A rapid dual staining procedure for the quantitative discrimination of prion amyloid from tissues reveals how interactions between amyloid and lipids in tissue homogenates may hinder the detection of prions

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases with no cure to this day, and are often associated with the accumulation of amyloid plaques in the brain and other tissues in affected individuals. The emergence of new variant Creutzfeldt-Jakob disease, an acquired TSE with a relatively long asymptomatic incubation period and unknown prevalence or incidence, which could potentially be iatrogenically transmitted, has prompted the need for sensitive and rapid methods of detection of the pathology indicator, the protease-resistant prion protein (PrP(Sc)), in tissues and on surgical instruments. To discriminate between common tissue proteins and amyloid-rich aggregates such as those formed by abnormal prion, we developed a quantitative thioflavin T/SYPRO Ruby dual staining procedure, used in combination with episcopic differential interference contrast/epifluorescence (EDIC/EF) microscopy for rapid scanning of samples. The detection limit of this direct observation technique applied to brain homogenates was greatly enhanced by the addition of Tween 20, as demonstrated in double-blind studies using various proportions of ME7-infected brain mixed with normal brain homogenate. The characteristic thioflavin T signal correlated with the relative amount of prion amyloid and proved at least 2-log more sensitive than the classic Western blot using the same prepared samples. This new sensitive microscopy procedure, which can be easily applied in instrument decontamination surveys, is likely to be more sensitive that Western blot in practice since it does not rely on the elution of resilient PrP(Sc) bound to the instrument surfaces. Our study also demonstrates how interactions between prion and lipid-rich tissue homogenates may reduce the sensitivity of such detection assays

Effect of drying time, ambient temperature and pre-soaks on prion-infected tissue contamination levels on surgical stainless steel: concerns over prolonged transportation of instruments from theatre to central sterile service departments

Iatrogenic transmission of prions through use of surgical instruments has been shown both experimentally and clinically. In addition, recent discoveries of prion protein accumulation in peripheral tissues such as appendix and muscle, and evidence suggesting human-to-human blood-borne transmission, have led to a concern that any residual soiling containing this agent may remain infectious even after sterile service processing. Removal of all proteinaceous material from surgical devices is extremely important for effective sterilization. This removal can be severely hampered if the contaminant is allowed to dry onto the instrument surface for any length of time. The current move to centralize sterile service centres and the inevitable lengthening of transport time between theatres and re-processing makes it necessary to minimize the amount of residual soiling adhering to an instrument before sterilization. This investigation simulates the period between the application of surgical instruments in theatre and their initial pre-wash by a washer/disinfector. The aim was to investigate the kinetics of drying at different temperatures, and the application of different commercially available pre-soak solutions in situ. The findings show that all pre-soaks significantly reduce (by up to 96%) the prion-infected tissue contamination, and that controlling the temperature whilst in transit between theatres and cleaning facilities may allow an increase in time before high protein adsorption levels occu

The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission

Regulations recommend the routine application of biochemical tests, such as the Ninhydrin or Biuret tests, to confirm the efficacy of hospital sterile service department (SSD) washer-disinfector cycles in removing proteinaceous material, particularly with respect to prions. The effectiveness of these methods relies on both the effective sampling of the instruments and the sensitivity of the tests employed. Two commercially available contamination assessment tests were evaluated for their sensitivity to ME7 brain homogenate on surgical-grade stainless steel surfaces. Controls were visualized by the application of episcopic differential interference contrast/Epi-fluorescence microscopy (EDIC/EF) combined with the sensitive fluorescent reagent, SYPRO Ruby, which has been shown previously to rapidly visualize and assess low levels of contamination on medical devices. The Ninhydrin test displayed a minimum level of detection observed by 75% of volunteers (MLD75) of 9.25 ?g [95% confidence interval (95% CI) 8.6–10.0 ?g]. The Biuret test provided better sensitivity, with a MLD75 of 6.7 ?g (95% CI 5.4–8.2 ?g). However, much lower concentrations of proteinaceous soiling (pg) were visualized using the EDIC/EF microscopy method. From these findings, it is clear that these approved colorimetric tests of cleaning are relatively insensitive. This investigation demonstrates how large amounts (up to 6.5 ?g) of proteinaceous brain contamination could remain undetected and the instruments deemed clean using such methods. The application of more sensitive cleanliness evaluation methods should be applied to reduce the risk of iatrogenic transmission of prion disease in ‘high-risk’ instruments such as neurosurgical devices

Are surgical stainless steel wires used for intracranial implantation of PrPsc a good model of iatrogenic transmission from contaminated surgical stainless steel instruments after cleaning?

Transmissible spongiform encephalopathies are a group of fatal, neurodegenerative diseases commonly known as prion diseases. Prion diseases can resist traditional inactivation strategies and may be iatrogenically transmitted by surgical instruments through the human population. These properties have led to the need for a suitable detection method of the prion infectious agent, and increased pressure regarding the development of anti-prion cleaning methodologies that would ensure the safety of surgical instruments. Although other techniques have been applied, the animal bioassay remains the 'gold standard' method for assessing infectivity. As the vast majority of surgical instruments are made of stainless steel, and in order to test this surface using the animal bioassay, the application of very thin surgical stainless steel wires has been widely adopted. These wires are easily inoculated and may be reimplanted into animals without the requirement for elution of the residual material. However, their comparability to the dimensions, shape and size of surgical instruments is questionable. This article shows how such contaminated wires (residual protein between 6.3 and 16.0 ng/ mm(2)) can be cleaned more easily than flat metal surfaces (residual protein between 63.9 and 89.3 ng /mm(2)) under comparable conditions using recommended cleaning agents. These results indicate that the application of wires as a realistic means of assessing the removal or inactivation of the prion infectious agent from surgical instruments should be treated with caution