1 P24 Transmissible spongiforme Enzephalopathien, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
2 FG 14 Angewandte Krankenhaushygiene und Infektionsprävention, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
3 ZBS1 Hochpathogene virale Erreger, Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany
Correspondence
Karin Lemmer
LemmerK{at}rki.de
Michael Beekes
BeekesM{at}rki.de
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ABSTRACT |
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INTRODUCTION |
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Following the emergence of BSE (Wells et al., 1987) and vCJD (Will et al., 1996
), substantial evidence has accumulated that the latter can most likely be attributed to transmission, presumably via contaminated food, of BSE from cattle to man (Bruce et al., 1997
; Hill et al., 1997
; Cousens et al., 1999
; Scott et al., 1999
). The countermeasures implemented in response to the BSE epidemic are expected effectively to prevent further spread of this disease to humans, thereby minimizing the risk of new primary vCJD infections. However, additional challenges for public health in the context of TSEs arise from the hypothetical as well as the established risks of human-to-human transmission of vCJD and classical CJD, respectively (Brown et al., 2001
; Taylor, 2003
; Beekes et al., 2004
; Llewelyn et al., 2004
). The experience with iatrogenic CJD, of which 267 cases were reported until July 2000 (Brown et al., 2000
), and the detection of infectivity or PrPSc in a variety of tissues from vCJD patients in addition to the brain and spinal cord (e.g. lymphatic system and peripheral nervous system; Bruce et al., 2001
; Wadsworth et al., 2001
; Hilton et al., 2002
; Haik et al., 2003
; Ramasamy et al., 2003
) have led to the formulation of national and international recommendations and guidelines aiming at the prevention of iatrogenic transmission of these diseases (Simon & Pauli, 1998
; World Health Organization, 1999
; Abschlussbericht der Task Force vCJK, 2002
).
In hospitals, it is of the utmost importance to avoid the spread of TSE infectivity via surgical instruments by effective and safe decontamination (e.g. cleaning, chemical disinfection, sterilization; Beekes et al., 2004; Sehulster, 2004
). This is highlighted by the fact that PrPSc has also been detected in skeletal muscles of CJD patients (Glatzel et al., 2003
) and is present in lymphatic tissues, and possibly blood, during preclinical phases of vCJD incubation (Hilton et al., 1998
, 2002
; Llewelyn et al., 2004
).
The high resistance of TSE agents to conventional methods of chemical or thermal inactivation and to UV or ionizing radiation (Alper et al., 1966, 1967
; Brown et al., 1982
, 1986
; Kimberlin et al., 1983
; Taguchi et al., 1991
; Tateishi et al., 1991
; Ernst & Race, 1993
; Taylor et al., 1994
; Manuelidis, 1997
; Taylor, 1999
; for review see Taylor, 2000
), as well as their high binding affinity to and tenacity on steel surfaces (Zobeley et al., 1999
; Flechsig et al., 2001
), warrant specific decontamination procedures in the reprocessing of surgical instruments (Rutala & Weber, 2001
). Treatments that are considered appropriate for decontamination include use of 12 M NaOH solution (for 24 h), 2·55 % NaOCl solution (for 24 h) as well as 3, 4 or 6 M GdnSCN solution (for 24 h, 1 h or 15 min, respectively), followed by steam sterilization at 134 °C for 18 min to 1 h (Simon & Pauli, 1998
; World Health Organization, 1999
; Hörnlimann et al., 2001
). Such stringent conditions, which are mandatory for the reprocessing of non-disposable instruments used in patients with CJD (or those with a recognizable risk of it), are hazardous to both equipment and operators, therefore they do not offer an option for the routine maintenance of surgical instruments used on patients without a recognizable risk of human TSE. Here, generally applicable decontamination strategies that take into account the theoretical risk of CJD and vCJD transmission from asymptomatic carriers on the one hand, without compromising the conventional processes for cleaning, disinfection and sterilization on the other, are required.
