Original article Evolution of adefovir-resistant HBV polymerase gene variants after switching to tenofovir disoproxil fumarate monotherapy

Transcription

1 Antiviral Therapy 0; :0 0 (doi: 0./IMP0) Original article Evolution of adefovir-resistant HBV polymerase gene variants after switching to tenofovir disoproxil fumarate monotherapy Florian van Bömmel *, Jörg Trojan, Katja Deterding, Heiner Wedemeyer, Hermann E Wasmuth, Dietrich Hüppe, Bernd Möller, Friederike J Bock, Heinz-Hubert Feucht, Thomas Berg Department of Hepatology, Clinic for Gastroenterology and Rheumatology, University Clinic Leipzig, Leipzig, Germany Department of Internal Medicine I, Johann Wolfgang Goethe-University Hospital, Frankfurt, Germany Abteilung für Gastroenterologie, Hepatologie und Endokrinologie, Medizinische Hochschule Hannover, Hannover, Germany Medical Department III, University Hospital Aachen, Aachen, Germany Gastroenterologische Schwerpunktpraxis, Herne, Germany Hepatologische Schwerpunktpraxis, Berlin, Germany Charité-Universitätsmedizin Berlin, Medizinische Klinik m.s. Hepatologie und Gastroenterologie, Berlin, Germany Laborgemeinschaft Hamburg, Hamburg, Germany *Corresponding author florian.vanboemmel@medizin.uni-leipzig.de Background: Tenofovir disoproxil fumarate (), an acyclic nucleotide analogue was shown to be effective in many HBV-infected patients with resistance to adefovir dipivoxil (ADV). This observation is intriguing because in vitro studies show that HBV mutations selected by ADV confer cross-resistance to. To assess the clinical relevance of this cross-resistance, we studied the evolution of HBV polymerase gene variants in patients with genotypic resistance against ADV (rtnt and/or rtav/t) during treatment. Methods: In 0 HBV-monoinfected patients ( male, mean age ± [range ] years, hepatitis B e antigen- positive) with virological breakthrough during ADV treatment associated with the mutations rtnt and/or rtat/v, HBV polymerase gene variants were studied during up to months of consecutive monotherapy with by population, line probe and clonal. Results: In all patients, switching to resulted in a continuous reduction of HBV DNA from a median of. (..) log 0 to. ( ) log 0, remaining in patients >00 at months. ADV-resistance mutations remained detectable throughout the whole observation period in most patients. Apart from an M0Q mutation in one sample, no new HBV polymerase gene mutations were found. In two patients with low level viraemia after weeks of, adding lamivudine led to a complete response within a few weeks. Conclusions: ADV-resistant HBV variants may further become selected during treatment, however they cause only a mild decrease in susceptibility. Introduction Currently, the nucleoside analogues lamivudine (LAM), telbivudine (LdT) and entecavir (ETV), as well as the two acyclic nucleotide analogues adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate () are licensed for the treatment of chronic HBV infections []. Nucleoside and acyclic nucleotide analogues represent different subclasses of reverse transcriptase inhibitors: while both are based on purines or pyrimidines, acyclic nucleotide analogues possess an open (acyclic) ribose ring which confers greater binding capacity to resistant HBV polymerase strains. At a therapeutic dose of 00 mg per day, shows significantly stronger antiviral efficacy as compared to ADV in a dose of 0 mg per day, as was recently demonstrated by two prospective, randomized trials in both hepatitis B e antigen (HBeAg)-positive and HBeAg-negative patients, most of whom were treatment-naive [,]. Importantly, was shown to successfully suppress viral replication in patients with genotypic LAM-resistance, as well as in patients who 0 International Medical Press - (print) 00-0 (online) 0

2 F van Bömmel et al. exhibited incomplete response to ADV without any evidence of genotypic resistance to ADV (that is, ADV primary non-responders) [ ]. To date, no selection of HBV polymerase gene mutations associated with breakthrough of HBV replication during treatment has been reported []. During treatment with ADV, by contrast, genotypic resistance with viral breakthrough does occur, namely in up to % of treatment-naive patients and in up to % of patients with prior LAM resistance after years of ADV monotherapy [ ]. and ADV possess closely related chemical structures, have equipotent antiviral activity against HBV on a molar basis and may also share some cross-resistance, as documented by in vitro studies revealing reduced susceptibility of ADV-resistant HBV strains to [ ]. Thus, for the mutations rtnt and rtat/v selected by ADV, a - to -fold and a - to -fold decrease in ADV susceptibility could be observed, as compared to a -fold and a.