DOI: 10.1111/ipd.12085
Effect of fluoride varnish and chlorhexidine–thymol varnish on mutans streptococci levels in human dental plaque: a double-blinded randomized controlled trial SANCHIT PAUL1, SUPRABHA BARANYA SHRIKRISHNA1, ETHEL SUMAN2, RAMYA SHENOY3 & ARATHI RAO1 1
Department of Paedodontics and Preventive Dentistry, Manipal College of Dental Sciences, Manipal University, India, Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, India, and 3Department of Public Health Dentistry, Manipal College of Dental Sciences, Manipal University, Mangalore, India
2
International Journal of Paediatric Dentistry 2014; 24: 399–408 Background. Recent systematic reviews on clinical
trials comparing the efficacy of chlorhexidine and fluoride varnish found that the evidence was inconclusive and further well-conducted randomized controlled clinical trials were advocated. Aim. To compare the effect of fluoride varnish (F) and Chlorhexidine–thymol varnish (CHX/T) with intensive application regimen on mutans streptococci (MS) levels in human dental plaque. Design. Seventy-five subjects between 6 and 10 years of age were allocated into three groups: Group 1-F varnish (n = 29); Group 2-CHX/T varnish(n = 29); Group 3-placebo varnish (n = 17) by stratified block randomization. After baseline plaque samples were obtained, varnish application
Introduction
Dental caries is a complex disease due to interaction of various factors such as host, agent, substrate, and time. Although, the cause of dental caries appears to be a complex interaction between genetic and environmental factors, it is important to understand that dental caries does not occur in the absence of dental plaque or dietary fermentable carbohydrates. Longitudinal studies have found a significant association between increased proportion of mutans streptococci (MS) in saliva and dental plaque and incidence of dental caries. Therefore, control of MS levels is an important target for caries prevention and control1. Correspondence to: Dr Suprabha B. S., ‘Shreyas’, 15/17/940-13, 5th Cross Road, Shivabagh, Kadri, Mangalore – 575 002, Karnataka, India. E-mail: [email protected]
was applied and repeated at an interval of 3 days in each group. Plaque samples were repeated at 48 h, 1 month, and 3 months. The samples were spread over mitis-salivarius-bacitracin (MSB) culture media, and the colony-forming units per ml (CFU) were measured. Results. In both Groups 1 and 2, Wilcoxon matched-pairs signed-rank test revealed significant differences in log CFU values of MS between baseline and 48 h, baseline and 1 month but no significant difference between baseline and 3 months. An intergroup comparison at different time intervals showed that the difference between three groups was statistically insignificant. Conclusion. F varnish and CHX/T varnish, with an intensive regimen application have equivocal effect on MS levels in dental plaque.
Among the possible strategies in MS control, chemotherapeutic treatment regimens have received much attention and have presented satisfactory results2. Fluoride over the time has emerged as a leading preventive agent for dental caries due to its ability to reduce acid formation in some bacterial species in dental plaque, including MS. Fluoride varnish is quick and easy to apply, well tolerated by young children3. It has been recommended for caries prevention in children with moderateto-high caries risk by a Cochrane database systematic review4 as well as the American Academy of Paediatric Dentistry5. Of all the antimicrobial agents available for dental use, chlorhexidine (CHX) is the most thoroughly researched in terms of ability to control cariogenic activity6. It is particularly effective in reducing the levels of MS in saliva and dental plaque7. Chlorhexidine is available in a variety of different formulations
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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(i.e., mouth rinse, gel, varnish, impregnated dental floss and toothpaste) and concentrations8,9. A review on the mutans streptococciinhibiting efficacy of CHX concluded that the most persistent reduction in mutans streptococci was achieved by CHX varnishes, followed by gels and mouthwashes7. In a systematic review by Twetman10, 16 trials of chlorhexidine varnish were evaluated and the evidence for the effectiveness of chlorhexidine varnish was rated as inconclusive due to conflicting results. In another systematic review, Zhang et al.9 concluded that chlorhexidine varnish had a moderate cariespreventive effect when applied at intervals of 3–4 months. Evidence for the effectiveness of chlorhexidine varnish for preventing caries in primary and permanent teeth of children and adolescents, however, is generally lacking.8 To enhance the effectiveness of chlorhexidine varnish, an intensive treatment regimen (2–4 applications at intervals of 2–7 days) has been recommended6. In an intensive treatment regimen study (two applications of 1% CHX during a 2-week period) by Petersson et al.11in 1991, a 3-month decrease was shown in dental biofilm Streptococcus mutans. Similar findings were observed by Twetman et al.12 in 1997. Variation was, however, observed in results in other studies, with the decrease lasting from 3 days to 2–3 weeks13,14. Zhang et al.9in his systematic review added that there was no evidence for effectiveness with longer intervals of application. Although the existing studies indicated improved efficacy with more frequent applications, the number was too small to be included in meta-analysis. A systematic review by Petersson et al.15also concluded that there is inconclusive evidence regarding the anticariogenic efficacy of fluoride varnishes based on application frequencies. Randomized trials are considered to provide the best evidence of effectiveness because they minimize selection bias. There have been only few randomized controlled trials comparing 1% chlorhexidine–thymol (CHX/T) and fluoride varnish (F)16–18. In these studies, dental caries was the outcome measure, and the effect on MS level was not evaluated. Evaluation of MS level in plaque may be important because of their association with dental caries and can serve
