Comparison of a commercial and ‘in house’ assay for B19 DNA SoGAT meeting XXI May 28-29 (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

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 2004 121, 7 2Hokynar et al J. of Clin. Microbiol. 2004 42, 2013 3Nuebling and Buchheit: PTS052 and 064; SOGAT Bern 06-2006 4Cohen et al J.Clin Virol 2006 36,152 5Report PTS096, EDQM, December 2008 24 February 2019

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 2004 121, 7 2Koppelman et al. Vox Sanguinis 2007 93, 208 24 February 2019

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

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 24 February 2019

Sequence primers/probe binding region Roche and In-house B19 DNA assay 24 February 2019

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

Phylogenetic analysis of 1550 bp fragment of B19 genome (NS1-VP1 region) AY903437-gt2 DQ333426-gt2 AY044266-gt2 EF216869-gt2 7087291-mrt07 AJ747293-gt2 7060774-sep07 6207458-dec06 AY064475-gt2 AY064476-gt2 AY083234-gt3 DQ234779-gt3 AY647977-gt3 DQ408305-gt3 AY582125-gt3 DQ234769-gt3 AJ249437-gt3 AX003421-gt3 NC004295-gt3 8419606-dec08 AF162273-gt1 NC 000883-gt1 M24682-gt1 AF161226-gt1 M13178-gt1 DQ293995-gt1 8083032-mei08 8060625-aug08 6163429-jun06 6903321-feb06 9087345-apr09 9088719-apr09 9106327-may09 0.01 B19 gt2 B19 gt3 B19 gt1 24 February 2019

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