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Given the loss of therapeutic efficacy associated with the development of resistance to lamivudine (LMV) and the availability of new alternative treatments for chronic hepatitis B patients, early detection of viral genotypic resistance could allow the clinician to consider therapy modification before viral breakthrough and biochemical relapse occur. To this end, 28 LMV-treated patients (44 +/- 12 years; 24 men), on their first therapy schedule, were monitored monthly at four Brazilian centers for the emergence of drug resistance using the reverse hybridization-based INNO-LiPA HBV DR assay and occasionally sequencing (two cases). Positive viral responses (HBV DNA clearance) after 6, 12, and 18 months of therapy were achieved by 57, 68, and 53% of patients, while biochemical responses (serum alanine aminotransferase normalization) were observed in 82, 82, and 53% of cases. All viral breakthrough cases (N = 8) were related to the emergence of YMDD variants observed in 7, 21, and 35% of patients at 6, 12, and 18 months, respectively. The emergence of these variants was not associated with viral genotype, HBeAg expression status, or pretreatment serum alanine aminotransferase levels. The detection of resistance-associated mutations was observed before the corresponding biochemical flare (41 +/- 14 and 60 +/- 15 weeks) in the same individuals. Then, if highly sensitive LMV drug resistance testing is carried out at frequent and regular intervals, the relatively long period (19 +/- 2 weeks) between the emergence of viral resistance and the onset of biochemical relapse can provide clinicians with ample time to re-evaluate drug therapy.

With the availability of more potent nucleotide/nucleoside analogues, the early detection of drug-resistant mutants of hepatitis B virus (HBV) is important for the strategic treatment of chronic hepatitis B. We studied 336 serum samples from 80 patients chronically infected with HBV who were receiving lamivudine treatment for the presence of lamivudine resistance mutations at codons 80, 173, 180, and 204 of the HBV polymerase. The sequencing data were compared with the results generated with the INNO-LiPA HBV DR (drug resistance) v2 strip, a line probe assay (LiPA) covering wild-type and mutant motifs, for resistance mutations to lamivudine and adefovir dipivoxil. This method provided at least the same information as sequencing for 99.1% of all codons analyzed. On the basis of the LiPA results, 20 of 80 patients developed a lamivudine resistance mutation after 1 year. In all 20 patients, the mutation occurred in the YMDD motif at reverse transcriptase position 204 (rt204; M204V/I) either with or without the compensatory mutation at position rt180 (L180M). A compensatory mutation at position rt80 (L80V/I) was detected in half of these patients. After 36 months, a compensatory mutation was seen at position rt173 (V173L) in 3/15 patients. Time-to-event survival analysis indicated a 2.8 times greater chance for LiPA to detect a given mutation than sequencing at any moment in time (hazard ratio, 2.8, 95% confidence interval, 1.79, 4.41; P < 0.0001). These results demonstrate that a highly sensitive and specific assay such as the INNO-LiPA HBV DR v2 can precociously detect and monitor the emergence of primary and compensatory lamivudine resistance mutations in patients chronically infected with HBV and is more sensitive than sequencing.

The factors determining the responsiveness of different hepatitis B virus (HBV) genotypes to interferon treatment are not fully understood. We investigated the relationship between HBV genetic characteristics and the outcome of short (16 weeks) or prolonged (32 weeks) treatment with standard interferon-alpha in a prospectively followed cohort of 103 patients across Europe with HBeAg positive chronic hepatitis B. INNO-LiPA assays and HBV DNA sequencing were used to determine HBV genotypes, mutations in the core promoter and precore/core regions. After 16-weeks interferon-alpha treatment, the rate of HBeAg clearance was higher in genotype A versus all other genotypes (P = 0.014), or genotype D alone (P = 0.05). The HBV genome analysis revealed that: (i) after 16-weeks treatment, an HBV subpopulation with core promoter mutations emerged or increased (P < 0.001) only in genotype A; (ii) the core gene of genotype A has the lowest number of amino acid variations in comparison with genotypes B, C, or D. Logistic regression analysis identified genotype A as a positive predictor of short (16 weeks) treatment response (P = 0.001; odds ratio 6.19, 95 confidence interval 1.94-19.8), having a greater impact than baseline HBV DNA or alanine aminotransferase (ALT) levels. In contrast, the response to prolonged interferon-alpha treatment was not different between HBV genotypes. These results suggest that HBV genotype A responds earlier to interferon treatment than other genotypes, which is associated with its molecular characteristics. The optimal duration of interferon-based therapies in chronic hepatitis B may vary between different HBV genotypes.

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.

