title: Real-time polymerase chain reaction detection of parvovirus B19 DNA in blood donations using a commercial and an in-house assay.
abstract: BACKGROUND
  European regulations require testing of manufacturing plasma for parvovirus B19 (B19) DNA to limit the load of this virus to a maximum acceptable level of 10 IU/µL. To meet this requirement, most manufacturers introduced a test algorithm to identify and eliminate high-load donations before making large manufacturing pools of plasma units. Sanquin screens all donations using a commercial assay from Roche and an in-house assay.
  
  
  METHODS
  Between 2006 and 2009, 6.2 million donations were screened using two different polymerase chain reaction (PCR) assays targeting B19 DNA. Donations with B19 DNA loads of greater than 1 × 10(6) IU/mL showing significant differences in viral load between the two assays were further analyzed by sequencing analysis.
  
  
  RESULTS
  A total of 396 donations with B19 DNA loads of greater than 1 × 10(6) IU/mL were identified. Fifteen samples (3.8%) had discordant test results; 10 samples (2.5%) were underquantified by the Roche assay, two samples (0.5%) were underquantified by the in-house assay, and three samples (0.8%) were not detected by the Roche assay. Sequencing analysis revealed mismatches in primer and probe-binding regions. Phylogenetic analysis showed that 12 samples were B19 Genotype 1. The three samples not detected by the Roche assay were B19 Genotype 2.
  
  
  CONCLUSIONS
  This study shows that 3.8% of the viremic B19 DNA-positive donations are not quantified correctly by the Roche or in-house B19 DNA assays. B19 Genotype 1 isolates showing incorrect test results are more common than B19 Genotype 2 or 3 isolates. Newly designed B19 PCR assays for blood screening should preferably have multiplexed formats targeting multiple regions of the B19 genome.
authors.affiliation: Sanquin Diagnostics, National Screening Laboratory of Sanquin and Viral Serology, Amsterdam, the Netherlands.
authors.forename: M H G M
authors.initials: MH
authors.lastname: Koppelman
authors.forename: P
authors.initials: P
authors.lastname: van Swieten
authors.forename: H T M
authors.initials: HT
authors.lastname: Cuijpers
mesh_terms: D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics*; D006801:Humans; D016732:Parvovirus B19, Human / Q000145:classification / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004279:DNA, Viral