peerj_json_files/PeerJ_Json_127.json

{
    "v1_Abstract": "This study aimed to characterize Staphylococcus aureus (S. aureus) strains isolated from human infections in Mongolia. Infection samples were collected at two time periods (2007-08 and 2011) by the National Center for Communicable Diseases (NCCD) in Ulaanbaatar, Mongolia. S. aureus isolates were characterized using polymerase chain reaction (PCR) for mecA, PVL, and sasX genes and tested for agr functionality. All isolates were also spa typed. A subset of isolates selected by frequency of spa types was subjected to antimicrobial susceptibility testing and multilocus sequence typing. Among 251 S. aureus isolates, genotyping demonstrated methicillin resistance in 8.8% of isolates (22/251). Approximately 28% of the tested S. aureus isolates were observed to be multidrug resistant (MDR). Sequence type (ST) 154 (spa t667) was observed to be a strain with high virulence potential, as all isolates for this spa type were positive for PVL, had a functional agr system and 78% were MDR. S. aureus isolates of ST239 (spa t037) were observed to cause infections and roughly 60% had functional agr system with a greater proportion being MDR. Additionally, new multilocus sequence types and new spa types were identified, warranting continued surveillance for S. aureus in this region.",
    "v2_Abstract": "This study aimed to characterize Staphylococcus aureus (S. aureus) strains isolated from human infections in Mongolia. Infection samples were collected at two time periods (2007-08 and 2011) by the National Center for Communicable Diseases (NCCD) in Ulaanbaatar, Mongolia. S. aureus isolates were characterized using polymerase chain reaction (PCR) for mecA, PVL, and sasX genes and tested for agr functionality. All isolates were also spa typed. A subset of isolates representing frequent spa types was subjected to antimicrobial susceptibility testing and multilocus sequence typing. Among 251 S. aureus isolates, genotyping demonstrated methicillin resistance in 8.8% of isolates (22/251). Approximately 28% of the tested S. aureus isolates were observed to be multidrug resistant (MDR). Sequence type (ST) 154 (spa t667) was observed to be a strain with high virulence potential, as all isolates for this spa type were positive for PVL, and 7/9 were MDR. S. aureus isolates of ST239 (spa t037) was observed to cause infections and mostly (80%) exhibited agr dysfunction with a high multidrug resistance profile. Additionally, a new multilocus sequence type ST2600 and new spa types (t10358, t10064, and t10066) were identified, warranting continued surveillance for S. aureus in this region.",
    "v2_col_introduction": "introduction  : In the past fifty years, Staphylococcus aureus (S. aureus) has established itself as one of the most frequent antibiotic resistant bacterial pathogens in hospitals and communities (Boucher & Corey 2008). S. aureus typically causes skin and soft tissue infections, but can also cause invasive infections such as bacteremia, sepsis, endocarditis, pneumonia, osteomyelitis, etc. (Hidron et al. 2008; Liu et al. 2011). In earlier years, S. aureus infections were commonly observed in individuals with a history of exposure to hospitals (David & Daum 2010). There has been a major epidemiologic transition since the mid-1990s when S. aureus was observed to cause infections in population with no known risk exposures (Bancroft 2007). The emergence of community-associated S. aureus has further magnified the challenge of S. aureus prevention and treatment practices (David & Daum 2010). Methicillin-resistant S. aureus (MRSA) is associated with the rise in attributable mortality due to staphylococcal infections (Cooper et al. 2004). Surveillance studies have observed a considerable difference in proportions of MRSA invasive infections in Europe ranging from < 1% in Denmark and the Netherlands to 44% in the United Kingdom and Greece (Cooper et al. 2004; Kock et al. 2010). Nationwide surveillance for invasive MRSA infections conducted in the United States reported about 94,000 cases resulting in approximately 18,000 deaths (Klevens et al. 2007). Worldwide, rates of MRSA have been increasing as observed from data obtained via surveillance initiatives by the National Nosocomial Surveillance System (NNIS) and the European Antimicrobial Resistance Surveillance System (EARSS) (Fridkin et al. 2002; Grundmann et al. 2006; Tiemersma et al. 2004; Turnidge & Bell 2000). Nevertheless, a major concern is the lack of data in many countries, particularly the developing countries, as this could potentially result in global transmission of undetected MRSA strains (Azeez-Akande 2010; Molton et al. 2013).\n2\n47 48\n49\n50\n51\n52\n53\n54\n55\n56\n57\n58\n59\n60\n61\n62\n63\n64\n65\n66\n67\n68\n69\nPeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013)\nR ev ie w in g M an\nus cr ip t\nS. aureus infections in Mongolia\nMongolia is a relatively small country in North-East Asia locked for the most part between China in the south and Russia in the north with a population of approximately 2.8 million (Bataar et al. 2010; Ider et al. 2010; Mongolia 2013). The capital city of Ulaanbaatar is home to roughly half of the country\u2019s population (Bank 2011). Infectious diseases still figure in the top 10 causes of death in the country, with sepsis being a common diagnosis among ICU patients (Bataar et al. 2010). The evolving political and economic changes in Mongolia have impacted the working of laboratory networks, data collection and management systems, and training of healthcare professionals in identification and prevention of hospital-acquired infections (Ider et al. 2010). There has also been a disruption of funds to hospitals to conduct surveillance for multidrug-resistant organisms. Due to insufficient laboratory capacity, hospitals in Mongolia use culture testing methods only when empiric therapy fails (Mongolia 2007). This process could potentially propagate antimicrobial resistance in pathogens such as S. aureus. In addition, a large herder population in Mongolia with a livestock population of 43 million animals increases the risk of transmission of zoonotic infections (Mongolia 2010). A community-based survey conducted by the World Health Organization (WHO) in Ulaanbaatar between March and April 2009 observed a prevalence of 42% in the use of non-prescription antibiotics among children less than 5 years of age. This proportion is much higher than other regions such as rural communities in Vietnam (12%) and a Chinese city (36%) (Togoobaatar et al. 2010). The study found approximately 50% of the children in participating households were prescribed antibiotics, of which roughly 51% children were given both prescribed and non-prescribed antibiotics by their caregiver (Togoobaatar et al. 2010). In a developing country such as Mongolia, unconditional use of antibiotics is of particular concern (Stefani & Goglio 2010). Selective pressures such as the unrestricted use of antibiotics and inadequate compliance to antibiotic regime in conjunction with inadequate surveillance for antimicrobial resistance are\n3\n70\n71\n72\n73\n74\n75\n76\n77\n78\n79\n80\n81\n82\n83\n84\n85\n86\n87\n88\n89\n90\n91\n92\n93\nPeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013)\nR ev ie w in g M an\nus cr ip t\nS. aureus infections in Mongolia\nsome of the important reasons for the emergence of highly resistant S. aureus strains (Grundmann et al. 2006; Stefani & Goglio 2010). High population density, urbanization, inadequate infection control policies, exploding antibiotic use, and lack of appropriate healthcare delivery are some of the established social risk factors for colonization and transmission of S. aureus strains in hospitals and communities (Charlebois et al. 2002; Chen et al. 2011; Clements et al. 2008; Henderson 2006; Rehm & Tice 2010). There are very few studies in the published literature on the epidemiology of S. aureus in Mongolia. A study conducted in 2006 in Ulaanbaatar analyzed S. aureus infection isolates obtained from four university hospitals (Orth et al. 2006). Analysis using molecular methods and antibiotic susceptibility testing in isolates from this study determined the prevalence of MRSA to be very low (2.9%) (Orth et al. 2006). However, this study only included isolates collected between 2000 and 2002 and characterized only the six MRSA isolates identified in their cohort of S. aureus isolates. The aim of our study is to bridge the gap in S. aureus literature from Mongolia, and determine the S. aureus molecular epidemiology and antimicrobial resistance patterns in Mongolia.",
    "v1_text": "materials and methods : This is an observational study conducted during two time periods (2007-08 and 2011) to investigate the prevalence of MRSA infections, and to characterize the S. aureus strains causing these infections in Mongolia. The University of Iowa IRB evaluated this project and determined that it did not qualify as human subjects research. To accomplish the study objective, we collaborated with the National Center for Communicable Diseases (NCCD) in Ulaanbaatar. The NCCD has an established Hospital Related Infection Surveillance and Research Unit (HRISRU) (Ider et al. 2010). This study characterized S. aureus isolated from human infections in a convenience sample obtained from the NCCD. In 2007-08, the NCCD transported a convenience sample of \u201cpotentially confirmed\u201d S. aureus isolates to the Centers for Emerging Infectious Diseases (CEID) at Coralville, IA as a collaborative project to characterize these infection isolates. Additionally, a study team member who traveled to Mongolia in 2011 tested potential S. aureus infection isolates banked in 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t Ulaanbaatar and shipped back frozen isolates to the CEID. Patient information such as age, gender, and sample type were available only for the 2007-08 infection samples. Isolates were labeled \u201cwound\u201d if collected from surgical site infections or wound samples. All urine samples collected in 2007-08 were obtained from voided urine i.e. none of these patients were catheterized. Biochemical testing and DNA isolation Isolates were grown and re-confirmed to be S. aureus at the CEID, as described previously (O'Brien et al. 2012). All S. aureus isolates were frozen and stored in glycerol broth solution at -800C for future use. S. aureus DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison WI) following manufacturer\u2019s instructions. Antimicrobial Susceptibility Testing (AST) The antimicrobial susceptibility of isolates were tested by the broth microdilution method in accordance with the Clinical Laboratory Standards Institute (CLSI) standards (CLSI 2012). Isolates were tested for susceptibility to the following 11 antimicrobials: oxacillin, gentamicin, erythromycin, clindamycin, tetracycline, trimethoprim/sulfamethaxozole (TMP/SMX), imipenam, levofloxacin, linezolid, vancomycin, and daptomycin. Resistance to high-level mupirocin and inducible clindamycin resistance (ICR) were also examined. All AST-confirmed MRSA isolates were considered to be multidrug resistant (MDR). MSSA isolates non-susceptible to \u2265 1 antimicrobial agent in \u22653 discrete antimicrobial categories were also classified as MDR, as per a recently published report on standardization of bacterial antimicrobial resistance profiles (Magiorakos et al. 2012). S. aureus genetic analysis Amplification of the spa fragment was performed using methods and primers described previously (Shopsin et al. 1999). Identification of the spa type for each isolate, the Based Upon Repeat Pattern (BURP) analysis to identify spa cluster complexes (spaCCs), and calculation of 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t the diversity index (Hunter & Gaston 1988) and corresponding