Ultrasonically nwater: a promiprogramme forcasts used in d

G. Wu, X. Yu, Z. Gu*

Department of Oral and MaxilPeoples Republic of China

pted008

Disinfection;

Bactericidal efficacy was examined using Staphylococcus aureus and Bacil-

* Corresponding author. Address: School of Stomatology, Zhejiang University, Department of Oral and Maxillofacial Surgery, No. 395,Yanan St, Hangzhou, Zhejiang Province 310006, Peoples Republic of China. Tel.: 0086 571 87217427.

E-mail address: noprint2000@zju.edu.cn

Journal of Hospital Infection (2008) 68, 348e354

Available online at www.sciencedirect.com

www.elsevierhealth.com/journals/jhinlus subtilis var. niger spores as indicators. Dimensional accuracy, surfacequality, and effect of corrosion were also evaluated for the different sam-ples. Results showed that except for B. subtilis var. niger spores on gypsumcasts, the bacterial reduction log10 values after 30e45 min treatment withUNEOW were all above 4. The impression dimensional changes showed nodifference between control and UNEOW groups, but both were significantlylower than the EOW and sodium hypochlorite groups (P< 0.05). The samewas true for the surface quality of impressions and gypsum casts. No assess-able corrosion was found on the titanium surface after a 45 min treatmentImpression; Gypsumcast; Dental metal

ultrasonically nebulised, electrolysed oxidising water (UNEOW) as a newinfection control programme. UNEOW was produced from freshly gener-ated electrolysed oxidising water (EOW). Samples of impressions, titaniumand gypsum were subjected to the following treatments: (1) immersion in1% sodium hypochlorite for 10 min; (2) immersion in EOW for 10 min; (3)exposure to UNEOW for 15, 30 and 45 min; (4) no disinfection (control).Received 30 July 2007; acceAvailable online 18 March 2

KEYWORDSElectronic oxidisingwater; Ultrasonicnebulisation;0195-6701/$ - see front matter 200doi:10.1016/j.jhin.2008.01.024ebulised electrolysed oxidisingsing new infection controlimpressions, metals and gypsumental hospitals

lofacial Surgery, School of Stomatology, Zhejiang University,

21 January 2008

Summary Controlling the transmission of infectious diseases by impres-sions, metals and dental casts in dental hospitals remains a challenge.Current disinfection methods have various drawbacks. This study intro-duced and provided a preliminary evaluation of the feasibility of using8 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

with UNEOW. The findings indicated that use of UNEOW is a feasible andormInf

fection requirements. For example, immersion in

sional accuracy of alginate imto ultraviolet light does not prantibacterial effect; and microfor metal disinfection. Thereuniversal disinfection proce

Electrolysed oxidising water (EOW) has been

Electrolysed oxidising water preparation

UNEOW for dental infection control 349pressions; exposureoduce a satisfactorywave is not suitablefore, finding a newss is a key goal

EOW was freshly generated using an EOWgenerator (SUNTECH-1000, ZH-Suntech, Zhuhai,China) and used within 2 h. The pH and oxida-tion-reduction potential (ORP) of the EOW werechemical solutions can compromise the dimen- and nebulisationpromising approach fby impressions, gypsu 2008 The Hospitalreserved.

Introduction

The control of infectious diseases transmitted bycontaminated dental impressions, metals andgypsum casts in dental hospitals and laboratoriesremains a challenge. The pathogens from patientssaliva and blood, such as cytomegalovirus, hepatitisB virus (HBV), hepatitis C virus (HCV), herpes simplexvirus types 1 and 2, human immunodeficiency virus(HIV)1, Mycobacterium tuberculosis, staphylococci,streptococci, and other viruses and bacteria especifically, those that infect the upper respiratorytract e can contaminate impressions, cross-infectresultant gypsum casts, and increase the risk ofinfection for dental healthcare workers (DHCWs). Arecent study confirmed that it is possible forimpression materials to transfer micro-organismsfrom patients mouths to DHCWs.2 Denture metalproduced in dental laboratories should also bedisinfected before use by patients. To control thetransmission of infections by dental impressions,gypsum casts and denture metals, each needs tobe disinfected before delivery and use.

The difficulties of this cross-infection control,however, arise from the special physical andchemical properties of the dental materials:instability of dimensional accuracy and surfacequality, which are crucial for subsequent treat-ment because impressions and gypsum casts areused to register or reproduce the form of the teethand oral tissues. Ideal disinfection processes notonly should have strong bactericidal properties butalso should not change the dimensional accuracyand surface quality of the disinfected materials.

