Relationship between HBsAg,HBcrAg and hepatocellular carcinoma in patients with undetectable HBV DNA under nucleos(t)ide therapy

We examined the relationship between hepatitis B surface and core‐related antigens (HBsAg, HBcrAg) and hepatocellular carcinoma (HCC) development in patients with undetectable serum HBV DNA receiving nucleos(t)ide analogue (NA). Seventy‐six HBV carriers with undetectable HBV DNA (<20 IU/mL) who subsequently developed HCC were compared with 152 matched controls. Clinical and laboratory parameters (including novel assays to measure linearized HBsAg [HQ‐HBsAg] and HBcrAg) were analysed. There were no significant differences in HBsAg/HQ‐HBsAg levels between the two groups. There was a significant difference in the median values of both pre‐ and post‐NA HBcrAg levels between the HCC and control groups (pre‐treatment: 279.0 vs 35.4 kU/mL, P=.005; post‐treatment: 10.2 vs 1.7 kU/mL, P=.005, respectively). For the whole HCC group, a cut‐off value of post‐treatment HBcrAg level ≥7.8 kU/mL yielded an area under receiver operating curve (AUROC) of 0.61 with a negative predictive value (NPV) of 77.0%. The OR of HCC development was 3.27. For noncirrhotic patients, the median values of post‐treatment HBcrAg level of HCC group and controls were 10.2 and 1.0 kU/mL, respectively (P=.001). A cut‐off value of HBcrAg level ≥7.9 kU/mL yielded an AUROC of 0.70 with a NPV of 80.6%. The OR of HCC development was 5.95. A higher pre‐ and post‐NA treatment HBcrAg level (but not HBsAg) was associated with an increased risk of HCC development in patients achieving undetectable serum HBV DNA while on NA therapy. HBcrAg may serve as a novel risk marker for HCC in this group of patients.     

乙型肝炎病毒共价闭合环状DNA的扩增与清除动力学

HBV基因组是一种松弛环状的双链DNA(relaxed circular DNA,rcDNA)分子,其两条链均不闭合,在病毒的复制过程中,病毒DNA进入宿主细胞核,两条链的缺口被补齐,形成超螺旋的共价闭合环状DNA(covalently dosed circular DNA,cccDNA).cccDNA是HBV在肝内进行复制的原始模板,虽然其含量较少,但对病毒在宿主细胞内感染状态的建立以及维持病毒在细胞内的复制过程具有十分重要的意义.     

Serum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients

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Intrahepatic hepatitis B virus covalently closed circular DNA can be a predictor of sustained response to therapy

BACKGROUND & AIMS: This study aimed to determine whether intrahepatic hepatitis B virus (HBV) covalently closed circular (ccc) DNA and total HBV DNA levels at the end of therapy would predict sustained response to therapy. METHODS: Hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients receiving either lamivudine monotherapy or combination of peginterferon and lamivudine had liver biopsy at the end of 1 year therapy and were followed for 52 more weeks after cessation of therapy. Serum HBV DNA, intrahepatic HBV ccc DNA, and total HBV DNA levels were determined. RESULTS: Forty-seven patients, including 34 males and 13 females, were studied. Twenty-seven patients received combination therapy, and 20 patients received lamivudine monotherapy. Twenty-nine patients had end-of-treatment virologic response, and 15 patients had sustained response 52 weeks after therapy. At the end of treatment, log serum HBV DNA levels correlated well with log intrahepatic HBV cccDNA and log intrahepatic total HBV DNA levels. Log intrahepatic cccDNA and log intrahepatic total DNA levels were significantly lower among patients with sustained virologic response. The adjusted odds ratio for log cccDNA was 5.3 (95% CI: 1.5-18.2, P = .009) and, for log intrahepatic HBV DNA, was 4.4 (95% CI: 1.3-14.7, P = .015) to predict sustained virologic response. Using log cccDNA at -0.80 copies/genome equivalent as cutoff, the sensitivity, specificity, and positive and negative predictive values and accuracy of predicting sustained virologic response were 73%, 78%, 56%, 86%, and 77% respectively. CONCLUSIONS: Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.     

Effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA in HBeAg-positive chronic hepatitis B patients

AIM： To evaluate the effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA （ccc DNA） in HBeAg-positive chronic hepatitis B patients.
METHODS： Seventy-one patients received lamivudine （n = 35）, or sequential therapy with lamivudine- interferon alpha 2b （IFN-α 2b, n = 24） for 48 wk, or IFN-α 2b （n = 12） for 24 wk. All subjects were followed up for 24 wk. Intrahepatic ccc DNA was measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP.
RESULTS： Sequential lamivudine- INF-α therapy, lamivudine and INF-α monotherapy reduced ccc DNA of 1.7 log, 1.4 log and 0.8 log, respectively （P 〈 0.05）. Seventeen out of the 71 patieots developed HBeAg seroconversion, the reduction of ccc DNA in the HBeAg seroconversion patients was more significant than that in the HBeAg positive patients （3.0 log vs 1.6 log, P = 0.0407）. Twenty-four weeks after antiviral therapy withdrawal, 16 patients had a sustained virological response, the baseline intrahepatic ccc DNA in the patients with a sustained virological response was significantly lower than that in the patients with virological rebound （4.6 log vs 5.4 log, P = 0.0472）. HBV genotype C accounted for 85.9% （n = 61）, and genotype B for 14.1% （n = 10）, respectively, in the 71 patients. There was no significant difference in the change of ccc DNA level between HBV genotypes C and B （2.1 log vs 1.9 log）.
CONCLUSION： Forty-eight week sequential lamivudine- INF-α therapy and lamivudine monotherapy reduce ccc DNA more significantly than 24-wk INF-α monotherapy. Low baseline intrahepatic ccc DNA level may predict the long-term efficacy of antiviral treatment. HBV genotypes C and B have no obvious influence on ccc DNA load.     

乙型肝炎病毒共价闭合环状DNA的研究进展

共价闭合环状DNA(covalently dosed circular DNA,cccDNA)在HBV的复制及感染状态的建立过程中起着非常重要的作用,现将cccDNA的研究进展总结如下. 一、HBV cccDNA的检测方法 1.PCR法:PCR法包括普通PCR和定量PCR.由于松弛环状DNA(relaxed circular DNA,rcDNA)在负链和正链上存在缺口,我们可以设计一对跨越两个缺口的引物,由于cccDNA有完整的双链,可以被有选择地扩增.但起始模板量不能太高,否则rcDNA也有可能被扩增,Kock等[1]发现,每个PCR反应管中HBV DNA量在6×105拷贝/ml时能达到最佳的灵敏度与选择性.     

Occult Hepatitis B Virus Infection: An Update

Occult hepatitis B virus (HBV) infection (OBI) refers to a condition in which replication-competent viral DNA is present in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for the HBV surface antigen (HBsAg). In this peculiar phase of HBV infection, the covalently closed circular DNA (cccDNA) is in a low state of replication. Many advances have been made in clarifying the mechanisms involved in such a suppression of viral activity, which seems to be mainly related to the host’s immune control and epigenetic factors. OBI is diffused worldwide, but its prevalence is highly variable among patient populations. This depends on different geographic areas, risk factors for parenteral infections, and assays used for HBsAg and HBV DNA detection. OBI has an impact in several clinical contexts: (a) it can be transmitted, causing a classic form of hepatitis B, through blood transfusion or liver transplantation; (b) it may reactivate in the case of immunosuppression, leading to the possible development of even fulminant hepatitis; (c) it may accelerate the progression of chronic liver disease due to different causes toward cirrhosis; (d) it maintains the pro-oncogenic properties of the “overt” infection, favoring the development of hepatocellular carcinoma.     

