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Detection of bacteria in blood products. Ineke Rood Blood cell Research, Sanquin research Medical Microbiology en Infection control , VU medical centre. Bacteria in platelet concentrates. ~1 in 300 PCs contain bacteria Storage conditions PCs 24 °C Shaking Sources of contamination

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detection of bacteria in blood products

Detection of bacteria in blood products

Ineke Rood

Blood cell Research, Sanquin research

Medical Microbiology en Infection control , VU medical centre

bacteria in platelet concentrates
Bacteria in platelet concentrates
  • ~1 in 300 PCs contain bacteria
  • Storage conditions PCs
    • 24°C
    • Shaking
  • Sources of contamination
    • Bacteria from skin
    • Bacteremia
slide3

BacT/Alert

  • 2001; nationwide screening for bacterial contamination with the BacT/Alert culture system
  • (+) High sensitivity, 1 to 10 CFU/ml
  • (+) Detection within 24 to 48 hours
  • (-) PC are issued as ‘negative to date’
slide4

Real time PCR assay

  • 2003; development of NAT
  • 16S rRNA gene
    • (+) Conserved in bacteria
    • (+) Multiple copies within the bacterial genome
    • (-) Presence of exogenous DNA in reagents
sensitivity real time pcr assay
Sensitivity Real time PCR assay
  • Sensitivity DNA assay;
    • E. coli 170 CFU/ml
    • S. epidermidis 750 CFU/ml

Background signal PC

sensitivity real time rt pcr assay
Sensitivity Real time RT-PCR assay
  • Sensitivity RNA assay;
    • E. coli 3 CFU/ml
    • S. epidermidis 80 CFU/ml
bact alert versus pcr
BacT/ALERT versus PCR
  • Spiking of PCs with bacteria (< 1 CFU/ml)
    • Staphylococcus epidermidis
    • Streptococcus pyogenes
    • Klebsiella pneumoniae
    • Eschericha coli
  • BacT/ALERT
    • Inoculation of culture bottles directly after spiking
  • PCR assay
    • Take samples 0, 24, 48, 72, 96 and 144 h after spiking
bact alert versus pcr1
BacT/ALERT versus PCR
  • AStaphylococcus epidermidis B Streptococcus pyogenes
  • C Klebsiella pneumoniae D Eschericha coli
  • N Controle (negative) PCs
conclusions
Conclusions
  • PCR assay
  • Better sensitivity with RNA detection
  • DNA/RNA extraction from Gram+ bacteria needs improvement
  • Background signal renders sensitivity
  • PCR assay versus BacT/ALERT
  • BacT/ALERT is very sensitive
  • BacT/ALERT can give false negative results when PCs are inoculated with low bacterial titers
  • Real Time RT-PCR detects PCs with low bacterial titers but at a later moment
  • PCR assay can be used shortly before transfusion