Monitoring antiviral resistance in patients receiving nucleos(t)ide analog therapies for hepatitis B: Which method should be used?
Authors: Bulent Degertekin, Anna S. Lok
Journal: Journal of Hepatology (2008)

Antiviral drug resistance is a major hurdle to the success of nucleos(t)ide analog therapy for hepatitis B. Among nucleoside-naïve patients, antiviral drug resistance had been reported in up to 70% of patients after 4 years of lamivudine (LVD), 29% after 5 years of adefovir, 1% after 4 years of entecavir (ETV), and 9–22% after 2 years of telbivudine [1Lok A.S. Lai C.L. Leung N. Yao G.B. Cui Z.Y. Schiff E.R. et al.Long-term safety of lamivudine treatment in patients with chronic hepatitis B.Gastroenterology. 2003; 125: 1714-1722Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 2Hadziyannis S.J. Tassopoulos N.C. Heathcote E.J. Chang T.T. Kitis G. Rizzetto M. et al.Long-term therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up to 5 years.Gastroenterology. 2006; 131: 1743-1751Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar, 3Colonno R.T. Rose R.E. Pokornowski K. Baldick C.T. Eggers B. Four year assessment of ETV resistance in nucleoside-naive and lamivudine refractory patients.J Hepatol. 2007; 46: S294Abstract Full Text PDF Google Scholar, 4Lai C. Gane E. Hsu C.W. Thongsawat S. Wang Y. Two-year results from the globe trial in patients with hepatitis B: greater clinical and antiviral efficacy for telbivudine (LdT) vs. lamivudine.Hepatology. 2007; 46: 63AGoogle Scholar]. Resistance rates are substantially higher in patients with prior resistance to LVD, with rates of up to 20% after 2 years of adefovir and 40% after 4 years of entecavir [3Colonno R.T. Rose R.E. Pokornowski K. Baldick C.T. Eggers B. Four year assessment of ETV resistance in nucleoside-naive and lamivudine refractory patients.J Hepatol. 2007; 46: S294Abstract Full Text PDF Google Scholar, 5Fung S.K. Chae H.B. Fontana R.J. Conjeevaram H. Marrero J. Oberhelman K. et al.Virologic response and resistance to adefovir in patients with chronic hepatitis B.J Hepatol. 2006; 44: 283-290Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar]. Sequential therapy had also been reported to result in the selection of multi-drug resistant HBV [6Villet S. Pichoud C. Villeneuve J.P. Trepo C. Zoulim F. Selection of a multiple drug-resistant hepatitis B virus strain in a liver-transplanted patient.Gastroenterology. 2006; 131: 1253-1261Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 7Yim H.J. Hussain M. Liu Y. Wong S.N. Fung S.K. Lok A.S. Evolution of multi-drug resistant hepatitis B virus during sequential therapy.Hepatology. 2006; 44: 703-712Crossref PubMed Scopus (222) Google Scholar]. A fundamental issue regarding antiviral resistance is the criteria for defining drug-resistant mutations and the method used to diagnose drug resistance [[8]Lok A.S. Zoulim F. Locarnini S. Bartholomeusz A. Ghany M.G. Pawlotsky J.M. et al.Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management.Hepatology. 2007; 46: 254-265Crossref PubMed Scopus (406) Google Scholar]. In clinical practice, virologic breakthrough (defined as a greater than 1 log increase in serum hepatitis B virus [HBV] DNA from nadir during treatment) is often the only method used to diagnose drug resistance. However, breakthroughs are not always due to drug resistance. Clinical trials found that 30–50% of patients with virologic breakthroughs had no detectable antiviral drug-resistant mutations indicating that non-adherence may be the reason for the breakthroughs [9Lai C.L. Chien R.N. Leung N.W. Chang T.T. Guan R. Tai D.I. et al.A one-year trial of lamivudine for chronic hepatitis B. Asia hepatitis lamivudine study group.N Engl J Med. 1998; 339: 61-68Crossref PubMed Scopus (1791) Google Scholar, 10Dienstag J.L. Schiff E.R. Wright T.L. Perrillo R.P. Hann H.W. Goodman Z. et al.Lamivudine as initial treatment for chronic hepatitis B in the United States.N Engl J Med. 1999; 341: 1256-1263Crossref PubMed Scopus (1342) Google Scholar, 11Chang T.T. Gish R.G. de Man R. Gadano A. Sollano J. Chao Y.C. et al.A comparison of entecavir and lamivudine for HBeAg-positive chronic hepatitis B.N Engl J Med. 2006; 354: 1001-1010Crossref PubMed Scopus (1276) Google Scholar, 12Lai C.L. Gane E. Liaw Y.F. Hsu C.W. Thongsawat S. Wang Y. et al.Telbivudine versus lamivudine in patients with chronic hepatitis B.N Engl J Med. 2007; 357: 2576-2588Crossref PubMed Scopus (713) Google Scholar, 13Lai C.L. Shouval D. Lok A.S. Chang T.T. Cheinquer H. Goodman