confidence intervals (Grundmann et al. 2001) was performed using the Ridom StaphType software (version2.2.1; Ridom GmbH, W rzburg, Germany) (\u1e7b Harmsen et al. 2003; Mellmann et al. 2008; Mellmann et al. 2007; Strommenger et al. 2008). All isolates were tested for the Panton-Valentine leukocidin (PVL) (lukS-PV and lukF-PV) (Lina et al. 1999), mecA (Bosgelmez-Tinaz et al. 2006), and the sasX gene as previously described (Holden et al. 2010; Li et al. 2012). Identified positive and negative controls were used in all molecular assays. Accessory Gene Regulator ( agr ) testing agr functionality (functional or dysfunctional) was measured using the level of \u03b4-hemolysin production, as described previously (Sakoulas et al. 2002; Schweizer et al. 2011; Traber & Novick 2006). agr positive and negative reference strains were used as controls to ensure validity of our findings. Multi-locus sequence typing (MLST) The allelic profile of S. aureus isolates were determined as described previously (Enright et al. 2000). At least one representative isolate from the most frequent spaCCs, rare spaCCs, spa types such as t037 that could potentially have two sequence types, or when available a MRSA isolate was selected for MLST analysis from each time period. Statistical analysis Data analysis was performed using the SAS statistical software (Version 9.3, SAS Institute Inc., Cary, NC). We used the 2-tailed Fisher\u2019s exact test, and the Wilcoxon signed-rank test to analyze categorical and continuous variables, respectively. P values \u2264 0.05 were considered statistically significant for associations between explanatory variables such as age, gender, and type of infection and S. aureus spa type, mecA, PVL and agr functionality. Antimicrobial susceptibility results were analyzed by year of data collection and methicillin-resistance status to observe trends in resistance for each tested antibiotic. Association between S. aureus antimicrobial susceptibility, 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t MDR, mecA, PVL, agr functionality, and spa types were also assessed. Odds ratio (OR) and 95% confidence interval (CI) was reported for significant associations. Results Patient and sample characteristics In total we analyzed 252 potential S. aureus isolates, 198 from 2011 and 54 from 2007-08 isolate collections. Of these, 251 were confirmed to be S. aureus isolates. Patient demographics and sample characteristics were available only for the 53 isolates collected in 2007-08. The age of patients ranged from 1day- 82 years (median: 24 years). Of the 53 patients, 31 (58.5%) were females and 22 (41.5%) males. Approximately 43% of the S. aureus were isolated from wound samples. Molecular typing We observed a greater proportion of isolates from the 2007-08 collection to be positive for the mecA gene. The cohort of S. aureus isolates from 2007-08 had greater proportion of PVL positivity in both the MSSA and MRSA isolates, with 59% of MRSA isolates being positive for the PVL gene (Table 1). S. aureus isolates were observed to have a greater proportion of functional agr in both time periods, albeit the proportions were higher for MSSA and MRSA in the 2011 collection. We observed that mecA positive S. aureus isolates had a 77% less likelihood of having a functional agr (OR=0.23, 95% CI 0.067, 0.79; p=0.033). A borderline significance was observed between age and PVL positivity in that most of our PVL-positive S. aureus isolates were obtained from older individuals (p=0.045, other data not shown). All isolates tested negative for the sasX gene. Antimicrobial susceptibility patterns A subset of S. aureus isolates (80/251, ~32%) were tested for antimicrobial susceptibility, based on the frequency of spa types. The proportion of S. aureus isolates that were MRSA (MDR) was greater in 2011 (Fig 1A). Interestingly, we did not observe any S. aureus isolates that belonged to 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t the MSSA-MDR category in 2011. Isolates in both years have comparable proportions of MSSA that do not meet the MDR criteria. There was no significant difference in the proportion of MDR isolates between the two study periods (p=0.092). Overall, the proportion of antibiotic resistance in tested S. aureus isolates was 38.8%. Greater proportion of MRSA isolates in 2011 was observed to be resistant to erythromycin, tetracycline, levofloxacin and TMP/SMX (Fig 1B). We also observed a wider spectrum of resistance in the 2011 MRSA isolates as there is additional resistance to gentamicin, imipenam, and ICR. MSSA isolates were observed to be proportionately more resistant to erythromycin and tetracycline in 2011 while resistance to levofloxacin, erythromycin and TMP-SMX was observed in 2007 isolates (Fig 1C). We did not identify high-level mupirocin resistance in our isolates. There was good concordance in mecA positivity and phenotypic expression of oxacillin resistance among tested S. aureus isolates. Three isolates were observed to have discordant oxacillin-resistance phenotype-genotype (data not shown). We observed that S. aureus isolates resistant to 3 or more antimicrobials had significantly lower odds of having a functional agr system (OR=0.166, 95% CI 0.046-0.59, p=0.014). Of the 22 MDR isolates, roughly 55% (12/22) were positive for the PVL gene, about 86% (19/22) positive for the mecA gene, and approximately 64% (14/22) had a functional agr phenotype. spa type distribution (BURP) and Multilocus sequence type (MLST) Eleven confirmed S. aureus isolates were identified as \u201cnon-typeable\u201d after at least two attempts to sequence the spa gene. Of these, 8 isolates demonstrated bands on amplification of the spa fragment with the published forward primer as well as an alternate forward primer (Molla et al. 2012). However, the RIDOM StaphType software could not assign spa types, possibly due to deviating repeats as demonstrated in previous studies (Baum et al. 2009; Sorum et al. 2013). The remaining 3 isolates demonstrated double bands for the spa gene as observed in a study 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t conducted by Shakeri et al (Shakeri et al. 2010), which could not give spa types even after three attempts on PCR or purification of bands from an agarose gel. We identified 63 distinct spa types among the 251 S. aureus isolates (Table 2). The most common spa types in 2007-08 were t589 (13%), t3465 (13%), and t435 (11%). spa types t435 (10%), t589 (8%), t5288 (7.5%), and 7% each t1460 and t8677 were the most frequently occurring strains in 2011. spa types belonging to spaCC 667 was observed to be the most frequently isolated MRSA strain while MSSA isolates were frequently of the spaCC 589 type. S. aureus isolates of spa types t589, t435, t037 and t667 were observed in both MRSA and MSSA groups (Table 2A & 2B). We identified 8 different spa types among the MRSA isolates and 58 spa types among the MSSA isolates. The diversity index was higher for MSSA isolates (0.952) compared to MRSA isolates (0.865), but due to the small sample size of MRSA isolates the confidence intervals overlap (Table 3). BURP analysis revealed clustering around putative founder spa types t589 (2011), t435 (2007-08 and 2011), t3465 (2007-08), and t8677 (2011) (Fig 2). We identified three new MSSA spa types (t10064, t10066, and t10358) in the 2011 collection. spaCC 667 constituted only 5% of all the strains. Nevertheless, isolates in this group had high PVL prevalence (100%), high mecA prevalence (72.7%), 100% functional agr isolates, and high multi-drug resistance. spaCC 037 also had high multidrug resistance but had lower presence of PVL (10%), mecA (50%) and functional agr (60%). Singleton spa types were t002, t126, t156, t521, t647, t803, t1451, t3329 and t8039. None of the tested singletons appeared to be MDR. A subset of S. aureus isolates from each time period was tested by MLST revealing the following information: t021 (ST30), t037 (ST239), t084 (ST15), t435 (ST121) and t667 (ST154). spa types t1460, t5288, and t589 were observed to be ST45 in our study. One t589 MRSA isolate from the 2011 collection was identified to be a new sequence type ST2600, a double-locus 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t variant (DLV) of ST45 as indicated by MLST. In addition, isolates belonging to spa types t8677 and t3465 in our sample set have novel arcC and aroE alleles and was categorized as ST2737 by the MLST database curator. Discussion The prevalence of MRSA among clinical S. aureus isolates obtained from Mongolia was 8.8%. A study conducted in 2006 in Mongolia analyzing S. aureus isolates collected between 2000-02 found a low prevalence of MRSA (2.9%) by susceptibility testing (Orth et al. 2006). Our study is based on a convenience sample of Mongolian S. aureus isolates. Nevertheless, we could infer with caution that there may be an increase in the prevalence of MRSA in Mongolia, reflected by the increase in MDR-MRSA with the relative absence of MDR-MSSA isolates in our sample set. Due to lack of published S. aureus data from Mongolia we compared our results to studies from China and Russia since there is a potential for transmission given its geographical proximity. China reported a prevalence of 50.4% for MRSA in 2005 with considerable variations even within the country (Chu et al. 2013; Song et al. 2013; Wang et al. 2008; Yu et al. 2012; Zhao et al. 2012). The proportion of methicillin resistance reported from S. aureus in Russia varied from 18% (Vorobieva et al. 2008) to 48% (Baranovich et al. 2010). Our study data observed a lower prevalence of MRSA relative to the neighboring countries. Nevertheless, given the convenience sample this may be an underestimate of the \u2018true\u2019 prevalence of MRSA in Mongolia. Our study observed genetic diversity in the MSSA isolates compared to the MRSA isolates. However, we note that the observed diversity could not be concluded to be statistically significant given the small sample size of MRSA isolates resulting in overlap of confidence intervals. A limited number of spa types were common in both groups and these are not genetically unrelated strains. This observation of common spa types is consistent with findings from other studies (Hallin et al. 2007; Strommenger et al. 2008), and supports the view that MRSA could potentially emerge from existing MSSA clones by acquisition of the SCCmec complex (Enright 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t et al. 2002; Robinson & Enright 2003). It is more likely, however, that in Mongolia, MRSA infections are increasing in prevalence due to strain replacement, not SCCmec acquisition, which is supported by the presence of MDR-MRSA without any MDR-MSSA in 2011. We are unable to conclude this hypothesis of strain replacement with certainty, hence it is crucial to implement surveillance protocols for S. aureus in Mongolia, as there are a myriad of factors contributing to antimicrobial resistance. We observed a significant association between antimicrobial resistance and functionality of the agr system suggesting a potential influence of antimicrobial resistance on the fitness of the pathogen via the agr system, or vice versa (Paulander et al. 2013). In addition, there appears to be a significant association between presence of the gene for methicillin resistance and having a dysfunctional agr system. Evidence from our study on the impact of drug resistance on the regulation of S. aureus virulence is consistent with findings from previous studies that observed changes in S. aureus cell wall induced by methicillin. This process was observed to affect the bacterial quorum sensing system leading to reduced virulence by suppression of toxins (Hao et al. 2012; Rudkin et al. 2012). These findings could potentially influence treatment options for S. aureus infections in Mongolia by considering