Redetection of HBV lamivudine-resistant mutations in a patient under entecavir therapy, who had been treated sequentially with nucleos(t)ide analogues

Development of hepatitis B virus (HBV)-resistant strains following nucleos(t)ide analog treatment is a major medical concern. This report describes a case of an adult patient with chronic HBV infection, sequentially treated with the nucleos(t)ide analogues, lamivudine, adefovir, and entecavir. During monotherapy with lamivudine, the patient developed lamivudine-resistant variants, which were undetectable during adefovir dipivoxil monotherapy. Twenty-two months after discontinuing lamivudine therapy, the resistant variants were again detected while the patient was receiving entecavir monotherapy. Genotypic analysis by sequencing the HBV polymerase was confirmed with the INNO-LiPA method. The results of this study suggest that entecavir treatment reselected residual lamivudine-resistant HBV variants, possibly because lamivudine-resistant HBV is less susceptible to entecavir than the wild-type virus. Despite the presence of these variants, the patient has had a complete virological response.     

Dynamics of lamivudine-resistant hepatitis B virus during adefovir monotherapy versus lamivudine plus adefovir combination therapy

Adefovir dipivoxil has been used alone or together with lamivudine to suppress lamivudine-resistant hepatitis B virus (HBV). However, the dynamics of HBV populations under different selection pressures and their impact on treatment outcome are poorly understood. Pyrosequencing was applied to quantify longitudinally the evolution of wild type and lamivudine/adefovir-resistant HBV. Eight patients, with lamivudine-resistant HBV, were randomized to receive adefovir monotherapy or adefovir/lamivudine combination therapy for a median of 79 and 71 weeks, respectively. Pyrosequencing proved highly sensitive with a lower limit of quantitation of minor HBV populations of 2% irrespective of viraemia levels. Adefovir/lamivudine treatment resulted in greater viraemia reduction than adefovir monotherapy. During combination therapy, lamivudine-resistant HBV populations (codons 180 and 204) remained dominant (>90%) and no adefovir-resistance developed. During adefovir monotherapy, reversion to wild-type HBV was detected in two patients with one patient accumulating rapidly adefovir-resistant HBV along with increased viraemia. In conclusion, the dynamics of drug-resistant HBV strains vary under different selection pressures which have a significant impact on the success of rescue therapy, as well as for the selection of new mutations. The use of techniques such as Pyrosequencing provides an evidence-based approach for successful management of drug-resistant HBV.     

Precore/core promoter mutations and genotypes of hepatitis B virus in chronic hepatitis B patients with fulminant or subfulminant hepatitis

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.     

Detection of YMDD motif mutants by oligonucleotide chips in lamivudine-untreated patients with chronic hepatitis B virus infection

Lamivudine, a nucleoside analogue, has been used widely as an effective antiviral agent for the treatment of patients with chronic hepatitis B virus (HBV) infection. However, the YMDD motif mutation of HBV polymerase resistant to lamivudine occurs very frequently after long term therapy. We developed an oligonucleotide chip for the detection of YMDD motif mutants resistant to lamivudine and investigated the prevalence of the mutants in patients with chronic HBV infection who had not been treated by lamivudine before. Forty patients who had not been treated with lamivudine were included in this study. Serum samples were tested by the oligonucleotide chips designed for detection of wild-type YMDD motif, M552V and M552I. Samples were confirmed by restriction fragment length polymorphism (RFLP) and direct sequencing. M552I mutants were detected by the oligonucleotide chips in 7.5% (3/40) of chronic HBV infected patients (2 chronic hepatitis and 1 cirrhosis). The results were in accordance with those of RFLP. YMDD motif mutants occur as natural genome variabilities in patients with chronic HBV infection who had not been treated with lamivudine before. Oligonucleotide chip technology is a reliable and useful diagnostic tool for the detection of mutants resistant to antiviral therapy in chronic HBV infection.     

Clinical and virological characteristics of lamivudine resistance in chronic hepatitis B patients: a single center experience