Z. et al.Entecavir versus lamivudine for patients with HBeAg-negative chronic hepatitis B.N Engl J Med. 2006; 354: 1011-1020Crossref PubMed Scopus (1071) Google Scholar]. Empiric modification of treatment based on presumed diagnosis of drug resistance may unnecessarily expose patients to sequential therapies and eventually exhaust all available treatments. On the other hand, failure to detect antiviral drug-resistant mutations does not exclude the possibility that breakthrough was related to drug resistance. Drug-resistant mutations may be present but were not detected due to lack of sensitivity of the method used. Direct (or population) sequencing of PCR-amplified HBV DNA is the most common method used for detecting antiviral drug-resistant mutations but these mutations need to reach approximately 20% of the total HBV quasispecies pool to be detected. Restriction fragment length polymorphism (RFLP) analyses and reverse hybridization assay (line probe, LiPA DR) are more sensitive and can detect drug-resistant mutations that comprise 5% of the total viral population [14Kim H.S. Han K.H. Ahn S.H. Kim E.O. Chang H.Y. Moon M.S. et al.Evaluation of methods for monitoring drug resistance in chronic hepatitis B patients during lamivudine therapy based on mass spectrometry and reverse hybridization.Antivir Ther. 2005; 10: 441-449PubMed Google Scholar, 15Lok A.S. Zoulim F. Locarnini S. Mangia A. Niro G. Decraemer H. et al.Monitoring drug resistance in chronic hepatitis B virus (HBV)-infected patients during lamivudine therapy: evaluation of performance of INNO-LiPA HBV DR assay.J Clin Microbiol. 2002; 40: 3729-3734Crossref PubMed Scopus (140) Google Scholar, 16Hussain M. Fung S. Libbrecht E. Sablon E. Cursaro C. Andreone P. et al.Sensitive line probe assay that simultaneously detects mutations conveying resistance to lamivudine and adefovir.J Clin Microbiol. 2006; 44: 1094-1097Crossref PubMed Scopus (50) Google Scholar, 17Allen M.I. Gauthier J. DesLauriers M. Bourne E.J. Carrick K.M. Baldanti F. et al.Two sensitive PCR-based methods for detection of hepatitis B virus variants associated with reduced susceptibility to lamivudine.J Clin Microbiol. 1999; 37: 3338-3347PubMed Google Scholar]. However, these assays can only detect a limited number of previously characterized mutations. Furthermore, RFLP analysis may not be possible for all resistant mutations as endonucleases specific for such sequences may not exist. Mutations may be missed because they are not captured by the assay e.g. mutations located outside the reverse transcriptase (RT) domain of the HBV polymerase gene and mutations not detected by any of the probes in the line probe assay. In this regard, sequencing using microchip-based technology using oligonucleotide microarrays has the advantage that the entire viral genome can be scanned in one assay [[18]Tran N. Berne R. Chann R. Gauthier M. Martin D. Armand M.A. et al.European multicenter evaluation of high-density DNA probe arrays for detection of hepatitis B virus resistance mutations and identification of genotypes.J Clin Microbiol. 2006; 44: 2792-2800Crossref PubMed Scopus (37) Google Scholar]. Furthermore, mutations may not be recognized if they have not been reported previously; this would particularly be difficult if baseline HBV sequences were not available for comparison. As an increasing number of patients with divergent HBV sequences are exposed to nucleos(t)ide analog treatments, additional mutations in the reverse transcriptase domain other than the signature resistance mutations may be identified, e.g. alanine to threonine substitution at position 181 (rtA181T) [[19]Yeh C.T. Chien R.N. Chu C.M. Liaw Y.F. Clearance of the original hepatitis B virus YMDD-motif mutants with emergence of distinct lamivudine-resistant mutants during prolonged lamivudine therapy.Hepatology. 2000; 31: 1318-1326Crossref PubMed Scopus (211) Google Scholar] and methionine to serine substitution at position 204 (rtM204S) [[20]Bozdayi A.M. Uzunalimoglu O. Turkyilmaz A.R. Aslan N. Sezgin O. Sahin T. et al.YSDD: a novel mutation in HBV DNA polymerase confers clinical resistance to lamivudine.J Viral Hepat. 2003; 10: 256-265Crossref PubMed Scopus (54) Google Scholar] had been reported to be associated with LVD resistance in addition to methionine to valine or isoleucine substitution at position 204 (rtM204V/I). Caution must be exercised in attributing changes in the HBV RT domain as the cause of antiviral drug resistance. The significance of several mutations such as rtA181T and secondary resistance to adefovir [21Borroto-Esoda K. Miller M.D. Arterburn S. Pooled analysis of amino acid changes in the HBV polymerase in patients from four major adefovir dipivoxil clinical trials.J Hepatol. 