Clinical characteristics and chronicity of acute hepatitis B induced by lamivudine-resistant strains

Whether resistant hepatitis B virus (HBV) strains are transmissible and can lead to chronic infection remains to be studied. The aim of this study was to investigate the clinical characteristics of patients with acute hepatitis B caused by lamivudine (LAM)‐resistant strains. Sera were collected from 234 Chinese patients with acute hepatitis B. LAM‐resistance mutations were identified by direct polymerase chain reaction (PCR) sequencing. LAM‐resistant HBV variants were detected in 11 of the 234 (4.7%) patients. Among these patients, six harbored the rtM204I mutation, two harbored the rtL180M + rtM204I mutations, one harbored the rtM204I + rtM204V mutations, one harbored the rtL80I + rtM204I mutations, and one harbored the rtV173L + rtL180M + rtM204V mutations. Three patients were infected with genotype B HBV and eight patients were infected with genotype C HBV. Two patients infected with viruses with LAM‐resistance mutations developed severe acute hepatitis. One patient developed chronic hepatitis B. This patient was infected with genotype C HBV that had LAM‐resistance mutations (rtL180M + rtM204I). The patient was diagnosed with an occult hepatitis B virus infection based on the presence of HBV DNA in the liver and the absence of detectable hepatitis B surface antigen (HBsAg) in the serum. LAM‐resistant HBV strains in China are transmissible, can cause acute hepatitis B, and can even progress to chronic infection in China. J. Med. Virol. 84:1558–1561, 2012. © 2012 Wiley Periodicals, Inc.     

Lamivudine and Famciclovir resistant hepatitis B virus associated with fatal hepatic failure.

BACKGROUND: Lamivudine (LMV) is the only nucleoside analogue approved for the treatment of chronic hepatitis B (CHB). LMV, as with other nucleoside analogues including Famciclovir (FCV), suppresses the replication of hepatitis B virus (HBV) by targeting the viral polymerase. However, prolonged antiviral therapy results in the emergence of drug resistance HBV which can contribute to virological breakthroughs and recurrent hepatitis flares. OBJECTIVES: A 38-year-old hepatitis B e antigen (HBeAg) positive Chinese female infected with genotype B HBV commenced treatment with FCV and LMV combination therapy but was later maintained on LMV monotherapy. The patient remained HBeAg positive throughout treatment. Virological breakthrough occurred with the emergence of drug resistant HBV. This coincided with worsening liver function and the patient died of subacute fulminant hepatitis. This study evaluated the virological factors that contributed to the clinical decline of the patient. STUDY DESIGN: Biochemical analysis and full-length HBV genomic sequencing were performed on serial serum samples collected from the patient before and during antiviral therapy. RESULTS: Virological analysis revealed that the pre-treatment dominant HBV quasispecies in the patient had a number of non-consensus genotype B mutations which were located in the basal core promoter (BCP), polymerase, X, core and S genes. Subsequent to the instigation of antiviral therapy, the dominant drug resistant HBV which caused virological breakthrough and was associated with hepatic failure displayed a series of unique mutations particularly in the BCP (A1762T and G1764A) and in the polymerase (rtL180M, rtM204V, rtA222T and rtL336V), core (cP5T, cS26A, cV85I and cP135A), surface (sI195M and sM213I) and X (xK95Q, xN118T, xK130M and xV131I) proteins. CONCLUSIONS: Monitoring for the accumulation of unique mutations within the genome of drug resistant HBV mutants isolated during long term antiviral therapy appears warranted in the clinical management of patients with CHB.     