the trade-off between fitness of the strain and its range of antimicrobial resistance. Our data suggests that the MRSA clone ST239-spa t037 is being transmitted amongst the population. This MLST type was also reported in the previous study from Mongolia, suggesting that ST239 could potentially be the dominant MRSA clone circulating in the country (Orth et al. 2006). ST239, a S. aureus bacterial hybrid formed by the admixture of MRSA clonal complexes ST30 and ST8 has been reported to be the dominant hospital clone in Asia (Aires de Sousa et al. 2003; Baranovich et al. 2010; Song et al. 2013; Xu et al. 2009; Yamamoto et al. 2012; Yu et al. 2012), Europe (Alp et al. 2009; Szczepanik et al. 2007; Wisplinghoff et al. 2005), South America (Carvalho et al. 2010; Vivoni et al. 2006), and the Middle East (Cirlan et al. 2005) and even 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t responsible for an outbreak of device-associated bacteremia in Europe (Edgeworth et al. 2007). In accordance with previous reports, all three identified ST239 S. aureus strains in our study were observed to be MDR (Smyth et al. 2010). We also observed S. aureus sequence types ST45 and ST121, which have previously been observed to be to be associated with S. aureus infections in other regions of the world, circulating in Mongolia. The proportion of multidrug resistance was observed to be higher in 2011 relative to 2007-08, albeit this difference was not statistically significant. Reports on S. aureus multidrug resistance observed variable rates ranging from ~29% - 100% from China (Chao et al. 2013; Chen et al. 2009; Wang et al. 2012) and about 90% in Russian MRSA isolates (Baranovich et al. 2010).. Studies observed that presence of the sasX gene in S. aureus potentially increased its virulence capacity by boosting the bacterial defense mechanism, particularly in the ST239 clones (Li et al. 2012). Furthermore, testing of S. aureus isolates from three teaching hospitals in eastern China demonstrated that the proportion of S. aureus strains that were non-ST239 and positive for the sasX gene increased from 5% to 28% between 2003-05 and 2009-11 (Holden et al. 2010; Li et al. 2012). None of our isolates, including the identified ST239 isolates, exhibited the presence of the sasX gene, suggesting its prevalence may be low or absent in Mongolia. We did not identify any known livestock-associated strains in our isolate collection, although several reports from China have observed the presence of these strains (Wagenaar et al. 2009; Zhao et al. 2012). Our study has several limitations. S. aureus isolates were collected as a convenience sample and could not be consistently linked to important patient information, particularly the 2011 collection that had a larger sample size for S. aureus isolates. This could have potentially resulted in inclusion of duplicate patient isolates and over-representation of S. aureus strains. In addition, isolates from both time periods were not collected in a systematic manner adding to the potential selection bias. Hence, results from this study may reflect only a snapshot of the \u2018true\u2019 estimate of S. aureus infections in Mongolia. Conclusions drawn from this study could be used as 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t preliminary results to design studies that are sufficiently powered to validate the observed associations. Nevertheless, there are not many studies from Mongolia and our study adds valuable information on the molecular epidemiology of S. aureus infections in Mongolia. Another drawback of our study was the inability to differentiate the potential origin of S. aureus strains as healthcare associated (HCA-) versus community associated (CA-) since we did not have access to the date of admission before S. aureus isolation from the infection. Given that in recent times there has been a gradual blurring in the origin of S. aureus strains, the reliability of this differentiation may be questionable (Mera et al. 2011). In addition, we also did not test all S. aureus isolates in our collection for antimicrobial susceptibility that could have resulted in loss of information on \u2018true\u2019 antimicrobial susceptibility trends. Conclusions In summary, our study observed an increasing prevalence of MRSA (8.8%) by AST, and recorded MDR rate of 28% in S. aureus isolates from Mongolia. We also observed the presence of previously identified S. aureus strains such as ST239 and ST30 adding its virulence potential to an existing burden of antimicrobial resistance. Regular surveillance and implementation of stricter policies for antimicrobial use is warranted to prevent further transmission of S. aureus in Mongolia. Acknowledgements: We thank Dr. Michael Otto and his lab for kindly providing the sasX positive control for molecular analysis, Dr. Marin L. Schweizer for sharing her expertise on the agr system and her time to train a graduate student on agr testing methods, and Dr. Brett Forshey for his assistance and inputs with statistical analysis. We also acknowledge and thank our collaborators at the NCCD in Mongolia for their assistance with sample collection, sample shipping, assimilation and communication of patient data, and review of the manuscript. 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t figure legends : Figure 1A: Illustration for multidrug resistance in Mongolia S. aureus isolates. MRSA = methicillin-resistant S. aureus, MSSA = methicillin-susceptible S. aureus, MDR = multidrug resistant, MSSA-not MDR category include MSSA isolates that are non-susceptible to \u2265 1 antimicrobial agent in < 3 discrete antimicrobial categories Figure 1B: Graph for antimicrobial resistance in MRSA isolates. *TMP-SMX = trimethoprim/sulfamethaxozole, MRSA = methicillin-resistant S. aureus, ICR = inducible clindamycin resistance Resistance to rifampicin and complete resistance to clindamycin were not observed Figure 1C: Graph for antimicrobial resistance in MSSA isolates. *TMP-SMX = trimethoprim/sulfamethaxozole, MSSA = methicillin-susceptible S. aureus No resistance was observed to gentamicin, rifampicin, imipenam, clindamycin or inducible clindamycin resistance Figure 2: Population snapshot for MRSA & MSSA Based-Upon Repeat Pattern (BURP) analysis. BURP grouping using default parameters resulted in 8 spaCCs and excluded 5 spa types (t026, t132, t517, t2493, and t10358). Each dot represents a unique spa type. Diameter of a dot is proportional to the quantity of corresponding spa type. Blue dots = group putative founders (i.e. spa type with the highest score within the CC). Yellow dots = putative subfounders with second highest score. If two or more spa types have the same highest founder score they are illustrated in blue. The distance between linked and/or unlinked spa types do not concern the genetic distance between them. pvl : Positive 40 (85.1) 5 (83.3) 1.00 55 (30.2) 8 (50) 0.16 Negative 7 (14.9) 1 (16.7) 127 (69.8) 8 (50) agr* Functional 37 (86.1) 2 (66.7) 0.39 175 (96.2) 13 (81.3) 0.037 Dysfunctiona l 6 (13.9) 1 (33.3) 7 (3.9) 3 (18.8) *Seven S. aureus isolates from 2007-08 did not grow for agr testing Significant if p\u22640.05 Data for 2x2 table presented as frequency (%) MRSA = methicillin-resistant S. aureus, MSSA = methicillin-susceptible S. aureus 1 2 3 4 5 6 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t Table 2(on next page) Distribution of spa types and spaCC among Mongolia MRSA and MSSA isolates *Bolded spa type is the putative founder for that spaCC. BURP clusters formed of 22 MRSA isolates. 3 non-typeable isolates and one spa type (2 isolates) excluded from BURP clusters. MRSA= methicillin-resistant S. aureus. *Bolded spa type is the putative founder for that spaCC. \u20ac Identified as new spa types. BURP clusters formed of 229 MSSA isolates. 8 non-typeable isolates and four spa types (6 isolates) excluded from BURP clusters. MSSA= methicillin-susceptible S. aureus. PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t spaCC Study assigned spaCC No. (%) of strains spa types* spaCC 589 CC1 2 (9.1) t589, t2397 spaCC 435 CC2 1 (4.5) t435 spaCC 037 CC4 5 (22.7) t037, t074 spaCC 667 CC7 8 (36.4) t667, t2832 spaCC 8 CC9 1 (4.5) t008 Total 17 *Bolded spa type is the putative founder for that spaCC BURP clusters formed of 22 MRSA isolates 3 non-typeable isolates and one spa type (2 isolates) excluded from BURP clusters MRSA= methicillin-resistant S. aureus (2B). MSSA spaCC Study assigned spaCC No. (%) of strains spa types* spaCC 589 CC1 76 (33.2) t5288, t3126, t1460, t589, t4153, t6242, t3103, t630, t073, t3219, t7043, t102, t550, t722, t908, t10064\u20ac spaCC 435 CC2 64 (27.9) t435, t270, t2392, t169, t272, t284, t159, t308, t8762, t6870, t2087, t1441 spaCC 8677 CC3 34 (14.8) t8677, t4049, t3156, t10066 \u20ac, t4473, t3465 spaCC 037 CC4 5 (2.2) t037, t021 spaCC 084 CC5 4 (1.7) t084, t085, t1038 spaCC 1194 CC6 3 (1.3) t1194, t1710 spaCC 667 CC7 3 (1.3) t667 spaCC 1010 CC8 2 (0.9) t1010, t081 spaCC 8 CC9 1 (0.4) t024 Total 192 *Bolded spa type is the putative founder for that spaCC \u20ac Identified as new spa types BURP clusters formed of 229 MSSA isolates 8 non-typeable isolates and four spa types (6 isolates) excluded from BURP clusters MSSA= methicillin-susceptible S. aureus 4 5 6 7 8 9 10 11 12 13 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t Table 3(on next page) Diversity index for Mongolia MRSA and MSSA isolates by spa typing and BURP CI: Confidence Interval, BURP: Based Upon Repeat Pattern analysis using the RIDOM StaphType. MRSA: methicillin-resistant S. aureus; MSSA: methicillin-susceptible S. aureus. PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t Table 3 Diversity index for Mongolia MRSA and MSSA isolates by spa typing and BURP Organism groups No. of isolates No. of isolates rejected by BURP Number of different spa types Typeability (%) Diversity/discriminatory index (95% CI) mrsa : (n=22) 19 3 8 95 0.865 (0.767-0.964) MSSA (n=229) 222 7 58 99.11 0.952 (0.942-0.962) MRSA+MSSA (n=251) 241 10 63 98.77 0.956 (0.947-0.965) CI: Confidence Interval, BURP: Based Upon Repeat Pattern analysis using the RIDOM StaphType MRSA: methicillin-resistant S. aureus; MSSA: methicillin-susceptible S. aureus 1 2 3 4 PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t Figure 2 Population snapshot for MRSA & MSSA Based-Upon Repeat Pattern (BURP) analysis BURP grouping using default parameters resulted in 8 spaCCs and excluded 5 spa types (t026, t132, t517, t2493, and t10358). Each dot represents a unique spa type. Diameter of a dot is proportional to the quantity of corresponding spa type. Blue dots = group putative founders (i.e. spa type with the highest score within the CC). Yellow dots = putative subfounders with second highest score. If two or more spa types have the same highest founder score they are illustrated in blue. The distance between linked and/or unlinked spa types do not concern the genetic distance between them. PeerJ reviewing PDF | (v2013:06:557:1:1:NEW 9 Sep 2013) R ev ie w in g M an us cr ip t",
