A Common Variant of Staphylococcal Cassette Chromosome mec Type IVa in Isolates from Copenhagen,Denmark, Is Not Detected by the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.Methicillin-resistant Staphylococcus aureus (MRSA) is a common nosocomial pathogen in countries all over the world. In recent years, community-associated MRSA (CA-MRSA) has become increasingly prevalent and has shown potential to cause health care-associated bloodstream infections (8, 26). Screening and isolation of MRSA-positive patients is essential to control the transmission of MRSA in hospitals (16, 24). However, conventional detection of MRSA by culture takes at least 48 h before a preliminary result is available, and as patients in many countries are only isolated when they are recognized as MRSA positive, the risk of having already transmitted MRSA is high. The real-time PCR BD GeneOhm MRSA assay (Becton Dickinson [BD] Diagnostics GeneOhm; San Diego, CA), formerly called IDI-MRSA, is one of a number of commercial kits for rapid MRSA detection directly from nasal swabs (7) and is based on primers developed by Huletsky et al. (18). The forward primers bind to the J3 region of the staphylococcal cassette chromosome mec (SCCmec), and the reverse primer binds in the orfX region that is specific for Staphylococcus aureus. At least seven SCCmec types are known (types I to VII) (3), and several subtypes, especially of type IV, have been described (21, 27).The BD GeneOhm MRSA assay has been tested in a number of studies (4, 5, 10, 11, 13-15, 22, 23, 25, 29-31). Most studies screened hospitalized patients, but only two studies described the SCCmec types of their MRSA isolates (15, 25). Therefore, it is possible that only a few predominant hospital clones with the same SCCmec types were tested. In Denmark, different CA-MRSA clones dominate and MRSA isolates mainly harbor SCCmec types IV (85%) and V (6%) (2). In-house testing with the Huletsky primers (18) revealed that they did not amplify a PCR fragment from our most-common MRSA clone, spa t024-sequence type 8 (ST8)-IVa. Based on this finding and with the knowledge of the high number of type IV subtypes known, we were interested in finding out whether the BD GeneOhm MRSA assay could detect MRSA isolates from a collection that included mainly CA-MRSA strains. We tested 349 MRSA isolates representing variants of SCCmec types I to V. Furthermore, we chose MRSA isolates of different staphylococcal protein A (spa) types to have a broad range of genetic backgrounds, testing the hypothesis that the same SCCmec type might have minor differences in different MRSA lineages and that these differences could be in the primer regions of the assay.     

Evaluation of the BD GeneOhm StaphSR Assay for Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Spiked Positive Blood Culture Bottles

To improve the clinical outcome of Staphylococcus aureus septicemia, the early selection of appropriate antibiotic treatment is crucial. Molecular diagnostics represents an attractive approach for the rapid identification of S. aureus and the determination of its methicillin (meticillin) resistance. In direct comparison to other molecular assays (sa442 and mecA real-time PCRs) and standard laboratory procedures, we evaluated the BD GeneOhm StaphSR assay for its use in the detection of S. aureus and methicillin-resistant S. aureus (MRSA) from spiked blood culture bottles (n = 134). In the case of detecting S. aureus (n = 90; for methicillin-susceptible S. aureus, n = 45; for MRSA, n = 45), the BD GeneOhm StaphSR assay had a sensitivity and a specificity of 100% each (95% confidence intervals [CIs], 96.0 to 100% and 82.4 to 100%, respectively). For MRSA (n = 45), the test was 95.6% (95% CI, 84.9 to 99.5%) sensitive and 95.3% (95% CI, 86.9 to 99.0%) specific. Overall, five discrepant results arose with this assay due to the presence of methicillin-susceptible, revertant MRSA strains (3/45) and MRSA strains that were not detected by the BD GeneOhm StaphSR assay (2/45). Compared to other real-time PCR-based molecular approaches and to conventional standard laboratory methods, the BD GeneOhm StaphSR turned out to be an appropriate diagnostic tool for a rapid (∼1.5 h), preliminary identification of S. aureus and MRSA from blood cultures.Staphylococcus aureus septicemia is associated with high mortality rates, prolonged hospitalization, and increased costs (3, 5). The prevalence of S. aureus septicemia is increasing, primarily due to infections caused by methicillin (meticillin)-resistant S. aureus (MRSA) (20). Several studies have shown that mortality rates among patients suffering from MRSA septicemia is significantly higher than those of patients suffering from infections caused by methicillin-susceptible S. aureus (MSSA) (5, 18, 19).The early selection of an appropriate antibiotic regime for the treatment of MSSA or MRSA is crucial for the patient''s outcome (4, 14, 15). However, bacterial identification and preliminary antibiotic susceptibility testing by standard microbiological procedures still requires 24 to 48 h after growth detection by automated incubation systems. In contrast, new real-time PCR-based methods that use samples directly from positive blood culture bottles allows differentiation of MSSA, MRSA, and coagulase-negative staphylococci (CoNS) within 1.5 to 3 h (7, 12, 13, 16). Such tests promote an early appropriate antibiotic selection, thus avoiding the unnecessary use of vancomycin, and they reduce mortality, the length of hospitalization, and costs associated with bloodstream infections caused by these bacteria (3).We described recently a real-time PCR method for the detection of MSSA, MRSA, and CoNS directly from positive blood cultures; it turned out to have 100% sensitivity and 100% specificity for the detection of MSSA and MRSA (7). In this study, the differentiation between MSSA and MRSA directly from signal-positive blood cultures was achieved by the separate detection of the S. aureus-specific chromosomal fragment sa442 and the mecA gene (encoding methicillin resistance). However, since this test is not a commercialized system and does not run on a common platform like, e.g., the SmartCycler (Cepheid, Sunnyvale, CA), its widespread application is limited. Moreover, in blood cultures containing a mixture of MSSA (sa442⁺ but mecA negative) and methicillin-resistant CoNS (MR-CoNS; sa442 negative but mecA⁺), the test is prone to lead to the incorrect identification of MRSA (sa442⁺ mecA⁺).The BD GeneOhm StaphSR assay (BD Diagnostics GeneOhm, Québec, Canada) provides a rapid, simple, commercially available diagnostic test that runs on the SmartCycler for the detection of S. aureus and MRSA from nasal swabs, wounds, and blood cultures. This multiplex real-time PCR amplifies an S. aureus-specific target sequence and a specific target near the staphylococcal cassette chromosome mec (SCCmec) insertion site and the orfX junction in MRSA, thereby differentiating between MSSA and MRSA (9, 17).Using the herein-described setting, we evaluated the BD GeneOhm StaphSR assay and the PCR that detects sa442 and mecA (designated sa442-mecA-PCR) for the detection of MSSA and MRSA directly from spiked blood cultures.     

Nasal Colonization of and Clonal Transmission of Methicillin-Susceptible Staphylococcus aureus among Chinese Military Volunteers

Military facilities provide unique opportunities for studying Staphylococcus aureus nasal colonization and transmission patterns. In this cross-sectional observational study, we assessed the prevalence of S. aureus nasal colonization among Chinese military volunteers in two camps in the Beijing area. Antimicrobial resistance patterns, risk factors for colonization, and transmission patterns using pulsed-field gel electrophoresis were also evaluated. From May to July 2007, 1,044 nasal swabs were collected from military volunteers from suburban (560) and urban (484) camps. A total of 209 S. aureus isolates were recovered, of which all were methicillin susceptible. Independent factors associated with methicillin-susceptible S. aureus (MSSA) nasal colonization included younger age (odds ratio [OR] = 1.51, 95% confidence interval [95% CI] = 1.03 to 2.21, P = 0.0347), higher education (OR = 1.38, 95% CI = 1.10 to 1.73, P = 0.0056), shorter length of service (OR = 1.74, 95% CI = 1.28 to 2.36, P = 0.0004), nonsmoking (OR = 1.61, 95% CI = 1.14 to 2.28, P = 0.0069), and inactive participation in social events (OR = 2.40, 95% CI = 1.25 to 5.49, P = 0.0082). Among 209 MSSA isolates, 126 (60.3%) were determined to be epidemic and a total of 12 genotypes were identified, of which four (90 isolates [71.4%]) represented the majority of strains. Length of service and camp location were statistically related to the four major MSSA genotype clonal transmissions. Our data indicated that MSSA, not methicillin-resistant S. aureus (MRSA), nasal colonization and clonal transmission occur in healthy military volunteers in Beijing. Younger, female, nonsmoking volunteers with higher education, little or no participation in social events, and less time in service are at higher risk for nasal MSSA carriage.Staphylococcus aureus is an important cause of skin and soft tissue infections, as well as invasive infections in humans (25). Since methicillin-resistant S. aureus (MRSA) was first reported, it has become endemic in hospitals and communities around the world (10). The recent emergence of a highly virulent community-associated MRSA (CA-MRSA) and vancomycin-resistant, intermediate-resistant, or heteroresistant S. aureus further heightens public health concerns (14, 17, 37, 46). Prevention of S. aureus infection and reduction of the spread of virulent and resistant strains are therefore of great importance.On the other hand, S. aureus is a member of the commensal microflora. The anterior nares of the nose are the primary reservoirs of S. aureus colonization in humans, and many S. aureus infections occur in persons with prior nasal bacterial carriage (47). Nasal colonization is an important step in the pathogenesis of S. aureus infection and is a risk factor for acquiring nosocomial infection (22). It has been shown that 80% of nosocomial S. aureus bacteremia episodes in carriers of this bacteria were attributed to an endogenous source (44). Nosocomial S. aureus bacteremia was three times more frequent in S. aureus carriers than in noncarriers (48). Numerous studies of S. aureus nasal carriage have been carried out in various geographic regions in the United States and the Netherlands (2, 5, 7, 21, 23, 27, 28, 41). Cross-section surveys of nasal carriage prevalence and transmission mechanisms in special healthy populations are beneficial in assessing risk factors associated with S. aureus infections (2, 8, 13, 26, 32-35). Military facilities provide unique opportunities for studying S. aureus nasal colonization and transmission (11, 19, 52).In China, MRSA was shown in 63% of S. aureus isolates, among which 77% nosocomial and 43% community isolates were MRSA (49). According to a study conducted in 2005, the mean incidence of MRSA across China was over 50%, and in Shanghai, the prevalence was over 80%, contributed to by two major epidemic MRSA clones with unique geographic distribution (24, 45, 50, 51). Therefore, understanding and controlling the spread of MRSA in both hospital and community settings in China are now of paramount importance. The majority of S. aureus isolates studied in China have been limited to clinical patients, and S. aureus isolates recovered from healthy populations or those from healthy military volunteers have not been previously reported.In this study, we reported a cross-sectional observational study conducted in two military camps in the Beijing area, People''s Republic of China. The prevalence of S. aureus nasal colonization and risk factors associated with colonization were assessed. Nasal carriage S. aureus isolates were genotyped to determine potential clonal transmission in military facilities and related transmission factors.(This study was presented in part at the 109th General Meeting of the American Society for Microbiology, Philadelphia, PA, 17 to 21 May 2009.)     