In the search for effective yet instrument-friendly and routinely applicable procedures complying with these requirements, we used an in vitro carrier assay to assess the decontamination efficacy exerted by various reagents on PrPSc and its proteinase K (PK)-resistant core PrP27-30 (referred to here as PrPSc/PrP27-30), attached to steel surfaces. The rationale of our experiments is based on the paradigm of a steel wire assay originally described by Zobeley et al. (1999) and Flechsig et al. (2001)
, as well as on the notion that a reduction of the amount of PK-resistant prion protein achieved by decontaminating reagents should, in principle, correlate with a decrease of infectivity (Beekes et al., 2004
). However, this correlation needs to be carefully validated for each individual setup of inactivation experiments, as highlighted by McLeod et al. (2004)
. Comprehensive studies in which PrPSc was visualized by Western blotting after immunolabelling with the monoclonal anti-PrP antibody 3F4 (mAb 3F4) have previously shown a close quantitative correlation between PrPSc/PrP27-30 and infectivity in the brains of hamsters infected with 263K scrapie agent (Beekes et al., 1996
; Baldauf et al., 1997
). In addition, studies have demonstrated an inactivation of 263K scrapie agent concomitant with the disappearance of PK-resistant PrP in hamster brain homogenates following incubation with an alkaline cleaning reagent (Baier et al., 2004
). We simulated contamination of surgical instruments using stainless steel wires incubated in 263K scrapie brain homogenate containing PrPSc/PrP27-30 (as well as a minor proportion of normal PrPC). Other than intact brain tissue, which is known to contain varying infectivity titres and concentrations of PrPSc in different cerebral areas, brain homogenate allows defined amounts of material to be dried onto carriers and facilitates the standardization and reproducibility of decontamination experiments within the same or in different laboratories. Scrapie brain homogenates have been used as the substrate of choice in a variety of inactivation studies (Taylor, 2000
, 2004
). Hard data formally proving that homogenized brain tissue dried onto wire surfaces provides an appropriate equivalent to intact brain tissue contaminating surgical instruments are limited, but there are reported findings that suggest this. First, inactivation of murine ME7 scrapie agent by formic acid was found to be similarly effective if applied to brain homogenate or intact, even fixed brain tissue (Taylor et al., 1997
). Second, under certain experimental conditions, homogenates were actually found to provide a more challenging substrate for inactivation than intact tissue: scrapie infectivity was inactivated in intact scrapie-infected mouse brain, but not in 10 % homogenates of brain tissue, by 2 % peracetic acid (Taylor, 1991
). One explanation for the presence of heat-or-chemical resistant subpopulations of scrapie agent might be the protective effect of aggregation which could occur in homogenates of infected tissue but not in undiluted tissue (Taylor, 2004
). Third, it has been observed that scrapie agent in partially smeared tissue dried onto glass or metal surfaces is more resistant to inactivation by autoclaving than in intact undisrupted tissue, which is thought to result from rapid PrPSc-fixation in the dried film (Taylor, 2000
, 2004
). Brain tissue, which provides one of the most challenging contaminations in the routine reprocessing of surgical instruments (Köhnlein et al., 2004
) is rich in lipids, compounds that are known to further increase the resistance to inactivation of TSE agents and the stability of PrPSc (Appel et al., 2001
). Thus, particularly when additionally fixed by air-drying as in our studies, scrapie brain homogenate attached to steel wires can be considered a relevant model for a worst-case scenario in nosocomial instrument decontamination.
With this paradigm, we reprocessed batches of steel wires for decontamination by exposure, under various conditions, to several different test reagents. Residual contamination of wires still present after reprocessing was then monitored by sensitive Western blot detection (Beekes et al., 1995; Thomzig et al., 2003
, 2004
) of full-length PrP (PrPSc and PrPC) and PrP27-30 which could be eluted from the carriers without or after PK digestion, respectively. Finally, the cleaning solutions in which the wires had been incubated were also scrutinized for their content of total PrP and PrP27-30.
A broad spectrum of reagents and processing conditions were screened for both efficacy of decontamination and the underlying active principles such as detachment, destabilization or degradation of surface-bound prion protein. This revealed several different, relatively mild candidate reagents which appear to be potentially suitable for the thorough decontamination of steel surfaces from PrPSc/PrP27-30.
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METHODS |
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In vitro carrier assay.
A stock of 10 % 263K scrapie brain homogenate, containing 108 50 % intracerebral lethal doses (LD50i.c.) of 263K agent and
10 µg pathological prion protein (PrPSc/PrP27-30) ml1 was prepared from brains of Syrian hamsters in the terminal stage of disease (Beekes et al., 1995
, 1996
) and stored at 70 °C in aliquots. Stainless steel wire (DIN-No. 1.4301, Forestadent, Pforzheim, Germany; diameter 0·25 mm) was cut into small pieces 5 mm long. These test bodies (here called wires) with a surface area of
4·0 mm2 (2
rh+2
r2), were washed in 2 % Triton X-100 for 15 min under constant ultrasonication (Sonorex RK 102 P; Bandelin Electronics), rinsed in distilled water, dried and sterilized in a steam autoclave at 121 °C for 20 min. In order to contaminate the carriers in vitro with PrPSc/PrP27-30, batches of 30 wires were incubated in 150 µl 10 % scrapie brain homogenate for 2 h under constant shaking at 37 °C and 700 r.p.m. in a thermomixer (Amersham Biosciences). Control batches of wires were similarly incubated in 10 % normal hamster brain homogenate. Following removal of the homogenate, wires were transferred to and placed separately from each other in Petri dishes, air-dried for 1 h, and stored overnight (
16 h) at room temperature. Subsequently, batches of 30 contaminated wires were incubated, in a volume of 1·5 ml, in the reagents to be tested for decontaminating activity (Table 1
). These incubations were performed in a thermomixer (400 r.p.m.) at the temperatures and for the times specified in Table 1
. Finally, the wire batches were rinsed under constant shaking five times, each time in 45 ml distilled water for 10 min at room temperature. To assess the influence of drying on the fixation of PrPSc/PrP27-30 to steel surfaces, a subset of wire batches was rinsed five times in 45 ml distilled water for 10 min at room temperature immediately after incubation in scrapie brain homogenate. After rinsing in distilled water, processing of wires was finished by air-drying in Petri dishes for 1 h, storing overnight (
16 h) at room temperature, and recollection of batches in test tubes. For each reagent tested four batches of 30 wires were processed independently. Residual prion protein contaminations of wires, which were still present after processing as outlined above, were eluted from the carrier surface as follows. Of the four batches independently processed per test reagent, two were boiled for 5 min in 52 µl electrophoresis sample loading buffer (62·5 mM Tris pH 6·8, 10 % glycerol, 5 % mercaptoethanol, 2 % SDS, 0·025 % bromophenol blue) containing 4 M urea; the remaining two batches were treated with 150 µg PK ml1 in a volume of 52 µl TBS-Sarkosyl (50 mM Tris/HCl, 150 mM NaCl pH 7·5, 1 % Sarkosyl) for 1 h at 37 °C, subsequently mixed with an equal volume of 2x sample loading buffer containing 8 M urea, and boiled for 5 min. Aliquots of wire eluates (20 µl) were removed (leaving the wires on the bottom of the tube) and analysed by SDS-PAGE and Western blotting for the presence of prion protein.
