In this study, we described the dynamics of HBV genomic changes and other related parameters (ALT level, HBeAg/antiHBe profile and viral load) during a 14-year period that included sequential monotherapy with lamivudine, adefovir, entecavir, and entecavir-tenofovir in a chronically HBV-infected patient. This patient initially treated with interferon, later consecutively resistant to lamivudine, adefovir, and entecavir -either as monotherapy or combined with tenofovir- showed viral and biochemical resistance with higher HBV DNA levels and only transient viral suppression. It was expected to find viral replication impairment when rtA181T mutation was detected under adefovir monotherapy, since this variant has a secretory defect and exerts a dominant negative effect on wild-type HBV virion secretion. Thus, this mutation is usually detected as a mixed population with wild-type .
This patient showed persistent HBV replication as the major determinant of the emerging genomic mutations [7, 25].
Entecavir is a 100-300 fold more potent inhibitor of the wild-type viral polymerase compared to lamivudine-resistant polymerase, when the sensitivity is decreased in a 20-150 fold [26, 27]. In this clinical setting, entecavir 1 mg once daily has been proven to be effective in the treatment of lamivudine-resistant patients [9, 11]. In presence of the rtL180M and rtM204V as well as entecavir-associated mutations, the susceptibility to entecavir decreased dramatically as seen by an increase in inhibitory concentration 50% (IC50) values from 280 to over 1500 fold .
Persistent viral replication and the error rate of HBV reverse transcriptase (~10-4 base/replication cycle) are major factors in the development of resistance .
The viral polymerase characterization in our patient at quasispecies level showed lamivudine-associated mutations rtL180M and rtM204V/I during adefovir, entecavir and entecavir-tenofovir therapies.
During the entecavir monotherapy we observed no viral suppression and tenofovir 300 mg once daily was added to the therapy resulting in a rapid 2 Log10 decline in viral load. This level was sustained for seven months until a viral rebound and biochemical breakthrough were observed. In spite of the fact that the A181T adefovir-resistance and the T184L entecavir-resistance mutations were present in the background, they were only minor components of the HBV quasispecies that emerged once tenofovir was added in two consecutive samples separated by a five-month interval. The alanine at position 181 seems to be critical in the development of resistance to nucleos(t)ide analog, since it is located in α-helix adjacent to the nucleotide binding site . Taking into account that the availability of free replication space is necessary for the spread of the mutant virus, the kinetics of emerging drug-resistant mutants is usually slow. Such HBV variants became neither dominant in the two highly replicative (~108 IU/ml) viral populations nor were detected three months later. The intrinsic resistance of the mutant and its replicative fitness could explain the time to its emergence as well as the ephemeral contribution to HBV quasispecies. In addition, taking into account that the clonal analysis was performed on some samples with low viremia levels, the consequent reduction in the number of viral species detected could hamper the interpretation of results.
This behaviour in HBV is remarkably different from that described in two recently reported cases showing entecavir resistance with a similar therapeutic background [31, 32]. The A194T mutation that appears to lead to an over ten-fold decrease in tenofovir sensitivity  was not detected in the HBV quasispecies composition of those previous isolates during entecavir-tenofovir therapy.
The combination of the rtL180M+ rtM204V mutations harbored by our patient results in a replicative fitness of about 50% of that shown by the wild-type virus [28, 31]. Additional mutations able to restore replicative fitness to similar levels in the wild type such as rtV173L and rtP177S were not found. These factors may explain, in part, the difference in resistance rates between entecavir and lamivudine, as more profound genetic changes are necessary for a mutant to become the dominant strain. The role of the rtA200V and rtI253V mutations found during lamivudine therapy is not clear. Their impact on sensitivity and replication fitness is unknown, but nevertheless, they remained present during entecavir monotherapy or combined entecavir-tenofovir therapy, which may imply a certain influence of these mutations . Future in vitro research should clarify their impact on drug sensitivity and replication fitness.
In spite of the fact that adefovir and tenofovir are active against lamivudine resistant mutants, their activities decrease [14, 27, 35, 36], and lamivudine mutations appear to have an impact on the therapeutic efficacy of adefovir in this clinical setting [6, 37]. Also, it is important to consider that adefovir monotherapy in lamivudine-experienced patients is a treatment modality that carries a significant risk of resistance in the long term. Current guidelines recommend that it could be overcome by the adefovir-lamivudine combination therapy .
Considering that HBV viral load exhibited intermittent long-term fluctuations under a constant therapy during the follow-up, an inadequate adherence appears to be a plausible cause for treatment failure since an inadequate absorption or altered metabolism does not seem to impact in this case in view of the concentration of drug found in plasma . The viral load decline was fast in the short term, which additionally supports a probable adequate drug metabolism but it needs to be determined by further accurate pharmacokinetic studies.
Based on our findings and the available data on HBV resistance, and in lieu of the fact that tenofovir is an appropriate therapeutic alternative for entecavir-refractory patients, in this case tenofovir was not efficacious.