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Quantitative measurement of the efficacy of protein removal by cleaning formulations; comparative evaluation of prion-directed cleaning chemistries
Journal of Hospital Infection (2010) 74, 144e151
Available online at www.sciencedirect.com
www.elsevierhealth.com/journals/jhin
Quantitative measurement of the efficacy of protein removal by cleaning formulations; comparative evaluation of prion-directed cleaning chemistries M. Ungurs a, J.R. Hesp a,*, T. Poolman a, G. McLuckie a, S. O’Brien a, H. Murdoch b, P. Wells c, N.D.H. Raven a, J.T. Walker a, J.M. Sutton a a
Centre for Emergency Preparedness and Response, Health Protection Agency, Porton Down, Salisbury, Wiltshire, UK b Health Protection Scotland, Infection Control, Glasgow, UK c Sterilisation and Disinfection Unit, Salisbury NHS Foundation Trust, Salisbury District Hospital, Salisbury, Wiltshire, UK Received 22 January 2009; accepted 24 July 2009 Available online 14 October 2009
KEYWORDS CreutzfeldteJakob disease; Decontamination; Detergent; Prion; Transmissible spongiform encephalopathy
Summary The stability of the infectious agent causing variant CreutzfeldteJakob disease (vCJD) has highlighted the importance of cleaning surgical instruments for controlling potential spread of iatrogenic CJD. In this study, thermostable adenylate kinases (tAKs) in test soil were coated on to stainless steel and these surrogate agents used to evaluate the efficacy of a range of cleaning chemistries in a bench-top washer disinfector (btWD), or as a pre-soak either with or without subsequent treatment by btWD. Two tAKs were tested initially for ease of removal, the most persistent being Sulfolobus acidocaldarius-derived tAK which was used for evaluating the cleaning chemistries. Conventional chemistries were generally more effective when used in a btWD than as pre-soaks. Cleaning efficacy improved when pre-soaks were followed by treatment with intermediate performing enzymes, demonstrating greater than additive effect on residual tAK activity. Three of the four prion-directed chemistries reduced residual tAK activity to below the limit of quantification (LoQ) by more than 4.8 log10; <175 pg tAK remaining as a pre-soak alone. A conventional alkaline cleaning product also reduced residual tAK activity to below the LoQ but only
* Corresponding author. Address: Technology Development Group, Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, Wiltshire SP4 0JG, UK. Tel.: þ44 1980 612643; fax: þ44 1980 612622. E-mail address: [email protected] 0195-6701/$ - see front matter ª 2009 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2009.07.023
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when used in a btWD. tAK soil dried on to the device was removed less efficiently than tAK soil still moist on the device, with a 320-fold and 28-fold increase in residual tAK activity for pre-soak and btWD, respectively. The study demonstrated the potential for a tAK indicator to describe the effectiveness of protein removal using different chemistries or treatment processes. ª 2009 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction The prion infectious agent responsible for variant CreutzfeldteJakob disease (vCJD) is highly resistant to inactivation by standard decontamination treatments. This includes methods currently used in hospitals, where autoclaving even at elevated temperatures and durations only partially inactivates transmissible spongiform encephalopathy agents.1 Currently, the World Health Organization recommends prolonged treatment with high concentrations of sodium hypochlorite or sodium hydroxide but these treatments are not generally suitable for the routine decontamination of surgical instruments.2,3 Treatments that can be used routinely to remove and inactivate prions that may otherwise be present as residual contaminants on surgical equipment have now been described and many are commercially available for use in sterile services departments (SSDs).4e8 The prion issue and its control have raised the profile of the cleaning component within the surgical instrument reprocessing cycle. Washer disinfectors in the UK are operated under the guidance of Health Technical Memorandum 2030 (currently under revision). The guidelines recommend annual validation of automated washer disinfectors, monthly protein residue tests and visual inspection of every load. Due to the significant gap in the sensitivity of these methods, working to these guidelines does not provide reassurance about potential prion removal. The effectiveness of residual protein tests, based on Ninhydrin and Biuret reactions, is in the range of micrograms of protein and this may be partly due to the recovery of protein from the instruments through swabbing.9 Approaches that measure residual protein on actual surgical instruments, such as episcopic differential interference contrast microscopy, avoid these sampling problems and can measure protein contamination down to 85 pg/mm2.10 Other methods which exploit in-situ labelling of bound proteins give similar levels of sensitivity.11 This study uses thermostable adenylate kinases (tAKs) to compare the efficacy of different cleaning chemistries as either a pre-soak or within an
automated washer disinfector cycle. By coupling ATP generation by tAK to a bioluminescent detection system it was possible to measure directly the residual enzyme remaining on surfaces after treatment, providing a quantifiable measure of cleaning performance. The study demonstrates the relative effectiveness of a range of cleaning chemistries for decontaminating stainless steel. It also establishes the method as both a research tool for quantifying parameters that affect the removal of protein from surfaces and potentially as a validation tool for use in hospital settings.
Methods Production of thermostable adenylate kinase (tAK) Synthetic genes encoding the native adenylate kinase proteins from Sulfolobus acidocaldarius (Sac) or Thermotoga maritima (Tma) were cloned into pMTL1015 (a pMTL1013 derivative).12 The recombinant plasmids were used to transform Escherichia coli RV308. Enzymes were purified as described previously and assessed by sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDSe PAGE).13 Protein concentration was measured using a standard Bio-Rad (Hercules, CA, USA) DC protein assay.
Contamination of stainless steel Aliquots (100 mL) of test soil were used to coat approximately 90 mm2 of one end of clean, 65 mm 5 mm, grade 304 stainless steel strips. The test soil consisted of 50% (v/v) Browne’s soil (Steris Corp., Mentor, OH, USA) spiked to include 0.1 mg/mL tAK. To represent a worst case scenario, the applied soil was allowed to dry on to the surface at 25 C for 2 h. For experiments requiring moist soil, strips were sealed in a humid environment after application of soil and maintained at 37 C for 30 min, to simulate exposure to blood and tissue during surgery and subsequent cleaning without
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allowing the contaminating material to dry on the surface. A standard curve of enzyme dried on to the strips ranging from 0.005 ng to 5000 ng per was prepared by diluting the enzyme preparation using 50% Browne’s soil and applying 100 mL to one end of a clean stainless steel strip. Blank controls were included that consisted of 100 mL of 50% (v/v) Browne’s soil applied to the end of the strips. The applied soil was allowed to dry on to the surface at 25 C for 2 h. The activity on the strips was measured using the Hygiena handheld ATP monitoring system (SystemSURE Plus; Hygiena, CA, USA) and a four-parameter logistic model (SigmaPlot v10; Systat Software) applied to the data.
