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Effective cleaning and decontamination of the internal air and water channels, heads and head-gears of multiple contra-angle dental handpieces using an enzymatic detergent and automated washer-disinfection in a dental hospital setting

  • E.C. Deasy
    Affiliations
    University of Dublin Trinity College, Microbiology Research Unit, Division of Oral Biosciences, Dublin Dental University Hospital, Dublin, Ireland
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  • T.A. Scott
    Affiliations
    Central Sterile Services Department, Dublin Dental University Hospital, Dublin, Ireland
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  • J.S. Swan
    Affiliations
    Facilities Department, Dublin Dental University Hospital, Dublin, Ireland
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  • M.J. O'Donnell
    Affiliations
    University of Dublin Trinity College, Microbiology Research Unit, Division of Oral Biosciences, Dublin Dental University Hospital, Dublin, Ireland
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  • D.C. Coleman
    Correspondence
    Corresponding author. Address: Microbiology Research Unit, Division of Oral Biosciences, Dublin Dental University Hospital, University of Dublin Trinity College, Lincoln Place, Dublin D02 F859, Ireland. Tel.: +353 1 6127276; fax: + 353 1 6127295.
    Affiliations
    University of Dublin Trinity College, Microbiology Research Unit, Division of Oral Biosciences, Dublin Dental University Hospital, Dublin, Ireland
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Open AccessPublished:August 06, 2022DOI:https://doi.org/10.1016/j.jhin.2022.07.019

      Summary

      Background

      Dental handpieces (DHPs) are reusable invasive medical devices that must be cleaned, decontaminated, lubricated and steam sterilized after use. DHPs have a complex internal design including narrow channels, contamination of which can compromise sterilization. DHPs are not designed for routine disassembly, making cleaning/decontamination efficacy difficult to monitor. Washer-disinfection is the preferred method of decontaminating DHPs, but few studies have investigated its direct effectiveness at reducing microbial contamination internally.

      Aims

      To use contra-angle DHPs as a model system to investigate the effectiveness of washer-disinfection at reducing microbial contamination of internal components of multiple DHPs.

      Methods

      The air and water channels and heads of 10 disassembled contra-angle DHPs (BienAir, Biel/Bienne, Switzerland) were inoculated separately with 108 colony forming units (cfu) of Pseudomanas aeruginosa, Staphylococcus aureus, Enterococcus hirae or Candida albicans in the presence of 0.3% bovine serum albumin (BSA) (clean conditions), 3.0% BSA or 10% artificial test soil (dirty conditions). After reassembly, all 10 DHPs underwent washer-disinfection simultaneously in a Míele (Míele Ireland Ltd., Dublin, Ireland) PG8528 washer-disinfector and were tested for reductions in micro-organisms and protein. Additional experiments were undertaken with three lubricated DHPs inoculated with S. aureus and 10% test soil. All experiments were repeated in triplicate.

      Findings

      On average, an approximate 5 log or greater reduction in microbial cfu and a >93% reduction in protein from DHP heads and channels was consistently recorded following washer-disinfection for all DHPs under all conditions tested.

      Conclusions

      The internal components of multiple DHPs can be effectively cleaned and decontaminated by washer-disinfection.

      Keywords

      Introduction

      Dental handpieces (DHPs) are among the most frequently used instruments in dentistry. DHPs are reusable invasive medical devices and must be cleaned, decontaminated, lubricated and sterilized after use [

      Regulation (EU) 2017/745 of the European Parliament and of the Council of 5 April 2017 on medical devices, amending Directive 2001/83/EC, Regulation (EC) No 178/2002 and Regulation (EC) No 1223/2009 and repealing Council Directives 90/385/EEC and 93/42/EEC.

