1 School of Chemistry, University of Edinburgh, Joseph Black Building, West Mains Road, Edinburgh EH9 3JJ, UK
2 School of Biological Science, University of Edinburgh, Darwin Building, Edinburgh, UK
3 Department of Orthopaedics and Trauma, University of Edinburgh, Edinburgh, UK
4 Sterile Services Department, Royal Infirmary of Edinburgh, Little France, Edinburgh, UK
5 Moredun Research Institute, Penicuik, Bush Loan, Edinburgh, UK
Correspondence
H. C. Baxter
h.baxter{at}ed.ac.uk
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ABSTRACT |
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INTRODUCTION |
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To date, there have been five authenticated incidents of surgical patients suffering from undiagnosed CJD, where the reuse of instruments from these surgical procedures represents a potential risk to other patients. Four of these incidences, in the UK (Mayor, 2003), the USA (Belkin, 2003
) and Australia (Zinn, 2000
), have exposed individuals within a total cohort of 43 patients to the risk of sporadic CJD (sCJD). A single instance in Canada exposed individuals within a cohort of 71 patients to a risk of variant CJD (vCJD) (Tapp, 2003
). The risk of iatrogenic transmission of CJD posed by neurosurgical interventions and invasive procedures involving the lymphoreticular system cannot be considered insignificant. These studies highlight the ongoing concerns about the level of residue removal that can be achieved by the types of decontamination methods currently employed and indicate that there is a need to develop more effective methods. Recent studies have suggested that a combination of protease and detergent treatments reduce TSE infectivity significantly. The infectivity of bovine spongiform encephalopathy (301V) mouse brain is reduced by a factor of 3 log doses by solution digestion with the modified subtilisin Properase (McLeod et al., 2004
). Reduction of steel-bound infectivity has proved more difficult. A three-stage process involving sequential digestion with protease K, pronase digestion and denaturation with hot SDS solution has recently been evaluated by using the mouse intracerebral wire-transmission model. This has been shown to reduce infectivity bound to stainless steel substantially (Jackson et al., 2005
).
A more radical generic approach to decontamination than enzymic digestions and chemical treatments would be one relatively unaffected by the chemical complexity or tenacity of adhesion of the contaminants, which could be used to remove all organic deposits. Radio-frequency (RF)-generated gas plasmas are commonly used for surface cleaning and decontamination in the electronics industry and can, in principle, degrade complex biomolecules completely to gaseous products without exposing the metal surfaces to high temperatures or corrosive chemicals (Sugawara et al., 1998). In a preliminary study using a low-pressure Ar : O2 plasma (procedure 1), we demonstrated that tenaciously bound organic material, which resisted conventional decontamination treatments, could be efficiently removed from the metal surfaces of contaminated endodontic files (Whittaker et al., 2004
).
In this study, we addressed two related questions. The first was whether RF gas-plasma treatment of surfaces contaminated with a TSE agent would effectively reduce the amount of TSE infectivity. To assess the method, we used intraperitoneally implanted stainless-steel spheres as surrogate surgical instruments, contaminated with the 263K strain of scrapie, to transmit the infection by the peripheral route in hamsters. We elected to use this route of infectivity transmission as it is arguably more relevant to transmission during general surgical interventions than the more sensitive wire brain-implantation method, which closely mimics direct transmission by neurosurgery procedures. We compared the gas-plasma method with standard cleaning procedures and showed that significant reductions in infectivity could be achieved by using procedures that included a gas-plasma treatment step.
Surgical instruments vary in structural complexity and in the degree and localization of soiling in use. The second question was whether RF gas-plasma treatment could indeed be practical as a routine method for the removal of contamination of instruments in a hospital setting. We evaluated the method on a series of reprocessed surgical instruments. These were intercepted directly after conventional cleaning and sterilization, and examined both before and after gas-plasma treatment by scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDX). Our results here showed that a gas-plasma cleaning step, as an adjunct to detergent cleaning, can achieve significant reductions in the amount of biological material adhering to instrument surfaces.