as an indicator of caries activity, thus redeeming it important in prevention of dental caries1,13. Recent systematic reviews advocated further well-conducted randomized and controlled clinical trials comparing chlorhexidine and fluoride varnish involving children and adolescents, as the evidence was inconclusive especially regarding application frequencies8,15,19. This study was undertaken to evaluate the effect of fluoride varnish (F) and chlorhexidine–thymol varnish (CHX/T) with intensive application regimen on growth and colonization of MS in human dental plaque using microbiological methods under randomized controlled clinical trial settings. The null hypothesis tested was that there is no statistically significant difference between the effect of fluoride varnish (F) and chlorhexidine–thymol varnish (CHX/T) on MS levels in human dental plaque. Materials and methods
The investigation was designed, analysed, and interpreted according to the Consolidated Standards of Reporting Trials (CONSORT). This was a single-centre, stratified block randomized, (6–10 years age, with imbalanced randomization [1.6 : 1]), double-blinded, placebo-controlled parallel-group study. All patients between 6 and 10 years of age, visiting the Paediatric Dentistry Department of Manipal College of Dental Sciences, Mangalore, India, were screened for inclusion and exclusion criteria. The area in which the study was carried out had high caries prevalence (82% in 5- to 7-year age group and 94% for 8- to 10-year age group)20. Inclusion criteria The inclusion criteria were as follows: DMFT/ deft > 1; subjects should be free of any systemic diseases; written informed consent from the parents/guardians; the agreement to comply with the study visits and procedures. Exclusion criteria Exclusion criteria were as follows: patient with relevant medical history; handicapped/
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Effect of fluoride and chlorhexidine–thymol varnishes
mentally challenged children; those taking xylitol chewing gums/undergone any fluoride treatment which could influence the conduct of the study; those who had received antimicrobial therapy in the previous 3 months; children with any intraoral device/developmental defect; poor cooperation reported by guardians of subjects. Ethical clearance was obtained from the institutional ethics committee prior to the study. Voluntary written informed consent was obtained from the parents, and a verbal informed consent was obtained from children, prior to the study. The varnishes used in this trial were Fluor Protector (IvoclarVivadent, Schaan, Liescstenstein Germany) which consists of 1% difluorosilane, with polyurethane as varnish base and ethyl acetate/isoamyl propionate as a solvent. The fluoride content is equivalent to 0.1% or 1000 parts per million (ppm) in solution; Cervitec Plus (IvoclarVivadent, Schaan, Liescstenstein, Germany) which consists of 1% chlorhexidine diacetate and 1% thymol with excipient as vinyl acetate copolymer and acrylate copolymer and ethanol as solvent. Sample size and selection The sample size was estimated to be 25 children for each varnish group(CHX/T and Fluoride varnish) considering a study power of 80% (1-b), to detect a specified difference (d) of 10%, statistical significance of 5% (a = 0.05), and effect size (d) 0.8 using GPower 3.1. A sample of 15 children was estimated for placebo varnish. Allowing for confounding bias and losses of 15% during the follow-up, 29 children were included in both the intervention groups at the outset and 17 in placebo group, thus giving a total sample of 75. The children were allocated into three groups by stratified block randomization method, with stratification carried out according to age. The groups were as follows: Group 1: Fluor Protector (F varnish) (n = 29); Group 2: Cervitec plus (CHX/T varnish) (n = 29); Group 3: placebo varnish (cavity varnish–copal rosin with diethyl ether as solvent; Namuvar Dental varnish, Deepti Dental Products of India Pvt. Ltd., Ratnagiri, India) (n = 17). The
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children were divided into five strata at an interval of one year based on age. A block size of six was used during randomization. Random allocation was done by an investigator who was neither involved in varnish application to study subjects nor measurement of outcomes, so as to achieve allocation concealment. The participants in this trial were blinded as to their group. Baseline information and treatment Oral hygiene practice and 24-h diet history of all the subjects were recorded using a questionnaire form, and oral hygiene instructions, diet counselling, and fluoridated toothpaste were given during the period of study. Routine dental treatment such as extractions and restoration of all open carious lesions were carried out prior to commencement of the study. Plaque sampling Plaque sampling was carried out at baseline, at 48 h after the varnish application, at the end of 1 month and at the end of the 3 months. The subjects were informed to refrain from oral hygiene procedures for 24 h, prior to plaque sampling. The adherent saliva was removed using water spray and then air-dried. Plaque was collected using a wooden toothpick from smooth surfaces (by streaking on buccal and lingual surfaces from occlusal to gingival one-third) of all the molars of the selected subjects and transferred to small plastic vials containing thioglycollate transport media. All the above-mentioned procedures were performed by a single investigator with proper aseptic measures. Microbiological processing Tip of the wooden toothpick was cut off and transported to microbiological laboratory containing thioglycollate transport media on the same day. Samples were homogenized manually by stirring using a stirrer. Inoculating loop with a diameter of 0.05 lL was used to collect the diluted plaque from transport vials. Plaque samples were spread over MSB culture media
