# Multidrug-resistantStaphylococcus aureus Colonization in Healthy Adults Is more Common in Bhutanese Refugees in Nepal than Those Resettled in Ohio

Authors: Jhalka Kadariya; Dipendra Thapaliya; Sabana Bhatta; Ram Lal Mahatara; Sandra Bempah; Nabin Dhakal; Tara C. Smith
Journal: BioMed Research International
 (2019)

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## Abstract

Although studies have shown that human migration is one of the risk factors for the spread of drug-resistant organisms such as methicillin-resistantStaphylococcus aureus (MRSA), surveillance studies examining MRSA among refugee populations in the US are lacking. This study aimed to assess the prevalence and molecular characteristics ofS. aureusamong Bhutanese refugees living in Nepal and resettled in Northeast Ohio (NEO). One hundred adult Bhutanese refugees from each geographic location were enrolled between August 2015 and January 2016. The participants were interviewed to collect demographic information and potential risk factors for carriage. Nasal and throat swabs were collected for bacterial isolation. AllS. aureus isolates were characterized byspa typing and tested for the presence of Panton-Valentine leukocidin (PVL) andmecA genes; selected isolates were tested by multilocus sequence typing (MLST). The overall prevalence ofS. aureus was 66.0% and 44.0% in NEO and Nepal, respectively. In Nepal, 5.8% (3/52) of isolates were MRSA and 1.1% (1/88) in NEO. Twenty-one isolates in NEO (23.9%) were multidrug-resistantS. aureus (MDRSA), while 23 (44.2%) in Nepal were MDRSA. In NEO, 41spatypes were detected from 88S. aureus isolates. In Nepal, 32spa types were detected from 52S. aureus isolates.spa types t1818 and t345 were most common in NEO and Nepal, respectively. The overall prevalence of PVL-positive isolates amongS. aureus in Nepal and NEO was 25.0% and 10.2%. ST5 was the most common sequence type in both locations. Bhutanese refugees living in Nepal and resettled in NEO had high prevalence ofS. aureus and MDRSA.The findings suggest a potential need for CA-MRSA surveillance among the immigrant population in the U S and among people living in Nepal, and a potential need to devise appropriate public health measures to mitigate the risk imposed by community-associated strains ofS. aureusand MRSA.


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## Body

## 1. Introduction

Staphylococcus aureusis a common opportunistic bacterium. This bacterium is an important pathogen due to combination of toxin-mediated virulence, invasiveness, and antibiotic resistance [1]. Although it may be part of the normal human microbiota, it can cause wide range of diseases from skin and soft-tissue infections (STIs) to severe invasive disease such as infective endocarditis, osteomyelitis, and toxic shock syndrome [2].S. aureusis also a major cause of food-borne illness worldwide [3]. An estimated 30% and 1.5% of the US population is colonized with methicillin-susceptibleStaphylococcus aureus(MSSA) and methicillin-resistantStaphylococcus aureus(MRSA), respectively [4, 5].S. aureuscan be recovered from many locations in and on the body, including the nose, throat, axillae, and groin, but the most important site for colonization is thought to be the anterior nares (nostrils) [4]. While colonization withS. aureusitself does not harm the host, colonization is a risk factor for developing subsequent symptomatic infections [6].S. aureusaccounts for nearly 20% of bloodstream infections in the hospital setting [7]. The treatment ofS. aureusinfections is often challenging due to the emergence of multidrug-resistant strains [8, 9]. Although drug resistance has been seen as a major risk in healthcare settings, a similar increasing trend is observed in community-acquired infections [10].Traditionally regarded as a nosocomial pathogen [11], MRSA infection outside of hospital has increased in incidence over the last decade [12] and has emerged as a major public health concern worldwide [13]. For example, community-acquired methicillin-resistantS. aureus(CA-MRSA) is the leading cause of identifiable skin and soft-tissue infections (SSTIs) observed in US emergency rooms [14]. An estimated 80,461 invasive MRSA infections occurred nationally in 2011. Of these, 16,560 were community-associated infections [15]. The economic impact of CA-MRSA is tremendous. A recent publication suggested these infections impose an annual burden of $478 million -$2.2 billion on third-party payers and $1.4 billion -$13.8 billion on society in the US [16]. Because of lengthy hospital stays, increased costs, and higher mortality, MRSA infections have imposed an increased disease burden in the last decade [12, 17].The disease burden caused byS. aureusis poorly understood in developing countries [18]. Although there have been some studies conducted in the context of Nepal, very little is known about the molecular epidemiology and transmission dynamics ofS. aureusin this country. Most of the studies conducted in Nepal have been focused on the hospital environment, examining only the prevalence and phenotypic characterization ofS. aureus [19–24]. As such, the molecular epidemiology ofS. aureus in community settings has not been investigated, andS. aureuscolonization parameters among the general population or specific groups such as Bhutanese refugees is unknown.In the US, little is known regarding the prevalence and epidemiology of MRSA in immigrant populations. Although some infections, including tuberculosis and syphilis, may be detected during the pre- or postarrival screening, MRSA colonization is not examined, as there has been no policy or protocol for such screening by the US health institutions.In the past several years, the US has granted asylum to Bhutanese immigrants of Nepalese origin that were displaced from Bhutan due to political reasons. As a result, Bhutanese of Nepali origin were one of the largest groups of refugees resettled in the US, accounting for 19% of the total 322,565 refugees admitted into the US between 2008 and 2012 [25]. This study investigated the prevalence and molecular epidemiology of S. aureuscarriage in adult Bhutanese refugees living in camps in eastern Nepal and resettled in Northeast Ohio (NEO), United States.


## 2. Materials and Methods

### 2.1. Study Population

A community-based cross-sectional study was conducted among Bhutanese refugees living in Nepal and NEO in between August 2015 and January 2016. The study sites were Beldangi refugee camp in Jhapa district of Nepal and Akron and Cleveland metropolitan area in NEO. A convenience sample of 100 Bhutanese refugees aged 18 years and older, upon their consent to participate in the study, was recruited in each site. In both study sites, participants were recruited in community settings. Most of the participants in Nepal were enrolled in their home. A small portion of participants were invited to come to Primary Health Center for interview and sampling for their convenience. However, a meeting room was used for sampling. In NEO, the participants were recruited by visiting Nepalese grocery stores, Nepali restaurants, and the households of the participants and via distribution of flyers in the community.


### 2.2. Survey Instrument and Study Variables

Data obtained from the questionnaire included sociodemographic information such as family size, education, income, and occupation; medical information such as presence or absence of asthma, heart disease, diabetes, autoimmune disease, and cancer; presence or absence of risk factors such as recent history of hospitalization, hospital visit, history of antibiotic treatment, time spent in jail, participation in team or contact sports, ownership of pets, and history ofS.aureusor MRSA infection in the respondent or his/her family members; occupational exposure; and some miscellaneous exposures such as exposure to manure, pork, chicken, turkey meats, live animals, and types of soap used in home. An outcome variable “S. aureusand MRSA colonization positive” was assessed in association with exposure variables such as environmental and occupational exposures.