We have investigated the characteristics of lamivudine-resistant strains in patients with chronic hepatitis B in Guangdong, China, where the predominant genotypes are B and C. Two hundred forty-seven patients treated with lamivudine in Nanfang Hospital were followed-up. Patients with hepatitis B e antigen (HBeAg) positive and hepatitis B virus (HBV)-DNA levels over 7.5 x 10(6) copies/ml at baseline had a shorter time to the selection of YMDD mutant (P = 0.02 and 0.00, respectively). The detection of YMDD mutant precedes HBV-DNA breakthrough and alanine transaminase (ALT) flare in about 2 and 3 months, respectively. The ALT flare after the appearance of YMDD mutants was more evident in HBeAg positive patients than HBeAg negative patients (P = 0.02). After emergence of YMDD mutant, the HBV-DNA level was significantly higher in genotype C patients compared with genotype B patients (P = 0.02). No significant difference of YMDD mutant pattern was found between patients with genotype B and C. Four kinds of new mutants were found in over two patients including rtL80I, rtG172E, rtG174C, and rtG172E/rtG174C. In vitro transfection and real-time analysis showed that rtG172E, rtG174C, and rtG172E/rtG174C mutants had a decreased replication competence compared with wild type (33%, 27%, and 15% of the wild type HBV, respectively). Our result suggest that genotypic monitoring of YMDD mutant is important for the management of patients treated with lamivudine.     

A novel accurate ACRS-PCR method with a digestion internal control for identification of wild type and YMDD mutants of hepatitis B virus strains

As a consequence of the point mutation in the YMDD motif of the hepatitis B virus (HBV) polymerase gene, lamivudine-resistant mutants have been reported in chronic hepatitis B patients who underwent lamivudine therapy. The objective of the study was to develop a novel accurate artificially created restriction site (ACRS) method with a digestion internal control for identification of YMDD, YIDD and YVDD HBV strains. Three conserved, specific and diagnostic primers introducing NdeI, SspI and AleI cleavage sites were designed in order to identify YMDD, YIDD and YVDD strains, respectively; while, their reverse primers also modified with the above recognition sites in order to enzyme correctness monitoring and false outcome avoiding. Thirty-two chronic hepatitis B patients who had taken lamivudine for 1-3 years and checked by the Inno-LiPA HBV DR kit, were evaluated by the ACRS method and then compared to sequencing data. The results of the ACRS method revealed the YMDD mutant strain in 20 patients, YMDD plus YIDD pattern in 1 patient, YMDD plus YVDD in 4 patients, the YIDD in 4 patients and mixed infection with each three strains in 1 patient. The sequencing and Inno-LiPA results were in agreement with the ACRS results. The novel ACRS method is a reliable, rapid and a cost-effective technique for determination of HBV strains with the wild type and YMDD mutant patterns.     

Genetic heterogeneity of the precore and the core promoter region of genotype C hepatitis B virus during lamivudine therapy

It has been reported that spontaneous or interferon (IFN)-induced hepatitis B e (HBe) seroconversion has usually been associated with the development of a stop codon in the precore region. However, the difference between lamivudine-induced seroconversion and spontaneous or IFN-induced seroconversion is not known. The aim of this study was to investigate the correlation between the evolution of the precore and core promoter mutations and lamivudine-induced seroconversion. Forty-five patients with chronic hepatitis B virus (HBV) infection who were treated with lamivudine for more than 1 year were enrolled. The nucleotide sequence of the precore and core promoter region was determined before and after treatment with lamivudine for 1 year. Among 29 patients who were hepatitis B e antigen (HBeAg)-positive before treatment, 12 (41.3%) lost HBeAg during the course of treatment for 1 year. Of these, eight patients (66.7%) still had precore wild type HBV after 1 year. After 1 year, reversion to precore wild type HBV was detected in 11 (64.7%) of 17 patients who had precore mutant HBV before treatment. Twelve (70.6%) of 17 patients who were persistently HBeAg-positive had precore wild type HBV before and after treatment for 1 year. Despite the loss of HBeAg, two thirds of the patients still had precore wild type HBV after the 1-year treatment. It is suggested that lamivudine-induced seroconversion differs from spontaneous or IFN-induced seroconversion in the change of nucleotides in the precore region. The reversion in the precore region may be caused by the difference of drug-susceptibility to lamivudine. The antiviral effect of lamivudine may be more effective in the precore mutant HBV than in the precore wild type HBV.     

Susceptibility of hepatitis B virus to lamivudine restored by resistance to adefovir

