Low HBsAg Levels Research Articles

F-17 Serum HBsAg decline is faster than previously estimated in long-term NUC responders with low treatment-induced HBsAg levels

F-17 Serum HBsAg decline is faster than previously estimated in long-term NUC responders with low treatment-induced HBsAg levels

Hepatitis B surface antigen serum level is associated with fibrosis severity in treatment-naïve, e antigen-positive patients

Little is currently known about the association between serum HBsAg or HBV DNA levels and the severity of liver disease in chronic hepatitis B (CHB) patients. Therefore, we investigated these relationships in a large cohort of unselected, well-characterized, treatment-naïve CHB patients. CHB patients were assessed at the Hôpital Beaujon in Paris, France, between 2000 and 2008. Serum samples and liver biopsies were obtained on the same day. HBsAg, HBV DNA, and HBV genotype were investigated using commercial diagnostic assays and liver histology was scored using the METAVIR system. 406 patients were included in this cross-sectional study. Serum HBsAg and HBV DNA levels in hepatitis B e antigen-positive (HBeAg[+]) patients showed strong correlation (r=0.44, p<0.0001), as did serum HBsAg levels and fibrosis severity (r=0.43, p<0.0001). HBeAg(+) patients with moderate to severe fibrosis exhibited significantly lower serum HBsAg and HBV DNA levels compared with patients with no or mild fibrosis. Modeling analysis suggested a serum HBsAg cut-off of 3.85 logIU/ml would provide a theoretical sensitivity of 100% (95% CI: 0-100), theoretical specificity of 86% (95% CI: 50-100), and a negative predictive value of 100% (95% CI: 67-100) in HBeAg(+) patients infected with HBV genotype B or C. We found an association between low serum HBsAg levels and moderate to severe fibrosis in HBeAg(+) CHB patients. Furthermore, we described a serum HBsAg cut-off for the prediction of fibrosis severity in CHB patients infected with HBV genotype B or C.

Hepatitis B virus basal core promoter mutations A1762T/G1764A are associated with genotype C and a low serum HBsAg level in chronically-infected HBeAg-positive Chinese patients

The present study was aimed to obtain baseline information of basal core promoter A1762T/G1764A and precore G1896A mutations of hepatitis B virus (HBV) in 192 HBeAg-positive chronically-infected Chinese patients, who were potential candidates for antiviral treatment. The detection of these mutations (including minor mutant subpopulations) was achieved by direct sequencing, whose sensitivity for minor mutant subpopulations identification was confirmed by clone sequencing. Patients enrolled were infected with either genotype B (46.35%) or C (53.65%) HBV identified by routine tests in our laboratory. The A1762T/G1764A or G1896A mutations were detected in 125specimens (125/192, 65.10%), in which 77 (77/125, 61.60%) existed as subpopulations. The A1762T/G1764A mutations were found to be more prevalent in genotype C than that in genotype B HBV [62.14% (64/103) vs. 20.22% (18/89), P<0.0001]. There is no statistically significant link between G1896A and genotypes. The emergence of A1762T/G1764A mutations was also found to be associated with an older age, an elevated ALT/AST level, and a lower HBsAg level in serum [wild-type vs. mutant: 4.57 (3.46–5.42) vs. 3.93 (2.51–5.36), P<0.0001]. In conclusion, HBV basal core promoter mutations A1762T/G1764A are associated with genotype C and a low serum HBsAg level in chronically-infected HBeAg-positive Chinese patients.