### 2.3. Data Collection

A set of pretested semistructured questionnaires was used to collect information from study participants. A face-to-face interview was conducted with each participant by the research team. Questionnaires, first developed in English language were translated into Nepali language, and the Nepali language questionnaire set was used to interview the participants in both study sites. The purpose of the study, its potential harm, and benefits were explained to each participant. Verbal and written consent was obtained from each participant to participate in the study. A copy of Nepali consent form was provided to each participant to take with them. The Institutional Review Board (IRB) of Kent State University and of the Nepal Health Research Council (NHRC) approved the study.


### 2.4. Sample Collection and Bacterial Culture

Biological specimens were collected from nares and throats using sterile swabs as described previously [26] and transported to the laboratory on blue ice packs. Samples were processed within 24 hours of collection. Samples were inoculated into 5ml (1X concentration) Baird Parker Broth with tellurite enrichment (Sigma products-Sigma-Aldrich, St. Louis, MO) in a 15 ml sterile plastic tubes by twisting the swab vigorously and incubated for 24 hours at 37°C. One microliter cultured broth was inoculated onto Baird Parker agar (BPA) with EY tellurite enrichment (BD) and was incubated for 48 hours at 37°C. Isolates were subcultured onto Columbia colistin-nalidixic agar with 5% sheep blood (Columbia CNA; Ramel) and tested via the catalase test, coagulase test, and using aS. aureus latex agglutination assay (Pastorex Staph-plus, Bio-Rad). AllS. aureusisolates were stored at -80°C.S. aureus prevalence was calculated as a percent of all individuals tested who were positive at either carriage site (nose or throat); MRSA and MDRSA prevalence were calculated as percentage of allS. aureus isolates who demonstrated these phenotypes.


### 2.5. Antimicrobial Susceptibility Testing (AST)

S. aureusisolates were tested for antibiotic susceptibility via Vitek-2 System version R06.01 (BioMérieux, Durham, NC) per manufacturer’s instructions. Isolates were tested for susceptibility to benzylpenicillin, oxacillin, tetracycline, erythromycin, ciprofloxacin, moxifloxacin, minocycline, clindamycin, trimethoprim-sulfamethoxazole (TMS), quinupristin/dalfopristin, gentamicin, levofloxacin, linezolid, daptomycin, vancomycin, rifampin, minocycline, tigecycline, and nitrofurantoin per Clinical and Laboratory Standards Institute guidelines [27]. Bacterial isolates resistant to three or more antibacterial or resistant to oxacillin were designated as multidrug-resistantS. aureus (MDRSA) [28].


### 2.6. Molecular Characterization

Genomic DNA was extracted using the Wizard Genomic DNA preparation kit (Promega, Madison, WI). The staphylococcus protein A (spa) gene was amplified as described [29, 30]. The detection of themecAand PVL genes was determined by PCR [31, 32].spatypes were assigned using Ridom® StaphType software (version2.2.1; Ridom GmbH, Wu-¨rzburg, Germany). The Based upon Repeat Pattern (BURP) algorithm was used to groupspa types into genetic clusters based on their genetic proximity [33]. A selected number of isolates were subjected to multilocus sequence typing (MLST) as described previously [34]. The most common, unique, and/or isolates of interest were chosen for MLST (e.g., those which were present in high numbers, low numbers, or unique in some manner including a novelspa type). Sequence types (STs) were assigned using organism specific MLST database (https://pubmlst.org/saureus/). PHYLOViZ software v2.0 was used to analyze Global optimal eBURST of STs and to draw minimum spanning tree and relatedness of STs as described by Francisco et al. [35, 36]. Positive (USA 300) and negative controls (reaction mixture without DNA template) were used inmecA, PVL, andspa PCR.


### 2.7. Statistical Analysis

Frequency distribution and proportions were calculated for the categorical variables while mean (± standard deviation), median, and range were calculated for continuous variables. Chi- square test or Fisher’s exact test and logistic regression were used to assess the association between outcome variables and the potential risk factors. The crude odds ratio (OR) was calculated with 95% confidence intervals. Statistical significance was assessed atα = 0.05 level. All statistical analyses were conducted using SAS statistical software (Version 9.3, SAS Institute Inc., Cary, NC).


## 2.1. Study Population

A community-based cross-sectional study was conducted among Bhutanese refugees living in Nepal and NEO in between August 2015 and January 2016. The study sites were Beldangi refugee camp in Jhapa district of Nepal and Akron and Cleveland metropolitan area in NEO. A convenience sample of 100 Bhutanese refugees aged 18 years and older, upon their consent to participate in the study, was recruited in each site. In both study sites, participants were recruited in community settings. Most of the participants in Nepal were enrolled in their home. A small portion of participants were invited to come to Primary Health Center for interview and sampling for their convenience. However, a meeting room was used for sampling. In NEO, the participants were recruited by visiting Nepalese grocery stores, Nepali restaurants, and the households of the participants and via distribution of flyers in the community.


## 2.2. Survey Instrument and Study Variables

Data obtained from the questionnaire included sociodemographic information such as family size, education, income, and occupation; medical information such as presence or absence of asthma, heart disease, diabetes, autoimmune disease, and cancer; presence or absence of risk factors such as recent history of hospitalization, hospital visit, history of antibiotic treatment, time spent in jail, participation in team or contact sports, ownership of pets, and history ofS.aureusor MRSA infection in the respondent or his/her family members; occupational exposure; and some miscellaneous exposures such as exposure to manure, pork, chicken, turkey meats, live animals, and types of soap used in home. An outcome variable “S. aureusand MRSA colonization positive” was assessed in association with exposure variables such as environmental and occupational exposures.


## 2.3. Data Collection

A set of pretested semistructured questionnaires was used to collect information from study participants. A face-to-face interview was conducted with each participant by the research team. Questionnaires, first developed in English language were translated into Nepali language, and the Nepali language questionnaire set was used to interview the participants in both study sites. The purpose of the study, its potential harm, and benefits were explained to each participant. Verbal and written consent was obtained from each participant to participate in the study. A copy of Nepali consent form was provided to each participant to take with them. The Institutional Review Board (IRB) of Kent State University and of the Nepal Health Research Council (NHRC) approved the study.


