|Year : 2013 | Volume
| Issue : 5 | Page : 575-581
|Efficacy of silver diamine fluoride as an antibacterial as well as antiplaque agent compared to fluoride varnish and acidulated phosphate fluoride gel: An in vivo study
Shalin Shah1, Vijay Bhaskar2, Karthik Venkataraghavan1, Prashant Choudhary1, M Ganesh2, Krishna Trivedi1
1 Department of Pedodontics and Preventive Dentistry, College of Dental Science and Research Centre, Bopal-Ghuma Road, Manipur, Ahmedabad, Gujarat, India
2 Department of Pedodontics and Preventive Dentistry, Ahmedabad Dental College and Hospital, Vivekanand Society, Bhadaj-Ranchhodpura Road, Santej, Gujarat, India
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|Date of Submission||23-Feb-2013|
|Date of Decision||12-Mar-2013|
|Date of Acceptance||18-Apr-2013|
|Date of Web Publication||21-Dec-2013|
| Abstract|| |
Background: Silver diamine fluoride (SDF) is already proven as an antibacterial agent in vitro. Present study was formulated to compare the efficacy of SDF as an antibacterial as well as antiplaque agent in vivo with fluoride varnish and acidulated phosphate fluoride (APF) gel.
Study Design: Total 123 children (male = 82, female = 41) were included in the study for a period of 18 months. Children were divided into three different groups-Group 1: SDF; Group 2: fluoride varnish; and Group 3: APF gel. All subjects were evaluated via plaque score at 6 th , 12 th , and 18 th months as well as Streptococcus mutans counts in saliva at 72 h, 6 th , 12 th , and 18 th months of follow-up.
Results: Significant reduction was found in plaque score as well as S. mutans counts irrespective of group division. On intergroup comparison, no statistically significant difference was found in plaque score, but significant reduction in S. mutans counts was found in Group 1 as compared with Groups 2 and 3, while no significant difference was found between Groups 2 and 3.
Conclusion: In vivo application of SDF on enamel significantly decreases S. mutans counts as compared to fluoride varnish and APF gel.
Keywords: Plaque index, silver diamine fluoride, Streptococcus mutans
|How to cite this article:|
Shah S, Bhaskar V, Venkataraghavan K, Choudhary P, Ganesh M, Trivedi K. Efficacy of silver diamine fluoride as an antibacterial as well as antiplaque agent compared to fluoride varnish and acidulated phosphate fluoride gel: An in vivo study. Indian J Dent Res 2013;24:575-81
Dental caries is still one of the most common chronic dental diseases affecting various age bars in all countries and all populations with varying degree of severity. Treatment of the dental caries may require advanced skills of clinicians and high cost of general anesthesia for patient management.  In 1942, Bibby  began era of topical fluorides with the use of a solution of 0.1% sodium fluoride (NaF). Subsequently over the years, various other topical fluoride agents have been evolved which in sequential order are stannous fluoride (SnF2) (1947), acidulated phosphate fluoride (APF) (1963), varnish containing fluoride (1964), and amine fluoride (1967). Fluorides have been proved to be the single most effective weapon in still limited arsenal of anticaries agents in last 60 years. Caries preventive programs can be effectively carried out using topical fluoride agents like fluoride mouth rinse and fluoride varnish. Studies also conclude that caries preventive effects of fluoride are almost exclusively topical. , Among them fluoride varnish and 1.23% APF gel were the most commonly used professionally applied topical fluoride agents till date  , still none of them have proved completely satisfactory.
