High prevalence of occult hepatitis B infection in an African urban population.

Occult hepatitis B infection (OBI), the presence of low hepatitis B virus (HBV) deoxyribonucleic acid (DNA) levels in patients without detectable hepatitis B surface antigen (HBsAg), has significant implications for understanding the natural history of hepatitis B infection. We determined the prevalence of OBI in African patients using a sensitive polymerase chain reaction (PCR) assay and describe here the characteristics of OBI in an urban African hospital population. Routine serological testing as well as molecular studies were performed on sera from 314 patients who were part of a previous study from an urban hospital emergency room in Kampala, Uganda, detecting HBV DNA using a nested PCR with amplification of two regions of the HBV genome. HBV viral loads (VL) were determined by real-time PCR (rtPCR) and sequencing performed to determine HBV genotype and S gene mutations. Among 314 subjects tested, 50 (16%) had chronic HBV infection, 94 (30%) had detectable HBV DNA despite testing HBsAg negative (OBI), and 170 (54%) were not infected. VLs of OBI subjects were relatively low although 19 (20%) had VL exceeding 10(4) IU ml(-) . Subjects with chronic HBV infection had a higher median VL compared to OBI patients (P < 0.001). All chronic HBV sequenced (10) and 83/89 OBI sequences were genotype A, the remaining six being genotype D. S-gene mutations were present in some but not all OBI patients (48%). OBI is more prevalent among African patients than previously thought. This may have implications for clinical management and transfusion-related HBV transmission.

Plasma osteopontin in acute liver failure.

BACKGROUND: Osteopontin (OPN) is a novel phosphoglycoprotein expressed in Kupffer cells that plays a pivotal role in activating natural killer cells, neutrophils and macrophages. Measuring plasma OPN levels in patients with acute liver failure (ALF) might provide insights into OPN function in the setting of massive hepatocyte injury. METHODS: OPN levels were measured using a Quantikine® ELISA assay on plasma from 105 consecutive ALF patients enrolled by the US Acute Liver Failure Study Group, as well as controls including 40 with rheumatoid arthritis (RA) and 35 healthy subjects both before, and 1 and 3 days after undergoing spine fusion (SF) surgery as a model for acute inflammation. RESULTS: Median plasma OPN levels across all etiologies of ALF patients were elevated 10- to 30-fold: overall median 1055ng/mL; range: 33-19,127), when compared to healthy controls (median in pre-SF patients: 41ng/mL; range 2.6-86.4). RA and SF post op patients had elevated OPN levels (37ng/mL and 198ng/mL respectively), well below those of the ALF patients. Median OPN levels were highest in acetaminophen (3603ng/mL) and ischemia-related ALF (4102ng/mL) as opposed to viral hepatitis (706ng/mL), drug-induced liver injury (353ng/mL) or autoimmune hepatitis (436ng/mL), correlating with the degree of hepatocellular damage, as reflected by aminotransferase values (R value: 0.47 for AST, p<0.001). CONCLUSIONS: OPN levels appeared to correlate with degree of liver necrosis in ALF. Very high levels were associated with hyperacute injury and good outcomes. Whether OPN exerts a protective effect in limiting disease progression in this setting remains uncertain.

Two distinct subtypes of hepatitis B virus-related acute liver failure are separable by quantitative serum immunoglobulin M anti-hepatitis B core antibody and hepatitis B virus DNA levels.

