key: cord-0968736-5swwc6kr
authors: Secker, Thomas.J.; Leighton, Timothy.G.; Offin, Douglas.G.; Birkin, Peter.R.; Hervé, Rodolphe.C.; Keevil, Charles.W.
title: Journal of Hospital Infection A cold water, ultrasonic activated stream efficiently removes proteins and prion-associated amyloid from surgical stainless steel
date: 2020-09-19
journal: J Hosp Infect
DOI: 10.1016/j.jhin.2020.09.021
sha: 8a3e44aadc27af8e3759262c6873156fbae1b21b
doc_id: 968736
cord_uid: 5swwc6kr

BACKGROUND: Sterile Service Department decontamination procedures for surgical instruments struggle to demonstrate efficient removal of the hardiest infectious contaminants, such as prion proteins. A recently designed novel system, which utilises a low pressure ultrasonic activated, cold water stream, has previously demonstrated efficient hard surface cleaning of several biological contaminants. AIM: To test the efficacy of an ultrasonically activated stream for the removal of tissue proteins, including prion-associated amyloid, from surgical stainless steel (SS) surfaces. METHODS: Test surfaces were contaminated with 22L, ME7 or 263K prion infected brain homogenates. The surfaces were treated with the ultrasonically activated water stream for contact times of 5 and 10 seconds. Residual proteinaceous and amyloid contamination were quantified using sensitive microscopic analysis, and immunoblotting was used to characterize the eluted prion residues before and after treatment with the ultrasonically activated stream. FINDINGS: Efficient removal of the different prion strains from the surgical SS surfaces was observed, and reduced levels of protease sensitive and resistant prion protein was detected in recovered supernatant. CONCLUSIONS: This study demonstrated that an ultrasonically activated stream has the potential to be a cost-effective solution to improve current decontamination practices and has the potential to reduce hospital acquired infections.

instruments struggle to demonstrate efficient removal of the hardiest infectious contaminants, 23 such as prion proteins. A recently designed novel system, which utilises a low pressure 24 ultrasonic activated, cold water stream, has previously demonstrated efficient hard surface 25 cleaning of several biological contaminants. 26

Aim: To test the efficacy of an ultrasonically activated stream for the removal of tissue 27 proteins, including prion-associated amyloid, from surgical stainless steel (SS) surfaces. 28

Methods: Test surfaces were contaminated with 22L, ME7 or 263K prion infected brain 29

homogenates. The surfaces were treated with the ultrasonically activated water stream for 30 contact times of 5 and 10 seconds. Residual proteinaceous and amyloid contamination were 31 quantified using sensitive microscopic analysis, and immunoblotting was used to characterize 32 the eluted prion residues before and after treatment with the ultrasonically activated stream. At present, the reprocessing of surgical instruments utilises; a pre-wash, washer/disinfector 44 cycle (run at elevated temperature with detergents) and sterilisation in high heat/pressure 45 autoclaves 1 . Decontamination protocols for reusable surgical instruments are very efficient 46 against microbiological contaminants. However, highly hydrophobic proteins such as prions, 47 responsible for the transmission of variant Creutzfeldt-Jakob disease (vCJD), are readily 48 adsorbed to surgical stainless-steel surfaces and poorly removed or inactivated by current 49 decontamination methods. This results in an impending risk of iatrogenic transmission of 50 vCJD 2-5 . A risk that has been experimentally demonstrated in both animal and cell-based 51 bioassays [6] [7] [8] [9] . 52

The latest estimated prevalence of asymptomatic carriers of the causative protein of vCJD 53 (PrP Sc ) in the UK is approximately 1/2000 10 . While the full impact of the genetic 54 susceptibility of the host remains unclear, the ostensibly long incubation periods and the 55 potential for disease transmission via infected blood [11] [12] [13] , imply that all surgical procedures 56 pose a risk of vCJD transmission. 57 Improvements in the methodologies used for reprocessing surgical instruments, potentially 58 contaminated with prions, are required to diminish the risk of iatrogenic vCJD transmission. 59

