Evaluation of cold atmospheric plasma for the decontamination of flexible endoscopes.

BACKGROUND: Despite adherence to standard protocols, residues including live micro-organisms may remain on the various surfaces of reprocessed flexible endoscopes. Prions are infectious proteins that are notoriously difficult to eliminate. AIM: To test the potential of cold atmospheric plasma (CAP) for the decontamination of various surfaces of flexible endoscopes, measuring total proteins and prion residual infectivity as indicators of efficacy. METHODS: New PTFE endoscope channels and metal test surfaces spiked with test soil or prion-infected tissues were treated using different CAP-generating prototypes. Surfaces were examined for the presence of residues using very sensitive fluorescence epimicroscopy. Prion residual infectivity was determined using the wire implant animal model and a more sensitive cell infectivity assay. FINDINGS: A CAP jet applied perpendicularly at close range on flat test surfaces removed soil within 3 min, but left microscopic residues and failed to eliminate prion infectivity according to the wire implant animal assay. The longitudinal gas flow from CAP prototypes developed for the treatment of long channels led to the displacement and sedimentation of residual soil towards the distal end, when applied alone. Observations of the plasma inside glass tubes showed temporal and spatial heterogeneity within a limited range. After the standard enzymatic manual pre-wash, 'CAP-activated' gas effluents prevented prion transmission from treated endoscope channels according to the prion infectivity cell assay. CONCLUSION: CAP shows promising results as a final step for decontamination of surgical surfaces. Optimizing CAP delivery could further enhance CAP efficacy, offering a safe, chemical-free alternative for the reprocessing of all luminal flexible endoscope surfaces.

Sensitive microscopic quantification of surface-bound prion infectivity for the assessment of surgical instrument decontamination procedures.

BACKGROUND: Pathogenic prions (PrPSc) are amyloid-rich hydrophobic proteins which bind avidly to surgical surfaces and represent some of the most difficult targets during the reprocessing of reusable surgical instruments. In-vitro methods to amplify and detect the presence of otherwise undetectable prion contamination are available, but they do not measure associated infectivity. Most of these methods rely on the use of proteinase K, however this can lead to the loss of a substantial portion of PrPSc, potentially producing false negatives. AIM: To develop a sensitive in-situ method without proteinase treatment for the dynamic quantification of amyloid accumulation in N2a #58 cells following 22L-prion infection from infected tissues and spiked stainless-steel surfaces. METHODS: We spiked cultures of N2a #58 cells with the 22L prion strain in solution or dried on stainless-steel wires and directly measured the accumulation of prion amyloid aggregates over several passages using highly sensitive fluorescence microscopy. FINDINGS: We demonstrated a 10-log dynamic range using our method to test residual prion infectivity, that was validated to show variable decontamination efficacy against prions from commercially available cleaning chemistries. CONCLUSIONS: The new cell-based infectivity method presented here avoids partial or possibly total proteinase K digestion of PrPSc in samples for greater sensitivity, in addition to low cost, no ethical concerns, and adaptability to detect different prion strains. This method can be used to test cleaning chemistries' efficacy with greater sensitivity than measuring total residual proteins, which may not correlate with residual prion infectivity.

A cold water, ultrasonically activated stream efficiently removes proteins and prion-associated amyloid from surgical stainless steel.

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 uses a low pressure ultrasonically 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 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 s. 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 stainless steel surfaces was observed, and reduced levels of protease-susceptible and -resistant prion protein was detected in recovered supernatant. CONCLUSION: 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.

Efficacy of humidity retention bags for the reduced adsorption and improved cleaning of tissue proteins including prion-associated amyloid to surgical stainless steel surfaces.

Increasing drying time adversely affects attachment of tissue proteins and prion-associated amyloid to surgical stainless steel, and reduces the efficacy of commercial cleaning chemistries. This study tested the efficacy of commercial humidity retention bags to reduce biofouling on surgical stainless steel and to improve subsequent cleaning. Surgical stainless steel surfaces were contaminated with ME7-infected brain homogenates and left to dry for 15 to 1,440 min either in air, in dry polythene bags or within humidity retention bags. Residual contamination pre/post cleaning was analysed using Thioflavin T/SYPRO Ruby dual staining and microscope analysis. An increase in biofouling was observed with increased drying time in air or in sealed dry bags. Humidity retention bags kept both protein and prion-associated amyloid minimal across the drying times both pre- and post-cleaning. Therefore, humidity bags demonstrate a cheap, easy to implement solution to improve surgical instrument reprocessing and to potentially reduce associated hospital acquired infections.

Cold water cleaning of brain proteins, biofilm and bone - harnessing an ultrasonically activated stream.

