HBV-DNA Concentration Research Articles

Management of hepatitis B in China

A randomised, multicentre, double-blind, placebo controlled trial was conducted in Chinese patients with chronic hepatitis B to compare the efficacy of once-daily lamivudine and placebo on serum HBV DNA, and to assess the long-term efficacy and safety of lamivudine. Patients received lamivudine 100 mg (n = 322) or placebo (n = 107) once daily for 12 weeks, and were then offered open-label lamivudine treatment for 2 years. Lamivudine therapy resulted in increased hepatitis B e antigen (HBeAg) loss and seroconversion (loss of HBeAg plus the development of antibodies to HBeAg) in patients with high baseline serum alanine aminotransferase (ALT) concentrations. At 2 years, loss of HBeAg was achieved by 27% (38/140), 38% (25/66) and 60% (9/15), and seroconversion was achieved by 17% (24/140), 24% (16/66) and 33% (5/15) of patients with baseline serum alanine aminotransferase (ALT) concentrations of >1 x upper limit of normal (ULN), >2 x ULN and >5 x ULN, respectively. With lamivudine treatment, serum HBV DNA decreased rapidly to very low concentrations and remained low throughout the 2 years of the study. At 1 year, 15% (43/295) of patients in the lamivudine group had developed YMDD (tyrosine-methionine-aspartate-aspartate amino acid motif of HBV polymerase) variant HBV. These patients derived clinical benefit with continued lamivudine therapy, demonstrated by serum HBV DNA and ALT concentrations below baseline, or normal serum ALT concentrations. Lamivudine was well tolerated and an effective once-daily oral therapy for Chinese patients with chronic hepatitis B with viral replication and liver disease.

Low level wild-type and pre-core mutant hepatitis B viruses and hbeAg negative reactivation of chronic hepatitis B

A qualitative and a quantitative mutation-site specific polymerase chain reaction assay (MSSA) was used to detect low level wild-type and pre-core mutant hepatitis B virus (HBV)-DNA. Serum samples from 11 anti-hepatitis B e (anti-HBe)-positive asymptomatic HBV carriers (Group A) and 10 anti-HBe-positive chronic hepatitis B patients who achieved alanine transaminase (ALT) normalization after antiviral therapy (Group B) were tested. Eleven patients had both wild-type and pre-core mutant HBV-DNA (52%, 4 from Group A and 7 from Group B), whereas 3 patients had only pre-core mutant HBV-DNA (14%, 2 from Group A and 1 from Group B) by qualitative MSSA assay. During a 3-year follow-up period, relapses were observed in 3 patients from Group B and intermittent ALT elevation was observed in 4 patients from Group A and 3 patients from Group B. The wild-type HBV-DNA concentration in the patients with reactivation was 10(2.06+/-2.62) copies/ml, whereas that in all patients without reactivation was below 10(2) copies/ml (P < .05). The pre-core mutant HBV-DNA concentration in the patients with reactivation was also significantly higher than that in the patients without reactivation (10(3.94+/-2.25) vs. 10(0.65+/-1.45) copies/ml, P < .001). All patients with both HBV-DNA concentrations below 10(2) copies/ml did not exhibit reactivation. Our result suggest that a high prevalence of coexistence of low level wild-type and pre-core mutant HBV-DNA has the potential for reactivation in anti-HBe-positive patients. Furthermore, quantification of wild-type and pre-core mutant HBV-DNA was useful to predict the prognosis of anti-HBe-positive infection and evaluate the efficacy of antiviral therapy.

Analysis of the Hepatitis B virus precore and ORF-X sequences in patients with antibody to hepatitis B e antigen with and without normal ALT levels

