Key Factors Influencing Caries Development in Preschoolers: A Focus on Socio-Demographic, Maternal Health, and Salivary Biomarkers in 3-Year-Olds

Introduction

Early childhood caries (ECC) refers to the presence of 1 or more decayed, extracted, or filled teeth in children up to 71 months of age [1]. The treatment of the disease is delicate, and if not treated on time, it causes pain, odontogenic infections, and premature loss of primary dentition, adversely affecting the quality of life of the child and the child’s family [2,3]. Furthermore, ECC is one of the strongest caries predictors in permanent dentition among school children and adolescents [4]. Unfortunately, epidemiological data show that it remains one of the most common childhood diseases, ranking 12th in children under 6 years of age [5,6]. The data from the World Health Organization (WHO) from 2019 indicate that over the past 30 years, there has been only a slight reduction of just 3% in the global prevalence of ECC and that as many as 514 million children worldwide have at least 1 primary tooth with untreated caries [7]. The global prevalence of ECC is 43%, and it is estimated that depending on the age and the population of children studied, it ranges from 12% to 98%, highest in countries with upper-middle income and lowest in high-income countries [7,8]. Interestingly, some children are caries-free for a long time, but at a certain point, they develop ECC. The period around the third year of life is particularly critical, as the primary dentition is complete, and significant cognitive, emotional, social, and personality developments occur [9,10]. Therefore, during early childhood, 3-year-olds are considered a high-risk subgroup that requires consideration in terms of primary caries prevention.

Identifying reliable early predictors of caries onset for subpopulations at risk is crucial, as it allows for selecting appropriate measures to prevent onset of the disease [11]. Unfortunately, this is far from simple in early childhood due to uniqueness of this developmental period, and also the complexity of caries etiology, which in addition to the tooth-microorganism-carbohydrate interaction, includes sociodemographic, economic, behavioral, and environmental factors, starting from factors at the individual level (micro level), through the family level (middle level), all the way to the influence of the state (macro level) [12,13]. Therefore, in addition to secondary factors, sociodemographic, economic, oral health behavioral, and environmental factors are of great importance for the etiology of caries.

Among other things, sociodemographic factors, such as gender, race, ethnicity, educational profile and age of parents, and living conditions affect the qualitative composition of the diet, eating habits, and maintenance of oral hygiene of children, and have a strong influence on the occurrence of ECC. Therefore, these factors are directly related to oral health behavior and environmental factors, forming a strong chain of factors that greatly influence ECC [14].

Research in the field of dental caries etiology remains highly relevant, with numerous studies examining the influence of various factors on the occurrence of this disease. It has been established that the presence of caries in children is significantly associated with sleep fragmentation [15]. Particular attention has been given to the analysis of properties and components of saliva in the etiopathogenesis of caries, as saliva is part of the biosystem in which the caries process takes place. Saliva serves as the medium for the ion exchange process, making its pH level critically important for demineralization and remineralization processes in dental caries [16]. However, due to the infectious nature of dental caries, the role of innate and acquired immunity in the etiology of caries has been particularly topical recently. It has been shown that proteinase 3 and interleukin 8 significantly influence the presence of ECC, suggesting their potential effectiveness as non-invasive indicators of caries risk [17]. Additionally, children with severe forms of ECC have been found to have significantly higher levels of matrix metalloproteinase 8 compared to caries-free children [18]. In recent decades, special attention has been directed toward small, cationic, antimicrobial peptides. In the oral cavity, their antimicrobial activity is primarily directed against bacteria, including cariogenic bacteria [19,20]. Defensins and cathelicidins are the most prominent representatives of this group of peptides. Through their antimicrobial properties, they contribute significantly to the biological control of dental caries [21]. Two subfamilies of human defensins have been identified: α45;-defensins and β-defensins [22]. Human α45;-defensins are primarily expressed in neutrophils and are therefore also known as human neutrophil peptides (HNPs). In the human oral cavity, only myeloid α45;-defensins have been identified: human neutrophil peptide 1 (HNP-1), human neutrophil peptide 2 (HNP-2), and human neutrophil peptide 3 (HNP-3). These peptides differ in their amino acid composition, whereas their antibacterial properties are very similar [23,24]. Human β-defensins are produced by oral epithelial cells and salivary glands. Human β-defensin 2 (hBD-2) has received the most attention in oral biology due to its strong antimicrobial activity against S. mutans [21]. The only biologically active form of cathelicidin in humans is the LL-37 peptide, which is derived from neutrophils, oral epithelial cells, and salivary glands [25].

Since the etiology of ECC is multicausal and quite complex, over 100 factors significant for assessing the risk of early childhood caries have been identified so far [26,27]. Cross-sectional studies have shown their significance as risk indicators of ECC, but longitudinal studies have produced conflicting data regarding their caries predictive value. Previous caries experience has the greatest predictive value, but it has no significance in the early prediction of caries [27,28]. High predictive value is also found in salivary microbiological tests [28,29]. On the other hand, the results regarding the importance of sociodemographic factors in the prediction of ECC are conflicting [27,30,31]. Similar results are seen with oral health behavioral and environmental factors [27,31]. To date, the significance of various components of saliva has been analyzed as caries risk factors. Due to the infectious nature of caries, considerable attention has been drawn to the potential significance of non-globulin antimicrobial proteins [32,33]. Significant focus has been directed at salivary HNP-1, hBD-2, and LL-37 cationic antimicrobial peptides. Cross-sectional studies have highlighted their potential as risk indicators of ECC [34–38], but their predictive role in the development of ECC remains insufficiently understood.

From the primary prevention perspective, the ability to predict the occurrence of caries in caries-free children is of key importance. Although it is well known that 3-year-olds are a subpopulation at high risk for rapid onset of dental caries, precise data on early predictors of caries in this age group remain limited. Moreover, existing data on the predictive significance of various risk factors are mostly derived from cross-sectional studies that included both caries-free children and those with ECC [39–41]. Prospective observational studies designed to follow caries-free 3-year-olds over time are rare. Only 1 study in the available literature has analyzed the caries predictive value of salivary defensins and cathelicidins in preschool children [42]. In addition, no studies have simultaneously analyzed sociodemographic, oral health behavioral, and maternal factors alongside salivary pH and salivary levels of cationic antimicrobial peptides. Therefore, the aim of this study was to examine and compare the significance of sociodemographic, oral health behavior, and maternal factors, as well as salivary pH and salivary levels of HNP-1, hBD-2, and LL-37 peptides, as early caries predictors in 3-year-olds.

Material and Methods

ETHICS STATEMENT:

This study was conducted in compliance with all principles of the Declaration of Helsinki and was approved by the Ethics Committee of the Faculty of Medicine, University of Niš, Serbia (decision number 12-14532-2/3). The study included patients from the Department of Preventive and Pediatric Dentistry at the Clinic of Dental Medicine, Faculty of Medicine, University of Niš, Serbia, as well as children from 3 kindergartens in the central area of the city of Niš, aged 36–48 months at the beginning of the study and their mothers. The directors of the institutions covered by the research were informed verbally and in writing about the aims of the study, the data collection process, and personal data protection, after which they gave written consent to participate in the study. After that, the mothers of the children were informed both orally and in writing about the aims of the study, data collection procedures, and measures taken to protect the identity of the children. The mothers gave special written informed consent for the clinical examination of their children and for their participation in a survey aimed at collecting sociodemographic, oral health behavior, and other data relevant to the study. Additionally, separate written consent was obtained for sampling unstimulated saliva to determine its pH value and salivary peptide levels. The study included only those children for whom written consent to participate had been obtained.

STUDY DESIGN AND SUBJECTS:

This study was designed as a 1year observational prospective study based on data collected at baseline (2017) and 12 months later.

INCLUSION AND EXCLUSION CRITERIA:

Subjects were selected based on data obtained by surveying mothers and conducting clinical examinations of the subjects. The basic inclusion criteria were: (1) caries-free children aged 36–48 months at the beginning of the study, with no structural tooth defects; (2) healthy children with no systemic diseases in their personal medical history; (3) children born and residing in the city of Niš, Republic of Serbia (long-term average fluoride concentration in drinking water ≤0.05 mg/mL); (4) children with complete primary dentition throughout the study, with no initiation of physiological tooth change; (5) children with whom it was possible to make contact during the clinical examination. The measurement of unstimulated saliva pH and salivary levels of HNP-1, hBD-2, and LL-37 peptides was performed in children who, in addition to meeting basic inclusion criteria, also met additional criteria: (1) subjects had not used antibiotics for at least 1 month before the onset of the study; (2) subjects without oral soft tissue diseases or any mechanical trauma to the oral tissues at the time of the examination; (3) subjects in whom saliva samples could be collected by spitting into sterile tubes; (4) written consent obtained from parents for saliva sampling from their children. Children with a history of any type of dental injury were excluded from the study.

