Munira Hussain

Evolution of multi-drug resistant hepatitis B virus during sequential therapy†

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 lamivudine‐resistant HBV mutations only, and ultimately all clones having multi‐drug resistant HBV mutations. In conclusion, mutations conferring resistance to multiple antiviral agents co‐locate 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.)

Sustained response after a 2-year course of lamivudine treatment of hepatitis B e antigen-negative chronic hepatitis B.

Summary.  Lamivudine has demonstrated efficacy for the treatment of hepatitis B e antigen‐negative chronic hepatitis B (e‐CHB). However, treatment withdrawal after 1 year has been associated with a high rate of relapse while long‐term treatment is associated with increasing risks of drug resistance. We report our treatment experience of 50 Chinese‐Canadian patients with e‐CHB. All patients received lamivudine for 2 years. Treatment was withdrawn at month 24 in patients who had undetectable hepatitis B virus (HBV) DNA by PCR and normal aminotransferases during the second year of therapy. All patients had HBV genotype B or C. Biochemical response at months 6, 12 and 24 was 74%, 71% and 66%, respectively. HBV DNA was undetectable at months 6, 12 and 24 by hybrid capture and PCR assays in 100%, 92% and 86%; and 94%, 88% and 74% patients, respectively. The cumulative rates of genotypic resistance (GR) after 1 and 2 years were 15% and 25%, respectively. Four (44%) patients with GR experienced a hepatitis flare. The probability of clinical and virological relapse 6, 12, and 18 months after treatment withdrawal were 12% and 30%, 18% and 50%, and 30% and 50%, respectively. Reinstitution of lamivudine resulted in prompt virological and biochemical responses. Our study demonstrates that a sustained response can be achieved after a 2‐year course of lamivudine in a subset of patients with e‐CHB.

Rapid and Sensitive Assays for Determination of Hepatitis B Virus (HBV) Genotypes and Detection of HBV Precore and Core Promoter Variants

ABSTRACT Hepatitis B virus (HBV) genotypes may influence HBeAg seroconversion rates, mutational patterns in the precore (PC) and core promoter (CP) regions, severity of liver disease, and response to antiviral treatment. Development of rapid, simple, and standardized assays to detect viral genotypes and common mutations in the PC and CP regions can accelerate research on the clinical significance of these variants. We aim to assess the accuracy of a line probe assay in determining HBV genotypes and detecting HBV PC and CP variants. HBV genotypes in 701 patients and PC and CP variants in 600 patients with chronic HBV infection from China and the United States were studied using the INNO-LiPA assay. All but one (99.9%) sample were classified by the genotyping assay. All eight genotypes, i.e., A to H, were found. The INNO-LiPA genotyping assay results were completely concordant with those of sequencing. Using the INNO-LiPA PC assay, 99.8 and 94.7% samples were classifiable in the PC and CP regions, respectively. The PC assay results were completely concordant with those of sequencing in all samples that showed either wild-type or variant sequence. The line probe assay was more sensitive in detecting mixtures than was direct sequencing. By INNO-LiPA, only 50 and 27% of the samples, with mixed wild-type and variant sequence in the PC and CP region, respectively, showed mixed sequence by direct sequencing. INNO-LiPA is rapid, sensitive, and reliable—thus enabling accurate determination of HBV genotypes and detection of PC and CP variants in a large population of patients.

Clinical outcome and virological characteristics of hepatitis B-related acute liver failure in the United States

Summary.  The role of hepatitis B virus (HBV) genotypes in the outcome of acute HBV infection is unclear. In this study, we aimed to evaluate the clinical and virological features of patients with hepatitis B‐related acute liver failure (HBV‐ALF) in the US. Clinical and laboratory features of consecutive patients with HBV‐ALF from the US ALF Study Group were analysed. Prevalence of HBV genotypes, precore stop (G1896A) and core promoter dual (T1762A, A1764T) variants among patients with HBV‐ALF were compared with a cohort of 530 patients with chronic HBV infection. Thirty‐four HBV‐ALF patients were studied: mean age 41 years, 56% men, 25 had detectable HBV‐DNA. HBV genotypes A, B, C and D were found in 36, 24, 8 and 32% patients, respectively. Precore stop and core promoter dual variants were detected in 32 and 44% of patients, respectively. Twenty‐three (68%) patients survived: 14 after liver transplant, nine without transplant. Older age was the only independent factor associated with poor outcome. Compared with patients with chronic HBV infection, patients with ALF were more likely to be non‐Asians (88%vs 44%, P = 0.005) and to have genotype D (32%vs 10%, P < 0.01). A higher prevalence of HBV genotype D persisted even after matching for race and HBeAg status (32%vs 16%, P = 0.007). We concluded that HBV genotype D was more frequently found in patients with HBV‐ALF than those with chronic HBV infection in the US. Further studies are needed to determine if HBV genotypes play a role in the outcome of acute HBV infection.