Population Structure of Staphylococcus aureus Strains Isolated from Intensive Care Unit Patients in The Netherlands over an 11-Year Period (1996 to 2006)

The genetic background and the presence of several virulence factors of Staphylococcus aureus isolates from intensive care unit (ICU) patients from 14 hospitals in The Netherlands isolated from 1996 until 2006 were investigated. In total, 936 methicillin-susceptible S. aureus (MSSA) and 7 methicillin-resistant S. aureus (MRSA) isolates were collected. The genetic background was determined by spa typing and multilocus sequence typing (MLST). The virulence determinants Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), and collagen adhesion (CNA) were detected with real-time PCR assays. On the MRSA isolates, mobile resistance staphylococcal cassette chromosome mec (SCCmec) typing was performed. Among the MSSA isolates, 313 different spa types were observed. A genetic background common to MRSA clones, e.g., MLST clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45, was observed among 62% of the isolates. The remaining isolates were associated with MSSA-related MLST CCs. MLST CC1, CC25, and CC30 were continuously present, and other MLST CCs fluctuated over time. Two percent of the MSSA isolates harbored PVL, 21% had TSST-1, and 46% were positive for CNA. There were no changes in the prevalence of the virulence factors over time. Four MRSA isolates were typed as ST8-MRSA-IV (where ST is the MLST sequence type and IV is the SCCmec type), two were ST5-MRSA-II, and one was ST228-MRSA-I. All MRSA isolates were PVL, CNA, and TSST-1 negative except for the two ST5-MRSA-II isolates, which were TSST-1 positive. No changes in the S. aureus genetic background and the prevalence of the virulence factors PVL, CNA, and TSST-1 were observed in ICU patients in The Netherlands over time.Around 20% of all patients in intensive case units (ICUs) acquire an ICU-related infection as a consequence of frequent use of antibiotics and intensive treatment procedures (1, 31). Of all ICU-related infections, 25% are caused by Staphylococcus aureus (31). Knowledge of the S. aureus population structure and of the prevalence of virulence factors has been proven crucial for the investigation of the epidemiology of S. aureus throughout the world (34).Methicillin-resistant S. aureus (MRSA) clones can emerge by horizontal transfer of the staphylococcal cassette chromosome mec (SCCmec) between methicillin-resistant coagulase-negative Staphylococcus or MRSA and methicillin-susceptible S. aureus (MSSA) (51). In the event of antibiotic pressure, the MSSA isolates have a high risk of SCCmec transfer and survive. As shown in the literature, MSSA lineages with a MRSA-unrelated background may not provide a stable genomic environment for the integration of SCCmec (4, 23, 30, 32, 36, 43). SCCmec transfer has been found to be stable in MSSA with a MRSA-related genetic background, i.e., multilocus sequence typing (MLST) clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45 (39). The MSSA lineages with a MRSA background possess certain characteristics that favor their persistence in the host as well as the transfer between hosts.As the highest antibiotic pressure in hospitals is found in ICUs, changes in the genetic background will be the most obvious among isolates from ICU patients. However, little is known about the genetic backgrounds of ICU isolates over time, and, therefore, this study investigates the genetic background and the virulence of S. aureus isolates obtained from 1996 to 2006 from ICU patients from 14 hospitals in The Netherlands.     

Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) in Specimens from Various Body Sites: Performance Characteristics of the BD GeneOhm MRSA Assay,the Xpert MRSA Assay,and Broth-Enriched Culture in an Area with a Low Prevalence of MRSA Infections

Universal surveillance upon patient admission is important in reducing the transmission of methicillin-resistant Staphylococcus aureus (MRSA) and associated disease in hospitals. High costs for the health care system in conjunction with MRSA have promoted the development of rapid screening methods to detect MRSA carriers. This study compared two real-time PCR methods, the BD GeneOhm MRSA assay (BDGO) and the Xpert MRSA assay, with broth-enriched culture to define their performance characteristics and rapidity in an area with low MRSA prevalence. In total, 414 swabs from the nose and 389 swabs from the groin from 425 patients were tested. Of those 425 patients, 378 had swabs from both the nose and groin in parallel. Two hundred thirty-one and 194 patients were randomly assigned to the BDGO group and the Xpert MRSA group, respectively. In general, sensitivity, specificity, and negative predictive value (NPV) were high for the BDGO (100%, 98.5%, and 100%, respectively) and the Xpert MRSA (100%, 98.2%, and 100%, respectively), irrespective of whether or not nasal and inguinal specimens were considered alone or combined. In contrast, the positive predictive value (PPV) was lower: before the resolution of discrepant results, the PPVs for nasal and inguinal specimens alone and combined were 87.5%, 86.7%, and 82.4% for the BDGO and 91.7%, 66.7%, and 92.9% for the Xpert MRSA, respectively. After the resolution of discrepant results, PPVs were 93.8%, 93.3% and 94.1% for the BDGO and 91.7%, 88.9% and 92.9% for the Xpert MRSA, respectively. With the BDGO, 4 of 16 carriers were each identified by nasal or inguinal swabs alone, whereas in the Xpert MRSA group, 4 of 13 carriers were exclusively identified by nasal swabs and 2 of 13 were identified by inguinal swabs alone. Both PCR methods showed no significant difference in the number of discrepant results (odds ratio, 0.70 [P = 0.789]), but specimens from wounds and other body sites (axilla, vagina, and throat) produced discrepancies more often than nasal and groin specimens (odds ratios, 4.724 [P = 0.058] and 12.163 [P < 0.001], respectively). The facts that no false-negative PCR results were detected and increased PPVs were found after the resolution of discrepant results point to PCR as the actual gold standard. Since both sensitivity and NPV were exceptionally high for PCR, backup cultures may, therefore, be unnecessary in an area with low prevalence and with a preemptive isolation strategy but may still be useful for PCR-positive specimens because of the lower PPV for both methods and the possibility of susceptibility testing. The median time for analysis, including extraction, hands-on time, and actual PCR was 2 h 20 min for the Xpert MRSA versus 5 h 40 min for the BDGO. Concerning reporting time, including administration and specimen collection, the Xpert MRSA was faster than the BDGO (7 h 50 min versus 17 h).Methicillin-resistant Staphylococcus aureus (MRSA) strains have become a major concern for health care systems. Prevention of the spread of MRSA has, therefore, become a main goal in the past decade, and active screening programs have been established worldwide (4, 27). Compared to infections caused by methicillin-susceptible S. aureus (MSSA), the organism causes severe infections with increased morbidity and mortality and prolonged hospitalization (9, 17). Unlike countries facing a high prevalence of MRSA, such as the United States and Japan, the prevalence in Switzerland has remained low to date (5, 13, 21, 32). In most parts of our country, prevalence rates between 4% and 7% are observed (19). Apart from its spread in the hospital environment, MRSA carriage in our community, as well as in other countries, seems to be more prevalent than previously assumed (31, 32, 37).To facilitate the rapid detection of colonized patients, real-time PCR assays have been developed. The first method to directly detect MRSA from clinical specimens was developed by Huletsky et al. (20). The principle of this method is used in two commercially available tests, the BD GeneOhm MRSA assay (BDGO) (BD, San Diego, CA) and the Xpert MRSA assay (Cepheid, Inc., Sunnyvale, CA).Recent studies have shown that universal admission surveillance for MRSA was associated with a reduction in MRSA disease (18, 28). Likewise, Cunningham et al. have reported a reduction in MRSA transmissions in a critical care unit. The authors attributed these findings, at least partially, to the availability of rapid PCR screening tests, apart from other measures like improved hygiene measures (10). PCR screening methods are cost efficient, especially in an area of low prevalence where high-risk patients are subjected to preemptive contact isolation (6). Our facility is a 1,000-bed tertiary care teaching hospital with a known low prevalence (<5%) of MRSA colonization of patients and follows a surveillance policy similar to that of the University Hospital of Berne, Switzerland (6). As reported in other studies, this means preemptive isolation on admission of all patients who (i) came from or had traveled to countries with known high prevalence rates for MRSA, (ii) were transferred from long-term care facilities, (iii) were transferred from another health care facility, (iv) were hospitalized within the previous 6 months, and/or (v) had a history of MRSA colonization or infection (6, 8, 23). As soon as PCR is negative for MRSA, patient isolation is ended. Under these circumstances, a rapid screening method with a high negative predictive value (NPV) is desirable, because the bulk of costs emerge mainly from noncolonized patients being unnecessarily isolated. In this study, we compared two real-time PCR methods, the BDGO and Xpert MRSA assays, with broth-enriched culture to assess their performance characteristics and rapidity in an area with a low prevalence of MRSA.     

