In vitro comparison of antifungal activity of conventional alcohol sprays and antimicrobial photodynamic therapy on acrylic denture resin

Abstract

BACKGROUND:

Traditionally, alcohol sprays are used for disinfection of acrylic-base denture surfaces. A limited number of studies have assessed the role of antimicrobial photodynamic therapy (aPDT) in this regard; however, it remains debatable whether conventional alcohol sprays are superior to aPDT in terms of antifungal activity or vis versa.

OBJECTIVE:

The aim of the present in vitro study is to compare the antifungal activity of conventional alcohol sprays and aPDT on acrylic denture resin.

METHODS:

Individuals wearing complete dentures at least on one arch were included. Dentures were randomly divided into three groups. Groups 1-3 were disinfected with an alcohol-based antiseptic spray and aPDT, respectively. Assessment of oral yeast growth was done using swab samples. The culture mediums were incubated at 37 ${}^{\circ }$ C for 72 hours and viewed through a microscope. The numbers of colony forming units (CFU/ml) were determined. $P<$ 0.05 were considered statistically significant.

RESULTS:

At baseline, the mean CFU/ml in Groups 1–3 were comparable. After disinfection, a statistically significant reduction in microbial CFU/ml was observed in Groups 1 ( $P<$ 0.05) and 2 ( $P<$ 0.05) compared with baseline. In Group 3, there was no difference in CFU/ml throughout the study. After disinfection, there was no difference in microbial CFU/ml in dentures in Groups 1 and 2.

CONCLUSION:

Conventional alcohol sprays are as effective as aPDT towards reducing oral yeasts CFU/ml on acrylic denture resin.

1. Introduction

Removable dental prostheses such as dentures are often used for oral rehabilitation of individuals with partial and/or complete edentulism [1, 2]. However, infection transmission between dentists, laboratory-technicians, and patients is possible in situations where dental prostheses such as dentures are fabricated, and/or repaired without efficient and effective disinfection protocols [3]. Prospero et al. [4] reported that oral fluids become aerosolized during dental procedures that may cause spread of pathogenic oral microbes. In other words, possibility of contamination with aerosols and pathogens cannot be overlooked during procedures including finishing, trimming, polishing of dental appliances [4, 5]. In this context, it is imperative to implement standardized and appropriate disinfection protocols that demonstrate antifungal activity against a broad spectrum of microbes and are simultaneously non-hazardous to health.

Immersion of removable dental prostheses in chemical disinfectants such as 1% sodium hypochlorite, 2% alkaline glutaraldehyde, 3% aqueous formaldehyde, cold plasma treatment (CPT) and 4% chlorhexidine gluconate is commonly performed for disinfection of acrylic-based denture surfaces [6, 7, 8, 9]. Cold plasma treatment (CPT) has emerged as a promising disinfection agent due to its antifungal properties [3]. In an in vitro study, Asnaashari et al. [3] compared the antifungal efficacy of CPT and traditional alcohol-based disinfectants on denture surfaces. According to the results, both chemical agents demonstrated reduction in microbial colony forming units (CFU/ml) [3]; however, the antifungal activity of conventional alcohol-based disinfection spray was significantly pronounced than CPT [3]. This study concluded that spraying of dentures with alcohol-based disinfection agents continues to be the most reliable denture infection protocol [3]. Antifungal photodynamic therapy (aPDT) is conventionally used as an adjunct therapy for reducing oral and subgingival microbial counts and facilitate healing [10, 11]. Nevertheless, aPDT can also be used to disinfect denture surfaces [12, 13]. Results from in vitro studies [12, 13] have shown that aPDT is more effective than chlorhexidine in terms of reducing counts of pathogenic bacteria such as Staphylococcus aureus, Streptococcus mutans, and Eischeria coli on polymethylmethacrylate denture-based polymers. It is also important to note that to date there are no clinical and/or experimental studies that have compared the antifungal activity of conventional alcohol sprays and aPDT on acrylic denture resin. The present experiment is based on the null hypothesis that there is no difference in the antifungal efficacy of conventional alcohol sprays and aPDT.

The aim of the present in vitro study is to compare the antifungal activity of conventional alcohol sprays and aPDT on acrylic denture resin.

2. Materials and methods

2.1 Ethical statement

The study protocol was reviewed and approved by the Institutional Review Board of Health Sciences Colleges Research on Human Subjects, College of Medicine, King Saud University, Riyadh, Saudi Arabia (Approval no. 22/0352/IRB). Individuals who had their dentures assessed in this investigation had previously provided a written informed consent and confirmed that they had no objections to have their dentures assessed in the present investigation. The individuals were aware that in case their denture was damaged (including but not limited to avulsion of acrylic tooth/teeth from the denture, fracture of denture or chipping of any denture surface) would be provided new dentures within 3 days with no costs.