UNLABELLED: Hepatitis B virus (HBV)-related acute liver failure (HBV-ALF) may occur after acute HBV infection (AHBV-ALF) or during an exacerbation of chronic HBV infection (CHBV-ALF). Clinical differentiation of the two is often difficult if a previous history of HBV is not available. Quantitative measurements of immunoglobulin M (IgM) anti-hepatitis B core antibody (anti-HBc) titers and of HBV viral loads (VLs) might allow the separation of AHBV-ALF from CHBV-ALF. Of 1,602 patients with ALF, 60 met clinical criteria for AHBV-ALF and 27 for CHBV-ALF. Sera were available on 47 and 23 patients, respectively. A quantitative immunoassay was used to determine IgM anti-HBc levels, and real-time polymerase chain reaction (rtPCR) was used to determine HBV VLs. AHBV-ALFs had much higher IgM anti-HBc titers than CHBV-ALFs (signal-to-noise [S/N] ratio median: 88.5; range, 0-1,120 versus 1.3, 0-750; P < 0.001); a cut point for a S/N ratio of 5.0 correctly identified 44 of 46 (96%) AHBV-ALFs and 16 of 23 (70%) CHBV-ALFs; the area under the receiver operator characteristic curve was 0.86 (P < 0.001). AHBV-ALF median admission VL was 3.9 (0-8.1) log10 IU/mL versus 5.2 (2.0-8.7) log10 IU/mL for CHBV-ALF (P < 0.025). Twenty percent (12 of 60) of the AHBV-ALF group had no hepatitis B surface antigen (HBsAg) detectable on admission to study, wheras no CHBV-ALF patients experienced HBsAg clearance. Rates of transplant-free survival were 33% (20 of 60) for AHBV-ALF versus 11% (3 of 27) for CHBV-ALF (P = 0.030). CONCLUSIONS: AHBV-ALF and CHBV-ALF differ markedly in IgM anti-HBc titers, in HBV VLs, and in prognosis, suggesting that the two forms are, indeed, different entities that might each have a unique pathogenesis.

Coordinated evolution among hepatitis C virus genomic sites is coupled to host factors and resistance to interferon.

Machine-learning methods in the form of Bayesian networks (BN), linear projection (LP) and self-organizing tree (SOT) models were used to explore association among polymorphic sites within the HVR1 and NS5a regions of the HCV genome, host demographic factors (ethnicity, gender and age) and response to the combined interferon (IFN) and ribavirin (RBV) therapy. The BN models predicted therapy outcomes, gender and ethnicity with accuracy of 90%, 90% and 88.9%, respectively. The LP and SOT models strongly confirmed associations of the HVR1 and NS5A structures with response to therapy and demographic host factors identified by BN. The data indicate host specificity of HCV evolution and suggest the application of these models to predict outcomes of IFN/RBV therapy.

Hepatitis B virus genotype G: prevalence and impact in patients co-infected with human immunodeficiency virus.

Relatively little is known about the role of hepatitis B virus (HBV) genotype G (HBV/G) in patients co-infected with human immunodeficiency virus (HIV) and HBV. This study examined the prevalence and association of HBV/G to liver fibrosis in co-infected patients. HBV genotypes were determined by direct sequencing of the HBV surface gene or Trugene® HBV 1.0 assay in 133 patients infected with HIV/HBV. Quantitative testing of HBV-DNA, HBeAg, and anti-HBe were performed using the Versant® HBV 3.0 (for DNA) and the ADVIA®Centaur assay. The non-invasive biomarkers Fib-4 and APRI were used to assess fibrosis stage. Genotype A was present in 103/133 (77%) of the cohort, genotype G in 18/133 (14%) with genotypes D in 8/133, (6%), F 2/133 (1.5%), and H 2/133 (1.5%). Genotype G was associated with hepatitis B e antigen-positivity and high HBV-DNA levels. Additionally, HBV/G (OR 8.25, 95% CI 2.3-29.6, P = 0.0012) was associated with advanced fibrosis score using Fib-4, whereas, being black was not (OR 0.19, 95% CI 0.05-0.07, P = 0.01). HBV/G in this population exhibited a different phenotype than expected for pure G genotypes raising the question of recombination or mixed infections. The frequent finding of HBV/G in co-infected patients and its association with more advanced fibrosis, suggests that this genotype leads to more rapid liver disease progression. Further studies are needed to understand why this genotype occurs more frequently and what impact it has on liver disease progression in patients with HBV/HIV.

Nonresponse to treatment for hepatitis C: current management strategies.

