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
Introduction
In 1999, a ‘snapshot’ survey of the decontamination services within the National Health Service (NHS) found that 109 of the 249 (44%) hospital sterile service departments (SSDs) in England did not meet acceptable decontamination standards.1 Following this sample study, a comprehensive survey of the NHS in England was commenced in October 2000 and a health service circular was issued.2 As a consequence of these reports, the UK Department of Health issued revised guidelines on the decontamination of instruments, and the British Government announced a £200 million investment programme to modernize all NHS decontamination/sterilization facilities.3, 4, 5
Over 6.5 million operations are performed each year in England alone.6 These procedures produce approximately 9.2 million surgical trays that require decontamination.7 With an average of 12 instruments/set, this means that approximately 110 million instruments require decontamination each year, or in real terms, two million instruments/week spread over the 249 hospitals with SSDs.8 Therefore, the average SSD, which handles approximately 50 000 trays/year, processes in excess of 1500 instruments/day. When surgical instruments have been taken into an SSD, either after surgery or new, they are cleaned and disinfected in a mechanical washer-disinfector with the application of an enzymatic or alkaline detergent, which may include sonication. Once dry, the instruments tend to be inspected by visual methods and passed for any residual soiling or mechanical failure before being packaged and sent for sterilization. It has been recommended that visual inspections should be performed daily in order to verify the efficacy of cleaning.9
Although it has been reported that haem pigment levels of 10 μg/cm2 can be detected, it has also been reported that bodily fluids without pigments are difficult to visualize, even in large quantities.9, 10 This is an important parameter because, for example, human cerebral spinal fluid is both colourless and odourless but has been shown to be a carrier of infection of prion diseases; a group of neurodegenerative and invariably fatal conditions that include variant Creutzfeldt-Jakob disease.11
As such, the need for sensitive assessment measures that ensure the highest standards of cleanliness are maintained is paramount. European guidelines ISO EN15883 and the British Health Technical Memorandum (HTM) 2030 provide recommendations for the assessment of instrument cleanliness. Two of the techniques outlined in these documents are based on the Biuret and Ninhydrin chemical reactions, requiring prior swabbing of instrument surfaces before chromogenic detection. The Biuret technique employs the reaction of copper ions to form a complex with peptide bonds under alkaline conditions; i.e. in the presence of sodium hydroxide, such ions are reduced from Cu2+ to Cu+. The addition of bicinchoninic acid (BCA) causes these copper ions to react and form a purple complex that can be readily visualized.
The Biuret test kit (Pro-tect M, Biotrace, Bridgend, UK) applied within this investigation consists of a swab and a tube containing both BCA and a copper sulphate solution separated by a thin film. After the test region has been swabbed, the swab is placed into the tube firmly, breaking the film thus allowing the two chemicals to mix and the Biuret reaction to start. The test requires incubation at 37 °C for 45 min to achieve the highest sensitivity.12
Application of the Ninhydrin reagent in the detection of latent fingerprints and the assessment of surface contamination is well documented.13, 14, 15 The methodology is based on the reaction of amino acids, peptides and proteins with 1,2,3-indantrione monohydrate. Classical Ninhydrin testing involves swabbing of the test surface followed by application of the reagent to the swab before incubation for 30 min at 110 °C in a dry oven.16 The Ninhydrin kit (Albert Browne Ltd, Leicester, UK) tested in this investigation utilizes Ninhydrin gel, which has fewer complications than the classical reagent and requires incubation at a lower temperature of 57 °C for approximately 60 min.
The present study was carried out to investigate the ability of the ISO-EN-15883- and HTM-2030-approved colorimetric tests to detect low levels of prion-infected brain material on surgical stainless steel surfaces.
Section snippets
Tissue
Prion-infected brain material was obtained from female C57BL/6J mice that had been injected with 1 μL of 10% (w/v) ME7-infected brain homogenate into the dorsal hippocampal region of the brain, as described elsewhere.17 All animals were killed 19–21 weeks post inoculation. The tissue was frozen in liquid nitrogen, subsequent ME7 10% (w/v) brain homogenate was produced, and the protein concentrations were assessed by the application of a total protein assay (Bio-Rad, Hercules, USA).