Serum samples from 20 anti-hepatitis B e antigen-positive patients with and without normal alanine aminotransferase (ALT) levels who had serum hepatitis B virus (HBV) DNA detectable only by polymerase chain reaction (PCR) were examined. Viral DNA was amplified by PCR, using primers that encompassed precore and ORF-X regions and sequenced directly, to investigate whether mutations in the nucleotide sequences of X and precore gene regions of HBV-DNA might be responsible for the difference in the activity of disease and in the levels of viral replication. The HBV-DNA concentration in patients with abnormal ALT levels was higher than in those with normal ALT. The amount of HBV-DNA correlated with the ALT levels (P < 0.05). Seventy-two percent of patients had HBV-DNA harboring the 1896 precore stop mutation, and there was a negative correlation between the percentage of precore mutant genotype and the HBV-DNA concentration (P < 0.05). Thirty percent of patients had mutations in ORF-X. Patients with ORF-X mutations had lower levels of HBV-DNA than those who had wild-type virus. The presence of mutations in precore and X regions may be related to a low HBV-DNA concentration and reduced biochemical activity in patients with anti-HBe.

Microfabricated disposable DNA sensor for detection of hepatitis B virus DNA

Abstract The authors report here a preliminary evaluation of a microfabricated disposable-type DNA sensor, based on photolithography technique, for the detection of hepatitis B virus (HBV) genome DNA. The DNA sensor was reacted with the HBV-DNA-inserted plasmid (pYRB259), immersed in a 100 μmol/l Hoechst 33258 solution and washed with a phosphate buffer. The electrochemical analysis showed that the anodic peak current derived from Hoechst 33258 bound to the formed hybrids on the electrode was increased with increasing the concentration of pYRB259 in the range from 10 4 to 10 6 copy/ml. Then, the authors evaluated the concentration of HBV-DNA extracted from patients' sera using a DNA sensor and competitive polymerase chain reaction (C-PCR). A correlation coefficient of 0.89 was obtained between the two assay methods. The variance for each sample in triplicate measurements ranged from 2 to 6% in the inter-assay using the DNA sensor. These results suggest that the microfabricated disposable DNA sensor can provide a specific and quantitative detection of HBV-DNA in sera.

Quantitative assessment of serum hepatitis B virus DNA: comparison between two different methods

Serum HBV DNA levels were quantified simultaneously by slot-blot hybridization using digoxigenin-labeled probe (S) and by Abbott HBV DNA assay (A). Samples, 69, from 64 patients seropositive for hepatitis B e antigen (HBeAg) were included and the percentages of hepatocytes containing hepatitis B core antigen (HBcAg) were also compared. The extent of HBcAg staining was only moderately correlated with HBV DNA levels detected by either Abbott (R2 = 0.41; P < 0.01) or slot-blot hybridization (R2 = 0.33; P < 0.01) assays. On the other hand, HBV DNA concentrations assessed by either method were well correlated although the levels differed markedly (S = 3.0 × A1.31; R2 = 0.85; P < 0.01). These results indicated that digoxigenin-labeled probe can be used to quantify serum HBV DNA concentration, although a different range of levels was obtained when compared with Abbott HBV DNA assay.

The value of quantiative detection of HBV-DNA amplified by PCR in the study of hepatitis B infection

Background/Aims: This study aimed to evaluate the usefulness of quantifying HBV-DNA amplified by polymerase chain reaction in chronic hepatitis B infection. Methods: Serum samples were obtained from 32 asymptomatic HBV carriers and 99 chronic hepatitis B patients (62 positive for anti-HBe and 37 positive for HBeAg). In addition, serial serum samples were analyzed from 15 HBeAg positive patients undergoing antiviral therapy and 17 anti-HBe positive patients with precore mutation. HBV-DNA quantification was carried out using an enzyme immunoassay with an HBV-DNA plasma standard. Results: The digoxigenin-incorporated polymerase chain reaction method detected HBV-DNA in 34.3% of the asymptomatic HBV carriers with a median HBV-DNA concentration of about 0.18×10 5 mol/ml (range 0.08–0.4), in 87%, of the anti-HBe positive chronic hepatitis cases with a range of 0.2 to >2×10 5 mol/ml and in 100% of the HBeAg positive patients, with a value in all cases over 2×10 5 mol/ml. We observed that after treatment, HBV-DNA tested negative in only two of the eight HBeAg positive chronic hepatitis patients who seroconverted to anti-HBe, and was positive in the seven remaining, with a median HBV-DNA value of about 0.2×10 5 mol/ml (0.09–0.4). In the precore mutants HBV-DNA values ranged from 0.2 to >×10 5 mol/ml. Conclusions: Polymerase chain reaction HBV-DNA quantification is a sensitive method for managing chronic hepatitis B patients, especially those with low viremia, and may be a valuable tool for evaluating the efficacy of antiviral therapy.

