Study period and locality
A cross-sectional study was conducted from February 7 to March 14, 2018, in primary schools, Harar town, Eastern Ethiopia. Harar is a capital town of Harari Regional State and it is located 515 km away from the capital city, Addis Ababa.
Population
A total of 5 government primary schools was selected and all children who were present in the school during data collection were included in the study.
Sample size determination
The sample size for the prevalence of dental caries was calculated by using a single population proportion formula. The sample size for associated factors was determined using Epi info (version 7) at a confidence interval (1-α) =95% and power (1-β) of 80%. Finally, a 10% non-response rate was added to the calculated sample size. Therefore, the largest sample size, i.e. 422 was used for the study.
Sampling
A total of 422 children was included from 5 schools, out of the total 20 government primary schools in Harar town. The selection of school children for the study was detrmined as follows. First, the five primary schools were selected based on the lottery method. Secondly, ten sections from both the first cycle and second cycle level were selected by simple random sampling. Thirdly, students were distributed proportionally amongst each section. Finally, participants were selected based on the list from the class teacher’s who is in - charge for the class using systematic random sampling. In the case of absenteeism and overlap, the next number was included in the study.
Data collection
A structured questionnaire was used to collect information about the socio-demographic characteristics and determinant factors relevant for dental caries amongst primary school children. Data were collected by face-to-face interview with the help of the children’s parents or guardians. Dietary data were collected using the Ethiopian demographic and health survey questionnaires as a baseline to estimate the habitual intake of foods and nutrients [16]. Interviewers collected information about the foods and drinks consumed during the preceding day by probing questions and cues that help the recollection of participant’s memory. Two new versions were developed for this survey: version C2 for children aged 3–11 years designed for completion by a parent/guardian with help from the child, and version C3 for young people aged 12–17 years for completion by the young person with help from their parent/guardian. Both questionnaires list 15 foods or drinks with a measure defined for each item. Participants were asked to estimate the frequency and amount of each food or drink consumed per day.
Clinical examination
A trained dental therapist (nurse) and a dentist (doctor) with assistant data recorder performed the clinical examination in the classrooms. A dental visual examination was performed on each child by one examiner using a light-emitting diode (LED), dental mirror, and explorer. The diagnostic criteria for caries were followed according to the WHO recommendations [17]. The DMFT (decayed, missing, and filled tooth number) scores were recorded and dental decay is measured using the count of the number of teeth or surfaces in a child’s that are decayed, missing or filled as a result of caries. A tooth was recorded as decayed when a lesion had an unmistakable cavity, undetermined enamel or detectably softened wall or floor. A tooth was recorded as missing when it was extracted due to caries. A tooth was recorded as filled when it was permanently filled without caries. The examiner was calibrated with an experienced dentist (doctor) in the same setting before the study. The intra-examiner agreement was assessed by re-examining a 10% random sample of the children on the same day. The dental assistant without the knowledge of the examiner performed the selection of children for duplicate examination.
Specimen collection, culture, and identification
Unstimulated whole saliva samples (2 mL) were collected from children [18] by a medical laboratory technologist. Children were requested to void saliva into a wide-mouthed disposable cup 2 h after their last meal in a mid-morning. The cup was then uniquely coded, kept in ice box, and transported for microbiological assay within 24 h.
Saliva (0.1 mL) was diluted with normal saline (0.9 mL) and tenfold serial dilutions of saliva were made up to 10− 6. The diluted sample (100 μL) was spread [19] evenly onto de Man, Rogosa and Sharpe agar (MRS agar) (HI Media Laboratories Pvt. Ltd., India) to obtain direct counts of Lactobacilli. Plates were incubated anaerobically using the anaerobic candle jar system at 37 °C. After 48 h of incubation, plates were observed for the presence of Lactobacillus like colonies. Additionally, Gram staining or catalase test [19,20,21] was performed.
Colony counting was performed using a colony counter and the number of colony-forming units (CFU) was multiplied by the number of times the original milliliter of sample diluted (the dilution factor of the plate counted) and expressed as CFU/mL [19] of saliva. Besides, the determination of the salivary pH for each sample was performed, using a special pH-test paper (Simplex Health pH test strips for urine and saliva: manufacturer part number sh003, United Kingdom). The threshold defined by the manufacturer made an interpretation.
Data quality control
A trained dental therapist performed clinical examination based on the WHO oral health assessment. Similarly, a trained medical laboratory technologist performed the laboratory diagnosis. A pretested and structured questionnaire used to collect data. The questionnaire was developed in the English language and translated to appropriate Ethiopian local languages (Amharic and Afan Oromo). Double entry was performed before data analysis for validation. Study participants were required to refrain from eating and drinking for a minimum of 2 h after arrival at school prior to sample collection.
Culture examinations were undertaken after checking growth-supporting characteristics, physical characteristics, gel strength and batch contamination of the media used. Prepared media quality was monitored by incubating 3–5% of the batch at 35–37 °C for overnight. The reference strain of Lactobacillus casei (ATCC 393) and Lactobacillus fermentum (ATCC 9338) obtained from Ethiopia Public Health Institution (EPHI), Addis Ababa were used for quality control of culture.
Data analysis
Data were checked for completeness, cleared and then entered and validated by Epi data, software version 3.1 and exported to the Statistical Package for Social Science (SPSS) software version 25.0 for analysis. Odds ratios (OR) and their 95% confidence intervals (CI) were estimated using bivariate and multivariate logistic regression analysis to identify possible explanatory variables on the occurrence of dental caries. The data were expressed as mean ± SD. The p-value < 0.05 was considered as statistically significant.