    "v2_text": "materials and methods : This is an observational study conducted during two time periods (2007-08 and 2011) to investigate the prevalence of MRSA infections, and to characterize the S. aureus strains causing these infections in Mongolia. The University of Iowa IRB evaluated this project and determined that it did not qualify as human subjects research. To accomplish the study objective, we collaborated with the National Center for Communicable Diseases (NCCD) in Ulaanbaatar. The NCCD has an established Hospital Related Infection Surveillance and Research Unit (HRISRU) 4 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia (Ider et al. 2010). This study characterized S. aureus isolated from human infections in a convenience sample obtained from the NCCD. S. aureus samples collected from various microbiology laboratories in 2011 was compared to banked S. aureus isolates in the period 2007-08. Patient information such as age, gender, and sample type were available only for the 2007-08 infection samples. Isolates were labeled \u201cwound\u201d if collected from surgical site infections or wound samples. All urine samples collected in 2007-08 were obtained from voided urine i.e. none of these patients were catheterized. Biochemical testing and DNA isolation Isolates were grown and confirmed to be S. aureus as described previously (O'Brien et al. 2012). All S. aureus isolates were frozen and stored in glycerol broth solution at -800C for future use. S. aureus DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega, Madison WI) following manufacturer\u2019s instructions. Antimicrobial Susceptibility Testing (AST) The antimicrobial susceptibility of isolates were tested by the broth microdilution method in accordance with the Clinical Laboratory Standards Institute (CLSI) standards (CLSI 2012). Isolates were tested for susceptibility to the following 11 antimicrobials: oxacillin, gentamicin, erythromycin, clindamycin, tetracycline, trimethoprim/sulfamethaxozole (TMP/SMX), imipenam, levofloxacin, linezolid, vancomycin, and daptomycin. Resistance to high-level mupirocin and inducible clindamycin resistance (ICR) were also examined. All AST-confirmed MRSA isolates and MSSA isolates non-susceptible to \u2265 1 antibiotic in \u22653 discrete antimicrobial categories were classified as multidrug resistant (MDR), as per a recently published report on standardization of bacterial antimicrobial resistance profiles (Magiorakos et al. 2012). S. aureus genetic analysis 5 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia Amplification of the spa fragment was performed using methods and primers, as described previously (Shopsin et al. 1999). Identification of spa type for each isolate and the Based Upon Repeat Pattern (BURP) analysis to identify spa cluster complexes (spaCCs) was performed using the Ridom StaphType software (version2.2.1; Ridom GmbH, W rzburg, Germany) (\u1e7b Harmsen et al. 2003; Mellmann et al. 2008; Mellmann et al. 2007; Strommenger et al. 2008). The allelic profile of S. aureus isolates was determined using the Multilocus sequence typing (MLST) (Enright et al. 2000). All isolates were tested for the Panton-Valentine leukocidin (PVL) (lukS-PV and lukF-PV) (Lina et al. 1999), mecA (Bosgelmez-Tinaz et al. 2006), and the sasX gene as previously described (Holden et al. 2010; Li et al. 2012). Studies observed that presence of the sasX gene in S. aureus potentially increased its virulence capacity by boosting the bacterial defense mechanism (Li et al. 2012). These studies observed the sasX gene in 95% and 72% of ST239 isolates in the year 2003-05 and 2009-11, respectively (Holden et al. 2010; Li et al. 2012). Identified positive and negative controls were used in all molecular assays. Accessory Gene Regulator ( agr ) testing agr functionality (functional or dysfunctional) was measured using the level of \u03b4-hemolysin production, as described previously (Sakoulas et al. 2002; Schweizer et al. 2011; Traber & Novick 2006). Statistical analysis Data analysis was performed using the SAS statistical software (Version 9.3, SAS Institute Inc., Cary, NC). We used the 2-tailed Fisher\u2019s exact test, and the Wilcoxon signed-rank test to analyze categorical and continuous variables, respectively. P values \u2264 0.05 were considered statistically significant for associations between explanatory variables such as age, gender, and type of infection and S. aureus spa type, mecA, PVL and agr functionality. Antimicrobial susceptibility results were analyzed by year of data collection to observe trends in resistance for each tested 6 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia antibiotic. Association between S. aureus MDR, and mecA, PVL, agr functionality, and spa types were also assessed. Results Patient and sample characteristics In total we analyzed 252 potential S. aureus isolates, 198 from 2011 and 54 from 2007-08 isolate collections. Of these, 251 were confirmed to be S. aureus isolates. Patient demographics and sample characteristics were available only for the 53 isolates collected in 2007-08. The age of enrolled patients ranged from 1day- 82 years (median: 24 years). Of the 53 patients, 31 (58.5%) were females and 22 (41.5%) males. Approximately 43% of the S. aureus were isolated from wound samples. Molecular typing We observed a high prevalence of the mecA gene in isolates from the 2007-08 collection (Table 1). All isolates tested negative for the sasX gene. The cohort of S. aureus isolates from 2007-08 had a significantly higher PVL prevalence (85%) with a significant difference between the two time periods (p=3.496E-12). There was a moderate statistically significant difference in agr function (p=0.023) with greater proportion of functional isolates in the 2011 isolate collection (Table 1). A borderline significance was observed between age and PVL positivity in that most of our PVL-positive S. aureus isolates were obtained from older individuals (p=0.045, other data not shown). A moderately significant association was observed between presence of mecA and agr functionality (p=0.033). Antimicrobial susceptibility patterns A subset of S. aureus isolates (80/251, ~32%) were tested for antimicrobial susceptibility, based on the frequency of spa types. The proportion of S. aureus isolates that were MRSA (MDR) was greater in 2011 (Fig 1). Interestingly, we did not observe any S. aureus isolates that belonged to 7 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia the MSSA-MDR category in 2011. Isolates in both years have comparable proportions of MSSA that do not meet the MDR criteria. There was no significant difference in the proportion of MDR isolates between the two study periods (p=0.092). There was a visible increase in the proportion of isolates resistant to oxacillin, tetracycline and gentamicin between the two time periods (Fig 1). Resistance to TMP/SMX appears to have decreased during the study periods. We also observed a wider spectrum of resistance in the 2011 isolates as there is additional resistance to clindamycin, imipenam, and ICR. We did not identify high-level mupirocin resistance in our isolates. Overall, the prevalence of antibiotic resistance in tested S. aureus isolates was 38.8% (71% vs. 29%).We observed good concordance in mecA positivity and phenotypic expression of oxacillin resistance among tested S. aureus isolates. Three isolates were observed to have discordant oxacillin-resistance phenotype-genotype (data not shown). A borderline significant association was observed between antimicrobial resistance in tested S. aureus isolates and agr functionality (p=0.0488). Of the 22 MDR isolates, roughly 55% (12/22) were positive for the PVL gene, about 86% (19/22) positive for the mecA gene, and approximately 64% (14/22) had a functional agr phenotype. spa type distribution (BURP) and Multilocus sequence type (MLST) Eleven confirmed S. aureus isolates were identified as \u201cnon-typeable\u201d after at least two attempts to sequence the spa gene. We identified 63 distinct spa types in our study (Table 2). The most common spa types in 2007-08 were t589 (13%), t3465 (13%), and t435 (11%). spa types t435 (10%), t589 (8%), t5288 (7.5%), and 7% each t1460 and t8677 were the most frequently occurring strains in 2011. 8 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia BURP analysis revealed clustering around founder spa types t589 (2011), t435 (2007-08 and 2011), t3465 (2007-08), and t8677 (2011) (Fig 2). We observed greater genetic diversity among the MSSA isolates and identified three new spa types (t10064, t10066, and t10358) in the 2011 collection. spa cluster complex 667 (spaCC 667) constituted only 5% of all the strains. Nevertheless, isolates in this group had high PVL prevalence (100%), high mecA prevalence (72.7%), 100% functional agr isolates, and high multi-drug resistance. spaCC 037 also had high multidrug resistance but had lower presence of PVL (10%), mecA (50%) and functional agr (60%). Singleton spa types were t002, t126, t156, t521, t647, t803, t1451, t3329 and t8039. None of the tested singletons appeared to be MDR. A subset of S. aureus isolates from the most frequent spa types in each time period were tested by MLST revealing the following information: t021 (ST30), t037 (ST239), t084 (ST15), t435 (ST121) and t667 (ST154). spa types t1460, t5288, and t589 were observed to be ST45 in our study. One t589 isolate from the 2011 collection was identified to be a new sequence type ST2600. Discussion The prevalence of MRSA among clinical S. aureus isolates obtained from Mongolia was 8.8%. A study conducted in 2006 in Mongolia analyzing S. aureus isolates collected between 2000-02 found a low prevalence of MRSA (2.9%) by susceptibility testing (Orth et al. 2006). Our study is based on a convenience sample of Mongolian S. aureus isolates. Nevertheless, we could infer with caution that there may be an increase in the prevalence of MRSA in 2011 reflected by the absence of MDR-MSSA isolates. These observations suggest that infections due to MRSA may be increasing in Mongolia, potentially replacing the MSSA strains while gaining resistance to a wide range of antibiotics. Due to lack of published S. aureus data from Mongolia we compared our results to studies from China and Russia since there is a potential for transmission given its 9 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia geographical proximity. China reported a mean rate of 50.4% for MRSA prevalence in 2005 with considerable variations even within the country (Chu et al. 2013; Song et al. 2013; Wang et al. 2008; Yu et al. 2012; Zhao et al. 2012). The proportion of methicillin resistance reported from S. aureus in Russia varied from 18% (Vorobieva et al. 2008) to 48% (Baranovich et al. 2010). Our study data observed a lower prevalence of MRSA relative to the neighboring countries. Nevertheless, given the convenience sample this may be an underestimate of the \u2018true\u2019 prevalence of MRSA in Mongolia. Our study observed greater genetic diversity in the MSSA isolates compared to the MRSA isolates. This is consistent with the finding that MRSA could potentially emerge from existing MSSA clones by acquisition of the SCCmec complex (Hanssen & Ericson Sollid 2006; Song et al. 2013). Hence it is crucial to implement surveillance protocols for S. aureus particularly in developing countries such as Mongolia that has myriad factors contributing to antimicrobial resistance. We observed a significant association between antimicrobial resistance and functionality of the agr system suggesting a potential influence of antimicrobial resistance on the fitness of the pathogen via the agr or vice versa (Paulander et al. 2013). In addition, there appeared to be an association between methicillin resistance and agr functionality consistent with previous findings on the regulation of drug resistance in MRSA (Hao et al. 2012). These findings could potentially influence treatment options for S. aureus infections in Mongolia by considering the trade-off between fitness of the strain and its range of antimicrobial resistance. Sequence types identified by MLST were consistent across time, regardless of the combinations of mecA, PVL and agr functionality suggesting that there may be only minimal or absent mutations in the S. aureus core genome. Our data suggests that the MRSA clone ST239-spa t037 is being transmitted amongst the population. This MLST type was also reported in the previous study from Mongolia, suggesting that ST239 could potentially be the dominant MRSA clone 10 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia circulating in the country (Orth et al. 2006). ST239, a S. aureus