Glass Bead Sterilizer Efficacy and Limitations for Aerobic

Efficacy of Glass Bead Sterilization
for Aerobic Bacterial
Decontamination of Surgical
Instruments in Serial Rodent
Surgeries
Karen A. Oppelt, DVM
East Carolina University
Department of Comparative Medicine
Overview
•
•
•
•
•
Introduction
Materials and Methods
Results
Conclusions and Discussion
Acknowledgements
Introduction
• Primarily used in human medicine and dentistry
• 1985 amendment to the AWARs and 1985 edition of
the Guide required aseptic technique for rodent
survival surgery
• Provided unique challenges to maintaining sterile
instruments in a practical and cost-effective manner
• Modified aseptic technique using a GBS first
described for serial rodent surgeries in 1993
• Eventually became an accepted Guide, ACLAM, NIH
and AAALAC standard
Introduction
• GBS studies performed between the 1950s and 1990s
indicated a wide variety of sterilization (or not)
temperatures and times
• With inconsistent sterilization, no standardization for
sterilization temperature or time, and no manufacturer
validation studies, the FDA and CDC banned GBS use in
1997 for the medical and dental professions
• No laboratory animal peer-reviewed study has
examined the efficacy, limitations, or the optimal
number of serial rodent surgeries that can be performed
when a GBS is used to decontaminate instruments
between the surgeries since the early 1990s
Materials and Methods
• Animals: 81 CD-1 (ICR)
retired sentinel females
≥16 weeks of age, cage
mate negative for
tested pathogens
Materials and Methods
• Experiment 1: two groups of 12 serial laparotomies
for nephrectomy using standard aseptic technique
(group 1 and 2)
• Experiment 2: four groups of 12 (groups 1-4)
surgeries performed as in Experiment 1 except
scissors cut the cecum prior to abdominal closure
(intentional contamination)
Materials and Methods
• Experimental Conditions
– Began w/sterile surgical and culture
supplies
– ACLAM board-certified veterinarian
with training and experience in serial
rodent surgery and GBS use
Materials and Methods
• Experimental Conditions
– GBS manufacturer instructions
followed for heating, periodic bead
stirring, and decontaminating small
metal instruments
• 30 mins prior to reach ~450°F
• After every third surgery
• Decontamination time/depth-1=15s and
2=30s with tips approximately 1.5” into
well
Materials and Methods
• Surgical procedure
– Laparotomy/nephrectomy
(aseptic)
– Laparotomy/nephrectomy/
cecal cut (contaminated)
– All mice euthanized
immediately following
abdominal closure
Materials and Methods
• Culture procedure
– Baseline/controls (pre-Sx)
– Instrument tips immediately
after Sx (pre-GBS)
– After surgeon wiped and
decontaminated tips (postGBS)
Materials and Methods
• Culture Method
– Swabs into TS broth media at 37°C for 96 hrs
– Broth aliquots onto TS w/5% SB and MacConkey agars at
37°C
– Plates observed for growth at 48 hrs and up to 96 hrs
– Minimal identification performed: Gram stain,
morphology, catalase test as appropriate
– Any growth post-GBS=failure to sterilize
Results
• Experiment 1-standard aseptic technique w/o
instrument contamination
– 0/29 positive pre- and post-GBS
– GBS failure rate=0%
– Bead temperatures ranged between 381.3-467.2°F for
group 1 and 2 surgeries with an average of 423.9°F
Results
• Experiment 2-aseptic technique w/intentional
instrument contamination
– 51/52 positive pre-GBS
– 2/51 positive post-GBS (group 1, Sx # 8 and 12)=4% GBS failure
rate
– Temperatures ranged between 400.4°-486.8°F with an average
of 430.2°F
– Pre-GBS cultures grew a variety of Gram positive and negative
organisms: rods, cocci, diplococci, Staphylococcus and
Streptococcus spp.
– Post-GBS Sx #8 (T=416.4°F) - Gram negative diplococci
– Post-GBS Sx#12 (T=414.8°F) - Gram and catalase positive cocci
consistent with Staphylococcus spp.
