Automated Scoring of Chromogenic Media for Detection of Methicillin-Resistant Staphylococcus aureus by Use of WASPLab Image Analysis Software

Recently, systems have been developed to create total laboratory automation for clinical microbiology. These systems allow for the automation of specimen processing, specimen incubation, and imaging of bacterial growth. In this study, we used the WASPLab to validate software that discriminates and segregates positive and negative chromogenic methicillin-resistant Staphylococcus aureus (MRSA) plates by recognition of pigmented colonies. A total of 57,690 swabs submitted for MRSA screening were enrolled in the study. Four sites enrolled specimens following their standard of care. Chromogenic agar used at these sites included MRSASelect (Bio-Rad Laboratories, Redmond, WA), chromID MRSA (bioMérieux, Marcy l''Etoile, France), and CHROMagar MRSA (BD Diagnostics, Sparks, MD). Specimens were plated and incubated using the WASPLab. The digital camera took images at 0 and 16 to 24 h and the WASPLab software determined the presence of positive colonies based on a hue, saturation, and value (HSV) score. If the HSV score fell within a defined threshold, the plate was called positive. The performance of the digital analysis was compared to manual reading. Overall, the digital software had a sensitivity of 100% and a specificity of 90.7% with the specificity ranging between 90.0 and 96.0 across all sites. The results were similar using the three different agars with a sensitivity of 100% and specificity ranging between 90.7 and 92.4%. These data demonstrate that automated digital analysis can be used to accurately sort positive from negative chromogenic agar cultures regardless of the pigmentation produced.     

Comparison of Chromogenic Media to BD GeneOhm Methicillin-Resistant Staphylococcus aureus (MRSA) PCR for Detection of MRSA in Nasal Swabs

To select a method for detecting methicillin-resistant Staphylococcus aureus (MRSA) in nasal swabs, we compared BD GeneOhm MRSA PCR and various culture media (mannitol salt agar with cefoxitin, MRSASelect, CHROMagar MRSA, and Spectra MRSA). While PCR detection of MRSA was more rapid, MRSASelect and Spectra MRSA demonstrated performance equivalent to that of PCR with maximal detection at 24 h.The most significant risk factor for development of a methicillin-resistant Staphylococcus aureus (MRSA) infection is hospitalization (6), and colonization of the nares by S. aureus is a risk factor for development of MRSA and methicillin-susceptible Staphylococcus aureus infections (10, 14). Our institution currently performs active nasal surveillance for MRSA in surgical patients in the intensive care unit and is instituting a broader surveillance program. We assessed the performance of four different culture media and a molecular diagnostic test for the detection of MRSA in nasal swabs in an attempt to find an approach that would perform effectively from both analytical and work flow perspectives.MRSA surveillance swabs of the anterior nares were collected on liquid Amies dual Bacti-swabs (Remel; Thermo Fisher Scientific, Lenexa, KS) by the care teams in the patient''s unit. For the first part of the study, both swabs were inoculated onto mannitol salt agar containing cefoxitin (5 μg/ml) (MSA-FX) (12) and then one swab was removed and vortexed in 1 ml of saline. Fifty microliters of the saline suspension was aliquoted onto each of the following culture media: CHROMagar MRSA (BBL; Becton Dickinson, Franklin Lakes, NJ), MRSASelect (Bio-Rad, Hercules, CA), Spectra MRSA (Thermo Fisher Scientific), and MSA-FX. Plates were incubated in the dark at 35°C. Twenty-eight MRSA-positive cultures were detected by direct plating of swabs to MSA-FX, and 29 MRSA-positive cultures were detected by at least two plates following swab extraction in saline, so this process did not lead to reduced detection. Fifty microliters of the saline suspension was added to 200 μl of achromopeptidase (1 U/μl) in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA buffer (Sigma-Aldrich, St. Louis, MO) for use in BD GeneOhm MRSA PCR (BD Diagnostics, San Diego, CA). For the second part of this study, one swab of the pair was inoculated onto MRSASelect medium, and the plates were read at 24 h. The other swab was added to a 1.5-ml tube. Either 200 μl or 300 μl of achromopeptidase (1 U/μl) was added, and the tube was vortexed for 10 s. Achromopeptidase samples from each part of the study were incubated at 37°C for 15 min, 99°C for 5 min, and on ice for at least 10 min. For BD GeneOhm MRSA PCRs, 2.8 μl of the lysate was used as template, and the assay was performed by following the manufacturer''s protocol. Thermal cycling was performed on a Cepheid SmartCycler (Sunnyvale, CA). Detection of the mecA gene by PCR with agarose gel analysis of isolated colonies was performed as described previously (11).     