Sensitive line probe assay that simultaneously detects mutations conveying resistance to lamivudine and adefovir

ABSTRACT The INNO-LiPA HBV DR v2 assay is designed to detect hepatitis B virus mutations conveying resistance to lamivudine and adefovir. Our study confirms that this assay can simultaneously detect the presence of lamivudine and adefovir resistance mutations in clinical samples, has a high degree of concordance with sequencing, and can detect mutants earlier.

Prevalence and significance of occult hepatitis B in a liver transplant population with chronic hepatitis C.

Occult hepatitis B virus (HBV) infection is defined as the detection of HBV deoxyribonucleic acid (DNA) in the serum or liver tissue of individuals who test negative for hepatitis B surface antigen (HBsAg). We undertook a prospective study to evaluate the significance and course of occult HBV in patients with hepatitis C virus (HCV) cirrhosis undergoing orthotopic liver transplantation (OLT). A sensitive real‐time polymerase chain reaction assay was utilized to test for serum HBV DNA at enrollment and for hepatic HBV DNA within the explant liver. Patients were followed with serum HBsAg and HBV DNA post‐OLT. A total of 56 patients with HCV cirrhosis were enrolled between October 2002 and July 2004; of these, 44 underwent OLT. The overall prevalence of occult HBV based on positive serum HBV DNA was 16 of 56 (28%), and based on positive hepatic HBV DNA (“occult HBV liver”) was 22 of 44 (50%). The presence of serum hepatitis B core antibody (anti‐HBc) and a past history of injection drug use correlated with occult HBV. Explant‐proven hepatocellular carcinoma (HCC) was found in 13 of 22 (59%) patients with occult HBV liver compared to 8 of 22 (36%) patients without occult HBV liver (P = 0.04, odds ratio = 3.1; confidence interval = 2.1‐5.4). Post‐OLT, no cases of HBV reactivation were noted, and there was no significant association between occult HBV and recurrent HCV. In conclusion, occult HBV is far more prevalent in patients with end‐stage HCV than would be expected from its prevalence in the general population. Occult HBV infection is strongly associated with the presence of anti‐HBc, history of injection drug use, and explant‐proven HCC. Liver Transpl 2007.

Presence of intrahepatic (Total and ccc) HBV DNA is not predictive of HBV recurrence after liver transplantation

Previous studies reported that hepatitis B virus (HBV) deoxyribonucleic acid (DNA) can be detected in livers of patients who received transplants for hepatitis B despite the absence of serological markers of HBV recurrence. Quantification of HBV DNA was not performed and presence of covalently closed circular (ccc) DNA was not analyzed in most studies. We aimed to quantify total and ccc HBV DNA in explant liver and post‐orthotopic liver transplantation (OLT) biopsies and to correlate the values with HBV recurrence post‐OLT. Frozen liver tissue from 34 patients (9 with explant liver only, 9 with explant liver and post‐OLT liver biopsies, and 16 with post‐OLT biopsies only) in the National Institutes of Health HBV‐OLT study was examined using real‐time polymerase chain reaction (PCR). Among the 18 patients with explant liver, 7 were hepatitis B e antigen (HBeAg)‐positive, 8 had detectable serum HBV DNA, and 10 received antiviral therapy prior to OLT. Total and ccc HBV DNA was detected in explant livers of 17 and 16 patients, respectively. Of the 10 patients who received antiviral therapy pre‐OLT, serum HBV DNA was undetectable in 8 at transplantation but 7 had detectable total and ccc HBV DNA in their explant liver. Of the 25 patients with post‐OLT biopsies, total HBV DNA was detected in 83% and ccc DNA in 17% of 47 biopsies, although only 2 patients had HBV recurrence. In conclusion, total and ccc HBV DNA could be detected in explant livers of most patients despite antiviral therapy pre‐OLT. Total but not ccc HBV DNA could be detected in post‐OLT liver biopsies of most patients despite undetectable serum HBV DNA and hepatitis B surface antigen (HBsAg). Our findings suggest that occult HBV reinfection occurs in most HBV patients after OLT and continued administration of appropriate prophylactic therapy is important in preventing overt HBV recurrence. Liver Transpl 13:1137–1144, 2007.