Decontamination using pre-soak treatments Each pre-soak decontamination treatment involved immersion of the soiled stainless steel strips Table I Cleaning agent
B C D E
F G H
I Water
Decontamination using washer disinfector procedures Soiled stainless steel strips were secured to the shelf of the btWD and submitted to washing using the agents and associated washing programmes (Table I). The btWD was commissioned and validated against the HTM2030 guidelines. The standard btWD cycle consisted of the following stages:
Comparison of efficacy of cleaning agents used in three process types Chemistry
Pre-soak/(btWD) wash cycle conditions Concentration (mL/L)
A
in cleaning agent maintained at the appropriate temperature in a stainless steel water-bath (Table I). After treatment with the cleaning agents, the strips were either rinsed with deionised water flowing at 8 L/min for 5 s, or transferred to a Medisafe (Bishop’s Stortford, UK) Pico Flush bench-top washer disinfector (btWD) and submitted to a standard cycle (see below) using cleaning agent A (Table I).
Enzymatic conventional Enzymatic conventional Enzymatic conventional Enzymatic conventional Enzymatic, neutral pH prion-directed
Alkaline prion-directed Alkaline prion-directed Enzymatic, alkaline prion-directed Alkaline conventional N/A
Log10 reduction in tAK activity
Temperature ( C)
Incubation time (min)
btWD
Pre-soak and rinse
Pre-soak and btWD
7.5 (7.5)
45 (43)
15 (15)
1.4
0.3
3.1
20 (3)
45 (43)
20 (15)
3.0
2.8
4.5
6 (3)
45 (43)
15 (43)
0.6
0.7
2.9
8 (3)
45 (43)
15 (15)
3.5
0.3
3.1
16.2 g/L component (a); 10 g/L component (b); 1.6 g/L component (c) (ND) 16 (16)
45 (ND)
15 (ND)
ND
2.8
4.8
45 (43)
15 (15)
4.8
4.8
4.8
10 (5)
55 (55)
15 (5)
4.8
4.8
4.8
20 (20)
60 (60)
30 (30)
4.8
4.8
4.8
4 (3)
45 (43)
15 (30)
4.8
0.8
3.1
N/A
45 (45)
15 (30)
0.3
0.4
2.7
Where possible the manufacturer’s instructions for dose, temperature and duration were followed. Cleaning agent E was not compatible with the bench-top washer disinfector (btWD). Indicator devices comprising 0.1 mg/mL of tAK in 50% Browne’s soil were processed using either a btWD cycle, or a pre-soak followed by a rinse or a btWD cycle. ATP generated by residual tAK was measured and converted into equivalent amount of residual protein using a standard curve (Figure 2A). ND, not done. Equivalent experiments carried out using Tma-based indicator strips gave values of 1.2 and 5.1 log10 reductions in activity for cleaning agent A, using either a pre-soak or btWD process, respectively. For each experiment (N ¼ 3) four stainless steel strips soiled with tAKspiked Browne’s test soil and four control stainless strips soiled with only Browne’s test soil were used.
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a 3 min cold water pre-wash, a main wash cycle (Table I); a rinsing cycle and a disinfect stage, which included heating to 80e85 C for 120 s.
4 Washer disinfector Control 3
Residual tAK activity was measured using a handheld ATP monitoring system (SystemSURE Plus; Hygiena). After treatment, stainless steel strips were transferred to an Ultrasnap sample tube (Hygiena), to which 200 mL of 67.5 mM ADP, 15 mM magnesium acetate solution was added, immediately after which the Ultrasnap enzyme reagent was added. The relative light units (RLUs) emitted by the reaction were measured after a 2 min incubation period at room temperature. For each experiment (N ¼ 3) four stainless steel strips soiled with tAK-spiked Browne’s test soil and four control stainless strips soiled with only Browne’s test soil were used.
Results The purification procedure resulted in enzymatically active protein of >99% purity as assessed by SDSePAGE and densitometry. The enzymes from both S. acidocaldarius (Sac) and T. maritima (Tma) on stainless steel were exposed to the conditions of 90 C, 2 min in a closed chamber to replicate the thermal element of an extended disinfection phase of the btWD cycle. There was minimal reduction in activity observed for Sac (5955 RLU 715 compared to non-heat-treated control, 6136 431) or Tma (7381 RLU 387 compared to non-heated control, 7305 333). There was also minimal reduction in activity observed for the Sac-based indicator following exposure to the thermal part of a complete btWD cycle (Figure 1). Standard curves for the enzymes are shown in Figure 2A. Initial studies explored the relative ease of removal of the two tAKs from stainless steel when processed using either a pre-soak protocol or within a small btWD. Using enzymatic cleaning product A, the indicators were processed under the manufacturer’s recommended conditions either as a pre-soak or in a btWD. Residual activity remaining on the steel surface was compared. Tma showed log10 reduction values of 1.2 and 5.1 for pre-soak and btWD, respectively, whereas the values for Sac were 0.3 and 1.4, respectively, demonstrating that Sac was more resistant to the chemical treatment, in either of the two processes. This suggested that Sac-based indicators
ATP (µM)
Measurement of remaining tAK activity after decontamination treatment
2
1
0 0.001
0.01
0.1
1 Sac (ng)
10
100
1000
Figure 1 Analysis of the effect of the thermal part of the washer disinfector cycle on the activity of Sulfolobus acidocaldarius-derived adenylate kinase (Sac). Sac standard curves were set up on metal tokens using 50% (w/v) Browne’s soil as described in Methods. The prepared tokens were sealed inside a vessel and subjected to a full standard washer disinfector cycle. Control samples were prepared as above but left at ambient temperature. Following processing the samples were assayed for activity as described previously. Relative light units values were converted to equivalent [ATP] using an ATP standard. Regression analysis was carried out using SigmaPlot (v10; Systat Software) with a four-parameter logistical fit applied to the data.
