Journal of the International Academy of Periodontology 2017 19/4: 138–144 Efﬁcacy of Two Pre-Procedural Rinses at Two Different Temperatures in Reducing Aerosol Contamination Produced During Ultrasonic Scaling in a Dental Set-up - A Microbiological Study Amruta Arun Joshi, Ashvini Mukul Padhye and Himani Swatantrakumar Gupta Department of Periodontics, MGM Dental College and Hospital, Navi Mumbai, India Abstract Aerosol has been considered one of the main concerns in the dental community because of possible risk of infection transmission. Antiseptics used in the form of pre-procedural rinses can reduce aerosol contamination during dental procedures. The aim of this study is to evaluate and compare the efﬁcacy of 0.05% cetylpyridinium chloride and 0.2% chlorhexidine pre-procedural rinses at 47ºC and 18ºC in reducing aerosol contamination during ultrasonic scaling procedures. Forty subjects were divided randomly and equally into four groups: A1 and A2 to receive cetylpyridinium chloride and B1 and B2 to receive chlorhexidine as a preprocedural rinses. Aerosol produced during the ultrasonic scaling procedure was collected on blood agar plates at three different locations, which were incubated at 37ºC for 48 hours and analysed for bacterial colony forming units (CFU). Cetylpyridinium chloride (0.05%) as a pre-procedural rinse was found to be equally effective in reducing aerosol contamination when compared with 0.2% chlorhexidine rinse (p > 0.05). Also, greater reduction of CFU was found with the use of tempered rinses at 47ºC with a highly statistically signiﬁcant difference (p < 0.001). Cetylpyridinium chloride (0.05%) can be considered as a promising alternative to the gold standard 0.2% chlorhexidine, with tempering the rinse showing the deﬁnite edge. Keywords: Aerosol, pre-procedural mouthrinse, cetylpyridinium chloride, chlorhexidine, ultrasonic scaling, temperature Introduction Aerosols emanating from human ﬂuids and medical procedures are solid or liquid particles, ranging in size from sub- to multi-micrometre, that are suspended in a gas (Occupation Safety and Health Administration). Harrel et al. (1998), Harrel (2004) and Saini (2015) stated that dental aerosols produced with the use of high speed hand pieces, ultrasonic scalers, air polishing devices and abrasion units, are complex and dynamic, wherein some particles are projected onto surfaces, Correspondence to: Amruta Arun Joshi, Department of Periodontics, MGM Dental College and Hospital, Junction of NH4 and Sion-Panvel Highway, Sector 1, Kamothe – 410209, Navi Mumbai, Maharashtra, India. Phone: +919619140326; E-mail: firstname.lastname@example.org © International Academy of Periodontology some settle due to gravity, and others can remain suspended in the air for long periods of time. Elevated levels of these contaminants present in the aerosol have been found during ultrasonic scaling procedures that may get inhaled and transported to alveoli. This process could result in respiratory problems and increase the risk of transmission of tuberculosis, severe acute respiratory syndrome, avian ﬂu and herpetic infections from patients to health care workers (Barbeau 2000; Harrel, 2004). Worrall et al. (1987), Harrel (1996) and Gupta et al. (2014) inferred that use of a pre-procedural antimicrobial mouthrinse may decrease the microbial aerosol contamination to a great extent. According to Lyle (2000), chlorhexidine (CHX) is considered the gold standard rinse due to its broad-spectrum antimicrobial activity and high substantivity. However, CHX presents side effects such as temporary loss of taste, staining of teeth, dryness and soreness of mucosa, and bitter taste. Joshi et al.: Effect of disinfectant temperature on the reduction of aerosol contamination during ultrasonic scaling Therefore, a need arises for evaluation of other equally effective antimicrobial rinses. Albert-Kiszely et al. (2007) and Silva et al. (2009) found that cetylpyridinium chloride (CPC), a member of the quaternary ammonium compound family, is an effective anti-plaque and anti-gingivitis agent. It is