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Presence (absence) of genotype 2 and 3 Erythrovirus DNA in manufacturing plasma

Presence (absence) of genotype 2 and 3 Erythrovirus DNA in manufacturing plasma. Theo Cuypers, Marco Koppelman, Margret Sjerps, Henk Reesink Sanquin Plasma Products and Diagnostic Services. Genotypes detection Erythroviruses by Parvo B19 DNA test (1).

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Presence (absence) of genotype 2 and 3 Erythrovirus DNA in manufacturing plasma

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  1. Presence (absence) of genotype 2 and 3 Erythrovirus DNA in manufacturing plasma Theo Cuypers, Marco Koppelman, Margret Sjerps, Henk Reesink Sanquin Plasma Products and Diagnostic Services SOGAT 25-05-2005

  2. Genotypes detection Erythroviruses by Parvo B19 DNA test (1) • Oct 2001 notification by EP (group 6B) for change Monograph Anti-D Immunoglobulin; NAT for Parvo B19 DNA, effective per January 2003. • Fractionation facilities anticipated on introduction in a relative short period (had to deal with plasma stocks). • No guidance on detection of other Erythroviruses than Parvo B19 virus. • Jan. 2003 official notification of Monograph Human Anti-D Immunoglobulin per January 2004. SOGAT 25-05-2005

  3. Genotypes detection Erythroviruses by Parvo B19 DNA test (2) • June 2003. Validation Guideline Parvo B19 NAT for Official Medicine Control laboratories (OMCL’s): test including detection of Erythrovirus sequences genotype 2 and 3 • July 2003. EDQM. Testing for Erythrovirus sequences is added as ‘important notice for information’ and not ‘mandatory’ • April 2004. Proficiency testing study (PTS) on B19 Virus DNA NAT of plasma pools Recommendation to OMCL’s: improve NAT methods to cover also B19 variant sequences (A6, V9) SOGAT 25-05-2005

  4. Erythrovirus variants • V9 (genotype 3) Nguyen et al.1999. Lancet 352: 1524. Heegaard et al. 2001 J. Med. Virol. 65: 362-367. • A6 (genotype 2) Nguyen et al. 2002. Virology 301: 374 • Belonging to the human Erythrovirus genus, genotype 1 (Parvo B19 virus), genotype 2 (prototype A6) en genotype 3 (prototype V9). SOGAT 25-05-2005

  5. Phylogenetic relationships SOGAT 25-05-2005

  6. Specificity of test kits for Erythrovirus genotype 2 and 3 quantification • Parvovirus B19 quantification kit RocheReliable quantification of genotype 1. Genotype 2 and 3 are not detected1,2,3. • RealArt Parvo B19 LC PCR Artus. Genotype 1 and 2 reliable quantified3. Genotype 3 under-quantified 1000x1. Lower limit of detection: 5000 kop/reaction2. • In house tests PTS study EDQM3. In house protocols detected genotype 1; genotype 2 missed by 50 % of the protocols. Genotype 3 was not included. • 1Baylis et al. J. of Virol. Methods2004 121, 7 • 2Hokynar et al J. of Clin. Microbiol. 2004 42, 2013 • 3 PTS052 “B19 Virus DNA Testing of Plasma Pools by NAT” September 2004 SOGAT 25-05-2005

  7. NAT testing for Erythroviruses genotype 2 and 3 sequences in plasma pools • Plasma pools ~ 100.000 Danish blood donations, no genotype 3 sequences1 • Plasma pools ~ 140.160 Finnish blood donations, no genotype 2 (and genotype 3) sequences2 • 52 plasma pools from 9 different manufacturers, no genotype 2 and 3 sequences detected3 • Plasma pools ~950.000 Dutch blood donations, no genotype 2 and 3 sequences4 • 1Heegaard et al. J. Med. Virol. 2001, 65 362-367 • 2Hokynar et al. J. of Clin. Microbiol. 2004, 42 2013-2019 • 3Baylis et al. J. of Virol. Methods 2004 121, 7-16 • 4Koppelman et al. 2004 manuscript in preparation SOGAT 25-05-2005

  8. Additional NAT testing manufacturing pool for genotype 2 and 3. • Sanquin Plasma-products • Specific NAT test for Erythrovirus genotype 2 and 3. • Test with no cross reactivity with Parvo B19 DNA (genotype 1). • Retested 321 manufacturing pools Sept 2003 Oct 2004 (~950.000 donations) • No genotype 2 and 3 sequences detected. SOGAT 25-05-2005

  9. Erythrovirus genotype 2 and 3 in coagulation factor concentrate1. • Haemophilia Centre Bonn Germany1. • 13 commercially available plasma-derived coagulation factor concentrates (181 lots). Period collected 2000-2003. • Analyzed with specific NAT tests for genotype 1 and 2 in final products. • Genotype 1 DNA 77/181 (42.5 %) and genotype 2 DNA 2/181 (1.1 %). • Genotype 2 always co-contaminated with genotype 1. • 1Schneider et al. 2004 Thromb Haemost 92 838-845 SOGAT 25-05-2005

  10. Conclusions (1) • Data on testing of plasma pools for Erythrovirus genotype 2 and 3 sequences do not indicate that the specificity of applied Parvo B19 tests should be extended to these new molecular variants. • Additional data on the prevalence and load of Erythroviruses genotype 2 and 3 in plasma pools and individual donations should be collected in a research setting. • Additional studies are dependent on publicly available reference materials for these molecular variants (WHO, EDQM, and commercial suppliers). SOGAT 25-05-2005

  11. Conclusions (2) • The new variant viruses are not classified by the International Committee on Taxonomy of Viruses. Terminology in literature is confusing: Family Parvoviridae, genus Erythrovirus, genotype 1 (Parvo B19), genotype 2 (A6 like), genotype 3 (V9 like)or Parvovirus B19 gt 1, gt 2, and gt 3. SOGAT 25-05-2005

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