DETERMINATION OF SAMPLE SIZE:

The sample size was determined based on data on the population of children aged 36–48 months in Nišava District, selected according to the described criteria, and the prevalence of caries in children aged up to 36 months in Serbia [43]. The sample size was calculated using the proportion method in the EPI INFO STATCALC program, version 7.2.2.6. The minimal calculated sample size was 164 subjects. Due to the possible dropout of 5% of the sample, slightly more children were selected.

DATA COLLECTION:

Data were collected at the beginning of the study (2017) and 12 months later by a well-trained examiner, who was a specialist in preventive and pediatric dentistry with long working experience. Data were collected in dental offices of the institutions included in the study (the kindergartens included in the study offer organized dental services as part of the Preventive and Pediatric Dentistry Department at the Health Center in Niš, Serbia).

BASELINE DATA COLLECTION:

Basic sociodemographic data about the children included in the study (gender, date and place of birth, permanent residence since birth, age and education level of parents), their eating habits (length of breastfeeding, number of meals per day, frequency of refined carbohydrate intake), oral hygiene habits (equipment, use of fluoride toothpaste, frequency of tooth brushing during the day), information related to experience with dental injuries and dental visits in the previous 12 months were collected by interviewing their mothers. Also, data on children’s systemic health and use of medications including the use of antibiotics at least 1 month before the onset of the study were collected by interviewing their mothers. Additionally, mothers were asked to do a self-assessment of the health of their own teeth.

CLINICAL EXAMINATION OF CHILDREN:

The children who met basic inclusion criteria were referred for further clinical dental examination, scheduled in the morning, 1 hour after the usual oral routine. Mothers were instructed that the examination would be conducted before breakfast on an empty stomach.

The clinical examination of the subjects was conducted in a dental chair under artificial lighting, using a dental mirror and dental probe. The modified DDE index was used to assess structural defects [44]. The study included only children with a DDE index of 0, meaning that children with any structural defects were excluded from further study. The assessment of oral hygiene status was performed using the modified simplified debris index (DI) of the Simplified Oral Hygiene Index, as in the study by Thwin et al [45]. Measurements were taken on the vestibular surfaces of teeth 55, 51, 65, 71, and the oral surfaces of teeth 75 and 85. Caries diagnostics was performed following the World Health Organization Oral Health Surveys Basic Methods criteria for epidemiological research [46]. During the examination, the presence of sound teeth was recorded, as well as the presence of decayed teeth (with or without cavitation), teeth missing due to caries, and filled teeth. A tooth is coded as sound if it shows no evidence of treated or untreated clinical caries including white-spot lesion. Caries diagnostics was performed to determine the dmft (decayed, missing, and filled teeth) and dmfs (decayed, missing, and filled surfaces) index. The study included only children with a dmft and dmfs index of 0 at the beginning of the study. The condition of the soft oral tissues was also registered according to the criteria of the World Health Organization [46].

UNSTIMULATED SALIVA SAMPLING AND MEASUREMENT OF PH:

In children who, in addition to meeting the basic criteria, also met additional inclusion criteria (N=35), the measurement of unstimulated saliva pH and salivary levels of HNP-1, hBD-2, and LL-37 was performed.

In children selected according to the additional criteria (N=35), the measurement of unstimulated saliva pH was performed right after the dental examination. Saliva samples were taken in the morning, 1 hour after the usual oral routine, approximately between 9: 00 and 10: 00 AM. Samples of saliva were taken on an empty stomach by spitting saliva into sterile tubes. Salivary pH was measured immediately after sampling a sufficient amount of saliva (around 1 mL) using a digital, portable pH meter (Hanna Instruments, USA).

