Caries outcome following an intensive fluoride varnish treatment

regimen for children at high risk for early childhood caries

Purpose. To evaluate the caries relapse rate for a cohort of 2- to 4-year-old children at high risk of early
childhood caries when treated with an intensive fluoride varnish (FV) regimen.
Methods. Eighty paediatric patients were recruited. Forty of these patients were high risk and received the
FV treatment (three applications within 2 weeks and additional applications at 1 and 3 months) during 2009–2010.
Mutans streptococci (MS) levels in the saliva were evaluated during the treatment period. A comparative group of 40
children, selected from an electronic record search at the New York University College of Dentistry to be of similar
age, gender, and ethnicity, but not at elevated risk for ECC, received the standard of care (semi-annual FV treatment).
Detailed caries examination and treatment records were obtained for all patients from 2009 to 2014.
Results. A significant reduction (P < 0.001) in MS levels was observed in the intensive FV treatment group
at the 3 month visit compared with baseline. There was no effect of the intensive FV treatment on caries outcome in
the anterior teeth, and the overall caries scores were significantly increased on the posterior teeth.
Conclusion. The intensive FV regimen appears insufficient to prevent caries relapse in children at high risk
for caries.

Introduction
Early childhood caries (ECC) is a serious disease affecting many young children today. Although
there is lack of consensus as to the definition of ECC among dental professionals at this time, the paediatric
dental community agrees that children under 3 years of age with at least one cavitation/carious lesion on a
smooth surface qualify as having severe ECC (S-ECC). Previous studies have shown that more children of
lower socio-economic status suffer from this disease when compared with the general paediatric population.
Other factors associated with ECC include a high level of mutans streptococci, saliva colonization by other
cariogenic bacteria, frequent sugary snacking, poor oral hygiene, mothers with active decay and a high level
of cariogenic microflora, and enamel defects (either genetic or acquired). If the disease is left untreated,
children with ECC can suffer from malnutrition due to eating difficulties and damage to the permanent
dental sac, resulting in space loss. Thus, it is important, whenever possible, to employ preventive measures
that delay, reduce, or prevent the onset of dental caries in young patients who may be particularly at risk
for this condition.
Fluoride has been a component of dental health in the USA since water fluoridation began in 1945.
Many studies have demonstrated the efficacy of frequent, low doses of fluoride in the treatment and
prevention of dental caries. Clinically, topical fluoride applications have been shown to remineralize enamel
surfaces, reverse initial carious lesion formation, and prevent cavities from further weakening tooth
structures. Fluoride treatment works by improving and strengthening enamel crystalline structure. In young
children with ECC, studies have shown that, topical applications of high fluoride concentrations, such as
fluoride varnishes (FV), form protective globules with calcium ions to prevent or arrest caries formation on
enamel surfaces in both permanent and primary teeth. Although it has been suggested that fluoride may
play an important antimicrobial role in reducing the risk for caries development, there is still uncertainty as
to how topical fluoride application contributes to caries prevention. Previous clinical studies have yielded
mixed results in which some reports have shown no direct correlation between fluoride release and
antimicrobial activity, whereas others have concluded that fluoride prevents caries in vivo when given in
sufficient quantities and under cariogenic conditions, including acid environments and excess
carbohydrates.
A study by Weintraub et al. showed a statistically significantly reduced percentage of children
with caries incidence (any decayed or filled surfaces at the last follow-up examination) in children who
received intended fluoride treatments compared with the control group who received no fluoride varnishes.
These study findings support the use of fluoride varnish to prevent ECC and reduce caries incidence in very
young children15. For children at high risk for caries development in the primary dentition, the standard
semi-annual application of FV may not be sufficient in preventing ECC. It has been suggested that an
intensive reapplication regimen (three doses in the initial 2 weeks) might provide greater protection from
caries progression beyond a single application.
The objective of this study was to determine the effect of an intensive FV regimen (three initial
applications within 2 weeks, one treatment at a 1-month recall visit, and one treatment at a 3-month recall
visit), designed by the Department of Pediatric Dentistry at the New York University College of Dentistry
(NYU Dentistry), on caries relapse rate and levels of mutans streptococci (MS), mainly Streptococcus
mutans. We also studied the effect of a semi-annual FV application in a cohort of children at nominal risk
for caries in the same dental clinic. Both cohorts were followed for 5 years.

