肝组织HBVDNA定量检查的临床意义

由于对HBV感染的认识正在不断深入，抗病毒治疗急待寻找判定效果可靠的方法，HBVDNA检测，特别是血液和肝组织定量检测显得极春重要。不加选择对HBV感染作肝活检时留取小粒肝组织，同时采血作HBVDNA定量检查。急慢性HBV感染病例均示肝组织内HBVDNA检查明显高于血内检出率，同时肝组织HBV含量明显高于血液的含量。肝组织检测HBVDNA可以明显提高HBV感染诊断率，血内HBVDNA阴转时不能说明肝组织内亦已阴转，血内HBVDNA阴转不宜随即停止治疗。     

血液和组织内HBVDNA定量检查的临床意义

HBV感染的治疗应以抗病毒为主,评价疗效应以HBVDNA消长来衡量.用PCR定量法不加选择地观察98例HBV急慢性感染病例的血液和肝、脾、肾、脑组织内HBVDNA含量.急性HBV感染血和肝组织内HBVDNA阳性率和平均含量均较低,慢性感染则均明显较高.治疗血内HBVDNA阴转后,肝组织内多数亦随之阴转.慢性HBV感染者血和肝、脾、肾、脑组织均可查见HBVDNA,肝、脾脏组织内含量较高,少数阴转可能晚于血液,不应忽略巩固治疗,防止复发.     

慢性乙型肝炎患者血清、肝内HBV DNA定量及肝组织HBV DNA载量与肝组织损害程度的关系

目的 了解慢性乙型肝炎患者血清、肝内HBV DNA定量及肝组织HBV DNA载量与肝组织损害程度的关系.方法 经血清学及肝穿刺病理证实为慢性乙型肝炎患者38例,分为2组(HBeAg阳性组及HBeAg阴性组).采用ELISA法测定HBV-M、实时荧光定量检测血清及肝内HBV DNA定量、肝组织HBV DNA载量.结果 HBeAg阳性组血清、肝内HBV DNA定量及肝组织HBV DNA载量均高于HBeAg阴性组(P均<0.001);血清HBV DNA定量随肝脏纤维化程度加重而升高(P<0.01),但与肝脏炎症程度未见明显相关;肝组织HBV DNA载量则随肝脏炎症及纤维化程度加重而降低(P<0.001).结论 血清HBV DNA定量与肝组织纤维化程度呈正相关,肝组织HBV DNA载量则与肝组织的炎症及纤维化程度呈明显负相关.     

乙型肝炎患者血清及肝组织中HBV DNA含量与肝脏炎症指标的相关性研究

目的 探讨慢性乙型肝炎患者血清HBV DNA与肝组织HBV DNA及其肝脏炎症指标的相关性.方法 随机选择慢性乙肝患者51例做血液HBV DNA检测,并行肝穿刺取肝组织,分别进行肝组织HBV DNA检测及组织学检测.结果 慢性乙型肝炎患者肝组织HBV DNA检出率明显高于血清内,肝组织内HBV DNA含量明显高于血液内.结论 乙型肝炎患者血清HBV DNA与肝脏炎症程度无显著性差异,无相关性,不能真实反映肝组织内HBV DNA感染及复制情况.肝组织内HBV DNA与肝脏炎症程度有显著性差异,呈反向相关.     

