Quantitative measurement of hepatitis B virus DNA in different areas of hepatic lobules in patients with chronic hepatitis B

Liver histology in chronic hepatitis B is marked by inflammatory infiltration involving the peripheral zones. The cause of such cellular infiltration remains unknown. The aim of the present study was to investigate the amounts of intrahepatic hepatitis B virus (HBV) DNA separately in the peripheral and central zones, using laser capture microdissection coupled with real-time quantitative polymerase chain reaction. Fourteen patients with chronic hepatitis B were included in the study. Liver biopsy samples were taken and hepatocytes were microdissected separately from peripheral and central zones. DNA was extracted from hepatocytes in each zone and evaluated the amounts of HBV-DNA. Immunohistochemical study for hepatitis B core antigen (HBcAg) was also performed. The amounts of total intrahepatic HBV-DNA in patients positive for hepatitis Be antigen (HBeAg) were greater than those in HBeAg-negative patients. There was no difference in HBV-DNA between the peripheral and central zones. Immunohistochemistry also showed that HBcAg-positive cells were distributed homogeneously in the hepatic lobules. In patients with peripherally predominant HBV-DNA, the serum alanine aminotransferase (ALT) level was lower than in patients with centrally predominant HBV-DNA. HBV-DNA was distributed homogeneously in the hepatic lobules. In patients with lower amounts of HBV-DNA in the peripheral zone, the serum ALT level tended to be higher than in other patients.     

Serum hepatitis B surface antigen is correlated with intrahepatic total HBV DNA and cccDNA in treatment‐naïve patients with chronic hepatitis B but not in patients with HBV related hepatocellular carcinoma

The aim of the study was to investigate correlations between intrahepatic hepatitis B virus total DNA, covalently closed circular DNA (cccDNA), and serum HBsAg in treatment‐naïve chronic hepatitis B and HBV related hepatocellular carcinoma (HCC). Liver tissues were taken from 42 HBV related HCC and 36 patients with chronic hepatitis B. A fraction of DNA extracted from liver tissue was digested with a plasmid‐safe ATP‐dependent DNase and used for HBV cccDNA detection. The remaining DNA was used for the detection of HBV total DNA and β‐globin, the latter of which is a housekeeping gene and quantified for normalization by real‐time PCR. Quantitation of serum HBsAg was performed by a chemiluminescence assay. Serum HBsAg had positive correlations with serum HBV DNA (r = 0.636, P < 0.001), intrahepatic HBV total DNA (r = 0.519, P = 0.001) and cccDNA (r = 0.733, P < 0.001) in 36 treatment‐naïve chronic hepatitis B, while HBsAg correlated poorly with DNA (r = 0.224, P = 0.210), intrahepatic total DNA and cccDNA in the tumor (r = 0.351, P = 0.031; r = 0.164, P = 0.324, respectively) and non‐tumor (r = 0.237, P = 0.152; r = 0.072, P = 0.667, respectively) liver tissues of 42 HCC. HBV cccDNA and total DNA were significantly higher in liver tissue from chronic hepatitis B than in tumor and non‐tumor of HCC (P < 0.001). Serum HBsAg and HBV DNA were also higher in chronic hepatitis B than in HCC (P < 0.001). It was concluded that levels of serum HBsAg and intrahepatic cccDNA and total DNA were significantly higher in chronic hepatitis B than in HCC, and significant correlations among them were observed in treatment‐naïve chronic hepatitis B but not in HCC. J. Med. Virol. 85:219–227, 2013. © 2012 Wiley Periodicals, Inc.     

慢性乙型肝炎患者HBV DNA与免疫球蛋白、补体的关系

目的 探讨慢性乙型肝炎患者HBV DNA检出情况与免疫球蛋白、补体的关系。方法 采用荧光定量-聚合酶链反应技术检测120例标本HBV DNA含量,同时速率散射比浊法检测免疫球蛋白IgG、IgA、IgM、补体C3、C4水平。结果 120例标本中,轻度、中度、重度慢性乙型肝炎患者HBV DNA含量差异有统计学意义。与对照组比较,轻、中、重度慢性乙型肝炎患者补体C3水平明显降低,重度慢性乙型肝炎患者补体C4水平也低于对照组;中、重度慢性乙型肝炎患者IgG、IgA含量明显增高,各组IgM差异无统计学意义。结论 FQ—PCR检测HBV DNA具有较高的灵敏度和特异性,结合免疫球蛋白、补体水平的检测结果对乙型肝炎的临床诊断与治疗方案的选择和疗效监测有重要意义。     

