High Genetic Variability of Hepatitis B Virus in Blood Donors With Surface Antigen Positive But DNA Negative in Southern China

ABSTRACT In our previous studies, 45.2% of donations with HBV HBsAg reactive (+) by ELISA but negative (−) by NAT were confirmed to have HBV infections in Shenzhen Blood Center, China. However, the serological and molecular characteristics of this type of HBV infection remain unclear, and several donations with indeterminate results require further investigation. In this current study, blood donations with HBsAg ELISA+/DNA NAT− results were collected and further characterized by various serological tests, quantitative PCR (qPCR) and nested PCR from January, 2019 to December, 2021. Molecular characterizations of HBV DNAs were performed by DNA sequencing. Additionally, donations with indeterminate results were classified through a follow‐up study to confirm whether they contain HBV DNA. Of 278 HBsAg ELISA+/DNA NAT− samples identified from the screening of 165 025 blood donations, 82 (82/278, 29.5%, including 52 males and 30 females) were confirmed HBsAg positive, leaving nine donations (9/278, 3.2%) with indeterminate results and 187 (187/278, 67.3%) as no HBV infections. Three serological patterns were observed among these 82 confirmed HBsAg positive donations: 74 samples were HBsAg+/anti‐HBe+/anti‐HBc+, seven samples were HBsAg+/anti‐HBc+, and one sample was HBsAg+ alone. Sequence analysis showed that 45 (45/50, 90%) were genotype B, while 4 (4/50, 8%) were genotype C and 1 (1/50, 2%) was genotype D. Notable mutations such as Q101R, Q129H, M133L/T, F134L, L175S, V177A, and N‐glycosylation mutations in S regions of HBV genotype B were observed. Additionally, high frequency mutations such as T1719G (33/35, 94.3%), A1752G/T (11/35, 31.4%), G1896A (26/35, 74.3%), and A1762T/G1764A (7/35, 20%) in the BCP/PC regions were also identified. All these mutations might contribute to low‐level HBsAg and/or extremely low viral loads. Six out of nine donations (6/9, 66.7%) with indeterminate results were tested positive for both HBsAg and HBV DNA during 65–192 days of follow‐up, and they were then confirmed as HBV infections. 31.7% donations with HBsAg ELISA+/NAT− results were confirmed as HBV infection. Various notable mutations identified in the BCP/PC and S regions may be responsible for the low viral loads and HBsAg level in these donations. Therefore, assays with high sensitivity, specificity, and ability to detect genetic variants (mutations) are essential for accurate blood screening in HBV‐endemic countries/regions to prevent the transmission of HBV through blood transfusion.