Background: Comprehensive study on viral factors predicting treatment responsiveness to lamivudine is lacking., Aims: To define the significance of various viral factors and changes of viral population with lamivudine treatment., Patients and Methods: Hepatitis B virus (HBV) DNA levels at baseline, week 24, 52 and year 3 were measured in 80 patients on continuous lamivudine therapy for 3 years. Genotypes, core promoter/precore mutations, YMDD mutations, polymorphic sequence of polymerase gene (rt91 I/L, rt256S/C) were determined at baseline, week 12, 24 and 52. YMDD mutations were also determined at year 3., Results: High alanine aminotransferase levels and presence of core promoter/ precore mutations at baseline were associated with higher chance of achieving HBV DNA <1,000 copies/ml (good response) and higher rate of hepatitis Be antigen (HBeAg) seroconversion at week 52. Achieving HBV DNA levels <1,000 copies/mi at week 24 as well as baseline core promoter/precore mutations were associated with higher chance of achieving good response, higher rate of HBeAg seroconversion and lower rate of YMDD mutations at year 3. Lamivudine reversed core promoter mutations to wild type in 25% of patients. All 5 patients with rt256C had poor HBV DNA response, persistent HBeAg and YMDD mutations by year 3. There was no difference in treatment response between patients with genotype B and C., Conclusions: Achieving HBV DNA levels <1,000 copies/ml at 24 week is the best target for short- and long-term treatment efficacy. Core promoter and precore mutations were associated with better treatment outcome, and rt256C polymorphism in the polymerase gene with poor response.

Background: The details of liver histology of patients with precore and core promoter mutations are still not clear., Aim: To determine the role of precore and core promoter mutations in liver histology in Chinese patients with chronic hepatitis B., Patients and Methods: Intrahepatic hepatitis B virus DNA (by COBAS Amplicor hepatitis B virus Monitor test) and precore and core promoter mutations (by a line probe assay) were measured in 54 chronic hepatitis B patients. Expression of hepatitis B core antigen, hepatitis B e antigen and hepatitis B surface antigen was determined by immunohistological staining. Histological activity index was scored according to Knodell's criteria., Results: Compared with patients without core promoter mutations, patients with core promoter mutations had more severe intrahepatic inflammation and fibrosis, and more cytoplasmic expression of hepatitis B core antigen (P = 0.028). No such differences were found in patients with and without precore mutations. Logistic regression showed that core promoter mutations were independently associated with cytoplasmic expression of hepatitis B core antigen (P = 0.026). Intrahepatic hepatitis B virus DNA levels correlated with serum hepatitis B virus DNA levels (r = 0.71, P < 0.001) and the percentage of hepatitis B core antigen-positive hepatocytes (r = 0.37, P = 0.047), but had no correlation with serum alanine aminotransferase levels nor the degree of inflammation and fibrosis., Conclusions: Patients with core promoter mutations had more severe inflammation and fibrosis, and more frequent cytoplasmic expression of hepatitis B core antigen. This suggested that core promoter mutations might cause more serious liver disease.

We studied the hepatitis B virus (HBV)-DNA levels below which the development of cirrhosis-related complications became unlikely in chronic hepatitis B (CHB). Seventy-nine Chinese CHB patients with cirrhosis-related complications and 158 age-, sex- and HBeAg status-matched patients without complications were enrolled. The precore and core promoter mutations were detected by the Line Probe assay (LiPA). HBVDNA levels were determined by Digene assay and Cobas Amplicor Monitor test. Patients with complications had higher HBVDNA levels than those without complications (P = 0.02). HBeAg-positive patients with complications had similar alanine transferase (ALT) and HBVDNA levels and frequency of precore mutations, but higher frequency of core promoter mutations (P = 0.003), compared with those without complications. Anti-HBe-positive patients with complications had higher ALT and HBVDNA levels (P < 0.01) but similar frequency of precore and core promoter mutations, compared with those without complications. Anti-HBe patients (24.5%) with complications had HBVDNA levels <10(4) copies/mL. The major factor for the development of cirrhotic complications was viral loads but cirrhotic complications continued to develop in patients with HBVDNA levels below 10(4) copies/mL.

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.

Background/aims: We conducted a population study to document the prevalence of hepatitis B virus (HBV) genotypes in Hong Kong., Methods: HBV genotypes, core promoter (CP) and precore mutations were determined in 776 asymptomatic patients., Results: 92.6% patients had single genotype [B (32.5%), C (62.5%)]. 99.1% of genotype B was subtype Ba. Patients with age <50 years had a lower prevalence of genotype B than patients with age >51 years (32.5% vs. 41%, respectively, P=0.028). Compared to patients with genotype C, patients with genotype B had a higher cumulative rate (P=0.018) and younger age (40.1 vs. 34.2 years, respectively, P=0.018) of HBeAg seroconversion. There were no differences in the HBV DNA levels between patients with genotypes B and C, and with wild-type and mutants of CP and precore regions. By multivariate analysis, patients with genotype C and with CP mutations had higher alanine aminotransferase (ALT) levels., Conclusions: B and C were the two most common HBV genotypes in Hong Kong. The former had a higher chance of earlier HBeAg seroconversion and lower ALT levels. The prevalence of genotype B was lower in patients with age <50, probably related to influx of immigrants from China since 1949.

