Fabrizia Pittaluga

COBAS AmpliPrep-COBAS TaqMan Hepatitis B Virus (HBV) Test: a Novel Automated Real-Time PCR Assay for Quantification of HBV DNA in Plasma

ABSTRACT Success in antiviral therapy for chronic hepatitis B is supported by highly sensitive PCR-based assays for hepatitis B virus (HBV) DNA. Nucleic acid extraction from biologic specimens is technically demanding, and reliable PCR results depend on it. The performances of the fully automatic system COBAS AmpliPrep-COBAS TaqMan 48 (CAP-CTM; Roche, Branchburg, NJ) for HBV DNA extraction and real-time PCR quantification were assessed and compared to the endpoint PCR COBAS AMPLICOR HBV monitor (CAHBM; Roche). Analytical evaluation with a proficiency panel showed that CAP-CTM quantitated HBV DNA levels in one single run over a wide dynamic range (7 logs) with a close correlation between expected and observed values (r = 0.976, interassay variability below 5%). Clinical evaluation, as tested with samples from 92 HBsAg-positive patients, demonstrated excellent correlation with CAHBM (r = 0.966, mean difference in quantitation = 0.36 log10 IU/ml). CAP-CTM detected 10% more viremic patients and longer periods of residual viremia in those on therapy. In lamivudine (LAM)-resistant patients, the reduction of HBV DNA after 12 months of Adefovir (ADF) was higher in the combination (LAM+ADF) schedule than in ADF monotherapy (5.1 logs versus 3.5 logs), suggesting a benefit in continuing LAM. CAP-CTM detected HBV DNA in liver biopsy samples from 15% of HBsAg-negative, anti-HBcAg-positive graft donors with no HBV DNA in plasma. The amount of intrahepatic HBV DNA was significantly lower in occult HBV infection than in overt disease. CAP-CTM can improve the management of HBV infection and the assessment of antiviral therapy and drug resistance, supporting further insights in the emerging area of occult HBV infection.

Hepatitis B Virus Reactivation and Efficacy of Prophylaxis with Lamivudine in Patients Undergoing Allogeneic Stem Cell Transplantation

Patients previously infected with hepatitis B virus (HBV) undergoing an allograft and recipients from HBV carrier donors are at risk of posttransplant viral reactivation. The role of prophylaxis with lamivudine remains unclear. One hundred seventeen patients, with a median age of 52 years (20-67 years), with various hematologic malignancies transplanted between 1999 and 2007 entered the study. Eighty-seven recipients negative for HBV surface antigen (HBsAg), antihepatitis B core antigen antibodies (anti-HBc), and HBV-DNA with HBsAg and HBV-DNA negative donors were defined as at low risk of HBV reactivation, whereas all the remaining 30 patients were defined as at high risk. Patients at high risk transplanted in 2005 or after received lamivudine to prevent HBV reactivation as per the Italian guidelines by the Associazione Italiana per lo Studio del Fegato (AISF). Patients at low risk did not experience HBV reactivation/hepatitis. Among the recipients at high risk, 11 of 25 anti-HBc positive, those HBsAg positive (2 of 2) or negative but transplanted from HBsAg positive donors (3 of 3) were treated with lamivudine. None of these developed HBV reactivation/hepatitis after a median follow-up of 40 months (17-55 months). Hepatitis developed in 3 anti-HBc positive untreated patients conditioned with a reduced-intensity regimen. Hepatitis B was not observed in recipients at low risk, transplanted from HBsAg negative/anti-HBc positive or negative donors. Lamivudine was effective in controlling reactivation in: HBsAg positive recipients, in patients transplanted from HBsAg positive donors and in HBsAg negative/antiHBc positive recipients, who showed a significant risk of reactivation if not given prophylaxis (NCT 00876148).

Valganciclovir as pre-emptive therapy for cytomegalovirus infection post-allogenic stem cell transplantation: implications for the emergence of drug-resistant cytomegalovirus

