High Prevalence of Occult Hepatitis B Virus Co-Infection Identified in Treponema Pallidum-Positive Blood Donations: Implications for HBV Risk Reduction

Over the past decade, the incidence of infectious syphilis has been on the rise in the general Chinese population. Consequently, Treponema Pallidum (TP) testing has been proposed as a surrogate marker for sexually transmitted pathogens and for monitoring risky sexual behaviors among blood donors globally. In addition, sexual contact with individuals chronically infected with hepatitis B virus (HBV) is recognized as one of the primary routes of HBV transmission. Blood donors may acquire HBV infection through sexual contact with chronically infected partners, particularly with occult hepatitis B infections (OBIs), which are characterized by intermittent and extremely low viral loads. Therefore, the prevalence of OBIs among syphilis-positive blood donations and the corresponding risks to blood safety require further investigation. This study aimed to investigate the prevalence of OBIs among syphilis-positive blood donors and assess the surrogate value of TP testing for evaluating OBI-related risks to blood supply. After routine screening using serological assays and nucleic acid testing (NAT), blood donation samples with positive anti-TP enzyme-linked immunosorbent assay (ELISA) results were collected and further confirmed by the Treponema Pallidum Particle Agglutination Assay (TPPA). For blood donations confirmed positive for syphilis, further tests were performed to characterize whether the donations had HBV co-infection, including electrochemiluminescence immunoassay (ECLI) for the detection of hepatitis B surface antigen (HBsAg), anti-hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg), anti-hepatitis B e antibody (anti-HBe), and anti-hepatitis B core antibody (anti-HBc). Additionally, quantitative real-time polymerase chain reaction (qPCR) was used for HBV DNA quantification, and nested PCRs for the S and basal core promoter/precore (BCP/PC) region were conducted in combination with high-volume nucleic acid extraction. Subsequently, molecular characterization of HBV DNA in these co-infected samples was carried out by DNA sequencing to analyze the viral genetic features. Of 252 anti-TP ELISA+ donations screened from 64,871 blood samples, 138 (138/250, 55.2%) donations were confirmed syphilis-positive but NAT−, among which 78 (78/138, 56.5%) were anti-HBc-positive, and 88 (88/138, 63.7%) had anti-HBs. Notably, seven donations (7/138, 5.1%) were diagnosed as OBI co-infections, and available sequence analysis revealed that three cases were genotype B and one case was genotype C. In addition, several mutations in the S region of the HBV genome were identified, including Q101R, K122R, Q129H, T131N, M133T, G145R, and Y161F mutations. Furthermore, nucleotide mutations such as T1719G, A1752T, G1896A, and A1762T/G1764A in the BCP/PC regions were also detected in these OBI donations. These mutations may contribute to the extremely low HBV viral loads and/or failure in HBsAg detection, collectively leading to OBIs. These data indicate that syphilis screening of blood donors has potential to serve as an additional safeguard measure for excluding donations co-infected with OBIs. The high prevalence of undetected OBIs in syphilis-positive blood donors further supports that syphilis screening has the potential to serve as a surrogate marker for HBV-related risks in the blood supply.