Song Shi, Su Qian, Yan Ying, Ji Huimin, Sun Huizhen, Feng Kaihao, Nuermaimaiti Abudulimutailipu, Halemubieke Shana, Mei Ling, Liu Xinru, Lu Zhuoqun, Chang Le, Wang Lunan
National Center for Clinical Laboratories, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing Hospital/National Center of Gerontology, Beijing, China.
Beijing Engineering Research Center of Laboratory Medicine, Beijing Hospital, Beijing, China.
J Clin Microbiol. 2025 May 14;63(5):e0207124. doi: 10.1128/jcm.02071-24. Epub 2025 Mar 31.
The significance of occult hepatitis B virus (HBV) infection (OBI) has been increasingly recognized while the underlying mechanisms remain incompletely understood. This study aimed to identify high-frequency OBI-related mutations in HBV surface antigen (HBsAg)-negative samples tested by the ultrasensitive Lumipulse G HBsAg-Quant assay. OBI samples were collected from 32 blood establishments across 14 provinces in China. Lumipulse G HBsAg-Quant assay was performed for the re-testing and reclassification of OBI. Mutations in genotypes B (GTB) and C (GTC) were analyzed to identify high-frequency single and combined mutations. Additionally, the efficacy of commercial reagents commonly employed in clinical diagnostics for detecting mutant HBsAg was evaluated. Western Blot was used for the confirmation of extracellular HBsAg as well as the detection of intracellular HBsAg. Hydrophilicity analysis and transmembrane distribution prediction of HBsAg were utilized for further validation. Single mutations at 17 sites and 9 combined mutations in GTB indicated a significantly elevated mutation frequency. In GTC, there were single mutations at 16 sites and 9 combined mutations. Several commercial reagents commonly demonstrated limited capacity toward mutant HBsAg with T123A/P, K141C, and P142R/I/K/L (GTB) and S114A/P (GTC). The findings indicated that mutations including T123A/C/K/S, S132G/Y, P142L/R/S/T, T143M, D144G, G145A, K160R+V168A, I4T+V168A, M103I+K122R, and M103I+Q181R (GTB), along with Q101H, M103I, R160K+C221Y (GTC), were associated with reduced levels of HBsAg both extracellularly and intracellularly. Additionally, K160R (GTB) and E2G (GTC) were associated with intracellular aggregation. This study elucidates the mutations associated with decreased extracellular HBsAg with ultrasensitive HBsAg assay, providing insight for further investigation into the mechanisms of OBI.
The sensitivity of HBsAg detection reagents directly impacts the identification of occult hepatitis B virus (HBV) infection (OBI). This study aims to identify high-frequency OBI-related mutations in HBV surface antigen (HBsAg)-negative samples evaluated using a Fujirebio-Lumipulse ultrasensitive HBsAg assay and to investigate the implications of these mutations on the antigenicity of HBsAg, the detection capacities of various HBsAg assays, and the effects on intracellular and extracellular levels of HBsAg. Generally, our study offers a new perspective on OBI-related mutations by ultrasensitive HBsAg assay and lays the groundwork for further research on the OBI mechanism and the enhancement of HBsAg detection reagents.
隐匿性乙型肝炎病毒（HBV）感染（OBI）的重要性已得到越来越多的认可，但其潜在机制仍未完全明确。本研究旨在通过超灵敏的Lumipulse G HBsAg定量检测法，鉴定HBV表面抗原（HBsAg）阴性样本中高频的OBI相关突变。从中国14个省份的32家血站收集OBI样本。采用Lumipulse G HBsAg定量检测法对OBI进行重新检测和重新分类。分析B基因型（GTB）和C基因型（GTC）中的突变，以鉴定高频的单突变和复合突变。此外，评估了临床诊断中常用的商业试剂检测突变型HBsAg的效能。采用蛋白质免疫印迹法确认细胞外HBsAg并检测细胞内HBsAg。利用HBsAg的亲水性分析和跨膜分布预测进行进一步验证。GTB中有17个位点的单突变和9个复合突变，其突变频率显著升高。在GTC中，有16个位点的单突变和9个复合突变。几种商业试剂对T123A/P、K141C和P142R/I/K/L（GTB）以及S114A/P（GTC）等突变型HBsAg的检测能力通常有限。研究结果表明，包括T123A/C/K/S、S132G/Y、P142L/R/S/T、T143M、D144G、G145A、K160R+V168A、I4T+V168A、M103I+K122R和M103I+Q181R（GTB），以及Q101H、M103I、R160K+C221Y（GTC）等突变与细胞外和细胞内HBsAg水平降低有关。此外，K160R（GTB）和E2G（GTC）与细胞内聚集有关。本研究阐明了与超灵敏HBsAg检测法检测到的细胞外HBsAg降低相关的突变，为进一步研究OBI机制提供了思路。
HBsAg检测试剂的灵敏度直接影响隐匿性乙型肝炎病毒（HBV）感染（OBI）的鉴定。本研究旨在通过富士瑞必欧Lumipulse超灵敏HBsAg检测法，鉴定HBV表面抗原（HBsAg）阴性样本中高频的OBI相关突变，并研究这些突变对HBsAg抗原性、各种HBsAg检测方法的检测能力以及对细胞内和细胞外HBsAg水平的影响。总体而言，我们的研究通过超灵敏HBsAg检测法为OBI相关突变提供了新的视角，为进一步研究OBI机制和改进HBsAg检测试剂奠定了基础。
