Valeria Ghisetti

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.

Transplantation of hepatitis B surface antigen–positive livers into hepatitis B virus–positive recipients and the role of hepatitis delta coinfection

The scarcity of liver donors requires consideration of grafts from sources not previously used. Allografts from hepatitis B surface antigen (HBsAg)‐carriers without a significant liver disease have been proposed for liver transplant recipients with hepatitis B virus (HBV)‐related cirrhosis and hepatocellular carcinoma (HCC). Combination prophylaxis schemes against HBV post‐liver transplantation (LT) recurrence are currently available; the efficacy of those schemes in HBV‐related cirrhosis and HCC must be assessed. This report describes the allocation of HBsAg‐positive grafts in three HBsAg‐positive recipients, with HBV‐related cirrhosis and evolving HCC lesions, two of them with hepatitis Delta virus (HDV) coinfection. Patients were administered anti‐hepatitis B immunoglobulins (HBIGs) and lamivudine in order to prevent HBV recurrence. In spite of anti‐HBV prophylaxis, HBV infection did persist after LT in all patients (no serum clearance of HBsAg). HBV replication assessed by serum HBV deoxyribonucleic acid (DNA) presence was detected in the first month after LT in the 3 recipients. A prompt HDV reinfection with a clinical and histological pattern of hepatitis was observed in the 2 HBV / HDV coinfected recipients. In 1 of them, an evolving chronic hepatitis required a second LT. The non‐HDV–infected patient showed an uneventful follow‐up, but the lack of the neutralizing effect of HBIGs and the high risk of escape mutants forced the addition of adefovir‐dipivoxil to lamivudine, in order to prevent viral variants and hepatitis recurrence. In conclusion, allografts from HBsAg‐positive donors in HBsAg‐positive recipients are associated with the persistence of the HBsAg after LT due to the failure of HBIG prophylaxis, even if lamivudine does inhibit virion production. This condition favors HDV replication and HDV hepatitis recurrence in coinfected patients. The allocation of HBsAg‐positive grafts in HBsAg‐positive recipients could be justified only in recipients without HDV coinfection and a combined prophylaxis with lamivudine and adefovir‐dipivoxil is currently the best way to manage escape mutants in these recipients. (Liver Transpl 2005;11:922–928.)

Correlation between Cytomegalovirus Infection and Raynaud’s Phenomenon in Lupus nephritis

Relationships between viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) are still elusive. Recent reports demonstrated the association of some viral infections with peculiar clinical events in the general population, such as cytomegalovirus (CMV) with arterial damage and Parvovirus B19 (PV-B19) with hematologic abnormalities. We planned to look for this kind of viral imprinting in SLE, hypothesizing that traces of specific features of some viral infections might be found in some subsets of seropositive SLE patients. In 60 SLE patients recruited at our nephrologic center, serology for CMV, PV-B19, Epstein-Barr virus viral capsid antigen (EBV-VCA), Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr virus early antigen (EBV-EA) was performed. χ2 and ANOVA were employed to compare the frequency and titers of antiviral antibodies in SLE patients with groups of transplant, hemodialysis and blood donor subjects. χ2, Fisher’s test, Bonferroni and Scheffe’s test were employed to compare the different biochemical/clinical features between seropositive and seronegative SLE patients. Univariate and multivariate analysis (logistic regression models) were employed to evaluate the odds ratio (OR) of different risk factors for vascular events (including Raynaud’s phenomenon, deep venous thrombosis) and hematologic abnormalities (including severe anemia, leukopenia and thrombocytopenia). Anti-CMV (82%), anti-PV-B19 (60%), anti-EBV-VCA (92%) and EBV-EA (45%) IgG antibodies were frequent in SLE, with higher prevalence in comparison with the blood donor group and higher titers in comparison with transplant and hemodialysis groups. CMV seropositivity was a highly significant risk factor for Raynaud’s phenomenon (OR +α in univariate and multivariate analysis = 13.51 using a correction of 0.5 in case of a zero event), but not for venous vascular events (OR = 1.31). An increased though not significant risk factor was found for antiphospholipid antibodies (OR = 2.71, p = 0.19), while the presence of nephrotic syndrome during the follow-up was a significant protective factor (OR = 0.15, p = 0.035). There was no significantly increased OR for PV-B19 seropositivity in cases with severe anemia (OR = 2.09, p = 0.29). No significant associations were found with the status of EBV reactivation. In conclusion, our results support the hypothesis that viral infection may imprint the course of SLE leading to specific clinical subsets (i.e. CMV and ‘vascular’ SLE, with more frequent Raynaud’s phenomenon and a less frequent typical histological renal picture responsible for nephrotic syndrome). Further prospective studies are justified to validate these correlations, mainly dealing with associations between acute viral infections and vascular events, thus eventually leading to a better understanding of mutual relationships between viruses and SLE.

