Comparison of Nasal Colonization of Methicillin-Resistant Staphylococcus aureus in HIV-Infected and Non-HIV Patients Attending the National Public Health Laboratory of Central Nepal

Background Staphylococcus aureus is a cardinal source of community- and hospital-acquired infection. HIV infection is a well-recognized risk factor for methicillin-resistant S. aureus (MRSA) carriage and infection. Intrinsically developed antibiotic resistance has sharply increased the burden of MRSA which is often associated with morbidity and mortality of the patients. Moreover, nasal carriage of S. aureus plays a significant role in spread of community-associated (CA) S. aureus infections. Methods This study was conducted from June 2016 to December 2016 at National Public Health Laboratory (NPHL), Kathmandu, with an aim to assess the rate of S. aureus nasal carriage and MRSA carriage among HIV-infected and non-HIV patients. A total of 600 nonrepeated nasal swabs were analyzed following standard microbiological procedures, where 300 swabs were from HIV-infected patients while remaining 300 were from non-HIV patients. The isolates were identified on the basis of colony characteristics and a series of biochemical tests. The antibiotic susceptibility test (AST) was performed by the modified Kirby–Bauer disc diffusion method. Inducible clindamycin resistance in isolates was confirmed by the D-test method. Results Overall, out of 600 nasal swabs of patients tested, 125 (20.8%) were S. aureus nasal carriers which included 80 out of 300 (26.66%) among HIV-infected patients and 45 (15%) out of 300 among non-HIV patients, and the result was statistically significant (p=0.0043). Among the isolated S. aureus, 11 (13.8%) MRSA were confirmed in HIV-infected while 3 (6.7%) MRSA were detected from non-HIV patients. A higher number of S. aureus carriers was detected among HIV-infected males 40 (26.49%), whereas MRSA carriage was more prevalent among HIV-infected females 7 (5.1%). Among the HIV-infected, patients of age group 31–40 years were the ones with highest carriage rate 36 (45%), while in non-HIV patients, the highest rate 13 (28.9%) of carriage was detected among the patients of age group 21–30 years. Statistically significant difference was found between S. aureus carriage and HIV infection in patients (p < 0.05). Higher rate 2/3 (66.7%) of inducible clindamycin resistance in MRSA was detected from non-HIV patients in comparison to HIV-infected patients 7/11 (63.63%) while the result was statistically insignificant (p > 0.05). All the MRSA isolates (100%) were resistant against co-trimoxazole while ciprofloxacin showed high rate of sensitivity towards both MSSA and MRSA. None of the isolates were detected as VRSA. The major factors associated with nasal colonization of S. aureus were close personal contact, current smoking habit, and working or living in a farm (p < 0.05). Conclusion Regular surveillance and monitoring of MRSA nasal carriage and antibiotic susceptibility pattern are of prime importance in controlling S. aureus infections especially in high risk groups like HIV-infected patients.


Introduction
Staphylococcus aureus is the most prevalent pathogen in community and health care setting and has been a serious threat to human health since its discovery [1]. S. aureus, especially methicillin-resistant S. aureus (MRSA), is responsible for varieties of maladies ranging from folliculitis to food poisoning. Moreover, it is responsible for causing life-threatening infections such as endocarditis, necrotizing pneumonitis, and osteomyelitis, among others [2]. Community-acquired MRSA (CA-MRSA) strain has emerged as a challenging pathogen which is frequently isolated from military personnel, drug users, athletes, and men who have sex with men [3]. e enduring threat and changing nature of S. aureus as a leading infectious pathogen has been well reported [4]. It is a common human skin colonizer, and pathogen armed with various classes of virulence factors including pore-forming toxins, superantigens, phagocytosis inhibitors, and biofilm forming capacity [5].
Some of the most severe infections caused by S. aureus include bacteremia, pneumonia, osteomyelitis, toxic shock syndrome, acute endocarditis, myocarditis, meningitis and abscesses in muscles, genitourinary tract infection, infection of central nervous system and various intraabdominal organs [6]. Methicillin-resistant S. aureus (MRSA) is a predominant cause of infection both in clinical and community settings which has amplified the disease burden to a major public health problem [7]. Colonizing feature of S. aureus is a potential factor for infection, an individual colonized with MRSA strain has a two to twelve fold more risk of subsequent infection [8]. Colonization and infection of MRSA has been recognized more in HIV-infected persons [9]. Moreover, higher prevalence of pathogenic MRSA strains have been documented among HIV-infected pediatric patients [10]. HIV infection and young age are considered as independent risk factors of MRSA infection [1]. High nasal carriage rate of MRSA in HIV infected persons may require early interventions. Hospitalized HIV-infected patients are nearly 17 times more likely to get S. aureus infection in comparison to non-HIV patients [11]. Not only this, the increasing prevalence of infections caused by MRSA has been difficult to treat due to the high rate of resistance to commonly prescribed antibiotics [12]. Determination of S. aureus nasal carriage rate and antibiotic resistance profiles along with molecular typing of nasal S. aureus isolates in healthy populations is necessary to identify risk factors associated with S. aureus infection [13][14][15].
In Nepal, reports on nasal carriage rate in vulnerable groups like HIV sero-positive individuals are very limited. erefore, this study was carried out to determine the rate of nasal carriage of S. aureus in HIV-infected patients which was compared with healthy carriers. Furthermore, the antibiotic resistance patterns of isolates to commonly prescribed antibiotics, was also investigated. is study could be beneficial in management of MRSA infections, particularly in HIV-infected patients, while suggesting appropriate measures for controlling the possible risks of S. aureus infections in HIV-infected patients.