Association of lamivudine‐resistant mutational patterns with the antiviral effect of adefovir in patients with chronic hepatitis B

Adefovir has a potent antiviral activity as a rescue treatment against lamivudine‐resistant strains. The aim of this study was to assess the patterns of lamivudine‐resistant mutations and their influence on the virologic response to adefovir rescue therapy in patients with lamivudine‐resistant chronic hepatitis B. Sixty‐seven patients with lamivudine‐resistant chronic hepatitis B were treated with adefovir monotherapy. Baseline blood samples were analyzed for lamivudine‐resistant mutations via restriction fragment mass polymorphism. Virologic responses, ALT normalization and loss of HBeAg were assessed. Serum HBV DNA levels were measured using real‐time PCR at baseline and 24 weeks of adefovir therapy. Of the 67 patients with chronic hepatitis B, 65 patients (97%) had lamivudine‐resistant mutations in the YMDD motif [27 (41%) rtM204I, 22 (34%) rtM204V, and 16 (25%) rtM204I/V]. In addition to the YMDD mutations, the rtL180M, rtL80I, and rtV173L mutations were also present in 78%, 43%, and 11% of patients, respectively. The rtM204V mutation always accompanied rtL180M, and rtL80I was always observed in conjunction with rtM204I. Decrease in mean serum HBV did not differ between patients carrying the rtM204I versus rtM204V mutant at week 24 (?3.3 vs. ?3.3 log₁₀ copies/ml, respectively; P = 0.303). The presence of the rtL180M, rtL80I, and rtV173L did not significantly affect viral load reduction during adefovir administration. These results demonstrate that the rtL80I mutant is co‐selected with rtM204I as a compensatory mutation in the same manner as rtL180M with rtM204V, and that adefovir shows similar antiviral efficacy against all of the evaluated patterns of lamivudine‐resistant HBV mutations. J. Med. Virol. 81:417–424, 2009. © 2009 Wiley‐Liss, Inc.     

Mutational patterns of hepatitis B virus genome and clinical outcomes after emergence of drug-resistant variants during lamivudine therapy: analyses of the polymerase gene and full-length sequences

It remains unclear whether mutational patterns of the hepatitis B virus (HBV) genome are associated with the development of severe hepatitis after the emergence of tyrosine-methionine-aspartate-aspartate (YMDD) variants during lamivudine treatment. Thirty patients with chronic hepatitis B who had YMDD variants during lamivudine therapy and were followed up subsequently while receiving lamivudine alone for at least 6 months were examined retrospectively. The lamivudine resistant mutations in the HBV polymerase gene were detected by a line probe assay, and the full-length sequences of HBV DNA were determined in some patients. Between months 5 and 33 of therapy, mutations from methionine to isoleucine at rt204 (rtM204I) were detected in 18 patients, and mutations from methionine to valine at rt204 (rtM204V) were detected in 12. The rtM204V mutations were always accompanied by mutations from leucine to methionine at rt180 (rtL180M), while rtM204I mutations were not. Baseline characteristics, alanine aminotransferase (ALT) levels, and HBV DNA levels within 6 months after the emergence of YMDD variants did not differ significantly between patients with rtM204I alone and those with rtL180M/rtM204V. No specific mutation was identified on full-length sequence analysis in three patients with a hepatitis flare. During long term follow-up, the addition of rtL180M to rtM204I was found in four patients 7-31 months after detecting the change at rt204 and was linked to increased ALT levels. In conclusion, mutational patterns of HBV DNA at the time of emergence of YMDD variants were apparently unrelated to the clinical outcomes in Japanese patients with chronic hepatitis B during lamivudine therapy.     

Evolution of drug-resistant mutations in HBV genomes in patients with treatment failure during the past seven years (2010–2016)

The objective of this study was to analyze the prevalence of drug-resistant HBV mutants in patients with treatment failure during the past seven years (2010–2016). 4055 HBV-infected patients who underwent HBV polymerase gene mutation test from 2010 to 2016 were enrolled. The nucleos(t)ide analogues (NAs) resistance mutation positions, including rtL180, rtA181, rtT184, rtS202, rtM204, rtI233, rtN236, rtI169, rtV173, and rtM250 were analyzed. Genotypic resistance mutations were detected in 30.8% (1248/4055) of the patients with treatment failure. Rates of drug-resistant mutations associated with LAM, ADV, ETV, and multidrug were 27.23% (1104/4055), 9.67% (392/4055), 3.69% (150/4055), and 0.79% (32/4055). Among the primary NA-resistant mutations, rtM204I (13.44%, 545/4055) occurred more frequently, followed by rtM204V, rtN236T, rtA181T, and rtA181V. For single-base mutations, rtL180M and rtA181V increased gradually during the past seven years, while rtM204I/V and rtN236T decreased after 2015. The development of drug-resistant mutations positively correlated with the consumption of ETV (r = 0.964, P = 0.002), and weakly correlated with that of LAM (r = 0.679, P = 0.109) and ADV (r = 0.429, P = 0.354). Moreover, single-base mutation rtA181V and multi-base mutations (rtL180M + M204I and rtL180M + M204V + M204I) were more common in HBV genotype C than those in genotype B (1.94% vs. 0.66%, 1.84% vs. 0.16%, 1.02% vs. 0.16%, respectively). NA-related mutations in HBV RT region increased in the past seven years, especially for LAM. Frequencies of rtL180M and rtA181T/V increased gradually in the past seven years, to which we should pay more attention.     