慢性乙型肝炎患者肝组织HBV cccDNA、总HBV DNA与血清HBV DNA的相关性及其与临床的关系

目的探讨慢性乙型肝炎患者肝组织HBV共价闭合环状DNA（cccDNA）、肝组织总HBV DNA（HBV tDNA）与血清HBV DNA之间的相关性及其与临床的关系。方法 78例慢性乙型肝炎患者入选本研究。肝组织β- globinDNA、HBV cccDNA和HBV tDNA采用实时荧光定量PCR方法检测,平均每个肝细胞HBV cccDNA和HBV tDNA含量（拷贝/cell）=HBV cccDNA（实测值）/β-globin DNA（实测值）和HBV tDNA（实测值）/β3-globin DNA（实测值）,肝组织HBV cccDNA和HBV tDNA含量的计算单位定义为log₁₀拷贝/10⁶cell;采用实时荧光定量PCR、ELISA法检测血清HBVDNA和HBV标志物;采用免疫组织化学方法检测肝细胞中HBsAg和HBcAg的表达。统计分析采用pearson相关分析及t检验。结果 （1）肝组织HBV cccDNA与HBV tDNA定量呈正相关（r=0.696,P<0.001）;肝组织HBV cccDNA与血清HBV DNA定量呈正相关（r=0.304,P<0.01）;肝组织HBV tDNA与血清HBV DNA定量呈正相关（r=0.341,P<0.01）;（2）肝细胞内HBcAg定性检测阳性患者的血清HBV DNA定量明显高于阴性患者,且差异有统计学意义（P<0.05）;肝细胞内HBcAg定性检测阳性患者与阴性患者的肝组织HBV cccDNA和HBV tDNA定量差异均无统计学意义和（P均>0.05）;（3）肝细胞内HBsAg定性检测阳性患者的血清HBV DNA定量明显高于阴性患者,且差异有统计学意义（P<0.05）;肝细胞内HBsAg定性检测阳性患者与阴性患者的肝组织HBV cccDNA和HBV tDNA定量差异均无统计学意义（P>0.05）;（4）HBeAg（+）/抗-HBe（-）患者血清HBV DNA定量明显高于HBeAg（-）/抗-HBe（+）患者,且差异有统计学意义（P<0.05）;HBeAg（+）/抗-HBe（-）患者肝组织HBV cccDNA和HBV tDNA定量与HBeAg（-）/抗-HBe（+）患者比较差异均无统计学意义（均P>0.05）;（5）肝组织HBV cccDNA、HBV tDNA以及血清HBV DNA三者与肝脏炎症活动度及纤维化程度均无显著相关性（P>0.05）。结论 （1）血清HBV DNA定量结果并不一定能完全反映患者肝组织中HBV cccDNA和HBV tDNA含量,尤其在血清HBV DNA<500拷贝/mL时,肝组织中仍存在HBV cccDNA和HBV tDNA,且含量大小不等。（2）肝细胞内HBcAg定性检测阳性或者HBsAg定性检测阳性患者的血清HBV DNA定量均明显高于阴性患者;而两者的肝组织HBV cccDNA和HBV tDNA定量均没有显著差异;（3）HBeAg（+）/抗-HBe（-）患者血清HBV DNA定量明显高于HBeAg（-）/抗-HBe（+）患者,而两者的肝组织HBV cccDNA和HBV tDNA均没有显著差异;（4）肝组织HBV cccDNA、HBV tDNA及血清HBV DNA与肝脏炎症活动度和纤维化程度均无显著相关性。     

乙型肝炎病毒核酸定量检测与临床的关系

目的：探讨血清HBV DNA水平与HBV标志（HBV M）表现模式，肝功能状态，肝内炎症的关系。方法：对219例排除甲，丙，丁和戊型肝炎病毒的混合与重叠感染患者中的HBsAg阳性的慢性乙型肝炎患者进行肝穿刺病理检查。HBV DNA定量采用荧光定量PCR分析系统，HBV M采用ELISA法。结果：血清HBV DNA水平与HBVM表现模式有关，HBsAg与HBeAg的存在影响HBV DNA水平变化。在HBsAg阳性患者中，HBV DNA水平与Scheuer分级无明显相关。血清谷丙转氨酶水平与HBV DNA水平无明显相关。结论HBeAg和HBV DNA有明显的相关，抗－HBe阳性者病毒未完全停止复制，只是复制水平降低。单抗HBe阳性，单抗HBc阳性，抗HBs阳性和抗HBc阳性未检出HBV DNA，肝内炎症活动程度与血清HBV DNA水平无明显关系。     

外周血单核细胞HBV cccDNA水平与乙型肝炎抗病毒疗效

乙型肝炎病毒共价闭合环状DNA（HBV cccDNA）,是HBV前基因组RNA复制的原始合成模板,cccDNA的存在是病毒复制及感染得以维持的根源。本研究对10例CHB患者进行外周血单核细胞HBV cccDNA定量分析,同时检测血清HBV DNA水平,探讨外周血HBV DNA阴转、HBeAg脏清学转换与肝组织cccDNA水平的关系,为临床寻求一种简易有效的监测方法。     