Material and methods

Study participants
This study consisted of two phases. The original clinical intervention was delivered during 2009–2010.
Then, a retrospective chart review was conducted during 2015–2016 to assess the caries relapse rate of this
cohort during a 5-year period after the intensive FV treatment. A total of 80 children, 2–4 years old who
were diagnosed with ECC and sought dental care at the Pediatric Dental Clinic of the New York University
College of Dentistry (NYU Dentistry) during 2009–2010, were included. The entire study was approved
by the Institutional Review Board of New York University School of Medicine (IRB 06-783 for Phase I,
IRB i15-00093 for Phase II).
Forty of the children who received an intensive FV treatment and returned for routine follow-up
care were assigned as the FV group. The inclusion criteria for children participating in the FV treatment
study at baseline were as follows: (1) children must be healthy without chronic diseases other than dental
caries; (2) children had at least one decayed tooth in their anterior teeth; (3) the carious lesions included
white spot lesions and no symptomatic pain or discomfort; (4) the cavitated lesions were not larger than 2
mm in diameter without approaching the pulp; and (5) children were not allergic to fluoride varnish
treatment.
The comparison group of 40 children of similar age, gender, ethnicity, and socio-economic status
was selected based on an electronic record search at NYU Dentistry. These children were seen at the same
dental clinic during the same period as the FV group with the same initial or recall examination (D0120 or
D0150), received semi-annual applications of fluoride varnish application (D1206 or D1208), and returned
for regular recall visits within 5 years after their initial dental visits.

Study procedure
All dental examinations were conducted by trained paediatric residents (ZM, PS, calibrated by NGH and
CC) in a dental clinic using a standard mouth mirror, explorers, and adequate illumination. The American
Academy for Pediatric Dentistry (AAPD) visual-tactile caries diagnostic criteria for ECC and WHO’s deft/s
index (cavitated decay, extracted due to caries, and filled teeth/tooth surfaces) were used for this study.
Caries were recorded as present based on a lesion in a pit or fissure or on a smooth tooth surface that had
an unmistakable cavity, undermined enamel, or a detectably softened floor or wall.
After examining the caries status and obtaining parental informed consent and paediatric assent,
children in the intensive FV treatment group received three topically applied fluoride varnishes (Durashield
CV 5% sodium fluoride clear varnish, SultanHealthcare, York, PA, USA) within a 2-week period followed
by additional applications at 1-month and 3-month visits. The children’s caries status and MS levels in the
saliva were assessed at each treatment visit.
All children were placed on 6-month recalls for 3 years after the initial dental visit. Caries relapse
was defined as the presence of at least one new cavitated caries lesion requiring a restoration that
necessitated a cavity preparation with a rotary instrument. A surface was considered to have experienced
new decay between two examinations if it was determined to be free of caries experience at the first
examination and demonstrated evidence of caries experience at the subsequent examination. To avoid
overstating the caries experience, if a tooth was observed to be missing due to caries or restored with a
crown, then this tooth contributed a total of three surfaces to our measurements. If a tooth had three or more
surfaces showing evidence of caries at one examination and then was found to be missing or crowned at a
later examination, this tooth was counted as one surface of new decay in the interim.

MS level evaluation
The Dentocult SM Strip mutans test (Orion Diagnostica, Finland) was used for the FV group.
Specifically, the children were asked to chew a piece of paraffin wax for 1 min under the close supervision
of the investigators. The strips were rotated on the tongue for ten rounds, and excess saliva was removed
according to the protocol by Jensen and Bratthall. These test strips were immediately placed into a test tube
containing a selective medium broth and bacitracin, incubated for 48 h at 37°C, and air-dried at room
temperature. The levels of MS colonization were examined and scored based on the presence of colony
forming units (CFUs) on the strips (Fig. S1). Compared with the standard chart provided by the
manufacturer, the following four categories were used, 0, 1, 2, and 3, which corresponded to <104 S.
mutans CFU, 104–105 S. mutans CFU, 105–106 S. mutans CFU, and >106 S. mutans CFU per ml of
saliva, respectively. The MS results were examined by three examiners (ZW, PO, and YL) and
independently scored for each patient. The Kappa values ranged from 0.85 to 0.92, indicating a high degree
of inter-examiner reliability among the investigators.

Statistical analysis
Analysis comparing the two groups of children regarding age, gender, ethnicity, and deft scores used an
independent samples t-test or Pearson’s chi-square test. Caries severity scores were compared over the 60-
month study period with a linear mixedeffects model, separately for each treatment group. Planned
comparisons of the mean caries scores at each follow-up period relative to the baseline values used a t-test
based on a pooled estimate of the residual error. In a similar way, mean SM Strip testing scores for the FV
group were compared before and after intensive fluoride varnish treatment. Associations between changes
in deft score over 1 year and MS levels over 3 months in the FV group were evaluated with Pearson’s
correlations. All analyses were conducted using SPSS (v24; IBM Corp., Armonk, NY, USA) with two sided
tests, and the significance levels were set to P < 0.05.