Serial monotherapy and add‐on regimes for treatment of chronic hepatitis B virus (HBV) infection may induce the accumulation of viral resistance mutations in patients, reducing the options for ongoing viral suppression. The induction of antiviral resistance by serial application of polymerase inhibitors does not necessarily imply that the subsequent combined use of the drugs will fail. Some HIV strains resistant to one polymerase inhibitor show increased susceptibility to another polymerase inhibitor. After failure of sequential lamivudine and adefovir monotherapy, two patients with hepatitis B changed to treatment with lamivudine plus adefovir and had renewed suppression of HBV. To study the mutational history of resistant HBV subpopulations in the two patients, a part of the HBV polymerase gene was amplified, cloned, sequenced, and analyzed for the presence of mutations, in sequential plasma samples. In both patients serial monotherapy caused the replacement in all HBV clones of wild‐type virus by classical lamivudine resistant mutants (L180M and M204V/I), which were replaced subsequently by adefovir resistant mutants (A181V and N236T). When finally lamivudine was added to adefovir, the A181V adefovir mutation persisted in all clones and lamivudine‐related mutations did not reappear. During 18 months of combination therapy, HBV‐DNA levels decreased 10,000, respectively, 1,000‐fold, despite the earlier resistance to lamivudine and adefovir. Although clinically insufficient, this effect indicates that HBV polymerase resistance mutations may be antagonistic, which is relevant if chronic HBV infection is to be treated by a combination of polymerase inhibitors. J. Med. Virol. 81:413–416, 2009. © 2009 Wiley‐Liss, Inc.     

Antiviral efficacy of combination therapy with entecavir and adefovir for entecavir/lamivudine-resistant hepatitis B virus with or without adefovir resistance

There is little clinical information on the management of hepatitis B virus (HBV) that is resistant to multiple drugs including entecavir (ETV). The present retrospective cohort study assessed the antiviral efficacy of ETV/adefovir dipivoxil (ADV) combination therapy for ETV-resistant HBV with prior lamivudine (LAM) resistance, and either with or without previous ADV resistance. The cumulative probability of achieving a virological response (undetectable serum HBV DNA) was compared by Kaplan-Meier analysis and the Breslow method. Seventeen patients with ETV-resistant HBV who were treated with ETV/ADV combination therapy for at least 6 months at a tertiary care center, were included; seven had dual resistance to ETV and LAM [ADV-r(-) group] and 10 had triple resistance to ETV, LAM, and ADV [ADV-r(+) group]. The median follow-up period was 9 months (range, 6-23). A virological response was noted in seven patients after a median of 3 months (range, 3-12) of treatment; five in the ADV-r(-) group and two in the ADV-r(+) group. The cumulative probability of a virological response was significantly higher in the ADV-r(-) group than in the ADV-r(+) group (6 months cumulative probability, 57.1% vs. 11.1%). In conclusion, ETV/ADV combination therapy led to virological responses in five of seven patients with resistance to ETV and LAM, but a significantly poorer response in patients with prior ADV resistance than in those without prior ADV resistance. Therefore, ETV/ADV combination therapy could be a useful therapeutic option for ETV- and LAM-resistant HBV without prior ADV resistance.     

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.     

Adefovir dipivoxil alone or in combination with ongoing lamivudine in patients with decompensated liver disease and lamivudine-resistant hepatitis B virus

The purpose of this prospective study was to evaluate the efficacy and safety of adefovir dipivoxil with or without ongoing lamivudine in decompensated lamivudine-resistant chronic hepatitis B patients. Forty-six hepatitis B e antigen (HBeAg)-positive patients with decompensated liver function and lamivudine-resistant hepatitis B virus (HBV) were assigned to adefovir dipivoxil monotherapy (n=18) or combination therapy with ongoing lamivudine (n=28) according to their own preference. After 24 weeks of treatment, 83% of monotherapy and 86% of combination therapy showed serum HBV DNA below detection limit (<0.5 pg/mL). Alanine aminotransferase (ALT) normalized in 78% and 82% respectively. Median Child-Pugh-Turcotte (CPT) score or Model for End-Stage Liver Disease (MELD) score reduced significantly by 3 or 5 point in monotherapy and 2 or 2 point in combination therapy respectively. There were no significant differences in rate of undetectable serum HBV DNA, median change of ALT and median reduction of CPT or MELD scores between the two groups. In conclusion, both adefovir dipivoxil monotherapy and combination therapy with ongoing lamivudine result in comparable virologic, biochemical, and clinical improvements in HBeAg-positive patients with decompensated liver function and lamivudine-resistant HBV. Combination with lamivudine showed no additional benefit over monotherapy during 24 weeks of treatment in these patients.     

