Comparison of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture by use of BBL CHROMagar MRSA for detection of MRSA in nasal surveillance cultures from an at-risk community population

We compared the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR assay to culture with BBL CHROMagar MRSA for nasal surveillance among 602 arrestees from the Baltimore City Jail. The sensitivity and specificity were 88.5% and 91.0%, respectively, and after secondary analysis using enrichment broth, they were 89.0% and 91.7%, respectively. Twenty-three of 42 false-positive PCR lysates contained methicillin-susceptible S. aureus.     

Performance of the BD GeneOhm MRSA achromopeptidase assay for real-time PCR detection of methicillin-resistant Staphylococcus aureus in nasal specimens

We evaluated the BD GeneOhm MRSA achromopeptidase (ACP) assay, which incorporates a new specimen preparation approach. A total of 1,216 leftover nasal samples were tested; using culture as the gold standard, the sensitivity and specificity were 92% and 94.6%, respectively. The new lysis method provides good sensitivity and simplifies specimen preparation.     

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).     

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.     

Reliability of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay in detecting MRSA isolates with a variety of genotypes from the United States and Taiwan

The BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay is a molecular screening test for detection of MRSA in nasal colonization. This assay coamplifies the extremity of staphylococcal chromosome cassette mec (SCCmec) and adjacent chromosomal DNA at the SCCmec insertion site. Increasing reports of novel SCCmec types and the diverse genetic backgrounds of MRSA strains prompted us to test the accuracy of the BD GeneOhm MRSA kit with 914 MRSA isolates with a variety of SCCmec types harbored in 21 genetic backgrounds, as determined by the multilocus sequence type (ST). The BD GeneOhm MRSA assay was performed on colony lysates; purified genomic DNA (0.2 pg/μl and 0.2 ng/μl) was tested to confirm negative results from lysates. Of 914 MRSA isolates tested, 911 tested positive (detection rate, 99.7%). The SCCmec types carried by assay-positive isolates were I, II, III, IV, V, V(5C2&5), VI, and VIII and SCCmec composite islands with mec class A and ccr complexes 2 and 4. One of the assay-negative isolates had a community-associated genotype: ST8, SCCmec type IV. However, this was an outlier among the 99.8% (434/435) ST8, SCCmec type IV-containing isolates that tested positive. The two other assay-negative isolates had a health care-associated genotype (ST5); both carried a distinct, uncommon, composite SCCmec type. In summary, the BD GeneOhm MRSA assay had a high rate of detection of MRSA isolates harboring common and uncommon SCCmec types from the United States and Taiwan.     

Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus

CHROMagar MRSA was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus (MRSA). A well-defined collection consisting of 216 MRSA strains and 241 methicillin-susceptible Staphylococcus aureus isolates was used. The sensitivity of CHROMagar MRSA after 24 h of incubation was 95.4%, increasing to 100% after 48 h. The specificity was already 100% after 24 h.     

A PCR method for the identification of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs

AIM: The aim of this study was to assess the discriminatory power and potential turn around time (TAT) of a PCR-based method for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from screening swabs. METHODS: Screening swabs were examined using the current laboratory protocol of direct culture on mannitol salt agar supplemented with oxacillin (MSAO-direct). The PCR method involved pre-incubation in broth for 4 hours followed by a multiplex PCR with primers directed to mecA and nuc genes of MRSA. The reference standard was determined by pre-incubation in broth for 4 hours followed by culture on MSAO (MSAO-broth). RESULTS: A total of 256 swabs was analysed. The rates of detection of MRSA using MSAO-direct, MSAO-broth and PCR were 10.2, 13.3 and 10.2%, respectively. For PCR, the sensitivity, specificity, positive predictive value and negative predictive values were 66.7% (95%CI 51.9-83.3%), 98.6% (95%CI 97.1-100%), 84.6% (95%CI 76.2-100%) and 95.2% (95%CI 92.4-98.0%), respectively, and these results were almost identical to those obtained from MSAO-direct. The agreement between MSAO-direct and PCR was 61.5% (95%CI 42.8-80.2%) for positive results, 95.6% (95%CI 93.0-98.2%) for negative results and overall was 92.2% (95%CI 88.9-95.5%). CONCLUSIONS: (1) The discriminatory power of PCR and MSAO-direct is similar but the level of agreement, especially for true positive results, is low. (2) The potential TAT for the PCR method provides a marked advantage over conventional methods. (3) Further modifications to the PCR method such as increased broth incubation time, use of selective broth and adaptation to real-time PCR may lead to improvement in sensitivity and TAT.     

