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Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 286-291

Effect of three different compositions of topical fluoride varnishes with and without prior oral prophylaxis on Streptococcus mutans count in biofilm samples of children aged 2–8 years: A randomized controlled trial

Department of Pedodontics and Preventive Dentistry, ITS-CDSR, Muradnagar, Ghaziabad, Uttar Pradesh, India

Date of Web Publication30-Sep-2019

Correspondence Address:
Dr. Sushma Yadav
C-108, Sec-9, New Vijay Nagar, Ghaziabad - 201 009, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JISPPD.JISPPD_62_19

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Background: Various strategies for controlling caries focus on disrupting the interaction between risk factors. Of these, fluoride varnish has been shown to reduce the colony-forming (CFU) units and water-insoluble extracellular polysaccharide amount. Casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) and xylitol-containing fluoride varnishes have recently gained importance as caries-protective fluoride varnishes. Aim: This study aims to assess and compare the reduction in Streptococcus mutans count in biofilm samples after topical application of three different fluoride varnishes and to evaluate the effect of oral prophylaxis prior to fluoride varnish application. Materials and Methods: Sixty healthy children with no active caries, in the age group of 2–8 years, were randomly divided into Group A = fluoride varnish containing CPP–ACP; Group B = fluoride varnish containing xylitol; and Group C = fluoride varnish with 0.9% difluorosilane; further, the groups were divided into two subgroups, namely A1, B1, and C1 with prior oral prophylaxis and A2, B2, and C2 without oral prophylaxis. Plaque samples were collected at baseline, 1st month, and 3rd month; cultured; and incubated, and CFU/ml was calculated. Results: Data were compiled, and CFU/ml was analyzed by independent t-test, paired t-test, and one-way ANOVA. There was no statistical difference between the fluoride groups. Furthermore, no statistically significant difference was seen between the subgroups. Conclusion: Fluoride varnish containing CPP–ACP showed higher reduction in S. mutans count followed by xylitol-containing fluoride varnish and Fluor Protector®. There was no effect of prior oral prophylaxis on the efficacy of fluoride varnish.

Keywords: Biofilm, casein phosphopeptide–amorphous calcium phosphate, Fluor Protector ®, MI Varnish™, oral prophylaxis, Profluorid ®, topical fluoride varnish, xylitol

How to cite this article:
Yadav S, Sachdev V, Malik M, Chopra R. Effect of three different compositions of topical fluoride varnishes with and without prior oral prophylaxis on Streptococcus mutans count in biofilm samples of children aged 2–8 years: A randomized controlled trial. J Indian Soc Pedod Prev Dent 2019;37:286-91

How to cite this URL:
Yadav S, Sachdev V, Malik M, Chopra R. Effect of three different compositions of topical fluoride varnishes with and without prior oral prophylaxis on Streptococcus mutans count in biofilm samples of children aged 2–8 years: A randomized controlled trial. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2022 Oct 1];37:286-91. Available from: http://www.jisppd.com/text.asp?2019/37/3/286/268185

   Introduction Top

Dental caries is a carbohydrate-modified, local infection that results from an interaction between oral flora and dietary carbohydrates on the tooth surface.[1] To adhere to the tooth structure, microorganisms utilize dietary sugars to create a sticky biofilm known as dental plaque. Acids produced by bacterial fermentation of carbohydrates reduce the pH of dental plaque to the point at which demineralization of the enamel occurs.[2]

Various strategies for controlling dental caries focus on disrupting the interaction between the risk factors (diet, microflora, and susceptible tooth). These measures include dietary modification, implementation of adequate oral hygiene practices, and use of fluorides, and pit-and-fissure sealants. All such measures directly or indirectly target the oral microflora.[3] Fluoride varnishes were originally developed to prolong the contact time between fluoride and dental enamel, as they adhere to the tooth surface for longer periods in a thin layer and prevent the immediate loss of fluoride after application, thus acting as slow-releasing reservoirs of fluoride. Sustained-release vehicles such as varnishes exert a long-term prophylactic effect.[4] Fluoride varnish has been shown to reduce the colony-forming unit (CFU) counts by inhibiting the carbohydrate metabolism of oral streptococci.[5]

