Genotypic and phenotypic characterization of methicillin-susceptible Staphylococcus aureus isolates misidentified as methicillin-resistant Staphylococcus aureus by the BD GeneOhm MRSA assay

Twenty-three nasal swab samples that tested positive for methicillin-resistant Staphylococcus aureus (MRSA) on initial testing by the BD GeneOhm MRSA assay (BD-MRSA PCR; BD GeneOhm, San Diego, CA) were culture positive only for methicillin-susceptible S. aureus (MSSA) from an enrichment broth. The 23 recovered isolates were confirmed as MSSA by a variety of phenotypic methods, including the BD Phoenix automated microbiology system (BD Diagnostics, Sparks, MD), oxacillin screening agar (BD Diagnostics), BBL CHROMagar MRSA (BD Diagnostics), and a PBP2' assay (Denka Seiken Co., Tokyo, Japan); susceptibilities were determined by using Mueller-Hinton agar with oxacillin. All were positive by nuc PCR, specific for S. aureus, but negative for mecA with one exception. Isolates were characterized by using multiplex PCR methodology to determine structural types and variants (SCCmec typing); additional PCRs were performed for the detection of the ccr and mec complexes, the junkyard regions as well as the Panton-Valentine leukocidin. Pulsed-field gel electrophoresis was used to determine clonality. One phenotypic MSSA isolate contained an intact SCCmec. Twelve MSSA isolates tested positive for MRSA by the BD-MRSA PCR because of amplification of the mec priming site flanking the SCC insertion point, although these isolates lacked mecA. The 10 remaining isolates were not MRSA and tested as MSSA by phenotypic and genotypic assays. In our patient population, diagnostic and surveillance testing and subsequent infection control practices may be impacted by the frequency of these excision events when using the BD-MRSA PCR for MRSA detection.     

The detection and differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus endocarditis by using the BD GeneOhm StaphSR Assay

We use the BD GeneOhm StaphSR Assay (BD Diagnostics, Oakville, Canada) to screen for Staphylococcus aureus nasal colonization and sought to evaluate this assay for the assessment of valve specimens from patients with endocarditis. We examined 23 paired fresh and formalin-fixed, paraffin-embedded cardiac valve tissue samples, 12 of which had S aureus endocarditis, using the BD GeneOhm StaphSR Assay for the detection and differentiation of methicillin-susceptible and methicillin-resistant S aureus. This assay appropriately characterized all specimens with respect to the presence or absence of S aureus. There was an 87.5% correlation between the presence or absence of the mecA gene and the oxacillin susceptibility results for the S aureus isolates studied. The GeneOhm StaphSR assay accurately detected S aureus in cardiac valve tissue samples. Rare discordances were observed between oxacillin susceptibility status and mecA gene detection by this assay.     

Multicenter evaluation of BBL CHROMagar MRSA medium for direct detection of methicillin-resistant Staphylococcus aureus from surveillance cultures of the anterior nares

Active surveillance for methicillin-resistant Staphylococcus aureus (MRSA) is among the strategies recommended by the Society for Healthcare Epidemiology of America for control of nosocomial MRSA infections. Infection control and laboratory personnel desire rapid, sensitive, and inexpensive methods to enhance surveillance activities. A multicenter study was performed to evaluate a new selective and differential chromogenic medium, BBL CHROMagar MRSA (C-MRSA) medium (BD Diagnostics, Sparks, MD), which enables recovery and concomitant identification of MRSA strains directly from nasal swab specimens taken from the anterior nares. Specimens were inoculated to C-MRSA and Trypticase soy agar with 5% sheep blood agar (TSA II, BD Diagnostics). Mauve colonies on C-MRSA at 24 h and 48 h and suspicious colonies on TSA II were confirmed as Staphylococcus aureus by Gram stain morphology and a coagulase test. In addition, the results of C-MRSA were compared to results of susceptibility testing (five different methods) of S. aureus strains isolated on TSA II. A total of 2,015 specimens were inoculated to C-MRSA and TSA II. Three hundred fifty-four S. aureus isolates were recovered; 208 (59%) were oxacillin (methicillin) susceptible and 146 (41%) were oxacillin resistant (MRSA). On C-MRSA, 139/146 or 95.2% of MRSA isolates were recovered, whereas recovery on TSA II was 86.9% (127/146) (P = 0.0027). The overall specificity of C-MRSA was 99.7%. When C-MRSA was compared to each susceptibility testing method, the sensitivity and specificity, respectively, were as follows: oxacillin MIC by broth microdilution, 94.4% and 96.7%; oxacillin screen agar, 94.3% and 96.7%; PBP2' latex agglutination, 93.7% and 98.5%; cefoxitin disk diffusion, 95.0% and 98.1%; and mecA PCR, 95.1% and 98.1%. In this study, C-MRSA was superior to TSA II for recovery of MRSA from surveillance specimens obtained from the anterior nares and was comparable to conventional, rapid, and molecular susceptibility methods for the identification of MRSA isolates.     

