NIBSC

Testing for Parvovirus B19 -Testing for Parvovirus B19 -Broadening the Assay to Cover Broadening the Assay to Cover

VariantsVariants

Sally Baylis, NIBSCSally Baylis, NIBSC

SoGAT 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

Flu

ores

cenc

e (F

1/F

2)

Cycle Number

Flu

ores

cenc

e (F

2/B

ack -

F1)

M 1 1 3 3 2 2 NTC MGenotype 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

Flu

ores

cenc

e (F

2/B

ack-

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

Lo

g C

on

cen

tra

tio

n I

U/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 Manufacturing plasma pools (Europe, North America)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

Flu

ores

cenc

e (F

1/F

2)NTC

Genotype 3 Genotype 2Genotype 1

NTC

Genotype 1

Genotype 3

Genotype 2

Temperature ºC

Flu

ores

cenc

e –d

(F1)

/ dT

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 Primer & probe design affects ability to detect and quantify variant virusesand quantify variant viruses

Compliance with EP threshold concentration of Compliance with EP threshold concentration of 101044 IU/ml may be compromised IU/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 What are the implications in detecting a pools with high titres of a variant erythrovirus?with 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