A comparison of a commercial and in house assay

1
A comparison of a commercial and in house assay

• CAF and Sanquin Plasma Products

Rand D Laboratory CAF Ruth Laub, Mario Di
Giambattista and Willy Verlinden Sanquin
Diagnostic Services Marco Koppelman, Margret
Sjerps, Mirjam de Waal
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

3
NAT testing for B19 genotype 2 and 3 sequences
in plasma pools

• Literature one report for the presence of a
  genotype 2 isolate1 and no reports for the
  presence of B19 genotype 3 isolates in blood
  donations and manufacturing pools in Europe2,3,4
• Sanquin data (covering the period 2004-2005)
• Plasma pools 950.000 Dutch blood donations, no
  genotype 2 and 3 sequences5
• Test pools screening 1,200,000 Dutch blood
  donations, no genotype 2 sequences6
• 1Blumel et al J, of Virol. 2005, 79 14197-14206
• 2Heegaard et al. J. Med. Vir. 2001, 65 362-367
• 3Hokynar et al. J. of Clin. Microbiol., 2004, 42
  2013-2019
• 4Baylis et al. J. of Vir. Methods 2004,121, 7-16
  
• 5Koppelman et al. 2004, type 2 and 3 specific
  PCR-assay
• 6Hybridization detection with Roche LightCycler
  Assay

4
Protocol for detection genotype 1, 2 and 3

• One nucleic acid extract, two Real-time PCR
  protocols
• Parvovirus B19 LightCycler kit from Roche.
  Reliable detection and quantification of
  genotype 1.
• In house B19 DNA real-time assay (LightCycler)
  essentially as described by Baylis et al. (Taqman
  probes)1.Reliable detection and quantification
  B19 genotypes 1, 2, and 3 genotype 2 validated,
  genotype 3 no reference material available, only
  tested on plasmid.

1Baylis et al. J. of Virol. Methods 2004 121, 7
5
Parvo B19 DNA tests with LightCycler (Roche) and
In house B19 DNA real-time assay

• 327 manufacturing pools 03-2005 12-2005
  450,000 donations no genotype 2 and 3
  sequences
• 576 manufacturing pools 12-2005 02-2007 2.15
  million donations no genotype 2 and 3 sequences
• 3502 test pools 12-2005 02-2007 1.76
  million donations
• one genotype 2 isolate discrepant between two
  tests
• two discrepant results between the two tests
  for genotype 1
• isolates

6
B19 DNA positive samples with discrepant results
in the Roche B19 DNA assay and in the B19 DNA in
house assay
B19 Sample Load Roche test Load in house test B19 variant Reason for detection failure
903321 7x105 IU/ml 3x107 IU/ml B19 gt1 mutation at 3-end of the reverse primer of Roche LC kit
207458 not detectable 3x107 IU/ml B19 gt2 mutation at 3-end of the reverse primer and mutations in probe binding site of Roche LC kit
163429 1x108 IU/ml not detectable B19 gt1 mutation in the middle of the probe binding site of in house test
7
Sequence primers/probe binding region Roche and
In-house B19 DNA assay
8
B19 DNA positive samples with discrepant results
in the Roche B19 DNA assay and in the B19 DNA in
house assay
B19 Sample Load Roche test Load in house test B19 variant Reason for detection failure
903321 7x105 IU/ml 3x107 IU/ml B19 gt1 mutation at 3-end of the reverse primer of Roche LC kit
207458 not detectable 3x107 IU/ml B19 gt2 mutation at 3-end of the reverse primer and mutations in probe binding site of Roche LC kit
163429 1x108 IU/ml not detectable B19 gt1 mutation in the middle of the probe binding site of in house test
9
Phylogenetic analysis of 1550 bp fragment of B19
genome (NS1-VP1 region)
B19 gt1
B19 gt2
B19 gt3
10
Conclusions

• Genotype 2 and 3 infections in blood donors are
  rare 2.6 million donations tested as
  manufacturing pools, no indication for genotype
  2 and 3 sequences 1.76 million donations
  analyzed in test pools (480-576 don.), one
  genotype 2 isolate identified
• 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
• The incidence of genotype 2 infection is
  comparable to genotype 1 infection missed by PCR
  due to a unique mutation.
• More sequence data on Parvo B19 virus isolates
  are necessary to design primers and probes
  sensitive for all variants and mutations in these
  variants.