Carriage rate of Neisseria meningitidis, antibiotic susceptibility pattern and associated risk factors among primary school children in Gondar town, Northwest Ethiopia



Globally, in 2012, about 1.2 million estimated cases were reported with ~ 135,000 deaths annually. In Ethiopia, specifically in our study area, limited information is found on the oropharyngeal carriage, antimicrobial resistance pattern, and associated risk factors for N. meningitidis among school children. So, the aim of this study was to assess oropharyngeal carriage rate of N. meningitidis, antibiotic susceptibility pattern and associated risk factors among primary school children in Gondar town, Northwest Ethiopia.


A cross sectional study was conducted from January to April, 2019 in Gondar town. Multi stage simple random sampling technique was used. A total of 524 oropharyngeal swabs were collected using sterile plastic cotton swabs. Modified Thayer Martin media was used for primary isolation. Antimicrobial susceptibility pattern was done based on Kirby-Bauer method on Muller-Hinton agar supplemented with 5% sheep blood. Multidrug resistance was defined as resistance of an isolate to two or more antimicrobial classes tested. Logistic regression model was used to see the association between dependent variables (Carriage rate of Neisseria meningitidis, Serogroups of Neisseria meningitidis and Antimicrobial susceptibility patterns) and independent variables (Socio-demographic data and risk factors). Variables with a P- value ≤0.2 during bivariable analysis was taken to multivariable analysis to check significant association of meningococcal carriage with risk factors. Finally, a P-value < 0.05 was considered as statistically significant. Data was summarized using numbers, percentages and tables.


A total of 53(10.1%) (CI: 7.6–12.8) N. meningitidis isolates were identified. Serogroup A 13 (24.5%) was the most prevalent followed by Y/W135 11(20.7%) whereas serogroup B 4(7.6%) was the least identified serotype. Meningococcal isolates were resistant to ciprofloxacin (45.3%) and trimethoprim-sulfamethoxazole (73.6%). Overall, most of meningococcal isolates showed about 32(60.4%) multidrug resistance. Meningococcal carriage rate was significantly associated with family size, tonsillectomy, passive smoking, number of students per class, sharing utensils, history of visiting healthcare institutions, and indoor kitchen.


This study highlights the need for reinforcement of case-based, laboratory confirmed surveillance of N. meningitidis carriage in Ethiopian elementary school students to enable mapping of distribution of serotypes of the causative organisms across the country and determine the current potential necessity of vaccination.



Background
Neisseria meningitidis is a Gram-negative diplococci with 13 distinct serotypes. It inhabits the mucosal surface of nasopharynx and oropharynx [1]. However, about 90% of human disease are caused by serogroup A, B, C, X, Y, and W135 [2]. About 5-10% of healthy people carry N. meningitidis in the nasopharynx and oropharynx and during epidemics, the carrier state rises to 70-80% [3]. Different factors can enhance carriage rate: Immunological susceptibility, travel, large population displacement, poor living condition, overcrowding, housing condition and climatic condition [4].
Meningococcal infection has been a big threat for the globe and exists as sporadic, hyper-sporadic, and epidemic disease. In 2012, an estimated 1.2 million cases of meningococcal infection per year was reported, with1 35,000 deaths worldwide [5]. The African meningitis belt is more affected area of bacterial meningitis characterized by distinct seasonal patterns. The disease incidence peaks in the dry season [6]. In Ethiopia, a major epidemic was recorded in 2001with 6964 cases and 330 deaths. Another epidemic was also recorded in 2003-2004 with 3326 cases and 160 deaths [7]. In 2017, a study was done in Addis Ababa, Ethiopia among school children and 20.4% of N. meningitidis carriage was documented [8].
Meningococcal infection can be prevented through taking different measures and vaccination can play a pivotal role. As the result, in 2010, scientists developed the new conjugate vaccine, MenAfrivac vaccine, and implemented in sub-Saharan Africa. This vaccine is developed from, N. meningitidis serogroup A capsular polysaccharide and vaccination was implemented among 1-29 years old individuals [9]. Mass vaccination campaign with the same vaccine was also practiced in Ethiopia, and vaccination has been implemented in 3 phases from 2013 to 2015 [10]. In the study area, limited information is found on the oropharyngeal carriage, antimicrobial resistance pattern and associated risk factors for N. meningitidis especially in school children. Thus, carriage studies are important to improve our understanding of the N. meningitidis serogroup distribution and also the epidemiology of meningococcal disease control. Thus, it is important to determine the percentage of carriage rates. If the rate of carriers were identified, then tools to reduce personal contacts could be provided in populations with a high carrier rate. This process may include avoidance of crowding, reconstruction of the air-condition systems of the dorms, personal health education, or the administration of vaccines. So, the aim of this study was to assess oropharyngeal carriage rate of N. meningitidis, antibiotic susceptibility pattern and associated risk factors among primary school children in Gondar town, Northwest Ethiopia.