Incidence,Risk Factors,and Outcomes of Panton-Valentine Leukocidin-Positive Methicillin-Susceptible Staphylococcus aureus Infections in Auckland,New Zealand

Panton-Valentine leukocidin (PVL) has been linked to invasive community-acquired methicillin-resistant Staphylococcus aureus infections. However, the association between disease and PVL-positive methicillin-susceptible Staphylococcus aureus (MSSA) has not been widely reported. We aimed to examine the epidemiology of PVL in clinical MSSA isolates from patients presenting to Auckland City Hospital. Four hundred eleven MSSA clinical isolates and 93 nasal carriage isolates were collected and tested for the presence of the lukSF-PV genes using PCR. The results were examined in light of host and disease factors. Multilocus sequence typing (MLST) was performed on a random subset of isolates to ensure that there was no single PVL-positive MSSA clone responsible for disease in Auckland. The prevalence of the lukSF-PV genes in MSSA isolates associated with disease (124/335; 37%) was not significantly different from the prevalence of the lukSF-PV genes in MSSA nasal carriage isolates (29/93; 31% [P = 0.33]). PVL-positive MSSA isolates in Auckland are genetically diverse and come from a number of different clonal complexes. PVL-positive infections peaked at between 10 and 20 years of age, with a subsequent decline. Pacific ethnicity, age, diagnosis of skin and soft tissue infection (SSTI), community-onset infection, and the need for surgical intervention were found by multivariate analysis to be independently associated with PVL-positive MSSA infection. More than one-third of MSSA infections in our patient population are caused by PVL-positive strains. Those patients with PVL-positive MSSA infection were more likely to be of Pacific ethnicity, be younger in age, have community-onset infection, have SSTI, and need surgical intervention.Staphylococcus aureus is a nasal commensal that can be detected in up to 20 to 30% of the general population, one-third of whom are persistently colonized (28). S. aureus produces a wide variety of virulence factors that contribute to its ability to colonize, invade, and evade the immune system, which includes Panton-Valentine leukocidin (PVL), a bicomponent, pore-forming toxin encoded by two contiguous genes, lukF-PV and lukS-PV. PVL can cause either neutrophil lysis or apoptosis and contributes to tissue necrosis (25). PVL has been linked to skin and soft tissue infections (SSTIs), necrotizing pneumonia, and bone and joint infections in humans (3, 11, 17). Rabbit and human leukocytes are highly sensitive to PVL-mediated leukocytosis (18), and animal studies have shown that PVL causes more-severe disease in dermonecrosis (7, 27), osteomyelitis (6), and necrotizing pneumonia models (B. A. Diep, L. Chan, and P. Tattevin, presented at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 2009).The presence of PVL has been extensively described for methicillin-resistant S. aureus (MRSA), specifically in association with staphylococcal cassette chromosome mec (SCCmec) type IV and also SCCmec type V (4, 25). The epidemiology of PVL-positive methicillin-susceptible S. aureus (MSSA) has not been reported as extensively, and the lukSF-PV genes are not exclusively linked to the presence of the SCCmec element. In the 1950s MSSA ST80 strains, which were associated with outbreaks of SSTI, harbored the lukSF-PV genes (22). There have also been recent reports of PVL-positive MSSA causing clusters of SSTI and necrotizing pneumonia (5, 15).The vast majority of S. aureus strains in New Zealand are methicillin susceptible (MSSA); the prevalence of methicillin-resistant S. aureus (MRSA) remains low, at about 5% (12). New Zealand has a high incidence of S. aureus disease; the incidence of S. aureus bacteremia in the late 1990s was 41 cases per 100,000 adults per year (12). We aimed to examine the prevalence of the lukSF-PV genes in MSSA isolates responsible for disease and asymptomatic nasal carriage, to determine risk factors for infection with PVL-positive MSSA, and to examine the association between PVL and severity of disease.     

Molecular Surveillance and Population Structure Analysis of Methicillin-Susceptible and Methicillin-Resistant Staphylococcus aureus in High-Risk Wards

In this study we report the results of analysis of 253 isolates of Staphylococcus aureus (132 methicillin [meticillin]-resistant S. aureus [MRSA] isolates and 121 methicillin-susceptible S. aureus [MSSA] isolates) from 209 patients admitted to 18 high-risk wards of six hospitals located in Florence, Italy, over an 8-month period during which a program of epidemiological surveillance of hospital-acquired infections was conducted. The majority (69%) of the 87 reported S. aureus infections were caused by MRSA. No outbreak events have been reported. All the isolates were typed by amplified fragment length polymorphism (AFLP), and AFLP profiles were analyzed in order to define similarity groups. The discriminatory power of AFLP is very high with MSSA (Simpson index of diversity [D], 95.9%), whereas its resolution capability with MRSA (D, 44.7%) is hampered by the well-known high clonality of these populations (the main MRSA group accounted for 74% of the MRSA isolates). Combining AFLP, improved by visual inspection of polymorphisms, with multiplex PCR greatly increases MRSA resolution (D, 85.5%), resolving the MRSA population to a level that is one of the highest reported in the literature. Widespread and sporadic clones of MSSA and MRSA were identified, and their diffusion in the different hospitals and wards over the surveillance period was studied. The understanding of MSSA and MRSA population structures should be the starting point for the design of a more rational surveillance program for S. aureus species, maximizing benefits and reducing the cost of infection control strategies.Surveillance of hospital-acquired infections (HAIs), as a critical part of any infection control program, is an indispensable instrument for identification of the dimensions of the problem, for early recognition of changes in infection patterns, and for monitoring of infection trends and rates. Furthermore, surveillance programs allow one to evaluate the effectiveness of interventions, reinforcing good practices and influencing key hospital staff and decision makers (3, 16).Molecular typing techniques greatly improve the quality of epidemiological information obtained by surveillance programs, allowing more-accurate differentiation of strains. Molecular typing techniques are very useful for recognizing sporadic, unrelated strains and endemic, persistent strains (1, 30) and for determining if a single strain or different unrelated strains are the cause of observed increases in the frequency of HAIs by a microbial species.Staphylococcus aureus is one of the main etiologic agents of HAIs, particularly in high-risk wards such as intensive care units (ICUs), and methicillin (meticillin)-resistant S. aureus (MRSA) strains are more frequently involved than methicillin-susceptible S. aureus (MSSA) strains (12, 35). This situation turns out to be particularly serious due to the diffusion of highly pathogenic and multidrug-resistant strains (6).The low degree of genetic variability reported for MRSA populations (30) is a major limitation to strain identification, especially when a short time period and a limited area, such as a single hospital, are monitored. Different molecular typing techniques have been used to point out minor but epidemiologically significant genetic differences between MRSA strains (7, 23, 32, 33, 34). No single technique is clearly superior to others in the resolution of MRSA populations, and a combination of two or more methods has been suggested to be the most efficacious approach (23, 34).Unlike MRSA strains, which have been the subject of several studies of virulence, pathogenesis, development of new antibiotic resistances, strain diffusion worldwide and in hospital settings, and genome analysis (5, 11, 14, 21, 28), MSSA strains, because of their susceptibility to first-line antibiotics, have only occasionally been the subject of molecular epidemiological studies in hospital settings (7, 38). Recent studies performed by multilocus sequence typing have shown a strong genetic relationship between MRSA and MSSA strains, suggesting that MRSA clones arise on multiple occasions from successful hospital MSSA clones by horizontal acquisition of the methicillin resistance (mec) gene (8).In this work, we report the results obtained from an extended molecular surveillance program for S. aureus carried out for 18 high-risk wards of six hospitals in Florence, Italy, over an 8-month period. Our aim was to study the population structure and the diffusion of the MRSA and MSSA strains that colonize and infect patients admitted to the wards under observation. With this aim, amplified fragment length polymorphism (AFLP) analysis was utilized to type MRSA and MSSA isolates, whereas multiplex PCR was used to subtype MRSA isolates falling into the same AFLP group. The Simpson index was employed to evaluate the discriminatory powers of the two molecular techniques and to analyze the structures of both the MRSA and the MSSA populations.     