-fold decrease in susceptibility, respectively [,]. In a recent clinical study, patients with genotypic ADV resistance and low-level HBV replication levels showed equal responsiveness to monotherapy compared to patients without ADV resistance []. However, in other studies, including some patients with ADV resistance and HBV replication levels > log 0, a significantly weaker responsiveness to was found during a treatment period of up to weeks [,,]. ADV as monotherapy has been largely abandoned in many recent treatment guidelines for HBV infections and it is now often replaced by monotherapy. For patients with ADV resistance, a combination treatment consisting of and a nucleoside analogue is recommended [,]. Since patients with prior ADV treatment now mostly receive, it remains an important question whether the loss of responsiveness observed in some patients is associated with ongoing selection of ADV resistance-associated mutations and whether different treatment strategies should apply for these patients. Therefore, we studied the evolution of HBV polymerase gene mutations during monotherapy in a group of patients with genotypic ADV resistance. Methods Patients From a previously published cohort of treatmentexperienced patients receiving monotherapy, patients with viral breakthrough associated with genotypic resistance to ADV monotherapy who were subsequently treated with monotherapy (00 mg/ day) were identified in German centres []. Patients were included in the study if serum samples stored at -0 C from the initial phase (months for patients b and i, month 0 for all other patients) after switching from ADV to treatment and at least one further follow-up sample during treatment were available, which was the case for 0 out of the patients. Mean HBV DNA and alanine aminotransferase (ALT) levels were similar in the 0 patients included and in the patients, who were not included (P= not significant, data not shown). All patients were treated with for a minimum duration of > months (range months) and all patients were adherent to the treatment regimen according to the study investigators reports. Patient characteristics Characteristics of the 0 patients at the start of treatment with are shown in Table. All patients had received ADV monotherapy for a median duration of (range ) months after they had developed resistance in the course of preceding LAM therapy (median duration [range ] months). Resistance to ADV in these patients was defined as viral breakthrough > log copies/ ml from nadir during ADV monotherapy and confirmed by detection of HBV polymerase gene mutations associated with resistance to ADV by direct. Treatment was switched promptly from ADV 0 mg/ day to 00 mg/day. Because was not approved for the treatment of HBV infection at the time of the study, informed consent was obtained from all patients before the start of treatment. End points The primary end point was the distribution of mutations associated with resistance to ADV during treatment. Secondary objectives were the proportion of patients who achieved HBV DNA levels <00 or < after months of monotherapy and the frequency of viral breakthrough due to HBV resistance as well as changes within the HBV polymerase gene, apart from mutations associated with ADV resistance during treatment. Evaluation of treatment During treatment, HBV DNA and ALT levels were measured every months. HBV DNA was measured using the Roche Amplicor (lower detection limit 00 ) or the COBAS TaqMan (Roche Diagnostic Systems, Branchburg, NJ, USA; lower detection limit IU/ml); samples with HBV DNA levels <00 measured by Roche Amplicor were re-measured with the more sensitive COBAS TaqMan. Results were expressed as by multiplication of the units/ml by factor., as suggested by the manufacturer, resulting in a lower limit of (or. log 0 ) or IU/ml, respectively International Medical Press

