Evaluation of CMV DNA in dried blood spot.

Cytomegalovirus is the primary viral cause of congenital infection. However, diagnosis may be difficult for clinical and technical reasons. Currently, evaluation of CMV DNA on dried blood spot (DBS) is an important instrument to define a congenital infection. The aim of this study was to identify a clinically and technically suitable diagnostic work-flow for CMV DNA evaluation on DBS. Sensitivity was not significantly influenced by storage time of up to 12 months and extraction technique; however, analysis in triplicate was crucial to obtain reliable results. Considering viral load in an infected foetus at risk of developing disease, a threshold value of approximately 104 copies/mL was characterized by high operating characteristics for detection of positivity at 12 months on DBS.

Detection of human cytomegalovirus in transbronchial biopsies from lung transplant recipients.

The role of human cytomegalovirus (HCMV) in lung transplantation (LT) and drawbacks related to viral quantification in bronchoalveolar lavage (BAL) underline the potential usefulness of investigating other specimens. Thirty-three LT recipients were prospectively studied by HCMV quantitative real time PCR on matched transbronchial biopsy (TBB), BAL, and whole blood specimens. Overall, 27/33 patients turned out HCMV-positive in at least one specimen: 7.1 %, 37.1 %, and 13.5 % of TBB, BAL, and blood samples, respectively. No significant association between HCMV on all types of specimens and acute rejection, lymphocytic bronchiolitis, bronchiolitis obliterans and bronchiolitis obliterans syndrome was found. HCMV pneumonia was associated to HCMV detection on TBB (p = 0.003) and whole blood (p = 0.008), not on BAL (p = 0.47). The highest mean viral load was detected in TBB from cases with HCMV pneumonia in comparison to all other cases, suggesting the potential use of HCMV investigation in TBB for evaluating posttransplant complications.

Evaluation of CMV-specific cellular immune response by EliSPOT assay in kidney transplant patients.

BACKGROUND: Immunological monitoring for CMV can be useful in transplant patients; however, few centers perform it on a routine basis. OBJECTIVES: In this study, CMV-specific cellular response was evaluated in a population of kidney transplant recipients and related to viral infection/reactivation and other demographic and clinical features. STUDY DESIGN: Three hundred and twenty-eight patients were studied by EliSPOT assay: 201 prospectively monitored in the first year posttransplantation, 127 with a single determination at >1 year. Clinical features, including occurrence of CMV-DNAemia, CMV serostatus, anti-viral strategies and immunosuppressive protocols, were evaluated. RESULTS: Overall, 66.5% of patients were CMV-responders at EliSPOT assay. No episode of infection occurred at follow-up (mean 24.5 months) in 73.4% responders versus 55.5% non-responders (p<0.005); CMV-free period was significantly longer in responders (p<0.001). Although no significant difference of peak viral load was found, prevalence of CMV-DNAemia values >10(5)copies/mL was significantly higher in non-responders versus responders (8.2% and 2.3%, p<0.05). Non-responder status was significantly associated to CMV-seronegativity (p<0.0001), anti-viral prophylaxis use (p<0.0001), and immunosuppression induction with basiliximab (p<0.005). No significant association was found for other clinical features and immunosuppressive protocols. CONCLUSIONS: Immunological data for CMV could be used in the clinical evaluation and decision-making process, in combination with virological monitoring, in kidney transplant recipients.

Comparison of two nucleic acid extraction and testing systems for HCMV-DNA detection and quantitation on whole blood specimens from transplant patients.

Quantitative detection of human cytomegalovirus (HCMV) DNA on whole blood is currently the primary choice for virological monitoring in transplant patients and for determining the appropriate antiviral strategy, however specific issues of variability remain in terms of extraction methods, amplification efficiency, and variability. This study compared the performance characteristics of two nucleic acid extraction and testing systems for HCMV-DNA quantitation, the artus® CMV QS-RGQ kit, associated with a fully automated DNA extraction and assay set up by Qiagen (system 1) and the Q-CMV Real Time Complete kit by Nanogen, associated with a semiautomated nucleic acid extraction system by Biomérieux (system 2) in 189 specimens from transplant patients and 10 from 2012 HCMV Quality Control for Molecular Diagnostics (QCMD). The two systems exhibited a 80.4% concordance. Differences between the two systems were within ±1 log10 copies/ml of the averaged log10 results for 88.9% of the tested specimens. For all qualitatively discordant specimens, mean viral load was ≤3 log10 copies/ml. Considering viral load measurement, system 1 gave earlier positives that system 2, with a 14.8% of specimens resulted positive at low viral loads with system 1 and negative with system 2. In QCMD specimens, difference was below 0.7 log10 copies/ml for both the systems. In conclusion, the two systems provided reliable and comparable results. Some specific performance characteristic and automation could be taken into account in terms of less hands of time, fewer errors and reliability.

Prevalence and follow-up of occult HCV infection in an Italian population free of clinically detectable infectious liver disease.

BACKGROUND: Occult hepatitis C virus infection (OCI) is a recently described phenomenon characterized by undetectable levels of HCV-RNA in serum/plasma by current laboratory assays, with identifiable levels in peripheral blood mononuclear cells (PBMCs) and/or liver tissue by molecular tests with enhanced sensitivity. Previous results from our group showed an OCI prevalence of 3.3% in a population unselected for hepatic disease. The present study aimed to evaluate OCI prevalence in a larger cohort of infectious liver disease-free (ILDF) subjects. Clinical follow-up of OCI subjects was performed to investigate the natural history of the infection. METHODS AND FINDINGS: 439 subjects referred to a Turin Blood Bank for phlebotomy therapy were recruited. They included 314 ILDF subjects, 40 HCV-positive subjects and 85 HBV-positive subjects, of whom 7 were active HBV carriers. Six subjects (4/314 ILDF subjects [1.27%] and 2/7 active HBV carriers [28%]) were positive for HCV-RNA in PBMCs, but negative for serological and virological markers of HCV, indicating OCI. HCV genotypes were determined in the PBMCs of 3/6 OCI subjects two had type 1b; the other had type 2a/2c. OCI subjects were followed up for at least 2 years. After 12 months only one OCI persisted, showing a low HCV viral load (3.73×10(1) UI/ml). By the end of follow-up all OCI subjects were negative for HCV. No seroconversion, alteration of liver enzyme levels, or reduction of liver synthesis occurred during follow-up. CONCLUSIONS: This study demonstrated the existence of OCI in ILDF subjects, and suggested a high OCI prevalence among active HBV carriers. Follow-up suggested that OCI could be transient, with a trend toward the decrease of HCV viral load to levels undetectable by conventional methods after 12-18 months. Confirmation studies with a longer follow-up period are needed for identification of the OCI clearance or recurrence rates, and to characterize the viruses involved.