慢性HBeAg阴性HBV感染的病毒生物标志物。

StephenW

Genes (Basel). 2018 Sep 27;9(10). pii: E469. doi: 10.3390/genes9100469.
Viral Biomarkers in Chronic HBeAg Negative HBV Infection.
Hadziyannis E1, Laras A2.
Author information

1
    Second Department of Medicine and Laboratory, Hippokrateio Hospital, National and Kapodistrian University of Athens, Athens 11527, Greece. [email protected].
2
    Second Department of Medicine and Laboratory, Hippokrateio Hospital, National and Kapodistrian University of Athens, Athens 11527, Greece. [email protected].

Abstract

Viral biomarkers are important tools for monitoring chronic hepatitis B virus (HBV) hepatitis B early antigen (HBeAg) negative infection, both in its natural course as well as during and after treatment. The biomarkers consist of antibodies against viral epitopes, viral proteins, and molecular surrogate markers of the quantity and transcriptional activity of the stable episomal HBV covalently closed circular DNA (cccDNA) which is located in the nuclei of the infected hepatocytes. HBV deoxyribonucleic acid (DNA) or else viral load measurement in plasma or serum is a marker of HBV replication of major clinical importance. HBV DNA is used for staging and treatment monitoring as described in international scientific guidelines. Quantification of HBV antigens, mainly hepatitis B surface antigen (HBsAg) as well as Hepatitis B core related antigen (HBcrAg), play an important yet secondary role, especially in cases of low or undetectable HBV DNA and has been evaluated for the classification of the inactive carrier state, as a predictor of subsequent HBsAg clearance, treatment outcome, and development of hepatocellular carcinoma (HCC). The measurement of the replicative intermediate HBV RNA in serum is currently evaluated and may also prove to be a significant biomarker particularly in patients treated with nucleot(s)ide analogs. This review focuses on the viral biomarkers mentioned above and their role in HBV, HBeAg negative, infection.
KEYWORDS:

HBV; HBV DNA; HBV RNA; HBcrAg; HBsAg; anti-HBe; biomarker

PMID:
    30262738
DOI:
    10.3390/genes9100469

StephenW

基因（巴塞尔）。 2018年9月27日; 9（10）。 pii：E469。 doi：10.3390 / genes9100469。
慢性HBeAg阴性HBV感染的病毒生物标志物。
Hadziyannis E1，Laras A2。
作者信息

1
    雅典国立和Kapodistrian大学Hippokrateio医院第二医学和实验室，雅典，11527，希腊。 [email protected]。
2
    雅典国立和Kapodistrian大学Hippokrateio医院第二医学和实验室，雅典，11527，希腊。 [email protected]。

抽象

病毒生物标志物是用于监测慢性乙型肝炎病毒（HBV）乙型肝炎早期抗原（HBeAg）阴性感染的重要工具，无论是在其自然过程中还是在治疗期间和之后。生物标志物由针对病毒表位，病毒蛋白和分子替代标志物的抗体组成，其稳定的游离型HBV共价闭合环状DNA（cccDNA）的数量和转录活性位于感染的肝细胞的细胞核中。 HBV脱氧核糖核酸（DNA）或血浆或血清中的病毒载量测量是HBV复制的标志物，具有重要的临床意义。如国际科学指南中所述，HBV DNA用于分期和治疗监测。 HBV抗原的定量，主要是乙型肝炎表面抗原（HBsAg）以及乙型肝炎核心相关抗原（HBcrAg），在HBV DNA低或不可检测的情况下发挥重要的次要作用，并已对其进行了分类评估。无活性载体状态，作为后续HBsAg清除，治疗结果和肝细胞癌（HCC）发展的预测因子。目前评估血清中复制型中间体HBV RNA的测量，并且还可以证明是特别是在用核苷酸类似物治疗的患者中的重要生物标志物。本综述着重于上述病毒生物标志物及其在HBV，HBeAg阴性，感染中的作用。
关键词：