High HBV Viral Loads in HIV-Infected Pregnant Women at a Tertiary Hospital, South Africa

High HBV Viral Loads in HIV-Infected Pregnant Women at a Tertiary Hospital, South Africa

Adding pegylated interferon to a current nucleos(t)ide therapy leads to HBsAg seroconversion in a subgroup of patients with chronic hepatitis B

BackgroundNucleos(t)ides effectively halt disease progression in hepatitis B but require long-term medication. ObjectivesTo determine whether add-on of peg-IFN to an ongoing nucleos(t)ide therapy accelerates decline of HBsAg and induces seroconversion. Study designWe observed HBsAg kinetics in 12 patients on a stable oral therapy with undetectable HBV-DNA who additionally received peg-IFN-alfa 2a as an individualized therapy. 3 patients were HBeAg positive. Mean baseline HBsAg was 4695 (range 16–15,120)IU/ml. ResultsA continuous decline of HBsAg was observed in 2 patients. The slope, respectively, became detectable at week 8 or 16. HBsAg had dropped by 2.90log10 or 4.25log10 fold at week 48, and anti-HBs appeared at week 40 or 32. Patient A – HBe-positive, genotype A, F3 fibrosis – had been HBV-DNA negative for 10 months receiving entecavir plus tenofovir. Previous therapy with peg-IFN had been unsuccessful, but now the patient experienced HBeAg seroconversion at week 24. Patient B – HBeAg negative, genotype D, cirrhosis – had a low initial HBsAg level of 16U/l. Receiving entecavir, his HBV-DNA had previously been non-detectable for 27 months. In the remaining 10 patients HBsAg declined only by a mean of 0.09log10 (range 0.01–0.25log10) after 8–24 (mean 16.4) weeks, and therefore, peg-IFN was stopped. No unexpected side effects were observed. DiscussionWe observed that the add-on of peg-IFN induced HBsAg seroconversion in 2 out of 12 patients. Response rates may have been higher with prolongation of therapy. The add-on concept merits to be evaluated in a clinical trial.

HDV-RNA and HBsAg Evolution during peg-IFN Treatment in Three HBV-HDV-HCV-HIV Coinfected Patients

Background: Patients coinfected with HBV, HDV, HCV and HIV are usually excluded from clinical trials. Data on pegylated interferon treatment in this setting are limited, with predictive factors for HDV virologic success being unknown. Objectives: In this study we analyzed the time course of HDV viral load and HBsAg in HBV-HDV-HCV-HIV patients, who underwent pegylated alfa-2a interferon (peg-IFN) therapy for HDV infection between 2005 and 2009, with different virologic outcomes (no response, relapse or sustained response). Methods: Three patients were selected for virologic analysis, since complete clinical and laboratory data and stored residual blood samples, collected before/during/after peg-IFN treatment were available. Plasma samples were retrospectively analyzed for HDV-RNA detection and quantitative HBsAg determination. Results: All patients were HCV-Ab positive, persistently HCV-RNA negative, and received a peg-IFN treatment curse (180 mcg/week) for 12 to 18 months. HIV and HBV viral loads remained undetectable due to underlying Tenofovir/Emtricitabine (TDF/FTC) treatment. Low baseline HDV-RNA and HBsAg levels were both observed in the patient with sustained viral response. A HDV-RNA decline greater than 2log10 at month 6 was observed in two of the three patients, both with compensated liver cirrhosis, achieving a viral clearance at the end of treatment. Conclusions: Although performed in few patients, this study suggests that a decline of HDV-RNA during treatment and low baseline quantitative HBsAg may be associated to HDV virologic response to peg-IFN in HIV-infected subjects, independently of fibrosis stage. If confirmed on larger patient number, these data may help to select those HDV-infected patients with a reliable chance to respond to prolonged peg-IFN treatment and suggest the importance of quantitative HBsAg monitoring in this setting.

P66 Low pre-treatment plasma HBsAg level and its decline during add-on interferon treatment predict response to combined lamivudine/interferon therapy in tolerant children with infancy-acquired chronic hepatitis B

IntroductionChanges in HBsAg plasma levels during antiviral therapy with pegylated interferon (IFN) predict response in adults with chronic hepatitis B (CH-B). Whether pre-treatment HBsAg plasma levels correlate with liver relaxed...