would be a tougher challenge test with which to evaluate the performance of cleaning processes, particularly those aimed at removing prions. The Sac indicator device was used to compare a series of cleaning chemistries, either with (products EeH) or without (AeD conventional enzymatic, I alkaline only) European Standard (CEmarked) prion inactivation claims (Table I). All of the chemistries, except products I and G, have instructions with suggested conditions for use as a pre-soak and these have been used for the majority of prion inactivation studies published to date. Overall, three of the four prion-directed chemistries (products F, G and H) were capable of reducing residual enzyme activity to below the LoQ of the assay, at greater than 4.8 log10 reduction of tAK activity (equivalent to <175 pg of residual tAK protein) (Figure 2C; Table I). The activity of the remaining chemistries ranged from 0.3 log10 reduction (conventional enzymatic products A and D) to 2.8 log10 (conventional enzymatic B and prion-directed, enzymatic E). In terms of tAK activity, this gives a range of 2.9 ng to 2.3 mg of residual protein remaining after processing. The
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A
B 10000 Tma Sac
6000
Remaining tAK (ng)
tAK activity (RLU)
8000
4000 2000
1000 100 10 1 0.1
0 0.001 0.01
C
0.1
1 10 tAK (ng)
100
0.01
1000 10000
D
10000
Remaining tAK (ng)
Remaining tAK (ng)
B
C
D F G Cleaning agent
H
I Water
D E F G Cleaning agent
H
I Water
10000 1000
1000 100 10 1 0.1 0.01
A
100 10 1 0.1
A
B
C
D E F G Cleaning agent
H
I Water
0.01
A
B
C
Figure 2 Comparison of the residual adenylate kinase (tAK) activity remaining after treatment with adenylate kinase derived from Sulfolobus acidocaldarius (Sac). The indicator devices comprised 0.1 mg/mL of tAK in 50% Browne’s soil. The wash conditions associated with the treatments are identified in Table I. ATP generated by residual tAK post processing was measured as relative light units (RLU) and converted into an equivalent amount of residual protein using a standard curve (A). The coated tokens were treated using either: a btWD cycle (B); a pre-soak followed by a rinse (C), or a pre-soak followed by a bench-top washer disinfector (btWD) cycle (D). Each experiment was repeated three times using four stainless steel strips soiled with tAK-spiked Browne’s test soil and four control stainless strips soiled with only Browne’s test soil. Using the standard curve data the limit of quantification was calculated as 175 pg per strip (dotted line).
only conventional alkaline product, I, gave a relatively low reduction in activity at 0.8 log10. When assessed in a btWD, run according to the manufacturer’s instructions, the prion-directed chemistries continued to clean at the LoQ. This was similar to the conventional alkaline product I, which shows greater than 4 log10 increase in cleaning performance (Figure 2B; Table I). The remaining cleaning products also showed an increase in cleaning efficacy in the btWD, except for the enzymatic cleaner C and water, which showed a small decrease. The increase in efficiency ranged from 0.2 log10 (product B increasing from 2.8 to 3.0 log10 reduction) to 3.2 log10 increase (product D increasing from 0.3 to 3.5 log10 reduction). The results demonstrate the lack of correlation between cleaning efficacy in the two processes,
with similar products that performed at equivalent levels in the pre-soak (e.g. products A and D) giving markedly different performances in the btWD. A great deal of interest has been expressed in understanding how different components of the surgical instrument cleaning cycle interact according to likely prion removal efficacy. The study explored the effectiveness of pre-soaks for improving the removal of soiling by subsequent btWD processing. The indicator devices were treated in a pre-soak protocol with each of the cleaning products. Cleaning product A was selected for subsequent treatment of the indicators in the btWD, as it provided a cleaning performance that was in the mid-range for conventional chemistries currently used in the profession (reducing activity by 1.4 log10). In each case that could be measured
Comparative evaluation of prion chemistries
Discussion In the light of vCJD, cleaning is assuming increasing importance as a component of the surgical instrument reprocessing cycle.8,14 This study looked at the potential of a new, rapid and sensitive enzyme-based assay to provide information on the effectiveness of different cleaning chemistries and other factors that might affect instrument reprocessing. Although the tAK enzymes that are used as the basis of the assay are not prions, they may share binding properties that, in combination with their exceptional thermostability, make them ideal for modelling and assessing prion removal characteristics. We showed that priondirected cleaning chemistries outperformed the majority of conventional chemistries in reducing tAK activity, supporting this conclusion. The study compared nine different cleaning chemistries for their ability to reduce the activity
10000 Remaining tAK (ng)
within the maximum sensitivity of the assay, the dual treatment resulted in a greater than additive reduction in activity of the two processes alone (Table I). This was the case even when cleaning product A was used for both pre-soak and btWD (0.3 ng of protein remaining), or when water was used as the pre-soak. Combinations with three of the four anti-prion chemistries could not be assessed due to their ability to clean to the LoQ of the assay as pre-soak alone. Only one additional combination reached the limit of detection of the assay, with prion-directed chemistry E as pre-soak followed by cleaning product A giving greater than a 4.8 log10 reduction in activity (<175 pg of tAK remaining). Another critical question that has been posed is the effect that extended drying has on effectiveness of removal of protein contamination. This was examined by comparing the removal of tAK soil from indicators that were allowed to dry at 25 C for 2 h (as used routinely for the comparison of cleaning products) against indicators that were held at 37 C for 30 min in a moist environment to simulate reasonable exposure to tissue during surgery and kept moist until decontamination. The condition of the soil when presented to cleaning treatment markedly affects cleaning efficacy (Figure 3). The residual tAK contamination after a soaking treatment for dried soil is about 320 times greater than that measured for moist soil. When using the washer disinfector treatment, tAK residual contamination after treatment is about 28 times greater with the dried soil compared with the residual contamination for moist soil.
149 Washer disinfector treatment
Sink soak treatment
1000 100 10 1
M
D M Soil condition
D
Figure 3 The effect of extended drying on the removal of adenylate kinase (tAK) as part of a test soil. 0.1 mg/mL of tAK was incorporated into 50% Browne’s soil and applied to steel strips. The strips were incubated at 25 C in an open container for 2 h or at 37 C within a closed chamber to simulate dried-on soil (D) or moist soil (M) respectively. The strips were treated using cleaning agent A as a pre-soak treatment or in the bench-top washer disinfector. The ATP generated by residual tAK on the indicator device was measured and converted into nanograms of protein remaining using a standard curve (Figure 2A). The experiment was repeated three times using four stainless steel strips soiled with tAK-spiked Browne’s test soil, and four control stainless strips soiled with only Browne’s test soil were used for each repeat. Log reduction values of 0.3 log10 and 2.8 log 10 (pre-soak) and 1.4 log 10 and 2.9 log 10 (btWD) were recorded for the dried-on soil and moist soil, respectively.