Novel, specialised prion decontamination protocols have been developed and in some cases 60 marketed for Sterile Service Departments (SSDs) 7, 14-22 . However, some of these protocols 61 are very aggressive and can be damaging to instrument surfaces and/or the 62 washer/disinfectors themselves 14 . Simple methods to adopt into SSD's have been researched 63 and demonstrated improved efficiency over current practices, such as preventing instruments 64 from drying once contaminated, i.e. keeping them in a moist environment prior to cleaning 23-65 27 . 66 J o u r n a l P r e -p r o o f Bjerknes forces 43 aid the scrubbing bubbles in efficiently removing contaminants from 92 microscopic crevices, such as those found on worn surgical instruments 44 , that are 93 traditionally difficult to clean by brushes, wiping, or by chemical means that rely on passive 94 diffusion for reagents to penetrate deep into the crevice 45 . The efficient removal of 95 contamination from crevices using a UAS system has been demonstrated previously 40 . 96 Furthermore, the microstreaming that radiates from the resonating bubbles can penetrate into 97 crevices present on the surfaces of the contaminant as shown in the insert in Figure 1 46 . The 98 fact that such results can be obtained in cold water without chemical additives warrants 99 investigation of UAS for the removal of infectious prion proteins from surgical surfaces. 100

High temperature decontamination using aggressive enzymatic or alkaline solutions, that are 101 currently adopted to clean expensive surgical items (such as intricate neurosurgical tools) are 102 ineffective at protein and prion removal, and can shorten the surgical item lifetime 47 . It is not 103 the purpose of this study to explore the replacement of such standard cleaning practices. 104 However, given the above properties of UAS, it is important to explore the possible benefits 105 of including an innovative cold-water UAS pre-wash (at the stage where SSDs conduct hand 106 brushing of instruments under a stream of water) that can be introduced with minimal 107 operator training. This would be particularly beneficial if it could be conducted immediately 108 after instrument use (e.g. before contaminated tissue dries on the instrument and becomes 109 harder to remove), although in this trial the contaminant is tested in a dried-on state. The 110 question is whether such a UAS pre-wash could remove a substantial proportion of the 111 contaminant, especially from microscopic crevices of the type associated with worn surgical 112 instrument surfaces, and break up aggregates in which the inner portion of biological 113 contaminant is partially protected from subsequent enzymatic cleaning chemistries. 114

A previous study demonstrated efficient tissue protein removal from surgical stainless steel 115 using the UAS 39 . However, due to the globular nature of the predominantly β-sheet 116 structured infectious prion protein, it adheres to surgical stainless steel far more rigorously 117 than do normal brain tissue proteins, and therefore the ability of UAS to remove brain tissue 118 protein cannot be taken as an indicator of any efficacy in reducing the iatrogenic transmission 119 risk of vCJD. Therefore, this study involved the contamination of surgical stainless-steel 120 surfaces with several amyloid-rich brain homogenates from prion infected rodents. Normal 121 tissue proteins and more hazardous prion-associated amyloid were differentially stained and 122 analysed using sensitive in situ microscopy, to compare the ability of UAS to remove both 123 during the same cleaning operation. inoculating, tokens were decontaminated and analysed to be deemed free of any 131 contamination following a previously described protocol 49 . 132

Murine scrapie ME7-infected brain homogenate produced from C57BL mice (TSE Resource 134

Centre, Roslin Institute, University of Edinburgh, Scotland, UK); murine scrapie 22L-135 infected brain homogenate produced from C57BL/6J mice (kindly donated from the 136 Neuroscience Department, School of Biological Sciences, University of Southampton) and 137

Syrian hamster scrapie 263K-infected brain homogenates (TSE Resource Centre, Roslin 138

Institute, University of Edinburgh, Scotland, UK) were standardized to 1 mg/ml (BSA 139 equivalent) in phosphate buffered saline (PBS, Gibco) with 0.1 % (v/v) Tween 20 (Sigma-140 Aldrich) as previously described 50 . 141

Pristine tokens were spiked with 1 µl (1 µg BSA equivalent) drops of 22L, ME7 or 263K 143 infected brain homogenate, and dried at 37 o C for 2 hours or room temperature for 24 hours. 144

Tokens were subjected to decontamination using a prototype recirculating UAS device (the 145 Mark I StarStream ® system (F0030001)) using fresh dH 2 O for each sample, running at 2.32 ± 146 0.02 L/min at room temperature with the ultrasound on for 5 and 10 s contact times, with the 147 sample being 10 mm from the nozzle ( Figure 1 ). Once processed the tokens were dried at 148 37 o C for 1 hour prior to staining and analysis. 149

