Ye Xianlin, Xu Xiaoxian, Dang Yinnan, Zeng Jinfeng, Xie He, Li Bin, He Baoren, Chen Limin
Department of Laboratory, Shenzhen Blood Center, Shenzhen, China.
Department of Laboratory, Qinghai Blood Center, Xining, China.
J Med Virol. 2025 May;97(5):e70396. doi: 10.1002/jmv.70396.
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.
在我们之前的研究中，在中国深圳血液中心，酶联免疫吸附测定（ELISA）检测乙肝表面抗原（HBsAg）呈反应性（+）但核酸检测（NAT）呈阴性（-）的献血者中，45.2%被确诊感染乙肝病毒（HBV）。然而，这类HBV感染的血清学和分子特征仍不清楚，一些结果不确定的献血样本需要进一步调查。在本研究中，收集了2019年1月至2021年12月期间HBsAg ELISA检测阳性/DNA NAT检测阴性的献血样本，并通过各种血清学检测、定量聚合酶链反应（qPCR）和巢式PCR进行进一步特征分析。通过DNA测序对HBV DNA进行分子特征分析。此外，通过随访研究对结果不确定的样本进行分类，以确认它们是否含有HBV DNA。在对165025份献血样本进行筛查时，共识别出278份HBsAg ELISA检测阳性/DNA NAT检测阴性的样本，其中82份（82/278，29.5%，包括52名男性和30名女性）被确诊为HBsAg阳性，9份（9/278，3.2%）结果不确定，187份（187/278，67.3%）未感染HBV。在这82份确诊为HBsAg阳性的样本中观察到三种血清学模式：74份样本为HBsAg+/抗-HBe+/抗-HBc+，7份样本为HBsAg+/抗-HBc+，1份样本仅为HBsAg+。序列分析表明，50份样本中有45份（45/50，90%）为B基因型，4份（4/50，8%）为C基因型，1份（1/50，2%）为D基因型。在HBV B基因型的S区观察到了显著突变，如Q101R、Q129H、M133L/T、F134L、L175S、V177A以及N-糖基化突变。此外，在基本核心启动子/前基因组（BCP/PC）区还发现了高频突变，如T1719G（33/35，94.3%）、A1752G/T（11/35，31.4%）、G1896A（26/35，74.3%）和A1762T/G1764A（7/35，20%）。所有这些突变可能导致低水平的HBsAg和/或极低的病毒载量。9份结果不确定的样本中有6份（6/9，66.7%）在随访65 - 192天期间HBsAg和HBV DNA检测均呈阳性，随后被确诊为HBV感染。31.7%的HBsAg ELISA检测阳性/NAT检测阴性的献血样本被确诊为HBV感染。在BCP/PC区和S区发现的各种显著突变可能是这些样本中病毒载量低和HBsAg水平低的原因。因此，对于HBV流行国家/地区的准确血液筛查，具有高灵敏度、高特异性以及能够检测基因变异（突变）的检测方法对于预防HBV通过输血传播至关重要。
