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Bacterial Screening of Platelet Concentrates by Real-Time PCR Theo Cuypers 1 also on behalf of, H.W. Reesink1,3 T. Mohammadi¹,² R.N.I. Pietersz¹ I.G.H. Rood1,2 P.H.M. Savelkoul² C.M.J.E. Vandenbroucke-Grauls² ¹ Sanquin, Amsterdam, The Netherlands ² VU University Medical Center, Dept. of Medical Microbiology & Infection Control, Amsterdam, The Netherlands ³ 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 - amplification - negative control : HLA – DQA DNA : Bordetella avium DNA 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) CT Log CFU/PCR Assay is linear from 1x100-1x105 CFU eq/PCR (R2:0.999) Mohammadi et al Transf (2005) 45: 731-6 Comparison Real Time-PCR and Automated Culturing source platelet pools* units (n) 2,146 positive result RT-PCR BacT/Alert n (%) n (%) 18 (0.8) 18 (0.8) * representing 10,739 donations BacT/Alert positive result = identified microorganism in culture bottle Mohammadi et al Transf (2005) 45: 731-6 Comparison Real Time-PCR and BacT/Alert Automated culturing Bacteria spp Propionibacterium Staphylococcus Bacillus Micrococcus Peptostreptococcus (n) (7) (6) (2) (2) (1) RT PCR Time to detection (hrs) (range) CT CFU/PCR (range) (range) 42 - 71 19 - 49 53 - 59 37 - 42 62 24.4 - 36.2 33.9 - 35.9 34.1 - 35.9 36.6 - 36.9 26.3 4 - 10 5 15 - 2x10 2 24 - 38 14 - 30 2x10 4 Mohammadi et al Transf (2005) 45: 731-6 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 days days days Bacterium spp 0 1 2 3 6 7 0 1 2 3 6 7 0 1 2 3 6 7 B. cereus + ND E. Coli - + S. epidermidis + ND P. acnes - - - - P. aeruginosa + ND ................. + ND ................. + ND ................. + ND ................. + ND ................. ................. + ND ................. + ND ................. ND ............ + ND - ................. + + ND 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 days days days Bacterium spp 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?