OBJECTIVES Valganciclovir is a well established drug for the management of cytomegalovirus (CMV) infection in haematopoietic stem cell transplantation (HSCT). Data concerning its safety regarding the development of drug resistance are required. The aim of the present study was to retrospectively investigate CMV drug resistance in a group of HSCT patients experiencing relapses of CMV infection after a first-line pre-emptive antiviral therapy. METHODS Thirteen adult HSCT patients out of 26 with asymptomatic CMV infection, experiencing relapsing infections 45-155 days after either intravenous (iv) ganciclovir (2 patients) or valganciclovir (11 patients), were studied. Genotypic assays for mutations in the viral phosphotransferase (UL97) and DNA-polymerase (UL54) genes were directly applied on patient specimens. Baseline CMV sequences were compared with those at the time of relapses to identify drug-resistant strains. RESULTS UL97 mutations A594V and M460V known to confer drug resistance developed in one relapsing patient who received iv ganciclovir as first-line therapy, corresponding to a rate of 7.7% of relapses due to drug-resistant strains and an overall 3.8% rate of infections due to CMV drug-resistant strains. UL54 drug resistance mutations were absent. No evidence of drug resistance was found in patients on valganciclovir either as first-line therapy or as treatment for relapses. CONCLUSIONS The safety profile of valganciclovir as anti-CMV pre-emptive therapy was confirmed, as well as that monitoring CMV drug resistance with genotypic tests on sequential isolates over the time-course of therapy offers guidance to tailor antiviral treatment in a clinically relevant time frame.

Prediction of occult hepatitis B virus infection in liver transplant donors through hepatitis B virus blood markers.

BACKGROUND Occult hepatitis B virus infection is defined as detectable HBV-DNA in liver of HBsAg-negative individuals, with or without detectable serum HBV-DNA. In deceased liver donors, results of tissue analysis cannot be obtained prior to allocation for liver transplantation. AIMS we investigated prevalence and predictability of occult hepatitis B using blood markers of viral exposure/infection in deceased liver donors. METHODS In 50 consecutive HBsAg-negative/anti-HBc-positive and 20 age-matched HBsAg-negative/anti-HBc-negative donors, a nested-PCR assay was employed in liver biopsies for diagnosis of occult hepatitis B according to Taormina criteria. All donors were characterized for plasma HBV-DNA and serum anti-HBs/anti-HBe. RESULTS In liver tissue, occult hepatitis B was present in 30/50 anti-HBc-positive (60%) and in 0/20 anti-HBc-negative donors (p<0.0001). All anti-HBc-positive donors with detectable HBV-DNA in plasma (n=5) or anti-HBs>1,000 mIU/mL (n=5) eventually showed occult infection, i.e, 10/30 occult hepatitis B-positive donors which could have been identified prior to transplantation. In the remaining 40 anti-HBc-positive donors, probability of occult infection was 62% for anti-HBe-positive and/or anti-HBs ≥ 58 mIU/mL; 29% for anti-HBe-negative and anti-HBs<58 mIU/mL. CONCLUSIONS In deceased donors, combining anti-HBc with other blood markers of hepatitis B exposure/infection allows to predict occult hepatitis B with certainty and speed in one third of cases. These findings might help refine the allocation of livers from anti-HBc-positive donors.

Sequential versus concomitant administration of ribavirin and interferon alfa-N3 in patients with chronic hepatitis C not responding to interferon alone. Results of a randomized controlled trial

We conducted a three-arm, randomized trial in 96 patients with chronic hepatitis C who did not respond to interferon alfa to compare treatments. Group 1 (33 patients) received ribavirin alone (1,000 mg/daily for 6 months) followed by interferon alfa n-3 alone (3 MU thrice weekly for 6 months); group 2 (33 patients) received ribavirin plus interferon alfa n-3 for 6 months at the above doses; and group 3 (30 patients) received interferon alfa n-3 alone (3 MU thrice weekly for 6 months). At the end of treatment, 3 patients (10%) in group 1, 13 (41%) in group 2, and 5 (17%) in group 3 had normal alanine transaminase (ALT) levels (group 2 vs. groups 1 and 3, P = .008). After 6 months of follow-up, only 4 patients (12.5%) in group 2 still had normal ALT values (P = .03). At the end of therapy, hepatitis C virus (HCV) RNA was no longer detectable by polymerase chain reaction in 4 (13%), 9 (27%), and 2 (7%) patients, respectively, in groups 1, 2, and 3 (P = NS). Six months posttherapy, only 5 (15%) patients in group 2 were still HCV RNA negative (P = .02). At the time of follow-up liver biopsy, performed 6 months after the end of treatment, a significant improvement of the necroinflammatory scores was observed among group 2 patients (P = .01) but not in the other two groups. Side effects reflected the profile of each drug as monotherapy; mild hemolytic anemia was the most frequent side effect caused by ribavirin. In conclusion, concomitant administration of ribavirin and interferon alfa n-3 was significantly superior to the sequential schedule or interferon alfa n-3 monotherapy in inducing a sustained response in patients with chronic hepatitis C who had not responded to interferon alone. However, combination therapy at the dose and duration adopted in this study is capable of modifying the natural course of the disease in only a minority of these patients.