Evaluation of a New Selective Medium,BD BBL CHROMagar MRSA II,for Detection of Methicillin-Resistant Staphylococcus aureus in Different Specimens

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.Controlling the spread of multidrug-resistant microorganisms and especially methicillin-resistant Staphylococcus aureus (MRSA) has become a major infection control objective in the United States (4) and many European countries (3, 4, 21). A part of most programs to control the spread of MRSA is screening of patients (4, 8, 14), and screening has even become mandatory in some countries (11, 31).Traditionally, MRSA screening included mainly the culturing of naris swabs. However, it has been demonstrated that up to 35% of MRSA carriers may be colonized only from sites other than the nares, for example, the throat or the rectum (1, 2, 16).Usage of chromogenic media can improve the sensitivity and pace of MRSA detection (5, 6, 9, 10, 12, 13, 15, 17,19, 20, 22-24, 26-30); however, currently available media that have been marketed at this time are recommended only for nasal specimens.This study was designed to compare the performance of BBL CHROMagar MRSAII (CMRSAII), a chromogenic medium which incorporates cefoxitin, with traditional culture media in the recovery and identification of MRSA isolates from clinical specimens, including respiratory, lower gastrointestinal, and skin specimens as well as wound cultures and blood culture bottles with Gram-positive cocci. In addition, it was designed to determine whether CMRSAII results may be reported as presumptive or definitive with no (or one) confirmatory test at 24 and 48 h of incubation.(These data were presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)     

Prevalence of and Risk Factors for Colonization by Methicillin-Resistant Staphylococcus aureus among Adults in Community Settings in Taiwan

In order to determine the prevalence of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) colonization among adults in community settings in Taiwan and identify its risk factors, we conducted the present study. For a 3-month period, we enrolled all adults who attended mandatory health examinations at three medical centers and signed the informed consent. Nasal swabs were taken for the isolation of S. aureus. For each MRSA isolate, we performed multilocus sequence typing, identification of the staphylococcal cassette chromosome mec, tests for the presence of the Panton-Valentine leukocidin gene, and tests for drug susceptibilities. Risk factors for MRSA colonization were determined. The results indicated that the MRSA colonization rate among adults in the community settings in Taiwan was 3.8% (119/3,098). Most MRSA isolates belonged to sequence type 59 (84.0%). Independent risk factors for MRSA colonization included the presence of household members less than 7 years old (P < 0.0001) and the use of antibiotics within the past year (P = 0.0031). Smoking appeared to be protective against MRSA colonization (P < 0.0001).Before the late 1990s, nearly all methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) infections occurred in patients with specific risk factors who were in health care facilities (31). However, the emergence of MRSA infections among previously healthy persons in community settings (without exposure to health care facilities) was noted thereafter (6, 31). Therefore, MRSA infections are now classified as health care-associated MRSA (HA-MRSA) infections and community-associated MRSA (CA-MRSA) infections (38).Strains responsible for CA-MRSA infections differ from those for HA-MRSA infections in several phenotypic and genetic features (1, 28). CA-MRSA strains carry type IV or V staphylococcal cassette chromosome mec (SCCmec) elements, are usually Panton-Valentine leukocidin (PVL) producing, and are not multidrug resistant; HA-MRSA strains carry type I, II, or III SCCmec elements, are usually not PVL producing, and are multidrug resistant (15, 22, 28).Initially, CA-MRSA infections were mostly reported in young children (36). However, as CA-MRSA infections became more common, infections were reported among people of all ages and contributed to the increase of community-associated S. aureus infections with significance (25, 29, 36). MRSA colonization is an important risk factor for subsequent MRSA infection (30), so several studies in the United States have characterized the MRSA colonization rate in a community setting (13, 16). These studies demonstrated that the nasal colonization rates among healthy children increased from 0.8% in 2001 to 9.2% in 2004 (13). The colonization rate was 0.84% among people participating in the 2001 to 2002 National Health and Nutrition Examination Survey (NHANES) (16).In Taiwan, MRSA strains of sequence type 59 (ST59), determined by multilocus sequence typing (MLST) and carrying type IV or V SCCmec elements, were recently found to be the major strains of CA-MRSA (5, 7, 27). Other studies demonstrated that these CA-MRSA strains were responsible for the rapid increase in the number of CA-MRSA infections among children and adults in Taiwan (7, 37). The MRSA colonization rates among Taiwanese children increased from 1.5% from 2001 to 2002 to 7.2% from 2005 to 2006 (18, 19). However, the MRSA colonization rate among adults in community settings in Taiwan is unclear. This study was conducted to determine the prevalence and risk factors for the colonization of MRSA among adults in community settings in Taiwan.     

Trends in Methicillin-Resistant Staphylococcus aureus Anovaginal Colonization in Pregnant Women in 2005 versus 2009

In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus. MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) (P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCCmec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) (P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.The rapid spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) across the United States has been associated primarily with the dissemination of a specific clone that has the pulsed-field gel electrophoresis (PFGE) pattern termed USA300 (20). CA-MRSA can cause infections in pregnant and postpartum women and outbreaks in newborn nurseries and neonatal intensive care units (NICUs) (5, 22, 26, 32, 35, 36, 38). CA-MRSA strains, including USA300, have replaced health care-associated (HA)-MRSA as the predominant strains isolated from infants in some NICUs (6, 35). CA-MRSA infections appear to be increasing in otherwise healthy neonates in the nursery (18, 38) who may acquire S. aureus from health care workers or from their mothers and other family members (19, 23, 25, 28).Vertical transmission from mothers to infants at delivery has also been proposed as a possible mechanism of acquisition of CA-MRSA (1, 2, 7, 28). S. aureus has been reported to colonize the vagina in 4 to 22% of pregnant women (2, 4, 9, 13, 14). In 2005, following an outbreak of USA300 in postpartum women at our medical center (Columbia University Medical Center), we determined that the prevalence of methicillin-susceptible S. aureus (MSSA) anovaginal colonization was 16.6% and the prevalence of MRSA colonization was 0.5% (9). Overall, 93% of MRSA strains were staphylococcal cassette chromosome mec (SCCmec) type IV or V, which is consistent with CA-MRSA (9). More recent studies conducted in other locales have suggested that the prevalence of anovaginal colonization with MRSA is increasing, with reported rates ranging from 3.5 to 10.4% (2, 13). The association of MRSA colonization with group B streptococcus (GBS) colonization is less clear; some reports have shown an increased rate of MRSA colonization in GBS-positive women (2), while others have shown a decreased rate (8, 32).Reports suggesting an increasing prevalence of MRSA among pregnant women, coupled with the recent emergence of USA300 in our NICU (6), led us to question whether the epidemiology of MRSA colonization was also changing in pregnant women in our population in New York City, NY. The objectives of this study were to determine the current prevalence of MRSA and MSSA colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to use molecular methods to characterize MRSA strains from the current study and compare those with strains from our 2005 study.     

