Comparison of the Efficacies of Disinfectants To Control Microbial Contamination in Dental Unit Water Systems in General Dental Practices across the European Union

Water delivered by dental unit water systems (DUWS) in generaldental practices can harbor high numbers of bacteria, includingopportunistic pathogens. Biofilms on tubing within DUWS providea reservoir for microorganisms and should be controlled. Thisstudy compared disinfection products for their ability to meetthe American Dental Association's guideline of <200 CFU ·ml^–1 for DUWS water. Alpron, BioBlue, Dentosept, Oxygenal,Sanosil, Sterilex Ultra, and Ster4Spray were tested in DUWS(n = 134) in Denmark, Germany, Greece, Ireland, The Netherlands,Spain, and the United Kingdom. Weekly water samples were testedfor total viable counts (TVCs) on yeast extract agar, and, wherepossible, the effects of products on established biofilm (TVCs)were measured. A 4- to 5-week baseline measurement period wasfollowed by 6 to 8 weeks of disinfection (intermittent or continuousproduct application). DUWS water TVCs before disinfection rangedfrom 0 to 5.41 log CFU · ml^–1. Disinfectants achievedreductions in the median water TVC ranging from 0.69 (Ster4Spray)to 3.11 (Dentosept) log CFU · ml^–1, although occasionalhigh values (up to 4.88 log CFU · ml^–1) occurredwith all products. Before treatment, 64% of all baseline samplesexceeded American Dental Association guidelines, compared toonly 17% following commencement of treatment; where tested,biofilm TVCs were reduced to below detectable levels. The antimicrobialefficacies of products varied (e.g., 91% of water samples fromDUWS treated with Dentosept or Oxygenal met American DentalAssociation guidelines, compared to 60% of those treated withSter4Spray). Overall, the continuously applied products performedbetter than those applied intermittently. The most effectiveproducts were Dentosept and Oxygenal, although Dentosept gavethe most consistent and sustained antimicrobial effect overtime.

INTRODUCTION

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Introduction

Dental unit water systems (DUWS) are used to irrigate the oralcavity during dental treatment. Water delivered from these devicesis not sterile and has been shown to contain high numbers ofbacteria (9, 38, 52). Biofilms accumulating on the inner surfaceof the tubing can be responsible for high levels of contaminationof water delivered by DUWS (21, 38, 51). A number of surveyshave reported that the majority of DUWS are supplied by tapwater (54). European Union (EU) guidelines recommend that tapwater should be delivered at <100 CFU · ml^–1(2); however, once the water enters the DUWS the number of bacteriacan increase, with numbers as high as 1.6 x 10⁸ CFU ·ml^–1 having been recovered in the outflow (12). Such highnumbers can result from numerous factors, including ambienttemperatures, stagnation, and the presence of biofilm (30).In the United States, the American Dental Association (ADA)and the Centers for Disease Control and Prevention have proposeda guideline for DUWS water of $≤$ 200 CFU · ml^–1 (equivalentto that required for dialysis water) (1). Currently, dentistsacross the world have little or no evidence-based guidelinesto control bacterial numbers in DUWS.

Typically, patients in the EU visit general dental practices(GDPs) every 6 months, with over 20 million visits per yearin 1998 in one large EU country alone (5). During almost everyvisit, the patient and the dental health care staff are exposedto the water from DUWS. These medical devices have the potentialto harbor opportunistic or frank pathogens, and Legionella pneumophila,Mycobacterium spp., Pseudomonas aeruginosa, and Candida spp.have been recovered from DUWS (4, 7, 28, 34, 46, 56). Exposureof dental personnel to such pathogens can be inferred from thefinding that dentists have significantly higher antibody titersto L. pneumophila than individuals in other, equivalent employmentsectors (10, 11, 24, 35), and asthma may be associated withoccupational exposure to endotoxin in aerosols from contaminatedDUWS (29). In addition, P. aeruginosa isolated from a DUWS wasfound to be responsible for the hospitalization of two patientsfollowing a visit to a dental surgery (19). The presence ofpathogens has further implications when one considers that failureof the three-in-one hand piece antiretraction valve (32, 37)could result in microorganisms being transferred among patients(cross-infection).

