Blood donor plasma samples were detected by the Ultrio Plus NAT system for HBV, HCV and HIV-1 in Shenzhen blood center, China. Reactive samples underwent further discriminatory testing of a single virus by the same methodology. A large number of cases of non-discriminated reactive (NDR) donors were found, leaving potential risk of transmitting HBV if not deferrals. This study identified those non-discriminated samples. The NDR plasma samples from blood donation screening were detected and classified by additional molecular and serological tests. Molecular characterizations of DNA+ NDR were determined by sequencing analysis. A number of 259 (0.21%) NDR plasma samples from screening of 123,280 eligible blood donors were detected, which presented a higher rate (91.1%) of anti-HBc reactivity and nearly half (46.7%) of HBV DNA+ that classified as occult HBV infection (OBI). Most OBI strains were wild-type HBV, but some substitutions V168A, S174 N, V177A, Q129R/L/H, G145A/R in S region of genotype B (OBIB) and T47K/V/A, P49H/L, Q101R/H/K, S174 N, L175S, V177A, T118 M/R/K, G145R/A/K/E, R160K/N in S region of genotype C (OBIC) strains were identified in high frequency. Nearly half of NDR blood samples were identified as OBI, in which a number of important mutations were detected. NDR donation might have potential risk for HBV transmission, but need to be further investigated.

Occult hepatitis B virus infection (OBI) represents a specific form of hepatitis B virus (HBV)infection characterized by the presence of replication-competent HBV DNA in the liver despite a negative blood test for hepatitis B surface antigen (HBsAg). Due to the incompletely-known mechanisms underlying its occurrence and the limitations of existing screening technologies, the viral loads in the blood of OBI patients are intermittent and often undetectable. Furthermore, lack of effective screening and shielding strategies in blood collection and supply institutions fail to prevent OBI individuals from donating blood, resulting in its susceptibility to transmission through blood transfusion, which poses a significant threat to blood safety. In this review, we summarize current understanding of OBI, challenges, and its implication in blood safety.

Immune control of various infectious diseases, particularly viral, was shown to be more efficient for females than males. Response to viral vaccines (HAV, HBV) was higher in females. Data on hepatitis B virus (HBV) markers accumulated over 15 years in blood donors was stratified according to sex, including HBsAg, HBV viral load and levels of anti-HBs in areas where genotypes B and C (China), genotype D (Iran, Lebanon, Tunisia) and genotype E (Ghana, Burkina Faso, Gabon) were prevalent. HBsAg was screened by either ELISA or rapid tests, anti-HBc and anti-HBs by ELISA, HBV DNA load by a standardized method across sites. In Ghanaian children less than 5 years, HBV DNA load was significantly lower in females than in males (p = 0.035). In China, Ghana, Burkina Faso and Gabon blood donors, median HBsAg prevalence was ~5% and 3% in China, ~8.5% and 4.5% in Gabon, ~16% and 11% in Burkina Faso and ~11% and 7% in Ghana for male and female donors, respectively (p < 0.001). In HBsAg+ Ghanaian blood donors, distribution and median viral load were not significantly different between sexes; occult hepatitis B infections (OBI) were significantly more frequent in males. In Chinese blood donor anti-HBc+ and anti-HBs+, anti-HBs levels tended to be higher in males but vaccinated donors’ anti-HBs+ only, while anti-HBs levels were females > males. In areas where genotypes B-E are dominant, the prevalence of chronic HBV infection (HBsAg+) seems better controlled before age 16–18 by females infected vertically or horizontally. OBIs appear considerably more frequent in men, suggesting lower efficacy of HBV infection control. Female blood donors appear significantly safer from HBV than males, and their donation should be encouraged.

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.