Candida species and selected behavioral factors co-associated with severe early childhood caries: Case-control study.

Severe Early Childhood Caries (sECC) is a multifactorial disease associated with the occurrence of specific oral microorganisms and other environmental, behavioral, and genetic factors. This study aimed to construct a multivariable model including the occurrence of Candida spp. and selected behavioral factors (length of breastfeeding, serving sweet beverages and beginning of brushing child's teeth) to determine their relationships to the occurrence of sECC. In this case-control study 164 children with sECC and 147 children without dental caries were included. MALDI-TOF MS and multiplex qPCR were used to identify Candida spp. and selected bacteria in dental plaque samples, respectively. A questionnaire on oral hygiene, diet, and children's health was filled in by the parents. The constructed multivariable logistic regression model showed an independent influence of the microbial and behavioral factors in sECC etiopathogenesis. The occurrence of C. albicans and C. dubliniensis was associated with higher odds of sECC development (odds ratio, OR: 9.62 and 16.93, respectively), together with breastfeeding of 6 months or less (OR: 2.71), exposure to sweet beverages (OR: 3.77), and starting to brush child's teeth after the 12th month of age (OR: 4.10), all statistically significant (p < 0.01). Considering the high occurrence of C. albicans and C. dubliniensis in dental plaque in children with sECC, we propose them as "keystone pathogens" and risk factors for sECC. The models showed that presence of specific species of Candida in dental plaque may be a better descriptor of sECC than the mentioned behavioral factors.

Association of the CD14 -260C/T polymorphism with plaque-induced gingivitis depends on the presence of Porphyromonas gingivalis.

BACKGROUND: Plaque-induced gingivitis is the most prevalent periodontal disease associated with pathogenic biofilms. The host immune system responds to pathogens through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and their co-receptor cluster of differentiation 14 (CD14). AIM: This study investigated the association between the functional polymorphism in the CD14 gene and the dental plaque microbiota in children with gingivitis. DESIGN: A total of 590 unrelated children (307 with plaque-induced gingivitis and 283 controls, aged 13-15 years) were enrolled in this case-control study. Dental plaque was processed using a ParoCheck® 20 detection kit. The CD14 -260C/T (rs2569190) polymorphism was determined with the PCR-RFLP method. RESULTS: Gingivitis was detected in 64.2% of boys and 35.8% of girls (P < .001). Children with gingivitis had a significantly higher occurrence of dental caries (P < .001). No significant differences in the CD14 -260C/T allele and genotype distribution among individuals with or without gingivitis in the whole cohort were found. Children with gingivitis and P gingivalis, however, were significantly more frequent carriers of the CT and TT genotypes than children with gingivitis without P gingivalis or healthy controls (P < .05). CONCLUSIONS: The CD14 -260C/T polymorphism acts in cooperation with P gingivalis to trigger plaque-induced gingivitis in Czech children.

Matrix metalloproteinases gene variants and dental caries in Czech children.

BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in tooth formation and the mineralization of dental tissue. The aim of the study was to analyse Czech children with primary/permanent dentition polymorphisms in those genes encoding MMP2, MMP3, MMP9, MMP13, MMP16, and MMP20, which had been previously associated with dental caries in other populations. METHODS: In total, 782 Czech children were included in this case-control study. DNA samples were taken from 474 subjects with dental caries (with decayed/missing/filled teeth, DMFT ≥ 1) and 155 caries free children (DMFT = 0) aged 13-15 years, as well as 101 preschool children with early childhood caries (ECC, dmft ≥ 1) and 52 caries free children (dmft = 0), were analyzed for nine MMPs single nucleotide polymorphisms (SNPs) using real time polymerase chain reaction TaqMan assays. RESULTS: There were no significant differences in the allele and/or genotype frequencies of all the studied MMPs SNPs among children with dental caries in primary/permanent dentition and the healthy controls (P > 0.05). In addition, similar allele or genotype frequencies of the studied MMPs SNPs were found in children with severe dental caries in their permanent teeth (children with DMFT ≥ 6) and the healthy controls (DMFT = 0, P > 0.05). CONCLUSIONS: This study demonstrated the lack of association between the selected SNPs in candidate genes of MMPs and the susceptibility to or severity of dental caries in both primary and permanent dentitions in Czech children.