## 2.4. Sample Collection and Bacterial Culture

Biological specimens were collected from nares and throats using sterile swabs as described previously [26] and transported to the laboratory on blue ice packs. Samples were processed within 24 hours of collection. Samples were inoculated into 5ml (1X concentration) Baird Parker Broth with tellurite enrichment (Sigma products-Sigma-Aldrich, St. Louis, MO) in a 15 ml sterile plastic tubes by twisting the swab vigorously and incubated for 24 hours at 37°C. One microliter cultured broth was inoculated onto Baird Parker agar (BPA) with EY tellurite enrichment (BD) and was incubated for 48 hours at 37°C. Isolates were subcultured onto Columbia colistin-nalidixic agar with 5% sheep blood (Columbia CNA; Ramel) and tested via the catalase test, coagulase test, and using aS. aureus latex agglutination assay (Pastorex Staph-plus, Bio-Rad). AllS. aureusisolates were stored at -80°C.S. aureus prevalence was calculated as a percent of all individuals tested who were positive at either carriage site (nose or throat); MRSA and MDRSA prevalence were calculated as percentage of allS. aureus isolates who demonstrated these phenotypes.


## 2.5. Antimicrobial Susceptibility Testing (AST)

S. aureusisolates were tested for antibiotic susceptibility via Vitek-2 System version R06.01 (BioMérieux, Durham, NC) per manufacturer’s instructions. Isolates were tested for susceptibility to benzylpenicillin, oxacillin, tetracycline, erythromycin, ciprofloxacin, moxifloxacin, minocycline, clindamycin, trimethoprim-sulfamethoxazole (TMS), quinupristin/dalfopristin, gentamicin, levofloxacin, linezolid, daptomycin, vancomycin, rifampin, minocycline, tigecycline, and nitrofurantoin per Clinical and Laboratory Standards Institute guidelines [27]. Bacterial isolates resistant to three or more antibacterial or resistant to oxacillin were designated as multidrug-resistantS. aureus (MDRSA) [28].


## 2.6. Molecular Characterization

Genomic DNA was extracted using the Wizard Genomic DNA preparation kit (Promega, Madison, WI). The staphylococcus protein A (spa) gene was amplified as described [29, 30]. The detection of themecAand PVL genes was determined by PCR [31, 32].spatypes were assigned using Ridom® StaphType software (version2.2.1; Ridom GmbH, Wu-¨rzburg, Germany). The Based upon Repeat Pattern (BURP) algorithm was used to groupspa types into genetic clusters based on their genetic proximity [33]. A selected number of isolates were subjected to multilocus sequence typing (MLST) as described previously [34]. The most common, unique, and/or isolates of interest were chosen for MLST (e.g., those which were present in high numbers, low numbers, or unique in some manner including a novelspa type). Sequence types (STs) were assigned using organism specific MLST database (https://pubmlst.org/saureus/). PHYLOViZ software v2.0 was used to analyze Global optimal eBURST of STs and to draw minimum spanning tree and relatedness of STs as described by Francisco et al. [35, 36]. Positive (USA 300) and negative controls (reaction mixture without DNA template) were used inmecA, PVL, andspa PCR.


## 2.7. Statistical Analysis

Frequency distribution and proportions were calculated for the categorical variables while mean (± standard deviation), median, and range were calculated for continuous variables. Chi- square test or Fisher’s exact test and logistic regression were used to assess the association between outcome variables and the potential risk factors. The crude odds ratio (OR) was calculated with 95% confidence intervals. Statistical significance was assessed atα = 0.05 level. All statistical analyses were conducted using SAS statistical software (Version 9.3, SAS Institute Inc., Cary, NC).


## 3. Results

### 3.1. Demographic Information

A total of 400 samples (200 nasal and 200 throat) were collected from 200 participants (100 from each study site). The mean age of the participants was 30.7 (SD 10.9) years in NEO and 36.8 (SD 13.3) years in Nepal. In NEO, 58% and 42 % of the participants were male and female, respectively, while the percentage of male and female participants in Nepal was 29% and 71%, respectively. In Nepal, the majority of the participants was from the Kirat religion (79%), married (86%), and had a formal education (60%). In NEO, the majority of the participants had formal education (86%) (Table1).Table 1
Socio-demographic characteristics from NEO and Nepal.
Category  NEO (n=100) Nepal (n=100) P-value Age in years (mean, SD)  30.7, 10.9 36.8, 13.3 Gender Female 42 71 <0.05 Male 58 29 Religion Kirat 45 79 <0.001 Others 55 21 Marital Status Married 45 86 <0.001 Others 55 14 Education No formal Ed 14 40 <0.001 Had formal Ed 86 60 ED, education.