3 Selection of HBV variants in ADV-resistant patients during treatment Table. Characteristics of 0 patients with genotypic resistance to ADV at the start of treatment with 00 mg/day Age, Weight, HBV HBe- Duration, Duration, HBV DNA level, ALT level, Patient Gender years kg genotype antigen months a months b Mutations cd Mutations de log 0 IU/ml a M 0 A Positive NT WT 0%; NT 0% b M D Positive AT, NT WT %; AT 0%;. AS+NT % c M 0 D Positive AT, NT WT %;. AT+NT % d M 0 D Positive 0 NT L0M+M0T 0%;. 0 AV 0%; AV+NT 0% e M 0 0 D Negative AV, NT WT 0%;. AV+NT 0% f F D Positive NT WT 0%; NT 0%. g M D Negative AT, NT AV+NT 00%. h M D Negative 0 M0Q, AT N/A. i M D Negative AV AV 00%. j M D Positive L0M, M0V, WT 0%; L0M 0%;. NT NT 0% a Lamivudine (LAM) treatment prior to adefovir dipivoxil (ADV) treatment. b ADV pre-treatment. c HBV polymerase gene mutations by direct. d The first available resistance for patients b and i was at month, for all other patients at month 0 of tenofovir disoproxil fumarate () treatment. e HBV polymerase gene mutations by clonal. ALT, alanine aminotransferase; F, female; M, male; N/A, not available; WT, HBV wild type. Determination of HBV polymerase gene variants and HBV genotype of the HBV polymerase gene as previously described was applicable in out of available serum samples stored at -0 C ( samples per patient) collected during the initial phase and at various intervals during monotherapy [0]. To do so, a PCR product bridging the region from codon rt to rt was amplified using nested PCR and the primers (_-AGACTCGTGGTGGACTTCTCT-_) and 0 (_-AGAATGTTTGCTCCAGACC-_) as external primers, and (_-GGATGTGTCTGCG- GCGTTT-_) and (_ACGTTGACAGACTTTC- CAATC-_) as internal primers. In seven samples, was not applicable due to low viral load. A total of serum samples were investigated for concomitantly occurring HBV variants by using a reverse hybridization line probe (Inno-LiPA HBV DR v, v and v, Innogenetics, Ghent, Belgium) as previously described []. Additionally, PCR products from serum samples were cloned into an E. coli cloning system (TOPO-TA cloning system, Invitrogen, Carlsbad, CA, USA), as previously described, and to colonies were sequenced [0]. Published nomenclature for amino acid positions in the HBV polymerase gene was used [,]. HBV variants and genotypes were determined by sequence alignment of the overlapping hepatitis B surface antigen with HBV sequences derived from GenBank, as previously described []. Prior to this, the derived sequences of the polymerase gene of both strands of the amplification products were compared with published wild-type sequences of the same genotype derived from GenBank and investigated for all described HBV mutations associated with resistance to nucleoside or nucleotide analogues, especially for the ADV resistance-associated mutations rtav/t and rtnt, as well as for LAM resistance-associated mutations (rtvl, rtl0m and rtm0i/v/s). Results Detection of ADV resistance-associated variants during monotherapy by population At the start of treatment the mutation rtnt was detectable in eight patients and the mutations rtat/v in six patients, and a combination of both mutations in four patients by population (Table ). One patient additionally showed the mutation rtm0v and one showed the mutation rtm0q, which has previously not been described. During treatment in seven patients in whom the mutation rtnt was detected at baseline, it remained detectable at all time points during treatment (mean, range months) when population was applicable (Figure A E, G and J) and became detectable for a period of months in one patient, who had initially shown the mutation rtat (Figure H). In one patient, the single mutation rtav remained detectable for a period of months from the initial treatment phase (Figure I). Three patients with one mutation associated with ADV resistance at the beginning of treatment showed a combination of rtav/t and rtnt during the course of treatment (Figure J, D and H). Antiviral Therapy. 0

4 F van Bömmel et al. Figure. Individual HBV DNA levels and changes within the HBV polymerase gene in ten patients with genotypic resistance to ADV during monotherapy with 00 mg per day A + LAM 0 HBV DNA, log 0 0 rtnt rtnt WT, % rtnt, % rtat rtnt, % Number B 0 + LAM 0 HBV DNA, log 0 rtat + (+) + + N/A rtnt N/A rtat + + N/A + + rtnt + + N/A + + WT, % N/A rtat, % N/A rtat N/A rtnt, % rtas N/A rtnt, % Number 0 N/A C HBV DNA, log rtat N/A rtnt N/A rtat + N/A + N/A (+) N/A rtnt + N/A + N/A + N/A WT, % N/A rtat N/A rtnt, % Number 0 N/A (A J) Data from patients a to j, respectively, is represented. The results from population and from of the HBV quasispecies by line probe and clonal at the corresponding time points are given in the tables below each graph. :. ADV, adefovir dipivoxil; LAM, lamivudine; N/A, not applicable;, tenofovir disoproxil fumarate; WT, wild type. 0 0 International Medical Press