HBV; HBV DNA; HBV RNA; HBcrAg;乙肝表面抗原;抗-HBe;生物标记物

结论：
    30262738
DOI：
    10.3390 / genes9100469

StephenW

3.2. Biomarkers for the Identification of the Inactive Carrier State
HBeAg negative patients with chronic HBV infection are categorized in two main states. In the inactive carrier state (recently renamed by EASL as ‘HBeAg negative chronic infection’), there is limited viral replication and liver inflammation [40]. The inactive HBV carrier presents with normal alanine aminotrasferase (ALT) levels (less than 35 U/L in males and less than 25 U/L in females) and low (<2000 IU/mL) or undetectable HBV DNA. Although the above cut-offs are based on international guidelines, the characterization of a patient as inactive carrier requires serial determinations of ALT and HBV DNA, every 3–4 months for the first year and subsequently every 6 months. This approach is imperative due to the fluctuations of HBV DNA and ALT that occur in patients at the second state of HBeAg negative infection with active hepatitis [39,40,76].
Since close follow up and serial testing of two biomarkers is essential for the identification of inactive carriers, the addition of a third biomarker has been evaluated. Given that the majority of inactive HBV carriers, have lower HBsAg levels in serum than patients with active inflammation [77,78], the first biomarker that was studied was qHBsAg. In a thorough study on HBV genotype D infection, qHBsAg values at a single time point of less than 1000 IU/mL in combination with HBV DNA < 2000 IU/mL and normal ALT demonstrated a diagnostic accuracy (DA) of 94.5% for the identification of the inactive carrier state, compared to monthly monitoring with the latter two biomarkers for one year. More specifically, the sensitivity of a single three-markers measurement was 91.1%, a specificity of 95.4%, a positive predictive value (PPV) of 87.9%, and a negative predictive value (NPV) of 96.7% [79]. Similar results have been observed in other HBV genotypes. In one study of 1068 Taiwanese HBeAg negative patients that had been diagnosed as HBV carriers, infected with HBV genotype B or C, the relationship between HBsAg level > 1000 IU/mL and the development of HBeAg-negative hepatitis in 13 years follow up was found to be significant (hazard ratio (HR) = 1.5, 95% CI = 1.2–1.9) and HBsAg < 1000 IU/mL in combination with low HBV DNA and ALT were found to be useful for identifying minimal-risk HBV carriers [80]. In the REVEAL cohort, in patients infected with B or C genotype, the combined testing with the same HBsAg cut-off, showed diagnostic accuracy for IC of 78% [81]. This HBsAg threshold of 1000 IU/mL seems to be the most reliable one for the differential diagnosis of CHB and the inactive carrier state [82]. The use of a lower qHBsAg cut-off e.g., 100 IU/mL for increased specificity, results in significant decrease in sensitivity (35%) [83]. Besides qHBsAg, the addition of liver stiffness measurement (LSM) as a fourth parameter, with a cut off of 6.2 kPa further improves the diagnostic accuracy of testing ALT, HBV DNA, and qHBsAg in a single time point, showing 100% specificity, 96% sensitivity, 100% PPV, 92% NPV, and 97% DA for the identification of ICs [84].