The current disinfection processes vary mark-edly in type, time and concentration. Chemicalmethods include immersion in sodium hypochlo-rite, glutaraldehyde etc.3 Physical methods in-clude microwave, ultraviolet light, etc. However,no single method has been able to fulfil all disin-reported to produce strong antimicrobial activityagainst many pathogens, such as Salmonella enter-itidis, L. monocytogenes and Staphylococcusaureus.4 EOW is generated from electrolysis of0.05e0.2% sodium chloride solution and iscollected from the anode. Unlike other chemicaldisinfectants, EOW is easy to collect and posesno harm to personnel or the environment becauseit ultimately reverts to NaCl solution. EOW hasbeen widely used in the food, medical and poultryindustries, among others.5 However, its directapplication for reducing the transmission of infec-tions by dental impressions, gypsum and metals islimited because, as with other chemical disinfec-tants, immersion in EOW will compromise thedimensional stability of the dental materials.

Some previous findings sparked the idea of usingultrasonic nebulisation to overcome this draw-back: spraying an impression with disinfectantleads to a more accurate cast than immersing it.6

In this study we investigated the use of ultra-sonic nebulisation to overcome EOWs drawback ofchanging dimensional accuracy as it was reportedthat spraying of disinfectant causes less change inthe dimensional accuracy of impressions thanimmersion does. With the aim of controlling thetransmission of infections in dental hospitals, weevaluated the feasibility of using ultrasonicallynebulised electrolysed oxidising water (UNEOW)as a universal new approach for disinfectingimpressions, dental metals and gypsum casts withhigh bactericidal efficacy, but without affectingdimensional accuracy, surface quality.

Methodscontrolling the transmission of infectious diseasescasts and denture metals in dental facilities.ection Society. Published by Elsevier Ltd. All rights

of infection control in dental hospitals andlaboratories.

measured with a pH meter (PH6000, Milwaukee

The following treatment groups (N 6 in each

9372, 3M, USA) 1.0 108 cfu/ml, to a final concen-

350 G. Wu et al.group) were established: (1) immersion in 1%sodium hypochlorite for 10 min; (2) immersion inEOW for 10 min; (3) exposure to UNEOW for 15,30 and 45 min; (4) control (no treatment, directlyto the next step).

Antibacterial efficacy

Alginate impressions, gypsum casts and titaniumwere formed into 1 1 0.5 cm discs to serve asmicro-organism vehicles. Discs were sterilised bygamma rays before use and inoculated by injectingand spreading 10 ml of S. aureus (ATCC 6538, Bacte-rial Laboratory, Pediatric Hospital, Zhejiang Uni-versity, China), 1.0 108 colony-forming units(cfu)/ml, or B. subtilis var. niger spores (ATCCInstruments Inc., Rocky Mount, NC, USA) and anORP meter (OR100, Yokogawa, Shanghai, China),respectively. The chlorine content in EOW was de-termined with a chlorine detection kit (Hach Com-pany, Loveland, CO, USA). The EOW had a pH of2.5, an ORP of 1150 mV, and about 50 mg/L freechlorine.

The fresh EOW was added into the drug con-tainer of the ultrasonic nebuliser (Multisonic Com-pact TM, Schill Company, Probstzella Germany),and the ultrasound frequency was adjusted to1.7 MHz, airflow to 40 L/min. The UNEOW spraywas guided into an airtight and transparent plasticbox (25 20 20 cm). The samples in the boxwere disinfected with UNEOW and kept for10 min until the fog in the box reached saturation.

Materials and experimental design

Irreversible hydrocolloid impression material (themost unstable but most commonly used impressionmaterial (Jeltrate, Dentsply, Tianjin, China), gyp-sum (Heraeus, Hangzhou, China) and pure titanium(representing dental metal) (Zheda Medical Appli-ance Company, Hangzhou, China) were selectedfor this study. The materials were manipulatedaccording to the manufacturers instructions.