Lymphoblastoid interferon alfa with or without steroid pretreatment in children with chronic hepatitis B: A multicenter controlled trial

The comparative efficacy of prednisolone followed by interferon alfa (IFN-alpha) versus IFN-alpha alone in enhancing the rate of antibody to hepatitis B e antigen (anti-HBe) seroconversion has not been evaluated in a large cohort of white children. To determine this, a multicenter-controlled trial was conducted in 95 hepatitis B virus (HBV)-DNA/hepatitis B e antigen (HBeAg)-positive children (median age, 9 years [range, 2-16 years]; 56 boys; 84 [89 percent] white), all having inflammatory changes on liver biopsy. Patients were randomized to receive either prednisolone followed by IFN-alpha (n = 34); placebo followed by IFN-alpha (n = 30); or no treatment (n = 31). The prednisolone/placebo was given on a double-blind basis. Lymphoblastoid IFN-alpha was given at 5 MU/m(2) three times a week for 12 weeks. Baseline clinical, biochemical, and histological features were similar for the three groups. The majority (85 percent) had a baseline aspartate aminotransferase (AST) level < or = 100 IU/L. On follow-up between 12 and 18 months (median, 15 months) after treatment, the loss of HBeAg with anti-HBe seroconversion was more common in patients pretreated with steroids (12 of 34 [35 percent]) or placebo [12 of 30 (40 percent)] as against controls (4 of 31 [13 percent], P< .05). Factors predictive of anti-HBe seroconversion were baseline HBV-DNA concentration of < or = 1,000 pg/mL and a greater degree of portal tract inflammation on pretrial biopsy. Our results show that in white children treatment with IFN-alpha, at the dose and duration used in this study, improves the rate of anti-HBe seroconversion. Steroid priming does not potentiate the effect of IFN-alpha.

Lymphoblastoid interferon alfa with or without steroid pretreatment in children with chronic hepatitis B: A multicenter controlled trial

The comparative efficacy of prednisolone followed by interferon alfa (IFN-alpha) versus IFN-alpha alone in enhancing the rate of antibody to hepatitis B e antigen (anti-HBe) seroconversion has not been evaluated in a large cohort of white children. To determine this, a multicenter-controlled trial was conducted in 95 hepatitis B virus (HBV)-DNA/hepatitis B e antigen (HBeAg)-positive children (median age, 9 years [range, 2-16 years]; 56 boys; 84 [89 percent] white), all having inflammatory changes on liver biopsy. Patients were randomized to receive either prednisolone followed by IFN-alpha (n = 34); placebo followed by IFN-alpha (n = 30); or no treatment (n = 31). The prednisolone/placebo was given on a double-blind basis. Lymphoblastoid IFN-alpha was given at 5 MU/m(2) three times a week for 12 weeks. Baseline clinical, biochemical, and histological features were similar for the three groups. The majority (85 percent) had a baseline aspartate aminotransferase (AST) level < or = 100 IU/L. On follow-up between 12 and 18 months (median, 15 months) after treatment, the loss of HBeAg with anti-HBe seroconversion was more common in patients pretreated with steroids (12 of 34 [35 percent]) or placebo [12 of 30 (40 percent)] as against controls (4 of 31 [13 percent], P< .05). Factors predictive of anti-HBe seroconversion were baseline HBV-DNA concentration of < or = 1,000 pg/mL and a greater degree of portal tract inflammation on pretrial biopsy. Our results show that in white children treatment with IFN-alpha, at the dose and duration used in this study, improves the rate of anti-HBe seroconversion. Steroid priming does not potentiate the effect of IFN-alpha. (Hepatology 1996 Apr;23(4):700-7)

Semiquantitative anti‐HBc IgM detection in children with chronic hepatitis B: A long‐term follow‐up study