Results
Average GBS temperatures and standard deviations for 6 groups of 12 serial surgeries
at each surgery in comparison to the manufacturer’s stated minimum
decontamination temperature
Average
Minimum
decontamination
temperature
490
480
470
Temperature °F
460
450
440
430
420
410
400
390
1
2
3
4
5
6
7
Surgery Number
8
9
10
11
12
Results
• Failure rates of the GBS under contamination
conditions calculated using 95% binomial exact
confidence intervals (Table 1)
• Binomial distributions based on the GBS failure rates
within the 95% confidence intervals were used to
determine the success probabilities of the GBS for
the serial mouse surgeries (Table 2)
Results
Using Table 1, find
applicable instrument
contamination and
corresponding GBS failure
rate.
Find your failure rate on
Table 2 and calculate
percent chance of success
for number of proposed
serial surgeries.
Decide if percent
chance of proposed
number of successful
serial surgeries is
acceptable.
Results
Table 1-Lower and Upper Limits of GBS Failure Rate in Relation to Instrument Contamination
Intra-operative
Contamination
GBS Failure
Rate
Rate
95% Confidence
Interval-Lower
95% Confidence
Interval-Upper
0%*
0%
0%
0%
3.125%
(0.1%)
(0.015%)
(0.4%)
6.25%
(0.2%)
(0.03%)
(0.8%)
12.5%
(0.5%)
(0.06%)
(1.7%)
25%
(1%)
(0.12%)
(3.4%)
33%
(1.3%)
(0.16%)
(4.4%)
50%
(2%)
(0.24%)
(6.7%)
66%
(2.6%)
(0.32%)
(8.9%)
75%
(3%)
(0.36%)
(10.1%)
100%**
(4%)
(0.5%)
(13.5%)
*Experiment 1 contamination and failure rates
**Experiment 2 contamination and failure rates
Results
Using Table 1, find
applicable instrument
contamination and
corresponding GBS failure
rate.
Find your failure rate on
Table 2 and calculate
percent chance of success
for number of proposed
serial surgeries.
Decide if percent
chance of proposed
number of successful
serial surgeries is
acceptable.
Table 2-Percent Chance* of Successful (Aseptic) Serial Rodent Surgeries Based on GBS Failure Rate
Failure
Number of consecutive successful surgeries
rate
1
2
3
4
(0.1%)
0.999
0.998
0.997
0.996
0.995 0.994 0.993 0.992 0.991 0.990 0.989
0.988
(0.5%)
0.995
0.990
0.985
0.980
0.975 0.970 0.966 0.961 0.956 0.951 0.946
0.942
(1%)
0.990
0.980
0.970
0.961
0.951 0.941 0.932 0.923 0.914 0.904 0.895
0.886
(1.5%)
0.985
0.970
0.956
0.941
0.927 0.913 0.900 0.886 0.873 0.860 0.847
0.834
(2%)
0.980
0.960
0.941
0.922
0.904 0.886 0.868 0.851 0.834 0.817 0.801
0.785
(2.5%)
0.975
0.951
0.927
0.904
0.881 0.859 0.838 0.817 0.796 0.776 0.757
0.738
(3%)
0.970
0.941
0.913
0.885
0.859 0.833 0.808 0.784 0.760 0.737 0.715
0.694
(3.5%)
0.965
0.931
0.899
0.867
0.837 0.808 0.779 0.752 0.726 0.700 0.676
0.652
(4%)
0.960
0.922
0.885
0.849
0.815 0.783 0.751 0.721 0.693 0.665 0.638
0.613
(4.5%)
0.955
0.912
0.871
0.832
0.794 0.759 0.724 0.692 0.661 0.631 0.603
0.575
(5%)
0.950
0.903
0.857
0.815
0.774 0.735 0.698 0.663 0.630 0.599 0.569
0.540
(5.5%)
0.945
0.893
0.844
0.797
0.754 0.712 0.673 0.636 0.601 0.568 0.537
0.507
(6%)
0.940
0.884
0.831
0.781
0.734 0.690 0.648 0.610 0.573 0.539 0.506
0.476
(6.5%)
0.935
0.874
0.817
0.764
0.715 0.668 0.625 0.584 0.546 0.511 0.477