5 Selection of HBV variants in ADV-resistant patients during treatment Figure. Continued D HBV DNA, log rtat N/A rtm0v N/A rtnt N/A rtl0v - N/A N/A rtav + N/A N/A rtiv + N/A N/A rtnt + N/A N/A rtl N/A N/A rtm0v, % rtav, % 0 0 N/A N/A rtav+ 0 0 N/A N/A rtnt, % Number 0 0 N/A N/A E HBV DNA, log 0 0 rtav + + N/A rtnt + + N/A rtav + + N/A rtnt + + N/A WT, % 0 0 N/A rtav+ 0 0 N/A rtnt, % Number 0 0 N/A 0 F 0 0 rtnt + N/A rtnt + + WT, % 0 N/A rtnt, % 0 N/A Number 0 HBV DNA, log 0 Antiviral Therapy. 0

6 F van Bömmel et al. Figure. Continued G HBV DNA, log rtat + + N/A rtnt + + N/A rtl0m rtat rtm0v rtnt WT, % 0 N/A rtav+ 00 N/A rtnt, % Number N/A H HBV DNA, log rtat N/A rtm0q + + (+) + N/A rtnt N/A rtl0m N/A N/A rtat N/A N/A rtm0v N/A N/A rtnt N/A + + (+) N/A WT, % N/A 00 N/A rtm0q, % N/A 0 N/A rtat, % N/A 0 0 N/A rtnt, % N/A 0 0 N/A rtat+ N/A 0 N/A rtnt, % Number N/A N/A I HBV DNA, log 0 0 rtav rtl0m rtav rtm0v rtav, % Number 0 0 International Medical Press

7 Selection of HBV variants in ADV-resistant patients during treatment Detection of HBV minor species by line probe and cloning At the start of treatment, the mutations rtnt, rtav and rtat were found in nine, three and four patients by line probe. Also, the mutations rtl0v, rtvl, rtm0 and rtm0v were detectable in some patients and the mutation rtiv was detected in one patient (Figure A J). Similar to population results, the mutations associated with ADV resistance remained detectable during treatment (Figure A J). Mutations associated with LAM resistance, which in some patients were simultaneously detectable with ADV resistant variants during the initial treatment phase, disappeared during the course of treatment (Figure G J). The proportion of HBV wild-type and HBV polymerase gene variants associated with HBV resistance concurrently present in one sample was assessed by clonal in of available serum samples from all 0 patients (Figure A J). In the initial phase of, treatment variants associated with resistance to ADV (rtnt, rtat/v) were found in 0% of the sequenced clones in all except patients e and h. In patient b, the rtas variant, which was recently described to be associated with resistance against LAM and ADV, was detected in one patient in combination with the mutation rtnt in out of the clones during the early treatment phase not in the follow-up samples taken during therapy []. As demonstrated with the line probe, mutations associated with LAM resistance were detectable in some patients during the initial treatment phase, but they disappeared during treatment (patients d and j). In addition, the relative percentage of wild-type HBV present in patients a, b, c and j at the first time point of clonal decreased or became undetectable. In patient h, a new variant, rtm0q, could be detected at baseline and remained detectable until month. At the same time points, the mutations rtat and rtnt were detected at months, and. A similar pattern, including the mutation rtm0q, was found by clonal at months and in out of and out of clones, respectively (Figure H). In contrast, at month, clonal showed HBV wild type in all clones sequenced. Antiviral efficacy of During ADV monotherapy and before the start of our study, all patients had suffered a resistance-associated virological breakthrough, and most patients had high HBV DNA levels at the start of treatment (as summarized in Table ). After switching to monotherapy, all patients showed a significant reduction in HBV DNA levels. Median HBV DNA levels decreased from. (..) log 0 at the start of Figure. Continued J HBV DNA, log rtat N/A rtm0v N/A rtnt N/A rtvl N/A rtl0m N/A rtav N/A rtat N/A rtm0v N/A rtnt N/A WT, % N/A rtl0m N/A rtm0v, % rtat N/A rtnt, % rtav N/A rtnt, % rtnt, % N/A Number 0 N/A treatment to a median of. (..) log 0 at months and. ( ) log 0 at months (Figure ). At month, HBV DNA was still > in eight and >00 in seven of the ten patients (median HBV DNA. [..] log 0 copies/ ml). At the end of the observation period (mean duration ±. [ ] months), five patients showed a complete virological response and had HBV DNA levels <00 and four of them had HBV DNA levels <. In the remaining six patients, HBV DNA levels were.,.0,.,.0,.0 and. log 0 (Figure A D, G and I). No breakthrough of HBV replication during monotherapy was observed in any of the patients. Median serum ALT levels decreased gradually over the first months of therapy. At the discretion of the attending physician, two patients with persistently measurable HBV replication Antiviral Therapy. 0