Improved Methods for Detection of Methicillin-Resistant Staphylococcus aureus

 In order to assess the performance of two detection methods, a set of 93 recent clinical isolates of Staphylococcus aureus, including a large number of strains that demonstrated low-level methicillin-resistance were evaluated using the MRSA-Screen (Denka Seiken, Japan), a commercial latex agglutination test to detect penicillin-binding protein 2′ (PBP2′), and a polymerase chain reaction assay using the LightCycler Instrument (Roche Diagnostics, Switzerland). The results show that the latex agglutination test is highly sensitive if performed after induction by cefoxitin. Inconclusive results can be rapidly confirmed on the same day by real-time polymerase chain reaction used to detect mecA and femA genes.     

Alternative Use for Spectra MRSA Chromogenic Agar in Detection of Methicillin-Resistant Staphylococcus aureus from Positive Blood Cultures

Spectra MRSA agar (Remel, Lenexa, KS), a novel chromogenic medium originally developed to detect methicillin-resistant Staphylococcus aureus (MRSA) from nasal swabs, was evaluated in this multicenter study for the detection of MRSA from positive blood cultures exhibiting Gram-positive cocci upon initial Gram staining.In recent decades, hospital-acquired (HA) and community-acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) infections have caused elevated morbidity and mortality rates and substantially increased health care costs (5, 8, 14). First isolated in the 1960s, HA-MRSA is now endemic in most U.S. hospitals and long-term-care facilities (9, 12). Classically associated with skin and soft tissue infections, CA-MRSA has been involved in an increasing number of cases involving bacteremia and sometimes fatal invasive disease (15). Regardless of origin, a major financial burden has been placed on the health care industry due to MRSA-related bacteremia that stems from extended hospital stays, the cost of isolation, and intensive-care-unit expenses (1, 5). This multicenter study compared the performance of a novel chromogenic medium, Spectra MRSA agar (Remel, Lenexa, KS), with that of traditional culture to screen for MRSA from positive blood cultures.Blood specimens were inoculated into aerobic and anaerobic blood culture bottles (site A, BacT/Alert FA [aerobic] and BacT/Alert FN [anaerobic]; site B, Standard Aerobic/F and Peds Plus/F [aerobic] and Lytic/10 Anaerobic/F [anaerobic]; site C, VersaTREK Redox 1 [aerobic] and VersaTREK Redox 2 [anaerobic]; site D, Plus Aerobic/F and Lytic/10 Anaerobic/F), followed by incubation in an automated blood culture system (site A, bioMérieux BacT/Alert; sites B and D, Becton Dickinson BACTEC; site C, TREK Diagnostics VersaTREK) for up to 5 days. Positive blood cultures, defined as Gram-positive cocci upon Gram staining, were subcultured onto Spectra MRSA agar and blood agar, the “gold standard.” Following aerobic incubation at 35°C, Spectra MRSA plates were observed for denim blue colony growth at 24- and 48 h-time intervals, indicating MRSA. Blood agar plates were observed for up to 48 h for suspected S. aureus colonies that were typically yellow, catalase positive, and composed of Gram-positive cocci often demonstrating beta-hemolysis (2).Suspected MRSA colonies from Spectra MRSA and blood agar plates were tested with the Oxoid PBP2′ latex agglutination test (Remel) and the oxacillin Etest (bioMérieux, Marcy l''Etoile, France) according to the protocol described by Peterson et al. (11) for MRSA confirmation. The MICs were determined as defined by the CLSI guidelines (3), and the values were interpreted as follows: ≤2 μg/ml, oxacillin sensitive; ≥4 μg/ml, oxacillin resistant; and 2 to 4 μg/ml, intermediate oxacillin resistance (3). PBP2′-negative S. aureus strains with an oxacillin MIC of 4 to 6 μg/ml were reported as oxacillin resistant as described in the CLSI guidelines (3). True positives (TP) are defined as denim blue colonies on Spectra MRSA agar that were identified as MRSA by the Oxoid PBP2′ test and oxacillin Etest (≥4 μg/ml) or S. aureus PBP2′-negative colonies with an oxacillin MIC of 4 to 6 μg/ml. False positives (FP) are defined as denim blue methicillin-susceptible S. aureus (MSSA) colonies on Spectra MRSA agar. True negatives (TN) are defined as the absence of denim blue colonies on Spectra MRSA agar that was confirmed negative for MRSA by blood agar and standard techniques. False negatives (FN) are defined as confirmed MRSA colonies on blood agar that did not grow on Spectra MRSA agar.The combined sensitivity and specificity of Spectra MRSA agar for the detection of MRSA were 96% and 99.6% at 24 h and 99.4% and 98.5% at 48 h (see Table ​Table11 for positive predictive values [PPV] and negative predictive values [NPV]). A total of seven FN isolates were reported at 24 h: four due to the absence of growth and three resulting from MRSA colonies expressing light-blue pigmentation. However, six of the seven FN isolates eventually produced denim blue colonies at 48 h. A total of two FP isolates were reported at 24 h: one isolate produced a single denim blue colony, and the other isolate produced pinpoint denim blue colonies (both confirmed as PBP2′-negative MSSA). Individual trial site analyses are shown in Table ​Table2.2. A total of six borderline oxacillin-resistant S. aureus (BORSA) strains were identified (S. aureus PBP2′-negative colonies with an oxacillin MIC of 4 to 6 μg/ml): three strains produced denim blue colonies at 24 h, and the remaining three strains (previously included with the FN results) demonstrated no growth at 24 h. However, two of the three strains eventually produced denim blue colonies at 48 h, and the remaining FN strain failed to produce any growth at 48 h.

