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Multi-drug resistant hepatitis B virus (HBV) has been reported in hepatitis B patients who received sequential antiviral therapy. In vitro studies showed that HBV constructs with mutations resistant to lamivudine and adefovir have marked reduction in sensitivity to combination of lamivudine and adefovir, whereas constructs with mutations resistant to either drug remain sensitive to the other drug. We conducted this study to determine whether mutations conferring resistance to multiple antiviral agents co-locate on the same HBV genome in vivo and to describe the evolution of these mutations. Sera from six patients who had been found to have multi-drug resistant HBV mutations to lamivudine ؉ adefovir, lamivudine ؉ hepatitis B immunoglobulin (HBIG), or lamivudine ؉ entecavir on direct sequencing were cloned after nested polymerase chain reaction (PCR). Analysis of 215 clones from 11 samples with multi-drug resistant mutations on direct sequencing showed that 183 (85%) clones had mutations to both therapies on the same genome; 31 clones had lamivudine-resistant mutants only. Clonal analysis of serial samples from three patients showed progressive evolution from all clones with lamivudine-resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations and clones that have lamivudineresistant HBV mutations only, and ultimately all clones having multi-drug resistant HBV mutations. In conclusion, mutations conferring resistance to multiple antiviral agents colocate on the same viral genome, suggesting that combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant HBV. De novo combination therapy may prevent the emergence of multi-drug resistant mutants. (HEPATOLOGY 2006;44:703-712.)

Mutations in the core promoter and precore regions are frequently found in hepatitis B e antigen (HBeAg)-negative patients, but precore stop codon mutation is restricted to hepatitis B virus (HBV) genotypes that have T at nucleotide 1858. The aims of this study were to determine the role of core promoter and/or precore mutations in HBeAg seroconversion and their impact on the subsequent course of liver disease, and to determine if core promoter mutations are more frequently selected in patients with HBV genotypes that preclude the development of precore stop codon mutation. Serial sera from 45 patients with chronic HBV infection were polymerase chain reaction (PCR)-amplified, and the HBV core promoter and precore regions were sequenced. Ninety-two percent of patients had core promoter or precore mutations after HBeAg seroconversion: 42% had core promoter changes only, 38% had precore stop codon mutations only, and 12% had changes in both regions. Seventy-three percent of the patients had persistently normal aminotransferases, and only 8% had multiple flares in aminotransferases after HBeAg seroconversion. Core promoter changes were significantly more common in patients infected with HBV who have C at nucleotide 1858 (91% vs. 27%; P F .01), while precore stop codon changes were exclusively found in patients infected with HBV who have T at nucleotide 1858 (87% vs. 0; P F .01). The vast majority of our patients had core promoter and/or precore mutations after HBeAg seroconversion. Nevertheless, most patients had sustained remission of liver disease. Our data suggest that core promoter changes are preferentially selected in patients infected with HBV genotypes that preclude the development of precore stop codon mutation. (HEPATOLOGY 1999;29:976-984.)

Hepatitis B virus (HBV) genotype and precore/core promoter mutations have been implicated in spontaneous and interferon alfa (IFN-+related hepatitis B e antigen (HBeAg) seroconversion. We performed a retrospective analysis of a previously reported randomized controlled trial to determine the effects of HBV genotype and precore/core promoter mutations on IFN-c~ response in patients with HBeAg-positive chronic hepatitis. Clinical data and stored sera from 109 (95%) patients in the original trial were analyzed. Seventy-three patients received IFN-a and 34 received no treatment (controls). Almost all patients had HBV genotypes B (38%) and C (60%). Antiviral response was achieved in 39% and 17% of IFN-a-treated patients (P = .03) and in 10% and 8% of untreated controls (1" = 38) with HBV genotype B and C, respectively. Multivariate analysis identified HBV genotype B, elevated pretreatment alanine aminotransferase (ALT) levels, and low pretreatment HBV-DNA levels but not IFN-a treatment as independent factors associated with antiviral response. Among the 66 patients with elevated pretreatment ALT level, antiviral response was achieved in 57% and 21% of IFN-a-treated patients (P = .019), and in 25% and 8% of untreated controls (P = .45) with HBV genotype B and C, respectively. Multivariate analysis showed that genotype B and low pretreatment HBV-DNA levels were independent predictors of antiviral response. In conclusion, our data showed that HBV genotype B was associ-

The goals of this retrospective study were to determine whether there is a threshold hepatitis B virus (HBV) DNA value associated with spontaneous or antiviral therapy-related hepatitis B e antigen (HBeAg) clearance. We also investigated whether there is an HBV DNA value that can be used for differentiating inactive carriers from patients with HBeAg-negative chronic hepatitis B. HBV DNA levels in sequential serum samples of 165 Chinese patients with different stages of chronic HBV infection were quantified by a polymerase chain reaction (PCR)-based assay. Our results showed that almost all of the patients (83%) who remained HBeAg-positive had HBV DNA levels that were persistently above lo5 copies/mL. Serum HBV DNA levels decreased by a mean of 3 loglo in patients with HBeAg loss, but 5 1% had levels above lo5 copies/mL at the time HBeAg first became undetectable. Mean serum HBV DNA levels were significantly lower in HBeAg-negative patients. HBV DNA value above lo5 copies/mL would exclude all inactive carriers, but 45% of patients with HBeAgnegative chronic hepatitis would also be excluded if testing were only performed at presentation and 30% would be excluded if testing were performed on 3 occasions. In conclusion, serum HBV DNA levels decreased significantly in patients with HBeAg loss, but there was no threshold HBV DNA level associated with HBeAg clearance. Given the fluctuating course of HBeAg-negative chronic hepatitis, it is not possible to define a single cutoff HBV DNA value for differentiating inactive carriers from patients with HBeAg-negative chronic hepatitis.

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Hepatitis B e antigen-negative chronic hepatitis B (eϪCHB) has been reported in Asia but its prevalence and clinical significance have not been determined. The aims of this study were to determine the prevalence of eϪCHB in Hong Kong and the frequency of precore and core promoter mutations in these patients. A cross-sectional study was performed in 350 consecutive Chinese patients (230 men and 120 women; mean age ؎SD, 42 ؎ 13 years) with chronic hepatitis B virus infection. A total of 243 (69%) patients were hepatitis B e antigen (HBeAg)-negative of whom 15% had clinical cirrhosis. In the remaining 85% of patients, 63% had normal and 22% had elevated transaminases. Serum hepatitis B virus (HBV) DNA was detectable using branched DNA assay in 46% of HBeAg-negative patients with clinical cirrhosis/elevated transaminases. Forty-five percent of the patients with eϪCHB had the precore stop codon mutation, and an additional 41% had core promoter changes. There was no correlation between the presence of precore/core promoter mutations and liver disease or HBV-DNA levels. Overall, 17% of HBeAg-negative patients were viremic and had evidence of chronic liver disease (eϪCHB) with mean HBV-DNA levels comparable with that in HBeAg-positive patients. In summary, we found that eϪCHB may be present in up to 17% of HBeAg-negative patients seen in a tertiary referral center in Hong Kong. eϪCHB may be a heterogenous condition and is not invariably associated with the precore HBV mutant. Population studies are needed to determine the true prevalence of eϪCHB in Asia and to assess its natural course and response to treatment. (HEPATOLOGY 2000;31:763-768.)