Evaluation of the BD GeneOhm assay using the rotor-gene 6000 platform for rapid detection of methicillin-resistant Staphylococcus aureus from pooled screening swabs

As health services move toward universal methicillin-resistant Staphylococcus aureus (MRSA) screening for hospital admissions, the most cost-effective approach is yet to be defined. In this study, one of the largest to date, we evaluated the performance of the BD GeneOhm MRSA assay on the Rotor-Gene 6000 thermal cycler, using samples taken directly from pooled MRSA screens. Results were compared with the same assay performed on the Smart-Cycler II platform and overnight broth culture. Samples yielding discrepant results were subjected to detailed analysis with an in-house PCR and patient note review. A total of 1,428 pooled MRSA screens were tested. Sensitivities and specificities of 85.3% and 95.8% for the Rotor-Gene and 81% and 95.7% for the Smart-Cycler were obtained, compared with broth enrichment. The sensitivity of the BD GeneOhm assay was increased to 100% when the results of in-house PCR and patient note review were taken into account. This study demonstrates that the Rotor-Gene 6000 thermal cycler is a reliable platform for use with the BD GeneOhm assay. It also proves that commercial PCR can be performed direct on pooled samples in selective broth, without the need for overnight incubation.     

Rapid Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) in Diverse Clinical Specimens by the BD GeneOhm MRSA Assay and Comparison with Culture