Prediction of response to entecavir therapy in patients with HBeAg-positive chronic hepatitis B based on on-treatment HBsAg, HBeAg and HBV DNA levels

Summary. Quantitative hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) assays are emerging as effective tools of on‐treatment predictors of response to antiviral agents, in addition to monitoring serum HBV DNA levels. However, the dynamic relationship between quantitative HBsAg, as well as HBeAg and HBV DNA, and the predictability of subsequent clinical outcomes during entecavir (ETV) therapy remain unclear. Eighty‐two patients with HBeAg‐positive chronic hepatitis B (CHB) received ETV therapy for ≥3 years. Virologic response (VR) after 3 years of ETV therapy was achieved in 73 (89.0%) patients. Among baseline and on‐treatment factors, on‐treatment HBV DNA levels performed better with respect to the prediction of response than HBsAg and HBeAg levels. Especially, the performance of absolute values of HBV DNA with respect to response was superior to HBV DNA decline from the baseline. The best predictive value was an absolute HBV DNA level of 2.3 log₁₀ IU/mL at month 6 (areas under the curve [AUROC], 0.977; 95% CI, 0.940–1.000; P < 0.001). HBeAg seroconversion after 3 years of therapy was achieved in 26 (31.7%) patients. On‐treatment HBeAg levels performed better with respect to the prediction of seroconversion than HBsAg and HBV DNA levels. The best cut‐off value for the HBeAg level at month 12 for the prediction of seroconversion was 0.62 log₁₀ PEIU/mL. Although the HBsAg level at baseline is often used to predict the antiviral potency of entecavir, on‐treatment HBV DNA and HBeAg levels are more helpful for prediction of subsequent clinical outcomes in HBeAg‐positive CHB patients with entecavir treatment.     

Dynamic analysis of hepatitis B virus DNA and its antigens in 2.2.15 cells

The 2.2.15 cells-derived from HepG2 cells transfected with a plasmid containing hepatitis B virus (HBV) DNA secrete surface antigen (HBsAg) particles, nucleocapsids and virions (Proc Natl Acad Sci U S A 1987; 84: 1005-1009). The latter elicit acute hepatitis in chimpanzees (Proc Natl Acad Sci U S A 1987; 84: 4641-4644). We studied the presence of intracellular and extracellular HBV covalently closed circular (ccc) DNA in this culture system by polymerase chain reaction (PCR), kinetically analysed HBsAg and hepatitis B e antigen (HBeAg) released in the culture media by quantitative enzyme-linked immunosorbent assay and quantitated by real-time PCR but HBV DNA from intracellular and extracellular HBV-DNA. HBV cccDNA was found both intracellularly and extracellularly. A significant correlation was seen between the extracellular HBV DNA levels and virus antigens (r = 0.833; P = 0.01 and r = 0.939; P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no statistical correlation between intracellular HBV DNA levels and virus antigen levels (r = 0.024; P = 0.955 and r = 0.177; P = 0.625 for HBsAg and HBeAg, respectively). These data would be valuable in studies of the HBV life cycle and of potential anti-viral agents.     

HBsAg and HBcrAg as predictors of HBeAg seroconversion in HBeAg‐positive patients treated with nucleos(t)ide analogues

