Study design and setting
We carried out a prospective cross-sectional study from September 1, 2016 to August 30, 2017. The study was carried out in pediatric emergency departments of two large hospitals in Addis Ababa, Ethiopia; Tikur Anbessa Specialized Hospital (TASH) and Yekatit 12 Hospital.
Participant selection and inclusion criteria
Among children between the ages of 0 to 15 years presenting to the two pediatric emergency departments (PED) during the study period, the ones that were included in the study were, those presenting with suspected CAS and who had not taken antibiotics within two weeks prior to presenting to the hospital. CAS was defined as a case of suspected sepsis in children with no hospital or health care admissions in the two weeks prior to the current admission 14 and identified from samples taken within 48 h of admission.15 Suspected sepsis was categorized based on the clinical decision of the attending physician and was defined as meeting the systemic inflammatory response syndrome (SIRS) criteria.16
Data collection and outcome measurement
Trained research nurses approached parents/guardians of 101 children, suspected with CAS during the study period. After obtaining informed consent, a structured questionnaire was used to obtain sociodemographic data and other relevant clinical data. List of antibiotics that were used for management of patients was extracted from medical records. Final outcome (discharge or death) was registered and length of stay in the hospital was recorded in days. The microbiological outcomes assessed were: culture positive CAS, pneumococcal CAS and antibiotic susceptibility pattern among isolates.
Laboratory procedures
Sample collection
At enrollment, venous blood (1 mL for < 1 month-olds and 2-5 mL for > 1 month-olds) was drawn aseptically and transferred into 20 ml Brain Heart Infusion (BHI) broth (Oxoid, Cheshire, England) bottles and mixed gently by inverting the bottle. In addition, 1-2 mL of blood, collected using Ethylenediaminetetraacetic acid (EDTA) vials was available for 69 children and was frozen at -80 °C for PCR.
Culture and identification
The inoculated BHI broth was cultured aerobically. After 24 hrs of incubation, Gram stain was done followed by subculture on Blood agar, Chocolate agar and MacConkey agar (Oxoid). Culture bottles that did not show growth were further incubated for 7 days and subcultured before being reported negative. Initial identification of bacteria was made by Gram stain, hemolytic activity on sheep blood agar plates, optochin sensitivity, bile solubility, coagulase test, colony morphology on MacConkey agar and growth on Mannitol salt agar at the Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia. Further identification was then performed on all isolates by Matrix- assisted laser desorption/ionization time of flight mass spectrometry at Ghent University, Ghent, Belgium. Coagulase negative staphylococci (CoNS), Micrococcus spp, and viridans streptococci were considered as contaminants when identified in the blood cultures.
Antibiotic susceptibility testing
Antibiotic susceptibility testing on a selected panel of antibiotics that are used locally was done using the Kirby-Bauer disk diffusion method.17 For pneumococcal isolates, penicillin resistance was initially measured using oxacillin discs and for isolates with zones ≤ 19 mm, minimum inhibitory concentrations (MICs) were determined using E- Test strips (bioMerieux, Marcy-l’Etoile, France). Test results of both disc diffusion and MICs were interpreted according to the Clinical and Laboratory Standard Institute (CLSI) criteria.18 American Type Culture Collection (ATCC) strains: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Streptococcus pneumoniae ATCC 49619 were used for quality control.
DNA extraction and amplification of lytA genes
DNA extraction was performed on available whole blood samples obtained from 69 children. Initial pretreatment of the samples was performed as described previously.19 DNA extraction was then carried out with the DNeasy® 96 Blood & Tissue Kit (Qiagen, Venlo, The Netherlands), following manufacturer’s instructions. Amplification of a 101-bp fragment of the lytA gene was carried out as previously described.20
PCRSeqTyping and serotyping
DNA extraction from S. pneumoniae isolates was carried out by alkaline lysis as described previously.21 Amplification and sequencing of the 1061 bp of the cpsB-gene (encoding for phosphotyrosine phosphatase) were done using PCRSeqTyping as previously described.22 Twenty μl of the amplicons were sent for sequencing to GATC Biotech (Constance, Germany) and sequencing was performed using the Sanger sequencing technique. cpsB sequences were used to interrogate GenBank database (http://www.ncbi.nlm.nih.gov/blast). Serotype was assigned based on a BLAST bit score of > 99% sequence identity of the query cpsB nucleotide sequence with the reference sequences from GenBank. Because correct serotype could not be assigned by PCRSeqTyping for two of the isolates, serotyping was performed on all isolates by the Quellung reaction 23 using pool and group antisera, obtained from the Statens Serum Institut (Copenhagen, Denmark).
Statistical analysis
Data were initially entered in ReDCap (Vanderbilt, USA), exported into Excel and analyzed using PASW Statistics 20 software (SPSS Inc., Chicago, Ill. USA). Age was stratified into four groups: < 28 days, 28 days-1 year, 2-5 years and ≥ 6 years. Continuous variables were reported as median and interquartile range (IQR). Descriptive statistics was used to analyze sociodemographic, clinical and laboratory characteristics. To identify prognostic factors of CAS, bivariate analysis using binary logistic regression was performed. Variables which were significant at P < 0.1 were then used in multivariable model. Variables in the multivariable analysis were considered as significant when P < 0.05. Results from the binary logistic regression analyses were reported as ORs and aORs with 95% CIs.
Ethical approval
The study procedures were in accordance with the Helsinki Declaration. The study was approved by the AHRI/All Africa Leprosy Rehabilitation and Training Hospital (ALERT) Ethics Review Committee, Addis Ababa University Institutional Review Board, Yekatit 12 Medical College Ethics Committee and the National Research Ethics Review Committee (No. 310/194/2017). The parents and/or guardians of all participants gave written informed consent.