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RESULTS |
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Efficacy and active principles of test reagents for decontamination of steel surfaces from PrPSc/PrP27-30
The efficacy of the decontamination of steel wires by various test reagents was assessed by comparing the initial load of contamination with the amount of total PrP and PrP27-30 residually attached to the carriers or released into the cleaning solution after processing.
This analytical approach also shed light on the active principles underlying the effects of the different reagents (degradation, detachment or destabilization of PrPSc). (i) If without PK treatment PrP could be detected only in a substantially reduced amount, or not at all, on the steel wires and in the cleaning solution, this indicated degradation of the protein; (ii) if without or after PK treatment PrP was found in the cleaning solution, the protein was at least in part detached from the wire surface; (iii) if prion protein visible in the Western blot prior to PK treatment was markedly reduced in its amount or completely disappeared upon digestion with PK, this showed that the reagent destabilized the protease-resistant core of PrPSc molecules in that it made this core more susceptible to enzymic degradation.
Table 1 summarizes the decontamination efficacy and active principles observed in our in vitro carrier assay for the test reagents examined.
Distilled water.
The mildest processing conditions were simulated by incubating wires in distilled water at different temperatures up to 90 °C. This treatment left massive contamination attached to the carriers. Compared with incubation at 23 and 90 °C for 60 min, or 55 °C for 10 min, exposure of wires to water at 90 °C for 10 min appears to exert a fixing effect (Fig. 2a and b, compare lanes 3, 4 and 6 against lane 5). However, even by simple processing in water, considerable proportions of PrP were detached from the wire surface and released into the aqueous phase (see Fig. 4a and b
, lanes 10, 11, 17 and 18), and after 60 min at 90 °C a proportion of the released PrPSc/PrP27-30 appeared to be destabilized and rendered susceptible to PK digestion (Fig. 4a and b
, lane 18). Such an effect was not observed with the fraction of PrPSc/PrP27-30 that remained attached to the wire surface under these processing conditions (Fig. 2b
, lane 6).
Sodium hydroxide and sodium hypochlorite.
In accordance with a wealth of previous findings on these reagents (Kimberlin et al., 1983; Brown et al., 1986
; Taylor et al., 1994
; Taylor, 2000
; Flechsig et al., 2001
; Rutala & Weber, 2001
), incubation of wires in 1 M NaOH or 2·5 and 1 % NaOCl (containing at least 20 000 and 8000 p.p.m. available chlorine, respectively) under the conditions specified in Table 1
led to apparently complete degradation of PrPSc/PrP27-30, as revealed by Western blotting (Figs 3a
, lane 1; 3b, lanes 12; 4a, lanes 1, 4 and 5). [The cross-reactive band visible after PK digestion in several samples (Fig. 3a
, lane 2; 3b, lane 3; 4b, lane 5) resulted from the protease; Korth et al., 2000
.] NaOH was similarly effective at a concentration of 0·5 M if applied at 55 °C. However, after incubation for 30 min at 23 °C with 0·5 M NaOH, some residual PrP remained detectable in several runs prior to PK digestion (Fig. 3a
, lane 3). Following treatment with 0·1 M NaOH, PK-sensitive residual PrP could be found attached to the wire surface as well as released into the cleaning solution (Fig. 3a
, lanes 5 and 6; Fig. 4a and b
, lane 3). This shows that 0·1 M NaOH exerts a detaching and destabilizing effect on the protein.
Guanidine thiocyanate.
After incubation in 4 M GdnSCN, strong Western blot signals for residual PrP contaminations of wires were observed (Fig. 3c, lanes 1 and 2). In wire eluates, PrP immunostaining could be detected down to a dilution of 1 : 100; these dilution samples represented 0·462 mm2 of wire surface and showed weak but still clearly discernible signals (not shown). Only a small proportion of the protein was detectable in the cleaning solution (Fig. 4a
, lane 12). Thus, there was no evidence for substantial degrading or detaching activity exerted by this reagent. However, complete disappearance of PrPSc/PrP27-30 after incubation with PK (Figs 3c
, lane 3; 4b, lane 12) indicated that the protease-resistant core of PrPSc was destabilized and rendered susceptible to enzymic degradation by this chaotropic reagent.