      ,
      ]. There are three basic types of DHPs including conventional or slow speed (contra-angle and straight), high speed turbine and surgical. DHPs are provided with compressed air and water supplies from the dental chair unit via a flexible hose. Compressed air is used to drive the air rotor of high-speed DHPs. In conventional DHPs, the movement of dental burs is mechanically transmitted through shafts and gears and is initiated by an electric or air powered motor. In conventional DHPs compressed air is used to cool the gears. Dental unit waterlines (DUWLs) provide water to cool and irrigate tooth surfaces as the heat generated during DHP use can harm dental pulp [
      • O'Donnell M.J.
      • Boyle M.A.
      • Russell R.J.
      • Coleman D.C.
      Management of dental unit waterline biofilms in the 21st century.
      ,
      • Coleman D.C.
      • O'Donnell M.J.
      • Shore A.C.
      • Russell R.J.
      Biofilm problems in dental unit water systems and its practical control.
      ].
      The internal components of DHPs are complex and consist of narrow water and air channels, the drive rotor, and the shafts and gears in slow-speed DHPs. DHPs become contaminated externally and internally during use [
      • Lewis D.L.
      • Boe R.K.
      Cross-infection risks associated with current procedures for using high-speed dental handpieces.
      ,
      • Chin J.R.
      • Miller C.H.
      • Palenik C.J.
      Internal contamination of air-driven low-speed handpieces and attached prophy angles.
      ,
      • Herd S.
      • Chin J.
      • Palenik C.J.
      • Ofner S.
      The in vivo contamination of air-driven low speed handpieces with prophylaxis angles.
      ,
      • Hu T.
      • Li G.
      • Zuo Y.
      • Zhou X.
      Risk of hepatitis B virus transmission via dental handpieces and evaluation of an anti-suction device for prevention of transmission.
      ,
      • Smith G.W.
      • Smith A.J.
      • Creanor S.
      • Hurrell D.
      • Bagg J.
      • Lappin D.F.
      Survey of the decontamination and maintenance of dental handpieces in general dental practice.
      ,
      • Smith G.
      • Smith A.
      Microbial contamination of used dental handpieces.
      ,
      • Smith A.
      • Smith G.
      • Lappin D.F.
      • Baxter H.C.
      • Jones A.
      • Baxter R.L.
      Dental handpiece contamination: a proteomics and surface analysis approach.
      ]. Contamination can originate from DUWLs, the compressed air supply and from the oral cavity [
      • O'Donnell M.J.
      • Boyle M.A.
      • Russell R.J.
      • Coleman D.C.
      Management of dental unit waterline biofilms in the 21st century.
      ,
      • Smith G.W.
      • Smith A.J.
      • Creanor S.
      • Hurrell D.
      • Bagg J.
      • Lappin D.F.
      Survey of the decontamination and maintenance of dental handpieces in general dental practice.
      ]. DUWLs are prone to contamination with microbial biofilm from micro-organisms in the supply water and from retraction of oral fluids into DHPs during use [
      • O'Donnell M.J.
      • Boyle M.A.
      • Russell R.J.
      • Coleman D.C.
      Management of dental unit waterline biofilms in the 21st century.
      ,
      • Coleman D.C.
      • O'Donnell M.J.
      • Shore A.C.
      • Russell R.J.
      Biofilm problems in dental unit water systems and its practical control.
      ]. Smith et al. demonstrated that the internal components of DHPs are frequently contaminated with human-derived proteins [
      • Smith A.
      • Smith G.
      • Lappin D.F.
      • Baxter H.C.
      • Jones A.
      • Baxter R.L.
      Dental handpiece contamination: a proteomics and surface analysis approach.
      ]. The external surfaces of handpieces get contaminated with oral fluids and tissue fragments during use, all of which harbour oral micro-organisms.
      Current guidelines stipulate that DHPs should be decontaminated and sterilized between patients by steam sterilization using a vacuum autoclave that has been commissioned appropriately and its various cycles validated independently [
      ]. The efficacy of steam sterilization can be compromised by organic material and therefore it is vital that DHPs are adequately cleaned prior to sterilization. The external surfaces of DHPs are commonly decontaminated by manually wiping with a cleaning solution followed by visual inspection [
      ,
      • Lewis D.L.
      • Boe R.K.
      Cross-infection risks associated with current procedures for using high-speed dental handpieces.
      ,
      • Smith G.W.
      • Smith A.J.
      • Creanor S.
      • Hurrell D.
      • Bagg J.
      • Lappin D.F.
      Survey of the decontamination and maintenance of dental handpieces in general dental practice.
      ]. The external surfaces can also be cleaned and thermally disinfected in a washer-disinfector [
      • Smith A.
      • Smith G.
      • Lappin D.F.
      • Baxter H.C.
      • Jones A.
      • Baxter R.L.
      Dental handpiece contamination: a proteomics and surface analysis approach.
      ,
      • Offner D.
      • Brisset L.
      • Musset A.M.
      Evaluation of the mechanical cleaning efficacy of dental handpieces.
      ,
      • Offner D.
      • Scholler J.
      • Musset A.M.
      Cleaning of dental handpieces and associated parameters: Internal and external cleaning, drying and rotation.
      ]. ISO-15883 details several methods for assessing the surface cleanliness of reusable medical devices following washer-disinfection; however, there is no specific procedure for evaluating the efficacy of washer-disinfection for the internal components of DHPs [
      International Organization for Standardization
      Washer-disinfectors — Part 1: general requirements, terms and definitions and tests 2006.
      ].
      Cleaning and decontaminating the internal components of DHPs is challenging because of their complex construction and because they are not designed for routine disassembly to ensure that internal components are free of contamination [
      • Smith A.
      • Smith G.
      • Lappin D.F.
      • Baxter H.C.
      • Jones A.
      • Baxter R.L.
      Dental handpiece contamination: a proteomics and surface analysis approach.
      ].
      Several manufacturers of devices developed to clean DHPs claim that their equipment can ensure adequate cleaning, however little independent direct evidence is available [
      • Offner D.
      • Brisset L.
      • Musset A.M.
      Evaluation of the mechanical cleaning efficacy of dental handpieces.
      ]. Spraying a cleaning solution into the channels and transmission components is one of the most widely used approaches to cleaning and disinfection of the internal elements of DHPs. Cleaning fluids often contain alcohols that denature proteins, which are very difficult to remove from metal surfaces [
      • Costa D.M.
      • Lopes L.K.O.
      • Hu H.
      • Tipple A.F.V.
      • Vickery K.
      Alcohol fixation of bacteria to surgical instruments increases cleaning difficulty and may contribute to sterilization inefficacy.
      ,
      • Pinto F.M.
      • Bruna C.Q.
      • Camargo T.C.
      • Marques M.
      • Silva C.B.
      • Sasagawa S.M.
      • et al.
      The practice of disinfection of high-speed handpieces with 70% w/v alcohol: an evaluation.
      ]. Furthermore, the process is very difficult to validate because of the inaccessibility of the internal components of DHPs. One study demonstrated that the use of 70% alcohol to disinfect the external surface of high-speed DHPs was ineffective [
      • Pinto F.M.
      • Bruna C.Q.
      • Camargo T.C.
      • Marques M.
      • Silva C.B.
      • Sasagawa S.M.
      • et al.
      The practice of disinfection of high-speed handpieces with 70% w/v alcohol: an evaluation.
      ]. DHPs are not suitable for immersion in disinfectants, which can lead to metal corrosion [
      • Offner D.
      • Brisset L.
      • Musset A.M.
      Evaluation of the mechanical cleaning efficacy of dental handpieces.
      ].
      Washer-disinfection is a reproducible process that can be validated for the external components of medical devices and is the preferred method of cleaning and decontaminating DHPs [
      Department of Health
      Decontamination: Health Technical Memorandum 01–05: decontamination in primary care dental practices London: Department of Health;.
      ]. Washer disinfectors are not mandatory for dental practices in all countries [
      Department of Health
      Decontamination: Health Technical Memorandum 01–05: decontamination in primary care dental practices London: Department of Health;.
      ,

      Dental Council. Code of practice relating to: infection prevention and control (2015). Dublin: Dental Council; 2015. Available at: www.dentalcouncil.ie/files/IPC%20Code%20-%20Final%20-%2020150402.pdf [last accessed May 2022].