The efficacy of the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay was assessed by analyzing nasal swabs and swabs from other body sites for the presence of MRSA in a low-prevalence area. From 681 patients with a high risk for MRSA carriage, 1,601 specimens were collected and transported in Amies agar. After discordant analysis, the sensitivity, specificity, positive predictive value, and negative predictive value of the BD GeneOhm MRSA assay were 84.3%, 99.2%, 88.4%, and 98.9%, respectively, compared to culture.Rapid availability of laboratory results is paramount for early detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers, implementation of efficient control measures, and adequate therapy. To date, detection of MRSA is conventionally done by culture. Several chromogenic culture media have contributed to a more rapid detection of MRSA (24 versus 72 h with conventional culture [6]). In contrast to former PCR procedures, which required previous isolation of the organism via culture (21), the commercial BD GeneOhm MRSA assay (formerly IDI-MRSA; BD-GeneOhm, San Diego, CA) discriminates mecA-positive Staphylococcus aureus from coagulase-negative staphylococci (CoNS) and detects MRSA in clinical specimens within a few hours. This real-time PCR assay has been used particularly in high-prevalence areas (3, 6, 9). In Switzerland, MRSA prevalence ranges between 4 and 7% with the exception of Geneva (>25% [4, 14]). Surveillance strategies involve screening for MRSA from the nose and other body sites. The BD GeneOhm MRSA assay has been approved by the United States Food and Drug Administration (FDA) for the detection of MRSA from nasal swabs stored in liquid Stuart''s medium. Analyzing specimens from other body sites by this test and transportation of the swab in media other than Stuart''s medium are currently not FDA approved for use with this test.In this study, we aimed at assessing (i) the performance of the BD GeneOhm MRSA assay compared to conventional culture in an extended spectrum of clinical specimens and (ii) the suitability of Amies agar as a transport medium for swabs in a low-prevalence setting.(The study has been presented at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007.)During 18 months, 1,601 single swabs from nose (n = 676), groin (n = 643), wound (n = 153), axilla (n = 61), throat (n = 37), rectum (n = 9), vagina (n = 5), and miscellaneous body sites (n = 17) were collected from 681 patients at high risk for MRSA carriage who were admitted to the Luzerner Kantonsspital, the major teaching hospital of central Switzerland, with approximately 1,300 beds, including a children''s hospital. High-risk patients were individuals (i) admitted from outbound hospitals/health care units of high-MRSA-prevalence areas in Switzerland or from hospitals/health care units of foreign countries; (ii) with skin lesions and intravenous drug abuse; or (iii) with a former positive MRSA test. Swabs were collected in Amies agar (Copanswab 108C; Copan, Brescia, Italy). If processing of the swabs was not possible on the same day, swabs were stored overnight at 4°C. Swabs were then broken off into the sample reagent buffer (blue-capped tubes supplied within the kit) and vortexed at high speed for 60 s. The full amount (approximately 1 ml) of buffer (without the swab) was transferred in another tube for cell lysis and DNA extraction according to the instructions of the manufacturer. PCR was performed with the Smart Cycler II instrument (Cepheid, Sunnyvale, CA). Positive and negative controls were included in each run. In the case of PCR inhibition, the sample was briefly frozen at −20°C to remove inhibitors and the run was repeated. Enrichment broth (1 ml; tryptic soy broth [Becton Dickinson, Allschwil, Switzerland] supplemented with 7.5% NaCl) was added to the sample buffer tube containing the swab and incubated at 35°C in ambient air for 24 h. The incubated enrichment broth (approximately 100 μl) was plated on chromogenic agar (Chrom ID MRSA agar; bioMérieux, Marcy l''Etoile, France) at 35°C in ambient air for 48 h. The chromogenic agar was screened for suspect colonies after 24 h and 48 h. Recovered blue colonies were then tested by the Staphaurex Plus coagulase test (Remel Europe Ltd., Dartford, Kent, United Kingdom) and confirmed as S. aureus by the Vitek 2 system (bioMérieux; GP colorimetric identification card; software version 04.03). Confirmation of methicillin resistance was done by disk diffusion testing with 30-μg cefoxitin (bioMérieux) according to the Clinical and Laboratory Standards Institute (7).Staphylococcal strains with discrepant results for the BD GeneOhm MRSA assay and culture were further studied. At first the BD GeneOhm MRSA assay was repeated from the DNA extract of the same specimen to exclude a confusion of samples. MRSA isolates from PCR-negative but culture-positive specimens were retested from subculture by the BD GeneOhm MRSA assay. For specimens with PCR-positive but culture-negative results, the patients'' medical history was studied (antibiotic therapy and previous carriage of MRSA). For these cases gel electrophoresis was performed to determine the size of amplicons in a 2% agarose gel (AgaroseUltraPured; Invitrogen Corporation, Carlsbad, CA). Discrepancies between the BD GeneOhm MRSA assay and culture were resolved according to at least one of the following criteria: PCR-positive but culture-negative results were considered true positive if (i) patients had been decolonized, (ii) the PCR product had the expected molecular size (as described previously by Huletsky et al. [15]), or (iii) there were PCR-positive and culture-positive results from other body sites of the same patient at the same time (Table ​(Table1).1). Sensitivity and specificity for the BD GeneOhm MRSA assay as well as the positive predictive value (PPV) and negative predictive value (NPV) including confidence intervals (CI; according to Wilson''s method [1]) were calculated and compared to culture (Table ​(Table22).

TABLE 1.

Resolution of discrepancies from PCR-positive and culture-negative cases

Clinical validation of the molecular BD GeneOhm StaphSR assay for direct detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in positive blood cultures