Recently, xylitol and casein phosphopeptide–amorphous calcium phosphate (CPP–ACP) have been added to potentiate the effect of fluoride varnishes. Xylitol is a nonfermentative sugar alcohol that inhibits the growth and metabolism of several bacterial species, but mutans streptococci appear to be the target organism.[6] In addition, the reduction of insoluble extracellular polysaccharides is probably of importance for the xylitol-induced decrease in both counts and transmission of mutans streptococci. CPP–ACP derived from milk protein casein has been reported to have antibacterial and buffering effects on plaque, which interfere in the growth of Streptococcus species.[7] The CPP–ACP also acts as a reservoir of bioavailable calcium and phosphate and maintains the solution supersaturated, thus facilitating remineralization.[8] A study done by Erdem et al. (2011) reported a reduction in the bacterial viability of Streptococcus mutans in biofilm after the application of CPP–ACP.[9]

The principal reason behind including prophylaxis as the preliminary step was that removed plaque might inhibit enamel fluoride uptake and reduce the caries-inhibitory potential. However, more than a dozen laboratory and clinical studies have shown that enamel fluoride uptake is not impeded by surface organic integuments and the clinical efficacy is unaffected by the type and degree of preliminary cleaning of the teeth.[10],[11],[12],[13]

Thus, this study evaluated the effect of three compositions of topical fluoride varnishes, i.e. 5% sodium fluoride with CPP–ACP (MI Varnish™), 5% sodium fluoride with xylitol (Profluorid ®), and 0.9% difluorosilane (Fluor Protector ®) [Figure 1], on the S. mutans count in the biofilm samples of children during primary and early mixed dentition periods with and without prior oral prophylaxis.
Figure 1: Topical fluoride varnishes used in this study

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   Materials and Methods Top

The present study was conducted at the Department of Pedodontics and Preventive Dentistry, Ghaziabad. Prior to the study, it was approved by the Institutional Ethical Committee. Children were screened and examined using a mouth mirror in daylight. The study was explained to the parents in detail. Personal details; details of past medical history, including any recent fluoride treatment; frequency of brushing; sweet/snack intake and consumption of sugared energy drinks; and the brand of toothpaste (to determine fluoride content) were obtained. Sixty healthy children of 2–8 years of age were screened through the International Caries Detection and Assessment System. Children with score 0 or no active caries were included in the study. Patients who were on artificial fluoride supplementation or antimicrobial therapy in the past 3 months, those having any systemic illness, or those wearing any orthodontic appliance were excluded from the study.

The statistician randomly assigned the children to the following three groups using Random Allocation Software version 2.0 (Isfahan University of Medical Sciences, Isfahan, Iran): Group A = MI Varnish; Group B = Profluorid; and Group C = Fluor Protector varnish, with twenty children in each group. For every group, two subgroups were assigned, ten children in each, namely subgroup 1 – with prior oral prophylaxis and subgroup 2 – without oral prophylaxis. In subgroup 1, oral prophylaxis was done before the application of fluoride varnish.

The children were instructed to abandon their regular oral care procedures, including fluoridated toothpaste, 1 day before plaque collection. Plaque sample was collected from the buccal surface of the maxillary right primary molar tooth (sample 1), with the help of an explorer, and was transferred to sterile tubes containing reduced transport fluid (RTF), and then it was sent for culturing and microbiologic analysis. The tubes were weighed before and after collection using a digital weighing machine in order to determine the weight of the plaque sample.