Recently, it was suggested that serum HBcrAg is more accurate than qHBsAg for the identification of inactive carriers, regardless of hepatitis B virus genotype [85]. In this study, the diagnostic accuracy of HBcrAg ≤ 3 logU/mL combined with HBV DNA ≤ 2000 IU/mL was 87% for genotype D, lower for genotypes F or H (73%) but higher for genotypes A and E (91 and 94%).
Some patients who display HBeAg negative serological profile with normal ALT, low viremia and/or qHBsAg higher than 1000 IU/mL, have a benign course of the infection and a proportion of them fulfill the criteria of IC in later time. In a recent study with such population included, the combined qHBsAg and HBV-DNA quantification had a diagnostic accuracy of 65.4% and 100% NPV for the identification of ICs. In this study HBV-DNA ≤ 2000 IU/mL and HBcrAg < 3 log or HBV DNA ≤ 2000 IU/mL and total anti-HBc ≤ 16,937 IU/mL had higher and almost the same DA around 86.5% and their diagnostic performance was improved by combing HBV-DNA ≤ 2000 IU/mL, HBcrAg ≤ 3 log and total anti-HBc ≤ 16,937 IU/mL (DA 89.5%, sensitivity 93%, specificity 84.8%, PPV 88.9%, NPV 90.3%) [86]. Total anti-HBc was quantified by a double-antigen sandwich immune-assay, calibrated using WHO standards.
The biological importance and clinical relevance of serum HBV RNA during the natural course of HBV infection and the differentiation between HBeAg negative carriers and active infection remain unclear. An early study suggested that HBV RNA in serum could be a useful marker for the recognition of the stages of chronic HBV infection [61]. Recently, HBV RNA levels were found to be independently associated with HBeAg status, serum ALT, HBV genotype, and the presence of BCP variants, in a large multiethnic cohort including 122 HBeAg(-) individuals (80% HBV genotype D) [87]. In another study including 24 HBeAg(-) patients (genotype B or C), it was shown that serum HBV RNA levels best correlated with intrahepatic HBV RNA levels, reflecting cccDNA transcriptional activity, rather than with intrahepatic cccDNA itself. The correlation with cccDNA, observed in HBeAg(+) patients, was not found in the HBeAg(-) group. No correlations between liver injury or histopathology and HBV-RNA levels were observed, nonetheless, the ratio of HBsAg to serum HBV-RNA was found to be the highest in IC patients [88]. Similarly, in a study of CHB patients, the correlation between serum HBV RNA and intrahepatic cccDNA levels was reported to be dependent on the serostatus of HBeAg and was not found in HBeAg(-) patients [89]. Furthermore, in an investigation that included HBeAg(-) negative subjects, the authors proposed the arithmetic addition of serum RNA on serum DNA levels in order to most accurately reflect intrahepatic cccDNA levels. However, this heterologous combination also failed to produce a correlation between serum RNA and liver cccDNA in the group HBeAg(-) chronically infected individuals [90]. Thus, concerning HBeAg(-) CHB, it is not clear whether or not and in what context serum HBV RNA can serve as a biomarker during the natural course of infection. Although correlation with intrahepatic cccDNA levels is poor, serum HBV RNA reflects viral activity at some level, specifically the transcriptional activity of cccDNA. However, its biological and clinical significance in the progression and pathogenesis of HBV infection require further investigation.