Evaluation of different methods in determination of low level HBsAg

To evaluate chemiluminescent immunoassay (CLIA), electrochemiluminescent immunoassay (ECLIA) and ELISA in determination of low level HBsAg. According to the standard of CLIA Architect i2000, 70 samples were divided into three groups by HBsAg concentration : <1 ng/ml, 1-5 ng/ml and >4 ng/ml. The samples were also determined by ECLIA MODULAR<E>170 and ELISA, and the results were compared with those measured by CLIA Architect i2000. The concordance rates of ECLIA MODULAR<E>170 with Architect i2000 was 79.2% for <1 ng/ml group, 100% for 1-5 ng/ml group, 100% for >4 ng/ml group. And the concordance rates of ELISA with Architect i2000 was 0 % for <1 ng/ml group, 45.5% for 1-5 ng/ml group and 100 % for >4 ng/ml group. For determination of low level HBsAg,CLIA Architect i2000 and ECLIA MODULAR<E>170 have high credibility. ELISA is also credible when HBsAg >5 ng/ml, but not for low level HBsAg, especially for HBsAg <1 ng/ml.

Analysis on clinical features and immunity in chronic hepatitis B virus infected patients with low-level HBsAg

To understand clinical features and immunity of chronic HBV-infected patients with high or low level HBsAg, the differences of serum two-component-determined circulating immune complexes (TCIC) and peripheral blood lymphocyte subsets between these patients were analyzed. 126 patients with chronic hepatitis B virus infection were divided into two groups including 44 with low-level HBsAg and 82 with high-level HBsAg, and three phases including non-active phase, immune active phase and immune tolerant phase by level of HBsAg and natural history of chronic hepatitis B virus infection. Antibody capture-ELISA method and flow cytometry were used to assay serum TCIC and peripheral blood lymphocyte subsets in 44 patients with low-level HBsAg (Group A), 82 patients with high-level HBsAg (Group B) and 22 healthy volunteers (Group C), respectively, and the results were analyzed and compared. Among these 44 patients in Group A, 40 patients (90.9%) were stable in non-active phase and the positive rate of HBsAg/IgG-CIC was the highest, accounting for 15.9% (7/44), while the positive rate of HBsAg/C3-CIC was the highest in Group B, accounting for 46.3% (38/82). The positive rates of serum TCIC, A value and non-active CD3+CD8+ percentages in Group A were all lower than those in Group B with corresponding phases (P<0.01-0.05), however, the percentages of CD4+/CD8+were higher than those in the corresponding phases of Group B(P<0.01). Patients with low-level HBsAg were correlated to low capability of TCIC synthesis and elimination and there was a low dose-induced immunotolerance phenomenon. Key words: Immune complex, chronic HBV infection, HBsAg, lymphocyte subsets, immunotolerance.