Stainless steel tokens
The tokens (25
Results
The prion-infected brain homogenate controls were placed on to stainless steel tokens and examined under the EDIC/EF microscope. The photomicrographs obtained show that low levels of proteinaceous contamination can be observed readily on the tokens, down to a level below 0.4 μg (Figure 1).
The Ninhydrin protein assessment techniques were then applied to known samples with the results obtained from the test, indicating a minimum level of detection observed by 75% of volunteers (MLD75) sensitivity
Discussion
There are approximately 6.5 million surgical procedures performed within England each year.19 These procedures are spread across the 182 acute NHS trusts, which themselves cover the 249 hospitals with SSDs in England.20 The emergence of evidence that highly robust infectious agents such as the prion protein, a characteristic of variant and sporadic Creuztfeldt-Jakob disease, may remain viable following standard hospital decontaminating procedures led the Department of Health to issue revised
Acknowledgements
The authors would like to thank Dr N. Khammo for her assistance. This work was funded by the Department of Health (Contract DH 0070073). The views expressed are not necessarily those of the Department of Health.
References (27)
The decontamination of surgical instruments in the NHS in England – update report. ‘A step change’
(2005)- Department of Health. Decontamination of medical devices. Health Service Circular HSC 2000/032. London, UK: Department...
- Department of Health. Variant Creutzfeldt-Jakob disease (vCJD): minimising the risk of transmission. Health Service...
- Department of Health. Controls assurance in infection control: decontamination of medical devices. Health Service...
- The Parliamentary Office of Science and Technology. v-CJD in the future. London, UK: The United Kingdom Parliament;...
Hospital Episode Statistics England: financial year 2003–04
(2004)NHS decontamination project, background and expectations
(2003)Paper on individual instrument and surgical tray identification
(2004)Cleaning (part 2) – validation of cleaning efficacy
Zentral Steril
(2002)Verification of the performance of washer-disinfectors used for thermal disinfection
Zentral Steril
(2003)
Transmissible spongiform encephalopathy agents: safe working and the prevention of infection
The development of novel ninhydrin analogues
Chem Soc Rev
Cited by (26)
Colloidal gel as an efficient process to treat Chemical, Biological, Radiological (CBR) and prion contaminated solid surfaces
2021, Chemical Engineering ScienceCitation Excerpt :Adding hypochlorite ions and sodium hydroxide to the decontaminating solution yields an innovative CBR gel that neutralizes both biological and chemical contaminations (Ludwig et al., 2014a,b). As a vacuumable gel, it should also be effective against radiological contaminations and in this study, we also investigated its decontamination efficiency against prions, which are problematic in healthcare and veterinary settings because of their transmissibility and wide spectrum of resistance (Georgsson et al., 2006; McDonnell et al., 2013; Lipscomb et al., 2007a; Lipscomb et al., 2007b; Lipscomb et al., 2006; Lahey et al., 2017; Bruna et al., 2020; Giles et al., 2008; Somerville and Gentles, 2011). After characterizing the physicochemical properties of the CBR gel, we tested the decontamination efficiency of fresh and aged gel samples, to evaluate its efficiency over time against a radiological agent (137Cs), the chemical agents soman, sulfur mustard, and VX, spores of Bacillus thuringiensis (Bt) and of Bacillus anthracis (Ba) as biological agents, and hamster prion protein (PrP) as a model disease-associated prion contamination.
Efficacy of current and novel cleaning technologies (ProReveal) for assessing protein contamination on surgical instruments
2019, Decontamination in Hospitals and HealthcareAn overview of the Choice Framework for local Policy and Procedures (CFPP) for decontamination of surgical instruments
2013, Decontamination in Hospitals and HealthcareEfficacy of current and novel cleaning technologies (ProReveal) for assessing protein contamination on surgical instruments
2013, Decontamination in Hospitals and Healthcare