慢性乙肝患者血清HBV DNA载量与HBV-M、ALT、AST含量的关系研究

目的 分析慢性乙型肝炎患者血清HBV DNA与乙肝标志物(HBV-M)、肝功能ALT、AST的关系,以期对慢性乙肝患者的防治提供借鉴.方法 选取在2011年4月至2012年6月入住我院慢性乙肝患者100例为研究对象,搜集乙肝六项、肝功能等资料,探讨HBV DNA载量与HBV-M、ALT、AST含量的关系.结果 各阳性模式的病毒载量比较,大三阳病毒载量高于其他组,差异有统计学意义(P＜0.05),其他各组比较,差异无统计学意义(P＞0.05).阳性率比较,大三阳高于其他组,差异有统计学意义(P＜0.05),其他各组比较,差异无统计学意义(P＞0.05).血清HBV DNA载量与血清HBeAg滴度无相关性(r=0.683,P＞0.05);血清HBV DNA载量与HBsAg含量无相关性(r=-0.483,P＞0.05);血清HBV DNA载量与ALT无相关性(r=-0.157,P＞0.05),血清HBV DNA载量与AST水平也无相关性(r=0.062,P＞0.05).结论 大三阳血清HBV DNA载量、病毒阳性率均高于其他阳性模式;但血清HBV DNA定量值的高低与HBV-M、ALT、AST含量无相关性,所以,HBV DNA可反映HBV在外周血的复制情况,并不能反映肝脏损伤程度及预后.     

Association of cytokines with hepatitis B virus and its antigen

To investigate the characteristics of cytokines in patients with different HBV infection status and their correlation with HBV DNA, HBsAg, and HBeAg levels. Peripheral blood samples were collected from patients with chronic HBV infection in immune tolerance phase (IT), HBeAg-positive chronic hepatitis B (CHB), and acute hepatitis B (AHB) groups, and levels of cytokines were detected by Luminex technique, and analyzed by FLEXMAP 3D analyzer. The correlation between cytokines and HBV DNA load, HBsAg, HBeAg, and alanine aminotransferase (ALT) level in patients with chronic HBV infection was analyzed. In total 312 subjects (184 males and 128 females) were enrolled in the study. There were significant differences among IT, CHB, and AHB groups in Flt-3L value (P = .003; H = 12.312), IFN-γ (P = .001; H = 11.723), IL-10 (P = .001; H = 18.736), IL-17A ((P = .001; H = 12.735), and TGF-β1 (P = .001; Z = 48.571). IFN-α2 levels in CHB group were significantly higher than those in IT and AHB groups (15.24 vs 35.78 pg/mL, P = .000; Z = 3.727; 13.88 vs 35.78 pg/mL, P = .024; Z = −2.258. In CHB group, the levels of HBsAg and ALT were positively correlated with the levels of IL-10 (r = .173; P = .006; r = 0.176; P = .006, respectively), while HBeAg level was positively correlated with the IFN-α2 level (r = .153; P = .016). In AHB group, the HBsAg level was positively correlated with Flt-3L, IFN-α2, IL-10, and IL-6 (r = .402; P = .023; r = .436; P = .016; r = .524, P = .002; r = .405; P = .022, respectively). HBeAg level was positively correlated with IFN-γ and IL-17A levels (r = .400; P = .023; r = .373; P = .036, respectively), and ALT level was positively correlated with IL-6 levels (r = .367; P = .039). In either AHB or CHB group, HBV DNA load was only related to TGF-β level (r = .493; P = .004; r = −.218, P = 0.009 respectively). The correlation between Flt-3L and HBsAg (F = 7.422; P = .007); IL-17, IL-6, and HBeAg (F = 5.757; P = .017; F = 6.156; P = .014) were statistically significant. There was significant correlation between TGF-β2 and HBV DNA (F = 11.795; P = .001), and between ALT and HBsAg, HBV DNA (F = 26.089; P = .000; F = 4.724; P = .031). HBsAg, HBeAg, and HBV DNA were correlated with cytokines and ALT in patients with HBV infection. The level of IFN-α2 was significantly higher in patients with CHB. HBV DNA load was only correlated with the level of TGF-β in acute or CHB.     

