Hepatitis B virus infection with high HBsAg and undetectable DNA levels in untreated blood donors: Viral mechanisms and transfusion safety implications

BACKGROUND: Blood donor screening occasionally identifies individuals with high HBV surface antigen (HBsAg) levels despite undetectable or extremely low HBV DNA. Molecular basis and transfusion risk of this discordant profile remain unclear. STUDY DESIGN AND METHODS: Seventy-five blood donors with HBsAg >100 IU/mL and undetectable or very low HBV DNA (<6 IU/mL), including 39 ID-NAT-nonreactive, were investigated. HBV DNA was further assessed using a second ID-NAT assay, confirmatory PCR following virion concentration, and sequencing. Sequences were compared with those from genotype-matched high-viremia controls. Selected basic core promoter (BCP) and core mutations were introduced into 1.18-mer replication-competent Hepatitis B virus (HBV) plasmids to assess effects on extracellular virion-associated DNA production. RESULTS: Four (5%) samples were nonreactive by the two ID-NAT assays. HBV DNA was confirmed in all donors, often at unquantifiable levels, and all were anti-HBc positive. No consistent molecular signature was identified. Heterogeneous, case-specific mutations were detected in BCP, core, and PreS1, including variants predicted to affect pregenomic RNA transcription, encapsidation, or virion assembly, with no evidence of impaired polymerase activity. In vitro, selected BCP mutations significantly reduced extracellular HBV DNA without affecting HBsAg production. Most core mutations had minimal impact, although two strains carrying cN92T/cM93T or cS170F showed reduced virion-associated DNA. Some constructs exhibited impaired virion secretion or increased naked capsid production. CONCLUSION: HBsAg-positive/low-DNA profile largely reflects chronic inactive infection with minimal viremia. Diverse regulatory and structural mutations may contribute in a multifactorial manner. Persistent viral genomes and residual replication support continued HBsAg screening where NAT sensitivity is suboptimal and anti-HBc testing cannot be implemented.