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).

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 24 h. 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.

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 2 h 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% CO 2 for 24 to 48 h. 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 gramnegative diplococcus with oxidase-positive, isolates were sub-cultured on a blood agar plate (BAP) (Oxoid, UK) with 5-10% CO 2 for 24 to 48 h, to guarantee the purity of colonies for the biochemical test. Plates were monitored every 24 h 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 nonserogroupable [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 37°c for 18-24 h [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- Total 8608 100 550 524 5 min; then discs were evenly distributed on the inoculated plate using sterile forceps and incubated in 5-10% CO 2 at 37°c for 20-24 h. The following routinely used antimicrobial agents were tested: cefotaxime (30 μg), minocycline (30 μg) meropenem (10 μg), azithromycin (15 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (1.25/ 23.75 μg), chloramphenicol (30 μg), and rifampin (5 μg). 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 Pvalue ≤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.

Demographic characteristics of study participants
A total of 524 school children (283 males and 241 females) were included in this study. The mean ± SD age of the participants was 12.2 ± 2.74 years. About 49% of the study participants were within the age group of 11-14 years ( Table 2).

Multidrug resistance pattern of N. meningitidis
Multidrug resistance pattern of N. meningitis isolates was also determined. Overall, most of the meningococcal isolates showed a high level of multidrug resistance with the rate of 32(60.4%). On the serogroup level, serogroup  (Table 6).

Associated risk factors of study participants
In this study, the average family size of students was 5.3 people per household and the average number of rooms per household was 2.3. From all study participants, 21% had a history of hospitalization at least for 1 day at health institutions. Of the total participants, the majority of (60.5%) had a history of tonsillectomy. Among the study participants who had a history of treatment before 2 weeks of the study period, 19.3% had poor treatment adherence. About 10.1% of the family of the study participants smokes cigarettes while 29.2% of the family had a history of living in crowded area (Table 7).

Risk factors analysis for oropharyngeal carriage of N. meningitidis
In bivariable logistic regression analysis associated factors with P-value < 0.2 were transferred to multivariable logistic regression to the significant association of these factors.

Discussion
Invasive meningococcal infection is a global problem occurring as sporadic, hyper-sporadic, and epidemic disease [15]. The problem has mainly occurred in the developing world especially in the African meningitis belt [16,17]. In Ethiopia, meningitis outbreaks have been occurred over several years, being responsible for morbidity and mortality [7]. Many researched evidences showed that people who are carriers of N. meningitidis are at high risk of developing invasive meningococcal disease in their life time specifically, if they are exposed to factors that wanes the immune system. However, in Ethiopia, there is only limited information regarding the N. meningitidis carriage rate, antimicrobial susceptibility pattern and associated factors [4,18]. Therefore, this study was intended to show the gap and fill the limited information on the oropharyngeal carriage, antimicrobial   resistance pattern and associated risk factors for N. meningitidis especially in school children in the study area. The overall N. meningitidis oropharyngeal carriage rate in this study was 10.1%. The predominant serogroup in our study was serogroup A (24.5%) and W135/Y (20.6%) while the least was serogroup B (7.6%). Despite the fact that menA vaccine mass vaccination campaign was implemented in 2012 in the study area, now it is 6 years after implementation and the prevalence of N. meningitidis serotype A will be reverted to high. The high prevalence of serotype W135/Y may be due to suppression of serotype A by the vaccine which in turn let serotype W135/Y to compete with other least prevalent serotypes. This overall carriage prevalence was markedly higher than a study conducted at Gondar University teaching hospital in 2012 (234 oropharyngeal swabs) among < 10 years OPD patients with 6% carriage [19]. The variation may result from the difference in the target population (asymptomatic vs symptomatic), time of investigation and sample size.
Our study also had a high prevalence of carriage than the studies conducted in Arba Minch, Southern Ethiopia among 7479 oropharyngeal samples with 6.6%   prevalence [20] and in Gurage Zone, Southern Ethiopia with 4.6% carriage rate [21]. Surveillance of invasive meningitis isolates in Ethiopia in 2012-2013 showed that in Hawassa, in the southern part of the country, serogroup A was the dominant cause of disease [22]. But in 2015, menA vaccine was given and this may be the reason for the decrement of the carriage rate. On those mentioned studies, no serogroup A was identified and serogroup B was the least identified. Implementation of menA mass vaccination campaign may be the reason for zero prevalence of serotype A. In contrast, our study had less carriage rate compared to three local studies conducted at Gondar university hospital (2019 CSF samples) in the year 2011 to 2013 with 18.4% prevalence [23], Addis Ababa (240 nasal swabs) with 20.4% carriage rate [8] and bacterial meningitis surveillance in Ethiopia, 2012-2013 (139 CSF samples) with 19.4% prevalence rate [24].
The antimicrobial susceptibility pattern of N. meningitidis was determined. In the present study, higher resistance was reported for cefotaxime (35.8%), ciprofloxacin (45.3%) and trimethoprim-sulfamethoxazole (73.6%). The increment of resistance may be due to the easy accessibility of drugs, the simplicity of taking drugs (oral route of administration) and the use of these antibiotics for a long period of time in the country especially ciprofloxacin and trimethoprim-sulfamethoxazole and on top of that, irrational drug use. Many studies done in developed countries showed that resistance to cefotaxime is rare. But, in the Southern parts of Ethiopia about 14% of cefotaxime resistance was reported which supports our findings [21].
Ciprofloxacin was another antimicrobial agent tested against N. meningitidis and high level of resistance (45%). In contrary to our study, in Addis Ababa [8] and Gondar [19], N. meningitidis was susceptible to ciprofloxacin with the rate of 83.7 and 78.6% respectively. This discrepancy might be due to the difference in antimicrobial usage practices.
In our study, associated risk factors like tonsillectomy (P = 0.006), large family size (P = 0.003), history of visiting health care institutions (P = 0.031), number of students per class greater than 40 (P = 0.048), indoor kitchen location (P = 0.009), sharing utensils (P = 0.007), and cigarette smoking (P = 0.004) were significantly associated with N. meningitidis carriage. In different studies determinants like family size [25], crowded living condition [8,26], the number of children per house [27], the number of positive household members [28], lower socioeconomic status [29], indoor kitchen [26], and overcrowding in the house [30] were significantly associated risk factors in which coincided with our study finding.

