Hepatitis B virus (HBV) infection is a major public health problem worldwide. The aim of this study was to document the dynamics of HBV viral load during the follow-up of chronic hepatitis B patients at the Saint Cami...Hepatitis B virus (HBV) infection is a major public health problem worldwide. The aim of this study was to document the dynamics of HBV viral load during the follow-up of chronic hepatitis B patients at the Saint Camille Hospital in Ouagadougou (HOSCO) from 2017 to 2021. This descriptive retrospective study was carried out in the Hepato-Gastro-Enterology Department of HOSCO and focused on patients who were undergoing treatment for chronic viral hepatitis B. A total of 260 cases of chronic hepatitis B were included in the study. The most affected age group was 21 to 30 years, accounting for 48.08% of the cases. Lifestyle factors included alcohol consumption (3.08%) and tobacco use (2.69%). Major risk factors for transmission included lack of vaccination (98.46%), family history of HBV infection (68.00%) and engagement in high-risk activities (28.00%). Patients requiring treatment were prescribed Tenofovir 300 mg tablets. FibroScan<sup>®</sup> showed the presence of stage F3-F4 fibrosis (2.14%) and S3 steatosis (13.33%). After one year of follow-up, 6.92% of patients achieved an undetectable viral load with normalized transaminase levels. The majority of other patients had a detectable viral load but below 20,000 IU/mL. The prevalence of viral hepatitis B remains significant worldwide. Although effective and well-monitored treatment can lead to undetectable viremia, prevention remains the most effective strategy for successful management of this disease.展开更多
AIM: To conduct a retrospective study in 400 chronic hepatitis B patients in order to identify hepatitis B viral factors associated with complications of liver disease or development of hepatocellular carcinoma. METH...AIM: To conduct a retrospective study in 400 chronic hepatitis B patients in order to identify hepatitis B viral factors associated with complications of liver disease or development of hepatocellular carcinoma. METHODS: The mean follow-up time was 83.6 ± 39.6 mo. Alpha-fetoprotein test and abdominal ultrasound were used for cancer surveillance. Hepatitis B basal core promoter mutants, precore mutants, genotypes, hepatitis B viral DNA (HBV DNA) level and hepatitis B e antigen (HBeAg) were measured. Univariate analysis and logistic regression were used to assess odds ratios for viral factors related to liver deaths and hepatocellular carcinoma development. RESULTS: During follow-up, 38 patients had liver deaths not related to hepatocellular carcinoma. On multivariate analysis, older age [odds ratio: 95.74 (12.13-891.31), P 〈 0.0001], male sex [odds ratio: 7.61 (2.20-47.95); P = 0.006], and higher Iogzo HBV DNA [odds ratio: 4.69 (1.16-20.43); P 〈 0.0001] were independently predictive for these liver related deaths. Also, 31 patients developed hepatocellular carcinoma. Multivariate analysis showed that older age [odds ratio: 26.51 (2.36-381.47); P = 0.007], presence of precore mutants [odds ratio: 4.23 (1.53-19.58), P = 0.02] and presence of basal core promoter mutants [odds ratio: 2.93 (1.24-7.57); P = 0.02] were independent predictors for progression to hepatocellular carcinoma. CONCLUSION: Our results show that high levels of baseline serum HBV DNA are associated with non- hepatocellular carcinoma-related deaths of liver failure, while genetic mutations in the basal core promoter and precore regions are predictive for development of hepatocellular carcinoma.展开更多
AIM: To develop a real-time PCR for detecting hepatitis B virus-(HBV) DNA based on TaqMan technology using a new MGB probe.METHODS: Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA s...AIM: To develop a real-time PCR for detecting hepatitis B virus-(HBV) DNA based on TaqMan technology using a new MGB probe.METHODS: Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA standard for quantitative analysis. A TaqMan-MGB probe between primers for amplification was designed to detect PCR products. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured.RESULTS: The detection limit of the assay for HBV DNA was 1 genome equivalent per reaction. A linear standard curve was obtained between 100 and 109 DNA copies/reaction (r>0.990). None of the negative control samples showed false-positive reactions in duplicate. HBV DNA was detected in 100% (50/50) of HBV patients with HbeAg, and in 72.0% (36/50) with HBsAg, HBeAb and HBcAb. The coefficient of variation for both intra- and inter-experimental variability demonstrated high reproducibility and accuracy.CONCLUSION: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.展开更多
