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Experience in Testing of Genotypes of B19

Experience in Testing of Genotypes of B19. B19 Virus Testing. NAT Testing Operations. Testing of plasma donation samples QC testing of plasma fractionation pools Example Talecris B19 virus testing system: Donation mini pool test - Qualitative NAT assay*

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Experience in Testing of Genotypes of B19

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  1. Experience in Testing of Genotypes of B19

  2. B19 Virus Testing NAT Testing Operations • Testing of plasma donation samples • QC testing of plasma fractionation pools • Example Talecris B19 virus testing system: • Donation mini pool test - Qualitative NAT assay* • Detects genotypes 2 and 3 at high-titer • QC test for fractionation pool - Qualitative NAT assay* • Detects genotypes 2 and 3 at high-titer • Investigational test - Quantitative NAT assay** * colorimetric readout ** fluorogenic readout

  3. B19V Testing at Talecris Biotherapeutics (formerly Bayer Biological Products) NAT development and technical operations activities • Assessment of B19 virus test performance • Quantitation of all elevated samples (2000-2002) against WHO standard (genotype 1) • Tracking and trending of B19 virus unit interdictions • Investigational assessment of viral loads in fractionation pools • Development of reagents for investigation and technology improvements • Archive of positive samples • Archive of screened mini-pools

  4. Potential for the Detection of B19V Genotypes During High-throughput NAT Testing Talecris Human B19 virus genotypes - Prevalence Study • Purpose • To determine the potential prevalence of non-genotypes 1 variants (e.g. genotypes 2 and 3) • Design • Differential detection of variants using NAT • Specific primer and fluorogenic probe combinations to include or exclude detection of genotypes • Secondary testing of archived materials previously screened for elevated B19 virus genotypes (including 340 individual reactive samples) • Archived samples with reduced B19 virus loads (elevated samples removed) • NAT testing using primers and detection probe specific for B19V variants • Demonstrated to detect synthetic A6, V9, and D91.1 targets • Does not detect B19V genotype 1 below 106 IU/ml

  5. B19Type 1-R B19 Universal Variant Probe B19 Type 1-F B19 Universal-R DST-F DST-R B19 Type 1 Probe 1611 1348 Promoter Non-structural proteins Structural proteins Parvovirus B19 5.6 kb Donor Sample Test Quantitative Test Potential for the Detection of B19V Genotypes during high-throughput NAT Testing Talecris Detection Strategy for B19 genotype 1 vs. variants

  6. Variant Target WT B19 Stds Potential for the Detection of B19V Genotypes during high-throughput NAT Testing • Detection using the B19 Universal Variant Probe • UVP can detect genotype 2 (A6) and genotype 3 (V9) DNA targets • UVP does not detect genotype 1 targets below 106 IU/ml

  7. Potential for the Detection of B19V Genotypes During High-throughput NAT Testing Study Materials • Manufacturing-scale plasma sample pools • Pools containing 3840 donations (40 X 96 donation sample minipools) • Combine 40 of the 96-donation sample minipools • Samples screened between 2000-2002 • high-titer samples removed and analyzed using genotype 1 specific test • 960 sample pools • Combine 10 of the 96-donation sample minipools • Samples screened between 2002-2003 • high-titer samples removed and analyzed using genotype 1 specific test

  8. Detection of B19 Virus Genotypes 2 and 3 During High-throughput NAT Testing Study Results

  9. Potential for the Detection of B19V Genotypes During High-throughput NAT Testing Study Conclusions • High-titer units containing B19 virus genotypes 2 and 3 are rare among U.S. source plasma donations • None of the study materials contained detectable levels of B19 virus genotypes 2 and 3 • Analysis of 340 reactive samples did not identify B19 virus genotypes 2 and 3 • The prevalence of B19V genotypes 2 and 3 in the U.S. source plasma supply chain appears to be low • If B19 virus genotypes 2 and 3 were present in fractionation pools, levels would have been below the detection limit of the utilized assay

  10. Potential for the Detection of B19V Genotypes During High-throughput NAT Testing Comments • Standardized material unavailable thus use of synthetic DNAs was required • Archived study material was representative of 1,5 million donations • To accelerate availability of results archived material collected for other purposes was used

  11. Additional Slides

  12. B19 Virus Genotypes Knowledge from the literature and collaborative communication • Prevalence • Genotypes 2 and 3 not frequently detected in blood or plasma donations;Hokynar et al., 2004, JClinMicrobiol.; 42(5):2013-9. • Genotype 1 (47%) and Genotype 2 (2.5%) detected in batches of clotting factor concentrates;Schneider et al. 2004, ThrombHaemost: 92(4):838-45 • Genotypes 2 and 3 identified in diagnostic clinical specimens;Sanabani et al., 2006, JClinMicrobiol. 44(2):604-6; Cohen, Gandhi, and Clewley, 2006 JClinVirol. ;36(2):152-5. • Genotype 3 endemic in Ghana;Parsyan et al., 2006, JClin Microbiol. 44(4):1367-75. • Profile of viremia • Persistence of variant genotypes (?) • Viral loads associated with variant genotypes (?) • Pathogenesis associated with variant genotypes (?)

  13. B19 Virus Parsimony Genotypes 2 and 3 vary ~ 5-15% from genotype 1 Servant et al., 2002 JVirol. 76(18):9124-34.

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