Povidone Iodine for Caries Prevention

Dental 'caries' is the term used to describe dental decay or 'cavities'. Dental caries can occur at any age but lesions prior to a child's 6th birthday qualify as 'early childhood caries' (ECC) and possibly as 'severe' early childhood caries (SECC) if the onset and severity meet definitions put forth by the American Academy of Pediatric Dentistry. ECC and SECC can have several unfortunate downstream consequences: pain, infection, nutritional deficiencies, poorer concentration in school, and social/cosmetic/financial concerns. Recent epidemiologic trends in ECC/SECC, reported by Kotha et al. (2022) based on data from the National Health and Nutrition Examination Survey (NHANES), have found little change in prevalence over the span from 2013 to 2018 with approximately 20% of children experiencing ECC by age 3 years with over half that percentage categorized as having SECC. Thus, there remains a critical need to improve prevention and treatment strategies.

Many variables can affect the likelihood of developing dental decay but prominent among these are excess consumption of sugary foods and drinks, inadequate oral hygiene, and poor access to dental care and oral health educational resources. Ultimately, microbes on the surface of teeth convert dietary sugar to acid that dissolves enamel thereby initiating and advancing decay. Brushing regularly with fluoridated toothpaste helps suppress the microbial population which decreases the acid challenge while fluoride promotes remineralization of enamel. Seeing a dentist regularly can ensure that any decay that develops is treated at its earliest stages thereby saving teeth.

While prevention strategies work well for many individuals, for a multitude of potential reasons they may prove inadequate for others. In particular, children with poor access to professional care or who are disadvantaged in other respects frequently fail to realize the benefits of the current standard of care.

The objective of this investigation is to test the effectiveness of povidone iodine (PI) as a home-administered preventive or treatment for dental caries that, unlike other treatment options, addresses the underlying cause - dental plaque that has an overabundance of strong acid-producing bacterial species. This imbalance of strong acid producers is referred to as a 'dysbiosis'. We will also test whether combining PI with probiotics - living microbes that can be used to boost health - will have a greater benefit than PI alone in maintaining or restoring a healthy microbial composition to dental plaque. We are in a unique position to leverage our access to children at high risk for decay to test multiple integrated hypotheses that we predict will result in more effective home dental care, and significant reductions in the incidence and severity of dental decay that is longer lasting than is possible with the current standard of care.

Despite tremendous advances in understanding how particular microbes in dental plaque lead to tooth decay, prevention and treatment options remain non-specific - dedicated to plaque control rather than to replacing the offending microbes with health-related species (correcting the dysbiosis). This limitation results in the chronic presence of an elevated microbial risk that likely proves most consequential in individuals with limited access to professional care and suboptimal oral hygiene habits. Importantly, PI is unique among current standard of care options because there is evidence that it not only can prevent caries but that it also attacks the microbial offenders that drive caries development. The addition of fluoride to water supplies and oral health care products was a game-changer. Protection appears to be due to improving the enamel remineralization/demineralization balance rather than any antimicrobial activity despite in vitro evidence of selective toxicity against S. mutans.

Consequently, fluoride is excellent at caries prevention (when available/applied in adequate amounts and frequency) but does not improve a dysbiotic plaque microbiome. Chlorhexidine, sometimes prescribed for individuals with rampant caries, is considered the 'gold-standard' of anti-plaque agents but a large NIDCR-sponsored study (Papas et al., 2012) found no greater protection against caries than the use of fluoride alone.

Additionally, chlorhexidine use does not appear to remedy a cariogenic dysbiosis. Silver products have a long history in dental treatment and have generated renewed interest in recent years. The strong staining properties of silver, however, limit its application. The preponderance of evidence does not support a role for silver in improving a cariogenic microbial dysbiosis but further investigation is necessary.

Iodine, like silver, has long been recognized for its antiseptic/antimicrobial properties. Combining iodine with povidone improves water solubility. Evidence to date suggests that PI offers the greatest breadth of desirable properties but additional investigation is necessary in order to make a strong case for widespread adoption by clinicians. This proposal seeks to document the effectiveness of home use of PI for providing long-term reduction in caries risk by reducing the microbial dysbiosis within cariogenic dental plaque.