Chronic hepatitis C affects >170 million people worldwide, causing cirrhosis and liver cancer in a sizeable proportion of patients. Substantial progress has been made in the treatment of chronic hepatitis C. More than 50% of patients can achieve sustained virological response after 24-48 weeks of interferon and ribavirin combination therapy, making chronic hepatitis C a potentially curable disease. However, a large proportion of patients with chronic hepatitis C do not clear the virus after current standard therapy. Hepatitis C virus develops two pathways to counteract the antiviral effect of interferon. Some chronic hepatitis C patients may have a virus that is more resistant to interferon therapy, while other patients appear to have defective immune responses or poor tolerance or compliance to interferon-based antiviral therapy. The possible strategies to improve antiviral efficiency in these nonresponders are to increase the dosage, prolong the duration of treatment and improve the compliance of patients. A total of 6-15% of prior nonresponders to standard interferon plus ribavirin therapy will respond to re-treatment with peginterferon plus ribavirin, while 32-50% of patients who have relapsed will respond to re-treatment. New small molecules are under development to treat chronic hepatitis C and may be important particularly in the treatment of prior nonresponders to current standard therapy.

Molecular mechanisms of resistance to antiviral therapy in patients with chronic hepatitis B.

Similar to the human immunodeficiency virus (HIV), the hepatitis B virus (HBV) replicates via reverse transcription, in this case, within infected hepatocytes. Substantial advances have been achieved in the past ten years in developing and utilizing nucleoside/nucleotide analog drugs to inhibit HBV replication. Most are chain terminators that interfere with one or more steps in the replication cycle. Four of them (lamivudine, adefovir dipivoxil, entecavir, and telbivudine), have been approved by the United States Food and Drug Administration (FDA) for the treatment of chronic hepatitis B (CHB). In clinical trials of HBeAg positive and negative CHB patients, 48-52 week of treatment with these drugs can induce a 4-7 log decrease of HBV viremia and histological improvement. Long-term suppression of active HBV replication has been found to be associated with decreased inflammation, reversal of liver fibrosis and a lower incidence of hepatocellular carcinoma. However, permanent clearance of HBV is rarely achieved with current available antiviral agents, maintenance therapy being required for continuous suppression of HBV replication. In patients on continuous therapy, drug resistant mutations develop with all four drugs. Combination therapy with different nucleos(t)ide analog drugs or nucleos(t)ide drugs and pegylated interferon needs further clinical study. Newer promising nucleotide analog drugs with more potent antiviral efficacy are also under development.

Role of hepatitis B virus genotypes in chronic hepatitis B exacerbation.

Hepatitis B virus (HBV) genotypes and precore and core promoter mutations were determined in 318 patients with HBV. Patients infected with HBV genotype B had a higher median alanine aminotransferase level and bilirubin level and a lower median albumin level during exacerbations of disease, compared with patients infected with HBV genotype C (all P<.001). By logistic regression analysis, HBV genotype B infection (P=.014) and low albumin levels (P=.006) were independently associated with a higher risk of hepatic decompensation during severe exacerbations of disease. Patients infected with genotype B had a significantly higher mortality due to hepatic decompensation than did patients with genotype C (70% vs. 27.8%; P=.05).

Prognostic factors in severe exacerbation of chronic hepatitis B.

Forty-seven patients with severe hepatitis B exacerbation were compared with patients who had mild exacerbation (n=96) or no exacerbation (n=96). Seventeen patients (36.2%) died or underwent liver transplantation. Preexisting cirrhosis and a prothrombin time (PT) of >30 s were associated with adverse outcome in 60.9% and 87.5% of patients, respectively. The rate of adverse outcome increased to 92.3% when albumin levels of < or =35 g/L and bilirubin levels of >200 microM were present. Other factors associated with adverse outcomes included peak bilirubin level, peak PT, time to reach peak PT, and the presence of encephalopathy and/or ascites. There was no difference in the frequency of precore mutations in patients with severe or mild exacerbation or without exacerbation. A significantly lower prevalence of core promoter mutants was found in patients with severe exacerbation (50%), compared with those who had mild exacerbation (81.3%; P=.004). Patients with severe exacerbation of hepatitis B with poor prognostic factors should be considered for early liver transplantation.

Hepatitis B virus genotypes B and C do not affect the antiviral response to lamivudine.