Presence of Genes Encoding the Panton-Valentine Leukocidin Exotoxin Is Not the Primary Determinant of Outcome in Patients with Complicated Skin and Skin Structure Infections Due to Methicillin-Resistant Staphylococcus aureus: Results of a Multinational Trial

The role of Panton-Valentine leukocidin (PVL) in determining the severity and outcome of complicated skin and skin structure infections (cSSSI) caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is controversial. We evaluated potential associations between clinical outcome and PVL status by using MRSA isolates from patients enrolled in two large, multinational phase three clinical trials assessing telavancin for the treatment of cSSSI (the ATLAS program). MRSA isolates from microbiologically evaluable patients were genotyped by pulsed-field gel electrophoresis (PFGE) and PCR for pvl and 31 other putative virulence determinants. A single baseline pathogen of MRSA was isolated from 522 microbiologically evaluable patients (25.1%) among 2,079 randomized patients. Of these MRSA isolates, 83.2% (432/519) exhibited the USA300 PFGE genotype and 89.1% (465/522) were pvl positive. Patients with pvl-positive MRSA were more likely than those with pvl-negative MRSA to be young, to be North American, and to present with major abscesses (P < 0.001 for each). Patients were significantly more likely to be cured if they were infected with pvl-positive MRSA than if they were infected with pvl-negative MRSA (91.6% versus 80.7%; P = 0.015). This observation remained statistically significant after adjustment for presence of abscess, fever, or leukocytosis; infection size; diabetes; patient age; and study medication received. The fnbA, cna, sdrC, map-eap, sed, seg, sei, sej, SCCmec type IV, and agr group II genes were also associated with clinical response (P < 0.05). This contemporary, international study demonstrates that pvl was not the primary determinant of outcome in patients with MRSA cSSSI.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a leading cause of complicated skin and skin structure infections (cSSSI) in the United States (4, 18, 23). In many regions of the United States, the genetically distinct community-associated MRSA (CA-MRSA) clone USA300 is now the predominant cause of cSSSI (13, 18, 25). Most CA-MRSA isolates isolated from cSSSI carry pvl, the gene encoding Panton-Valentine leukocidin (PVL) (6, 19, 26). PVL is a pore-forming, bicomponent exotoxin known to induce cell death by necrosis or apoptosis (8). Infections caused by S. aureus strains carrying pvl are commonly thought to be associated with worse clinical outcome (6, 16). For example, the presence of the PVL toxin has been shown to cause necrotizing pneumonia in animal models (14) and is associated with necrotizing S. aureus pneumonia in humans (9). However, other laboratories have found that PVL is not a virulence determinant in murine models of CA-MRSA infection (1, 27). In addition, recent studies by our group have suggested that the presence of pvl was associated with a better clinical outcome in patients with cSSSI (2) or bacteremia (15) due to S. aureus. Thus, the role of PVL in determining the clinical severity and outcome of MRSA cSSSI remains unresolved.As part of two phase 3 clinical trials, we have established a large collection of contemporary, geographically diverse S. aureus isolates from a clinically well-characterized population of cSSSI patients. Using this resource, the current study sought to accomplish two objectives, (i) to validate our previous observation that the presence of pvl is not the primary determinant of clinical outcome in patients with cSSSI due to MRSA and (ii) to identify potential associations between other putative bacterial virulence genes and clinical outcome in patients with cSSSI due to MRSA.(These data were presented, in part, at the 48th Annual ICCAC/IDSA 46th Annual Meeting, 25 to 28 October 2008, Washington, DC.)     

Comparison of a Rapid Molecular Method,the BD GeneOhm Cdiff Assay,to the Most Frequently Used Laboratory Tests for Detection of Toxin-Producing Clostridium difficile in Diarrheal Feces

Six hundred diarrheal stool specimens were collected from inpatients and outpatients at local university hospitals for the detection of toxigenic Clostridium difficile using three parallel methods, the BD GeneOhm Cdiff assay, the tissue culture cytotoxicity assay, and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux). Toxigenic C. difficile culture was also performed to further clarify discordant results. During a 3-month study period, 58 (9.7%) of the 600 diarrheal samples examined were positive by the BD GeneOhm Cdiff assay, while the Vidas C. difficile toxin A and B assay and the cytotoxicity assay performed directly on stool samples gave 4.7% and 6.3% positivity rates, respectively. In the case of four samples, BD GeneOhm Cdiff assay results were not evaluable at first because of the presence of PCR inhibitors, but upon repeat testing from the frozen lysates, all of these samples proved to be negative. After resolution with toxigenic culture, the cytotoxicity assay proved to be positive in 55 samples (9.2%), while the ELFA was positive in 37 samples (6.2%). Results of culture and repeated cytotoxicity assays emphasized the importance of the culture method, because the use of ELFA or enzyme immunoassay without a culture method may lead to a substantial portion of toxigenic C. difficile strains being missed.Toxin-producing Clostridium difficile strains are important pathogens among patients who are treated with antibiotics or chemotherapeutic agents not only in the hospital environment but also in the community (3, 6, 10). Since the recognition of outbreaks of C. difficile infection (CDI) caused by C. difficile PCR ribotype 027 in Canada, the United States, and several European countries, rapid and accurate diagnosis of CDI is very important to stop the spread of these strains (7, 8, 19). In addition, the increasing morbidity and mortality rates associated with CDI and the increasing number of recurrences and therapeutic failures also highlight the need for the development of a rapid and reliable detection method for toxigenic C. difficile in diarrheal feces (12).Only a few laboratories routinely use the tissue culture cytotoxicity and toxin neutralization assays for the detection of toxigenic C. difficile in feces, because they are labor-intensive and time-consuming and standardization is very difficult. Due to their rapid turnaround time, enzyme immunoassays (EIAs) that detect toxin A and/or toxin B in stool are used in most laboratories (11, 16). To increase the sensitivity of these tests and in some instances to facilitate epidemiological investigations, culture of C. difficile has become essential. In spite of this, most laboratories use a single toxin detection test on feces for detection of toxigenic C. difficile (4). In the last 10 years, in-house PCR and real-time PCR assays have been developed to detect C. difficile toxin genes. These assays have shown very good sensitivity and specificity and short turnaround times (1, 17). However, widespread use of PCR methods in routine clinical microbiology is limited because these tests require special DNA extraction procedures to eliminate PCR inhibitors from fecal specimens and they cost more than do traditional testing methods.The BD GeneOhm Cdiff assay provides a rapid method for the qualitative detection of the C. difficile toxin B gene (tcdB) in diarrheal specimens from patients suspected of having CDI. This test is based on the amplification of the tcdB gene and the detection of the amplified DNA using fluorogen-labeled probes. Amplification, detection, and interpretation of the results are done automatically by the SmartCycler instrument (Cepheid, Sunnyvale, CA).Our aims were to compare the performance of the BD GeneOhm Cdiff assay to those of the tissue culture cytotoxicity assay and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux, Marcy-l''Etoile, France), for the direct detection of toxins A and B from fecal samples.     

Clinical Application of Real-Time PCR to Screening Critically Ill and Emergency-Care Surgical Patients for Methicillin-Resistant Staphylococcus aureus: a Quantitative Analytical Study

The clinical utility of real-time PCR screening assays for methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) colonization is constrained by the predictive values of their results: as MRSA prevalence falls, the assay''s positive predictive value (PPV) drops, and a rising proportion of positive PCR assays will not be confirmed by culture. We provide a quantitative analysis of universal PCR screening of critical care and emergency surgical patients using the BD GeneOhm MRSA PCR system, involving 3,294 assays over six months. A total of 248 PCR assays (7.7%) were positive; however, 88 failed to be confirmed by culture, giving a PPV of 65%. Multivariate analysis was performed to compare PCR-positive culture-positive (P+C+) and PCR-positive culture-negative (P+C−) assays. P+C− results were positively associated with a history of methicillin-sensitive Staphylococcus aureus infection or colonization (odds ratio [OR], 3.15; 95% confidence interval [CI], 1.32 to 7.54) and high PCR thresholds of signal intensity, indicative of a low concentration of target DNA (OR, 1.19 per cycle; 95% CI, 1.11 to 1.26). P+C− results were negatively associated with a history of MRSA infection or colonization (OR, 0.19; 95% CI, 0.09 to 0.42) and male sex (OR, 0.40; 95% CI, 0.20 to 0.81). P+C+ patients were significantly more likely to have subsequent positive MRSA culture assays and microbiological evidence of clinical MRSA infection. The risk of subsequent MRSA infection in P+C− patients was not significantly different from that in case-matched PCR-negative controls. We conclude that, given the low PPV and poor correlation between a PCR-positive assay and the clinical outcome, it would be prudent to await culture confirmation before altering infection control measures on the basis of a positive PCR result.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is endemic in hospitals and health care facilities in most countries of the world (5). It is frequently carried into the community on colonized, discharged patients, forming a reservoir which then returns to the health care facility when asymptomatic carriers are readmitted (9). Admitted carriers may then develop endogenous infection or become sources of nosocomial transmission to other patients. Screening for MRSA carriage on admission has therefore become a component of many infection control programs (2, 8, 26, 27, 38, 42). This facilitates targeted treatment and infection control measures for MRSA-positive patients while avoiding unnecessary isolation and treatment of noncarriers.Conventional culture-based screening may take several days to produce a result, and it is widely hypothesized that a reduced turnaround time would allow faster implementation of appropriate patient management. PCR-based MRSA screening assays have the potential to provide a result in 2 to 3 h of laboratory time and have a total turnaround time from specimen collection to ward report of approximately 20 h (1, 16, 21). The BD GeneOhm MRSA assay (previously known as IDI-MRSA; BD Diagnostics, San Diego, CA) and the Cepheid GeneXpert MRSA assay (Cepheid, Sunnyvale, CA) are two such PCR tests that detect the presence of characteristic MRSA DNA sequences bridging the SCCmec resistance cassette and the S. aureus-specific orfX open reading frame gene (18). However, concerns have been raised over false-positive and -negative results with these tests (11, 36).Previous estimates of the sensitivities, specificities, negative predictive values (NPV), and positive predictive values (PPV) of the BD IDI-MRSA test and other PCR-based assays have varied considerably (for a summary, see Table S1 in the supplemental material). In our own cluster-randomized crossover study of adult general ward patients admitted during 2006 and 2007, the overall prevalence of MRSA carriage on admission was 6.7% (4.9% of assays) and, in comparison with parallel culture screenings, the BD IDI-MRSA PCR test had a sensitivity of 88%, a specificity of 96%, an NPV of 99%, and a PPV of 55%. (21). With low carriage rates, a high NPV is to be expected, but the relatively low PPV exacerbated concerns about high rates of false positivity with this test.Our previous study did not demonstrate a difference in MRSA acquisition rates between admitted patients screened by PCR and by culture on general medical and surgical wards (21). Therefore, we now limit the use of PCR assays to the high-risk populations of patients in adult and pediatric critical care units and those admitted as surgical emergencies. In the present study, we investigate the performance of the BD GeneOhm MRSA PCR test for admission screening of these groups and consider its usefulness in guiding patient management.     