慢性HBV感染者肝脏HBV cccDNA含量相关因素分析

目的探讨慢性HBV感染者肝组织中HBV cccDNA含量与血清病毒标志物、HBV DNA及肝脏病理分级的关系,为临床评价抗病毒治疗效果及疗程确定提供理论依据。方法以2007年5月-2008年2月住院的30例慢性HBV感染者为研究对象,应用实时荧光定量聚合酶链反应（RT-PCR）方法检测患者肝组织中HBV cccDNA、肝组织总HBV DNA（HBV tDNA）和血清HBVDNA,同时用化学发光免疫分析法检测HBsAg、HBeAg定量,分析感染者肝组织内HBV cccDNA与肝组织内HBV DNA、血清HBVDNA、HBsAg及HBeAg定量水平之间的关系,并比较肝组织中HBV cccDNA含量与肝脏病理炎症和纤维化分级的关系。采用Pear-son简单相关和Spearman等级相关法进行相关性分析。结果 30例慢性HBV感染者肝组织中均可检出HBV cccDNA,范围在3.15×103～1.06×107拷贝/mg;肝组织cccDNA定量与肝组织总HBV DNA定量呈正相关（r=0.375,P〈0.05）,与血清HBV DNA无相关性（r=0.174,P〉0.05）;肝组织中HBV cccDNA水平与血清HBsAg定量呈高度正相关（r=0.562,P〈0.001）,而与血清HBeAg定量无相关性（r=0.152,P〉0.05）。肝组织cccDNA定量与肝组织炎症活动度（G）及纤维化程度（S）无相关性（r=0.082,P〉0.05）。结论慢性HBV感染者肝组织内HBV cccDNA成稳定的中等水平复制;血清HBV DNA载量不能直接代表其肝组织中的HBV cccDNA水平;血清HBsAg定量可作为反映肝组织中HBV cccDNA水平的指标。     

乙肝病毒感染者血清HBeAg模式与HBVDNA定量关系的研究

探讨乙肝病毒感染者血清HBeAg模式与HBV DNA定量之间的关系。采用酶联免疫技术和实时荧光定 量PCR法,分别检测随机选择的80例乙肝病毒感染者血清HBeAg模式及HBV DNA定量。结果显示,当乙肝病毒 感染者血清HBV DNA含量为A、C级时,血清HBeAg模式与HBV DNA定量之间亦无相关性(P>0．05)。当乙肝病 毒携带者血清HBV DNA含量为B级时,HBeAg阳性模式HBV DNA含量显著高于HBeAg阴性者,血清HBeAg模式 与HBV DNA定量之间有显著相关性(r=0．28,t=1．19,P<0．05)。由此可推测,只有当机体内乙型肝炎病毒水平在 一定的范围内,机体的免疫应答能力才与乙型肝炎病毒水平相呼应。     

乙型肝炎病毒cccDNA定量与乙型肝炎临床及病理关系

目的探讨慢性乙型肝炎（CHB）肝组织HBVcccDNA定量与乙型肝炎的关系。方法分别采用荧光定量PCR、酶联免疫吸附分析法（ELISA）检测48例CHB肝组织HBVcccDNA定量、肝组织和血清HB VDNA定量、乙型肝炎病毒标志物。同时用链霉菌抗生素蛋白-过氧化物酶连接法（SP）检测肝细胞中HBcAg表达。分析肝组织HBVcccDNA与组织和血清HBV DNA、HBeAg、肝细胞内HBcAg水平及肝脏炎症活动度的关系.结果1．肝组织HBVcccDNA定量与组织和血清HBV DNA定量呈正相关（r=0．837，P〈0．001；r=0．627，P〈0．005）；2．肝组织HBV cccDNA定量与肝细胞内HBcAg半定量呈正相关（r=0．618，P〈0．005）；3．肝组织HBV cccDNA定量与肝脏炎症活动度尤明显相关（P〉0．05）：4．HBeAg阳性较抗-HBe阳性患者肝组织HBV cccDNA定量、肝组织和血清HBV DNA定量高（P〈0．05）。结论荧光定量PCR法检测肝组织HBV cccDNA定量是评价HBV复制最直接可靠的指标，在CHB的诊断和抗病毒治疗中有重要意义。但与肝组织炎症无明显相关。     

