Molecular Assays for Quantitative and Qualitative Detection of Influenza Virus and Oseltamivir Resistance Mutations Erhard van der Vries, Jeer Anber,

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Presentation transcript:

Molecular Assays for Quantitative and Qualitative Detection of Influenza Virus and Oseltamivir Resistance Mutations Erhard van der Vries, Jeer Anber, Anne van der Linden, Yingbin Wu, Jolanda Maaskant, Ralph Stadhouders, Ruud van Beek, Guus Rimmelzwaan, Albert Osterhaus, Charles Boucher, Martin Schutten The Journal of Molecular Diagnostics Volume 15, Issue 3, Pages 347-354 (May 2013) DOI: 10.1016/j.jmoldx.2012.11.007 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Linearity of the quantification assay. The linearity of the quantification assay was addressed by analyzing two series, each with 10-fold dilutions of influenza A (A/PR/8/34, white squares) and B (B/Lee/40, black squares) virus stocks counted using electron microscopy. CT values were plotted as a function of the viral load. Standard curves for influenza A and B virus were obtained by linear curve fitting. One influenza A virus dilution (asterisk) fell below the LLOQ and was excluded from the standard curve. The Journal of Molecular Diagnostics 2013 15, 347-354DOI: (10.1016/j.jmoldx.2012.11.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 LLOD and LLOQ of the quantification assay. A: LLOD values for influenza A and B virus were obtained by testing replicates (n = 48) of low-titer virus dilutions and 95% hit-rate probit analysis. The horizontal dotted line indicates the 95% confidence limit (CL). LLOD = 1.7 (CL = 1.6–2.2) log10 vp/mL for influenza A virus and LLOD = 2.2 (CL = 2.1–2.7) log10 vp/mL for influenza B virus. B: The coefficient of variation was plotted as a function of the viral load. LLOQ = 2.1 log10 vp/mL for influenza A virus (black line and white squares) and LLOQ = 2.3 log10 vp/mL for influenza B virus (dashed line and black squares), as determined by nonlinear curve fitting. The Journal of Molecular Diagnostics 2013 15, 347-354DOI: (10.1016/j.jmoldx.2012.11.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Effect of mismatches on performance of quantification assay as the approximated LLOD of the quantification assay if the matched RNA (white symbols) or the most dissimilar RNA (black symbols) was targeted. Probability of a positive result, as determined by 95% hit-rate analysis, was plotted as a function of the viral particle count per milliliter for influenza A (squares) and B (circles). The Journal of Molecular Diagnostics 2013 15, 347-354DOI: (10.1016/j.jmoldx.2012.11.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Longitudinal internal and external assay controls. A: CT values (white circles) of the universal internal standard (PDV), from analysis of 114 respiratory specimens isolated in 26 consecutive runs. B: CT values of the high (A−4, black squares), midrange (A−6, white squares), and low (A−8, black circles) viral load external standards for influenza A virus. The horizontal dashed line indicates the mean of the datapoints in the series; the gray region encompasses to ±2 SD from the mean. The Journal of Molecular Diagnostics 2013 15, 347-354DOI: (10.1016/j.jmoldx.2012.11.007) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions