This was a laboratory based cross-sectional study whereby biodata and midstream urine samples were collected from febrile children attending Mulago hospital from February to May, 2019.
Mulago National Referral Hospital was founded in 1913 and is located on Mulago hill in the northern part of Kampala, Uganda’s Capital city. The coordinates of the hospital are 0020’16.0’’N, 32034’32.0’’E (latitude: 0337786, longitude: 32.575550). It is the largest public hospital in the country with an estimated 1,500 beds. It is the National Regional Hospital for the entire country and a teaching hospital for Makerere College of Health Sciences. It also serves as a general hospital for Kampala metropolitan area.
Sample size determination
The sample size was determined using a formula by Thrusfield as follows (12);
Where; n was the calculated sample size., z the desired level of confidence(1.96), i the standard sampling error (10%), p the estimated prevalence from previous studies 20.3% (13). Although a minimum sample size required was 62, up to 160 febrile children from Mulago hospital who met the inclusion criteria were recruited in the study to increases precision.
Collection of samples
A mid-stream urine sample was collected from each child enrolled into the study. A sterile wide mouthed plastic urine sample container was used. The sample container was labeled with the sample number, sex, patient code, age and time of collection. Care takers of the children were informed on how to collect the sample without contamination. The collected urine sample was then transported to the laboratory and processed within one hour.
Febrile children from two to ten years, presenting with or without any other clinical signs of urinary tract infections were enrolled into the study. Children who were critically ill and needed special care and a sample could not be collected and those who were currently on antibiotic therapies were not enrolled in the study.
Laboratory examination of the samples
The samples were subjected to the routine macroscopy, biochemistry, microscopy, culture and sensitivity according to the standard practice.
Biochemistry of the urine was carried out using a dipstick so as to assess the proteins, nitrites, glucose and white blood cells. A dipstick was dipped into the urine so as to wet it after which it was removed and results read within 60 seconds.
Microscopic examination of the sample
The samples were examined using a microscope for pus cells, epithelial cells and cellular casts. A wet preparation was made by aseptically transferring a drop of urine onto a slide and covered with a coverslip. The preparation was then be examined using the 10X and 40X objective (14).
Culturing the specimen
The urine samples were inoculated onto Cystine Lactose Electrolyte Deficient (CLED) and Blood agar culture media using a 1 µl wire loop. Culture plates were incubated at 370 C for 24 hours. Colonies of the isolated organism were counted, a positive culture was defined as a urine sample containing ≥ 105 CFUs/mL of a uropathogen (14).
The colonies of the cultured organism were categorized morphologically so as to identify the organism. Morphology analyses included; size, shape, elevation, margin and surface.
A Gram stain was performed on the cultured organisms so as to determine their Gram reaction. Using an applicator stick, one colony was picked and smeared onto a clean glass slide and then air dried, fixed smear was covered with crystal violet stain for 60 seconds and then rapidly washed off with clean water. All the water was tipped off and then the smear was covered with Lugol’s iodine for 60 seconds, the iodine was then washed off with clean water. The smear was decolorized with acetone–alcohol and then washed off immediately with clean water. The smear was then covered dilute carbol-fuscin stain for 1 minutes. The stain was washed off with clean water , the back of the slide was then wiped clean and placed on a draining rack to air dry. The smear were examined microscopically, first with the 40X objective to check the staining and to see the distribution of material, and then with the oil immersion objective to report the bacteria and cells (14).
Biochemical tests were performed so as to identify the genus and species of the causative agents, the process involved the use conventional biochemical tests.
Biochemical tests included;
Indole test was be used to distinguish E.coli from Klebsiella spp. Colonies of the microorganism were inoculated in a bijou bottle containing about 3 mLs of sterile peptone water, they were incubated at 37 oC for 18 hrs. Thereafter 0.5mls of Kovac’s reagent were added to the bijou bottle and shaken gently. A positive test was indicated by a red ring while a negative test was indicated by absence red ring.
This test was used to distinguish between Staphylococcus spp. and Streptococcus spp. 4 drops of 3% hydrogen peroxide were added to the test tube and a colony of the organism was placed in the tube using an applicator stick. The tube was then observed for immediate bubble formation. If the isolate contained catalase enzyme, bubbles of oxygen were produced. Staphylococcus aureus was used as a positive control and Streptococcus pneumoniae was used as a negative control.
Paper oxidase test was performed on all Gram negative bacteria. It was used to differentiate between Pseudomonadaceae and Enterobacteriaceae. A filter paper was placed in a clean Petri dish and three drops of freshly prepared oxidase reagent were added to it. Using an applicator stick, a colony of the test organism was picked from the Petri dish and was smeared on the wet filter paper. Changes in the color of the filter paper were observed in 5 seconds. A positive test was shown by formation of a purple color while the negative test left the reagent colorless. Pseudomonas aeruginosa was used as a positive control while E coli were used as a negative control.
This test was used to identify Proteus spp. which produce urease enzyme. Urea agar slant was streaked on the surface with a colony from the culture plate and then incubated at 37 for 18 hours. Positive test was indicated by bright pink color while negative test was indicated by no color change.
This test was performed to differentiate Staphylococcus aureus from other Staphylococcus spp.
Slide method: Two drops of normal saline were put onto a slide. Using an applicator stick, the test organism was emulsified in the normal saline. A drop of plasma was then placed on the slide mixed well and then rocked gently for 10 seconds. Clamping on the slide indicated a positive test. If the slide coagulase test was negative, a tube coagulase test was done for confirmation.
Tube method: This test was performed on slide coagulase negative organisms or when the slide results were not conclusive. Freshly cultured organisms were used. 0.2ml of plasma were pipetted into a test tube and then 0.8ml of test broth culture was added to the test tube. The contents were mixed gently and then incubated at 37 oC for an hour, if no clotting occurred, the tube was examined again after 3 hours and if the test was still negative, the tube was left at room temperature overnight and examined again. Clotting of the tube contents indicated Staphylococcus aureus while no clotting of the tube contents meant a negative test.
Other biochemical tests included; Triple sugar Iron (TSI), Sugars (lactose, glucose, mannitol, and sucrose).
Antibiotic susceptibility testing
Antibiotic susceptibility was determined by the modiﬁed Kirby-Bauer disc diffusion technique. Commonly prescribed antibiotics for urinary tract infections at Mulago Hospital were tested. A pure isolate and identified colon of bacteria was be picked and inoculated in peptone water to form a bacterial cell suspension. An appropriate volume of peptone water which contains the cells was uniformly inoculated on Mueller Hinton Agar media. The antibiotic drug discs were then placed onto the culture media and then incubated for 18 hours aerobically. The diameter of the Zone of inhibition was measured. The results were interpreted according to the guidelines of The Clinical and Laboratory Standards Institute (14). The antibiotics used included; Amoxicillin-clavulanate, Ampicillin, Ceftazidime, Ciprofloxacin, Cotrimoxazole, Imipenem, Nalidixic-acid and Nitrofurantoin. For statistical analysis, bacteria with intermediate susceptibility were considered as resistant (15). Multiple drug resistance (MDR) was defined as an isolate resistant to 3 or more unrelated antibiotics tested. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as a control strains.
The collected data were entered in Microsoft Excel, cleaned and then exported to Statistical Package for Social Scientists (SPSS) software, Version 20.0. The Chi-square test was used to test for associations between potential factors with prevalence of UTIs.