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Testing for Parvovirus B19 - Broadening the Assay to Cover Variants

Testing for Parvovirus B19 - Broadening the Assay to Cover Variants. Sally Baylis, NIBSC SoGAT XVII. Screening Plasma Pools for Parvovirus B19 - an OMCL Perspective. European Pharmacopoeia Monographs:

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Testing for Parvovirus B19 - Broadening the Assay to Cover Variants

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  1. Testing for Parvovirus B19 -Broadening the Assay to Cover Variants Sally Baylis, NIBSC SoGAT XVII

  2. Screening Plasma Pools for Parvovirus B19 - an OMCL Perspective • European Pharmacopoeia Monographs: • “Human anti-D immunoglobulin” & “Human anti-D immunoglobulin for intravenous administration” (Jan. 2004) • “Human plasma (pooled and treated for virus inactivation)” (July 2004) • Plasma pools should contain not more than 104 IU/ml parvovirus B19 DNA

  3. Variant Erythroviruses • V9 isolated from a child with transient aplastic crisis (Nguyen et al., 1998, 1999) • LaLi, K71 dermal isolates (Hokynar et al., 2002) • A6 isolated from an anaemic HIV-positive patient (Nguyen et al., 2002) • D91.1 isolated from a child with transient aplastic crisis (Servant et al., 2002) • Classification proposed by Servant et al., (2002) based upon sequence analysis of the NS1/VP1 region

  4. Genetic Diversity of Erythroviruses: Analysis of the NS1/VP1 Region A6 Servant et al., J. Virol., 2002

  5. Roche Parvovirus B19 Quantification Kit F1) - Genotype 1 Fluorescence (F2/Back Fluorescence (F1/F2) Genotype 2 NTC Genotype 3 M 1 1 3 3 2 2 NTC M Cycle Number Cycle Number Region amplified: NS1

  6. F1) - Fluorescence (F2/Back Cycle Number Artus RealArtTM Parvo B19 LC Kit Genotype 3 Genotype 2 Genotype 1 NTC M 1 1 3 3 2 2 NTC M Region amplified: VP1

  7. Sensitivity of Detection of Different Erythrovirus Genotypes

  8. In-house Erythrovirus TaqMan Assay • Consensus assay, primers & probe directed to the NS1 gene • Manufacturing plasma pools (Europe, North America) • Extraction using the MagNA Pure (Total Nucleic Acid, large volume) & real-time PCR on the LightCycler • Standard curve – WHO International Standard for parvovirus B19 (99/800)

  9. Genotype 3 dT d(F1)/ Genotype 1 – Genotype 2 Fluorescence NTC Temperature º C In-house Erythrovirus TaqMan Assay Genotype 3 Genotype 2 Genotype 1 Fluorescence (F1/F2) NTC Cycle Number

  10. Conclusions • The commercial assays have limitations in the detection and quantification of the variant erythroviruses • Of 58 plasma pools screened with the Roche & in-house TaqMan assays, results for parvovirus B19 are in agreement • No evidence currently for the presence of variant erythroviruses in manufacturing pools examined by NIBSC

  11. Discussion • Primer & probe design affects ability to detect and quantify variant viruses • Compliance with EP threshold concentration of 104 IU/ml may be compromised • Clinical significance, prevalence & geographical distribution of erythrovirus variants is still largely unknown • What are the implications in detecting a pools with high titres of a variant erythrovirus?

  12. Acknowledgements Kevin Brown, NIH Daniel Candotti & Jean-Pierre Allain, Cambridge Kati Hokynar & Klaus Hedman, University of Helsinki Annabelle Servant & Antoine Garbarg-Chenon, Paris

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