Pneumococcal nasopharyngeal carriage and antimicrobial susceptibility profile in children under five in southern Ethiopia

Ethics approval and consent to participate

Ethical approval was obtained from the Institutional Review Board (IRB) of College of Medicine and Health Sciences, Hawassa University (IRB reference number: IRB/006/11). The purpose and importance of the study was explained to each study participants. To ensure confidentiality of participants, data collection tools were anonymous with no participant identifiers. Participants were interviewed alone to maintain privacy. All participants were not paid for the test. Informed written consent was obtained from a parent or guardian for children to participate in the study. The study incurs no cost to the study participants and were interviewed free of charge.

Study location and sampling technique

The study was conducted between November 2018 and March 2019 at outpatient departments (OPD) of two public Hospitals – Adare and Hawassa University comprehensive specialized Hospitals (HUCSH) in Hawassa City, which were purposively selected to represent primary healthcare and referral facilities in the region; Adare General Hospital is a primary care facility while HUCSH is the main referral hospital in southern Ethiopia. In the study district the coverage of three doses of pneumococcal conjugate vaccine (PCV) coverage was at 61% in 2019¹⁶, which showed significant improvement from the 2016 reported coverage of 53%¹⁷.

Sample size (n) was calculated using single proportion formula (Equation 1) assuming a prevalence (p) of 43% based on data reported in North West Ethiopia (43.8%)¹⁸ with 95% confidence interval (z=1.96) and 5% precession (d), and 10% non-response rate which resulted in a sample size of 417. A systematic random sampling method was used to select participants – every k^th child was selected from a total of OPD attendees every day. The list of all children who presented to the OPDs everyday was used as a sampling frame to decide the value of k. Parents or legal authorized representatives were invited to participate in the study. The informed consent process was administered to those who agreed to participate in the study.

Inclusion and exclusion criteria

Inclusion were all under-five children who visit OPDs of the two hospitals during the study period and who consent to participate in the study.

Exclusion criteria included subjects who had an illness that made nasal swabbing difficult, and those with severe respiratory problems (for example acute attack of bronchial asthma), had anatomical abnormalities of the nose (e.g. cleft palate) and who were on antibiotics in the two weeks prior to the start of the study.

Data collection

Structured questionnaires developed for the purpose of the study, pilot tested on 5% of the sample before implementation, were used to collect information on socio-demographics, clinical data, and associated factors. Based on the results of the pilot testing of whether questions were correctly understood by the interviewers and respondents or not, the questionnaires were revised to improve clarity. The pilot testing did not reveal significant errors in the questionnaires. The tools were first developed in English (see extended data¹⁹), translated to local language (see extended data²⁰), and back translated to English by an independent translator to ensure internal validity. In addition to interviews of parents/guardians, medical records of participants were reviewed to abstract past medical history. PCV vaccination status was also assessed through interviews with parents/guardians and vaccination card. Trained data collector healthcare professionals administered the questionnaires to the parents or LAR in a quiet room; data collectors also measured child’s weight to the nearest 0.1kg and height/length to the nearest 1cm using electronic weighing scale and length/height board. Anthropometrics were then interpreted using WHO Z scores, where a score of <-2 is considered to indicate undernutrition.

Sample collection and processing

Nasopharyngeal specimens were collected using sterile swabs in two replicates. One NP specimen was collected per child by gentle insertion of sterile flexible plastic applicator rayon tipped swab (Copan, Brescia, Italy, catalogue number: 26061), which was done by tilting slightly backwards and immobilize child’s head while gently restraining the child’s body. Once in place, the swab was rotated and left in place for five seconds to saturate the tip before slowly removing it. After collection, the sample was immediately placed in 1ml skimmed milk tritons glucose glycerol (STGG) transport media in tubes. Any excess samples were cut off before inoculating in the transport medium in tubes, after which the caps were tightened securely. The NP specimen was processed within 8 hours of collection and in cases where delay was encountered, it was stored at -20°C. Culturing the NP swab-STGG specimens was done on tryptone soy agar base (Oxoid, Basingstoke, Hampshire, England; Catalogue number: 105459). Briefly, the NP swab-STGG specimens were mixed thoroughly by vortexing for 30 seconds, 10 µl of the sample was then used to inoculate the plates and streaked using a sterile wire loop. The streaked plates were incubated into 5% CO2 incubator at 37°C for 24 hours. Plates were fully examined for any growth and the plates displaying no growth were re-incubated before being reported as negative. Identification of positive culture results was performed based on the appearance of colonies and the hemolytic pattern – small and watery growth surrounded by a greenish zone of alpha-hemolysis on the media.

Optochin susceptibility and a bile solubility biochemical test

We employed similar microbiological methods to those reported by Gebre et al.¹⁸. Briefly, to isolate pneumococci, suggestive colonies were sub-cultured and tested for optochin susceptibility and bile solubility. Optochin susceptible strains with ≥14mm in diameter zone of inhibition were identified as S. pneumonia. Next, alpha hemolytic strains with zone of inhibition <14 mm underwent bile solubility test using 2% sodium deoxycholate or bile salt base (Oxoid, Basingstoke, Hampshire, England; Catalogue number: 89904).

Bacterial cell suspension samples were prepared from freshly streaked presumed positive colonies of S. pneumonia in sterile normal saline. An adjusted 1ml of suspension was divided into two equal amounts of 0.5 ml in each tube. Then 0.5 ml of normal saline was added to one tube and 0.5 ml of 2% bile salt to the other tube as a test followed by incubation in 5% CO2 incubator at 37°C for up to 2 hours. A loss of turbidity in the bile tube but not in the saline control tube was considered as a positive test.