Hepatitis B virus (HBV) infection is a global public health problem that concerns 350 million people worldwide. Individuals with chronic hepatitis B (CriB) are at increased risk of developing liver cirrhosis, hepa...Hepatitis B virus (HBV) infection is a global public health problem that concerns 350 million people worldwide. Individuals with chronic hepatitis B (CriB) are at increased risk of developing liver cirrhosis, hepatic de-compensation and hepatocellular carcinoma. To maintain undetectable viral load reduces chronic infection complications. There is no treatment that eradicates HBV infection. Current drugs are expensive, are associated with adverse events, and are of limited efficacy. Current guidelines try to standardize the clinical practice. Nevertheless, controversy remains about management of asymptomatic patients with CriB who are hepatitis B e antigen (HBeAg)-positive with normal alanine aminotransferase, and what is the cut-off value of viral load to distinguish HBeAg- negative CriB patients and inactive carriers. We discuss in detail why DNA level alone is not sufficient to begin treatment of CriB.展开更多
AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted...AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 10^4/mL, 6.3 × 10^2/mL and 1.6 × 10^3/ mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 10^9/mL, 2.08 × 10^6/mL and 4.40 × 10^7/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10^S/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.展开更多
AIM To investigate whether hepatitis viral DNA load at 24 wk of treatment predicts response at 96 wk in patients with chronic hepatitis B.METHODS A total of 172 hepatitis B envelope antigen(HBe Ag)-positive chronic he...AIM To investigate whether hepatitis viral DNA load at 24 wk of treatment predicts response at 96 wk in patients with chronic hepatitis B.METHODS A total of 172 hepatitis B envelope antigen(HBe Ag)-positive chronic hepatitis B patients who received initial treatment at 16 tertiary hospitals in Hunan Province, China were enrolled in this study. All patients received conventional doses of lamivudine and adefovir dipivoxil, telbivudine, entecavir dispersible tablets, or entecavir tablets for 96 wk. Patients who used other antiviral drugs or antitumor and immune regulation therapy were excluded. Patients were stratified into three groups according to their viral DNA load at 24 wk: < 10 IU/m L(group 1), 10-103 IU/m L(group 2), and > 103 IU/m L(group 3). Correlations of 24-wk DNA load with HBe Ag negative status and HBe Ag seroconversion at 96 wk were analyzed. Receiver operating characteristic curve analysis was used to test the predictive value of the HBV DNA load at 24 wk for long-term response.RESULTS The rates of conversion to HBe Ag negative status and HBe Ag seroconversion rates were 53.7% and 51.9%, respectively, in group 1; 35.21% and 32.39% in group 2; and 6.38% and 6.38% in group 3. The receiver operating characteristic curves for the three subgroups revealed that the lowest DNA load(< 10 IU/m L) was better correlated with response at 96 wk than a higher DNA load(10-103 IU/m L). Nested PCR was used for amplifying and sequencing viral DNA in patients with a viral DNA load > 200 IU/m L at 96 wk; resistance mutations involving different loci were present in 26 patients, and three of these patients had a viral DNA load 10-103 IU/m L at 96 wk. CONCLUSION Hepatitis B viral DNA load at 24 wk of antiviral treatment in patients with chronic hepatitis B is a predictor of the viral load and response rate at 96 wk.展开更多
In DNA-guided hepatitis B treatment, viral load is insufficient, and requires other viral markers for treatment of hepatitis B patients as in patients with acute exacerbation of chronic hepatitis B, end-stage renal di...In DNA-guided hepatitis B treatment, viral load is insufficient, and requires other viral markers for treatment of hepatitis B patients as in patients with acute exacerbation of chronic hepatitis B, end-stage renal disease on dialysis, human immunodeficiency virus co-infected patients. There are exceptions to this rule: a residual level hepatitis B virus (HBV) DNA at 24 wk predicts beneficial outcome and reduced resistance at i year. The genotypic viral resistance to antiviral agents and occult HBV infection can be determined by HBV-DNA levels.展开更多
To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA (eccDNA) of duck hepatitis B virus (DHBV), a pair of primers was designed from both sides of ...To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA (eccDNA) of duck hepatitis B virus (DHBV), a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5' end and Tamra at its 3' end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln(x) + 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.展开更多
AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of dir...AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of direct sequencing of PCR amplified products, a 16-mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASOn) directed against the HBV U5-like region was synthesized and then linked with one live-targeting ligand, the galactosylated poly-L-lysine. Their effect on the expression of HBV gene was observed using the 2.2.15 cells.RESULTS HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw1 by sequencing so that antisense oligonucleotides could bind specifically to the target sequence through base piring. Under the same experimental conditions, the inhibitory rates of PS-ASON to HBsAg and HBeAg were 70% and 58% at a concentration of 10μmol/L, while by ligand-PS-ASON they were 96% and 82%, the amount of HBV DNA in cultured supernatant and cells was reduced significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotides had no cytotoxicity.CONCLUSION Antisense oligonucleotides complexed by the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors, resulting in supecific inhibition of HBV gene expression and replication.展开更多
AIM:To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. METHODS: The reference sera of the Chinese Na...AIM:To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. METHODS: The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS: The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION: The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.展开更多
AIM: To determine whether new cut-off values for aianine aminotransferase (ALT) and baseline hepatitis B virus (HBV) DNA levels better differentiate HBeAg(-) chronic hepatitis B (CriB) patients from inactive ...AIM: To determine whether new cut-off values for aianine aminotransferase (ALT) and baseline hepatitis B virus (HBV) DNA levels better differentiate HBeAg(-) chronic hepatitis B (CriB) patients from inactive chronic carriers. METHODS: Ninety-one patients [32 HBeAg(+) CriB, 19 inactive carriers and 40 HBeAg(-) CriB] were followed up for 2 years and were tested for HBV DNA levels by a PCR-based assay. ALT was tested twice during the last 6 mo using new cut-off values: ULN (upper limit of normal) 30 IU/L for males, 19 IU/L for females. Diagnostic accuracy, sensitivity, specificity, positive and negative predictive values were calculated by discriminant analysis. RESULTS: When using the revised ALT cut-off values, the lowest optimal HBV DNA level that differentiated HBeAg(-) CHB patients from inactive carriers was 50000 copies/mL. The diagnostic accuracy of HBV DNA to determine inactive carriers with a cut-off of 50000 copies/mL was similar to the previously recommended cut-off of 100000 copies/mL (91%). HBV DNA levels were lower than the cut-off value in 95% of inactive carriers and in 28% of HBeAg(-) CHB patients. With ALT 〈 30 IU/L in men and 〈 19 IU/L in women and HBV DNA levels 〈 100000 copies/mL, the risk of CHB is 5%. On the other hand, if ALT values were 〉 30 IU in men and 〉 19 IU in women and baseline HBV DNA levels were 〉 100000 copies/mL, the risk is 86%. CONCLUSION: New cut-off values for ALT together with HBV DNA levels proposed by AASLD (American Association for the Study of Liver Diseases) and NIH (National Institute of Health) consensus seem appropriate to characterize inactive carriers.展开更多
AIM:To estimate the amount of apoptosis among healthy HBsAg carriers,patients with chronic HBV infection treated wibh lamivudine and patients with chronic HCV infection treated with interferon alpha and ribavirin.Acti...AIM:To estimate the amount of apoptosis among healthy HBsAg carriers,patients with chronic HBV infection treated wibh lamivudine and patients with chronic HCV infection treated with interferon alpha and ribavirin.Activity of apoptosis was evaluated by serum sFas/sFasL concentration measurement. Moreover dependence between apoptosis and HBV-DNA or HCV-RNA levels was studied. METHODS:Eighty-six persons were included into study:34 healthy HBsAg carders,33 patients with chronic HBV infecl^on and 19 patients with chronic HCV infection.Serum levels of sFas/sFasL were measured by ELISA assay.HBV-DNA and HCV-RNA were measured by RT-PCR assay.Levels of sFas/sFasL were determined before and 2 and 12 wk after therapy in patients with chronic hepatitis B and C infection. HBV-DNA or HCV-RNA was detected before treatment and 6 mo after treatment. RESULTS:Twenty-four (71%) healthy HBsAg carders showed HBV-DNA over 10~5/mL,which was comparable to the patients with chronic hepatitis B.independently from HBV-DNA levels, the concentration of sFas among healthy HBsAg carders was comparable to healthy persons.Among patients with chronic hepatitis B and C,the concentration of sFas was significantly higher in comparison to healthy HBsAg carriers and healthy persons.In chronic hepatitis B patients the concentration of sFas was decreased during lamivudine treatment.Among chronic hepatitis C patients the concentration of sFas was increased during IFN alpha and ribavirin treatment,sFasL was not detected in control group.Furbhermore sFasL occurred more frequently in chronic hepatitis C patients in comparison to chronic hepatitis B patients. CONCLUSION:There are no correlations between apoptosis and HBV-DNA levels.However ther is an association between apoptosis and activity of inflammation in patients with chronic HBV infection.Apoptosis can be increased in patients with chronic hepatitis C by effective treatment which may be a result of apoptosis stimulation by IFN-α.展开更多
INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant ...INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].展开更多
AIM To investigate and compare the analytical and clinical performance of Tian Long automatic hypersensitive hepatitis B virus(HBV) DNA quantification system and Roche CAP/CTM system.METHODS Two hundred blood samples ...AIM To investigate and compare the analytical and clinical performance of Tian Long automatic hypersensitive hepatitis B virus(HBV) DNA quantification system and Roche CAP/CTM system.METHODS Two hundred blood samples for HBV DNA testing, HBV-DNA negative samples and high-titer HBV-DNA mixture samples were collected and prepared. National standard materials for serum HBV and a worldwide HBV DNA panel were employed for performance verification. The analytical performance, such as limit of detection, limit of quantification, accuracy, precision, reproducibility, linearity, genotype coverage and cross-contamination, was determined using the Tian Long automatic hypersensitive HBV DNA quantification system(TL system). Correlation and Bland-Altman plot analyses were carried out to compare the clinical performance of the TL system assay and the CAP/CTM system. RESULTS The detection limit of the TL system was 10 IU/m L, and its limit of quantification was 30 IU/m L. The differences between the expected and tested concentrations of the national standards were less than ± 0.4 Log10 IU/m L, which showed high accuracy of the system. Results of the precision, reproducibility and linearity tests showed that the multiple test coefficient of variation(CV) of the same sample was less than 5% for 102-106 IU/m L; and for 30-108 IU/m L, the linear correlation coefficient r2 = 0.99. The TL system detected HBV DNA(A-H) genotypes and there was no cross-contamination during the "checkerboard" test. When compared with the CAP/CTM assay, the two assays showed 100% consistency in both negative and positive sample results(15 negative samples and 185 positive samples). No statistical differences between the two assays in the HBV DNA quantification values were observed(P > 0.05). Correlation analysis indicated a significant correlation between the two assays, r2 = 0.9774. The Bland-Altman plot analysis showed that 98.9% of the positive data were within the 95% acceptable range, and the maximum difference was-0.49.CONCLUSION The TL system has good analytical performance, and exhibits good agreement with the CAP/CTM system in clinical performance.展开更多
PCR technique was used to detect HBV DNA in liver tissue samples for study of the prevalence of HBV DNA in tumorous and nearby nontumorous liver tissues from 16 hepatocellular carcinoma(HCC) patients.Three primer pair...PCR technique was used to detect HBV DNA in liver tissue samples for study of the prevalence of HBV DNA in tumorous and nearby nontumorous liver tissues from 16 hepatocellular carcinoma(HCC) patients.Three primer pairs, Sl/S2, C1/C2 and X1/X2, used in this study were selected from S region, pre C and C region,and X region of HBV DNA, respectively. The detecting with agarose gel electrophoresis and ethidium bromide staining(PCR-EB) was 10-2 pg, and that with Southern blot hybridization was 10-6 pg. The positive rates in amplification of HBV DNA by S, C and X region primer pairs in liver samples were 43.8%(14/32), 71.9%(23/32)and 71.9%(23/32), respectively. There was significant difference between the positive rates in amplification with S primer and with C Primer(P<0.05), but no significant difference between the C primer and the X primer(P<0.05), and between the S primer and the X primer(0.10>P>0.05). HBV DNA fragments were detected in the livers from all 16 cases. The results indicated that X gene integration inducing hepatocellular carcinogenesis and arrest of C gene expression causing escape from host immune surveillance are the possible mechanisms of HCC development in patients with perisistent HBV infection.展开更多
