Statistical correlation between enterovirus genome copy numbers and infectious viral particles in wastewater samples

Aims: Classic virological tests are time consuming and labour-intensive; realtime RT-PCR has proven to be a fast method to detect and quantify enterovirus genomes in clinical and environmental samples. This method is unable to discriminate between infective and noninfective enterovirus particles; few clinical studies have compared real-time RT-PCR and viral culture. We wondered if the enterovirus genome quantification could be correlated to the infectivity. Methods and Results: We used the statistical approach to verify our hypotheses to correlate data, obtained by the standard method (most probable number of cytopathic units—MPNCU) and molecular test (real-time RT-PCR), on wastewater treatment plant samples. Chi-squared test was used, considering several cut-off values (‘50’-‘100’-‘200’ genome copy numbers), to determine statistical significance in comparison of the two methods. Chi-square value was not significant when cut-off of 50 (P = 0Æ103) and 100 (P = 0Æ178) was assumed but was significant with cut-off of 200 (P = 0Æ044). Conclusion: This limit, 200 genome copy, could be used as cut-off value to indicate enterovirus survival in environmental monitoring. Significant and Impact of the Study: To introduce a fast procedure that is able to compensate for disadvantages of cell culture method for viral environmental analyses

Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

<p>Abstract</p> <p>Background</p> <p>Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.</p> <p>Results</p> <p>EBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 10²to 1.3 × 10⁸copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10³to 2.0 × 10⁵copies/ml in infectious mononucleosis (n = 7), 7.5 × 10⁴to 1.1 × 10⁵copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10²to 5.6 × 10³copies/ml in HIV-infected patients (n = 12), and 2.0 × 10²to 9.1 × 10⁴copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).</p> <p>Conclusion</p> <p>Two sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.</p