A wide range of disinfectant products are now being developedfor use in DUWS, and these have been evaluated using a varietyof approaches (39, 48, 49), although rarely have these productsbeen compared in a general dental practice setting. The aimof this study was therefore to evaluate and compare the levelsof control achievable by the application of different disinfectantsto DUWS in GDPs.

MATERIALS AND METHODS

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Materials and Methods

Selection of GDPs.
GDPs in seven European countries (Denmark, Germany, Greece,Ireland, Spain, The Netherlands, and the United Kingdom) werecontacted by mail and invited to participate in the study. Representativedental practices that (i) were willing to participate in trialsof disinfectants, (ii) would allow sampling to evaluate theefficacy of the product, and (iii) would also comment on theirease of use were selected from each country. The selected practicesused units supplied by bottled water, mains water, or headertank systems. All the samples were collected and processed inthe same time frame and according to the same protocols in allcountries.

Modification of DUWS.
In the case of mains water systems, dental units were modifiedto facilitate the addition of a disinfectant to the water usedin the DUWS by fitting an externally mounted purge system (MicryliumLaboratories, Inc., Phoenix, AZ). This was carried out by aqualified dental unit installer following the manufacturer'sinstructions. The system consisted of a 700-ml bottle and twoswitches; one to toggle between municipal and bottle water suppliesand the other to turn the air purge on or off. Using this system,the municipal water supply could be bypassed and the bottleused to add a disinfectant intermittently to the DUWS, e.g.,overnight or during the weekend, after which time the municipalwater supply could be reconnected for use during treatment.Alternatively, if a disinfectant was to be used continuously,it was added to the bottle supply, which was subsequently usedthroughout patient treatment sessions. Air purging of the waterlines,if recommended by the disinfectant's manufacturer, could alsobe carried out using this system.

Sampling of DUWS.
A test period consisted of 4 to 5 weeks of baseline value measurementfollowed by 6 to 8 weeks of disinfection. This period was alwayspreceded by a nondisinfecting period of at least 14 days toallow biofilm formation (48) and washout of residual productwhen multiple products were tested serially in one DUWS. Inorder to measure DUWS contamination, samples (100 ml) were takenweekly for 4 to 5 weeks before and for 6 to 8 weeks after disinfection.Samples were collected at approximately mid-morning from thedistal outlets of the three-in-one syringes of all DUWS. Wherepossible, a 1-centimeter length of the waterline tubing supplyingthe three-in-one syringe was cut out for biofilm analysis (48).One tubing sample was taken during pretreatment baseline measurement,and one was taken after the disinfection period was finished.All the staff performing the disinfecting and sampling procedureswere instructed to use the same methodology.

Water samples were processed as described previously (45). Approximately100 ml of water sample was passed through a sterile nozzle intoa sterile water bottle containing 0.1 g sodium thiosulfate toremove any residual disinfectant (Abinghurst Ltd., Northampton,United Kingdom). Samples were returned to the laboratory ina cool box (4 to 8°C) within 3 h and then filtered through100-ml capacity, 0.2-µm analytical test filter funnels(Techware, Poole, United Kingdom) in order to recover the waterbornemicroorganisms. The membrane was removed from the funnel withsterile forceps and placed in a screw cap sterile container(Elkay Products Inc., Shrewsbury, MA). Microorganisms were washedfrom the membrane by vortexing the container for 1 min in 10ml of sterile phosphate-buffered saline (PBS).

