This was a descriptive cross-sectional study carried out between January and April 2018 at Mbale Regional Referral Hospital on both in and Outpatients.
The study was conducted at both the Outpatient and In-patient departments (wards) of Mbale Regional Referral Hospital located in Mbale municipality. The hospital serves as both a regional referral and a tertiary teaching hospital for the medical school of Busitema University and School of clinical officers. It has a bed capacity of 400 and serves a population of approximately 4 million people.
The study targeted all patients seeking medical care at Mbale RRH who are asymptomatic carriers of MRSA. These included those from the inpatient (medical, surgical and maternity wards) and outpatient departments.
We included Patients aged 15 years and above, seeking medical care at either the In-patient or the Outpatient department at MRRH and had consented to take part in the study. Written informed consent for children aged 16 and 15 years was sought from their parents or guardians.
Sample size and recruitment of study participants
The sample size estimation was calculated using Kish Leslie formula. Consent was sought prior to recruiting of the participants. A cluster random sampling technique was used whereby participants were selected randomly with respect to the two broad clusters i.e. OPD and in-patients to ensure equal representative sampling. A questionnaire was administered through interviewing of participants to capture both demographic factors and predictor factors for MRSA nasal carriage.
Sample collection and transportation
From each participant, specimens for S. aureus culture were collected from both the anterior nares using sterile broth moistened dry swabs. The swabs were immediately placed into a tube with Cary-Blair transport media (BBLTM, BD bioscience), labelled with the participants' study number, initials, date and time of sample collection. Samples were immediately delivered to the Clinical Microbiology Laboratory, Mbale RRH for processing.
Isolation and identification of S. aureus
The swabs were first inoculated on Blood Agar and incubated at 37°C aerobically for 24 hours after which plate reading was done. Gram staining was performed on discrete colonies displaying the culture characteristics of S. aureus to confirm for Gram-positive cocci in clusters. A series of conventional biochemical tests such as catalase test using 3% hydrogen peroxide, bound and free coagulase using reconstituted rabbit lyophilized plasma (Remel, Europe, ltd. Dartford, Kent DA2 6PT, UK) were performed to identify the S. aureus. Further confirmation was done by sub-culturing of the isolated colonies of S. aureus onto Mannitol Salt Agar (MSA, Oxoid, CM0085. Thermo-scientific, US) and incubated at 37℃ for 24 hours aerobically. After 24 hours, the plates were examined for growth with interest in mannitol fermenting colonies that appeared as yellow colonies, measuring 1-2 mm in diameter and slightly raised.
Preservation of the Isolates
By use of a sterile pre-flamed wire loop, pure growing colonies were scraped off the Blood agar purity plates and suspended in 1ml of 15% glycerol broth the frozen at -80℃ till needed for MRSA phenotypic testing.
Phenotypic detection for MRSA
Agar disk diffusion (Kirby Bauer) technique on 4% sodium chloride Mueller Hinton Agar (BBLTM, BD) was employed. The inoculum was prepared by picking distinct colonies from a fresh pure loon culture on Blood agar and suspended into 5mls of a 0.85% saline water to make a bacterial suspension. The suspension was vortexed for 15 seconds and the turbidity adjusted visually by adding sufficient saline water to achieve a 0.5 McFarland standard. A sterile cotton swab was dipped into the suspension, rotated several times and the excess fluid removed by pressing on the sidewalls of the tube. The dried surface of the agar was evenly streaked with the sterile swab aseptically rotating the plate at 60° to ensure uniform distribution. A cefoxitin disc (30 µg, HIMedia, Mumbai, India) was placed on the surface of the inoculated plate using sterile forceps. The setup was incubated at 37℃ for 24 hours aerobically. The plates were read after 24 hours of incubation and the zone diameters of inhibition were measured using a Vernier calliper. The measured zones of inhibition were recorded and interpretations were done following the interpretive cut-offs (MRSA_ ≤ 21 mm and MSSA _ ≥ 22 mm) as per the Clinical Laboratory Standard Institute (CLSI) guidelines (22). The positively screened isolates were subjected to cefoxitin MICs (>8mg/mL) to confirm for MRSA carriage on the Phoenix M50 instrument using PMIC panels (BD) for confirmation.
Data analysis was done using Stata Corp. version 13. Descriptive statistics including proportions, means were used to describe the participants and to determine prevalence. We considered a univariate analysis at a level of significance of 0.2 to describe the categorical variables by sex and obtain baseline characteristics. The P-value was adjusted to allow for multiple logistic regression. Associations were generated using Odds ratio at 95% CI and a P-value of ≤0.05. We performed a logistic regression to determine any association between the factors collected as predictors and MRSA nasal carriage as the outcome.
Standard Operating procedures were followed, aseptic transfer techniques were performed, reagents were kept at 2-8℃, and the questionnaire was pretested before the commencement of the study. S. aureus ATCC 25923 was used as the positive control strain for the identification of biochemical tests and susceptibility tests on 4% sodium chloride Muller Hinton Agar. Commercially available control strain of S. aureus ATCC 700699 was used as a live positive control for MRSA on the PMIC panels.