2007; 47: 492-498Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 22Lacombe K. Ollivet A. Gozlan J. Durantel S. Tran N. Girard P.M. et al.A novel hepatitis B virus mutation with resistance to adefovir but not to tenofovir in an HIV-hepatitis B virus-co-infected patient.Aids. 2006; 20: 2229-2231Crossref PubMed Scopus (40) Google Scholar], rtI233V and primary resistance to adefovir [23Schildgen O. Sirma H. Funk A. Olotu C. Wend U.C. Hartmann H. et al.Variant of hepatitis B virus with primary resistance to adefovir.N Engl J Med. 2006; 354: 1807-1812Crossref PubMed Scopus (196) Google Scholar, 24Curtis M. Zhu Y. Borroto-Esoda K. Hepatitis B virus containing the I233V mutation in the polymerase reverse-transcriptase domain remains sensitive to inhibition by adefovir.J Infect Dis. 2007; 196: 1483-1486Crossref PubMed Scopus (37) Google Scholar] and rtA194T and secondary resistance to tenofovir [25Sheldon J. Camino N. Rodes B. Bartholomeusz A. Kuiper M. Tacke F. et al.Selection of hepatitis B virus polymerase mutations in HIV-coinfected patients treated with tenofovir.Antivir Ther. 2005; 10: 727-734PubMed Google Scholar, 26Delaney 4th, W.E. Ray A.S. Yang H. Qi X. Xiong S. Zhu Y. et al.Intracellular metabolism and in vitro activity of tenofovir against hepatitis B virus.Antimicrob Agents Chemother. 2006; 50: 2471-2477Crossref PubMed Scopus (204) Google Scholar] remain controversial. Thus, phenotypic assays are necessary to confirm that a newly identified mutation(s) is associated with drug resistance and to define the spectrum of mutations associated with resistance to new therapies. In this issue of the Journal, Baldick et al. present a comprehensive phenotypic analysis of 511 patient isolates collected from clinical trials of ETV therapy in nucleoside-naïve and in LVD-refractory hepatitis B patients [[27]Baldick C.J. Eggers B.J. Fang J. Levine S.M. Pokornowski K.A. Rose R.E. et al.Hepatitis B virus quasispecies susceptibility to entecavir confirms the relationship between genotypic resistance and patient virologic response.J Hepatol. 2008; 48: 895-902Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar]. In previous studies, the authors demonstrated that mutations associated with resistance to LVD decrease susceptibility to ETV by 8-fold and addition of mutations at rtT184, S202, and M250 further decrease susceptibility to ETV by approximately 10-fold [[28]Tenney D.J. Levine S.M. Rose R.E. Walsh A.W. Weinheimer S.P. Discotto L. et al.Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to lamivudine.Antimicrob Agents Chemother. 2004; 48: 3498-3507Crossref PubMed Scopus (499) Google Scholar]. In the current study, the authors first investigated the correlation between genotypic resistance and ETV susceptibility using laboratory clones. Then, they repeated the same studies using patient-derived clones. The authors found that laboratory clones with rtM204V and rtL180M substitutions displayed 8-fold reduced ETV susceptibility, and addition of rtT184L, rtS202G or rtM250V substitutions further reduces ETV susceptibility by approximately 100-fold relative to wild-type HBV. They also showed that patient isolates displayed a similar relationship between genotypic and phenotypic resistance as compared to laboratory clones although the variability in ETV susceptibility was greater with patient derived clones. These findings are important as phenotypic assays are time-consuming [8Lok A.S. Zoulim F. Locarnini S. Bartholomeusz A. Ghany M.G. Pawlotsky J.M. et al.Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management.Hepatology. 2007; 46: 254-265Crossref PubMed Scopus (406) Google Scholar, 29Zoulim F. In vitro models for studying hepatitis B virus drug resistance.Semin Liver Dis. 2006; 26: 171-180Crossref PubMed Scopus (48) Google Scholar]. That the same results can be obtained by introducing a variety of mutations to laboratory clones versus creating separate clones derived from individual patient isolates enable phenotypic confirmation of newly identified putative drug-resistant mutations to be performed more expeditiously. The authors noted that while the results of individual patient clones generally correlated with ‘population phenotyping’ in which the entire patient isolate quasispecies is cloned en masse to mimic the circulating virus pool, a decrease in ETV susceptibility was observed only when clones with ETV-resistant mutations approach 25% of the virus population. Previous reports from these authors [28Tenney D.J. Levine S.M. Rose R.E. Walsh A.W. Weinheimer S.P. Discotto L. et al.Clinical emergence of entecavir-resistant hepatitis B virus requires additional substitutions in virus already resistant to lamivudine.Antimicrob Agents Chemother. 