|How to cite this URL:|
Shah S, Bhaskar V, Venkataraghavan K, Choudhary P, Ganesh M, Trivedi K. Efficacy of silver diamine fluoride as an antibacterial as well as antiplaque agent compared to fluoride varnish and acidulated phosphate fluoride gel: An in vivo study. Indian J Dent Res [serial online] 2013 [cited 2021 Aug 4];24:575-81. Available from: https://www.ijdr.in/text.asp?2013/24/5/575/123374
Silver diamine fluoride (38% w/v) [molecular formula: Ag (NH 3 ) 2 F. E.g. Saforide solution (J Morita Company, Japan) was introduced in Japan in 1970s. Since then it was used in Japan as an effective caries arresting agent.  Different in vivo studies done by Chu et al.,  Llodra et al.,  and Yee et al.,  mentioned that SDF was found to be effective in arresting as well as in reducing dental caries in primary teeth and also it provides an alternative when restorative treatment for primary teeth is not an option. On the contrary, in vitro trials done by Knight et al., , found that dentinal discs treated with silver diamine fluoride (SDF) shows more reduction in Streptococcus mutans Till date no in vivo study has been reported to check its applicability as an antibacterial as well as antiplaque agent when applied on enamel surface.
| Aims and Objectives|| |
Keeping the above-mentioned discussion in mind, this study was formulated to compare the efficacy of SDF (38% w/v) (Saforide-J. Morita company, Japan) as an anticarious agent with fluoride varnish (6% NaF, 6% CaF2) (Bifluoride 12 Voco, Germany) and APF gel 1.23% (Fluocal, Septodont, France) with the following objectives:
- To evaluate the changes in plaque score after application of SDF, fluoride varnish, and APF gel.
- To compare the effect of SDF, fluoride varnish, and APF gel on S. mutans counts.
| Materials and Methods|| |
The study was conducted in the Department of Pedodontics and Preventive Dentistry. It was a randomized, controlled, prospective in vivo study. Study protocol was approved from ethical committee of the Dental College and Hospital.
A total of 419 children were screened from four primary and secondary government-funded schools in Gandhinagar district, Gujarat, India. Children in these schools are from a low socioeconomical status. At the time of screening, type of oral hygiene practice was also evaluated and children who regularly brush their teeth twice daily with fluoridated paste were included in the study. The screening was done with mouth mirror and explorer under good illumination (natural daylight). At the end of screening, 123 children (male = 82, female = 41) with the mean age of 8.38 ± 0.75 years who fulfilled the inclusion criteria given below were selected. Before the commencement of the study, the parents were clearly explained the purpose of the study and informed consent from both parents was obtained for the subjects participating in the study. The trial was conducted for a period of 18 months.
Subjects fulfilling the following inclusion criteria were included in the study [Figure 1]:
- All permanent first molars fully erupted.
- Subjects with decayed missing filled surface + DMFS (decayed, missing, filled surface - Primary teeth + Decayed Missing, Filled Surface - Permanent teeth) score equal to or more than 1. Analysis of variations (ANOVA) shows no statistical significant difference between study groups at baseline. [Table 1].
- Subjects with all deciduous molars present.
- No known history of allergy against silver particles or colophonium.
Group 1: Children receiving application of SDF (38% w/v) (Saforide, J. Morita Corporation, Japan) on all deciduous canines and molars and first permanent molars (n = 41).
Group 2: Children receiving application of fluoride varnish (6% NaF, 6% CaF 2 ) (Bifluoride 12, Voco, Germany) on all deciduous canines and molars and first permanent molars (n = 41).
Group 3: Children receiving application of APF gel 1.23% (Fluocal, Septodont, France) on all deciduous canines and molars and first permanent molars (n = 41).
Patients were first evaluated for baseline examinations of following criteria:
- Plaque index (Silness and Loe). 
- For S. mutans count in the saliva inoculated on tryptone-yeast-cysteine-sucrose-bacitracin agar (TYCSB) media.
Method for saliva collection
The saliva sample was taken with the help of cotton swab 2 h after brushing or before meals. The swab was also wiped from buccal and lingual surfaces of all teeth. From each swab, 0.1 mL of saliva was placed into a calibrated sterile vial containing phosphate buffered saline (pH = 7.3) and was transported to the microbiology laboratory at 4°C in icebox where it was processed within 4 h.