Detection of hepatitis B viral DNA by polymerase chain reaction in patients with hepatitis B surface antigen

Hepatitis B virus (HBV) DNA was assayed using the polymerase chain reaction in serum samples of 116 hepatitis B surface antigen (HBsAg) carriers, including 30 positive for hepatitis B e antigen (HBeAg) and 86 negative for HBeAg. In the HBeAg-positive group, all were positive for HBV DNA. In the HBeAg-negative group, 80.2% were positive for HBV DNA (80.0% in the healthy carrier group, 90.0% in the chronic active liver disease group, and 69.2% in patients with cirrhosis). This study indicated that every HBeAg-positive carrier as well as the majority of HBeAg-negative carriers were infectious and, in the latter group, that viral replication is most active in patients with chronic active liver disease.     

Serum hepatitis B surface antigen levels correlate with high serum HBV DNA levels in patients with chronic hepatitis B: a cross-sectional study

Purpose: The hallmark of chronic hepatitis B (CHB) infection is the presence of hepatitis B surface antigen (HBsAg) positivity for at least 6 months. Recently, serum levels of HBsAg have been compared with serum HBV DNA as a surrogate marker to monitor CHB patients. However, data correlating these two markers are scarce. Hence, the present study was done to correlate HBV DNA with HBsAg in CHB patients. Materials and Methods: Consecutive patients of CHB were included. HBV DNA was measured by real-time polymerase chain reaction (PCR). Serum HBsAg was measured by Architect HBsAg. Results: Of the 198 patients enrolled, 166 fulfilled the inclusion criteria (mean age 43 ± 14 years, 87% males) and the median HBV DNA was 1.7 × 10³ (range 6.0–1.1 × 10⁸) IU/ml. Median HBsAg was 8.7 × 10³ (range 5.0–3.2 × 10⁵) IU/ml. Overall correlation between HBV DNA and HBsAg was weak but significant (Spearman ρ = 0.443, P < 0.01). Correlation in HBe antigen-positive group was better (ρ = 0.402, P < 0.01) in comparison to HBe antigen-negative group (ρ = 0.193 P = 0.05). Good correlation existed in treatment-naïve group (ρ = 0.538, P < 0.01). Correlation was regardless of normal or raised alanine transaminase (ALT). Eighty (48%) patients had high HBV DNA (≥2000 IU/ml). Correlation in high DNA group was significant (P < 0.01). The best cut-off of HBsAg for diagnosing high DNA is 3.36 ×10³ IU/ml. Conclusions: Serum HBsAg correlates with HBV DNA in CHB patients, especially in high serum HBV DNA, HBe antigen-positive and treatment-naïve group. HBsAg levels can be used for predicting high serum HBV DNA levels.     

Comparison of the COBAS TaqMan HBV test with the COBAS Amplicor monitor test for measurement of hepatitis B virus DNA in serum

Quantitation of low hepatitis B virus (HBV) DNA levels in patients with chronic hepatitis B is important for monitoring natural history of disease and treatment efficacy. This study aimed to compare the quantitation range and analytical sensitivity of the newly developed COBAS TaqMan HBV test (TaqMan test) with the COBAS Amplicor HBV Monitor Test (Amplicor test), using the Eurohep HBV reference plasma and serum samples from patients. Serial dilutions (2.7x10(1)-2.7x10(8) copies/ml) of the Eurohep HBV reference plasma and 50 serum samples from chronic hepatitis B patients were tested by both assays. The TaqMan test could detect seven (2.7x10(2)-2.7x10(8) copies/ml) of eight dilutions of the reference plasma, while the Amplicor test could only detect three of them (2.7x10(3)-2.7x10(5) copies/ml). The HBV DNA values measured by the TaqMan test correlated very well with the theoretical Eurohep standard values (r=0.998, P<0.001). There were good correlations between the HBV DNA levels measured by the two assays on both the Eurohep reference plasma (r=0.993, P<0.001) and serum samples from patients (r=0.904, P<0.001). Compared to the Amplicor test, the TaqMan test had a higher sensitivity (50 vs. 300 copies/ml), shorter assay time (6 vs. 10 hr), and wider dynamic range (8 vs. 3 logs), and was more cost-effective in a clinical setting. These data indicate that the TaqMan test is an excellent tool for HBV DNA quantitation.     