Serum anti-HBc IgM titres were monitored monthly by a semiquantitative method in 14 children with HBeAg positive chronic hepatitis B followed up for 18-65 months. All patients, but one, were treated with alfa-interferon (IFN) at different times. On the whole, 12 flare-up episodes were observed and 7 patients cleared HBV-DNA and seroconverted to anti-HBe. Seroconversion occurred only in patients with pretreatment anti-HBc IgM index greater than 0.15 and serum HBV-DNA concentration below 100 pg/ml; the pretreatment alanine aminotransferase (ALT) value was not predictive of response. Combining anti-HBc IgM results and serum HBV-DNA levels observed during the pre-IFN period allowed a precise identification of patients who were likely to respond to IFN therapy. Patients who seroconverted to anti-HBe showed a progressive reduction in serum anti-HBc IgM titres within 6 months. Interestingly, one child, in whom HBV-DNA reappeared and who reconverted to HBeAg 7 months after treatment, showed no anti-HBc IgM decrease after the transient clearance of HBV-DNA and anti-HBe seroconversion. Semiquantitative anti-HBc IgM detection is a useful tool in the decision making process for children with chronic hepatitis B.

Therapy of chronic hepatitis B with a 6-month course of ribavirin

Ribavirin is a nucleoside analogue with broad spectrum antiviral activity that has been shown to inhibit viral replication in the woodchuck model of hepatitis B virus infection. We studied the effect of ribavirin on viral replication in 18 patients with chronic hepatitis B who were positive for hepatitis B e antigen. Patients were randomized to receive a 24-week course of oral ribavirin at a dose of either 800, 1000, or 1200 mg/kg per day. All patients completed 24 weeks of treatment and an additional 24 weeks of follow up without significant side effects except for mild, reversible hemolytic anemia. Response to ribavirin was similar among all three dosage groups (p > 0.5); hence the data were pooled and analyzed together. Mean hepatitis B virus DNA levels decreased from 162.7 (95% confidence interval, 106 to 219) pg/ml before treatment to its lowest level of 114.3 (95% confidence interval, 53 to 175) pg/ml at week 20 (p < 0.05). Two patients became negative for HBV DNA and lost hepatitis B e antigen. Mean serum alanine aminotransferase activity decreased markedly from 131.1 (95% confidence interval, 84 to 178) U/l before treatment to 62.4 (95% confidence interval, 48 to 77) U/l at the end of 24 weeks of ribavirin (p < 0.05) and became normal in four patients (22%). Aminotransferase levels returned to baseline within 4 weeks once ribavirin was discontinued, while HBV DNA concentrations remained below baseline even at the end of 24 weeks of follow up.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatitis B Virus in the Colostra of HBeAg-Positive Carrier Mothers

To investigate the amount of hepatitis B virus (HBV) in colostra of hepatitis B e antigen (HBeAg)-positive carrier mothers and the relationship of HBV amount between colostrum and maternal blood, 50 HBeAg-positive carrier mothers were recruited and studied for hepatitis B surface antigen (HBsAg) titer, HBeAg titer, and HBV-DNA concentration in their sera and colostra. According to the presence or absence of seral HBV-DNA determined by dot hybridization, these 50 HBeAg-positive carrier mothers could be divided into two groups: group 1 (n = 28, HBV-DNA < 0.04 ng/ml), and group 2 (n = 22, HBV-DNA > or = 0.04 ng/ml). The colostral HBsAg and HBeAg titers were both significantly higher in group 2 than in group 1. In addition, both colostral HBsAg and HBeAg titers had positive correlation with each corresponding maternal blood. Although the colostral HBV-DNA was undetectable by dot hybridization, all were positive by polymerase chain reaction with Southern blot hybridization. Because HBV-DNA can be detected in all HBeAg-positive carrier mother's milk, it reinforces the necessity of hepatitis B vaccination for the neonates born to these carrier mothers, particularly in countries with a high carrier rate.

