A Randomized Clinical Trial of Silver Diamine Fluoride to Arrest Early Childhood Caries in Young Children
Phase: NA
Conditions: Early Childhood Caries
Sponsor: University of Manitoba

A Clinical Trial of Silver Diamine Fluoride to Arrest Early Childhood Caries in Young Children 
NCT04054635 
Study Protocol and Statistical Analysis Plan 
15 April 2019

Purpose:  
To investigate the effectiveness of using silver diamine fluoride (SDF) to arrest early childhood 
caries in very young children randomized to different application frequency regimens. We will 
also study potential oral microbiome changes in children receiving SDF treatment. 
Hypotheses: 
1. Two applications of SDF at different frequencies will yield similar arrest rates.  
2. SDF will negatively influence the population of cariogenic bacteria in the oral microbiome. 
Objectives (*primary objective, †secondary objectives): 
1. Determine the effectiveness of various application regimens of SDF to arrest caries*;  
2. Determine potential changes to the oral microbiome in young children†; and 
3. Determine the impact on early childhood oral health related quality of life†.  
Methods: 
Design: We propose a RCT to study the use of SDF to arrest cavitated caries lesions in primary 
teeth at different application regimens. Regimen 1 will be two applications of SDF four months 
apart, which is the protocol frequency adopted by the Winnipeg Regional Health Authority’s 
Clinical Guideline on SDF. This frequency was used in a pilot study. Regimen 2 will be two 
applications of SDF six months apart (American Dental Association’s [ADA] recommendation). 
Regimen 3 will be two applications of SDF one month apart, which is proposed in the American 
Academy of Pediatric Dentistry’s clinical practice guideline. The ADA recently indicated that 
SDF should be prioritized over 5% NaFV for nonrestorative management of cavitated lesions. 
Thus, we will not include a control group to receive fluoride varnish as this would now be 
considered unethical and substandard care. However, we will attempt to follow as a comparison 
group those children whose parents/caregivers do not consent to SDF to manage their child’s 
caries and only choose 5% NaFV. 
Arrest of caries lesions will be determined by assessing clinical hardness, colour change, and size 
of lesions at baseline, at second visit, and at the final study visit. Children will be recruited and 
we will perform block randomization by site in order to achieve equal proportions in each 
regimen by clinic site of recruitment. Study visits will occur at the Children’s Hospital Research 
Institute of Manitoba (CHRIM) or at community-based dental clinics. Following informed 
consent the child’s parent/caregiver will complete a short questionnaire (via interview) on 
general and dental health, oral hygiene, dietary intake of sugars, and family demographics along 
with the ECOHIS to assess OHRQL. SDF will be applied on the day of recruitment to cavitated 
lesions involving dentin followed by 5% NaFV. Depending on which frequency regimen 
children are randomized to, they will return for a second visit. During this second visit, caries 
lesions treated with SDF at baseline will be assessed to see if caries is arrested as determined 
through measures of hardness, colour change, and size. At this same visit a second application of 
SDF will be applied to these initially treated caries lesions followed by 5% NaFV. Children and 
parents will return for the third and final study visit according to the schedule of their 
randomized grouping. Parents/caregivers will complete a follow-up questionnaire similar to the 

