|Year : 2007 | Volume
| Issue : 4 | Page : 157-163
Effect of fluoride varnish on Streptococcus mutans counts in plaque of caries-free children using dentocult SM strip mutans test: A randomized controlled triple blind study
J Jeevarathan1, A Deepti1, MS Muthu1, V Rathna Prabhu1, GS Chamundeeswari2
1 Department of Pediatric Dentistry, Meenakshi Ammal Dental College and Hospital, Chennai, Tamilnadu, India
2 Department of Microbiology, Meenakshi Ammal Dental College and Hospital, Chennai, Tamilnadu, India
Meenakshi Ammal Dental College and Hospital, Alapakkam Main Road, Maduravoyal, Chennai - 600 095, Tamilnadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Dental caries is one of the most prevalent infectious diseases and although of multifactorial origin, Streptococcus mutans is considered the chief pathogen in its development. Fluoride is one of the most effective agents used for the reduction of dental caries apart from oral hygiene maintenance.
Aims: The aim of this study was to estimate the counts of Streptococcus mutans and to evaluate the effect of Fluor Protector fluoride varnish on these counts in the plaque of caries-free children using Dentocult SM Strip Mutans.
Materials and Methods: Thirty caries-free subjects were selected for the study based on the information obtained from a questionnaire and were randomly assigned to the control group consisting of ten subjects and the study group consisting of twenty subjects. Plaque samples were collected on the strips from the Dentocult SM kit and after incubation, the presence of Streptococcus mutans was evaluated using the manufacturer's chart. The study group was subjected to a Fluor Protector fluoride varnish application following which the samples were collected again after 24 hours.
Results: The average Streptococcus mutan s counts in the primary dentition of caries-free children before and after the application of Fluor Protector fluoride varnish were 10 4 -10 5 colony forming units (CFU)/ml and < 10 4 CFU/ml respectively.
Conclusion: The results showed that the study group had a statistically significant reduction in the plaque Streptococcus mutans counts than the control group.
Keywords: Dentocult SM strips, fluor protector, fluoride varnish, plaque, Streptococcus mutans
|How to cite this article:|
Jeevarathan J, Deepti A, Muthu M S, Rathna Prabhu V, Chamundeeswari G S. Effect of fluoride varnish on Streptococcus mutans counts in plaque of caries-free children using dentocult SM strip mutans test: A randomized controlled triple blind study. J Indian Soc Pedod Prev Dent 2007;25:157-63
|How to cite this URL:|
Jeevarathan J, Deepti A, Muthu M S, Rathna Prabhu V, Chamundeeswari G S. Effect of fluoride varnish on Streptococcus mutans counts in plaque of caries-free children using dentocult SM strip mutans test: A randomized controlled triple blind study. J Indian Soc Pedod Prev Dent [serial online] 2007 [cited 2019 Aug 19];25:157-63. Available from: http://www.jisppd.com/text.asp?2007/25/4/157/37010
Dental caries is a carbohydrate-modified, local infection which destroys the hard tissues of a tooth.  It is a complex disease which is expressed as an interaction of various factors including the host, agent, substrate and time.  Modern concepts consider caries as an interaction between genetic and environmental factors in which social, behavioral, psychological and biological factors are expressed in a highly complex interactive manner.  Most important in the understanding of the caries process is that dental caries does not occur in the absence of dental plaque or dietary fermentable carbohydrate, hence, it is considered a dietobacterial disease. 
Plaque is a soft, translucent and tenaciously adherent material accumulating on the surface of teeth. A number of endogenous oral microorganisms found in dental plaque are considered crucial to the initiation and progression of dental caries. These microorganisms include mutans streptococci ( Streptococcus mutans , Streptococcus sobrinus ), Lactobacillus species, Actinomyces species, nonmutans streptococci and yeast.  The microbial traits strongly associated with caries include:
a. The ability to produce acid and to sustain acid production at low pH levels that result in the demineralization of the calcified structure.
b. The formation and use of extra- and intracellular storage polysaccharides that permit microorganisms to continuously produce acid even after dietary carbohydrates have been depleted.
c. The formation of water-insoluble glucans that aid in the accumulation of mutans streptococci in plaque and modification of its diffusion characteristics, allowing the substrate to diffuse to a deeper layer of plaque adjacent to the tooth surface. 