8 F van Bömmel et al. Figure. Follow-up of median HBV DNA levels and ALT levels in ten patients with genotypic ADV resistance after switching to monotherapy with 00 mg per day 0 00 HBV DNA, log 0 HBV DNA ALT ALT, U/L a 0 0 Month of treatment Patients under observation, n: Standard deviation is indicated by error bars. a :. ADV, adefovir dipivoxil; ALT, alanine aminotransferase;, tenofovir disoproxil fumarate. had LAM 00 mg/day added to their ongoing 00 mg/day therapy at month. This resulted in a prompt and complete HBV DNA response (Figure A and B). Factors associated with response to The initial slope of viral decline was comparable in all patients, irrespective of the pattern of viral variants (Figure ). There was no correlation between patient characteristics, HBV genotype, HBe-antigen status and the virological response pattern to. Also, the duration of previous treatment with ADV, as well as with LAM, did not differ for patients with complete and patients with partial response to (Student s t-test; P=0.). However, the level of baseline HBV DNA was significantly associated with response to. Thus, ADV-resistant patients with HBV DNA levels 0 at baseline (patients e, f and j) were more likely to achieve HBV DNA levels < after months of monotherapy, compared with patients with HBV DNA>0 (patients a d, g and h; P=0.0, Fisher s exact test). Discussion To date, HBV polymerase gene mutations conferring resistance to have not been described, although the chemical structures of and ADV are very similar. However, the HBV variants rtav and rtnt were found to lower the susceptibility to both substances in vitro and some patients in whom these mutations were selected during ADV treatment showed reduced susceptibility to [,,]. In order to further evaluate this phenomenon we studied the evolution of ADV resistance-associated mutations in 0 patients after switching to a monotherapy with. Interestingly, although all of the 0 patients studied showed a pronounced decline in hepatitis B viraemia during treatment, we found a persistence of the HBV variants rtnt or rtav/t as single or as combined mutations provided the methods used for sequence were applicable. Generally, during treatment, a homogenization of the HBV subspecies was observed in terms of persistence of variants associated with ADV resistance and decrease or loss of mutations associated with LM resistance and wildtype HBV. In some patients, ADV resistance-associated mutations even became selected as the dominant population, as was the case in patients a e (Figure ). Our observation of ongoing selection of mutations associated with ADV resistance, especially the mutation rtnt are in accordance with phenotypic in vitro studies showing a mild decrease in susceptibility caused by these variants [,]. While this relatively mild decrease in susceptibility may be sufficient to cause a viral rebound in patients treated with a dosage of 0 mg ADV per day, might still be largely effective 0 0 International Medical Press

9 Selection of HBV variants in ADV-resistant patients during treatment when administered at higher dosages of 00 mg per day, as indicated by the marked decrease in HBV DNA observed in all patients, and suppression to undetectable HBV DNA levels in four of the patients studied. Importantly, neither viral breakthrough nor selection of new mutations was observed during the follow-up, including the previously discussed rtat mutation []. However, after a median duration of months of treatment, HBV DNA levels remained detectable in most of the patients when measured by highly sensitive s. This is of concern because ongoing viral replication during treatment with nucleoside/nucleotide analogues (even at low levels) can be an important risk factor for the selection of resistance-associated variants and probably also for disease progression. Due to persistent measurable HBV replication during monotherapy in patients a and b, combination treatment with and LAM was initiated after and months, respectively, resulting in a complete suppression of HBV DNA replication. Before LAM was added to, in patient a the detectable HBV quasispecies had completely evolved to variants associated with ADV resistance (rtnt, rtav/t or a pattern of both mutations). The late initiation of combination treatment in these two patients is not in-line with recent HBV treatment guidelines which demand a direct addon