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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[mitis salivarius supplemented with bacitracin (0.2 U/mL), and sucrose (200 g/litre)], a selective medium for MS, by spreading method21 and incubated for 48 h at 37°C in an incubator. After 48 h of incubation period, MS appeared on the culture plate as small, rough, raised and adherent colonies. The colonyforming units per ml (CFU) of diluted plaque were measured based on values obtained per 100 lL of diluted plaque. All the microbiological procedures were performed by two microbiology personnel who were blinded with respect to the group from which the plaque was collected. Culture plates were examined for identification of colonies of MS and counted manually by both the microbiology personnel to obtain the surface viable count21. Interexaminer variability was assessed by intraclass coefficient correlation analysis, which was found to be 0.92, reflecting a high degree of conformity in observational judgments between the two examiners. Varnish application procedure After baseline plaque samples were obtained from each of the subject, varnish application procedure was carried out for each of the subject in all three groups. Before application of varnish, oral prophylaxis was carried out for subjects. The teeth were then isolated with cotton rolls and saliva ejector and dried with gentle blow of air for 30 s using threeway air syringe. Approximately 0.1 mL of the designated varnish was applied to all the teeth of subjects in all the three groups. A small brush supplied by the manufacturer was used for Cervitec group. For Fluor Protector and placebo varnish groups, applicator tips were used for application of varnish. Varnish was allowed to dry, and after one minute, the cotton rolls were removed. Subjects in all the three groups were instructed not to rinse their mouth, not to drink or eat anything for three hours, and not to brush till the next day. Follow-up The varnish application was repeated at an interval of 3 days for all the three groups in
the same manner as above. The follow-up plaque samples from all the three varnish groups were taken at the end of 48 h, at one month and at the end of three months and assessed for MS counts in the same manner as described before. During the study period, the subjects were asked to report any adverse effects noticed by them immediately to the parents and the investigator; however, none of the subjects reported any adverse effect. Statistical analysis The obtained data were compiled systematically, and the colony-forming units per ml (CFU) were transformed to logarithmic values. Statistical data were analysed using SPSS for Windows release 19.0 (SPSS Inc., Chicago, IL, USA). Because of the high degree of skewness of the microbiological data (Kolmogorov–Smirnov test P < 0.001), nonparametric methods were used for analysis of all microbiological data. Comparison of plaque MS log CFU values at baseline, 48 h, at the end of 1 month and 3 months for each group was made by Friedman’s test, and intergroup comparisons were made with Wilcoxon matched-pairs signed-rank test. Comparison between the groups at various time intervals was made by Kruskal–Wallis test. Results and Observations
At the end of the study, of 75 subjects recruited at baseline level, seven subjects did not report on the days of follow-up plaque sampling due to illness or migration to another city. This amounts to 9.33% of dropouts which was well within our estimated dropout percentage. For the final analysis of the data, the dropouts were eliminated, and the rest of the data was considered as depicted in trial profile (Fig. 1). Chi-square test revealed that at baseline, there was no statistically significant difference between the groups with respect to age, gender, oral hygiene practices, and sugar frequency in the diet (Table 1 and Table 2). No significant difference was also seen in DMFT, deft (Table 2), and log CFU values at baseline (P = 0.49) (Fig 2).
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Effect of fluoride and chlorhexidine–thymol varnishes
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ASSESSED FOR ELIGIBILITY
(N = 90) Excluded: (n = 15) Reasons: Parents’ consent not obtained
ENROLLMENT
RANDOMIZATION
(N = 75)
ALLOCATION
Allocated to intervention GROUP-1 FLUORPROTECTOR (n = 29)
Allocated to intervention GROUP-2 CERVITEC PLUS (n = 29)
Allocated to intervention GROUP-3 PLACEBO (n = 17)
FOLLOW UP for 3 months
GROUP-1 FLUORPROTECTOR Lost to follow up: n = 3
GROUP-2 CERVITEC PLUS Lost to follow up: n = 3
GROUP-3 PLACEBO Lost to follow up: n = 1
REASONS FOR DROP OUT: ABSENCE ON THE DAY OF EXAMINATION; REFUSAL; ILLNESS; MIGRATION
ANALYSIS
GROUP-1 FLUORPROTECTOR Analyzed: n = 26
GROUP-2 CERVITEC PLUS Analyzed: n = 26
GROUP-3 PLACEBO Analyzed: n = 16
Figure 1. Trial profile.
Friedman’s test revealed statistically significant difference over time in both Fluor Protector group (Group 1) and Cervitec Plus group (Group 2) (P < 0.001). Wilcoxon matched-pairs signed-rank test revealed significant differences in MS log CFU between baseline and 48 hours and between baseline and 1 month in both Group 1 and Group 2. The statistically significant difference was, however, no longer demonstrable between baseline and 3 month in both the groups. Significant difference was also observed when compared between 48 h and 1 month, 48 h and 3 month and between 1 month and 3 month (Table 3). In Group 3 (placebo varnish group), Friedman’s test revealed that there was no statistically sig-
nificant difference over three months time (P = 0.19). When intergroup comparison was made, at all the time intervals, the difference between three groups was found to be statistically insignificant with almost similar values at the end of 3 month (Table 4 and Fig 2). Although there was decrease in log CFU values in both the intervention groups (Group 1 and 2) at 48 h, there was no statistically significant difference between the groups at 48 h (P = 0.40). Similarly, there was no difference at 1 month and 3 months also. Also, the decrease at 48 h in the intervention groups was not statistically significant as compared with the placebo varnish group. Similarly, the increase in log CFU values at one
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Table 1. Comparison of descriptive statistics at baseline levels.
Variables Gender Male Female Toothbrush use Yes No Brush frequency Once/day Twice/day Fluoridated toothpaste Yes No Sugar exposure 4/day Total
Group 1 no. (%)
Group 2 no. (%)
Group 3 no. (%)
19 (73.1) 7 (26.9)
15 (57.9) 11 (42.1)
24 (92.3) 2 (7.7)
v2
P value
10 (62.5) 6 (37.5)
1.39
0.49
25 (96.1) 1 (3.8)
15 (93.2) 1 (6.2)
0.35
0.84
22 (84.6) 4 (15.4)
20 (76.9) 6 (23.1)
13 (81.2) 3 (18.8)
0.49
0.78
22 (84.6) 4 (15.4)
25 (96.2) 1 (3.8)
15 (93.8) 1 (6.2)
7.35
0.12
18 7 1 26
17 7 2 26
11 4 1 16
0.38
0.98
(69.3) (26.9) (3.8) (100)
(65.4) (26.9) (7.7) (100)
(68.75) (25.0) (6.25) (100)
Table 2. Comparison of mean age and mean deft/DMFT values of different groups. Variable
Group 1 (26)
Group 2 (26)
Group 3 (16)
v2
P value
Age deft DMFT
8.81 1.44 2.04 1.69 0.50 1.10
8.54 1.52 2.85 1.73 0.46 0.95
8.56 1.54 3.44 1.86 0.25 0.58
0.36 11.29 5.63
0.89 0.50 0.69
Group 1 4.4
Group 2
Group 3
4.36
4.35
4.33
log Cfu values
4.3
4.3 4.31
4.28
4.28
4.2
4.2
4.2
4.13 4.1
4.1 4.03 4 3.9 3.8
Baseline
48 h
1 month
3 months
Time interval
Figure 2. Median log colony-forming units values of mutans streptococci (MS) among the three groups at various time intervals.
month and 3 months in the intervention groups was statistically not different from the placebo varnish group. Discussion
The present study compared the effect of F varnish (Fluor Protector) and CHX/T varnish
(Cervitec Plus) with intensive application regimen on MS levels in human dental plaque using microbiological methods under randomized controlled clinical trial settings. Children between 6 and 10 years of age were chosen for the study population because at this age, they are highly susceptible to dental caries and are undergoing multiple permanent tooth eruption22. Various researchers have found that the most frequent variable in caries risk assessment models is one that describes past caries experience. Thus, in this trial, DMFT/ deft > 1 was considered as one of the inclusion criteria indicating child at moderate-to-high caries risk. This is in accordance with caries risk assessment tool given by AAPD in 201123. In the study, the estimated sample size for CHX/T and F varnish was 25 children per group and 15 children for placebo varnish group. This unequal allocation ratio is justified on the grounds of ethics, economic efficiency, and to maximize available participants for trial. At the same time, the statistical power is not compromised as the allocation ratio is more than 3:124.
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Table 3. Intragroup comparison at different time intervals.