HBeAg seroconversion marks an important spontaneous change and treatment end‐point for HBeAg‐positive patients and is a pre‐requisite for HBsAg loss or functional cure. In this retrospective analysis, we aimed to identify predictors of seroconversion using serum quantitative HBsAg and HBcrAg, in HBeAg‐positive patients treated with nucleos(t)ide analogues (NA). Data and samples from 118 HBeAg‐positive adults (genotypes A‐G) started on NA between Jan 2005 and Sept 2016 were retrospectively analysed at several time‐points. The predictive power of on‐treatment levels of HBsAg and HBcrAg was determined using receiver operating curve (ROC) analysis and cut‐off values determined by maximized Youden's index. About 36.4% of patients achieved HBeAg seroconversion after a median of 39 months’ treatment. On treatment kinetics of HBV DNA, HBsAg and HBcrAg differed between HBeAg seroconverters and nonseroconverters. A combination of HBsAg and HBcrAg had the greatest predictive value for HBeAg seroconversion: at 6 months, HBsAg of 3.9 log₁₀ IU/mL and HBcrAg of 5.7 log₁₀ U/mL had a sensitivity of 71.4%, specificity of 79.5%, positive predictive value (PPV) of 65.2% and negative predictive value (NPV) of 83.8%, with AUROC of 0.769 (0.668, 0.869; 95%CI), and at 12 months, HBsAg 3.8 log₁₀ IU/mL and HBcrAg 5.5 log₁₀ U/mL had a sensitivity of 73.7%, specificity of 79.5%, PPV of 63.6% and NPV of 86.1%, with AUROC 0.807 (0.713, 0.901; 95% CI). In conclusion, our results may be used to identify patients who are unlikely to achieve treatment end‐points, which will be important as the future management of chronic hepatitis B looks to therapies that offer functional cure.     

HBV感染者血清中HBVcccDNA、HBeAg及HBV DNA的关系

探讨HBV感染者血清HBVcccDNA与血清HBV DNA及HBeAg的关系。分别以PER分子信标技术和ELISA方法对非HBV相关肝炎、HBV健康携带者、急性乙型肝炎（AHB）、慢性乙型肝炎（CHB）、乙肝肝硬化、乙肝患者血清中HBVcccDNA HBV DNA含量及HBeAg进行了检测。HBVcccDNA仅见于HBV DNA阳性血清中；HBeAg阳性组的HBVcccDNA阳性率显著高于HBeAg阴性组（P〈0．05）；145例HBV DNA阳性患者中，HBVcccDNA阳性组HBVD—NA水平显著高于HBVcccDNA阴性组（P〈0．01）。血清HBVcccDNA可能是乙肝病毒在患者体内大量复制的血清标志。     

肝组织cccDNA水平与血清病毒学应答后治疗时间的关系

目的 探讨慢性乙型肝炎(CHB)患者肝组织cccDNA水平与外周血HBV DNA<1000 拷贝/ml后继续治疗时间的关系.方法 分别采用荧光定量PCR、酶联免疫吸附分析法检测58例CHB患者肝组织HBV cccDNA水平、肝组织和血清HBV DNA载量、HBV标志物,分析肝组织HBV cccDNA水平、肝组织总HBV DNA水平、HBeAg血清学转换与血清病毒学应答后继续治疗时间的关系.组间比较采用Nemenyi法,相关分析采用Spearman法.结果 肝组织HBVcccDNA水平在血清HBV DNA两阳性组间尢明显差异,而阴性组明显低于阳性组(χ2=9.6948,P<0.01;χ2=9.2824,P<0.01).35例达到血清病毒学应答后行肝活组织检查的患者,肝组织cccDNA水平随继续治疗时间的延长而降低(χ2≥6.4674,P<0.05),肝组织cccDNA水平在抗-Hbe(+)组明显低于HBeAg(+)组、HBeAg(-)/抗-Hbe(-)组(χ2=10.7482,P<0.01;χ2=11.7549,P<0.01).14例肝组织cccDNA水平低十检测限的患者,有12例已经发生HBeAg血清学转换,占抗-Hbe(+)组的2/3,其在血清病毒学应答后继续治疗时间平均为35个月,发生HBeAg血清学转换后继续治疗时间平均为30个月.结论 当患者发生血清病毒学应答后,肝组织cccDNA水平随继续治疗时间延长而降低;继续治疗35个月以上且血清抗-Hbe持续(+)30个月以上时,有2/3的患者肝组织cccDNA定量低于检测限水平.     

