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

BACKGROUND: Epstein-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). RESULTS: The 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 (R(2 )= 0.7789; p < 0.0001), with the systematic overestimation by EBV RQ-PCR possibly linked to different amplification efficiency in calibration standards. CONCLUSION: Both 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

Heterogeneity and penetration of HIV-1 non-subtype B viruses in an Italian province: Public health implications

SUMMARYThis study assessed changes in prevalence and distribution of HIV-1 non-subtype B viruses in Italian and immigrant patients over two decades in a province in Italy. All HIV-positive patients who underwent genotypic resistance testing were selected. Prevalence of non-subtype B viruses in 3-year periods was calculated. All sequences of non-subtype B and those provided by REGA as unassigned were analysed for phylogenetic relationships. In total, 250/1563 (16%) individuals were infected with a non-subtype B virus. Prevalence increased over time, reaching a peak (31·5%) in 2004–2006. In Italian patients, the most frequent subtypes were B (92·5%) and F1 (4%). F1 subtype was also prevalent in patients from South America (13·6%); in patients of African origin, CRF02_AG (54·9%) and G (12·3%) were the most frequent. HIV-1 non-subtype B infections in Italians were mostly found in patients who acquired HIV sexually. A phylogenetic relationship between F subtypes in Italian and representative HIV-1 sequences from Brazil was found. C subtypes in Italians were phylogenetically related to subtypes circulating in Brazil. Inter-subtype recombinants were also found in the latest years. The HIV-1 epidemic in Brescia province evolved to the point where about 1/3 patients recently diagnosed harboured non-B HIV subtypes. The distribution of HIV-1 non-B subtypes in Italian patients resembled that in South American patients and phylogenetic relatedness between some Italian and South American HIV-1 strains was found. The possible epidemiological link between these two populations would have been missed by looking only at risk factors for HIV acquisition declared by patients. The evidence of inter-subtype recombinants points to significant genetic assortment. Overall our results support phylogenetic analysis as a tool for epidemiological investigation in order to guide targeted prevention strategies

HIV Infection among Illegal Migrants, Italy, 2004–2007

To determine HIV prevalence and place of exposure for illegal migrants in Italy, we tested 3,003 illegal adult migrants for HIV; 29 (0.97%) were HIV positive. Antibody avidity index results (indicators of time of infection) were available for 27 of those persons and showed that 6 (22.2%) presumably acquired their infection after migration

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

<p>Abstract</p> <p>Background</p> <p>Quantitative 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.</p> <p>Methods</p> <p>475 consecutive samples from 156 immunosuppressed patients were tested for HCMV by pp65 antigenemia and Real-time PCR assay.</p> <p>Results </p> <p>136 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.</p> <p>Conclusion</p> <p>Both 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.</p

Comparison of HIV-1 Genotypic Resistance Test Interpretation Systems in Predicting Virological Outcomes Over Time

Background: Several decision support systems have been developed to interpret HIV-1 drug resistance genotyping results. This study compares the ability of the most commonly used systems (ANRS, Rega, and Stanford's HIVdb) to predict virological outcome at 12, 24, and 48 weeks. Methodology/Principal Findings: Included were 3763 treatment-change episodes (TCEs) for which a HIV-1 genotype was available at the time of changing treatment with at least one follow-up viral load measurement. Genotypic susceptibility scores for the active regimens were calculated using scores defined by each interpretation system. Using logistic regression, we determined the association between the genotypic susceptibility score and proportion of TCEs having an undetectable viral load (<50 copies/ml) at 12 (8-16) weeks (2152 TCEs), 24 (16-32) weeks (2570 TCEs), and 48 (44-52) weeks (1083 TCEs). The Area under the ROC curve was calculated using a 10-fold cross-validation to compare the different interpretation systems regarding the sensitivity and specificity for predicting undetectable viral load. The mean genotypic susceptibility score of the systems was slightly smaller for HIVdb, with 1.92±1.17, compared to Rega and ANRS, with 2.22±1.09 and 2.23±1.05, respectively. However, similar odds ratio's were found for the association between each-unit increase in genotypic susceptibility score and undetectable viral load at week 12; 1.6 [95% confidence interval 1.5-1.7] for HIVdb, 1.7 [1.5-1.8] for ANRS, and 1.7 [1.9-1.6] for Rega. Odds ratio's increased over time, but remained comparable (odds ratio's ranging between 1.9-2.1 at 24 weeks and 1.9-2.