Conclusion
Neisseria meningitidis prevalence in the present study had a high carriage rate among males than females. Serogroup A and Y/W135 were predominantly circulating meningococcal isolates in the community. Meningococcal carriage rate among primary school students was significantly associated with larger family size, students with tonsillectomy, parental cigarette smoking, students with greater than 40 per class, sharing utensils, history of visiting healthcare institutions and indoor kitchen.
The antibiotics markedly resisted by meningococcal isolates were trimethoprim-sulfamethoxazole, ciprofloxacin, and cefotaxime. The effective antibiotics identified in this study were minocycline, azithromycin, meropenem and chloramphenicol. Most of the meningococcal isolates were identified as multidrug resistance, with serogroup B and serogroup X had markedly higher resistance. We recommend the scientific community as well as the health sector to perform continuous surveillance of N. meningitidis carriage to control any possible diversity and emerging virulent strains in high-risk populations as well as to predict the epidemiology of meningococcal infections and the clinical spectrum of affected populations. Especially, molecular identification is essential for identification of which genotypes is circulating. Health education should be strengthened to reduce N. meningitidis carriage and possible risk factors. Moreover, antibiotic stewardship should be well strengthened at all health facility level to reduce the expanding of drug resistance problem. Authors' contributions ZT did conceptualization, analyzing the data, methodology designing, investigation during the laboratory work, writing original draft and review the final manuscript. FM did conceptualization, methodology designing, writing original draft and review the final manuscript. MT did conceptualization, analyzing the data, methodology designing, writing original draft and review the final manuscript. TB did conceptualization, methodology designing, investigation during the laboratory work, writing original draft and review the final manuscript. All authors have read and approved the manuscript.

Funding
This study was not funded.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate An ethical clearance letter was obtained from the Departmental Research and Ethics Review Committee of school of biomedical laboratory science. The reference number of the ethical letter was "Ref no-SBMLS/2123/11". This ethical letter was obtained from Mr. Mekonnen Girma (mekonnen2302 @cmail.com), Markos Negash (markosnegash@yahoo.com) and Bamilaku Enawgaw (bamlak21@gmail.com). Written consent was obtained from participants and informed as their participation was voluntary. Study participants were also informed about the purpose of the study. Confidentiality was maintained at all levels of the study. In addition, study participants involvement was based on a voluntary basis and participants who were unwilling to take part in the study and those who need to quit their participation at any stage were informed to do so without any restriction. Consent to participate was obtained from the parents/guardians of study participants under the age of 16.