A quantitative assessment of residual protein levels on dental instruments reprocessed by manual, ultrasonic and automated cleaning methods.

OBJECTIVE: To assess residual protein on dental instruments cleaned in general dental practice by manual, manual plus ultrasonic and automated washer disinfector (AWD) processes. DESIGN AND SETTING: Instruments submitted by 30 dental surgeries in the South West of England. SUBJECTS (MATERIALS) AND METHODS: Instruments analysed were matrix bands, associated retaining clips, diamond and stainless steel burs, extraction forceps and hand scalers. Each instrument was visually assessed under magnification for residual debris. Residual protein was extracted by immersion in detergent and sonication. A collection of used but uncleaned instruments of each type (n = 177) was also analysed for adherent protein using ophthalaldehyde/N-acetylcysteine reagent. MAIN OUTCOME MEASURES: Residual protein levels allowed comparisons to be made on the effectiveness of different cleaning processes. RESULTS: One thousand, three hundred and four instruments were analysed. Observational data demonstrated several shortcomings in cleaning chemistries and operation of the AWD. For uncleaned instruments, median residual protein levels ranged from 0.4 µg (stainless steel burs) to 462 µg (extraction forceps). Following manual washing, median protein levels ranged from 0.3-78 µg; for manual plus ultrasonic washing, levels ranged from 9-39 µg and AWD levels ranged from 0.3-27 µg. Manual washing combined with ultrasonic cleaning was significantly less effective than the other two processes (p <0.008). AWDs reduced the variability in the cleaning process. No correlation was found between visual scoring and residual protein determination. CONCLUSION(S): There was a wide variation in residual protein levels both within and between different methods and instruments and this underlines the complexity of this process.

Thermostable adenylate kinase technology: a new process indicator and its use as a validation tool for the reprocessing of surgical instruments.

Adenylate kinase (tAK), a thermostable enzyme, was assessed as a possible means of providing a quantitative measure of cleaning efficacy suitable for validating the performance of an automated washer disinfector (AWD) during routine use. Two indicator formulations were developed using either a commercially available washer disinfector soil or a protein-based soil. Each indicator consisted of 100 microg (in test soil) of tAK dried on to a steel or plastic surface. These indicators were placed in each basket of a washer disinfector and processed alongside soiled surgical instruments during a standard day's operation. After processing, remaining tAK activity was detected using a rapid enzyme assay (2 min detection time) in a handheld hygiene monitor. The amount of tAK remaining on each indictor after a full AWD cycle was found to range from 0.1 to 0.4 ng, which represented a mean log(10) removal of 5.8+/-0.3. There was no statistical difference in the residual tAK activity between individual runs or the position of the indicator in the machine. The tAK indicator was also used to analyse the protein removal within each component of the wash cycle. These results demonstrated that all phases of the wash process contributed to the removal of the protein load, with the main wash alone being responsible for 3.6-4.0 log(10) reductions in protein activity. We propose that a quantitative cleaning index using such rapid readout indicator devices would provide a valuable addition to the methodologies for validating cleaning processes.

Quantitative measurement of the efficacy of protein removal by cleaning formulations; comparative evaluation of prion-directed cleaning chemistries.

The stability of the infectious agent causing variant Creutzfeldt-Jakob disease (vCJD) has highlighted the importance of cleaning surgical instruments for controlling potential spread of iatrogenic CJD. In this study, thermostable adenylate kinases (tAKs) in test soil were coated on to stainless steel and these surrogate agents used to evaluate the efficacy of a range of cleaning chemistries in a bench-top washer disinfector (btWD), or as a pre-soak either with or without subsequent treatment by btWD. Two tAKs were tested initially for ease of removal, the most persistent being Sulfolobus acidocaldarius-derived tAK which was used for evaluating the cleaning chemistries. Conventional chemistries were generally more effective when used in a btWD than as pre-soaks. Cleaning efficacy improved when pre-soaks were followed by treatment with intermediate performing enzymes, demonstrating greater than additive effect on residual tAK activity. Three of the four prion-directed chemistries reduced residual tAK activity to below the limit of quantification (LoQ) by more than 4.8 log(10); <175pg tAK remaining as a pre-soak alone. A conventional alkaline cleaning product also reduced residual tAK activity to below the LoQ but only when used in a btWD. tAK soil dried on to the device was removed less efficiently than tAK soil still moist on the device, with a 320-fold and 28-fold increase in residual tAK activity for pre-soak and btWD, respectively. The study demonstrated the potential for a tAK indicator to describe the effectiveness of protein removal using different chemistries or treatment processes.

Decontamination of prion protein (BSE301V) using a genetically engineered protease.