Steps for the study
After baseline evaluations and saliva collection, full mouth ultrasonic scaling was done. Additionally, polishing rubber cup in a slow speed handpiece were used while applying a constant stream of water. All carious lesions present in the mouth were restored with intermediate restorative material (IRM) (Kalzinol, DPI, India). The above-mentioned procedure was same for all subjects. Afterward, they were divided in to three different groups on a random basis using computerized randomizationTables (GraphPad Software, Inc, CA, USA). Fluoride application was done on primary canine, first and second molars as well as permanent first molar based on the individual group. All the children had received fluoride application at the baseline as well as at 6 th and 12 th month of follow-up.
Anterior teeth were excluded because patient selected in this study have transition period for incisors at this stage and also SDF can cause staining of the tooth surfaces, so it is not indicated for anterior teeth.
Procedure for fluoride application
Application of SDF
Before starting the procedure, the whole mucosal surface in the oral cavity was covered with the Vaseline, to protect it from mild burning sensation due to SDF. Isolation of the teeth was done with the help of cotton rolls and high volume suction [Figure 2]. Lid of the bottle was removed just before the application and drop of solution was squeezed on the cotton pellet. Then application was done for 3-4 min on all surfaces of four teeth in single quadrant at one time.
- This procedure was repeated on all quadrants in similar manner.
- As per manufacturer's instructions, after 3-4 min of application the patient was allowed to clean his mouth by gargling with distilled water or normal saline.
Application of fluoride varnish and APF gel was also done as per manufacturer's instructions in their respective group [Figure 3] and [Figure 4]. Also, the patients were instructed not to rinse, drink, or eat for at least 30 min, take liquid and semisolid diet for that day and do not brush the teeth for that day. The similar procedure was repeated in all three groups at 6 th month of follow-up, when subjects received second fluoride application.
All the patients were followed-up and evaluated at end of 6 th , 12 th , and 18 th months. If any new carious lesion or loss of restoration was detected, it was restored at the same visit. Dropout rate of the subjects was around 9% at the end of study. The reason for dropout is mainly due to migration of the family to other place.
Plaque index 
The baseline as well as follow-up plaque scoring was done by two different examiners blinded to the group division at 6 th , 12 th and 18 th months of intervals. Prior to the study, diagnostic criteria were carefully standardized as per indices given by Silness and Loe. Both the examiners calibrated around 25 children (20% of sample size) before starting the study. Interexaminer reproducibility was very good throughout the study. The values of the κ statistic were at least 0.95. The plaque scoring was done on selected teeth (selected mouth basis). Only plaque of the cervical 3rd of the tooth was evaluated with no attention to plaque that has extended to the middle of the incisal thirds. The surfaces examined were the four gingival areas of the tooth, that is, the distal-facial, facial, mesial-facial, and lingual surfaces. Six teeth were evaluated: 16, 52, 12, 64, 36, 72, 32, and 84.
Streptococcus mutans count
S. mutans counts in the saliva were checked with the help of TYCSB media after 72 h of application (3 rd day), at 6 th , 12 th , and 18 th month of follow-up.
Method of microbiological analysis of salivary samples
Samples were vortexed for 30 s to disperse bacteria. A total of 100-μL aliquots of tenfold dilutions were plated onto S. mutans-selective TYSCB media. Plates were inoculated and were incubated at 37°C for 72 h. Colonies were counted by colony counter by two different individuals at different time to reduce the interpersonal bias and the mean colony-forming units/mL (CFUs/mL) saliva was calculated for S. mutans.
After incubation, the colonies were randomly selected to study the characters like color, size, shape, convexity, surface margins, consistency, opacity, hemolysis on blood agar, pigmentation, gram staining, mannitol and sorbitol fermentation, esculin hydrolysis and catalase test at Supratech Micropath Laboratory and Research Institute, Ahmedabad, India and it was confirmed that the organisms were S. mutans.
Estimation of sample size was based on the expected amount of reduction in S. mutans counts on the basis of pilot study. A total of 30 children were included in pilot study (10 per each group). S. mutans counts were obtained before and 1 month after the application of topical fluoride agent. On the basis of difference in mean number between groups and standard deviation obtained from pilot study, the sample size was estimated to be around 110 using the nomogram given by Altman.  Keeping the estimated dropout rate in mind around 125 sample size was decided, among them total 123 subjects were included in the study.