TABLE 1.

Combined clinical trial site data for sensitivity, specificity, PPV, and NPV analysis of Spectra MRSA agar for the detection of MRSA^a

Evaluation of a Modular Multiplex-PCR Methicillin-Resistant Staphylococcus aureus Detection Assay Adapted for mecC Detection

A mecC (mecA_LGA251)-adapted multiplex PCR-based methicillin-resistant Staphylococcus aureus (MRSA) detection assay was evaluated using an international, spa-typed Staphylococcus aureus collection comprising 51 mecC-positive MRSA, 240 mecA-positive MRSA, and 50 mecA- and mecC-negative methicillin-susceptible S. aureus (MSSA) isolates. The assay showed 100% sensitivity and specificity for S. aureus species identification as well as for mecA and mecC detection.     

Multicenter Evaluation of MRSASelect II Chromogenic Agar for Identification of Methicillin-Resistant Staphylococcus aureus from Wound and Nasal Specimens

Hospitals strive to reduce methicillin-resistant Staphylococcus aureus (MRSA) prevalence via active surveillance of inpatient populations. Rapid and inexpensive screening methods are utilized when molecular methods are not operationally feasible. In this multisite clinical trial, the utility of Bio-Rad''s MRSASelect II was evaluated for MRSA identification from remnant nares and wound swabs. The prevalence of MRSA was 11.1% (n = 1,384) from nares samples and 18.1% (n = 842) from wound samples. MRSASelect II had an overall concordance of 95.4% (confidence interval [CI] = 94.5% to 96.2%) compared to a broth-enriched reference standard. Comparisons between results, stratified by examination times, exhibited a nonsignificant trend toward increased positivity at prolonged incubation times. Cefoxitin screening of colonies directly from MRSASelect II was 96.7% (95.8% to 97.3%) concordant compared to testing of colonies following broth enrichment. A comparison of MRSASelect and MRSASelect II revealed no statistical differences; however, the latter exhibited earlier positivity, greater selectivity, and more intense indicator staining, which resulted in facilitated differentiation of positive results. MRSASelect II agar is a simple, rapid, and robust method to routinely screen patients for MRSA colonization without the need for additional testing.     

Spectra MRSA,a New Chromogenic Agar Medium To Screen for Methicillin-Resistant Staphylococcus aureus

