Ann Neurol 2011;70:742C750. JCPyV-infected patients, and JCPyV DNA was detected in 50% of the patients without anti-JCPyV antibodies. When JCPyV DNA was investigated in blood and urine the frequency of contamination was higher than previously described. Conclusion: Under these experimental conditions, with respect to the observed frequency of JCPyV contamination, the sensitivity of the anti-JCPyV antibody assay was lower than expected. Progressive multifocal leukoencephalopathy (PML) caused by JC polyomavirus (JCPyV) reactivation is the most severe adverse event observed in patients with relapsing-remitting multiple sclerosis (RRMS) treated with natalizumab.1 In order to predict PML risk, a biological marker of JCPyV infection is needed. Because of the intermittent nature of JCPyV productive contamination and because JCPyV DNA detection by PCR assay may be elusive, the presence of anti-JCPyV WIN 55,212-2 mesylate antibodies (Abs) seems to be a marker of contamination that is more stable over time. Based on this assumption, an assay that is highly sensitive and specific for the detection of anti-JCPyV Abs in serum (Stratify) was developed.2 In addition, an algorithm based on this assay has been developed and validated for PML risk stratification in patients with multiple sclerosis (MS) treated with natalizumab.3 However, the actual frequency of JCPyV-infected patients in MS patient populations is still controversial, mainly because it depends around the JCPyV DNA identification method. Here the sensitivity of the WIN 55,212-2 mesylate Stratify test in patients with MS was evaluated by comparing the frequency of anti-JCPyV Abs with the frequency of JCPyV DNA in different body fluid compartments. METHODS Patients. Paired blood and urine samples from 39 patients who were consecutively referred to the outpatient clinic of the Regional Reference Center for Multiple Sclerosis of the Careggi University Hospital, Florence, Italy, were prospectively collected. The patients were recruited according to the following inclusion criteria: definite diagnosis of MS; relapsing-remitting course; fulfillment WIN 55,212-2 mesylate of the European Medicines Agency indications for natalizumab administration in MS; and written informed consent to participate in a program for PML risk stratification established in collaboration with the Marketing Authorization Holder (MAH) of natalizumab and based on a centralized anti-JCPyV Ab assay in sera and on a locally organized JCPyV DNA analysis in blood and urine. The results included in the present study were generated by analyzing the diagnostic data reported in the clinical records. The study was approved by the local Ethic Committee (Comitato Etico Azienda Ospedaliero-Universitaria Careggi, Largo Brambilla 3, Firenze) according to the Italian legislation requirements (n. OSS 14.073). The authors obtained preventive unrestricted permission for analyzing the data provided by the MAH of the anti-JCPyV Ab assay. JCPyV PCR quantification. The JCPyV DNA was extracted from peripheral blood mononuclear cells (PBMC) obtained from enriched buffy coats after density gradient separation of 8 mL of whole blood, 200 L of plasma, and 200 L of urine, and was quantified by JCPyV-specific real-time PCR targeting the large T gene performed in triplicate using 100 ng of DNA (forward primer 5-GCAGCTTAGTGATTTTCTTAGG-3, reverse primer 5-GAACACAGGTGTTTCCACCTGG-3, and TaqMan MGB probe Fam-5-GGCACTGAATATTCATTCATGG3). Each assay was carried out with negative controls (no template) and DNA 10-fold dilutions (101C106 copies) of a plasmid carrying the amplicon cloned (standard curve plot was 101C106 copies vs 36-21 cycle threshold). The low limit of quantification from the JCPyV DNA was 100 copies per mL of plasma Rabbit Polyclonal to U12 and urine or per g of PBMC DNA. JCPyV antibody assay. Anti-JCPyV Abs had been recognized in serum with a 2-stage virus-like particle-based ELISA assay (Stratify, Concentrate Diagnostics, supplied by Biogen Idec).4 Individuals who tested positive for JCPyV DNA in bloodstream or urine but who had undetectable serum antibodies had their serum examples retested for anti-JCPyV Abs. Outcomes Demographic and medical features and JCPyV disease status from the 39 individuals with RRMS are reported in desk 1. The bloodstream and urine examples had been gathered at baseline and through the therapy biannually, from 2008 to Sept 2012 July. Individuals received between 0 and 35 infusions and added between 1 and 8 examples. We contained in the research the 1st positive isolation of JCPyV DNA as well as the anti-JCPyV Ab assay examined in the serum test from the same patient gathered concurrently or.