StephenW

3.2。用于识别非活动载体状态的生物标志物
HBeAg慢性HBV感染的阴性患者分为两个主要状态。在非活动携带者状态（最近由EASL重命名为'HBeAg阴性慢性感染'），病毒复制和肝脏炎症有限[40]。无活性的HBV携带者呈现正常的丙氨酸氨基转移酶（ALT）水平（男性小于35 U / L，女性小于25 U / L）和低（<2000 IU / mL）或检测不到的HBV DNA。尽管上述临界值基于国际指南，但将患者定性为非活性载体需要连续测定ALT和HBV DNA，第一年每3-4个月一次，随后每6个月一次。这种方法势在必行，因为HBV DNA和ALT的波动发生在HBeAg活动性肝炎第二状态的患者[39,40,76]。
由于两种生物标志物的密切随访和连续测试对于鉴定非活性载体是必不可少的，因此已经评估了添加第三种生物标志物。鉴于绝大多数非活动性HBV携带者，血清中HBsAg水平低于活动性炎症患者[77,78]，研究的第一个生物标志物是qHBsAg。在对HBV基因型D感染的彻底研究中，qHBsAg值在单个时间点小于1000 IU / mL，与HBV DNA <2000 IU / mL和正常ALT相结合，证明诊断准确度（DA）为94.5％与每月监测后两种生物标志物相比，不活跃的携带者状态为一年。更具体地，单个三标记物测量的灵敏度为91.1％，特异性为95.4％，阳性预测值（PPV）为87.9％，阴性预测值（NPV）为96.7％[79]。在其他HBV基因型中观察到类似的结果。在一项对1068名被诊断为HBV携带者，感染HBV基因型B或C的台湾HBeAg阴性患者的一项研究中，发现HBsAg水平> 1000 IU / mL与HBeAg阴性肝炎在13年随访中发展的关系被发现显着（风险比（HR）= 1.5,95％CI = 1.2-1.9）和HBsAg <1000 IU / mL与低HBV DNA和ALT相结合被发现可用于鉴定最小风险的HBV携带者[80] 。在REVEAL队列中，在感染B或C基因型的患者中，使用相同HBsAg截止值的联合检测显示IC的诊断准确率为78％[81]。该HBsAg阈值为1000 IU / mL似乎是CHB鉴别诊断和非活动载体状态最可靠的阈值[82]。使用较低的qHBsAg截止值（例如100IU / mL）以提高特异性，导致灵敏度显着降低（35％）[83]。除qHBsAg外，增加肝硬度测量（LSM）作为第四个参数，截止值为6.2 kPa，进一步提高了在单个时间点测试ALT，HBV DNA和qHBsAg的诊断准确性，显示100％特异性，96用于识别IC的灵敏度％，100％PPV，92％NPV和97％DA [84]。
最近，有人提出，无论乙肝病毒基因型如何，血清HBcrAg都比qHBsAg更准确，可用于鉴定非活动携带者[85]。在本研究中，HBcrAg≤3logU/ mL与HBVDNA≤2000IU / mL的诊断准确率对于基因型D为87％，对于基因型F或H（73％）较低，但对于基因型A和E较高（91和94％）。
一些显示HBeAg阴性血清学特征且ALT正常，低病毒血症和/或qHBsAg高于1000 IU / mL的患者具有良性感染过程，并且其中一部分在以后的时间内满足IC的标准。在最近的一项包含此类人群的研究中，qHBsAg和HBV-DNA的组合定量诊断准确率分别为65.4％和100％NPV，用于IC的鉴定。在这项研究中，HBV-DNA≤2000IU / mL和HBcrAg <3 log或HBVDNA≤2000IU / mL和总抗-HBc≤16.637IU / mL具有更高且几乎相同的DA约86.5％，并且它们的诊断性能得到改善通过梳理HBV-DNA≤2000IU / mL，HBcrAg≤3log和总抗-HBc≤16.637IU / mL（DA 89.5％，灵敏度93％，特异性84.8％，PPV 88.9％，NPV 90.3％）[86]。通过双抗原夹心免疫测定法定量总抗-HBc，使用WHO标准校准。
血清HBV RNA在HBV感染自然过程中的生物学重要性和临床意义以及HBeAg阴性携带者与活动性感染的分化仍不清楚。早期研究表明，血清中的HBV RNA可能是识别慢性HBV感染阶段的有用标志[61]。最近，在一个包括122个HBeAg（ - ）个体（80％HBV基因型D）的大型多种族群体中，发现HBV RNA水平与HBeAg状态，血清ALT，HBV基因型和BCP变异体的存在独立相关[87]。 。在包括24名HBeAg（ - ）患者（基因型B或C）的另一项研究中，显示血清HBV RNA水平与肝内HBV RNA水平最佳相关，反映了cccDNA转录活性，而不是肝内cccDNA本身。在HBeAg（+）组中观察到的与cccDNA的相关性在HBeAg（ - ）组中未发现。没有观察到肝损伤或组织病理学与HBV-RNA水平之间的相关性，然而，发现HBsAg与血清HBV-RNA的比率在IC患者中最高[88]。同样，在一项CHB患者的研究中，血清HBV RNA与肝内cccDNA水平之间的相关性据报道依赖于HBeAg的血清状态，而在HBeAg（ - ）患者中未发现[89]。此外，在一项包括HBeAg（ - ）阴性受试者的调查中，作者提出了血清RNA水平的血清RNA的算术添加，以便最准确地反映肝内cccDNA水平。然而，这种异源组合也未能在HBeAg（ - ）慢性感染个体组中产生血清RNA和肝cccDNA之间的相关性[90]。因此，关于HBeAg（ - ）CHB，尚不清楚血清HBV RNA在感染的自然过程中是否可以作为生物标志物。尽管与肝内cccDNA水平的相关性较差，但血清HBV RNA在某种程度上反映了病毒活性，特别是cccDNA的转录活性。然而，其在HBV感染的进展和发病机理中的生物学和临床意义需要进一步研究