Residual tissue protein and prion-associated amyloid on the control and processed surfaces 151 was quantified, in situ, using the total protein blot stain SYPRO Ruby (SR; Invitrogen, UK) 152 and the amyloid specific stain Thioflavin T (ThT [0.2% (w/v) in 0.01M HCl]; Sigma-153 Aldrich), as described elsewhere 50, 51 . Fluorescent signal was visualised using Episcopic 154 Differential Interference Contrast (EDIC) microscopy coupled with Epifluorescence (EF -155 Best Scientific, Wroughton, UK) 50, 52 . Full X/Y scans of the contaminated areas were 156 acquired at x100 magnification showing the SYPRO Ruby (excitation: 470nm; emission: 157 618nm) and ThT (0.2% (w/v) in 0.01M HCl Sigma-Aldrich) signals. The captured images 158 were analysed using ImageJ software (National Institutes of Health). 159

To analyse the effects of the UAS treatment on infectious prion proteins, immunoblot 161 analysis was used to determine the presence of PrP c and proteinase K (PK) resistant PrP Sc in 162 both 22L-spiked distilled water, as an untreated control, and the effluent taken from the UAS system post cleaning of 22L-spiked stainless-steel tokens. Controls were prepared by spiking 164 1 L of sterile distilled water with 15 µg of 22L-infected brain homogenate. UAS positive 165 samples were prepared from capturing the 1 L UAS effluent post cleaning of 15 surgical 166 stainless-steel tokens contaminated with 1 µg 22L-infected brain homogenates each (dried for 167 24 hours at room temperature) as described above. The control and effluent solutions were 168 filtered through nitrocellulose membranes to capture the suspended protein aggregates. 169 After 24 h drying at room temperature the 22L brain homogenate, again demonstrated the 203 highest affinity for the stainless steel with the highest attachment of protein and prion-204 associated amyloid observed. When compared with 2 hours drying, the 263K-contaminated 205 homogenate resulted in higher protein attachment after 24 h drying and the ME7 infected 206 brain homogenates demonstrated similar protein attachment but higher prion-associated 207 amyloid attachment (Figure 3 ). The removal of 22L and ME7 tissues was slightly more 208 difficult using a 5 s UAS treatment with 91 and 90 % protein and 97 and 99 % amyloid 209 removal, respectively (Figure 3 ). After 10 s UAS treatment the removal was improved with 210 98 and 99 % protein and 99 -100 % amyloid removal, respectively. The 263K was harder to 211 remove after 24 hours drying with only 56 % protein and 90 % amyloid removal after the 5 s 212 UAS treatment, however, after the 10 s UAS treatment the cleaning was improved with 74 % 213 protein and 87 % amyloid removal (Figure 3 ). The percentage of amyloid within the total 214 residual contamination was again very low with 4 -8 % amyloid remaining for all the 215 samples after 10 s UAS contact time (Figure 3) . 216

The effluent from the UAS system after decontaminating the 22L spiked surfaces was filtered 218 and labelled for residual prion protein (both non-resistant and PK-resistant) and compared to 219 control samples of distilled water spiked with the equivalent amount of 22L brain 220 homogenate. A clear reduction of both the PK-sensitive and PK-resistant prion protein from 221 the tokens was observed (as demonstrated by the protein capture on nitrocellulose membranes 222 following the previously demonstrated 98 -99 % protein and 99 -100 % amyloid removal, 223 described above) after 10 s UAS treatment (Figure 4) . The reduction in immuno-labelled 224 prion proteins post UAS treatment could be demonstrating that the UAS treatment is 225 destructive to the antibody specific epitopes of the prion protein, therefore reducing the 226 immunochemical detection post UAS treatment. Furthermore, small protein aggregates could 227 be observed in the control samples but not in the samples post UAS treatment, suggesting that 228 the UAS may degrade and/or solubilize these aggregates. 229 230

Current practices for the decontamination and sterilisation of surgical instruments within 232