Emotional stimuli candidates for behavioural intervention in the prevention of early childhood caries: a pilot study.

BACKGROUND: Oral diseases, such as early childhood caries (ECC), have a complex etiology with common, behaviour-related risk factors. Appropriately targeted behavioural intervention using effective tools can help to eliminate risk behaviour leading to ECC. The aim of this study was to ascertain which visual stimuli with a supporting text evoke the strongest emotional response in infants' mothers and, therefore, are suitable candidates for inclusion in behavioural interventions within the prevention of ECC. METHODS: Thirty-nine mothers of one-year-old children who filled out an originally designed electronic questionnaire, containing 20 visual stimuli with accompanying texts related to dental caries (10/10 with positive/negative intended emotional response), were included in this cross-sectional study. The emotional impact of each stimulus in the mothers was evaluated using the Self-Assessment Manikin (SAM) technique, which represents three emotional dimensions: valence, arousal, and dominance. RESULTS: Each of the stimuli was assessed by the mothers of infants based on its emotional impact. The real emotional response (evaluated according to the median of valence) was in line with the primarily intended response in 90% of cases (p < 0.05). The text with a warning evoked a greater emotional response (evaluated according to the median of arousal) in mothers than only the informative instruction (p < 0.05). The relationship between arousal and valence (r = - 0.99; p < 0.05) indicates that the more aversive stimuli raise higher arousal. The significant correlation between valence and dominance shows that the more positive the stimuli, the higher feeling of control over the evoked emotion the mothers have (r = 0.83; p < 0.05), and, on the contrary, the lowest control over emotion is correlated with higher arousal (r = - 0.85; p < 0.05). Generally, mothers rated themselves as in high control of their emotions over the individual stimuli. CONCLUSIONS: This pilot study proved that negative pictorial and text warnings about the risks of developing caries had the potential to evoke strong emotional responses in the mothers of infants. We identified three visual stimuli that could be included in future extensive motivation material in an attempt to affect the preventive behaviour of mothers, and thus the oral health of their infants.

Lack of association between ENAM gene polymorphism and dental caries in primary and permanent teeth in Czech children.

OBJECTIVES: The enamelin gene (ENAM) polymorphism (rs12640848) was recently associated with dental caries in primary teeth in Polish children. The aims of the present study were to prove this association in primary dentition and to find a possible effect of this variant on caries development in permanent dentition in Czech children. MATERIALS AND METHODS: This study comprised 905 Czech children. Totally, 187 children aged 2-6 years with primary dentition [78 healthy subjects (with decayed/missing/filled teeth, dmft = 0) and 109 patients with early childhood caries (ECC; dmft ≥ 1)] were included in this case-control study. In addition, 177 subjects aged 13-15 years without caries (DMFT = 0) and 541 children with dental caries (DMFT ≥ 1) in permanent dentition were selected from the ELSPAC study. Genotype determination of the ENAM polymorphism (rs12640848) was based on the TaqMan method. RESULTS: No significant differences in the allele or genotype frequencies between the caries-free children and those affected by dental caries were observed in both primary and permanent dentitions. CONCLUSIONS: Lack of association between the ENAM polymorphism (rs12640848) and dental caries in Czech children was detected. CLINICAL RELEVANCE: Although ENAM is considered as a candidate gene for dental caries, the presence of the ENAM variant (rs12640848) cannot be used as a risk factor of this multifactorial disease in the Czech population.

Lack of Association between BMP2/DLX3 Gene Polymorphisms and Dental Caries in Primary and Permanent Dentitions.