16 PREVALENCE AND RISK FACTOR OF HBV INFECTION AMONG 4959 IMMIGRANTS FROM NON EU COUNTRIES IN ITALY

16 PREVALENCE AND RISK FACTOR OF HBV INFECTION AMONG 4959 IMMIGRANTS FROM NON EU COUNTRIES IN ITALY

Molecular analysis on chronic hepatitis B patients with low-level HBsAg

Objective To investigate the molecular characteristics and epidemiological signification of patients with low-level HBsAg. Methods PCR and gene sequencing were used to detect HBV DNA and Tyr-Met-Asp-Asp(YMDD) mutant in 136 serum samples with low-level HBsAg and 44 sernm samples with high-level HBsAg. Genotyping was performed in 47 cases with HBV DNA 105 copies/L by concentration method and 37 cases with high-level HBsAg. S gene sequences and serotypes were analyzed in 14 cases with HBV DNA 105 copies/L and 29 cases with high-level HBsAg. S gene sequences were compared with the consensus sequence of Chinese strain by BioEdit software. Results The HBV DNA-positive rate, YMDD mutation rate and HBV DNA load (logarithm) in low-level and high-level HBsAg group were 34.6% (47/136), 0% (0/136), 6.5±1.4 and 84.1% (37/44), 9.1% (4/44), 8.9±1.8, respectively. There was statistically significant differences between two groups (for concentration method,χ2 = 30.8, P 0.05). S gene sequencing results showed no S gnne variation was detected, but there were 6 single nucleotide polymorphisms in 16 cases, which would not result in the alternation of amino acid. Conclusions Low-replication phenomenon of HBV DNA was present in patients with low-level HBsAg. The major genotyps and serotype was type B and adw/ayw, respectively. Polymorphic variants have been found in the S gene. The existence of low-level HBsAg might be related with its own molecular characteristics resulting in low expression of HBsAg or immune tolerance induced by low-level HBsAg after HBV infection. Key words: Hepatitis B; chronic; Hepatitis B surface antigen; Polymerase chain reaction; Genotype

Negative correlation between viral load and HBsAg levels in chronic HBV-infected patients

The objective of this study is to reveal the relationship between viral load (as HBV DNA) and HBsAg levels. Ninety-two chronically HBV-infected patients were included in the study. The patients were divided in two different groups: the cirrhotic group (n = 32) and the non-cirrhotic group (n = 60). The correlation between study groups was also examined with regard to HBeAg status. Hepatitis B viral markers (HBsAg, HBeAg, Anti-HBs, anti-HBc and anti-HBe) and HBV viral load of the patients were measured. A significant negative correlation between HBV DNA and HBsAg levels was found in the non-cirrhotic group (p < 0.01). The anti-HBc level was higher in the non-cirrhotic group than in the cirrhotic group (p < 0.016). The viral load was significantly higher in HBeAg (+) patients in comparison with HBeAg (-) cases (p < 0.0001). The HBsAg level was low in HBeAg (+) patients, whereas it was higher in HBeAg (-) cases (p < 0.001). In conclusion, a significant negative correlation between viral load and HBsAg levels was detected in the non-cirrhotic chronically HBV-infected group. Therefore, concomitantly low HBsAg and HBV DNA levels may indicate a better prognosis compared to high HBsAg and low HBV DNA levels.

Low pretreatment serum HBsAg level and viral mutations as predictors of response to PEG-interferon alpha-2b therapy in chronic hepatitis B

Background Viral genomic mutations have become increasingly recognized as being associated with the outcome of chronic HBV infection. However, the role of viral mutations as a predictor of response to pegylated-interferon (PEG-IFN) therapy has so far remained unclear. Study design Viral mutations in the enhancer II/basal core promoter (BCP)/precore and the pre-S regions were characterized by direct sequencing in pretreatment serum samples of 50 patients with chronic hepatitis B (33 HBeAg-positive and 17 HBeAg-negative), who were treated for 48 weeks with PEG-IFN alpha-2b. Results Sustained virological response at 48 weeks post treatment, defined as HBeAg seroconversion and HBV DNA < 2000 IU/mL for HBeAg-positive patients, and HBV DNA < 200 IU/mL for HBeAg-negative patients, was achieved in 12 (36.4%) and 6 (35.3%) of HBeAg-positive and HBeAg-negative patients, respectively. Response to PEG-IFN therapy correlated to low pretreatment HBsAg level but did not correlate with HBV genotype, pretreatment alanine transaminase and HBV DNA levels. In HBeAg-positive hepatitis, PEG-IFN response correlated with the appearance of double BCP mutations (A1762T/G1764A) at baseline ( P = 0.041). In the HBeAg-negative group, response to PEG-IFN therapy was associated with the presence of pre-S mutation/deletions ( P = 0.028). Multivariate analysis identified low pretreatment HBsAg level as an independent factor associated with SVR in both groups. Conclusions Pretreatment quantitative HBsAg determination is useful for predicting response to PEG-IFN therapy. The presence of double BCP and pre-S mutation/deletions at entry may be associated with a high rate of antiviral response in HBeAg-positive and HBeAg-negative hepatitis, respectively.