      ,

      Department of Health, Social Services and Public Safety. (2013) Updated Northern Ireland guidance on decontamination in primary care dental practices: HTM 01-05 2013 edition. Belfast: Department of Health, Social Services and Public Safety; 2013. Available at: https://hscbusiness.hscni.net/pdf/PEL%2013-13%20UPDATED%20NI%20GUIDANCE.pdf [last accessed May 2022]

      ,
      Scottish Dental Clinical Effectiveness Programme
      Cleaning of dental instruments.
      ]. Some studies demonstrated the effectiveness of washer disinfectors at cleaning the outside surfaces of DHPs and a few have demonstrated its efficacy at reducing organic contamination on internal components [
      • Smith A.
      • Smith G.
      • Lappin D.F.
      • Baxter H.C.
      • Jones A.
      • Baxter R.L.
      Dental handpiece contamination: a proteomics and surface analysis approach.
      ,
      • Offner D.
      • Brisset L.
      • Musset A.-M.
      Cleaning of dental handpieces: a method to test its efficiency, and its evaluation with a washer-disinfector lubricator-dryer.
      ]. However, little published data is available on the direct effectiveness of washer disinfectors at significantly reducing microbial contamination from the internal components of DHPs, especially in a dental hospital setting where large numbers of DHPs must be decontaminated daily.
      The purpose of this study was to use contra-angle DHPs as a model system to directly investigate the effectiveness of washer-disinfection at reducing microbial bioburden of internal components of multiple DHPs deliberately contaminated with each of four challenge micro-organisms in the presence/absence of organic soil in a dental hospital central decontamination unit.

      Methods

      DHPs

      BienAir Dental SA (Biel/Bienne, Switzerland) CA 1:1 L contra-angle DHPs were used throughout this study and were never used for patient treatment. These DHPs consist of a head, a neck, and a sheath (Figure 1). The head houses the head-gear which contains a dental bur orifice. Burs are held in place by a latch grip integrated in the head-gear.
      Figure 1
      Figure 1Photograph showing an example of the contra-angle dental handpiece (DHP) model used in this study and some of its internal components. (a) Frontal view of a DHP showing the angled main body and the head, neck and sheath. The head contains an opening into which a dental bur is fitted. The bur is driven by internal gears powered by an electric motor. (b) View of a DHP with the head removed showing the openings of the narrow-compressed air and water channels at the top of the image. (c) Image showing components of a disassembled DHP including the head with the bur opening and water and air outlets at the 1 o'clock (marked with a white arrow), 5 o'clock and 8 o'clock positions (top left), the press button plate that closes the back of the DHP head (top centre), the DHP head-gear (top right) into which a dental bur fits and the middle gear shaft that powers the head-gear (bottom).
      The back of the head unit is sealed by a push-button plate (Figure 1). The head-gear drives the dental bur during operation and is driven by the middle gear located in the neck of the DHP (Figure 1). These DHPs are supplied with compressed air and water, and they contain narrow air and water channels and are powered by an electric motor attached to a flexible arm connected to a dental chair unit (DCU). Three pairs of small water and air outlets surround the dental bur orifice in the DHP head (Figure 1). At the Dublin Dental University Hospital (DDUH) compressed air is provided to each DCU from a central source. Water containing very low levels of micro-organisms is provided to DUWLs from a central supply treated continuously with residual electrochemically generated hypochlorous acid [
      • O'Donnell M.J.
      • Boyle M.
      • Swan J.
      • Russell R.J.
      • Coleman D.C.
      A centralised, automated dental hospital water quality and biofilm management system using neutral Ecasol maintains dental unit waterline output at better than potable quality: a 2-year longitudinal study.
      ].
      One of the researchers was trained to disassemble and reassemble the DHPs for microbial inoculation and recovery experiments.

      Washer-disinfector

      A Míele (Míele Ireland Ltd., Dublin, Ireland) PG8528 washer-disinfector was used throughout this study. In the DDUH the equipment is connected to a variable-speed water pump that modulates between 1 and 5 bar. The equipment is fitted with Míele E919 dental modules for cleaning and decontaminating DHPs (Figure 2a). Up to six modules can be accommodated in the washer, each containing adapters for 10 DHPs (Figure 2b). W&H (W&H, Bürmoos, Austria) A803 DHP adapters (Figure 2a) were used throughout the study. The enzymatic detergent Endozime Xtreme Power (0.1% v/v) (The Ruhof Corporation, Mineola, NY, USA) was used for all washer-disinfection experiments.
      Figure 2
      Figure 2Photographs showing Míele (Dublin, Ireland) PG8528 washer-disinfector E919 dental modules. (a) The image shows a Míele E919 dental module equipped with 10 W&H (Bürmoos, Austria) DHP adapters with contra-angle DHPs in situ. The white arrow shows the dental module water inlet, and the smaller black arrows show the direction of flow of water within the module. The DHPs are numbered 1–10 and show the relative positions of the 10 DHPs subjected to washer-disinfection throughout this study ( and Supplementary Table S1). During washer-disinfection, water/cleaning solution is injected under pressure up into the internal lumens and channels of each DHP via the adapter as well as on to the outsides of each DHP by the washer-disinfector spray arms. (b) The Míele PG8528 washer-disinfector can accommodate up to six E919 dental modules, each of which contains adapters for up to 10 DHPs. The dental module in the foreground is fitted with W&H DHP adapters. The other five modules are fitted with other DHP adapters that were not used in this study.
      The parameters for each washer-disinfection cycle were as follows: (i) prewash with mains water at 22 °C for 6 min, (ii) cleaning with enzymatic detergent at 55 °C for 8 min, (iii) rinsing with reverse osmosis purified water for 5 min, (iv) thermal disinfection at 92 °C for 2 min and (v) drying 25 min.

