BackgroundProtease inhibitors (PIs) to treat hepatitis C (HCV) virus infection have been approved and others are under development.ResultsThe aims of this study were to illustrate natural polymorphisms in the HCV protease and measure the frequency of PI resistance mutations in different HCV genotypes from PI-naïve patients.Direct sequencing of HCV NS3/4A protease was performed in 156 HCV patients naïve to PIs who were infected with genotype 1a (n = 31), 1b (n = 39), 2 (n = 30), 3 (n = 33) and 4 (n = 23).Amino acid (aa) substitutions associated with HCV PI resistance were found in 17/156 (10.8%) sequences. Mutations V36L, T54S, V55A/I, and Q80K/L were observed in 29% of patients with genotype 1a, and V55F, Q80L/N and M175L in 10% of patients with genotype 1b. The mutation V158M was found in 3% of patients with genotype 2, D168Q was present in 100% of patients with genotype 3 and D168E was observed in 13% of patients with genotype 4. In addition, multiple aa polymorphisms not associated with PI resistance were detected in patients with genotypes 1a, 1b and 4.ConclusionsAlthough major PI resistance mutations were not detected, other resistance mutations conferring low level resistance to PIs together with a number of natural polymorphisms were observed in proteases of PI naïve HCV patients. A more extensive analysis is needed to better evaluate the impact of baseline resistance and compensatory mutations in the efficacy of HCV PI treatment.

Epstein-Barr virus (EBV) DNA levels in whole-blood samples of 54 pediatric patients receiving T-celldepleted haploidentical hematopoietic stem cell transplantation (HSCT) in 2003 to 2007 were retrospectively compared with EBV DNA loads in peripheral blood mononuclear cells (PBMC). Determination of EBV DNA in whole blood missed 1 of 19 patients (5.2%), who tested positive for EBV DNA in PBMC. The analytical sensitivity of EBV DNA detection in whole-blood samples relative to that in PBMC was 94.7%. Regression analysis showed a significant correlation between DNA levels in PBMC and whole blood (r ‫؍‬ 0.81; P < 0.001). Relative to that in PBMC, the appearance of EBV DNA in whole blood was delayed in 9/18 patients (median, 49 days; range, 6 to 226 days), while peak levels and clearance were reached simultaneously. Following peak levels, EBV DNA showed a slower decline in whole blood than in PBMC. In conclusion, (i) EBV DNA levels in PBMC were significantly correlated with those in whole blood; (ii) a differential kinetics of EBV DNA in the two blood compartments was observed; and (iii) monitoring of EBV DNA levels in whole blood appears to be a valuable alternative to PBMC in the follow-up of pediatric recipients of haploidentical T-cell-depleted HSCT.Epstein-Barr virus (EBV)-associated posttransplantation lymphoproliferative disorders (PTLD) represent a life-threatening complication for recipients of solid-organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT) (2, 11, 16). The heterogeneous clinical manifestations of PTLD are mostly due to B-lymphocyte proliferation driven and sustained by EBV latency products (5,11,22). In immunocompetent individuals, uncontrolled EBV-driven B-cell proliferation is prevented mainly by virus-specific cytotoxic T lymphocytes (CTL), while in transplant recipients, impaired CTL control of EBV infection due to pharmacological immune suppression or physical removal of T lymphocytes favors the development of PTLD (11,16).Recipients of T-cell-depleted, HLA-haploidentical HSCT are exposed to the highest risk of developing PTLD (8), because they cannot benefit from adoptive transfer of virus-specific CTL, and a timely diagnosis of EBV-driven lymphoproliferation is mandatory for prompt therapeutic intervention in this severe complication. Preemptive treatment of PTLD in HSCT includes infusion of an anti-CD20 monoclonal antibody (MAb), tapering of immune-suppressive regimens (whenever possible), and adoptive infusion of donor-derived EBV-specific CTL in patients at risk for PTLD (6, 26).The EBV DNA load in blood is considered to reflect EBVinduced cell proliferation, and quantitative determination of EBV DNA levels in peripheral blood mononuclear cells (PBMC), plasma, or whole blood has been proposed as a means to identify SOT or HSCT recipients at risk for developing PTLD (1, 4, 7, 10, 12, 14-17, 19-21, 24, 25). While it is accepted that EBV DNA levels in the blood of patients with EBV-related PTLD are significantly higher than those in healthy EBV-seropositive indivi...

Transplantation Centers using human cytomegalovirus (HCMV) antigenemia-based preemptive therapy will need to replace in the near future the antigenemia assay with a more standardized and automatable assay, such as a molecular assay quantifying HCMV DNA in blood (DNAemia). Thus, in view of replacing antigenemia with clinically safe cutoff values, DNAemia levels corresponding to antigenemia cutoffs guiding HCMV preemptive therapy were determined retrospectively in solid organ and hematopoietic stem cell transplant recipients (HSCTR) using an "in-house" quantitative PCR (QPCR) method. Since preemptive therapy had prevented appearance of HCMV disease in all patients tested, DNA cutoffs determined retrospectively had to be considered as safe clinically as antigenemia cutoffs used prospectively. However, in solid organ transplant recipients (SOTR), initiating preemptive therapy upon an antigenemia cutoff of 100 pp65-positive leukocytes, a DNAemia cutoff of 300,000 copies/ml blood had positive and negative predictive values of >90%, indicating that a DNAemia cutoff could achieve, in terms of prevention of HCMV disease, the same clinical results as the antigenemia cutoff. In HSCTR, initiating preemptive therapy upon first antigenemia positivity, a DNAemia cutoff of 10,000 copies/ml blood had a positive predictive value of >90%, indicating that the great majority of patients treated under the antigenemia guidance would have been treated also using this DNA cutoff. On the other hand, the negative predictive value of 28.6% indicated that two out of three HSCTR had been treated under the antigenemia guidance having the same levels of viral DNA as the untreated patients. The data suggest that a quantitative cutoff could be adopted as a guiding criterion for preemptive therapy also in HSCTR. Regression analysis allowed to determine the DNAemia (corresponding to QPCR) cutoff values for two commercial assays tested both in solid organ and HSCTR. Retrospective DNAemia cutoff values will be verified for safety in prospective trials.

Summary. Post-transplant lymphoproliferative disorders (PTLDs) are heterogeneous severe complications occurring in 1-10% of transplanted patients. In most cases, PTLDs are associated with Epstein-Barr virus (EBV) infection but, recently, some clinical studies have reported an increasing number of EBV-negative PTLDs. Several studies have emphasized the critical role of the early identification of patients at risk for PTLD, in prompting the adoption of either pre-emptive strategies or timely treatment. To this purpose, monitoring of EBV DNA load in peripheral blood mononuclear cells is considered to be a useful test. Moreover, recently, the role of interleukin (IL)-10 in EBV-related diseases has been remarked, and high levels of IL-10 have been detected in PTLD patients. In this study, both EBV load and IL-10 were monitored in 38 PTLD patients at diagnosis and during follow-up, as well as in a control group, in order to establish the diagnostic role of the two tests, their relationship with the different PTLD subsets (EBV-positive and EBV-negative) and their behaviour during treatment. Results of our study suggest that the usefulness of IL-10 assay for early diagnosis of PTLD is similar to that of EBV load quantification, and its clinical diagnostic value is lower in EBV-negative than in EBV-positive PTLDs.