StephenW

3.3. Biomarkers for the Prediction of Spontaneous HBsAg Clearance
Since 0.4–2.3% of HBe Ag negative inactive carriers clear HBsAg yearly [91,92,93], AASLD in 2018 suggested as practice guidance that HBeAg negative ICs should be tested annually for HBsAg [76].
In mostly Asian studies of patients infected with HBV genotypes B and C, it has been shown that the levels and kinetics of HBsAg in serum are predictors of subsequent spontaneous HBsAg loss. In one of the first studies, a threshold of baseline qHBsAg ≤ 100 IU/mL had 75% sensitivity and 91% specificity to predict subsequent HBsAg seroclearance, which was not associated with baseline serum HBV DNA [94]. Very low qHBsAg < 10 IU/mL was found to be an excellent predictor of its clearance, with a hazard ratio (HR) 13.2 compared to levels above 1000 IU/mL [80]. In another study, a baseline qHBsAg < 200 IU/mL resulted in a sensitivity of 84.2% and specificity 73.4% and qHBsAg kinetics, that is an annual 0.5 log reduction, in a sensitivity of 62.8% and 88.7% specificity for the prediction of HBsAg clearance, in a three year follow-up period [95]. More recently, in a cohort of patients with 3.08% annual HBsAg clearance rate, baseline qHBsAg levels predicted HBsAg loss (AUROC 0.965 (95% CI, 0.947–0.980)), with baseline levels < 10 IU/mL showing diagnostic an accuracy of 93.4%, a sensitivity of 87.2%, a specificity of 94.8%, a positive predictive value of 79.1%, and a negative predictive value of 97.0% [96]. In addition, a scoring system for the prediction of HBsAg seroclearance in HBeAg-seronegative chronic hepatitis B patients with genotype B or C infection has been proposed incorporating baseline qHBsAg and HBV DNA levels [97].
In a recent study, in patients infected mainly with HBV genotype D and 4.6% annual serum HBsAg clearance, the combination of qHBsAg ≤ 100-IU/mL with HBV-DNA ≤ 200-IU/mL exhibited good performance (87.5% DA, 84.2% sensitivity, 88.2% specificity, 66.7% PPV, and 95.2% NPV) for the prediction of HBsAg loss. Baseline q HBsAg levels were independently correlated with HBsAg clearance and were significantly lower (median 0.75 vs. 2.81 log10 IU/mL, p < 0.001) in patients who cleared HBsAg. Yearly decline of qHBsAg was also found to be a predictive factor being higher in patients who cleared HBsAg (median 0.22 vs. 0.020 log10 IU/mL/year, p < 0.001). HBcrAg was not found to be a predictor of HBsAg loss [86].

StephenW

3.3。用于预测自发性HBsAg清除率的生物标志物
由于0.4-2.3％的HBe Ag阴性非活性携带者每年清除HBsAg [91,92,93]，2018年的AASLD建议作为实践指导，每年应对HBsAg检测HBeAg阴性IC [76]。
在大多数感染HBV基因型B和C的患者的亚洲研究中，已经显示血清中HBsAg的水平和动力学是随后自发性HBsAg消失的预测因子。在最初的一项研究中，基线qHBsAg≤100IU / mL的阈值具有75％的敏感性和91％的特异性来预测随后的HBsAg血清清除，这与基线血清HBV DNA无关[94]。非常低的qHBsAg <10 IU / mL被发现是其清除率的极好预测因子，与高于1000 IU / mL的水平相比，风险比（HR）为13.2 [80]。在另一项研究中，基线qHBsAg <200 IU / mL导致灵敏度为84.2％，特异性为73.4％，qHBsAg动力学，即每年0.5个对数减少，灵敏度为62.8％，特异性为88.7％，用于预测HBsAg通关，在三年的随访期内[95]。最近，在一组HBsAg年清除率为3.08％的患者中，基线qHBsAg水平预测HBsAg消失（AUROC 0.965（95％CI，0.947-0.980）），基线水平<10 IU / mL显示诊断准确度为93.4 ％，敏感性为87.2％，特异性为94.8％，阳性预测值为79.1％，阴性预测值为97.0％[96]。此外，已经提出了用于预测基因型B或C感染的HBeAg血清阴性慢性乙型肝炎患者中HBsAg血清清除率的评分系统，其结合了基线qHBsAg和HBV DNA水平[97]。
在最近的一项研究中，在主要感染HBV基因型D且年血清HBsAg清除率为4.6％的患者中，qHBsAg≤100-IU / mL与HBV-DNA≤200-IU / mL的组合表现出良好的表现（87.5％DA，84.2）预测HBsAg消失的敏感性％，特异性88.2％，PPV 66.7％，NPV 95.2％。基线q HBsAg水平与HBsAg清除率独立相关，并且在清除HBsAg的患者中显着降低（中位数0.75对2.81 log10 IU / mL，p <0.001）。 qHBsAg的年度下降也被发现是清除HBsAg的患者中较高的预测因素（中位数0.22对0.020 log10 IU / mL /年，p <0.001）。 HBcrAg未被发现是HBsAg丢失的预测因子[86]。

StephenW

https://www.mdpi.com/2073-4425/9/10/469/htm