Study Design.
is study was a prospective crosssectional study. e study was carried out at bacteriology laboratory of National Public Health Laboratory, Teku, Kathmandu. Study population consisted of HIV-infected and non-HIV infected patients from whom nasal swab was collected. A total of 600 nasal swabs were collected for laboratory diagnosis, of which 300 swabs each were from HIV and non-HIV patients. e study duration was 6 months from June 2016 to December 2016. Preformed questionnaire was used to obtain the clinical history and demographic information of each patient. We also collected the information about health conditions and environmental factors of the subjects which were hypothesized to be major factors associated with MRSA colonization in the study population.

Inclusion and Exclusion Criteria.
Patients with known history of HIV infection (seropositive) and noninfection (seronegative) were included in the study. Nasal swabs collected with standard operating procedure were accepted where complete label and strict sterile condition was maintained. ose samples that were improperly labeled and those from the patients who were non-HIV but having other immunodeficiency conditions like renal transplant, cancer, diabetes, liver cirrhosis, malignancy, chronic cardiovascular diseases, or consuming any immunosuppressant medicine were excluded from the study. Additionally, patients who had received any kind of antibiotics within the previous two weeks were also excluded from this study.

Specimen Collection and
Transport. Sampling procedure was done by a well-trained laboratory technician. Nasal swabs were collected by using sterile cotton swabs moistened with sterile normal saline. Each nasal swab was obtained by rotating 2-3 times in the anterior nares of patients' and was transported to bacteriology department quickly for further processing [16]. All the collected swabs were transported in a zip lock bag to the laboratory. No transport medium was used because the bacteriology department and sample collection sites were adjacent to each other and additionally, drying of swab was prevented.

Microscopic Observation.
e nasal swabs were evenly smeared on clean, dry, and grease-free glass slide. en smear was heat fixed and stained by Gram stain procedure [17]. e stained smear was observed microscopically using the 100x objective for presence of Gram positive cocci in grape-like clusters.

2.4.2.
Culture of Nasal Swabs. Primary culture was done on 5% sheep blood agar, Mac-Conkey agar, and Mannitol salt agar under aseptic conditions. Identification of isolated colonies was done by colony morphology, Gram staining, catalase test, slide coagulase test, and tube coagulase test [18].
e diameter of zone of inhibition (ZOI) was measured and the results were interpreted. Antimicrobials doses were selected on the basis of prescription frequency by physician. Minimum inhibitory concentration (MIC) values of used antibiotics were unable to determine due to unavailability of all antibiotics powder at the time of study period.

Screening of Methicillin-Resistant S. aureus (MRSA).
Screening for methicillin resistance in S. aureus was done by using cefoxitin (30 µg) antibiotic disc following modified Kirby-Bauer disc diffusion technique. ose S. aureus that showed zone size of ≤21 mm around cefoxitin disk were confirmed as MRSA strain.

Screening of Inducible Clindamycin-Resistant S. aureus.
e macrolide-inducible clindamycin resistance in S. aureus was detected by D test method [20]. 0.5 McFarland standard bacterial suspensions of the test isolate was inoculated on MHA plate and then clindamycin (2 µg) and erythromycin (15 μg) discs were kept 15 mm edge to edge on the same plate. en the plates were aerobically incubated overnight at 37°C. After the incubation period, flattening of zone (Dshaped) around the clindamycin disc was considered as inducible clindamycin resistance positive in S. aureus. Results of erythromycin-resistant S. aureus after D-test were interpreted into three phenotypic categories; MS B (macrolide-streptogramin B), inducible MLS B ( i MLS B ), and constitutive c MLS B phenotype.

