Study design and setting
A hospital-based cross-sectional study was conducted at Hawassa University Comprehensive Specialized Hospital (HUCSH) from February to May, 2016. The hospital is situated at Hawassa, the capital city of the Southern Nation, Nationalities and Peoples’ Region. The hospital is the largest in the administrative region with bed capacity of 400. The antiretroviral therapy (ART) clinic in the facility offers treatment service for new and follow-up HIV-infected clients. Clinical and immunological assessments (CD4+ T cell count) at enrolment and at three-monthly intervals help determine patients’ eligibility for HAART. HIV-infected patients with clinical indications of GIT diseases are routinely investigated for intestinal parasites in the hospital. Stool bacterial culturing is not performed on routine basis.
The study population consisted of HIV-infected patients attending the ART clinic at HUCSH during the study period and presenting with signs and symptoms of GIT disease. Patients aged <18 years, or who could not provide a stool sample or who had taken antimicrobial treatment (except trimethoprim-sulphamethoxazole (SXT) prophylaxis) within two weeks prior to the time of data collection were excluded.
Sample size and sampling technique
The sample size was estimated using a single proportion formula, and assuming a prevalence of enteric bacterial infection in HIV- infected patients of 16% , 95% level of confidence and 5% margin of error. A convenient sampling technique was used to enroll study participants, in which consecutive HIV-infected patients eligible for enrollment were invited to participate.
Socio- demography and clinical data
A structured questionnaire was used to collect socio-demographical (age, sex, residence, educational status) and other related factors (hand washing practice, habit of consuming raw food, own domestic animals, availability and usage of latrine, source and treatment of water for drinking). Patients were also asked for complaints of diarrhea (passing three or more loose or liquid stools over a 24-h period). Data on recent level of CD4+ T cell count, HAART status and prophylactic usage of SXT was obtained from patients’ medical records.
Bacterial isolation and characterization
A single stool sample was collected from each study participant using a sterile and disinfectant-free container. Stool samples were cultured on MacConkey (MAC) and Xylose Lysine Deoxychocolate (XLD) agar media after enrichment on Selenite-F Broth (Oxoid, United Kingdom). Further, blood free Campylobacter selective agar with Cefoperazone, Amphotericin B and Teicoplanin (CAT) selective supplement (Himedia, Ltd) was inoculated for isolation of Campylobacter species XLD and MAC agar plates were incubated aerobically at 37 °C for 24 h. Agar plates for isolation of Campylobacter species were incubated under microaerophilic conditions (5–10% O2 and 10% CO2 concentration) at 37 °C for 48 h. To create microaerophilic condition, the inoculated agar plates were kept in a gas jar containing gas generating kit (Campy Gen™, Oxoid, United Kingdom). Isolates were characterized using biochemical tests. Serotyping was performed for Salmonella, Shigella and E. coli using commercial anti-sera.
Antimicrobial susceptibility test
Antibiotic susceptibility testing was performed using the Kirby-Bauer disc diffusion method according to the recommendation of the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2011). In brief, 1–3 similar bacterial colonies were inoculated into a nutrient broth to prepare inoculums; broths were incubated at 37 °C for 4 h. Turbidity of broths was standardized at 0.5 McFarland using sterile phosphate buffered saline (pH, 7.2). The standardized suspension was inoculated on Mueller-Hinton agar plates. Defibrinated sheep blood (5%) was added for susceptibility testing of Campylobacter isolates. Antibiotic discs representing commonly prescribed antimicrobials in the study area were tested; these included chloramphenicol (30 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), erythromycin (15 μg), gentamicin (10 μg), nalidixic acid (30 μg), norfloxacin (30 μg), tetracycline (30 μg), and ceftriaxone (30 μg). Plates were incubated aerobically at 37 °C for 16–18 h; the incubation for Campylobacter species was done under microaerophilic condition for 48 h. The diameter of inhibition zone was measured using a caliper and interpreted according to the standard (CLSI, 2011). A reference strain of E. coli (ATCC-25922) was tested as a control.
Data entry and analysis were performed using SPSS Version 20 software. Results were summarized using percentages and frequencies. Binary logistic regression analyses were performed and crude odds ratio with 95% confidence interval (CI) were calculated to measure the strength of association between the dependent and independent variables.