Comparison of the Next-Generation Xpert MRSA/SA BC Assay and the GeneOhm StaphSR Assay to Routine Culture for Identification of Staphylococcus aureus and Methicillin-Resistant S. aureus in Positive-Blood-Culture Broths

A bloodstream infection with Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a serious condition that carries a high mortality rate and is also associated with significant hospital costs. The rapid and accurate identification and differentiation of methicillin-susceptible S. aureus (MSSA) and MRSA directly from positive blood cultures has demonstrated benefits in both patient outcome and cost-of-care metrics. We compare the next-generation Xpert MRSA/SA BC (Xpert) assay to the GeneOhm StaphSR (GeneOhm) assay for the identification and detection of S. aureus and methicillin resistance in prospectively collected blood culture broths containing Gram-positive cocci. All results were compared to routine bacterial culture as the gold standard. Across 8 collection and test sites, the Xpert assay demonstrated a sensitivity of 99.6% (range, 96.4% to 100%) and a specificity of 99.5% (range, 98.0% to 100%) for identifying S. aureus, as well as a sensitivity of 98.1% (range, 87.5% to 100%) and a specificity of 99.6% (range, 98.3% to 100%) for identifying MRSA. In comparison, the GeneOhm assay demonstrated a sensitivity of 99.2% (range, 95.2% to 100%) and a specificity of 96.5% (range, 89.2% to 100%) for identifying S. aureus, as well as a sensitivity of 94.3% (range, 87.5% to 100%) and a specificity of 97.8% (range, 96.1% to 100%) for identifying MRSA. Five of six cultures falsely reported as negative for MRSA by the GeneOhm assay were correctly identified as positive by the Xpert assay, while one culture falsely reported as negative for MRSA by the Xpert assay was correctly reported as positive by the GeneOhm assay.     

Real-time PCR can rapidly detect methicillin-susceptible and methicillin-resistant Staphylococcus aureus directly from positive blood culture bottles

We developed, validated, and implemented real-time polymerase chain reaction (PCR) detection of the femA gene for Staphylococcus aureus and the mecA gene for methicillin resistance directly from BACTEC (Becton Dickinson, Sparks, MD) blood culture bottles showing gram-positive cocci in clusters. For the 332 positive blood cultures tested, the assay had 100% sensitivity and specificity for identifying methicillin-susceptible (n=28) and methicillin-resistant (n=28) S aureus, and overall was 98% sensitive and 94% specific, with 3 uninterpretable test results when identification of coagulase-negative staphylococci was included. PCR detection yields rapid (2-3 hours) results and accurate identification of S aureus directly from signal-positive blood culture bottle samples.     

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.     

Evaluation of the BBL Crystal MRSA ID System for detection of oxacillin resistance in Staphylococcus aureus.

