Elisabetta Cariani

Relationship between pp65 antigenemia levels and real-time quantitative DNA PCR for Human Cytomegalovirus (HCMV) management in immunocompromised patients

BackgroundQuantitative real-time PCR assays, which are more rapid and practical than pp65 antigenemia determination, are progressively becoming the preferred method for monitoring Human Cytomegalovirus (HCMV) reactivation. However, the relationship between HCMV DNA and antigenemia levels is still under investigation. The aim of this study was to analyse the relationship between HCMV DNA and pp65 antigenemia levels in order to identify clinically useful threshold values for the management of patients.Methods475 consecutive samples from 156 immunosuppressed patients were tested for HCMV by pp65 antigenemia and Real-time PCR assay.Results136 out of 475 consecutive samples derived from 48 patients showed evidence of HCMV infection. HCMV DNA was detected in 106 samples, pp65 antigen in 3, and both markers in 27. pp65 antigen detection was associated with higher HCMV DNA levels. The cut-off HCMV DNA level that best predicted pp65 antigenemia in this series of samples was 11,500 copies/ml, but different threshold levels could be observed for specific groups of patients. HCMV disease was observed in 5 out of 48 patients with active HCMV infection. The presence of clinical symptoms was associated with positive pp65 and with higher antigenemia levels. Higher HCMV DNA load at the onset of viral replication was correlated to the development of clinical symptoms.ConclusionBoth pp65 antigenemia and HCMV DNA load can be useful for the prospective monitoring of immunocompromised subjects. Specific cut-off levels capable of triggering preemptive antiviral treatment should be determined in accordance to the type of test used and the characteristics of patients and prospectively validated.

Analysis of the hepatitis B virus genome and immune response in HBsAg, anti-HBs positive chronic hepatitis

Although the development of antibodies against the hepatitis B virus surface antigen generally leads to the clearance of the infecting virus, anti-HBs reactivity has been reported in patients with chronic hepatitis. In the present study we analyzed the viral genome and the antibody specificity in a series of serum samples collected from a patient who seroconverted to anti-HBs during interferon therapy without clearing HBsAg. The appearance of an anti-HBs response was accompanied by the emergence of a pre-S1 defective viral genome. However, the wild-type adw2 molecular species remained largely dominant during follow up. The patients antibody response to the surface viral antigens was directed towards the heterologous y subdeterminant and the pre-S1 fragment deleted in the variant hepatitis B virus. These results suggest that the selection of the escape viral mutant does not play a major role in viral persistence.

Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma

BackgroundEpstein-Barr virus (EBV) DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy) and an in-house assay (EBV RQ-PCR).ResultsThe range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R2 = 0.7789; p < 0.0001), with the systematic overestimation by EBV RQ-PCR possibly linked to different amplification efficiency in calibration standards.ConclusionBoth the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement.

Differential pattern of sequence heterogeneity in the hepatitis C virus E1 and E2/NS1 proteins

The E1 and E2/NS1 genes, encoding the putative hepatitis C virus envelope proteins, show a high rate of sequence variations. We analyzed the degree and distribution of sequence heterogeneity in serum samples from hepatitis C virus-infected subjects. The mutations in the E1 region were mainly type-specific and the rate of variability was apparently not linked to the clinical phase of the infection. The sequence evolution of the E1 region during interferon treatment was low, regardless of the response to therapy. In contrast, an increased degree of variation, apparently related to the stage of viral replication, was present in E2 region derived from patients undergoing interferon treatment. These results are consistent with the hypothesis that the E2 protein represents a major target of the immune response.

Diagnosis of viral hepatitis with a nonisotopic hybridization assay

The DNA enzyme immunoassay is an efficient method for the screening of PCR products derived from different hepatitis virus genomes, and allows to bypass both agarose gel electrophoresis and Southern blot hybridization with radioactively labeled probes. A wider application of this method will disclose new perspectives for the introduction of PCR in clinical laboratories.