Microbiological procedures

Microbiological procedures were conducted by an institutional microbiologist. The plaque samples collected which were placed in RTF were processed within 3 h. For the determination of the prevalence of S. mutans in samples, the micromethod described by Westergren and Krasse [14] was used. The samples were dispersed for 30 s on a Whirlimixer ® and diluted in 10-fold steps, in 0.05 M phosphate buffer (pH 7.3) containing 0.4% KCl. From appropriate dilutions, 25-μl aliquots were spotted in duplicate on Mitis Salivarius (MS) agar, selective for S. mutans. The MS agar plates were incubated for 48 h at 37°C in jars filled with 93% N2 and 5% CO2. The total number of colonies of S. mutans as CFU/ml was counted on MS agar using a colony counter [Figure 2].
Figure 2: Streptococcus mutans colony on Mitis Salivarius agar base at baseline, 1st month, and 3rd month

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On the same day, after the baseline sample collection, full-mouth ultrasonic scaling and polishing was done for children assigned in the subgroup A1, B1, and C1.

Application of fluoride varnish

First, the tooth surfaces were cleaned and then dried with an air syringe, and isolation of the teeth was done with the help of cotton rolls, a high-volume evacuator with a saliva ejector, and a cheek retractor. A thin layer of the mentioned fluoride varnish was applied on all the tooth surfaces with the help of a microbrush, according to the manufacturer's guidelines [Figure 3]a, [Figure 3]b, [Figure 3]c. The cotton rolls were removed after 1 min. The patients were instructed not to rinse or drink anything for 1 h and were also asked to avoid hard food, brushing, or flossing till the next morning after application.
Figure 3: (a) Caries-free individual. (b) Plaque sample collection before fluoride varnish application. (c) Fluoride varnish application using applicator

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The children were recalled after 1 month and 3 months for the collection of plaque sample (sample 2 and sample 3, respectively). The plaque was collected, cultured, and incubated for the same period as described earlier. Then, the CFUs/ml of S. mutans were counted using the colony counter.

Statistical analysis

The mean, mean difference, and standard deviation of the data were calculated. The data were normally distributed, so parametric tests were used, i.e., one-way ANOVA and paired t-test. The level of significance and confidence interval were 5% and 95%, respectively. Data were analyzed using SPSS 16.0 software (IBM SPSS Statistics, IBM Corporation, US).

   Results Top

The mean S. mutans (CFU/ml) in plaque sample at baseline, 1st month, and 3rd month after a single topical application of three fluoride varnishes is depicted in [Graph 1]. The results showed that from baseline, there were 23.95% and 37.70% reduction in S. mutans (CFU/ml) at 1st month and 3rd month, respectively. Through data analysis, there was no statistically significant difference seen in the reduction of S. mutans count in biofilm samples. However, among the groups, MI Varnish group showed maximum reduction (41.20%) in the S. mutans (CFU/ml) from baseline to the 3rd month, followed by Profluorid (37.90%) and Fluor Protector (33.29%) [Table 1] and [Table 2].

Table 1: Comparison of mean difference±standard deviation of Streptococcus mutans colony-forming units/ml in different fluoride varnish groups with and without prior oral prophylaxis between two time intervals

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Table 2: Percentage Streptococcus mutans reduction in fluoride varnish groups at the follow-up period

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There was no effect of prior oral prophylaxis on the efficacy of fluoride varnish. There was no statistically significant difference between all the three fluoride varnish groups at baseline, 1st month, and 3rd month with and without oral prophylaxis (P > 0.05) [Table 3] and [Table 4].
Table 3: Comparison of mean Streptococcus mutans count in biofilm of three fluoride varnish groups with oral prophylaxis at different time intervals

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Table 4: Comparison of mean Streptococcus mutans count in biofilm of three fluoride varnish groups without prior oral prophylaxis at different time intervals

