Study area, design & period
Hawassa University College of Medicine and Health Sciences Comprehensive Specialized Hospital is a tertiary level teaching hospital found in Hawassa city, Southern Ethiopia. Hawassa is the capital city of Southern Nations Nationalities and Peoples Regional State (SNNPRs) and is located 273 km to South of Addis Ababa. HUCSH has an estimated catchment of more than 5 million. According to information from hospital HMIS unit, the pediatric outpatient department (OPD) receives about 9,000 cases annually. The Pediatric Department of HUCSH has a total of 65 beds with average monthly admission rate of 75 children. We employed a cross sectional study design from February 1/ 2018 to July 30/ 2018 to enroll children with suspected UTI at the Pediatrics Department of HUCSH.
Children below the age of 15 years who were clinically suspected to have UTI and presenting to Hawassa University Comprehensive Specialized Hospital (HUCSH) were enrolled. Written informed assent was obtained from guardians or patients before data collection.
Patients who had at least one of the signs and symptoms of urinary tract infection like; frequency, urgency, dysuria, abdominal pain, back pain, fever (>38.0°C).
Patients who received antibiotics within two weeks before presentation to the hospital were excluded.
Sample size determination and Sampling techniques
Sample size calculated using single-population proportion formulae with assumptions based on a previous study done at Tikur Anbessa Specialized Hospital, which showed prevalence of extended spectrum β-lactamase (ESBL) of 78.6%  gave the largest sample size. The final sample size for a population survey considering a 10% non-response rate gave 284 subjects suspected of UTI.
Formulae: (Sample size) n = Z2α/2 p (1-p)/ d2
Where: n = the number of study subjects from which samples will be taken
P = anticipated population proportion. Prevalence of ESBL taken as 0.786
Z = Standard normal distribution value at 95% CI, which is 1.96
d = Degree of precision taken as 5%.
Socio-demographic and clinical data:
Socio-demographic characteristics like; age, sex, residence, paternal and maternal educational and occupational status, family living standard and clinical history of participants such as hospital admission and history of UTI within the past 12 months were recorded.
Laboratory data collection:
A total of 284 early morning mid-stream (MSU) and catheterized urine samples was collected using sterile, clean, transparent, screw-capped, wide-mouth plastic cups. All urine samples were properly labeled with unique code, age, sex, and time of collection and the sample was transported to microbiology laboratory within two hours of collection. By using the standard wire loop which has 1µl diameter approximately 0.001 ml urine was inoculated on 5% sheep Blood agar (OXOID Ltd. England) and MacConkey agar (OXOID Ltd., Basingstoke, United Kingdom) plate and the plates were incubated aerobically at 37°C for 24hrs.
A significant bacterium was determined on Blood agar plate (BAP) if single midstream and catheterized urine culture yields at least 105 CFU/mL . All types of colonies on the primary plates were examined macroscopically for hemolysis, changes in physical appearance of differential media, and the colony characteristics were recorded.
Morphology, Gram reaction and arrangement of the microorganisms were noted to identify the bacteria by Gram’s staining method. Biochemical tests were used for further identifications of Enterobacteriaceae isolates like Indole production, sugar fermentation, H2S and gas production, citrate utilization, motility test, mannitol test, and urease and oxidase test. In case of delay, the isolated bacteria were kept at 2–8°C in the nutrient broth for not more than 24hrs until the antimicrobial sensitivity test was done.
Detection of ESBL producing Enterobacteriaceae
ESBL production of Enterobacteriaceae were first screened by the standard disk diffusion
method and then were confirmed by using the double disc diffusion method as set by Clinical and Laboratory Standards Institute guideline (CLSI) .
Screening test for ESBL producing Enterobacteriaceae
Screening test for ESBL detection was done according to the CLSI guidelines . A 0.5 McFarland’s standard suspension of the test isolates was streaked by using a sterile cotton swab on the surface of a Muller Hinton Agar plates (MHA) (OXOID Ltd. England). Cefotaxime (CTX: 30μg), ceftazidime (CAZ: 30μg) and ceftriaxone (CTR: 30μg) were placed on the MHA using an automated dispenser and lightly pressed on to make sure they are firmly placed to the media. These plates were incubated for 18-24 hours at 370C. Isolates showing inhibition zone size below the CLSI stated break points were considered a potential ESBL-producer when the inhibition zone size for: ceftazidime < 22 mm, cefotaxime < 27 mm and ceftriaxone < 25 mm.