Urea.
Following treatment with 4 M urea, substantial amounts of PrP were retained on the wire surface (Fig. 3d, lanes 1 and 2) as well as being released into the cleaning solution (Fig. 4a
, lanes 15 and 16). Large proportions of the residual PrPSc/PrP27-30 on the wires and in the cleaning solution were resistant to digestion with PK (Figs 3d
, lane 3; 4b, lanes 15 and 16). PrP immunostaining in wire eluates examined after or without PK digestion could be detected down to a dilution of 1 : 100, with very weak signals in the samples representing 0·462 mm2 of wire surface (not shown). These findings demonstrate that 4 M urea, under the conditions used, exerted a detaching but no prominent destabilizing or degrading activity on PrPSc/PrP27-30.
Alkaline cleaner.
The commercially available alkaline cleaner considerably reduced the load of prion protein attached to the wires (Fig. 3e, lanes 1 and 2), apparently at least in part by mediating substantial release of PrP into the cleaning solution (Fig. 4a
, lanes 6 and 7). Digestion with PK led to a complete disappearance of visible residual PrPSc/PrP27-30 contamination (Figs 3e
, lane 3; 4b, lanes 6 and 7). Taken together, these observations show that the alkaline cleaner exerted a detaching and strong destabilizing effect.
SDS.
SDS at 5 and 2·5 % achieved marked decontamination of the wires when applied at 90 °C for 1 h or longer, with no residual PrP detectable on the carrier surface without or after PK digestion (Fig. 3f, lanes 1 and 2, 4 and 5). These processing regimes caused release of a massive proportion of wire-attached PrP into the SDS solution (Fig. 4a
, lanes 13 and 14). On digestion with PK, all residual PrP immunoreactivity disappeared completely (Fig. 4b
, lanes 13 and 14). Thus processing of contaminated wires in SDS, as specified above, exerted a strong detaching and destabilizing effect on PrPSc/PrP27-30. However, exposure of wires to 5 % SDS at 23 °C, even for 16 h, resulted in incomplete decontamination (Fig. 3f
, lanes 3 and 6).
SDS/sodium hydroxide.
When contaminated steel wires were processed in a mixture of 0·2 % SDS and 0·3 % NaOH (previously used for decontamination purposes in the laboratory of Professor H. Diringer at the Robert Koch-Institut) for 2·5, 5 or 10 min at 23 °C, only very weak shadows in the molecular mass range of PrP immunoreactivity (Fig. 3g, lanes 1 and 3), or no PrP at all (Fig. 3g
, lane 5), could be detected by Western blotting of carrier eluates. However, substantial amounts of PrP were released into the cleaning solution (Fig. 4a
, lanes 8 and 9). After digestion with PK, no residual PrPSc could be found on the wires or in the cleaning solution (Figs 3g
, lanes, 2, 4 and 6; 4b, lanes 8 and 9). These observations indicate a marked detaching and destabilizing activity exerted by this reagent mixture on PrPSc/PrP27-30.
Peracetic acid.
Following treatment of contaminated wires in 0·25 % peracetic acid for 1 h at 23 °C, very strong Western blot signals (Fig. 3h, lanes 1 and 2) indicated massive residual contamination of carriers. On exposure to PK, the residual surface-bound PrP showed strong resistance to enzymic degradation (Fig. 3h
, lane 3). In the eluates of wires treated with peracetic acid, it was possible to detect PrP both before and after PK digestion down to a dilution of 1 : 100, with prominent Western blot signals observed in samples representing 0·462 mm2 of wire surface. Only minute traces of immunoreactive material were released into the peracetic acid solution, displaying an apparent molecular mass between that of the 45 and 66 kDa MW marker proteins (Fig. 4a
, lane 19). This material was apparently sensitive to digestion with PK (although the lower amount of material represented in Fig. 4b
, lane 19 compared with that in 4a, lane 19, has to be taken into consideration). In any case, peracetic acid, a potent oxidizing biocide, had no visible decontaminating effect and did not exert significant detaching, destabilizing or degrading effects on PrPSc/PrP27-30 if applied at a concentration of 0·25 %.
Peracetic acid/sodium hydroxide.
The disinfectant containing peracetic acid and NaOH reduced the load of PrP to the threshold of detection if applied for 2 h at 23 °C (Fig. 3i, lane 3). The reagent mixture detached substantial amounts of immunoreactive material from the carriers, displayed predominantly as a broad band from the top of the gel down to 45 kDa (Fig. 4
, lane 20). This smear possibly resulted from reagents present in the sample that severely interfered with the electrophoretic separation of proteins. After incubation with PK a weak immunostaining signal, possibly indicating PrP dimers, could still be detected in the cleaning solution at a molecular mass between
60 and 66 kDa (Fig. 4b
, lane 20). Taken together, these findings suggest that the protease-resistant core of PrPSc was substantially destabilized by the disinfectant used, which also appeared to exert a strong degrading activity.
