乙型肝炎病毒标志物临床应用专家共识 2023
乙型肝炎病毒标志物临床应用专家共识
本共识在新发布的《慢性乙型肝炎防治指南（2022年版）》[1]基础上，对经典及新型HBV实验室检测相关标志物的临床应用进行证据总结和要点推荐，旨在指导其规范、合理的临床应用。
特殊人群（慢性肝病、结核病和风湿免疫病患者）新型冠状病毒疫苗接种专家建议
《慢性乙型肝炎防治指南（2022年版）》更新要点及解读（PPT课件）
非一线核苷（酸）类似物经治慢性乙型肝炎患者治疗策略调整专家共识
丙氨酸氨基转移酶（ALT）持续正常的慢性乙型肝炎诊疗专家共识
乙型肝炎病毒现症感染肝功能异常患者初诊时的诊断思维及处理策略
阻断乙型肝炎病毒母婴传播临床管理流程（2021年）| 指南与规范
我被同行医生问倒了：乙型肝炎病毒三抗体阳性，是否具有免疫力？
本文中共识要点的证据等级分为A、B和C三个级别，推荐强度分为1和2两个级别，见表1 （根据GRADE分级修订）。
一、HBV标志物的产生及其在自然史分期中的应用
（一）HBV生命周期及其标志物
由此可见，HBV标志物如HBsAg、HBcrAg、HBV DNA和HBV pgRNA等均为包含多组分的混合体（图1）。
当前以"临床治愈"为目标的抗病毒新药研发方兴未艾[11]，包括病毒进入抑制剂、衣壳组装调节剂、RNA干扰和免疫调节剂等，针对复制周期的不同阶段。
（二）HBV标志物在慢性HBV感染自然史分期中的应用
可人为将慢性HBV感染自然史划分为4个阶段[12]，即免疫耐受期、免疫清除期、免疫控制期和再活动期，不同分期的病毒标志物（HBV pgRNA、HBcrAg检测见文献[13,14]）有不同的结果组合（表2和图2）。
血清HBsAg、HBV DNA、pgRNA和HBcrAg处于最高水平，且彼此呈正相关[15,16,17,18]。
HBsAg、HBV DNA、pgRNA和HBcrAg水平大幅下降，但仍较高，当发生HBeAg阴转或血清学转换后，HBsAg、HBV DNA、pgRNA和HBcrAg水平进一步下降。
qAnti-HBc较免疫清除期有所下降，但高于免疫耐受期。
血清HBsAg、HBV DNA、pgRNA、HBcrAg和qAnti-HBc均较免疫控制期有反弹，同时，多数患者存在前核心（precore，前C）区和/或基本核心启动子（basal core promoter，BCP）的基因突变。
HBV标志物在不同分期中有不同的结果组合，有助于区分免疫耐受期和免疫清除期、免疫控制期和再活动期，对指导启动治疗有一定价值。
联合应用HBV定量标志物如HBsAg、HBV DNA和qAnti-HBc等，有助于进一步明确慢性HBV感染自然史分期，判断是否符合抗病毒治疗适应证（B2）。
二、经典HBV标志物及其临床意义
（一）HBV血清学标志物
1．HBsAg和抗-HBs：
约有5%的感染者HBsAg和抗-HBs同时阳性，可能是病毒持续感染或疫苗接种等因素形成的免疫压力导致HBV突变和免疫逃逸毒株的产生，从而造成这种特殊血清学结果模式。此时抗-HBs并无保护作用，反而可能是与肝纤维化和肝硬化相关的高危因素[23,24]。
2．HBeAg和抗-HBe：
值得注意的是，前C区和/或BCP突变可导致HBeAg表达降低甚至转阴，但HBV DNA仍为阳性，此时病毒复制仍相对活跃。
3．抗-HBc：
抗-HBc IgG出现较迟，但长期存在甚至终身维持阳性，是现症或既往感染的标志；在HBsAg阴性的个体，其阳性也提示可能存在隐匿性乙型肝炎病毒感染（occult hepatitis B virus infection, OBI）。
数学模型分析提示，在我国18~70岁的人群中广泛开展HBsAg、抗-HBs、HBeAg、抗-HBe和抗-HBc联合筛查，符合成本效益，可提高HBV感染的诊断率，助力实现世界卫生组织提出的2030年消除HBV感染所导致的重大公共卫生危害这一目标[25]。
HBV血清学标志物检测方法有酶联免疫吸附试验、免疫层析技术（如胶体金技术）及化学发光技术等[26]，化学发光技术具有灵敏度高、易于实现自动化等优势，逐渐占据了主导地位[27]。
（二）HBV分子生物学标志物
1．HBV DNA：
HBV DNA检测以实时荧光定量PCR技术为主。
2．HBV基因分型：
基因型检测主要基于DNA杂交、PCR产物Sanger测序或新一代测序（NGS）或实时荧光PCR技术等。
3．耐药突变：
若使用Sanger测序或NGS技术，可同时提供耐药突变和基因型结果。
4．前C区/BCP突变：
前C区/BCP突变检测技术与HBV基因型和耐药突变类似。
5．cccDNA：
DNA印迹（Southern blot）是检测cccDNA的经典方法，但灵敏度有限，操作繁琐，无法临床常规开展；实时荧光定量PCR等技术检测cccDNA时，通过精巧设计引物和探针，可与rcDNA等进行区分，但这类方法缺乏标准化，不同实验室间的结果有很大差异[49]，因此，在临床上常规开展cccDNA检测仍面临很大挑战。
慢性HBV携带状态和非活动性HBsAg携带状态患者，需6~12个月检测HBV DNA；NAs治疗中，应每3~6个月检测HBV DNA；Peg-IFN-α治疗时，应每3个月检测HBV DNA（A1）。
三、经典HBV标志物的技术改进及其临床意义