Hypothesis(es) and objective(s): We hypothesize that home use of PI will reduce the incidence and severity of dental decay in a high-risk population of children. Though not a substitute for proper oral hygiene such as brushing twice a day for two minutes each, its application is so quick that compliance is more likely than engaging in routine, effective oral hygiene and it would likely boost the effectiveness of imperfect oral hygiene practices. We further hypothesize that PI has selective toxicity for oral pathogens and its use would improve the microbial make-up of dental plaque thereby helping sustain beneficial effects. Finally, we hypothesize that this latter property of PI can be combined with probiotic administration to elevate the beneficial effect.

Study Protocol:

Patient Pool: The patient pool will be children of ages 1 to 3 years old who receive care

through the Infant Oral Health clinic that is part of the Iowa City Women, Infant, Children (WIC) Program in Iowa City. The University of Iowa Department of Pediatric Dentistry has an ongoing collaboration with the IC-WIC to provide oral health care and sees 16 to 24 children per week (Weber-Gasparoni et al., 2010). The children are typically from low-income families and all are at high risk for decay. We estimate that we could recruit 3 to 5 subjects/week. To estimate the number of subjects necessary to obtain statistically significant results, we extrapolated from the results of Lopez et al. (2002) who used 10% povidone iodine and an outcome measure of new caries. Based on the

differences between treatment and placebo groups, 126 subjects (42 Group 1, PI; 42 Group 2, PI + probiotics; and 42 Group 3, placebo) would give 80% power to determine a statistical difference in caries outcome with an alpha error of 0.05. To meet this number, we will enroll 174 subjects to account for an estimated 27% annual attrition.

Subjects will be randomly assigned to Groups 1, 2 or 3. However, if a subject consents to use PI but not probiotics, their assignment will be to either Group 1 or Group 3. We anticipate completing enrollment during year 1 of the study but have reserved 18 months to meet the recruitment goal. We also want to make clear that Group 3 will be the placebo group for the Group 1 iodine treatment. After considerable contemplation, we decided against including a strict placebo group for the probiotic administration. We considered having Group 3 use both the PI placebo and probiotic placebo but that would 'muddy' the analytical comparison with Group 1 (PI alone). Including another distinct placebo group would add considerable expense to the project that might not be justified if compliance with a daily regimen turned out to be low. As designed, we feel this project will yield strong data for the efficacy of home use of PI. The inclusion of a probiotic group has the potential to add significant insight into the mechanism by which PI exerts any observed benefit, or it may offer a lesson in the limits of compliance for any sort of daily oral health promoting regimen.

Enrollment and Visit Protocols: Pediatric Dental Residents will be the practitioners

treating the prospective participants. They will be calibrated with respect to caries scoring (ICDAS) prior to initiating the study. Once it is determined that the subjects satisfy the inclusion criteria (child of appropriate age; no imminent plans to relocate), they will follow an IRB-approved recruitment/enrollment protocol that will also determine

if any exclusion criteria (thyroid condition; antibiotic usage in the prior 3 months) apply. Once informed consent is obtained, the current caries status will be recorded and a pooled plaque sample will be taken using a sterile foam-tipped applicator (Puritan®) and swabbing all exposed tooth surfaces. The study coordinator will provide the practitioner with PI (Povi-One povidone iodine unit dose kit; Elevate Oral Care®), PI and probiotic drops (Prodentis by BioGaia), or placebo (colored tea for PI). The practitioner will apply the PI treatment onto the teeth of the participant and instruct them in home use. For participants given probiotic, verbal instructions will be provided to administer the drops once a day after brushing before bedtime. While this protocol precludes keeping practioners blinded with respect to the intervention, only the study coordinator will keep a record of the participants' group assignment so that practioners will not have access to group assignment records at follow-up appointments thereby minimizing the possibility of bias in subsequent caries evaluations. The PI protocol will be one application per week for one month and then once a month thereafter. Probiotic application will be daily. The concentration of the povidone iodine will be 5% and the maximum dose of probiotic will be 5 drops. The amount disbursed in an application and the frequency of application will be well within the safety margins for iodine exposure (Frank et al., 2020). Subjects will receive reminders ahead of a scheduled home application of PI via text message or communication method of choice to help ensure compliance. Subjects will also have the opportunity to reply to the messaging so that compliance can be tracked and/or keep a home diary of compliance and personal oral hygiene habits (How many times per day/week do the children brush? Themselves or by parents? Does the toothpaste contain fluoride?). At each 6-month follow-up