TVCs and detection of Pseudomonas aeruginosa in water samples.
Total viable counts (TVCs) and counts of Pseudomonas aeruginosa(as a marker of the presence of opportunistic pathogens) werecarried out as described previously (45), using decimal dilutionsof the water samples (in sterile PBS) on (i) yeast extract agarfor environmental microorganisms (3), incubated at 37°Cfor up to 3 days, and (ii) CFC supplemented with SR103 for Pseudomonasspp. (26), incubated at 37°C for up to 48 h. Colonies werecounted on day 3, and the TVC was calculated as CFU ·milliliter^–1. Colonies on the Pseudomonas agar were confirmedas P. aeruginosa according to color, ammonia production, andoxidase activity. TVCs were used as the definitive measure oftotal microbial contamination of the water passing through theDUWS. This was compared with the U.S. guideline for DUWS of $≤$ 200 CFU · ml^–1 as recommended by the American DentalAssociation (1). All of the participants used the same microbiologicalmethods (46). The limit of detection was 10 CFU · ml^–1.

Analysis of biofilm accumulation.
Biofouling was assessed by measuring the TVCs of defined areasof the inner surface of the tubing as previously described (49).The DUWS tubing was sectioned horizontally and rinsed twicein nonflowing sterile PBS to remove nonadhered cells, and thesurface biofilm was removed by scraping it into 1 ml of sterilePBS with a sterile dental probe. The TVC was then determinedas described above, and the limit of detection was 10 CFU ·cm^–2.

Evaluation of disinfectant agents in GDP DUWS.
Disinfectants (Table 1) were selected from a previous laboratorymodel evaluation study according to their ability to kill microbialcells and remove biofilm from the inner surfaces of DUWS tubing(47, 48). Products were supplied and used either intermittentlyor continuously (see below) according to the manufacturers'instructions. Treatment was administered each evening priorto the closing of the surgery, and on weekends, for up to 8weeks. Where necessary, the disinfectant was removed from thesystem by flushing the following morning before patient treatmentbegan. The removal of disinfectant was confirmed either by observingthe loss of product color from the outflowing water or froma negative potassium iodide detection test for hydrogen peroxide-basedproducts.

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TABLE 1. Summary of active agents, concentrations, type of application, and sources of the products evaluated

Intermittent application of disinfectants.
At the end of the working day, 200 to 250 ml of disinfectantwas placed in the reservoir bottle that supplied the dentalunit with water, and the solution was run through the systemfor 2 minutes. The unit was then turned off, and the disinfectantwas left overnight in situ. At the beginning of the next workingday, the bottle was disconnected and the residual solution pouredfrom the bottle without rinsing. The bottle was filled withwater and connected to the unit, after which the system wasflushed until all of the remaining solution had been dischargedfrom the outflow of the DUWS (residual product was detectedas described above). The dental unit was then ready for dailyuse.

For intermittent disinfection of mains water-supplied systems,the unit was disconnected from the mains water supply and connectedto the bottle with disinfectant by an externally mounted purgesystem (see "Modification of DUWS" above). The product was administeredas described above, and at the beginning of the next workingday the DUWS was reconnected to the mains supply for use indaily practice. The disinfectants applied intermittently wereBioBlue, Sanosil, Sterilex Ultra, and Ster4Spray (Table 1).

Continuous application of disinfectants.
The DUWS water reservoir bottle was filled with disinfectantand connected to the unit, and the disinfectant was run throughthe system for 2 minutes. The dental unit was then ready fordaily use, and the disinfecting agent was present in the outflowingwater throughout the day. The disinfectants used for continuoususe were Alpron, Oxygenal, and Dentosept (Table 1). The productswere left in situ overnight.

Statistical analysis.
The results for pre- and postdisinfection for each product werecompared using a Mann-Whitney test for two independent groupsof samples. To test for statistical differences between allproducts, a Kruskall-Wallis test was used. Mann-Whitney testswere subsequently used to compare products with all other productsseparately (SPSS 12.0.1 for Windows; SPSS, Chicago, IL).