of the trimeric S. acidocaldarius tAK enzyme from surgical steel. The reduction in activity is likely to be a function of both protein removal from surfaces and inactivation of the enzyme on the surface. The standard wire-implantation model used in the majority of bioassay studies is similarly multifactorial, with no direct measure of prion removal being able to approach the sensitivity required to demonstrate complete removal from the surface.4,6 A second monomeric tAK derived from T. maritima was used in the development of the indicator and, although tolerant to extremes of pH, it was found to be much more susceptible to the mechanical cleaning action of the btWD than the enzyme from S. acidocaldarius. It could be argued on this basis that the Tma enzyme reflects the surface-binding behaviour of proteins in general, whereas the behaviour of the Sac enzyme reflects the ability of certain proteins, such as prions, to bind tenaciously to stainless steel.15 This is supported by evidence that the Sac enzyme binds preferentially to plastic and metals even when these surfaces are pre-bound with commonly used blocking agents such as skimmed milk and Tween-20 (results not shown). The difference in
150 the surface-binding properties may reflect a partial unfolding of the hydrophobic core of the protein and its interaction with the surface of the steel.13 The majority of prion inactivation studies have been based on conditions similar to the pre-soak treatments used in this study. The comparative ability of the cleaning chemistries to reduce the levels of tAK activity are reminiscent of these studies, where no single class of chemistry was universally effective against prions.4,16 In the presoak model only three of the four prion-targeted chemistries were able to clean to the limits of quantification of the assay, with an additional conventional alkaline chemistry matching this performance in the btWD only. The results with this latter product, I, demonstrate the difficulties of extrapolating from relatively static wash conditions to those encountered even within small btWDs. Other studies that have explored this phenomenon with regards to the sensitivity of soil tests have also showed that mechanical action played a pivotal role in cleaning.17 Although not seen in this study, combinations of treatments can pose compatibility issues between pre-soaking agent and cleaning agent used in the WD.17 The comparison between manual and automated studies is well-described in endoscope reprocessing, with mechanical cleaning producing better results than manual methods.18 It would be expected that the cleaning performance of the cleaning agents used in this study would be better when used in a WD operating in an SSD with more powerful mechanical action being available. Decontamination in the btWD used in this study is, therefore, more reliant on the action of the cleaning agent used. The combination of a pre-soak with subsequent treatment in a btWD proved the most effective process with high log reductions in almost all cases. In all the combinations tested, where cleaning was within the dynamic range of the assay, a greater than additive increase in log reduction was observed. This might have been expected where the combination of the chemistries favours removal of proteins attached via different surface interactions. However, the observed increase due to the combination of pre-soak and btWD, even with the same product or class of products, may reflect the effects of rehydration of the soil favouring its subsequent removal. More direct evidence for the role in soil hydration in efficacy of removal was evaluated using the soiled indicators in either dried or moist environments. The greater reduction in activity on the
M. Ungurs et al. indicators not allowed to dry echoes those observed previously.19 With the increased awareness of the importance of the cleaning component of the instrument reprocessing cycle, a number of methods have been developed to assess the relative removal of protein soiling, to complement the visual inspection and colorimetric protein assays in current use. Some of these highly sensitive methods have been able to provide valuable information on the mechanisms of protein binding and removal, including those related to prion decontamination.8,10,11 The advantages of the method used in this study are its ease and speed of use in combination with its sensitivity, meaning that it could be used for the real-time monitoring of the cleaning of instrument batches in high throughput reprocessing situations. A companion paper to this study describes the use of the same type of indicator device within a hospital SSD in a comprehensive comparison of different presentations.20 The combination of a single experimental model and an indicator for in-process validation provides a useful platform for understanding the dynamics of protein removal and supporting front-line staff engaged in the day-to-day practicalities of instrument cleaning.
Acknowledgements We acknowledge the assistance of M. Johnson, Medisafe, UK for technical assistance on the set-up of the Pico bench-top washer disinfector and the manufacturers of the cleaning chemistries for the supply of reagents. Conflict of interest statement The views expressed in the publication are those of the authors and not necessarily those of the Department of Health. Funding source The project was funded by the Department of Health, New and Emerging Applications of Technology (NEAT; reference G021).
References 1. Taylor D. Practical aspects of decontamination of the unconventional transmissible agents that cause sporadic and variant CreutzfeldteJakob disease and other similar human diseases. Ig Sanita Pubbl 2004;60:141e150. 2. World Health Organization. WHO infection control guidelines for transmissible spongiform encephalopathies.
Comparative evaluation of prion chemistries
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
Report of a WHO consultation. Geneva, Switzerland: WHO; 23e26 March 1999. McDonnell G, Burke P. The challenge of prion decontamination. Clin Infect Dis 2003;36:1152e1154. Fichet G, Comoy E, Duval C, et al. Novel methods for disinfection of prion-contaminated medical devices. Lancet 2004;364:521e526. Baier M, Schwarz A, Mielke M. Activity of an alkaline ‘cleaner’ in the inactivation of the scrapie agent. J Hosp Infect 2004;57:80e84. Dickinson J, Murdoch H, Dennis MJ, et al. Decontamination of prion protein (BSE301V) using a genetically engineered protease. J Hosp Infect 2009;72:65e70. Lemmer K, Mielke M, Kratzel C, et al. Decontamination of surgical instruments from prions. II. In vivo findings with a model system for testing the removal of scrapie infectivity from steel surfaces. J Gen Virol 2008;89:348e358. Stephenson J. Halting the spread of human prion diseasee exceptional measures for an exceptional problem. J Hosp Infect 2007;65(Suppl. 2):14e18. Lipscomb IP, Pinchin HE, Collin R, Harris K, Keevil CW. 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. J Hosp Infect 2006;64:288e292. 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;62:141e148. Baxter RL, Baxter HC, Campbell GA, et al. Quantitative analysis of residual protein contamination on reprocessed surgical instruments. J Hosp Infect 2006;63:439e444. Brehm JK, Chambers SP, Brown KJ, Atkinson T, Minton NP. Molecular cloning and nucleotide sequence determination
151
13.
14.
15.
16.
17.
18. 19.
20.
of the Bacillus stearothermophilus NCA 1503 superoxide dismutase gene and its overexpression in Escherichia coli. Appl Microbiol Biotechnol 1991;36:358e363. Vonrhein C, Bonisch H, Schafer G, Schulz GE. The structure of a trimeric archaeal adenylate kinase. J Mol Biol 1998; 282:167e179. Murdoch H, Dickinson J, Walker J, Sutton J, Raven N. Surface decontamination of surgical instruments: an ongoing dilemma. J Hosp Infect 2006;63:432e438. Flechsig E, Hegyi I, Enari M, Schwarz P, Collinge J, Weissmann C. Transmission of scrapie by steel-surfacebound prions. Mol Med 2001;7:679e684. Yan ZX, Stitz L, Heeg P, Pfaff E, Roth K. Infectivity of prion protein bound to stainless steel wires: a model for testing decontamination procedures for transmissible spongiform encephalopathies. Infect Control Hosp Epidemiol 2004;25: 280e283. Edouard S, Bassin G, Corvaisier S. Investigation of the cleaning performance in washer-disinfectors: sensitivity of three soil tests to the cleaning parameters. Zentr Steril 2006;14: 197e215. Bradley CR, Babb JR. Endoscope decontamination: automated vs. manual. J Hosp Infect 1995;30(Suppl.):537e542. Lipscomb IP, Pinchin H, Collin R, Keevil CW. 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. J Hosp Infect 2007;65:72e77. Hesp JR, Poolman T, Budge C, et al. Thermostable adenylate kinase technology: a new process indicator and its use as a validation tool for the reprocessing of surgical instruments. J Hosp Infect 2010;74:137e143.
