Detection of bacteria in blood products

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Presentation transcript:

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

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

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’

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

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

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

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 PCR A Staphylococcus epidermidis B Streptococcus pyogenes C Klebsiella pneumoniae D Eschericha coli N Controle (negative) PCs

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