Microsphere-based IgG avidity assays using as model human parvovirus B19 and CMV

Abstract

No: 1625 Presentation at ESCV 2015: Poster 2 Microsphere-based IgG avidity assays using as model human parvovirus B19 and CMV Yilin Wang1, L. Hedman1, V. Nurmi1, I. Ziemele2, M. Lappalainen3, M. Soderlund-Venermo1, K. Hedman1,3 1 Virology, University of Helsinki, Finland 2 Riga Stradins University, Riga, Latvia 3 HUSLAB, Helsinki University Hospital, Finland Background: Human parvovirus B19 (B19V) and cytomegalovirus (CMV) are ubiquitous pathogens that may lead to severe intrauterine infections. Thus, the serological status of the mother is important in counselling and recognition of acute infection. IgG-aviditymeasurement differentiates recent from past infection and it is useful in timing of primary infections. A bead based-Suspension Immuno Assay (SIA; Luminex®) simultaneously detects multiple analytes in a single test. This multiplex array could be a feasible approach to high throughput diagnostics with its related efficiency, in terms of time, cost and sample consumptions compared to the currently used methods based en enzyme immune-assay. We developed singleplex and duplex IgG-avidity SIAs based on low-avidity antibody elution by urea. Methods: B19V and CMV IgG titres before and after urea treatment were measured by SIAs. We used as antigens (i) prokaryotic fusion protein containing the B19 VP1 unique region and (ii) HCMV (AD169 stain) purified viral lysate conjugated onto colourcoded Luminexmagneticmicrospheres. Captured antibodies by the microspheres were visualized with secondary antibodies bound with Streptavidin-phycoerythrin (SA-PE). Avidity indices were calculated from end-point titres of IgG by use of three different mathematical methods. The new methods were evaluated in relation to in-house and commercial reference assays, using serum samples from well-characterized cohorts. Results: The B19V and CMV IgG avidity SIAs both as singleplex, or in a duplex format, efficiently excluded acute infection from past immunity. The three different mathematical methods showed similarly good performances. Conclusion: B19V and CMV IgG-avidity SIAs were successfully set up in singleplex and duplex formats. The IgG-avidity SIAs were highly sensitive and specific in the identification of primary infection and differentiation of acute from past infection. The newly developed avidity SIAs relative to corresponding commercial or in-house EIAs are cost-beneficial. http://dx.doi.org/10.1016/j.jcv.2015.07.240 Abstract No: 1629 Presentation at ESCV 2015: Poster 2 Aanalytical performance of VERIS MDx system HCV assay for quantifying HCV RNA K. Saune ∗, C. Hasle, J. Boineau, F. Abravanel, C. Mengelle, J. Izopet Department of Virology, Federative Institute of Biology, CHU Toulouse, France Background: To evaluate the performances of the VERIS/MDx System HCV Assay (Beckman–Coulter), a new fully automated system for HCV RNA quantitation in plasma. Results were compared to the COBAS® Ampliprep/COBAS® TaqmanTM (CAPCTM) HCV Test (Roche). Methods: Precision was performed on 5 QC controls, at different HCV RNA levels (7.9 log IU/ml; 5.0 log IU/ml; 3.4 log IU/ml; 1.6 log IU/ml; 0 IU/ml), tested in duplicate for 20 days. Analytical specificitywas assessed on 180 plasma fromnegative anti-HCV andHCV RNA blood donors. The limit of detection was determined using serial dilutions ofHCVWHOstandard run in replicates of 12per day for3daysusingProbit analysis. Linearitywasassessedusing4 replicate of a serially diluted clinical plasma from 6.4 to 0.6 log IU/ml. Method comparison between VERIS MDx HCV assay and CAPCTM HCV assay was analyzed using Passing–Bablok linear regression on 198patient sampleswithknowngenotype infection for140of them (G1, n=69; G2, n=16; G3, n=27; G4, n=24; G5, n=2; G6, n=2). Bland–Altman for overall bias and Spearman correlation was also performed on positive samples. Results: For precision, the standard deviation was ≤0.11 log IU/ml. The analytical specificity was 100%. The limit of detection was 16.4 IU/ml (CI95%: 13.1–23.6 IU/ml). The linearity ranged from 1.5 to 6.4 log IU/ml. The Passing–Bablok regression showed VERIS log IU/ml =−0.33+1.04×CAPCTM log IU/ml with bias of −0.18, −0.10 and −0.06 log IU/ml for 25th, 50th, 75th percentiles respectively. The Spearman correlation showed a good correlation between the two assays (r=0.92, p 0.5 log IU/ml were observed more frequently for genotype 4 (59%) than for the others (G1: 7%; G2: 6%; G3: 15%). http://dx.doi.org/10.1016/j.jcv.2015.07.241