SSDs are not entirely efficient at removing all potentially infectious material, especially, 233 hardy prion proteins. Therefore, surgical instruments which may have come in contact with 234 CJD-infected tissues cannot be deemed safe post cleaning 3, 7, 16, 53 and are subsequently 235 quarantined. Simple, cost effective methods to prevent the initial attachment of bioburden to 236 surgical surfaces have been demonstrated [25] [26] [27] . Ultrasonic baths provide efficient cleaning 237 using water alone, however; the limitations associated with water baths was described earlier 238 in this manuscript. This study has tested the efficacy of UAS technology for the removal of 239 total protein and prion-amyloid from stainless steel, which is considered the most difficult 240 contaminant to decontaminate in the surgical field. 241

The UAS technology demonstrated significant removal of the three prion strains tested after 242 differing drying and UAS treatment times; however, increased UAS treatment times are 243 required to further improve the efficacy of the UAS treatment. The efficient removal of ME7 244 and 22L, both murine adapted scrapie strains, was very similar following both drying and 245 UAS treatment times. However, 263K, a hamster adapted scrapie strain, was harder to 246 remove and would require a longer UAS treatment to reduce to the levels observed with the 247 two murine strains. This observation suggests that the hamster brain constituents and PrP Sc 248 conformation is different to the mouse brains and showed increased affinity to stainless steel. 249

This highlights the importance of studying different prion strains, from different hosts when 250 determining the efficacy of hospital decontamination tools. For comparison of the efficacy of 251 the UAS system to that of cleaning chemistries used in SSDs, the removal of ME7-infected 252 brain homogenate from stainless steel tokens using the same methodology as this study have 253 been previously published 3, 25 . Hervé et al (2010) , tested four different cleaning chemistries 254 marketed for proteinaceous decontamination which demonstrated total protein removals of 255 39%, 97.9%, 98.9% and 99.85%, respectively 3 . Secker et al (2012) , tested two cleaning 256 chemistries, also marketed for proteinaceous decontamination, which demonstrated total 257 protein removal of 0% and 90.1%, respectively 25 . All the cleaning chemistries tested in these 258 studies required heating of the cleaning solution, whereas the UAS system tested here 259 removed 97% total protein with cold water and only a 10s contact time. A recent NIHR 260

Health Technology Assessment (HTA) has extensively compared studies quantifying the 261 efficacy of interventions to reduce the surgical transmission of vCJD 54 . The other important 262 observation was that the UAS system favourably removed the prion-associated amyloid 263 (infectious prion proteins in the aggregated form) from the surfaces. Demonstrated by the low 264 percentages of the total residual proteinaceous contamination being ThT positive amyloid, 265 compared to the comparative treatment using commercially available cleaning chemistries 3, 266 25 . 267

Immunoblot analysis of both PK-sensitive and -resistant residues of PrP was carried out to 268 determine the presence and state of prion aggregates post UAS decontamination. Following 269 the predetermined 98 -99 % protein and 99 -100 % prion-amyloid removal, described 270 above, the supernatant from the UAS treatment was filtered and the prion proteins were 271 labelled. The PK resistant and sensitive aggregates observed in the control immunoblots were 272 not present in the UAS treated samples; suggesting that the UAS mechanism of action is 273 causing the breakdown of the PrP aggregates, reducing the available epitopes for antibody 274 binding, and therefore a reduction in antibody positive PrP residues. Furthermore, this would 275 explain why an increase in the removal of prion-amyloid using the UAS system was 276 observed, as described earlier. Further work is required to confirm and determine if the 277 breakdown of PrP caused by the action of UAS correlates with a reduction in prion 278

The results from this study demonstrated efficient removal of tissue proteins, and more 280 importantly prion-associated amyloid from surgical stainless-steel harnessing the power of 281 water at ambient temperature. While the cleaning efficacy demonstrated by this system is 282 improved compared to that of the best currently available cleaning chemistries tested on the 283 same contaminants, interestingly the UAS appeared more effective at removing prion-284 amyloid as well as the total proteinaceous contamination. 285

This study has demonstrated the efficacious ability of the UAS to clean with just cold water. 286

However, the UAS system could work also with chemical cleaners, so that you can get a 287 synergistic effect of mechanical (acoustically activated bubbles) and chemical cleaning. 288