Quality Control.
Media preparation, inoculation, and culture were performed in strict aseptic conditions. e prepared media were checked for the appearance of pure growth of organisms by using ATCC control strain. Stable strain of S. aureus ATCC 25923 was used as the control organism for the laboratory procedures. e thickness of MHA plates was maintained at 4 mm and the pH at 7.2-7.4. Microscope, incubator, centrifuge, refrigerator, water bath, autoclave, and hot air oven were checked regularly to ensure the correct functioning of equipment for the reliability of results. e temperature for all equipment was monitored and recorded.
2.6. Data Analysis. All data were analyzed using SPSS version 21.0. Chi square test was calculated where p value of <0.05 was considered statistically significant at 95% of confidence level.

Results
Among 600 nasal swabs investigated for determination of S. aureus and MRSA nasal carriage, 125 patients were found to be colonized with S. aureus among which 80 specimens were from HIV-infected and 45 from non-HIV individuals. e total prevalence of S. aureus colonization in HIV was found to be 26.66% while colonization in non-HIV was 15%. e MRSA colonization among the HIV patients was 13.8% (11/80) among 80 S. aureus isolates, whereas the MRSA colonization in non-HIV was 6.7% (3/45) among 45 S. aureus isolates.
Out of 300 nasal swabs analyzed from HIV-infected individuals, S. aureus isolates were 80, of which 26.5% (40/151) were male carriers whereas 23.2% (32/138) were female carriers and among third gender 72.7% (8/11) were S. aureus carriers (Table 1). Similarly, out of 45 S. aureus isolates from the 300 nasal swabs collected from non-HIV patients; 17.5% (27/154) were isolated from males whereas 12.3% (18/146) were from females. e study reveals that the S. aureus carrier percentage was higher among males in HIV-infected as well as in non-HIV patients ( Table 2). e nasal carriage rate was not statistically significant with gender of patients (p > 0.05).
In HIV-infected patients, a high rate of S. aureus nasal carriage was found among the patients of age group 31-40 years with 36 (45%) followed by 21-30 years with 15 (18.8%), 41-50 years with 13 (17.5%), 11-20 years with 8 (10%) whereas S. aureus was not detected from the patients of age group 61 year or above (Table 1). Similarly, in non-HIV patients the high rate of S. aureus nasal carriage was found among the patients of age group 31-40 years with 12 (26.7%) followed by 11 (24.4%) in age group 41-50 years, 7 (15.6%) in age group 51-60 years, 5 (11.1%) in patients of age group 61 year or above whereas S. aureus was not detected from the patients of age group 10 years or less ( Table 2). S. aureus carrier rate was statistically insignificant in relation to age groups of patients (p > 0.05).
Among the 300 HIV-infected patients, the growth of S. aureus was found in 80 (26.6%), of which 11 (13.8%) were confirmed as MRSA strains and remaining 69 (86.2%) were methicillin-sensitive Staphylococcus aureus (MSSA) ( Table 1). On the other hand, from the 300 non-HIV patients 45 (15%) S. aureus were isolated where 3 (6.7%) isolates were confirmed as MRSA strain whereas 42 (93.3%) were MSSA strains ( Table 2). e results claim higher percentage of MRSA among HIV-infected patients as compared to non-HIV patients.
e association between MRSA isolates and HIVinfected patients was found to be statistically significant (p < 0.05). More MRSA isolates were isolated from HIVinfected male patients 6(7.5%) than from females 4 (5%) whereas 1 (1.3%) was isolated from third gender patients with known HIV infection. MSSA was found higher 34 (42.5%) in male patients as compared to female patients 28 (35%). Similarly, more MRSA 2 (4.4%) was found among male non-HIV patients whereas one MRSA was reported from female patients. MSSA isolates rate was also higher among male 25 (55.6%) as compared to female 17 (37.8%) patients ( Figure 1). MRSA and MSSA carrier rate was statistically insignificant in relation with gender of patients (p > 0.05).   Among the S. aureus isolates, the highest rate of antibiotic susceptibility was observed against vancomycin (100%) followed by tetracycline (94.4%), cefoxitin (88.8%) and gentamycin (80.8). High rate of resistance was seen towards erythromycin (41.6%) followed by co-trimoxazole (40.8%). Most 10 (71.4%) of the MRSA were resistant towards ciprofloxacin (Table 3).
Among 125 S. aureus isolates from HIV-infected and non-HIV patients, percentage of both inducible and constitutive clindamycin resistance was higher in MRSA as compare to MSSA strains. Inducible MLS B -(iMLS B ) resistance, constitutive MLS B resistance, and macrolidestreptogramin B (MS B ) resistance were detected in 30 (24%), 19 (15.2%), and 34 (27.2%) isolates of S. aureus, respectively. Higher rate of iMLS B and cMLS B was seen among S. aureus isolated from HIV-infected patients while the rate of inducible MLS B resistance was statistically insignificant as to the origin of S. aureus isolates i.e. HIV-infected and non-HIV patients (p > 0.05) ( Table 4).
Nasal colonization rate of S. aureus was found significantly higher among the patients who used to work or live on a farm (p < 0.05). Similarly, inpatients showed higher rate 69 (55.2%) of S. aureus carriage as compared to outpatients 56 (44.8%), which was statistically significant data (p � 0.005). Additionally, patients with current smoking habit showed higher rate 76 (60.8%) of S. aureus carriage as compare to non-smokers 49 (39.2%) (p � 0.0051). Patients who had a job with close human contact were more prone to being a S. aureus carrier 89 (71.2%) as compared to those not having close human contact jobs 36 (28.8%) (p � 0.0076) ( Table 5).