AIMS: To evaluate the BBL Crystal MRSA ID System for detection of oxacillin resistance in Staphylococcus aureus. METHODS: 52 methicillin resistant S aureus (MRSA) from five different countries and 85 methicillin susceptible S aureus (MSSA) were included. The species was confirmed by tube coagulation and detection of the clumping factor using the Staphaurex Plus. Clonal non-identity of the MRSA isolates was shown by pulsed field gel electrophoresis. MIC values (oxacillin) were determined using the Etest. Polymerase chain reaction was carried out to detect the mecA gene. The BBL Crystal MRSA ID System was carried out according to the manufacturer's instructions. RESULTS: The BBL Crystal MRSA ID System showed fluorescence in 45 of 52 MRSA (sensitivity 86.5%; negative predicitive value 92.2%), and the specificity was 97.6% (positive predicitive value 95.7%). Two of seven MRSA that failed to show fluorescence had MIC values (oxacillin) of 4 mg/litre. CONCLUSIONS: The BBL Crystal MRSA ID System is a valuable test for detecting oxacillin resistance in S aureus. Its major advantage is the short time (4-5 hours) required to perform the test when organisms are grown on tryptic soy agar or sheep blood agar. Difficulties may arise in borderline resistant isolates.     

Comparison of the BBL CHROMagar Staph aureus agar medium to conventional media for detection of Staphylococcus aureus in respiratory samples

Screening for Staphylococcus aureus has become routine in certain patient populations. This study is the first clinical evaluation of the BBL CHROMagar Staph aureus agar (CSA) medium (BD Diagnostics, Sparks, Md.) for detection of S. aureus in nasal surveillance cultures and in respiratory samples from cystic fibrosis (CF) patients. S. aureus colonies appear mauve on CSA. Other organisms are inhibited or produce a distinctly different colony color. S. aureus was identified from all media by slide coagulase, exogenous DNase, and mannitol fermentation assays. Susceptibility testing was performed using the agar dilution method. A total of 679 samples were evaluated. All samples were inoculated onto CSA. Nasal surveillance cultures were inoculated onto sheep blood agar (SBA) (BD Diagnostics), and samples from CF patients were inoculated onto mannitol salt agar (MSA) (BD Diagnostics). Of the 679 samples cultured, 200 organisms produced a mauve color on CSA (suspicious for S. aureus) and 180 were positive for S. aureus on SBA or MSA. Of 200 CSA-positive samples 191 were identified as S. aureus. Nine mauve colonies were slide coagulase negative and were subsequently identified as Staphylococcus lugdunensis (one), Staphylococcus epidermidis (three), Staphylococcus haemolyticus (one), and Corynebacterium species (four). CSA improved the ability to detect S. aureus by recovering 12 S. aureus isolates missed by conventional media. Of the 192 S. aureus isolates recovered, 122 were methicillin susceptible and 70 were methicillin resistant. Overall, the sensitivity and specificity of CSA in this study were 99.5 and 98%, respectively. There was no difference in the performance of the slide coagulase test or in susceptibility testing performed on S. aureus recovered from CSA compared to SBA or MSA. Our data support the use of CSA in place of standard culture media for detection of S. aureus in heavily contaminated respiratory samples.     

Evaluation of three techniques for detection of low-level methicillin-resistant Staphylococcus aureus (MRSA): a disk diffusion method with cefoxitin and moxalactam,the Vitek 2 system,and the MRSA-screen latex agglutination test

Very-low-level methicillin-resistant Staphylococcus aureus (MRSA), or class 1 MRSA, is often misdiagnosed as methicillin-susceptible S. aureus (MSSA). We evaluated the performances of three methods for detection of low-level methicillin resistance: the disk diffusion method using the cephamycin antibiotics cefoxitin and moxalactam, the Vitek 2 system (bioMérieux), and the MRSA-screen test (Denka). Detection of the mecA gene by PCR was considered to be the "gold standard." We also determined the sensitivity of the oxacillin disk diffusion method with 5- and 1-microg disks and that of the Oxascreen agar assay with 6 mg of oxacillin liter(-1) for detection of MRSA. We compared the distributions of MICs of oxacillin and cefoxitin by the E-test (AB Biodisk), and those of moxalactam by dilutions in agar, for MRSA and MSSA isolates. The 152 clinical isolates of S. aureus studied were divided into 69 MSSA (mecA-negative) and 83 MRSA (mecA-positive) isolates, including 63 heterogeneous isolates and 26 class 1 isolates (low-level resistance). The cefoxitin and moxalactam disk diffusion tests detected 100% of all the MRSA classes: cefoxitin inhibition zone diameters were <27 mm, and moxalactam inhibition zone diameters were <24 mm. The Vitek 2 system and the MRSA-screen test detected 94 and 97.6% of all MRSA isolates, respectively. The sensitivities of the 5- and 1-microg oxacillin disks were 95.2 and 96.4%, respectively, whereas that of the Oxascreen agar screen assay was 94%. All of the tests except the 1-microg oxacillin disk test were 100% specific. For the class 1 MRSA isolates, the sensitivity of the Vitek 2 test was 92.3%, whereas those of the MRSA-screen test and the disk diffusion method with cefoxitin and moxalactam were 100%. Therefore, the cefoxitin and moxalactam disk diffusion methods were the best-performing tests for routine detection of all classes of MRSA.     