Prediction of Treatment-Related HBsAg Loss in HBeAg-Negative Chronic Hepatitis B: A Clue from Serum HBsAg Levels

Quantification of HBsAg in serum may be of clinical importance in predicting HBsAg seroconversion and complete response to treatment. Serum HBsAg was quantified by ADVIA Centaur in 63 patients with HBeAg-negative chronic hepatitis B (CHBe-). A total of 42 had received interferon-alpha2b (IFN-alpha2b) (median 12.1 months; 19 sustained responders including 12 HBsAg-seroconvertors; 23 non-sustained responders) and 21 were on lamivudine (LAM) (median 33.0 months). Measurements were done at baseline, during and at the end of treatment, and during and at the end of follow-up. Baseline median [interquartile range (IQR)] HBsAg levels in all patients were 3286 (1602-7458) IU/ml, not different between IFN- and LAM-treated (P = 0.139). IFN significantly depressed HBsAg in all patients except IFN non-responders, but HBsAg decline persisted only in sustained responders. Low pretreatment HBsAg level was the only significant prognostic variable of HBsAg seroconversion by multivariate analysis. LAM treatment also suppressed HBsAg levels but at a significantly slower rate compared with IFN (P = 0.022). The median (IQR) estimated time to HBsAg undetectability (ETU-HBsAg), derived from best curve fitting, was 127 (87.6-263.5) months for LAM virological responders and 65.3 (36.3-95.0) months for IFN sustained responders (P = 0.002). In 12 HBsAg seroconvertors, ETU-HBsAg was similar to the real time of HBsAg loss (P = 0.525) and seroconversion (0.056). In CHBe-, IFN induces a sharper decrease in serum HBsAg compared with LAM and low pretreatment levels are significantly associated with HBsAg serocon-version. Serial HBsAg measurements may be useful for prediction of HBsAg loss and our data suggest that to achieve this, 5.4 years of sustained response to IFN or 10.6 years of effective LAM therapy are probably needed.

O.170 Characterization and functional analyses of hepatitis B virus (HBV) isolated from liver tissue of patients with occult HBV infection

The present study was aimed to obtain baseline information of basal core promoter A1762T/G1764A and precore G1896A mutations of hepatitis B virus (HBV) in 192 HBeAg-positive chronically-infected Chinese patients, who were potential candidates for antiviral treatment. The detection of these mutations (including minor mutant subpopulations) was achieved by direct sequencing, whose sensitivity for minor mutant subpopulations identification was confirmed by clone sequencing. Patients enrolled were infected with either genotype B (46.35%) or C (53.65%) HBV identified by routine tests in our laboratory. The A1762T/G1764A or G1896A mutations were detected in 125 specimens (125/192, 65.10%), in which 77 (77/125, 61.60%) existed as subpopulations. The A1762T/G1764A mutations were found to be more prevalent in genotype C than that in genotype B HBV [62.14% (64/103) vs. 20.22% (18/89), P < 0.0001]. There is no statistically significant link between G1896A and genotypes. The emergence of A1762T/G1764A mutations was also found to be associated with an older age, an elevated ALT/AST level, and a lower HBsAg level in serum [wild-type vs. mutant: 4.57 (3.46–5.42) vs. 3.93 (2.51–5.36), P < 0.0001]. In conclusion, HBV basal core promoter mutations A1762T/G1764A are associated with genotype C and a low serum HBsAg level in chronically-infected HBeAg-positive Chinese patients.

