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Improved solution methods, gas-plasma decontamination and gas phase inactivation substantially reduce TSE infectivity, but a strategy combining several methods may meet needs better than any one-step universal process
Helen C Baxter
BSc PhD Senior Research Scientist
Anita C Jones
BSc PhD Reader in Physical Chemistry
Robert L Baxter
BSc PhD Professor of Chemical Biology, MIDAS Unit
School of Chemistry
University of Edinburgh
Edinburgh
UK
The infectious material of prion-related disorders is thermostable, binds strongly to steel and is highly resistant to conventional decontamination methods. Recent years
have seen the development of new solutions and gas-based methods for inactivating prion infectivity. Here, we focus on the development of radio-frequency gas-plasma procedures which inactivate prion infectivity and improve decontamination by over 1,000-fold. New methods of measuring cleaning efficacy, which are necessary to monitor decontamination to these levels, are briefly described.
Sterile service provision
The overriding focus of sterile service provision in healthcare is the prevention of iatrogenic infection. Secondary, but important, considerations are the prevention of foreign material contamination and minimisation of immune reactions. The instrument reprocessing specialist is faced with a requirement for high-volume throughput of increasingly complex surgical instrumentation and the ever-present threat of new disease vectors.
In recent years, the biggest challenge has come from the potential for transmission, by surgical intervention, of human transmissible encephalopathies (TSEs) – principally
Creutzfeldt–Jakob disease (CJD) in its various forms. The infectious agents of TSEs are misfolded cellular PrP proteins (prions).[1] The tenacity of adhesion of prion proteins to surfaces and their resistance to thermal denaturation is such that only limited removal and inactivation is possible using normal cleaning and autoclaving procedures.[2]
The sporadic and familial forms of CJD occur at a frequency of about one in a million of the population, but cases of variant CJD (vCJD) are rarer. Although this was once thought to be a UK problem, the appearance of confirmed vCJD cases in other European countries has proven otherwise. It is now acknowledged that the disease may have been more widely spread by blood products than previously supposed and there are various estimates of the size of a latent reservoir of (as yet) asymptomatic vCJD carriers.[3] Thus, there may always be a small pool of potential donors – and current evidence suggests the risk of iatrogenic transmission could be significant, particularly in neurosurgical, posterior eye and tonsillectomy procedures.
The most obvious way to prevent iatrogenic transmission of prion diseases is exclusive use of “single-use” instruments. This is neither costeffective nor practical in many situations but has been adopted for certain procedures such as tonsillectomy in some countries. Allied to this prophylactic approach are the UK policies of withdrawing instruments used on potentially infected individuals from use and the segregation of instruments used for the cohort of children born after the advent of vCJD.[4]
Current technology
The essential elements of instrument reprocessing involve: decontaminating (or cleaning) as soon after use as practicable, checking cleanliness, packaging (where appropriate) and sterilisation either by chemical means or by steam autoclaving. The first key step is decontamination, which includes the removal of tissue and adventitious postsurgical microbial growth from the instrument.
It has been pointed out by several authors that determination of the efficacy of the cleaning processes is one of the major challenges in decontamination practice. This is normally carried out by visual examination, although testing for gross protein contamination on surfaces using ninhydrin swabs and for microbiological contamination by measuring ATP are both used.
Another method of quality control in automated washing processes relies on soil detection in the final rinsing solutions rather than that remaining on the instruments. The second active part of the process is sterilisation, by which remaining viruses and microorganisms are physically or chemically inactivated. Quality assurance of the effectiveness of sterilisation equipment is generally monitored by testing the viability of yeast spores using a test strip.
The relatively small number of cases of iatrogenic transmission of the most common infectious diseases bears tribute to the effi ciency of modern medical decontamination practice.
Recent developments in prion decontamination
There are various strategies by which instrument
reprocessing may be improved to reduce the threat posed by prion-type infection. The first involves the inactivation of the infectious material during the wet cleaning process. Historically, soaking in hypochlorite or strong alkali has been recommended for prion inactivation. And while these can be highly effective, the damage that these reagents cause to metal surfaces renders them unsuitable for surgical instrument processing.
[[HHE.T21a]]
Recently, several detergent-protease (for example, Prionzyme M, Rely+On) and detergentalkali cocktails (for example, Hamo 100 Prion Inactivating Detergent, SeptoClean, Deconex) have come on to the market.[5] These are designed to solubilise and denature or cleave the proteins adhering to instrument surfaces. These solution methods frequently require long soaking times but have been shown to markedly reduce TSE infectivity in rodent transmission models. A point worth considering here is that solution deactivation methods may show signifi cantly different inactivation kinetics with different TSE strains.
Two very different processes have been described in which instruments are exposed in a chamber to vapour-phase hydrogen peroxide[6] or ozone (the TSO3 process). Both reagents oxidise and denature proteins directly on the surfaces and the hydrogen peroxide process results in some fragmentation. Both have proven effective in inactivating experimental TSE infectivity. It should be noted, however, that the reduction in infectivity achieved by these processes is due to chemical inactivation of the infectious agent and not to the removal of biological molecules from the treated surfaces.
Radio-frequency gas-plasma treatment, which we have studied in our laboratory, is a radically different approach to decontamination which combines both the destruction of
contaminating molecules and the removal of the products. Gas-plasmas are generated by passing radio-frequency energy through a mixture of oxygen and argon at low pressure. The gases are highly energised, producing excited atoms, radicals and ions. These oxidise and fragment the biomolecules contaminating the surface (Figure 1). Complex macromolecular structures are broken down into simple gaseous molecules such as CO2, H2O, SO2 and NO2.