### 3.2. Prevalence ofS. aureus

The overall prevalence ofS. aureus was 66.0% (66/100; 95% CI 56.7%–75.3%) and 44.0% (44/100; 95% CI 34.3%–53.7%) in NEO and Nepal, respectively, for individuals colonized at either biological site. A statistically significant difference (p=0.002) was observed in the prevalence ofS. aureus between the two sampling sites.In Nepal, 39.0% (39/100; 95% CI 29.4%–48.5%) and 13.0% (13/100; 95% CI 6.4%–19.6%) of the participants were colonized withS. aureus in their throat and nose, respectively, while the prevalence ofS. aureus colonization in nose and throat in NEO was 25.0% (25/100; 95% CI 16.5%–33.5%) and 63.0% (63/100; 95% CI 53.5%–72.5%), respectively. The difference ofS. aureus colonization in nose and throat between Nepal and NEO was significant (p = 0.04 for nose and 0.001 for throat). The prevalence of positive isolates from both locations (nose and throat) from the same participants was 16% (7/44; 95% CI 5.1% - 26.7%) in Nepal and 33% (22/66; 95% CI 22% - 44.7%) in NEO. This difference was significant (= 0.04).The overall prevalence of MRSA (based on the presence of themecA gene) was 3.0% (3/100; 95% CI 0 – 6.3%) in Nepal and 1.0% (1/100; 95% CI 0 – 2.9%) in NEO. No significant difference was observed in MRSA prevalence between Nepal and NEO (p = 0.62, OR 0.32; 95% CI 0.03%–3.19%).Tables2 and 3 show participants’ demographics as well as medical, occupational, and environmental risk factors previously associated with the presence or absence ofS. aureus. In NEO, a higher prevalence ofS. aureus was found among males, participants who handled meat (pork and poultry) in the last six months, and participants who had skin infections in the last six months. In Nepal, a higher prevalence ofS. aureus was found among females, participants who had children attending day care, and found who were diagnosed with skin infections and autoimmune diseases (Tables 2 and 3).Table 2
Exposure variables in association withS. aureuscolonizationin NEO (n=100).
Variables SA+ (%) SA – (%) P-value Gender Male 42 (72.4) 17 (27.6) 0.13 Female 24 (57.1) 18 (42.9) Daycare going children Yes 10 (52.6) 9 (47.4) 0.18 No 56 (69.1) 25 (30.9) Skin infections (within 6 months) Yes 12 (75.0) 4 (25.0) 0.56 No 54 (64.3) 30 (35.7) Hospital admission(within 6 months) Yes 26 (61.9) 16 (38.1) 0.52 No 40 (69.0) 18 (31.0) Hospital visits (within 6 months) Yes 41 (60.3) 27 (39.7) 0.11 No 25 (78.1) 7 (21.9) Volunteers in health Institution Yes 3 (50.0) 3 (50.0) 0.40 No 63 (67.0) 31 (33.0) Pork handle (within 6 months) ≥1times per week 40 (71.4) 16 (28.6) 0.21 Do not handle 26 (59.1) 18 (40.9) Poultry handle (within 6 months) ≥1times per week 52 (71.2) 21 (28.8) 0.09 Do not handle 14 (51.8) 13 (48.2) Length of stay in the U.S. Less than 5 years 45 (70.3) 19 (29.7) 0.27 5 or more years 21 (58.3) 15 (41.6) Family size ≤5 members 29 (64.4) 16 (35.6) 0.83 >5 members 37 (67.3) 18 (32.7) Occupation Daily wages labor 35 (74.5) 12 (25.5) 0.13 Others 31 (58.5) 22 (41.5) Antibiotics use (within 6 months) Yes 7 (58.3) 5 (41.7) 0.52 No 59 (67.1) 29 (32.9) Skin infections (within 6 months) Yes 12 (75.0) 4 (25.0) 0.56 No 54 (64.3) 30 (35.7) Participated in Sports (within 6 months) Yes 26 (62.0) 16 (38.0) 0.52 No 40 (69.0) 18 (31.0)Table 3
Exposure variables in association withS. aureus colonization in Nepal.
Variables (n=100) SA+ (%) SA – (%) P-value Gender Female 34 (47.9) 37 (52.1) 0.27 Male 10 (34.5) 19 (65.5) Religion Kirat 37 (46.9) 42 (53.1) 0.32 Others 7 (33.3) 14 (66.7) Education Formal Education 27 (45) 33 (55) 0.83 No formal Education 17 (42.5) 23 (57.5) Marital status Married 36 (41.9) 50 (58.1) 0.38 Others 8 (57.1) 6 (42.9) Family size ≤5 members 26 (46.4) 30 (53.6) 0.68 >5 members 18 (41.0) 26 (59.0) Daycare Yes 11 (47.8) 12 (52.8) 0.81 No 33 (42.9) 44 (57.1) Nearest farm < 50meter 24 (41.4) 34 (58.6) 0.54 ≥50meters 20 (47.6) 22 (52.4) Hand wash frequency After each work 39 (46.4) 45 (53.6) 0.28 Before meal 5 (31.2) 11 (68.8) Skin infections (within 6 months) Yes 16 (51.6) 15 (48.4) 0.38 No 28 (40.6) 41 (59.4) Autoimmune disease (within 6 months) Yes 9 (60.0) 6 (40.0) 0.17 No 34 (41.0) 49 (59.0) Ear Infection(within 6 months) Yes 13 (39.0) 20 (61.0) 0.51 No 31 (46.0) 36 (54.0) Antibiotics use (within 6 months) Yes 8 (32.0) 17 (68.0) 0.16 No 36 (48.0) 39 (52.0) Skin disease (within 6 months) Yes 16 (51.6) 15 (48.4) 0.30 No 28 (40.6) 41 (59.4) Hospital admission(within 6 months) Yes 3 (30.0) 7 (70.0) 0.34 No 41 (45.6) 49 (54.4) Hospital visits (within 6 months) Yes 4 (25.0) 12 (75.0) 0.09 No 40 (47.6) 44 (52.4) Volunteers in health Institution Yes 12 (42.9) 16 (57.1) 0.88 No 32 (44.4) 40 (55.6) Pork handle (within 6 months) Yes 7 (31.8) 15 (68.2) 0.19 No 37 (47.4) 41 (52.6) Poultry handle (within 6 months) Yes 24 (40.0) 36 (60.0) 0.32 No 20 (50.0) 20 (50.0)


### 3.3. Antibiotic Susceptibility Testing

All isolates were tested for antibiotic susceptibility. All isolates in Nepal and NEO were resistant to penicillin. Oxacillin resistance was observed for 1.1% (1/89) and 5.8% (3/52) of the isolates in NEO and Nepal, respectively. Resistance to other antibiotics was higher for Nepal than Ohio. Additionally, MDRSA rates were higher in Nepal than Ohio (44.2% and 23.6%, respectively) (Figure1).Figure 1
Antibiotic resistance profile of isolates. MDR, multidrug-resistant.