To date, there have been no studies examining the role of hepatitis B virus (HBV) genotypes on the response to lamivudine therapy and the development of YMDD mutations. The present study aimed at determining any differences in the antiviral response and risk of YMDD mutations between lamivudine-treated patients with HBV genotype B and genotype C. Eighty-two patients receiving lamivudine were recruited. HBV genotypes at baseline and YMDD mutations at week 52 were determined by line probe assays (LiPA). HBV DNA levels were determined by the Cobas Amplicor HBV Monitor Test. Seventeen (20.7%) and sixty-four (78%) patients had single genotypes of B and C, respectively. At both week 24 and 52 there were no differences in the median reduction of HBV DNA levels (median 4 logs drop), the median reduction of alanine aminotransferase (ALT) levels, and the proportion with normalization of ALT [8/8 (100%) vs 26/37 (70.3%), P=0.19] between patients with genotypes B and C. The rate of HBeAg seroconversion [3/17 (17.6%) vs 6/64 (9.4%), P=0.39] and the chance of YMDD mutation development [3/17 (17.6%) vs 12/64 (18.8%), P=1.0] at week 52 were also similar between patients with genotype B and C, respectively. In conclusion, there was no difference in the antiviral response and the rate of development of YMDD mutations in Chinese patients with genotype B and C after 1 year of lamivudine. Determination of HBV genotypes before lamivudine therapy was probably not an important pretreatment investigation to predict antiviral responses in Chinese patients.

Change in fibrosis score as a predictor of mortality among HIV-infected patients with viral hepatitis.

Noninvasive markers of liver fibrosis, measured at baseline, have been shown to predict liver-related mortality. It remains unknown if a change in the value of the scores over time predicts mortality in patients with HIV and viral hepatitis. In this retrospective study, survival in HIV/hepatitis B virus (HBV; n = 67), HIV/hepatitis C virus (HCV; n = 43), and HIV/HBV/HCV (n = 41) patients was examined using Kaplan-Meier life table analysis. Aspartate aminotransferase (AST)-to-platelet ratio index (APRI) and FIB-4 scores, two noninvasive markers of liver fibrosis, were calculated at baseline and at last available clinical follow-up to determine the change in fibrosis score. Factors associated with mortality were assessed by Cox proportional hazards, including the change in the noninvasive marker score between the two time points. All-cause mortality was determined by Social Security Death Index and chart review. Sixty-seven were coinfected with HIV/HBV, 43 with HIV/HCV, and 41 were triply infected (HIV/HBV/HCV). Kaplan-Meier analysis showed similar survival for the three groups at 7 years of follow-up (p = 0.10). However, median length of follow-up was lower in HIV/HCV (60.5; range 0-102) compared to HIV/HBV (75.7; 12.3-126.5) and HIV/HBV/HCV (80.0; 2.7-123) months, respectively, p = 0.02. Baseline fibrosis score (p = 0.002), an increase in the value for noninvasive measurements for fibrosis (p < 0.001), and the presence of HIV/HCV coinfection (p = 0.041) were each associated with higher risk for mortality. Baseline fibrosis score (p = 0.03) and an increase in FIB-4 score (p = 0.05) were independent predictors of all-cause mortality, but liver-related mortality was not evaluated. In this study, baseline fibrosis score was predictive of 7-year all-cause mortality. Further studies are needed in a prospective cohort to evaluate the predictive value of monitoring changes in fibrosis scores over time to predict mortality in patients with viral hepatitis.

Pegylated interferon and ribavirin promote early evolution of nonstructural 5A protein in individuals with hepatitis C who demonstrate a response to treatment.

BACKGROUND: Hepatitis C virus (HCV) quasispecies diversity is more likely to affect early viral decline during treatment of hepatitis C than is having human immunodeficiency virus (HIV) infection. We evaluated the influence of HCV therapy on changes in the nonstructural 5A (NS5A) protein. METHODS: Fifteen patients with HCV genotype 1 infection with or without HIV infection were recruited for the present study, and the decrease in the HCV RNA level was measured at early time points. The evolution of HCV NS5A quasispecies within the first week was analyzed by comparing the clones observed at later times in the study with the baseline consensus sequence of individual patients. The response to therapy was defined as an early response (ER; ie, an HCV RNA level <615 IU/mL at week 4) or a slow response (SR; ie, a detectable HCV RNA level at week 4). RESULTS: HIV infection did not affect early viral kinetics. At baseline, lower diversity was seen in NS5A and in the amino and carboxyl termini of patients with an ER, compared with those with an SR. Rapid evolution of the NS5A genetic region occurred in patients with an ER (P = .01) but not in those with an SR (P = .73). The evolution was the result of an increase in the number of amino acid substitutions in the carboxyl region (P = .02) in patients with an ER. CONCLUSIONS: Selective pressure appears to result in more-marked changes in individuals with an ER than in those with an SR. The carboxyl terminus was subject to the most change and may be an important determinant of phenotypic resistance to interferon-based therapy.