RESULTS

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Results

Dental unit water systems and general dental practices.
Seven different disinfection products were tested in seven Europeancountries in a total of 134 dental units (Table 2). The averageperiod of disinfection was 6.9 weeks; due to methodologicalissues, some GDPs had to withdraw units from the disinfectionperiod earlier than the intended 8 weeks. Alpron was testedin 37 units (28% of total units), Sanosil in 30 units (22%),BioBlue in 26 units (19%), Oxygenal in 15 units (11%), and Dentosept,Sterilex, and Ster4Spray in 11 (8%), 10 (7%), and 5 (4%) units,respectively (Table 2). Source water TVCs were measured previouslyand ranged from 0.77 to 2.31 log CFU · ml^–1 (46).In the general dental practices visited, the TVCs of the waterflowing from the three-in-one syringe ranged from 0 (i.e., nodetectable colonies) to 5.41 log CFU · ml^–1 duringbaseline pretreatment measurements. Before treatment, 64% ofall baseline TVCs measured in samples from all countries (n= 585) were above the ADA recommendation of $≤$ 200 CFU ·ml^–1, while during treatment, 83% of all measurements(n = 926) were below the ADA guideline. Baseline values fromunits in Germany were excluded from this analysis because theseDUWS had an integrated disinfection system which provided anautomatic continuous disinfection of the unit, making it impossibleto determine nontreated baseline values.

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TABLE 2. Criteria used for evaluation of efficacies of the disinfection products in all countries

Effect of disinfection.
The following products were tested for their ability to produceTVCs compliant with the ADA recommendation of $≤$ 200 CFU ·ml^–1 ( $≤$ 2.3 log CFU · ml^–1) in outflow waterof the DUWS (Table 1).

(i) Alpron.
Alpron was tested in a continuous mode in 37 DUWS (Table 2).The use of Alpron resulted in a significant decrease in microbialcounts to below 200 CFU · ml^–1 (P < 0.01) incomparison to the baseline values prior to treatment (Fig. 1).Eighty-seven percent of treated water samples achieved the ADAguideline, and the change in median TVC between the baselineand treated water samples was 2.10 log CFU · ml^–1(Table 2).

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FIG. 1. Median water TVCs before ( ${blacksquare}$ ) and after ( ${square}$ ) the start of disinfection of DUWS for all products. Median values are shown as black lines in the boxes. The boxes represent the interquartile ranges, and the whiskers indicate the minimum and maximum values up to 1.5 times the IQR (made with SPSS 12.0.1 for Windows). ${circ}$ , outlier value (between 1.5 and 3 times the IQR); *, extreme value (more than 3 times the IQR); ${dagger}$ , statistically significant change.

(ii) BioBlue.
BioBlue was tested in intermittent mode in 26 DUWS (Table 2),and a significant decrease in microbial counts (P < 0.01)compared to the baseline results was found (Fig. 1). The meanpercentage of treated water samples delivering water containingless than 200 CFU · ml^–1 was 74%, and the changein median TVC between the baseline and treated water sampleswas 1.79 log CFU · ml^–1 (Table 2). The error barsin Fig. 1 representing the range of values demonstrate thata number of samples (mainly in Ireland) showed high values duringtreatment, indicating an inconsistent effect of the disinfectant.

(iii) Dentosept.
Dentosept was tested in 11 dental units (Table 2) in a continuousmode. There was a significant difference (P < 0.01) betweenthe baseline and treatment values (Fig. 1), with 91% of thetreated water samples having a TVC of $≤$ 200 CFU · ml^–1and a change of 3.11 log CFU · ml^–1 between thebaseline and treated water samples (Table 2). Extreme and outliervalues that were measured (Fig. 1) indicate the occurrence ofoccasional high values during treatment. An outlier value wasdefined as being between 1.5 and 3 times the interquartile ranges(IQR), while extreme values were more than 3 times the IQR.

(iv) Oxygenal.
Oxygenal was tested in 15 DUWS (Table 2) in a continuous mode.A significant difference (P < 0.05) between pre- and posttreatmentTVCs was found, and 91% of the treated water samples had a TVCof $≤$ 200 CFU · ml^–1, with a change between the medianbaseline and treated water samples of 1.24 log CFU ·ml^–1 (Table 2). One extreme value that was measured inThe Netherlands (Fig. 1) was due to a problem with the applicationof the product to the DUWS, and the dentist stopped participatingin the study after this high value was measured.

