Detection of bacteria in blood products

1. Detection of bacteria in blood products Ineke Rood Blood cell Research, Sanquin research Medical Microbiology en Infection control , VU medical centre

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

3. 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’

4. 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

5. Sensitivity Real time PCR assay • Sensitivity DNA assay; • E. coli 170 CFU/ml • S. epidermidis 750 CFU/ml Background signal PC

6. Sensitivity Real time RT-PCR assay • Sensitivity RNA assay; • E. coli 3 CFU/ml • S. epidermidis 80 CFU/ml

7. 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

8. BacT/ALERT versus PCR • AStaphylococcus epidermidis B Streptococcus pyogenes • C Klebsiella pneumoniae D Eschericha coli • N Controle (negative) PCs

9. 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