Characteristics of lamivudine-resistant hepatitis B virus (HBV) strains with and without breakthrough hepatitis in patients with chronic hepatitis B evaluated by serial HBV full-genome sequences

Lamivudine therapy often causes breakthrough of hepatitis B virus (HBV) DNA and breakthrough hepatitis. The aim of this study was to determine the viral factors that relate to HBV-DNA breakthrough with and without breakthrough hepatitis. Among 82 patients with chronic hepatitis B (CHB) who received lamivudine at a dose of 100 mg daily for more than 24 months, 23 patients had HBV-DNA breakthrough induced by a lamivudine-resistant mutant. Of these 23 patients, 16 had breakthrough hepatitis and 7 had only HBV-DNA breakthrough. Serial HBV-DNA full-genome sequences during therapy were examined in 10 (7 had breakthrough hepatitis and 3 did not) of these 23 patients by direct sequencing. Mutations in the S region were examined by cloning in representative patients. There were no significant differences in the baseline clinical backgrounds and virus marker between patients with and without breakthrough hepatitis. The HBV amino acid substitutions at breakthrough hepatitis were identical to those at HBV-DNA breakthrough. Cloning analysis revealed that monoclonal mutational strain appeared at breakthrough and no such mutations existed at baseline. Regarding HBV amino acid substitutions in the polymerase region, S region, X region, and precore-core region with breakthrough compared to baseline, there was no significant differences of the numbers of amino acid substitution between breakthrough hepatitis and non-breakthrough hepatitis. There were no common amino acid changes in patients with breakthrough hepatitis. Although monoclonal lamivudine-resistant strain emerged at HBV-DNA breakthrough in patients with CHB, there were no common amino acid changes, suggesting viral factor may have insignificant role in breakthrough hepatitis.     

阿德福韦酯单药及其联合拉米夫定治疗拉米夫定耐药型HBeAg阳性慢性乙型肝炎的疗效比较

目的比较阿德福韦酯（ADV）单药及其联合拉米夫定治疗拉米夫定耐药型HBeAg阳性慢性乙型肝炎（chronic hepatitis B,CHB）的临床疗效。方法对24例ADV单药及28例ADV联合拉米夫定治疗拉米夫定耐药型HBeAg阳性慢性乙型肝炎患者的疗效进行回顾性分析,比较两组患者HBVDNA、ALT水平及HBV DNA检测不到率、ALT复常率的差异。结果两组患者的性别、年龄、治疗前的HBV DNA及ALT水平差异均无统计学意义。两组的HBV DNA、ALT水平在治疗48周、72周时分别与同组治疗前比较均有明显降低（P〈0．05）。治疗48周时,联合组的HBV DNA检测不到率虽高于单药组,但差异无统计学意义（50％vs25％,P〉0．05）;ALT复常率两组间无明显差别（67．9％vs75％,P〉0．05）。治疗72周时,联合组的HBV DNA检测不到率为68％,要显著高于单药组的33．3％（P〈0．05）;联合组的ALT复常率为93％,显著高于单药组的70．8％（P〈0．05）。结论ADV单药或联合拉米夫定均是治疗拉米夫定耐药HBeAg阳性CHB的有效方法,联合治疗的疗效要优于单药治疗。     

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.     

Virologic response is not durable after adefovir discontinuation in lamivudine-resistant chronic hepatitis B patients

Background/Aims

We investigated the durability of the biochemical and virologic responses after adefovir (ADV) discontinuation in lamivudine-resistant (LMV-R) chronic hepatitis B (CHB) patients, and the outcomes of ADV discontinuation compared to that of ADV maintenance.

Methods

The indication for ADV treatment cessation was an undetectable level of hepatitis B virus (HBV) DNA documented on two occasions at least 6 months apart. All patients received additional ADV for at least 12 months after the confirmation of undetectable HBV DNA (Cobas TaqMan PCR assay, <70 copies/mL). Of 36 patients who had a sufficient ADV therapeutic effect, 19 discontinued ADV treatment, while the others maintained it. A virologic rebound was arbitrarily defined as the redetection of HBV DNA at a level higher than 10⁵ copies/mL.

Results

In the ADV discontinuation group, ADV treatment and additional therapy were administered for medians of 33 months (range, 12-47 months) and 18 months, respectively. The patients were followed for a median of 12 months (range, 3-30 months) after ADV cessation. During that period, 18 of 19 patients (95%) experienced viral relapse. Viral rebound was observed in six patients (32%). However, 12 of 18 patients (67%) exhibited serum HBV DNA levels of less than 10⁵ copies/mL. Biochemical relapses were observed in four of the six patients with viral rebound. In the ADV maintenance group, patients were treated for a median of 53 months (range, 31-85 months), and 9 patients (53%) experienced viral breakthrough.

Conclusions

During short-term follow-up after ADV discontinuation, most patients (95%) exhibited viral relapse, whereas and viral breakthrough occurred in about half of patients (53%) maintained on ADV therapy. Therefore, the durability of virologic response after ADV discontinuation in LMV-R patients was unsatisfactory. In addition, and viral breakthrough was not infrequent in the ADV continuation group.