(v) Sanosil.
Sanosil was tested in intermittent mode in 30 dental units (Table2). A significant decrease (P < 0.01) between results forbaseline and treated water samples was found. The percentageof treated water samples that achieved the ADA guideline was83%, and the change in median TVC between the baseline and testwater samples was 1.57 log CFU · ml^–1 (Table 2).The broad range of the error bars (Fig. 1), indicating a variableeffect of the product, was caused mainly by two units in Spainin which high values were found during treatment; these valueswere defined statistically as outliers in the longitudinal study(Fig. 2).

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FIG. 2. Longitudinal analysis of water TVCs during 8 weeks of disinfection of DUWS. Disinfection starts at week 1; week 0 represents the median baseline value. Median values are shown as black lines in the boxes. The boxes represent the interquartile ranges, and the whiskers indicate the minimum and maximum values up to 1.5 times the IQR (made with SPSS 12.0.1 for Windows). ${circ}$ , outlier value (between 1.5 and 3 times the IQR); *, extreme value (more than 3 times the IQR).

(vi) Sterilex Ultra.
Sterilex Ultra was tested in 10 DUWS in intermittent mode (Table2). The overall reduction in median TVC values of the baselineand the treated water samples was statistically significant(P < 0.05) (Fig. 1). Seventy-eight percent of the treatedwater samples were found to have less than 200 CFU ·ml^–1, with a change of 1.50 log CFU · ml^–1between the median values of the baseline and treated watersamples (Table 2). There was a considerable difference in effectbetween countries, and the broad range of the data indicatedby the error bars was caused mainly by TVCs from units in Ireland(one value was defined as an extreme outlier [Fig. 2]). However,the median TVC during treatment in Ireland was below 200 CFU· ml^–1, with 68% of the treated water samples beingbelow this limit.

(vii) Ster4Spray.
Ster4Spray was tested in intermittent mode in five units (Table2). The median TVC was lower than the ADA guideline after treatment,but this difference was not statistically significant (Fig.1). The distance between the error bars, representing the rangeof the values, indicates an inconsistent effect of the product(Fig. 1). Sixty percent of treated water samples were foundto have TVCs of less than 200 CFU · ml^–1, witha change of 0.69 log CFU · ml^–1 between the medianvalues of baseline and treated water samples (Table 2).

Longitudinal analysis of total viable counts during treatment.
A week-by-week comparison of products was performed to see ifproducts differed in their patterns of disinfection, as it washypothesized that some products might show a more rapid or sustainedeffect on TVC reduction than others (Fig. 2). Dentosept gavea marked and consistent reduction in median TVC values duringthe treatment period. The use of Alpron and, to a lesser extent,Oxygenal also caused a steady decline in median TVCs over time,whereas the other products did not have this effect (Fig. 2).

Dental units in which a product was tested for 6 weeks or morewere included in a longitudinal analysis of the weekly TVCsto assess the presence of solitary high values in an otherwiselow-value series. Samples treated with products in a seriesthat yielded a median TVC of greater than 200 CFU · ml^–1were excluded from this analysis. A solitary high TVC valueflanked by low values in the weeks before and after such anoccurrence was found for 26 of 111 samples (23%) (Table 2).Of these DUWS, seven units (6%) showed a second high value duringthe same testing period. The analysis showed that this phenomenonoccurred with all products tested. Alpron was the product leastprone to incidental high values, with 26 out of 29 units showingno evidence of high solitary TVC values. BioBlue, Sanosil, andOxygenal all suffered from these high values in about 20% ofdental units. Single high TVCs were present in almost half ofthe units that used Sterilex and Dentosept and in three of thefive units that used Ster4Spray (Table 2).

Biofilm.
Biofilm samples from 28 DUWS in two countries (Greece and theUnited Kingdom) were evaluated before and after disinfectionwith the products Alpron, BioBlue, and Sanosil. Comparison ofbaseline and posttreatment values showed a significant decreasein median TVCs (P < 0.01) for all products tested. Baselinemedian TVCs were 1.45 log CFU · cm^–2 for Alpronand 1.04 log CFU · cm^–2 for both Bioblue and Sanosil.All products reduced the median TVCs after the treatment periodto levels below the detection limit, corresponding to a medianpercent reduction of >99% for all three products (data notshown).