UNSTIMULATED SALIVA SAMPLING AND MEASUREMENT OF SALIVARY LEVEL OF HNP-1, HBD-2, AND LL-37 PEPTIDES:

Immediately after determining the pH of unstimulated saliva, sampling was performed to determine salivary levels of HNP-1, hBD-2, and LL-37 peptides. It was performed by spitting into sterile tubes for 5 to 10 minutes to collect 2 mL of unstimulated saliva. The samples were stored at +2°C and transported within 1 hour of collection to the Scientific Research Center for Biomedical Research at the Faculty of Medicine, University of Niš, Republic of Serbia, for further analysis, where they were centrifuged at 10 000 rpm, at +4°C for 10 minutes. The supernatant was separated, and the samples were divided and frozen at −82°C until the next phase. The salivary level of HNP-1, hBD-2, and LL-37 peptides was determined using enzyme-linked immunosorbent assay (ELISA) with commercial ELISA kits according to the manufacturer’s instructions (Human alpha-Defensin 1 - DuoSet ELISA, DuoSet^® Ancillary Reagent Kit 2, R&D Systems, Minneapolis, USA; Human β-Defensins 2 ELISA Kit, Cusabio Biotech, Houston, USA; Human LL-37 ELISA kit, HyCult Biotech, Uden, The Netherlands). The detection range for the HNP-1 ELISA kit was 0.50–32 ng/mL, for the hBD-2 ELISA kit 62.5 pg/mL - 4000 pg/mL, and for the LL-37 ELISA kit 0.1–100 ng/mL. The optical density of the samples was read on a Spark Multimode Plate Reader (Tecan Trading AG, Männedorf, Switzerland) at a wavelength of 450 nm, and HNP-1, hBD-2, and LL-37 concentrations (ng/mL) were calculated using TableCurve 2D v5.0 software (Grafiti LLC, Palo Alto, CA, USA). The concentration of all tested peptides was expressed in ng/mL.

MONITORING CHANGES IN CARIES STATUS OF SUBJECTS:

A systematic dental examination of the subjects was repeated after 12 months to monitor caries incidence. Caries diagnostics was performed following the same diagnostic procedure as at the beginning of the study.

STATISTICAL ANALYSIS:

Data were processed using the R software package [47] and are presented as arithmetic means, standard deviations and medians, minimum and maximum values, and absolute and relative numbers. The 1-year caries incidence was calculated by following changes in the dmft index after 12 months. The comparison of numerical variables between children diagnosed with caries after 1 year and those who remained caries-free during the same period was performed using the t test or Mann-Whitney test, depending on the data distribution. The categorical variables were compared using the chi-square test or Fisher’s exact test. Logistic regression analysis (univariate and multivariate analysis) was used to assess the potential caries predictive significance of sociodemographic, oral health behavior, and maternal factors, as well as clinical and salivary parameters in 3-year-olds. The calibration of the multivariable model was tested using the Hosmer-Lemeshow test. The null hypothesis was tested with a significance level of P<0.05

Results

SOCIODEMOGRAPHIC, ORAL HEALTH BEHAVIOR, MATERNAL, CLINICAL, AND EXAMINED SALIVARY CHARACTERISTICS OF CHILDREN:

The study included 76 caries-free boys (46.1%) and 89 caries-free girls (53.9%). The average age of the subjects was 40.5±4.2 months. The average length of breastfeeding was 9.7±6.4 months.

The highest percentage of subjects (46.1%) consumed refined carbohydrates once a day. In addition, most of the examined children (63.0%) brushed their teeth once a day. All children used a toothbrush and fluoride toothpaste for oral hygiene, but most mothers (95%) were unaware of the fluoride concentration in their children’s toothpaste. All mothers stated that their children brushed their teeth with parental assistance. We found that 68.5% of the children did not have a personal dentist, and 48.5% had never visited a dentist by the time of the examination. Most mothers (72.1%) reported that all of their own teeth had been repaired.

CARIES INCIDENCE IN THE STUDIED GROUP OF CHILDREN: After 1 year, caries was diagnosed in 48 subjects (29.1%). In the group of children who developed caries during the year, the dmft and dmfs indices were 3.27±1.84 and 4.33±2.54, respectively. Caries incidence and the parameters of caries prevalence among children diagnosed with caries after 1 year are shown in Table 2.

SOCIODEMOGRAPHIC, ORAL HEALTH BEHAVIOR, MATERNAL, CLINICAL, AND EXAMINED SALIVARY CHARACTERISTICS OF CHILDREN REGARDING 1-YEAR CARIES INCIDENCE:

The comparison of sociodemographic, oral health behavior, maternal, clinical and examined salivary characteristics of children revealed that the children diagnosed with caries after 1 year were breastfed significantly longer (P<0.01), that their DI values were significantly higher (P<0.001), that they visited a dentist significantly less often (p=0.016), and that their mothers, based on self-assessment, had significantly worse dental health (P<0.001) (Table 3).

