Presentation Theo Cuypers 2nd presentation

							  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 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     : 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)




         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

                                          positive result
                                       RT-PCR BacT/Alert
  source                units (n)       n (%)       n (%)

  platelet pools*         2,146        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               RT PCR
                              Time to          CT         CFU/PCR
Bacteria spp         (n)     detection
                           (hrs) (range)     (range)       (range)
Propionibacterium    (7)     42 - 71       24.4 - 36.2    4 - 10 5
Staphylococcus       (6)     19 - 49       33.9 - 35.9   15 - 2x10 2
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        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
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?