Testing for Parvovirus B19 - Broadening the Assay to Cover Variants

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
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
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

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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