Prevalence of methicillin-resistant Staphylococcus aureus in a tertiary hospital in Nepal

SETTING: Patan Hospital, Lalitpur, Nepal. OBJECTIVES: To describe 1) the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and its antibiotic sensitivity pattern; 2) the demographic and clinical characteristics associated with MRSA infections; and 3) the treatment outcomes of in-patients with MRSA infection among patients with S. aureus infection between January 2018 and December 2020. DESIGN: This was a cross-sectional study using electronic and paper-based hospital records of patients with S. aureus infection. RESULTS: Of the 1,804 patients with S. aureus infection, 1,027 patients (57%, 95% CI 55–59) had MRSA. The MRSA were susceptible to vancomycin (100%), linezolid (96%), doxycycline (96%), chloramphenicol (86%) and cotrimoxazole (70%), and resistant to erythromycin (68%), clindamycin (56%), gentamycin (58%), ciprofloxacin (92%) and ofloxacin (91%). The prevalence of MRSA was higher in 2019, among out-patients, and in respiratory samples, and lower in blood samples. Of the 142 in-patients with MRSA, 93% had a successful clinical outcome (cured/improved). CONCLUSION: More than 50% of patients with S. aureus infection had MRSA that were resistant to commonly available antibiotics. This calls for strengthening surveil-lance and good infection control practices in this hospital.

M ethicillin-resistant Staphylococcus aureus (MRSA) are genetic sub-variants of the S. aureus that are resistant to β-lactam antibiotics. 1 Part of the normal bacterial flora of the skin and anterior nares in humans, S. aureus can cause different types of difficult-to-treat infections such as pyogenic skin infections, wound infections, bacteraemia, urinary tract infections and respiratory tract infections. 2,3 Although MRSA infections can be acquired in community settings, it most commonly occurs in hospital settings, especially in those places where infection prevention and control measures are poorly implemented. These bacteria usually spread accidentally through direct contact with an infected wound or from the contaminated hands of healthcare providers. Also, people who are colonised with MRSA may not have signs of infection, and they can spread the bacteria to others. 4 Mortality rates in patients with MRSA are known to be significantly higher than in those with methicil-lin-susceptible S. aureus (MSSA) infections (15.6% vs. 6.2%). 5,6 Addressing public health challenges associated with MRSA requires surveillance, good infection control practices, especially in hospitals, and access to life-saving antibiotics that are effective against MRSA. 7 The prevalence of MRSA among those with S. aureus ranges from 13% to 74% in different parts of the world. 8 According to the literature and data from various countries in the South-East Asian and the Western Pacific regions, MRSA prevalence among those with S. aureus infection is reported to be between 2% to 69%. 9 In Nepal, a country in South Asia, MRSA prevalence among those with S. aureus infections ranges between 15% and 69% and varies widely across geographic locations. 10 Given the public health significance of MRSA infections, there is a need to monitor MRSA prevalence (as a part of surveillance for antimicrobial resistance [AMR]) in various settings, although the studies on MRSA or the results are not novel in our setting. Furthermore, there is minimal information on clinical management and treatment outcomes of inpatients with MRSA infection. We therefore conducted a hospital-based operational research study to determine the prevalence of MRSA in patients with S. aureus infection based on various documented demographic and clinical characteristics, antimicrobial sensitivity patterns to commonly used antibiotics and the treatment outcomes of inpatients with MRSA infection at Patan Hospital, a large tertiary care hospital in Lalitpur, Nepal.
The specific objectives were 1) to determine the number and proportion of specimens with MRSA among those who are culture-positive for S. aureus infections during January 2018 to December 2020; 2) to describe the drug resistance patterns of those with MRSA; and 3) to describe demographic, clinical characteristics and treatment outcomes of in-patients with MRSA infection.

Study design
This is a descriptive cross-sectional study involving a review of secondary data from electronic databases and paper-based patient records at Patan Hospital.