Mupirocin Resistance among Methicillin-Resistant Staphylococcus aureus-Colonized Patients at Admission to a Tertiary Care Medical Center

All patients admitted to our tertiary care hospital from 1 December 2007 to 10 June 2008 were screened for methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) nasal colonization, and the isolates were tested for mupirocin susceptibility by using Etest. Mupirocin resistance (MR) was noted to occur in 3.4% of MRSA carriers, and high-level MR was noted to occur in 0.62% of carriers.Mupirocin is an antimicrobial that inhibits the synthesis of bacterial proteins by competitive inhibition of bacterial isoleucyl-tRNA synthetase (2). Mupirocin was approved for decolonization of the anterior nares as part of a comprehensive program to control the introduction and spread of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) in health care facilities (Bactroban Nasal package insert; GlaxoSmithKline, Research Triangle Park, NC).MRSA infections continue to increase in incidence and have become endemic in several U.S. hospitals (5, 11). Recently in Illinois, Public Act 095-0312 was passed, mandating MRSA screening for all patients admitted to an intensive care unit as well as for non-intensive care unit patients at high risk for MRSA carriage (9). Nasal colonization with MRSA has been shown to be a risk factor for nosocomial transmission and MRSA infection (8). With the increased use of mupirocin, two different groups of mupirocin-resistant MRSA clones have emerged: those with high-level resistance (≥512 μg/ml) and those with low-level resistance (8 to 256 μg/ml) (7, 11, 12). High-level resistance is believed to be the result of a mutated gene on a plasmid, while low-level resistance is thought to be caused by a chromosomal mutation for a gene coding for the tRNA synthetase (15). Currently, little is known about the prevalence of mupirocin-resistant strains among MRSA-colonized patients upon admission to hospitals throughout the United States. Our study sought to determine the rate of mupirocin resistance among patients testing positive for MRSA nasal colonization upon admission to our tertiary care center.All patients admitted to Loyola University Health Systems from 1 December 2007 to 10 June 2008, with consent, had dual nasal swab specimens collected upon admission. The first nasal swab was screened for MRSA nasal colonization via PCR using the Cepheid Xpert MRSA assay. If the first nasal swab yielded a positive or indeterminate screen result, the second swab was automatically cultured, and MRSA isolates were tested for mupirocin susceptibility by using Etest (AB Biodisk, Solna, Sweden). Quality control for mupirocin Etest strips was performed weekly throughout the course of the study by using Staphylococcus aureus ATCC 29213. All quality control results fell within the tentative quality control range of 0.064 to 0.25 g/ml provided in the Etest package insert. The Etest that was used has not been cleared by the FDA. Presence of resistance was defined as ≥8 μg/ml. High-level resistance was defined as a MIC of ≥512, and low-level resistance was defined as a MIC of 8 to 256 μg/ml.During the study period, 14,840 patients were screened and 948 (6.3%) patients tested positive for MRSA by the PCR screen. Five hundred ninety-one of the patients with positive screen results had positive MRSA cultures. Dual swabs were not available on all patients with positive screen results, and the culture positivity rates among patients with positive screen results ranged from 69 to 78% per month. Twenty of 591 (3.4%) isolates were resistant to mupirocin; 17 (2.9%) isolates had low-level mupirocin resistance (MIC, 8 to 256 μg/ml), and 3 (0.5%) had high-level mupirocin resistance (MIC, ≥512 μg/ml). Of the 20 patients with mupirocin-resistant MRSA, 9 were females and 11 were males (P = 0.821). These patients ranged from 2 years to 95 years of age. Twelve patients were older than 60 years of age. One of the 40 pediatric patients colonized with MRSA had a low-level-mupirocin-resistance MRSA isolate. One of the three patients with high-level resistance had a previous history of multiple MRSA abscesses. None of these three patients had a history of mupirocin therapy in the past.Despite the reported increased incidence of mupirocin-resistant S. aureus from various regions of the world, the literature on this topic is limited to relatively isolated areas (4, 16, 18, 20, 21). The first large study of universal surveillance for MRSA in the United States, published in 2008, reported the use of mupirocin for nasal decolonization; however, it did not address the incidence of mupirocin resistance among the MRSA isolates (17). In a subsequent letter, the authors of this study reported that mupirocin resistance occurred in 5.6% of isolates before the surveillance began and 4.1% at the end of program''s first year of universal surveillance (16). Studies addressing the true prevalence of mupirocin-resistant MRSA throughout hospitals in United States are lacking.Mupirocin-resistant MRSA is present in our Chicago area patient population at admission, but it comprised only 3.4% of MRSA isolates, and high-level resistance was distinctly uncommon. Although 60% of mupirocin-resistant MRSA isolates were found in patients >60 years of age, it does not appear that increased age is a risk factor for mupirocin-resistant MRSA. Our data, in conjunction with previous published studies, indicate that low-level mupirocin resistance was more prevalent than high-level mupirocin resistance among MRSA isolates (1, 10, 14). The occurrence of high-level-mupirocin-resistance MRSA in patients without past histories of mupirocin exposure is cause for concern.The clinical significance of low- or high-level mupirocin resistance upon the efficacy of mupirocin decolonization remains unclear. Our study was not designed to address this issue. The published studies offer disparate results. Topical mupirocin achieves remarkably high concentrations (20,000 μg/ml) which far exceed the MICs of even high-level-mupirocin-resistance strains (3, 6). Semret and Miller found that there was no significant difference in MRSA clearance of all infected sites, regardless of whether the patients were colonized with mupirocin-sensitive MRSA (68% clearance) or mupirocin-resistant MRSA (52% clearance) (P = 0.365) but that when patients were colonized with MRSA only in the nares, clearance rates were slightly better in patients colonized with mupirocin-sensitive strains (85.7%) than in those colonized with mupirocin-resistant strains (44.4%) (P = 0.145) (19).The induction of mupirocin resistance through sporadic nasal decolonization of MRSA-colonized patients in the preoperative setting or for hospital admissions is also not well defined. Studies have shown the emergence of mupirocin-resistant isolates in some settings where mupirocin-containing MRSA decolonization regimens were used (13). Mupirocin-resistant MRSA has also been associated with an increase in in-hospital mortality, compared to the level associated with mupirocin-susceptible MRSA (10). Large-scale studies and epidemiologic data on mupirocin use, trends in mupirocin resistance, and eventual correlation with clinical outcomes are lacking.Our data indicate that high-level mupirocin resistance is distinctly uncommon in our patient population. Periodic monitoring would be useful for detecting changing trends in mupirocin resistance, especially since recently enacted state legislations mandate active MRSA surveillance programs, in turn helping to drive increased use of mupirocin decolonization treatment to curb MRSA transmission.     