2.2 Eligibility criteria

Individuals wearing complete dentures at least on one arch were included. Self-reported tobacco-smokers, smokeless tobacco chewers, and immunosuppressed individuals such as patients with diabetes mellitus (DM) and acquired immune deficiency syndrome were excluded.

2.3 Study samples and groups

In the present in vitro study, maxillary dentures from 36 completely edentulous individuals were utilized as microbial sources. The dentures ( $n=$ 36) were randomly divided into three groups. Randomization was done by picking a paper from an opaque bag that was labelled Group 1, Group 2 or Group 3. Dentures in Groups 1 ( $n=$ 12) and 2 ( $n=$ 12) were disinfected with an alcohol-based antiseptic spray (Aseptoprint ${}^{\mathrm{\circledR }}$ , OCC, Switzerland) and aPDT, respectively. Dentures in the control group (Group 3) were washed with distilled water.

2.4 Microbial assessment

Each of the 36 maxillary complete dentures were placed on a sterile petri-dish (GPJYYDS, Part # YYDS-O220730J-0185, USA), which were 90 mm and 15 mm in depth and width, respectively. Assessment of oral yeast growth was done using swab samples that were collected and deposited on tubes containing 0.9% Sodium chloride (NaCl) solution. The solution was further diluted by adding 90 ml 0.9% NaCl from which, ten lambda were spread on culture plates that were precoated with blood agar (Merck, Darmstadt, Germany). The culture mediums were incubated at 37 ${}^{\circ }$ C for 72 hours following which microbes were viewed through a microscope (Stereoscopic, Nikon, Japan). The numbers of colony forming units (CFU/ml) were determined using a colony counter (AMETEK, Inc. and Reichert, Inc., Canada).

2.5 Disinfection

In Group 1, 10 milliliters of the alcohol-based spray (Aseptoprint ${}^{\mathrm{\circledR }}$ , OCC, Switzerland) was applied to each denture in the control group and sealed in an airtight bag for three minutes. The dentures were then removed from the bag and washed with distilled water. In the test group, aPDT was performed in accordance with the protocol described in a recent experimental study by Alhenaki et al. [12]. In summary, denture surfaces were treated with 500 mg/L methylene blue (MB) (Vetec-Fine Chemicals Ltd., NSW 1765, Australia). A single coating of the photosensitizer was added to all surfaces of the denture using the brush technique. After 60 seconds of MB application, the photosensitizer was activated using a diode laser of wavelength 600 nm and an outpower and power density of 200 mW and 25 mW/cm ${}^2$ . The denture surfaces were exposed to the laser for a duration three minutes. In Group 3, the dentures were sprayed with 10 ml distilled water, placed in a sterile bag and sealed for three minutes. In all groups, swab sampling was redone and CFU/ml were re-determined according to the technique described above.

2.6 Statistical analysis

A computer software (SPSS version 20; IBM Corp., Chicago, IL, USA) was used to perform statistical analysis. The statistician compared the microbial CFU/ml in each group and was blinded to the identity of the study groups. Data normality was assessed using the Sharpio Wilk test and group comparisons were done using the one-way analysis of variance. For multiple comparisons, the Bonferroni post-hoc adjustment test was performed. Level of significance was set at $P$ -values, which were less than 0.05.

3. Results

3.1 Baseline

The mean CFU/ml were 5.06 $\pm$ 0.96, 5.12 $\pm$ 0.58 and 5.2 $\pm$ 0.78 CFU/ml in Groups 1–3, respectively. There was no statistically significant difference in CFU/ml in Groups 1–3.

3.2 After denture disinfection

In Groups 1 ( $P<$ 0.05) and 2 ( $P<$ 0.05), there was a statistically significant reduction in microbial CFU/ml was in comparison with their respective CFU/ml at baseline. In Group 3, there was no difference in microbial CFU/ml before and after denture disinfection ( $P=$ 0.14). The microbial CFU/ml were significantly higher in Group 3 ( $P<$ 0.05) compared with Groups 1 and 2. There was no difference in microbial CFU/ml in dentures in Groups 1 and 2. These results are shown in Table 1.

