Combined use of Pastorex Staph-Plus and either of two new chromogenic agars, MRSA ID and CHROMagar MRSA, for detection of methicillin-resistant Staphylococcus aureus

We describe the search toward a fast and reliable strategy to detect and confirm the presence of methicillin-resistant Staphylococcus aureus (MRSA) in screening samples. First, we evaluated the sensitivities and specificities of oxacillin resistance screening agar (ORSA) with enrichment (tryptic soy broth [TSB] and ORSA [TSB-ORSA]) and without enrichment (ORSA), MRSA ID (MRSA_ID) plates, and CHROMagar MRSA (C_MRSA) plates, all of which were inoculated with equal volumes of a suspension made by emulsifying screening swabs. Whereas the sensitivities after 48 h were similar for all media tested (77% for MRSA_ID and ORSA; 73% for C_MRSA and ORSA after enrichment [TSB-ORSA]), the specificities of MRSA_ID (98% after 24 h and 94% after 48 h) and C_MRSA (98% after 24 h and 90% after 48 h) were superior to the specificities of ORSAs (92% after 24 h and 83% after 48 h) and TSB-ORSA (86% after 24 h and 81% after 48 h). Subsequently, the performance of the Pastorex Staph-Plus agglutination test with presumptive MRSA isolates taken directly from chromogenic agars (direct_Pastorex agglutination) was compared to that of the Pastorex Staph-Plus agglutination test with isolates from blood agar subcultures (conventional_Pastorex agglutination). When the direct_Pastorex agglutination test on MRSA_ID plates was combined with Gram staining, the direct_Pastorex agglutination test with samples from MRSA_ID plates was as reliable as the conventional_Pastorex agglutination test with samples from blood agar subcultures from MRSA_ID plates. In contrast, the direct_Pastorex agglutination test with samples from C_MRSA plates gave false-negative results. Finally, we calculated the processing times of the four different strategies, namely, (i) enrichment in TSB supplemented with NaCl, subsequent culture on ORSA, and the conventional_Pastorex agglutination test; (ii) direct inoculation of ORSA combined with conventional_Pastorex agglutination test; (iii) direct inoculation of MRSA_ID plates combined with Gram staining and the direct_Pastorex agglutination test; and (iv) direct inoculation of C_MRSA plates combined with Gram staining and the direct_Pastorex agglutination test. We concluded that the use of MRSA_ID in combination with Gram staining and the direct_Pastorex agglutination test is faster and more specific than the other strategies tested.     

Comparison of MRSASelect Agar,CHROMagar Methicillin-Resistant Staphylococcus aureus (MRSA) Medium,and Xpert MRSA PCR for Detection of MRSA in Nares: Diagnostic Accuracy for Surveillance Samples with Various Bacterial Densities

