A dichotomous separation of hepatitis B viral DNA and hepatitis B surface antigen (HBsAg) concentrations occurs during the natural history and treatment of chronic hepatitis B. We have evaluated the ability of hepatitis B virus (HBV) RNA and hepatitis B core‐related antigen (HBcrAg) as surrogates of silencing of covalently closed circular DNA (cccDNA), to characterize this dissociation, and virological outcomes.

Three cohorts of hepatitis B e antigen (HBeAg)‐negative patients were studied: cohort A: 66 HBeAg‐negative patients on long‐term nucleos(t)ide analogue (NA) therapy; cohort B: 23 antibodies against hepatitis B e antigen (anti‐HBe)‐positive patients who stopped treatment; and Cohort C: 19 anti‐HBe‐positive patients on long‐term NA treatment who achieved HBsAg loss and in whom treatment was withdrawn. Concentrations of HBV serological/virological biomarkers (HBV DNA, HBsAg, HBcrAg, and HBV RNA) were measured in sequential samples at different time points on/off therapy. Cohort A: After 3 years of antiviral therapy, 33% and 30% had detectable HBcrAg and HBV RNA, respectively, despite all being HBV‐DNA negative. After 5 years’ therapy with NA, 27% and 14% had detectable HBcrAg and HBV RNA. Detectable HBcrAg and HBV RNA at the time of treatment withdrawal was only observed in those patients who developed a severe aminotransferase flare. Only those patients with HBV reactivation in cohort C had detectable HBV RNA at treatment withdrawal, but HBcrAg and HBV DNA were not detected.

HBcrAg and HBV RNA are sensitive biomarkers of continued transcription of cccDNA in HBeAg‐negative patients despite marked HBV‐DNA suppression by NA. These markers were predictors of severe alanine transaminase flares, after treatment withdrawal, and HBV‐DNA reactivation. Their measurement during the natural history of hepatitis B, and on treatment with current and new agents, could characterize residual HBV‐RNA transcription from cccDNA and assist drug development and disease management.