      Challenge micro-organisms

      The three bacterial strains used as process challenge micro-organisms are those specified in BS-EN-14561:2006 [
      International Organization for Standardization. BS EN ISO 14561
      Chemical disinfectants and antiseptics – quantitative carrier test for the evaluation of bactericidal activity for instruments used in the medical area – test method and requirements (phase 2, step 2).
      ] including Pseudomanas aeruginosa ATCC15442, Staphylococcus aureus ATCC6538 and Enterococcus hirae ATCC10542. The laboratory yeast strain Candida albicans SC5314 (ATCCMYA-2876) was also used [
      • Jones T.
      • Federspiel N.A.
      • Chibana H.
      • Dungan J.
      • Kalman S.
      • Magee B.B.
      • et al.
      The diploid genome sequence of Candida albicans.
      ]. All strains were purchased from the American Type Culture Collection and were used separately to inoculate DHP air and water channels and heads/head-gears to monitor the decontamination efficacy of washer disinfection. To prepare challenge inocula, bacterial strains were cultured on tryptone soya agar (TSA) (Oxoid Ltd./ThermoFisher Scientific, Basingstoke, UK) at 37 °C for 24 h and a single colony was inoculated into 25 mL of tryptone soya broth (Oxoid) in a 250-mL conical flask and grown at 37 °C in a shaking incubator at 200 rpm to 109 colony forming units (cfu)/mL. C. albicans strain SC5314 was cultured on YPD agar (MP Biomedicals, Solon, OH, USA) at 30 °C for 24 h and a single colony was inoculated into 25 mL of YPD broth (MP Biomedicals) in a 250-mL conical flask and grown in a 30 °C shaking incubator at 200 rpm to 109 cfu/mL.

      Recovery of micro-organisms from DHP channels and heads/head-gears

      Before each experiment, DHPs were sterilized in a vacuum steam sterilizer at 134 °C. Prior to inoculation, the small press-button plate sealing the DHP head was removed, followed by removal of the head, head-gear and middle gear, providing access to the openings of the air and water channels (Figure 1). The partially disassembled DHP was then positioned horizontally and 100 μL of culture inoculum supplemented with 0.3% (w/v) bovine serum albumin (BSA) (clean conditions), 3.0% (w/v) BSA (dirty conditions) or 10% artificial test soil (dirty conditions) (Edinburgh test soil, Cúram Medical, Dublin, Ireland, compliant with ISO-15883-5-2021 [

      International Organization for Standardization. BS EN ISO 15883-5. Washer-disinfectors – Part 5: performance requirements and test method criteria for demonstrating cleaning efficacy. London: British Standards Institute; 2021.

      ]) was inoculated into both channels using a 0.3-mL insulin syringe with a 30-gauge Micro-Fine needle (Becton Dickson and Company, Franklin Lakes, NJ, USA) and allowed to dry for 30 min. The angle in the body of the DHP ensured the head and neck were horizontal, permitting retention of the inocula in the channels. After drying, the inoculated channels were sampled by inserting sterile, tapered periopoints (02 Absorbent Points, Dentsply Sirona, Charlotte, NC, USA). Periopoints are used for sampling periodontal pockets and are ideal for sampling narrow lumens [
      • O'Connor A.M.
      • McManus B.A.
      • Kinnevey P.M.
      • Brennan G.I.
      • Fleming T.E.
      • Cashin P.J.
      • et al.
      Significant enrichment and diversity of the staphylococcal arginine catabolic mobile element ACME in Staphylococcus epidermidis isolates from subgingival peri-implantitis sites and periodontal pockets.
      ]. Periopoints were placed in 1 mL phosphate-buffered saline (PBS) (Oxoid) in a sterile 1.5-mL tube and vortexed for 1 min to release micro-organisms. Serial dilutions were prepared in PBS and 100-μL aliquots spread in triplicate on to TSA agar for bacteria and YPD agar for C. albicans and incubated as described above. Following incubation, the bacterial/yeast colonies were counted and the total number of bacteria/yeasts recovered from the channels determined.
      For each challenge micro-organism, 100 μL of culture inoculum supplemented with 0.3%, 3.0% BSA or 10% test soil was inoculated into the head of a non-disassembled DHP placed horizontally through the dental bur orifice and allowed to dry for 30 min. The DHP head was then aseptically removed and the press-button plate, the head-gear and the head were placed in 5 mL of PBS in a sterile 25-mL tube and agitated for 1 min to release bacterial/yeast cells into solution (Figure 1). Serial dilutions were prepared in PBS and 100-μL aliquots plated in triplicate on TSA/YPD media and the total number of bacteria/yeasts recovered from the DHP head, head-gear and button determined.
      Additional experiments were undertaken with four DHPs that were lubricated with W&H Service Oil F1 MD-500 using an Assistina 301 plus DHP maintenance unit (W&H) according to the manufacturer's instructions prior to sterilization at 134 °C. Then the heads and channels of three DHPs were inoculated with S. aureus ATCC6538 in the presence of 10% test soil as described above, followed by reassembly of the DHPs and washer-disinfection. The fourth DHP was retained as a control. Following washer-disinfection, the DHPs were disassembled and the reduction in bacterial counts and protein recovered from DHP head/head-gears and channels calculated relative to the control DHP. Experiments were repeated on three separate occasions.

      Micro-organism counts in DHP channels and heads/head-gears following washer-disinfection

      Each challenge micro-organism preparation was inoculated separately into the heads and channels of 11 DHPs as described above. After drying, DHPs were reassembled and 10 were subjected to a washer-disinfection. The remaining inoculated DHP served as a control. Following washer-disinfection, all 11 DHPs were disassembled, sampled as described above and the log reduction in bacterial/yeast counts calculated relative to the control inoculated DHP in each case. Experiments were repeated in triplicate with all 10 DHPs for each challenge organism under clean (0.3% BSA) and two sets of dirty conditions (3.0% BSA and 10% artificial test soil).

