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Bacterial Screening of Platelet Concentrates by Real-Time PCR

Bacterial Screening of Platelet Concentrates by Real-Time PCR. Theo Cuypers 1 also on behalf of, H.W. Reesink 1,3 I.G.H. Rood 1,2 T. Mohammadi¹ , ² P.H.M. Savelkoul² R.N.I. Pietersz¹ C.M.J.E. Vandenbroucke-Grauls².

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Bacterial Screening of Platelet Concentrates by Real-Time PCR

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  1. 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. Savelkoul² R.N.I. Pietersz¹ C.M.J.E. Vandenbroucke-Grauls² ¹ 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

  2. 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

  3. 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

  4. 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

  5. Comparison Real Time-PCR and Automated Culturing * representing 10,739 donations BacT/Alert positive result = identified microorganism in culture bottle Mohammadi et al Transf (2005)45: 731-6

  6. Comparison Real Time-PCR and BacT/Alert Mohammadi et al Transf (2005)45: 731-6

  7. 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

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

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

  10. 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)

  11. 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?

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