1. Comparison of a commercial and ‘in house’ assay for B19 DNA
SoGAT meeting XXI May (2009), Brussels, Belgium
Theo Cuypers1, Peter van Swieten1 and Marco Koppelman2
Sanquin Diagnostic Services, Amsterdam, The Netherlands
Sanquin Diagnostics, Amsterdam, the Netherlands
1. Viral Diagnostic Services
2. National Screening Laboratory Sanquin (NSS)
24 February 2019

2. Specificity of commercial test kits for parvovirus B19 genotype 2 and 3
RealArt Parvo B19 LC PCR (Qiagen-Artus). Genotype 1 and 2 reliable quantified3. Genotype 3a 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 EDQM:
- 2004: 56% participants missed genotype 23
- 2005: 41% participants missed genotype 2, this included 25% of the ‘in house’ assays3
- 2008: Several labs missed cloned Parvo B19 gt 2 and 3 samples in the auxiliary study to PTS0965
1Baylis et al. J. of Virol. Methods , 7
2Hokynar et al J. of Clin. Microbiol , 2013
3Nuebling and Buchheit: PTS052 and 064; SOGAT Bern
4Cohen et al J.Clin Virol ,152
5Report PTS096, EDQM, December 2008
24 February 2019

3. Protocol for detection B19 genotype 1, 2 and 3
One nucleic acid extract, two Real-time PCR protocols
Parvovirus B19 LightCycler kit from Roche. Detection and quantification of genotype 1
‘In house’ B19 DNA real-time assay (LightCycler) essentially as described by Baylis et al.1 and Koppelman et al.2 (Taqman probes) Detection and quantification B19 genotypes 1, 2, and 3; genotype 2 validated, genotype 3 tested on plasmid
1Baylis et al. J. of Virol. Methods , 7
2Koppelman et al. Vox Sanguinis , 208
24 February 2019

4. Screening for B19 virus DNA in the Netherlands and Belgium
Year
# donations tested
# acute infections (> 100,000 IU/ml)
Acute infection/10,000 donations
2006
1.44 x 106
116
0.81
2007
1.58 x 106 65
0.41
2008
1.57 x 106 67
0.43
2009*
0.53 x 106 70 --
Total
5.12 x 106
318
0.62
*until April
24 February 2019

5. Discrepant samples between the Roche and the ‘in-house’ B19 DNA assay
Plasma
unit
Load Roche assay (IU/ml)
Load in house assay (IU/ml)
Affected assay
Discrepancy
419606
8.5 x 107
4.1 x 105
‘In house’
under-quantification (207x)
903321
7.0 x 105
3.0 x 107
Roche
under-quantification (43x)
082223
6.0 x 105
1.0 x 109
under-quantification (1,667x)
085146
0.7 x 103
1.3 x 106
under-quantification (1,857x)
087345
1.0 x 1010
1.5 x1012
under-quantification (150x)
088719
1.1 x 105
3.3 x 107
under-quantification (300x)
106327
7.9 x 104
1.1 x 106
under-quantification (14x)
207458
not detectable
Lack of detection
087291
5.0 x 107
060774
9.0 x 108
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6. Sequence primers/probe binding region Roche and In-house B19 DNA assay
24 February 2019

7. Discrepant samples; reason under quantification or failure detection
Plasma
unit
Load Roche assay (IU/ml)
Load ‘in house’ assay (IU/ml)
Affected assay
Reason under quantification or failure detection
419606
8.5 x 107
4.1 x 105
‘In house’
Forward primer mutation 3’-end
903321
7.0 x 105
3.0 x 107
Roche
Reverse primer mutation 3’-end
082223
6.0 x 105
1.0 x 109
085146
0.7 x 103
1.3 x 106
087345
1.0 x 1010
1.5 x1012
088719
1.1 x 105
3.3 x 107
106327
7.9 x 104
1.1 x 106
207458
not detectable
Reverse primer 3’-end and probe hybridization
087291
5.0 x 107
060774
9.0 x 108
24 February 2019

8. Phylogenetic analysis of 1550 bp fragment of B19 genome (NS1-VP1 region)
AY gt2
DQ gt2
AY gt2
EF gt2
mrt07
AJ gt2
sep07
dec06
AY gt2
AY gt2
AY gt3
DQ gt3
AY gt3
DQ gt3
AY gt3
DQ gt3
AJ gt3
AX gt3
NC gt3
dec08
AF gt1
NC gt1
M24682-gt1
AF gt1
M13178-gt1
DQ gt1
mei08
aug08
jun06
feb06
apr09
apr09
may09
0.01
B19 gt2
B19 gt3
B19 gt1
24 February 2019

9. Conclusions
Identification of two mutations in B19 genotype 1 isolates with implications for the quantification of the isolates
- Six isolates with substantial (14x-1800x) under quantification in the Roche LightCycler assay
- One isolate with substantial (200x) under quantification in the ‘in house’ test
To address the high genetic variability of B19 virus, two regions of the genome are targeted for amplification and detection
New developed B19 virus DNA tests should preferable include two regions of the genome that are simultaneously targeted for amplification and detection in one assay
In the Netherlands and Belgium, B19 genotype 2 isolates are detected in plasma units out of specification for fractionation with frequency of 1/100 compared to genotype 1 isolates
B19 genotype 3 isolates are not detected until now
24 February 2019