Precore and core promoter mutations at the time of HBeAg seroclearance in Chinese patients with chronic hepatitis B.

OBJECTIVE: To determine the prevalence of precore and core promoter (CP) mutations before, at and after HBeAg seroclearance in Chinese patients. METHODS: Precore and CP mutations were determined in 93 patients with chronic hepatitis B 12-24 months before, at and 12 months after the time of HBeAg seroclearance. RESULTS: No significant changes were found in the prevalence of precore or CP mutations before, at and after HBeAg seroclearance. Seven patients (7.8%) had HBeAg seroreversion within 1 year of HBeAg seroclearance. There was no significant difference in the prevalence of precore and CP mutations between patients with and without HBeAg seroreversion. 68.3% and 48.7% of patients harbored the same precore and CP genotypes throughout. 32.0% patients with precore mutations and 8.9% patients with CP mutations before HBeAg seroclearance had reversion to wild type within 1 year of HBeAg seroclearance. Patients with genotype C patients had a higher prevalence of CP mutations before HBeAg seroclearance compared with patients with genotype B (82.4% vs. 44%, P=0.001). CONCLUSIONS: Precore and CP mutations existed in a substantial proportion of Chinese patients before HBeAg seroclearance. The replication of precore and, to a lesser extent, CP mutants could be suppressed around the time of HBeAg seroclearance.

Determinants for the occurrence of acute exacerbation of hepatitis B virus infection in Chinese patients after HBeAg seroclearance.

This study was performed to determine the factors for predicting the occurrence of acute exacerbation of hepatitis B virus infection in HBeAg-negative patients. Two hundred and sixteen patients with known times of HBeAg seroclearance were recruited. Liver biochemistry and virologic markers were monitored. Precore and core promoter mutations were determined by a line probe assay. The median age at HBeAg seroclearance was 34.5 years. The median follow-up duration was 26.4 months. Fifty-six (27.9%) patients had acute exacerbations. By Cox regression analysis, male gender, older age, and core promoter mutations at the time of HBeAg seroclearance were independently associated with the occurrence of acute exacerbation after HBeAg seroclearance (P = 0.025, 0.018, and 0.001, respectively). Fourteen (7.0%) patients had HBeAg seroreversion within a median follow-up period of 11.6 months after HBeAg seroclearance. By Cox regression analysis, older age at HBeAg seroclearance was independently associated with the chance of HBeAg seroreversion (P = 0.01). We concluded that male patients with core promoter mutations and delayed HBeAg seroclearance had a higher cumulative chance of acute exacerbation in the HBeAg-negative phase. Patients with delayed HBeAg seroclearance had a higher frequency of HBeAg seroreversion.

Effect of lamivudine therapy on the serum covalently closed-circular (ccc) DNA of chronic hepatitis B infection.

OBJECTIVE: To determine the effect of 1-yr lamivudine treatment on serum covalently closed-circular DNA (cccDNA) level. PATIENTS AND METHOD: Serum total HBV DNA and cccDNA levels at baseline, week 24, and week 52 were measured in 82 lamivudine-treated patients, 17 of whom received 1-yr placebo and acted as controls. RESULTS: There was a significant reduction in the cccDNA levels from baseline (median 3.0 x 10(6) copies/ml) to week 24 (33,476 copies/ml) and week 52 (48,694 copies/ml) (p < 0.001 for both). The median reduction in cccDNA level at week 24 and 52 were 2.21 and 2.12 logs, respectively, which were significantly greater than those of controls (0.31 log, p < 0.001; 0.2 log, p < 0.001, respectively). Fifteen patients (18.3%) developed YMDD mutations by week 52. Compared to patients without YMDD mutations, patients with YMDD mutations had significantly less median reduction of total HBV DNA level (4.44 vs 3.65 logs, respectively, p= 0.02) and cccDNA level (2.27 vs 1.65 logs, respectively, p= 0.016) at week 24 and significantly less median reduction of cccDNA at week 52 (2.35 vs 0.8 logs respectively, p < 0.001). CONCLUSIONS: One-year lamivudine treatment decreased serum cccDNA level by 2 logs. The chance of YMDD mutations at week 52 was related to the magnitude of viral suppression at week 24.