Available online at www.sciencedirect.com
www.elsevierhealth.com/journals/jhin
Quantitative measurement of the efficacy of protein removal by cleaning formulations; comparative evaluation of prion-directed cleaning chemistries M. Ungurs a, J.R. Hesp a,*, T. Poolman a, G. McLuckie a, S. O’Brien a, H. Murdoch b, P. Wells c, N.D.H. Raven a, J.T. Walker a, J.M. Sutton a a
Centre for Emergency Preparedness and Response, Health Protection Agency, Porton Down, Salisbury, Wiltshire, UK b Health Protection Scotland, Infection Control, Glasgow, UK c Sterilisation and Disinfection Unit, Salisbury NHS Foundation Trust, Salisbury District Hospital, Salisbury, Wiltshire, UK Received 22 January 2009; accepted 24 July 2009 Available online 14 October 2009
KEYWORDS CreutzfeldteJakob disease; Decontamination; Detergent; Prion; Transmissible spongiform encephalopathy
Summary The stability of the infectious agent causing variant CreutzfeldteJakob disease (vCJD) has highlighted the importance of cleaning surgical instruments for controlling potential spread of iatrogenic CJD. In this study, thermostable adenylate kinases (tAKs) in test soil were coated on to stainless steel and these surrogate agents used to evaluate the efficacy of a range of cleaning chemistries in a bench-top washer disinfector (btWD), or as a pre-soak either with or without subsequent treatment by btWD. Two tAKs were tested initially for ease of removal, the most persistent being Sulfolobus acidocaldarius-derived tAK which was used for evaluating the cleaning chemistries. Conventional chemistries were generally more effective when used in a btWD than as pre-soaks. Cleaning efficacy improved when pre-soaks were followed by treatment with intermediate performing enzymes, demonstrating greater than additive effect on residual tAK activity. Three of the four prion-directed chemistries reduced residual tAK activity to below the limit of quantification (LoQ) by more than 4.8 log10; <175 pg tAK remaining as a pre-soak alone. A conventional alkaline cleaning product also reduced residual tAK activity to below the LoQ but only
* Corresponding author. Address: Technology Development Group, Health Protection Agency, Centre for Emergency Preparedness and Response, Porton Down, Salisbury, Wiltshire SP4 0JG, UK. Tel.: þ44 1980 612643; fax: þ44 1980 612622. E-mail address: [email protected] 0195-6701/$ - see front matter ª 2009 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jhin.2009.07.023
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when used in a btWD. tAK soil dried on to the device was removed less efficiently than tAK soil still moist on the device, with a 320-fold and 28-fold increase in residual tAK activity for pre-soak and btWD, respectively. The study demonstrated the potential for a tAK indicator to describe the effectiveness of protein removal using different chemistries or treatment processes. ª 2009 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.
Introduction The prion infectious agent responsible for variant CreutzfeldteJakob disease (vCJD) is highly resistant to inactivation by standard decontamination treatments. This includes methods currently used in hospitals, where autoclaving even at elevated temperatures and durations only partially inactivates transmissible spongiform encephalopathy agents.1 Currently, the World Health Organization recommends prolonged treatment with high concentrations of sodium hypochlorite or sodium hydroxide but these treatments are not generally suitable for the routine decontamination of surgical instruments.2,3 Treatments that can be used routinely to remove and inactivate prions that may otherwise be present as residual contaminants on surgical equipment have now been described and many are commercially available for use in sterile services departments (SSDs).4e8 The prion issue and its control have raised the profile of the cleaning component within the surgical instrument reprocessing cycle. Washer disinfectors in the UK are operated under the guidance of Health Technical Memorandum 2030 (currently under revision). The guidelines recommend annual validation of automated washer disinfectors, monthly protein residue tests and visual inspection of every load. Due to the significant gap in the sensitivity of these methods, working to these guidelines does not provide reassurance about potential prion removal. The effectiveness of residual protein tests, based on Ninhydrin and Biuret reactions, is in the range of micrograms of protein and this may be partly due to the recovery of protein from the instruments through swabbing.9 Approaches that measure residual protein on actual surgical instruments, such as episcopic differential interference contrast microscopy, avoid these sampling problems and can measure protein contamination down to 85 pg/mm2.10 Other methods which exploit in-situ labelling of bound proteins give similar levels of sensitivity.11 This study uses thermostable adenylate kinases (tAKs) to compare the efficacy of different cleaning chemistries as either a pre-soak or within an
automated washer disinfector cycle. By coupling ATP generation by tAK to a bioluminescent detection system it was possible to measure directly the residual enzyme remaining on surfaces after treatment, providing a quantifiable measure of cleaning performance. The study demonstrates the relative effectiveness of a range of cleaning chemistries for decontaminating stainless steel. It also establishes the method as both a research tool for quantifying parameters that affect the removal of protein from surfaces and potentially as a validation tool for use in hospital settings.
Methods Production of thermostable adenylate kinase (tAK) Synthetic genes encoding the native adenylate kinase proteins from Sulfolobus acidocaldarius (Sac) or Thermotoga maritima (Tma) were cloned into pMTL1015 (a pMTL1013 derivative).12 The recombinant plasmids were used to transform Escherichia coli RV308. Enzymes were purified as described previously and assessed by sodium dodecyl sulphateepolyacrylamide gel electrophoresis (SDSe PAGE).13 Protein concentration was measured using a standard Bio-Rad (Hercules, CA, USA) DC protein assay.
Contamination of stainless steel Aliquots (100 mL) of test soil were used to coat approximately 90 mm2 of one end of clean, 65 mm 5 mm, grade 304 stainless steel strips. The test soil consisted of 50% (v/v) Browne’s soil (Steris Corp., Mentor, OH, USA) spiked to include 0.1 mg/mL tAK. To represent a worst case scenario, the applied soil was allowed to dry on to the surface at 25 C for 2 h. For experiments requiring moist soil, strips were sealed in a humid environment after application of soil and maintained at 37 C for 30 min, to simulate exposure to blood and tissue during surgery and subsequent cleaning without
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allowing the contaminating material to dry on the surface. A standard curve of enzyme dried on to the strips ranging from 0.005 ng to 5000 ng per was prepared by diluting the enzyme preparation using 50% Browne’s soil and applying 100 mL to one end of a clean stainless steel strip. Blank controls were included that consisted of 100 mL of 50% (v/v) Browne’s soil applied to the end of the strips. The applied soil was allowed to dry on to the surface at 25 C for 2 h. The activity on the strips was measured using the Hygiena handheld ATP monitoring system (SystemSURE Plus; Hygiena, CA, USA) and a four-parameter logistic model (SigmaPlot v10; Systat Software) applied to the data.
