Evolution and phenotypic characterization of whole HBV genome in compliant patients experiencing unexplained entecavir treatment failure.

Entecavir treatment failure can be observed in compliant patients despite an absence of detectable resistance mutations by Pol/RT Sanger sequencing. We hypothesized that these unexplained treatment failures could rely on other mechanisms of viral resistance, especially on mutations selected outside of the Pol/RT domain. Partial virological response to entecavir was observed in three patients treated with immunosuppressive drugs, without selection of Pol/RT resistance mutations. Mutations selected in the whole HBV genome during entecavir treatment and potentially associated with resistance were searched for using deep sequencing and characterized using a phenotypic resistance assay. Mutations Q206K (pre-core/core), Q120K (pre-S1/pre-S2, T-cell epitope) and A300E (spacer domain) were selected during entecavir treatment in patient #1 but were not associated with an increased level of resistance to entecavir or an increase in HBV replication capacity. Core promoter mutations T1753G, A1762T and G1764A were present as major mutations before and after treatment in patient #1. HBs Ag immune escape mutations were present as major mutations before and after treatment in patients #2 (sK122R, sT126I, sP127S and sG145R) and #3 (sM133I). We demonstrated that PVR to entecavir does not require selection of any resistance mutation in the whole HBV genome. Our results demonstrate that major mutations can be selected outside of the Pol/RT domain before or during entecavir treatment. These mutations could contribute to entecavir treatment failure by other mechanisms than an increased level of resistance.

Revisiting HBV resistance to entecavir with a phenotypic approach.

Treatment adaptation after hepatitis B virus (HBV) treatment failure relies on genotypic resistance testing. However, the results of such tests are not always consistent with treatment response. These discrepancies may be due to differences in resistance levels between isolates with the same genotypic resistance testing profiles. We explored this hypothesis by investigating six cases of entecavir treatment failure with an integrative strategy combining genotypic and phenotypic resistance testing, medical record review and therapeutic drug monitoring. Among isolates with genotypic reduced susceptibility to entecavir, one displayed a higher level of resistance to entecavir (mean fold change in entecavir IC50 of 1 508 ± 531 vs. 318 ± 53, p = 0.008). This isolate harbored a substitution (rt250L) at a position reported to be associated with resistance (rt250V). Reversion to wild-type amino acid at this position partially restored susceptibility to entecavir, confirming that the rt250L mutation was responsible for the high level of resistance to entecavir. This is the first description of entecavir treatment failure associated with selection of the rt250L mutation without other entecavir resistance mutations. One isolate with genotypic resistance to entecavir, harboring the rt173L mutation, displayed a lower level of resistance than the other, harboring the rt202G mutation (mean fold change of 323 ± 124 vs. 6 036 ± 2 100, p = 0.20). These results suggest that isolates harboring the rt250L mutations should be considered resistant to entecavir, whereas isolates harboring the rt173L mutations should be considered to display reduced susceptibility to entecavir. An integrative approach to antiviral drug resistance in HBV would provide a more accurate assessment of entecavir treatment failures and help to improve the accuracy of genotypic testing algorithms.