Colonization rate of Streptococcus pneumoniae, its associated factors and antimicrobial susceptibility pattern among children attending kindergarten school in Hawassa, southern Ethiopia

Objective The aim of this study was to determine the colonization rate of Streptococcus pneumoniae, antimicrobial susceptibility pattern and associated risk factors among children attending kindergarten school in Hawassa, Ethiopia. Results Out of 317 study participants, 68 (21.5%) were colonized with S. pneumoniae. Colonization rate was significantly associated with factors such as age (3 to 4 years old) (P = 0.01), having a sibling whose age was less than 5 years (P = 0.011), sharing a bed with parents (P = 0.005), cooking within bedroom (P = 0.002), and previous hospitalization (P = 0.004). Forty-four (64.6%), 33 (48.5%), and 2942.6%) of S. pneumoniae isolated were resistant to cotrimoxazole, penicillin, and tetracycline respectively.


Introduction
Streptococcus pneumoniae (S. pneumoniae) is the major cause of childhood morbidity and mortality in the world [1,2]. In Africa, S. pneumoniae caused 1-4 million episodes of disease among under-five children in 2007 [3]. In Ethiopia, 21.4% of invasive disease among children and adults were caused by S. pneumoniae [4,5]. Even though pneumococcal conjugate vaccine (PCV-10) was introduced in Ethiopia since 2011 as part of childhood immunization, 43.3% of meningitis and sepsis were caused by S. pneumoniae among children in 2015 [6].
Nasopharyngeal colonization of S. pneumoniae always precedes disease and serves as a reservoir for the transmission of the pathogen within the community [1]. The colonization rate varies depending on age, vaccination status, and other factors [7]. The rate of S. pneumoniae colonization is particularly high among young children attending kindergarten schools [8,9]. Vaccination status is assumed to reduce the colonization rate among children; however, its effect on colonization was not assessed in most low-income countries including Ethiopia. Factors such as environmental [10], socio-demographic [11] and previous health condition [8] can increase the prevalence of S. pneumoniae among school children. Moreover, nasopharyngeal colonization with antibiotic-resistant S. pneumoniae has been increasing in different parts of the world [10,12,13]. The rise of colonization rate with antibiotic-resistant S. pneumoniae creates a challenge to treat the disease caused by S. pneumoniae among carriers [10,14].
Most of the studies focusing on S. pneumoniae in Ethiopia were conducted among sick children [4,5]. Data on the S. pneumoniae colonization rate, associated risk factors, and antimicrobial susceptibility pattern among kindergarten school children were scarce in the study area. Therefore the present study was aimed to determine

Study area and period
Hawassa is the capital city of the Southern Nations, Nationalities and People Regional State. The city is located on the shores of Lake Hawassa in the Great Rift Valley and is located 275 km to the south of Addis Ababa, the capital of Ethiopia [15].

Study design
A community-based cross-sectional study was conducted from March 17, 2018, to May 20, 2018, at Tabor sub city, Hawassa, Ethiopia.

Study population
All children who attended kindergarten schools found in Tabor sub city, Hawassa, Ethiopia during the study period.

Sample size determination
Sample size was calculated by using a single proportion formula.
where n: sample size; Z: reliability coefficient (confidence level) which is 95% = 1.96; P: anticipated population proportion; D: the margin of error, which is 5% = 0.05. By using the anticipated population proportion of 44.8% from a previous study [8], the sample size was calculated as follows: Since the source population was less than 10,000, the sample size was recalculated using the correction factor as follows: After adding 10% of non-respondent the final the sample size was 324.

Sampling technique
At Tabor sub city, there are seven primary schools with a total of 1302 children attending kindergarten. Out of the total children, 324 were selected randomly with an equal proportion from each school. From 324 selected children, seven refused to participate in the study.

Inclusion criteria
Children who attended kindergarten school during the study period, those whose age was less than or equal to 6 years, and children whose parents had accepted the consent to participate in the study were included.

Exclusion criteria
Children who were on an antibiotic for the last 3 weeks, those with any sign and symptoms of respiratory disease and those who were not brought to school by their parents or guardians were excluded from the study.

Data collection
At each selected school, the parents of the children were consented and interviewed for sociodemographic, the environmental and previous health condition of the children by the trained data collectors. Data was collected by using pre-structured and pre-tested questionnaire.

Sample collection, handling, and transport
The nasopharyngeal specimen was collected by passing sterile rayon-tipped swab gently back from one nostril along the floor of the nasal cavity until it touches the posterior wall of the nasopharynx. One nasopharyngeal specimen per child was collected by a trained nurse.