bacterial hybrid formed by the admixture of MRSA clonal complexes ST30 and ST8 has been reported to be the dominant hospital clone in Asia (Aires de Sousa et al. 2003; Baranovich et al. 2010; Song et al. 2013; Xu et al. 2009; Yamamoto et al. 2012; Yu et al. 2012), Europe (Alp et al. 2009; Szczepanik et al. 2007; Wisplinghoff et al. 2005), South America (Carvalho et al. 2010; Vivoni et al. 2006), and the Middle East (Cirlan et al. 2005) and even responsible for an outbreak of device-associated bacteremia in Europe (Edgeworth et al. 2007). In accordance with previous reports, all three identified ST239 S. aureus strains in our study were observed to be MDR (Smyth et al. 2010). The proportion of multidrug resistance was observed to be higher in 2011 relative to 2007-08, albeit this difference was not statistically significant. Reports on S. aureus multidrug resistance observed variable rates ranging from ~29% - 100% from China (Chao et al. 2013; Chen et al. 2009; Wang et al. 2012) and about 90% Russian MRSA isolates (Baranovich et al. 2010). Our results suggest that there may be a surge in S. aureus antimicrobial resistance in the endogenous strains in Mongolia, potentially triggered by unrestricted use of antibiotics. None of our isolates, including the identified ST239 isolates, exhibited the presence of the sasX gene, suggesting its prevalence may be low or absent in Mongolia. We did not identify any livestock-associated strains in our isolate collection, although several reports from China have observed the presence of these strains in human and animal population (Zhao et al. 2012). Our study has several limitations. S. aureus isolates were collected as a convenience sample and could not be consistently linked to important patient information, particularly the 2011 collection that had a greater sample size for S. aureus isolates. We were also unable to identify any duplicate isolates. In addition, isolates from both time periods were not collected in a systematic manner adding to the selection bias. Hence, results from this study may reflect only a snapshot of the \u2018true\u2019 estimate of S. aureus infections in Mongolia. Conclusions drawn from this study could be 11 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia used as preliminary results to develop further studies with a stronger prospective study design. Nevertheless, there are not many studies from Mongolia and our study adds valuable information on the molecular epidemiology of S. aureus infections in Mongolia. Another drawback of our study was the inability to differentiate the potential origin of S. aureus strains as healthcare associated (HA-) versus community associated (CA-) since we did not have access to the date of admission before S. aureus isolation from the infection. Given that in recent times there has been a gradual blurring in the origin of S. aureus strains the reliability of this differentiation may be questionable (Mera et al. 2011). In addition, we also did not test all S. aureus isolates in our collection for antimicrobial susceptibility. Conclusion In summary, our study observed an increasing prevalence of MRSA by AST, and recorded an antimicrobial resistance rate of 38.8% and a multidrug resistance rate of 28% in S. aureus isolates from Mongolia. We also observed the presence of previously identified S. aureus strains such as ST239 and ST30 adding its virulence potential to an existing burden of antimicrobial resistance. Regular surveillance and implementation of stricter policies for antimicrobial use is warranted to prevent further transmission of S. aureus in Mongolia. Acknowledgements: We thank Dr. Michael Otto and his lab for kindly providing the sasX positive control for molecular analysis and Dr. Brett Forshey for his assistance and inputs with statistical analysis. We also acknowledge and thank our collaborators at the NCCD in Mongolia for their assistance with sample collection, sample shipping, assimilation and communication of patient data, and review of the manuscript. Funding: This study was funded by start-up funds from the University of Iowa (TCS) with assistance from the Stanley Foundation. 12 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia Potential conflicts of interest: All authors declare no potential conflicts of interest. 13 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia References Aires de Sousa M, Crisostomo MI, Sanches IS, Wu JS, Fuzhong J, Tomasz A, and de Lencastre H. 2003. Frequent recovery of a single clonal type of multidrug-resistant Staphylococcus aureus from patients in two hospitals in Taiwan and China. J Clin Microbiol 41:159-163. 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Organisation of hospital infection control in Mongolia. J Hosp Infect 75:209-213. Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, Harrison LH, Lynfield R, Dumyati G, Townes JM et al. . 2007. Invasive methicillin-resistant Staphylococcus aureus infections in the United States. JAMA 298:1763-1771. Kock R, Becker K, Cookson B, van Gemert-Pijnen JE, Harbarth S, Kluytmans J, Mielke M, Peters G, Skov RL, Struelens MJ et al. . 2010. Methicillin-resistant Staphylococcus aureus (MRSA): burden of disease and control challenges in Europe. Euro Surveill 15:19688. Li M, Du X, Villaruz AE, Diep BA, Wang D, Song Y, Tian Y, Hu J, Yu F, Lu Y et al. . 2012. MRSA epidemic linked to a quickly spreading colonization and virulence determinant. Nat Med 18:816-819. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, Vandenesch F, and Etienne J. 1999. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 29:1128-1132. Liu C, Bayer A, Cosgrove SE, Daum RS, Fridkin SK, Gorwitz RJ, Kaplan SL, Karchmer AW, Levine DP, Murray BE et al. . 2011. Clinical practice guidelines by the infectious diseases society of america for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children. Clin Infect Dis 52:e18-55. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B et al. . 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18:268-281. Mellmann A, Weniger T, Berssenbrugge C, Keckevoet U, Friedrich AW, Harmsen D, and Grundmann H. 2008. Characterization of clonal relatedness among the natural population of Staphylococcus aureus strains by using spa sequence typing and the BURP (based upon repeat patterns) algorithm. J Clin Microbiol 46:2805-2808. Mellmann A, Weniger T, Berssenbrugge C, Rothganger J, Sammeth M, Stoye J, and Harmsen D. 2007. Based Upon Repeat Pattern (BURP): an algorithm to characterize the long-term evolution of Staphylococcus aureus populations based on spa polymorphisms. BMC Microbiol 7:98. Mera RM, Suaya JA, Amrine-Madsen H, Hogea CS, Miller LA, Lu EP, Sahm DF, O'Hara P, and Acosta CJ. 2011. Increasing role of Staphylococcus aureus and community-acquired methicillin-resistant Staphylococcus aureus infections in the United States: a 10-year trend of replacement and expansion. Microb Drug Resist 17:321-328. Molton JS, Tambyah PA, Ang BS, Ling ML, and Fisher DA. 2013. The Global Spread of Healthcare-Associated Multidrug-Resistant Bacteria: A Perspective From Asia. Clin Infect Dis. Mongolia D. 2013. Geographical location. Available at http://www.discovermongolia.mn/country/where_is_mongolia.html. Mongolia MoHo. 2007. An overview of hospital laboratory services in Mongolia. 16 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia Mongolia WHOMoH. 2010. WHO country cooperation strategy for Mongolia 2010-2015. O'Brien AM, Hanson BM, Farina SA, Wu JY, Simmering JE, Wardyn SE, Forshey BM, Kulick ME, Wallinga DB, and Smith TC. 2012. MRSA in conventional and alternative retail pork products. PLoS One 7:e30092. Orth D, Grif K, Erdenechimeg L, Battogtokh C, Hosbayar T, Strommenger B, Cuny C, Walder G, Lass-Florl C, Dierich MP et al. . 2006. Characterization of methicillin-resistant Staphylococcus aureus from Ulaanbaatar, Mongolia. Eur J Clin Microbiol Infect Dis 25:104-107. Paulander W, Nissen Varming A, Baek KT, Haaber J, Frees D, and Ingmer H. 2013. Antibiotic-mediated selection of quorum-sensing-negative Staphylococcus aureus. MBio 3:e00459-00412. Rehm SJ, and Tice A. 2010. Staphylococcus aureus: methicillin-susceptible S. aureus to methicillin-resistant S. aureus and vancomycin-resistant S. aureus. Clin Infect Dis 51 Suppl 2:S176-182. Sakoulas G, Eliopoulos GM, Moellering RC, Jr., Wennersten C, Venkataraman L, Novick RP, and Gold HS. 2002. Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin. Antimicrob Agents Chemother 46:1492-1502. Schweizer ML, Furuno JP, Sakoulas G, Johnson JK, Harris AD, Shardell MD, McGregor JC, Thom KA, and Perencevich EN. 2011. Increased mortality with accessory gene regulator (agr) dysfunction in Staphylococcus aureus among bacteremic patients. Antimicrob Agents Chemother 55:1082-1087. Shopsin B, Gomez M, Montgomery SO, Smith DH, Waddington M, Dodge DE, Bost DA, Riehman M, Naidich S, and Kreiswirth BN. 1999. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J Clin Microbiol 37:3556-3563. Smyth DS, McDougal LK, Gran FW, Manoharan A, Enright MC, Song JH, de Lencastre H, and Robinson DA. 2010. Population structure of a hybrid clonal group of methicillin-resistant Staphylococcus aureus, ST239-MRSA-III. PLoS One 5:e8582. Song Y, Du X, Li T, Zhu Y, and Li M. 2013. Phenotypic and molecular characterization of Staphylococcus aureus recovered from different clinical specimens of inpatients at a teaching hospital in Shanghai between 2005 and 2010. J Med Microbiol 62:274-282. Stefani S, and Goglio A. 2010. Methicillin-resistant Staphylococcus aureus: related infections and antibiotic resistance. Int J Infect Dis 14 Suppl 4:S19-22. Strommenger B, Braulke C, Heuck D, Schmidt C, Pasemann B, Nubel U, and Witte W. 2008. spa Typing of Staphylococcus aureus as a frontline tool in epidemiological typing. J Clin Microbiol 46:574-581. Szczepanik A, Koziol-Montewka M, Al-Doori Z, Morrison D, and Kaczor D. 2007. Spread of a single multiresistant methicillin-resistant Staphylococcus aureus clone carrying a variant of staphylococcal cassette chromosome mec type III isolated in a university hospital. Eur J Clin Microbiol Infect Dis 26:29-35. Tiemersma EW, Bronzwaer SL, Lyytikainen O, Degener JE, Schrijnemakers P, Bruinsma N, Monen J, Witte W, Grundman H, and European Antimicrobial Resistance Surveillance System P. 2004. Methicillin-resistant Staphylococcus aureus in Europe, 1999-2002. Emerg Infect Dis 10:1627-1634. 17 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t S. aureus infections in Mongolia Togoobaatar G, Ikeda N, Ali M, Sonomjamts M, Dashdemberel S, Mori R, and Shibuya K. 2010. Survey of non-prescribed use of antibiotics for children in an urban community in Mongolia. Bull World Health Organ 88:930-936. Traber K, and Novick R. 2006. A slipped-mispairing mutation in AgrA of laboratory strains and clinical isolates results in delayed activation of agr and failure to translate delta- and alpha-haemolysins. Mol Microbiol 59:1519-1530. Turnidge JD, and Bell JM. 2000. Methicillin-resistant Staphylococcal aureus evolution in Australia over 35 years. Microb Drug Resist 6:223-229. Vivoni AM, Diep BA, de Gouveia Magalhaes AC, Santos KR, Riley LW, Sensabaugh GF, and Moreira BM. 2006. Clonal composition of Staphylococcus aureus isolates at a Brazilian university hospital: identification of international circulating lineages. J Clin Microbiol 44:1686-1691. Vorobieva V, Bazhukova T, Hanssen AM, Caugant DA, Semenova N, Haldorsen BC, Simonsen GS, and Sundsfjord A. 2008. Clinical isolates of Staphylococcus aureus from the Arkhangelsk region, Russia: antimicrobial susceptibility, molecular epidemiology, and distribution of Panton-Valentine leukocidin genes. APMIS 116:877-887. Wang H, Liu Y, Sun H, Xu Y, Xie X, and Chen M. 2008. In vitro activity of ceftobiprole, linezolid, tigecycline, and 23 other antimicrobial agents against Staphylococcus aureus isolates in China. Diagn Microbiol Infect Dis 62:226-229. Wang L, Liu Y, Yang Y, Huang G, Wang C, Deng L, Zheng Y, Fu Z, Li C, Shang Y et al. . 