Rapid laboratory methods provide optimal support for active surveillance efforts to screen for methicillin-resistant Staphylococcus aureus (MRSA). Most laboratories struggle to determine the optimal use of resources, considering options to balance cost, speed, and diagnostic accuracy. To assess the performance of common methods, the first comparison of MRSASelect agar (MS) and CHROMagar MRSA (CA), with and without broth enrichment followed by a 24-h subculture to MS, was performed. Results were compared to those of the Xpert MRSA assay. For direct culture methods, the agreement between MS and CA was 98.8%. At 18 h, direct MS identified 93% of all positive samples from direct culture and 84% of those identified by the Xpert MRSA. For Trypticase soy broth-enriched MS culture, incubated overnight and then subcultured for an additional 24 h, the agreement with Xpert MRSA was 96%. The agreement between direct MS and Xpert MRSA was 100% when semiquantitative culture revealed a bacterial density of 2+ or greater; however, discrepancies between culture and Xpert MRSA arose for MRSA bacterial densities of 1+ or less, indicating low density as a common cause of false-negative culture results. Since 1+ or less was established as the most common MRSA carrier state, broth enrichment or PCR may be critical for the identification of all MRSA carriers who may be reservoirs for transmission. In this active-surveillance convenience sample, the use of broth enrichment followed by subculture to MS offered a low-cost but sensitive method for MRSA screening, with performance similar to that of Xpert MRSA PCR.The spread of methicillin-resistant Staphylococcus aureus (MRSA) is a major concern in healthcare settings because human “carriers” can spread MRSA to others, resulting in increased morbidity, mortality, and costs (13, 14, 17). One strategy to help prevent and control MRSA infections is the use of active surveillance cultures to screen patients for nasal carriage of MRSA, a practice coupled with appropriate barrier precautions for colonized or infected patients. Active surveillance programs are growing in number in the United States (10, 20, 23, 24), despite differences in opinions about the practice and reported gaps in the literature (19). Therefore, clinical microbiology laboratories must respond to provide support for active surveillance screening methods.There is continued debate about the practicality and cost benefit of different MRSA detection methods. Typically, agar culture or PCR methods are used (4); however, additional workload and costs cause laboratories to struggle with resource allocation issues, which often depend on the interplay between assay accuracy, turnaround time, cost per test, and workforce availability. While PCR results can be available in as little as 2 h and are known to provide excellent sensitivity and specificity for MRSA screening (4, 25, 28), PCR methods are more costly than culture methods (6). Alternately, selective and differential chromogenic agars have gained popularity because of lower cost and mid-range speed. Because of their resistance to antimicrobials in the agar, MRSA colonies grow and subsequently produce distinct color changes caused by the cleavage of a chromogenic substrate by a specific enzyme of S. aureus. Results can be achieved in as little as 18 to 24 h of incubation, depending on the agar used (1, 4, 5, 15, 27; BBL CHROMagar MRSA package insert no. 8012632, 2006 [Becton Dickinson and Company]; MRSASelect package insert nos. 63747 and 63749, 2007 [Bio-Rad Laboratories]). Although less costly, direct agar cultures are relatively insensitive in comparison to PCR and broth enrichment cultures. Broth enrichment cultures can improve sensitivity in comparison to that of nonenriched (direct) cultures (2, 9, 16, 21); however, enrichment delays results and adds to workload and costs. Thus, a discussion of the advantages and limitations of each methodological approach become critical. Decisions about the type of screening method used must be made based on the performance of the methods and resources available for each health care system. Clearly, the most sensitive screening system is desirable; however, at times, financial realities may affect our clinical practices.Because of the critical balance between speed, accuracy, cost, and hands-on time associated with each screening method, we designed and evaluated a broth enrichment algorithm that relies on the use of chromogenic agar as the subculture medium for broth enrichment in order to maximize both the speed and sensitivity of agar-based approaches. We compared the method''s performance to that of a PCR reference method, the Cepheid Xpert MRSA assay.To our knowledge, the study is the first clinical assessment of Bio-Rad''s MRSASelect agar (MS) and Becton Dickinson''s BBL CHROMagar MRSA (CA) combined with broth enrichment and compared to the Xpert MRSA assay. It is also the first study to describe the performance of the methods relative to the various bacterial densities of MRSA which are found in active surveillance samples from hospitalized patients. The study goal was to validate an off-label use of MS as a subculture medium, in combination with tryptic soy broth (TSB) enrichment, to improve the recovery of MRSA bacteria with minimum impact to workload and cost.(This information was presented, in part, at the 108th General Meeting of the American Society for Microbiology, Boston, MA, 1 to 5 June 2008 [18].)     

Prospective comparison of a new chromogenic medium, MRSASelect, to CHROMagar MRSA and mannitol-salt medium supplemented with oxacillin or cefoxitin for detection of methicillin-resistant Staphylococcus aureus

MRSASelect agar was compared to CHROMagar, mannitol-salt agar with oxacillin, and mannitol-salt agar with cefoxitin (MSA-CFOX) for the isolation of methicillin-resistant Staphylococcus aureus (MRSA). The sensitivities and specificities were 97.3% and 99.8%, 82.9% and 99.1%, 80.2% and 79%, and 99.1% and 84.8%, respectively. MSA-CFOX and MRSASelect had a high sensitivity. MRSASelect, however, was more specific and proved to be a more reliable and rapid medium for the detection of MRSA.     

Comparison of BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR versus the CHROMagar MRSA assay for screening patients for the presence of MRSA strains

We compared the BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) real-time PCR assay with the CHROMagar MRSA assay for the detection of MRSA in 286 nasal surveillance specimens. Compared with the CHROMagar MRSA assay, PCR had sensitivity, specificity, positive predictive value, and negative predictive values of 100%, 98.6%, 95.8%, and 100%, respectively. The mean PCR turnaround time was 14.5 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.     

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.     

Evaluation of three chromogenic media (MRSA-ID, MRSA-Select and CHROMagar MRSA) and ORSAB for surveillance cultures of methicillin-resistant Staphylococcus aureus

Screening specimens were homogenised in saline 0.9% w/v before either direct inoculation or following enrichment in broth on three chromogenic media (MRSA-ID, CHROMagar MRSA and MRSA Select) and ORSAB medium for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In total, 102 of 466 specimens yielded MRSA on at least one medium. After incubation for 16-18 h, the sensitivity was 51%, 59%, 47% and 65% on MRSA-ID, CHROMagar MRSA, ORSAB and MRSA Select, respectively, compared with 82%, 75%, 67% and 80%, respectively, after 42 h, and 93%, 95%, 79% and not tested, respectively, following broth enrichment. There were significantly more MRSA colonies on MRSA-Select after 16-18 h than on ORSAB or MRSA ID (p 0.001 and 0.0022, respectively), whereas there were more MRSA colonies after 42 h on MRSA-ID and MRSA-Select than on ORSAB (p 0.0004 and 0.012, respectively). The specificity of the media for identifying MRSA based on the colour of colonies after incubation for 16-18 h was 100%, 99%, 99% and 100%, respectively, compared with 98%, 97%, 98% and 98%, respectively, after 42 h, and 100%, 99%, 100% and not tested, respectively, following broth enrichment. The speed of detection (mean time to report a positive result) was 1.65, 1.72, 2.31 and 1.35 days, respectively. For each of the three media tested following enrichment, the use of an enrichment broth increased the detection rate of MRSA by 16-24%.     