Antimicrobial susceptibility test

Disk diffusion (modified Kirbye-Bauer) method on Mueller Hinton agar (Oxoid, Basingstoke, Hampshire, England, Catalogue number: 105437) supplemented with 5% sheep blood was employed for AST²¹. Standard disks of commonly used antibiotics including Tetracycline – 30µg, Trimethoprim/sulfamethoxazole – 1.25+23.75µg, Oxacillin – 1µg, Chloramphenicol – 30µg, and Erythromycin – 15µg (Oxoid, Basingstoke, Hants RG24 8PW, UK) were used antimicrobial susceptibility testing of all the isolates.

Following inoculation of the bacteria suspension on the Mueller-Hinton agar plate, which is supplemented with 5% sheep blood agar, and then air drying, the antibiotic disks were dispensed aseptically using an automatic disk dispenser. Next, the plates were incubated in a 5% CO2 incubator at 37°C for 24 hours. Finally, zone diameters of growth inhibition were measured to the nearest millimeters using a ruler and were interpreted using cut-off points for each antibiotic disk, which range from 0.5µg/mL for vancomycin to 8µg/mL for gentamicin and tetracycline, in the Clinical and Laboratory Standard Institute (CLSI) result interpretive standards²¹. Categorically, results were interpreted as susceptible, intermediate, or resistant²². S. pneumonia ATCC 49619 provided by the Ethiopian National Quality Assurance Directorate (Catalogue number: 0947L) was used as a positive quality control strain for all procedures.

Data analysis

Bivariate and multivariate binary logistic regression models containing sociodemographic and clinical variables to assess independent predictors of pneumococcal NP carriage were produced. Variables with p-value <0.2 in the bivariate model were included in the multivariate model. Odds ratios and 95% confidence intervals (CI) were used to measure the association between potential risk factors and occurrence of NP carriage at the individual level. Level of significance for the multivariate models was set at p-value < 0.005. Anthropometrics were assessed following standard procedures²³, Z-score of <-2.0 was used as a cut off to define wasting, stunting, and underweight for weight-for-height, height/length-for-age and weight-for-age assessments respectively²⁴. All the statistical tests were performed using Stata version 14.0 (StataCorp, Texas, USA).

Socio-demographic characteristics

A total of 413 children participated in the study with 99.04% response rate; 226 (54.7%) were female. Age of the children ranged from 3–59 months with mean age (standard deviation – SD) of 36.63 (18.85) months. The majority, 308 (74.6%) of the children were from an urban setting; 157 (38.0%) of the parents/guardians attended primary education; and 230 (55.7%) of the parents/guardians were housewives. More than half, 215 (52.1%), of participants were from a family who had an average monthly income of USD 30 to 60 (Table 1²⁵).

Prevalence of pneumococcal nasopharyngeal carriage

The overall prevalence of pneumococcal NP carriage rate was 39% [95% confidence interval (CI): 34.4–43.8]. The highest prevalence of NP carriage was observed in those aged 3 – 23 months (49.8%). More boys than girls had NP colonization (42.0% in girls versus 35.3% in boys). Of the study participants who lived in urban settings, 46 (43.8%) were carrier for S. Pneumonia (Table 1²⁵).

Predictors of nasopharyngeal carriage

In bivariate analysis, sociodemographic variables including sex, place of residence, and age of the child had statistically significant association with pneumococcal NP carriage. Similarly, family factors including larger family size, presence of other siblings, and co-sleeping with other family members were predictors of NP pneumococcal colonization. Attendance at day care centers and presence of acute and chronic malnutrition were associated with an increased probability of NP Streptococcal colonization. Interestingly, level of PCV vaccination and lack of any vaccination were not associated with probability of NP pneumococcal colonization.

Next, we constructed a multivariable regression model including variables with a p-value < 0.2 in the bivariate analysis. Being under two years of age (AOR 2.31: 95% CI: 1.07, 4.98), those living with one or more siblings (AOR 1.95: 95% CI: 1.01, 3.76), history of co-sleeping with family members (AOR 2.09, 95% CI: 1.16, 3.79) and attendance at kindergarten/day care (AOR 1.84: 95% CI: 1.09, 3.11) were found to result in an increased probability of pneumococcal NP colonization (Table 1²⁵). Children who were stunted, 2.17(1.07,4.34); and wasting, 2.68(1.58,4.55) had a higher probability of pneumococcal colonization.

Antimicrobial susceptibility bacterial isolates

Antimicrobial susceptibility was determined for all 161 isolates of S. pneumonia to six commonly prescribed antimicrobial agents: Oxacillin, tetracycline, Erythromycin, TMP-SMX, and chloramphenicol. Among tested antimicrobial agents, higher rates of S. pneumonia resistance was reported in Oxacillin, 62 (38.5%); Tetracycline, 60 (37.3%) and Trimethoprim-sulfamethoxazole, 55 (34.2%). Comparatively, the lowest resistance rate was exhibited by Erythromycin, 11 (6.8%); chloramphenicol, 117(10.6%); and Vancomycin, 13(8.1). multidrug resistance to two or more antimicrobials was identified in 68 (42.2%) isolates (Table 2²³).

Underlying data

Figshare: Last Pneum Referal2.sav siraj Dem F.sav. https://doi.org/10.6084/m9.figshare.13297724.v1²⁵

This project contains the following underlying data:

Extended data

Figshare: Questionnaire (English Version).docx. https://doi.org/10.6084/m9.figshare.13297781.v1¹⁹

This project contains the following extended data:

Figshare: Amharic version of the questionnaire. https://doi.org/10.6084/m9.figshare.13356641.v1²⁰

This project contains the following extended data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).