ArticlesNovel methods for disinfection of prion-contaminated medical devices
Introduction
The occurrence in the UK in 1996 of variant Creutzfeldt-Jakob disease (vCJD), linked to the consumption of bovine spongiform encephalopathy (BSE)-tainted meat products, raised concerns that human beings might have been exposed to secondary infections by the BSE/vCJD agent via medical procedures or the administration of human derived biological products, including blood. Many peripheral tissues from patients with vCJD have been shown to be infectious, and by contrast with sporadic Creutzfeldt-Jakob disease (sCJD), the biochemical marker of prion diseases (PrPres, the protease-resistant, pathological form of the prion protein) is detectable in lymphoid organs like spleen, tonsils, thymus, and appendix.1, 2 We have also shown that PrPres is present in the mucosa of the intestine and in peripheral nerves in a non-human primate model of vCJD.3 The iatrogenic risk of vCJD has recently been substantiated by the report of a probable case of transfusion-related disease.4
As a consequence, precautionary measures have been implemented with regard to blood products, tissue grafts, and the decontamination of surgical instruments. Several iatrogenic transmissions of sCJD due to neurosurgical instrument and electrode contamination have been identified in the past.5, 6 To avoid such events in the future, regulatory measures have been taken concerning the re-use and decontamination of neurosurgical instruments.7, 8, 9 These measures consist mainly of recommendations to use disposable instruments or harsh decontamination procedures—ie, immersion in 1N NaOH for 1 h followed by porous load autoclaving at 134°C for 18 min.9 Similar measures should, if practicable, be taken for all surgical instruments and for endoscopic devices, but are hindered by the corrosive effect of either NaOH or NaOCl and the incompatibility of autoclaving with all devices containing plastic, gum, joints, or electronic components.10
There is thus an urgent need to explore new procedures and chemical formulations that are both effective and practical for use on instruments and surfaces. So far, most inactivation studies have used residues from tested materials as inocula in standard infectivity bioassays,7, 8 although a strong correlation between infectivity and PrPres has been documented in a study of Cohn-fractionated plasma.11
A system based on the use of steel wires has been previously described that mimics the contact of surgical instruments in living organisms.12, 13 We applied this method to inactivation studies on prions bound to surfaces. We tested several decontamination procedures and chemical formulations, and devised three new inactivation protocols applicable to fragile devices and surfaces that were equal to or better than autoclaving at 134°C for 18 min.
Section snippets
Animals
6-week-old female Syrian golden hamsters (Charles River, France) were used in this study.14, 15 All animals were housed in level 3 care facilities officially registered for prion experimental studies on rodents (agreement number A 92-032-02 for animal care facilities, agreement number 92-189 for animal experimentation).
Infectious material
The hamster-adapted scrapie strain 263K was stabilised and propagated in the Syrian golden hamster.14, 15 Brains of hamsters at the terminal stage of the disease, typically
Bioassays
Wires were individually implanted into the prefrontal subcortical region of anesthetised hamsters. Animals were regularly monitored for clinical signs of transmissible spongiform encephalopathy (TSE), and killed at the terminal stage of the disease. LD50 values were determined according to Reed and Muench's method.17 Diagnosis of TSE was confirmed by detection of PrPres in brain by ELISA and western blot techniques, according to a previously described protocol.18
In-vitro analysis of mechanisms of action
For in-vitro analysis, the dried inoculum was treated, and, after removal from glass slides, resuspended in 120 μL of water. Samples were treated or not with increasing amounts of proteinase K for 10 min at 37°C, 2× Laemmli buffer was added, and 20 μl of each extract was used for PrPres detection.19
In the cases of LpH and LpHse, the product was added 4:1 to 20% brain homogenate in 5% glucose solution and incubated for 30 min at 20°C. Since phenol inhibits proteases, we had to extract PrPres
Immunohistochemistry
Immunochemistry analyses were done as previously described.3 Each brain containing the inserted wire was fixed by immersion in Carnoy's fluid, and then transferred to butanol until paraffin embedding with removal of the wire before microtome section. 5-μm-thick sections were cut and mounted on polylysine-coated slides. After treatment with proteinase K (2 μg/mL, 10 mins at 37°C), PrP was detected with a monoclonal antibody coupled to biotine (SAF-32, raised against hamster PrP, codon 79–92, 1
Role of the funding source
This work has been partly supported (in-vivo studies) by Steris Ltd. Steris had no role in the collection and analysis of data, and the role of Steris in study design was limited to protocols of wire decontamination.
Results
Wire contamination with serial dilutions of brain homogenates from hamsters terminally ill with scrapie (strain 263K), and implantation into recipient animals, established a relation between the infectivity titre (endpoint at 1×105·6 LD50), the transmission rate, and the incubation period (table 1). The progressive decrease in the infection rate, along with an increase in the incubation period as the dilution of the infective material increases, is similar to previous findings with direct
Discussion
Using WHO reference treatments9 as decontamination controls, we confirmed the effectiveness of 1N NaOH and 20 000 ppm NaOCl to decontaminate wires coated with hamster-adapted scrapie, as had been previously shown for tissue suspensions.7, 8 We also confirmed that inactivation by autoclaving at 134°C for 18 min was only complete when the wires were immersed in water: incomplete inactivation of wires placed in the autoclave on a dry support underlines the protective or fixing effect which can
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