Real-time quantification of hepatitis B virus core-promoter and pre-core mutants during hepatitis E antigen seroconversion.

BACKGROUND/AIMS: Detection of hepatitis B virus (HBV) core-promoter A(1762)T-G(1764)A and pre-core G(1896)A mutants has relied on qualitative assays. We tested the hypothesis that the quantity of A(1762)T-G(1764)A and G(1896)A mutants might have clinical impact, by quantifying these mutants before and after HBe antigen (HBeAg) seroconversion in 58 patients. METHODS: A real-time quantitative-polymerase chain reaction (Q-PCR) was developed, using minor groove binder (MGB)-conjugated TaqMan probes to impart reaction specificity for wildtype/mutant HBV populations. RESULTS: Significant quantities (>20%) of core-promoter A(1762)T-G(1764)A mutant existed in 65% of patients before and after HBeAg seroconversion, and were significantly changed (>20% increase/decrease) in 13% of patients after seroconversion. Quantity of A(1762)T-G(1764)A mutants was positively correlated with alanine aminotransferase (ALT) (P<0.001) and HBV DNA (P<0.001) levels, both before and after HBeAg seroconversion. Significant quantities of pre-core G(1896)A mutant existed in about 90% of patients before and after HBeAg seroconversion, and were changed in 16% of patients after seroconversion. Quantity of G(1896)A mutant was negatively correlated with ALT (P=0.044) and HBV DNA (P=0.007) levels. CONCLUSIONS: The A(1762)T-G(1764)A and G(1896)A mutants existed in a high proportion of patients before and were unaffected after HbeAg seroconversion. The quantities of A(1762)T-G(1764)A mutant were positively and G(1896)A mutant negatively correlated with liver inflammation and viral replication.

Clinical evaluation of the digene hybrid capture II test and the COBAS AMPLICOR monitor test for determination of hepatitis B virus DNA levels.

The measurement of hepatitis B virus (HBV) DNA is important for the assessment of liver disease and treatment efficacy. Most commercially available assays for the determination of HBV DNA levels have limited linear ranges. This study was performed to evaluate the clinical performance of the Digene Hybrid Capture II (Digene HC II assay) and the COBAS AMPLICOR Monitor test (COBAS-AM assay), with special emphasis on anti-HBV e antigen (HBeAg)-positive patients with low HBV DNA levels. A total of 425 Chinese patients with chronic hepatitis B were recruited. A total of 107 patients were HBeAg positive, and 318 patients were HBeAg negative. The Digene HC II assay and the COBAS-AM assay had similar intra-assay and interassay variabilities. A total of 264 patients (62.1%) had HBV DNA levels undetectable by the Digene HC II assay, and 47 patients (11.1%) had HBV DNA levels undetectable by the COBAS-AM assay (P < 0.001). For the 161 patients with HBV DNA levels detectable by the Digene HC II assay, the HBV DNA levels obtained by the Digene HC II assay and by the COBAS-AM assay showed an excellent correlation (r = 0.95; P < 0.001). The linear ranges of the Digene HC II assay and the COBAS-AM assay marginally overlapped. Before HBV DNA levels could be determined by the COBAS-AM assay, predilution had to be performed for 158 of 161 patients (98.1%) with HBV DNA levels detectable by the Digene HC II assay and for 10 of 264 patients (3.8%) with HBV DNA levels undetectable by the Digene HC II assay. The cost for assaying each serum sample by using different strategies was calculated. The COBAS-AM assay was more sensitive than the Digene HC II assay and more suitable for monitoring low levels of HBV viremia.