SIAMOC position paper on gait analysis in clinical practice: General requirements, methods and appropriateness. Results of an Italian consensus conference

Gait analysis is recognized as a useful assessment tool in the field of human movement research. However, doubts remain on its real effectiveness as a clinical tool, i.e. on its capability to change the diagnostic-therapeutic practice. In particular, the conditions in which evidence of a favorable cost-benefit ratio is found and the methodology for properly conducting and interpreting the exam are not identified clearly. To provide guidelines for the use of Gait Analysis in the context of rehabilitation medicine, SIAMOC (the Italian Society of Clinical Movement Analysis) promoted a National Consensus Conference which was held in Bologna on September 14th, 2013. The resulting recommendations were the result of a three-stage process entailing i) the preparation of working documents on specific open issues, ii) the holding of the consensus meeting, and iii) the drafting of consensus statements by an external Jury. The statements were formulated based on scientific evidence or experts' opinion, when the quality/quantity of the relevant literature was deemed insufficient. The aim of this work is to disseminate the consensus statements. These are divided into 13 questions grouped in three areas of interest: 1) General requirements and management, 2) Methodological and instrumental issues, and 3) Scientific evidence and clinical appropriateness. SIAMOC hopes that this document will contribute to improve clinical practice and help promoting further research in the field

Evaluation of a new quantitative test for HCV core Ag

Introduction.The quantitative determination of viral antigens is a diagnostic innovation. The availability of an automated test for detecting the “core” antigen of the hepatitis C virus (HCV) allowed to assess its characteristics and potential applications. Methods.The Abbott ARCHITECT HCV core Ag assay is a fully automated CMIA measuring HCV core antigen at concentrations between 3 and 180,000 fmol / L. The evaluation has been conducted on anti-HCV positive samples at various levels: on samples for which the quantitative (bDNA) and / or qualitative (TMA) HCV-RNA have been tested and on samples of hemodialysis patients. Results.A positivity for HCV Ag was detectable in 10/142 (7.0%) sera with single anti-core reactivity at additional RIBA 3 test while 58 samples reactive to NS3 or NS4 or NS5 were all HCV Ag negative.The frequency of Ag positivity was correlated with the anti-HCV signal (at anti-HCV ARCHITECT), ranging from 71% in samples with S / CO&gt; 5 to 6% in samples with S / CO &lt;5. The antigen was detectable at low concentrations (average 50±1.17 fmol / L) in 8 / 53 samples (15.1%) with viremia below 500 IU / mL. On samples with quantifiable HCV-RNA, the correlation between bDNA and HCV Ag was very good. Of the 65 examined hemodialysis patients, 46 were positive for both antibodies and antigen, 15 for only antibodies, 3 negative for both and one was highly positive for HCV Ag (and HCV-RNA), but anti-HCV negative. Conclusion. The sensitivity and the practicality of the new quantitative test for HCV Ag allow to envisage the use for the evaluation of active HCV infection in anti-HCV positive patients, monitoring patients at risk of infection and as a complement of quantitative viremia

Performance evaluation of the automated NucliSens EasyMAG nucleic acid extraction platform in comparison to QIAamp Mini Kit from clinical specimens.

The performance of the NucliSens easyMAG platform for the extraction of nucleic acid from different clinical specimens was compared with a manual procedure. A total of 308 specimens were analyzed: 209 plasma samples collected for virus detection and quantification of cytomegalovirus (CMV) and Epstein–Barr virus (EBV) (n = 70), and 29 for HIV genotyping for drug resistance. Linearity of extraction was tested on dilution series of CMVand EBV; the correlation coefficient (R2) for standard curves based on repeated extraction runs was 0.99 for CMV and EBV. Inter- and intrarun variability was in accordance with previous studies, and the correlation between automated and manual extraction was very high. The concordant results were 95.7% for CMV and 100% for EBV. The results of sequence analysis for HIV drug resistance showed a concordance in 24 of 29 specimens. The NucliSens easyMAG is extremely easy to perform, is automated, and resulted in a strong reduction of hands-on time compared with manual protocol