Pre-existing YMDD mutants in treatment-naïve patients with chronic hepatitis B are not selected during lamivudine therapy

Although the rate at which mutations in the tyrosine‐methionine‐aspartate‐aspartate (YMDD) motif of hepatitis B virus polymerase form is high during prolonged lamivudine (LAM) therapy, these mutations sometimes occur naturally in treatment‐naïve patients with chronic hepatitis B. The prevalence of natural YMDD mutants differs geographically, and its clinical significance during LAM therapy is unknown. This study aimed to investigate whether pre‐existing YMDD mutants were selected during LAM therapy. It included 14 treatment‐naïve patients who were treated with LAM for at least 9 months. LAM resistance was evaluated before and at 3‐month intervals during treatment. Mutations were analyzed by direct sequencing, restriction fragment mass polymorphism (RFMP) assays, and a single‐step multiplex polymerase chain reaction (PCR) test using dual‐priming oligonucleotide (DPO) primers. DPO‐based multiplex PCR showed two YMDD mutations in two patients before LAM therapy; rtM204V and rtL180M + rtM204V/I. Further, two patients had an rtL180M mutation without an accompanying rtM204V/I mutation. No mutant was detected in any patient by direct sequencing or the RFMP assay before LAM therapy. A virological response was observed at 3 months in all patients with pre‐existing YMDD mutants. All mutations disappeared after 3 months of LAM therapy, and during the follow‐up period, no re‐emergence was detected by any of the three methods. Further, the viral load was suppressed optimally. In conclusion, pre‐existing YMDD mutants were cleared early during the course of LAM therapy, which produced a consistent virological response, and the mutants were not selected by LAM therapy. J. Med. Virol. 84:217–222, 2012. © 2011 Wiley Periodicals, Inc.     

Oligonucleotide chip for detection of Lamivudine-resistant hepatitis B virus

Hepatitis B virus (HBV) is one of the major causes of liver disease worldwide. It is important to conduct antiviral therapy against chronic hepatitis B to minimize the amount of liver damage. Lamivudine has been known to be an effective antiviral agent for the treatment of HBV infection. However, the emergence of viral mutants resistant to lamivudine is the main concern during the treatment of HBV-infected patients. Therefore, the detection of lamivudine-resistant mutants is of clinical importance. We have developed an oligonucleotide chip for the detection of lamivudine-resistant HBV which is rapid and accurate. The oligonucleotide chip consists of quality control probes, negative control probes, and specific oligonucleotide probes for the detection of lamivudine-resistant HBV. The specific probes consist of five probes for the detection of wild-type rtL180, rtM204, and rtV207 sequences and seven probes for the detection of HBV mutations. We tested 123 serum samples from patients with chronic HBV infection who had received lamivudine therapy. Eighty samples contained mutants with YMDD mutations. Among these, 17 contained rtM204V (YVDD), 24 contained rtM204I3 (YIDD3), 3 contained rtM204I2 (YIDD2), and 36 contained mixed types. We compared the results obtained with our oligonucleotide chip with those obtained by PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing. The rate of concordance between the assay with the oligonucleotide chip and PCR-RFLP analysis for detection of the YMDD motif was 96.7%. The rate of concordance between the results obtained with the oligonucleotide chip for the detection of rtL180 and rtV207 and the results obtained by sequencing was 100%. Thus, the oligonucleotide chip is a reliable and useful tool for the detection of antiviral-resistant HBV.     