All the collected data were evaluated using SPSS VERSION 13 software for windows. For plaque score as well as for S. mutans counts, paired sample t-test was done for intragroup comparison and ANOVA test was done followed by post hoc test Multiple comparisons Tukey's honestly significant difference (HSD) for intergroup comparison. Mann-Whitney test was used to compare the significant difference for new caries development between each group.
| Results|| |
Following variables were evaluated during study
Age and sex wise distribution of the subjects are mentioned in [Figure 5] and [Figure 6] respectively. [Table 2] shows mean plaque score as well as total number of subjects available at baseline, 6 th month, 12 th month, and finally at 18 th month. Irrespective of group division, statistically significant reduction was found between baseline mean plaque score with that of 6 th , 12 th , and 18 th months follow-up (P < 0.05). Intragroup comparison for plaque score was also mentioned in [Table 2] and for intergroup comparison ANOVA shows no significant difference in plaque score between any of the group (P > 0.05).
Streptococcus mutans counts in saliva
[Table 3] shows mean value of S. mutans counts at baseline, on 3 rd day (After 72 h), 6 th month, 12 th month, and finally at 18 th month. Irrespective of group division clear statistically significant reduction in S. mutans counts was found between baseline with that of 3 rd day, 6 th month, 12 th month, and 18 th month (P < 0.05). Intragroup comparison was also mentioned in [Table 3]. ANOVA test was done followed by post hoc test multiple comparisons Tukey's HSD for intergroup comparison which was mentioned in [Table 4].
| Discussion|| |
The present study was conducted as a randomized, controlled prospective in vivo trial with SDF as an experimental material, against fluoride varnish and APF gel as comparative groups. In this study, effects of extraneous variables such as the age or sex of the patient, severity in terms of caries activity, food and oral hygiene habits, and so on might influence the response to treatment. So, to promote comparability of the treatment groups, randomization of the subjects was done via computer-generated randomization GraphPad software. Also, it was attempted to reduce the confounding factors as low as possible by including the children from almost similar socioeconomic status, food and oral hygiene habit, and statistically insignificant caries distribution.
Age group selected for the study was 6-9 years, with first permanent molars fully erupted. As the second window of infectivity opens at this age,  first permanent molars were at highest risk to be affected by dental caries. The present study also supports the proposal by Johnston and Lewis  that professionally applied topical fluorides may be practical preventive treatments that allow more high-risk children (included in present study) to be intervened at early age. To maintain the uniformity of the subject selection, only those subjects were included who had all primary molars present. As it was unethical to leave the open carious lesion and proceed with study, all existing carious lesions were restored and defective restorations treated by IRM before instituting the study protocol. This criteria was similar to Bellini et al.,  and Zickert and Emilson. 
An expert panel of the American Dental Association in 2006 concluded that "fluoride varnish applied every 6 months is effective in preventing caries in the primary and permanent dentition of children and adolescents."  Also, biannual applications of APF gel were used by Hawkins and Locker  and Agrawal and Pushpanjali  and found significant caries reduction. On the contrary, there are no published recommendations for the frequency of SDF applications. Chu et al.,  applied it annually, while Llodra et al.,  applied 6-monthly. So, considering the frequency of application of fluoride varnish and APF gel, in this study 6-monthly application of SDF was used to maintain the uniformity of application.
