[Show abstract][Hide abstract] ABSTRACT: Hepatitis B virus (HBV) and hepatitis delta virus (HDV) interplay was investigated by examining liver and serum samples from 21 coinfected and 22 HBV-monoinfected patients with chronic liver disease. Different real-time PCR assays were applied to evaluate intrahepatic amounts of HBV DNA, covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA), pre-S/S RNAs, and HDV RNA. Besides HBV DNA and HDV RNA levels, HBsAg concentrations in the sera were also determined. HDV-coinfected cases showed significantly lower median levels of serum HBV DNA (-5 log), intrahepatic relaxed-circular DNA (-2 log), and cccDNA (-2 log) than those of HBV-monoinfected cases. Interestingly, pgRNA and pre-S/S RNA amounts were significantly lower (both -1 log) in HDV-positive patients, whereas serum HBsAg concentrations were comparable between the two patient groups. Pre-S/S RNA and HBsAg amounts per cccDNA molecule were higher in HDV-positive patients (3-fold and 1 log, respectively), showing that HBV replication was reduced, whereas synthesis of envelope proteins was not specifically decreased. The ratios of cccDNA to intracellular total HBV DNA showed a larger proportion of cccDNA molecules in HDV-positive cases. For these patients, both intrahepatic and serum HDV RNA amounts were associated with cccDNA but not with HBsAg or HBV DNA levels. Finally, HBV genomes with large deletions in the basal core promoter/precore region were detected in 5/21 HDV-positive patients but in no HDV-negative patients and were associated with lower viremia levels. These findings provide significant information about the interference exerted by HDV on HBV replication and transcription activities in the human liver.
Journal of Virology 10/2010; 85(1):432-9. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study assess prevalence, risk factors, and clinical and virological features of dual hepatitis B virus (HBV)/hepatitis C virus (HCV) infection.
We evaluated 837 hepatitis B surface antigen positive patients, prospectively enrolled in 14 Italian units.
Anti-HCV was present in 59 cases (7%); age specific prevalences were 4.5% (0-30 years), 4.4% (>30-50) and 14% (>50). Independent predictors of dual infection were age >42 years, history of I.V. drug use (IDU), blood transfusion and residence in the South of the country. The strength of the association with IDU was high, but this exposure accounted for five coinfection cases only. Cirrhosis was present in 107 of the 709 patients with HBV alone (15.1%), in 30 of 69 with hepatitis D virus coinfection (43%) and in 17 of 59 with HCV coinfection (28.8%); a light alcohol use was marginally associated with cirrhosis. Of 36 B/C coinfected patients, 16 (44.4%) had only HBV-DNA in serum, (median age=47.5 years) five (13.9%) had both HBV-DNA and HCV-RNA (age=53), nine (25%) had HCV-RNA alone (age=59) and six (16.7%) tested negative for both.
This study depicts the epidemiological and clinical burden of dual HBV/HCV infection in Italy.
Journal of Hepatology 12/2003; 39(6):1036-41. · 9.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The virological profiles of hepatitis B and C viruses (HBV and HCV) and their interplay in cases of coinfection are undefined. A suppressed and occult HBV infection may occur in hepatitis B surface antigen (HBsAg) negative patients with chronic hepatitis C. The HCV core protein is able to inhibit HBV "in vitro," and serines at positions 99 and 116 are essential for such inhibition. We aimed to assess the HBV and HCV virological profiles in cases of coinfection and to evaluate the relationship between HCV core gene variability and HBV activity.
Eighty-two anti-HCV positive patients were examined: 35 cases were HBsAg positive, 24 were HBsAg negative with "occult" HBV infection, and 23 were HBV negative. HBV and HCV viremia levels were evaluated in all cases. HCV genomic region coding for the aminoacid sequence 99-116 of core protein was amplified and sequenced in all HCV RNA positive cases. The entire core gene was amplified and sequenced in three randomly selected cases.