In the absence of sufficient cleaning of medical instruments, contamination and infection can result in serious consequences for the health sector and remains a significant unmet challenge. In this paper we describe a novel cleaning system reliant on cavitation action created in a free flowing fluid stream where ultrasonic transmission to a surface, through the stream, is achieved using careful design and control of the device architecture, sound field and the materials employed. Cleaning was achieved with purified water at room temperature, moderate fluid flow rates and without the need for chemical additives or the high power consumption associated with conventional strategies. This study illustrates the potential in harnessing an ultrasonically activated stream to remove biological contamination including brain tissue from surgical stainless steel substrates, S. epidermidis biofilms from glass, and fat/soft tissue matter from bone structures with considerable basic and clinical applications.

Doped diamond-like carbon coatings for surgical instruments reduce protein and prion-amyloid biofouling and improve subsequent cleaning.

Doped diamond-like carbon (DLC) coatings offer potential antifouling surfaces against microbial and protein attachment. In particular, stainless steel surgical instruments are subject to tissue protein and resilient prion protein attachment, making decontamination methods used in sterile service departments ineffective, potentially increasing the risk of iatrogenic Creutzfeldt-Jakob disease during surgical procedures. This study examined the adsorption of proteins and prion-associated amyloid to doped DLC surfaces and the efficacy of commercial cleaning chemistries applied to these spiked surfaces, compared to titanium nitride coating and stainless steel. Surfaces inoculated with ME7-infected brain homogenate were visualised using SYPRO Ruby/Thioflavin T staining and modified epi-fluorescence microscopy before and after cleaning. Reduced protein and prion amyloid contamination was observed on the modified surfaces and subsequent decontamination efficacy improved. This highlights the potential for a new generation of coatings for surgical instruments to reduce the risk of iatrogenic CJD infection.

Adsorption of prion and tissue proteins to surgical stainless steel surfaces and the efficacy of decontamination following dry and wet storage conditions.

Iatrogenic transmission of the infectious prion protein (PrP(Sc)) is a potential threat due to its resistance to many chemical and enzymatic decontamination protocols and its strong adhesive properties to stainless steel. The conditions in which surgical instruments are handled during and after surgery may affect the level of tissue protein, prion attachment and the efficacy of subsequent decontamination regimes. This study investigated the adhesion of tissue protein and prion-associated amyloid to surgical stainless steel with respect to time and various storage conditions, and the subsequent outcome on the efficacy of enzymatic cleaning chemistries. Surfaces were contaminated with ME7-infected brain homogenate and left to dry between 0 and 120 min at room temperature or 24 h, in dry or moist conditions. Residual contamination before and after cleaning was visualised using sensitive fluorescent staining and episcopic differential interference contrast/epifluorescence microscopy. Longer drying times increased both protein and prion amyloid adsorption and affected the efficacy of the cleaning chemistries tested. A moist environment post-contamination significantly reduced the attachment of both protein and prion amyloid to the surgical stainless steel surface. Maintaining moist conditions could potentially improve the subsequent decontamination of reusable surgical instruments, also reducing process time and cost.

Current risk of iatrogenic Creutzfeld-Jakob disease in the UK: efficacy of available cleaning chemistries and reusability of neurosurgical instruments.

The initial cleaning of reusable surgical devices is critical to ensure the efficacy of the subsequent sterilisation process. Transmissible spongiform encephalopathies (TSEs) are incurable and fatal neurodegenerative diseases apparently transmitted simply by the absorption or ingestion of self-aggregating protease-resistant prions (PrP(Sc)), which are very resilient to most standard cleaning chemistries and heat-based decontamination techniques. Therefore there is a risk of iatrogenic transmission from reusable surgical devices if these are allowed to retain potentially infectious material after standard reprocessing through sterile service departments (SSDs). We aimed to assess the current state of surgical instrument decontamination with the collaboration of anonymous SSDs. Surgical stainless steel surfaces were spiked with prion-infected brain homogenates, and episcopic differential interference contrast/epifluorescence (EDIC/EF) microscopy was applied to quantify the amount of residual prion amyloid and other proteins remaining after decontamination with enzymatic cleaners currently employed by SSDs. Reusable instruments deemed 'clean and ready to use' were also stained for comparison with our findings in the laboratory. All cleaning chemistries were only partially effective under the recommended conditions. More importantly, PrP(Sc) constituted the main fraction of the remaining contamination left on these surfaces. The neurosurgery instruments also harboured amyloid and general protein contamination. This study shows that currently marketed cleaning chemistries and recent decontamination protocols do not completely suppress the threat from iatrogenic CJD. These findings should be taken into account for risk assessment purposes and re-evaluating instrument handling and decontamination practices.