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   Discussion Top

The most important anticaries effect of fluoride is considered to result from its action on the tooth/plaque interface, through the promotion of remineralization of early caries lesions and by reducing tooth enamel solubility.[15] Varnishes were originally developed to prolong the contact time between fluoride and dental enamel, as they adhere to the tooth surface for longer periods in a thin layer, and prevent the immediate loss of fluoride after application, thus acting as slow releasing reservoirs of fluoride. The time required to apply the varnish varies from 1 to 4 min per child, depending on the number of teeth present, and immediately following application, the child can close his/her mouth because the varnish hardens on contact with saliva and forms a film that adheres to tooth surfaces. The fluoride varnish has less potential for harms than other forms of high-concentration topical fluoride varnishes because the amount of fluoride in the fluoride varnish is approximately one-tenth that of other professionally applied products.[16]

In the present study, fluoride varnish containing CPP–ACP showed reduction in S. mutans counts, attributing to the fact that CPP–ACP and calcium compete for the same binding sites on S. mutans, where CPP–ACP binds with twice the affinity to bacterial cells compared to calcium. This enhanced availability of calcium in the plaque has anticaries effects by promoting remineralization and hampering demineralization. Furthermore, there is evidence that a high concentration of free calcium has bactericidal or bacteriostatic effects, also due to the additive anticariogenic effect of CPP–ACP and fluoride, attributable to the localization of ACP fluoride at the tooth surface by the CPP.[17] Similar results were seen in a study by Erdem et al. who reported a reduction in bacterial viability of S. mutans after the application of varnish containing CPP–ACP.[9] A study by Li et al. also stated that CPP–ACP has a long-term remineralizing effect on early caries lesions.[18] The reduction of S. mutans on 1 month is more when compared to that of the 3rd month. Studies by Shen et al. suggest that fluoride ion in MI Varnish exhibited the highest release at 24 h (1149 μmol/g), which represented close to full release (96%) of the added fluoride and 1183.27 μmol/g of fluoride ion release at 48 h.[19]

The presence of xylitol in Profluorid varnish causes reduced synthesis of insoluble polysaccharides by S. mutans, causing decrease in the adhesivity of plaque to the tooth surface,[20] thus reducing the CFUs of S. mutans. Jafari et al. also suggested that the application of xylitol-based varnishes not only inhibited demineralization and enhanced remineralization but also reduced the level of S. mutans in the oral cavity.[21] Mäkinen et al.,[22] Milgrom et al.,[23] Haresaku et al.,[24] and Badet et al.[20] also demonstrated a xylitol-associated decrease of S. mutans counts in plaque.

Zickert and Emilson who compared Duraphat ® and Fluor Protector suggested that Fluor Protector has a lower fluoride concentration and caries-inhibiting activity than Duraphat, but the amount of fluoride deposited in teeth was more after the use of Fluor Protector when compared to that of Duraphat. This fluoride that could have leached out from the teeth could have been taken up by the plaque to inhibit the growth of bacteria.[25] Ekenbäck et al. suggested that hydroxyapatite crystals pretreated with Fluor Protector showed a statistically significant reduction in lactic acid formation in S. mutans.[26]

Fluoride varnish containing CPP–ACP was seen to have the highest inhibitory effect against S. mutans, followed by xylitol-containing varnish; while difluorosilane showed the least inhibitory effect. This might be attributed to the greater fluoride content of MI Varnish and Profluorid and the comparatively low fluoride content of Fluor Protector. The difference in their composition may account for their varied antimicrobial effects. Babu et al. found MI Varnish to be a more potent remineralizing agent than Fluor Protector.[27]

The principal reason behind including oral prophylaxis as the preliminary step in a topical fluoride treatment was that removed plaque and pellicle might inhibit enamel fluoride uptake and reduce the caries-inhibitory potential of the procedure. However, more than a dozen laboratory and clinical studies have shown that enamel fluoride uptake is not impeded by surface organic integuments and that the clinical efficacy of the procedure is unaffected by the type and degree of preliminary cleaning of the teeth. In our study also, there was no significant difference seen with respect to prior oral prophylaxis done in all the fluoride groups in S. mutans reduction. Houpt et al.,[10] Ripa,[11] Katz,[12] Steele et al.,[13] and McNee et al.,[28] through various in vivo and in vitro studies of fluoride diffusion through plaque and fluoride uptake by uncleaned enamel, concluded that the presence or absence of prior oral prophylaxis does not inhibit the uptake of topically applied fluoride by tooth enamel.