Comparison of the BD Max Methicillin-Resistant Staphylococcus aureus (MRSA) Assay and the BD GeneOhm MRSA Achromopeptidase Assay with Direct- and Enriched-Culture Techniques Using Clinical Specimens for Detection of MRSA

We evaluated the new, fully automated molecular BD Max methicillin-resistant Staphylococcus aureus (MRSA) assay for detection of methicillin-resistant S. aureus in a low-prevalence (4.1%) setting. Sensitivity, specificity, and positive and negative predictive values were 93.9%, 99.2%, 83.8%, and 99.7%, respectively. The assay reported fewer unresolved results than the BD GeneOhm MRSA ACP assay.     

Methicillin-Resistant Staphylococcus aureus: Comparison of Susceptibility Testing Methods and Analysis of mecA-Positive Susceptible Strains

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for an increasing number of serious nosocomial and community-acquired infections. Phenotypic heterogeneous drug resistance (heteroresistance) to antistaphylococcal beta-lactams affects the results of susceptibility testing. The present study compared the MRSA-Screen latex agglutination test (Denka Seiken Co., Ltd., Tokyo, Japan) for detection of PBP 2a with agar dilution, the VITEK-1 and VITEK-2 systems (bioMérieux, St. Louis, Mo.), and the oxacillin agar screen test for detection of MRSA, with PCR for the mecA gene used as the "gold standard" assay. Analysis of 107 methicillin-susceptible S. aureus (MSSA) isolates and 203 MRSA isolates revealed that the MRSA-Screen latex agglutination test is superior to any single phenotype-based susceptibility testing method, with a sensitivity of 100% and a specificity of 99.1%. Only one isolate that lacked mecA was weakly positive by the MRSA-Screen latex agglutination test. This isolate was phenotypically susceptible to oxacillin and did not contain the mecA gene by Southern blot hybridization. The oxacillin agar screen test, the VITEK-1 system, the VITEK-2 system, and agar dilution showed sensitivities of 99.0, 99.0, 99.5, and 99%, respectively, and specificities of 98.1, 100, 97.2, and 100%, respectively. The differences in sensitivity or specificity were not statistically significant. Oxacillin bactericidal assays showed that mecA- and PBP 2a-positive S. aureus isolates that are susceptible to antistaphylococcal beta-lactams by conventional methods are functionally resistant to oxacillin. We conclude that the accuracy of the MRSA-Screen latex agglutination method for detection of PBP 2a approaches the accuracy of PCR and is more accurate than any susceptibility testing method used alone for the detection of MRSA.     

Comparison of the BD GeneOhm VanR assay to culture for identification of vancomycin-resistant enterococci in rectal and stool specimens

Active screening for vancomycin-resistant enterococci (VRE) in rectal and stool specimens has been recommended to limit the spread of antimicrobial resistance within certain high-risk populations. Directly from 502 rectal swabs and stool specimens, we evaluated the diagnostic performance of the BD GeneOhm VanR assay (BD GeneOhm, San Diego, CA), a rapid real-time PCR test that detects the presence of vanA and/or vanB genes. The VanR assay was compared to culture consisting of both bile-esculin-azide agar with 6 mug/ml vancomycin (BEAV agar) (BD Diagnostics, Sparks, MD) and BEAV broth with 8 mug/ml vancomycin (Hardy Diagnostics, Santa Maria, CA). Enterococci were identified to the species level using standard biochemical tests and a Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). The susceptibility of the enterococci to vancomycin and teicoplanin was determined using an Etest (AB Biodisk, Solna, Sweden). VRE were initially isolated from 147 cultures, and the VanR assay detected 142 of the 147 positive cultures for a sensitivity of 96.6%. The specificity was 87.0% (309/355) largely due to false positives seen with the vanB portion of the assay. The sensitivity when testing rectal swabs was 98.3%, and the sensitivity for stool samples was 95.4% (P = 0.643). The specificity of rectal swabs was comparable to that of the stool specimens (87.5% and 86.5%, respectively). When used only to detect VanA resistance, the VanR assay was 94.4% (136/144) sensitive and 96.4% (345/358) specific, with positive and negative predictive values of 91.3% and 97.7%, respectively. In summary, the BD GeneOhm VanR assay is a good screening test for VRE in our population of predominantly vanA-colonized patients. However, patient samples testing only vanB positive should be confirmed by another method for the presence of VRE.     

