55 CHB patients were enrolled between August 2015 and May 2017. The peripheral blood samples were collected from all individuals. The levels of interferon (IFN)-α2, interleukin (IL)-10, transforming growth factor (TGF)-β1, HBV-DNA, HBsAg, and HBeAg and liver function were measured. The quantitative determinations of cytokines levels, including IFN-α2, IL-10, and TGF-β1 were performed using Luminex multiplex technology. The correlation of cytokines to ALT, HBV-DNA, HBsAg, and HBeAg was analyzed by linear regression analysis. Results: IFN-α2 levels were similar between HI and IT groups (15.35 [5.70, 67.65] pg/ml vs. 15.24 [4.07, 30.73] pg/ml, Z = -0.610, P = 0.542), while it elevated significantly in CHB group (35.29 [15.94, 70.15] pg/ml vs. 15.24 [4.07, 30.73] pg/ml; Z = -2.522, P = 0.012). Compared with HI group (3.73 [2.98, 11.92] pg/ml), IL-10 concentrations in IT group (5.02 [2.98, 10.11] pg/ml), and CHB group (7.48 [3.10, 18.00] pg/ml) slightly increased (χ = 2.015, P = 0.365), and there was no significant difference between IT and CHB group (Z = -1.419, P = 0.156). The TGF-β1 levels among HI (3.59 ± 0.20 pg/ml), IT (3.62 ± 0.55 pg/ml), and CHB groups (3.64 ± 0.30 pg/ml) were similar (χ = 2.739, P = 0.254). In all chronic HBV-infected patients (including patients in IT and CHB groups), the elevation of IFN-α2 level was significantly associated with ALT level (β= 0.389, t = 2.423, P = 0.018), and was also negatively correlated to HBV-DNA load (β = -0.358, t = -2.308, P = 0.024), HBsAg (β = -0.359, t = -2.288, P = 0.025), and HBeAg contents (β = -0.355, t = -2.258, P = 0.027). However, when both ALT level and cytokines were included as independent variable, HBV-DNA load, HBsAg, and HBeAg contents were only correlated to ALT level (β = -0.459, t = -4.225, P = 0.000; β = -0.616, t = -6.334, P = 0.000; and β = -0.290, t = -2.433, P = 0.018; respectively). Conclusions IFN-α2 elevation was associated with ALT level in patients with chronic HBV infection. However, in CHB patients, only ALT level was correlated to HBV-DNA, HBsAg and HBeAg contents.
Adult ; Alanine Transaminase ; blood ; Antigens, Surface ; Case-Control Studies ; Cytokines ; blood ; DNA, Viral ; Female ; Hepatitis B ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; Hepatitis B virus ; Hepatitis B, Chronic ; blood ; immunology ; Humans ; Male ; Young Adult

BACKGROUND/AIMS: Clinical characteristics of patients with chronic hepatitis B (CHB) who developed genotypic resistance to entecavir (ETV) were compared to those without resistance. METHODS: Two hundred fifty eight CHB patients who underwent ETV treatment in our institution from July 2007 to May 2013 were included. RESULTS: Eight (3.1%) patients developed genotypic resistance to ETV during the follow-up period. The patterns of genotypic resistance to ETV were as follows: L180M + M204V + S202G (n=3); M204I + V173M (n=1); I169V + V173M (n=1); L180M + M204V + V173L (n=1); L180M + M204V + V173L + M250V (n=1); M204I + V214A + P237H (n=1). The cumulative occurrence rates of genotypic resistance to ETV were not significantly different between CHB patients with prior nucleos(t)tide analogues (NA) exposure (NA experienced, n=56) and NA naïve patients (n=202, P=0.823 by log rank comparison). Older age, higher baseline log10hepatitis B virus-deoxynucleic acid (log10HBV-DNA), higher log10HBV-DNA at 3, 6, 12 and 24 months after baseline, and complete virologic response (CVR, undetectable serum HBV-DNA by polymerase chain reaction 6 months after ETV treatment) were significant contributors to the development of genotypic resistance to ETV. Multivariate analyses showed higher log10HBV-DNA 6 months after baseline and absence of CVR were independent and significant contributors to the development of ETV resistance. CONCLUSIONS: Clinical characteristics of patients who developed ETV resistance were higher log10HBV-DNA 6 months after baseline and absence of CVR during the ETV treatment.
Follow-Up Studies ; Hepatitis B e Antigens ; Hepatitis B, Chronic* ; Hepatitis, Chronic* ; Humans ; Multivariate Analysis ; Polymerase Chain Reaction

<b>BACKGROUNDb>To establish a new assay for detecting the quantity of HBV DNA with PCR and enzyme-linked immunosorbent assay(ELISA).

<b>METHODSb>The products of PCR using primers pre-labeled with biotin were hybridized with the capture probes that were immobilized on the microtiter strips and then bond with Sav-Ap. The quantity of DNA was detected by measuring the yellow color at 405 nm wave length.

<b>RESULTSb>Totally 125 sera from patients with hepatitis B were tested for HBV DNA by this method,the sera were also tested for HBV immunological markers by solid phase radio immuno-assay (SPRIA). The HBV positive rate with PCR-ELISA was 64.9% (24/37) in samples which were positive for HBsAg, HBeAg and HBcAb; and 34.2% (13/38) in sera which were positive for HBsAg, HBeAb and HBcAb; in sera positive for HBsAg and HBcAb or only HBcAb, the positive rate was 6.7% (1/15) and 5.9% (2/34) respectively.

<b>CONCLUSIONSb>The PCR ELISA assay is simple and suitable for clinical laboratory in quantitative determination of HBV DNA.

DNA Probes ; DNA, Viral ; analysis ; biosynthesis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; Sensitivity and Specificity