Detection of pseudomonads.
Pseudomonads were detected in Germany only. Three units werepositive before the product evaluation period, and two of thoseunits tested positive during treatment as well. Counts rangedfrom 5 to 125 CFU · ml^–1 (data not shown). Allpseudomonads found were confirmed to be Pseudomonas aeruginosa.

Ease of use and adverse events.
A number of practical issues associated with the use of particularproducts were identified. These issues included dissolutionof tubing and a lightweight nonpolypropylene plastic bottleon one unit (Sterilex Ultra), blocking of the tubing in thedental unit (Sterilex Ultra, three units; Ster4Spray, two units),and foaming and brownish discoloration of the dental unit waterand staining of dental equipment surfaces in four units (Alpron).All Micrylium purge dispenser systems fitted to mains-fed DUWSthat participated in this study had to be repaired at leastonce due to mechanical failures.

Assessment of overall efficacy of disinfectants.
A statistically significant difference in median TVC reduction(P < 0.01) was found between products used either continuouslyor intermittently (Table 1). The baseline median values forthese two groups showed no significant difference, with 2.54log CFU · ml^–1 for continuously used products and2.49 log CFU · ml^–1 for intermittently used products.Treatment median values were 0.83 log CFU · ml^–1for continuously used products and 1.25 log CFU · ml^–1for intermittently used products.

There were significant differences between agents in terms ofwater TVCs (P value from Kruskal-Wallis-H). Further statisticaltests showed that Dentosept was significantly more effectivethan all other agents (P = 0.0001). Oxygenal was significantlymore effective than all agents except Dentosept (P = 0.001).

DISCUSSION

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Discussion

The biofilm on the inner surface of the tubing of dental unitsprovides a continuous reservoir for microorganisms (6, 33, 41,50-52). Not only patients but also dentists and dental personnelare at risk of being infected with opportunistic pathogens suchas Pseudomonas or Legionella species by means of cross-infectionor following aerosol formation from water emanating from DUWS(8, 27, 55).

Various products have now been developed to reduce the numberof bacteria delivered to the patient by decontamination of thewater used in DUWS (12, 13, 17, 18, 20, 31, 36, 38, 40, 49),and although these have been tested in a variety of settings,no multiproduct comparison in GDP surgeries has been done untilnow. In this trial, seven antimicrobial products approved foruse in dental units were tested in seven EU countries for theirability to reduce TVCs to meet the ADA guideline of 200 CFU· ml^–1 in the outflowing water (1) (Tables 1 and2). Other criteria examined were the presence of occasionalhigh TVC values in a series of low values, material incompatibilityissues, the extent of the reduction of water TVCs during treatmentcompared to baseline TVCs in the same unit, and, where possible,the effect of disinfectants on biofilm TVCs.

Alpron.
Alpron has been tested in a number of studies. Smith et al.(39) found that Alpron reduced the TVC to a level similar tothat in drinking water when used continuously over a periodof 6 to 13 weeks. In the present study, during continuous useof Alpron, the bacterial counts of the outflowing water werereduced to below the ADA threshold of 200 CFU · ml^–1in 87% of samples. Biofilm samples also showed a significantreduction in TVCs when measured before disinfection and after8 weeks of disinfection with Alpron. This agrees with a previousfinding that Alpron completely removes the biofilm in dentaltubing (48). Others have shown that Alpron is also effectivein reducing TVCs to acceptable levels (i.e., <200 CFU ·ml^–1) when used intermittently (25).

BioBlue.
Kettering et al. (15) tested BioBlue (then named Bio2000) usinga discontinuous regimen and concluded that it was not able toreduce the TVC to below 200 CFU · ml^–1 when usedin combination with tap water. Although BioBlue achieved levelsof less than 200 CFU · ml^–1 in 74% of samples inthe present study, in some DUWS the measurements fluctuated,indicating an inability of the product to consistently maintainthe TVC at below 200 CFU · ml^–1 between disinfectionperiods. A factor that might explain these findings could bethe presence of weakened biofilm remnants after overnight treatmentthat slough off during the day. In this study, BioBlue reducedbiofilm TVCs by >99%, although in a previous study in a laboratorymodel, BioBlue achieved only a 53% reduction of the tubing'sinner surface biofilm coverage (48).

