Hepatitis B virus (HBV) genome detection and genotyping in virally suppressed patients using nested polymerase chain reaction-based Sanger sequencing

Hepatitis B virus (HBV) genotypes have important clinical implications. Current genotyping methods are less sensitive in patients with ultra-low HBV viral load. We report a highly sensitive and specific nested polymerase chain reaction (PCR) assay for genotyping patient HBV. Total DNA derived from plasma of 14 (HBsAg+ and/or HBsAg-) HBcAb+ patients was used for HBV-specific nested PCRs targeting the preC/C, X/BCP/preC, and surface regions. All patients were treated with long-term nucleos(t)ide analogues (NAs), and 12/14 have undetectable viremia (clinical PCR: sensitivity >10 IU/mL). Surface amplicons were sequenced, aligned with reference genomes, and used in phylogenetic tree construction to determine genotype. HBV DNA was detected in 14/14, including 3 occult (HBsAg-/HBcAb+) cases. Genotypes identified were 6/14 B, 6/14 C, and 2/14 D. This assay in virologically suppressed patients may be useful for future studies requiring genotype prior to assessment of immunomodulatory and/or direct acting anti-viral therapeutics in patients on potent NAs.