Candidate reagents for thorough decontamination of steel surfaces from PrPSc/PrP27-30
In the test assay used, failure to detect residual prion protein in wire eluates which were not subjected to prior PK digestion indicated an efficacy of surface decontamination in the range of at least 500- to 1000-fold compared with the initial load of PrP bound to the carriers. Such thorough decontamination efficacy the highest possible measurable in our assay or at least a reduction of contamination to the threshold of detection (as indicated by faint shadows in the Western blot which may potentially, but not necessarily, point to the presence of minute traces of residual PrP) could be achieved with the following reagents and minimum incubation times/temperatures: 1 M NaOH (30 min/23 °C; 5 min/55 °C); 0·5 M NaOH (60 min/23 °C; 5 min/55 °C); 2·5 and 1 % NaOCl (5 min/23 °C); 5 and 2·5 % SDS (60 min/90 °C); 0·2 % SDS/0·3 % NaOH (2·5 min/23 °C); and the disinfectant containing peracetic acid and NaOH (120 min/23 °C).
Of these treatments, only those based on highly concentrated NaOH and NaOCl did not cause any detectable contamination of the cleaning solution. With the disinfectant containing peracetic acid and NaOH, substantial amounts of immunoreactive material extending from the top of the gel down to 45 kDa were found in the cleaning solution, and on treatment with 0·2 % SDS/0·3 % NaOH, or highly concentrated SDS alone, strong PrP signals occurred in this analyte. However, with the possible exception of the disinfectant containing peracetic acid and NaOH, any PrPSc/PrP27-30 detached by these reagents under the specified conditions from the surface of contaminated wires was substantially destabilized, as revealed by apparently complete disappearance after digestion with PK.
The alkaline cleaner routinely used cleared the carriers to a very large extent, although not completely, from PrP at concentrations of 1 % (60 min/23 °C; 5 min/55 °C) and 0·5 % (60 min/23 °C; 10 min/55 °C), and rendered the residual protein, which remained attached to the wires, susceptible to degradation by PK. The latter was also observed for 4 M GdnSCN (10 min/23 °C), although this chaotropic reagent did not show prominent detaching or degrading activity. PrPSc/PrP27-30 released into the cleaning solution during incubation of wires in the alkaline cleaner and 4 M GdnSCN showed marked sensitivity to PK due to the strong destabilizing effects exerted by these reagents.
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DISCUSSION |
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Assuming that a specific misfolded state of PrPSc or its protease-resistant core PrP27-30 is more or less essential for maintaining TSE infectivity, virtually all strategies to inactivate TSE agents aim at modifying the conformation, folding or aggregation of PrPSc so that it becomes accessible to complete enzymic degradation by PK (Beekes et al., 2004). Therefore, and in the light of the experimentally established close correlation between TSE infectivity and PK-resistant PrPSc/PrP27-30, testing for the latter in an in vitro carrier assay such as that used in our study appears to be a suitable rapid screening method for identification of candidate decontamination reagents. Probing the detaching, destabilizing and degrading activity of test reagents on PrPSc in the wire assay is easily accomplished by Western blotting with and without PK digestion of samples eluted from the wire surface and taken from the cleaning solution. Precise knowledge of how much PrPSc remains invisibly attached to the wire surface after boiling in sample loading buffer containing urea would further specify the sensitivity of our in vitro carrier assay. However, as crude scrapie brain homogenate was used for the contamination of wires with PrPSc (and for a variety of other experimental/technical reasons) this information is difficult to obtain by conventional protein and amino acid analyses.
In any case, bioassays still provide the most sensitive and accurate method for detection and titration of TSE agents, and constitute the ultimate touchstone for measuring the efficiency of decontamination procedures for TSEs. Therefore, any loss of infectivity suggested by the reduction of PK-resistant prion protein in the in vitro carrier assay needs to be confirmed by validating the decontamination of carriers in reporter animals. As recent reports have consistently shown, monitoring the efficacy of wire reprocessing in vivo is feasible (Flechsig et al., 2001; Yan et al., 2004
). If carried out sequentially in two steps (first in an in vitro then in an in vivo format) the steel wire assay can contribute considerably to the reduction of animal experiments in TSE research, which in addition to ethical considerations are time-consuming and cost-intensive. Apart from these aspects, the carrier assay provides further advantages: (i) it models specific problems of instrument reprocessing more realistically than inactivation studies on liquid tissue homogenates; and (ii) it allows the examination of substances that are not suitable for bioassays using tissue homogenates because they are toxic and cannot be adequately removed or neutralized.