Serum HCV RNA was detected in all cases but 13, all HBsAg positive individuals; HCV viremia levels of the other 22 HBsAg positive subjects were similar to those detected in HBsAg negative patients with or without occult HBV infection. Among the 35 HBsAg positive patients both HBV DNA and HCV RNA were detected in five cases, HCV RNA alone in 17, and HBV DNA alone in six, whereas seven cases had undetectable levels of both viruses. Sequencing analyses showed that the HCV core gene was highly preserved in all patients.
A wide spectrum of HCV and HBV virological patterns may occur in a case of coinfection. HCV core variability is not related to HBV activity "in vivo."
The American Journal of Gastroenterology 07/2002; 97(6):1518-23. · 7.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Covalent attachment of a 40-kilodalton polyethylene glycol moiety to interferon alpha2a (peginterferon alpha2a) results in sustained delivery and reduced clearance compared with standard interferon alpha2a. The aim of the study was to compare viral kinetics in patients treated with standard or peginterferon alpha2a.
Patients with chronic hepatitis C were randomly assigned to receive either standard interferon alpha2a thrice weekly (n = 16) or 180 microg peginterferon alpha2a once weekly (n = 17) for 48 weeks. HCV RNA was quantitated before and frequently during treatment.
The extent of the second-phase decline of HCV RNA, representing the degradation rate of infected cells during therapy for responding patients, was 0.02 +/- 0.03 day(-1) (HCV-1), 0.88 +/- 0.64 day(-1) (HCV non-1), 0.06 +/- 0.08 day(-1) (HCV-1), and 0.44 +/- 0.33 day(-1) (HCV non-1) in patients treated with standard or peginterferon alpha2a, respectively. The second-phase decline was low (<0.05 day(-1)) in most patients without a virological end-of-treatment response, and the second-phase decline was high (>0.25 day(-1)) in all patients with sustained virological response.
The degradation rate of infected cells is HCV genotype dependent. Treatment with peginterferon alpha2a may reinforce the death rate of infected cells (particularly in HCV-1-infected patients) or stabilize the therapeutic effect on viral production. The second-phase decline of HCV RNA is predictive of virological sustained response.
[Show abstract][Hide abstract] ABSTRACT: Background & Aims: Covalent attachment of a 40-kilodalton polyethylene glycol moiety to interferon α2a (peginterferon α2a) results in sustained delivery and reduced clearance compared with standard interferon αa. The aim of the study was to compare viral kinetics in patients treated with standard or peginterferon α2a.
Methods: Patients with chronic hepatitis C were randomly assigned to receive either standard interferon α2a thrice weekly (n = 16) or 180 μg peginterferon μ2a once weekly (n = 17) for 48 weeks. HCV RNA was quantitated before and frequently during treatment.
Results: The extent of the second-phase decline of HCV RNA, representing the degradation rate of infected cells during therapy for responding patients, was 0.02 ± 0.03 day-1 (HCV-1), 0.88 ± 0.64 day-1 (HCV non-1), 0.06 ± 0.08 day-1 (HCV-1), and 0.44 ± 0.33 day-1 (HCV non-1) in patients treated with standard or peginterferon α2a, respectively. The second-phase decline was low (<0.05 day-1) in most patients without a virological end-of-treatment response, and the second-phase decline was high (>0.25 day-1) in all patients with sustained virological response.
Conclusions: The degradation rate of infected cells is HCV genotype dependent. Treatment with peginterferon α2a may reinforce the death rate of infected cells (particularly in HCV-1-infected patients) or stabilize the therapeutic effect on viral production. The second-phase decline of HCV RNA is predictive of virological sustained response.