High frequency of lamivudine resistance mutations in Brazilian patients co‐infected with HIV and hepatitis B

This study analyzed the genotype distribution and frequency of lamivudine (LAM) and tenofovir (TDF) resistance mutations in a group of patients co‐infected with HIV and hepatitis B virus (HBV). A cross‐sectional study of 847 patients with HIV was conducted. Patients provided blood samples for HBsAg detection. The load of HBV was determined using an “in‐house” real‐time polymerase chain reaction. HBV genotypes/subgenotypes, antiviral resistance, basal core promoter (BCP), and precore mutations were detected by DNA sequencing. Twenty‐eight patients with co‐infection were identified. The distribution of HBV genotypes among these patients was A (n = 9; 50%), D (n = 4; 22.2%), G (n = 3; 16.7%), and F (n = 2; 11.1%). Eighteen patients were treated with LAM and six patients were treated with LAM plus TDF. The length of exposure to LAM and TDF varied from 4 to 216 months. LAM resistance substitutions (rtL180M + rtM204V) were detected in 10 (50%) of the 20 patients with viremia. This pattern and an accompanying rtV173L mutation was found in four patients. Three patients with the triple polymerase substitution pattern (rtV173L + rtL180M + rtM204V) had associated changes in the envelope gene (sE164D + sI195M). Mutations in the BCP region (A1762T, G1764A) and in the precore region (G1896A, G1899A) were also found. No putative TDF resistance substitution was detected. The data suggest that prolonged LAM use is associated with the emergence of particular changes in the HBV genome, including substitutions that may elicit a vaccine escape phenotype. No putative TDF resistance change was detected after prolonged use of TDF. J. Med. Virol. 82:1481–1488, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of a new variant in the YMDD motif of the hepatitis B virus polymerase gene selected during lamivudine therapy

A new hepatitis B virus variant selected during lamivudine treatment was detected, in which the methionine (rtM204) in the so-called YMDD motif in the C domain of the catalytic site of the polymerase gene was replaced by a serine (rtM204S). This change simultaneously resulted in a tyrosine-195 into valine variant (sY195V) in the surface protein HBsAg. The detection of this YSDD variant was initially observed, after an increase of HBV DNA levels, by sequencing of amplification products from day 586. A specific RFLP assay was developed that could identify 10% of YSDD-containing variants in the virus pool, which enabled detection of this new variant virus at day 506. However, by cloning several PCR products and sequencing individual recombinant clones, the mutation was first identified at day 477, before a significant increase of HBV DNA was observed in serum. The mutation was followed by a leucine to methionine change at position 180 (rtL180M). The consequences of this mutation for disease management and diagnostic strategies are discussed.     

Real-time PCR detection of multiple lamivudine-resistant mutations with displacing probes in a single tube.

BACKGROUND: Detection of lamivudine-resistant hepatitis B virus (HBV) is essential to clinical diagnosis and treatment. OBJECTIVES: To establish a single tube, real-time PCR assay for simultaneous detection of multiple lamivudine-resistant mutations in serum samples. STUDY DESIGN: By using four sequence-specific displacing probes labeled with different fluorophores, a single real-time PCR reaction can tell whether a sample contains any of the following HBV variants: wild-type, rtM204 mutant; mixtures of wild-type and rtM204 mutant; mixtures of rtM204 and rtL180 mutant; mixtures of wild-type, rtM204 mutant and rtL180 mutant. The assay was evaluated with 50 HBV mutation(s)-containing samples and 36 HBeAg-positive samples. RESULTS: The results of the real-time PCR assay were consistent with the DNA sequencing, but with much higher sensitivity for detecting a mixture of quasispecies. As few as 10(2)-10(3)copies/ml HBV of all four sequences in pure population and as little as 5% mutant DNA in the presence of wild-type DNA can be detected. CONCLUSIONS: Application of this high throughput assay into clinical use should enable earlier diagnosis and better treatment of lamivudine-resistant HBV.