Household smoking impact on the oral health of 5- to 7-years-old children

The ethical approval of this study was obtained from the local institutional scientific research ethical committee, Umm AlQura University, KSA (HAPO-02-K-012-1412), and conducted according to the Declaration of Helsinki. A written informed consent was received from each participant’s guardian following a comprehensive explanation of the research methodology and any anticipated inconveniences.

The study sample of this cross sectional study consisted of healthy children aged 5- to 7-years-old attending the outpatient clinics at the Faculty of Dentistry, Umm AlQura University, Saudi Arabia; during the period from May to September, 2022.

For sample size determination, the risk of dental caries progression was 14% with anticipated increase of 30% for children not exposed to household smoking and children exposed to household smoking respectively [15]. To acquire 80% study power with a two-sided α equal to 0.05, a minimum of 104 participants per group were needed.

Participating children were matched between case and control groups regarding age, gender, parental education and social class to guard against the confounder effect.

The administered questionnaire tool was created based on formerly validated questionnaire [16, 17]. The questionnaire consisted of 26 closed questions regarding their sociodemographic characteristics and parental smoking habits. The selection criteria for children to be enrolled in the present study were as follows; If a child had been subjected to regular household smoking since birth regardless the amount or the frequency of smoking; he/she was grouped into the household smoking HS group (n = 105). The control group consisted of children who had not been exposed to household smoking by either parents since birth (n = 105). Children who suffered from any systemic illness or reported chronic use of medication were not included in the study. The enrolled children were only whom parent/legal guardian accepted voluntary participation in this study.

All children were examined clinically by the same examiner, a consultant of Pediatric Dentistry; using a sterile disposable diagnostic kit for each participant while sitting on a regular chair. Each diagnostic kit consisted of plane mouth mirror, sickle explorer No.23, spoon excavator, and tweezer.

The examiner received a training process followed by calibration exercise using photographic slides of clinical cases of each criterion of ICDAS and HSPM assessment to reach acceptable level of agreement and consistency of findings. The data of 18 patients were recorded at the baseline then re-calibrated after one-week interval [18]. Intra-examiner reliability was evaluated using SPSS program where almost perfect agreement level was achieved (Kappa = 85.9%).

Dental caries assessment was carried out according to the International Caries Detection and Assessment System (ICDAS) criteria; code 1 was assigned when the enamel surface showed early visual changes confined to pits and fissures following air drying, code 2 was assigned for: apparent visual changes in enamel, code 3 was assigned for: demarked breakdown in opaque enamel surface without evidence of dentinal effect, code 4 was assigned when: dark shadow became evident in dentin, code 5 was assigned for: distinct cavitated lesion in dentin, and code 6 was assigned for: extensive cavity involving more than half of the dentin surface [19]. According to the highest ICDAS score, the participants were divided as follows: low caries activity involving only enamel (codes 1–3), moderate caries activity involving enamel and dentine (code 4), and high caries activity involving cavitated (codes 5 and 6) caries lesions [20].

Assessment of the hypomineralized second primary molar (HSPM) was carried out using the MIH/HSPM index following the European Academy of Paediatric Dentistry (EAPD) molar incisor hypomineralization (MIH) diagnostic criteria modified for HSPM assessment. The tooth was considered hypomineralized if any of the following characteristics were observed; demarked opacities of enamel, post eruptive enamel breakdown (PEB), atypical carious lesions or restorations or extractions that do not match the dental caries pattern of the child [21, 22].

The gingival Index (GI) was used to assess gingival health status [23]. The gingival inflammation severity was scored on a scale from 0 to 3 following gingival inspection per surface according to the following criteria; score 0; no signs of inflammation, score 1; mild inflammation, slight redness, slight oedema, probing with a blunt probe did not elicit bleeding, score 2; moderate inflammation, oedema, redness, glazing, with swollen marginal gingiva and bleeding accompanying probing using a blunt explorer, and score 3; severe inflammation, marked redness and oedema, spontaneous bleeding and/or ulceration. The tooth score was calculated by dividing the sum of all surface scores, by four. Only fully erupted teeth were examined. The individual GI score was calculated for every participant via dividing the sum of all assessed teeth divided by the total number of examined teeth. According to the GI score, the participants were categorized to; score 0 for not inflamed gingiva, score 0.1–1 for mild inflammation, score 1.1–2 for moderate gingival inflammation and score 2.1–3 for severe gingival inflammation [24].