Furthermore, previous studies have demonstrated that the UAS efficiently removes microbial 289 contamination from rough, etched surfaces 40 . Thus demonstrating that a UAS has the ability 290 to clean items, such as surgical instruments, that contain dynamic differences in surface 291 topography 40 . In its current form, the UAS system is designed as a hand-held device, and the 292 plan is to include this in a pilot to test as a pre-clean before the surgical instruments proceed 293 on to washer-disinfectors (i.e. at the stage where currently SSDs conduct washing by hand, 294 brushing and pre-cleaning of surgical instruments). The mechanical removal by UAS of 295 prion-associated amyloid embedded in dried-on brain homogenate, demonstrates an 296 interesting parallel with the problem of removing the SARS-CoV-2 virus responsible for the 297 current COVID-19 from touch-surfaces. Lacking an appropriate attachment mechanism, the 298 virus relies on the stickiness of respiratory secretions in which it resides (that are composed 299 mainly of mucin glycoproteins, surfactant and intercellular fluid) to attach to abiotic surfaces. 300 Therefore, the efficient ability of the UAS system for removing prion-associated amyloid by 301 cleaning away the biological material, in the case of this study brain homogenate, as well as 302 bacteria and lubricant contamination, previously published 40, 46 , highlights the importance of 303 testing this system against viruses. If viruses can also be removed by UAS, then incorporation 304 of UAS in society to clean these surfaces with just water could aid infection prevention, 

The datasets generated and analysed during this study will be openly available from the 322 University of Southampton repository at http://dx.doi.org/10.5258/SOTON/[ Note to editors: 323

The policy of the University of Southampton is that they will grant a link for insertion once 324 the paper is accepted to avoid their repository referring to papers that were not published]. 325

TS carried out laboratory experimentation, data analysis interpretation, participated in the 327 design of the study and drafted the manuscript; TL conceived the study, coordinated across 328 disciplines, and helped draft the manuscript; DO and PB helped conceive the study and 329 provided support for the set-up and running of the UAS; RH helped with experimental design 330 and drafting the manuscript; CK oversaw the microbiological components and helped draft 331 the manuscript. All authors gave final approval for publication. 332

One of the authors (T.G.L.) is Director and Inventor-in-Chief of the company (Sloan Water 334 Technology, Ltd.) that holds the patent to this technology but has drawn no salary from this. 335 J o u r n a l P r e -p r o o f Tissue protein (Dark grey bars) and prion-associated amyloid (light grey bars) attachment 545 from different prion-infected brain homogenates (22L, ME7 and 263K) to surgical stainless 546 steel pre and post treatment with an ultrasonically activated stream (UAS) (Graph A). Brain 547 homogenate was initially dried for 2h at 37 o C prior to cleaning (Pos). The orange dashes 548 represent percentage protein removal and the blue dashes represent percentage prion-549 associated amyloid removal (graph A). Graph B has an expanded y-axis scale to distinguish 550 the lower levels of contamination. Data shows mean ± SEM (N=9), however, in 551 decontamination and other research areas 55 , outliers are also important to assessing outcomes, 552

whether it be risk of infection, or the response of the most sensitive individuals to some 553 stimulus; ***: p=≤0.001 for total proteins; † †: p=≤0.01 for amyloid, when compared to the 554 corresponding positive controls, respectively. 555 556 557

Tissue protein (Dark grey bars) and prion-associated amyloid (light grey bars) attachment 560 from different prion-infected brain homogenates (22L, ME7 and 263K) to surgical stainless 561 steel pre and post treatment (5 and 10s contact times) with an ultrasonically activated stream 562 (UAS) (Graph A). Brain homogenate was initially dried for 24h at room temperature prior to 563 cleaning (Pos). The orange dashes represent percentage protein removal and the blue dashes 564 represent percentage prion-associated amyloid removal (graph A). Graph B has an expanded 565 y-axis scale to highlight the lower levels of contamination. Data shows mean ± SEM (N=9); 566 however, in decontamination and other research areas 55 , outliers are also important to 567 assessing outcomes, whether it be risk of infection, or the response of the most sensitive 568 individuals to some stimulus; *: p=≤0.05 and ***: p=≤0.001 for total proteins; † †: p=≤0.01 569 and † † †: p=≤0.001 for amyloid, when compared to the corresponding positive controls, 570 respectively. 571 572

Immunoblot films showing captured proteins from 1 L of 22L-spiked solution containing 15 574 µg of 22L homogenate in distilled water (A and B) and from the UAS system effluent after 575 treating surfaces contaminated with the equivalent amount of 22L homogenate (C and D). 576

Proteins were detected using the primary antibody 6H4, without (A and C) or with PK 577 digestion (B and D). 578

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