The aim of this study was to analyze the association between BMP2 (rs1884302) and DLX3 (rs2278163) gene polymorphisms and dental caries in primary and permanent dentitions. A total of 914 subjects were genotyped by the TaqMan methods: 176 caries-free children (with Decayed/Missing/Filled Teeth, DMFT = 0), 542 patients with dental caries in permanent dentition (DMFT ≥1), 83 caries-free children with primary teeth (with decayed/missing/filled teeth, dmft = 0), and 113 children with early childhood caries (ECC, dmft ≥1). There were no significant differences in allele/genotype frequencies between patients with caries in permanent dentition/ECC and caries-free children or between patients with very low (DMFT = 0-2), low (DMFT = 3-5), moderate (DMFT = 6-8), or high (DMFT ≥9) caries experience. Variability in BMP2 and DLX3 was not associated with caries in the Czech population.

Vitamin D Receptor TaqI Gene Polymorphism and Dental Caries in Czech Children.

AIM: We analyzed the VDR TaqI (rs731236) gene polymorphism in children with and those without dental caries. METHODS: A total of 388 subjects, 153 caries-free (with decayed/missing/filled teeth [DMFT] = 0) and 235 children with dental caries (DMFT ≥1), were genotyped by the TaqMan method. RESULTS: Although no significant differences in VDR TaqI allele and genotype frequencies between caries-free and caries-affected children were detected, a significant association between this polymorphism and gingivitis was found (p < 0.05). CONCLUSIONS: In contrast to previous studies from China and Turkey, the VDR TaqI gene variant cannot be used as a marker for identification of Czech children with increased dental caries risk.

Detection of selected periodontal bacteria in preschool children affected by early childhood caries.

The aim of this study was to compare the detection frequency of periodontal bacteria in dental plaque in children with early childhood caries (ECC) with and without gingival inflammation. A convenience sample of 25 preschool children (mean age 3.61 years, SD 1.42) was recruited. Dental plaque was taken from periodontal areas with and without visible signs of inflammation and processed using the StomaGene® (Protean s.r.o. Czech Republic) and ParoCheck® 20 (Greiner Bio-one GmbH, Germany) detection kits. The two sample t tests between percents for differences between inflammatory and healthy sites and kappa statistics for the agreement of both systems were used. At the inflammatory sites, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were significantly more frequently detected by StomaGene® while Fusobacterium nucleatum, A. actinomycetemcomitans, Tanarella forsythia and Prevotella intermedia were significantly more frequently identified by ParoCheck® test. The agreement between the two detection systems was substantial for A. actinomycetemcomitans and F. nucleatum in the samples collected from inflamed sites and only for F. nucleatum from clinically healthy sites. Therefore, we recommend that the same system should be used when the same patient is examined repeatedly.

ACE Insertion/Deletion Polymorphism Associated with Caries in Permanent but Not Primary Dentition in Czech Children.

OBJECTIVE: Dental caries is a multifactorial, infectious disease where genetic predisposition plays an important role. Insertion/deletion (I/D) polymorphism of angiotensin-converting enzyme (ACE) has very recently been associated with caries in Polish children. The aim of this study was to analyze ACE I/D polymorphism in a group of caries-free children versus subjects affected by dental caries in the Czech population. MATERIALS AND METHODS: In this case-control study, 182 caries-free children (with decayed/missing/filled teeth, DMFT = 0), 561 subjects with dental caries (DMFT ≥1) aged 13-15 years and 220 children aged 2-6 years with early childhood caries (ECC, dmft ≥1) were included. Genotype determination of ACE I/D polymorphism in intron 16 was based on the TaqMan method. RESULTS: Although no significant differences in the allele or genotype frequencies between the caries-free children and those affected by dental caries were observed, statistically significant differences between the children with DMFT = 0 and the subgroup of 179 patients with high caries experience (DMFT ≥4; p < 0.01 and p < 0.05, respectively) were detected. The comparison of DD versus II+ID genotype frequencies between the patients with DMFT ≥1 or DMFT ≥4 and healthy children also showed significant differences (31.5% or 35.6% vs. 23.6%, p < 0.05 or p < 0.01, respectively). A gender-based analysis identified a significant difference in the DD versus II+ID genotype frequencies only in girls (p < 0.05). In contrast, no significant association of ACE I/D polymorphism with ECC in young children was found (p > 0.05). CONCLUSIONS: ACE I/D polymorphism may be associated with caries in permanent but not primary dentition, especially in girls in the Czech population.