monocationic at oral pH that permits dual retention in the oral environment, as both surfactant chains and cationic charges may adsorb to intraoral surfaces, which are lipophilic and anionic. CPC acts primarily by penetrating the bacterial cell membrane, causing leakage of cell components, disruption of bacterial metabolism, inhibition of cell growth and ﬁnally cell death (Quirynen et al., 2005) Complementarily, Konig (2002) demonstrated that temperature appears to be a key determinant in altering the kinetics of the active ingredient in a mouthrinse. Tempering 0.2% CHX rinse to 47ºC demonstrated signiﬁcantly more intensive anti-plaque effect as compared to the solution cooled at 18ºC. In a previous study by Reddy et al. (2012), tempered 0.2% CHX demonstrated reduced bacterial counts in dental aerosols when compared to that of non-tempered CHX and sterile water. A vista that remains to be explored is modulating the temperature/concentration of CPC for enhancing its antimicrobial activity when used as a pre-procedural rinse. Hence, this study attempts to evaluate and compare the efﬁcacy of two pre-procedural mouth rinses containing 0.05% CPC and 0.2% CHX at two different temperatures of 47ºC and 18ºC in reducing aerosol contamination during ultrasonic scaling procedure. Materials and methods This single-centre, double-masked, randomized, prospective, four-group parallel designed study was conducted over a period of 60 days. From September 2015 to October 2015, 40 patients (28 males and 12 females, mean age 32.5 years) were recruited. The patients were included if they had a minimum of 20 natural teeth present, excluding third molars, diagnosed with chronic gingivitis having a sulcus probing depth of ≤ 3 mm, modiﬁed gingival index ≥ 1 (Lobene et al., 1986) and gingival bleeding index > 30% of the sites examined (Ainamo and Bay, 1975). Exclusion criteria were history of known allergies to constituents found in conventional mouthrinses, patients with untreated/grossly carious teeth, those who had undergone non-surgical or surgical periodontal therapy and antibiotic and/or anti-inﬂammatory therapy within the past 6 months. Systemically compromised patients and pregnant and lactating women were also excluded. The study was carried out in accordance with ‘The Code of Ethics of the World Medical Association’ (Declaration of Helsinki, 64th WMA General Assembly, Fortaleza, Brazil, October 2013) for experiments involving humans and the protocol was approved by the Institutional Ethics Review Committee of Mahatma Gandhi 139 Mission’s Dental College and hospital, Navi Mumbai and a detailed informed consent was obtained from the 40 empirically selected patients. Two commercially available solutions of 0.05% of CPC mouthwash (Colgate Plax® Colgate Palmolive Ltd, Mumbai, India) and 0.2% CHX mouthwash (Hexidine® ICPA Health Products Ltd, Ankleshwar, India) were procured from manufacturers and were transferred into identical opaque white bottles labelled as A1, B1, A2 and B2 for the purpose of blinding, by an investigator not involved in the study. The identity of the samples was revealed at the completion of the study. To calculate the sample size at 80% power, the level of signiﬁcance was set at 0.05, to detect a standard deviation (SD) in CFU counts of 18.80 (Konig, 2002) with a ratio of sample sizes in both groups equal to 1. These data, when analyzed by MedCalc Statistical Software version 13.3.1 (MedCalc Software bvba, Ostend, Belgium; http://www.medcalc. org; 2015) yielded a sample size of 10 per group. Computer-generated random numbers were used for randomization, and 40 patients were divided into four groups of 10 each as follows: Group A1 - warm 0.05% CPC (47ºC); Group B1 - warm 0.2% CHX (47ºC); Group A2 - cold 0.05% CPC (18ºC); Group B2 - cold 0.2% CHX (18ºC). For tempering the rinses to 47ºC, 10 mL of speciﬁed mouthwash was heated in a calibrated beaker in a thermostatically regulated water bath. Similarly, the solutions were cooled to 18ºC using a portable cooler (Lab Hosp Corporation, Kalbadevi, Mumbai, India). All patients were asked to rinse