High Levels of mecA DNA Detected by a Quantitative Real-Time PCR Assay Are Associated with Mortality in Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is known to be a poor prognostic factor. While several PCR assays for the detection of MRSA in various clinical samples were recently reported, the possibility that a quantitative PCR assay could be used to quantify and monitor MRSA bacteremia has not been explored. In this study, we established a quantitative real-time PCR assay for the mecA gene using known copy numbers of a plasmid containing mecA DNA as a standard and the previously described mecA-specific primers and probe (P. Francois et al., J. Clin. Microbiol. 41:254-260, 2003). We employed this assay to examine 250 sequential whole-blood samples from 20 adult patients, including 13 survivors and 7 nonsurvivors, with culture-proven MRSA bacteremia at the intensive care units of National Taiwan University Hospital between 1 July 2006 and 31 January 2007. The levels of mecA DNA in the nonsurvivors were significantly higher than those in the survivors during the three periods of bacteremia examined (days 0 to 2, 3 to 5, and 6 to 8) (P = 0.003 by two-tailed Mann-Whitney U test). Moreover, the nonsurvivors had higher mecA DNA levels than the survivors after 3 days and 7 days of anti-MRSA therapy (medians for nonsurvivors and survivors at 3 days, 5.86 and 4.30 log copies/ml, respectively; medians for nonsurvivors and survivors at 7 days, 5.21 and 4.36 log copies/ml, respectively; P = 0.02 and P = 0.04, respectively, by two-tailed Mann-Whitney U test). Together, these findings suggest that the level of mecA DNA in blood could potentially be used to monitor MRSA bacteremia and evaluate responses to therapy.Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that is one of the most common causes of both community-acquired and nosocomial infections worldwide, especially in intensive care units (ICUs) (5, 8, 33, 49). In the past two decades, the proportion of MRSA infections has increased dramatically, and up to 60% of S. aureus isolates from ICUs were reported to be methicillin resistant (26). Compared with cases of bacteremia caused by methicillin-susceptible S. aureus (MSSA) strains, cases of bacteremia caused by MRSA strains have been shown to be associated with more persistent infections, more recurrent episodes, longer hospital stays, and higher rates of mortality (3, 6, 11, 13, 14, 27). Because of the high rates of mortality and the refractoriness of MRSA bacteremia to treatment, MRSA bacteremia has become a very challenging infectious disease (18, 21, 38, 39, 42).It has recently been demonstrated that high bacterial loads in blood correlate with disease severity in patients with infections caused by Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis (4, 22, 35, 41), as well as in a murine model of Serratia marcescens infection (25). In the case of S. aureus-related infections, it was reported that positive blood cultures during follow-up and the persistence of fever were suggestive of a complicated course (6, 15, 37). However, the drastic decrease in the sensitivity of blood culture after the initiation of antimicrobial therapy, even when special culture media are used, has made the use of blood cultures to monitor responses to therapy and outcomes very difficult (17, 19, 31, 36). On the other hand, most of the real-time PCR assays used for the detection of MRSA have been qualitative in nature and have used specimens from blood culture bottles or nasal swabs rather than blood samples directly and therefore have provided little information regarding the MRSA load in blood (16, 20, 24, 43, 48).In this study, we established a quantitative real-time PCR assay to quantify the mecA DNA load in blood by using a previously described mecA-specific primer pair and probe and known copy numbers of a plasmid containing the mecA gene as a standard (16). We then used this assay to investigate the mecA DNA load during the course of infection in 20 patients with culture-proven MRSA bacteremia. We applied this assay to monitor patients with MRSA bacteremia and demonstrated that high mecA DNA loads after 3 days and 7 days of therapy correlate with poor outcomes.     

Emergence of a Community-Associated Methicillin-Resistant Staphylococcus aureus Strain with a Unique Resistance Profile in Southwest Nigeria

Phenotypic, genotypic, and toxin gene analyses have not yet been done all in one for the Nigerian Staphylococcus aureus population. This study provides a comprehensive overview of the molecular epidemiology and genetic diversity of S. aureus strains at the largest university clinic in Ibadan, Nigeria. From 1,300 patients'' clinical samples collected at the University Teaching Hospital in Ibadan, Nigeria, during a 1-year-surveillance in 2007, 346 nonduplicate S. aureus isolates were obtained. All isolates underwent antibiotic susceptibility testing, toxin gene analysis, multilocus sequence typing, agr group typing, and spa typing. For methicillin (meticillin)-resistant S. aureus (MRSA), staphylococcal cassette chromosome mec (SCCmec) typing was also performed. Of the 346 isolates, 20.23% were methicillin resistant. Thirty-three patients'' isolates (47.15%) fulfilled the definition criteria for community-associated MRSA (CA-MRSA) according to a review of the medical charts. The majority of MRSA strains analyzed were isolated from surgical or pediatric patients. The commonest types of MRSA infection identified were surgical-site infections (>70%), whereas those for CA-MRSA were conjunctivitis and otitis (19 patients [57.6%]) and accidental skin and subcutaneous tissue infections (14 patients [42.4%]). The methicillin-susceptible S. aureus strains (ST1, ST5, ST15, ST7, ST8, ST25, ST30, ST72, ST80, ST121, and ST508) were heterogeneous by phenotypic and genotypic analyses. The first report of a Panton-Valentine leukocidin-positive ST88 strain (agr III, SCCmec IV) in Nigeria, as well as genetic analyses of this strain, is presented in this study. The ST88 strain was resistant to trimethoprim-sulfamethoxazole as well as to penicillin and oxacillin. CA-MRSA infections are increasing rapidly among young patients with ophthalmologic and auricular infections. Urban regions with populations of lower socioeconomic status and evidence of overcrowding appear to be at high risk for the emergence of this clone.Staphylococcus aureus is an important human pathogen and is implicated in a wide variety of infections (13, 19, 28). In Nigeria, S. aureus causes significant epidemiologic and therapeutic problems. Nigeria is the most densely populated African country, and Ibadan is the capital of the Southwest province Oyo, which has a population of 3.6 million and is the largest geographical area. Over the past 20 years, the incidences of both community-acquired (CA) and hospital-acquired (HA) S. aureus infections have increased, while antibiotic treatment is increasingly hampered by the spread of S. aureus strains that are resistant to multiple antibiotics, including methicillin (meticillin) (10, 11, 19, 32).The African data on S. aureus, particularly on antibiotic susceptibility, are extremely limited (3, 27), although methicillin-resistant S. aureus (MRSA) has disseminated in African countries. Between 1996 and 1997, the prevalences of MRSA, determined in eight African countries, were relatively high in Nigeria, Kenya, and Cameroon (21 to 30%) and were below 10% in Tunisia and Algeria, although in Algeria this rate increased to 14% (16, 26). All MRSA isolates were sensitive to vancomycin. The isolates were also highly sensitive to ciprofloxacin, except in Kenya, Morocco, and Tunisia, where relative resistance to this drug has been reported (16). Moreover, the results of 4 years of studies from a number of hospitals in Kenya have shown that 90% of patients admitted to burn units were colonized or infected with MRSA (20). The increasing prevalence of MRSA infections among nonhospitalized patients due to the emergence of unique community-associated S. aureus strains has become a Nigerian problem as well as a global problem. Because the genetic analysis of indigenous S. aureus strains is limited in Nigeria, we aimed to study the genetics, prevalence, and dissemination of such strains in Ibadan, where one of the largest university hospitals in Nigeria is located (2, 23).The objectives of this study were (i) to determine the antibiotic susceptibility profiles, genotypes, and toxin profiles of methicillin-susceptible S. aureus (MSSA) and MRSA strains from two hospitals in Ibadan, Nigeria, (ii) to determine the prevalence of MRSA, and (iii) to characterize the genetic determinants of CA-MRSA strains upon hospital admission.(Part of this work was presented at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, September 2007.)     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Predominance and Emergence of Clones of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus in Malaysia

We define the epidemiology of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in a central teaching and referral hospital in Kuala Lumpur, Malaysia. This is done on the basis of spa sequencing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and virulence gene profiling. During the period of study, the MRSA prevalence was 44.1%, and 389 MRSA strains were included. The prevalence of MRSA was found to be significantly higher in the patients of Indian ethnicity (P < 0.001). The majority (92.5%) of the isolates belonged to ST-239, spa type t037, and possessed the type III or IIIA SCCmec. The arginine catabolic mobile element (ACME) arcA gene was detected in three (1.05%) ST-239 isolates. We report the first identification of ACME arcA gene-positive ST-239. Apart from this predominant clone, six (1.5%) isolates of ST-22, with two related spa types (t032 and t4184) and a singleton (t3213), carrying type IVh SCCmec, were detected for the first time in Asia. A limited number of community-acquired (CA) MRSA strains were also detected. These included ST-188/t189 (2.1%), ST-1/t127 (2.3%), and ST-7/t091 (1%). Panton-Valentin leukocidin (PVL) was detected in all ST-1 and ST-188 strains and in 0.7% of the ST-239 isolates. The majority of the isolates carried agr I, except that ST-1 strains were agr III positive. Virulence genes seg and sei were seen only among ST-22 isolates. In conclusion, current results revealed the predominance of ST-239-SCCmec III/IIIA and the penetration of ST-22 with different virulence gene profiles. The emergence in Malaysia of novel clones of known epidemic and pathogenic potential should be taken seriously.Methicillin-resistant Staphylococcus aureus (MRSA) is an established human pathogen that causes both health care-associated (HA) and community-acquired (CA) infections. Modern MRSA has evolved from several successful clonal lineages of methicillin-susceptible S. aureus strains via acquisition of a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). This element contains the mecA gene, which encodes penicillin-binding protein 2′ (PBP2′) with significantly reduced affinity for β-lactams (36).Increased emergence of multidrug resistance among MRSA strains has become a major concern in the hospital environment, as it invokes a tremendous financial burden and enhanced morbidity and mortality due to hard-to-treat systemic infections (7). Genotyping data from large international studies have shown that a limited number of major clones of MRSA display an enhanced propensity to spread and cause opportunistic human infections in various parts of the world (5, 13, 33). MRSA was introduced into Malaysia in the early 1970s (26), and a review of the records of the microbiology laboratories of all state hospitals in Malaysia showed that the proportion of MRSA isolates from S. aureus-infected individuals had been approximately 21% throughout the last few years.Phenotypic MRSA typing methods are not suited to detailed epidemiological surveillance (20, 40). Several superior molecular typing methods can be used for supporting infection control and for tracing the nosocomial sources and transmission routes of bacterial pathogens. Among these methods, multilocus sequence typing (MLST) has recently been proven to be the best for long-term global epidemiological and bacterial population genetics studies. It is a discriminatory genotypic method where isolates are typed by sequencing variable regions of housekeeping genes. A combination of alleles from the seven loci forms a sequence type (ST). Similar STs are grouped into clonal complexes (13). spa typing has been shown to be as discriminatory as the current gold standard method, pulsed-field gel electrophoresis (PFGE) (10). spa types can be determined via amplification and sequencing of the X region of the protein A gene (spa), which contains polymorphic direct repeats. StaphType (version 1.4; Ridom GmbH, Würzburg, Germany) provides a software tool enabling straightforward sequence analysis and designation of spa types by synchronization via a central server (42). spa typing is useful in analyzing hospital outbreaks and in identifying genetic changes that occur over a relatively short time span (14, 43). Molecular typing data on HA MRSA isolates in Malaysia are sparse in comparison with those on strains deriving from Europe, the United States, or Japan. This is notwithstanding the steadily increasing but already high rates of MRSA infections in Malaysia (38). Pilot data hinted at the predominance of a single MRSA genotype in Malaysia. This is similar to the situation in many other Asian countries (1, 23, 31).For the present study, clinical MRSA isolates associated with various infections were collected from the largest public tertiary referral hospital in Kuala Lumpur, the capital of Malaysia. The epidemiology of MRSA in this hospital (HKL) will most likely reflect the nation''s epidemiology as it is the major government referral hospital for patients from all states in Malaysia. Each year at least 1 million people (∼3.8% of the total Malaysian population) are treated here. HKL records an annual MRSA prevalence over 40%. Many of these MRSA strains are multidrug resistant. Additional epidemiological studies are clearly warranted in order to increase the insight into the dynamics of MRSA epidemiology in Malaysia.The aims of the present study were to characterize the current Malaysian MRSA isolates and determine their molecular epidemiology by MLST, spa typing, SCCmec typing, and virulence gene profiling.     