Time interval Group 1 Group 2
48 h–baseline
1 month– baseline
Z
Z
P value 4.10 3.92
Effect of fluoride varnish and chlorhexidine–thymol varnish on mutans streptococci levels in human dental plaque: a double-blinded randomized controlled trial SANCHIT PAUL1, SUPRABHA BARANYA SHRIKRISHNA1, ETHEL SUMAN2, RAMYA SHENOY3 & ARATHI RAO1 1
Department of Paedodontics and Preventive Dentistry, Manipal College of Dental Sciences, Manipal University, India, Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, India, and 3Department of Public Health Dentistry, Manipal College of Dental Sciences, Manipal University, Mangalore, India
2
International Journal of Paediatric Dentistry 2014; 24: 399–408 Background. Recent systematic reviews on clinical
trials comparing the efficacy of chlorhexidine and fluoride varnish found that the evidence was inconclusive and further well-conducted randomized controlled clinical trials were advocated. Aim. To compare the effect of fluoride varnish (F) and Chlorhexidine–thymol varnish (CHX/T) with intensive application regimen on mutans streptococci (MS) levels in human dental plaque. Design. Seventy-five subjects between 6 and 10 years of age were allocated into three groups: Group 1-F varnish (n = 29); Group 2-CHX/T varnish(n = 29); Group 3-placebo varnish (n = 17) by stratified block randomization. After baseline plaque samples were obtained, varnish application
Introduction
Dental caries is a complex disease due to interaction of various factors such as host, agent, substrate, and time. Although, the cause of dental caries appears to be a complex interaction between genetic and environmental factors, it is important to understand that dental caries does not occur in the absence of dental plaque or dietary fermentable carbohydrates. Longitudinal studies have found a significant association between increased proportion of mutans streptococci (MS) in saliva and dental plaque and incidence of dental caries. Therefore, control of MS levels is an important target for caries prevention and control1. Correspondence to: Dr Suprabha B. S., ‘Shreyas’, 15/17/940-13, 5th Cross Road, Shivabagh, Kadri, Mangalore – 575 002, Karnataka, India. E-mail: [email protected]
was applied and repeated at an interval of 3 days in each group. Plaque samples were repeated at 48 h, 1 month, and 3 months. The samples were spread over mitis-salivarius-bacitracin (MSB) culture media, and the colony-forming units per ml (CFU) were measured. Results. In both Groups 1 and 2, Wilcoxon matched-pairs signed-rank test revealed significant differences in log CFU values of MS between baseline and 48 h, baseline and 1 month but no significant difference between baseline and 3 months. An intergroup comparison at different time intervals showed that the difference between three groups was statistically insignificant. Conclusion. F varnish and CHX/T varnish, with an intensive regimen application have equivocal effect on MS levels in dental plaque.
Among the possible strategies in MS control, chemotherapeutic treatment regimens have received much attention and have presented satisfactory results2. Fluoride over the time has emerged as a leading preventive agent for dental caries due to its ability to reduce acid formation in some bacterial species in dental plaque, including MS. Fluoride varnish is quick and easy to apply, well tolerated by young children3. It has been recommended for caries prevention in children with moderateto-high caries risk by a Cochrane database systematic review4 as well as the American Academy of Paediatric Dentistry5. Of all the antimicrobial agents available for dental use, chlorhexidine (CHX) is the most thoroughly researched in terms of ability to control cariogenic activity6. It is particularly effective in reducing the levels of MS in saliva and dental plaque7. Chlorhexidine is available in a variety of different formulations
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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(i.e., mouth rinse, gel, varnish, impregnated dental floss and toothpaste) and concentrations8,9. A review on the mutans streptococciinhibiting efficacy of CHX concluded that the most persistent reduction in mutans streptococci was achieved by CHX varnishes, followed by gels and mouthwashes7. In a systematic review by Twetman10, 16 trials of chlorhexidine varnish were evaluated and the evidence for the effectiveness of chlorhexidine varnish was rated as inconclusive due to conflicting results. In another systematic review, Zhang et al.9 concluded that chlorhexidine varnish had a moderate cariespreventive effect when applied at intervals of 3–4 months. Evidence for the effectiveness of chlorhexidine varnish for preventing caries in primary and permanent teeth of children and adolescents, however, is generally lacking.8 To enhance the effectiveness of chlorhexidine varnish, an intensive treatment regimen (2–4 applications at intervals of 2–7 days) has been recommended6. In an intensive treatment regimen study (two applications of 1% CHX during a 2-week period) by Petersson et al.11in 1991, a 3-month decrease was shown in dental biofilm Streptococcus mutans. Similar findings were observed by Twetman et al.12 in 1997. Variation was, however, observed in results in other studies, with the decrease lasting from 3 days to 2–3 weeks13,14. Zhang et al.9in his systematic review added that there was no evidence for effectiveness with longer intervals of application. Although the existing studies indicated improved efficacy with more frequent applications, the number was too small to be included in meta-analysis. A systematic review by Petersson et al.15also concluded that there is inconclusive evidence regarding the anticariogenic efficacy of fluoride varnishes based on application frequencies. Randomized trials are considered to provide the best evidence of effectiveness because they minimize selection bias. There have been only few randomized controlled trials comparing 1% chlorhexidine–thymol (CHX/T) and fluoride varnish (F)16–18. In these studies, dental caries was the outcome measure, and the effect on MS level was not evaluated. Evaluation of MS level in plaque may be important because of their association with dental caries and can serve