According to Mellberg et al.,  topically applied fluoride may result in less plaque accumulation and ultimately decrease in dental caries. Suzuki et al.,  in their in vitro study also mentioned that SDF offers a possible antiplaque action. So, in present study, it was decided to record plaque score at baseline and at every follow-up visits to evaluate the efficacy of fluoride application. For plaque score, standard criteria given by Silness and Loe  were used, as mentioned in different studies. ,
Considering the group-wise plaque reduction, generalized reduction was found in all groups compared to baseline score. In case of SDF group, significant reduction was found only at 18 th month of follow-up compared to baseline(P < 0.05). This could be due to the follow-up application, which was done at 6 th and 12 th months leading to cumulative effect being seen at 18 th month. Suzuki et al.,  in their in vitro study mentioned that silver ions in combination with fluoride offer a possible antiplaque action of the agent. Results of this study only partially supported this statement. Also SDF has basic pH, which might be the possible factor for enhanced plaque accumulation. In case of fluoride varnish group, significant reduction in plaque score was found at all the follow-ups (P < 0.05). This finding was similar to study done by Klimek et al.,  in which significant reduction in plaque score was found after application of fluoride varnish. In case of APF gel group also, significant reduction was found only at 18 th month of follow-up (P < 0.05). This might be associated with comparatively low fluoride content (12,300 ppm) in APF gel. On intergroup comparison, no significant difference was found between any groups at follow-up visits (P > 0.05). As, some authors like Bellini et al.,  in their study mentioned that preventive effect on plaque and gingival score was essentially attributed to the regular professional tooth cleaning rather than fluoride application, the reduction in plaque score obtained in present study might be due to combined effect of professional tooth cleaning and fluoride application.
S. mutans counts
Ever since Bibby  demonstrated that carbohydrate metabolism in pure cultures of oral Streptococci were inhibited by fluoride, many reports have been published on direct and indirect effects of fluoride on the metabolism of oral bacteria and dental plaque ecology. One of the most important advantages of SDF is an additive effect of silver in reducing S. mutans counts. Different in vitro studies ,, found SDF was highly effective in reducing the S. mutans counts. Till date, no reported study had compared the effect of SDF on reduction of S. mutans counts in vivo. The criteria fixed for saliva sampling in the present study was to wait for at least 2 h after tooth brushing, so that normal bacterial count can be reestablished. The method of saliva sample collection was according to Wan et al.  The TYCSB media was used for detection of S. mutans counts, which is the most selective of the media tested for S. mutans, and was the least supportive of non-S. mutans. ,
Ekenback et al.,  studied the colonization of cariogenic bacteria in plaque after application of four different fluoride varnishes found no statistically significant difference between baseline and over time (1 week, 1 and 6 months). Considering this Jeevarathan et al.,  choose to evaluate S. mutans count only after 24 h. As professional tooth cleaning was done before fluoride application, it would difficult to judge effect of fluoride on S. mutans counts within the period of 24 h; therefore, it was decided to evaluate the S. mutans count after 3 days of application in present study and as the follow-up protocol of study was set at 6 th , 12 th and 18 th months, the S. mutans counts were also evaluated at that time.
In present study, in vivo effect on S. mutans counts was checked comparing three different topically applied fluorides. Compared to baseline S. mutans counts decreased at all the follow-up visits in all the three groups. Only in APF gel group, no significant reduction was found at 12 th month compared to baseline. The results obtained in the APF gel group could be due to fact that amount of fluoride uptake after single application of APF gel may not have significant antibacterial effect considering that APF gel has least fluoride available (12,300 ppm) considering all three groups.
Considering the intergroup significance, significant reduction was found in subjects who received SDF application compared with those subjects who had received fluoride varnish and APF gel application. SDF seems to have higher potential in reducing of S. mutans counts compared to other two agents due to high fluoride content and also the antibacterial potential of silver providing an additive effect on reduction of S. mutans counts. ,, The results of present in vivo study support the finding of previous in vitro studies who found significant decrease in S. mutans counts after application of SDF.
| Conclusion|| |
The following conclusions were drawn:
- Plaque score decreased in all the subjects that may be attributed to preventive protocol used in study as well as fluoride application, but no significant difference between groups.
- SDF can produce significant reduction in the S. mutans counts in saliva compared to fluoride varnish and APF gel.
- It can be derived that SDF might be used effectively as a topical fluoride agent for caries prevention due to its greater antibacterial efficacy as compared to other professionally applied topical fluoridated agents fluoride varnish and APF gel, still additional research is required.
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Department of Pedodontics and Preventive Dentistry, College of Dental Science and Research Centre, Bopal-Ghuma Road, Manipur, Ahmedabad, Gujarat
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4]
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