Transmission of Methicillin-Resistant Staphylococcus aureus to Household Contacts

The frequency of and risk factors for methicillin-resistant Staphylococcus aureus (MRSA) transmission from a MRSA index person to household contacts were assessed in this prospective study. Between January 2005 and December 2007, 62 newly diagnosed MRSA index persons (46 patients and 16 health care workers) and their 160 household contacts were included in the study analysis. Transmission of MRSA from an index person to household contacts occurred in nearly half of the cases (47%; n = 29). These 29 index persons together had 84 household contacts, of which two-thirds (67%; n = 56) became MRSA positive. Prolonged exposure time to MRSA at home was a significant risk factor for MRSA transmission to household contacts. In addition, MRSA colonization at least in the throat, younger age, and eczema in index persons were significantly associated with MRSA transmission; the presence of wounds was negatively associated with MRSA transmission. Furthermore, an increased number of household contacts and being the partner of a MRSA index person were household-related risk factors for MRSA acquisition from the index person. No predominant pulsed-field gel electrophoresis (PFGE) type was observed to be transmitted more frequently than other PFGE types. To date, screening household contacts and providing MRSA eradication therapy to those found positive simultaneously with the index person is not included in the “search-and-destroy” policy. We suggest including both in MRSA prevention guidelines, as this may reduce further spread of MRSA.Methicillin-resistant Staphylococcus aureus (MRSA) is currently the most prevalent antibiotic-resistant pathogen in hospitals in many parts of the world, and there are a growing number of reports describing its increasing prevalence in various community populations (10-12). MRSA is an important cause of infections, and MRSA infections are increasing in both health care centers and the community. Compared to methicillin-sensitive Staphylococcus aureus (MSSA), infections with MRSA are more difficult to treat and tend to have a poorer outcome (2, 8).Carriage of MRSA is a prerequisite for most MRSA infections and plays an important role in the dissemination of this organism within health care facilities and into the community (3, 6, 7, 9). In the Netherlands, due to the “search-and-destroy” infection control policy and a strict antibiotic policy, the number of patients colonized with MRSA is still very limited (13, 31, 34). The “Destroy” part of this policy is important, as it eliminates two out of the three known reservoirs, carriage in patients and carriage in health care workers (HCWs), whereas the third reservoir is the environment. But even in low-prevalence countries like the Netherlands, the emergence of community-acquired MRSA has caused a change in MRSA epidemiology and an increasing number of MRSA cases (13).In the past, it has been shown that carriers of Staphylococcus aureus and MRSA can be a source of transmission of these pathogens to their household contacts (5, 17, 18, 21, 26). The exact risk factors for transmission of MRSA to household contacts have not been studied properly, but close contact, the environment, or being an HCW are thought to be plausible risk factors for transmission (28, 29, 32).The contribution of transmission in households to the MRSA burden has not yet been studied, and because of lack of data and well-calculated scenarios, no evidence-based policy for this reservoir has been developed. For this reason, being a household contact of a MRSA carrier has not yet been established as a risk group for MRSA under the Dutch “search-and-destroy” policy.The aims of this study are to gain insight in the frequency of and risk factors for transmission of MRSA to household contacts and therefore into the community.(The work was presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy—Infectious Diseases Society of America [ICAAC/IDSA], 24 to 28 October 2008, Washington, DC [24a].)     

Patterns of Susceptibility of Aspergillus Isolates Recovered from Patients Enrolled in the Transplant-Associated Infection Surveillance Network

We analyzed antifungal susceptibilities of 274 clinical Aspergillus isolates from transplant recipients with proven or probable invasive aspergillosis collected as part of the Transplant-Associated Infection Surveillance Network (TRANSNET) and examined the relationship between MIC and mortality at 6 or 12 weeks. Antifungal susceptibility testing was performed by the Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth dilution method for amphotericin B (AMB), itraconazole (ITR), voriconazole (VOR), posaconazole (POS), and ravuconazole (RAV). The isolate collection included 181 Aspergillus fumigatus, 28 Aspergillus niger, 27 Aspergillus flavus, 22 Aspergillus terreus, seven Aspergillus versicolor, five Aspergillus calidoustus, and two Aspergillus nidulans isolates and two isolates identified as Aspergillus spp. Triazole susceptibilities were ≤4 μg/ml for most isolates (POS, 97.6%; ITR, 96.3%; VOR, 95.9%; RAV, 93.5%). The triazoles were not active against the five A. calidoustus isolates, for which MICs were ≥4 μg/ml. AMB inhibited 93.3% of isolates at an MIC of ≤1 μg/ml. The exception was A. terreus, for which 15 (68%) of 22 isolates had MICs of >1 μg/ml. One of 181 isolates of A. fumigatus showed resistance (MIC ≥ 4 μg/ml) to two of three azoles tested. Although there appeared to be a correlation of higher VOR MICs with increased mortality at 6 weeks, the relationship was not statistically significant (R² = 0.61; P = 0.065). Significant relationships of in vitro MIC to all-cause mortality at 6 and 12 weeks for VOR or AMB were not found.Invasive aspergillosis (IA) is an important problem in immunocompromised patients, especially in persons who have received hematopoietic stem cell or solid organ transplantation. On the basis of recent treatment guidelines, voriconazole is recommended as the primary therapy for IA, with alternatives including lipid preparations of amphotericin B (AMB), caspofungin, micafungin, itraconazole (ITR), and posaconazole (POS) (28). In vitro resistance among Aspergillus species is uncommon and may be increasing (5, 9, 27, 30). Several studies report prevalence of triazole resistance of up to 4.2% among Aspergillus isolates, or as much as 2.1% among Aspergillus fumigatus isolates (13, 14, 23). Triazole cross-resistance has been reported in several studies (14, 18, 19). In contrast, other studies have described triazole resistance in <1% of isolates, even in the postvoriconazole era (8, 19). Because of the potential of increasing MICs to triazoles and widespread use of triazoles for IA treatment, surveillance of Aspergillus susceptibility, especially among isolates causing IA, is warranted.Examining the influence of antifungal resistance on clinical outcomes has been challenging because of the difficulty in establishing large cohorts of IA patients with available isolates and because of the low frequency of resistant isolates. Moreover, a myriad of factors besides isolate susceptibility may contribute to patient outcomes. Recent reports describe the challenges of in vitro-in vivo correlations of Aspergillus spp. with current susceptibility testing methodologies (2, 17, 21). This challenge is especially daunting when considering the number of host- and transplant-related variables that impact outcomes of this infection (16, 25). While the impact of resistant Aspergillus isolates on outcomes has been demonstrated in animal models for triazoles, echinocandins, and AMB, a paucity of human data is available (2, 7, 12, 13, 17). Herein, we describe in vitro susceptibility patterns of Aspergillus isolates from transplant recipients with proven or probable IA collected as part of the Transplant-Associated Infection Surveillance Network (TRANSNET). In addition, the in vitro MIC in relationship to all-cause mortality is examined.