Loss of intrahepatic HBsAg expression predicts sustained response to peginterferon and is reflected by pronounced serum HBsAg decline

There is a lack of knowledge regarding the effect of peginterferon (PEG‐IFN) on the expression of intrahepatic hepatitis B core and surface antigen (HBcAg and HBsAg) in chronic hepatitis B (CHB) and its relation with response to therapy. Fifty‐two HBeAg‐positive and 67 HBeAg‐negative CHB patients with paired liver biopsies taken at baseline and after 1 year of PEG‐IFN therapy were studied. After PEG‐IFN therapy, HBeAg‐negative patients showed a significant reduction in both intrahepatic HBcAg (P = 0.04) and HBsAg expression (P < 0.001). In contrast, a reduction in intrahepatic HBcAg expression was not observed in HBeAg‐positive patients, while a trend in reduction of intrahepatic HBsAg staining was found (P = 0.09). Post‐treatment, 7 (13%) HBeAg‐positive and 9 (14%) HBeAg‐negative patients had no expression of intrahepatic HBsAg. Patients without any intrahepatic HBsAg expression post‐treatment were more likely to achieve a combined response (HBeAg loss with hepatitis B virus (HBV) DNA <2000 IU/mL for HBeAg ‐positive and HBV DNA <2000 IU/mL and normal alanine aminotransferase for HBeAg‐negative CHB): 71% vs 5% for HBeAg‐positive (P < 0.001) and 60% vs 16% for HBeAg‐negative patients (P = 0.004), respectively. Moreover, a more profound decline of serum HBsAg was observed in patients with absence of intrahepatic HBsAg staining (3.1 vs 0.4 log IU/mL, P < 0.001 and 1.7 vs 0.4 log IU/mL, P = 0.005 for HBeAg‐positive and HBeAg‐negative CHB, respectively). In conclusion, PEG‐IFN reduces expression of intrahepatic HBsAg. Loss of HBsAg as assessed by immunohistochemistry from the liver predicts a sustained response and is reflected in a pronounced serum HBsAg decline.     

Correlation between HBsAg quantitative assay results and HBV DNA levels in chronic HBV

Background

Viral load has been used to diagnose and monitor patients who are being treated for chronic hepatitis B (CHB). The Diagnosis methods are molecular-based and expensive. Quantitation of hepatitis B surface antigen (HBsAg) by automated chemiluminescent micro-particle immunoassay has been proposed to be a surrogate marker. Quantitating HBV DNA levels molecularly is expensive; thus, a cheaper laboratory test as a surrogate diagnostic marker might simplify our management.

Objectives

We determined whether quantitative HBsAg levels correlate with HBV DNA levels in CHB.

Patients and Methods

In this cross-sectional study, all CHB patients who were referred by a gastroenterologist to undergo quantitative HBV DNA assay in a qualified laboratory in Mashhad, Iran in 2009 were enrolled, and blood samples was obtained. Patients who were positive for antibodies to HCV and HDV were excluded. HBV DNA was measured by real-time polymerase chain reaction, and serum HBsAg was quantified byelectrochemiluminescence assay (Roche Diagnostic).

Results

Of 97 patients, 70 were male (72%) and 27 were female (28%); the mean age was 39 ± 11 years. Eighty-seven percent wasHBeAg-negative. By Mann-Whitney test,HBSAg titer differed significantly between HBeAg-positive and -negative patients (P = 0.001), as did HBV DNA levels (P = 0.009). By Spearman test, there was no significant correlation between HBsAg and HBV DNA levels (P= 0.606 and r = 0.53).

Conclusions

HBeAg-negative patients have higher levels of HBsAg and lower levels of HBV DNA. By electrochemiluminescence assay,HBsAg has no significant correlation with HBV DNA levels in CHB with predominant genotype D and HBeAg negativity in Iran.     

