## 4 Discussion
The RENO-S130 sterilizer can operate in non-lumen mode, which is suitable for medical devices that require surface sterilization, or Eco mode, which is used for medical devices containing an inner compartment. The results from the present study suggest the RENO-S130 device is effective at inactivating scrapie prion. There are, however, a few limitations to this study. Firstly, quantitative PMCA methods such as real-time quaking-induced conversion (qRT-QuIC) should be performed to fully elucidate the effect of RENO-S130 treatment on prions. Secondly, the observed reduced levels of infectivity caused by treatment with the RENO-S130 device should be clarified in a quantitative manner. Additional analyses, including endpoint titration [42] and an incubation time interval assay [43], would be required to determine the reduced levels of infectivity resulting from RENO-S130 treatment. Nonetheless, because the observed inactivation effect of RENO-S130 was considerable, it can be concluded that treatment with the RENO-S130 device clearly does inactivate scrapie prion. Noteworthy, the brain sample homogenates comprised about 2 mg of proteins. It is estimated that protein contamination levels resulting from surgical procedures are typically much lower (8 to 91 ug per medical device) [44]. In addition, pretreatment washing of medical devices using enzyme or alkaline detergents will achieve 4-6 log reduction in microorganisms and proteins [45]. Hence, pretreatment alongside exposure to the RENO-S130 device in the sterilization process may enhance the total reduction of prion infectivity to achieve complete inactivation. Our previous studies have shown that reactive chemical species generated by the gas plasma may be major contributing factors responsible for the inactivation of pathogens including prions [34; 46; 47]. Nevertheless, the main reactive chemical species besides hydrogen peroxide gas contributing to the inactivation of prions by the RENO-S130 device remains to be clarified. Our preliminary studies using an ozone analyzer UV-100 (Eco Sensors Division of KW) Engineering Inc., Santa Fe, NM, USA) have shown that the concentration of ozone in the sterilization chamber box of RENO-S130 was 10-50 ppm. Ozone is generated when air is introduced into the DBD plasma region and then moved into the sterilization chamber. A recent study has shown that hydrogen peroxide gas and ozone gas synergistically decrease the infectivity of prion [48], and ozone generated by the DBD plasma in RENO-S130 may contribute to the enhanced sterilization effect. Further studies are required to ascertain which of these factors contribute to reducing the in vitro propagation activity and infectivity of prions. Additional studies using optical emission spectroscopy (OES) and electron spin resonance (ESR) spectroscopy would be also necessary to determine the reactive chemical species in hydrogen peroxide gas plasma generated by the RENO-S130 device that are involved in the mechanism of prion inactivation. Taken together, our results indicate that scrapie prion can be rendered harmless by treatment with a hydrogen peroxide gas plasma sterilizer combined with DBD and corona discharge using a RENO-S130 device. PMCA analysis and mouse bioassays demonstrate that the RENO-S130 treatment decreased the in vitro propagation activity of scrapie prion as well as the infectivity of the prions. Therefore, this sterilization procedure is potentially applicable to the decontamination of prion-contaminated medical devices. However, for medical applications, this disinfection/sterilization method will need to be fully evaluated using human prions to establish whether it is effective at preventing iatrogenic transmission. Susceptibility to physical and chemical treatment may vary between different animal prions [11]. Indeed, the heat susceptibility of prion depends on the specific prion strains [49]. Therefore, prions derived from different strains and animals may behave differently to the treatment with the RENO-S130 device.