Bacterial Screening of Platelet Concentrates by Real-Time PCR

Theo Cuypers 1

also on behalf of,H.W. Reesink1,3 I.G.H. Rood1,2

T. Mohammadi¹,² P.H.M. Savelkoul²R.N.I. Pietersz¹ C.M.J.E. Vandenbroucke-Grauls²¹ Sanquin, Amsterdam, The Netherlands² VU University Medical Center, Dept. of Medical Microbiology & Infection Control, Amsterdam, The Netherlands³ Academic Medical Center, Dept. of Gastroenterology and Hepatology, Amsterdam, The Netherlands

Method (1)Extraction (MagNa Pure)- Filtration MagNa Pure DNA isolation chemicals

(filtration Gen Elute Plasmid Maxiprep (Sigma))- 200 µL PC- Extraction according to manufacturer

Real-Time PCR (ABI-PRISM 7000)

- pre-treatment 25 µL Taqman PCR mixture with Sau3AI digestion

- amplification- detection

Mohammadi et al, 2003, 2004, 2005

Method (2)

Controls- DNA extraction : HLA – DQA DNA- amplification : Bordetella avium DNA- negative control

Standard calibration curve (quantification)- Staphylococcus aureus DNA (ATCC 25923)- serial dilutions with known CFUs

Mohammadi et al, 2003, 2004, 2005

Standard Curve with DNA Extracted from Serial Dilutions of S. aureus (ATCC 25923)

Assay is linear from 1x100-1x105 CFU eq/PCR (R2:0.999)

Log CFU/PCR

CT

Mohammadi et al Transf (2005) 45: 731-6

Comparison Real Time-PCR and Automated Culturing

Mohammadi et al Transf (2005) 45: 731-6

* representing 10,739 donations

BacT/Alert positive result = identified microorganism in culture bottle

positive result

source

units (n)

RT-PCR n (%)

BacT/Alert n (%)

platelet pools* 2,146 18 (0.8) 18 (0.8)

Comparison Real Time-PCR and BacT/Alert

Mohammadi et al Transf (2005) 45: 731-6

Automated

culturing

RT PCR

Bacteria spp (n)

Time to

detection

(hrs) (range)

CT

(range)

CFU/PCR

(range)

Propionibacterium (7) 42 - 71 24.4 - 36.2 4 - 105

Staphylococcus (6) 19 - 49 33.9 - 35.9 15 - 2x102

Bacillus (2) 53 - 59 34.1 - 35.9 24 - 38

Micrococcus (2) 37 - 42 36.6 - 36.9 14 - 30

Peptostreptococcus (1) 62 26.3 2x104

Analysis of spiked PCs• Bacteria spp (B. cereus, E. coli, S. epidermidis,

P. acnes, P. aeruginosa)• Ten fold serial dilutions plated on agar• BacT/Alert culture 20 mL (aerobic and anaerobic

bottles)• time = 0 : < 2 hrs after spiking• time = 1, 2, 3, 6 and 7 : days after spiking• Real Time PCR at all time points• BacT/Alert at t=0, and other time points only

when earlier samples were negative

Mohammadi et al. Vox Sang(2005) 89: 208-214

Results BacT/Alert

1 CFU/mL 10 CFU/mL 100 CFU/mL

Bacterium spp days days days

0 1 2 3 6 7 0 1 2 3 6 7 0 1 2 3 6 7

B. cereus + ND ................. + ND ................. + ND .................

E. Coli - + ND ............ + ND ................. + ND .................

S. epidermidis + ND ................. + ND ................. + ND .................

P. acnes - - - - + ND - + ND ............ - + ND ............

P. aeruginosa + ND ................. + ND ................. + ND .................

All spiked dilutions were tested in triplicate Mohammadi et al. Vox Sang(2005) 89: 208-214

Results Real Time PCR

1 CFU/mL 10 CFU/mL 100 CFU/mL

Bacterium spp days days days

0 1 2 3 6 7 0 1 2 3 6 7 0 1 2 3 6 7

B. cereus - + + + + + + + + + +

+

+ + + + + +

E. Coli - + + + + + + + + + +

+

+ + + + + +

S. epidermidis - + + + + + - + + + + +

+ + + + + +

P. acnes - + + + + + - + + + + +

+ + + + + +

P. aeruginosa - ND + + + + - ND + + + +

+ ND + + + +

All spiked dilutions were tested in triplicate Mohammadi et al. Vox Sang(2005) 89: 208-214

Conclusion Real Time-PCR

• improvement sensitivity/specificity by filtration and digestion of reagents

• screening > 2000 PCs (~10,000 donors) indicate equal sensitivity and specificity with BacT/Alert

• time to detection: 4 hrs (BacT/Alert ± 24 hrs)• sensitivity improved when PCs stored for

> 24 hrs (after preparation, i.e. 48 hrs after collection)

Biological standards to compare NAT test protocols for bacterial contamination

• Application of both methods to prevent endogenous contamination of reagents; not perfect with all reagent batches. Selection of batches is necessary.

• Alternative PCR assays and methods in development to get around this problem.

• Problem is direct comparison of the sensitivity of assays. CFU is to imprecise to determine small differences.

• Develop ideas to prepare and characterize standards. Biomatrix, panels with dilution series of representative strains?