文摘Hepatitis B virus (HBV) infection is a major public health problem worldwide. The aim of this study was to document the dynamics of HBV viral load during the follow-up of chronic hepatitis B patients at the Saint Camille Hospital in Ouagadougou (HOSCO) from 2017 to 2021. This descriptive retrospective study was carried out in the Hepato-Gastro-Enterology Department of HOSCO and focused on patients who were undergoing treatment for chronic viral hepatitis B. A total of 260 cases of chronic hepatitis B were included in the study. The most affected age group was 21 to 30 years, accounting for 48.08% of the cases. Lifestyle factors included alcohol consumption (3.08%) and tobacco use (2.69%). Major risk factors for transmission included lack of vaccination (98.46%), family history of HBV infection (68.00%) and engagement in high-risk activities (28.00%). Patients requiring treatment were prescribed Tenofovir 300 mg tablets. FibroScan<sup>®</sup> showed the presence of stage F3-F4 fibrosis (2.14%) and S3 steatosis (13.33%). After one year of follow-up, 6.92% of patients achieved an undetectable viral load with normalized transaminase levels. The majority of other patients had a detectable viral load but below 20,000 IU/mL. The prevalence of viral hepatitis B remains significant worldwide. Although effective and well-monitored treatment can lead to undetectable viremia, prevention remains the most effective strategy for successful management of this disease.
文摘AIM: To conduct a retrospective study in 400 chronic hepatitis B patients in order to identify hepatitis B viral factors associated with complications of liver disease or development of hepatocellular carcinoma. METHODS: The mean follow-up time was 83.6 ± 39.6 mo. Alpha-fetoprotein test and abdominal ultrasound were used for cancer surveillance. Hepatitis B basal core promoter mutants, precore mutants, genotypes, hepatitis B viral DNA (HBV DNA) level and hepatitis B e antigen (HBeAg) were measured. Univariate analysis and logistic regression were used to assess odds ratios for viral factors related to liver deaths and hepatocellular carcinoma development. RESULTS: During follow-up, 38 patients had liver deaths not related to hepatocellular carcinoma. On multivariate analysis, older age [odds ratio: 95.74 (12.13-891.31), P 〈 0.0001], male sex [odds ratio: 7.61 (2.20-47.95); P = 0.006], and higher Iogzo HBV DNA [odds ratio: 4.69 (1.16-20.43); P 〈 0.0001] were independently predictive for these liver related deaths. Also, 31 patients developed hepatocellular carcinoma. Multivariate analysis showed that older age [odds ratio: 26.51 (2.36-381.47); P = 0.007], presence of precore mutants [odds ratio: 4.23 (1.53-19.58), P = 0.02] and presence of basal core promoter mutants [odds ratio: 2.93 (1.24-7.57); P = 0.02] were independent predictors for progression to hepatocellular carcinoma. CONCLUSION: Our results show that high levels of baseline serum HBV DNA are associated with non- hepatocellular carcinoma-related deaths of liver failure, while genetic mutations in the basal core promoter and precore regions are predictive for development of hepatocellular carcinoma.
文摘AIM: To develop a real-time PCR for detecting hepatitis B virus-(HBV) DNA based on TaqMan technology using a new MGB probe.METHODS: Plasmid containing the sequence of X gene (1414-1744 nt) was constructed as HBV-DNA standard for quantitative analysis. A TaqMan-MGB probe between primers for amplification was designed to detect PCR products. The interested sequence contained in the plasmid and in clinical specimens was quantitatively measured.RESULTS: The detection limit of the assay for HBV DNA was 1 genome equivalent per reaction. A linear standard curve was obtained between 100 and 109 DNA copies/reaction (r>0.990). None of the negative control samples showed false-positive reactions in duplicate. HBV DNA was detected in 100% (50/50) of HBV patients with HbeAg, and in 72.0% (36/50) with HBsAg, HBeAb and HBcAb. The coefficient of variation for both intra- and inter-experimental variability demonstrated high reproducibility and accuracy.CONCLUSION: Real-time PCR based on TaqMan-MGB probe technology is an excellent method for detection of HBV DNA.