      Protein assay

      Inoculated DHP heads/rotors and channels were tested for residual protein following washer-disinfection. Tests were undertaken on samples recovered as described above. Protein was detected using the QuantiPro BCA assay kit (Sigma-Aldrich/Merck, Arklow, Ireland) according to the manufacturer's instructions. The relative reduction in protein in DHP channels and heads/head-gears from washer-disinfected DHPs was determined relative to unwashed controls.
      The external surfaces of 10 DHPs were painted with 10% test soil and left to dry for 30 min followed by washer-disinfection. One additional painted DHP was retained as a control. The DHPs were visually inspected for residual test soil immediately following washer-disinfection and the surfaces were swabbed with sterile swabs soaked in 1% (w/v) sodium dodecyl sulphate (pH 11.0) and tested for protein using the QuantiPro BCA assay kit. Surfaces were also tested using the Pyromol-Test for residual protein (PEREG GmbH, Waldkraiburg, Germany) according to the manufacturer's instructions.

      Results

      Decontamination of DHP internal components by washer-disinfection

      The internal surfaces of the head, press-button plate and head-gear (all three hereafter referred to as the head) and air and water channels of contra-angle DHPs were used as a model system for monitoring the efficacy of decontamination by washer-disinfection. The internal surfaces of 11 DHP heads and both channels were inoculated with one of four challenge micro-organisms under clean and two sets of dirty conditions. Ten of the inoculated DHPs were then inserted into a Míele E919 dental module (Figure 2a) and subjected to washer-disinfection (see Methods). The remaining DHP in each case acted as a control to establish a baseline for recovery of micro-organisms and protein in the absence of washer-disinfection. Experiments were undertaken in triplicate for each DHP under each set of conditions. Following washer-disinfection, DHPs were disassembled, and the head and channels sampled for micro-organisms and residual protein. During each washer-disinfection cycle, the same DHP was consistently placed in the same position in the Míele E919 dental module (Figure 2a).