Current status of interferon alpha in the treatment of chronic hepatitis B

Interferon alpha is the only available therapy for patients with chronic hepatitis B. With interferon alpha 3-15 MU thrice weekly or 5 MU daily during 3-6 months one-third of the patients achieve seroconversion of HBeAg and HBV-DNA together with normalization of aminotransferases and slight improvement of histology. Loss of HBsAg is reported in a minority of responders during treatment, but increases during follow-up. Patients with baseline alanine aminotransferase of at least twice the upper limit of normal and low HBV-DNA concentration achieve the best response rates. HIV-positive patients with low CD4 counts and Asians are poor responders. As side-effects influenza-like symptoms are experienced by almost all patients. Mild leukopenia, thrombocytopenia and decreased hairgrowth are frequently reported. Severe depression, depersonalization and psychosis are reported in a small number of patients but tend to be poorly recognized in some studies. The decision whether dose reduction is indicated seems strongly related to the opinion of the investigator. Although long-term effects on the occurrence of cirrhosis and the development of hepatocellular carcinoma are not available yet, the achieved results are promising.

Early predictor of the efficacy of immunoprophylaxis against perinatal hepatitis B transmission: analysis of prophylaxis failure

To research early predictors of the efficacy of the ongoing mass immunoprophylaxis against perinatal hepatitis B virus (HBV) transmission in Taiwan and to analyse the possible causes of immunoprophylaxis failure, 52 hepatitis Be antigen (HBeAg)-positive carrier mothers were recruited for the study. Maternal blood samples were taken at the first and third trimesters and delivery. Umbilical blood was collected and venous blood samples were taken at 4, 7, 11 and 14 months of age. Serum hepatitis B surface antigen (HBsAg) and antibody titres, HBeAg titre and HBV-DNA concentration were analysed. All the umbilical cord blood samples were negative for HBsAg. Among the 52 vaccinated infants, four were poor responders (anti-HBs <10 mIU ml −1 before vaccine booster). Another five infants became HBsAg-positive by 4 months of age and remained carriers. All these five carrier mothers were HBV-DNA-positive and three of them had risk factors related to maternal-fetal haemorrhage during pregnancy or delivery. The remaining 43 infants showed protective anti-HBs level (>10 mIU ml −1) by 4 months of age. Three mothers out of these 43 cases also had the same haemorrhage risk factors during pregnancy or delivery, but were HBV-DNA-negative. Therefore, the anti-HBs level at 4 months is a predictor of the success of immunoprophylaxis. It may be helpful to distinguish HBV-DNA-positive carrier mothers among HBeAg-positive ones, to avoid inducing more maternal-fetal haemorrhage in such cases during pregnancy or delivery. Otherwise, additional hepatitis B immune globulin may be indicated in such cases to raise the successful prevention rate.

Advantage of PCR for detecting low amounts of HBV DNA in patients' sera

Serum hepatitis B virus DNA (HBV DNA) is now the most important and reliable marker for monitoring viral replication. Quantitative detection of HBV DNA in serum is based on a commercial standardized solution hybridization assay (Genostics). In this work, we studied the sensitivity and specificity of this method, in comparison with the polymerase chain reaction (PCR) technique, for low-value HBV DNA serum samples. Fifty-four patients with or without HBV serological markers were divided into 4 groups according to their HBV DNA values. Genomic amplication was found to affect 2 conserved regions of the viral genome, the S and C regions. Samples with an HBV DNA concentration equal to or greater than 1.5 pg/ml were considered positive in the “Genostics” test. A total of 38% of patients considered negative in the quantitative assay (< 1.5 pg/ml) were found to be positive for HBV DNA in serum after PCR. Only 26% of patients with an HBV DNA concentration of between 1.5 and 10 pg/ml in the Genostics test had PCR-detectable viral DNA in serum. Some 56% of patients with HBV DNA values between 10 and 20 pg/ml were found to be positive after amplification. All patients whose HBV DNA values were above 20 pg/ml had PCR-detectable viral DNA in serum. Our PCR results suggest that the positive limit level of the Genostics test has to be re-evaluated. Indeed, for low values of HBV DNA (under 20 pg/ml and especially under 10 pg/ml), it is not possible to conclude about the positivity from the quantitative assay, and results have to be estimated according to the clinical and serological status of the patients. Moreover, PCR can be falsely negative because of methodological problems. Nevertheless, this study confirms that PCR does enable detection of the viral genome in HBV-seronegative patients and in “old” and “cured” HBV-infection marker carriers.