Mutans streptococci possess all of these virulent traits which support their role in the caries process. They become pathogenic only under conditions that lead to frequent and prolonged acidification of the dental plaque. Mutans streptococci and lactobacilli gain a selective advantage over other microorganisms as a result of their aciduric properties. Streptococcus mutans adapt to the low pH of this environment and thus, increase their rate of acid production and drive the pH still lower resulting in a cariogenic plaque.  The exact extent of their role in the development of dental caries has been disputed as according to Van Houte, Streptococcus mutans play a significant role whereas Loesche claimed that it is the chief pathogen. , Longitudinal studies have shown a relative rise of Streptococcus mutans counts in plaque samples from tooth surfaces that become carious at a later stage. ,,,
Caries activity is a compound diagnosis derived from immediate past experience, lesion progression and the clinical appearance of the lesion or cavities.  Caries activity is evaluated on the basis of data obtained from clinical examination and assessment of factors associated with the pathogenesis of the disease. This data on dental caries can be collected by traditional visual inspection and probing or by some objective detection methods which rely on the mineral changes as a basis for evaluation of caries activity and risk assessment.  None of these methods aim at the estimation of the chief pathogen - Streptococcus mutans . Microbial monitoring has been considered as an alternative method for evaluating current caries activity and future caries risk. According to Shi et al. , the Dentocult SM kit is a reliable method for measuring the status of dental caries in preschool children and also a valuable tool in the prevention and treatment of dental caries.  In 2003, they added that Dentocult SM is a test which is helpful for the diagnosis of caries and its progression based on Streptococcus mutans counts. 
As dental caries is of multifactorial etiology, preventive measures usually involve a combination of dietary counseling, oral hygiene measures and fluoride application.  Oral hygiene measures aim at the removal of plaque from the tooth surfaces which contain Streptococcus mutans and other bacteria. Fluoride acts primarily via topical mechanisms including inhibition of demineralization, enhancement of remineralisation at the crystal surfaces and inhibition of bacterial enzymes.  Marsh and Bradshaw found that 19 ppm of fluoride in an in vitro mixed culture study inhibited the growth of mutans streptococci.  Hamilton found a high fluoride concentration in the oral cavity might inhibit acid production by bacteria and may reduce the number of certain species.  Songpaisan et al . reported low counts of Streptococcus mutans in children using 0.5% hydrofluoride solution.  Berg et al. and Forss et al . have found that the plaque adjacent to fluoride-releasing glass ionomers demonstrate lower levels of Streptococcus mutans . , Yoshiara et al . found that the long-term use of a fluoride mouth rinse might contribute to the reduction of mutans streptococci.  This present study was designed to estimate the Streptococcus mutans counts and evaluate the effect of Fluor Protector fluoride varnish on these counts in the plaque of caries-free children.
| Materials and Methods|| |
This study was planned and carried out in the Department of Pediatric Dentistry in association with the Department of Microbiology in Meenakshi Ammal Dental College. All kindergarten children in Arulmigu Meenakshi Amman Matriculation Higher Secondary School, Chennai were screened by examiner A using a mouth mirror and probe in daylight. Forty-eight caries-free children with a full set of primary dentition were selected. The parents of these children were asked to report to the department. The study was explained to the parents in detail. A child's personal details, details of past medical history including any recent antibiotic exposure, past dental history including recent fluoride treatment, frequency of brushing, sweets/snacks intake and consumption of sugared/energy drinks and the brand of toothpaste (to determine fluoride content) were obtained through a questionnaire from parents. Thirty subjects were selected for the study with the following inclusion criteria:
- Caries-free primary dentition
- No history of intake of antibiotics for the past 3-4 weeks
- No history of fluoride treatment for the past two weeks.