      Reduction in microbial bioburden in inoculated DHP heads and channels

      For each of the three challenge bacterial strains tested under clean conditions (0.3% BSA), on average an approximate 5 log or greater reduction in bacterial cfu recovered from DHP heads and channels was observed consistently for all 10 DHPs tested (Table I). Similar reductions were observed under both sets of dirty conditions. The average log reduction in S. aureus cfu from DHP heads was 5.27 ± 0.23 (3% BSA) and 5.11 ± 0.58 (10% test soil) and from channels was 5.57 ± 0.14 (3% BSA) and 5.59 ± 0.16 (10% test soil). The average log reduction in E. hirea cfu from DHP heads was 5.32 ± 0.38 (3% BSA) and 5.37 ± 0.08 (10% test soil) and from channels was 5.48 ± 0.18 (3% BSA) and 5.58 ± 0.13 (10% test soil). The average log reduction in P. aeruginosa cfu from DHP heads was 6.07 ± 0.05 (3% BSA) and 5.57 ± 0.48 (10% test soil) and from channels was 5.87 ± 0.22 (3% BSA) and 5.72 ± 0.33 (10% test soil).
      Table IReduction in the density of four challenge micro-organisms recovered from internal components of 10 contra-angle dental handpieces (DHPs) under clean and dirty conditions following washer-disinfection relative to inoculated DHPs not subjected to washer-disinfection
      Challenge micro-organismConditions
      The artificial test soil (Edinburgh test soil, Cúram Medical, Dublin, Ireland) used was compliant with ISO-15883-5-2021 [25].
      DHP
      Bacterial recovery data shown for channels represent the average recovery data from both air and water channels for each DHP tested with each challenge micro-organism. Bacteria recovered from heads include organisms recovered from the DHP head, press button plate, head-gear and middle gear of each DHP.
      site
      Log10 reduction in bacterial count
      Average reduction in bacterial count from three separate experiments.
      (± standard deviation)
      DHP1DHP2DHP3DHP4DHP5DHP6DHP7DHP8DHP9DHP10Overall average
      Staphylococcus aureus ATCC 6538Clean (0.3% BSA)Head5.06 (0.98)5.40 (0.41)5.33 (0.53)5.46 (0.31)5.57 (0.17)5.48 (0.28)5.43 (0.36)5.22 (0.56)5.36 (0.47)5.46 (0.31)5.38 (0.43)
      Channels5.50 (0.05)5.50 (0.05)5.44 (0.90)5.50 (0.05)5.50 (0.05)5.50 (0.05)5.50 (0.05)5.50 (0.05)5.39 (0.24)5.33 (0.34)5.46 (0.09)
      Dirty (3% BSA)Head5.73 (0.07)5.51 (0.02)5.73 (0.07)4.79 (0.85)4.45 (0.77)5.24 (0.16)5.68 (0.16)4.75 (1.27)5.59 (0.16)5.22 (0.52)5.27 (0.23)
      Channels5.61 (0.23)5.33 (0.47)5.79 (0.07)4.94 (1.42)5.66 (0.28)5.67 (0.27)5.79 (0.07)5.48 (0.50)5.79 (0.07)5.70 (0.15)5.57 (0.14)
      Dirty (10% test soil)Head5.53 (0.23)4.82 (0.89)5.14 (0.56)4.82 (0.89)5.16 (0.59)4.86 (0.79)5.14 (0.75)5.17 (0.55)5.39 (0.53)5.13 (0.59)5.11 (0.58)
      Channels5.62 (0.23)5.71 (0.20)5.61 (0.38)5.24 (0.86)5.77 (0.10)5.71 (0.10)5.22 (0.76)5.82 (0.00)5.49 (0.59)5.77 (0.10)5.59 (0.16)
      Enterococcus hirea ATCC 10542Clean (0.3% BSA)Head5.06 (0.67)5.12 (0.76)5.58 (0.09)5.04 (0.90)5.52 (0.23)5.63 (0.15)5.63 (0.15)5.49 (0.13)5.58 (0.09)4.94 (0.69)5.36 (0.28)
      Channels5.82 (0.00)5.82 (0.00)5.51 (0.41)5.77 (0.10)5.67 (0.27)5.71 (0.19)5.77 (0.10)5.77 (0.10)5.51 (0.14)5.70 (0.22)5.70 (0.07)
      Dirty (3% BSA)Head5.57 (0.17)5.52 (0.08)5.57 (0.17)5.52 (0.23)5.57 (0.17)4.47 (1.24)4.98 (1.12)5.43 (0.36)5.57 (0.17)4.96 (0.57)5.32 (0.38)
      Channels5.57 (0.21)5.57 (0.21)5.62 (0.21)5.62 (0.21)5.62 (0.21)5.49 (0.50)5.62 (0.21)5.02 (0.21)5.06 (0.98)5.62 (0.21)5.48 (0.18)
      Dirty (10% test soil)Head5.38 (0.67)5.77 (0.00)5.28 (0.85)5.65 (0.21)4.77 (0.77)5.32 (0.40)4.92 (0.88)5.32 (0.40)5.55 (0.39)5.77 (0.00)5.37 (0.08)
      Channels5.75 (0.41)5.69 (0.31)5.45 (0.10)5.65 (0.25)5.45 (0.10)5.52 (0.03)5.75 (0.41)5.36 (0.25)5.63 (0.22)5.55 (0.08)5.58 (0.13)
      Pseudomonas aeruginosa ATCC 15442Clean (0.3% BSA)Head5.71 (0.27)5.38 (0.83)5.83 (0.10)5.73 (0.19)5.83 (0.10)5.74 (0.22)5.19 (1.03)5.78 (0.17)5.78 (0.17)5.78 (0.17)5.67 (0.24)
      Channels5.96 (0.23)5.96 (0.23)5.96 (0.23)5.82 (0.40)5.72 (0.40)5.52 (0.53)5.96 (0.23)5.96 (0.23)5.96 (0.23)5.89 (0.29)5.87 (0.18)
      Dirty (3% BSA)Head5.91 (0.32)6.12 (0.09)6.12 (0.09)6.17 (0.00)6.06 (0.09)6.12 (0.09)6.12 (0.09)6.17 (0.00)5.84 (0.08)6.12 (0.09)6.07 (0.05)
      Channels5.77 (0.06)5.86 (0.18)5.91 (0.27)5.91 (0.27)5.91 (0.27)5.91 (0.27)5.79 (0.37)5.91 (0.27)5.91 (0.27)5.75 (0.07)5.87 (0.22)
      Dirty (10% test soil)Head5.49 (0.71)5.10 (1.15)5.09 (1.03)5.85 (0.14)5.85 (0.29)5.85 (0.23)5.90 (0.23)5.90 (0.23)5.54 (0.47)5.10 (1.52)5.57 (0.48)
      Channels5.90 (0.29)5.90 (0.13)5.90 (0.13)5.34 (1.21)5.96 (0.23)5.54 (0.70)5.50 (0.92)5.91 (0.29)5.45 (0.24)5.81 (0.15)5.72 (0.33)
      Candida albicans ATCC MYA-2876Clean (0.3% BSA)Head5.17 (0.32)5.26 (0.40)5.26 (0.40)5.26 (0.39)5.26 (0.39)5.26 (0.36)5.26 (0.36)5.26 (0.37)5.26 (0.37)5.26 (0.37)5.25 (0.36)
      Channels4.95 (0.23)4.95 (0.23)4.95 (0.23)4.95 (0.23)4.95 (0.23)4.95 (0.23)4.95 (0.23)4.95 (0.23)4.95 (0.23)4.95 (0.23)4.95 (0.23)
      Dirty (3% BSA)Head4.65 (0.84)4.81 (0.61)5.21 (0.44)4.61 (0.90)5.21 (0.44)5.21 (0.44)5.21 (0.44)5.21 (0.44)5.21 (0.44)5.21 (0.44)5.06 (0.22)
      Channels
      No viable Candida cells were recovered by culture following washer-disinfection, thus all of the readings for the 10 DHPs are identical.
      4.93 (0.09)4.93 (0.09)4.93 (0.09)4.93 (0.09)4.93 (0.09)4.93 (0.09)4.93 (0.09)4.93 (0.09)4.93 (0.09)4.93 (0.09)4.93 (0.09)
      Dirty (10% test soil)Head4.98 (0.61)5.27 (0.17)5.27 (0.17)4.92 (0.22)4.97 (0.34)5.27 (0.17)5.27 (0.17)4.66 (1.15)5.16 (0.3104.55 (1.35)5.03 (0.25)
      Channels4.73 (0.85)4.81 (0.71)5.09 (0.23)4.91 (0.53)5.09 (0.23)5.03 (0.33)5.09 (0.23)5.09 (0.23)4.80 (0.74)5.09 (0.23)4.97 (0.43)
      Each challenge micro-organism was inoculated separately into the head and air and water channels of 11 DHPs. Ten of these DHPs were processed by washer-disinfection. For each washer-disinfector cycle, one inoculated DHP was left untreated as a control. Following washer-disinfection, all 11 DHPs were tested for recovery of micro-organisms using periopoints as described in the Methods and the log reduction in bacterial/yeast counts calculated relative to the untreated control inoculated DHP in each case. The results shown are the average of three separate experiments for each DHP with each challenge micro-organism. BSA, bovine serum albumin.
      a Average reduction in bacterial count from three separate experiments.
      b The artificial test soil (Edinburgh test soil, Cúram Medical, Dublin, Ireland) used was compliant with ISO-15883-5-2021 [25].
      c Bacterial recovery data shown for channels represent the average recovery data from both air and water channels for each DHP tested with each challenge micro-organism. Bacteria recovered from heads include organisms recovered from the DHP head, press button plate, head-gear and middle gear of each DHP.
      d No viable Candida cells were recovered by culture following washer-disinfection, thus all of the readings for the 10 DHPs are identical.
      In the case of the C. albicans strain, on average an approximate 5 log reduction in cfu recovered from DHP heads was recorded under clean conditions (0.3% BSA) (average 5.25 ± 0.36) with a slightly lower log reduction recorded for DHP channels (average 4.95 ± 0.23) (Table I). Similar results were obtained for DHP heads and channels under both sets of dirty conditions with an average log reduction in C. albicans cfu from DHP heads of 5.06 ± 0.22 (3% BSA) and 5.03 ± 0.25 (10% test soil) and from channels of 4.93 ± 0.09 (3% BSA) 4.97 ± 0.43 (10% test soil).
      For all four challenge micro-organisms used under clean or dirty conditions, consistent log reductions in microbial count were recovered for all 10 DHPs, regardless of their position in the Míele dental module during washer-disinfection (Figure 2a). During washer-disinfection DHP1 was positioned closest to the water inlet, where the water pressure is at its highest, whereas DHP10 was furthest away (Figure 2a, Table I).