of a nucleoside analogue to ongoing treatment with a nucleotide analogue in patients with partial response or resistance to ADV [,]. However, the relative homogenisation of the HBV quasispecies towards ADVresistant variants, which was observed during monotherapy, might have optimized the efficacy of the combination treatment. We are still faced with the intriguing observation that in most patients with ADV resistance, complete suppression of HBV replication can be achieved with monotherapy, while in some patients HBV replication remains measurable at low levels [,,]. The observation, that some patients carrying the mutation rtnt alone or in combination with the mutation rtav/t only show partial response to during up to months was recently confirmed in another study in patients with suboptimal response or resistance to ADV []. In this study, persistence of ADV resistance-associated mutations was also associated with the level of HBV DNA at the start of treatment. Most interestingly, in of the patients, substitutions in the HBV polymerase associated with resistance to ADV newly emerged during treatment []. In contrast, in another recent study evaluating in patients with a suboptimal response to ADV, the presence of mutations associated with ADV resistance did not influence the response to subsequent monotherapy []. However, none of the patients with genotypic ADV-resistant variants had experienced a virological breakthrough during treatment with ADV, probably because the proportion of ADV-resistant variants within the quasispecies population may have been relatively small. Moreover, the duration of ADV treatment before switching to was limited to a maximum of months in these patients, whereas many of the patients in our study had received ADV for a longer period. This suggests that not only the presence of genotypic ADV resistance but the occurrence of a viral breakthrough during ADV treatment, as well as the level of HBV replication in patients with ADV-resistance, may influence the efficacy of subsequent monotherapy. However, since in our study none of the patients with ADV resistance experienced a viral breakthrough during monotherapy the current general recommendation of add-on combination treatment with a nucleoside analogue in patients with incomplete response to monotherapy is challenged and should be further studied. Another possible approach for treating patients with a partial response or genotypic resistance to ADV has been presented in two recent studies, in which patients were directly switched from ADV to monotherapy with entecavir [,]. However, entecavir monotherapy can be impaired by the presence of LAM resistance mutations, which can persist even on long-term ADV therapy as documented in our study (patients d and j). In conclusion, in most patients with genotypic resistance to ADV and intermediate or high HBV replication during monotherapy, continuous selection of the rtnt and rtav/t mutation was observed. It seems plausible that these mutations may have reduced susceptibility in some patients. Nevertheless, all patients showed a strong and lasting decrease in HBV replication without a virological breakthrough. Other treatment options such as ETV monotherapy or combination treatment with and LAM on the selection of mutations associated with ADV resistance are certainly of interest and should be examined in future studies comprising a larger number of patients. Acknowledgements This work was supported by the German Competence Network for Viral Hepatitis (Hep-Net), funded by the German Ministry of Education and Research (BMBF, grant number 0 KI 0, project number 0.., core project number 0.), Genetic Host Factors in Viral Hepatitis and Genetic Epidemiology Group in Viral Hepatitis, the BMBF project: Host and viral determinants for susceptibility and resistance to hepatitis C virus infection (grant number 0KI0) and the BMBF Project: HOPE (grant number 000-) DLR 0ES0 and by the EU-Vigilance network of excellence combating viral resistance (VIRGIL, Antiviral Therapy. 0