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vices are offered by both public and private providers. Infectious diseases remain among the top causes of death. As of 2017, these are highly prevalent. 12 Antibiotics are sold over the counter and are readily available to the general public. 13 However, the importance of the problem has been recognised, and an integrated response to combat AMR has been included in the Nepal Health Sector Strategy Plan. The National Action Plan (NAP) to fight AMR is still under development and is being adapted from the five strategic objectives (awareness, surveillance, infection prevention and control, antimicrobial usage, and research and innovation) of the global action plan for AMR. The incorporation of AMR activities in the existing programme is currently very limited, or none in some cases. 14 Nepal was enrolled in the Global Antimicrobial Surveillance System (GLASS) in 2018, with 21 sentinel laboratories performing antimicrobial susceptibility testing (AST). These sites perform routine culture and AST for different types of specimens. The test result data are recorded in local databases and reported to the National Public Health Laboratory (NPHL). NPHL acts as a national coordinating centre by validating, combining and uploading all data to GLASS annually, and provides external quality control to participating laboratories. In 2018, only 15 (out of 21) provided data for GLASS, and the microbiology laboratory at Patan Hospital was one of those. 15 Patan Hospital is located in the Lalitpur District of Nepal. It is a 650-bed, tertiary-care teaching hospital attached to the Patan Academy of Health Sciences (PAHS). As one of the largest hospitals in Nepal, it provides quality care to nearly 320,000 outpatients and 20,000 in-patients per year.
The hospital's microbiology laboratory, one of the sentinel laboratories for AMR surveillance, has internal and external quality control programmes operated by the NPHL and Christian Medical College, Vellore, India. Blood specimens were collected in BD BACTEC TM (BD, Franklin Lakes, NJ, USA) aerobic bottles and incubated until bacterial growth, which was detected using BD BACETC TM FX blood culture system. Specimens with growth were inoculated onto blood agar, chocolate agar and MacConkey agar. Other biological specimens such as pus, respiratory secretions, urine samples were inoculated onto blood, chocolate or MacConkey agar depending on the type of specimen. Culture samples were aerobically incubated at 37°C for 24-48 h. S. aureus were identified using Gram's stain, catalase and coagulase tests. AST was performed using the modified Kirby Bauer disk diffusion method as recommended in the Clinical and Laboratory Standards Institute (CLSI; Wayne, PA, USA) guidelines. 16 MRSA was detected using Cefoxitin disc (30 μg). Results of the performed tests were entered into the electronic hospital information system.

Study population and study period
For objectives 1 and 2, we included all patients who submitted biological specimens for culture and AST from January 2018 to December 2020 and were posi-tive for S. aureus. These patients were selected by reviewing the electronic hospital information system. From this patient population, we selected in-patients whose biological specimens yielded MRSA for objective 3.

Data sources and collection
Patients' demographic profiles and laboratory results were obtained from the electronic database of the hospital information system and entered into a Microsoft Office Excel spreadsheet (Microsoft, Redmond, WA, USA). A unique patient encounter number (EncID) recorded in the electronic database was used to trace the hospital number of admitted patients. A permanent hospital number was assigned to patients on admission to the hospital. This permanent number was used to trace the inpatient's record files. Data on clinical characteristics and antibiotics usage profile were retrieved from these files held at the archives of Patan Hospital. Data were first entered in a proforma, and these data were later entered into EpiData software v3.1 (EpiData Association, Odense, Denmark).

Data analysis
Data entered into EpiData were exported and analysed using the statistical software Stata v15.1 (StataCorp, College Station, Tx, USA). AWaRe classification ('Access', 'Watch' and 'Reserve' group of antibiotics) was used to group antibiotics for AST. 17 Data are summarised using frequencies and percentages. We used bivariable binomial log models to study the association between demographic and clinical factors with demographic and clinical characteristics. These associations are presented as prevalence ratios; P0.05 was considered statistically significant.

Ethics considerations
The study obtained ethics approval from the Institutional Review Committee of Patan Academy of Health Sciences, Lalitpur, Nepal (Ref: bss2102231481/Date: 021-02-23) and from the Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease, Paris, France (EAG number: 04/20, Date 2020-02-05). As this study involved analysing of retrospective data from routine records, the need for informed consent was waived; data confidentiality was maintained throughout the study.

RESULTS
Of the 114,137 samples received for culture between 2018 and 2020, 1,804 showed S. aureus growth. The antibiotics used for AST, the number of samples tested for each antibiotic and the susceptibility/resistance pattern is given in Table 1

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erythromycin belonging to the 'Watch' group of antibiotics. For these seven antibiotics, resistance levels ranged from 22% for cotrimoxazole to 72% for ciprofloxacin. About 24% of the S. aureus isolates that were multidrug-resistant were tested for linezolid, the antibiotic belonging to the 'Reserve' group; 3% of the samples were found to be resistant to it. The AST pattern of MRSA samples is given in Table 2.
Although not all samples were tested for antibiotic resistance, 100% of those tested were susceptible to vancomycin, 97% to linezolid and 96% to doxycycline. In addition to these, MRSA were also susceptible to chloramphenicol (89%) and cotrimoxazole (71%). MRSA were highly resistant (90% of the samples) to ciprofloxacin and ofloxacin.
The demographic and clinical characteristics of the patients from whom S. aureus and MRSA were isolated are given in Table 3. In those with S. aureus infection, the prevalence of MRSA was similar in both the sexes in different age groups. Almost all samples from in-patients had MRSA when compared to ~50% among samples from outpatients. The prevalence of MRSA was higher in 2019 than in 2018 (prevalence ratio [PR] 1.10, 95% CI 1.00-1.20], in outpatients than in-patients (PR 2.38, 95% CI 2.05-2.75) and among S. aureus isolated from respiratory specimens when compared to pus (PR1.33, 95% CI 1.10-1.62). MRSA prevalence was lower among S. aureus isolated from blood when compared to pus (PR 0.80, 95% CI 0.65-0.98).
The demographic and clinical characteristics of these 142 in-patients with MRSA infection and their treatment outcomes are given in Table 4. Although not all patients in this group were tested to the various groups of antibiotics, the MRSA was susceptible to vancomycin (100%), linezolid (95%), doxycycline (94%), chloramphenicol (86%) and cotrimoxazole (72%) in those tested. Based on the laboratory AST report, the initial antibiotics used for treatment in this group were changed in 74% of cases. The median duration of hospitalisation of these in-patients was 7 days (interquartile range: 5-13), and 93% had a successful treatment outcome (cured/improved).