CARIES PREDICTIVE SIGNIFICANCE OF THE EXAMINED VARIABLES:

The univariate logistic regression analysis determined that the length of breastfeeding (OR 1.017, P=0.001), DI values >1 (OR 6.324, P<0.001), not having a personal dentist at this age (OR 2.454, P=0.012), and poor dental health of mothers (OR 10.521, P<0.001) were the significant early caries predictors in the studied group of children (Table 4).

The multivariate model confirmed that length of breastfeeding duration (OR 1.088, P=0.017), DI values >1 (OR 6.667, P<0.001), lack of a personal dentist (OR 2.850, P=0.036) and poor maternal dental health (OR 9.110, P<0.118) remained significant early caries predictors (Table 4).

The other tested variables were significant predictors of caries (Table 4).

Discussion

This study determined that the most significant early predictors of caries onset in 3-year-olds are debris index, breastfeeding duration, lack of a personal dentist at this age, and poor dental health of mothers.

This study confirmed that the period around age years is a particularly high-risk period for rapid onset of caries. The study initially included 165 caries-free 3-year-olds. It is important to emphasize that the respondents were from one of the countries with higher middle income [48], where, as previously mentioned, the prevalence of ECC is the highest. After 1 year, caries was diagnosed in 29.1% of the participants, with average dmft and dmfs indices of 3.27±1.84 and 4.33±2.54, respectively. The incidence values of caries that were obtained can be considered relatively high. It is well known that due to the anatomical-morphological and histological characteristics of primary teeth, the progression of caries in them is twice as fast as in permanent teeth [49]. However, the explanation for such high caries incidence values should also be sought clinical, biological, socio-economic, behavioral, and environmental factors, starting at the individual level, through the family level, all the way to the influence of the state.

Studies indicate that microbiological tests and previous caries experience have the highest predictive significance of ECC [27–29]. Microbiological tests are important biological factors and could be significant early predictors of caries onset in 3-year-olds. However, their application incurs additional costs and requires special equipment; thus, their use in routine clinical practice is limited, especially in low- and middle-income communities, where the problem of early childhood caries is particularly emphasized. Therefore, this study did not consider their significance as predictors of ECC. On the other hand, clinical variables (previous caries experience, presence of structural defects, oral hygiene index values) have also shown a high predictive value for ECC [27,28,50–51]. The best caries predictive factor to date is previous caries experience [27,28], but since this is not relevant for primary prevention of ECC, this study initially focused on caries-free 3-year-olds. To more accurately identify predictors of ECC in 3-year-olds, children with structural defects were also excluded. Finally, the study found that DI>1 values are important clinical predictors of ECC in 3-year-olds, which is consistent with numerous studies [28,31,52,53]. DI values are direct indicators of proper oral hygiene – its frequency, regularity, duration, and technique. Regarding oral hygiene, children aged 3 are completely dependent on their parents, as confirmed by all the mothers, who reported that their children brushed their teeth with the help of their parents.

Examined sociodemographic variables (gender, parents’ age, and education) were not did good predictors of caries. Similar results were obtained with some oral health behavior determinants such as tooth brushing frequency, number of meals a day, frequency of refined carbohydrate intake, and number of visits to the dentist in the last 12 months. However, the study showed that breastfeeding length, lack of a personal dentist, and poor dental health status of the mother are significant early predictors of caries onset in 3-year-olds. These results suggest that, in addition to oral health behavior-related factors, environmental factors are also important early predictors of caries onset in 3-year-olds, primarily at the middle and macro levels. Since the period around the third year of life is a specific developmental stage, individual-level factors at this age are of little significance, as children are still dependent on parents regarding factors important for preserving oral health. Therefore, parents have a great responsibility, particularly in terms of maintaining their children’s oral health.

Despite contradictory results regarding the effect of the length of breastfeeding on the dental health of children [54,55], the length of breastfeeding proved to be an important early predictor of caries in the studied group of children. This finding is rather interesting, as breastfeeding was completed in all subjects, yet it still showed predictive importance. Although the study did not analyze this, it could be explained by the fact that long-term breastfeeding can contribute to developing other bad habits later in childhood, such as night-time meals.

