Staphylococcus aureus is among the most significant human bacterial pathogens worldwide (1), with Methicillin-Resistant Staphylococcus aureus (MRSA) currently being the most common antibiotic-resistant strain in most of the hospital and community settings (2)(3). Staphylococcus aureus is also the second leading cause of nosocomial bacteremia (4) and has been highlighted as a priority organism of interest by the WHO (5). Asymptomatic nasal carriage of Methicillin-Resistant Staphylococcus aureus (MRSA) can predispose the host to a wide array of infections that would pose a challenge in the management of the cases in the current era of antibiotic resistance (6)(7).
The magnitude of MRSA remains greatly unknown in developing countries yet surveillance systems to guide interventions require expertise and resources, which are inadequate(8). Studies conducted elsewhere by Conceiçao et al.,, and Aiken et al.,, documented MRSA prevalence rates of 26.9% and 7.0% respectively (9)(10). However, in resource-limited settings, not much has been done to explore on MRSA nasal carriage as reported by Bebell et al., 2016 and yet MRSA is currently recognized as the leading cause of hospital-acquired infections (11). Empirical and inappropriate use of antibiotics, multiple-pathology and use of invasive devices have attributed to a high rate of MRSA hence extended hospital duration and high treatment costs among patients (12).
Identifying the source, reservoirs and vectors for the spread of antibiotic-resistant bacteria poses significant challenges in tracking antimicrobial resistance. The patient’s carried endogenous microflora may all play a vital role in the cause of nosocomial infections (13)(14). In a study conducted on surgical wound infections in the hospital indicated that 65.9% of the cases were caused by MRSA strains (15). There are currently no documented reports on nasal colonization and carriage rates of MRSA among patients and associated carriage factors in Mbale Regional Referral Hospital. With most patients coming from different settings, our study aimed to determine the prevalence of Staphylococcus aureus and MRSA nasal colonization, and further identify the risk factors associated with asymptomatic carriage of MRSA.
Methods
Study design
This was a descriptive cross-sectional study carried out between January and April 2018 at Mbale Regional Referral Hospital on both in and Outpatients.
Study area
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.
Target population
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.
Inclusion Criteria
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.
Recruitment of study participants
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 to capture both demographic factors and predictor factors for MRSA nasal carriage.
Sample collection and transportation
From each participant, specimens for Staphylococcus 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 Staphylococcus 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 Staphylococcus 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 Staphylococcus aureus. Further confirmation was done by sub-culturing of the isolated colonies of Staphylococcus 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 (31).
Data Analysis
Raw data were entered into Microsoft Excel and later exported to Stata Corp. version 13 for analysis. Descriptive statistics including proportions, means were used to describe the participants and to determine prevalence. 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. We considered a univariate analysis at a level of significance of 0.2 to describe the categorical variables by sex and obtain baseline characteristics.
Quality Control
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. Staphylococcus 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.