Evaluation of a new radioimmunoassay for detecting hepatitis B e antigen and its antibody

A new commercially available radioimmunoassay (RIA) (Abbott-HBe^TM) was used for determination of hepatitis B e-antigen (HBeAg) and its antibody (anti-HBe). Serial serum samples from 20 transiently HBsAg-positive patients with acute hepatitis were tested. In nearly all patients HBeAg could be shown for a short period with subsequent development of antiHBe. From 24 chronic HBsAg carriers serial serum samples collected during several years were tested. In 18 of 19 initially HBeAg-positive patients the HBeAg was lost after 6 months to 6 years; in 14 anti-HBe developed. A correlation was seen between the seroconversion and normalization of elevated alanine transferase levels. From another 22 chronic HBsAg carriers single serum samples were assayed. These samples were selected because neither HBeAg nor anti-HBe could be detected by the immunodiffusion (ID) technique. They had previously been examined for HBV-associated DNA-polymerase activity. In 20 patients HBeAg or anti-HBe could be detected by RIA. Those who were DNA-polymerase negative had anti-HBe, and 3 of 4 who were DNA-polymerase positive had HBeAg. When compared to earlier results by ID in these materials a higher frequency of HBeAg and, in particular, anti-HBe was detected by the RIA. By this test, HBeAg or anti-HBe was found in nearly all patients. The usefulness of HBeAg/anti-HBe in the evaluation of infectivity and prognosis in hepatitis B has been limited by the low sensitivity of the earlier test systems. Thus, this new RIA is a valuable addition to the diagnostic tests for patients with hepatitis B.     

Characteristics of lamivudine-resistant hepatitis B virus (HBV) strains with and without breakthrough hepatitis in patients with chronic hepatitis B evaluated by serial HBV full-genome sequences

Lamivudine therapy often causes breakthrough of hepatitis B virus (HBV) DNA and breakthrough hepatitis. The aim of this study was to determine the viral factors that relate to HBV-DNA breakthrough with and without breakthrough hepatitis. Among 82 patients with chronic hepatitis B (CHB) who received lamivudine at a dose of 100 mg daily for more than 24 months, 23 patients had HBV-DNA breakthrough induced by a lamivudine-resistant mutant. Of these 23 patients, 16 had breakthrough hepatitis and 7 had only HBV-DNA breakthrough. Serial HBV-DNA full-genome sequences during therapy were examined in 10 (7 had breakthrough hepatitis and 3 did not) of these 23 patients by direct sequencing. Mutations in the S region were examined by cloning in representative patients. There were no significant differences in the baseline clinical backgrounds and virus marker between patients with and without breakthrough hepatitis. The HBV amino acid substitutions at breakthrough hepatitis were identical to those at HBV-DNA breakthrough. Cloning analysis revealed that monoclonal mutational strain appeared at breakthrough and no such mutations existed at baseline. Regarding HBV amino acid substitutions in the polymerase region, S region, X region, and precore-core region with breakthrough compared to baseline, there was no significant differences of the numbers of amino acid substitution between breakthrough hepatitis and non-breakthrough hepatitis. There were no common amino acid changes in patients with breakthrough hepatitis. Although monoclonal lamivudine-resistant strain emerged at HBV-DNA breakthrough in patients with CHB, there were no common amino acid changes, suggesting viral factor may have insignificant role in breakthrough hepatitis.     

慢性乙型肝炎患者NK细胞水平与HBV DNA的关系

我们将HBV DNA阴性的慢性乙型肝炎(CHB)患者(50例)与HBV DNA阳性的CHB患者(128例)比较NK细胞水平,并进行肝功能的比较,探讨CHB患者的NK细胞水平与HBV DNA的关系.     

Performance evaluation of ExiStation HBV diagnostic system for hepatitis B virus DNA quantitation

The performance of a recently developed real-time PCR system, the ExiStation HBV diagnostic system, for quantitation of hepatitis B virus (HBV) in human blood was evaluated. The detection limit, reproducibility, cross-reactivity, and interference were evaluated as measures of analytical performance. For the comparison study, 100 HBV-positive blood samples and 100 HBV-negative samples from Korean Blood Bank Serum were used, and the results of the ExiStation HBV system showed good correlation with those obtained using the Cobas TaqMan (r² = 0.9931) and Abbott real-time PCR systems (r² = 0.9894). The lower limit of detection was measured as 9.55 IU/mL using WHO standards and the dynamic range was linear from 6.68 to 6.68 × 10⁹ IU/mL using cloned plasmids. The within-run coefficient of variation (CV) was 9.4%, 2.1%, and 1.1%, and the total CV was 11.8%, 3.6%, and 1.7% at a concentration of 1.92 log₁₀ IU/mL, 3.88 log₁₀ IU/mL, and 6.84 log₁₀ IU/mL, respectively. No cross-reactivity or interference was detected. The ExiStation HBV diagnostic system showed satisfactory analytical sensitivity, excellent reproducibility, no cross-reactivity, no interference, and high agreement with the Cobas TaqMan and Abbott real-time PCR systems, and is therefore a useful tool for the detection and monitoring of HBV infection.     