appointment, subjects will again have their caries status recorded and provide a pooled-plaque sample. They will also be asked questions related to use of antibiotics during the period between baseline and follow-up appointments. Maximal duration of participation for any subject will be 1 year.

Processing of Plaque Samples for Microbiome Analysis: Pooled plaque samples will be

collected as described above. The foam tip of the applicator will be broken off into 300ul RNAlater™ (Invitrogen) for transport to lab and isolation of DNA using the Qiagen DNeasy PowerLyzer PowerSoil Kit. The DNA that is recovered contains strands from all the microbes present in the sample. Technology allows the DNA to be sequenced and computational software is capable of analyzing the sequences and providing a breakdown of the proportional representation of the different types of microbes present. To accomplish this, the DNA samples will be sent to LC Sciences (Houston, TX) for the sequencing and microbial data analysis. [For those technically inclined, the 16S sequencing primers will target the V3 and V4 regions. The resulting 465bp amplicon will be sequenced on a NovaSeq platform with 250bp paired-end reads. Raw data files will be obtained in FASTQ format, overlapping sequences merged, screened for data quality and chimera filtered. The algorithm DADA2 will then be applied to generate representative sequences and construction of Amplicon Sequence Variant tables that will be the basis for analyses of diversity and differential abundance analyses.

Taxonomy will be assigned using the QIIME2 naïve bayes classifier (Bokulich et al., 2018) trained on the Human Oral Microbiome Database (HOMD) v15.23].

Previous studies have demonstrated that treatment with 10% PI can reduce salivary/plaque levels of the highly acidogenic bacterial species Streptococcus mutans (Amin et al., 2004; Simratvir et al., 2010) but further substantiation including effects on the totality of the oral microbial population are warranted. We recently completed a longitudinal study focusing on the microbial changes that preceded a diagnosis of ECC (unpublished data) and found that S. mutans appeared to be the main driver or major contributor in about three-quarters of the children. In the other 25% who developed caries, it appeared that non-mutans acidogenic species or the loss of base-producing species were the major drivers. Therefore, our microbiome analysis will focus on S. mutans as well as on changes in the representation of key genera that are health related (Neisseria, Haemophilus) or caries related (Actinomyces, Prevotella, Kingella, Candida).

Data Analyses: Our data analyses will focus on answering three main questions: 1) did

home use of PI reduce caries incidence and severity?; 2) did home use of PI reduce the presence in dental plaque of microbial species most associated with dental decay?; and 3) did a daily regimen of probiotics enhance the effectiveness of the PI treatments by further reducing caries incidence/severity and the presence of decay-associated microbes? To answer each of these questions, caries rates and microbial composition outcomes from Group 3 (placebo) will be compared with Groups 1 (PI) and 2 (PI + probiotic), individually and in combination. Outcomes from Groups 1 and 2 can also be compared directly. We expect to use ANOVA for statistical analysis but will consult with a biostatistician to ensure that all analyses are experimentally appropriate and valid.

The analyses described above will provide answers to our three main questions and directly address the hypotheses being tested. Our data on compliance will provide perspective for these outcomes. We anticipate that home use of PI is quick and simple enough to elicit good compliance. We are less sure of what to expect with the probiotic. On the one hand, administration (5 drops) is quicker than brushing one's teeth (though recommended use is after brushing). On the other hand, our study groups may not be particularly proficient in adhering to recommended oral hygiene practices. Overall, we expect the PI to be protective to a significant degree regardless of personal daily oral hygiene habits. However, we expect that the magnitude of protection will improve in direct proportion to the frequency of personal oral hygiene, including compliance of probiotic use.