      Influence of DHP position in the washer-disinfector on microbial burden reduction

      Experiments were undertaken with seven DHPs inoculated with the S. aureus challenge micro-organism and 10% test soil. Following inoculation and reassembly, one DHP was placed at position 10 (Figure 2) in each of six separate Míele E919 dental modules and subjected to washer-disinfection. The remaining DHP served as a control. Following washer disinfection, the DHPs were disassembled and tested for micro-organisms and protein. All experiments were repeated three times. For each of the six DHPs, an average of >5 log reduction in S. aureus cfu recovered from DHP heads and channels was observed relative to control DHPs, regardless of which of the six washer-disinfection dental modules was used (Supplementary Table S2, Figure 2b).

      Influence of DHP lubrication on microbial bioburden reduction

      Three DHPs were lubricated using the Assistina 301 plus automated system prior to sterilization and inoculation of the heads and channels with S. aureus ATCC6538 in the presence of 10% test soil followed by washer-disinfection. One additional lubricated and inoculated DPH served as a control. Following washer-disinfection, the log reduction in bacterial cfu from heads and channels was calculated relative to the control DHP. In three separate experiments, the average log reduction in bacterial cfu was 5.96 ± 0.1 (heads) and 5.69 ± 0.2 (channels).

      Reduction in protein in DHP heads and channels

      For each of the 10 DHPs inoculated with 10% test soil in the absence of challenge micro-organisms, on average a >95% reduction in protein recovered from DHP heads and channels was observed following washer-disinfection relative to unwashed inoculated control DHPs (Supplementary Table S1). Similar reductions in protein levels in heads and channels were obtained with DHPs inoculated with challenge micro-organisms under both sets of dirty conditions following washer-disinfection (Supplementary Table S1). No protein was detected in heads and channels inoculated under clean conditions following washer-disinfection (data not shown).
      The average reduction in protein from the 10 DHP heads and channels inoculated with S. aureus and (i) 3% BSA was 97.6 ± 1.9% and 94.9 ± 1.4%, respectively, and (ii) 10% test soil was 98.6 ± 0.5% and 92.7 ± 1.8%, respectively. The average reduction in protein from the 10 DHP heads and channels inoculated with E. hirea and (i) 3% BSA was 99.2 ± 0.1% and 93.6 ± 2.4%, respectively, and (ii) 10% test soil was 99.4 ± 0.3% and 93.1 ± 3.1%, respectively. The average reduction in protein from the 10 DHP heads and channels inoculated with P. aeruginosa and (i) 3% BSA was 98.5 ± 0.3% and 98.6 ± 0.6%, respectively, and (ii) 10% test soil was 97.9 ± 1.4% and 94.2 ± 5.1%, respectively. The average reduction in protein from the 10 DHP heads and channels inoculated with C. albicans and (i) 3% BSA was 98.8 ± 0.2% and 96.3 ± 0.3%, respectively, and (ii) 10% test soil was 99.4 ± 0.1% and 93.2 ± 5.4%, respectively.
      For all four challenge micro-organisms used under both sets of dirty conditions, consistent reductions in protein levels in heads and channels were observed for all 10 DHPs tested regardless of their position in the dental module used to retain the DHPs in the washer-disinfector (Figure 2a). Similar results were obtained with the six DHPs inoculated with S. aureus and 10% test soil placed at position 10 in each of six separate Míele E919 dental modules (Supplementary Table S2, Figure 2).
      In the case of the three DHPs that were lubricated with oil and sterilized prior to inoculation with S. aureus and 10% test soil followed by washer-disinfection, an average of 99.69% ± 0.1% and 98.98% ± 0.3% reduction in protein was recorded for DHP heads and channels, respectively, on three separate occasions relative to inoculated but unwashed controls.

      Test soil removal from the outside surfaces of DHPs by washer-disinfection

      The exterior surfaces of 10 DHPs that were painted with 10% test soil and left to dry were free from visible contamination following washer-disinfection. All the DHPs were negative for residual protein using the Pyromol test. There was a 99.98% ± 0.02% reduction in protein on the DHP surfaces using the DHPs QuantiPro BCA assay relative to controls.

