1. Detection of bacteria in blood products
12-nov-18
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 (+) Conserved in bacteria 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
Background signal PC
Sensitivity DNA assay;
E. coli CFU/ml
S. epidermidis 750 CFU/ml

6. Sensitivity Real time RT-PCR assay
Sensitivity RNA assay;
E. coli 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
A Staphylococcus epidermidis B Streptococcus pyogenes
C Klebsiella pneumoniae D Eschericha coli
N Controle (negative) PCs

9. Conclusions PCR assay PCR assay versus BacT/ALERT
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