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METHODS |
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We also examined surgical instruments in regular use in a teaching hospital surgical unit. The instruments had been cleaned stringently by conventional hospital procedures, were fully compliant with Quality Management System EN ISO 9002 and judged suitable for reuse for surgical procedures. On examination by SEM, out of a total of 17 randomly selected instruments from a single tray of instruments, 14 showed significant levels of contamination. A further 35 instruments were selected from random trays for detailed examination.
Surface contamination of stainless-steel spheres.
The inoculum was prepared as a 20 % (w/v) brain homogenate of the 263K strain of hamster scrapie in 0·32 M sucrose, and had a titre of 107 infectious units in 50 µl of a 1 : 100 dilution by the intercranial route (Kimberlin & Walker, 1978), a pH of 7·5 and a total protein concentration of 22·5 mg ml1.
Pre-weighed stainless-steel spheres (type 316, 2 mm diameter; Alfa Aesar) were immersed in 20 µl volumes of freshly prepared inoculum and allowed to dry at room temperature to a constant weight for approximately 3 days. The mean weight of the homogenate dried onto the spheres was 1·1 mg.
Treatment of the stainless-steel spheres.
Spheres in group 1 were left untreated. In group 2, the spheres were autoclaved at 137 °C for 18 min, followed by a TriGene disinfectant wash and then rinsed with water. In group 3, they were washed rigorously in TriGene disinfectant and then rinsed in water. In group 4, they were subjected to the RF gas-plasma treatment defined in procedure 3 (see below).
Bioassays.
Single spheres were implanted intraperitoneally into individual 6-week-old female hamsters by using an implant needle. Each group comprised five hamsters. The inoculated hamsters were monitored for symptoms of scrapie infection and euthanized once clinical scrapie disease was established (Marsh & Kimberlin, 1975).
Gas-plasma decontamination procedures.
Gas-plasma treatments were carried out by using a Plasma-Etch PE-200 (Plasma Etch). Three separate gas-plasma procedures were used in this study. In procedure 1, the instrument temperature was held at 25 °C and an Ar : O2 (1 : 2) mixture (at 66·7 Pa) was subjected to RF excitation (13·5 MHz) at a power density of >6 mW cm3 for 1 h. In procedure 2, the SSD-cleaned instruments were subjected to procedure 1 and then sonicated in distilled water. In procedure 3, the SSD-cleaned instruments were soaked in distilled water for 30 min and subjected to procedure 1 while still wet (Baxter et al., 2005).
Detection and analysis.
SEM inspection of the instruments was carried out by using a Philips XL30CP Microscope, operating at 20 kV, with resolution of greater than 5 nm. Secondary electron (SE) and backscatter electron (BE) imaging enabled regions with a mean atomic number difference >0·1 to be resolved.
EDX was carried out in the SEM by using an Oxford Instruments Isis 300 X-ray analyser, capable of detecting elements of atomic number greater than 6. The imaged area (5 µm) was subjected to elemental analysis to a depth of approximately 3 µm (V=6x1017 m3) by using an X-ray fluorimeter capable of detecting elements comprising 0·1 % of the mass of the sample volume.
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RESULTS |
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No residual contamination on the RF gas-plasma treated spheres could be detected by SEM (Fig. 1g and h). EDX analysis with a detection limit of 56 fmol carbon (0·5 amol of a typical 30 kDa protein) within the sample volume indicated that the treatment had removed all of the experimental contamination, and analysis by bioassay showed no transmission of infectivity (Table 1
, group 4).