Confirmatory test for ESBL producing Enterobacteriaceae: The ESBLs detection was carried out by double disc synergy test using third generation cephalosporins and modified double disc synergy test using cefepime along with the third generation cephalosporins. All the strains which were positive for ESBL screening test was selected for checking for ESBLs production.
Double disk synergy test (DDST)
The double disc synergy test (DDST) is used as primary isolation method to identify the ESBL producing organisms. Antibiotic discs of amoxicillin / clavulanic acid (20/10μg) at the center; cefotaxime (30μg) and ceftazidime were placed at a distance of 15 mm apart center to center and incubated at 37˚C for 18-24 hours. A clear extension of cefotaxime and ceftazidime inhibition zones towards the disc containing clavulanic acid was considered as ESBL producer. Isolates which were screened and found positive for ESBL production may be negative for confirmatory test using DDST due to the coproduction of other beta lactamases like AMPC lactamase. In such cases, modified double disk synergy test were used using Cefepime antibiotics for inhibiting other beta lactamases (AMPC lactamase).
Modified Double disk synergy test (MDDST)
The test is performed by using a disc of amoxicillin-clavulanate (20/10 μg) along with four cephalosporins (cefotaxime, ceftriaxone, ceftazidime and cefepime). A disc which contained amoxicillin clavulanate (20/10 μg) was placed in the centre of the plate. The discs of third generation cephalosporin (cefotaxime, ceftriaxone and ceftazidime) and fourth generation cephalosporin (cefepime) were placed 15 mm and 20 mm apart respectively, centre to centre to that of the amoxicillin clavulanate disc on the surface of MHA plate, as recommend by CLSI, 2017. The plates were incubated at 37˚C for 24 hours. Any distortion or increases in the zone toward the disc of amoxicillin-clavulanate were considered as positive for ESBLs production.
Antimicrobial Susceptibility Testing
Antibiotic susceptibility testing was done by Kirby Bauer disk diffusion method on MHA as recommended by the CLSI guidelines [9, 10] for the following antimicrobial discs: ampicillin (AMP: 10 μg), amoxicillin-clavulanic acid (AMC: 20/10μg), ciprofloxacin (CEP: 30 μg), gentamicin (G: 10 μg), trimethoprim sulfamethoxazole (STX: 1.25/23.75 µg) cefotaxime (CTX: 30 µg), ceftriaxone (CTR: 30 μg), cefoxitin (FOR: 30 μg), tetracycline (TE: 30 μg), nitrofurantoin (NIF: 300 μg, BD), norfloxacin (NOR: 10 μg), and meropenem (MEM: 10 μg). The selections of antimicrobial agents for Enterobacteriaceae depend on the availability and recommendations from CLSI 2017. After overnight incubation of the Mueller–Hinton agar plate with antimicrobial discs at 37°C, the zone of inhibition was measured by using a ruler and interpreted by comparing the Kirby–Bauer chart. The antibiotic discs used were from Abtek Biologicals Ltd., Liverpool, United Kingdom product. Control strain ATCC-25922 E. coli used to monitor quality of antibiotic discs during antimicrobial susceptibility testing. During ESBL detection methods, E. coli ATCC 25922 (ESBL negative) and K. pneumoniae ATCC 700603 (ESBL positive) were used. Multidrug resistance was defined as simultaneous non-susceptible to three or more drugs from different classes of antibiotics.
The data were analyzed by using SPSS version 20 software. All variables with a P-value <0.25 in bivariate analysis were taken to multivariable logistic regression analysis. On multivariable analysis, variables with P-value <0.05 at 95% confidence interval were considered as statistically significant. Wealth status of the family used as a variables using principal component analysis (PCA).
Written informed assent were obtained from study participants or caretakers of children. Patient privacy was protected by de-identification of records. Names of patients were coded. All data obtained in the course of the study were kept confidential, and was used solely for the purpose of the study. Positive laboratory result from the study participant was communicated to their physicians for appropriate treatment or management.