文摘Hepatitis B virus (HBV) infection is a global public health problem that concerns 350 million people worldwide. Individuals with chronic hepatitis B (CriB) are at increased risk of developing liver cirrhosis, hepatic de-compensation and hepatocellular carcinoma. To maintain undetectable viral load reduces chronic infection complications. There is no treatment that eradicates HBV infection. Current drugs are expensive, are associated with adverse events, and are of limited efficacy. Current guidelines try to standardize the clinical practice. Nevertheless, controversy remains about management of asymptomatic patients with CriB who are hepatitis B e antigen (HBeAg)-positive with normal alanine aminotransferase, and what is the cut-off value of viral load to distinguish HBeAg- negative CriB patients and inactive carriers. We discuss in detail why DNA level alone is not sufficient to begin treatment of CriB.
基金Supported by the National Natural Science Foundation of China(No. 30371328), the Key Project of Natural Science Foundationof Shandong Province (No. Z2002C01), and the Key Project ofShandong Academy of Medical Sciences (No. 2005007)
文摘AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA. METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified. RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and X regions was 5.7 × 10^4/mL, 6.3 × 10^2/mL and 1.6 × 10^3/ mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 × 10^9/mL, 2.08 × 10^6/mL and 4.40 × 10^7/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis, which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A, B and C was around 10^S/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate. CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.
基金Supported by the National High Technology Research and Development Program(863 Program)No.2012AA022605
文摘AIM To investigate whether hepatitis viral DNA load at 24 wk of treatment predicts response at 96 wk in patients with chronic hepatitis B.METHODS A total of 172 hepatitis B envelope antigen(HBe Ag)-positive chronic hepatitis B patients who received initial treatment at 16 tertiary hospitals in Hunan Province, China were enrolled in this study. All patients received conventional doses of lamivudine and adefovir dipivoxil, telbivudine, entecavir dispersible tablets, or entecavir tablets for 96 wk. Patients who used other antiviral drugs or antitumor and immune regulation therapy were excluded. Patients were stratified into three groups according to their viral DNA load at 24 wk: < 10 IU/m L(group 1), 10-103 IU/m L(group 2), and > 103 IU/m L(group 3). Correlations of 24-wk DNA load with HBe Ag negative status and HBe Ag seroconversion at 96 wk were analyzed. Receiver operating characteristic curve analysis was used to test the predictive value of the HBV DNA load at 24 wk for long-term response.RESULTS The rates of conversion to HBe Ag negative status and HBe Ag seroconversion rates were 53.7% and 51.9%, respectively, in group 1; 35.21% and 32.39% in group 2; and 6.38% and 6.38% in group 3. The receiver operating characteristic curves for the three subgroups revealed that the lowest DNA load(< 10 IU/m L) was better correlated with response at 96 wk than a higher DNA load(10-103 IU/m L). Nested PCR was used for amplifying and sequencing viral DNA in patients with a viral DNA load > 200 IU/m L at 96 wk; resistance mutations involving different loci were present in 26 patients, and three of these patients had a viral DNA load 10-103 IU/m L at 96 wk. CONCLUSION Hepatitis B viral DNA load at 24 wk of antiviral treatment in patients with chronic hepatitis B is a predictor of the viral load and response rate at 96 wk.
文摘In DNA-guided hepatitis B treatment, viral load is insufficient, and requires other viral markers for treatment of hepatitis B patients as in patients with acute exacerbation of chronic hepatitis B, end-stage renal disease on dialysis, human immunodeficiency virus co-infected patients. There are exceptions to this rule: a residual level hepatitis B virus (HBV) DNA at 24 wk predicts beneficial outcome and reduced resistance at i year. The genotypic viral resistance to antiviral agents and occult HBV infection can be determined by HBV-DNA levels.
文摘To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA (eccDNA) of duck hepatitis B virus (DHBV), a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5' end and Tamra at its 3' end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln(x) + 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.