A previous study has demonstrated the potential of alkaline proteases to inactivate bovine spongiform encephalopathy (BSE301V). Here we explored the use of MC3, a genetically engineered variant of Bacillus lentus subtilisin. MC3 was used to digest BSE301V infectious mouse brain homogenate (iMBH). MC3 eliminated all detectable 6H4-immunoreactive material at pH 10 and 12; however, Proteinase K was only partially effective at pH 12. When bioassayed in VM mice, MC3- and Proteinase K-digested iMBH gave respectively 66.6% and 22.7% survival rates. Using a titration series for disease incubation, this equates to a >7log reduction in infectivity for MC3 and >6log reduction for Proteinase K. This study demonstrates the potential for thermostable proteases to be developed as effective inactivation processes for prion agents in healthcare management.

Implications for Creutzfeldt-Jakob disease (CJD) in dentistry: a review of current knowledge.

This review explores our current understanding of the risks of (variant) Creutzfeldt-Jakob disease transmission via dental practice, and whether they merit the rigorous enforcement of improved standards of instrument cleaning and decontamination. The recognition of prions as novel infectious agents in humans has caused significant concern among the public and medical professionals alike. Creutzfeldt-Jakob disease (CJD) in humans has been shown to be transmissible via several routes, including transplantation, contaminated medical products, and via neurosurgery. While the likelihood of transmission via dentistry is undoubtedly very low, this may be amplified considerably by unknown risk factors, such as disease prevalence (particularly in the UK), altered tissue distribution of vCJD, and the failure of decontamination processes to address the inactivation of prions adequately. Since current diagnostic techniques are unable to detect PrP(Sc) in human dental tissues, there is limited evidence for the presence of infectivity. Given these uncertainties, the control of risk by reinforced and improved decontamination practices seems the most appropriate response.

Cleanability of dental instruments--implications of residual protein and risks from Creutzfeldt-Jakob disease.

Cleaning of dental instruments is the first line of control in reducing the adherent bioburden. The threat of vCJD and the difficulty in removing prion protein has provided a new challenge for cleaning surgical and dental instruments. Prion proteins are also more resistant to many disinfection and sterilisation techniques. A number of different methods are currently available in primary care for cleaning instruments including manual washing, ultrasonic cleaners and washer disinfectors. Manual cleaning of dental instruments is time-consuming, introduces operator error and the risk of puncture wounds, is not reproducible and does not completely remove debris from instruments. Ultrasonic baths are significantly more effective than hand cleaning alone and are currently used by the majority of dental surgeries (often as an adjunct to manual cleaning). Automated washer-disinfectors appear to provide a validated, reliable and reproducible procedure for disinfection and sterilisation of dental instruments to ensure both the safety of patients and dental staff. Dental instruments that are difficult to clean are frequently contaminated with tissue debris after routine reprocessing and cannot be excluded as a potential transmission risk for infectious agents, including prions. The transmission of vCJD via dentistry is considered to be low risk, however, the Department of Health (DoH) has recently advised dentists to ensure that endodontic reamers and files are treated as single-use as a precautionary basis in order to further reduce any risk of vCJD transmission.

Surface decontamination of surgical instruments: an ongoing dilemma.

The issues of cross-infection and the survival of variant Creutzfeldt Jakob disease (vCJD) on surgical instruments have highlighted the importance of cleanliness of multiple-use surgical instruments. The aim of this study was to assess the levels of total protein contamination on a wide range of surgical instruments as an indication of the effectiveness of routine cleaning and disinfection in hospitals. Anonymized trays of wrapped and autoclaved instruments were supplied to two laboratories for analysis at the stage where they would normally be returned to operating theatres. Instruments were assessed for residual protein and total organic matter. Laboratory A showed that 17% (35/206) of instruments were above a threshold that equated to 200 microg. The worst examples, a McIvor gag, a Draffin rod (child) and a Yankaur sucker, had 1.028, 1.286 and 2.228 mg of extractable protein, respectively. The median (25th, 75th percentiles) amount of protein from instruments from different hospitals assessed in Laboratory B ranged from 8 (3, 30)mug (Hospital C) to 91 (35, 213) mug (Hospital D) (P=0.044). The residual matter washed from instruments varied from 0.62 (0.32, 0.81) mg (Hospital E) to 3.5 (3.5, 4.0) mg (Hospital A) (P=0.0001). In one case, 45 mg of residual organic matter was washed from an instrument (split stem). In conclusion, this study demonstrated that a proportion of instruments at the point of use show levels of protein that could pose a direct cross-infection risk via prion agents and other organic contamination that may reduce the effectiveness of cleaning/disinfection strategies targeted against either prions or traditional infectious agents.