2004; 48: 3498-3507Crossref PubMed Scopus (499) Google Scholar, 30Colonno R.J. Rose R. Baldick C.J. Levine S. Pokornowski K. Yu C.F. et al.Entecavir resistance is rare in nucleoside naive patients with hepatitis B.Hepatology. 2006; 44: 1656-1665Crossref PubMed Scopus (321) Google Scholar] and some sections of the current paper indicated that the backbone for ETV resistance is a combination of rtL180M and rtM204V. However, data in this current study clearly showed that rtM204I alone has the same effect (Figs. 1B and 2, and Table 2). Clarification of this point is important as patients with telbivudine resistance which is associated with rtM204I but not rtM204V [[31]Lai C.L. Leung N. Teo E.K. Tong M. Wong F. Hann H.W. et al.A 1-year trial of telbivudine, lamivudine, and the combination in patients with hepatitis B e antigen-positive chronic hepatitis B.Gastroenterology. 2005; 129: 528-536Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar] would also have diminished response to ETV and increased risk of ETV resistance. The authors observed a range in ETV resistance in the phenotypic studies with rtS202G/C substitutions, some rtT184 substitutions, and multiple ETV resistance substitutions being associated with the highest level of resistance. These data need to be verified as only 1 or 2 clones from patient isolates were tested for some substitutions. Until further data are available, all the reported substitutions including those involving rtM250 should be considered to be associated with ETV resistance. An important contribution of Baldick et al.’s study is the careful analysis of isolates from patients with virologic breakthrough but no detectable substitutions at rt184, 202 and 250. The authors performed phenotypic studies using full-length patient isolates and recombinants created with the patients’ RT domain obtained at the time of virologic breakthrough and found similar ETV susceptibility as wild-type HBV clones as well as isolates from these patients obtained at baseline. By contrast, studies using isolates from patients with LVD or ETV resistance substitutions showed the expected ETV susceptibility profiles whether patient RT or full-length virus sequence was used. These data indicate that virologic breakthrough in patients with no detectable substitutions at rt184, 202 and 250 is likely related to non-compliance with medications and not to unidentified mutations in the RT domain or elsewhere in the HBV genome. How would the results of this study influence antiviral resistance monitoring of hepatitis B patients receiving nucleos(t)ide analog therapies? Serum HBV DNA monitoring to detect virologic breakthrough remains the most practical method in clinical practice. Testing for genotypic resistance should be performed in patients with virologic breakthrough. Treatment should be modified in patients found to have signature resistance mutations. Medication compliance should be reinforced in patients with no detectable resistance mutations. Additional testing using more sensitive methods for genotypic resistance may be performed. Alternatively, genotypic resistance can be repeated in patients with persistent virologic breakthrough despite medication compliance. Substitutions in the HBV RT domain that had not been reported to be associated with resistance to the antiviral drug administered should be tested in phenotypic assays. Laboratory strains with the identified substitutions introduced by site directed mutagenesis would suffice in most instances but confirmation of phenotypic resistance using selected patient isolates should still be performed when evaluating resistance to new antiviral drugs. Only substitutions shown to reduce susceptibility in carefully conducted phenotypic studies should be reported as resistance-associated mutations. These recommendations are in line with those of an international panel [[8]Lok A.S. Zoulim F. Locarnini S. Bartholomeusz A. Ghany M.G. Pawlotsky J.M. et al.Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management.Hepatology. 2007; 46: 254-265Crossref PubMed Scopus (406) Google Scholar].