### 3.4. Molecular Characterization ofS. aureus

spa typing was carried out on all positive isolates. In NEO, a total of 41spatypes were detected from 88S. aureus isolates. Overall, t1818 was the most commonspa type (12/88; 13.6%), followed by t002 (7/88; 8.0%), t104 (6/88; 6.8%), t008, t084, t085, t091 (4/88 each; 4.5%), and t521 (3/88; 3.4%). All otherspa types (t005, t10559, t1094, t11906, t122, t127, t131, t16153, t164, t1654, t1818, t1839, t190, t1911, t1931, t2119, t213, t2379, t2663, t273, t345, t4371, t442, t491, t521, t6127, t616, t688, t7264, t774, t803, t818, t878, t934, and t9432) were ≤ 2.2% ofS. aureus isolates.In Nepal, a total of 32spatypes were detected from 52S. aureus isolates. Overall, t345 was the most commonspa type (5/52; 9.6%), followed by t311, t2119 (4/52 each; 7.7%), t442, t127 (3/52 each; 5.8%), t021, t084, t091, t159, t164, and t934 (2/52 each; 3.8%). All otherspa types (t002, t008, t085, t1149, t11917, t12219, t1427, t15347, t15578, t15579, t15580, t15581, t1839, t2663, t304, t3175, t376, t4188, t701, t7766, t878) were less than 2.0% of the isolates.spa types were grouped based on their genetic proximity tospa types associated with specific cluster complexes. BURP grouping using default parameters (“exclude parameters that are shorter than 5 repeats” and “spa types are clustered if costs are less or equal than 4”) resulted in 7spa CCs and 11 singletons (t021, t127, t159, t164, t878, t1149, t2663, t11917, t15579, t15580, and t15581) in Nepal and 6spa CCs and 16 singletons (t005, t091, t104, t122, t127, t164, t190, t213, t616, t818, t878, t1654, t2663, t7264, t10559, and t16153) in NEO (Supplemental Figures 1-3).Thirty-nineS. aureus isolates from NEO and 29 from Nepal were subjected to MLST. A total of 26 different STs were detected from NEO. The most common ST was ST5 (17.9%; 7/39), followed by ST8 (10.3%; 4/39), ST1, ST15, ST3206, and ST96 (5.1% each; 2/39). No other STs constituted more than one testedS. aureus isolates (Table 4). A total of 22 different STs were detected from Nepal. The most common ST was ST5 (10.3%; 3/29), followed by ST15, ST3206, ST6, ST672, and ST80 (6.9 % each; 2/29). No other STs constituted more than one testedS. aureus isolate (Table 5).Table 4
Molecular characteristics of selectedS. aureus isolates from NEO (N=38).
Isolates Source mecA PVL AST spa MLST 27NEOT Throat – – P t002 ST5 89NEOT Throat – + P, G, C, L, T t005 ST672 33NEON Nose + – P, O, E t008 ST8 3NEOT Throat – – P, C, L, TS t084 ST2885 11NEON Throat – – P t085 ST845 38NEOT Throat – – P, TS t091 ST789 9NEOT Throat – – P, T t104 ST8 68NEOT Throat – – P t10559 ST2733 23NEOT Throat – + P, TS t1094 ST2112 80NEOT Throat – + P, C, L t11906 ST2884 63NEOT Throat – – P, E, CL t122 ST2102 8NEON Nose – – P t127 ST573 69NEON Nose – – P t131 ST1290 72NEOT Throat – – P t164 ST20 48NEOT Throat – – P t1654 ST667 30NEOT Throat – – P, T t1818 ST96 1NEOT Throat – + P, C, L, TS t1839 ST3206 82NEOT Throat – – P t190 ST8 70NEOT Throat – – P, TS t1931 ST1 84NEOT Throat – – P t2119 ST2871 88NEOT Throat – – P t2379 ST5 39NEOT Throat – + P, C, L, E, CL t2663 ST2233 18NEOT Throat – – P t273 ST1 1NEON Nose – + P, C, L, TS t345 ST3206 59NEOT Throat – – P t4371 ST5 4NEOT Throat – – P, TS t442 ST5 13NEOT Throat – – P, C, L, TS t491 ST199 9NEON Nose – – P, C, L, TS t521 ST96 96NEOT Throat – – P t6127 ST8 50NEOT Throat – – P t616 ST944 12NEOT Throat – – P t688 ST5 8NEOT Throat – – P t7264 ST5 55NEOT Throat – – P t774 ST15 79NEOT Throat – – P t803 ST15 28NEOT Throat – – P t818 ST5 29NEOT Throat – – P t878 ST2849 58NEOT Throat – – P, G, E, CL t934 ST80 16NEOT Throat – – P, C, L t9432 ST2128 P, benzylpenicillin; C, ciprofloxacin; E, erythromycin; CL, clindamycin; G, gentamicin; T, tetracycline; TS, trimethoprim-sulfamethoxazole; L, levofloxacin; O, oxacillin; AST, antibiotic susceptibility testing. Inclusion of antibiotic name denotes resistance.Table 5
Molecular characteristics of selectedS. aureus isolates from Nepal (N=29).
Isolates Source mecA PVL AST Spa ST 68T5 Throat – – P, TS t002 ST5 26T5 Throat – – P, C t11917 ST72 71T5 Throat – – P t15579 ST2885 74N Nose – – P, C, L, E, CL t15581 ST3206 19N Nose – – P t008 ST8 101N Nose – + P, L, M, TS t021 ST1482 102T Throat – – P, TS t084 ST15 76T Throat – – P t085 ST15 12T Throat – – P t091 ST2081 24N Nose – – P, C, L t1149 ST291 105T Throat – + P, G, C, L, E t12219 ST672 83T Throat + – P, O, C, E, CL, Mi, T, R t127 ST1 48T Throat – – P, G, E, CL t1427 ST361 70T Throat – – P t15578 ST2990 1T Throat – – P, E, CL, TS t159 ST800 85T Throat – – P, G, TS t164 ST20 67T Throat – + P, TS t1839 ST573 65T Throat – – P, TS t2119 ST2871 106T Throat – + P, G, C, L, E t2663 ST2233 15T Throat + – P, O t304 ST6 32N Nose – + P, TS t311 ST5 42T Throat – – P, G, T t3175 ST672 77N Nose + + P, O, CL, R t345 ST3206 97T Throat – – P, E, CL t376 ST80 82T Throat – – P, C t4188 ST199 33T2 Throat – – P, TS t442 ST5 107T Throat – – P t701 ST6 56T Throat – – P t878 ST2849 109N Nose – – P, G, E, CL t934 ST80The overall prevalence of PVL genes amongS. aureus isolates in Nepal and NEO was 25.0% (13/52; 95% CI 13.2%–36.7%) and 10.2% (9/88; 95% CI 3.8%-16.4%), respectively. A statistically significant higher prevalence of PVL-positive isolates was observed in Nepal compared to NEO (p=0.02). All PVL-positiveS. aureus (t1839, t345, t1094, t2663, t008, t213, t11906, and t005) from NEO were MSSA. Two-thirds of the PVL-positive isolates from NEO (66.7%; 6/9) and 46.2% (6/13) from Nepal were MDRSA. Only one PVL-positive isolate from NEO (t345) wasmecA-positive. ThreemecA-positive isolates from NEO belonged to ST1 (t127), ST6 (t304), and ST3206 (t345). OnemecA-positive isolate from Nepal belonged to ST8 (t008). AllmecA-positive isolates were also phenotypically resistant to oxacillin.


## 3.1. Demographic Information

A total of 400 samples (200 nasal and 200 throat) were collected from 200 participants (100 from each study site). The mean age of the participants was 30.7 (SD 10.9) years in NEO and 36.8 (SD 13.3) years in Nepal. In NEO, 58% and 42 % of the participants were male and female, respectively, while the percentage of male and female participants in Nepal was 29% and 71%, respectively. In Nepal, the majority of the participants was from the Kirat religion (79%), married (86%), and had a formal education (60%). In NEO, the majority of the participants had formal education (86%) (Table1).Table 1
Socio-demographic characteristics from NEO and Nepal.
Category  NEO (n=100) Nepal (n=100) P-value Age in years (mean, SD)  30.7, 10.9 36.8, 13.3 Gender Female 42 71 <0.05 Male 58 29 Religion Kirat 45 79 <0.001 Others 55 21 Marital Status Married 45 86 <0.001 Others 55 14 Education No formal Ed 14 40 <0.001 Had formal Ed 86 60 ED, education.