Changes of hepatitis B virus DNA in liver and serum caused by recombinant leukocyte interferon treatment: analysis of intrahepatic replicative hepatitis B virus DNA

Twenty patients with HBeAg-positive chronic liver disease were given large doses of recombinant leukocyte interferon for 4 weeks. Changes of hepatitis B virus DNA in livers and sera were analyzed by the molecular hybridization technique in paired biopsies obtained before and 2 weeks after treatment. Serum hepatitis B virus DNA was examined before, during and after the treatment until 4 weeks post-interferon. Analysis of hepatic hepatitis B virus DNA revealed species that appeared to represent various forms of replicative hepatitis B virus DNA, i.e., relaxed circular, linear, supercoiled and single-stranded hepatitis B virus DNA, respectively. No evidence of integration of hepatitis B virus DNA in genomic DNA was obtained. Of 15 cases which were positive for hepatic hepatitis B virus DNA before treatment and in which paired biopsies were obtained, hepatic hepatitis B virus DNA became negative in 4, decreased in 5 and unchanged in 6. Among several types of replicative viral DNA in liver tissue, supercoiled hepatitis B virus DNA tended to remain after other forms were reduced. A close correlation between hepatic and serum hepatitis B virus DNA was found in 37 liver biopsy samples and corresponding sera. These results indicate that interferon treatment reduces serum hepatitis B virus levels by inhibiting viral replication in the liver and that persistence or reappearance of hepatitis B virus in serum after interferon is associated with replication.     

乙型肝炎病毒前S1蛋白的检测及临床意义

目的分析前S1(Pre-S1)蛋白在诊断慢性乙型肝炎病毒复制及肝脏炎症及纤维化程度中的作用。方法收集慢性乙型肝炎患者103例,均经肝活组织检查证实。检测其Pre-S1蛋白,HBV标志物与HBV DNA,按病理学将肝组织炎症分为G1、G2、G3、G4级,纤维化程度分为S1、S2、S3、S4期。结果以HBV DNA定量103拷贝/毫升为诊断标准,Pre-S1蛋白与HBV DNA的符合率为88.1%;在肝组织不同炎症程度G1、G2、G3、G4级,HBV DNA与Pre-S1蛋白的符合率分别为88.1%、96.3%、92.9%、83.3%;在不同肝纤维化程度S1、S2、S3、S4期,符合率分别为84.2%、91.7%、90.9%、75.0%。结论Pre-S1蛋白对HBV复制的情况其检测灵敏性(与HBV DNA的相关性)和检测有效率都优于HBeAg检测;Pre-S1蛋白对肝脏炎症及纤维化程度的判断有较好的临床价值。     

Status of hepatitis B virus DNA in alcoholic liver disease: a study of a large urban population in the United States

Two reports have shown hepatitis B virus DNA in serum and liver tissue in alcoholic liver disease with negative serum HBsAg, suggesting a pathogenetic role for hepatitis B virus. We studied hepatitis B virus DNA in serum and liver from three groups of alcoholic patients; (Group 1) 50 patients without liver disease, (Group 2) 108 patients with alcoholic liver disease and (Group 3) five patients with alcoholic liver disease and hepatocellular carcinoma. Serum was tested for HBsAg, anti-hepatitis B core and anti-hepatitis B surface by radioimmunoassay and hepatitis B virus DNA by direct spot hybridization. Liver tissue from Groups 2 and 3 (113 patients) was examined by Southern blot analysis using 32P-labeled hepatitis B virus DNA clone from pBR322. Controls were 21 patients with chronic hepatitis B virus (14 patients with chronic active hepatitis, seven patients with cirrhosis and hepatocellular carcinoma). Serum and tissue were analyzed for hepatitis B virus DNA. Hepatitis B virus DNA was not detected in either serum or liver tissue in any of the 163 patients (Groups 1 to 3). In contrast, among the controls, hepatitis B virus DNA was present in the serum of 15 of the 21. Tissue DNA in those with chronic active hepatitis revealed 10/14 with free hepatitis B virus DNA, two with integrated sequences and two with no viral sequences. All seven patients with hepatocellular carcinoma had integrated viral DNA sequences in the tumor tissues. From these results, it appears that hepatitis B virus does not play a role in the pathogenesis of alcoholic liver disease.     