Candidate reagents for decontamination identified in the in vitro carrier assay
Using the approach described here, several different reagents were found to exert a potent decontaminating activity on PrPSc/PrP27-30 attached to steel surfaces. The strong decontaminating activities of NaOH and NaOCl previously established by comprehensive infectivity studies (Kimberlin et al., 1983; Brown et al., 1986
; Taylor et al., 1994
; Taylor, 2000
; Flechsig et al., 2001
; Rutala & Weber, 2001
) and their underlying active principles were mirrored in the in vitro carrier assay, which confirmed the suitability of the assay for pre-assessment of candidate reprocessing procedures. The alkaline cleaner biochemically identified in the in vitro carrier assay as a promising candidate reagent for surface decontamination has recently been shown in tissue homogenates to achieve efficient destabilization of PrPSc and inactivation of 263K scrapie agent (Baier et al., 2004
). Furthermore, 4 M GdnSCN, which displayed a strong destabilizing activity in our in vitro assay, has already been shown completely to inactivate the infectivity of murine Rocky Mountain laboratory scrapie agent bound to steel wires (Flechsig et al., 2001
). Finally, again consistent with our findings, a high decontaminating efficiency has been reported for 5 % SDS used at higher temperatures (Taylor et al., 1999
; Taylor, 2004
). On the other hand, water and 4 M urea (applied for 1 h), both of which are suggested by our assay to exert no significant effect of decontamination, are well known for not substantially inactivating TSE infectivity (Brown et al., 1986
; Prusiner et al., 1993
). Thus for the range of reference reagents examined here, our biochemical screening assay has provided reliable information for the preselection of candidate decontaminants as well as insights into their active principles. The results observed after reprocessing wires with 0·2 % SDS/0·3 % NaOH or the disinfectant containing peracetic acid and NaOH suggest that further testing of these reagents under various conditions in the in vivo format of the carrier assay would be of interest. Such in vivo validations are currently under way after implantation of reprocessed wires into reporter animals.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Alper, T., Haig, D. A. & Clarke, M. C. (1966). The exceptionally small size of the scrapie agent. Biochem Biophys Res Commun 22, 278284.[Medline]
Alper, T., Cramp, W. A., Haig, D. A. & Clarke, M. C. (1967). Does the agent of scrapie replicate without nucleic acid? Nature 214, 764766.[Medline]
Appel, T., Wolff, M., von Rheinbaben, F., Heinzel, M. & Riesner, D. (2001). Heat stability of prion rods and recombinant prion protein in water, lipid and lipidwater mixtures. J Gen Virol 82, 465473.
Baier, M., Schwarz, A. & Mielke, M. (2004). Activity of an alkaline cleaner in the inactivation of the scrapie agent. J Hosp Infect 57, 8084.[CrossRef][Medline]
Baldauf, E., Beekes, M. & Diringer, H. (1997). Evidence for an alternative direct route of access for the scrapie agent to the brain bypassing the spinal cord. J Gen Virol 78, 11871197.[Abstract]
Beekes, M., Baldauf, E., Caßens, S. & 7 other authors (1995). Western blot mapping of disease-specific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method. J Gen Virol 76, 25672576.[Abstract]
Beekes, M., Baldauf, E. & Diringer, H. (1996). Sequential appearance and accumulation of pathognomonic markers in the central nervous system of hamsters orally infected with scrapie. J Gen Virol 77, 19251934.[Abstract]
Beekes, M., Mielke, M., Pauli, G., Baier, M. & Kurth, R. (2004). Aspects of risk assessment and risk management of nosocomial transmission of classical and variant CreutzfeldtJakob disease with special attention to German regulations. In Prions. A Challenge for Science, Medicine and the Public Health System. Contributions to Microbiology, vol. 11, pp. 117135. Edited by H. F. Rabenau, J. Ciantl & H. W. Doerr. Basel: Karger.
Brown, P., Gibbs, C. J., Jr, Amyx, H. L., Kingsbury, D. T., Rohwer, R. G., Sulima, M. P. & Gajdusek, D. C. (1982). Chemical disinfection of CreutzfeldtJakob disease. N Engl J Med 306, 12791282.[Medline]
Brown, P., Rohwer, R. G. & Gajdusek, D. C. (1986). Newer data on the inactivation of scrapie virus or CreutzfeldtJakob disease virus in brain tissue. J Infect Dis 153, 11451148.[Medline]
Brown, P., Preece, M., Brandel, J. P. & 12 other authors (2000). Iatrogenic CreutzfeldtJakob disease at the millennium. Neurology 55, 10751081.
Brown, P., Cervenakova, L. & Diringer, H. (2001). Blood infectivity and the prospects for a diagnostic screening test in CreutzfeldtJakob disease. J Lab Clin Med 137, 513.[CrossRef][Medline]
Bruce, M. E., Will, R. G., Ironside, J. W. & 10 other authors (1997). Transmissions to mice indicate that new variant CJD is caused by the BSE agent. Nature 389, 498501.[CrossRef][Medline]
Bruce, M. E., McConnell, I., Will, R. G. & Ironside, J. W. (2001). Detection of variant CreutzfeldtJakob disease infectivity in extraneural tissues. Lancet 358, 208209.[CrossRef][Medline]
Cousens, S. N., Linsell, L., Smith, P. G., Chandrakumar, M., Wilesmith, J. W., Knight, R. S., Zeidler, M., Stewart, G. & Will, R. G. (1999). Geographical distribution of variant CJD in the UK (excluding Northern Ireland). Lancet 353, 1821.[CrossRef][Medline]
Ernst, D. R. & Race, R. E. (1993). Comparative analysis of scrapie agent inactivation methods. J Virol Methods 41, 193202.[CrossRef][Medline]
Flechsig, E., Hegyi, I., Enari, M., Schwarz, P., Collinge, J. & Weissmann, C. (2001). Transmission of scrapie by steel-surface-bound prions. Mol Med 7, 679684.[Medline]
Glatzel, M., Abela, E., Maissen, M. & Aguzzi, A. (2003). Extraneural pathologic prion protein in sporadic CreutzfeldtJakob disease. N Engl J Med 349, 18121820.
