**3.5 Viral efficacy indicated through bacterial spore validation**

The EPA and the Centers for Disease Control and Prevention (CDC) recognize that certain microorganisms can be ranked with respect to their tolerance to chemical disinfectants [7]. As a result, efficacy against less susceptible bacterial spores can be extrapolated to indicate efficacy against more susceptible microorganisms, including enveloped and non-enveloped viruses [8, 9, 52].

#### **Figure 6.**

*Viral Reductions Post Hybrid Hydrogen Peroxide (HHP) Fogging. Data table demonstrating the efficacy of HHP fogging for reducing tested viruses and bacteriophage to below the limit of detection–not detected (ND)– measured as either plaque-forming units (PFU) or median tissue culture infectious dose (TCID50) [19].*

### *3.5.1 Bacterial spore efficacy data: necropsy, laboratories, and interstitial spaces*

To assess efficacy within various Biosafety Level 3 Agricultural (BSL-3Ag) environments, Kansas State University challenged the HHP system within their Biosecurity Research Institute, a BSL-3Ag facility. Testing was performed in three laboratories representing a range of sizes: 2,281 ft3 (65 m<sup>3</sup> ), 4,668 ft<sup>3</sup> (132 m<sup>3</sup> ), and 44,212 ft3 (1,252 m<sup>3</sup> ). Each of the two smaller laboratories were tested over a series of three disinfection cycles with biological indicators of *Geobacillus stearothermophilus* (6.2 log10 spores) encased in Tyvek/Tyvek and placed throughout the laboratories, in laboratory equipment such as biological safety cabinets, and in the overhead interstitial space (drop ceiling). Testing in these laboratories resulted in a greater than 6-log10 reduction of all 252 challenged indicators, including those placed in the difficult to access interstitial space.

Within the largest space tested, the 44,212 ft3 (1,252 m<sup>3</sup> ) necropsy laboratory, four HHP devices were used for the disinfection cycle. The smart technology of the HHP system automated the connection of multiple Pulse fogging devices for a synchronized, custom-calibrated, HHP cycle. A total of 206 biological indicators were tested over two HHP cycles in locations throughout the laboratory, including at the 21-ft (6.4 m) ceiling height, soft-sided anteroom, walk-in cooler, and change rooms. All 206 challenged indicators were negative for spore growth, demonstrating a greater than 6-log10 reduction of *G. stearothermophilus*. This BSL-3Ag testing provides real-life results within the targeted environment for the HHP system. The smart controls and automation allowed this testing to be performed in house by the laboratory personnel [41].

#### *3.5.2 Bacterial spore efficacy data: sterilization study on porous surfaces*

The BMBL (6th edition) defines sterilization as; "a physical or chemical process that kills or inactivates all microbial life forms including highly resistant bacterial spores." The importance of sterilization is well understood in life science, pharmaceutical, and healthcare industries. Through the process of sterilization, researchers and physicians alike establish the basis for reliable and safe protocols and procedures. Standards for fogging sterilization testing are developed by the Association of Official Analytical Chemists (AOAC International), a globally recognized, third party not-for-profit, that provides education and facilitates the development of test methods and standards.

The HHP system was challenged with the Fogging Devices Sterilant Test (OCSPP 810.2100) for efficacy against *B. subtilis* (strain 19615) spores, an opportunistic pathogen, which is tolerant of ultraviolet light and high temperatures, and *Clostridium sporogenes* (strain 3584) spores, a strain of *Clostridium botulinum*. These two spores are designated for this test due to their enhanced survivability compared to other spore types. Two carrier formations were used for both spore types, porcelain Penicylinders and Dacron™ suture loops. Each carrier type was saturated with the substrate, distributing spores throughout these materials. Half of each type of carrier was placed inside Tyvek/Tyvek pouches, with the remaining carriers placed in glass petri dishes. Carriers with these bacterial spores were placed throughout the 9<sup>0</sup> <sup>11</sup>″ � <sup>14</sup><sup>0</sup> <sup>6</sup>″ � <sup>12</sup><sup>0</sup> <sup>9</sup>″ (1,833 ft<sup>3</sup> / 51 m<sup>3</sup> ) testing room. A total of 151 carriers were tested, with only three carriers being found positive for spore growth, all on porcelain Penicyliner carriers enclosed in Tyvek/Tyvek pouches (1 *B. subtilis*, 2 *C. sporogenes*) [50]. This testing method is designed to challenge a fogging system's penetration and subsequent disinfection of spores within these porous carriers. These results demonstrated the HHP system's ability to penetrate through two forms of porous surfaces to inactivate the resistant spores.

*Hybrid Hydrogen Peroxide for Viral Disinfection DOI: http://dx.doi.org/10.5772/intechopen.100237*

### *3.5.3 Bacterial spore efficacy data: sporicidal study in a tripartite soil load*

*Clostridioides difficile* is a bacterium responsible for causing almost half a million infections in the United States alone each year, with fatal outcomes for 1 in 11 people over the age of 65 within one month of infection [53]. *Clostridioides difficile (C. diff)* is considered one of the most epidemiologically important pathogens, as its environmental persistence, antibiotic resistance, and low infectious dose have led to this bacterium plaguing hospitals and long-term care facilities alike [54]. Precisely due to the hardiness of this bacterium in spore form, *C. diff* has become a standard against which to measure disinfectant efficacy and forms the basis of the EPA's Emerging Viral Pathogen efficacy and approval [12]. With the understanding that pathogens in the environment do not exist in a vacuum, but rather are more likely to be found within a soil load consisting of physiological fluids such as blood, purulent material, or feces, the EPA updated testing requirements for sporicidal classification to challenge not only against hardy *C. diff* spores, but to test such spores within three protective materials (tripartite load; bovine serum albumin, yeast extract, mucin). In 2018, the HHP system was awarded sporicidal classification in the EPA's most stringent *C. diff* test; elimination of *C. diff*spores in a tripartite soil load. A total of 63 carrier plates over three testing lots were exposed to the HHP cycle, resulting in the inactivation of all 63 carriers and an average log10 reduction of 6.6 for this difficult to kill bacterial spore. This testing confirmed the HHP system's ability for high-level disinfection with sporicidal classification [46].