The role of the hepatitis B virus infection in patients with chronic hepatitis in argentina

Over a seven-year period, we monitored 221 patients with chronic hepatitis from two medical centers. By using the counterelectrophoresis (CEP) test to detect the presence of HBsAg and anti-HBc, or both, we established that 87.7% of them had hepatitis B infection. Serum specimens originally found negative for HBsAg by CEP were further tested by reversed passive hemagglutination (RPH), and those originally found negative for anti-HBc by CEP were further tested by radioimmunoassay (RIA). Five patients were anti-HBc-positive and HBsAg-negative. No sex predominance was observed, but HBsAg incidence increased with increasing age. The HBeAg antigen was detected in 46.8% of the 161 cases tested for it; the most frequent subtype found was adw (63.7%). The present findings indicate that HBV infection largely contributes to the development of chronic hepatitis in Argentinian patients.     

Preponderance of hepatitis B virus genotype B contributes to a better prognosis of chronic HBV infection in Okinawa,Japan

The present study was designed to examine the distribution of hepatitis B virus (HBV) genotypes among patients at various stages of chronic liver disease type B in Okinawa Prefecture, Japan, where the prevalence of hepatitis B surface antigen is the highest in Japan despite the lowest mortality rate from primary liver cancer. Serum samples from 227 HBV carriers were determined for HBV genotype by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Five of 227 sera were negative for HBV DNA by nested PCR and were excluded from the genotype analysis. Genotype B was predominant in asymptomatic carriers (45/67, 67%), whereas genotype C was predominant in chronic liver disease: 49% (50/103) in patients with chronic hepatitis, 63% (20/32) in patients with cirrhosis, and 60% (12/20) in patients with hepatocellular carcinoma. The distribution of genotype B decreased with increasing liver disease severity. However, this tendency was seen among patients aged less than 50 years old, whereas the prevalence of genotype B was similar among carriers with various liver diseases who were older than age 50. In conclusion, HBV genotype B was prevalent and less frequent among patients with advanced liver disease, particularly in patients aged less than 50 years. These findings suggest that the preponderance of genotype B is responsible for the low mortality rate of primary liver cancer associated with HBV seen in Okinawa Prefecture, despite having the highest HBV carrier rate in Japanese.     

Outcome in Caucasian patients with hepatitis B e antigen negative chronic infection: A long-term observational cohort study

Sensitive polymerase chain reaction assays to measure hepatitis B virus (HBV) DNA became only available the last decade. Hence, the long-term outcome of Caucasian patients in Western Europe with hepatitis B e antigen (HBeAg)-negative chronic infection, especially with a baseline HBV DNA level ⩾2000 IU/mL, is still unclear. Out of a cohort of 1936 chronic HBV patients, 413 Caucasian individuals were identified with HBeAg-negative chronic infection, defined as persistently normal alanine aminotransferase (ALT) levels and HBV DNA levels <20 000 IU/mL. During a mean follow-up of 12 years, 366 (88.6%) maintained an HBeAg-negative chronic infection status, whereas 25 (6.1%) developed chronic active hepatitis (CAH). In total, Nine of these 25 CAH cases were related to immunosuppression. In total, 22 (5.3%) individuals had ALT > 2 × upper limit of normal due to non-HBV-related causes. The cumulative probability of spontaneously developing CAH after 10 years was almost exclusively seen in patients with baseline HBV DNA level ⩾2000 IU/mL (11.7% vs 1.2%; P < .001). Advanced liver disease developed significantly more in patients with baseline HBV DNA level ⩾2000 IU/mL (5.2% vs 1.5%; P = .018) and occurred especially in patients with obesity (16.7% vs 4.2%; P = .049). The incidence of hepatocellular carcinoma was 0.0%. Caucasian patients with HBeAg-negative chronic infection and baseline HBV DNA level <2000 IU/mL have an excellent long-term prognosis in the absence of immunosuppressive therapy. However, patients with baseline HBV DNA level ⩾2000 IU/mL are at risk to develop advanced liver disease.     

Quantitative relationship between HBeAg and DNA polymerase activity in sera from patients with chronic active hepatitis during a three-month period

In eight patients with biopsy-confirmed chronic active hepatitis (CAH), hepatitis B e antigen (HBeAg) levels and DNA polymerase (DNA-P) activity were assayed three times a week for six weeks and once a week for another six weeks. HBeAg levels were rather constant, whereas DNA-P activity fluctuated. No correlation was observed between the quantities of HBeAg and DNA-P activity. An unexpected fluctuation in DNA-P activity was noted in all patients after an influenza vaccination.