**3.2 Validating the HHP process**

When targeting pathogens invisible to the eye, there must be some way to measure the efficacy of disinfection. Employing validation tools gives the ability to verify a disinfection process using living organisms and giving results rooted in science. Though several types of chemical and pH indicators exist, indicators of *Geobacillus stearothermophilus* bacterial spores (1 <sup>10</sup><sup>6</sup> organisms) are used as the international standard for validation of sterilization by hydrogen peroxide [44, 45]. These 6-log10 indicators consist of a verified population of approximately 1 million bacterial spores. The evolutionary hardiness of bacterial spores has led to them being used as a standard of measurement for sterilization [2]. Inactivation of these difficult-to-penetrate spores also represents confirmation of efficacy in disinfecting lower-level pathogens, such as non-enveloped viruses, gram-negative and gram-positive bacteria, molds, yeasts, and enveloped viruses (**Figure 5**) [8, 9, 45]. Likewise, proving inactivation of these robust organisms predicts successful disinfection of more susceptible pathogens [11, 12].

**Figure 5.**

*Microbiological Disinfection Hierarchy. Described in* chemical disinfection of medical and surgical materials, *EH Spaulding ranked the microbiological hierarchy of disinfectants, listing organisms from least susceptible to most susceptible, according to their vulnerability to disinfectants [8, 9, 45].*

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

Recognizing a disinfectant's ability to kill less susceptible pathogens as an indicator of broader effectiveness, the EPA offers a variety of specific designations a chemical or system can claim. In 2018, the HHP system was approved for sporicidal classification by the EPA for a 6-log10 reduction of *Clostridioides difficile* (*C. diff*) in a tripartite soil load [46]. The EPA's Emerging Viral Pathogens claim was additionally approved for the HHP system on the basis of this sporicidal data [37]. Granting of this classification may further support the validity that efficacy against bacterial spores will likely conclude efficacy against enveloped and non-enveloped viruses. Targeting a 6-log10 or greater reduction of bacterial spores for validation is a key component of achieving a successful high-level disinfection [47]. Achieving this 6-log10 sporicidal kill will enable confidence against more susceptible organisms, such as enveloped or non-enveloped viruses [9] which may exist in a soil load or biofilm, making them more difficult to inactivate [13, 43].

### **3.3 Viral efficacy data: norovirus**

Norovirus, a single stranded non-enveloped virus of the Caliciviridae family, is a leading cause of acute gastroenteritis in humans. The most common genogroup GII is responsible for 95% of infections, which can have severe and even fatal outcomes in at-risk populations such as young children or the elderly. Norovirus, once present, can become a pervasive problem due to the environmental stability of the virus, low infectious dose, resistance to alcohol and chlorine-based disinfectants, and the potential for prolonged asymptomatic shedding of infected individuals. Norovirus is also used as a target organism for testing, as it is considered to be a non-enveloped virus with relatively low susceptibility to disinfectants [48].

In 2018, a 1,600-bed assisted living facility had a norovirus outbreak affecting 1/4 of the residents within a 2-week period with an average of 40 new cases a day, despite protective measures such as the quarantine of afflicted individuals. A biodecontamination company employing HHP technology was brought into the facility for outbreak response and control. HHP fogging was implemented as part of a 5-point process including continued quarantine and enhanced staff education. After a four-day implementation period, no new cases were reported, effectively ending the outbreak [49].

The HHP system was also tested under GLP conditions for efficacy against the norovirus testing surrogate feline calicivirus [20]. In this testing, 21 inoculated glass agar carrier plates were placed throughout the test room, ranging from floor level to 12 feet (3.6 m) in height, and exposed to the HHP fogging protocols. There was no recovered virus from the challenged plates for an overall reduction of 7.6 log10 (**Table 1**). Interestingly, efficacious results were also noted in GLP compliant testing when a carrier plate lid was accidentally left on during the HHP fogging cycle. This protocol deviation allowed for the observation that, even under these challenging conditions, the HHP fog migrated underneath the lid and achieved inactivation of viral particles [20].

The combination of these two studies demonstrates that the HHP system effectively disinfects complex spaces contaminated with norovirus or its surrogates in both laboratory and real-world conditions. Though the assisted living facility case study did not measure a numerical reduction of viral burden, the effective outbreak control of 100% reduction in new cases leads to the conclusion that norovirus was reduced to levels less than the infectious dose.

#### **3.4 Viral efficacy data: within porous materials**

In the spring/summer of 2020, the COVID-19 pandemic triggered a scarcity, and subsequent shortage of personal protective equipment (PPE) used by hospitals and


#### **Table 1.**

*Efficacy. Summary table of data presented within this chapter demonstrating efficacy of the HHP system against a range of pathogens and substrates. Sporicidal results show inactivated (negative) carriers by log reduction, viral results show either log reduction or limit of detection (LOD) where applicable. \* indicates where log10 reduction is the starting log titer and the LOD = log titer.*

other healthcare facilities. In an attempt to find ways to mitigate this emergency, researchers at Pennsylvania State University (Penn State) employed HHP to disinfect expired N95 respirators to assess the applicability of the HHP system for this use. Respirators were tested both for any physical degradation effects of the treatment on the respirator material and for efficacy of disinfection of respirator components via inoculation with three viral pathogens and one bacteriophage. Viral work performed at the Eva J Pell Biosafety Level 3 laboratory at Penn State used viruses of different characteristics, as well as a bacteriophage, to represent the range of physical characteristics of pathogens to which healthcare workers may be exposed (**Table 1**) [19]. Three viruses: herpes simplex virus (HSV-1; enveloped

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

virus; family Herpesviridae), coxsackievirus (CVB3; non-enveloped virus; family Picornaviridae), and SARS-CoV-2 (isolate USA-WA1/2020; enveloped virus; family Coronaviridae), as well as pseudomonas bacteriophage (phi6; enveloped), were chosen for testing (**Figure 6**). The inside, outside, and strap materials of the respirators were used as inoculation sites. While the majority of these surfaces are made up of porous materials, at least one type of respirator had an outer layer of hydrophobic material which caused the inoculation droplet to dry into a 'coffee ring' pattern on the respirator. This testing of porous materials is significant because it presents a more difficult challenge to disinfection than non-porous surfaces, since the materials which absorb the pathogen may also provide a degree of protection, at least temporarily [51]. Disinfectant efficacy testing is commonly done on nonporous surfaces, which does not reflect the difficulty and variables that porous surfaces present.

Testing performed at Penn State also included the use of biological indicators as validation of the protocol for a successful HHP cycle. For each HHP cycle, 6 to 12 biological indicators (*Geobacillus stearothermophilus* ATCC® 7953) with a mean spore count 2.4 <sup>10</sup><sup>5</sup> on stainless steel carriers encased in Tyvek®/Glassine pouches were placed throughout the room. In the total of 14 disinfection cycles, only 2 of 138 indicators returned positive for spore growth. These included preliminary cycles, which were intended to establish optimal cycle parameters [19].
