1 / 12

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:

landen
Télécharger la présentation

Testing for Parvovirus B19 - Broadening the Assay to Cover Variants

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related