Development of rapid sensitive neuroblastoma and stem cell culture systems to assess the efficacy of prion decontamination technologies

Transmissible spongiform encephalopathies (TSE), otherwise known as prion diseases, are fatal degenerative brain diseases involving conversion from the normal alpha helical PrP protein to the beta-pleated sheet PrPsc isoform. Current epidemiological and research evidence suggests that the risk of PrPsc transmission from TSE patients to other humans may be very low; nevertheless, TSE agents constitute a serious bio-medical hazard and the current concern of blood borne infection is increasing the perceived risk. A proven route of infection involves contaminated surgical instruments, particularly those in contact with neural tissues. As such, prions are a significant concern to the life sciences, veterinary and medical professions, and there is an urgent need for safely removing and deactivating them on a range of surfaces. Although government agencies, WHO and other institutions have distributed some guidance for safe working and prevention of infection, these guidelines are not compulsory and are not harmonised globally. In the UK, guidelines recommending relatively strict standards for decontamination of medical instruments have been proposed by the ACDP, SEAC and Department of Health. Resistance of PrPsc to sterilisation cleaning has raised concerns about decontaminating surgical instruments such as endoscopes that usually involves a preliminary treatment, rinsing, actual disinfection, final rinsing and storage. PrPsc destruction requires prolonged autoclaving, which is only possible at the present time with the most recent rigid endoscopes. Indeed, there are general concerns about the efficacy of autoclaving, chemical disinfectants and the temperatures and contact times required. Many decontamination studies carried out thus far have involved solutions or suspensions of PrPsc agent and may not reflect the behaviour of surface-bound infectivity. In one notable study, Zobeley et al. used Scrapie mouse brain homogenate bound to stainless steel to show that PrPsc was not removed after repeated washing with phosphate buffered saline; moreover, there was a 30-fold reduction in prion after treating with 10% formaldehyde solution for one hour. The prion contaminated steel still caused infection when introduced into normal mouse brain. The majority of labs rely on detection of PrPsc on surfaces by swabbing the surface or immersion of small areas in detergent before quantification using Proteinase K pre-treatment (to eliminate PrP) and Western blots. This approach is relatively insensitive and may underestimate the amount of prion present if the antibody-binding epitopes have been modified by the decontamination or preparation procedures used. However, Prof Keevil's group at Southampton have developed a patented procedure for the rapid, sensitive detection of general contamination of medical instruments and specific PrPsc attachment, in situ, before and after cleaning. Their epifluorescence microscopy approach, coupled with episcopic differential interference contrast microscopy to visualise highly curved or serrated surfaces, permits detection of sub-micron, sub-picogram amounts of amyloid plaque on surfaces. Studies using ME7 infected mice show that some detergent or enzyme solutions can give good but not complete removal of brain material containing PrPsc dried on stainless steel. The biochemical methods developed at STERIS, Southampton and CEA Saclay, France have identified interesting chemical solutions to prion decontamination. Currently this research uses a modification of the stainless steel microwire inoculation into mouse brain technique to demonstrate infectivity. It is proposed to complement the work on prion visualization and decontamination by investigating the use of neuroblastoma and stem cell models at Southampton to rapidly assess the infectivity of contaminated steel surfaces following decontamination procedures. The student will be seconded to CEA Saclay to compare cell culture with the live model assay