Written consent was obtained from these parents.
Each subject was assigned a specific number by examiner B by asking them to draw lots. A statistician randomized the numbers into a control group and a study group. Group I (study group) consisted of 20 subjects and Group II (control group) consisted of ten subjects. The subjects were blinded as to their group.
The counts of Streptococcus mutans in plaque was determined using the simple chairside method (Dentocult SM Strip mutans orion Diagnostica, Espoo, Finland) [Figure - 1]. For the baseline status of Streptococcus mutans , plaque samples were collected using different toothpicks for the control and study group from the following four sites:
a. buccal surface of the maxillary right molar [Figure - 2]
b. labial surface of the maxillary incisor
c. labial surface of the mandibular incisor and
d. lingual surface of the mandibular left molar
These samples were spread thoroughly but gently on the four sites of the rough surface of the strip. The strips were then placed in a selective culture broth with the smooth surfaces clipped and attached to the cap. The vials were then labeled with the lot numbers and incubated in an upright position at 37°C for 48 h with the cap opened one quarter of a turn to allow growth of the organisms.
After the collection of the plaque samples from all patients, the fluoride varnish was applied to the subjects of the study group by examiner B on the same day. First, the tooth surfaces were completely cleaned, dried with an air syringe and then isolated with cotton rolls as per the manufacturer's instructions. A high-volume evacuator with saliva ejector and cheek retractor was also used. A thin layer of Fluor Protector (Ivoclar, Vivadent) [Figure - 1] fluoride varnish was applied on all the tooth surfaces using a suitable brush. The cotton rolls were removed after one minute and the patient was asked not to rinse the mouth immediately and not to eat or brush their teeth for 45 min. After 24 h, plaque samples were again collected from the subjects of both the groups. These were also incubated for the same period as before and the same interpreters evaluated the results again.
After incubation, the presence of Streptococcus mutans was confirmed by detecting light-blue to dark-blue, raised colonies on the inoculated surface of the strip. Colonies suspended in the culture broth were excluded from the evaluation. The results were evaluated according to the manufacturers' chart [Figure - 3].
Class 0: <10,000 CFU/ml
Class 1: <100,000 CFU/ml
Class 2: 100,000-1000 000 CFU/ml
Class 3: >1000 000 CFU/ml
The results were interpreted by two independent interpreters (examiners A and C) who were also blinded about the group division [Figure - 4]. Inspection of growth was done sideways against light or with a magnifying glass to look for raised colonies. The presence of epithelial cells on the strip surface should be differentiated from the mutans colonies, which was done by passing a gloved finger along the strip. The epithelial cells on the strip surface were smooth while the streptococci colonies were rough. Hence, only the rough colonies were considered to estimate Streptococcus mutans growth.
The subjects in the experiment group, the examiner who collected the plaque and the interpreter of the results were blinded about the division of groups. Hence, this study can be considered a randomized, controlled, triple-blind study.
| Results|| |
[Table - 1] shows the distribution of the subjects and their mean age in the study and control groups. There were 20 subjects in the study group and ten in the control group. The mean ages for the study and control groups were 4.17± 0.80 and 4.49± 1.12 years (mean± standard deviation, SD) respectively.
[Table - 2] shows the distribution of the pretreatment bacterial counts in the plaque of caries-free primary dentition. The median bacterial count was in the range of 10 4 -10 5 CFU/ml with the mean bacterial scores being approximately 1.33 ± 0.55.
[Table - 3],[Table - 4] show the distribution of the different variables along with pretreatment and post treatment bacterial counts for the study group. While all the subjects of both the groups used only fluoridated toothpaste, the frequency of brushing, sweets/snacks intake and sugared drinks varied with the subjects of both groups. There was no statistically significant effect of the above variables on the pretreatment and posttreatment bacterial counts in both groups (Chi-square p > 0.05).