Postpartum subsidence of hepatitis B viral replication in HBeAg-positive carrier mothers.

To elucidate the effects of pregnancy and delivery on hepatitis B e antigen (HBeAg)-positive carrier mothers, 31 HBeAg-positive carrier mothers were followed-up postpartum 1 year, with 30 HBeAg-positive nonpregnant female carriers as controls. Serum hepatitis B surface antigen (HBsAg), HBeAg titer, and hepatitis B virus (HBV)-DNA concentration were studied at defined intervals. The results revealed that in the control group HBeAg titers and HBV-DNA concentrations fluctuated, whereas the HBsAg titers showed little change, but HBeAg clearance or seroconversion to anti-HBe were not noted on follow-up. In contrast, one carrier mother seroconverted to anti-HBe during pregnancy and the antibody persisted thereafter. Five of the remaining 30 carrier mothers cleared HBeAg postpartum, and among these five cases, one also seroconverted to anti-HBe. In addition, in another five of the 30 cases, the HBV-DNA fell to undetectable level (less than 0.04 ng/ml). All these ten cases had a common tendency of showing a decrease in HBeAg titers and/or HBV-DNA concentrations 1-2 months after delivery. The HBeAg titers and HBV-DNA concentrations in the other 11 cases remained unchanged, whereas the remaining nine cases had increased levels. It is concluded that subsidence of HBV replication is precipitated by delivery in one-third of HBeAg-positive carrier mothers in Taiwan, and this occurs most frequently 1-2 months postpartum.

Treatment of Patients with Chronic Type B Hepatitis and Concurrent Human Immunodeficiency Virus Infection with a Combination of Interferon Alpha and Azidothymidine: A Pilot Study

Six patients with chronic type B hepatitis and concurrent infection with the immunodeficiency virus were treated with 600 mg azidothymidine (AZT)/day and 3 X 10(6) units of interferon-alpha (IFN-alpha) every other day for a total of 4 months. None of the patients treated lost the hepatitis B virus (HBV). HBV-DNA concentrations were not significantly influenced by this treatment. Human immunodeficiency virus (HIV) infection was also not affected except for a transient rise in CD 4-positive cells in 2 individuals, who had initially low CD 4-positive cells. Treatment did not influence the presence of HIV-Ag in the serum. In conclusion, a combination therapy of IFN and AZT does not seem to be beneficial at the doses given and the time involved.

Assessment of hepatitis B virus DNA levels in chronic HBsAg carriers with or without hepatitis delta virus superinfection

In order to document the incidence of hepatitis delta virus (HDV) replication markers and their relationship to HBV replication, 91 HBsAg chronic carriers were studied. Of these, 51 were anti-HD-positive (19 HBeAg-positive and 32 anti-HBe-positive). Liver HDAg was found in 75% of anti-HD-positive patients. Of the 19 patients who had anti-HD and HBeAg, 13 were HBV-DNA-positive. None of the anti-HBe patients were HBV-DNA-positive. No differences with respect to HBV-DNA concentration were observed between anti-HD-positive and -negative patients. Liver HDAg was detected with similar frequency in patients who were HBeAg- and HBV-DNA-positive (63.6%) and in those who were anti-HBe-positive (78.5%), with no statistically significant difference. HBcAg and HDAg were simultaneously detected in 36% of the anti-HD cases. Patients with anti-HD and HBV-DNA had the highest levels of transaminases (SGPT). Our results suggest that in certain patients HDV and HBV replication coexists without mutual inhibition.

Effects of HIV superinfection on HBV replication in a chronic HBsAg carrier with liver disease

A case of HIV superinfection observed in an HBsAg/HBeAg-positive male homosexual with chronic persistent hepatitis is described. Soon after the appearance of clinical and serological features of acute HIV infection, a rapid fall to a normal value of ALT was noted with simultaneous recrudescence of HBV replication lasting for several months as detected by an increase of the HBV-DNA concentration in the serum. Our observations suggest that the reduction of hepatocyte necrosis and the increase in HBV replication were a consequence of impaired T cell function during acute HIV infection.