Discussion
Colonization of MRSA significantly increases the risk of S. aureus infection during hospital stay and even after discharge [21]. Furthermore, nasal carriage of S. aureus is a cardinal source of infection and nasopharynx is considered as the main reservoir of S. aureus [1]. e outbreaks of MRSA infection especially in group of people like security shelters, health care centers and refugee camps strongly supports the theory that the spread of S. aureus requires close personal contact [22]. e rate of nasal carriage depends on various factors like clinical environment, geographical area, occupation of patients, and patient's immune status among others. Previous studies have reported up to 33% nasal colonization of S. aureus among HIV-infected population [12].
Total of 600 nasal swabs were analyzed in this study where 300 nasal swabs were from HIV-infected patients and remaining 300 were from non-HIV patients. e prevalence of S. aureus in HIV-infected patients was found to be more than the rate in non-HIV patients. is study shows the total prevalence rate of nasal carriage of S. aureus to be 20.8% (125/600). HIV-infected patients have higher rate of S. aureus nasal carriage than non-HIV infected patients and the result was statistically significant (p < 0.05). High rate of S. aureus colonization in HIV infected individuals can lead to severe infections in this group of people [23]. Immune status along with an individual behavioral features can play a positive role in S. aureus colonization and subsequent infection [24]. In a similar study carried out by Gonsu et al. (2013) in Cameroon, 40.6% of S. aureus nasal carriage was reported, whereas 34.6% of isolated S. aureus were MRSA among medical staffs and adult hospitalized patients [25]. Lilian et al. (2013) from Brazil have published a report of 27.2% as the prevalence of nasal colonization with S. aureus in patients with HIV/AIDS whereas the rate of MRSA was 21.8% [26]. Furthermore, the study has reported that nasal colonization of S. aureus was found to be more among the patients with CD4 cell count <200 cells/mm 3 and who had a high HIV viral load. Hence, HIV-infected patients are more vulnerable group to develop infections due to MRSA. On the other hand, S. aureus nasal colonization rate was found 18.4% among the healthy secondary school going students in Iraq whereas only 2.04% isolates were found to be MRSA [27]. e nasal carriage rate of S. aureus can be directly linked with underlying diseases, surrounding environment, age of patients and close contact activities [28]. In a study conducted among health care workers of Pakistan, 18.2% were nasal carriers of S. aureus where the count was lead my midwives (30%) then maintenance staffs of the hospital (28.6%) where only 1.5% of isolates were MRSA [29]. e real reason behind higher rate of S. aureus nasal colonization in midwives is unclear but this could be due to frequent contact with hospitalized patients and longer stay in hospital environment [30]. In a result reported by Khanal et al. (2015) the highest rate of MRSA nasal carriage was noted among hospital nurses which was 7.8% followed by hospital staffs of surgical wards and operating department whereas high number of S. aureus isolates (20.8%) were reported from doctors [31].
is study reveals higher rate of S. aureus colonization among patients of age group 31-40 years both in HIVinfected and no-HIV patients with prevalence rate 45% and 26.7%, respectively. Similarly, in a study carried out by Reinato et al., they have reported 36.1% of S. aureus colonization rate among the HIV/AIDS patients of age group 30-39 years [26]. e risk factors that have been associated with the emergence of MRSA infections among HIV infected individuals are not practically understood well but may be due to some possible risk factors, such as living conditions, prior hospitalization, use of fluoroquinolones and third generation cephalosporin antibiotics, intravenous drug usage, other secondary coinfections and low level of CD4 cells [32]. Some studies have suggested that participation in high risk sexual behaviors, use of public bath, anal intercourse, men who have sex with men (MSM), and sex with multiple partners may flourish the transmission of S. aureus and MRSA [33][34][35].
In this study, more S. aureus isolates were reported from males as compared to females both in HIV-infected and non-HIV patients though the result was not statistically significant (p < 0.05). e nasal carriage of S. aureus is found to be higher among male of active age groups who are mostly involved in outdoor activities, frequently visit to health care centers, and regular exposure to mass of people [31]. In contrast, higher rate of S. aureus nasal colonization was reported in female ). e association between type of patients (inpatients/out-patients) and MSSA carriers rate was statistically significant (p � 0.005). ese results suggest that alcohol consumption, working/living in farm area and job with human contact environment positively contribute towards the spread of MSSA and MRSA. Moreover, hospitalized patients are at greater risk of having CA-MRSA colonization. Individual behavioral factors, environmental, social, HIV-host factors, and all these in united form play a positive role in S. aureus colonization and subsequent series of infection [30].
ere was 100% sensitivity to the antibiotic vancomycin.
e rate of co-trimoxazole resistance was higher among S. aureus isolated from HIV-infected patients than non-HIV patients. Co-trimoxazole is being used as a major chemoprophylaxis agent for HIV-infected patients for the treatment of various bacterial infections. Increasing rate of antibiotic resistance among MRSA isolates may increase the burden of infections in the community and clinical setting especially in patients with HIV/AIDS [37]. Similar result was reported on antibiotic susceptibility pattern of   is variation could be due to different clinical settings, type of patients, environment, S. aureus with different susceptibility patterns, and behavioral factors of patients [41]. Clindamycin is a drug of choice for the treatment of S. aureus infections but constitutive or inducible resistance to this antibiotic has been a major cause of treatment failure. Both MRSA and inducible clindamycin resistance S. aureus are serious challenge to health care management of Nepal especially in immunosuppressed groups like patients with HIV/ AIDS, tuberculosis, renal transplant, cancer, diabetes, liver cirrhosis, malignancy, and chronic cardiovascular diseases.
High prevalence of nasal colonization along with increased rate of antibiotic resistance in S. aureus has posed a great threat to public health of the country and beyond. Compromised immune system and other risk factors including behavioral characters of HIV patients have led to emergence of MRSA which ultimately results in increase burden of community and hospital acquired infections. Increasing treatment failure due to high level of antibiotic resistance of S. aureus has expanded the burden of diseases in developing countries. Routine surveillance, proper monitoring of antibiotic resistance pattern and implementation of control strategies to prevent circulation of S. aureus strains in both clinical and hospital settings along with early detection of pathogenic MRSA isolates is very crucial for the reduction of morbidity and mortality due to diverse forms of S. aureus infections. Hence, routine screening, investigation of S. aureus nasal carriage particularly in immune deficient individuals, monitoring of their antibiotic susceptibility and control of S. aureus transmission are preventive me2asures for proper management of infections due to S. aureus.