2012. Multidrug-resistant clones of community-associated meticillin-resistant Staphylococcus aureus isolated from Chinese children and the resistance genes to clindamycin and mupirocin. J Med Microbiol 61:1240-1247. Wisplinghoff H, Ewertz B, Wisplinghoff S, Stefanik D, Plum G, Perdreau-Remington F, and Seifert H. 2005. Molecular evolution of methicillin-resistant Staphylococcus aureus in the metropolitan area of Cologne, Germany, from 1984 to 1998. J Clin Microbiol 43:5445-5451. Xu BL, Zhang G, Ye HF, Feil EJ, Chen GR, Zhou XM, Zhan XM, Chen SM, and Pan WB. 2009. Predominance of the Hungarian clone (ST 239-III) among hospital-acquired meticillin-resistant Staphylococcus aureus isolates recovered throughout mainland China. J Hosp Infect 71:245-255. Yamamoto T, Takano T, Higuchi W, Iwao Y, Singur O, Reva I, Otsuka Y, Nakayashiki T, Mori H, Reva G et al. . 2012. Comparative genomics and drug resistance of a geographic variant of ST239 methicillin-resistant Staphylococcus aureus emerged in Russia. PLoS One 7:e29187. Yu F, Li T, Huang X, Xie J, Xu Y, Tu J, Qin Z, Parsons C, Wang J, Hu L et al. . 2012. Virulence gene profiling and molecular characterization of hospital-acquired Staphylococcus aureus isolates associated with bloodstream infection. Diagn Microbiol Infect Dis 74:363-368. Zhao C, Sun H, Wang H, Liu Y, Hu B, Yu Y, Sun Z, Chu Y, Cao B, Liao K et al. . 2012. Antimicrobial resistance trends among 5608 clinical Gram-positive isolates in China: results from the Gram-Positive Cocci Resistance Surveillance program (2005-2010). Diagn Microbiol Infect Dis 73:174-181. 18 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t Table 1(on next page) Prevalence of S. aureus genes PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t Frequency (%) p-value Gene tested 2007-08 (N=53) 2011(N=198) mecA Positive 6 (11.3) 16 (8.1) 0.459Negative 47 (88.7) 182 (91.9) PVL Positive 45 (84.9) 63 (31.8) <0.001Negative 8 (15.1) 135 (68.2) agr* Functional 39 (84.8) 188 (95.0) 0.015Dysfunctional 7 (15.2) 10 (5.1) *Seven S. aureus isolates from 2007-08 did not grow for agr testing, significant if p\u22640.05 PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t Table 2(on next page) Distribution of spa types and spaCC among Mongolia S. aureus isolates PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t spaCC type Study assigned spaCC No. (%) of strains spa types* spaCC 589 CC1 78 (37.9) t5288, t3126, t1460, t589, t4153, t6242, t2397, t3103, t630, t073, t3219, t7043, t102, t550, t722, t908, t10064 spaCC 435 CC2 62 (30.1) t435, t270, t2392, t169, t272, t284, t159, t308, t8762, t6870, t2087, t1441 spaCC 8677 CC3 34 (16.5) t8677, t4049, t3156, t10066, t4473, t3465 spaCC 037 CC4 10 (4.9) t037, t021, t074 spaCC 084 CC5 4 (1.9) t084, t085, t1038 spaCC 1194 CC6 3 (1.5) t1194, t1710 spaCC 667 CC7 11 (5.3) t667, t2832 spaCC 1010 CC8 2 (1.0) t1010, t081 spaCC 8 CC9 2 (1.0) t008, t024 Total 206 *Bolded spa type is the founder for that spaCC BURP clusters formed of 240 S. aureus isolates 25 singleton and 11 non-typeable isolates excluded from BURP clusters PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t Figure 1 Antimicrobial resistance and MDR trends in Mongolia S. aureus isolates *TMP/SMX = trimethoprim/sulfamethaxozole, MDR = multi-drug resistant, MSSA = methicillin-susceptible S. aureus, ICR = Inducible Clindamycin Resistance PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t Figure 2 Population snapshot for Based-Upon Repeat Pattern (BURP) analysis BURP grouping using default parameters resulted in 8 spaCCs and excluded 5 spa types (t026, t132, t517, t2493, and t10358). Each dot represents a unique spa type. Diameter of a dot is proportional to the quantity of corresponding spa type. Blue dots = group founders (i.e. spa type with the highest score within the CC). Yellow dots = subfounders with second highest score. If two or more spa types have the same highest founder score they are illustrated in blue. The distance between linked and/or unlinked spa types do not concern the genetic distance between them. PeerJ reviewing PDF | (v2013:06:557:0:0:NEW 2 Jun 2013) R ev ie w in g M an us cr ip t",
    "url": "https://peerj.com/articles/177/reviews/",
    "review_1": "Paul Appelbaum \u00b7 Sep 16, 2013 \u00b7 Academic Editor\nACCEPT\nWith this new version of the paper, I think you have successfully addressed the reviewer's comments. In particular, as you now make clear, this case illuminates the complexity of integrating WGS data into clinical care.",
    "review_2": "Paul Appelbaum \u00b7 Sep 9, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nWe have now received a re-review of your paper from one of the original reviewers. As you can see from the comments, the reviewer has indicated that further major revision is required, and I agree. The problem that remains with this paper is the disjunction between your assertions that the case study demonstrates the utility of integrating WGS into this patient\u2019s treatment and the difficulty in identifying how the WGS findings impacted that treatment. As has been the case from the beginning, your challenge is to demonstrate what your title promises, i.e., that precision medicine in fact has been integrated into and made a difference in this patient\u2019s treatment. If you would like to have another shot at supporting that claim, we would entertain another round of revisions. However, if you choose to seek publication elsewhere, we would certainly understand.",
    "review_3": "Reviewer 1 \u00b7 Sep 7, 2013\nBasic reporting\nNo comments\nExperimental design\nWGS is not clearly justified: what was the goal / research question of WGS?\nValidity of the findings\n\u2022 The conclusions should be appropriately stated, should be connected to the original question investigated, and should be limited to those supported by the results.\n\n\u201cTo our knowledge, this is the first study in the clinical neurosciences that integrates detailed neuropsychiatric phenotyping, deep brain stimulation for OCD and clinical-grade WGS with management of genetic results in the medical treatment of one person with severe mental illness.\u201d (Conclusions section of the Abstract).\nIt is not clear what the authors mean by \u201cmanagement of genetic results in the medical treatment\u201d; there is no mention of any influence of genetic study on diagnosis, prognosis or medical treatment. And \"integration\" of \"clinical-grade WGS\" with other parts of the study is overstated; the authors barely try making reasonable connection between sequencing results and the phenotype.\n\n\u201cWe offer this as an example of precision medicine in neuropsychiatry including brain-implantable devices and genomics-guided preventive health care.\u201d\nGenomics did not guide the authors to any specific medical decisions, and they did not describe how genomics helped them in the prevention of any health-related issues of the patient (see also my remarks about Conclusions section of the manuscript). The authors actually offer opinions within the manuscript which one could consider as not fully agreeing with the statement above: \u201cWGS cannot act as a diagnostic and prognostic panacea, but instead could act to elucidate potential risk factors for some illnesses\u201d. It is not clear how the authors make a clear distinction between \u201cpotential risk factors\u201d and prognosis, and if they think knowing \u201cpotential risk factors\u201d provides sufficient information to guide preventive health care.\n\nFirst paragraph of Introduction criticizes current standardized approaches in current medicine and psychiatry in particular, and contrasts it with individual-focused medical care. It sounds like a manifesto making bold general statements, most of which could be considered controversial. It is inappropriate for a case study, and does little to introduce specific problems addressed in the manuscript.\nThere are no objections to the description and short discussion of genetic variants found in the patient and their possible involvement in the phenotype. Personal genome interpretation is still in its infancy, and every case is an interesting teaching point. However, more general discussion of WGS and conclusions are sometimes irrelevant to the case. The motivation of using WGS for the patient is unclear.\nThe examples of use of whole genome or exome sequencing for identification of disease cause (Introduction) are deceiving, as they are related to monogenic disorders when only one mutation in one gene is causative. This paradigm is not applicable to OCD. OCD is a complex disorder, with at least several genetic factors involved in the etiology. Based on genome-wide association studies, it is quite possible that some genetic variations predisposing to the disease are not missense or nonsense mutations in protein-coding genes, but are located outside known genes and involved in molecular pathology through yet unknown mechanisms.\nAnother misleading example is presented in the Conclusion section, where the authors refer to data of other groups using WGS as a part of the evaluation of patients with undiagnosed diseases or diseases with unclear etiology. It was expected that the disease in every particular case could be caused by either one or a combination of very few genetic variants in the genes known to be involved in diseases with similar phenotypes. The motivation of referred articles was to improve diagnosis of, or the risk to develop, a particular phenotype by identification of such specific DNA variants or mutations. It does not seem to be a goal in the described study, and again raises the question: what was the reason of WGS in this particular patient?\n\n\u201c\u2026our data also does not allow us to exclude the possibility of polygenic and epistatic modes of inheritance\u201d (397-398) \u2013 I would exclude this phrase; although the statement is formally correct, there is a notion that the authors tried or wanted to exclude polygenic mode of inheritance; I hope they did not.\n\n\u201cWe provide our study as a cautionary one: WGS cannot act as a diagnostic and prognostic panacea, but instead could act to elucidate potential risk factors for some illnesses\u201d (398-400). The authors discussed using WGS for different kinds of disorders, including monogenic disorders. For many of monogenic disorders WGS may provide all the necessary information for diagnosis and, in many cases, prognosis of the disease and treatment. The authors make this statement in a context which may assume only neuropsychiatric disease, but it would be more appropriate to specifically state it.\n\nThe authors provide the only example of usefulness of WGS for the patient: \u201chealth information stemming from these data were nevertheless immediately useful in the care of this person, as a variant associated with his ophthalmologic phenotype did indeed inform and enrich his care\u201d (Conclusions, 477-479). However, in the manuscript (without consideration of supplemental material) there is no mentioning of any mutation involved in ophthalmologic phenotype, and there is no information about how these data were used in the care of the patient.\n\nOverall, the manuscript describes an interesting study which deserves publication. However, it contains several confusing discussion points and still lacks clarity in the goal of WGS of the genome of the patient. The manuscript would benefit tremendously from shortening the introduction, discussion and conclusions to strictly relevant points, and making the manuscript more focused and concise.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Integrating precision medicine in the study and clinical treatment of a severely mentally ill person (v0.2)\". PeerJ https://doi.org/10.7287/peerj.177v0.2/reviews/1",