with 10 mL of the assigned rinse for 60 seconds, 10 minutes prior to the ultrasonic scaling procedure. Prior to each appointment, a closed operatory was fumigated using 34% formaldehyde and all surfaces were disinfected with 70% isopropyl alcohol (A.B Enterprises, Mumbai). Only one patient was treated per day by the same right handed operator for the entire span of the study, so as to allow the room to be free of aerosols. At the beginning and end of the treatment, the ultrasonic scaler unit (SUPRASSON® P5 Booster, France) was ﬂushed with 0.5% sodium hypochlorite (Clorox disinfectant cleaner) for 10 minutes to ensure disinfection of water lines. Oral prophylaxis for all of the study participants was carried out in a standardized dental chair using distilled water with controlled frequency (30 KHz) and water pressure (0.3 MPa). No person other than the patient, the operator and the assistant was allowed in the vicinity of the operatory within the diameter of four feet to avoid contamination of the operating ﬁeld. Blood agar plates, used to collect airborne microorganisms were prepared as instructed by the manufacturer (Micro Master Labs Pvt Ltd, Thane, Maharashtra, India). Brieﬂy, blood agar base was sterilized at 121ºC for 15 minutes and then cooled to 50ºC in a water bath to which 5% sterile sheep blood was added aseptically. 140 Journal of the International Academy of Periodontology (2017) 19/4 This was dispensed in sterile petri plates and stored at 2 - 8ºC until further use. The blood agar plates mounted ﬁrmly on a board were suspended with strings around the necks of the patient, the assistant and the operator, such that it rested on their chests at a standardized distance of 12 inches from the patient’s mouth with the philtrum of the upper lip serving as a ﬁxed reference point, as shown in Figure 1. These plates were labelled as P, A and O respectively. The labelled plates were exposed at the start of the scaling procedure performed for a duration of 30 minutes, and were left uncovered on the operator’s stool, assistant’s stool and the back rest of the dental chair for additional 30 minutes to collect samples of any aerosolized bacteria. These were then closed with a lid and placed upside down to prevent contamination of blood agar with the moisture on the under surface of the lid. The plates were incubated at 37º C for 48 hours to facilitate the growth of micro-organisms, following which they were analyzed for bacterial colony forming units (CFUs) using a colony counter (Lab Line Stock Centre, Mumbai, India). Statistical analysis Data analysis was done using ‘MedCalc Statistical Software’ version 13.3.1 (MedCalc Software Bvba, Ostend, Belgium). Scores were averaged for age, number of teeth present, MGI and GBI across all groups. All variables were expressed as mean with standard error. The averaged values were tested for normality using the Kolmogorov Smirnov test. The data presented normality and the parametric test of analysis of co-variance (ANCOVA) was used to find which pairs differed signiﬁcantly with respect to temperature difference. Intergroup comparisons were done using unpaired ttests at p < 0.05. Results The non-compliance rate for our study was zero and there were no dropouts. Table 1 represents demographic characteristics of the sampled population. Figure 2 demonstrates mean ± standard error of the mean (SEM) CFU counts for all groups at three different locations, namely the chest areas of patient (P), assistant (A) and operator (O). Among all the groups, the patients’ chest area of group A2 demonstrated maximum mean CFU counts (103.60 ± 4.08) while the assistant’s chest area of group B1 showed the lowest counts (38.50 ± 2.20). On applying an unpaired t-test to compare groups A2 and B2, a statistically signiﬁcant difference was found for the assistant’s chest area location (p = 0.02). However, no statistical difference (p > 0.05) was found on intergroup comparison for all other locations, thereby proving that both mouthrinses were equally effective irrespective of the temperature difference (Table 2). Table 