## 3.2. Prevalence ofS. aureus

The overall prevalence ofS. aureus was 66.0% (66/100; 95% CI 56.7%–75.3%) and 44.0% (44/100; 95% CI 34.3%–53.7%) in NEO and Nepal, respectively, for individuals colonized at either biological site. A statistically significant difference (p=0.002) was observed in the prevalence ofS. aureus between the two sampling sites.In Nepal, 39.0% (39/100; 95% CI 29.4%–48.5%) and 13.0% (13/100; 95% CI 6.4%–19.6%) of the participants were colonized withS. aureus in their throat and nose, respectively, while the prevalence ofS. aureus colonization in nose and throat in NEO was 25.0% (25/100; 95% CI 16.5%–33.5%) and 63.0% (63/100; 95% CI 53.5%–72.5%), respectively. The difference ofS. aureus colonization in nose and throat between Nepal and NEO was significant (p = 0.04 for nose and 0.001 for throat). The prevalence of positive isolates from both locations (nose and throat) from the same participants was 16% (7/44; 95% CI 5.1% - 26.7%) in Nepal and 33% (22/66; 95% CI 22% - 44.7%) in NEO. This difference was significant (= 0.04).The overall prevalence of MRSA (based on the presence of themecA gene) was 3.0% (3/100; 95% CI 0 – 6.3%) in Nepal and 1.0% (1/100; 95% CI 0 – 2.9%) in NEO. No significant difference was observed in MRSA prevalence between Nepal and NEO (p = 0.62, OR 0.32; 95% CI 0.03%–3.19%).Tables2 and 3 show participants’ demographics as well as medical, occupational, and environmental risk factors previously associated with the presence or absence ofS. aureus. In NEO, a higher prevalence ofS. aureus was found among males, participants who handled meat (pork and poultry) in the last six months, and participants who had skin infections in the last six months. In Nepal, a higher prevalence ofS. aureus was found among females, participants who had children attending day care, and found who were diagnosed with skin infections and autoimmune diseases (Tables 2 and 3).Table 2
Exposure variables in association withS. aureuscolonizationin NEO (n=100).
Variables SA+ (%) SA – (%) P-value Gender Male 42 (72.4) 17 (27.6) 0.13 Female 24 (57.1) 18 (42.9) Daycare going children Yes 10 (52.6) 9 (47.4) 0.18 No 56 (69.1) 25 (30.9) Skin infections (within 6 months) Yes 12 (75.0) 4 (25.0) 0.56 No 54 (64.3) 30 (35.7) Hospital admission(within 6 months) Yes 26 (61.9) 16 (38.1) 0.52 No 40 (69.0) 18 (31.0) Hospital visits (within 6 months) Yes 41 (60.3) 27 (39.7) 0.11 No 25 (78.1) 7 (21.9) Volunteers in health Institution Yes 3 (50.0) 3 (50.0) 0.40 No 63 (67.0) 31 (33.0) Pork handle (within 6 months) ≥1times per week 40 (71.4) 16 (28.6) 0.21 Do not handle 26 (59.1) 18 (40.9) Poultry handle (within 6 months) ≥1times per week 52 (71.2) 21 (28.8) 0.09 Do not handle 14 (51.8) 13 (48.2) Length of stay in the U.S. Less than 5 years 45 (70.3) 19 (29.7) 0.27 5 or more years 21 (58.3) 15 (41.6) Family size ≤5 members 29 (64.4) 16 (35.6) 0.83 >5 members 37 (67.3) 18 (32.7) Occupation Daily wages labor 35 (74.5) 12 (25.5) 0.13 Others 31 (58.5) 22 (41.5) Antibiotics use (within 6 months) Yes 7 (58.3) 5 (41.7) 0.52 No 59 (67.1) 29 (32.9) Skin infections (within 6 months) Yes 12 (75.0) 4 (25.0) 0.56 No 54 (64.3) 30 (35.7) Participated in Sports (within 6 months) Yes 26 (62.0) 16 (38.0) 0.52 No 40 (69.0) 18 (31.0)Table 3
Exposure variables in association withS. aureus colonization in Nepal.
Variables (n=100) SA+ (%) SA – (%) P-value Gender Female 34 (47.9) 37 (52.1) 0.27 Male 10 (34.5) 19 (65.5) Religion Kirat 37 (46.9) 42 (53.1) 0.32 Others 7 (33.3) 14 (66.7) Education Formal Education 27 (45) 33 (55) 0.83 No formal Education 17 (42.5) 23 (57.5) Marital status Married 36 (41.9) 50 (58.1) 0.38 Others 8 (57.1) 6 (42.9) Family size ≤5 members 26 (46.4) 30 (53.6) 0.68 >5 members 18 (41.0) 26 (59.0) Daycare Yes 11 (47.8) 12 (52.8) 0.81 No 33 (42.9) 44 (57.1) Nearest farm < 50meter 24 (41.4) 34 (58.6) 0.54 ≥50meters 20 (47.6) 22 (52.4) Hand wash frequency After each work 39 (46.4) 45 (53.6) 0.28 Before meal 5 (31.2) 11 (68.8) Skin infections (within 6 months) Yes 16 (51.6) 15 (48.4) 0.38 No 28 (40.6) 41 (59.4) Autoimmune disease (within 6 months) Yes 9 (60.0) 6 (40.0) 0.17 No 34 (41.0) 49 (59.0) Ear Infection(within 6 months) Yes 13 (39.0) 20 (61.0) 0.51 No 31 (46.0) 36 (54.0) Antibiotics use (within 6 months) Yes 8 (32.0) 17 (68.0) 0.16 No 36 (48.0) 39 (52.0) Skin disease (within 6 months) Yes 16 (51.6) 15 (48.4) 0.30 No 28 (40.6) 41 (59.4) Hospital admission(within 6 months) Yes 3 (30.0) 7 (70.0) 0.34 No 41 (45.6) 49 (54.4) Hospital visits (within 6 months) Yes 4 (25.0) 12 (75.0) 0.09 No 40 (47.6) 44 (52.4) Volunteers in health Institution Yes 12 (42.9) 16 (57.1) 0.88 No 32 (44.4) 40 (55.6) Pork handle (within 6 months) Yes 7 (31.8) 15 (68.2) 0.19 No 37 (47.4) 41 (52.6) Poultry handle (within 6 months) Yes 24 (40.0) 36 (60.0) 0.32 No 20 (50.0) 20 (50.0)


## 3.3. Antibiotic Susceptibility Testing

All isolates were tested for antibiotic susceptibility. All isolates in Nepal and NEO were resistant to penicillin. Oxacillin resistance was observed for 1.1% (1/89) and 5.8% (3/52) of the isolates in NEO and Nepal, respectively. Resistance to other antibiotics was higher for Nepal than Ohio. Additionally, MDRSA rates were higher in Nepal than Ohio (44.2% and 23.6%, respectively) (Figure1).Figure 1
Antibiotic resistance profile of isolates. MDR, multidrug-resistant.


