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

Summary

Regulations recommend the routine application of biochemical tests, such as the Ninhydrin or Biuret tests, to confirm the efficacy of hospital sterile service department (SSD) washer-disinfector cycles in removing proteinaceous material, particularly with respect to prions. The effectiveness of these methods relies on both the effective sampling of the instruments and the sensitivity of the tests employed. Two commercially available contamination assessment tests were evaluated for their sensitivity to ME7 brain homogenate on surgical-grade stainless steel surfaces. Controls were visualized by the application of episcopic differential interference contrast/Epi-fluorecence microscopy (EDIC/EF) combined with the sensitive fluorescent reagent, SYPRO Ruby, which has been shown previously to rapidly visualize and assess low levels of contamination on medical devices. The Ninhydrin test displayed a minimum level of detection observed by 75% of volunteers (MLD₇₅) of 9.25 μg [95% confidence interval (95% CI) 8.6–10.0 μg]. The Biuret test provided better sensitivity, with a MLD₇₅ of 6.7 μg (95% CI 5.4–8.2 μg). However, much lower concentrations of proteinaceous soiling (pg) were visualized using the EDIC/EF microscopy method. From these findings, it is clear that these approved colorimetric tests of cleaning are relatively insensitive. This investigation demonstrates how large amounts (up to 6.5 μg) of proteinaceous brain contamination could remain undetected and the instruments deemed clean using such methods. The application of more sensitive cleanliness evaluation methods should be applied to reduce the risk of iatrogenic transmission of prion disease in ‘high-risk’ instruments such as neurosurgical devices.

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.