[Show abstract][Hide abstract] ABSTRACT: A clinical evaluation of an automated quantitative PCR assay, the COBAS AMPLICOR HCV MONITOR test, version 2.0 (v2.0), was carried out to assess the performance of this test in comparison with that of the previous, manual version, the AMPLICOR HCV MONITOR test, and with that of nested PCR. Serial dilutions of serum samples infected with genotype 1b, 2a, or 3, as well as synthetic RNA transcripts and serum samples derived from 87 patients with chronic hepatitis C and infected with genotype 1a, 1b, 2a, 2b, 3a, 3b, 4, or 5, were analyzed to determine the ability of the system to efficiently quantify various hepatitis C virus (HCV) genotypes. These experiments showed that the COBAS AMPLICOR HCV MONITOR test, v2.0, has mean intra-assay, interassay, and interoperator coefficients of variation that range from 22 to 34.5% and a 3-logarithm dynamic range, which spans from 10(3) to 10(6) copies/ml. Compared to the previous, manual version of the test, the COBAS AMPLICOR HCV MONITOR test, v2.0, showed an improved efficacy for all genotypes, especially genotypes 2, 3, and 4, whose estimated concentrations were on average 1 logarithm higher. When used to monitor patients under treatment, however, both versions showed the same patterns of viremia, indicating that the COBAS AMPLICOR HCV MONITOR test, v2.0, and the AMPLICOR HCV MONITOR test were equally effective at detecting relative viremia changes in serial samples. As expected, the automated test was less sensitive than nested PCR; among specimens from a cohort of patients treated with interferon, nested PCR identified three more viremic specimens, which probably contained very low concentrations of HCV RNA.
Journal of Clinical Microbiology 07/2000; 38(6):2210-4. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: No data are available about the amount of hepatitis B virus (HBV) genomes in liver of patients with chronic HBV infection. The aim of this study was to quantify the intrahepatic HBV DNA in hepatitis B surface antigen (HBsAg)-positive patients with either active or suppressed viral replication and in HBsAg-negative subjects with occult HBV infection. We optimized the Roche "Amplicor HBV Monitor" kit for quantifying liver HBV DNA and analyzed hepatic DNA extracts and serum samples from 19 HBs-Ag-positive and 43 HBsAg-negative individuals. Eight of the HBsAg carriers had active HBV replication, and for 3 of them we analyzed samples obtained before and at the end of 1 year of lamivudine treatment. Five hepatitis Delta virus (HDV) coinfected patients and 6 healthy HBsAg carriers had inhibited HBV activity. Among the HBsAg-negative subjects 21 had occult HBV infection and 22 had no evidence of HBV infection. The median of HBV genomes per microgram of liver DNA milliliter of serum was 34,500 to 2,620,000 in patients with active viral replication, 20,000 to 3,900, 000 before and 10,000 to 2,800 at the end of therapy in lamivudine-treated individuals, 9,800 to 600 in HDV-infected individuals, and 7,450 to 17,400 in healthy HBsAg carriers. These data indicate that cases with suppressed HBV activity, despite the very low levels of viremia, maintain a relatively high amount of intrahepatic viral genomes. This virus reservoir is likely involved in HBV reactivation, which is usually observed after stopping lamivudine treatment. Finally, the analysis of cases with occult HBV infection showed that the assay we used was able to specifically detect and quantify as few as 100 copies of viral genomes per microgram of liver DNA.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether the measurement of HBV DNA by quantitative polymerase chain reaction (PCR) is helpful in monitoring response to interferon treatment in chronic hepatitis B virus infection, we have determined sequentially serum levels of HBV DNA during and up to 18 months after treatment, in 10 patients with a sustained response (all anti-HBe positive, five also HBsAg negative and anti-HBs positive) and, as controls, in 12 non-responders.
Serum HBV DNA was measured by standard hybrisation assay (Genostics, Abbott) and by quantitative PCR (Amplicor HBV Monitor test, Roche Diagnostic Systems).
