S gene mutations may lead to undetectable HBsAg, contributing to occult HBV infection (OBI). Nucleic acid testing (NAT) has been widely implemented to reduce transfusion-transmitted OBI. However, distinguishing between false positives and true OBI among NAT+/HBsAg– donors remains challenging, as these individuals frequently exhibit inconsistent or fluctuating test results. This study aims to improve infection status confirmation and donor management by analyzing detection patterns and investigating S gene mutations. Blood donors were detected for HBV using individual-NAT and HBsAg enzyme-linked immunosorbent assays. Routine discriminatory test (dHBV), additional three repeated dHBV (3-repeated dHBV), and serological assays were conducted to confirm the infection status of NAT+/HBsAg– donors. Serial NAT results were analyzed to assess temporal fluctuations. S gene mutations related with OBI were identified through molecular characterization and sequencing analysis. A total of 336,537 blood donors were detected, and 556 (0.16%) NAT+/HBsAg– cases were identified. Among 297 donors undergoing confirmation, 112 (37.71%) were confirmed by routine dHBV, 152 (51.18%) by 3-repeated dHBV, and 43 (14.48%) by multiple serial NATs. In total, 187 donors (62.96%, 187/297) were confirmed HBV-infected, significantly higher than routine dHBV alone (P < 0.01). Among the 187 confirmed HBV-infected donors, only 59 (31.55%) were positive in all four dHBV tests, while 128 (68.45%) showed missed detections, suggesting extremely low viral loads. In serial NAT testing, 83 (73.45%, 83/113) of donors confirmed by dHBV showed no further NAT-positive results, and an average of 5.6 tests was needed to detect one extra positive case. Molecular analysis revealed that OBI samples had more mutations in both the major hydrophilic region (MHR) and non-MHR compared to HBsAg + donors (P < 0.05), with a significantly higher prevalence of the E2G mutation. OBI is a major contributor to inconsistent NAT results. Comprehensive measures, especially repeated testing, are recommended to improve confirmation rates and reduce the risk of re-entry of potentially infectious donors. Additionally, further molecular research on S gene mutations is essential for elucidating OBI mechanisms and enhancing assay sensitivity.
Du et al. (2025) studied this question.