NIBSC
Testing for Parvovirus B19 -Testing for Parvovirus B19 -Broadening the Assay to Cover Broadening the Assay to Cover
VariantsVariants
Sally Baylis, NIBSCSally Baylis, NIBSCSoGAT XVIISoGAT XVII
Screening Plasma Pools for Screening Plasma Pools for Parvovirus B19 - an OMCL PerspectiveParvovirus B19 - an OMCL Perspective
European Pharmacopoeia Monographs:European Pharmacopoeia Monographs:
““Human anti-D immunoglobulin” & “Human anti-D Human anti-D immunoglobulin” & “Human anti-D immunoglobulin for intravenous administration” (Jan. immunoglobulin for intravenous administration” (Jan. 2004)2004)
““Human plasma (pooled and treated for virus Human plasma (pooled and treated for virus inactivation)” (July 2004)inactivation)” (July 2004)
Plasma pools should contain not more than 10Plasma pools should contain not more than 1044 IU/ml parvovirus B19 DNAIU/ml parvovirus B19 DNA
Variant ErythrovirusesVariant Erythroviruses
V9V9 isolated from a child with transient aplastic isolated from a child with transient aplastic crisis (Nguyen crisis (Nguyen et alet al., 1998, 1999)., 1998, 1999)
LaLi, K71LaLi, K71 dermal isolates (Hokynar dermal isolates (Hokynar et alet al., 2002)., 2002) A6 A6 isolated from an anaemic HIV-positive patient isolated from an anaemic HIV-positive patient
(Nguyen (Nguyen et alet al., 2002)., 2002) D91.1 D91.1 isolated from a child with transient aplastic isolated from a child with transient aplastic
crisis (Servant crisis (Servant et alet al., 2002)., 2002)
Classification proposed by Servant Classification proposed by Servant et alet al., (2002) ., (2002) based upon sequence analysis of the NS1/VP1 based upon sequence analysis of the NS1/VP1 regionregion
Genetic Diversity of Erythroviruses: Genetic Diversity of Erythroviruses: Analysis of the NS1/VP1 RegionAnalysis of the NS1/VP1 Region
Servant et al., J. Virol., 2002
A6
NIBSC
Fluo
resc
ence
(F
1/F2
)
Cycle Number
Fluo
resc
ence
(F2/
Bac
k -F1
)
M 1 1 3 3 2 2 NTC M Genotype 3
Genotype 1
Genotype 2 NTC
Cycle Number
Region amplified: NS1
Roche Parvovirus B19 Quantification Roche Parvovirus B19 Quantification KitKit
Artus Artus RealArtRealArtTMTM Parvo B19 LC Kit Parvo B19 LC Kit
Cycle Number
Fluo
resc
ence
(F2/
Bac
k-F1
)
M 1 1 3 3 2 2 NTC M
Genotype 3
Genotype 1Genotype 2
NTC
Region amplified: VP1
Sensitivity of Detection of Different Sensitivity of Detection of Different Erythrovirus GenotypesErythrovirus Genotypes
0
1
2
3
4
5
6
7
8
Roche Artus
Log
Conc
entra
tion
IU/m
l
Genotype 1 (B19)
Genotype 2 (A6)
Genotype 3 (V9)
Genotype 3 (D91.1)
In-house Erythrovirus TaqMan AssayIn-house Erythrovirus TaqMan Assay
Consensus assay, primers & probe directed to the Consensus assay, primers & probe directed to the NS1 geneNS1 gene
Manufacturing plasma pools (Europe, North America)Manufacturing plasma pools (Europe, North America)
Extraction using the MagNA Pure (Total Nucleic Acid, Extraction using the MagNA Pure (Total Nucleic Acid, large volume) & real-time PCR on the LightCyclerlarge volume) & real-time PCR on the LightCycler
Standard curve – WHO International Standard for Standard curve – WHO International Standard for parvovirus B19 (99/800)parvovirus B19 (99/800)
Cycle Number
Fluo
resc
ence
(F1/
F2)
NTC
Genotype 3 Genotype 2Genotype 1
NTC
Genotype 1
Genotype 3
Genotype 2
Temperature ºC
Fluo
resc
ence
–d(F
1)/ d
T
In-house Erythrovirus TaqMan AssayIn-house Erythrovirus TaqMan Assay
ConclusionsConclusions
The commercial assays have limitations in the The commercial assays have limitations in the detection and quantification of the variant detection and quantification of the variant erythroviruseserythroviruses
Of 58 plasma pools screened with the Roche & in-Of 58 plasma pools screened with the Roche & in-house TaqMan assays, results for parvovirus B19 house TaqMan assays, results for parvovirus B19 are in agreementare in agreement
No evidence currently for the presence of variant No evidence currently for the presence of variant erythroviruses in manufacturing pools examined erythroviruses in manufacturing pools examined by NIBSCby NIBSC
DiscussionDiscussion
Primer & probe design affects ability to detect and Primer & probe design affects ability to detect and quantify variant virusesquantify variant viruses
Compliance with EP threshold concentration of 10Compliance with EP threshold concentration of 1044 IU/ml may be compromisedIU/ml may be compromised
Clinical significance, prevalence & geographical Clinical significance, prevalence & geographical distribution of erythrovirus variants is still largely distribution of erythrovirus variants is still largely unknownunknown
What are the implications in detecting a pools with What are the implications in detecting a pools with high titres of a variant erythrovirus?high titres of a variant erythrovirus?
AcknowledgementsAcknowledgements
Kevin Brown, NIH
Daniel Candotti & Jean-Pierre Allain, Cambridge
Kati Hokynar & Klaus Hedman, University of Helsinki
Annabelle Servant & Antoine Garbarg-Chenon, Paris