Few studies have examined Chinese patients with chronic hepatitis B who exhibit hepatitis B surface antigen (HBsAg) seroclearance. We comprehensively studied the biochemical, virological, histological, and clinical aspects of 92 patients with HBsAg seroclearance (median follow-up, 126 months). Ninety-two HBsAg-positive controls matched for age, sex, and duration of follow-up were also recruited. Liver biochemistry, serum hepatitis B virus (HBV) DNA levels, and development of clinical complications were monitored. Intrahepatic total and covalently closed circular (ccc) HBV DNA were measured quantitatively in 16 patients. HBV genotype was determined in 30 patients. The mean age at HBsAg seroclearance was 48.8 (+ 13.81) years. There was a significant improvement in serum alanine aminotransferase levels after HBsAg seroclearance (p<0.0001). Patients with genotype B had a higher chance of HBsAg seroclearance than those with genotype C (P =.014). Ninety-eight percent of patients had undetectable serum HBV DNA. Thirty-seven percent of patients had low titer of intrahepatic HBV DNA, mainly in the form of cccDNA (71%-100%). All 14 patients with liver biopsies had near normal histology. There was no difference in the risk of development of hepatocellular carcinoma (HCC) between patients with and without HBsAg seroclearance. However, the mean age of HBsAg seroclearance was significantly older in patients with HCC than in patients without HCC (P =.016). In conclusion, patients with HBsAg seroclearance had favorable biochemical, virological, and histological parameters. Intrahepatic HBV DNA level was low and predominantly in the form of cccDNA. However, HCC could still develop, particularly in patients with cirrhosis who had HBsAg seroclearance at an older age.

A prototype line probe assay (LiPA) for identifying hepatitis B virus (HBV) precore variants (INNO-LiPA HBV precore) was evaluated using a panel of 50 sera from 46 patients with HBV infection. The assay detected sequence variations detected commonly in the precore promoter region and in amino acid codons 28 and 29 of the precore gene. There was strong agreement between INNO-LiPA HBV precore results and those of a codon 28 point mutation assay (PMA), with identical results obtained in 40 of 43 sera (93%) typeable by both assays (kappa coefficient (kappa)=0.90). In addition, the precore codon 29 sequence identified by the INNO-LiPA HBV precore was confirmed by nucleotide sequencing in all seven samples analysed. However, the INNO-LiPA HBV precore identified precore promoter sequences much less efficiently. The prototype assay could identify codon 28/29 sequences from as little as 10 HBV genome equivalents in 10 microl serum, and in experiments using artificially prepared mixtures of variants could identify a minor component constituting 2.5% of the total viral DNA population. The INNO-LiPA HBV precore was also straightforward technically and rapid, and is therefore likely to be useful for epidemiological investigations into the prevalence, distribution and clinical significance of HBV precore variants.

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).

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.

To assess whether extended treatment with interferon improves the outcome of hepatitis B e antigen (HBeAg)-negative chronic hepatitis B, 101 consecutive patients were treated with 6 MU of interferon alfa 2b 3 times weekly for 24 months. During the 68-month study, 30 patients (30%) had a sustained response (i.e., normal serum transaminase levels and undetectable hepatitis B virus DNA by non-polymerase chain reaction [PCR] assays), and 15 cleared serum surface antigen. Twenty-five nonresponders, 16 relapsers, and 30 who discontinued treatment were considered treatment failures. Multivariate analysis predicted a sustained response for young age (odds ratio, 0.94; 95% confidence interval, 0.89-0.99; P =.041) and high pretreatment serum levels of immunoglobulin M (IgM) anti-hepatitis B core antigen (HBc) (odds ratio, 4.52; 95% confidence interval, 1.63-12.5; P =.004). Liver disease progressed in none of the sustained responders but in 16 with treatment failure (0% vs. 22%, P =.002); hepatocellular carcinoma (HCC) developed with similar frequency in both groups (7%). Overall, estimated 8-year complication-free survival was longer for the 30 sustained responders than the 71 patients with treatment failure (90% vs. 60%, P <.001), but 8-year patient survival was similar in the 2 groups (100% and 90%). Short complication-free survival was predicted by failure to respond to interferon (hazard ratio, 7.8; 95% confidence interval, 1.8-34.0; P =.006) and high scores for liver fibrosis (hazard ratio, 1.71; 95% confidence interval, 1.17-2.50; P =.005). In conclusion, 24 months of treatment with interferon alfa 2b led to sustained disease suppression in a significant proportion of patients with HBeAg-negative chronic hepatitis B.

