The hospital performs a wide range of surgical procedures. All surgical instruments at the facility are reprocessed in a centralized Sterile Processing Department. The first step in reprocessing includes cleaning and disinfection in a machine washer/disinfector (Reliance 444 or Reliance Synergy Washer/Disinfector, STERIS Corporation, Mentor, Ohio). During this step, instruments pass through 5 chambers, including 1) pre-washing with water and enzymatic solution for 1 minute, 2) washing using detergent solution at 150°F for 4 minutes, 3) ultrasonic cleaning with detergent for 4 minutes, 4) thermal and lubricant rinse with water at 180°F for 1 minute followed by instrument mild lubricant at 180°F, and 5) drying for 4 minutes at 180°F. The final step in reprocessing is steam sterilization. For instruments sterilized inside Steriset Containers, the standard operating procedure for the facility indicates that the autoclave (Amsco Eagle, STERIS Corporation) should be set on the pre-vacuum cycle as recommended by the manufacturer.
Evaluation of the effectiveness of the washer/disinfector
The evaluation was not conducted as a research project and no human subjects were involved. The Cleveland VA Medical Center’s Research and Development Committee reviewed the manuscript and approved submission for publication. We evaluated the effectiveness of the washer/disinfector for removal of vegetative bacteria and spores from surgical instruments. The instruments used for this evaluation were identical to those that were in the Steriset Containers when the failure to follow recommended procedures occurred; all of the instruments were non-hollow and without channels (e.g., retractors, clamps, forceps, needle holders, and scissors). For assessment of removal of vegetative bacteria, the ends of surgical instruments (e.g., retractors, clamps) that contact patients’ tissues were suspended for 1 minute in overnight cultures of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) (pulsed-field gel electrophoresis type USA 300) or vancomycin-resistant Enterococcus (VRE) (C68, a VanB-type VRE isolate) containing 9 log10 colony-forming units (CFU) of bacteria per mL. For assessment of removal of spores, the instruments were suspended for 1 minute in suspensions containing 106 CFU per milliliter of non-toxigenic Clostridium difficile (American type culture collection #43593) spores. The contaminated instruments were processed in the washer/disinfector as recommended by the manufacturer; control instruments were contaminated in an identical fashion but were not processed in the washer/disinfector.
Survival of C. difficile spores was assessed using broth enrichment cultures in C. difficile brucella broth (CDBB)  and survival of VRE and MRSA was assessed using broth enrichment cultures in brain-heart infusion broth. The inoculated instruments were suspended for 1 minute in the nutrient broth with vortexing and growth was assessed after 48 hours of incubation by plating onto C. difficile brucella agar (CDBA)  for C. difficile or 5% sheep blood agar plates for MRSA and VRE. The limit of detection for the organisms was ~1 log10CFU per mL, as determined by assessing recovery of serially diluted preparations of the test organisms applied to instruments.
Evaluation of the effectiveness of sterilization inside the Steriset Container on the gravity cycle
We evaluated the effectiveness of sterilization inside the Steriset Container when run on the gravity cycle. The autoclave settings were identical to those for the two autoclave runs that had occurred in the incident when the integrators indicated inadequate sterilization (i.e., 270°F for 15 minutes, pressure 28 to 30 psig). Temperature and pressure monitoring equipment are included features of the Amsco autoclave and records of temperature and pressure are maintained for each run of the autoclave; review of the monitoring strips from the time of the incident confirmed that a temperature of 270°F had been achieved for 15 minutes at a pressure of 28 to 30 psig. In addition, the number of instruments and loading pattern inside the container was the same as for the incident (i.e., the Steriset Containers ranged from half full to completely full with non-hollow instruments including forceps, needle holders, scissors, retractors, clamps). The loading pattern of the autoclave for the test runs was designed to replicate the loading pattern during the incident (i.e., fully loaded autoclaves with 9 to 12 Steriset Containers per load). In 4 of the 5 experimental runs included in the analysis, an Attest Rapid Readout Steam Pack (3M, Saint Paul, MN) containing 105Geobacillus stearothermophilus spores wrapped in paper was included inside the autoclave but outside the Steriset Containers.
Four methods were used to assess sterilization inside the Steriset Containers. These included: 1) 3M Comply (SteriGage) Steam Chemical Integrators, 2) Biological indicator (BI): 3M Attest1292 Rapid Readout BI/Steam containing Geobacillus stearothermophilus spores, 3) C. difficile spores (105 CFU in 0.1 mL of sterile water), and 4) MRSA (9 log10CFU in 1 mL of brain-heart infusion broth). In addition to the suspensions of MRSA and C. difficile in capped glass tubes, multiple instruments were suspended for 1 minute in bacterial suspensions of MRSA and C. difficile spores as described previously and placed into Steriset Containers. Survival of C. difficile spores or MRSA was assessed using broth enrichment cultures in C. difficile brucella broth (CDBB)  or brain-heart infusion broth, respectively (i.e., sterile nutrient broth was added directly to the tubes that contained the organisms or the inoculated instruments were suspended for 1 minute in the nutrient broth with vortexing and growth was assessed after 48 hours of incubation by plating onto C. difficile brucella agar (CDBA)  for C. difficile or 5% sheep blood agar plates for MRSA).
To address the question that was raised regarding the possibility that there might be “cold pockets” of air inside the Steriset Container, we used thermal probes (Max Temperature Tester, American Dental Accessories (Minneapolis, MN), to compare the temperature inside versus outside the container in the autoclave and placed biological indicators (i.e., 3M Attest 1292 Rapid Readout BI/Steam containing G. stearothermophilus spores) in multiple locations within the Steriset Container (i.e., each corner of the container and in the middle). Acceptance of the steam chemical integrator placed inside the Steriset Container was interpreted as an indication that some steam had entered the container (i.e., the pellet in the integrator has a melting point of 285˚F and will only melt at the 270˚F operating temperature of the autoclave if some steam has entered because steam lowers the melting point).
Calculation of the risk of transmission of MRSA and hepatitis B virus
We used the method of Rutala and Weber  to calculate the risk of transmission of MRSA by the surgical instruments that were inadvertently autoclaved on the gravity cycle. The reductions in MRSA for the washer/disinfector (1:1,000,000) and for the gravity autoclave cycle inside a Steriset Container (1:10,000,000) were based on the data presented here. The likelihood of MRSA contamination of the instruments prior to reprocessing was based on a 15% prevalence of MRSA carriage among inpatients at the hospital. MRSA was chosen because it is a common cause of surgical site infections and the usual perioperative prophylaxis regimen used in the hospital (i.e., cefazolin) would not be protective against this organism. We also estimated the risk of transmission of hepatitis B virus using the same methods.
Fisher’s exact test was used to compare categorical data.