as an indicator of caries activity, thus redeeming it important in prevention of dental caries1,13. Recent systematic reviews advocated further well-conducted randomized and controlled clinical trials comparing chlorhexidine and fluoride varnish involving children and adolescents, as the evidence was inconclusive especially regarding application frequencies8,15,19. This study was undertaken to evaluate the effect of fluoride varnish (F) and chlorhexidine–thymol varnish (CHX/T) with intensive application regimen on growth and colonization of MS in human dental plaque using microbiological methods under randomized controlled clinical trial settings. The null hypothesis tested was that there is no statistically significant difference between the effect of fluoride varnish (F) and chlorhexidine–thymol varnish (CHX/T) on MS levels in human dental plaque. Materials and methods
The investigation was designed, analysed, and interpreted according to the Consolidated Standards of Reporting Trials (CONSORT). This was a single-centre, stratified block randomized, (6–10 years age, with imbalanced randomization [1.6 : 1]), double-blinded, placebo-controlled parallel-group study. All patients between 6 and 10 years of age, visiting the Paediatric Dentistry Department of Manipal College of Dental Sciences, Mangalore, India, were screened for inclusion and exclusion criteria. The area in which the study was carried out had high caries prevalence (82% in 5- to 7-year age group and 94% for 8- to 10-year age group)20. Inclusion criteria The inclusion criteria were as follows: DMFT/ deft > 1; subjects should be free of any systemic diseases; written informed consent from the parents/guardians; the agreement to comply with the study visits and procedures. Exclusion criteria Exclusion criteria were as follows: patient with relevant medical history; handicapped/
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Effect of fluoride and chlorhexidine–thymol varnishes
mentally challenged children; those taking xylitol chewing gums/undergone any fluoride treatment which could influence the conduct of the study; those who had received antimicrobial therapy in the previous 3 months; children with any intraoral device/developmental defect; poor cooperation reported by guardians of subjects. Ethical clearance was obtained from the institutional ethics committee prior to the study. Voluntary written informed consent was obtained from the parents, and a verbal informed consent was obtained from children, prior to the study. The varnishes used in this trial were Fluor Protector (IvoclarVivadent, Schaan, Liescstenstein Germany) which consists of 1% difluorosilane, with polyurethane as varnish base and ethyl acetate/isoamyl propionate as a solvent. The fluoride content is equivalent to 0.1% or 1000 parts per million (ppm) in solution; Cervitec Plus (IvoclarVivadent, Schaan, Liescstenstein, Germany) which consists of 1% chlorhexidine diacetate and 1% thymol with excipient as vinyl acetate copolymer and acrylate copolymer and ethanol as solvent. Sample size and selection The sample size was estimated to be 25 children for each varnish group(CHX/T and Fluoride varnish) considering a study power of 80% (1-b), to detect a specified difference (d) of 10%, statistical significance of 5% (a = 0.05), and effect size (d) 0.8 using GPower 3.1. A sample of 15 children was estimated for placebo varnish. Allowing for confounding bias and losses of 15% during the follow-up, 29 children were included in both the intervention groups at the outset and 17 in placebo group, thus giving a total sample of 75. The children were allocated into three groups by stratified block randomization method, with stratification carried out according to age. The groups were as follows: Group 1: Fluor Protector (F varnish) (n = 29); Group 2: Cervitec plus (CHX/T varnish) (n = 29); Group 3: placebo varnish (cavity varnish–copal rosin with diethyl ether as solvent; Namuvar Dental varnish, Deepti Dental Products of India Pvt. Ltd., Ratnagiri, India) (n = 17). The
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children were divided into five strata at an interval of one year based on age. A block size of six was used during randomization. Random allocation was done by an investigator who was neither involved in varnish application to study subjects nor measurement of outcomes, so as to achieve allocation concealment. The participants in this trial were blinded as to their group. Baseline information and treatment Oral hygiene practice and 24-h diet history of all the subjects were recorded using a questionnaire form, and oral hygiene instructions, diet counselling, and fluoridated toothpaste were given during the period of study. Routine dental treatment such as extractions and restoration of all open carious lesions were carried out prior to commencement of the study. Plaque sampling Plaque sampling was carried out at baseline, at 48 h after the varnish application, at the end of 1 month and at the end of the 3 months. The subjects were informed to refrain from oral hygiene procedures for 24 