baseline tool. Caries lesions previously treated by SDF will be assessed for clinical hardness, 
colour change and size to determine if they have arrested.  
Population: As ECC is age specific, participants will be < 72 months of age with active caries 
and their parents/caregivers. The majority will be recruited from community-based dental clinics 
in Winnipeg (i.e., Access Downtown, Mount Carmel Clinic, and SMILE Plus Children’s Dental 
Clinic) or who are currently on a wait list for dental surgery under general anesthesia in 
Winnipeg. We found that these were ideal sites for our recently completed feasibility trial of 
SDF. We will restrict participation to children living in the Winnipeg region or within a one hour 
drive of Winnipeg to minimize the risk of loss-to-follow-up. Eligibility criteria are designed to 
select young children who have active caries, and thus are at an increased risk of morbidity from 
existing caries and onset of new caries. 
Inclusion Criteria: 1) Child is < 72 months of age with ECC with active caries lesions; 2) Child 
has ≥ 1 primary tooth with caries that is eligible to receive SDF. Eligible primary teeth must: a) 
have soft cavitated caries lesions extending into dentin (ICDAS 5 or 6); b) the cavitated lesions 
must allow for direct application of SDF. Teeth that meet any of the PUFA criteria (i.e. 
spontaneous pain due to caries, pulp exposure, mobility, signs of pulpal infection such as 
abscess, fistula, or swelling) will be excluded. However, a child would still be eligible even if 
they have at least one tooth that meets PUFA criteria, but other eligible teeth with caries do not. 
Exclusion Criteria: 1) Child is allergic or has sensitivity to silver or other heavy metal ions; 2) 
Child has hereditary generalized developmental defects of enamel (e.g., amelogenesis 
imperfecta, dentinogenesis imperfecta); 3) Child has severe medical problems that limit 
participation; 4) Child requires immediate rehabilitation under general anesthesia because of 
severe infection or pain; 5) Antibiotic use within the last 2 weeks (for the microbiome sub-
study). 
Sample and Justification of Sample Size: Sample size for this pilot study has been determined in 
discussion with Dr. Robert Tate (collaborator, Professor & Biostatistician). Our intent is to 
compare arrest rates between groups and to see whether the three different regimens will yield 
arrest rates within a range of what we will determine to be clinically acceptable. Based on our 
recent pilot feasibility study of SDF with 40 children (with 239 lesions) we reported an arrest 
rate of approximately 96% following two applications of SDF. Results from a recent systematic 
review state that 80% of lesions can arrest because of SDF. We believe that a range of arrest 
rates from 80% to 96% yield similar beneficial outcomes in the clinical setting. Based on our 
pilot study, 40 children had 239 lesions (average 6 lesions/child). With our pilot sample of 239 
lesions, it is possible to estimate an arrest rate with a 95% confidence interval to be accurate 
within ± 6.5%. With 400 lesions anticipated in our proposed recruitment sample, the 95% 
confidence interval for the arrest rate would be ± 5.0%. We propose three SDF regimen groups 
and anticipate that each group will have 22 children anticipating an average of six lesions. This 
would mean 396 lesions would be followed in this study. We anticipate that with 400 lesions the 
percent of arrest will be within ± 5% if we have 22 children in each group. To deal with potential 
drop-outs and loss-to-follow-up of up to 20% we will over recruit by into each group and recruit 
28 per group. We anticipate that we can successfully recruit 10 children each month. 
Instrumentation and Outcome Measures: Baseline and follow-up visit questionnaires will be a 
modified versions of those used in our SDF pilot feasibility study (developed by Dr. Margherita 
Fontana [collaborator] in her SDF efficacy study.The questionnaire collects child and family 

demographic information, oral hygiene and use of toothpaste, dietary habits (i.e., snack foods 
and drinks), consumption of fluoridated water, appearance of the teeth, and symptoms (e.g., 
pain). The tools also include sections for the parent/caregiver to complete with their child on how 
the child believes their teeth look and feel and how they rated their visit and SDF treatment. 
The Early Childhood Oral Health Impact Scale (ECOHIS) will be used to assess changes in 
OHRQL from baseline to completion of study. ECOHIS is a validated tool for use with 
parents/caregivers of children < 72 months of age. It consists of 13 questions grouped into two 
sections: 1) child impact, and 2) family impact. The child impact section includes nine questions 
in four areas: 1) symptoms, 2) functions, 3) psychology, and 4) self-image and social interaction. 
The family impact component covers two areas: 1) parental distress and 2) family function.  
Each question is scored from 0 to 5 [0= never, 1= hardly ever, 2= occasionally, 3 = often, 4= 
very often, 5= don’t know]. A total score for each child is calculated by summing up response 
codes. The ECOHIS will allow us to calculate and compare mean ECOHIS scores at each visit 
and between groups. Our team has experience with analyzing ECOHIS.  
Clinical outcomes assessed will include the proportion of teeth arrested (i.e., “arrest rate” = total 
number teeth arrested / total number of teeth treated), the proportion of children with any 
arrested caries, associated morbidity (dental pain and, dental abscess assessed using the PUFA 
index), and proportion of children requiring dental surgery under GA. The PUFA index is a score 
of the consequences of severely decayed teeth with visible pulpal involvement (P/p), ulceration 
caused by dislocated tooth fragments (U/u), fistula (F/f) and abscess (A/a). We will also record 
dmft and dmfs scores for each child at baseline and at subsequent study visits. The dmft and 
dmfs scores are cumulative counts of the total number of primary teeth or surfaces decayed, 
missing due to, or filled because of caries.  
Caries activity will be determined according to hardness and colour of the caries lesion. 
Hardness is the best indicator of dentinal tissue caries activity. We will assess the hardness of 
caries lesions by applying light force to the caries lesion with a ball ended probe and will classify 
the lesion into one of three hardness categories: 1) very soft; 2) medium; or 3) very hard. 
Hardness is associated with arrested caries. We will record these ratings of hardness of lesions on 
the Clinical Record Forms at the baseline visit, second visit, and third (final) clinical visit. The 
Clinical Record Forms will be those used in our pilot feasibility study of SDF. The colour of 
caries lesions will also be assessed. Dentin colour of each caries lesion will be classified as: 1) 
yellow; 2) brown; or 3) black. The colour black is associated with arrested caries. The size of 
caries lesions will also be measured (in mm) at the baseline, second, and third clinical visits.  
Problems and adverse events will be recorded reported to the University of Manitoba’s ethics 
office. 
Implementation: Research staff will regularly visit community-based dental clinics in the 
Winnipeg region to recruit participants. Clinical dental staff at these sites will also be briefed on 
the study, its objectives, and eligibility criteria so that they can inform parents/caregivers of the 
study and make referrals to the research team. We will also recruit participants who are on the 
Children’s Hospital dental clinic’s wait list for pediatric dental surgery. Recruitment posters will 
also be placed in patient waiting areas of the various dental clinics and the study advertisements 
will also be distributed to all dentists in the Winnipeg region via the Manitoba Dental 
Association’s weekly email distribution list. From our past study experiences we know that we 
can recruit at least 10 participants each month. 