Dentosept.
Overall, Dentosept was very effective in reducing TVCs and maintainingthe microbial load to levels well below 200 CFU · ml^–1during this study, although occasional incidental high valueswere found. This product delivered the greatest reduction inTVCs, which resulted in the combined lowest number of sampleswhich failed to meet the ADA guidelines during the treatmentperiod. This is consistent with previous data which showed thatdaily application of Dentosept reduced TVCs in dental unitsto meet the ADA guideline (14).

Oxygenal.
Oxygenal has been evaluated previously in a laboratory DUWSbiofilm model (48) and demonstrated a 99.2% reduction of biofilmcoverage of dental tubing, while no TVC could be detected inthe outflowing water after a single treatment. In the presentstudy, Oxygenal achieved a statistically significant reductionin TVC, resulting in only a small percentage of samples thatexceeded the ADA guidelines during the longitudinal study; theextent of the decrease was not very large, probably as a resultof the low baseline values (Fig. 1).

Sanosil and Sterilex Ultra.
Tuttlebee et al. (43) compared Sanosil (5 to 8 weeks) and SterilexUltra (7 to 20 weeks) in GDPs using once-weekly disinfectionof the DUWS, and both products reduced TVCs significantly tobelow the ADA guideline of $≤$ 200 CFU · ml^–1. Usingmicroscopy imaging techniques, a significant reduction in biofilmcoverage has also been observed (43, 48). Similar results werereported for both products in laboratory models of DUWS (22,48). In the present study, Sanosil and Sterilex Ultra significantlyreduced or maintained TVCs below ADA guidelines; Sanosil alsohad a significant effect on biofilm TVC, as described previously(48). Unfortunately, the application of Sterilex Ultra was stoppedin three DUWS due to material incompatibility issues. The problemof blocking of DUWS tubing when using Sterilex Ultra has beendescribed before (43) and may be caused by sedimentation ofexcess Sterilex Ultra that has not dissolved settling onto thetubing's narrow inner surface. In a previous study, SterilexUltra initially reduced bacterial counts to levels below theADA guideline but failed to maintain low TVCs during an 8-weekfollow-up period (16). This finding concurs with the relativelyhigh prevalence of incidental high values that was found inthe present study (Table 2), which may have been caused by regrowthand detachment of the biofilm.

Ster4Spray.
Ster4Spray was tested in only five units and showed variableTVCs. The product seemed prone to incidental high values, butfurther research with more DUWS should be carried out in orderto obtain more conclusive data. A previous study which testedsix units reported that Ster4Spray reduced the TVC to below200 CFU · ml^–1 in 90% of samples collected over10 days (23). However, the number of units tested was also small,and the testing period was too short to be able to conclusivelyassess the long-term efficacy of the product.

Low median baseline values and percent reduction.
The antimicrobial efficacy of a disinfectant should be assessedby both the reduction in TVC (log CFU · milliliter^–1)and the mean percent reduction in absolute TVC (Table 2). Thisis because even a relatively small reduction in log CFU ·ml^–1 can correspond to a high percent reduction in absoluteTVC when the initial (baseline) median TVCs are low. This effectwas clearly visible in the Oxygenal data in Table 2, where amean reduction in log CFU · milliliter^–1 of 1.24corresponds with an absolute TVC reduction of 97%. Median baselineTVC values in both countries where Oxygenal was tested (Irelandand The Netherlands) were lower than 200 CFU · ml^–1,and in this case the percent reduction was a more accurate indicatorof the efficacy of the product than the absolute reduction ofmedians in log CFU · milliliter^–1.