The rapid detection of Staphylococcus aureus bacteremia and a swift determination of methicillin susceptibility has serious clinical implications affecting patient mortality. This study evaluated the StaphSR assay (BD GeneOhm, San Diego, CA), a real-time PCR assay, for the identification and differentiation of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from 300 positive blood cultures. The BD GeneOhm StaphSR assay was performed and interpreted according to the manufacturer's recommendations. Positive blood cultures (containing predominantly gram-positive cocci in clusters) were subcultured on 5% sheep blood agar plates. After 18 to 24 h of incubation, isolates morphologically consistent with S. aureus were presumptively identified by latex agglutination (Staphaurex Plus; Remel, Lenexa, KS). Susceptibility testing was initially performed with the Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). Additional susceptibility testing of samples with discrepant results was done using BBL oxacillin screen agar (BD Diagnostics, Sparks, MD), oxacillin and cefoxitin Etests (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar, an immunoassay for penicillin binding protein 2' (Denka Seiken Co., Tokyo, Japan), and mecA PCR. The sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm StaphSR assay for MSSA detection were 98.9, 96.7, 93.6, and 99.5%, respectively. For the detection of MRSA, the BD GeneOhm StaphSR assay was 100% sensitive and 98.4% specific; positive and negative predictive values for MRSA detection were 92.6 and 100%, respectively. Inhibition was seen with only one sample, and the issue was resolved upon retesting. The BD GeneOhm StaphSR assay appears to be a valuable diagnostic tool for quickly differentiating bacteremia caused by MSSA and MRSA from that caused by other gram-positive cocci.     

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.     

Evaluation of the Xpert methicillin-resistant Staphylococcus aureus (MRSA) assay using the GeneXpert real-time PCR platform for rapid detection of MRSA from screening specimens

The need for rapid methods to accurately detect methicillin-resistant Staphylococcus aureus (MRSA) is widely acknowledged, and a number of molecular assays are commercially available. This study evaluated the Xpert MRSA assay, which is run on the GeneXpert real-time PCR platform (Cepheid) for use in a clinical laboratory. The following parameters were investigated: (i) the limits of detection (LoDs) for four MRSA strains; (ii) the ability to detect isolates of MRSA from a collection representative of MRSA in Ireland since 1974 (n = 114) and the ability to detect control strains with staphylococcal cassette chromosome mec types IVa (IV.1.1.1), IVb (IV.2.1.1), IVc (IV.3.1.1), IVd (IV.4.1.1), V (V.1.1.1), V_T, and VI; and (iii) performance in a clinical trial with swabs from nose, throat, and groin/perineum sites from 204 patients, where results were compared with those obtained by direct and enrichment cultures. The average LoD of the four test strains was 610 CFU/ml (equivalent to 58 CFU/swab). All 114 MRSA isolates and 7 control strains tested were detected. Sensitivity, specificity, and positive and negative predictive values for clinical specimens from all sites investigated were 90%, 97%, 86%, and 98%, respectively, but throat specimens yielded poor sensitivity (75%). Sensitivity, specificity, and positive and negative predictive values for nasal specimens were 95%, 98%, 90%, and 99%, respectively. Overall, the assay was rapid and easy to perform, but performance might be enhanced by the inclusion of an equivocal interpretive category based on analysis of all available amplification data.     

Comparison of the Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Assay,BD GeneOhm MRSA Assay,and Culture for Detection of Nasal and Cutaneous Groin Colonization by MRSA

