Molecular characteristics of HBV infection among blood donors tested HBsAg reactive in a single ELISA test in southern China

According to the blood screening strategies in China, hepatitis B surface antigen (HBsAg) screening using two different enzyme-linked immunosorbent assay (ELISA) kits and a nucleic acid test (NAT) are employed for hepatitis B virus (HBV) screening to ensure blood safety for future transfusions. Blood donations are considered suitable for clinical blood transfusions when all three tests yield negative results. However, inconsistencies between HBsAg and HBV DNA results have been reported for 6–9% of blood donations [1]. Previously, we found that of 307,740 seronegative blood samples, 80 were classified as occult HBV infection (OBI), characterized by HBsAg non-reactive (NR) and NAT reactive (R) [2].

Another consideration is HBsAg R and NAT NR results. More specifically, blood with HBsAg ELISA R-NR and NAT NR results may place recipients at risk of HBV transmission, due to the extremely low levels of HBsAg and HBV DNA, respectively. Previous studies have shown that mutations in the S region and basic core promoter (BCP) and pre-core (PC) of HBV can affect the detection of HBsAg and viral nucleic acid [3, 4].

To accurately determine the presence of HBV infection in blood donors, we conducted blood screening for HBV DNA and HBsAg using ELISA, chemiluminescent microparticle immunoassay (CLIA), and electrochemiluminescence immunoassay (ECLIA). Furthermore, quantitative real-time PCR (qPCR) and nested PCR were performed to investigate the molecular characteristics of HBV with low viral loads among blood donors that tested HBsAg R in a single ELISA test, and to identify the potential molecular mechanisms underlying this mode of HBV infection.

HBV infection remains a major threat to public health and blood safety. In China, approximately 50% of the individuals have a history of HBV infection, and 7.2% are chronic carriers of HBsAg [8]. HBsAg and HBV DNA screening were implemented among blood donors to decrease the risk of HBV transmission through blood transfusion [9]. Complicated infectious modes, such as OBI, HBsAg ELISA R-NAT NR, and ELISA R for HBV-associated markers, have been reported in Shenzhen blood donors [7, 10]. The occurrence of such complications restricts the reliability of HBV detection in China. Notably, samples with HBsAg ELISA R-NR and NAT NR results may have a risk of HBV transmission, due to the low levels of HBsAg and HBV DNA. Due to the blood screening protocol in China, limited data on the molecular diversity and mutation analysis of HBV in blood donors is currently available. Hence, we investigated the molecular characteristics of HBV in blood donors that were missed by initial NAT screening but detected as HBsAg ELISA R-NR through two different ELISA kits.

In this study, 79 samples with HBsAg ELISA R-NR and NAT NR results were found in 100,252 blood donations. After a series of confirmatory tests, of which 17 of 79 (21.9%) samples were confirmed as HBsAg-positive, accounting for 0.017% (17 of 100,252) of the overall sample pool (95%CI: 0.0097–0.026%). A previous study reported that 10% of blood samples with HBsAg ELISA R-NR results were transferred to ELISA R-R results after follow-up studies [11], and HBV DNA was detected in 13.3% (4/30) of such ELISA R-NR samples using qPCR [12], which was lower than that in our results (21.9%). This discrepancy may be due to manufacturer differences.

Seventeen samples were missed by one of the two ELISA kits, but most of them were detected by CLIA (16) and ECLIA (15). Currently, ELISA is the only serological assay approved for HBV blood screening in China. Automated CLIA and ECLIA have been used in clinical laboratories but not for HBV screening among blood donors. Furthermore, of the 17 HBsAg-positive samples with NR results for initial NAT screening (Grifols), most HBV infections were detected by nested PCR (16) and qPCR (14). Previously, we identified 14 OBI patients (HBsAg ELISA NR and NAT R) out of 490 donors, who tested ELISA reactive for hepatitis B core antibodies (HBcAb), after the application of more sensitive assays [13]. Hence, more sensitive serological and NAT assays are needed for blood screening.

In addition to the different capabilities available for HBV detection, HBV genetic diversity and various mutations should be considered when investigating inconsistencies in HBV screening assays. The majority of HBV-positive blood donors in this study were genotype B (11/13, 84.6%), which is similar to our previous report in Shenzhen in 2015 (14/15, 93.3%) [8]. A nationwide survey reported that HBV-B was predominant in the southern areas of China [14]. Genotype D is mainly found in the Mediterranean region and in certain Asian countries [15]. One genotype D strain observed in our study highlights that increased international travel and immigration may have contributed to the input of the epidemic.

HBV is prone to mutations in various gene regions. Typically, mutations in the HBsAg region can cause immune escape, resulting in the failure to detect HBsAg [16]. Furthermore, HBV immune escape mutants are highly contagious and pathogenic in immunodeficient patients [17]. In vivo studies have found that certain mutations in the S region of HBV can lead to reduced, or even inhibited, secretion of viral particles, and reduced HBsAg synthesis [18]. Mutations in the MHR and non-MHR in the S region, such as Y100C, Y103I, Q129H, T131I, M133L/S/T, F134L, T143M, G145R, and L175S, were found in this study, and could reduce the affinity of monoclonal antibodies for “a” epitopes by altering the structure of HBsAg protein [19], which may contribute to the failure of commercial reagents.

Mutations in the BCP/PC region can regulate the secretion of the hepatitis e antigen (HBeAg), leading to the cessation of antigen production. Typically, BCP mutations (A1762T and G1764A) can reduce the mRNA synthesis of the pre-C region, which is reflected as a very low level of HBV DNA [20]. G1896A is another typical mutation in the pre-C region, which generates a stop codon at the 28th amino acid position of the HBeAg sequence, resulting in the inhibition of protein synthesis [21]. These three mutations in the BCP/PC region found in this study are closely associated with the development of hepatocellular carcinoma [20]. Fifteen cases of T1719G mutations in the BCP/PC region were identified in 16 samples in the present study, which can inhibit in vitro HBV replication [22], and may lead to a low HBV DNA viral load.

It is worth noting that among the 17 samples with HBV infection, two samples (ID: 83, 97) had high viral loads (> 1000 IU/mL) detected by qPCR, but were reported as NR in routine NAT testing (Grifols). The N40S mutation in the S region was identified in both samples. Whether this has an impact on NAT requires further investigation. Some studies have reported false-negative results and underestimated viral load among blood donors with HBV infection using various commercial kits [23, 24]. Therefore, NAT with two targets in different gene regions is recommended to avoid mismatch due to possible mutations.

In summary, we analyzed the molecular characteristics of HBV among blood donors using routine ELISA R-NR and NAT NR results. Most positive cases were identified by CLIA and ECLIA, as well as qPCR and nested PCR. Analysis of the S gene sequence revealed a large number of mutations, which are associated with disrupting HBsAg detection. Some mutations in the BCP/PC regions were identified in this study, which may lower the viral load of HBV DNA and cause false-negative NAT results. Our results highlight the need to improve the sensitivity of HBsAg screening and NAT methods, to aid in the detection of low HBV viral loads and mutated HBV strains.