0.446
(7%)
0.930
0.865
0.804
0.748
0.696 0.647 0.602 0.560 0.520 0.484 0.450
0.419
(7.5%)
0.925
0.856
0.791
0.732
0.677 0.626 0.579 0.536 0.496 0.459 0.424
0.392
(8%)
0.920
0.846
0.779
0.716
0.659 0.606 0.558 0.513 0.472 0.434 0.400
0.368
(8.5%)
0.915
0.837
0.766
0.701
0.641 0.587 0.537 0.491 0.450 0.411 0.376
0.344
(9%)
0.910
0.828
0.754
0.686
0.624 0.568 0.517 0.470 0.428 0.389 0.354
0.322
(9.5%)
0.905
0.819
0.741
0.671
0.607 0.549 0.497 0.450 0.407 0.369 0.334
0.302
(10%)
0.900
0.810
0.729
0.656
0.590 0.531 0.478 0.430 0.387 0.349 0.314
0.282
(10.5%)
0.895
0.801
0.717
0.642
0.574 0.514 0.460 0.412 0.368 0.330 0.295
0.264
(11%)
0.890
0.792
0.705
0.627
0.558 0.497 0.442 0.394 0.350 0.312 0.278
0.247
(11.5%)
0.885
0.783
0.693
0.613
0.543 0.480 0.425 0.376 0.333 0.295 0.261
0.231
(12%)
0.880
0.774
0.681
0.600
0.528 0.464 0.409 0.360 0.316 0.279 0.245
0.216
(12.5%)
0.875
0.766
0.670
0.586
0.513 0.449 0.393 0.344 0.301 0.263 0.230
0.201
(13%)
0.870
0.757
0.659
0.573
0.498 0.434 0.377 0.328 0.286 0.248 0.216
0.188
(13.5%)
0.865
0.748
0.647
0.560
0.484 0.419 0.362 0.313 0.271 0.235 0.203
0.175
*Percent chance = Number X 100
5
6
7
8
9
10
11
12
Conclusions and Discussion
• The GBS can be used to effectively and successfully
sterilize instrument tips for up to 12 serial mouse
laparotomies for nephrectomy with a skilled
veterinary surgeon using aseptic technique
(experiment 1)
– Effectiveness is dependent on adherence to standard
aseptic surgical technique, following the instructions for
GBS use, and a surgeon skilled, trained, and experienced in
using a GBS for instrument decontamination in serial
rodent surgeries
Conclusions and Discussion
• When instrument tip contamination occurs, the GBS
is less effective for instrument decontamination and
only up to 7 surgeries could reliably (successfully) be
performed by a skilled veterinary surgeon using the
same aseptic technique (experiment 2 with
intentional contamination)
– In our study, sterilization failure could not be directly
attributed to bead temperature
• The risk of instrument contamination increases in the
research setting when inadequate aseptic technique
is practiced, the GBS instructions are not followed, or
the surgeon is inadequately skilled or trained
Conclusions and Discussion
• Risk-based recommendations that we developed can be
used on an individual basis to determine an appropriate
optimum number of serial rodent surgeries
• Rather than one size fits all, this approach customizes to the
individual performing the serial surgeries, minimizing crosscontamination (infection) and maximizing animal welfare
• Instruments and/or instrument packs should be replaced
when they become contaminated with fecal material and we
do not recommend using a GBS for instrument
decontamination in serial rodent intestinal surgeries
Acknowledgements
•
Many thanks to the ACLAM Foundation for supporting our work and to Corinne
Buck, Peggy Pittman, Qiang Wu, Marty Roop, Matt Rosenbaum, Robin Alligood
Anita Coburn, and Dorcas O’Rourke for their valuable assistance and input!