The collection of saliva samples was carried out using the masticatory stimulated saliva method between 9:00 AM and 12:00 PM, 2 hours after breakfast. Each participant was kindly guided to set motionless in an upright position and swallow (time of start), then was asked to chew on a piece of paraffin wax for 30 sec. and lean his/her head foreword to collect the stimulated saliva volume over 5 min. in a sterile graduated container. The saliva flow rate was calculated by dividing the volume of the collected saliva over time (ml/min) [25]. Immediately following the collection of saliva samples, saliva pH was measured via a precalibrated digital pH meter (AD1000, ADWA Instruments Kft., Szeged, Hungary). Collected saliva samples were stored at − 80 °C (Thermo Fisher Scientific LLC Model No. UXF40086D62, Asheville, NC USA) until utilized for analysis of secretory IgA levels. At the time of testing, the samples were brought to room temperature and then placed in the centrifugation machine (Multifuge X1R Centrifuge, USA) at 3000 rpm for 15 min. The supernatant from each sample was used to measure the level of sIgA (μg/ml). A qualified lab technician under the supervision of specialist of microbiology ran the ELISA tests for sIgA using DRG® IgA Salivary ELISA (SLV-4636, DRG International, Inc. USA) following the manufacturer’s instructions. The sIgA kit was provided with IgA standards S0 – S4 (5 vials, 1 mL each) and control reagents (1 vial, 1 mL) ready for use. Concentration of Control is Lot-specific. The standard concentrations are 1000 times lower than the values reported in the reference range because the samples are diluted 1:1000 while the standards are not. The Standard concentrations to be used for calculation were: S0 S1 S2 S3 S4 at 0, 6.9, 62, 132, and 400 μg/mL. The controls should be treated as unknowns and values determined as the test procedure conducted.

Each participant was provided with a 120 ml clean plastic container to collect a urine sample at the end of the clinical examination visit. Collected urine samples were coded and stored at − 20 °C refrigerator (Thermo Fisher Scientific LLC Model No. UXF40086D62, Asheville, NC USA) until analysis for cotinine (ng/ml) using DRG® Cotinine (Urine) ELISA (EIA-1377, DRG International, Inc. USA) following the manufacturer’s instructions. The calibrators were included when the assay was conducted. The negative calibrator consisted of I mL of urine matrix negative for cotinine. The positive calibrators consisted of 1 mL of urine matrix containing 50, 500 (Cut-off Calibrator), and 5000 ng/mL cotinine. The negative control should have greater absorbance than the cut-off while the positive control should have lower absorbance than the cut-off calibrator. Reading the stopped assay was carried within 15 minutes at 450 nm.

The concentrations of sIgA in saliva and cotinine in urine were read using SPECTROstar®^Nano (Nano microplate reader, BMG LABTECH, Germany). All data were displayed using Multi-user Reader Control and MARS data analysis software (BMG LABTECH, Germany).

The statistical analysis was carried out using SPSS software version 25 (SPSS Inc., IBM Crop, Armonk, NY, USA) to test for significance at p ≤ 0.05. The collected data were explored for normality using Kolmogorov Smirnov statistical test (sample size ≥50), and the data were found approximate normal (kurtosis excess between − 1 and + 1). The z-test was applied for normality where at absolute z-value ±3.29 (medium-sized sample 50 ≤ n = 105/gp ≤300), normal distribution of the sample was detected [26, 27]. Student’s t-test was used to find significant differences between continuous values, such as participants’ age, gingival index score, saliva pH, flow rate, sIgA level, and cotinine level. The chi-square test was used to test for the significance of categorical and binomial data, such as parental employment, number of rooms, gender, sweets consumption, brushing frequency, and HMPM. The correspondence analysis was used to test for significance if one of variables has more than two values as with the variables of parents’ levels of education, type of house ventilation, ICDAS score, smoking form, frequency of smoking per day, and smoking pattern. The correlation between cotinine levels in urine and sIgA was tested for association using Bivariate Pearson correlation coefficient test.

The assessment of sociodemographic characteristics revealed that no statistically significant difference can be identified between the HS group and the controls regarding the level of parental education, employment, number of children in family, number of existing rooms and presence or absence of adequate ventilation (Table 1).

The comparison between the HS and the control group revealed the absence of gender predilection or any statistically significant difference in sweets consumption habits or brushing frequency. The HS group presented with statistically significant higher score of ICDAS, HSPM and GI (Table 2). Smoking frequency higher than 20 cigarettes/day showed a significant association with moderate to high caries activity while smoking anywhere in the house was found to be significantly associated with increased caries severity (Table 3). Smoking cigarette only and smoking anywhere in the house showed limited impact on occurrence of HSPM and found associated with absence of HSPM (Table 4). Smoking frequency more than 20 cigarettes/day showed statistical significant association with moderate severity of gingival inflammation in HS group (Table 5).

Children subjected to household smoking showed statistically significant lower mean values of saliva flow rate, pH, and sIgA with higher mean value of cotinine in urine (Table 2). An inverse relationship has been detected between the mean value of cotinine and sIgA (r = − 0.888, p < 0.000). The increase in cotinine level in urine denoting exposure to tobacco smoke found to be associated with statistically significant with reduced sIgA in saliva (Fig. 2).