HBsAg seroclearance in Chinese patients receiving lamivudine therapy for chronic hepatitis B virus infection.

We report two Chinese patients in whom lamivudine treatment resulted in HBsAg seroclearance. One patient received lamivudine, and another patient received 12-week famciclovir treatment followed by lamivudine. Lamivudine was maintained after HBeAg seroconversion. These two patients lost HBsAg at 24 and 27 months (ages, 23 and 19.3 years, respectively) and developed measurable titer of anti-HBs after 65 and 71 months of therapy, respectively. The liver biochemistry was normal after HBeAg seroconversion. The serum hepatitis B virus (HBV) DNA levels were undetectable (<200 copies/ml) both at the time of HBeAg seroconversion and at the last follow-up. Liver biopsy of one patient showed nearly normal histology, with undetectable intrahepatic total HBV DNA and covalently closed circular DNA. In conclusion, lamivudine therapy can result in HBsAg seroclearance at an early age even though the phenomenon is rare.

Long-term follow-up study of Chinese patients with YMDD mutations: significance of hepatitis B virus genotypes and characteristics of biochemical flares.

We sought to examine the role of hepatitis B virus (HBV) genotypes in virological breakthroughs and biochemical flares in patients with YMDD mutations during lamivudine therapy. Virologic breakthroughs (i.e., the reappearance of HBV DNA as determined by bDNA assay) and biochemical flares (mild flares = alanine aminotransferase [ALT] between 2 and 10 times the upper limit of normal [ULN]; severe flares = ALT >10 times ULN) were monitored in 154 hepatitis B e antigen-positive patients receiving long-term lamivudine. The HBV genotypes and YMDD mutations were determined. Forty-three patients had virological breakthroughs with YMDD mutations (median follow-up of 29.6 months [range, 22.3 to 61.4]). Twenty patients (47%) patients had mild biochemical flares; seven (16%) had severe flares. Two patients showed an elevation of bilirubin level that is >2 times the ULN. All patients recovered spontaneously. The cumulative risks for biochemical flares were 28, 47, and 58% for the first 3 years, respectively. Patients with biochemical flares compared to those without flares had a significantly higher median pretreatment ALT level (61 U/liter versus 34.5 U/liter [P = 0.012]). There were no differences in the cumulative risk of virological breakthroughs, risk, and severity of biochemical flares between patients with genotypes B (n = 11) and C (n = 32). There was an increase in the percentage of patients with single YMDD mutant at last follow-up compared to that at the time of virological breakthroughs (74% [n = 32] versus 47% [n = 20], respectively; P = 0.015). The chances of YMDD mutations with virological breakthroughs and biochemical flares were similar in patients with genotypes B and C. Biochemical flares were common, with 16% being severe in nature. High pretreatment ALT levels were associated with a higher chance of biochemical flares.

Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA.

Levels of hepatitis B virus (HBV) DNA in the blood serve as an important marker in monitoring the disease progression and treatment efficacy of chronic HBV infection. Several commercial assays are available for accurate measurement of HBV genomic DNA, but many of them are hampered by relatively low sensitivity and limited dynamic range. The aim of this study was to develop a sensitive and accurate assay for measuring HBV genomic DNA using real-time PCR with a molecular beacon (HBV beacon assay). The performance of this assay was validated by testing serial dilutions of the two EUROHEP HBV DNA standards (ad and ay subtypes) of known concentrations. The assay showed low intra-assay (<7%) and interassay (<5%) variations for both subtypes. Its dynamic range was found to be 10(1) to 10(7) copies per reaction (1.0 x 10(2) to 1.0 x 10(9) copies ml(-1)). The assay was further evaluated clinically using serum samples from 175 individuals with chronic hepatitis B. The HBV DNA level measured by this assay showed good correlation with that measured by the commercially available COBAS AMPLICOR HBV Monitor test (r = 0.901; P < 0.001). The higher sensitivity and broader dynamic range of this assay compared to the existing commercial assays will provide an ideal tool for monitoring disease progression and treatment efficacy in HBV-infected patients, in particular for those with low levels of HBV viremia.