Occult hepatitis B virus infection in HBsAg negative patients undergoing liver transplantation: Clinical significance

Occult Hepatitis B virus (o‐HBV) infection has been reported in HB surface antigen (HBsAg)‐negative liver donors whose risk of transmitting HBV justifies a specific prophylaxis in liver recipients. The clinical significance of o‐HBV infection in HBsAg‐negative recipients and their need for prophylaxis is unknown. Liver samples collected during surgery from 23 HBsAg‐negative patients (9 liver donors and 14 recipients) and 20 HBsAg‐positive recipients (controls) were studied by polymerase chain reaction with an independent set of primers mapping the core and surface HBV genes. Intrahepatic HBV DNA was detected as core and surface genes in all the HBsAg‐positive recipients, in none of the HBsAg‐negative donors and in 9/14 (64%) of the HBsAg‐negative recipients (2 HCV negative, 7 HCV positive). The intrahepatic amount of HBV was significantly lower in HBsAg‐negative than in HBsAg‐positive livers (median values 1.36 Log10/μg DNA vs. 3.66 Logs, p<0.0001, core gene, and 1.13 vs. 6.21 Logs p<0.0001, surface gene). No HBV DNA was detected in plasma from o‐HBV recipients; one of them tested positive in lymphocytes. No correlation was found between o‐HBV and serologic markers of previous HBV exposure, response to vaccination, acute rejection, hepatitis D and G virus‐infections. None of o‐HBV carriers experienced a de novo hepatitis B after transplantation (median follow‐up: 477 days). Occult HBV is frequent in HBsAg‐negative liver recipients. It is not associated with increased episodes of acute rejection, coinfection with hepatotropic viruses, different responses to HBV vaccination, or the development of de‐novo hepatitis B. In o‐HBV infection a particular virus‐host interaction can explain the low intrahepatic HBV content and the lack of extrahepatic HBV replication, thus justifying the low risk of hepatitis B reactivation, in absence of specific prophylaxis, once the recipient liver is removed. (Liver Transpl 2004;10:356–362.)

Declining prevalence of HIV-1 drug resistance in antiretroviral treatment-exposed individuals in Western Europe

HIV-1 drug resistance represents a major obstacle to infection and disease control. This retrospective study analyzes trends and determinants of resistance in antiretroviral treatment (ART)-exposed individuals across 7 countries in Europe. Of 20 323 cases, 80% carried at least one resistance mutation: these declined from 81% in 1997 to 71% in 2008. Predicted extensive 3-class resistance was rare (3.2% considering the cumulative genotype) and peaked at 4.5% in 2005, decreasing thereafter. The proportion of cases exhausting available drug options dropped from 32% in 2000 to 1% in 2008. Reduced risk of resistance over calendar years was confirmed by multivariable analysis.

Evaluation of a rapid antigen and antibody combination test in acute HIV infection.

BACKGROUND New strategies at implementing HIV testing including rapid HIV assays are highly recommended to avoid late diagnosis. To shorten the diagnostic window period, the first point-of-care HIV assay, Determine HIV ½ Ag/Ab Combo (D4G, Alere, I) for the combined detection of p24 and anti-HIV antibody has been recently marketed and mainly tested in high prevalence setting. OBJECTIVES To establish D4G performances in acute HIV infection (AHI) in a setting at low HIV-1 prevalence. STUDY DESIGN D4G performances were compared with HIV-1 RNA levels in a panel of well-characterized serum specimens from 17 patients with AHI. For specificity, 124 anti-HIV negative serum specimens from patients seeking HIV testing were studied. RESULTS D4G detected HIV infection in 15/17 patients. D4G antigen was positive in only 5 patients (29.4%), 4 of them with a viral load >10 million copies/mL. D4G antibody was reactive in other 10 patients (sensitivity: 58.8%, viral load from 70,161 to 8,120,000 copies/mL). Combined D4G sensitivity for acute HIV-1 infection was 88.2%; no false positive or invalid result was recorded (100% specificity, positive and negative predictive values: 100% and 98.4%, respectively). CONCLUSION In spite of a poor antigen sensitivity with optimal performances only for viral load >10 million copies/mL, D4G performances in acute HIV-1 infection were enhanced by the addition of p24 testing to the antibody. Improved HIV rapid testing to shorten the window period is important as rapid tests play a major role in expanding access to HIV testing and preventing HIV transmission.