DISCUSSION
This is the first study on AST from Patan Hospital; we report that more than half of the patients (1,804 biological samples with S. aureus infection) had MRSA that was resistant to several 'Access'

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and 'Watch' group antibiotics, and more than 90% of the in-patients with MRSA infection had successful treatment outcomes. Study findings provide the following perspectives on S. aureus and MRSA infection in our hospital setting. First, the prevalence of MRSA among those with S. aureus was 57% in our study, which is higher than that of a study conducted by Kshetry et al., which reported the prevalence of MRSA to be 37.6%. 18 In a similar study conducted by Sapkota et al., MRSA prevalence was found to be 70.6%, which was higher than that of our study. 19 Other studies conducted at different time points in the same geographical area have shown relatively lower prevalences of MRSA than that of our study. 10 As our hospital is a tertiary referral hospital, prevalence in our setting may have been higher due to the selective referral of patients who had not responded to antibiotics, or due to rising MRSA infections in the community. Increasing levels of MRSA infection in the community is a cause for concern and calls for strengthening infection prevention and control measures and improved access to second-line antibiotics to treat such infections. In our study, most MRSA were sensitive to chloramphenicol (89%) and cotrimoxazole (71%) in the 'Access' group of antibiotics, and this is a positive sign, as both these antibiotics are easily accessi-ble and cheap. Most MRSA infections in our setting may therefore not need costly second-line antibiotics (vancomycin, linezolid) for treatment.
Second, the prevalence of MRSA was found to be higher in 2019 than in 2018. The decline in the number of samples in 2020 may have been due to the reduction in hospital visits because of COVID-19-induced travel restrictions. Furthermore, MRSA prevalence was higher among respiratory samples than in pus samples, and lower among blood samples. Previous studies have shown that a higher prevalence of MRSA is associated with sex (higher among males than in females), 20 age (higher among the elderly than in the young), 20 and among in-patients than in out-patients. 20 We did not find such associations in our study despite the large sample size.
Third, despite fewer MRSA in-patient records studied for treatment outcomes, we noted that treatment outcomes were good in more than 93% of the patients, with a 0% mortality rate. This contrasts with the studies conducted elsewhere, where mortality rates have been up to 15% in those with MRSA infection. 5,6 Apart from possible selection bias, we believe that the factor contributing to high treatment success levels was perhaps MRSA susceptibility to a large number of commonly available antibiotics, such

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as chloramphenicol and cotrimoxazole. However, because of the low proportion of in-patients studied, our study findings on treatment outcomes may not be generalisable to other patients whose records were not studied. This is an area for future prospective research.
The major strengths of the study were as follows: 1) we reviewed a large dataset of S. aureus samples; 100% of these samples assessed for oxacillin resistance in a microbiology laboratory with good laboratory practices (in accordance with CLSI guidelines).
We therefore believe that our study provides a reliable estimate of the resistance of S. aureus to various antibiotics and MRSA prevalence in our hospital setting. 2) We conducted the study using routine hospital data; therefore, the study reflects the ground reality under routine conditions. The major limitations of the study are as follows: 1) we did not have information on the referral practices of clinicians' bacterial culture and AST. Therefore, we do not know whether all patients likely to have S. aureus underwent bacterial culture and AST. This limits our ability to generalise the study findings beyond our study population. 2) As not all samples of MSSA and MRSA were tested to all antibiotics, we are unable to assess multidrug resistance and compare the multidrug resistance profiles for MSSA and MRSA. 3) Information on MRSA treated in the outpatient department was not available in the hospital records; future research into treatment outcomes of such patients is warranted.
In conclusion, more than half of the patients with S. aureus infection at Patan Hospital had MRSA. The MRSA prevalence among S. aureus infection was higher in 2019, and in respiratory samples, but lower in blood samples. The most common antibiotics to which MRSA was susceptible were vancomycin, linezolid, doxyclinine, chloramphenicol and cotrimoxazole; treatment outcomes of in-patients with MRSA were good, with more than 90% having a favourable outcome.