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Year : 2021  |  Volume : 19  |  Issue : 4  |  Page : 320-324

Levels of Streptococcus Mutans in mother–child pairs following application of fluoride varnish containing casein phosphopeptide: Amorphous calcium phosphate

Department of Pedodontics and Preventive Dentistry, The Oxford Dental College and Hospital, Bengaluru, Karnataka, India

Date of Submission13-Aug-2021
Date of Decision12-Oct-2021
Date of Acceptance08-Nov-2021
Date of Web Publication15-Dec-2021

Correspondence Address:
Priya Subramaniam
Department of Pedodontics and Preventive Dentistry, The Oxford Dental College and Hospital, Hosur Road, Bommanahalli, Bengaluru, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jiaphd.jiaphd_146_20

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Background and Objectives: Dental caries is an infectious transmissible disease resulting from tooth-adherent specific bacteria, primarily Mutans streptococci. Fluoride varnish is an effective antimicrobial agent against Streptococcus mutans (S. mutans). The aim of the study was to evaluate the levels of S. mutans in children following application of a fluoride varnish containing CPP-ACP in mother–child pairs. Materials and Methods: Sixty children aged 18–36 months, at high caries risk along with their mothers formed the study group. Baseline levels of S. mutans in saliva of mothers and dental plaque of children was microbiologically assessed for S. mutans using the culture method. They were divided into three groups, consisting of 20 mother–child pairs. Group 1: Application of varnish to mother and saline as a placebo to child; Group 2: Applying varnish to child and saline as a placebo to mother; Group 3: Applying varnish to both mother and child. S. mutans levels in dental plaque of children were assessed at 3, 6, 9, and 12 months. Data obtained were subjected to statistical analysis using one way analysis of variance, paired t-test and multiple Post hoc tukey. The level of Significance was considered at 5% Results: In all the groups, there was a significant reduction of S. mutans levels in dental plaque of children at 3, 6, 9, and 12 months. The highest reduction in S. mutans was in Group 3 (95.33%) followed by (95.12%) in Group 2 and 40.6% in Group 1 (P < 0.05). Conclusion: Application of fluoride varnish containing CPP-ACP to the children gave equivocally significant reduction in S. mutans levels of dental plaque in children, as compared to applying in both mother and child.

Keywords: Casein phosphopeptide-amorphous calcium phosphate, dental plaque, fluoride varnish, Streptococcus mutans

How to cite this article:
Subramaniam P, Raj N S, Papulwar SM. Levels of Streptococcus Mutans in mother–child pairs following application of fluoride varnish containing casein phosphopeptide: Amorphous calcium phosphate. J Indian Assoc Public Health Dent 2021;19:320-4

How to cite this URL:
Subramaniam P, Raj N S, Papulwar SM. Levels of Streptococcus Mutans in mother–child pairs following application of fluoride varnish containing casein phosphopeptide: Amorphous calcium phosphate. J Indian Assoc Public Health Dent [serial online] 2021 [cited 2023 Feb 5];19:320-4. Available from: https://www.jiaphd.org/text.asp?2021/19/4/320/332524

  Introduction Top

Mutans Streptococci (MS) comprise the major microorganisms implicated in the etiology of dental caries occurring on smooth surfaces.[1] Caufield et al. reported that the initial acquisition of MS occurred at the age of 19–31 months, with a median age of 26 months, during a discrete period termed as “window of infectivity.”[2] Streptococcus mutans (S. mutans) is a predominant etiologic agent of dental caries. Mothers with high levels of MS tend to have children with high levels of MS.[1]

Professionally applied topical fluoride treatment is known to be efficacious in reducing prevalence of dental caries.[3],[4] Fluoride varnish is suitable for application on tooth surfaces of children.[5] Its ease of application makes it attractive for use with young or un-cooperative patients.[6]

The ability of fluoride to prevent demineralization and promote remineralization has been enhanced by the incorporation of casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP). These nanocomplexes are derived from casein, a natural protein extracted from milk. CPP-ACP has been shown to prevent demineralization and to promote remineralization of subsurface enamel lesions in animal and human in situ studies.[7],[8] Studies have shown a synergistic effect of CPP-ACP and fluoride in reducing caries.[9],[10]

However, oral antibacterial agent as a single treatment in mothers may not be adequate to prevent S. mutans transmission and subsequent caries in children.[11] Similarly, oral antimicrobial preventive measures in infants and young children may not be adequate to reduce dental caries if maternal salivary levels of S. mutans remain high.[12] The transmission of S. mutans from mother to child should be prevented. Studies are lacking on the efficacy of preventive measures applied to both mother–child pairs.[11],[12] Simultaneous applications of caries preventive measures in mothers and children could probably be more beneficial, rather than individual use in either mother or child only. The hypothesis of this study is that application of a fluoride varnish containing CPP-ACP to mother–child pairs, and either child or mother will reduce S. mutans in the child.

Therefore, the present study was carried out to evaluate the levels of S mutans in dental plaque in children following application of a fluoride varnish containing CPP-ACP in mother–child pairs.

