Staphylococcus xylosus PCR-validated Decontamination of Murine Individually Ventilated Cage Racks and Air Handling Units by Using 'Active-Closed' Exposure to Vaporized Hydrogen Peroxide

Abstract

Vaporized hydrogen peroxide (VHP) is used to decontaminate clinical, biocontainment, and research animal rooms and equipment. To assist with its implementation in a murine facility, we developed a safe and effective method of VHP sterilization of IVC racks and air handling units (AHU). Safety of VHP decontamination was assessed by ensuring VHP levels dissipated to less than 1 ppm in the room prior to personnel reentry and inside the primary enclosure prior to the return of mice; this condition occurred at least 18 h after the VHP cycle. Efficacy of VHP sterilization was assessed by using chemical indicators, biologic indicators, and PCR testing for Staphylococcus xylosus, a commensal organism of murine skin and an opportunistic pathogen, which was present in 160 of 172 (93%) of specimens from occupied IVC racks and the interior surfaces of in-use AHU. Neither mechanized washing nor hand-sanitizing eradicated S. xylosus from equipment airway interiors, with 17% to 24% of specimens remaining PCR-positive for S. xylosus. 'Static-open' VHP exposure of sanitized equipment did not ensure its sterilization. In contrast, 'active-closed' VHP exposure, in which IVC racks were assembled, sealed, and connected to AHU set to the VHP cycle, increased the proportion of chemical indicators that detected sterilizing levels of VHP inside the assembled equipment, and significantly decreased PCR-detectable S. xylosus inside the equipment. Supplementing bulk steam sterilization of the primary enclosure with VHP sterilization of the secondary housing equipment during room change-outs may help to mitigate opportunistic agents that jeopardize studies involving immunodeficient strains.

Figure 1.

Room preparation prior to VHP exposure. Room volume was calculated and programmed via the (A) electronic console outside of the room which controlled the (B) VHP generator and aeration catalytic accelerator, which were positioned inside the room with housing equipment to be decontaminated. HVAC supply and exhaust vents and ceiling thimble connections were covered and the (C) room door sealed with tape.

Figure 2.

To assess the efficacy of each VHP cycle, indicators were placed in the room before VHP exposure, including (A) CI (arrow) on the room wall, and inside equipment airways, including (B) CI inside vertical manifolds and (C) BI inside exhaust plenums of IVC racks. In addition, specimens were collected (D) from soiled, washed, or VHP-exposed equipment surfaces and then PCR-analyzed for the presence of S. xylosus.

Figure 3.

Completion of VHP exposure was assessed first from outside the room by using (A) the electronic console, ensuring that it reported less than 1 ppm inside the sealed room and then after unsealing the door (B) leading into the room containing equipment after VHP exposure and assessing H₂O₂ levels at (C) the door threshold and (D) various locations throughout the room by using a handheld H₂O₂ monitor.

Figure 4.

Static–open VHP exposure, with ceiling thimble connections and HVAC supply and exhaust vents covered, all AHU off, hoses left disconnected, and the IVC rack left unassembled and unconnected to an AHU.

Figure 5.

Active–closed VHP exposure, with ceiling thimble connections and HVAC supply and exhaust vents covered, the IVC rack fully assembled, plenums capped, and hoses connecting each IVC rack to an AHU set to the VHP cycle.

Figure 6.

VHP levels detected inside hoses that connected the AHU to either the IVC rack supply (green) or exhaust (red) plenum or to the ceiling thimble (blue) before and after active–closed VHP exposure.

Figure 7.

Mean quantitative PCR copy numbers of S.xylosus detected in the exhaust plenum following VHP active–closed exposure and the return of murine inventory.