Haik, S., Faucheux, B. A., Sazdovitch, V., Privat, N., Kemeny, J. L., Perret-Liaudet, A. & Hauw, J. J. (2003). The sympathetic nervous system is involved in variant CreutzfeldtJakob disease. Nat Med 9, 11211123.[CrossRef][Medline]
Hill, A. F., Desbruslais, M., Joiner, S., Sidle, K. C., Gowland, I., Collinge, J., Doey, L. J. & Lantos, P. (1997). The same prion strain causes vCJD and BSE. Nature 389, 448450.[CrossRef][Medline]
Hilton, D. A., Fathers, E., Edwards, P., Ironside, J. W. & Zajicek, J. (1998). Prion immunoreactivity in appendix before clinical onset of variant CreutzfeldtJakob disease. Lancet 352, 703704.[CrossRef][Medline]
Hilton, D. A., Ghani, A. C., Conyers, L., Edwards, P., McCardle, L., Penney, M., Ritchie, D. & Ironside, J. W. (2002). Accumulation of prion protein in tonsil and appendix: review of tissue samples. BMJ 325, 633634.
Hörnlimann, B., Pauli, G., Harbarth, S., Widmer, H.-R. & Simon, D. (2001). Die Prävention von Prionkrankheiten im medizinischen Bereich. In Prionen und Prionkrankheiten, pp. 415422. Edited by B. Hörnlimann, D. Riesner & H. Kretzschmar. Berlin, New York: de Gruyter.
Ironside, J. W. (2000). Pathology of variant CreutzfeldtJakob disease. Arch Virol Suppl 16, 143151.[Medline]
Jendroska, K., Heinzel, F. P., Torchia, M., Stowring, L., Kretzschmar, H. A., Kon, A., Stern, A., Prusiner, S. B. & DeArmond, S. J. (1991). Proteinase-resistant prion protein accumulation in Syrian hamster brain correlates with regional pathology and scrapie infectivity. Neurology 41, 14821490.[Abstract]
Kascsak, R. J., Rubenstein, R., Merz, P. A., Tonna-DeMasi, M., Fersko, R., Carp, R. I., Wisniewski, H. M. & Diringer, H. (1987). Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins 7. J Virol 61, 36883693.[Medline]
Kimberlin, R. H., Walker, C. A., Millson, G. C., Taylor, D. M., Robertson, P. A., Tomlinson, A. H. & Dickinson, A. G. (1983). Disinfection studies with two strains of mouse-passaged scrapie agent. Guidelines for CreutzfeldtJakob and related agent. J Neurol Sci 59, 355369.[CrossRef][Medline]
Köhnlein, J., Schmidt, V., Staffeldt, J. & Werner, H.-P. (2004). Analysis of different test soilings for verification of cleaning performance. Hyg Med 29, 1319.
Korth, C., Kaneko, K. & Prusiner, S. B. (2000). Expression of unglycosylated mutated prion protein facilitates PrPSc formation in neuroblastoma cells infected with different prion strains. J Gen Virol 81, 25552563.
Llewelyn, C. A., Hewitt, P. E., Knight, R. S., Amar, K., Cousens, S., Mackenzie, J. & Will, R. G. (2004). Possible transmission of variant CreutzfeldtJakob disease by blood transfusion. Lancet 363, 411412.[CrossRef][Medline]
Manuelidis, L. (1997). Decontamination of CreutzfeldtJakob disease and other transmissible agents. J Neurovirol 3, 6265.[Medline]
McKinley, M. P., Bolton, D. C. & Prusiner, S. B. (1983). A protease-resistant protein is a structural component of the scrapie prion. Cell 35, 5762.[Medline]
McLeod, A. H., Murdoch, H., Dickinson, J. & 7 other authors (2004). Proteolytic inactivation of the bovine spongiform encephalopathy agent. Biochem Biophys Res Commun 317, 11651170.[CrossRef][Medline]
Prusiner, S. B. (1982). Novel proteinaceous infectious particles cause scrapie. Science 216, 136144.[Medline]
Prusiner, S. B. (1998). Prions. Proc Natl Acad Sci U S A 95, 1336313383.