[[HHE.T21b]]
In essence, molecules are “burnt” off the surface but the process occurs at room temperature and so there is no collateral damage to metal surfaces. This technique reduces tissue protein loading on surfaces to levels 1,000 to 10,000 times lower than conventional detergent cleaning methods. We have shown that radio-frequency gas-plasmas destroy the infectivity of 263K scrapie adsorbed on stainless steel to below the level of detection using interperitoneal hamster bioassays.[7]
An example of radio-frequency gas-plasma decontamination
Apart from prion inactivation, the most obvious benefit of radio-frequency gas-plasma decontamination lies in the improved cleaning of instruments – which may also have wide-reaching advantages in other areas. An example from
our work, carried out in collaboration with the decontamination unit at the Royal Infirmary of Edinburgh and workers at Plasma-Etch Inc, using a PE-BT1 instrument, is discussed below. The pair of ophthalmic microforceps shown in Figure 2 are a typical example of a visually clean reprocessed instrument. Scanning electron microscopy (SEM) showed that these still had substantial amounts of residual contamination. Elemental analyses of patches of contamination on the forcep tips show a composition typical of protein.
Gas-plasma treatment of the instrument resulted in removal of the areas of organic contamination and elemental analysis of sampled areas of the surface show no indication of any residual contaminating material.
Monitoring decontamination
An important issue raised by these studies is that the sensitivity of the procedures currently being used to evaluate the amount of residual tissue left after decontamination – normally visual inspection augmented sporadically by ninhydrin swab tests – is woefully inadequate. After conventional processing, visually clean instruments normally have averaged residual surface protein loadings of ca 0.5 mg/mm2, which is well below the typical sensitivity of ninhydrin swab detection.[8] SEM and EDX analysis, while useful in a laboratory environment, would be too expensive and time-consuming to be used by a healthcare professional to monitor the efficacy of decontamination.
In recent years, methods using fluorescent reagents, coupled with detection by surface fluorescence scanning[9] or epifluorescence differential interference contrast (EFDIC) microscopy,[10] have been developed that lower the limit for
protein detection to a few picograms/mm.[2]
In our laboratory, we have developed a routine method involving derivatisation of the proteins of the residues and scanning using a surface scanning fluorimeter for determining protein contamination on instruments. This enables us to monitor and define the level of contamination
before and after the decontamination procedure.
It is hoped that this type of detection instrumentation, which is fast, sensitive and efficient, can be successfully developed for routine use for quality control in decontamination facilities.
Future prospects in prion removal
There is no doubt that all of the newer processes – improved solution methods, gas phase inactivation and gas-plasma decontamination – substantially reduce TSE infectivity, but it seems unlikely that a one-step universal process will emerge that will meet all needs. The most effective way to deal with potential CJD contamination may prove to be a multiple stepwise strategy combining several new methods. This could involve the use of the newer detergent–chemical inactivator cocktails in initial cleaning and, especially for high-risk instruments such as those used for neurosurgical or anterior eye procedures, the introduction of a final radio-frequency gas-plasma decontamination step after cleaning but before wrapping.
The synergistic effect of adding these procedures to decontamination unit practice could potentially reduce the risk of CJD infection by over a millionfold.
Acknowledgements
This work was undertaken with funding from the Department of Health, UK. The views expressed in the publication are those of the authors and not necessarily those of the DH. We wish to thank the staff of the decontamination unit, Royal Infirmary of Edinburgh, and Greg Delarg and John Woods of Plasma-Etch Inc.
References
1. Chesebro B. Introduction to the transmissible spongiform
encephalopathies or prion diseases. Br Med Bull 2003;66:1-20.
2. Zobeley E, Flechsig E, Cozzio A, Enari M, Weissmann C. Infectivity of scrapie prions bound to a stainless steel surface. Mol Med 1999;5(4):240-3.
3. Ironside JW. Variant Creutzfeldt– Jakob disease: risk of transmission by blood transfusion and blood therapies.
Haemophilia 2006;12 Suppl 1:8-15.
4. National Institute for Health and Clinical Excellence. IP Guideline 196. Patient safety and reduction of risk of transmission of Creutzfeldt–Jakob disease (CJD) via interventional procedures. London: NICE; 2006.
W: www.nice.org.uk/IPG196
5. Sutton JM, Dickinson J, Walker JT, Raven ND. Methods to minimize the risks of Creutzfeldt–Jakob disease ransmission by surgical procedures: where to set the standard? Clin Infect Dis 2006;43(6):757-64.
6. Fichet G, Antloga K, Comoy E, Deslys JP, McDonnell G. Prion inactivation using a new gaseous hydrogen peroxide
sterilisation process. J Hosp Infect 2007;67(3):278-86.
7. Baxter HC, Campbell GA, Whittaker AG, Aitken A, Simpson AH, Casey M, et al. Elimination of TSE infectivity and
decontamination of surgical instruments using RF gas-plasma treatment. J Gen Virol 2005;86:2393-9.
8. Baxter RL, Baxter HC, Campbell GA, Grant K, Jones AC, Richardson P, et al. Quantitative analysis of residual
protein contamination on reprocessed surgical instruments. J Hosp Infect 2006;63(4):439-44.
9. Kovalev VI, Barton JS, Richardson PR, Jones AC. Highly sensitive rapid fluorescence detection of protein
residues on surgical instruments (ultrasensitive and single-molecule detection technologies). Proc SPIE 2006;6092:0J/1-8.
10. Lipscomb IP, Sihota AK, Botham M, Harris KL, Keevil CW. Rapid method for the sensitive detection of protein
contamination on surgical instruments.
J Hosp Infect 2006;62(2):141-8. Reno, Nevada, for helpful discussions