（一）定量HBsAg（quantitative HBsAg，qHBsAg）和高灵敏度HBsAg检测
HBsAg < 1 000 IU/ml联合HBV DNA < 2 000 IU/ml，区分非活动性HBsAg携带状态和HBeAg阴性CHB的准确性为78%[51]。
HBeAg阴性CHB患者在停药前HBsAg水平低于100~200 IU/ml，提示持续应答甚至HBsAg消失的可能性较高，而qHBsAg高水平意味着较高的停药复发风险[54,55,56,57,58]。
NAs序贯联合PEG-IFN-α治疗，基线低HBsAg水平（< 1 500 IU/ml）且HBeAg阴转，或治疗早期（12或24周）HBsAg < 200 IU/ml或HBsAg下降> 1个log10 IU/ml，可预测最有可能获得HBsAg阴转的患者；而治疗24周时HBsAg仍然≥200 IU/ml的患者获得HBsAg阴转的可能性小，应考虑停用PEG-IFN-α,继续NAs治疗。
当前主流的基于化学发光技术的HBsAg试剂LOD一般为0.05 IU/ml，而高敏HBsAg试剂LOD可达0.005~0.000 5 IU/ml，其应用有助于进一步缩短急性HBV感染的诊断窗口期，提高OBI的检出率，但也对"临床治愈"的标准提出了新挑战[63,64]，同时，对检测平台的稳定性和抵抗样本交叉污染等性能也提出了更高的要求。
此外，CHB患者（特别是HBeAg阴性CHB患者）HBsAg还可能源自整合HBV DNA的表达，整合HBsAg的临床意义并不明确，且当前试剂均无法区分HBsAg是由病毒合成还是源于整合[68,69,70]。
PEG-IFN-α治疗中应监测qHBsAg，HBeAg阳性CHB患者治疗12周qHBsAg > 20 000 IU/ml或水平不降可作为停用干扰素指征，NPV接近100%（A1）；而NAs治疗中，qHBsAg往往下降缓慢，低水平qHBsAg提示停药后复发风险较低（A1）。
检测qHBsAg有助于甄别CHB临床治愈的优势人群。基线低HBsAg水平（< 1 500 IU/ml）且HBeAg阴转,或治疗早期（12或24周）HBsAg < 200 IU/ml或HBsAg下降> 1个log10 IU/ml，是获得临床治愈的积极因素（B2）。
（二）HBeAg定量（qHBeAg）检测
qHBeAg也有助于预测NAs治疗的HBeAg血清学转换，恩替卡韦治疗24周时qHBeAg出现明显下降，对治疗2年后获得HBeAg血清学转换的阳性预测值可达83.3%[73]，应鼓励患者继续治疗。
PEG-IFN-α治疗12或24周时检测qHBeAg，其高水平提示不太可能获得HBeAg血清学转换，对治疗结束后获得HBeAg血清学转换的NPV很高；而NAs治疗早期qHBeAg下降则是获得HBeAg血清学转换的积极因素（A2）。
（三）核心抗体定量（qAnti-HBc）检测
在接受免疫抑制治疗人群中，高水平的qAnti-HBc提示较高的HBV再激活风险[18,81,82,83]。
高水平qAnti-HBc提示肝脏存在炎症和纤维化，是ALT的补充，有助于判断是否需要启动抗病毒治疗（A2）；若治疗前qAnti-HBc水平较高，或抗病毒治疗中大幅下降，提示较高的应答率和较低的停药后复发风险（A2）。
（四）高灵敏度HBV DNA检测
另外，OBI患者血清病毒载量往往很低（一般< 200 IU/ml），使用高灵敏度HBV DNA监测也有助于及时发现免疫缺陷人群OBI的再激活[86]。
高灵敏度HBV DNA检测要求对极低病毒载量检测结果有很好的重复性，要避免出现样本交叉污染或气溶胶污染引起的低值、假阳性结果，因此，对检测技术和操作者的要求也更提高。
建议使用高灵敏度HBV DNA检测试剂（LOD为5~20 IU/ml）监测和管理NAs治疗中的LLV患者，也有助于监测OBI患者HBV再激活（A2）。
四、新型HBV标志物及其临床意义
1．HBcrAg：
新一代试剂灵敏度可提高8倍，但仍存在检测假阳性的问题[94,110]。
2．pgRNA：
国内外已有多种商品化pgRNA定量试剂，但缺乏国际标准品，不同试剂的灵敏度也有较大差异。
血清HBcrAg和HBV pgRNA水平均可反映cccDNA水平或转录活性，二者相关性较好且不受治疗的影响。NAs治疗过程中HBV DNA低于检测下限时，可进一步检测pgRNA或HBcrAg，如果"阳性"则提示应继续治疗以规避停药后复发风险（B2）。
五、HBV标志物检测的质量控制
qHBsAg、抗-HBs、qHBeAg、qAnti-HBc和HBV DNA等定量标志物均有国际定量标准品，结果单位为IU/ml或mIU/ml，实验室推广应用国际标准品，有助于提高结果的量值溯源性和不同实验室间结果的可比性。
六、HBV标志物的特点见表4。
七、尚待研究和解决的问题
6．建立规范的cccDNA检测技术，并探索外周血脱落肝细胞或血清cccDNA的检测技术，提高cccDNA检测的临床可及性；或继续探索能更好反映肝内cccDNA数量或转录活性的标志物，使其能更好地用于指导临床。
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引用：中华医学会肝病学分会基础医学与实验诊断协作组.乙型肝炎病毒标志物临床应用专家共识[J].中华肝脏病杂志, 2023, 31(4): 389-400.