Table 1

Mean colony forming units on Sabouraud dextrose agar at baseline in Groups 1–3

Microbial assessment	Before disinfection			After disinfection
	Group 1	Group 2	Group 3	Group 1	Group 2	Group 3
Dentures ${}^{\ast }$ assessed ( $n$ )	12	12	12	12	12	12
Colony forming units (CFU/ml)	5.06 $\pm$ 0.96	5.12 $\pm$ 0.58	5.2 $\pm$ 0.78	1.27 $\pm$ 0.3	0.8 $\pm$ 0.07	5.26 $\pm$ 0.75
	CFU/ml ${}^{†}$	CFU/ml ${}^{‡}$	CFU/ml	CFU/ml ${}^{\mathrm{\S }}$	CFU/ml ${}^{\mathrm{\S }}$	CFU/ml

${}^{\ast }$ Maxillary complete dentures. ${}^{†}$ Compared with Group 1 after disinfection ( $P<$ 0.05). ${}^{‡}$ Compared with Group 2 after disinfection. ${}^{\mathrm{\S }}$ Compared with Group 2 after disinfection ( $P<$ 0.05).

4. Discussion

Polymers used in the fabrication of dentures (predominantly methyl methacrylate-based resins) often act as reservoirs for microbial colonization and proliferation [14, 15]. Poor denture and oral hygiene and irregular disinfection of denture surfaces are risk factors for oral inflammatory conditions such as denture stomatitis (DS) [16, 17]. Traditionally, manual brushing of denture surfaces is recommended by clinicians for maintaining domestic denture hygiene [18, 19]. However, professional cleansing of dentures is often carried by healthcare providers at patients’ routine dental visits; and antiseptic solutions including but not limited to chlorhexidine and alcohol sprays are often used in this regard [20]. In recent years, aPDT has emerged as a promising antifungal therapeutic strategy for the management of oral diseases such as periodontitis, peri-implantitis and oral cancer [21]. However, there is mounting evidence supports the theme that aPDT can successfully be used for disinfection of dental prostheses including dentures [12, 22].

In the present in vitro experiment, we compared the antifungal effectiveness of aPDT and conventional alcohol towards microbial reduction n $=$ on acrylic-based complete denture surfaces. The present results showed no statistically significant difference in Groups 1 and 2 (alcohol spay and aPDT, respectively) in terms of microbial reduction. However, the results clearly showed that there was a marked reduction in the microbial CFU/ml in Groups 1 and 2 compared with the control group (Group 3). Our experimental results support the outcomes reported in a recent clinical study by de Senna et al. [23] in which, the effectiveness of APDT towards fungal reduction on denture surfaces. In this aPDT was performed using MB as photosensitizer and at a power of 100 mW and 28 J/cm ${}^2$ energy fluence [23]. The 15 days follow-up assessment of dentures showed a significant reduction in fungal load in dentures that were treated with aPDT compared with controls that were treated with topical 2% miconazole gel [23]. Vlahova et al. [24] used photodynamic infection to eradicate Candida albicans from acrylic-based prostheses. According to the results aPDT was 100% efficient in disinfecting acrylic-based dental prostheses and the authors concluded that aPDT is an effective protocol for denture disinfection [24]. Nevertheless, it is noteworthy that results of the present investigation showed no difference in the antifungal efficacy of alcohol-spray and aPDT. This outcome reflects that although aPDT is a reliable disinfection protocol for denture disinfection, it is not superior or inferior to alcohol sprays in terms of antifungal disinfection efficacy. From the authors perspective, alcohol-based sprays are more “user friendly” whereas aPDT warrants additional armamentarium and is technique sensitive. In this context, alcohol-sprays can be considered as a reliable therapeutic protocol over aPDT for disinfection of dentures.

The major most limitation of this study is that the outcomes were based on laboratory-based investigations. From a clinical perspective, it is notable that patients with systemic diseases such as DM and tobacco product users (such as cigarette-smokers) were excluded. It is well established that oral yeast carriage is significantly higher among immunosuppressed individuals and habitual nicotinic product users in comparison with medially healthy individuals and non-smokers [25, 26]. It is therefore likely that in immunosuppressed individuals and tobacco-smokers, aPDT is more effective than alcohol sprays in reducing oral yeasts counts on denture surfaces. Moreover, aPDT was only performed once in this experiment. It is therefore possible that in patients with oral diseases such as DS, multiple sessions of aPDT and/or alcohol spray applications are needed to significantly reduce microbial counts on denture surfaces. Further studies are needed to test these hypotheses. From a clinical standpoint, the present results showed that conventional alcohol sprays continue to be a cost as well as therapeutically effective protocol for disinfection of denture surfaces. However, in patients with oral diseases such as DS adjunct disinfection protocols such as aPDT may help microbial elimination from contaminated denture surfaces. Further studies are needed to test this hypothesis.

5. Conclusion

Conventional alcohol sprays are as effective as aPDT towards reducing oral yeasts CFU/ml on acrylic denture resin.