Diagnostic impact of the genetic variability of the hepatitis B virus surface antigen gene

The genetic variability of hepatitis B virus (HBV) represents a challenge for the sensitivity of immunologic and molecular based assays. Genotyping studies show that the genetic diversity of HBV is very high even in industrialized countries. The analytical sensitivity of HBsAg and anti-HBs assays may be dependent on HBV genotype or subtype and could possibly lead to false negative results in samples with low-level HBsAg. It is possible that the recognition of genotypes E and F may be impaired. Immunoassays based on polyclonal capture antibody show the highest sensitivity for the recognition of recombinant mutants or serum samples harboring mutant forms of HBsAg. However, they do not guarantee full sensitivity, especially for the detection of the G145R mutation and amino acid insertions or substitutions in positions 120-123. Detection of HBsAg needs to be improved by the introduction of new HBsAg assays able to recognize so far described S gene mutants and with a lower detection threshold than current immunoassays in order to detect smallest amounts of HBsAg in low level carriers. There is also a need for more complete epidemiological data on the prevalence of HBsAg mutants especially for G145R and assays for the (differential) screening of mutants need to be developed and evaluated.

Variation in the sensitivity of HBsAg screening kits.

Fifteen HBsAg kits from 14 manufacturers were assessed. Their sensitivity was evaluated by testing 150 HBsAg-positive sera, sera from four donors who were low-level HBsAg carriers, and sequential specimens from 22 seroconverting individuals together with dilutions of six of these specimens. The British HBsAg Working Standard (0.5 IU mL-1) and the NIBSC/UKBTS HBsAg Monitor Sample (0.125 IU mL-1) were also tested. Five assays failed to detect one of the 150 routine HBsAg-positive sera. Four assays (Auszyme Monoclonal; Monolisa Ag HBs 2nd generation; Murex HBsAg; Ortho HBsAg Test Systems 3) were able to detect HBsAg in all but one of the six sera from low-level carriers, whereas one assay (MicroTrak II HBsAg) detected only one of the six. The most sensitive kit (Monolisa Ag HBs 2nd generation) detected HBsAg in 79 specimens from the seroconversion panels; four other kits detected HBsAg in at least 70 specimens, seven in 60-69, two in 50-59 and the least sensitive in 31. Further analysis of the findings on seroconverters indicated a median reduction in the duration of HBsAg detection of 5 days or more for four assays when compared with the most sensitive assay. One kit (Auszyme Monoclonal) detected HBsAg in 15 of the 18 dilutions prepared from the seroconversion specimens, whereas three kits detected HBsAg in fewer than 10 dilutions. Two kits gave negative reactions with the British HBsAg Working Standard on all of five occasions and six were consistently unreactive with the NIBSC/UKBTS HBsAg Monitor Sample; only three kits (Bioelisa, Enzygnost, Murex) were always reactive. There is therefore substantial variation in sensitivity among the HBsAg kits currently available.

Properties of Hepatitis B Virus Pre-S1 Deletion Mutants

Deletion mutants of hepatitis B virus (HBV) pre-S proteins were detected in serum in 2 of 10 (20%) individuals with chronic hepatitis B infection following the initiation of interferon treatment. The size of these deletions was up to one-half of the entire pre-S1 region. In vivo, all HBV deletion mutants were found to coexist with a full-length "wild-type" viral genome. The functional properties of a HBV-deleted mutant were studied in detail and revealed a stop codon in the pre-S2 open reading frame in all 20 of the clones sequenced. Several of the deleted mutants produced low-level HBsAg in culture supernatants compared to wild-type virus due to a putative loss of transcription factor binding sites. Transfection experiments in human hepatoma cells (HuH-7) demonstrated that the polymerase gene function was not affected by the large pre-S1 deletions and mutant viral genomes were capable of replication. However, secretion of incapsidated mutant viral genomes was blocked in HuH-7 cells. Cotransfection studies with a plasmid expressing only the HBV pre-S1, pre-S2, and S proteins resulted in complete restoration of vital particle secretion. Our findings suggest that an in vivo trans-complementation phenomenon would have had to occur to permit secretion from the liver into serum of the nucleocapsids containing these deleted viral genomes.