The study was conducted at the Tikur Anbesa Specialized Hospital (TASH) which is the teaching hospital for the College of Health Science at Addis Ababa University. TASH is the largest specialized hospital in Ethiopia with over 700 beds, and serves as a training center for undergraduate and postgraduate medical students, dentists, nurses, midwives, pharmacists, medical laboratory technologists, radiology technologists, and others who lead the solution for health problems of the community and the nation. With more than 70 percent of childhood deaths attributable to communicable diseases and malnutrition, Ethiopia’s healthcare resources have been primarily focused on the treatment and prevention of diseases such as malaria and diarrhea .
Study design and period
A cross-sectional study was conducted from September 2017 to June 2018 to identify the bacterial profiles and antimicrobial susceptibility patterns among septicemia in under five patients with acute febrile illness in Tikur Anbessa Specialized Hospital in Addis Ababa.
Inclusion and Exclusion criteria
Children aged under five years including neonates with fever, those diagnosed with sepsis, severe sepsis and septic shock. All children who gave blood samples were volunteers with parental permission to participate in the study. Those patients who were a febrile under five years and patients who had taken antibiotics within the last 7 days were excluded.
Sample size calculation
The sample size for the study was determined using a single population proportion formula. The study considered prior prevalence and antibiotic resistance data in septicemia patients at Tikur Anbesa Specialized Hospital which demonstrated 27.9% bacterial isolation (19) with a 95% level of confidence and 5% margin of error. n = (Zα) 2 (pq)/ d2 where: n = sample size, Zα/2 = level of confidence, P = diarrhea prevalence q = 1-p d2 = margin of error (0.05): n= z² * p * q / d² ,
, p=0.279, q=0.721, d=0.05, Zα/2=1.96; 1.962*0.279*0.721/0.052=309. Considering a 10% non- response rate, a total of 340 children patients were enrolled in the study.
The study subjects were selected using convenient sampling technique from all patients attending Tikur Anbessa Specialized Hospital among children under five with febrile illness clinically diagnosed at pediatric OPD, ICU and inpatient pediatric wards admitted during the study period. Venipuncture was employed for those children that fulfilled the inclusion criteria.
Data collection procedure
A standardized questionnaire was used to collect socio-demographic characteristics such as, gender, age, and clinical presentation (fever, vomiting), and household income. Patients visiting outpatient departments (pediatric and general medicine) and those admitted through inpatient units were investigated for bloodstream infections by their unit physicians. Selection criteria included the onset of fever (>37∘C) or the presence of any clinical symptoms compatible with infection.
Blood sample collection
A venous blood culture specimen was taken using aseptic technique by cleansing the collection site with 70% alcohol followed by 10% povidone-iodine solution and were collected by trained laboratory personnel. About 2.5-5ml of blood was collected and inoculated in an aerobic 30ml BacT/ALERT PF Plus pediatric bottles with a blood to broth ratio of 1: 10-1:30. At least 2 sets of blood cultures were collected from a patient with suspected bacteremia prior to the initiation of antimicrobial therapy.
Culture Isolation and Identification
The initial BacT/ALERT culture bottles were incubated in automated BacT/ALERT® 3D instrument at 37oC with 5% CO2 for 5 days for the primary isolation. Two aerobic blood culture bottles were used for each patient and growth in both bottles were considered a positive culture. Microbial growth was detected by the instrument and subsequently sub cultured on 5% sheep blood agar, chocolate agar, and MacConkey Agar plates (Oxoid Ltd, UK) and incubates for bacterial isolation at 37oC for 18-24 hours. The MacConkey agar plate was incubated aerobically while chocolate and blood agar plates were incubated in a microaerophilic atmosphere (5-10% CO2) using a candle jar. A negative result was checked by a gram stain and a final subculture at the end of 5th day before reporting as a negative result. Growth was examined for colonial morphology including size, consistency, shape, hemolysis and ability to ferment lactose. For gram negative isolates, convectional biochemical test were performed .
Antibiotic Susceptibility Test
A pure colony of the bacterial isolate was mixed with 0.85% normal saline and adjusts to obtain a 0.5 McFarland standard density required for antibiotic susceptibility testing. Bacterial isolates were tested against the following antibiotic panel commonly used - for gram negative bacteria: tobramycin (10μg), amoxicilin-clavulanate (20/10μg), amikacin (30μg), gentamycin (10μg), ampicillin (10μg), piperacillin-tazobactam (100/10μg), cefotaxime (30μg), cefepime (30μg), ceftriaxone (30μg),ciprofloxacin (5μg), impenem/meropinem (10μg),trimethoprim- sulfamethoxazole (1.25/23.75μg) were tested. The Kirby-Bauer disk diffusion method was used for susceptibility testing using Muller Hinton agar and the standard interpretative zone size criteria from the current CLSI standards .
Detection of Carbapenem Resistance
All carbapenem resistance or intermediate isolates were phenotypically confirmed for the presence of carbapenemase using the modified carbapenem inactivation test (mCIM) to identify carbapenem resistant Enterobacteriacae (CRE) isolates as recommended by CLSI .
Detection of extended spectrum beta-lactamase
Initial screening for ESBL used the diameters of zones of inhibition produced by ceftazidime (30µg), ceftriaxone (30µg) and cefotaxime (30µg) measured within the CLSI screening criteria. These breakpoints that indicate ESBL production are ceftazidime ≤22mm, ceftriaxone ≤ 25 mm and cefotaxime ≤ 27mm. Phenotypic detection of ESBL production was confirmed by conducting a double disk synergy test and combined disk test according to CLSI guideline .
Combined disk (double disk potentiate) test (CDT)
Ceftazidime (30 µg) disk and cefotaxime (30µg) disks were used alone and in combination with clavulanic acid (30 µg/10 µg) for phenotypic confirmation of the presence of ESBLs. A ≥5 mm increase in zone diameter for either of the cephalosporin disks and their respective cephalosporin/clavulanate disks were interpreted as an ESBL producer. According to CLSI, this method is used as a reference phenotypic method for comparing double disk synergy method .
Double Disk Synergy test (DDST)
The test isolate was spread onto a Mueller–Hinton agar plate. Ceftriaxone (30 µg), cefotaxime (30 µg), ceftazidime (30µg), aztreonam (30µg) and amoxicillin/ clavulanic acid (20/10 µg disks were placed at distances of 20 mm (edge to edge) from the amoxicilin-clavulanate acid disk placed in the middle of the plate. After 24-h incubation, an enhanced zone of inhibition between either of the cephalosporin antibiotics and the Amoxicillin/Clavulanic acid disk is interpreted as a positive test .
Data quality assurance
Media was prepared as per manufacturer instructions and the laboratory Standard Operating Procedures (SOP) was strictly followed. Media were within expiration date and quality control parameters were used as defined in the CLSI standards.
Use ATCC control strain for each isolated bacterium included E. coli 25922, Pseudomonas aeruginosa 27853, and H. influenzae 10479, K.pnuemoniae 700603, S.typhimurium 13311. The results were reported on a log sheet and isolates were stored at -80 0c in skim milk.
Data analysis and interpretation
Statistical package for social science (SPSS) versions 20.0 was employed to analyze the work and to make inferences on the frequency of occurrence of the bacterial pathogens associated with febrile illness and to show resistance patterns. Descriptive statistics to analysis used frequency, proportions graphs, and crosstabs and odds ratio. Bivariate analysis was performed for each factor associated with bloodstream infection. Regression analysis was conducted to identify associated factors and how they are associated with dependent variables .The strength of association was presented by odds ratio and 95% confidence interval and p-value of <0.05 was considered as statistical significant association.