      Discussion

      Oral biomaterial and micro-organisms can be retracted into DHPs during use and contaminate internal components [
      • Herd S.
      • Chin J.
      • Palenik C.J.
      • Ofner S.
      The in vivo contamination of air-driven low speed handpieces with prophylaxis angles.
      ,
      • Smith G.
      • Smith A.
      Microbial contamination of used dental handpieces.
      ,
      • Dreyer A.G.
      • Hauman C.H.
      Bacterial contamination of dental handpieces.
      ]. Microbial biofilm in DUWLs provides an additional source of contamination [
      • O'Donnell M.J.
      • Boyle M.A.
      • Russell R.J.
      • Coleman D.C.
      Management of dental unit waterline biofilms in the 21st century.
      ,
      • Coleman D.C.
      • O'Donnell M.J.
      • Shore A.C.
      • Russell R.J.
      Biofilm problems in dental unit water systems and its practical control.
      ]. The use of validated automated washer-disinfectors is currently the gold standard for cleaning and decontaminating dental instruments. A number of studies have shown that washer-disinfection is effective at cleaning the exterior of DHPs, and a few have shown its efficacy at reducing organic contamination on internal components [
      • Smith A.
      • Smith G.
      • Lappin D.F.
      • Baxter H.C.
      • Jones A.
      • Baxter R.L.
      Dental handpiece contamination: a proteomics and surface analysis approach.
      ,
      • Offner D.
      • Brisset L.
      • Musset A.-M.
      Cleaning of dental handpieces: a method to test its efficiency, and its evaluation with a washer-disinfector lubricator-dryer.
      ]. However, there is very little published data on the direct efficacy of washer disinfectors at significantly reducing microbial bioburden from the internal channels and other components of DHPs, mainly due to difficulties in accessing the internal components, as DHPs are not designed to be routinely disassembled. The present study set out to address this deficit by deliberately inoculating the channels and heads of multiple contra-angle DHPs with four challenge micro-organisms under clean and dirty conditions and monitoring the reduction in microbial bioburden and protein following washer-disinfection. The very high densities of challenge micro-organisms inoculated into the DHPs was deliberately far in-excess of the levels of micro-organisms contaminating the internal components of DHPs following clinical use. Sterilized DHPs were disassembled to facilitate inoculation of the internal components followed by reassembly, washer-disinfection, disassembly and sampling for micro-organisms and residual protein.
      An approximate 5 log reduction in S. aureus, E. hirea and P. aeruginosa cfu recovered from DHP heads and channels was consistently observed for all 10 DHPs tested following washer-disinfection under clean and both sets of dirty conditions (Table I). On average a >93% reduction in protein was recorded for DHP heads and channels under all test conditions (Supplementary Table S1). Similar reductions in microbial cfu and protein were obtained with C. albicans SC5314 (Table I and Supplementary Table S1). Similar reductions in microbial cfu and protein were recorded for all 10 DHPs regardless of each DHP's position in the module holding the DHPs during washer-disinfection (Figure 2, Table I and Supplementary Table S1). DHP10, which was furthest away from the water inlet in the washer-disinfector module, yielded similar results to DHP1 (closest to the water inlet). A series of experiments with six DHPs in which the channels and heads were inoculated with S. aureus and 10% test soil were undertaken with six separate dental modules, with each DHP located at position 10 (i.e. furthest away from the water inlet of each module) (Figure 2) followed by washer-disinfection. In each case, a >5 log reduction in bacterial count and a >93% reduction in protein recovered from heads and channels was consistently recorded, regardless of dental module (Supplementary Table S2). Only one of the 10 adapters for DHPs was occupied in each of the six dental modules used and water freely discharged from the nine unoccupied adapters in each module during washer-disinfection.
      All these findings demonstrated that the Míele PG8528 washer-disinfector with the enzymatic detergent used was consistently effective at significantly reducing microbial and protein contamination of internal components and channels of multiple DHPs simultaneously. Up to 60 individual DHPs can be decontaminated simultaneously using the Míele PG8528 washer-disinfector, which is ideal for dental hospitals where large numbers of DHPs must be decontaminated daily.
      The internal components of DHPs must be lubricated regularly. Winter et al. [
      • Winter S.
      • Smith A.
      • Kirk B.
      • Lappin D.
      The influence of lubricating oil on the efficacy of steam sterilization processes used to decontaminate dental handpieces.
      ] postulated that the presence of lubricating oil in DHPs can be detrimental to the efficacy of steam sterilization. To determine whether the presence of lubrication oil in DHPs affected the efficacy of washer-disinfection at significantly reducing microbial counts and protein levels in DHPs, three DHPs were lubricated with maintenance oil prior to sterilization followed by inoculation of both channels and heads with S. aureus ATCC6538 in the presence of 10% test soil. In three separate experiments with the three DHPs, a >5 log reduction in bacterial cfu and a >98% reduction in protein was recorded for both DHP heads and channels. These findings demonstrate that oil lubrication of DHPs did not adversely affect decontamination of internal components of DHPs by washer-disinfection, at least under the conditions used. Winter et al. [
      • Winter S.
      • Smith A.
      • Kirk B.
      • Lappin D.
      The influence of lubricating oil on the efficacy of steam sterilization processes used to decontaminate dental handpieces.
      ] commented that many dentists use spray cans to lubricate DHPs and that if they are used incorrectly, oil will be located throughout the internal surfaces and channels, which is a challenge for steam penetration. In the present study, lubrication was undertaken with the Assistina 301 plus automated system, which ensures correct lubrication of DHPs.
      This study was limited to contra-angle DHPs. The internal design of turbine DHPs is different, as they lack gears and usually have fewer bearings. Because of the higher rotational speeds of turbine DHPs, there are greater opportunities for suck-back via the bur orifice when the devices are stopped resulting in internal contamination. Nonetheless, the internal architecture of the contra-angle DHPs used here is complex, and all were consistently decontaminated by washer-disinfection.
      In conclusion, in a dental hospital setting multiple DHPs can simultaneously be effectively decontaminated internally and externally by washer-disinfection using an enzymatic detergent.

      Acknowledgements

      We wish to acknowledge the staff of the Dublin Dental University Hospital Central Sterile Supplies Department for facilitating this study. We wish to thank Edgar Schönbächler, CEO Bien-Air Dental SA, Bienne, Switzerland for providing the contra-angle handpieces used in this study.

      Conflict of interest statement

      D.C.C. received partial funding for this project from Bien-Air Dental SA, Bienne, Switzerland. Bien-Air had no role in the decision to publish the study or in the manuscript contents. All other authors have no conflicts of interest to declare.

      Funding sources

      This study was supported by the Dublin Dental University Hospital Microbiology Research Unit and by Bien-Air Dental SA , Bienne, Switzerland.

      Appendix A. Supplementary data

      The following are the Supplementary data to this article:

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