Study design, period, study area, and population
A prospective cross-sectional study was conducted from May 1 to July 14, 2022 at the University of Gondar Comprehensive Specialized Hospital, and Kallen Bnakafl and Menna Geriatrics Support Centers Clinics. Both the hospital and geriatrics support centers are located in Gondar town, Amhara regional state, Northwest Ethiopia, situated at 742 km from the capital city of Ethiopia, Addis Ababa. The hospital is a tertiary level teaching and referral hospital catering more than 500 beds for inpatients and rendering referral health services for more than 5 million inhabitants in Northwest Ethiopia. Kallen Bnakafl and Menna Geriatrics Support Centers Clinics provides care and support for more than 110 versus 200 geriatrics permanently within the centers and for more than 100 versus 160 geriatrics outside the center, respectively. All geriatric patients who attended at the University of Gondar Comprehensive Specialized Hospital and lived in Kallen Bnakafl and Menna Geriatrics Support Centers were used as the source population while all UTI-confirmed geriatrics admitted at the University of Gondar Comprehensive Specialized Hospital and attended in Kallen Bnakafl and Menna Geriatrics Support Centers Clinics were used as the study population.
Inclusion criteria: Geriatric patients admitted to the University of Gondar Comprehensive Specialized Hospital and attending the Kallen Bnakafl and Menna Geriatrics Support Centers Clinics, who presented with urinary symptoms, were diagnosed with a UTI, and provided informed consent, were included in the study.
Exclusion criteria: Geriatric patients with cognitive impairments and those who had used antibiotics in the two weeks prior to or during the data collection period were excluded.
Study variables: The dependent variable was prevalence of multi-drug resistant bacterial isolates whereas the independent variables were previous history of UTI, recurrent UTI, catheterization, antibiotic use without prescriptions, diabetes Miletus, and frequent hospitalization of UTI-confirmed geriatrics.
Operational definitions
Geriatricwas defined as age group equal and above 65 years23. UTI–confirmed geriatrics were defined as those geriatrics having significant bacteriuria from microbiological analysis of urine in the hospital adult wards (C and D) and geriatric support center clinics. Significant bacteriuria was defined as when a properly collected midstream urine specimen shown to contain > 105CFU per ml24. Symptomatic UTI was associated with the patients complaining of dysuria, fever, urgent and frequent urination, flank pain, along with malodorous and/or cloudy urine and the number of bacteria on the urine is greater than 105CFU/ml of midstream urine10. Mid-stream urine was defined as a specimen obtained from the middle part of urine flow that is free of contamination, the pre-urethral area is cleansed and the patient is requested to discard the initial flow of urine before collecting the specimen in a sterile container. Multi-drug resistancewas defined as non-susceptibility to at least one agent in three or more antimicrobial classes25. Extensive-drug resistancewas defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories tested for a particular microorganism. (i.e., bacterial susceptibility to one or two antimicrobial classes) in this study22. Pan-drug resistancewas defined as non-susceptibility all to agents in all antimicrobial classes for each bacterium in this study26.
Sample size determination and sampling technique
The sample size was determined by using a single population proportion formula. The proportion was taken at 50%, with 95% of confidence intervals (CI) and 5% of margin error.
$$\:n=\frac{{\left(Z\alpha\:/2\:\right)}^{2}P\left(1-p\right)}{{\left(d\right)}^{2}}$$
$$\:n=\frac{{\left(1.96\right)}^{2}*0.5\left(1-0.5\right)}{{\left(0.05\right)}^{2}}=384$$
Using reduction formula as the total UTI-confirmed geriatric population based on the same period of the last year’s record, estimated population was 440.
$$\:{n}_{f}=\frac{n*N}{n+N}$$
$$\:{n}_{f}=\frac{384*440}{384+440}=205$$
By considering a 10% non-response rate: 205 × 0.1 = 20.5 ~ 21,
\(\:{\text{n}}_{\text{f}}\) = 205 + 21 = 226
Therefore, the final calculated sample size was equal to 226.
Where N is the total population, \(\:{\text{n}}_{\text{f}}\) = the final sample size, Zα/2 = the standard normal deviation, at 95% confidence level = 1.96, the prevalence (p) = 0.5; 1−p = 0.5 and the desired degree of accuracy (d) = 0.05.
A total of 226 study participants having UTI were recruited in the study using systematic sampling technique.