Comparison between quantitative hepatitis B surface antigen,hepatitis B e‐antigen and hepatitis B virus DNA levels for predicting virological response to pegylated interferon‐α‐2b therapy in hepatitis B e‐antigen‐positive chronic hepatitis B

Aim: The aim of this study was to compare the clinical applicability of quantitative serum hepatitis B surface antigen (HBsAg), hepatitis B e‐antigen (HBeAg) and hepatitis B virus (HBV) DNA for predicting virological response (VR) to pegylated interferon (PEG‐IFN) therapy. Methods: Thirty HBeAg‐positive chronic hepatitis B patients who received PEG‐IFN‐α‐2b for 48 weeks were enrolled. Quantitative HBsAg, HBeAg and HBV DNA were measured before, during and after the therapy. Paired liver biopsies were performed before and after treatment for covalently closed circular (ccc)DNA and intrahepatic HBV DNA analysis. Results: VR at 48 weeks post‐treatment, defined as HBeAg seroconversion and HBV DNA less than 10 000 copies/mL was achieved in 10 (33.3%) patients. Responders had significantly lower baseline HBsAg, HBeAg, cccDNA and intrahepatic HBV DNA levels than non‐responders. Baseline and reduced levels of log₁₀ HBsAg and log₁₀ HBeAg correlated well with those of log₁₀ cccDNA and log₁₀ total intrahepatic HBV DNA. Responders showed consistent decrease in serum HBsAg, HBeAg and HBV DNA levels during therapy. HBeAg level of 2.0 log₁₀ sample to cut‐off ratio at week 24 on therapy provided the best prediction of sustained virological response, with sensitivity and negative predictive values of 85% and 92%, respectively. One patient (3.3%) who cleared HBsAg at follow up exhibited a more rapid decline in serum HBsAg during therapy than those who developed VR without HBsAg clearance. Conclusion: Quantitative measurement of serum HBeAg during therapy may be superior to serum HBsAg and HBV DNA as a prediction of HBeAg seroconversion. Kinetics of HBsAg levels on therapy may help predict HBsAg clearance after treatment.     

血清HBsAg、HBcrAg和HBV DNA预测慢性乙型肝炎肝组织病理状态的性能评价

目的评价血清HBsAg、HBcrAg、HBV DNA预测慢性乙型肝炎肝组织病理状态的性能。方法将324例HBeAg阳性和255例HBeAg阴性慢性乙型肝炎患者随机分为3组匹配的训练集和验证集。血清HBsAg和HBcrAg分别采用Abbott Architect I2000和Fujirebio Lumipulse G1200全自动化学发光免疫系统检测,血清HBV DNA采用Bio-Rad Icycler荧光定量PCR仪检测。肝组织病理学诊断采用Scheuer评分系统,其中病理学分级包括G0~G4五级,分期包括S0~S4五期。结果无论HBeAg阳性或阴性患者,3个训练集与3个匹配的验证集性别比例和平均年龄、病理学分级和分期构成比之间的差异均无统计学意义(P0.05)。HBeAg阳性患者,血清HBsAg、HBcrAg、HBV DNA预测全集病理学分级≥G3和分期≥S4的ROC曲线下面积最大;参照预测3个训练集病理学分级≥G3和分期≥S4的最佳截断值,血清HBsAg、HBcrAg、HBV DNA预测3个匹配的验证集病理学分级≥G3和分期≥S4的灵敏度极差分别为37%和9%、30%和16%、17%和14%,特异度极差分别为12%和5%、13%和3%、15%和6%。HBeAg阴性患者,血清HBcrAg、HBV DNA预测全集病理学分级≥G2和分期≥S2的ROC曲线下面积最大;参照预测3个训练集病理学分级≥G2和分期≥S2的最佳截断值,血清HBcrAg、HBV DNA预测3个匹配的验证集病理学分级≥G2和分期≥S2的灵敏度极差分别为11%和20%、46%和19%,特异度极差分别为15%和2%、38%和16%。结论 HBeAg阳性患者,血清HBsAg、HBcrAg、HBV DNA可预测的最佳病理状态为病理学分级≥G3和分期≥S4,其预测理学分期≥S4的稳定性高于预测病理学分级≥G3;HBeAg阴性患者,血清HBcrAg和HBV DNA可预测的最佳病理状态为病理学分级≥G2和分期≥S2,血清HBcrAg预测病理学分级≥G2和分期≥S2的稳定性高于HBV DNA。