[Table - 5] shows the distribution and average pre- and posttreatment bacterial counts in both groups. There was no statistically significant difference in the distribution of bacterial scores in the control group (Mann-Whitney U test p = 0.455), the average pre- and posttreatment bacterial counts being in the range of 10 4 -10 5 CFU/ml. There was a statistically significant difference in the bacterial counts (Mann-Whitney U test p = 0.000) for the study group. The average pretreatment bacterial count was about 10 4 -10 5 CFU/ml whereas it is <10 4 CFU/ml after the fluoride varnish application.
[Table - 6] shows the inter-examiner relationship between the pre- and posttreatment bacterial counts in all the subjects. The bacterial scores value was 0.926 and 0.821 using Cohen's kappa.
| Discussion|| |
Fluoride has been found to be the most effective cariostatic agent in the field of dentistry especially in pediatric dentistry. The cariopreventive action of fluoride is reported to be due to its effects on the teeth, bacteria and plaque. Fluoride alters the physiochemical properties of teeth by making them more resistant to acid dissolution due to the formation of fluorapatite or fluorhydroxyapatite. It also increases the posteruptive maturation, enhances remineralization and inhibits demineralization.  Fluoride inhibits various bacterial enzymes like enolases, phosphatases, proton-extruding ATPases and pyrophosphatases.  It also influences the bacterial composition and alters the plaque ecosystem.  Plaque fluid, an aqueous phase within the plaque, has a higher fluoride concentration than any other oral fluid. , The sources of this plaque fluoride are the calcium fluoride on the enamel beneath the plaque, calcium fluoride in plaque and fluoride in saliva and gingival fluid.  Numerous in vitro and animal studies have shown that fluoride affects the carbohydrate metabolism of mutans streptococci. 
In our study, the average CFU number in the plaque of caries-free children with primary dentition was in the range of 10 4 -10 5 with the average bacterial score being about 1.33± 0.55. S. mutans CFU levels considered a risk for caries in adults and older children per milliliter of stimulated saliva were about 10 5 -10 6 . ,,,
There was no statistical significance of the relationships between pretreatment bacterial scores and frequency of brushing or sweets/snacks intake and sugared/energy drinks consumption for the study group [Table - 3]. Yoshiara et al . compared variables like sealants, DFS, DMFS, frequency of sweet drinks, sweets/snacks and brushing, fluoridated and nonfluoridated toothpaste with bacterial counts while evaluating the effect of fluoride mouth rinse on these counts.  Only the frequency of sweets/snacks, DFS and sealants were found to have a significant effect on the bacterial count. The reason for this significance could be the larger sample size of their study and the higher (class 2 and 3) and lower (class 0 and 1) bacterial score grouping as compared to our study. In our study, all the subjects of the study and control groups used only fluoridated toothpaste and hence, no statistical analysis was done to evaluate its effect on the bacterial count. But according to Peterson et al ., there is no difference in the level of mutans streptococci between subjects using or not using fluoridated toothpaste.  Yoshiara and his co-workers also did not find any significant effect of fluoridated toothpaste on Streptococcus mutans counts. 
Plaque samples were collected from the previously mentioned sites with the aim of assessing the overall effect of the fluoride varnish on Streptococcus mutans counts. However, Vogel and Ekstrand found that there is a large variation between plaque fluid fluoride concentrations at various sites of the oral cavity.  They collected plaque samples from the upper and lower molars and incisors after rinsing with 10 ml of 0.2% NaF (sodium fluoride) mouth rinse. Plaque fluid collected in the maxillary incisor region had a much higher concentration than any other sites. Considering the above fact and the feasibility to sample four sites simultaneously in a plaque strip from the Dentocult SM kit, we decided to take samples from these four sites. According to Shi et al. , Dentocult SM is the best test for the diagnosis of the presence of caries and its prognosis with a high statistical significance. Dentocult SM is better than Dentocult LB for caries risk assessment. , The advantages of this test include that it is chairside assuring greater patient compliance especially for young subjects, it needs minimal armamentarium, is less time-consuming and facilitates sample collection. Davenport et al. , compared the Dentocult SM kit with a conventional method and found that this dip-slide test provides a simple and suitable method of screening salivary Streptococcus mutans levels, which may have a useful role in caries risk assessment.  In a similar study by Karjalainen and his co-workers, the sensitivity, specificity and accuracy of Dentocult SM were found to be better than those of conventional methods. In their study, the plaque test surpassed the salivary strip test in terms of sensitivity and accuracy when both were compared. 