Hepatitis B Virus Core Promoter Mutations Contribute to Hepatocarcinogenesis by Deregulating SKP2 and its Target, p21

BACKGROUND & AIMS Clinical studies have associated hepatitis B virus core promoter (CP) mutations with an increased risk of hepatocellular carcinoma. The CP region overlaps with the HBV X (HBx) gene, which has been implicated in hepatocarcinogenesis. The cyclin kinase inhibitor p21WAF1/CIP1 is an important regulator of cell cycle progression and proliferation. We determined whether HBx mutants that result from mutations in the CP deregulate p21 and these processes. METHODS We constructed a series of HBx mutants with changes in the CP region that correspond to A1762T/G1764A (TA), T1753A, T1768A, or a combination of these (combo) and expressed them, along with wild-type HBx under control of its endogenous promoter, in primary human hepatocytes (PHHs) and HepG2 cells. We then analyzed the effects of CP mutations on expression and degradation of p21 and the effects on cell cycle progression and proliferation. RESULTS The combo mutant decreased levels of p21 and increased cyclin E expression in PHHs and HepG2 cells. The combo mutant, but not HBx with single or double CP mutations, accelerated p21 degradation in HepG2 cells. The combo mutant increased expression of S-phase kinase-associated protein 2 (SKP2) in PHHs and Huh7 cells. Silencing of SKP2 abrogated the effects of CP mutations on p21 expression. The kinetics of p21 expression correlated with changes in cell cycle distribution. The combo mutant accelerated cell cycle progression; p21 overexpression restored G1 arrest. CONCLUSIONS HBx mutants with changes that correspond to a combination of CP mutations up-regulate SKP2, which then down-regulates p21 via ubiquitin-mediated proteasomal degradation. CP mutations might increase the risk of hepatocellular carcinoma via this pathway.

Sensitivity and accuracy of an updated line probe assay (HBV DR v.3) in detecting mutations associated with hepatitis B antiviral resistance.

BACKGROUND/AIMS Early detection of antiviral drug-resistant mutations enables prompt initiation of rescue therapy. The aim of this study was to determine the accuracy and sensitivity of a new line probe assay in the detection of antiviral drug-resistant HBV mutations. METHODS One-hundred samples from 54 patients with virologic breakthrough during entecavir, lamivudine or adefovir treatment and 21 samples from 21 nucleoside-naïve patients were tested by direct sequencing and an updated line probe assay (Innogenetics, HBV DR v.3) which incorporates probes that can detect mutations at 11 positions of the reverse transcriptase region of the HBV polymerase gene. RESULTS Complete concordance between line probe and sequencing results was observed for 90/121 samples (74.3%) and 1291/1331 amino acid positions (96.9%). Testing of follow-up samples and clonal analysis of discordant samples confirmed the presence of mutations where line probe assay but not direct sequencing detected mutations. HBV DR v.3 assay consistently detected mutations present in > or = 5% of the virus population when HBV DNA concentration was > or = 4 log10copies/mL. CONCLUSIONS The updated version of the line probe assay (HBV DR v.3) has high concordance with direct sequencing in detecting antiviral drug-resistant mutations but its sensitivity in detecting mutations at some positions needs to be improved.

Occult and previous hepatitis B virus infection are not associated with hepatocellular carcinoma in United States patients with chronic hepatitis C.

Previous studies have suggested that prior exposure to hepatitis B virus (HBV) infection may increase the risk of development of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C. The aim of this study was to compare the prevalence of previous or occult HBV infection in a cohort of hepatitis B surface antigen–negative patients with histologically advanced chronic hepatitis C in the United States who did or did not develop HCC. Stored sera from 91 patients with HCC and 182 matched controls who participated in the Hepatitis C Antiviral Long‐term Treatment against Cirrhosis (HALT‐C) Trial were tested for hepatitis B core antibody (anti‐HBc), hepatitis B surface antibody, and HBV DNA. Frozen liver samples from 28 HCC cases and 55 controls were tested for HBV DNA by way of real‐time polymerase chain reaction. Anti‐HBc (as a marker of previous HBV infection) was present in the serum of 41.8% HCC cases and 45.6% controls (P= 0.54); anti‐HBc alone was present in 16.5% of HCC cases and 24.7% of controls. HBV DNA was detected in the serum of only one control subject and no patients with HCC. HBV DNA (as a marker of occult HBV infection) was detected in the livers of 10.7% of HCC cases and 23.6% of controls (P = 0.18).