h, prior to plaque sampling. The adherent saliva was removed using water spray and then air-dried. Plaque was collected using a wooden toothpick from smooth surfaces (by streaking on buccal and lingual surfaces from occlusal to gingival one-third) of all the molars of the selected subjects and transferred to small plastic vials containing thioglycollate transport media. All the above-mentioned procedures were performed by a single investigator with proper aseptic measures. Microbiological processing Tip of the wooden toothpick was cut off and transported to microbiological laboratory containing thioglycollate transport media on the same day. Samples were homogenized manually by stirring using a stirrer. Inoculating loop with a diameter of 0.05 lL was used to collect the diluted plaque from transport vials. Plaque samples were spread over MSB culture media
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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[mitis salivarius supplemented with bacitracin (0.2 U/mL), and sucrose (200 g/litre)], a selective medium for MS, by spreading method21 and incubated for 48 h at 37°C in an incubator. After 48 h of incubation period, MS appeared on the culture plate as small, rough, raised and adherent colonies. The colonyforming units per ml (CFU) of diluted plaque were measured based on values obtained per 100 lL of diluted plaque. All the microbiological procedures were performed by two microbiology personnel who were blinded with respect to the group from which the plaque was collected. Culture plates were examined for identification of colonies of MS and counted manually by both the microbiology personnel to obtain the surface viable count21. Interexaminer variability was assessed by intraclass coefficient correlation analysis, which was found to be 0.92, reflecting a high degree of conformity in observational judgments between the two examiners. Varnish application procedure After baseline plaque samples were obtained from each of the subject, varnish application procedure was carried out for each of the subject in all three groups. Before application of varnish, oral prophylaxis was carried out for subjects. The teeth were then isolated with cotton rolls and saliva ejector and dried with gentle blow of air for 30 s using threeway air syringe. Approximately 0.1 mL of the designated varnish was applied to all the teeth of subjects in all the three groups. A small brush supplied by the manufacturer was used for Cervitec group. For Fluor Protector and placebo varnish groups, applicator tips were used for application of varnish. Varnish was allowed to dry, and after one minute, the cotton rolls were removed. Subjects in all the three groups were instructed not to rinse their mouth, not to drink or eat anything for three hours, and not to brush till the next day. Follow-up The varnish application was repeated at an interval of 3 days for all the three groups in
the same manner as above. The follow-up plaque samples from all the three varnish groups were taken at the end of 48 h, at one month and at the end of three months and assessed for MS counts in the same manner as described before. During the study period, the subjects were asked to report any adverse effects noticed by them immediately to the parents and the investigator; however, none of the subjects reported any adverse effect. Statistical analysis The obtained data were compiled systematically, and the colony-forming units per ml (CFU) were transformed to logarithmic values. Statistical data were analysed using SPSS for Windows release 19.0 (SPSS Inc., Chicago, IL, USA). Because of the high degree of skewness of the microbiological data (Kolmogorov–Smirnov test P < 0.001), nonparametric methods were used for analysis of all microbiological data. Comparison of plaque MS log CFU values at baseline, 48 h, at the end of 1 month and 3 months for each group was made by Friedman’s test, and intergroup comparisons were made with Wilcoxon matched-pairs signed-rank test. Comparison between the groups at various time intervals was made by Kruskal–Wallis test. Results and Observations
At the end of the study, of 75 subjects recruited at baseline level, seven subjects did not report on the days of follow-up plaque sampling due to illness or migration to another city. This amounts to 9.33% of dropouts which was well within our estimated dropout percentage. For the final analysis of the data, the dropouts were eliminated, and the rest of the data was considered as depicted in trial profile (Fig. 1). Chi-square test revealed that at baseline, there was no statistically significant difference between the groups with respect to age, gender, oral hygiene practices, and sugar frequency in the diet (Table 1 and Table 2). No significant difference was also seen in DMFT, deft (Table 2), and log CFU values at baseline (P = 0.49) (Fig 2).
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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ASSESSED FOR ELIGIBILITY
(N = 90) Excluded: (n = 15) Reasons: Parents’ consent not obtained
ENROLLMENT
RANDOMIZATION
(N = 75)
ALLOCATION
Allocated to intervention GROUP-1 FLUORPROTECTOR (n = 29)
Allocated to intervention GROUP-2 CERVITEC PLUS (n = 29)
Allocated to intervention GROUP-3 PLACEBO (n = 17)
FOLLOW UP for 3 months
GROUP-1 FLUORPROTECTOR Lost to follow up: n = 3
GROUP-2 CERVITEC PLUS Lost to follow up: n = 3
GROUP-3 PLACEBO Lost to follow up: n = 1
REASONS FOR DROP OUT: ABSENCE ON THE DAY OF EXAMINATION; REFUSAL; ILLNESS; MIGRATION
ANALYSIS
GROUP-1 FLUORPROTECTOR Analyzed: n = 26
GROUP-2 CERVITEC PLUS Analyzed: n = 26
GROUP-3 PLACEBO Analyzed: n = 16
Figure 1. Trial profile.