Staphylococcus spp. and spores of Bacillus sp.are commonly used as test micro-organisms forlow-level and high-level hospital disinfectants,respectively. To show the efficacy of UNEOW inthe disinfection of the three materials, we alsoused S. aureus and Bacillus subtilis var. niger sporesas indicators. We also evaluated the dimensionalaccuracy of impressions, the surface quality of im-pressions and gypsum casts, and whether UNEOWhad a corrosive effect on titanium.tration of about 1.0 106 cfu/disc under sterilisa-tion. The discs were kept in 24-well cell cultureplates at room temperature for 1 h to allow the bac-teria to attach. After disinfection the discs weretransferred into sterile centrifuge tubescontaining 5 ml 0.5% Na2S2O3 and 0.03 mol/L phos-phate-buffered saline, pH 7.2e7.4, and left for1 min to neutralise the EOW before analysis. Thesolution underwent vortex agitation for 40 s andthen was serially diluted. Of each dilution, 1 mlwas inoculated on MuellereHinton agar (BD, SanJose, CA, USA), which was incubated for 48 h at37 C. The numbers of colonies were counted andcalculated to cfu/ml. The following formula wasused to express the bactericidal efficacy:

log10reduction log10cfu=ml of the control group log10cfu=ml of the disinfection group

Dimensional accuracy

A custom-made stainless steel edentulousmaxillary arch was used as a master model. Inthe approximate position of the left and right firstpremolar and second molar, grooves of smallpositive cone shape (4 mm diameter round base,3 mm height) were scored into the occlusion sur-face. The top points of the positive cones wereused as reference points. Customised trays anduniform special seating were used in subsequentsteps to ensure the homogeneity and reproducibil-ity of the impressions. The preparation of impres-sion and resultant gypsum cast were described ina previous study.3

All measurements were completed by a singlewell-trained operator using a caliper (HangzhouMeasurement Appliance Co., Hangzhou, China)accurate to 0.02 mm. Four lines (AeB, BeC,CeD, DeA) were measured, and all the readingswere repeated three times for each linear mea-surement. The mean reading was taken as the finalfor the cast. In order to calculate the percentagedeviation of the cast compared to the mastermodel, the following formula was used:

deviation % cast readingmaster modelreading=master model reading 100

Surface quality

To examine the effect of the disinfection protocolon the surface quality of impressions and gypsumcasts, a surface profilometer (Federal Surfana-lyzer-5000 Mechanical Stylus Profiler, FederalProducts, Providence, RI, USA) was used to record

the surface roughness after different treatments. titanium, alginate impressions and gypsum casts.

UNEOW for dental infection control 351Customised metal boxes (3 cm 4 cm 0.5 cm)with covers were used to form fine surfaces on gyp-sum cast and impression samples. The inner wallsof the boxes were well polished by a polishing sys-tem (Saphir 360, Mager Scientific, Inc., Dexter, MI,USA). The impression material and gypsum weremixed with water and carefully poured into thebox with vibration to avoid air trapping. The boxwas covered, turned over, and loaded with a 1 kgweight. The impression material was allowed toset for 5 min and the gypsum for 40 min beforethe cover was opened. The samples were sub-jected to different disinfection protocols andthen measured. The parameters of surface instru-ments were set as described in a previous study.6

In order to calculate the percentage roughnesschanges compared to the well-polished coversurface, the following formula was used:

%changesample roughness cover roughness=cover roughness 100%

Metal corrosion

A scanning electron microscope (SEM; PhillipsXL30-FEG, Phillips, Eindhoven, The Netherlands)was used to examine the corrosive effect ofUNEOW on the surface of dental titanium. Tita-nium discs (1.5 1.5 0.2 cm) were well-polishedwith a polishing system (Saphir 360, Mager Scien-tific, Inc., Dexter, MI, USA) and then washed withacetone, 70% ethanol and distilled water for15 min before disinfection. The discs were driedin an oven at 50 C overnight and then stuck tostubs and gold-sputtered before analysis.

Statistical analysis

The results of dimensional accuracy and surfaceroughness were analysed with one-way analysis ofvariance. Significant differences between treat-ments were evaluated using the SPSS 11.5 statisticalsoftware package (SPSS Inc., Chicago, Illinois, USA).Significance was set at P 0.05. Post-hoc compari-sons were made using the Bonferroni correction.

Results

Antibacterial efficacy

The antibacterial efficacy of each disinfectiontreatment was calculated and expressed by killrate (%) and log10 reduction (Figure 1). Immersion inEOW for 10 min resulted in a 100% kill rate forS. aureus and B. subtilis var. niger spores onlittle lower than 4. UNEOW for 45 min also resultedin a 100% kill rate for S. aureus, and above 99.99%for B. subtilis var. niger spores. Immersion in 1%sodium hypochlorite also resulted in log10 reductionsof around 4 for S. aureus and around 3.5 for B. subtilisvar. niger spores (metal 3.46, impressions 3.62,gypsum 3.56); these were all lower than for UNEOW30 min. Except for the EOW group, the kill ratesof various treatments for B. subtilis var. nigerspores were all lower than those for S. aureus.