Culture and identification of S. pneumoniae
The specimens were inoculated onto sheep blood agar (Oxoid Ltd, CM0271) supplemented with 5 μg/ml gentamycin by rolling the swab over a small area of the plate and stretching the sample using a sterile loop within 3 h of collection. The inoculated media was incubated in 5% CO 2 enriched atmosphere at 37 °C for 18 to 20 h. Suspected colonies, which appears as a greenish colony (alpha hemolytic), were subcultured on blood agar into which 5 μg Optochin disks (Mumbai, India) was placed, and then incubated within 5% CO 2 enriched atmosphere at 37 °C for 24 h. After overnight incubation, the sensitivity of Optochin was checked by measuring the diameter of the inhibition zone. If the diameter was ≥ 14, it was assumed to be S. pneumoniae; if the diameter was < 14, bile solubility tests (tube method) was performed by using 2% sodium deoxycholate (Oxoid Ltd) [16].

Data management and quality control
Quality of the data was ensured by using a pre-structured questionnaire. For laboratory analysis, the sterility of the prepared media was checked by incubating 5% of prepared media within a 5% CO 2 enriched atmosphere at 37 °C for 24 h before using it. A quality control strain of S. pneumoniae was used as a positive control for each test.

Data processing and analysis
Data were analyzed by using SPSS version 22. The frequency of variables, the prevalence of S. pneumoniae, and antibiotic susceptibility pattern was determined by using SPSS version 22. The association between risk factors and S. pneumoniae colonization was determined by using logistic regression. A P-value less than 0.05 at 95% confidence interval (CI) was considered statistically significant.

Discussion
The S. pneumoniae colonization rate among children who attended kindergarten school in the present study was 21.5%. The finding of this study was higher than the study conducted in the northern part of Ethiopia (10.3%) [17], Gambia (7.6%) [18] and Tanzania (12.3%) [19]. S. pneumoniae colonization rate found in this study was low compared to report from Gondar, Ethiopia (41%) [10], Jimma, Ethiopia (43%) [14] and Kenya (35%) [20]. The possible explanation for the variation might be due to vaccination status and age differences. This indicates that children whose age is below 6 years old are more colonized compared with those who are above 6 years old. Additional explanations for the differences observed are sample size, seasonal variation and method used. Even though the nasopharyngeal colonization rate of S. pneumoniae among children varies throughout the world, the result of this study was in line with the colonization rate reported from Nigeria [21], Morocco [22] and Kenya [23].
In the present study, we assessed different factors that could possibly increase the colonization rate of S. pneumoniae. Children whose age was in between 3 and 4 years were 3.1 times at risk to be colonized with S. pneumoniae (P = 0.01). This finding was in line with studies conducted in Belgium, Spain, and Ethiopia [10,24,25]. The decline in S. pneumoniae colonization rate as age increases could be due to the gradual acquisition of mucosal immunity and reduction of exposure. This indicates children whose age was in between 3 and 4 year were at high risk of acquiring S. pneumoniae colonization than those whose age was in between 5 and 6 years. Children who lived together with a sibling(s) whose age was less than 5 years old had 2.9 times chance to be colonized with S. pneumoniae compared to children who did not have a sibling whose age was not less than 5 years old (P = 0.001). This finding was comparable with a report from other parts of Ethiopia [10,14].
Unlike previous studies from Ethiopia [10,14], children who did not have their own separate bed in the present study had 3.5 times chance to be colonized with S. pneumoniae (P = 0.005). Children who sleep in the cooking room had 3.6 times chance to be colonized with S. pneumoniae compared with children who sleep in a bedroom which was free from cooking (P = 0.002). This finding was comparable with a report from Kenya [26]. Children with a history of hospital admission had 3.5 times chance to be colonized with S. pneumoniae (P = 0.004). This finding was consistent with a study conducted in France and Uganda [27,28].
Factors such as attending daycare centers and being passive smoker were not significantly associated with S. pneumoniae colonization during multivariable analysis. In contrast to the present study, a significant association of daycare attendance and passive smoking with S. pneumoniae colonization rate was reported from other countries [14,29,30]. In the current study, a high proportion (21%) of vaccinated children were colonized with S. pneumoniae even though it was not statistically significant (P < 0.05).
Out of the total S. Pneumoniae strains collected in the present study, 64.6% and 42.6% were resistant to cotrimoxazole and tetracycline respectively. This finding was comparable with previous studies conducted in different parts of Ethiopia [8,10,14] and Morocco [31]. The prevalence of tetracycline resistant S. pneumoniae reported from Iran (22.6%) was low compared to our study [32]. Unlike the current study, the majority of S. pneumoniae isolated from Kenya (98.6%) were resistant to cotrimoxazole [33]. Wide usage of antibiotics could be one of the reasons for high resistance rate observed in different countries. The prevalence of penicillin (oxacillin) resistant S. pneumoniae we found (48.5%) was low compared to the report Kenya (82%) [33].
According to our observation, there is wide use of cotrimoxazole in the study area for the treatment of pneumonia. Moreover, most of S. pneumoniae isolates collected in this study were resistant to cotrimoxazole indicating the importance of revising the treatment guideline for pneumonia.

Limitations
Penicillin resistance was performed by using a modified disk diffusion method. Not able to type S. pneumoniae isolates.