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23-mei-17
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
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
40
Background signal PC
Ct
30
20
10
0
-1
0
1
2
3
4
LOG(CFU/ml)
5
6
7
8
Sensitivity DNA assay;
• E. coli
170 CFU/ml
• S. epidermidis
750 CFU/ml
40
40
30
30
Ct
Ct
Sensitivity Real time RT-PCR assay
20
20
10
10
0
0
-1
0
1
2
3
4
LOG(CFU/ml)
5
6
7
8
-1
0
1
2
3
4
LOG(CFU/ml)
Sensitivity RNA assay;
• E. coli
• S. epidermidis
3 CFU/ml
80 CFU/ml
5
6
7
8
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
BacT/Alert Dag 2 (24h) Dag 3 (48h) Dag 6 (144h)
Pos dag
A 3/3
0.9
B 0/3
DNA RNA Plaat
DNA RNA Plaat
DNA RNA Plaat
3+ 3+ 3+
3+ 3+ 3+
2+ 2+ 2+
2+ 2+ 2+
2+ 2+ 2+
-
-
-
C 3/3
0.5
3+ 3+ 3+
3+ 3+ 3+
3+ 3+ 3+
D 2/3
0.75
1+ 3+ 1+
3+ 3+ 3+
3+ 3+ 3+
-
-
-
N 0/3
-
-
A Staphylococcus epidermidis
C Klebsiella pneumoniae
N Controle (negative) PCs
-
-
-
-
B Streptococcus pyogenes
D Eschericha coli
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