Limitations of the study

  1. The present in vivo study needs long-term clinical follow-up
  2. The study had smaller sample size
  3. Socioeconomic status of the participants was not considered.

   Conclusion Top

Based on the results of the study, the following can be concluded:

  1. Fluoride varnish containing CPP–ACP had good clinical success in reducing S. mutans count
  2. Use of fluoride varnish containing CPP–ACP and xylitol as a preventive intervention is effective in preventing caries in children
  3. Prior oral prophylaxis had no significant effect on the efficacy of topical fluoride varnish application.

Thus, the present study demonstrates the reduction in S. mutans counts by fluoride varnishes containing CPP–ACP and xylitol to a greater extent than conventional fluoride varnish. The procedure takes less time, renders less patient discomfort, and can be used in uncooperative pediatric patients. It is important, however, that further prospective researches be carried out to support the use of fluoride varnishes in order to expand the body of evidence so that clinicians can justify decisions with evidence-based dentistry.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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Houpt M, Koenigsberg S, Shey Z. The effect of prior tooth cleaning on the efficacy of topical fluoride treatment. Two-year results. Clin Prev Dent 1983;5:8-10.  Back to cited text no. 10
Ripa LW. Effect of prior tooth – Cleaning on biannual professional APF topical fluoride gel-tray treatments: Results after two years. Clin Prev Dent 1984;5:3-7.  Back to cited text no. 11
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Steele RC, Waltner AW, Bawden JW. The effect of tooth cleaning procedures on fluoride uptake in enamel. Pediatr Dent 1982;4:228-33.  Back to cited text no. 13
Westergren G, Krasse B. Evaluation of a micromethod for determination of Streptococcus mutans and Lactobacillus infection. J Clin Microbiol 1978;7:82-3.  Back to cited text no. 14
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Rose RK. Binding characteristics of Streptococcus mutans for calcium and casein phosphopeptide. Caries Res 2000;34:427-31.  Back to cited text no. 17
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Shen P, Bagheri R, Walker GD, Yuan Y, Stanton DP, Reynolds C, et al. Effect of calcium phosphate addition to fluoride containing dental varnishes on enamel demineralization. Aust Dent J 2016;61:357-65.  Back to cited text no. 19
Badet C, Furiga A, Thébaud N. Effect of xylitol on an in vitro model of oral biofilm. Oral Health Prev Dent 2008;6:337-41.  Back to cited text no. 20
Jafari K, Hekmatfar S, Fereydunzadeh M.In vitro comparison of antimicrobial activity of conventional fluoride varnishes containing xylitol and casein phosphopeptide-amorphous calcium phosphate. J Int Soc Prev Community Dent 2018;8:309-13.  Back to cited text no. 21
Mäkinen KK, Isotupa KP, Mäkinen PL, Söderling E, Song KB, Nam SH. Six-month polyol chewing-gum programme in Kindergarten-age children: A feasibility study focusing on mutans streptococci and dental plaque. Int Dent J 2005;55:81-8.  Back to cited text no. 22
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Haresaku S, Hanioka T, Tsutsui A, Yamamoto M, Chou T, Gunjishima Y. Long-term effect of xylitol gum use on mutans streptococci in adults. Caries Res 2007;41:198-203.  Back to cited text no. 24
Zickert I, Emilson CG. Effect of a fluoride-containing varnish on Streptococcus mutans in plaque and saliva. Scand J Dent Res 1982;90:423-8.  Back to cited text no. 25
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Babu KL, Subramaniam P, Teleti S. Remineralization potential of varnish containing casein phosphopeptides-amorphous calcium phosphate with fluoride and varnish containing only fluoride: A comparative study. Saudi J Oral Sci 2018;5:35-40.  Back to cited text no. 27
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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3], [Table 4]

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