1. Experience in Testing of Genotypes of B19

2. Testing of plasma donation samples
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*
Investigational test - Quantitative NAT assay**
* colorimetric readout
** fluorogenic readout

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

6. 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
Variant Target
WT B19 Stds

7. Manufacturing-scale plasma sample pools
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
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

8. Number of Donations Represented
Detection of B19 Virus Genotypes 2 and 3 During High-throughput NAT Testing
Study Results
Pool Size
(samples)
Number of Pools Tested
Detection Frequency
Number of Donations Represented
3840
242
0/242
929,280
960
609
0/609
584,640
Total samples/donations tested without detection of high-titer variants
1,513,920

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. Archived study material was representative of 1,5 million donations
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 Prevalence Profile of viremia
Knowledge from the literature and collaborative communication
Prevalence
Genotypes 2 and 3 not frequently detected in blood or plasma donations; Hokynar et al., 2004, J Clin Microbiol.; 42(5):
Genotype 1 (47%) and Genotype 2 (2.5%) detected in batches of clotting factor concentrates; Schneider et al. 2004, Thromb Haemost: 92(4):838-45
Genotypes 2 and 3 identified in diagnostic clinical specimens; Sanabani et al., 2006, J Clin Microbiol. 44(2):604-6; Cohen, Gandhi, and Clewley, 2006 J Clin Virol. ;36(2):152-5.
Genotype 3 endemic in Ghana; Parsyan et al., 2006, J Clin Microbiol. 44(4):
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 J Virol. 76(18):