Evaluation of MRSA-Screen, a simple anti-PBP 2a slide latex agglutination kit, for rapid detection of methicillin resistance in Staphylococcus aureus

The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.     

Performance of BD Max StaphSR for Screening of Methicillin-Resistant Staphylococcus aureus Isolates among a Contemporary and Diverse Collection from 146 Institutions Located in Nine U.S. Census Regions: Prevalence of mecA Dropout Mutants

This study determined the performance of BD Max StaphSR and the rate of methicillin-resistant Staphylococcus aureus (MRSA) with an unrecognized staphylococcal cassette chromosome mec (SCCmec) right-extremity junction (MREJ) region among 907 methicillin-resistant S. aureus (MRSA) and 900 methicillin-susceptible S. aureus (MSSA) isolates. The rate of mecA/mecC dropout mutants was also evaluated. Only three MRSA isolates (99.7% sensitivity; 904/907) were classified as MSSA by the BD Max StaphSR assay, due to negative results for MREJ. Eight MSSA isolates (99.1% sensitivity; 892/900) were assigned as MRSA. However, six of these MSSA isolates had the mecA gene confirmed by PCR and sequencing (99.8% sensitivity; 898/900). Overall, 7.1% (64/900) of MSSA isolates showed results compatible with a mecA dropout genotype.     

Early Screening of oxacillin-resistant Staphylococcus aureus and Staphylococcus epidermidis from blood culture

The timely detection of blood-borne pathogens is one of the most important functions of the microbiology laboratory. Recently, methicillin-resistant staphylococci have become the most important pathogens seen by the laboratory. The purpose of this study was to evaluate Staphy agar, a novel screening medium, for the detection methicillin-resistant Staphylococcus aureus, S. epidermidis, or other coagulase-negative staphylococci (CNS) from positive blood cultures showing Gram-positive cocci in clusters. Eighty-six blood cultures that yielded Gram-positive cocci in clusters were included in this study. The organisms were finally identified by the Vitek system, and oxacillin resistance was confirmed by polymerase chain reaction (PCR)-based mecA gene detection. The identification and oxacillin resistance of all S. aureus strains showed complete agreement with the Vitek and PCR results. The presumptive detection of S. epidermidis and other CNS were consistent with the Vitek system in 94.7%, and the screening of oxacillin resistance was consistent with the result of PCR in 92.1% of 38 strains. The Staphy agar method is reliable and rapid for differentiating Gram-positive cocci in clusters in blood and for determining their methicillin resistance.     

Performance of the BD GeneOhm methicillin-resistant Staphylococcus aureus test before and during high-volume clinical use

We evaluated the use of the BD GeneOhm MRSA real-time PCR assay (BD Diagnostics, San Diego, CA) for the detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA). The initial evaluation consisted of 403 paired nasal swabs and was done using the specimen preparation provided with the kit and an in-house lysis method that was specifically developed to accommodate large-volume testing using a minimal amount of personnel time. One swab was placed in an achromopeptidase (ACP) lysis solution, and the other was first used for culture and then prepared according to the kit protocol. PCR was performed on both lysates, and results were compared to those for culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay were 98%, 96%, 77%, and 99.7% with the kit lysate and 98%, 95%, 75%, and 99.7% with the ACP lysate (P, not significant), respectively. The second evaluation was done after implementation of all-admission surveillance using PCR with ACP lysis and a sampling of 1,107 PCR-negative samples and 215 PCR-positive samples that were confirmed by culture. The results of this sampling showed an NPV of 99.9% and a PPV of 73.5% (prevalence, 6%), consistent with our initial findings. The BD GeneOhm MRSA assay is an accurate and rapid way to detect MRSA nasal colonization. When one is dealing with large specimen numbers, the ACP lysis method offers easier processing without negatively affecting the sensitivity or specificity of the PCR assay.     