A clear difference in HBV viral load between responders and non-responders was observed from the fourth week of treatment and was maintained throughout the study period. At the last follow up 16-26 (median 21) months after starting treatment, all the 10 responders were HBV DNA negative by hybridisation. By PCR, however, five (one anti-HBs and four anti-HBe positive) were still HBV DNA positive. In addition, one anti-HBs positive patient HBV DNA negative by PCR at last follow up, had fluctuating levels of HBV DNA by PCR during the observation period, only intermittently falling below the threshold of the assay.
The measurement of HBV DNA by quantitative PCR provides early prediction of response to interferon, allowing prompt modification of treatment. With this technique, HBV DNA is detected in a high proportion of sustained responders, suggesting that HBV may never be completely eliminated by interferon treatment, even after anti-HBs seroconversion.
Journal of Hepatology 07/1999; 30(6):965-9. · 9.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fluctuations of hepatitis C virus (HCV)-RNA serum levels were monitored in a multicenter study in 76 chronic HCV carriers who had been followed longitudinally without receiving antiviral therapy to assess their relation with the course of liver disease activity. Forty-four patients had normal transaminases over more than 2 years, while 32 additional patients had fluctuating levels. Viral load was measured in serial serum samples prospectively collected for 10 to 12 months in 54 patients and in sera stored yearly up to 8 years in an additional 22 patients. In patients tested monthly, a lesser extent of fluctuations was detected in cases with constantly normal transaminases as compared with those with fluctuating transaminases. In the former group, the mean difference between maximum and minimum values observed in each individual patient was 0.7 Log, while in the latter group, it was 1.3 Log (P =.0004). Most of these patients experienced, on average, three peaks of viremia over 1 year. The range of variation observed upon yearly testing was between 0.2 and 2.2 Log and did not reach statistical significance between the two groups. In conclusion, a careful viral replication profile can be achieved only by monthly testing, because longer time intervals could miss viremia fluctuations. HCV-RNA levels are more stable in asymptomatic HCV carriers than in patients with biochemical activity of liver disease.
[Show abstract][Hide abstract] ABSTRACT: Viral load has emerged recently as a reliable marker of disease progression and therapeutic efficacy in chronic infections, including AIDS and hepatitis C. The clinical management of type B hepatitis could also be improved by monitoring viremia levels in patients with chronic liver disease undergoing anti-viral treatment. To address this question we evaluated the performance of a newly developed, quantitative PCR assay (Amplicor HBV Monitor test, Roche Diagnostic Systems) in the assessment of viremia changes over time in a group of 45 patients with chronic active hepatitis (CAH) who received interferon treatment. Of the 45 patients, 14 were HBsAg and anti-HBeAg positive and 31 HBsAg, HBeAg positive. Follow-up extended up to 24 months. An average of ten samples per patient were analyzed for levels of ALT, IgM anti-HBc (Abbott Laboratories), HBV DNA by in-house dot-blot hybridization and hybridization-capture assays (HBV-DNA hybrid capture kit, Murex Diagnostics) and by Amplicor HBV Monitor. A sustained biochemical response was observed at the end of treatment in 12 HBeAg-positive and in seven anti-HBeAg positive patients. This was accompanied by the disappearance of HBeAg and of HBV DNA (hybridization assays) in all cases and by the clearance of IgM anti-HBc in 70% of the cases. Viremia (quantitative PCR assay) became undetectable only in 25-30% of cases and was associated with the loss of HBsAg. A good correlation was observed between the time course of IgM anti-HBc, quantitative PCR and dot-blot hybridization although the latter missed 33% of viremic samples. Together, these results indicate that the Amplicor HBV Monitor test is a robust and standardized assay for quantifying HBV viremia levels in the range from 10(2) to 10(7) copies/ml. Compared to other current markers, viral load may provide additional clinical information by predicting long term virologic response and HBsAg clearance in patients with normal ALT at the end of interferon therapy.
Journal of Virological Methods 11/1998; 74(2):155-65. · 1.90 Impact Factor