3 represents multiple comparisons using ANCOVA performed to ﬁnd out which pairs differed signiﬁcantly at the 5% level of signiﬁcance with respect to temperature. A highly statistically signiﬁcant difference was found on comparing group A1 with A2 and B1 with B2 (p < 0.001). Table 1: Baseline demographic and clinical characteristics of the four groups. Subjects Group Group Group Group p value A1 A2 B1 B2 Age 32.5 Males/females 7/3 Number of 26.52 teeth MGI 2.5 GBI 46% 32.7 32.64 33.0 NS 6/4 7/3 8/2 NS 27.24 27.11 26.83 NS 2.7 49% 2.3 47% 2.5 43% NS NS NS, not statistically signiﬁcantly different; MGI, modiﬁed gingival index; GBI, gingival bleeding index. Figure 1. The position of the blood agar plates at three different locations, namely the chest areas of the patient (P), operator (O) and assistant (A). Joshi et al.: Effect of disinfectant temperature on the reduction of aerosol contamination during ultrasonic scaling 141 Figure 2. Comparison of mean ± standard error of the mean (SEM) colony forming units (CFU) counts at three different locations in all four groups. Table 2: Results of unpaired t-tests comparing mean colony forming unit (CFU) counts between groups. Location and rinse Patient Group A1 (Warm CPC) Group B1 (Warm CHX) Group A2 (Cold CPC) Group B2 (Cold CHX) Assistant Group A1 (Warm CPC) Group B1 (Warm CHX) Group A2 (Cold CPC) Group B2 (Cold CHX) Operator Group A1 (Warm CPC) Group B1 (Warm CHX) Group A2 (Cold CPC) Group B2 (Cold CHX) t-test p value Signiﬁcance 1.712 0.104 NS 0.095 0.9249 NS 0.083 0.9348 NS 2.383 0.0284* S 0.655 0.5208 NS 1.177 0.2547 NS *p value < 0.05; NS, non-signiﬁcant; S, signiﬁcant; CPC, 0.05% cetylpyridinium chloride; CHX, 0.2% chlorhexidine; 142 Journal of the International Academy of Periodontology (2017) 19/4 Table 3: Results of ANCOVA test comparing mean CFU counts within groups with respect to temperature difference Location and rinse Patient Group A1 (Warm CPC) Group A2 (Cold CPC) Group B1 (Warm CHX) Group B2 (Cold CHX) Assistant Group A1 (Warm CPC) Group A2 (Cold CPC) Group B1 (Warm CHX) Group B2 (Cold CHX) Operator Group A1 (Warm CPC) Group A2 (Cold CPC) Group B1 (Warm CHX) Group B2 (Cold CHX) F value p value Signiﬁcance 53.79 < 0.001* HS < 0.001* HS < 0.001* HS < 0.001* HS < 0.001* HS < 0.001* HS 35.31 25.56 *p value < 0.05; HS, highly signiﬁcant; CPC, 0.05% cetylpyridinium chloride; CHX, 0.2% chlorhexidine This analysis revealed that the groups A1 and B1 (47ºC) showed the maximum reduction in bacterial counts in all three areas as compared to their cold counterparts A2 and B2 at 18ºC. Discussion Under the traditional paradigm, a dental health care worker would be considered to be at high risk for droplet transmission. Legnani et al. (1994) and Bennett et al. (2000) showed that use of ultrasonic scaling procedures resulted in peak concentrations of microbial aerosols in dental treatment rooms. Harrel and Molinari (2004) enumerated three levels of defence in the reduction of aerosols as personal protective barriers, routine use of pre-procedural rinses and high volume evacuation devices. Personal protective equipment, such as a surgical mask, face shield, or eyewear prevents the projection of microorganism-laden particles onto the mucosal membranes. However, they do not counter an ever present hazard of inhaling particulate aerosols when near an infectious patient. CHX has already proven its efﬁcacy as a pre-procedural rinse in reducing bacterial aerosol contamination with the use of an air polisher, as studied by Logothetis and Martinez-Welles (1995) and ultrasonic scaler as demonstrated by Sawhney et al. (2015). Konig (2002) inferred that increasing the temperature of 0.2% CHX to 47ºC was effective in reducing vital plaque content, as assessed by the vital ﬂuorescence technique. A 25% increase in bacteria kill rate was observed following irrigation with the tempered CHX solution. This effect was not solely due to the physical parameter “temperature,” as the combination of heat and water resulted in unchanged vitality rate of micro-organisms cultured from plaque samples. The biologic basis