Detection of methicillin (meticillin)-resistant Staphylococcus aureus colonization was assessed using combined nose and groin swabs in two commercial PCR assays (the Xpert MRSA assay and the BD GeneOhm MRSA assay). Compared to routine culture, both had similar sensitivities (87.0% versus 84.8%, respectively) and specificities (93.8% versus 92.7%, respectively). Combined PCR assays provide a rapid and more-complete assessment of colonization at a cost similar to that of single-site analysis.The Xpert methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) assay (GXP-MRSA; Cepheid, Sunnyvale, CA), performed in the GeneXpert system (Cepheid), has the advantage that individual specimens can be rapidly processed without the need for batching of tests (4, 15). GXP-MRSA has FDA approval for direct detection of MRSA in nasal specimens only, and its performance on swabs from other sites has been assessed only on single swabs (11). However, processing combined nose and groin (CNG) swabs potentially provides a more complete assessment of MRSA colonization at an assay cost similar to that of assessing nasal colonization alone (3). We assessed the accuracy of GXP-MRSA for detecting MRSA colonization by the use of CNG swabs compared to that of processing both swabs separately and by culture assay (study A). Second, we prospectively compared the accuracy levels of GXP-MRSA, BD GeneOhm MRSA assay (BD-MRSA; Becton Dickinson Diagnostics, San Diego, CA), and culture for the detection of MRSA colonization by the use of CNG swabs (study B).Both studies were conducted at the Austin Hospital, Melbourne, Australia, a 450-bed tertiary care university teaching hospital with known high (12 to 15%) rates of patient MRSA colonization (3, 9). The studies were approved by the hospital''s human ethics committee. All participating patients gave informed consent, were ≥18 years old, and had their anterior nares (N) and the cutaneous area in their groin (G) swabbed with two sets of Copan Liquid Stuart double swabs (Venturi Transystem; Copan Diagnostics, Corona, CA). Both swabs from the first collected set (N1 and N2; G1 and G2) were used for PCR testing. A swab from the second collected set was used for culture (N3 and G3), and the other (N4 and G4) was stored at 4°C for later use if required for repeat PCR.All nose and groin swabs were cultured onto chromogenic MRSA agar (Oxoid, Basingstoke, England), and then each swab was placed into 1 ml of tryptone soya broth containing 6.5% sodium chloride. Chromogenic MRSA agar plates were incubated at 35°C and read after 24 and 48 h. Broth cultures were incubated for 48 h at 35°C and then subcultured onto chromogenic MRSA agar. All potential MRSA colonies were subcultured prior to routine identification and antibiotic susceptibility tests (1, 5). MRSA isolates were defined as nonmultiresistant (nmMRSA) if they were resistant to less than three non-β-lactam antibiotics (6). A patient was defined as “MRSA colonized” if, at the time of sample collection, the agar and/or broth cultures from the nose and/or groin were positive.In study A, the GXP-MRSA assay was performed on specimens collected from patients who, based on our hospital''s routine screening program, were suspected of being MRSA colonized or noncolonized. Each patient had separate nose (N1), groin (G1), and CNG swabs (N2 and G2 together) tested according to the manufacturer''s instructions for handling a nose swab (4). If any of the GXP-MRSA results were invalid, the assay was repeated (when possible) using the spare nose (N4) and/or groin (G4) swab, which was frozen (−20°C for 30 min) then thawed (unpublished manufacturer''s recommendations) prior to insertion in the elution reagent. We defined a specimen as “unresolved” if both the initial and repeat PCR assays were invalid or if the initial assay was invalid and no repeat swab was available for retesting. Unresolved specimens were excluded from final analysis. Crystalline material was noted when the elution reagent was removed from storage at 4°C during study A. Following discussions with the company representative, the elution reagent was routinely warmed for 15 to 30 min at 35°C after removal from storage at 4°C in study B. This aimed to dissolve any crystalline material and reduce the potential risk of an invalid result.The results of study A are shown in Table ​Table1.1. Among the selected 43 patients, 22 were MRSA colonized and 21 noncolonized. Initially, 10 patients (12 specimens) had invalid results with GXP-MRSA assays of nose specimens, groin specimens, or CNG swabs, resulting in “invalid/inhibited” rates of 1/43 (2.3%), 2/43 (4.7%), and 9/43 (20.9%), respectively. After repeat testing, 37/43 patients (86%) had valid PCR results for all three assays (N1, G1, and CNG [N2 and G2]), and the unresolved rates for the respective specimen assays were 1/43 (2.3%), 2/43 (4.7%), and 4/43 (9.3%) (Table ​(Table1).1). The explanation for the CNG specimens having higher initial “invalid/inhibited” and subsequent unresolved rates than the individual specimens is unclear, but we believe the need to dissolve the crystalline material (see above) may have been a contributing factor. In study B (see below), in which the crystalline material was dissolved, the “invalid/inhibited” rates for CNG specimens were comparable to those reported by the manufacturer for assessment of nasal specimens alone.

TABLE 1.

