An enzyme–detergent method for effective prion decontamination of surgical steel

Abstract

Prions, transmissible agents that cause Creutzfeldt-Jakob disease (CJD) and other prion diseases, are known to resist conventional sterilization procedures. Iatrogenic transmission of classical CJD via neurosurgical instruments is well documented and the involvement of lymphoreticular tissues in variant CJD (vCJD), together with the unknown population prevalence of asymptomatic vCJD infection, has led to concerns about transmission from a wide range of surgical procedures. To address this problem, conditions were sought that destroy PrP Sc from vCJD-infected human tissue and eradicate RML prion infectivity adsorbed onto surgical steel. Seven proteolytic enzymes were evaluated individually and in pairs at a range of temperatures and pH values and the additional effects of detergents, lipases and metal ions were assessed. A combination of proteinase K and Pronase, in conjunction with SDS, was shown to degrade PrP Sc material from highly concentrated vCJD-infected brain preparations to a level below detection. When RML prion-infected wires were exposed to the same enzymic treatment, intracerebral bioassay in highly susceptible hosts showed virtually no infectivity. The prion-degrading reagents identified in this study are readily available, inexpensive, non-corrosive to instruments, non-hazardous to staff and compatible with current equipment and procedures used in hospital sterilization units.

Feasibility of infectious prion digestion using mild conditions and commercial subtilisin

“…The observation that pH dependent dose response greatly decreases with increasing enzyme concentration suggests that a resistant core of PrP res infectivity may exist below the limit of quantitation (LOQ) of the ELISA assay, as raw absorbance is still slightly above background. As others have observed, in vitro results are not predictive of in vivo results (Jackson et al, 2005;Kocisko et al, 1995). Therefore, to conclude if the conditions used in our in vitro digestions conditions removed all detectable CWD PrP res infectivity, a bioassay using transgenic cervidized mice will be required.…”

“…Bioassays in live animals are more sensitive, test actual infection, are representative of natural infection, and can detect low-level residual infectivity that may reside below the limit of detection of traditional immunoassays. Experiments utilizing mouse bioassays to investigate decontamination of PrP res material with proteinase K (Jackson et al, 2005;Yakovleva et al, 2004), Properase (McLeod et al, 2004), and Rapid Multi-Enzyme cleaner 3 M (Lawson et al, 2006), suggests that enzymatic approaches to PrP res contamination is achievable. Although the references cited were somewhat successful, the difficulty in achieving a robust PrP res decontamination method is also revealed in the published data.…”

Disinfection and Sterilization of Prion-Contaminated Medical Instruments

“…Observations of analogous regimens by others yielded similar results. 4,5 Yan et al 3 also concluded that even the combination of NaOH and autoclaving at 134°C for 18 minutes may not be completely effective, so that decontamination protocols combining chemical and heat inactivation should be carefully selected and experimentally validated. 3 Furthermore, we believe that the paucity of reported cases of iatrogenic TSE attributed to contaminated surgical instruments processed by conventional techniques may not be as reassuring as Rutala and Weber 1 assert.…”

“…We and others have repeatedly demonstrated that all disinfection or sterilization processes can be made to fail by altering the test method or parameters for disinfection and/or sterilization. 4 " 11 In fact, we can make any Environmental Protection Agencyregistered disinfectant and any US Food and Drug Administration-cleared sterilization process fail to inactivate even easy-to-kill microorganisms, such as bacteria, on the basis of the methodology used. It is crucial, especially when assessing the efficacy of protocols for the disinfection of prions, that one place greater weight on studies using disinfection protocols representative of current and standard instrument reprocessing procedures.…”

Rapid chemical decontamination of infectious CJD and scrapie particles parallels treatments known to disrupt microbes and biofilms

“…[18][19][20] A variety of other proteases also has shown little detrimental effect on 263K or RML scrapie agent brain titers in different material contexts. 21,22 Indeed, PK C Pronase yielded negligible destruction of RML infectivity without first pre-heating samples at 100 C for 15 min in 4% SDS. 22 In a physiological context, TSE agents, like many viruses, can survive proteolytic digestive juices whereas PrP does not.…”