GLUT2 and TAS1R2 Polymorphisms and Susceptibility to Dental Caries.

OBJECTIVE: Dental caries is one of the most frequent multifactorial diseases. Among the numerous factors influencing the risk of caries, genetics plays a substantial role, with heritability ranging from 40 to 60%. Gene variants affecting taste preference and glucose transport were recently associated with caries risk. The aim of this study was to analyze two common polymorphisms in the sweet taste receptor (TAS1R2) and glucose transporter (GLUT2) genes in children with dental caries and healthy controls in the Czech population. METHODS: A total of 637 unrelated Caucasian children, aged 11-13 years, were included in this case-control study. One hundred and fifty-five subjects were caries-free (with decayed/missing/filled teeth, DMFT = 0) and 482 children were caries-affected (DMFT ≥ 1). The TAS1R2 (Ile191Val, rs35874116) and GLUT2 (Thr110Ile, rs5400) genotypes were determined using the 5' nuclease TaqMan® assay for allelic discrimination. RESULTS: Compared with subjects with the common Thr allele, carriers of the Ile allele of GLUT2 had significantly more frequently dental caries (p < 0.05, OR = 1.639, 95% CI: 1.089-2.466). Similarly, children with the Val allele for the TAS1R2 Ile191Val polymorphism were more frequently affected by caries than children who carried the Ile allele (p < 0.05, OR = 1.413, 95% CI: 1.014-1.969). In contrast, no significant associations between GLUT2 and/or TAS1R2 polymorphisms and fillings were found, but allele frequencies of the TAS1R2 variant were marginally significantly different between children with DMFT = 0 and DMFT ≥1 (p = 0.053, OR = 1.339, 95% CI: 0.996-1.799). However, no significant interaction between both genes and risk of dental caries was found. CONCLUSIONS: In conclusion, GLUT2 and TASR1 polymorphisms may influence the risk of caries in the Czech population.

Evaluation of the strain identity between isolates from caries lesions and root canals in early childhood caries cases.

Early childhood caries (ECC) has become a serious medical problem worldwide in the last decade. Bacterial microflora of the dental plaque and oral cavity is considered an important factor in the formation and progression of dental caries. The aim of this study was strain typing and comparison of bacterial isolates retrieved from caries lesions and root canal contents of the same teeth. In total, 18 pairs of presumptive streptococci and lactobacilli retrieved from dental caries and root canals isolated from ECC-affected children, were selected on the basis of biotyping results and rep-PCR fingerprinting with (GTG)5 primer. Strain typing was further done using the RiboPrinter microbial characterization system (DuPont Qualicon). The automated ribotyping determined 14 pairs of the strains (77.8 %) to be identical. The results obtained confirmed that identical bacterial strains colonized both the decayed dental surface and the necrotic content of the dental pulp cavity during the cariogenesis. Our finding supports the assumption that bacteria could penetrate through the damaged dental surface to the inner parts of the teeth.

Comparative evaluation of automated ribotyping and RAPD-PCR for typing of Lactobacillus spp. occurring in dental caries.

A group of 67 Lactobacillus spp. strains containing Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus salivarius species isolated from early childhood caries and identified to the species level in a previous study (Svec et al., Folia Microbiol 54:53-58, 2009) was characterized by automated ribotyping performed by the RiboPrinter microbial characterization system and by randomly amplified polymorphic DNA fingerprinting (RAPD-PCR) with M13 primer to evaluate these techniques for characterization of lactobacilli associated with dental caries. Ribotyping revealed 55 riboprints among the analysed group. The automatic identification process performed by the RiboPrinter system identified 18 strains to the species level, however cluster analysis divided obtained ribotype patterns into individual clusters mostly corresponding to the species assignment of particular strains. RAPD-PCR fingerprints revealed by the individual Lactobacillus spp. showed higher variability than the ribotype patterns and the fingerprint profiles generated by the analysed species were distributed among one to four clusters. In conclusion, ribotyping is shown to be more convenient for the identification purposes while RAPD-PCR fingerprinting results indicate this method is a better tool for typing of Lactobacillus spp. strains occurring in dental caries.