Friedman’s test revealed statistically significant difference over time in both Fluor Protector group (Group 1) and Cervitec Plus group (Group 2) (P < 0.001). Wilcoxon matched-pairs signed-rank test revealed significant differences in MS log CFU between baseline and 48 hours and between baseline and 1 month in both Group 1 and Group 2. The statistically significant difference was, however, no longer demonstrable between baseline and 3 month in both the groups. Significant difference was also observed when compared between 48 h and 1 month, 48 h and 3 month and between 1 month and 3 month (Table 3). In Group 3 (placebo varnish group), Friedman’s test revealed that there was no statistically sig-
nificant difference over three months time (P = 0.19). When intergroup comparison was made, at all the time intervals, the difference between three groups was found to be statistically insignificant with almost similar values at the end of 3 month (Table 4 and Fig 2). Although there was decrease in log CFU values in both the intervention groups (Group 1 and 2) at 48 h, there was no statistically significant difference between the groups at 48 h (P = 0.40). Similarly, there was no difference at 1 month and 3 months also. Also, the decrease at 48 h in the intervention groups was not statistically significant as compared with the placebo varnish group. Similarly, the increase in log CFU values at one
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Table 1. Comparison of descriptive statistics at baseline levels.
Variables Gender Male Female Toothbrush use Yes No Brush frequency Once/day Twice/day Fluoridated toothpaste Yes No Sugar exposure 4/day Total
Group 1 no. (%)
Group 2 no. (%)
Group 3 no. (%)
19 (73.1) 7 (26.9)
15 (57.9) 11 (42.1)
24 (92.3) 2 (7.7)
v2
P value
10 (62.5) 6 (37.5)
1.39
0.49
25 (96.1) 1 (3.8)
15 (93.2) 1 (6.2)
0.35
0.84
22 (84.6) 4 (15.4)
20 (76.9) 6 (23.1)
13 (81.2) 3 (18.8)
0.49
0.78
22 (84.6) 4 (15.4)
25 (96.2) 1 (3.8)
15 (93.8) 1 (6.2)
7.35
0.12
18 7 1 26
17 7 2 26
11 4 1 16
0.38
0.98
(69.3) (26.9) (3.8) (100)
(65.4) (26.9) (7.7) (100)
(68.75) (25.0) (6.25) (100)
Table 2. Comparison of mean age and mean deft/DMFT values of different groups. Variable
Group 1 (26)
Group 2 (26)
Group 3 (16)
v2
P value
Age deft DMFT
8.81 1.44 2.04 1.69 0.50 1.10
8.54 1.52 2.85 1.73 0.46 0.95
8.56 1.54 3.44 1.86 0.25 0.58
0.36 11.29 5.63
0.89 0.50 0.69
Group 1 4.4
Group 2
Group 3
4.36
4.35
4.33
log Cfu values
4.3
4.3 4.31
4.28
4.28
4.2
4.2
4.2
4.13 4.1
4.1 4.03 4 3.9 3.8
Baseline
48 h
1 month
3 months
Time interval
Figure 2. Median log colony-forming units values of mutans streptococci (MS) among the three groups at various time intervals.
month and 3 months in the intervention groups was statistically not different from the placebo varnish group. Discussion
The present study compared the effect of F varnish (Fluor Protector) and CHX/T varnish
(Cervitec Plus) with intensive application regimen on MS levels in human dental plaque using microbiological methods under randomized controlled clinical trial settings. Children between 6 and 10 years of age were chosen for the study population because at this age, they are highly susceptible to dental caries and are undergoing multiple permanent tooth eruption22. Various researchers have found that the most frequent variable in caries risk assessment models is one that describes past caries experience. Thus, in this trial, DMFT/ deft > 1 was considered as one of the inclusion criteria indicating child at moderate-to-high caries risk. This is in accordance with caries risk assessment tool given by AAPD in 201123. In the study, the estimated sample size for CHX/T and F varnish was 25 children per group and 15 children for placebo varnish group. This unequal allocation ratio is justified on the grounds of ethics, economic efficiency, and to maximize available participants for trial. At the same time, the statistical power is not compromised as the allocation ratio is more than 3:124.
© 2013 BSPD, IAPD and John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Table 3. Intragroup comparison at different time intervals.
Time interval Group 1 Group 2
48 h–baseline
1 month– baseline
Z
Z
P value 4.10 3.92
