## 4 Discussion  
The prion agent is a pathogen that is highly resistant to inactivation using standard chemical and physical disinfection methods. Prion-related iatrogenic disease can be caused by prion-contaminated medical devices [13; 17]. Thus, there is a requirement to develop effective methods to inactivate prion agents from medical instruments. The usefulness of a novel hydrogen peroxide gas plasma sterilizer RENO-S130 for prion inactivation has been evaluated. Several studies have reported that gas plasma has an inactivating effect on prions [36]. For example, gas plasma generated by corona discharge in open air was shown to inactivate scrapie prions [37]. In the study, [PERSON] et al. used a cell infectivity assay and Western blot analysis and found a reduction of both PrP\({}^{\text{Sc}}\) and PrP\({}^{\text{C}}\) after exposure to gas plasma. In addition, [PERSON] et al. [38] contaminated stainless steel spheres with scrapie prion before treatment with RF plasma generated using an Ar/O\({}_{2}\) gas mixture for 1 h, which prolonged the incubation time of hamsters infected with the scrapie prion. In a study of the European project BIODECON, prions were shown to be inactivated by Ar-plasma more efficiently than a mixed Ar/N\({}_{2}\) plasma [39]. At present, the hydrogen peroxide gas plasma sterilization device STERRAD(r) is primarily employed to disinfect medical instruments that cannot be exposed to extreme heat [40]. An earlier study demonstrated that a stainless steel wire contaminated with Scrapie 263K could be efficiently sterilized by treatment with a hydrogen peroxide gas plasma sterilizer such as STERRAD(r) NX or STERRAD(r) 1005 [41]. The results showed that STERRAD(r) NX (90% hydrogen peroxide gas, 53 \({}^{\circ}\)C, 7 min) had an inactivating effect i.e., none of the 8 test animals exposed to the treated prion developed the disease, and the infectious titer was reduced by at least 5 to 6 logs. However, STERRAD(r) 100S (59% hydrogen peroxide gas, 50 \({}^{\circ}\)C, 20 min) showed almost no prion inactivating effect. This observation indicated that hydrogen peroxide gas may only have a prion inactivating effect under specific conditions (e.g., incubation temperature, concentration). However, other studies suggested that the sterilization effect of STERRAD(r) occurs irrespective of the presence or absence of gas plasma and that only hydrogen peroxide gas appears to contribute to the inactivation [16; 18]. This hypothesis is supported by the results of experiments that indicate hydrogen peroxide gas itself elicits a prion inactivating effect [29; 30]. Thus, it is necessary to analyze the prion inactivation effect of each device in turn because conditions, such as the sterilization process, hydrogen peroxide concentration, and incubation temperature, differ depending on the specific hydrogen peroxide gas plasma sterilizer. Here, we examined the effect of hydrogen peroxide gas plasma generated by the combination of DBD and corona discharge using a new sterilizer RENO-S130 as a potential disinfection/sterilization method for the scrapie prion agent and confirmed the inactivation effect. In the RENO-S130 device, hydrogen peroxide is vaporized as a sterilizing agent and plasma is generated for processing [35]. RENO-S130 differs from other hydrogen peroxide gas plasma sterilizers in terms of temperature, humidity, hydrogen peroxide gas concentration, etc. during sterilization. The temperature of the sterilization chamber was maintained below 55 \({}^{\circ}\)C. The RENO-S130 sterilizer has a decompression process in which the inside of the chamber is vacuum depressurized to 0.5 Torr. Hydrogen peroxide (50%) is injected into the chamber, and gas plasma is generated by DBD for sterilization at 760 Torr (non-lumen mode) or at 760-988 Torr (Eco mode). The gas plasma generated by corona discharge degrades the hydrogen peroxide gas during decompression to 0.5 Torr, which is then further replaced with air to completely remove any residual hydrogen peroxide gas.