1. Distributions of parvovirus B19 genotype 1-3 in blood donations
15-jan-19
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 , 7
2Hokynar et al J. of Clin. Microbiol , 2013
3PTS052 and 064 “B19 Virus DNA Testing of Plasma Pools by NAT”
4Cohen et al J.Clin Virol ,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 , 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 , 4500 assays):
Plasma pools ~ 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, ; 2Hokynar et al. J. of Clin. Microbiol. 2004, ; 3Baylis et al. J. of Vir. Methods , 7-16; 4Koppelman et al. 2004, type 2 and 3 specific PCR-assay; 5Hybridization detection with Roche LightCycler Assay;

5. Discrepancy between Roche B19 LightCycler assay and ‘in house’ B19 DNA real time assay in a test pool
Roche B19 DNA assay
In house B19 DNA assay
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 : 12169

7. 15-jan-19 B19 gt1 B19 gt2 B19 gt3 B19 gt1 B19 gt2 B19 gt3 B19 gt1

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 : 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
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