Chinese patients with chronic hepatitis B virus (332 with and 44 without cirrhosis-related complications) were studied. Fifty percent of patients <30 years old had precore mutations. The prevalence of precore mutations among hepatitis B e antigen (HBeAg)-positive patients, although lower than that among anti-HBe-positive patients (P=.031), was already high (44.2%). Median HBV DNA level in anti-HBe-positive patients was 1.5 x 10(6)-1.55 x 10(6) copies/mL, irrespective of the presence or absence of precore mutations. There was no difference in the prevalence of precore mutations between patients with and without complications (P, not significant). However the prevalence of core promoter mutations was higher among patients with complications than among those without complications (90.5% vs. 69.3%, respectively; P=.003). In conclusion, precore mutations occurred in a large proportion of Chinese patients with chronic hepatitis B virus before HBeAg seroconversion. The development of complications was not related to precore mutations but was probably due to the persistence of significant viremia after HBeAg seroconversion.

Sensitive and early detection of emerging hepatitis B virus (HBV) drug resistance may not only help monitor the viral dynamics associated with lamivudine treatment but could also improve therapeutic decision making. This is especially important when new antivirals effective against lamivudine-resistant HBV become available. A total of 159 serum samples from 33 chronic HBV patients receiving lamivudine treatment were analyzed at four centers for the presence of lamivudine-resistant mutations at codons 528 [180] (proposed revised nomenclature according to Stuyver et al. [Hepatology 33:751-757, 2001] shown in brackets), 552 [204], and 555 [207] of the HBV polymerase. Sequencing data were compared with results generated by the INNO-LiPA HBV DR line probe assay (LiPA), an assay based on reverse hybridization of amplified HBV DNA fragments with specific nucleotide probes immobilized on nitrocellulose strips. LiPA provided at least the same information as sequencing for 97.5% of all codons analyzed for codon 528 [180], 95% for codon 552 [204], and 100% for codon 555 [207]. The most common reason for discrepant or indeterminate results (0.4% and 1.5%, respectively) in a small percentage of the population tested could be attributed to polymorphisms not yet covered by LiPA probes. In at least five patients, a mutant could be detected earlier by LiPA than by sequencing. In 15 patients, LiPA detected mixed wild-type and mutant virus populations before viral breakthrough. These results demonstrate that INNO-LiPA HBV DR is a highly sensitive and easily applicable assay for the detection and monitoring of lamivudine-resistant mutations in chronic hepatitis B patients and that the assay is more sensitive than sequencing in detecting mixed mutant and wild-type sequences.

Factors associated with hepatitis B virus (HBV) DNA breakthrough and the significance of YMDD variants without the presence of wild-type YMDD during prolonged lamivudine treatment are unknown. We studied the amino acid sequence of codon 552 (YMDD motif) and codon 528 by means of a line probe assay in 159 chronic HBV patients (median follow-up 29.6 months). Pretreatment HBV DNA levels and alanine transaminase (ALT) levels correlated inversely with the time to HBV DNA breakthrough with YMDD variants (r = -0.46, P =.001; r = -0.45, P =.001 respectively). Patients harboring YMDD variants 3 months before HBV DNA breakthroughs had higher HBV DNA breakthrough levels compared with those without YMDD variants 3 months before HBV DNA breakthroughs (18.9 x 10(6) vs. 5.4 x 10(6) copies/mL, P =.007). Patients with HBV DNA breakthroughs had higher percentages of YMDD variants without the presence of wild-type YMDD compared with patients without HBV DNA breakthrough (25.6% vs. 9%, P =.007 for single M552I variant; 20.9% vs. 8.1%, P =.026 for single M552V variant; 30.2% vs. 9.9%, P =.004 for M552I/M552V variants). Patients with HBV DNA levels of more than 10(3) copies/mL after 6 months of lamivudine therapy had a 63.2% chance of subsequently developing YMDD variants. HBeAg seroconversion occurred in 2 patients after the emergence of YMDD variants. Only one patient developed YMDD variant after HBeAg seroconversion. There was no increase in the rate of development of YMDD variants or L528M mutation in patients receiving lamivudine 25 mg daily or famciclovir 500 mg 3 times a day before being given lamivudine 100 mg daily.