    "review_4": "Paul Appelbaum \u00b7 Jul 31, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nAs the comments from both reviewers indicate, they see a significant conceptual problem with your paper. You indicate that you see this case as constituting \"an advance in the field of applying genomics to the individualized care of one person with severe mental illness.\" However, as the reviewers suggest--and I agree--it's not clear how the genomic findings contributed in any way to this patient's treatment. If this paper is ultimately to be acceptable to PeerJ, you will need to demonstrate that contribution clearly. Otherwise, as Reviewer 1 indicates, the genomic findings appear simply to be epiphenomenal data in a case report on DBS.",
    "pdf_1": "https://peerj.com/articles/177v0.3/submission",
    "pdf_2": "https://peerj.com/articles/177v0.2/submission",
    "review_5": "Reviewer 1 \u00b7 Jul 31, 2013\nBasic reporting\nThe report is much narrated based on the experience of one of the authors, rather than on explaining objectively the project, the work performed, and the results obtained. The discussion is subjective and repetitive in different parts and although some issues regarding availability of genomic testing and return of results are raised, these should be discussed in a more objective way.\nExperimental design\nNo comments on the methods as most of the data production was done in an external certified clinical laboratory, data analysis was performed using commercially available software only. Maybe, more details on how the deep brain stimulation device operates and the procedures needed to implement this kind of treatment should be included.\nValidity of the findings\nThe main and most interesting findings of this report are the favorable results obtained from the deep brain stimulation device used in the patient and the reportedly dramatic changes and improvements in the patient\u2019s behavior. The authors would better focus on reporting, documenting, and following up in more detail the effects, both favorable and potential unfavorable, of the device in the patient\u2019s behavior and how can this type of devices be used to treat other neuropsychiatric disorders.\nThis is not a genomics or whole-genome article, as the results obtained from the genomic sequencing of the patient did not informed, guided or helped in any significant way in the diagnosis or treatment of the patient. The authors do not provide any additional evidence that any of the variants associated to behavioral disorders has a contribution to the patient\u2019s phenotype, except for the already published associations of these variants. Many \u201cnormal\u201d individuals in the general population carry these variants and they do not present with clinical neuropsychiatric disorders, therefore the relative contribution of these variants to this patient\u2019s phenotype is not attainable. The authors do not provide any further information, rather than speculation, on the Refsum disease variant that would support or discard the contribution of this variant to the ophthalmologic phenotype in the patient. All other genomic information from the whole-genome approach seems to be not significant for the purposes of this report and therefore treated as supplementary data. The whole-genome analysis seems to have been done superficially and although some findings can be useful for the patient\u2019s health care in the future, such as pharmacological variants, the results obtained are not significant for this report.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Integrating precision medicine in the study and clinical treatment of a severely mentally ill person (v0.1)\". PeerJ https://doi.org/10.7287/peerj.177v0.1/reviews/1",
    "pdf_3": "https://peerj.com/articles/177v0.1/submission",
    "review 6": "Reviewer 2 \u00b7 Jul 8, 2013\nBasic reporting\nThe manuscript describes one of the first studies attempted involving whole genome sequencing as a part of \u201cindividualized medicine\u201d. This aspect will surely attract many readers, but I believe that the manuscript in its current form would leave readers disappointed. The authors did not conclude about the usability of whole genome sequencing for diagnosis or for choosing a treatment strategy, nor did they provide personal impressions, advantages, shortcomings or prognoses of clinical use of WGS. The list of polymorphisms selected by the authors for discussion does not explain the phenotype and does not lead to any specific decisions in treatment. A possible exception is the homozygous mutation in CYP2C9, which may explain the lack of response of the patient to fluoxetine, but it needs more detailed analysis or functional assay to confirm that resistance to the drug is indeed caused solely by this variant of CYP2C9. However, even if conclusive, it does not justify WGS by itself. The conclusion section, presented in the abstract, does not contain any specific conclusion.\nThe introduction is written emotionally and criticizes current approaches to diagnosis (\u201ccoarse categorizations in psychiatry which bear little resemblance to reality\u201d) and monetary incentives in clinical trials contrasting them to individualized medicine. However, it does not provide a fair introduction to the following sections.\nExperimental design\nResearch question is not clearly defined.\nValidity of the findings\nNeither the original question nor the conclusions are appropriately stated.\nOverall, the methods used in the study are reasonable. However, I think that the description of methods for detection of deleterious mutations creates a false feeling of reliability of the distinction between harmful and benign mutations. So far, different approaches result in lists of predicted deleterious mutations with a relatively low degree of overlap. A statement that for an Omicia Score of 0.85 the analyses results in a 1% false positive rate could be misleading without knowing the false negative rate and without specifying how these rates were calculated.\nAdditional comments\nThe usability of WGS data for clinical psychiatry at the current state of knowledge is not clear, and it is assumed that the authors will discuss it. The idea of looking for deleterious mutations is reasonable, but it was not justified or explained in the manuscript. We know that OCD is not a monogenic disorder, so identification of one deleterious mutation even in a very good candidate gene will not \u201cexplain\u201d the disease. So what was the purpose of WGS, what were the expectations, and what models could be used for the analysis? I think that the manuscript should be revised. I recommend clearly stating the aim of the study and conclusions. WGS data needs a different angle of discussion, specifically how sequencing can help in diagnosis and selection of an optimal treatment strategy.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Integrating precision medicine in the study and clinical treatment of a severely mentally ill person (v0.1)\". PeerJ https://doi.org/10.7287/peerj.177v0.1/reviews/2",
    "all_reviews": "Review 1: Paul Appelbaum \u00b7 Sep 16, 2013 \u00b7 Academic Editor\nACCEPT\nWith this new version of the paper, I think you have successfully addressed the reviewer's comments. In particular, as you now make clear, this case illuminates the complexity of integrating WGS data into clinical care.\nReview 2: Paul Appelbaum \u00b7 Sep 9, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nWe have now received a re-review of your paper from one of the original reviewers. As you can see from the comments, the reviewer has indicated that further major revision is required, and I agree. The problem that remains with this paper is the disjunction between your assertions that the case study demonstrates the utility of integrating WGS into this patient\u2019s treatment and the difficulty in identifying how the WGS findings impacted that treatment. As has been the case from the beginning, your challenge is to demonstrate what your title promises, i.e., that precision medicine in fact has been integrated into and made a difference in this patient\u2019s treatment. If you would like to have another shot at supporting that claim, we would entertain another round of revisions. However, if you choose to seek publication elsewhere, we would certainly understand.\nReview 3: Reviewer 1 \u00b7 Sep 7, 2013\nBasic reporting\nNo comments\nExperimental design\nWGS is not clearly justified: what was the goal / research question of WGS?\nValidity of the findings\n\u2022 The conclusions should be appropriately stated, should be connected to the original question investigated, and should be limited to those supported by the results.\n\n\u201cTo our knowledge, this is the first study in the clinical neurosciences that integrates detailed neuropsychiatric phenotyping, deep brain stimulation for OCD and clinical-grade WGS with management of genetic results in the medical treatment of one person with severe mental illness.\u201d (Conclusions section of the Abstract).\nIt is not clear what the authors mean by \u201cmanagement of genetic results in the medical treatment\u201d; there is no mention of any influence of genetic study on diagnosis, prognosis or medical treatment. And \"integration\" of \"clinical-grade WGS\" with other parts of the study is overstated; the authors barely try making reasonable connection between sequencing results and the phenotype.\n\n\u201cWe offer this as an example of precision medicine in neuropsychiatry including brain-implantable devices and genomics-guided preventive health care.\u201d\nGenomics did not guide the authors to any specific medical decisions, and they did not describe how genomics helped them in the prevention of any health-related issues of the patient (see also my remarks about Conclusions section of the manuscript). The authors actually offer opinions within the manuscript which one could consider as not fully agreeing with the statement above: \u201cWGS cannot act as a diagnostic and prognostic panacea, but instead could act to elucidate potential risk factors for some illnesses\u201d. It is not clear how the authors make a clear distinction between \u201cpotential risk factors\u201d and prognosis, and if they think knowing \u201cpotential risk factors\u201d provides sufficient information to guide preventive health care.\n\nFirst paragraph of Introduction criticizes current standardized approaches in current medicine and psychiatry in particular, and contrasts it with individual-focused medical care. It sounds like a manifesto making bold general statements, most of which could be considered controversial. It is inappropriate for a case study, and does little to introduce specific problems addressed in the manuscript.\nThere are no objections to the description and short discussion of genetic variants found in the patient and their possible involvement in the phenotype. Personal genome interpretation is still in its infancy, and every case is an interesting teaching point. However, more general discussion of WGS and conclusions are sometimes irrelevant to the case. The motivation of using WGS for the patient is unclear.\nThe examples of use of whole genome or exome sequencing for identification of disease cause (Introduction) are deceiving, as they are related to monogenic disorders when only one mutation in one gene is causative. This paradigm is not applicable to OCD. OCD is a complex disorder, with at least several genetic factors involved in the etiology. Based on genome-wide association studies, it is quite possible that some genetic variations predisposing to the disease are not missense or nonsense mutations in protein-coding genes, but are located outside known genes and involved in molecular pathology through yet unknown mechanisms.