10 F van Bömmel et al. project number LSHM-CT-00-0). The authors acknowledge Bernhard Zöllner for assistance with laboratory work. Disclosure statement FvB and TB have received grant support from Gilead Sciences. TB and DH are members of the Gilead advisory board. All remaining authors declare no competing interests. References. Wiegand J, van Bömmel F, Berg T. Management of chronic hepatitis B: status and challenges beyond treatment guidelines. Semin Liver Dis 00; 0:.. Heathcote E, Gane EJ, De Man RA, et al. Long-term ( year) efficacy and safety of tenofovir disoproxil fumarate () treatment in HBeAg-positive patients (HBeAg+) with chronic hepatitis B (study 0). Hepatology 00; :A.. Marcellin P, Buti M, Krastev Z, et al. Continued efficacy and safety through years of tenofovir disoproxil fumarate () treatment in HBeAg-negative patients with chronic hepatits B (study 0). Hepatology 00; :A.. Snow-Lampart A, Chappell B, Curtis M, et al. No resistance to tenofovir disoproxil fumarate detected after up to weeks of therapy in patients monoinfected with chronic hepatitis B virus. Hepatology 0; :.. van Bömmel F, Zöllner B, Sarrazin C, et al. Tenofovir for patients with lamivudine-resistant hepatitis B virus (HBV) infection and high HBV DNA level during adefovir therapy. Hepatology 00; :.. Tan J, Degertekin B, Wong SN, et al. Tenofovir monotherapy is effective in hepatitis B patients with antiviral treatment failure to adefovir in the absence of adefovir-resistant mutations. J Hepatol 00; :.. Berg T, Marcellin P, Zoulim F, et al. Tenofovir is effective alone or with emtricitabine in adefovir-treated patients with chronic-hepatitis B virus infection. Gastroenterology 00; :0.. Levrero M, Cimino L, Lampertico P, et al. Tenofovir () for chronic hepatitis B patients with suboptimal response to adefovir (ADV) or ADV/LAM treatment: results of the OptiB Italian multicenter prospective open label study. Hepatology 00; :A.. Angus P, Vaughan R, Xiong S, et al. Resistance to adefovir dipivoxil therapy associated with the selection of a novel mutation in the HBV polymerase. Gastroenterology 00; :. 0. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Longterm therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B. N Engl J Med 00; :.. Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, et al. Longterm therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up to years. Gastroenterology 00; :. Accepted 0 January 0; published online August 0. Lada O, Benhamou Y, Cahour A, et al. In vitro susceptibility of lamivudine-resistant hepatitis B virus to adefovir and tenofovir. Antivir Ther 00; :.. Delaney WE, IV, Ray AS, Yang H, et al. Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus. Antimicrob Agents Chemother 00; 0:.. Qi X, Xiong S, Yang H, et al. In vitro susceptibility of adefovir-associated hepatitis B virus polymerase mutations to other antiviral agents. Antivir Ther 00; :.. Brunelle MN, Jacquard AC, Pichoud C, et al. Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir. Hepatology 00; :.. Patterson SJ, George J, Strasser SI, et al. Tenofovir disoproxil fumarate rescue therapy following failure of both lamivudine and adefovir dipivoxil in chronic hepatitis B. Gut 0; 0:.. van Bömmel F, de Man RA, Wedemeyer H, et al. Long-term efficacy of tenofovir monotherapy for hepatitis B virusmonoinfected patients after failure of nucleoside/nucleotide analogues. Hepatology 00; : 0.. European Association For The Study Of The Liver. EASL Clinical Practice Guidelines: management of chronic hepatitis B. J Hepatol 00; 0:.. Lok ASF, McMahon BJ. Chronic hepatitis B. Hepatology 00; :0. 0. Zöllner B, Petersen J, Puchhammer-Stöckl E, et al. Viral features of lamivudine resistant hepatitis B genotypes A and D. Hepatology 00; : 0.. Stuyver L, Van Geyt C, De Gendt S, et al. for monitoring drug resistance in hepatitis B virus-infected patients during antiviral therapy. J Clin Microbiol 000; :0 0.. Stuyver LJ, Locarnini SA, Lok A, et al. Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region. Hepatology 00; :.. Lok AS, Zoulim F, Locarnini S, et al. Antiviral drug-resistant HBV: standardization of nomenclature and s and recommendations for management. Hepatology 00; :.. Karatayli E, Karayalçin S, Karaaslan H, et al. A novel mutation pattern emerging during lamivudine treatment shows cross-resistance to adefovir dipivoxil treatment. Antivir Ther 00; :.. Sheldon J, Camino N, Rodes B, et al. Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir. Antivir Ther 00; 0:.. Reijnders JG, Pas SD, Schutten M, et al. Entecavir shows limited efficacy in HBeAg-positive hepatitis B patients with a partial virologic response to adefovir therapy. J Hepatol 00; 0:.. Lai CL, Elion R, Sherman M, et al. Entecavir (ETV) therapy in chronic hepatitis B patients previously treated with adefovir (ADV) with incomplete response on-treatment or relapse off-treatment. J Hepatol 00; 0:S. 0 0 International Medical Press