Decontamination using pre-soak treatments Each pre-soak decontamination treatment involved immersion of the soiled stainless steel strips Table I Cleaning agent
B C D E
F G H
I Water
Decontamination using washer disinfector procedures Soiled stainless steel strips were secured to the shelf of the btWD and submitted to washing using the agents and associated washing programmes (Table I). The btWD was commissioned and validated against the HTM2030 guidelines. The standard btWD cycle consisted of the following stages:
Comparison of efficacy of cleaning agents used in three process types Chemistry
Pre-soak/(btWD) wash cycle conditions Concentration (mL/L)
A
in cleaning agent maintained at the appropriate temperature in a stainless steel water-bath (Table I). After treatment with the cleaning agents, the strips were either rinsed with deionised water flowing at 8 L/min for 5 s, or transferred to a Medisafe (Bishop’s Stortford, UK) Pico Flush bench-top washer disinfector (btWD) and submitted to a standard cycle (see below) using cleaning agent A (Table I).
Enzymatic conventional Enzymatic conventional Enzymatic conventional Enzymatic conventional Enzymatic, neutral pH prion-directed
Alkaline prion-directed Alkaline prion-directed Enzymatic, alkaline prion-directed Alkaline conventional N/A
Log10 reduction in tAK activity
Temperature ( C)
Incubation time (min)
btWD
Pre-soak and rinse
Pre-soak and btWD
7.5 (7.5)
45 (43)
15 (15)
1.4
0.3
3.1
20 (3)
45 (43)
20 (15)
3.0
2.8
4.5
6 (3)
45 (43)
15 (43)
0.6
0.7
2.9
8 (3)
45 (43)
15 (15)
3.5
0.3
3.1
16.2 g/L component (a); 10 g/L component (b); 1.6 g/L component (c) (ND) 16 (16)
45 (ND)
15 (ND)
ND
2.8
4.8
45 (43)
15 (15)
4.8
4.8
4.8
10 (5)
55 (55)
15 (5)
4.8
4.8
4.8
20 (20)
60 (60)
30 (30)
4.8
4.8
4.8
4 (3)
45 (43)
15 (30)
4.8
0.8
3.1
N/A
45 (45)
15 (30)
0.3
0.4
2.7
Where possible the manufacturer’s instructions for dose, temperature and duration were followed. Cleaning agent E was not compatible with the bench-top washer disinfector (btWD). Indicator devices comprising 0.1 mg/mL of tAK in 50% Browne’s soil were processed using either a btWD cycle, or a pre-soak followed by a rinse or a btWD cycle. ATP generated by residual tAK was measured and converted into equivalent amount of residual protein using a standard curve (Figure 2A). ND, not done. Equivalent experiments carried out using Tma-based indicator strips gave values of 1.2 and 5.1 log10 reductions in activity for cleaning agent A, using either a pre-soak or btWD process, respectively. For each experiment (N ¼ 3) four stainless steel strips soiled with tAKspiked Browne’s test soil and four control stainless strips soiled with only Browne’s test soil were used.
Comparative evaluation of prion chemistries
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a 3 min cold water pre-wash, a main wash cycle (Table I); a rinsing cycle and a disinfect stage, which included heating to 80e85 C for 120 s.
4 Washer disinfector Control 3
Residual tAK activity was measured using a handheld ATP monitoring system (SystemSURE Plus; Hygiena). After treatment, stainless steel strips were transferred to an Ultrasnap sample tube (Hygiena), to which 200 mL of 67.5 mM ADP, 15 mM magnesium acetate solution was added, immediately after which the Ultrasnap enzyme reagent was added. The relative light units (RLUs) emitted by the reaction were measured after a 2 min incubation period at room temperature. For each experiment (N ¼ 3) four stainless steel strips soiled with tAK-spiked Browne’s test soil and four control stainless strips soiled with only Browne’s test soil were used.
Results The purification procedure resulted in enzymatically active protein of >99% purity as assessed by SDSePAGE and densitometry. The enzymes from both S. acidocaldarius (Sac) and T. maritima (Tma) on stainless steel were exposed to the conditions of 90 C, 2 min in a closed chamber to replicate the thermal element of an extended disinfection phase of the btWD cycle. There was minimal reduction in activity observed for Sac (5955 RLU 715 compared to non-heat-treated control, 6136 431) or Tma (7381 RLU 387 compared to non-heated control, 7305 333). There was also minimal reduction in activity observed for the Sac-based indicator following exposure to the thermal part of a complete btWD cycle (Figure 1). Standard curves for the enzymes are shown in Figure 2A. Initial studies explored the relative ease of removal of the two tAKs from stainless steel when processed using either a pre-soak protocol or within a small btWD. Using enzymatic cleaning product A, the indicators were processed under the manufacturer’s recommended conditions either as a pre-soak or in a btWD. Residual activity remaining on the steel surface was compared. Tma showed log10 reduction values of 1.2 and 5.1 for pre-soak and btWD, respectively, whereas the values for Sac were 0.3 and 1.4, respectively, demonstrating that Sac was more resistant to the chemical treatment, in either of the two processes. This suggested that Sac-based indicators
ATP (µM)
Measurement of remaining tAK activity after decontamination treatment
2
1
0 0.001
0.01
0.1
1 Sac (ng)
10
100
1000
Figure 1 Analysis of the effect of the thermal part of the washer disinfector cycle on the activity of Sulfolobus acidocaldarius-derived adenylate kinase (Sac). Sac standard curves were set up on metal tokens using 50% (w/v) Browne’s soil as described in Methods. The prepared tokens were sealed inside a vessel and subjected to a full standard washer disinfector cycle. Control samples were prepared as above but left at ambient temperature. Following processing the samples were assayed for activity as described previously. Relative light units values were converted to equivalent [ATP] using an ATP standard. Regression analysis was carried out using SigmaPlot (v10; Systat Software) with a four-parameter logistical fit applied to the data.