文摘AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of direct sequencing of PCR amplified products, a 16-mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASOn) directed against the HBV U5-like region was synthesized and then linked with one live-targeting ligand, the galactosylated poly-L-lysine. Their effect on the expression of HBV gene was observed using the 2.2.15 cells.RESULTS HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw1 by sequencing so that antisense oligonucleotides could bind specifically to the target sequence through base piring. Under the same experimental conditions, the inhibitory rates of PS-ASON to HBsAg and HBeAg were 70% and 58% at a concentration of 10μmol/L, while by ligand-PS-ASON they were 96% and 82%, the amount of HBV DNA in cultured supernatant and cells was reduced significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotides had no cytotoxicity.CONCLUSION Antisense oligonucleotides complexed by the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors, resulting in supecific inhibition of HBV gene expression and replication.
文摘AIM:To compare the clinical performance of a real-time PCR assay with the COBAS Amplicor Hepatitis B Virus (HBV) Monitor test for quantitation of HBV DNA in serum samples. METHODS: The reference sera of the Chinese National Institute for the Control of Pharmaceutical and Biological Products and the National Center for Clinical Laboratories of China, and 158 clinical serum samples were used in this study. The linearity, accuracy, reproducibility, assay time, and costs of the real-time PCR were evaluated and compared with those of the Cobas Amplicor test. RESULTS: The intra-assay and inter-assay variations of the real-time PCR ranged from 0.3% to 3.8% and 1.4% to 8.1%, respectively. The HBV DNA levels measured by the real-time PCR correlated very well with those obtained with the COBAS Amplicor test (r = 0.948). The real-time PCR HBV DNA kit was much cheaper and had a wider dynamic range. CONCLUSION: The real-time PCR assay is an excellent tool for monitoring of HBV DNA levels in patients with chronic hepatitis B.
文摘AIM: To determine whether new cut-off values for aianine aminotransferase (ALT) and baseline hepatitis B virus (HBV) DNA levels better differentiate HBeAg(-) chronic hepatitis B (CriB) patients from inactive chronic carriers. METHODS: Ninety-one patients [32 HBeAg(+) CriB, 19 inactive carriers and 40 HBeAg(-) CriB] were followed up for 2 years and were tested for HBV DNA levels by a PCR-based assay. ALT was tested twice during the last 6 mo using new cut-off values: ULN (upper limit of normal) 30 IU/L for males, 19 IU/L for females. Diagnostic accuracy, sensitivity, specificity, positive and negative predictive values were calculated by discriminant analysis. RESULTS: When using the revised ALT cut-off values, the lowest optimal HBV DNA level that differentiated HBeAg(-) CHB patients from inactive carriers was 50000 copies/mL. The diagnostic accuracy of HBV DNA to determine inactive carriers with a cut-off of 50000 copies/mL was similar to the previously recommended cut-off of 100000 copies/mL (91%). HBV DNA levels were lower than the cut-off value in 95% of inactive carriers and in 28% of HBeAg(-) CHB patients. With ALT 〈 30 IU/L in men and 〈 19 IU/L in women and HBV DNA levels 〈 100000 copies/mL, the risk of CHB is 5%. On the other hand, if ALT values were 〉 30 IU in men and 〉 19 IU in women and baseline HBV DNA levels were 〉 100000 copies/mL, the risk is 86%. CONCLUSION: New cut-off values for ALT together with HBV DNA levels proposed by AASLD (American Association for the Study of Liver Diseases) and NIH (National Institute of Health) consensus seem appropriate to characterize inactive carriers.
