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Testing for Parvovirus B 19 Broadening the Assay to Cover Variants NIBSC Sally Baylis, Testing for Parvovirus B 19 Broadening the Assay to Cover Variants NIBSC Sally Baylis, NIBSC So. GAT XVII

Screening Plasma Pools for Parvovirus B 19 - an OMCL Perspective n European Pharmacopoeia Screening Plasma Pools for Parvovirus B 19 - an OMCL Perspective n European Pharmacopoeia Monographs: n n n “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 B 19 DNA

Variant Erythroviruses n n n V 9 isolated from a child with transient aplastic Variant Erythroviruses n n n V 9 isolated from a child with transient aplastic crisis (Nguyen et al. , 1998, 1999) La. Li, K 71 dermal isolates (Hokynar et al. , 2002) A 6 isolated from an anaemic HIV-positive patient (Nguyen et al. , 2002) D 91. 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 NS 1/VP 1 region

Genetic Diversity of Erythroviruses: Analysis of the NS 1/VP 1 Region A 6 Servant Genetic Diversity of Erythroviruses: Analysis of the NS 1/VP 1 Region A 6 Servant et al. , J. Virol. , 2002

Fluorescence (F 2/Back -F 1) Roche Parvovirus B 19 Quantification Kit Fluorescence (F 1/F Fluorescence (F 2/Back -F 1) Roche Parvovirus B 19 Quantification Kit Fluorescence (F 1/F 2) Genotype 1 Genotype 2 NTC NIBSC M 1 1 3 3 2 2 NTC M Cycle Number Cycle Genotype 3 Number Region amplified: NS 1

Fluorescence (F 2/Back -F 1) Artus Real. Art. TM Parvo B 19 LC Kit Fluorescence (F 2/Back -F 1) Artus Real. Art. TM Parvo B 19 LC Kit Genotype 1 Genotype 2 Genotype 3 NTC M 1 1 3 3 2 2 NTC M Cycle Number Region amplified: VP 1

Sensitivity of Detection of Different Erythrovirus Genotypes Sensitivity of Detection of Different Erythrovirus Genotypes

In-house Erythrovirus Taq. Man Assay n n Consensus assay, primers & probe directed to In-house Erythrovirus Taq. Man Assay n n Consensus assay, primers & probe directed to the NS 1 gene Manufacturing plasma pools (Europe, North America) Extraction using the Mag. NA Pure (Total Nucleic Acid, large volume) & real-time PCR on the Light. Cycler Standard curve – WHO International Standard for parvovirus B 19 (99/800)

Fluorescence (F 1/F 2) In-house Erythrovirus Taq. Man Assay Genotype 3 Genotype 2 Genotype Fluorescence (F 1/F 2) In-house Erythrovirus Taq. Man Assay Genotype 3 Genotype 2 Genotype 1 NTC Cycle Number Fluorescence –d(F 1)/d. T Genotype 3 Genotype 1 Genotype 2 NTC Temperature ºC

Conclusions n n n The commercial assays have limitations in the detection and quantification Conclusions n n n The commercial assays have limitations in the detection and quantification of the variant erythroviruses Of 58 plasma pools screened with the Roche & inhouse Taq. Man assays, results for parvovirus B 19 are in agreement No evidence currently for the presence of variant erythroviruses in manufacturing pools examined by NIBSC

Discussion n n Primer & probe design affects ability to detect and quantify variant Discussion n n 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 Acknowledgements Kevin Brown, NIH Daniel Candotti & Jean-Pierre Allain, Cambridge Kati Hokynar & Klaus Hedman, University of Helsinki Annabelle Servant & Antoine Garbarg-Chenon, Paris