Long-term histologic and virologic outcomes of acute self-limited hepatitis B

The long-term impact of acute self-limited hepatitis B on the liver is unknown. Fourteen patients were recalled at a median of 4.2 years (range, 1.8-9.5 years) after the onset of acute hepatitis B. All showed clinical and serologic recovery with circulating hepatitis B surface antigen (HBsAg) clearance. Antibody to HBsAg (anti-HBs) had developed in 12 patients. Nine underwent liver biopsies at a median of 7.2 years, and histologic findings were evaluated using Ishak scores. Serum samples and frozen liver tissue were subjected to real-time detection polymerase chain reaction (PCR) to quantify the surface and X regions of the hepatitis B virus (HBV) genome and qualitative PCR to detect the covalently closed circular (ccc) HBV DNA replicative intermediate. Three patients had low levels of circulating HBV DNA up to 8.9 years after the onset, whereas both HBV DNA surface and X regions were found in the liver of all 9 patients examined, including 7 negative for serum HBV DNA. Liver viral loads assessed by the 2 regions showed a significant correlation (r = 0.946; P =.008), and all patients tested positive for ccc HBV DNA. Liver fibrosis and mild inflammation persisted in 8 patients. The fibrosis stage had relation to peak serum HBV DNA in the acute phase (P =.046) but not to liver viral loads in the late convalescent phase. In conclusion, occult HBV infection persists in the liver and is accompanied by abnormal liver histology for a decade after complete clinical recovery from acute self-limited hepatitis B.     

Evaluation of the in situ assay for HBV DNA: An observational real-world study in chronic hepatitis B

The visualization of intrahepatic hepatitis B virus (HBV) DNA by in situ hybridization (ISH) has uncovered some interesting aspects of HBV life cycle at the single-cell level. In the current study, we intend to evaluate the reliability and robustness of this assay in the real-world clinical scenario and its relationship with currently available clinical biomarkers in chronic hepatitis B (CHB) patients.In this cross-sectional study, 94 CHB patients and 10 patients with non-HBV related liver diseases were enrolled. Liver biopsies and routine histopathology analysis were performed. Intrahepatic HBV DNA and viral antigens (HBsAg and HBcAg) were detected by ISH and immunohistochemistry (IHC), respectively. The basic biochemical and virological parameters such as alanine transaminase, serum HBV DNA, and serum HBsAg were measured.The HBV DNA-ISH assay showed 55.8% (53/94 cases) positive rate in CHB patients, no false positive was found in non-HBV related hepatitis. The IHC of HBsAg and HBcAg showed a positive rate of 94.7% (89/94 cases) and 19.5% (17/87 cases), respectively. Quantification of HBV DNA-ISH signal showed a significant correlation with serum HBV DNA (r_s = 0.6223, P < .0001). In addition, the staining pattern of HBV DNA in situ in the context of collagen deposition informed the histopathological progression of chronic liver disease.The application of this ISH assay in evaluating intrahepatic viral replication in real-world CHB patients showed favorable performance. It can be a complementation to conventional liver histopathology examination and IHC detection of viral antigens. This methodology provides an intuitive assessment of virological and pathological state of CHB patients, and further supports clinical diagnosis and management.     

Hepatitis C and B viruses in hepatitis B surface antigen-negative hepatocellular carcinoma.

The relative role of hepatitis C virus and hepatitis B virus in hepatitis B surface antigen-negative hepatocellular carcinoma was evaluated by polymerase chain reaction in 31 patients from Taiwan. Twenty-one were positive for antibody to hepatitis C virus (group 1) and 10 were negative (group 2). Of the group 1 patients, hepatitis C viral RNA was detected in the serum by polymerase chain reaction in 16 and in the liver tissue in 17, whereas hepatitis B viral DNA was found in the liver tissue in only 4, and none were found in the serum. In group 2 patients, hepatitis C viral RNA was detected in the serum of 1 and in the liver tissue of another. In contrast, hepatitis B viral DNA was found in the serum of 4 patients and in the liver tissues of 5. It was concluded that hepatitis C virus plays an important role in hepatocarcinogenesis in hepatitis B surface antigen-negative patients in Taiwan, especially in those who had antibody to hepatitis C virus; in those without antibody to hepatitis C virus, hepatitis B virus might still be associated with the development of hepatocellular carcinoma in a significant proportion of such patients.     