Our research staff will contact interested parents/caregivers and will inform them of the study 
objectives, eligibility criteria, and procedures. Children will undergo a dental screening by the 
principle investigator or a calibrated and trained dental examiner to determine whether children 
meet the clinical criteria for this study by having cavitated caries lesions involving dentin (i.e. 
ICDAS category 5 or 6). Since ECC is more common in disadvantaged populations, we will 
arrange for transportation (i.e., taxis) for participants. We also anticipate that some participants 
will be newcomers and will require interpreters. We will arrange for WRHA interpreters when 
needed. Visits will occur at the principle investigator’s dental suite at CHRIM or at one of the 
community-based clinics.  
Baseline visit: Following written informed consent, children will be randomly allocated into one 
of three groups using sealed envelopes to ensure random allocation. Parents/caregivers will 
complete the baseline questionnaire administered by interviewer. Children will undergo a dental 
examination (without x-rays) to record the number of teeth and tooth surfaces with caries 
meeting ICDAS 5 or 6 criteria and the specific tooth numbers. We will record the location, size, 
hardness, colour, and activity status of each eligible caries lesion. We will also assess the total 
number of primary teeth and tooth surfaces affected by caries (i.e., dmft and dmfs). We will also 
determine the PUFA index. SDF will then be applied with a microbrush for one minute to each 
cavitated lesion meeting the eligibility criteria followed by 5% NaFV. 
Second visit: Depending on the regimen children will return 4 months (Regimen 1), 6 months 
(Regimen 2), or 1 month (Regimen 3) later for their first follow-up visit and second application 
of SDF. Parents/caregivers will complete the ECOHIS tool. Prior to the second application of 
SDF, caries lesions that received the first application of SDF will be assessed to determine if 
caries is arresting. Lesions will be assessed for colour change, size, and hardness. Clinical data 
will be recorded including dmft, dmfs, and PUFA scores will be recorded. Following the dental 
assessment of the lesions under investigation, SDF will be applied to these lesions for one minute 
followed by 5% NaFV.  
Third visit: Depending on the regimen children will return 4 months (Regimen 1), 6 months 
(Regimen 2), or 1 month (Regimen 3) later for their final follow-up visit. Parents/caregivers will 
complete a follow-up questionnaire administered by interviewer. Caries lesions that were treated 
with applications of SDF will be assessed to see if caries is arrested. Caries lesions will be 
assessed for colour change, size, and clinical hardness. We will also record dmft, dmfs, and 
PUFA scores. Clinical data will be recorded on the Clinical Record Form. 
Clinical data from baseline and each follow-up visit will be linked with questionnaire data. Data 
will be entered into a REDCap database and saved on the secure server at CHRIM.  
Oral Microbiome Sub-Study: SDF may inactivate cariogenic bacteria in vitro. There was only 
one study that conducted RNA analysis of plaque in six children before and after treatment (3 
receiving SDF and 3 placebo) reporting no changes in relative abundance of cariogenic bacteria. 
In that study, not only was sample size a major limitation, but the lead author is a director of 
Advantage ArrestTM. Our proposal to assess 30 children would provide statistical power and help 
elucidate if SDF leads to changes in the oral bacteriome and mycobiome. This will provide novel 
insights into SDF’s mechanism of action, whether antimicrobial, remineralization, or both. Ten 
children from each regimen will have plaque samples collected. Samples will be obtained at 
baseline (prior to SDF application), at the first follow-up visit (prior to SDF application), and at 
the final visit. Nucleic acid isolation from plaque samples will be done using the DNeasy 

