Selection of multiple mutations in precore, core promoter and polymerase in a chronic HBV infected patient during long-term course of antiviral therapy

We report a case of a chronically HBV-infected, HBeAg+, genotype D male patient with histological evidence of advanced liver disease. At baseline, HBV-DNA titres and HBsAg level in serum were 2×106 copies/ml and 10µg/ml(Laurell), respectively. Levels of intrahepatic DNAs (total 8,2 copies/cell, cccDNA 0,04 copies/cell) indicated moderate viral activity. Coexistence of variants harbouring precore (pc) and core promoter (cp) mutations along with wild-type (wt) strains was shown using Innolipa hybridisation assay. Antiviral therapy was started with PEG-IFN a2b (1,5ug/kg) and Adefovir 10mg (ADV). After 48 weeks HBeAg became undetectable and HBV-DNA and HBsAg serum levels decreased to 103 copies/ml and 0,5µg/ml, respectively and intrahepatic totalDNA and cccDNA lowered significantly (0,13 total DNA/cell and cccDNA <LLoD). Treatment was continued per study protocol with ADV monotherapy and 105 weeks after treatment initiation ADV resistant mutant rtN236T was detected. HBV-DNA and HBsAg increased to 2×108 copies/cell and 20µl/ml, respectively, and liver biopsy revealed viral activity exceeding baseline values (total DNA 1062 copies/cell, cccDNA 13 copies/cell). Mutations for pc and cp became the predominant viral strain with wt virions undetectable in hybridisation assays (LLoD<5%). Add-on therapy with Lamivudine 100mg (LAM) and ADV was initiated but the patient stopped taking ADV at his own risk and continued only on LAM mono-therapy. Viremia dropped after six months of LAM mono-therapy to 105 copies/ml but plateaued. The patient was switched to Entecavir (ETV) 1mg/day and viremia dropped to 2×103 copies/ml after another six months of ETV and transaminases normalised. The patient is continuing on ETV and is followed by 3 months intervals of HBV-DNA PCR.

Discussion: Selection of variants harbouring pc and cp and viral polymerase mutations during antiviral therapy enhanced replicative activity in this patient permitting reestablishment of significantly higher intrahepatic cccDNA and serum HBV-DNA levels and demonstrating the biological fitness of this multi-mutant viral strain and highlights the difficulties in long-term HBV treatment, especially in patients with advanced liver disease. Pc and cp mutation status during ETV therapy will be reported during DGVS.