  Materials and Methods Top

The present double-blinded placebo controlled in vivo study was conducted over a period of 12 months. Approval to conduct the study was taken from the institution's ethics committee (Ref. No. 222/2014-15). Written permission was taken from the authorities of various play homes and pediatric clinics of south Bangalore. Healthy children aged 18–36 months along with their mothers were selected for the study.

Sample size estimation was based on 5% significance level with a power of 80%.

n = Estimation of sample size.

zα = 1.96 for 5% significant level.

z1−β = 0.841 for 80% power.

σ = Standard deviation.

= Mean.

By substituting the values we get n = 14.

Assuming 20% attrition (drop out) the required sample size becomes 14 + 4 = 18

The sample size was rounded off to 20 in each group.

Eighty healthy children aged 18-36 months along with their mothers were initially screened from various play homes and pediatric clinics of south Bangalore. Prior to the study, nature of the study was explained to the parents and their written informed consent was taken for participation in the study. A questionnaire was distributed to the mothers to record only the demographic details.

Inclusion criteria[5],[13]

For mothers

  • Healthy mothers with high salivary S mutans levels (≥105 CFU/ml of saliva)
  • DMFT score ≥2.

For children (any two)[5]

  • Child having visible plaque on teeth
  • Presence of incisors and at least 2 molars
  • Child having dmfs ≥3
  • Child having sugar-containing snacks more than 3 times between their meals
  • Child using a bottle containing natural or added sugar while sleeping
  • Child having one or more white spot lesions or enamel defects.

Exclusion criteria

  • Medically compromised mother/child or both
  • Mother wearing any removable/fixed prosthesis
  • Mother wearing any orthodontic appliance.

Sixty mother–child pairs who fulfilled the inclusion criteria formed the study group. Oral examination of mother–child pairs was carried out under adequate natural day light using a sterile mouth mirror to record dental caries according to the WHO criteria.[14] Oral examination was carried out by a single examiner to avoid any inter examiner variability.

From each mother, samples of saliva were collected. The mothers were instructed to sit in a relaxed, upright position by keeping the mouth open continuously for 5 min and to spit into a sterile graduated plastic tube. To avoid any diurnal variation, the salivary samples were collected between 10–11:30 am after breakfast. From the graduated tube, 1 ml of collected unstimulated salivary samples were transferred to an eppendorf tube using a dropper and transported to the laboratory for microbiological analysis within 1 h.[15]

From each child, dental plaque samples were collected. The mothers were instructed not to brush or clean their child's teeth on the day of sample collection. The dental plaque samples were collected from the interproximal surfaces and cervical region of all the teeth present using autoclaved wooden tooth picks. The collected samples were transferred to the sterile eppendorf tubes containing 1 ml of saline.[2],[16] In case of young un-cooperative children, the child was made to lie down in a lap to lap position between mother and the examiner for the collection of plaque samples.[17] All the collected samples were transported to the laboratory for microbiological analysis. The plaque samples were homogenized manually using a stirrer, where the sample from Eppendorf tube in saline was serially diluted aseptically by using 0.1 ml inoculum up to 10−5. Direct sample and diluted samples were pour plated out using 1 ml inoculum on Miti salivarius bacitracin agar. Incubation was done for 24 h at 37°C. At the end of incubation period, colonies showing blue color and a granular frosted-glass appearance are considered as S. mutans of dental plaque in children within one hour. Colonies of S. mutans were counted with the help of a digital colony counter.[15]

Following collection of baseline samples in children, complete oral rehabilitation of all mothers and their children was carried out including oral prophylaxis, restorative procedures and extraction of teeth.

The mother–child pairs were then randomly allocated to three groups by lottery method, consisting of 20 pairs in each as follows:

  • Group 1: Application of fluoride varnish to mother and saline as a placebo to child
  • Group 2: Application of fluoride varnish to child and saline as a placebo to mother
  • Group 3: Application of fluoride varnish to both mother and child.

Fluoride varnish (MI Varnish™, GC Corporation, Tokyo, Japan) was applied to mothers and children according to the manufacturer's instructions.[18] The fluoride varnish was applied between 10 am and 11am and both mothers and children were instructed not to eat, drink for at least 1 h and not to brush their teeth till the next morning. In Group 1 and 2, saline was applied as a placebo to children and mothers, respectively. Mother–child pairs were advised to refrain from tooth brushing. Oral hygiene instructions were given to all mother and child pairs on the day of application.[19]

During the study, all mothers were asked to report immediately to the day care authorities/clinics or investigator if any adverse effects were noticed by them. Fluoride varnish was applied at baseline, 3, 6, and 9 months. Dental plaque samples were collected only from children at 3, 6, 9, and 12 months and processed for S. mutans levels as described earlier.