There was no statistically significant difference between the posttreatment bacterial score and frequency of brushing, sweets/snacks intake and sugared/energy drinks consumption [Table - 4]. According to Ekenbach et al . who studied the colonization of cariogenic bacteria in the plaque of exposed root surfaces after application of various varnishes, found no statistically significant difference between the baseline and over time (one week, one and six months) samples with the Fluor Protector varnish.  Hence, a 24 h plaque sample was collected after which the fluoride varnish was applied even to the control group.
There was a statistically significant effect in relationship between the pre- and posttreatment bacterial scores in the study group (Mann Whitney test, p = 0.000) [Table - 5]. The average pretreatment bacterial count of the study group was in the range of 10 4 -10 5 CFU/ml whereas the posttreatment count is <10 4 CFU/ml. According to Killian et al., naturally occurring fluoride does not significantly influence the bacterial composition of plaque but higher levels of fluoride could eliminate susceptible micro-organisms and modify the plaque ecosystem. , Skold-Larsson and his co-workers compared the fluoride concentrations of three varnishes in plaque and found some of the members of the Fluor Protector group showed increased levels of fluoride in plaque even after 30 days.  Munshi et al. evaluated the demineralizing inhibitory and antibacterial effects of Fluor Protector in an in vitro study and found that it has the highest demineralizing inhibitory effect and the lowest antibacterial effect when compared to Bifluoride-12 and Fluoritop SR.  Ekenbach and co-workers found that hydroxyapatite crystals pretreated with Fluor Protector showed a statistically significant reduction in lactic acid formation in Streptococcus mutans .  However, Zickert and Emilson found that Duraphat did not have any significant effect on plaque or salivary levels of Streptococcus mutans on the 4 th , 10 th and 21 st days after treatment in preschool children. They did not however, evaluate the 24 h action of Duraphat on plaque samples.  Even though FluorProtector has a lower fluoride concentration and caries inhibiting activity than Duraphat, the amount of fluoride deposited in teeth was more after the use of Fluor Protector than after 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. Brown et al . showed that Streptococci were eliminated in 10/30 patients whose plaque had 115 ppm of fluoride.  Enolase activities in strains of mutans streptococci were inhibited by 50% by fluoride concentrations ranging from 16 to 54 mM.  Hence, the reduction of bacterial counts in this study could be due to the high concentration of fluoride from Fluor Protector (1000 ppm) that might have entered the bacterial cell and resulted in the inhibition of various cellular processes.
| Conclusion|| |
The conclusions derived from the results of this study are:
- Fluor Protector fluoride varnish causes a statistically significant reduction in Streptococcus mutans counts in plaque after 24 h.
- The average Streptococcus mutans count in the primary dentition of caries-free children is in the range of 10 4 -10 5 CFU/ml.
- The average Streptococcus mutans count in the primary dentition of caries-free children after Fluor Protector fluoride varnish application is below 10 4 CFUml.
- Frequency of brushing, sweets/snacks intake, sugared/energy drinks consumption and the use of fluoridated toothpaste do not have any statistically significant effect on Streptococcus mutans counts in plaque.
| Acknowledgment|| |
We would like to thank Dr. Raghuram M.Sc, Ph.D., M.D., F.A.B.M.S., Professor and Head, Department of Microbiology, for his guidance and for allowing us to use his laboratory to carry out this study.
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4]
[Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5], [Table - 6]
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