would be a tougher challenge test with which to evaluate the performance of cleaning processes, particularly those aimed at removing prions. The Sac indicator device was used to compare a series of cleaning chemistries, either with (products EeH) or without (AeD conventional enzymatic, I alkaline only) European Standard (CEmarked) prion inactivation claims (Table I). All of the chemistries, except products I and G, have instructions with suggested conditions for use as a pre-soak and these have been used for the majority of prion inactivation studies published to date. Overall, three of the four prion-directed chemistries (products F, G and H) were capable of reducing residual enzyme activity to below the LoQ of the assay, at greater than 4.8 log10 reduction of tAK activity (equivalent to <175 pg of residual tAK protein) (Figure 2C; Table I). The activity of the remaining chemistries ranged from 0.3 log10 reduction (conventional enzymatic products A and D) to 2.8 log10 (conventional enzymatic B and prion-directed, enzymatic E). In terms of tAK activity, this gives a range of 2.9 ng to 2.3 mg of residual protein remaining after processing. The
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A
B 10000 Tma Sac
6000
Remaining tAK (ng)
tAK activity (RLU)
8000
4000 2000
1000 100 10 1 0.1
0 0.001 0.01
C
0.1
1 10 tAK (ng)
100
0.01
1000 10000
D
10000
Remaining tAK (ng)
Remaining tAK (ng)
B
C
D F G Cleaning agent
H
I Water
D E F G Cleaning agent
H
I Water
10000 1000
1000 100 10 1 0.1 0.01
A
100 10 1 0.1
A
B
C
D E F G Cleaning agent
H
I Water
0.01
A
B
C
Figure 2 Comparison of the residual adenylate kinase (tAK) activity remaining after treatment with adenylate kinase derived from Sulfolobus acidocaldarius (Sac). The indicator devices comprised 0.1 mg/mL of tAK in 50% Browne’s soil. The wash conditions associated with the treatments are identified in Table I. ATP generated by residual tAK post processing was measured as relative light units (RLU) and converted into an equivalent amount of residual protein using a standard curve (A). The coated tokens were treated using either: a btWD cycle (B); a pre-soak followed by a rinse (C), or a pre-soak followed by a bench-top washer disinfector (btWD) cycle (D). Each experiment was repeated three times using four stainless steel strips soiled with tAK-spiked Browne’s test soil and four control stainless strips soiled with only Browne’s test soil. Using the standard curve data the limit of quantification was calculated as 175 pg per strip (dotted line).
only conventional alkaline product, I, gave a relatively low reduction in activity at 0.8 log10. When assessed in a btWD, run according to the manufacturer’s instructions, the prion-directed chemistries continued to clean at the LoQ. This was similar to the conventional alkaline product I, which shows greater than 4 log10 increase in cleaning performance (Figure 2B; Table I). The remaining cleaning products also showed an increase in cleaning efficacy in the btWD, except for the enzymatic cleaner C and water, which showed a small decrease. The increase in efficiency ranged from 0.2 log10 (product B increasing from 2.8 to 3.0 log10 reduction) to 3.2 log10 increase (product D increasing from 0.3 to 3.5 log10 reduction). The results demonstrate the lack of correlation between cleaning efficacy in the two processes,
with similar products that performed at equivalent levels in the pre-soak (e.g. products A and D) giving markedly different performances in the btWD. A great deal of interest has been expressed in understanding how different components of the surgical instrument cleaning cycle interact according to likely prion removal efficacy. The study explored the effectiveness of pre-soaks for improving the removal of soiling by subsequent btWD processing. The indicator devices were treated in a pre-soak protocol with each of the cleaning products. Cleaning product A was selected for subsequent treatment of the indicators in the btWD, as it provided a cleaning performance that was in the mid-range for conventional chemistries currently used in the profession (reducing activity by 1.4 log10). In each case that could be measured
Comparative evaluation of prion chemistries
Discussion In the light of vCJD, cleaning is assuming increasing importance as a component of the surgical instrument reprocessing cycle.8,14 This study looked at the potential of a new, rapid and sensitive enzyme-based assay to provide information on the effectiveness of different cleaning chemistries and other factors that might affect instrument reprocessing. Although the tAK enzymes that are used as the basis of the assay are not prions, they may share binding properties that, in combination with their exceptional thermostability, make them ideal for modelling and assessing prion removal characteristics. We showed that priondirected cleaning chemistries outperformed the majority of conventional chemistries in reducing tAK activity, supporting this conclusion. The study compared nine different cleaning chemistries for their ability to reduce the activity
10000 Remaining tAK (ng)
within the maximum sensitivity of the assay, the dual treatment resulted in a greater than additive reduction in activity of the two processes alone (Table I). This was the case even when cleaning product A was used for both pre-soak and btWD (0.3 ng of protein remaining), or when water was used as the pre-soak. Combinations with three of the four anti-prion chemistries could not be assessed due to their ability to clean to the LoQ of the assay as pre-soak alone. Only one additional combination reached the limit of detection of the assay, with prion-directed chemistry E as pre-soak followed by cleaning product A giving greater than a 4.8 log10 reduction in activity (<175 pg of tAK remaining). Another critical question that has been posed is the effect that extended drying has on effectiveness of removal of protein contamination. This was examined by comparing the removal of tAK soil from indicators that were allowed to dry at 25 C for 2 h (as used routinely for the comparison of cleaning products) against indicators that were held at 37 C for 30 min in a moist environment to simulate reasonable exposure to tissue during surgery and kept moist until decontamination. The condition of the soil when presented to cleaning treatment markedly affects cleaning efficacy (Figure 3). The residual tAK contamination after a soaking treatment for dried soil is about 320 times greater than that measured for moist soil. When using the washer disinfector treatment, tAK residual contamination after treatment is about 28 times greater with the dried soil compared with the residual contamination for moist soil.
149 Washer disinfector treatment
Sink soak treatment
1000 100 10 1
M
D M Soil condition
D
Figure 3 The effect of extended drying on the removal of adenylate kinase (tAK) as part of a test soil. 0.1 mg/mL of tAK was incorporated into 50% Browne’s soil and applied to steel strips. The strips were incubated at 25 C in an open container for 2 h or at 37 C within a closed chamber to simulate dried-on soil (D) or moist soil (M) respectively. The strips were treated using cleaning agent A as a pre-soak treatment or in the bench-top washer disinfector. The ATP generated by residual tAK on the indicator device was measured and converted into nanograms of protein remaining using a standard curve (Figure 2A). The experiment was repeated three times using four stainless steel strips soiled with tAK-spiked Browne’s test soil, and four control stainless strips soiled with only Browne’s test soil were used for each repeat. Log reduction values of 0.3 log10 and 2.8 log 10 (pre-soak) and 1.4 log 10 and 2.9 log 10 (btWD) were recorded for the dried-on soil and moist soil, respectively.
