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: “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

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

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

Roche Parvovirus B19 Quantification Kit - 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

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

Sensitivity of Detection of Different Erythrovirus Genotypes

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)

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

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

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?

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