PowerSoil Kit (Qiagen). It allows the isolation of genomic DNA (gDNA) from fungi and Gram 
(+/-) bacteria, which will be used for downstream analyses. For the 16S rRNA (bacteriome) and 
ITS1 rRNA (mycobiome) amplicon sequencing, isolated gDNA will be used for PCR 
amplification of the V4 region of the bacterial 16S rRNA gene, and for PCR amplification of the 
fungal Internal Transcribe Spacer 1 (ITS1) rRNA gene. gDNA will be sequenced at Génome 
Québec using paired-end Illumina MiSeq PE250 sequencing. Data analysis will be performed in 
the Chelikani lab using established methods. Briefly, demultiplexed raw sequencing results will 
be filtered and clustered into operational taxonomic units (OTUs) and alpha and beta diversity 
analyses performed using QIIME2 (Quantitative Insights into Microbial Ecology) v. 2018.11. 
16S sequences will be aligned and taxonomy assigned with the HOMD database. ITS1 sequences 
will be aligned and taxonomy assigned with the UNITE database.  
Data Analysis:  
This study will estimate the success of different SDF. Descriptive statistics will be used (e.g. 
frequencies, proportions, and means ± standard Deviations). The “arrest rate” (primary outcome) 
will be calculated for the second visit and again at the third and final visit. Bivariate analyses will 
assess changes in clinical measures and survey data from baseline to the follow-up. McNemar’s 
test will be used to compare the arrest rate between the second and third clinical visits and to 
compare dichotomous data from the baseline questionnaire with that from the follow-up 
questionnaire within each regimen. Chi-squared analysis will compare arrest rates between the 
regimen groups. Paired t-test will be used to compare normally distributed interval data from the 
baseline questionnaire with that from the follow-up questionnaire within regimens (Mann-
Whitney test if not normally distributed). We will rely on repeated measures analysis of variance 
to assess changes in ECOHIS scores and other continuous data over time (i.e. baseline vs. second 
visit vs. final study visit) within each regimen group and to assess differences between regimen 
groups. Although this is a pilot RCT, we will explore potential changes in the oral microbiome of 
those randomized to different regimens. This will provide insights into potential mechanisms of 
SDF action, whether it has antimicrobial activity and influences the oral microbiome 
composition. A p value ≤ 0.05 will be significant.  
Ethics:  
An application will be submitted to the University of Manitoba’s Biomedical Research Ethics 
Board by the June 2019. Additional approvals are being sought from the WHRA and Mount 
Carmel Clinic. Considering some participants will be First Nations, we will notify 
Nanaandawewigamig before starting this RCT, although First Nations status will not be a 
covariate in our data analysis. Written informed consent will be obtained from parents or legal 
guardians of the child. The consent form will state that parents/caregivers are free to withdraw at 
any time, without any effect on the child’s dental treatment at the dental clinic they attend. 
Patients of the PI are eligible to participate. However, he will not obtain consent from 
parents/caregivers of his own patients. At the end of this RCT we will offer SDF to parents to 
manage any new caries lesions that have developed on their child’s other teeth or surfaces or 
those lesions not eligible in this RCT (i.e., ICDAS 3 and 4).  
Knowledge Translation (KT):  
Study outcomes will be disseminated at conferences and in publications. Dr. Robert J. Schroth is 
a Canadian Institute for Health Research Embedded Clinician Researcher and is integrated into 

the Winnipeg Regional Health Authority and Shared Health, providing opportunity to share 
findings with stakeholders responsible for oral health policy and programs. We will also work 
with our knowledge users (Drs. James Taylor, Alyssa Hayes, and Khalida Hai-Santiago). We 
will rely on established networks to share our findings. Dr. Robert J. Schroth co-leads (and Dr. 
Michael E. K. Moffatt is a member of) the Healthy Smile Happy Child initiative, which 
promotes early childhood oral health in Manitoba. Telehealth and Zoom conferencing are used 
for these activities. Drs. Schroth and Moffatt are part of Nanaadawewigamig’s Canadian 
Institutes for Health Research Partners for Engagement and Knowledge Exchange, which will 
disseminate evidence to communities. Dr. Schroth also leads the Oral Health and the Aboriginal 
Child Knowledge Transfer website where findings will be disseminated (> 35,500 page views). 
The Canadian Dental Association’s Knowledge Networks will assist with knowledge translation 
to inform dental professionals, stakeholders, researchers, and the public. Dr. Schroth is 
strategically positioned as chair of the Candian Dental Association’s Committee on Clinical and 
Scientific Affairs, which provides another avenue to disseminate findings to inform policy and 
clinical guidelines on SDF.  
Significance and Future Directions:  
SDF may be a simple and non-invasive agent to arrest caries in children. Before SDF can be 
implemented in dental public health programs and Indigenous communities it is important to 
study which application regimen is most effective and appropriate. CHRIM funding will provide 
essential information on the ability for SDF to arrest ECC and will inform a future CIHR 
application to conduct a scaled-up RCT of SDF with high-risk children, including those from 
Indigenous, newcomer, rural, and disadvantaged communities.