Dimensional accuracy

For the selected alginate impression material, thedimensional accuracy changes in the EOW and 1%sodium hypochlorite groups were significantlyhigher (P< 0.05) than in the UNEOW and controlgroups (Figure 2). The largest deviation (%) occur-ring in the EOW group was 0.136, a little greaterthan with sodium hypochlorite treatment. The di-mensional changes in the 15, 30 and 45 min UNEOWgroups were 0.058, 0.0528 and 0.0666, whichshowed no significant differences from the controlgroup (P> 0.05). There were no differencesbetween UNEOW treatment groups, although therewas a mild increase for the 45 min time-point.

Surface quality

The mean roughness was measured to show thereproducibility of impressions and gypsum casts.The mean roughness of immersion in EOW and 1%sodium hypochlorite for 10 min was significantlyhigher (P< 0.05 and P< 0.01, respectively) thanfor the other groups (Figure 3). There were nosignificant differences between the three UNEOWgroups or between the three UNEOW groups andthe control group.

Metal corrosion

The surface of titanium treated by UNEOW showed novisible corrosion within 45 min. The stripes after pol-ishing were still clear and successive in each group.

Discussion

Disinfection of saliva-contaminated dental impres-sions, denture metals and gypsum casts is one ofThe reductions of the two bacteria in the UNEOW15 min group were all around or below 3 log10,reductions in the UNEOW 30 min group were allabove 4 (99.99%), except that the reductions ofB. subtilis var. niger spores on gypsum casts were a

9A

352 G. Wu et al.2

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tionthe important aspects of infection control indental facilities; however, no available method isuniversally applicable. The use of UNEOW fordisinfection was evaluated in this study.

Bactericidal efficacy is the first requirement fora disinfection process. Our results showed thatimmersion in EOW resulted in a 100% kill rate forS. aureus and B. subtilis var. niger spores after10 min. The strong bactericidal properties ofEOW were also indicated in previous studies.5,7

B. subtilis var. niger spores, which are much moredifficult to kill than normal bacteria, have fre-quently been used as an indicator for a disinfectantof high efficacy. Our results also showed that

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Figure 1 The antibacterial efficacy of different disinfectiogypsum casts and metal discs: (A) Staphylococcus aureus; (UNEOW 30 min; UNEOW 45 min; UNEOW 10 min; 1%except for 10 min EOW treatment (100% killrate), the average kill rates of other treatmentswere all lower for B. subtilis var. niger sporesthan for S. aureus.

The disinfection efficacy of sodium hypochloritevaried a lot in previous studies.3 This was due tothe different concentrations, immersion timesand pH values of sodium hypochlorite used. Ourresults showed that the log10 reduction of S. aureuswas around 4, which is the gold standard fora dental disinfectant, but it was unsatisfactoryfor B. subtilis var. niger spores. The kill rates ofsodium hypochlorite were all a little lower thanthe 30 min UNEOW treatment for both S. aureus

ssion Gypsum

ession Gypsum

n protocols used on the surfaces of dental impressions,B) Bacillus subtilis var. niger spores. UNEOW 15 min;sodium hypochlorite 10 min.

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Figure 2 The mean percentage deviation in dimensional accuracy of hydrocolloid impressions after differentdisinfection protocols (*P< 0.001).

UNEOW for dental infection control 353and B. subtilis var. niger spores. The high ORP andlow pH in EOW contributed to the difference be-tween EOW and sodium hypochlorite, whichrelied only on its chemical components.

Except for B. subtilis var. niger spores ongypsum, the log10 reductions of UNEOW 30 mintreatment were all above 4, and the kill rate ofUNEOW 45 min for S. aureus even reached up to100%. This suggests that the UNEOW disinfectiontime should be set at 30e45 min.0

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Figure 3 The mean roughness of hydrocolloid impressi(**P< 0.001, *P< 0.05). Impression; Gypsum.The impression dimensional accuracy changes ofthe UNEOW and control groups were significantlylower than for the 10 min EOW and 1% sodiumhypochlorite immersion groups. The loss of waterfrom alginates is due to a major decrease inentropy consequent on the cross-linking reaction,and a change of Gibbs free energy to a positive.8