According to the study results, the lack of a personal dentist is a significant early predictor of caries onset in 3-year-olds. Such results clearly show the necessity of intensive health education of parents, especially mothers, from pregnancy and the earliest stages of their child’s life to provide them with information on the importance of maintaining the oral health of their children, as well as with detailed instructions for maintaining oral hygiene, with constant motivation and re-motivation to persist. This also includes information on fluoride use, as many mothers have pointed out that they did not know the fluoride concentration in the toothpaste they use for their children. On the other hand, the study showed that poor dental health of mothers is a significant predictor of ECC in 3-year-olds, confirming the influence of oral health behavior of mothers on their children’s oral health. Finally, the study showed that 48.5% of the children had never visited a dentist by the time of the study and that 68.5% of the children did not have a personal dentist. These results suggest the importance of mandatory preventive dental check-ups from birth through early childhood. The implementation of preventive check-ups is usually legally regulated, but in many countries worldwide it is only recommended and not mandatory. Therefore, it is necessary to reconsider the frequency of such examinations in the health systems of these communities, and they should be aligned with the needs of children. With these preventive check-ups, it is necessary to continuously assess caries risk, with mandatory correction of risk factors for the occurrence of this disease. Only in this way can primary prevention of ECC be achieved, thereby reducing the prevalence of this disease. Therefore, in addition to parents, the state also bears significant responsibility through regulations on pediatric dental healthcare.

Also, this study analyzed the importance of cationic antimicrobial peptides HNP-1, hBD-2, and LL-37, as well as the pH of unstimulated saliva, as early predictors of caries onset in 3-year-olds. Unfortunately, no significant findings were made in this regard. The obtained results agree with the study by Simon Sor et al (2018) and with the results of our previous study that analyzed the caries predictive significance of these peptides in early adolescents [42,56]. However, the importance of these peptides in predicting ECC should not be dismissed. This study analyzed only a small number of cationic peptides in a small sample, which is an important limitation of this part of the study. Cationic antimicrobial peptides are important components that are the first line of defence of the oral cavity against cariogenic microorganisms. Their salivary level depends on numerous factors, and these peptides have a complex antimicrobial mechanism that stimulates cytokine production, IgA, and IgG, and synergistic action with other antimicrobial peptides [20,57]. Therefore, the obtained results should not discourage future research into the significance of cationic antimicrobial peptides and other non-immunoglobulin antimicrobial peptides as early predictors of ECC.

The main limitation of this study is that salivary pH and peptide levels were not analyzed in all subjects. Additionally, the study did not consider changes in dietary habits or oral hygiene maintenance, which change over time. Furthermore, the mothers’ dental health status was based on self-assessment, which is subjective and was not verified by a dental professional. Finally, the study did not consider many other potentially important socio-economic, behavioral, and other factors, as well as a broad range of non-immunoglobulin antimicrobial peptides. Outcome data were not available for 5.17% of participants; therefore, we were unable to compare their baseline characteristics with those who completed the study. While this is a limitation, the relatively low attrition rate is unlikely to have introduced major bias. Therefore, well-designed prospective observational studies are required to accurately identify early predictors of caries in 3-year-olds, who are highly vulnerable to the rapid onset of this disease. To address the issue of time-dependent variables, such studies require both entry and follow-up questionnaires and should specifically analyze the predictive value of variables that changed over time in comparison to those that remained stable. Moreover, the dental status of parents should be evaluated by a qualified dental professional.

Conclusions

This 1-year observational prospective study showed that the most significant early predictors of caries onset in 3-year-olds are debris index, breastfeeding length, lack of a personal dentist at this age, and poor dental health of mothers. These results suggest that key factors in preventing caries in 3-year-olds include parents’ behavior regarding the maintenance of their own and their children’s oral health, as well as macro-level factors, such as state-level factors related to the organization of dental healthcare from early childhood. Longitudinal studies are needed to accurately determine the significance of these variables in predicting early childhood caries by monitoring changes in the oral hygiene and dietary habits of children of this age. Our study did not determine the caries-predictive significance of the tested cationic salivary peptides, and further research in this field encompassing a variety of antimicrobial peptides found in saliva is still needed. This would make a significant step forward in caries prediction and, consequently, in caries risk assessment.