and rationale for this anti-plaque effect could be attributed to enhanced bactericidal activity at an elevated temperature. Also, CHX has been found to have an increase in sporicidal effect when the temperature was increased to 60 - 70ºC (Shaker, 1986). However, use of CHX is not free of undesired side effects. The potential of CPC as an anti-plaque agent is well documented in the literature (Silva et al., 2009; Garcia et al., 2011). But there exists lacunae regarding the role of tempered 0.05% CPC as a pre-procedural rinse to control aerosol contamination. The Scientiﬁc Committee on Consumer Safety (2015) reported that degradation of pure CPC occurs at 130ºC, with complete thermal decomposition at 234ºC. Konig (2002) also reported that tempering rinses to 47ºC exhibits neither painful sensations nor permanent pulpal damage. To the best of our knowledge, this study is the ﬁrst to simultaneously assess the efﬁcacy of two commercially available mouthrinses, 0.05% CPC and 0.2% CHX, when used as pre-procedural rinses at two different temperatures, in reducing aerosol contamination. Various investigators (Bentley, 1994; Chiramana, 2013) have studied the spread of aerosols within the range of 2 to 6 feet. In the present study, the perimeter of the operatory was limited to a diameter of 4 feet and the aerosols were collected on blood agar plates, which are considered as a valid non-selective medium for culturing airborne bacteria (Johnston et al., 1978). These plates were positioned at three different locations at a distance of 12 inches from the patient’s philtrum as performed previously by Gupta et al. (2014). Further exposure of these plates for an additional 30 min after ultrasonic scaling was done so as to allow gravitational settling of airborne bacteria (Larato et al., 1966). Joshi et al.: Effect of disinfectant temperature on the reduction of aerosol contamination during ultrasonic scaling The results of our study suggest that the pre-procedural rinse containing 0.05% CPC is as effective as 0.2% CHX in reducing aerosol contamination, which is in agreement with the study conducted by Feres et al. (2010). Our results are also in accordance with the study conducted by Reddy et al. (2012), where the tempered solutions heated to 47ºC were found to be signiﬁcantly more effective in reducing aerosol contamination at the chest areas of patient, operator, and assistant as compared to the cold solutions used at 18ºC. Nevertheless, on comparing mean bacterial counts at the assistant chest area after rinsing with only cold CPC and CHX solutions, a statistically signiﬁcant difference was noted, indicating CPC was more effective than CHX at 18ºC. The CFU estimation in our study includes only aerobic bacteria capable of growth on blood agar plates; anaerobic bacteria and viruses that require specialized media were not isolated, which needs to be addressed in further investigations. No attempt was made to differentiate these bacteria based on cultural characteristics. Within the limits of our study, the emerging evidence beckons towards the role of tempered CPC in effectively reducing aerosol contamination. Nonetheless, these data need to be interpreted with caution, as sample size was small. Hence, there is a need for longitudinal studies of a single cohort with a larger sample size demonstrating efﬁciency of tempered CPC in mitigating aerosol bacteria and viruses. Conclusion Overall, the results of our investigation clearly indicate that a pre-procedural rinse containing 0.05% cetylpyridinium chloride can be considered as a promising alternative in reducing aerosol contamination during ultrasonic scaling procedures when compared to the gold standard 0.2% chlorhexidine, with tempering the rinse showing the deﬁnite edge. Also, it can be concluded that the amount of viable bacteria in aerosol is maximum at the patient’s chest area followed by the operator and assistant in a descending manner, thus reinforcing the use of personal protective barriers to minimize the risk to dental professionals. 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