Results
A total of 40 children were scheduled to receive the intensive FV treatment during November 2007 to
December 2008. Thirtyseven of children completed the treatment, 22 of them were followed up to 12
months; 12 of them were followed up to 60 months. During the same time period, 40 children received the
standard-care fluoride varnish treatment were included in the study; 33 of them were followed up to 60
months. Therefore, observational data of the 73 children were included in the final data analysis.
Among the 73 children examined, 55 (75%) children were of Hispanic descent, and 47% were
female. The mean age of the children from the FV group was 32.2 months (range, 14–56 months), and the
mean age in the control group was 36.6 months (range, 19–50 months). There were no significant
differences in the distribution of the children regarding age, gender, and ethnicity between the two groups
(Table 1). According to the study design, children in the FV group were high risk for caries with a mean
deft score of 4.86. Children in the comparison group were low risk for caries with a mean deft score of 0.47.
The differences were present for both anterior and posterior teeth between the two groups at the baseline
and the 60-month visits (Table 1).
As illustrated in Figure 1, the early caries lesions on anterior teeth were arrested 1 week after the
FV treatment in additional to professional prophylaxis procedures and oral hygiene instruction. The
progression of caries lesions development on the anterior teeth appears reduced at the 4- to 12-month visit,
but an analysis failed to show an effect over time (MIXED analysis, F = 1.98, P = 0.09). The cohort began
and ended the observation interval with anterior deft scores of about 4. In the meanwhile, the mean deft
scores of the posterior teeth increased from less than 1 to more than 4 after the intensive FV treatment
(MIXED analysis, F = 18.43, P < 0.001; Fig. 2a). Post hoc tests showed that the mean scores at the 13- to
24-month, 25- to 36-month, and 37- to 60-month visits were significantly higher than the baseline caries
score (P = 0.002 to P < 0.001). For the comparison group, the overall caries scores increased steadily with
time in both anterior and posterior teeth (MIXED analysis, F = 5.84, and F = 10.09, respectively, both P <
0.001), from a mean score of <0.5 to about 1 (Fig. 2b). The deft scores at all intervals after 13 months were
different from baseline in both anterior and posterior teeth (P = 0.009 to P < 0.001). Parallel analyses that
included a reduced sample with complete data over the observation interval produced similar results,
indicating the reported results were not biased by attrition.
The MS levels were determined using the Strip SM test for the FV group at baseline and at the 1-
week, 2-week, 1-month, and 3-month visits, and the distribution of scores over time is presented in Table
2. Analysis showed a significant shift towards lower MS levels (score ‘0’ and ‘1’) over time (mixed models
F = 95.8, P < 0.001). Post hoc tests showed a reduction within 1 week of the intensive fluoride varnish
application (P < 0.001), and then another reduction between 1 and 2 weeks (P < 0.001). MS levels then
stabilized at this lower level for the remainder of the study (P > 0.5). Changes in deft score and changes in
MS over 1 year were uncorrelated (r = 0.09 and r = -0.12, P > 0.45, for anterior and posterior teeth,
respectively), suggesting that the reduction in MS levels was not related to changes in caries experience.

Discussion
Currently, professionally applied fluoride varnish to the primary teeth at least twice per year is highly
recommended by the American Dental Association, the American Academy of Pediatrics, the American
Academy of Pediatric Dentistry, and the US Preventive Services Task Force. This treatment regimen is
used extensively in both public and private dental practice settings to reduce the risk for ECC. Although
clinical studies have shown a moderate level of effectiveness for FV application in preventing dental caries
in children, a wide range of caries-preventive efficacy rates have been reported with different application
protocols, and caries relapses occurs frequently. A frequent FV treatment regimen has been suggested as a
vehicle to prolong the period of fluoride contact with the enamel surface, enhance the formation of
fluoridated hydroxyapatite, and reduce the solubility of enamel in an acidic environment, hence, promoting
the benefits from FV treatment. Two in vitro studies using an intensive fluoride varnish protocol showed
that the multifluoride application provided a high fluoride deposit on the enamel surface. In superficial,
microscopic cavities, the regimen was able to nearly stop all carious processes.
In this study, however, intensive FV applications resulted in no change in deft score on the anterior
teeth and caries progressed on the posterior teeth. By contrast, MS levels decreased significantly following
intensive FV application, but greater reductions in MS levels were not associated with greater reductions
in deft. These findings are in agreement with a previous study by Weinstein et al. in which an annual
application of an intensive FV regimen (three applications over 2 weeks) was compared with single FV
applications that were applied semi-annually for up to 3 years. As the caries-preventive fluoride
concentrations in the saliva diminished 6 h after topical FV application, the authors concluded that there
was no practical advantage of using the intensive treatment. It remains possible that intensive FV treatment
had some clinical benefit, in that caries might have progressed even further without such treatment.
Evaluation of this question would require a future study that randomized high-risk children to standard and
intensive FV treatment regimens.
These findings raise some questions as to whether it is necessary to provide this treatment option
to low-income families with children at high risk for ECC due to the unsatisfactory long-term outcomes.
To make an FV intervention programme effective requires parents or families to commit to multiple follow-
up recalls. Parents of children from low-income families, such as head start families in the United States,
constantly battle with numerous social, cultural, or occupational obstacles in presenting their children for
the completion of multiple-year-length preventive programmes. A future study to determine the long-term
effects of an annual intensive FV regimen could be of major interest.
Children with ECC have higher levels of MS and other oral bacteria than do children without this
disease. If levels of these micro-organisms can be reduced, dental caries prevalence can be significantly
reduced. In early studies by Marquis et al. have shown that FV application in combination with other caries-
preventive measures could not only cause remineralization of early carious lesions but also reduce the level
of cariogenic bacteria, mainly MS, in saliva, and prevent the disease progression. It has also been shown
that MS levels increase significantly, and a high proportion of children develop new lesions shortly after
FV treatments are discontinued. As expected, we observed an alteration in the distribution of children in
each MS category after the intensive FV treatment during the 3 months after the FV treatment. One
limitation of the study was that the microbiological evaluation was conducted only for the FV intensive
treatment group but not for the comparative group. Although the overall MS changes were statistically
significant compared with baseline for the FV treatment group, the study was unable to demonstrate the
effect of the intensive regimens on MS levels in all young children. Nevertheless, the findings were
preliminary and support previous studies showing that FV treatment is most effective for reducing caries
risk on smooth surfaces. To effectively reduce these microbial burdens, a controlled fluoride application
regimen is needed for children at high risk of caries development. Currently limited clinical reports are
available regarding FV treatment regimens with respect to MS levels in young children, although carious
lesions on smooth surfaces have been reported to harden after FV applications.
Here, we presented a clinical observational study with a number of interesting findings and some
limitations. The study was a retrospective study; the comparative group, although carefully selected based
on electronic chart reviews and matched age, gender, and ethnicity, was not an ideal control group for the
intensive FV treatment. The diagnosis of ECC (inclusion criteria), patient risk assessment, and semi-annual
fluoride varnish applications could vary over time relative to the FV treatment group. In addition, most of
the children in the comparative group received semiannual fluoride varnish treatment, and some of them
returned in 3 months or after 1 year. The inconsistence in clinical visits could increase the variability in the
caries outcome assessment and comparison between the two groups. Those issues represent challenges to
conducting clinical intervention studies on caries, particularly in paediatric patients.

Conclusions
Based on this study’s findings, the following conclusions can be drawn:
1) Even though an intensive fluoride varnish regimen had a beneficial effect on MS levels 3 months post-
treatment, there was no significant reduction in caries progression one or more years later. Thus, the
regimen alone might not be sufficient to prevent caries relapse in children at high risk for ECC.
2) It remains possible that intensive FV treatment had some benefit, in that caries might have progressed
even further without such treatment. Therefore, the longterm effect of fluoride varnish application on
cariogenic bacteria, S. mutans, and total oral microbiota requires a future study that randomizes high-risk
children to standard and intensive FV treatment regimens.

Why this paper is important to paediatric dentists

• Professional administering FV treatment at least twice a year is highly recommended by the American Dental Association, and
the American Academy of Pediatric Dentistry for children with an increased caries risk, the study failed to demonstrate that
additional FV applications increase the treatment effectiveness and reduce caries progression in primary teeth.
• For children at high risk for ECC, more comprehensive measures are needed to prevent caries development or reoccur in addition
to rigorous FV treatment approach.

Acknowledgements
The authors wish to thank the following individuals at the New York University College of Dentistry, New
York, NY, USA, who provided assistance with this project: Dr. Mark Wolff, Professor and Chairman of
the Department of Cariology and Comprehensive Care, for providing all the medical charts of the
participants; Dr. Amr Moursi; and the staff members of the Department of Pediatric Dentistry for assisting
in patient care.

Conflict of interest
The authors declare no conflict of interest related to the present study.
Author contributions
Dr. Agelina E. Paek, Dr. Zhemeng Wang, Dr. Patrick So, and Dr. Neal G. Herman carried out the research;
Dr. Neal G. Herman and Dr. Yihong Li designed the research; Dr. Courtney Chinn mentored and assisted
the clinical investigators; Dr. Agelina E. Paek and Dr. Yihong Li wrote the manuscript; Mr. Andrew
Hopkins edited the manuscript; Dr. Malvin N. Janal assisted with the statistical analysis; Dr. Neal G.
Herman and Dr. Courtney Chinn critically edited the manuscript.