Study design and settings
This was a cross sectional study conducted from May to July 2017 in three hospitals, in Dar es Salaam, Tanzania. The study sites included Muhimbili National hospital (MNH) the main specialized tertiary hospital with 1500-bed capacity, admitting 150–180 patients per day of which 2% are due to diarrheal disease. In addition, Amana and Temeke, regional referral hospitals with 250 –300 bed capacity each, admitting 50–70 patients per day of which 6–7% are due to diarrheal diseases.
Study population, sample size and sampling procedure
A total of 196 adult patients aged 18 years and above who were admitted in medical wards, Intensive Care Unit (ICU) and isolation wards for more than 24 hours were randomly selected and consecutively enrolled in this study.
Structured questionnaires were used to collect study participants’ clinical and demographic information. Information recorded included, age, sex, admission unit, diarrhea status, antibiotic use in the past three months, hospitalization history in the past three months, history of invasive procedures in the past three months and co-morbidities.
Trained nurses collected rectal swabs from consented participants and immediately put in Cary Blair transport media. The specimens were transported in a cool box with ice packs to Microbiology and Immunology Bacteriology research laboratory at Muhimbili University of Health and Allied Sciences (MUHAS) for processing.
In the laboratory, rectal swabs were cultured immediately on MacConkey agar supplemented with Ceftazidime 1mg/L and incubated aerobically at 37 ºC for 24 hours as a screening test for ESBL-PE (14). Isolated bacteria were identified based on colonial morphology, Gram staining and a set of conventional biochemical tests which included, Indole, Citrate, Sulphur Indole Motility (SIM) and API 20E tests.
Isolated organisms in screening test were potentially considered as ESBL-PE; however, they were further confirmed by double disk diffusion method (16). Briefly, both ceftazidime (30 μg) and cefotaxime (30 μg), alone and in combination with clavulanate (10 μg) were placed into inoculated Muller Hinton Agar (MHA) plate with test organism and incubated at 370C aerobically for 18 hours. The zones of inhibition were observed and interpreted according to Clinical and Laboratory Standards Institute (CLSI) 2015 guidelines. ESBL-PE was confirmed when there was ≥5 mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanate versus when tested alone (16).
Antibiotic susceptibility test
The confirmed ESBL-PE isolates were tested for antimicrobial susceptibility using Kirby-Bauer disk diffusion method according to CLSI guidelines (16). Briefly, homogenous colonial suspensions were prepared using 3- 5 colonies from a pure culture comparable to 0.5 McFarland turbidity standard. Standardized suspensions were inoculated on MHA, and then incubated at 37 ºC aerobically for 24 hours. The zones of inhibition were interpreted according to CLSI guidelines. E. coli ATCC 25922 was used as a control organism. The disks used included: chloramphenicol (20 μg), gentamicin (10 μg), ciprofloxacin (5μg), sulphamethoxazole/trimethoprim (1.25/23.75µg), tetracycline (30 μg) (Oxoid, UK).
Descriptive analysis was performed using statistical package for social science (SPSS) version 20. Categorical variables were summarized in a form of frequencies and percentages. Fisher’s exact test was employed to compare the associated factors for ESBL-PE. P-value < 0.05 was considered as statistically significant. Logistic regression was done to determine the factors associated with ESBL-PE, odds ratio was obtained at 95% confidence intervals.