Development of a functional and disinfectable sewage vent high-efficiency particulate air filter system and its validation using vaporized hydrogen peroxide

Airborne disinfection of high-containment facilities is mandatory for safe operations. However, sewage vent air—particularly in large-animal experimental units—can represent a critical gap and is typically not considered in the design of high-efficiency particulate air (HEPA) filter systems. Here, we present an engineering design enabling HEPA filtration of sewage vent air from primary biological containments, including large-animal facilities and necropsy suites. We additionally evaluated vaporized hydrogen peroxide (vH 2 O 2 ) fumigation as an airborne disinfection approach. Disinfection efficacy was determined by quantitative carrier testing using commercial spore carriers (CSC) coated with 10 6 spores of Geobacillus stearothermophilus and compared with germ carriers coated with surrogate viruses, mycobacteria, and other spores. Temperature, relative humidity (RH), and vH 2 O 2 concentration were monitored in real time to define process parameters and validate protocols targeting ≥4 log 10 reduction. Inactivation efficacy differed substantially among challenge agents: CSC were least resistant to inactivation, whereas murine norovirus (MNV) on germ carriers was most challenging to inactivate. At medium-high vH 2 O 2 concentrations (up to 520 parts per million [ppm]), increasing RH was the dominant driver of inactivation. Based on these data, we developed a biphasic disinfection cycle maintaining a saturated humidity environment with ~35% H 2 O 2 for ≥10 h, achieving ≥4 log 10 reduction of MNV infectivity and G. stearothermophilus spore viability on germ carriers. Overall, vH 2 O 2 under high-to-saturated RH is an effective means to disinfect technical infrastructure in biological containment settings handling high-consequence pathogens. IMPORTANCE Vent air from sewage systems in biological containment facilities where large animals are kept should be considered a contaminated waste stream. Therefore, effective high-efficiency particulate air (HEPA) filter systems that prevent mold or other moisture-related deterioration of the filters need to be installed. Airborne vaporized hydrogen peroxide (vH 2 O 2 ) is an appropriate method to decontaminate these complex HEPA filter systems. High to saturated humidity is required for virucidal and sporicidal efficacy under medium to high concentration of vH 2 O 2 . Commercially available spore carriers are artificial in design, resulting in lower resistance to inactivation compared to custom germ carriers coated with non-enveloped viruses and thus should be avoided as read-out (validation means) by the biosafety community working with high-consequence pathogens in primary containments.