Hepatitis B virus DNA in liver and peripheral blood mononuclear cells during reduction in virus replication

Changes in hepatitis B virus DNA in blood cells and liver were studied in 34 patients (12 controls, 22 under therapy). There were no basal differences in the presence of the viral genome in blood cells between treated and control patients. A significant decrease in the percentage of patients with viral genome in blood cells was observed in patients who lost this marker in serum. Replicative intermediates of the viral genome were observed in the basal liver samples from all patients. In patients who lost the viral DNA in serum, hepatitis B virus genome became undetectable in the second liver sample in all but two patients who had viral sequences integrated in the host genome. Replicative intermediates were found in all patients with serum viral DNA. The results of this study suggest that there may be a relation between the disappearance of hepatitis B virus DNA in liver and blood cells.     

Multicenter study of lamivudine therapy for hepatitis B after liver transplantation. Lamivudine Transplant Group

Hepatitis B after liver transplantation is often fatal, and no proven medical therapy exists for this condition. We chose to study the potential efficacy of lamivudine therapy for patients with chronic hepatitis B after liver transplantation. Fifty-two patients with chronic hepatitis B after liver transplantation were treated in an open label, multicenter study. Each had detectable hepatitis B virus (HBV) DNA in serum and 45 (87%) had detectable serum hepatitis B e antigen before treatment. Patients were treated for 52 weeks with lamivudine (100 mg daily). The primary endpoint was undetectability of HBV DNA; secondary endpoints included normalization of serum alanine transaminase (ALT) levels, disappearance of hepatitis B e antigen, and improvement in liver histology. After treatment, 60% of patients had undetectable HBV DNA by solution hybridization assay, 14 (31%) of the initially positive patients lost hepatitis B e antigen; hepatitis B surface antigen was undetectable in 3 (6%); and serum ALT levels normalized in 71%. Blinded histological assessments showed improvement in the histological activity index (P =.007 for periportal necrosis,.001 for lobular necrosis, and.013 for portal inflammation). YMDD variants of HBV, potentially associated with drug resistance, were detected in 14 (27%) of the patients. Repeat liver biopsies in 7 patients with the mutated virus were unchanged in 2, improved in 2, and worse in 3. We conclude that lamivudine is a potentially effective therapy for hepatitis B after liver transplantation.     

Characterization of serum activin-A and follistatin and their relation to virological and histological determinants in chronic viral hepatitis

BACKGROUND/METHODS: Hepatocyte proliferation in viral hepatitis is regulated by a number of growth factors. Activin-A inhibits hepatocyte DNA synthesis while follistatin, a potent activin-A antagonist, promotes liver regeneration. We report the first study of activin-A and follistatin in human viral hepatitis. Sera from 15 normal subjects, 22 hepatitis B and 47 hepatitis C patients were analysed for activin-A and follistatin and correlated with serological and histological markers of liver injury and with specific immunohistochemistry. RESULTS: All groups showed immunoreactivity for activin with hepatocyte localisation. Serum activin-A was significantly increased in viral hepatitis patients compared to controls, was greater in hepatitis B compared to hepatitis C, and correlated with serum aminotransferase and hepatitis B viral replication. A concurrent rise in serum follistatin was not observed in either group, but serum follistatin correlated inversely with hepatitis B DNA levels. Although hepatocyte apoptosis in hepatitis C and proliferation in both groups was significantly elevated compared to controls, there was no correlation with serum activin-A or follistatin. CONCLUSIONS: Activin-A and follistatin are constitutively expressed in human liver and serum concentrations are increased in viral hepatitis. Dysregulation of the activin/follistatin axis may be linked to hepatitis B replication but does not correlate with hepatocyte apoptosis.