A novel chromogenic medium, Spectra MRSA (Remel, Lenexa, KS), was designed to detect methicillin-resistant Staphylococcus aureus (MRSA) rapidly and more efficiently than traditional media (i.e., tryptic soy agar with 5% sheep blood [SBA] and mannitol salt agar [MSA]). A multicenter study (including four clinical trial sites and the Medical College of Wisconsin [MCW] Milwaukee, WI) compared the performance characteristics of Spectra MRSA to those of the traditional media for the detection of MRSA. For this study, 767 nasal swab specimens from the multicenter study (traditional medium used, SBA) and 667 nasal swab specimens from MCW (traditional medium used, MSA) were plated on each test medium and examined after 24 and 48 h of incubation. At 24 h, the sensitivity and the specificity of each medium were as follows: in the multicenter study, 95.4% and 99.7%, respectively, for Spectra MRSA and 93.6% and 100%, respectively, for SBA; at MCW, 95.2% and 99.5%, respectively, for Spectra MRSA and 88.7% and 94.0%, respectively, for MSA. The positive predictive values of each medium at 24 h were as follows: in the multicenter study, 98.1% for Spectra MRSA and 100% for SBA; at MCW, 95.2% for Spectra MRSA and 60.4% for MSA. In our evaluation, we found that Spectra MRSA was able to rapidly identify and differentiate methicillin-resistant S. aureus from methicillin-susceptible S. aureus on the basis of the utilization of chromogens that result in denim blue colonies, thus eliminating the need for biochemical analysis and antimicrobial susceptibility testing. Extending the incubation beyond 24 h did not significantly improve the recovery of MRSA and resulted in decreased specificity.A significant effort has been put forth to determine effective infection control practices that may be used to limit the spread of methicillin-resistant Staphylococcus aureus (MRSA) and minimize its impact on patient care and hospital budgets in response to the increasing rates of occurrence of MRSA in health care settings. Although most hospitals adhere to a policy of contact isolation and attempt to limit inappropriate antimicrobial usage, there is strong evidence that active surveillance cultures (ASCs) for patients at risk for MRSA colonization can increase the chance of identifying occult MRSA reservoirs and further limit the nosocomial spread of the organism (16). Recent reports by Salgado and Farr (17) and Lucet et al. (13) have shown that the organisms in positive cultures of clinical specimens routinely submitted from patients represent only a small fraction of the reservoir of antibiotic-resistant pathogens, and the largest source for nosocomial spread was attributed to asymptomatic, colonized patients who went unrecognized and unisolated in the absence of ASCs. Additionally, consensus suggests that a restricted formulary alone is unlikely to prevent the emergence and persistence of MRSA and that the use of contact precautions is important to prevent the spread of MRSA from colonized patients (5).While the anterior nares are the most common site of S. aureus colonization and the most frequently screened and recommended site for specimen collection due to the satisfactory sensitivities of tests with that type of specimen and the ease with the specimen may be obtained (15, 18, 20), other recent studies have shown that sampling of other body sites (the oropharynx, perianal region, and groin) may enhance the sensitivity of screening for MRSA and predict the likelihood of S. aureus infection (3, 7, 11, 14, 22). Most importantly, it is agreed that the culture of specimens from more body sites would yield higher rates of recovery, but the increased resources required for the screening of specimens from multiple sites outweigh the incremental increase in yield that would be attained (5). In order to reduce the economic burden related to longer patient stays and higher costs associated with therapy and infection control, rapid and accurate tests for screening for MRSA are needed to guide intervention and decrease delays in the implementation of contact precautions. A lengthy turnaround time would further augment the risk for nosocomial transmission; thus, any effective screening protocol should be deemed to have a turnaround time within 24 h.The methods currently available for screening for MRSA include standard culture, methods that use chromogenic media, and molecular-based testing. Although PCR methods have been highly acclaimed in the recent literature for their sensitivity and speed, the cost per test and the up-front laboratory cost of implementation are very high and may generate significant pressure to demonstrate cost savings in an unrealistic time frame (5). Culture methods that employ a selective chromogenic medium have been described to be less costly, more reliable, and faster for screening for MRSA than traditional culture (5, 8). The purpose of the multicenter study described here was to compare the performance of a novel chromogenic medium, Spectra MRSA, with that of traditional culture and validate the use of this product for screening for MRSA from nasal swab specimens.     

Prospective Two-Center Comparison of Three Chromogenic Agars for Methicillin-Resistant Staphylococcus aureus Screening in Hospitalized Patients

Three chromogenic media, chromID MRSA SMART (SMART), chromID MRSA first generation (chromID), and Brilliance MRSA (OX2), were evaluated for methicillin-resistant Staphylococcus aureus (MRSA) screening using 1,220 samples. The sensitivity at 24 h was significantly better with the SMART agar (66.4%) than that with chromID agar (50.5%). Enrichment and incubation until 48 h are still needed for an optimal yield.     