Surgical instruments
To test the feasibility of using gas plasma alone as a cleaning method for heavily contaminated instruments, we examined a number of single-use, disposable orthopaedic bone-saw blades. These had been used in conventional surgery and, for reasons of safety of handling within the laboratory, the instruments were autoclaved before being washed stringently in a manner similar to that used for washing the stainless-steel spheres. By using SE imaging, we observed large deposits of material (Fig. 2a). EDX analysis indicated that the material contained carbon, nitrogen, oxygen, sulfur, calcium and phosphorus. As calcium and phosphorus were detected only as trace components of residues on the reprocessed instruments, it seemed likely that this was due to the mineral matrix of microscopic bone fragments adhering to the blades. The RF gas-plasma treatment removed all of the organic deposits, although calcium, phosphorus and some carbon remained in some surface residues (Fig. 2b
). This was reasonable, as we would expect a residue of calcium carbonate and phosphates to remain after oxidation of the bone matrix. These salt deposits were removed easily by brief sonication of the instruments in sterile water (Fig. 2c
), although sonication alone, without a prior plasma treatment, had little effect (data not shown).
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DISCUSSION |
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In this study, we have shown that both stringent washing procedures and RF gas-plasma treatment (using procedure 3) removed contamination from spheres coated experimentally with the hamster-adapted strain of scrapie, reducing the infectivity to below the level detectable by this bioassay. However, by using backscatter SEM and EDX analysis, we found that the washed spheres still showed visible levels of residual contamination and that traces of organic material still adhered to the surfaces. Conversely, the plasma-cleaned spheres appeared, at least to the detection limits of our analysis, to be clear of all traces of contamination. We concluded that, whilst both procedures removed the TSE infectivity, RF gas-plasma treatment appeared to be more effective in the removal of residual debris from stainless steel.
As the washing protocol that we used here for the decontamination of the spheres was effectively that currently in use in clinical environments for the decontamination of surgical instruments, we conducted a study on a sample set of 49 reprocessed instruments (Table 2). These instruments were removed from clinical use after conventional hospital decontamination and examined by SEM and EDX both prior to and after Ar : O2 plasma treatment. Our results indicated that a significant amount of adhered organic residue remained on the instruments after conventional cleaning. Typically, these residues varied from spots of <1 µm in diameter to irregular areas of several square millimetres. Some indication of the significance of the level of contamination observed may be gleaned from the approximation that a 1 µm2 spot of a monolayer of a pure globular protein with a molecular mass of 30 kDa (roughly that of PrPC) would contain in the order of 104 protein molecules. This gives cause for concern, particularly for instruments that are reused in neurosurgery and ophthalmic procedures, as an estimate for a PrPSc unit of infectivity measured by end-point titration is approximately 105 molecules (Prusiner, 1991
). When the instruments were subjected to the first RF gas-plasma cleaning procedure (Table 2
), a single exposure to low-pressure Ar : O2 plasma substantially diminished the levels of contaminating organic residues on exposed surfaces, but the RF gas plasma alone did not remove inorganic residues. However, when instruments were treated by using RF gas-plasma procedure 3 (Table 2
) where the residual contaminants were hydrated by soaking prior to plasma treatment, removal of the debris to below the level of our detection methods could readily be achieved. Studies are currently under way to determine the nature of the physical interaction of the RF gas plasma with the hydrated residues.
This work has shown that gas-plasma treatment can be used effectively to remove residual contamination from the surfaces of metal surgical instruments without apparent damage to the instruments themselves. At present, there is no simple, practical, cost-effective way of distinguishing between PrPSc and other proteins on the surfaces of surgical devices. The most effective way to prevent CJD transmission is therefore to remove all traces of residual contamination from medical instruments. We have demonstrated convincingly, both experimentally and on surgical instruments, that soaking together with gas-plasma treatment can decrease residual contamination to a level undetectable by SEM and EDX. Furthermore, gas-plasma treatments may prove especially valuable in the routine cleaning of complex instruments where a build-up of tissue and salt residues in areas inaccessible to normal cleaning processes may create protective sites that could render routine sterilizing procedures ineffective. In this context, we conclude that it is timely to consider the implementation of gas-plasma treatment for the effective decontamination of surgical instruments.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 8 March 2005;
accepted 10 May 2005.
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