A humidity of 100% was thought to be the best con-dition for storage, rather than immersion in fluid,and UNEOW provided that condition. This couldEOW 10 min 1% Sodiumhypochlorite

10 min

Control

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**

*

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ons and gypsum after different disinfection protocols

be the mechanism through which UNEOW leads tolower dimensional accuracy changes. The dimen-sional changes in the UNEOW treatment groupwere a little higher than the control group, butnot significantly so. The deviation levels of thetwo immersion groups were a little differentfrom levels in other studies, which might be dueto different material combinations, operation con-ditions and measuring methods.3

The surface roughness changes of impressionsand gypsum casts in the 10 min EOW and 1% sodiumhypochlorite groups were much higher than in theUNEOW and control groups; no significant differ-ence was found between the UNEOW groups andcontrol groups. These changes were due to thedirect contact seen with EOW, with high ORP andlow pH values, whereas nebulisation reduced the

Conflict of interest statementNone declared.

Funding sourceThe work is supported by Health Bureau ofZhejiang Province, China through Contract No.2005B158.

References

1. Mehtar S, Shisana O, Mosala T, Dunbar R. Infection controlpractices in public dental care services: findings from oneSouth African Province. J Hosp Infect 2007;66:65e70.

2. Al-Jabrah O, Al-Shumailan Y, Al-Rashdan M. Antimicrobialeffect of 4 disinfectants on alginate, polyether, and polyvinyl

354 G. Wu et al.contact chance and the effect dramatically. Thesame was true for UNEOWs corrosive effect ontitanium. SEM images did not show visible corro-sion dents on titanium surfaces after treatmentwith UNEOW for 45 min. The results indicatedthat UNEOW treatment could not cause markeddeterioration on titanium within 45 min. In fact,EOW is commonly used to disinfect endoscopes,which are also made of metal.9

The results indicated that the use of UNEOW isa feasible and promising approach for controlling thetransmission of infectious diseases by impressions,gypsum casts and denturemetals in dental facilities.Certainly some issuesdsuch as UNEOWs effect onother kinds of impression materials and metals,salivas effect on the bactericidal efficacy of UN-EOW, and UNEOWs effect on viruses such as HIV andHBVdstill need to be investigated before disinfec-tion with UNEOW can be generally recommended.

In conclusion, UNEOW used for 30e45 min withthree dental materials showed satisfactory disin-fection efficacy without compromising dimensionalaccuracy and surface quality.siloxane impression materials. Int J Prosthodont 2007;20:299e307.

3. Taylor RL, Wright PS, Maryan C. Disinfection procedures:their effect on the dimensional accuracy and surface qualityof irreversible hydrocolloid impression materials and gypsumcasts. Dent Mater 2002;18:103e110.

4. Liu C, Duan J, Su YC. Effects of electrolyzed oxidizing wateron reducing Listeria monocytogenes contamination onseafood processing surfaces. Int J Food Microbiol 2006;106:248e253.

5. Shama RR, Demirci A. Treatment of Escherichia coli O157: H7inoculated alfalfa seeds and sprouts with electrolyzedoxidizing water. Int J Food Microbiol 2003;86:231e237.

6. Johnson GH, Chellis KD, Gordon GE, Lepe X. Dimensionalstability and detail reproduction of irreversible hydrocolloidand elastomeric impressions disinfected by immersion.J Prosthet Dent 1998;79:446e453.

7. Kiura H, Sano K, Morimatsu S, et al. Bactericidal activity ofelectrolyzed acid water from solution containing sodiumchloride at low concentration, in comparison with that athigh concentration. J Microbiol Methods 2002;49:285e293.

8. Nallamuthu N, Braden M, Patel MP. Dimensional changes ofalginate dental impression materials. J Mater Sci: MaterMed 2006;17:1205e1210.

9. Lee JH, Rhee PL, Kim JH, et al. Efficacy of electrolyzed acidwater in reprocessing patient-used flexible upper endo-scopes: comparison with 2% alkaline glutaraldehyde.J Gastroenterol Hepatol 2004;19:897e903.

Ultrasonically nebulised electrolysed oxidising water: a promising new infection control programme for impressions, metals and gypsum casts used in dental hospitalsIntroductionMethodsElectrolysed oxidising water preparation and nebulisationMaterials and experimental designAntibacterial efficacyDimensional accuracySurface qualityMetal corrosionStatistical analysis

ResultsAntibacterial efficacyDimensional accuracySurface qualityMetal corrosion

DiscussionConflict of interest statementFunding source

References