文摘AIM:To estimate the amount of apoptosis among healthy HBsAg carriers,patients with chronic HBV infection treated wibh lamivudine and patients with chronic HCV infection treated with interferon alpha and ribavirin.Activity of apoptosis was evaluated by serum sFas/sFasL concentration measurement. Moreover dependence between apoptosis and HBV-DNA or HCV-RNA levels was studied. METHODS:Eighty-six persons were included into study:34 healthy HBsAg carders,33 patients with chronic HBV infecl^on and 19 patients with chronic HCV infection.Serum levels of sFas/sFasL were measured by ELISA assay.HBV-DNA and HCV-RNA were measured by RT-PCR assay.Levels of sFas/sFasL were determined before and 2 and 12 wk after therapy in patients with chronic hepatitis B and C infection. HBV-DNA or HCV-RNA was detected before treatment and 6 mo after treatment. RESULTS:Twenty-four (71%) healthy HBsAg carders showed HBV-DNA over 10~5/mL,which was comparable to the patients with chronic hepatitis B.independently from HBV-DNA levels, the concentration of sFas among healthy HBsAg carders was comparable to healthy persons.Among patients with chronic hepatitis B and C,the concentration of sFas was significantly higher in comparison to healthy HBsAg carriers and healthy persons.In chronic hepatitis B patients the concentration of sFas was decreased during lamivudine treatment.Among chronic hepatitis C patients the concentration of sFas was increased during IFN alpha and ribavirin treatment,sFasL was not detected in control group.Furbhermore sFasL occurred more frequently in chronic hepatitis C patients in comparison to chronic hepatitis B patients. CONCLUSION:There are no correlations between apoptosis and HBV-DNA levels.However ther is an association between apoptosis and activity of inflammation in patients with chronic HBV infection.Apoptosis can be increased in patients with chronic hepatitis C by effective treatment which may be a result of apoptosis stimulation by IFN-α.
基金Project supported by the grant from Science Foundation of Ministry of Health of China, No. 96-1-347.
文摘INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].
文摘AIM To investigate and compare the analytical and clinical performance of Tian Long automatic hypersensitive hepatitis B virus(HBV) DNA quantification system and Roche CAP/CTM system.METHODS Two hundred blood samples for HBV DNA testing, HBV-DNA negative samples and high-titer HBV-DNA mixture samples were collected and prepared. National standard materials for serum HBV and a worldwide HBV DNA panel were employed for performance verification. The analytical performance, such as limit of detection, limit of quantification, accuracy, precision, reproducibility, linearity, genotype coverage and cross-contamination, was determined using the Tian Long automatic hypersensitive HBV DNA quantification system(TL system). Correlation and Bland-Altman plot analyses were carried out to compare the clinical performance of the TL system assay and the CAP/CTM system. RESULTS The detection limit of the TL system was 10 IU/m L, and its limit of quantification was 30 IU/m L. The differences between the expected and tested concentrations of the national standards were less than ± 0.4 Log10 IU/m L, which showed high accuracy of the system. Results of the precision, reproducibility and linearity tests showed that the multiple test coefficient of variation(CV) of the same sample was less than 5% for 102-106 IU/m L; and for 30-108 IU/m L, the linear correlation coefficient r2 = 0.99. The TL system detected HBV DNA(A-H) genotypes and there was no cross-contamination during the "checkerboard" test. When compared with the CAP/CTM assay, the two assays showed 100% consistency in both negative and positive sample results(15 negative samples and 185 positive samples). No statistical differences between the two assays in the HBV DNA quantification values were observed(P > 0.05). Correlation analysis indicated a significant correlation between the two assays, r2 = 0.9774. The Bland-Altman plot analysis showed that 98.9% of the positive data were within the 95% acceptable range, and the maximum difference was-0.49.CONCLUSION The TL system has good analytical performance, and exhibits good agreement with the CAP/CTM system in clinical performance.
文摘PCR technique was used to detect HBV DNA in liver tissue samples for study of the prevalence of HBV DNA in tumorous and nearby nontumorous liver tissues from 16 hepatocellular carcinoma(HCC) patients.Three primer pairs, Sl/S2, C1/C2 and X1/X2, used in this study were selected from S region, pre C and C region,and X region of HBV DNA, respectively. The detecting with agarose gel electrophoresis and ethidium bromide staining(PCR-EB) was 10-2 pg, and that with Southern blot hybridization was 10-6 pg. The positive rates in amplification of HBV DNA by S, C and X region primer pairs in liver samples were 43.8%(14/32), 71.9%(23/32)and 71.9%(23/32), respectively. There was significant difference between the positive rates in amplification with S primer and with C Primer(P<0.05), but no significant difference between the C primer and the X primer(P<0.05), and between the S primer and the X primer(0.10>P>0.05). HBV DNA fragments were detected in the livers from all 16 cases. The results indicated that X gene integration inducing hepatocellular carcinogenesis and arrest of C gene expression causing escape from host immune surveillance are the possible mechanisms of HCC development in patients with perisistent HBV infection.