Distributions of parvovirus B19 genotype 1-3 in blood donations

1. Distributions of parvovirus B19 genotype 1-3 in blood donations MHGM Koppelman and HTM Cuijpers

2. Specificity of commercial test kits for Parvo B19 virus genotype 2 and 3 • RealArt Parvo B19 LC PCR (Artus). Genotype 1 and 2 reliable quantified3. Genotype 3 under-quantified 1,000x1 or missed2,4. • Parvovirus B19 LightCycler quantification kit (Roche).Reliable quantification of genotype 1. Genotype 2 and 3 are not detected1,2,3. • Reflected in results proficiency studies EDQM3: • - 2004: 56% participants missed genotype 2 • - 2005: 41% participants missed genotype 2, this included 25% of the ‘in house’ assays. • 1Baylis et al. J. of Virol. Methods 2004 121, 7 • 2Hokynar et al J. of Clin. Microbiol. 2004 42, 2013 • 3PTS052 and 064 “B19 Virus DNA Testing of Plasma Pools by NAT” • 4Cohen et al J.Clin Virol 2006 36,152

3. Additional protocol for detection genotype 2 and 3 • One nucleic acid extract, two Real-time PCR protocols • 1. Parvovirus B19 LightCycler kit from Roche • Reliable detection and quantification of genotype 1. • 2. ‘In house’ B19 DNA real-time assay with Taqman probes (LightCycler) essentially as described by Baylis et al1. • Reliable detection and quantification B19 genotypes 1, 2, and 3; genotype 2 validated, genotype 3 no reference material available. 1Baylis et al. J. of Virol. Methods 2004 121, 7

4. NAT testing for B19 genotype 2 and 3 sequences • Literature: no indication for the presence of B19 genotypes 2 and 3 in blood donations and plasma for fractionation1,2,3 • Sanquin data (covering the period 2004-2006, 4500 assays): • Plasma pools ~950.000 Dutch blood donations, no genotype 2 and 3 sequences4 • Plasma pools ~1,200,000 Dutch blood donations, no genotype 2 sequences5 • Plasma pools ~1.650,000 Dutch blood donations, no genotype 2 and 3 sequences using the ‘in house’ B19 assay3 • 1Heegaard et al. J. Med. Vir. 2001, 65 362-367; 2Hokynar et al. J. of Clin. Microbiol. 2004, 42 2013-2019; 3Baylis et al. J. of Vir. Methods 2004 121, 7-16; 4Koppelman et al. 2004, type 2 and 3 specific PCR-assay; 5Hybridization detection with Roche LightCycler Assay;

5. Roche B19 DNA assay In house B19 DNA assay Discrepancy between Roche B19 LightCycler assay and ‘in house’ B19 DNA real time assay in a test pool 104 IU/mL 750 IU/mL 3x104 IU/mL 104 IU/mL

6. Phylogenetic tree based on sequence of 863 bp NS1-VP1 fragment according to Candotti et et al.1 B19 gt 3 B19 gt 2 B19 gt 1 1Candotti et al. J.Virol. 2004 78: 12169

7. B19 gt1 B19 gt2 B19 gt3 B19 gt1 B19 gt2 B19 gt3 B19 gt1 B19 gt2 B19 gt3

8. Discrepancy between Roche B19 LightCycler assay and ‘in house’ B19 DNA real time assay in a donation 104 IU/mL 108 IU/mL Roche B19 DNA LightCycler assay ‘In house’ B19 DNA assay 104 IU/mL

9. Phylogenetic tree based on sequence of 863 bp NS1-VP1 fragment according to Candotti et et al.1 B19 gt 3 B19 gt 2 B19 gt 1 1Candotti et al. J.Virol. 2004 78: 12169

10. Sequence ‘In house’ PCR fragment with unique mutation in the Taqman probe binding site EVF Taqman Probe EVR

11. Conclusions • No evidence for the presence of B19 genotypes 2 and 3 in Dutch blood donations during period 2004-2006 • Identification of two new ‘unique’ mutations in B19 genotype 1 isolates • one sample missed by the Roche LightCycler assay • one sample missed by the ‘in house’ assay • Validation of commercially available and ‘in house’ quantitative B19 tests for genotype specificity is dependent on publicly available reference materials for B19 genotype 2 and 3 variants (WHO, EDQM, and commercial suppliers of reference materials).

12. Effect of lowering the annealing/extension temperature of 60°C to 58°C or 56°C 10,000 IU/ml B19 DNA control Annealing/extension at 58°C Discrepant sample (with mutation in the probe binding site) NC Annealing/extension at 56°C