Data obtained were tabulated and subjected to statistical analysis using one way analysis of variance, paired t-test and multiple Post-hoc tukey. Significance was considered at 5% (P < 0.05)

  Results Top

There was a significant reduction of S mutans levels in dental plaque of children at 3, 6, 9 and 12 months in all groups [Table 1]. Intra-group comparisons, showed significant difference between baseline and subsequent evaluations [Table 2]. At 12 months, there was 40.65% of reduction in Group 1 (P = 0.001), 95.12% in Group 2 (P = 0.0001) and 95.33% in Group 3 (P = 0.0001), which were highly significant (P < 0.05). The pair wise comparison of reduction in S mutans levels between Group 1 and the other two groups at all periods of evaluation is given in [Table 3] (P = 0.0001) (P < 0.05).
Table 1: Levels of Streptococcus mutans in dental plaque in children

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Table 2: Intra group reduction in Streptococcus mutans at different time intervals

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Table 3: Pairwise comparison of groups in reduction of plaque Streptococcus mutans levels

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  Discussion Top

The American Academy of Pediatric Dentistry (AAPD) encourages the use of professional preventive measures in mothers to reduce the risk of developing early childhood caries in their children.[5] Fluoride varnish is a well-established effective antimicrobial agent in reducing S. mutans.[3] The application of fluoride varnish is well accepted by young children, as it is easy, safe and it does not take long time. Since the varnish sets in contact with intraoral moisture, thorough drying is not required prior to application and wiping with a gauze or cotton rolls is adequate.[20] Weintraub et al. stated that fluoride varnish and parental counseling should be recommended as part of caries prevention programs targeting infants and toddlers.[21]

The focus of preventive measures has shifted to remineralizing agents such as CPP-ACP.[22],[23] Remineralization of early carious lesions is of paramount importance in children at high caries risk. Therefore, in this study, a varnish (MI Varnish™) having a synergistic effect of both CPP-ACP and fluoride was used.[9],[10],[24] The varnish was applied at an interval of every 3 months as per the recommendations of AAPD.[5]

In our study, the pour plate method was used for cultivation of S mutans because this method allows for growth of all viable bacterial cells.[16] Culturing has an advantage that contamination during laboratory manipulation may be more readily recognized than with molecular procedures.[25]

Following application of the varnish, S. mutans levels in dental plaque significantly reduced in all the children at 3 months and remained low until the end of the study. This reiterates the importance of applying fluoride varnish at frequent intervals in children at high risk of caries. Various studies also reported the four pediatric applications of fluoride varnish combined with oral hygiene instructions were an effective method.[26],[27]

The reduction in acid production by S. mutans is due to the inhibitory action of fluoride ions on enolase and other enzymes in the Emden-Meyerhof Pathway.[15] CPP-ACP nanocomplexes bind to adhesin molecules on S. mutans and thus impair their incorporation into dental plaque.[28] Elevation of plaque calcium and phosphate ions has a buffering effect and can suppress fermentation by aciduric species, even in the presence of fermentable sugars.[16] Cochrane et al. suggested that the ion-release profile of MI varnish™ had greater fluoride and calcium ion release compared with other varnishes containing calcium fluoride, tricalcium phosphate, ACP, and fluoride.[9] In this varnish, the bioavailability of fluoride ions in plaque increases and therefore prolongs its anti-microbial effect.[8],[19]

CPP and ACP nanocomplexes have been demonstrated to have anticariogenic activity.[7] CPP has shown to not only increase fluoride incorporation into plaque, but it also increases the incorporation of fluoride into subsurface enamel and substantially increases remineralization of subsurface layer of enamel compared with fluoride alone.[10],[28]

In the present study, application of varnish to only the children (Group 2) gave equivocally significant reduction in S mutans, as compared to applying in both mother and child (Group 3). During the initial months of life, S. mutans get transmitted to infants mainly from their mothers through vertical transmission.[2] It is then followed by both vertical and horizontal transmission through interaction with siblings and other family members.[15] However, in urban communities there are a higher number of nuclear families and “single child” families. In Bengaluru, due to the increasing number of working mothers, children spend considerable time in play homes and crèches.[2],[29] Thus, their interactions with other children and caretakers would have probably exposed them to a higher risk of horizontal transmission of S. mutans from sources other than their family. Therefore, the benefit to the children was <50%, when only their mothers received fluoride application.

It is important to educate working mothers and health professionals on the transmission of S. mutans to infants and toddlers in the community. Implementation of oral hygiene practices and preventive measures including periodic application of fluoride varnish in children at caries risk is necessary.[30] There is a possibility that certain mothers did not follow the oral hygiene instructions regularly, which could be a limitation of this study.

In this study, the frequent application of MI varnish™ in mother–child pairs was effective in reducing S. mutans in children at high caries risk. Further studies on the progress of dental caries in these children can be carried out. The use of fluoride varnish containing CPP-ACP can be recommended for remineralization of early carious lesions in young children.

  Conclusion Top

  1. In mother–child pairs, the application of fluoride varnish containing CPP-ACP only to the child gave highly significant reduction in S mutans levels of dental plaque
  2. Application of the varnish to both mother–child also significantly reduced S mutans levels.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3]


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