Study Area and Period
The study was conducted in Jigjiga, a city in the Somali Region of Ethiopia from 12 April to 27 May 2021. Located in the Fafan Zone with 60 km west of the border with Somalia. Jigjiga is 630 km east far from the capital city, Addis Ababa There are currently ninety-nine (99) primary schools, seventy-four (74) private schools, and twenty-five (25) government schools, with a total of 36,507 students, 20,056 males and 16,451 females (16).
Study Design and population
A cross-sectional study was conducted. School children from governmental and private primary schools who were randomly selected from twenty schools using a lottery system.
Inclusion and Exclusion Criteria
All students in selected schools aged 7–14 years old who attend the class during the study period were included in the study. All children on antibiotics for the previous two weeks, as well as those with any signs or symptoms of respiratory diseases such as fever, sore throat, cough, or watery nasal discharge, were excluded from the study.
Sample Size Determination and sampling technique
A single proportion proportion formula was used to determine the sample size for this quantitative study, considering the following assumptions: A 95% confidence level, margin of error (0.05) carriage rate among school children, prevalence study of asymptomatic pharyngeal carriage prevalence of S. pyogenes, antimicrobial pattern and related risk factors among school children in Hawassa, southern Ethiopia 12.2 % (15). To compute for non-response rate, 10% of the total sample = 16 was added. Thus, a total of 178 study subjects were included.
sample size was determined by considering different factor-associated GAS or S. pyogenes colonization using double population proportion formula with the assumption of a two-sided confidence level of 95%, the margin of error 5%, and power of 80%. Finally, 231 study participants were calculated. So, the sample size for a single population proportion was smaller than the sample size calculated for the second calculated double population proportion, which is a sample size of 231 was used. Because we used a multi-stage sampling method, we introduced a design effect. As a result, 231 was multiplied by 2, resulting in a sample size of 462.
Sampling Procedure
In order to include study participants in the study, a multistage sampling technique was used. There was a total of 99 primary schools (74 private and 25 government). Through a lottery system, twenty (20) schools were chosen at random from both government and private schools. Then the calculated sample size for the study was proportionally allocated to each selected school. A simple random sampling method was used to enroll children. Samples were taken from children whose parents agree to participate until the sample size attend from each selected school.
Sample Collection and Transportation Methods
Data on the socio-demographic characteristics of the parents/guardians and children, as well as the children's clinical history, were collected using a structured questionnaire adopted from previous studies (15, 17). Two professional nurses administered the questionnaires, and two trained laboratory personnel used cotton swabs to collect a throat sample from a selected child. The throat swab samples were placed in Amie's transport media. Within two hours, the sample was sent in a cold chain to Jigjiga University Sultan Sheik Hassan referral hospital laboratory for investigation.
Laboratory investigation
The throat sample was cultured on 5% sheep blood agar plates (Himedia, India) by rolling the swab over a small area of the plate and streaking the sample with a sterile loop, then incubated at 37°C with 5% CO2 atmosphere for 24 hours. A catalase test and gram staining were performed on colonies having ß-hemolysis. All catalase-negative and gram-positive cocci were subcultured for 24 hours at 37°C on 5% fresh blood agar plates with a Bacitracin disk in a 5% CO2 atmosphere to differentiate colonies suspected to be S. pyogenes. Any zone of inhibition around the bacitracin disk was a candidate for Pyrrolidonyl arylamidase (PYR) tests, change of color to red /purple was confirmed positive for S. pyogenes. (18, 19).
Antimicrobial Susceptibility Testing
The drug susceptibility test was done by a disk diffusion method by using Muller Hinton Agar (MHA) supplemented with 5% sheep’s blood. Colony suspension was made using normal saline (0.85% NaCl) equivalent to 0.5% McFarland standard from grown overnight colonies (18-24 hours) on sheep blood agar plate.
The suspension was inoculated to an MHA plate with 5% sheep’s blood using a culture swab and incubated at 5% CO2 for 18 to 24 hours. Drug disks containing penicillin (10 IU), erythromycin (15 g), azythromycin (15 g), amoxicillin (10 g), chloramphenicol (30 g), ceftriaxone (30 g), vancomycin (30 g), and tetracycline (10 g) were utilized. The drugs are selected in accordance with the Ethiopian Drug Administration standard treatment guidelines for health centers and Control Authority's and the Clinical Laboratory Standards Institute's (CLSI). The zone of inhibition was measured with a ruler, then recorded and compared to the Clinical and Laboratory Standards Institute (20).
Data Quality Control
The questionnaire was written in English, then translated into Amharic and Somali, and finally back to English to ensure uniformity. The questionnaire was pre-tested before actual data collection begins. For laboratory testing, the sterility of each batch of produced media was determined by incubating 5% of the culture media in a 5% CO2 enriched atmosphere at 37 °C for 24 hours before using it. Streptococcus pyogenes (ATCC 12696) and Streptococcus agalactae (ATCC 13813) were used as a positive and negative control respectively. Quality assurance in antimicrobial susceptibility was done by repeating the selected tests on the same day as the original.
Method of Data Analysis
The data was entered and coded into the Epi-Data version 3.1 upon creating the questionnaire template. The entered data was cleaned to ensure the validity of all recorded data. The analysis was then carried out using SPSS version 26.0. Descriptive statistics and frequency tables were used to summarize the data. The magnitude of the association between the different variables with the outcome variable was measured by the adjusted odds ratio with a 95% confidence interval. Bivariate and multivariable logistic regression analysis was made to obtain the odds ratio and the confidence interval of statistical associations. All variables that are significant at p-value < 0.25 in the bivariate analysis were considered for multivarible analysis. The strength of statistical association was measured by adjusted odds ratios and 95% confidence intervals. Statistical significance was declared at p<0.05.