The association of MMP-9 and IL-18 gene promoter polymorphisms with gingivitis in adolescents.

OBJECTIVE: Interleukin 18 (IL-18) is shown to be a proinflammatory cytokine that regulates the expression of matrix metalloproteinase 9 (MMP-9). The aim of this study was to test for differences between Czech adolescents with and without gingivitis in relation to MMP-9 and IL-18 polymorphisms. DESIGN: A total of 298 Caucasian children, aged 11-13 years, were examined to assess gingival health. DNA for genetic analysis was obtained from buccal epithelial cells, and the MMP-9 -1562C/T and IL-18 -607A/C variants were identified with PCR-RFLP. RESULTS: Gingivitis was present in 49.3% of the adolescents examined, the rest of the group was considered healthy. The IL-18 -607C and MMP-9 -1562T alleles were found in 58.9% and 8.3% of the healthy subjects, and in 62.2% and 15.0% of the patients with gingivitis, respectively. Although differences in allele frequencies were not significant for IL-18 variant, they were significant for MMP-9 polymorphism (p=0.01, p(corr) < 0.05). Furthermore, a highly significant association of the composite genotype (formed by the variants of the both genes) with gingivitis was found (p=0.004, p(corr) < 0.05). CONCLUSIONS: The -1562 T allele of MMP-9 gene could have a role in gingivitis in adolescents. In addition, interaction of the MMP-9 and IL-18 genes could be considered a risk factor for the development of gingivitis in children.

Evaluation of (GTG)5-PCR for rapid identification of Streptococcus mutans.

Repetitive sequence-based polymerase chain reaction (PCR) fingerprinting using the (GTG)(5) primer was applied for fast screening of bacterial strains isolated from dental plaque of early childhood caries (ECC)-affected children. A group of 29 Gram-positive bacteria was separated into a homogeneous cluster together with Streptococcus mutans reference strains and constituted an aberrant branch after the numerical analysis of (GTG)(5)-PCR fingerprints. Automated ribotyping with EcoRI restriction enzyme (RiboPrinter microbial characterization system) revealed high genetic heterogeneity among the tested group and proved to be a good tool for strain-typing purposes. Further characterization of the studied strains was achieved by extensive phenotyping and whole-cell protein fingerprinting and confirmed all the strains as S. mutans representatives. Obtained results showed rep-PCR fingerprinting with the (GTG)(5) primer to be a fast and reliable method for identification of S. mutans.

Association of interleukin-6 (IL-6) haplotypes with plaque-induced gingivitis in children.

OBJECTIVES: The proinflammatory cytokine interleukin-6 (IL-6) is a key regulator of the host response to microbial infection and major modulator of extracellular matrix catabolism and bone resorption. The aim of this case-control study was to test differences between children with and without gingivitis in the distribution of IL-6 alleles at positions -174, -572, and -597 and their haplotypes. MATERIAL AND METHODS: A total of 455 Caucasian children, aged 11 to 13 years, were enrolled in this study. According to gingival bleeding on probing indices, 183 were classified as healthy subjects and 272 as children with plaque-induced gingivitis. DNA for genetic analysis was obtained from buccal epithelial cells and PCR-RFLP methods were used for genotyping three selected IL-6 promoter polymorphisms. RESULTS: Complex analysis revealed significant differences in haplotype frequencies between patients and healthy subjects (p<0.01). The CGA haplotype was significantly more frequent in children with gingivitis than in healthy subjects (41.5% versus 34.1%). In subanalyses, we found that IL-6 -174C allele was more frequent in patients (44.3%) than in healthy children (36.1%, p=0.016, P(corr)<0.05). Multivariate logistic regression analysis showed that allele C remained a risk factor for gingivitis in children (p=0.03) regardless of plaque or gender. However, the proportions of the IL-6 -597 and -572 genotypes were comparable between the two groups. CONCLUSIONS. Our results indicate that the three promoter polymorphisms in the IL-6 gene act in a cooperative fashion and suggest that IL-6 haplotypes could play a role in the pathogenesis of gingivitis in Caucasian children.