Data collection and laboratory methods
The important socio-demographic and clinical history of the study participants such as age, gender, education level, income status, residence, occupation, history of UTI and antibiotic use, hospitalization, catheterization and presence of comorbidities like history of diabetes were collected by trained nurses using a structured questionnaire via face-to-face interviews with geriatrics. Clean catch mid-stream urine was collected in a sterile screw-capped, wide-mouthed urine cup with prior adequate instruction of the participants.
Culture and bacterial identification
Early morning mid-stream urine sample was collected and transported to Medical Bacteriology Laboratory; inoculated in Cysteine Lactose Electrolyte Deficient (CLED) agar (BIOMARKR Laboratories, India) within two hours and incubated for 18–24 h at 35–37 °C. A 0.001 ml inoculating loop was used to inoculate the urine specimen. Colonies with significant bacteriuria (> 105 CFU/ml) were sub-cultured on MacConkey agar and mannitol salt agar (MSA) for isolation of a single species and further characterization by lactose fermentation on MacConkey agar and mannitol fermentation on MSA. Identification of bacteria was done using colony characteristic, Gram staining, biochemical tests such as triple sugar iron, motility, indole, citrate, urease, lysine decarboxylase tests, and novobiocin antibiotic test.
Antimicrobial susceptibility testing
The antimicrobial susceptibility test of all identified bacterial isolates were performed in vitro according to the criteria of clinical and laboratory standards institute (CLSI) using the Kirby-Bauer disk diffusion method on Muller-Hinton agar (MHA). A loop full of bacteria (3–5 identical colonies) were taken from a pure culture colony and transferred to a tube containing 5 ml of normal saline and mixed gently until it forms a homogeneous suspension. The turbidity of the suspension then adjusted to the turbidity of McFarland 0.5 in order to standardize the inoculums size and swabbed on MHA using a sterile cotton swab. The following antimicrobials were used with their respective concentration: ampicillin (10 µg), amoxicillin-clavulanate (20/10 µg), tobramycin (10 µg), tetracycline (30 µg), ciprofloxacin (5 µg), ceftazidime (30 µg), cefazolin (30 µg), cefotaxime (30 µg), meropenem (10 µg), nitrofurantoin (300 µg) and nalidixic acid (30 µg) for Enterobacteriaceae. For Pseudomonasspecies the antimicrobial disks were gentamycin (10 µg), tobramycin (10 µg), norfloxacin (10 µg), meropenem (10 µg). Whereas, vancomycin (30 µg), norfloxacin (10 µg), penicillin (10 units), cefoxitin (30 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), gentamycin (10 µg) and doxycycline (30 µg) were for pathogenic Gram-positive cocci. The criteria used to select the antimicrobial agents were based on both their availability for the management of UTIs and the 2022 CLSI guideline27. The plates then incubated at 37 °C for 18–24 h. Diameters of the zone of inhibition around the discs were measured to the nearest millimeter using a ruler and the isolates classified as susceptible and resistant. Isolates intermediate between susceptible and resistant were considered as resistant28.
Methicillin resistant Staphylococcus aureus detection
A lawn culture of S. aureus was made on the MHA plate and cefoxitin (30 µg) disc applied and incubated at (35–37 °C) for 24 h in ambient air. According to CLSI criteria < 10, 11–12 mm, and > 13 mm were categorized as methicillin resistant, intermediate and susceptible, respectively.
Quality control
The questionnaire was checked for its completeness and validity. It was also assured for its consistency by develop first in English, translated to the local language (Amharic), and then re-translated back to English. The culture media were checked for sterility prior to inoculation by incubating 5% of the newly prepared media at (35–37 °C) overnight. Gram’s staining reagents, culture media, and antimicrobial discs were also assured for their expiry date. Turbidity of the suspension was standardized against 0.5 McFarland standards. All antimicrobial discs and culture plates were stored at recommended refrigeration temperature (2–8 °C). Reference strains of E. coli (ATCC-25922), P. aeruginosa (ATCC-27853), and S. aureus (ATCC-25923) were used to check the performance of the media and antimicrobials.
Statistical analysis
Data was coded, entered into Epi Data version 4.6.0.0 and its completeness and clearance was checked to ensure the validity of all recorded data. Then it was exported to Stata/IC version 14.0 software for analysis. The study population was described by using summary statistics. Texts, tables, and figures were used to present the data. Logistic regression model was used to see the association between each independent and the outcome variable. All variable with P-value < 0.05 in the bivariate analysis were included in the final model of multivariate analysis in order to identify associated factors for the prevalence of MDR UTI. The direction and strength of the statistical association were measured using an odds ratio with a 95% confidence interval. An adjusted odds ratio along with a 95% CI was estimated, and a P-value < 0.05 was considered statistically significant.
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