Prusiner, S. B., Groth, D., Serban, A., Stahl, N. & Gabizon, R. (1993). Attempts to restore scrapie prion infectivity after exposure to protein denaturants. Proc Natl Acad Sci U S A 90, 27932797.[Abstract]
Ramasamy, I., Law, M., Collins, S. & Brooke, F. (2003). Organ distribution of prion proteins in variant CreutzfeldtJakob disease. Lancet Infect Dis 3, 214222.[CrossRef][Medline]
Rutala, W. A. & Weber, D. J. (2001). CreutzfeldtJakob disease: recommendations for disinfection and sterilization. Clin Infect Dis 32, 13481356.[CrossRef][Medline]
Scott, M. R., Will, R., Ironside, J., Nguyen, H. O., Tremblay, P., DeArmond, S. J. & Prusiner, S. B. (1999). Compelling transgenetic evidence for transmission of bovine spongiform encephalopathy prions to humans. Proc Natl Acad Sci U S A 96, 1513715142.
Sehulster, L. M. (2004). Prion inactivation and medical instrument reprocessing: challenges facing healthcare facilities. Infect Control Hosp Epidemiol 25, 276279.[Medline]
Simon, D. & Pauli, G. (1998). Krankenversorgung und Instrumentensterilisation bei CJK-Patienten und CJK-Verdachtsfällen. Bundesgesundheitsblatt 7, 279285.
Taguchi, F., Tamai, Y., Uchida, K., Kitajima, H., Kojima, H., Kawaguchi, T., Ohtani, Y. & Miura, S. (1991). Proposal for a procedure for complete inactivation of the CreutzfeldtJakob disease agent. Arch Virol 119, 297301.[Medline]
Tateishi, J., Tashima, T. & Kitamoto, T. (1991). Practical methods for chemical inactivation of CreutzfeldtJakob disease pathogen. Microbiol Immunol 35, 163166.[Medline]
Taylor, D. M. (1991). Resistance of the ME7 scrapie agent to peracetic acid. Vet Microbiol 27, 1924.[CrossRef][Medline]
Taylor, D. M. (1999). Inactivation of prions by physical and chemical means. J Hosp Infect 43, S69S76.[CrossRef][Medline]
Taylor, D. M. (2000). Inactivation of transmissible degenerative encephalopathy agents: a review. Vet J 159, 1017.[CrossRef][Medline]
Taylor, D. M. (2003). Preventing accidental transmission of human transmissible spongifom encephalopathies. Br Med Bull 66, 293303.
Taylor, D. M. (2004). Resistance of transmissible spongiform encephalopathy agents to decontamination. Contrib Microbiol 11, 136145.[Medline]
Taylor, D. M., Fraser, H., McConnell, I., Brown, D. A., Brown, K. L., Lamza, K. A. & Smith, G. R. (1994). Decontamination studies with the agents of bovine spongiform encephalopathy and scrapie. Arch Virol 139, 313326.[Medline]
Taylor, D. M., Brown, J. M., Fernie, K. & McConnell, I. (1997). The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue. Vet Microbiol 58, 167174.[CrossRef][Medline]
Taylor, D. M., Fernie, K., McConnell, I. & Steele, P. J. (1999). Survival of scrapie agent after exposure to sodium dodecyl sulphate and heat. Vet Microbiol 67, 1316.[CrossRef][Medline]
Thomzig, A., Kratzel, C., Lenz, G., Krüger, D. & Beekes, M. (2003). Widespread PrPSc accumulation in muscles of hamsters orally infected with scrapie. EMBO Rep 4, 530533.
Thomzig, A., Schulz-Schaeffer, W., Kratzel, C., Mai, J. & Beekes, M. (2004). Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie. J Clin Invest 113, 14651472.
Wadsworth, J. D. F., Joiner, S., Hill, A. F., Campbell, T. A., Desbruslais, M., Luthert, P. J. & Collinge, J. (2001). Tissue distribution of protease resistant prion protein in variant CreutzfeldtJakob disease using a highly sensitive immunoblotting assay. Lancet 358, 171180.[CrossRef][Medline]
Wells, G. A., Scott, A. C., Johnson, C. T., Gunning, R. F., Hancock, R. D., Jeffrey, M., Dawson, M. & Bradley, R. (1987). A novel progressive spongiform encephalopathy in cattle. Vet Rec 31, 419420.
Will, R. G., Ironside, J. W., Zeidler, M. & 7 other authors (1996). A new variant of CreutzfeldtJakob disease in the UK. Lancet 347, 921925.[Medline]
World Health Organization (1999). WHO Infection Control Guidelines for Transmissible Spongiform Encephalopathies. Report of a WHO consultation, Geneva, Switzerland, 2326 March. WHO/CDS/CSR/APH/2000.3.
Yan, Z. X., Stitz, L., Heeg, P., Pfaff, E. & Roth, K. (2004). Infectivity of prion protein bound to stainless steel wires: a model for testing decontamination procedures for transmissible spongiform encephalopathies. Infect Control Hosp Epidemiol 25, 280283.[Medline]
Zobeley, E., Flechsig, E., Cozzio, A., Enari, M. & Weissmann, C. (1999). Infectivity of scrapie prions bound to a stainless steel surface. Mol Med 5, 240243.[Medline]
Received 5 June 2004;
accepted 21 August 2004.
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