Comparison of results for the GXP-MRSA assay versus culture using individual and CNG swabs for detection of MRSA colonization

Comparison of culture screening protocols for methicillin-resistant Staphylococcus aureus (MRSA) using a chromogenic agar (MRSA-Select)

To compare the culture screening protocols for methicillin-resistant Staphylococcus aureus (MRSA), a total of 300 duplicate nasal swabs (233 initial cultures and 67 weekly follow-up cultures) were collected consecutively from 233 patients in the Intensive Care Unit (ICU). One swab was plated directly on MRSA-Select agar (D-MRSA-Select) and observed at 24 hr. The duplicate swab was incubated in tryptic soy broth (TSB) with 6.5% NaCl for 24 hr, and then subcultured on MRSA-Select (B-MRSA-Select), BAP (B-BAP), and mannitol salt agar with 4 mg/L oxacillin (B-MSA(OXA)), and observed at 24 hr. MRSA was detected in 13.7% (32/233) of the initial and 22.4% (15/67) of the follow-up specimens. A patient was classified as MRSA-positive if any of the media grew colonies that were tested and confirmed to be MRSA. In the initial screening samples, the sensitivities of D-MRSA-Select, B-MRSA-Select, B-BAP, and B-MSA(OXA) were 78.1%, 84.4%, 78.1%, and 65.6%, respectively, and the specificities were 100%, 98.0%, 83.1%, and 93.5%, respectively. The sensitivities of all but the B-MRSA-Select protocol were significantly lower (p <0.05). In follow-up screening, the sensitivities of D-MRSA-Select, B-MRSA-Select, B-BAP, and B-MSA(OXA) were 66.7%, 86.7%, 66.7%, and 53.3%, respectively, and the specificities were 100%, 98.1%, 90.4%, and 90.4%, respectively. D-MRSA-Select protocol was considered useful in screening for MRSA because it was fast, highly specific, and showed sensitivity comparable to B-BAP. Salt-containing enrichment broth in conjunction with MRSA-Select (B-MRSA-Select) provides a promising way to increase sensitivity in initial and follow-up screening for MRSA.     

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.     

Methicillin-resistant Staphylococcus aureus (MRSA) detection: comparison of two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) with three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA) for use with infection-control swabs

Methicillin-resistant Staphylococcus aureus (MRSA) is an increasing problem. Rapid detection of MRSA-colonized patients has the potential to limit spread of the organism. We evaluated the sensitivities and specificities of MRSA detection by two molecular methods (IDI-MRSA PCR assay and GenoType MRSA Direct PCR assay) and three selective MRSA agars (MRSA ID, MRSASelect, and CHROMagar MRSA), using 205 (101 nasal, 52 groin, and 52 axillary samples) samples from consecutive known MRSA-infected and/or -colonized patients. All detection methods had higher MRSA detection rates for nasal swabs than for axillary and groin swabs. Detection of MRSA by IDI-MRSA was the most sensitive method, independent of the site (94% for nasal samples, 80% for nonnasal samples, and 90% overall). The sensitivities of the GenoType MRSA Direct assay and the MRSA ID, MRSASelect, and CHROMagar MRSA agars with nasal swabs were 70%, 72%, 68%, and 75%, respectively. All detection methods had high specificities (95 to 99%), independent of the swab site. Extended incubation for a further 24 h with selective MRSA agars increased the detection of MRSA, with a corresponding decline in specificity secondary to a significant increase in false-positive results. There was a noticeable difference in test performance of the GenoType MRSA Direct assay in detection of MRSA (28/38 samples [74%]) compared with detection of nonmultiresistant MRSA (17/31 samples [55%]) (susceptible to two or more non-beta-lactam antibiotics). This was not observed with selective MRSA agar plates or IDI-MRSA. Although it is more expensive, in addition to rapid turnaround times of 2 to 4 h, IDI-MRSA offers greater detection of MRSA colonization, independent of the swab site, than do conventional selective agars and GenoType MRSA Direct.     

Use of BBL CHROMagar MRSA medium for identification of methicillin-resistant Staphylococcus aureus directly from blood cultures

We evaluated the ability of BBL CHROMagar MRSA medium (Becton Dickinson, Sparks, MD) to identify methicillin-resistant Staphylococcus aureus (MRSA) directly upon subculture from positive blood culture bottles. There were 124 MRSA isolates recovered from blood cultures in the study. BBL CHROMagar MRSA medium was highly sensitive (97.6% [121/124] at 18 to 24 h of incubation and 100% [124/124] at 48 h) and 99.9% specific for identifying MRSA from positive blood cultures.