Rapid Identification of Methicillin-Resistant Staphylococcus aureus from Positive Blood Cultures by Real-Time Fluorescence PCR

Methicillin-resistant Staphylococcus aureus septicemia is associated with significant morbidity and mortality and requires treatment with intravenous glycopeptides. For blood cultures positive for gram-positive cocci, 24 to 48 h is required for the detection of S. aureus bacteremia and the provision of antibiotic susceptibility testing results. We describe a molecular biology-based assay that requires 2 h from the time of initial positivity of blood cultures. The assay correctly detected 96% of the S. aureus isolates including all methicillin-resistant S. aureus isolates. Clinical data collected during the study suggest that 28% of patients with S. aureus bacteremia do not receive early and appropriate treatment and that 10% of patients may initially be receiving inappropriate glycopeptide treatment.     

Validation of multiplex PCR strategy for simultaneous detection and identification of methicillin resistant Staphylococcus aureus

Multiplex polymerase chain reaction (PCR) strategy is described for rapid identification of clinically relevant methicillin resistant Staphylococcus aureus (MRSA) that targets mecA and coagulase genes. In this study, 150 staphylococcal clinical isolates were used that included 40 isolates of MRSA, 55 isolates of methicillin susceptible S. aureus (MSSA), 44 isolates of methicillin susceptible coagulase negative Staphylococcus spp. (MS-CoNS) and 11 isolates of methicillin resistant coagulase negative Staphylococcus spp. (MR-CoNS). Out of 55 S. aureus strains, three strains demonstrated mecA gene, which appeared to be oxacillin sensitive by disc diffusion. When (MS-CoNS) were evaluated, 10 isolates classified as oxacillin sensitive phenotypically, yielded positive results in PCR method. The results for mecA detection by PCR were more consistent with disk susceptibility tests in case of MRSA (100%) and MSSA (95%) isolates. In contrast to above results with MRSA and MSSA, mecA detection by PCR in MS-CoNS showed less correlation with disk susceptibility tests (77%). The results for coag detection by PCR were consistent with phenotypic tests in all isolates.     

Three-Way Comparison of BBL CHROMagar MRSA II,MRSASelect, and Spectra MRSA for Detection of Methicillin-Resistant Staphylococcus aureus Isolates in Nasal Surveillance Cultures

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired and life-threatening infections. Active surveillance programs for MRSA utilize either molecular or culture-based methods. A prospective study was performed to compare the performance of selective and differential chromogenic media, BBL CHROMagar MRSA II (CMRSA II; BD Diagnostics, Sparks, MD), MRSASelect (Bio-Rad Laboratories, Redmond, WA), and Spectra MRSA (Remel, Lenexa, KS), for the detection of MRSA in nasal swab specimens. A total of 515 compliant remnant nasal swab specimens were sequentially used to inoculate BBL Trypticase soy agar with 5% sheep blood (TSA II) and each chromogenic medium. After 24 h of incubation, colony color reactions and morphology on chromogenic media were compared to suspicious colonies on nonselective TSA II. MRSA on TSA II was confirmed by Gram staining, a coagulase test, and a cefoxitin disk test. The overall prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) on TSA II was 12.4% (64/515) and 9.7% (50/515), respectively. When each chromogenic medium was compared to the standard culture method, the sensitivity and specificity, respectively, were as follows: CMRSA II, 87.7% and 98.6%; MRSASelect, 89.0% and 93.4%; and Spectra MRSA, 83.6% and 92.1%. The positive predictive values were highest for CMRSA II (91.4%), followed by MRSASelect (69.1%) and Spectra MRSA (63.5%). False-positive results on chromogenic media were mainly due to color interpretation. The negative predictive values for all three media were greater than 97%. In conclusion, CMRSA II gave the best overall results for detecting MRSA from nasal specimens.