## 3.4. Molecular Characterization ofS. aureus

spa typing was carried out on all positive isolates. In NEO, a total of 41spatypes were detected from 88S. aureus isolates. Overall, t1818 was the most commonspa type (12/88; 13.6%), followed by t002 (7/88; 8.0%), t104 (6/88; 6.8%), t008, t084, t085, t091 (4/88 each; 4.5%), and t521 (3/88; 3.4%). All otherspa types (t005, t10559, t1094, t11906, t122, t127, t131, t16153, t164, t1654, t1818, t1839, t190, t1911, t1931, t2119, t213, t2379, t2663, t273, t345, t4371, t442, t491, t521, t6127, t616, t688, t7264, t774, t803, t818, t878, t934, and t9432) were ≤ 2.2% ofS. aureus isolates.In Nepal, a total of 32spatypes were detected from 52S. aureus isolates. Overall, t345 was the most commonspa type (5/52; 9.6%), followed by t311, t2119 (4/52 each; 7.7%), t442, t127 (3/52 each; 5.8%), t021, t084, t091, t159, t164, and t934 (2/52 each; 3.8%). All otherspa types (t002, t008, t085, t1149, t11917, t12219, t1427, t15347, t15578, t15579, t15580, t15581, t1839, t2663, t304, t3175, t376, t4188, t701, t7766, t878) were less than 2.0% of the isolates.spa types were grouped based on their genetic proximity tospa types associated with specific cluster complexes. BURP grouping using default parameters (“exclude parameters that are shorter than 5 repeats” and “spa types are clustered if costs are less or equal than 4”) resulted in 7spa CCs and 11 singletons (t021, t127, t159, t164, t878, t1149, t2663, t11917, t15579, t15580, and t15581) in Nepal and 6spa CCs and 16 singletons (t005, t091, t104, t122, t127, t164, t190, t213, t616, t818, t878, t1654, t2663, t7264, t10559, and t16153) in NEO (Supplemental Figures 1-3).Thirty-nineS. aureus isolates from NEO and 29 from Nepal were subjected to MLST. A total of 26 different STs were detected from NEO. The most common ST was ST5 (17.9%; 7/39), followed by ST8 (10.3%; 4/39), ST1, ST15, ST3206, and ST96 (5.1% each; 2/39). No other STs constituted more than one testedS. aureus isolates (Table 4). A total of 22 different STs were detected from Nepal. The most common ST was ST5 (10.3%; 3/29), followed by ST15, ST3206, ST6, ST672, and ST80 (6.9 % each; 2/29). No other STs constituted more than one testedS. aureus isolate (Table 5).Table 4
Molecular characteristics of selectedS. aureus isolates from NEO (N=38).
Isolates Source mecA PVL AST spa MLST 27NEOT Throat – – P t002 ST5 89NEOT Throat – + P, G, C, L, T t005 ST672 33NEON Nose + – P, O, E t008 ST8 3NEOT Throat – – P, C, L, TS t084 ST2885 11NEON Throat – – P t085 ST845 38NEOT Throat – – P, TS t091 ST789 9NEOT Throat – – P, T t104 ST8 68NEOT Throat – – P t10559 ST2733 23NEOT Throat – + P, TS t1094 ST2112 80NEOT Throat – + P, C, L t11906 ST2884 63NEOT Throat – – P, E, CL t122 ST2102 8NEON Nose – – P t127 ST573 69NEON Nose – – P t131 ST1290 72NEOT Throat – – P t164 ST20 48NEOT Throat – – P t1654 ST667 30NEOT Throat – – P, T t1818 ST96 1NEOT Throat – + P, C, L, TS t1839 ST3206 82NEOT Throat – – P t190 ST8 70NEOT Throat – – P, TS t1931 ST1 84NEOT Throat – – P t2119 ST2871 88NEOT Throat – – P t2379 ST5 39NEOT Throat – + P, C, L, E, CL t2663 ST2233 18NEOT Throat – – P t273 ST1 1NEON Nose – + P, C, L, TS t345 ST3206 59NEOT Throat – – P t4371 ST5 4NEOT Throat – – P, TS t442 ST5 13NEOT Throat – – P, C, L, TS t491 ST199 9NEON Nose – – P, C, L, TS t521 ST96 96NEOT Throat – – P t6127 ST8 50NEOT Throat – – P t616 ST944 12NEOT Throat – – P t688 ST5 8NEOT Throat – – P t7264 ST5 55NEOT Throat – – P t774 ST15 79NEOT Throat – – P t803 ST15 28NEOT Throat – – P t818 ST5 29NEOT Throat – – P t878 ST2849 58NEOT Throat – – P, G, E, CL t934 ST80 16NEOT Throat – – P, C, L t9432 ST2128 P, benzylpenicillin; C, ciprofloxacin; E, erythromycin; CL, clindamycin; G, gentamicin; T, tetracycline; TS, trimethoprim-sulfamethoxazole; L, levofloxacin; O, oxacillin; AST, antibiotic susceptibility testing. Inclusion of antibiotic name denotes resistance.Table 5
Molecular characteristics of selectedS. aureus isolates from Nepal (N=29).
Isolates Source mecA PVL AST Spa ST 68T5 Throat – – P, TS t002 ST5 26T5 Throat – – P, C t11917 ST72 71T5 Throat – – P t15579 ST2885 74N Nose – – P, C, L, E, CL t15581 ST3206 19N Nose – – P t008 ST8 101N Nose – + P, L, M, TS t021 ST1482 102T Throat – – P, TS t084 ST15 76T Throat – – P t085 ST15 12T Throat – – P t091 ST2081 24N Nose – – P, C, L t1149 ST291 105T Throat – + P, G, C, L, E t12219 ST672 83T Throat + – P, O, C, E, CL, Mi, T, R t127 ST1 48T Throat – – P, G, E, CL t1427 ST361 70T Throat – – P t15578 ST2990 1T Throat – – P, E, CL, TS t159 ST800 85T Throat – – P, G, TS t164 ST20 67T Throat – + P, TS t1839 ST573 65T Throat – – P, TS t2119 ST2871 106T Throat – + P, G, C, L, E t2663 ST2233 15T Throat + – P, O t304 ST6 32N Nose – + P, TS t311 ST5 42T Throat – – P, G, T t3175 ST672 77N Nose + + P, O, CL, R t345 ST3206 97T Throat – – P, E, CL t376 ST80 82T Throat – – P, C t4188 ST199 33T2 Throat – – P, TS t442 ST5 107T Throat – – P t701 ST6 56T Throat – – P t878 ST2849 109N Nose – – P, G, E, CL t934 ST80The overall prevalence of PVL genes amongS. aureus isolates in Nepal and NEO was 25.0% (13/52; 95% CI 13.2%–36.7%) and 10.2% (9/88; 95% CI 3.8%-16.4%), respectively. A statistically significant higher prevalence of PVL-positive isolates was observed in Nepal compared to NEO (p=0.02). All PVL-positiveS. aureus (t1839, t345, t1094, t2663, t008, t213, t11906, and t005) from NEO were MSSA. Two-thirds of the PVL-positive isolates from NEO (66.7%; 6/9) and 46.2% (6/13) from Nepal were MDRSA. Only one PVL-positive isolate from NEO (t345) wasmecA-positive. ThreemecA-positive isolates from NEO belonged to ST1 (t127), ST6 (t304), and ST3206 (t345). OnemecA-positive isolate from Nepal belonged to ST8 (t008). AllmecA-positive isolates were also phenotypically resistant to oxacillin.


