Study settings, design and period
A hospital based cross-sectional study was conducted from February 15, 2019 to June 15, 2019 at ART clinic of Felege-Hiwot Referral Hospital Bahir Dar, North West Ethiopia. The hospital has 13 wards, 430 beds, and about 531 health professionals. The daily outpatient clients are more than 600. The ART clinic at Felege-Hiwot referral hospital is one of the largest in Amhara region, which has been serving more than 400 patients per week.
Sample size and sampling technique
A total of 165 HIV patients diagnosed with pneumonia were included in the study, based on the inclusion/exclusion criteria. Convenience sampling technique was employed to include study participants who meet the inclusion criteria until the required sample size is achieved.
Inclusion and Exclusion Criteria
Human immunodeficiency virus positive, antiretroviral therapy users and non-users aged ≥18
with a diagnosis of pneumonia as described by the clinician were included in the study. Patients who were critically ill, who cannot produce sputum, who took antibiotics during the past two week period except cotrimoxazole prophylaxis, patients whose age <18 years were excluded from the study.
Data collection procedures
Data on socio-demographic characteristics and associated factors were collected using a structured and pretested questionnaire.
Specimen collection, transport and processing
Pneumonia suspected individuals showing clinical symptoms such as shortness of breath, chest
pain, fever, chills, tiredness, and cough were examined by clinicians and the sputum samples
were collected using clean, dry, wide-necked, leak proof container. The patients were requested
to take a deep breath, cough deeply and vigorously to produce about 2ml of purulent sputum
specimen. Then it was labeled with screening ID number and/or patient ID number
and transported to Flege Hiwot Referral Hospital microbiology laboratory for processing within 2hrs or stored at 4∘C until further processing. Macroscopic examination was done to determine the sample integrity and Gram staining was performed from the purulent part of the sputum. Slides with more than 25 leucocytes and fewer than 10 epithelial cells per low power field (10x) were accepted for culture. Samples that had more than 10 epithelial cells and less than 25 leukocyte counts per low power field were discarded .
Cultivation and Identification of Isolates
Blood, Chocolate, and MacConkey agar plates were prepared as per the manufacturers‟ instructions. Using a sterile wire loop, purulent sputum sample was streaked onto each agar plate. Chocolate agar plates were incubated at 37ºC for 24 hours in a candle jar.
MacConkey agar and blood agar plates were incubated aerobically at 37ºC for 24 hours. After
incubation, the plates were inspected for any growth and negative plates were incubated for an
additional 24 hours. In order to get pure colony the samples were streaked in four quadrants of the plate . Following the standard microbiological procedure, the bacterial isolates were characterized using colony morphology, hemolysis, gram stain, and by means of a panel of biochemical tests. For gram positive bacteria we used catalase, coagulase, optochin, bile solubility test and for gram negative bacteria motility, indole, urea, lysine decarboxylase (LDC), oxidase, triple sugar iron agar (TSI) and citrate utilization tests and growth in blood agar with S. aureus (satellitizm test) for H. influenzae .
Antimicrobial susceptibility test
Antibiotic sensitivity test for the isolated organism was done by using Kirby Bauer Disc
Diffusion Method. Bacterial inoculums were prepared from 3-5 pure colonies by suspending the
freshly grown bacteria in 25ml of sterile nutrient broth (Oxoid, Ltd.,England) and mixed thoroughly to make the suspension homogenous. The suspension was compared with turbidity equivalent to 0.5 McFarland standards and was streaked on entire Muller-Hinton agar plate for
those organisms that are not fastidious. For fastidious organisms like Haemophilus spp., it was
streaked on to chocolate agar and for Streptococcus species; it was streaked on to blood agar . The disks containing the antimicrobial agents were applied within 15 minutes of inoculation on Muller-Hinton agar plate. The discs were about 25mm apart to each other. They were pressed down firmly to ensure complete level contact with the agar and incubated overnight at 35ºC. Diameter of zone of inhibition was measured and CLSI zone diameter criterion was used to interpret the level of susceptibility to each antibiotic [16, 17]. The antimicrobial agents that were used for gram positive bacteria include, oxacillin(1μg), chloramphenicol(30μg), ciprofloxacin(5μg), clindamycin(2μg), cotrimoxazole (1.25/23.75μg), tetracycline (30μg), erythromycin (15μg), and Cefoxitin (30μg). For gram negative bacteria, Gentamycin (10μg), amoxicillin (25μg), agumentin(20/10μg), chloramphenicol(30μg), ciprofloxacin(5μg), cotrimoxazole (1.25/23.75μg), tetracycline(30μg) and ceftazidime(30μg) were used. All antibiotics were obtained from Abtek Biologicals Ltd., UK.
The filed questionnaires were checked for their completeness. Media preparations were made based on the manufacturer’s instruction. Standard operating procedures were followed during specimen collection, handling, transportation, microbiological analysis, and interpretation. Ten percent of media per batch were incubated overnight for sterility check. Standard reference strain of American type culture collection S. aureus (ATCC-25923), E. coli (ATCC-25922) and P. aeruginosa (ATCC-27853) were used as control bacteria strains to evaluate the support of media for bacterial growth and for evaluation of efficacy of antimicrobial discs. The interpretation of zone of inhibition diameter was performed based on the updated 2019 CLSI guideline.
Data processing and analyses
Data was entered into EpiData and exported to SPSS statistical software version 23 for analysis.
Descriptive statistics was calculated to visualize the distribution of the outcome variable in
relation to socio-demographic characteristics and other possible associated factors. A univariate logistic regression model was used to determine the possible association of each variable with
the outcome variable. All covariates that were associated with the outcome variable in the
univariate analysis were subsequently included in the multivariable analysis. Multivariable
analysis was done to those independent variables with p-value < 0.05 to identify factors that are
independently associated with the dependent variable. The strength of the association was
interpreted using an odds ratio in a 95% confidence interval. In all cases, P-value less than 0.05
were considered as statistically significant.