Occult HBV among anti-HBc–reactive blood donors in Colombia: low HBsAg prevalence, genotype F3, and small-S variation relevant to HBsAg detectability

PURPOSE: Occult hepatitis B infection (OBI) can sustain residual transfusion risk because HBV DNA may persist at very low levels while HBsAg remains undetectable by routine serology. We assessed OBI among anti-HBc-reactive, HBsAg-negative (anti-HBc+/HBsAg-) donors in Bucaramanga, Colombia and characterized small-S variation within genotype F3. METHODS: In 2021, we analyzed 61,188 donations in three licensed blood banks using routine serology. From anti-HBc+/HBsAg- donations, 219 randomly selected sera underwent nested PCR targeting the small-S/reverse-transcriptase overlap, followed by Sanger sequencing. Genotyping and phylogeny used a curated 960-nt small-S fragment. Associations with OBI were estimated by logistic regression. RESULTS: HBsAg reactivity was 0.21% and anti-HBc prevalence was 1.77%. OBI occurred in 16/219 anti-HBc+/HBsAg- donations (7.3%; 95% CI, 4.5-11.5), approximately 130 per 100,000 donations (95% CI, 80-204). Among the 16 OBI cases, 12 were anti-HBs reactive. Sex, age, and anti-HBs were not associated with OBI after adjustment. All sequences clustered within genotype F3. Small-S amino acid variation at positions previously linked to altered HBsAg detectability was observed in 11/16 (68.8%): A166G (5/16) outside "a"; "a" substitutions L127R, K141N, N146Y, C147S; variants at the pre-"a" boundary K122R/T123A; loop-proximal cysteine changes C149S/C149F and S117C; stop codons W156 and L216*; additional MHR changes included V106D, T118S, I150V, P151R, I152S, K160I. CONCLUSIONS: All OBI cases belonged to genotype F3 and showed small-S variants at positions previously associated with HBsAg detectability, which may contribute to reduced detection rather than classical antigenic escape. Among anti-HBc+/HBsAg- donations, targeted NAT identified donors with detectable HBV DNA within this serologic profile that routine screening alone could not resolve, indicating a residual risk that may be reduced through selective NAT where universal implementation is not feasible.