Longitudinal analysis of TVCs.
The incidental high TVC values found in the longitudinal analysisof the data during disinfection may have been caused eitherby contamination of the water sample by biofilm detaching fromthe inner surface of the dental tubing or by a regrowth of bacteriadue to a lack of compliance by dental staff in applying thedisinfectant according to the manufacturer's instructions. Compliancehas been shown to be an issue when nonautomated procedures arein place (53). Several chair manufacturers, e.g., KaVo and Siemens,now integrate an automated water disinfection system with electronicmonitoring in their DUWS. This reduces the occurrence of problemsthat may be the result of accidental noncompliance. The occurrenceof the aforementioned occasional high TVC values has not beendescribed before and can go unnoticed if a unit is tested onlyperiodically. Such levels of bacteria might pose a threat todentists and patients. They also indicate that the water qualityof units should be tested at several time points so that anaccurate assessment of the numbers of microorganisms in theoutflowing water can be made.

The nature of this multicountry, multiproduct study using dentalunits of various design did not make it possible to have equalnumbers of identical units using each disinfectant. However,the longitudinal analysis gave insights into the extent andconsistency of the antimicrobial effects of particular agents.Continuous use of Dentosept (and to some extent Alpron and Oxygenal)produced a marked and sustained reduction in water TVC (Fig.2).

Ease of use and adverse events.
A number of practical issues associated with the use of particularproducts were reported by the dentists. Alpron required a 3-hourtime commitment for the three-stage initial application phase,and in some practices, its use resulted in foaming, staining,and a brown discoloration of the water. Ster4Spray and SterilexUltra caused blocking of the tubing in some DUWS. However, materialcompatibility issues are often recognized by manufacturers,who may subsequently recommend specific products to be usedwith their equipment; for example, the manufacturers of SterilexUltra advise against the use of nonpolypropylene bottles. Therefore,dental practitioners must consult with the manufacturer of theirDUWS prior to introducing any chemical agent, as this may otherwiseinvalidate their warranty. Other studies have raised additionalconcerns. Brass coupling connectors used in domestic water systemscan be corroded by too harsh a chemical treatment, and thismay lead to leaks and failure of the DUWS (44). Disinfectantscould come into contact with the oral cavity, and this mightaffect the adhesion of resins to both enamel and dentin, sothat restorations could fail prematurely (36, 42). The riskof chemical exposure of the patient and health care workersalso has to be considered when using disinfectant products inDUWS (17).

In GDPs supplied by the mains water supply, independent waterdispensers (Micrylium, Toronto, Canada) were retrofitted tothe mains supply line in order for disinfectants to be addedto the DUWS. All dispensers had to be repaired or replaced atsome time during the study. Bottle-fed units needed no adaptation,and few problems were reported.

Evaluation of product efficacy.
In conclusion, this study, in which a number of disinfectantshave been compared under realistic conditions of use in GDPsin several countries, has identified products that reduced theTVCs in DUWS to levels recommended by the ADA. When furtherstatistical analyses were performed, in which all agents werecompared with each other, the most effective products were Dentoseptand Oxygenal. However, Dentosept gave the most consistent andsustained antimicrobial effect over time. Overall, the continuouslyapplied products performed better than the intermittently appliedproducts.

ACKNOWLEDGMENTS

This investigation was supported by (i) the Primary Dental CareR&D Programme and National Research Register research grantPDC97-213 from the NHS Executive, North West, Warrington, UnitedKingdom, and (ii) the European Commission, specific RTD program"Quality of Life and Management of Living Resources 4.1 Environmentand Health."

We acknowledge the assistance of the disinfectant manufacturersfor the supply of disinfectants and of the dental teams fortheir time and for allowing access to the DUWS.

No conflicts of interest are reported by any of the authors.

FOOTNOTES

* Corresponding author. Mailing address: Centre for Emergency Preparedness and Response, Health Protection Agency, Porton Down, Salisbury SP4 0JG, United Kingdom. Phone: 44 (0) 1980 612643. Fax: 44 (0) 1980 612731. E-mail: jimmy.walker{at}hpa.org.uk.

Present address: Biosciences, Quest International, Ashford,Kent TN24 0LT, United Kingdom.

REFERENCES

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