Rapid Detection of Epidemic Strains of Methicillin-Resistant Staphylococcus aureus

Fifty methicillin-resistant Staphylococcus aureus (MRSA) initial isolates obtained from patients hospitalized in the orthopedic clinic of the Frankfurt University Hospital and 150 methicillin-sensitive Staphylococcus aureus (MSSA) isolates were investigated in this study to determine whether the Slidex Staph-Kit is capable of differentiating between MRSA and MSSA owing to its unique performance characteristics. The Slidex Staph-Kit is a combined latex hemagglutination test designed to detect clumping factor, protein A, and a specific surface immunogen for S. aureus. Clumping factor-positive strains cause erythrocytes sensitized with fibrinogen to hemagglutinate, thereby resulting in visible red clumps. S. aureus strains deficient in clumping factor agglutinate latex particles sensitized with specific antibodies against surface proteins of S. aureus, thereby resulting in visible white clumps. Our results demonstrate that white clumping has a 99% specificity as well as a 98% positive predictive value for MRSA. Clumping factor-negative MRSA, which have been reported to occur in several countries, are epidemic in the Frankfurt area and account for 80% of all MRSA initial isolates in the orthopedic clinic of the Frankfurt University Hospital. Genotyping of all MRSA isolates by macrorestriction analysis of chromosomal DNA revealed that 83% of clumping factor-negative MRSA are closely related to the “southern-German” epidemic strain. This is the first study demonstrating the Slidex Staph-Kit’s capability for identifying epidemic clumping factor-negative S. aureus strains as methicillin resistant even prior to antimicrobial susceptibility testing.     

Evaluation of the mecA femB Duplex Polymerase Chain Reaction for Detection of Methicillin-Resistant Staphylococcus aureus

 This study systematically evaluated a recently described duplex polymerase chain reaction test for methicillin-resistant Staphylococcus aureus with 25 different German epidemic strains of methicillin-resistant Staphylococcus aureus and 66 staphylococci other than methicillin-resistant Staphylococcus aureus, including 17 different coagulase-negative staphylococcal species and subspecies, that were either oxacillin susceptible or oxacillin resistant. The results were compared with those of conventional cultural identification and susceptibility testing. Of the 91 isolates tested, all 25 confirmed strains of methicillin-resistant Staphylococcus aureus were identified correctly. None of the remaining strains of methicillin-susceptible Staphylococcus aureus was misidentified as methicillin-resistant Staphylococcus aureus. It was concluded that the duplex polymerase chain reaction appears to offer a time-saving and accurate method of detection of methicillin-resistant Staphylococcus aureus.     

Evaluation of Brilliance MRSA 2 Agar for Detection of Methicillin-Resistant Staphylococcus aureus in Clinical Samples

We compared 2 chromogenic media (Oxoid Brilliance MRSA 2 agar [Thermo Fisher Scientific] and MRSA-ID [bioMérieux]) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in 1,368 hospital samples. For both media, broth enrichment was essential to obtain satisfactory diagnostic performance. Although with direct cultures only, the diagnostic performance (particularly sensitivity) of Brilliance MRSA 2 agar appears better than that of MRSA-ID, no difference in sensitivity or specificity between the media was detected after inclusion of an enrichment step.     

Community-Associated Methicillin-Resistant Staphylococcus aureus Mediastinitis

Community-associated methicillin (meticillin)-resistant Staphylococcus aureus (CA-MRSA) continues to emerge as a cause of serious infections, chiefly of the skin and soft tissues. We present the first documented case of CA-MRSA mediastinitis in an adult. Blood and mediastinal isolates were characterized as CA-MRSA by pulsed-field gel electrophoresis and susceptibility testing.     

Performance of a New Chromogenic Medium,BBL CHROMagar MRSA II (BD), for Detection of Methicillin-Resistant Staphylococcus aureus in Screening Samples

