通过液滴数字PCR定量抗-HBc阳性肝脏供体中的HBV cccDNA：检测隐

StephenW

J Hepatol. 2018 Apr 2. pii: S0168-8278(18)31963-9. doi: 10.1016/j.jhep.2018.03.021. [Epub ahead of print]
Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: a new tool to detect occult infection.Caviglia GP1, Abate ML2, Tandoi F3, Ciancio A2, Amoroso A4, Salizzoni M3, Saracco GM2, Rizzetto M2, Romagnoli R3, Smedile A2.
Author information
1Department of Medical Sciences, University of Turin, Gastroenterology Division of Città della Salute e della Scienza of Turin, University Hospital, Turin, Italy. Electronic address: [email protected].2Department of Medical Sciences, University of Turin, Gastroenterology Division of Città della Salute e della Scienza of Turin, University Hospital, Turin, Italy.3General Surgery 2U, Liver Transplant Center - A.O.U. Città della Salute e della Scienza di Torino, Molinette Hospital, Department of Surgical Sciences, University of Turin, Turin, Italy.4Regional Transplant Center, Piedmont, A.O.U. Città della Salute e della Scienza di Torino, Molinette Hospital, Turin, Italy.

AbstractBACKGROUND & AIMS: The accurate diagnosis of occult HBV infection (OBI) requires the demonstration of HBV DNA in liver biopsies of HBsAg-negative subjects. However, in clinical practice a latent OBI is deduced by the finding of the antibody to the HB-core antigen (anti-HBc). We investigated the true prevalence of OBI and the molecular features of intrahepatic HBV in anti-HBc-positive subjects.
METHODS: The livers of 100 transplant donors (median age 68.2 years; 64 males, 36 females) positive for anti-HBc at standard serologic testing, were examined for total HBV DNA by nested-PCR and for the HBV covalently closed circular DNA (HBV cccDNA) with an in-house droplet digital PCR assay (ddPCR) (Linearity: R2 = 0.9998; lower limit of quantitation and detection of 2.4 and 0.8 copies/105 cells, respectively).
RESULTS: A true OBI status was found in 52% (52/100) of the subjects and cccDNA was found in 52% (27/52) of the OBI-positive, with a median 13 copies/105 cells (95% confidence interval 5-25). Using an assay specific for anti-HBc of IgG class, the median antibody level was significantly higher in HBV cccDNA-positive than negative donors (5.7 [3.6-9.7] vs. 17.0 [7.0-39.2] COI, p = 0.007). By multivariate analysis, an anti-HBc IgG value above a 4.4 cut-off index (COI) was associated with the finding of intrahepatic HBV cccDNA (OR = 8.516, p = 0.009); a lower value ruled out its presence with a negative predictive value of 94.6%.
CONCLUSIONS: With a new in-house ddPCR-based method, intrahepatic HBV cccDNA was detectable in quantifiable levels in about half of the OBI cases examined. The titer of anti-HBc IgG may be a useful surrogate to predict the risk of OBI reactivation in immunosuppressed patients.
LAY SUMMARY: The covalently closed circular DNA (cccDNA) form of the Hepatitis B virus (HBV) sustains the persistence of the virus even after decades of resolution of the florid infection (Occult HBV infection=OBI). In the present study we developed an highly sensitive method based on droplet digital PCR technology for the detection and quantitation of HBV cccDNA in the liver of subjects with OBI .We observed that the amount of HBV cccDNA may be inferred from the titer in serum of the IgG class antibody to the hepatitis B core antigen (anti-HBc IgG). The quantitation of anti-HBc IgG may represent a surrogate to discriminate the patients at the highest risk of HBV reactivation following immunosuppressive therapies.

Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

KEYWORDS: HBcrAg; Hepatitis B virus; Occult HBV infection

PMID:29621551DOI:10.1016/j.jhep.2018.03.021

StephenW

J Hepatol。 2018年4月2日。pii：S0168-8278（18）31963-9。 doi：10.1016 / j.jhep.2018.03.021。 [电子版提前打印]
通过液滴数字PCR定量抗-HBc阳性肝脏供体中的HBV cccDNA：检测隐匿性感染的新工具。
Caviglia GP1，Abate ML2，Tandoi F3，Ciancio A2，Amoroso A4，Salizzoni M3，Saracco GM2，Rizzetto M2，Romagnoli R3，Smedile A2。
作者信息

1
    都灵大学医学系，意大利都灵大学医院都灵Cittàdella Salute e della Scienza胃肠病科。电子地址：[email protected]。
2
    都灵大学医学系，意大利都灵大学医院都灵Cittàdella Salute e della Scienza胃肠病科。
3
    普通外科2U，肝移植中心 - A.O.U. Cittàdella Salute e della Scienza di Torino，Molinette医院，意大利都灵都灵大学外科学系。
4
    区域移植中心，Piedmont，A.O.U. Cittàdella Salute e della Scienza di Torino，Molinette Hospital，Turin，Italy。

抽象
背景＆目标：

对隐匿性HBV感染（OBI）的准确诊断需要在HBsAg阴性受试者肝活组织检查中证实HBV DNA。然而，在临床实践中，通过发现针对HB-核心抗原的抗体（抗-HBc）推断潜在的OBI。我们调查了抗-HBc阳性受试者中OBI的真正普遍性和肝内HBV的分子特征。
方法：

在标准血清学检测中，100名移植供体的肝脏（中位年龄68.2岁; 64名男性，36名女性）抗-HBc阳性，通过巢式PCR和HBV共价闭合环状DNA（HBV cccDNA）检测总HBV DNA， （ddPCR）（线性度：R2 = 0.9998;定量下限和分别检测2.4和0.8拷贝/ 105细胞）。
结果：

在52％（52/100）的受试者中发现了真正的OBI状态，在OBI阳性的52％（27/52）中发现了cccDNA，中位数为13 copies / 105 cells（95％置信区间5- 25）。使用针对IgG类抗HBc的特异性测定，HBV cccDNA阳性中值抗体水平显着高于阴性供体（5.7 [3.6-9.7]比17.0 [7.0-39.2] COI，p = 0.007）。通过多变量分析，高于4.4临界指数（COI）的抗HBc IgG值与肝内HBV cccDNA的发现相关（OR = 8.516，p = 0.009）;一个较低的值排除了它的存在，其阴性预测值为94.6％。
结论：

在新的内部基于ddPCR的方法中，在检查的约一半的OBI病例中，肝内HBV cccDNA在可定量水平中可检测到。抗-HBc IgG的滴度可能是预测免疫抑制患者中OBI再激活风险的有用替代物。
总结：

即使经过数十年的华盛顿感染（隐匿性HBV感染= OBI）的解决，乙型肝炎病毒（HBV）的共价闭合环形DNA（cccDNA）形式仍维持病毒的持久性。在本研究中，我们开发了一种基于液滴数字PCR技术的高灵敏度方法，用于检测和定量OBI受试者肝脏中的HBV cccDNA。我们观察到HBV cccDNA的量可以从血清中的效价推断出针对乙型肝炎核心抗原的IgG类抗体（抗-HBc IgG）。抗-HBc IgG的定量可以代表区分在免疫抑制治疗后HBV再激活风险最高的患者。

版权所有©2018年欧洲肝脏研究协会。由Elsevier B.V.出版。保留所有权利。
关键词：

HBcrAg;乙型肝炎病毒;隐匿性HBV感染

结论：
    29621551
DOI：
    10.1016 / j.jhep.2018.03.021