Study Setting, Design, and Period
A community-based cross-sectional study was conducted among primary school children in Gondar town, North West Ethiopia, from January to April 2019. This study was conducted in six primary schools. Gondar is found 737 km from Addis Ababa, the capital city of Ethiopia, and 180 km from Bahir Dar, the capital of Amhara national regional state. Gondar town and its surroundings have 44 elementary schools, 11 secondary schools and 30 kindergartens.
Sample size and Sampling technique
The sample size (524) was determined by using a single population proportion formula by considering the prevalence of 20.4% [8], with a 95% confidence interval, and a 5% margin of error, with 10% no-response rate and design effect. The multistage sampling technique was used to select schools. Then schools were stratified to grades and sections. The total number of study participants were allocated proportionally to each school, grades and sections based on the school sampling frame and the study subjects were selected by simple random sampling technique (Table 1).
Table 1: List of selected elementary schools and number of selected students in Gondar town, Northwest Ethiopia, January to April, 2019
S.no
|
Elementary school
|
Students per school
|
Proportion
|
Proportionally allocated number of students per school
|
number of students taken (sample)
|
1
|
Abiwotfire
|
2010
|
23.4
|
128
|
120
|
2
|
Hubret
|
1090
|
12.7
|
70
|
66
|
3
|
AtseBekafa
|
1146
|
13.2
|
73
|
64
|
4
|
TsadikuYohanis
|
1450
|
16.8
|
92
|
92
|
5
|
Meseret
|
1512
|
17.6
|
97
|
94
|
6
|
Chechela
|
1400
|
16.3
|
90
|
88
|
Total
|
8608
|
100
|
550
|
524
|
Data collection and laboratory methods
Data collection procedures
A pre-tested questionnaire based on postulated or known risk factors was developed and modified to explore the objectives of the study. Then, it was checked on school children who were not included in the study. It was prepared in English and translated to Amharic then translated back into English to check the accuracy of the translation. The questionnaire design included two parts; socio-demographic characteristics and associated risk factors.
The questionnaire and assent/consent form were distributed to the selected students at school after informing the purpose of the study and the right of the study participants. Questionnaire and assent/consent form were also distributed to guardians and emphasis was given to return the questionnaire and assent/consent form after twenty-four hours. Students living alone (with the age range of 17-18 years) were considered as adults and informed to fill the questionnaire and sign the consent form at school. Socio-demographic characteristics and other relevant information filled by the parents/guardians and students were collected at school by trained laboratory technologists before sample collection.
Laboratory methods
Oropharyngeal sample collection
Oropharyngeal swabs were collected by a trained medical microbiologist using a plain cotton swab (Unison Narula, India) using tongue depressor (Unison Narula group, India) at the posterior pharyngeal wall behind the uvula and tonsils of each volunteer participant. After collection, samples were transported by using Amies transport media (Bio mark, India) to the University of Gondar teaching hospital laboratory within two hours of collection within a cold box.
Culture and identification
Once the specimens reached to Gondar University teaching laboratory, it was inoculated on Modified Thayer Martin (MTM) culture media (Oxoid, UK). The inoculated MTM plates were incubated at 37°C with 5-10% CO2 for 24 to 48 hours. A presumptive diagnosis was done by gram stain and colony characteristics on the agar plate. Medium to large, round, smooth, convex, colorless-to-grey, opaque colonies on the MTM was further confirmed by the oxidase test (Deben Diagnostics Ltd, UK). After confirmation, the presence of gram-negative diplococcus with oxidase-positive, isolates were sub-cultured on a blood agar plate (BAP) (Oxoid, UK) with 5-10% CO2 for 24 to 48 hours, to guarantee the purity of colonies for the biochemical test. Plates were monitored every 24 hours for the growth of typical colonies.
Carbohydrate utilization test (glucose, maltose, lactose, and sucrose) was performed by cystine trypticase agar (CTA) (SRL, India) to further differentiate Neisseria meningitidis from Moraxella species and other nonpathogenic Neisseria species. Isolates with gram-negative diplococci, oxidase-positive, glucose fermenter, maltose fermenter, lactose and sucrose none- fermenter were interpreted and confirmed as N. meningitidis. Once the species are known, the serogroup of isolates was determined with the slide agglutination method while using commercially prepared antiserum A, B, C, W135/Y, and X (Bio-Rad, France) and antiserum X (BD Difco, USA). Negative for these six serogroups was classified as non-serogroupable[8]
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was carried out on isolates of N. meningitidis by using disc diffusion technique as per the standard Kirby-Bauer method on Mueller-Hinton agar (Bio mark, India) supplemented with 5% sheep blood at 37ºc for 18-24 hours [11]. A suspension of the test organism was prepared equivalent to 0.5 McFarland. The surface of Mueller-Hinton agar supplemented with 5% sheep blood was completely covered by rotating the swab. The plates were allowed to dry for 3-5 minutes; then discs were evenly distributed on the inoculated plate using sterile forceps and incubated in 5-10% CO2 at 37ºc for 20-24 hours. The following routinely used antimicrobial agents were tested: cefotaxime (30μg), minocycline (30μg) meropenem (10µg), azithromycin (15µg), ciprofloxacin (5μg), trimethoprim-sulfamethoxazole (1.25/ 23.75 µg), chloramphenicol (30μg), and rifampin (5μg). Diameters of the zone of inhibition around the disc was measured to the nearest millimeter using a graduated caliper in millimeters and results were classified as sensitive, intermediate and resistant based on CLSI-2018 guideline[12]. Multidrug resistance was defined as resistance of an isolate to two or more antimicrobial classes tested [13].
Laboratory data quality assurance
Preanalytical, analytical and post analytical quality assurance was maintained [14].
Data analysis and interpretation
All data was entered to EPI info version 7 for data clearance and consistency and exported to SPSS version 20.0 for analysis. Descriptive statistics was computed to calculate frequencies. The magnitude of the association between different variables and oropharyngeal meningococcal carriage was assessed using bivariate and multivariate analysis. Variables which had a P- value ≤0.20 for bivariate analysis was taken to multivariate analysis to check real association of meningococcal carriage rate with risk factors and expressed by adjusted odds ratio at 95% confidence interval. A P-value < 0.05 was considered as statistically significant. Data was summarized using numbers, percentages and tables.
Ethical considerations
The study was conducted after obtaining institutional ethical clearance (“Ref No-SBMLS/2123/11”) from University of Gondar. Support letter was sought from Gondar town educational office. Assent from the parents/guardians of youth students and assent/consent from the study participants was obtained.