## 4. Discussion

In this cross-sectional study, we investigated the prevalence and molecular epidemiology ofS. aureus among Bhutanese refugees living in Nepal (n=100) and in NEO (n=100). The overall prevalence ofS. aureus was lower in Nepal (44.0%) compared to NEO (66.0%). TheS. aureus and MRSA prevalence of this study in Nepal is similar to previous studies that documented between 15.7%–43.8%S. aureus carriage rates and 2.3%–7.5% MRSA nasal colonization among healthcare workers and patients in Nepal [37–42]. However, the nasal carriage ofS. aureus (13.0%) and MRSA (1.0%) in Nepal is lower than in a prior study conducted in a community setting in Nepal [43]. A previous study examining the prevalence of MRSA in a school children in Pokhara City of Nepal via nasal swab documented 17.4% (32/184) overall MRSA prevalence [43]. Such differences inS. aureus and MRSA prevalence may be due to geographical, population, and methodological differences. For example, we defined MRSA based on the presence ofmecA gene, our sampling took place in eastern Nepal, and participants had to be at least 18 years old to be eligible for our study. Work by Rijal et al. (2008) took place in western Nepal, enrolled only minors (younger than 15 years old), and used a phenotypic method (Kirby-Bauer) for MRSA designation. Although the prevalence of MRSA in NEO is consistent with a previous report that suggested 0.84% of the noninstitutionalized US population were colonized with MRSA, the prevalence ofS. aureus is significantly higher in this study (31.6% versus 66.0%) compared to that report [5].S. aureus prevalence in our study was not associated with demographic factors such as age, gender, marital status, occupation, education, and income in both locations, nor with reported previous antibiotic exposure. The odds ofS. aureus colonization among participants in NEO was 2.5 times higher than colonization in Nepal (OR = 2.58, 95% CI: 1.45%–4.58%, p <0.001). However, variability in participants’ demographic characteristics as well as length of residency in the US may have contributed to these differences. For example, the demographic composition of participants from Nepal was different from those in NEO in regard to gender, religion, marital status, and education. No significant difference was observed inS. aureus colonization among participants who lived less than 5 years in NEO.The prevalence ofS. aureus in the nose and throat was higher in NEO than in Nepal (p=<0.05). Although the anterior nares are considered as the most common site ofS. aureus colonization [6], the result of this study showed that the colonization ofS. aureus was higher in throat compared to the nose in both locations of the study. This result is in consistent with a previous study done in Sweden that found throat as more common site of colonization [44].Of the 18 drugs tested, resistance was observed to 9 and 12 different antibiotics in NEO and Nepal, respectively (Figure1). Compared to NEO, a significantly higher proportion of isolates (23.6% versus 44.2%) were MDRSA in Nepal. The higher proportions of isolates were resistant to different classes of antibiotics in Nepal as compared to NEO.S. aureus were frequently resistant to drugs that are used commonly in Nepal. For instance, ciprofloxacin is one of the most commonly prescribed antibiotics in Nepal [45], and trimethoprim/sulfamethoxazole is one of the most preferred antimicrobial in Nepal due to its wide spectrum and low cost [46]. One of the studies done in a tertiary care hospital in western Nepal demonstrated that approximately 56% and 27% of the MRSA isolates were resistant to trimethoprim/sulfamethoxazole and ciprofloxacin, respectively [24]. In Nepal, 42% and 27% of the isolates were resistant to trimethoprim/sulfamethoxazole and ciprofloxacin, respectively. Antibiotics overuse is a common drug use problem in Nepal [47]. For example, a study conducted in eastern Nepal documented that 84% of the prescriptions contained antibiotics comprising 42.8% of the total number of drugs [48]. In NEO, 22.5% and 18.0% of the isolates were resistant to trimethoprim/sulfamethoxazole and ciprofloxacin, respectively.The prevalence of PVL-positive isolates was significantly higher among refugees living in Nepal compared to NEO (p=0.02). This is consistent with prior publications demonstrating a high prevalence PVL-positive isolates in clinical specimens in Nepal [49, 50].spa type t345 (ST 3206) was the most commonspa type in Nepal.spatype t345 was previously isolated from patients infected withS. aureus in Banglore, India [51].spa type t1818 (ST96) was most common in NEO.spa type t1818 has been previously isolated from clinical specimens in France [52], clinical samples from intensive care units of a tertiary hospital in Korea [53], and clinical samples from intensive care unit patients in the Netherlands [54].Spa type t345 was found in both locations. However,spa type t1818 was found only in NEO. Only one isolate ofspa types t002 (ST5) and t008 (ST8) each was found in Nepal. In NEO, 7 (8.0%; 7/88) and 4 (4.5%; 4/88) isolates were t002 and t008, respectively.spa type t002 (ST5/USA100) and t008 (ST8/USA300) are common hospital-associated and community-associatedS. aureus stain, respectively. ST5 was the most common sequence type in both locations.There are number of limitations to this study. This study employed a convenience sampling method to collect a limited number of human samples from populations in narrow geographic locations. As such, the generalizability of these findings is limited. Since the study design is cross-sectional, it does not allow for causal inference between exposures and outcomes. Although most of the medical and occupational risk factors were associated withS. aureuscolonization, the association was not statistically significant. Hence, the study was not able to establish the demographic as well as medical and occupational exposures potentially implicated withS. aureus colonization. Since the study participants in Nepal and NEO are different and resettled refugees had varying duration of time regarding their residency in NEO, there might be other confounding factors affectingS. aureus colonization. The lack of longitudinal follow-up limits the understanding of the dynamics of persistent colonization ofS. aureus and MRSA in the study population as well as the effects of environmental and occupational exposure over time.The strengths of the study include the acquisition of samples from two locations that enrolled participants from the same ethnocultural heritage. The inclusion of survey questions in addition to human samples provided information concerning exposure variables. To our knowledge, this is the first study conducted to assess the prevalence and molecular characterization ofS. aureusin immigrant population in the United States and in a community setting in Nepal. Follow-up studies could examine changes in colonization in a longitudinal manner as immigrants are resettled in Ohio or other areas in the US.


## 5. Conclusions

In conclusion, the findings of this study indicate that Bhutanese refugees living in Nepal aand resettled in NEO had high prevalence ofS. aureus and MDRSA carriage.S. aureus colonization in throat was significantly higher than nasal colonization in both locations. The prevalence of MSSA was higher among resettled Bhutanese refugee living in NEO as compared to those living in Nepal. Information aboutS. aureus epidemiology in developing countries is important, as almost 1 billion people cross international boundaries each year. Population mobility imposes threats to the distribution of antimicrobial drug-resistant organisms [10]. MRSA has been isolated from immigrant people in Europe [55, 56]. For example, a total of 56 persons who had recently arrived in Sweden between 2002 and 2003 as immigrants had cases of MRSA, giving an overall risk of 15.9 cases/100,000 immigrants [56]. In Spain, of the 19 patients infected with CA-MRSA, 15 were immigrants from South America [55]. The findings of this study emphasize the potential need for surveillance among immigrant population in the US and among people living in Nepal and a potential need to devise public health interventions to mitigate the risk imposed byS. aureusinfections.



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*Source: 5739247-2019-07-01.xml*