Conclusion
e findings of this study show high rates of S. aureus colonization (mainly MRSA) and subsequent infections among HIV-infected patients. Smoking and alcohol drinking habits pose an individual at higher risk of S. aureus infection. Inducible clindamycin resistance (iMLS B ) S. aureus in HIV patients is increasing. D-test for the detection of iMLS B can be included as a screening test in routine laboratory investigation. Special attention is urgent to keep transmission and emergence of MRSA strains under control in people living with HIV/AIDS. S. aureus isolates showed high frequency of sensitive to tetracycline but we should take in account geographic differences in AST pattern when selecting antibiotic. Control strategies and interventions are very crucial to stop the spread to this "superbug" beyond the border.

Limitations
We cannot reveal the exact figure of S. aureus colonization without investigating swabs of other probable body sites like groin, pharynx, axillae, and anus. Furthermore, we could not collect the other epidemiological risk factors which could directly affect the MSSA and MRSA colonization and infection pattern. We could not perform/provide the CD4 count data to correlate with the immune status of HIV infected individuals too. Due to limitation of laboratory resources we could not run molecular procedures for confirmation of mecA gene in MRSA isolates. Further investigation in molecular level with collection of broad risk factors of large number of participants from different parts of the nation is required to generalize the result.

Data Availability
e data used to support the findings of this study are included within the article.