Methicillin Resistant Staphylococcus aureus nasal carriage and associated factors in a rural tertiary hospital in Eastern Uganda: A prospective cross-sectional study

Background: Asymptomatic 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 a current era encompassed with antibiotic resistance. To inform public health strategies, the study sought to describe MRSA nasal carriage frequencies and the associated factors concerning nasal carriage among patients attending Mbale Regional Referral Hospital (MRRH). Methods: Two hundred eighteen consented participants presenting to the hospital for medical care between January and April 2018 were recruited to participate in this study. Sampling was done on both anterior nares using a pre-moistened swab and then transported to the laboratory at Room temperature for the detection of MRSA. Culture was performed on blood agar and plates incubated at 37℃ for 24 hours. Identification of Staphylococcus aureus was done using conventional biochemical tests . MRSA was detected phenotypically using cefoxitin (30µg) as a surrogate test as per the Clinical Laboratory Standard Institute guidelines 2017 version. Patient demographic characteristics and the MRSA nasal carriage factors were collected using a pre-tested questionnaire. The collected raw data was entered into excel and later imported into STATA software for analysis. Results: Overall, Majority of the participants were in-patients (138, 63.3%) with the proportions of both females and males among the participants being 154/218 (70.6%) and 64/218 (29.3%) respectively. Mean age for both female and male participants was 40.16 (SD± 17.04) years respectively. Staphylococcus aureus nasal carriage rate among the participants was 22.9% (50/218), with 72% (36/50) of the harboured strains phenotypically expressing methicillin resistance. Colonization with Methicillin Resistant Staphylococcus aureus did not show any significant relationship with all the studied factors.

Conclusion: There was a moderate Staphylococcus aureus nasal carriage among the participants in Mbale Regional Referral Hospital. We also observed a highly displayed phenotypic expression of methicillin resistance among the isolated Staphylococcus aureus strains. The studied factors indicated an independent influence on the rate of nasal carriage. For surveillance purposes to combat future outbreaks, there is a need to do a larger study with better power to better draw generalizable conclusions of carriage in the population.

Background
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 (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.

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 Inpatient 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 (BBL TM , 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 (BBL TM , 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.

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.

Demographic characteristics of patients
The study recruited 218 participants aged 15 years and above presenting to Mbale  (table 3).

Distribution of Methicillin resistance among Staphylococcus aureus Isolates.
Of the 50 studied Staphylococcus aureus isolates, 72% (36/50) of the isolates were found to be phenotypically expressing methicillin resistance while 28% (14/50) were methicillin sensitive as seen in figure 1.

Multivariate analysis of studied factors for association with Methicillin Resistant
Staphylococcus aureus nasal colonization.   (24). However, our finding was higher than a prevalence rate of 2.8% reported by Bebell et al from Mbarara Hospital, Uganda (11). Our observed high (72%) methicillin resistance expression among the isolated Staphylococcus aureus strains was contrary to reports from three regional hospitals in Tanzania that is Muhimbili National Hospital (10.5%), Mwananyamala and Amana regional hospitals (24.7%) (25) (26). This could have been attributed to the high numbers of in-patients who were included in the study and duration of hospital stay could have had an effect of acquisition.

Colonization with Methicillin Resistant
In addition, the choice of the participants included could have affected the prevalence since our study did not consider the children age group.

Factors associated with Methicillin Resistant Staphylococcus aureus nasal carriage
In our study, colonization by Methicillin Resistant Staphylococcus aureus did not indicate any significant statistical association with all the tested variables. Nonetheless, we observed different levels of consistency with the findings of previous studies. Our study indicated that antibiotic usage did not increase the likelihood of colonization by Methicillin Resistant Staphylococci aureus (OR = 0.7516896; 95% CI = 0.3804775-1.485974; P-value = 0.411), while HIV status was found to have no effect on the nasal carriage rate even with low immunity (OR = 0.7215959; 95% CI = 0.2871594-2.107922; P-value = 0.551).