Two chromogenic media for the detection of MRSA were compared: BBL CHROMagar MRSA II (BD) and MRSA ID agar (bioMérieux). Following overnight nonselective enrichment, 1,919 screening samples were inoculated on both chromogenic agars. After 24 h, the sensitivities of both media were high and comparable. Both media showed an important decrease in specificity after 48 h of incubation (decreases of 8% for MRSA II and 10% for MRSA ID), but MRSA II was significantly more specific at both time points.Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infections worldwide. Infection with MRSA is associated with important clinical and financial implications (7). Screening of high-risk populations and subsequent isolation of carriers are a cost-effective measure to prevent transmission in the hospital, at least if screening results are reliable and readily available (14). A wide range of culture methods and, more recently, molecular methods have been used for MRSA screening (1, 10, 13). Selective media such as mannitol salt agar supplemented with oxacillin have shown limited sensitivity (5) and specificity (4). Broth enrichment culture has been recommended, resulting in a significantly higher yield (15). The incorporation of cefoxitin in culture media has proven to be superior to the incorporation of oxacillin in culture media in the detection of MRSA (12). Recently, chromogenic media became available, permitting the direct detection and identification of MRSA through the presence of specific chromogenic substrates and incorporated antibiotics (6, 8, 9, 11, 12, 16).The purpose of this study was the evaluation of a new chromogenic medium, BBL CHROMagar MRSA II (Becton Dickinson) (hereafter, MRSA II), in comparison to a second, already established chromogenic agar, MRSA ID (bioMérieux), for the detection of MRSA in screening samples after nonselective enrichment. On this new medium, MRSA is visualized as mauve colonies by the presence of a specific chromogenic substate (proprietary formulation) and cefoxitin (5.2 mg/liter). Additional selective agents are added for the inhibition of Gram-negative organisms, yeasts, and other Gram-positive cocci. MRSA II is a modified version of the existing BBL CHROMagar MRSA (cefoxitin, 6 mg/liter). On MRSA ID, MRSA is detected as green colonies through the presence of a chromogenic substrate targeting the α-glucosidase enzyme of S. aureus and cefoxitin (4 mg/liter). Previous studies have shown that MRSA ID is a highly valuable medium for the detection of MRSA, with sensitivities up to 96.4% and specificities up to 99.5% after 24 h of incubation (5, 6, 11).A multicenter prospective evaluation was set up in 5 microbiology laboratories. A total of 1,919 samples submitted for MRSA screening were included, consisting of 588 nares swabs, 394 perianal swabs, 320 throat swabs, 430 pooled swabs (throat, nose, and perianal), and 187 wound swabs. All specimens were enriched overnight in 5 ml nonselective tryptic soy broth (TSB) at 35°C in ambient air. After plating 10 μl of the TSB broth on MRSA II and MRSA ID medium using a three-streak dilution method, all media were incubated at 35°C in ambient air. All plates were read at 24 h and 48 h of incubation, and colony color, size, and growth intensity were scored. Identification of suspicious colonies (characteristic color and morphology) was assessed with at least a coagulase tube test with rabbit plasma or slide agglutination in combination with the detection of DNase. Screening tests for MecA-mediated oxacillin resistance were used according to CLSI guidelines (3). When confirmation tests of suspicious colonies showed no growth of MRSA, the sample was considered false positive. No growth or the absence of suspicious colonies was considered negative. In case of MRSA detection on only one chromogenic medium, the sample was inoculated again on both media starting from TSB to check the reproducibility of the obtained data (n = 8). Quality control testing was successfully performed on each lot of chromogenic media before use in the study (plating a standardized inoculum of ATCC 43300 or ATCC 29213, an in-house methicillin-susceptible S. aureus [MSSA] strain with a cefoxitin MIC of 4 μg/ml).MRSA was detected in 274 samples (14.3%) on one or both chromogenic media (Table ​(Table1).1). There was no difference in sizes and growth intensities of positive colonies on both chromogenic media. After 24 h, MRSA was detected in 261 samples on MRSA II and in 257 samples on MRSA ID. After 48 h, 271 samples were positive on MRSA II and 269 on MRSA ID. This resulted in comparable sensitivities after 24 h of incubation for MRSA II and MRSA ID (95.3% and 93.8%, respectively; McNemar P = 0.42) as well as after 48 h (98.9% and 98.2%, respectively; McNemar P = 0.72). For both chromogenic media, the sensitivities were significantly higher after 48 h of incubation versus 24 h of incubation (MRSA II, P = 0.002; MRSA ID, P = 0.0005). However, when we excluded the results of one center, we observed nearly identical sensitivities after 24 and 48 h of incubation (98.3% and 99.1%, respectively, for MRSA II, P = 0.5; 97.8% and 99.1%, respectively, for MRSA ID, P = 0.25), with a positivity rate of 13.2% in 1,759 samples. This one center included 160 samples (positivity rate of 26.2%), with significantly higher sensitivities for both chromogenic media after 48 h compared to 24 h. Sensitivity for MRSA II increased from 78.5% to 95.2% (P = 0.0078), and that for MRSA ID increased from 71.4 to 92.8% (P = 0.0039). It was stressed that the same procedure was followed in the 5 centers, with extra care for a complete 24-h incubation before the first reading of the chromogenic agars. However, this center reported a possible too cautious early assignment of suspected colonies after 24 h, as they use selective instead of TSB enrichment in their daily routine.