Immune Reconstitution and Early Infectious Complications Following Nonmyeloablative Hematopoietic Stem Cell Transplantation

Abstract Non-myeloablative stem cell transplantation (NMT) has been increasingly used in compromised patients who would otherwise have been unable to undergo allotransplant. There is little understanding of the kinetics of immune reconstitution and its influence on infective complications following NMT. The aim of present study was to evaluate lymphocyte subset reconstitution over the first 12 months post-transplant in 15 adult patients receiving NMT with comparison to that of 30 patients grafted with a conventional hemopoietic stem cell transplantation (HSCT). NMT recipients were conditioned with fludarabine-based conditioning regimens. Peripheral blood stem cell (PBSC) was the source of stem cells in 13 NMT recipients and in 24 conventional HSCT recipients. Absolute numbers of helper (CD4+) T cells, naive (CD4+ CD45RA+) and memory (CD4+ CD45RO+) T cells as well as suppressor (CD8+) T cells, CD19+ B cells and NK cells were comparable in the two groups at all time points after transplantation. A median value of 200 CD4+ T cells/μl was achieved at 2 months post-transplant by the NMT and HSCT recipients. The CD4:CD8 ratio remained severely depressed throughout the study period. Almost all CD4+ lymphocytes expressed CD45RO antigen in the both groups of patients B lymphocytes showed low counts throughout the entire study period in both groups. Bacteremia and CMV antigenemia occurred respectively in 13 and 36% of the patients in the NMT group and in 15 and 39% of the patients in the HSCT group. Our preliminary data indicate that patients receiving a NMT have a lymphocyte reconstitution similar to that observed in patients who received a conventional HSCT. The incidence of bacteremia and CMV infection were not significantly different between the groups. Nevertheless, due to the small sample size, these results should be considered suggestive rather than definitive.

Delayed Acquisition of High-Avidity Anti-Cytomegalovirus Antibody Is Correlated with Prolonged Antigenemia in Solid Organ Transplant Recipients

Previous studies have demonstrated that maturation of cytomegalovirus (CMV)-specific antibodies in solid organ transplant recipients is delayed after primary CMV infection. To investigate the clinical significance of this finding, the avidity indices of anti-CMV antibody were determined in parallel with other serologic and virologic parameters in serial serum samples from 24 solid organ transplant recipients who had primary CMV infection that began during the first 3 months after transplantation. The data obtained show that a delay in antibody maturation is significantly correlated with a long persistence of positive antigenemia.

Multicenter comparative study of Epstein–Barr virus DNA quantification for virological monitoring in transplanted patients

BACKGROUND EBV-related post-transplant lymphoproliferative diseases are usually accompanied by increased EBV DNA in peripheral blood. Monitoring EBV DNAemia is the basis for weighing decisions regarding initiation of pre-emptive or anti-EBV-related tumor therapy. However, the definition of clinically relevant cut-off values is hampered by the lack of standardization in EBV DNA testing. OBJECTIVES To estimate inter-laboratory variability and to evaluate the impact of different matrices in EBV DNA load determination in Italian laboratories involved in monitoring of virus infections in transplanted patients. STUDY DESIGN Two different proficiency panels were distributed among seven centers: the first contained cell-associated and cell-free EBVs; the second was prepared by spiking both cell-associated and cell-free EBVs in EBV DNA-negative whole blood from EBV seropositive healthy donors. Samples were extracted and amplified with different methods. Intra-laboratory and inter-laboratory variabilities was evaluated. RESULTS 337 EBV DNA determinations were performed. Sensitivity was 100% for both panels, specificity was 100% for the first and 74% for the second panel, where whole blood was utilized as the matrix. Discrepant results in the second panel were restricted to samples containing low copy numbers. Quantification fell within ±0.5 log in 73% of the determinations. Values for cell-associated samples tended to be more heterogeneous than those obtained from cell-free samples. Good overall linearity was observed for each sample type; inter-laboratory variability ranged from 4.71% to 12.86%. CONCLUSIONS The results of this multicenter study indicate that EBV DNAemia may be reliably quantified by different laboratories using a variety of commercial and in-house molecular assays.

Kinetics and prediction of HBsAg loss during therapy with analogues in patients affected by chronic hepatitis B HBeAg negative and genotype D

In patients affected by chronic hepatitis because of HBV infection, long‐term suppressive therapy with nucleos(t)ides analogues in the HBeAg− patients has shown low effects on HBsAg titre (qHBsAg) decrease, and HBsAg loss is difficult to achieve. Thus, in this type of patients the main goals of antiviral therapy is the suppression of HBV‐DNA and ALT normalization.