\nAnother misleading example is presented in the Conclusion section, where the authors refer to data of other groups using WGS as a part of the evaluation of patients with undiagnosed diseases or diseases with unclear etiology. It was expected that the disease in every particular case could be caused by either one or a combination of very few genetic variants in the genes known to be involved in diseases with similar phenotypes. The motivation of referred articles was to improve diagnosis of, or the risk to develop, a particular phenotype by identification of such specific DNA variants or mutations. It does not seem to be a goal in the described study, and again raises the question: what was the reason of WGS in this particular patient?\n\n\u201c\u2026our data also does not allow us to exclude the possibility of polygenic and epistatic modes of inheritance\u201d (397-398) \u2013 I would exclude this phrase; although the statement is formally correct, there is a notion that the authors tried or wanted to exclude polygenic mode of inheritance; I hope they did not.\n\n\u201cWe provide our study as a cautionary one: WGS cannot act as a diagnostic and prognostic panacea, but instead could act to elucidate potential risk factors for some illnesses\u201d (398-400). The authors discussed using WGS for different kinds of disorders, including monogenic disorders. For many of monogenic disorders WGS may provide all the necessary information for diagnosis and, in many cases, prognosis of the disease and treatment. The authors make this statement in a context which may assume only neuropsychiatric disease, but it would be more appropriate to specifically state it.\n\nThe authors provide the only example of usefulness of WGS for the patient: \u201chealth information stemming from these data were nevertheless immediately useful in the care of this person, as a variant associated with his ophthalmologic phenotype did indeed inform and enrich his care\u201d (Conclusions, 477-479). However, in the manuscript (without consideration of supplemental material) there is no mentioning of any mutation involved in ophthalmologic phenotype, and there is no information about how these data were used in the care of the patient.\n\nOverall, the manuscript describes an interesting study which deserves publication. However, it contains several confusing discussion points and still lacks clarity in the goal of WGS of the genome of the patient. The manuscript would benefit tremendously from shortening the introduction, discussion and conclusions to strictly relevant points, and making the manuscript more focused and concise.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Integrating precision medicine in the study and clinical treatment of a severely mentally ill person (v0.2)\". PeerJ https://doi.org/10.7287/peerj.177v0.2/reviews/1\nReview 4: Paul Appelbaum \u00b7 Jul 31, 2013 \u00b7 Academic Editor\nMAJOR REVISIONS\nAs the comments from both reviewers indicate, they see a significant conceptual problem with your paper. You indicate that you see this case as constituting \"an advance in the field of applying genomics to the individualized care of one person with severe mental illness.\" However, as the reviewers suggest--and I agree--it's not clear how the genomic findings contributed in any way to this patient's treatment. If this paper is ultimately to be acceptable to PeerJ, you will need to demonstrate that contribution clearly. Otherwise, as Reviewer 1 indicates, the genomic findings appear simply to be epiphenomenal data in a case report on DBS.\nReview 5: Reviewer 1 \u00b7 Jul 31, 2013\nBasic reporting\nThe report is much narrated based on the experience of one of the authors, rather than on explaining objectively the project, the work performed, and the results obtained. The discussion is subjective and repetitive in different parts and although some issues regarding availability of genomic testing and return of results are raised, these should be discussed in a more objective way.\nExperimental design\nNo comments on the methods as most of the data production was done in an external certified clinical laboratory, data analysis was performed using commercially available software only. Maybe, more details on how the deep brain stimulation device operates and the procedures needed to implement this kind of treatment should be included.\nValidity of the findings\nThe main and most interesting findings of this report are the favorable results obtained from the deep brain stimulation device used in the patient and the reportedly dramatic changes and improvements in the patient\u2019s behavior. The authors would better focus on reporting, documenting, and following up in more detail the effects, both favorable and potential unfavorable, of the device in the patient\u2019s behavior and how can this type of devices be used to treat other neuropsychiatric disorders.\nThis is not a genomics or whole-genome article, as the results obtained from the genomic sequencing of the patient did not informed, guided or helped in any significant way in the diagnosis or treatment of the patient. The authors do not provide any additional evidence that any of the variants associated to behavioral disorders has a contribution to the patient\u2019s phenotype, except for the already published associations of these variants. Many \u201cnormal\u201d individuals in the general population carry these variants and they do not present with clinical neuropsychiatric disorders, therefore the relative contribution of these variants to this patient\u2019s phenotype is not attainable. The authors do not provide any further information, rather than speculation, on the Refsum disease variant that would support or discard the contribution of this variant to the ophthalmologic phenotype in the patient. All other genomic information from the whole-genome approach seems to be not significant for the purposes of this report and therefore treated as supplementary data. The whole-genome analysis seems to have been done superficially and although some findings can be useful for the patient\u2019s health care in the future, such as pharmacological variants, the results obtained are not significant for this report.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #1 of \"Integrating precision medicine in the study and clinical treatment of a severely mentally ill person (v0.1)\". PeerJ https://doi.org/10.7287/peerj.177v0.1/reviews/1\nReview 6: Reviewer 2 \u00b7 Jul 8, 2013\nBasic reporting\nThe manuscript describes one of the first studies attempted involving whole genome sequencing as a part of \u201cindividualized medicine\u201d. This aspect will surely attract many readers, but I believe that the manuscript in its current form would leave readers disappointed. The authors did not conclude about the usability of whole genome sequencing for diagnosis or for choosing a treatment strategy, nor did they provide personal impressions, advantages, shortcomings or prognoses of clinical use of WGS. The list of polymorphisms selected by the authors for discussion does not explain the phenotype and does not lead to any specific decisions in treatment. A possible exception is the homozygous mutation in CYP2C9, which may explain the lack of response of the patient to fluoxetine, but it needs more detailed analysis or functional assay to confirm that resistance to the drug is indeed caused solely by this variant of CYP2C9. However, even if conclusive, it does not justify WGS by itself. The conclusion section, presented in the abstract, does not contain any specific conclusion.\nThe introduction is written emotionally and criticizes current approaches to diagnosis (\u201ccoarse categorizations in psychiatry which bear little resemblance to reality\u201d) and monetary incentives in clinical trials contrasting them to individualized medicine. However, it does not provide a fair introduction to the following sections.\nExperimental design\nResearch question is not clearly defined.\nValidity of the findings\nNeither the original question nor the conclusions are appropriately stated.\nOverall, the methods used in the study are reasonable. However, I think that the description of methods for detection of deleterious mutations creates a false feeling of reliability of the distinction between harmful and benign mutations. So far, different approaches result in lists of predicted deleterious mutations with a relatively low degree of overlap. A statement that for an Omicia Score of 0.85 the analyses results in a 1% false positive rate could be misleading without knowing the false negative rate and without specifying how these rates were calculated.\nAdditional comments\nThe usability of WGS data for clinical psychiatry at the current state of knowledge is not clear, and it is assumed that the authors will discuss it. The idea of looking for deleterious mutations is reasonable, but it was not justified or explained in the manuscript. We know that OCD is not a monogenic disorder, so identification of one deleterious mutation even in a very good candidate gene will not \u201cexplain\u201d the disease. So what was the purpose of WGS, what were the expectations, and what models could be used for the analysis? I think that the manuscript should be revised. I recommend clearly stating the aim of the study and conclusions. WGS data needs a different angle of discussion, specifically how sequencing can help in diagnosis and selection of an optimal treatment strategy.\nCite this review as\nAnonymous Reviewer (2013) Peer Review #2 of \"Integrating precision medicine in the study and clinical treatment of a severely mentally ill person (v0.1)\". PeerJ https://doi.org/10.7287/peerj.177v0.1/reviews/2\n",
    "LLM_extracted_review": "[\"**Review 1:** The paper successfully addresses the reviewer's comments and highlights the complexity of integrating WGS data into clinical care.\\n\\n**Review 2:** The paper requires major revisions due to a disjunction between claims of WGS utility in treatment and the actual impact of WGS findings. The authors need to demonstrate how precision medicine has been integrated into the patient's treatment.\\n\\n**Review 3:** \\n- WGS is not clearly justified; the research question is unclear.\\n- Conclusions should be connected to the original question and limited to results supported by data.\\n- The claim of integrating WGS with treatment is overstated; no influence of genetic study on diagnosis or treatment is mentioned.\\n- The introduction makes bold, controversial statements inappropriate for a case study.\\n- The motivation for using WGS is unclear, and examples provided are misleading.\\n- The manuscript lacks clarity on the goal of WGS and should be more focused and concise.\\n\\n**Review 4:** There is a significant conceptual problem; it is unclear how genomic findings contributed to the patient's treatment. The paper needs to clearly demonstrate this contribution.\\n\\n**Review 5:** \\n- The report is subjective and repetitive, lacking objective discussion on genomic testing.\\n- The main findings focus on deep brain stimulation rather than genomics.\\n- Genomic sequencing did not significantly inform the diagnosis or treatment.\\n- The whole-genome analysis appears superficial and lacks significant findings relevant to the report.\\n\\n**Review 6:** \\n- The manuscript does not conclude on the usability of WGS for diagnosis or treatment strategy.\\n- The introduction is emotionally charged and does not fairly introduce the study.\\n- The research question is not clearly defined, and the conclusions are not appropriately stated.\\n- The reliability of distinguishing harmful mutations is misleading.\\n- The purpose of WGS and its expectations are unclear; the manuscript needs revision to clarify the study's aim and conclusions.\"]"
}