TABLE 1.

Number of MRSA strains isolated from 1,919 screening samples

Evaluation of the LightCycler Methicillin-Resistant Staphylococcus aureus (MRSA) Advanced Test for Detection of MRSA Nasal Colonization

Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization is crucial for the prevention and control of MRSA infections in health care settings. The LightCycler MRSA Advanced Test (Roche Diagnostics) is a commercially available real-time PCR assay for direct detection of MRSA nasal colonization by targeting of the staphylococcal cassette chromosome mec (SCCmec)-orfX junction. The diagnostic performance of the assay was compared with that of ChromID MRSA agar (bioMérieux) culture and an in-house duplex real-time PCR assay. Among 1,246 nasal swab specimens collected from 2 general hospitals in Hong Kong, 174 (14%) were considered true positive for MRSA. Chromogenic culture and the in-house real-time PCR assay identified 147 (84.5%) and 133 (76.4%) true-positive cases with specificities of 100% and 98.6%, respectively. Based on the target melting temperature (T_m) values (57.0 to 62.0°C) defined by the manufacturer, the LightCycler MRSA Advanced Test identified only 85 (48.9%) true-positive specimens. Interestingly, an additional 60 (34.5%) true-positive specimens were detected despite atypical T_m values of 55°C, providing overall sensitivity and specificity values of 83.3% and 99%, respectively. Among isolates with T_m values of 55°C, most were typed as clonal complex 45 (CC45). By sequence analysis of the SCCmec-orfX junction, characteristic single-nucleotide polymorphisms (SNPs) were identified only in isolates with T_m values of 55°C and not in those with typical T_m values. It is conceivable that those SNPs were located inside the target region of the proprietary hybridization probes, which resulted in a T_m shift in the melting curve analysis. Our study highlights the importance of a global evaluation of commercial kits so that the interpretation algorithm covers different lineages of MRSA clones prevalent in various geographical regions.     

Evaluation of a Chromogenic Biplate Medium (ChromID MRSA/ChromID S. aureus) for the Simultaneous Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus in Preoperative Screening Samples from the Anterior Nares

We evaluated the performance of the ChromID MRSA/ChromID S. aureus biplate for the simultaneous detection of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in preoperative screening samples. The sensitivity and specificity were 94.2% and 93.6%, respectively, for the S. aureus compartment and 92.9% and 99.7% for the MRSA compartment after 48 h incubation.     

Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay

Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) contribute to significant hospitalization costs. We report here a droplet digital PCR (ddPCR) assay, which is a next-generation emulsion-based endpoint PCR assay for high-precision MRSA analysis. Reference cultures of MRSA, methicillin-susceptible S. aureus (MSSA), and confounders were included as controls. Copan swabs were used to sample cultures and collect specimens for analysis from patients at a large teaching hospital. Swab extraction and cell lysis were accomplished using magnetic-driven agitation of silica beads. Quantitative PCR (qPCR) (Roche Light Cycler 480) and ddPCR (Bio-Rad QX100 droplet digital PCR system) assays were used to detect genes for the staphylococcal protein SA0140 (SA) and the methicillin resistance (mecA) gene employing standard TaqMan chemistries. Both qPCR and ddPCR assays correctly identified culture controls for MRSA (76), MSSA (12), and confounder organisms (36) with 100% sensitivity and specificity. Analysis of the clinical samples (211 negative and 186 positive) collected during a study of MRSA nasal carriage allowed direct comparison of the qPCR and ddPCR assays to the Cepheid MRSA GeneXpert assay. A total of 397 clinical samples were examined in this study. Cepheid MRSA GeneXpert values were used to define negative and positive samples. Both the qPCR and ddPCR assays were in good agreement with the reference assay. The sensitivities for the qPCR and ddPCR assays were 96.8% (95% confidence interval [CI], 93.1 to 98.5%) and 96.8% (95% CI, 93.1 to 98.5%), respectively. Both the qPCR and ddPCR assays had specificities of 91.9% (95% CI, 87.5 to 94.9%) for qPCR and 91.0% (95% CI, 86.4 to 94.2%) for ddPCR technology.