of the trimeric S. acidocaldarius tAK enzyme from surgical steel. The reduction in activity is likely to be a function of both protein removal from surfaces and inactivation of the enzyme on the surface. The standard wire-implantation model used in the majority of bioassay studies is similarly multifactorial, with no direct measure of prion removal being able to approach the sensitivity required to demonstrate complete removal from the surface.4,6 A second monomeric tAK derived from T. maritima was used in the development of the indicator and, although tolerant to extremes of pH, it was found to be much more susceptible to the mechanical cleaning action of the btWD than the enzyme from S. acidocaldarius. It could be argued on this basis that the Tma enzyme reflects the surface-binding behaviour of proteins in general, whereas the behaviour of the Sac enzyme reflects the ability of certain proteins, such as prions, to bind tenaciously to stainless steel.15 This is supported by evidence that the Sac enzyme binds preferentially to plastic and metals even when these surfaces are pre-bound with commonly used blocking agents such as skimmed milk and Tween-20 (results not shown). The difference in
150 the surface-binding properties may reflect a partial unfolding of the hydrophobic core of the protein and its interaction with the surface of the steel.13 The majority of prion inactivation studies have been based on conditions similar to the pre-soak treatments used in this study. The comparative ability of the cleaning chemistries to reduce the levels of tAK activity are reminiscent of these studies, where no single class of chemistry was universally effective against prions.4,16 In the presoak model only three of the four prion-targeted chemistries were able to clean to the limits of quantification of the assay, with an additional conventional alkaline chemistry matching this performance in the btWD only. The results with this latter product, I, demonstrate the difficulties of extrapolating from relatively static wash conditions to those encountered even within small btWDs. Other studies that have explored this phenomenon with regards to the sensitivity of soil tests have also showed that mechanical action played a pivotal role in cleaning.17 Although not seen in this study, combinations of treatments can pose compatibility issues between pre-soaking agent and cleaning agent used in the WD.17 The comparison between manual and automated studies is well-described in endoscope reprocessing, with mechanical cleaning producing better results than manual methods.18 It would be expected that the cleaning performance of the cleaning agents used in this study would be better when used in a WD operating in an SSD with more powerful mechanical action being available. Decontamination in the btWD used in this study is, therefore, more reliant on the action of the cleaning agent used. The combination of a pre-soak with subsequent treatment in a btWD proved the most effective process with high log reductions in almost all cases. In all the combinations tested, where cleaning was within the dynamic range of the assay, a greater than additive increase in log reduction was observed. This might have been expected where the combination of the chemistries favours removal of proteins attached via different surface interactions. However, the observed increase due to the combination of pre-soak and btWD, even with the same product or class of products, may reflect the effects of rehydration of the soil favouring its subsequent removal. More direct evidence for the role in soil hydration in efficacy of removal was evaluated using the soiled indicators in either dried or moist environments. The greater reduction in activity on the
M. Ungurs et al. indicators not allowed to dry echoes those observed previously.19 With the increased awareness of the importance of the cleaning component of the instrument reprocessing cycle, a number of methods have been developed to assess the relative removal of protein soiling, to complement the visual inspection and colorimetric protein assays in current use. Some of these highly sensitive methods have been able to provide valuable information on the mechanisms of protein binding and removal, including those related to prion decontamination.8,10,11 The advantages of the method used in this study are its ease and speed of use in combination with its sensitivity, meaning that it could be used for the real-time monitoring of the cleaning of instrument batches in high throughput reprocessing situations. A companion paper to this study describes the use of the same type of indicator device within a hospital SSD in a comprehensive comparison of different presentations.20 The combination of a single experimental model and an indicator for in-process validation provides a useful platform for understanding the dynamics of protein removal and supporting front-line staff engaged in the day-to-day practicalities of instrument cleaning.
Acknowledgements We acknowledge the assistance of M. Johnson, Medisafe, UK for technical assistance on the set-up of the Pico bench-top washer disinfector and the manufacturers of the cleaning chemistries for the supply of reagents. Conflict of interest statement The views expressed in the publication are those of the authors and not necessarily those of the Department of Health. Funding source The project was funded by the Department of Health, New and Emerging Applications of Technology (NEAT; reference G021).
References 1. Taylor D. Practical aspects of decontamination of the unconventional transmissible agents that cause sporadic and variant CreutzfeldteJakob disease and other similar human diseases. Ig Sanita Pubbl 2004;60:141e150. 2. World Health Organization. WHO infection control guidelines for transmissible spongiform encephalopathies.
Comparative evaluation of prion chemistries
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
Report of a WHO consultation. Geneva, Switzerland: WHO; 23e26 March 1999. McDonnell G, Burke P. The challenge of prion decontamination. Clin Infect Dis 2003;36:1152e1154. Fichet G, Comoy E, Duval C, et al. Novel methods for disinfection of prion-contaminated medical devices. Lancet 2004;364:521e526. Baier M, Schwarz A, Mielke M. Activity of an alkaline ‘cleaner’ in the inactivation of the scrapie agent. J Hosp Infect 2004;57:80e84. Dickinson J, Murdoch H, Dennis MJ, et al. Decontamination of prion protein (BSE301V) using a genetically engineered protease. J Hosp Infect 2009;72:65e70. Lemmer K, Mielke M, Kratzel C, et al. Decontamination of surgical instruments from prions. II. In vivo findings with a model system for testing the removal of scrapie infectivity from steel surfaces. J Gen Virol 2008;89:348e358. Stephenson J. Halting the spread of human prion diseasee exceptional measures for an exceptional problem. J Hosp Infect 2007;65(Suppl. 2):14e18. Lipscomb IP, Pinchin HE, Collin R, Harris K, Keevil CW. 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. J Hosp Infect 2006;64:288e292. 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;62:141e148. Baxter RL, Baxter HC, Campbell GA, et al. Quantitative analysis of residual protein contamination on reprocessed surgical instruments. J Hosp Infect 2006;63:439e444. Brehm JK, Chambers SP, Brown KJ, Atkinson T, Minton NP. Molecular cloning and nucleotide sequence determination
151
13.
14.
15.
16.
17.
18. 19.
20.
of the Bacillus stearothermophilus NCA 1503 superoxide dismutase gene and its overexpression in Escherichia coli. Appl Microbiol Biotechnol 1991;36:358e363. Vonrhein C, Bonisch H, Schafer G, Schulz GE. The structure of a trimeric archaeal adenylate kinase. J Mol Biol 1998; 282:167e179. Murdoch H, Dickinson J, Walker J, Sutton J, Raven N. Surface decontamination of surgical instruments: an ongoing dilemma. J Hosp Infect 2006;63:432e438. Flechsig E, Hegyi I, Enari M, Schwarz P, Collinge J, Weissmann C. Transmission of scrapie by steel-surfacebound prions. Mol Med 2001;7:679e684. Yan ZX, Stitz L, Heeg P, Pfaff E, Roth K. Infectivity of prion protein bound to stainless steel wires: a model for testing decontamination procedures for transmissible spongiform encephalopathies. Infect Control Hosp Epidemiol 2004;25: 280e283. Edouard S, Bassin G, Corvaisier S. Investigation of the cleaning performance in washer-disinfectors: sensitivity of three soil tests to the cleaning parameters. Zentr Steril 2006;14: 197e215. Bradley CR, Babb JR. Endoscope decontamination: automated vs. manual. J Hosp Infect 1995;30(Suppl.):537e542. Lipscomb IP, Pinchin H, Collin R, Keevil CW. 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. J Hosp Infect 2007;65:72e77. Hesp JR, Poolman T, Budge C, et al. Thermostable adenylate kinase technology: a new process indicator and its use as a validation tool for the reprocessing of surgical instruments. J Hosp Infect 2010;74:137e143.