Rapid confirmation of suspected methicillin-resistant Staphylococcus aureus colonies on chromogenic agars by a new commercial PCR assay, the GenomEra MRSA/SA Diagnose

A new automated closed tube PCR assay, the GenomEra(?) MRSA/SA Diagnose (Abacus Diagnostica Oy, Finland) was evaluated for rapid confirmation of methicillin-resistant Staphylococcus aureus (MRSA) from cultured screening specimens. The ability of the assay to detect genotypically different MRSA strains was studied with a collection of 304 MRSA isolates covering 68 spa types. The specificity was investigated with a collection of 146 non-MRSA staphylococcus isolates. The usefulness of the assay for clinical purposes was assessed by a sequential combination of MRSA screening culture and confirmation of the colonies with the GenomEra MRSA/SA Diagnose assay. A total of 145 suspected MRSA colonies on chromogenic plates were analyzed this way. All MRSA isolates from the culture collection and from the clinical screening specimens were confirmed as MRSA with the GenomEra MRSA/SA Diagnose assay and none of the non-MRSA staphylococci caused false-positive results, which indicates both sensitivity and specificity of 100%. The combination of GenomEra MRSA/SA Diagnose with preceding culture on selective MRSA agar permitted MRSA confirmation within 24 h. This practice offers a reliable and quick detection of MRSA that is also suitable in areas where several strain types cause epidemics.     

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.     

Detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood with the EVIGENE MRSA detection kit

A total of 200 blood cultures containing putative staphylococci were analyzed by a commercial gene probe hybridization assay (EVIGENE; Statens Serum Institut, Copenhagen, Denmark), and 18 were identified as methicillin-resistant Staphylococcus aureus (MRSA) positive. Of these, 17 were positive by PCR and 16 were positive by culture. Detailed analysis of the discrepant results showed that the EVIGENE kit allowed specific identification of MRSA in blood cultures without any of the drawbacks associated with PCR.     

Real-time multiplex PCR for direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples enriched by broth culture

A real-time multiplex PCR using the orfX and staphylococcal cassette chromosome (SCC) mec of Staphylococcus aureus was developed. The aim was to achieve a rapid and sensitive high-throughput method for direct detection of heterogeneous methicillin-resistant S. aureus (MRSA) in clinical samples, present in a low-endemic population, such as in Sweden. Consecutive broth enriched pooled clinical screening samples (nares, throat and/or perineum/groin) (n = 541 pools), broth enriched clinical samples showing growth of methicillin-sensitive S. aureus (MSSA) (n = 95 pools), clinical MRSA isolates (n = 173), MRSA reference strains (n = 43) and various coagulase-negative staphylococcal isolates (n = 33) were analyzed. The multiplex PCR detected all heterogeneous MRSA strains (n = 173) obtained in our area as well as all pooled consecutive broth enriched clinical samples with MRSA, i. e. 36 of 541 pools. None of the CoNS were positive. However, 18 out of 541 pools (3.3%) were positive in the multiplex PCR but no growth of MRSA could be detected by subculture and were regarded as false positive. Furthermore, the assay is rapid and reliable negative results can be delivered to the clinician within 18 h that will facilitate the infection control management of patients and hospital staff.     

Methicillin-resistant Staphylococcus aureus (MRSA) ST398 in a farmer with skin lesions and in pigs of his farm: clonal relationship and detection of lnu(A) gene

Skin infection associated with methicillin-resistant Staphylococcus aureus (MRSA)-ST398 was detected in a pig-farmer, and MRSA-ST398 isolates were also detected in nasal samples of the patient and of 11/12 pigs on his farm. Twelve MRSA isolates were obtained from skin lesions (n = 6) and nasal samples (n = 6) of the patient in two sampling moments and 11 MRSA isolates from nasal samples of pigs. They were typed as t011-SCCmecIVa-agrI and t108-SCCmecV-agrI (patient and pigs) and t588-SCCmecV-agrI (patient). The following resistance genes were detected (number isolates): tet(K) (1), tet(L) (23), tet(M) (13), erm(A) (13), erm(C) (13), msr(A) (11), lnu(A) (21), aph(2″)-acc(6′) (3), ant(4′) (13), aph(3′) (12), dfrS1 (15) and dfrK (22). Seventeen human and animal MRSA-ST398 isolates showed indistinguishable PFGE patterns (A1-spa-t011 or B2-spa-t108) and similar phenotypic-genotypic characteristics, including the presence of the lnu(A) gene, associated with lincomycin resistance. Potential pig-to-human transference of ST398 is suggested in this study. The first detection of the lnu(A) gene in MRSA-ST398 is reported.     

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.     

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.     

Novel multiplex PCR assay for simultaneous identification of community-associated methicillin-resistant Staphylococcus aureus strains USA300 and USA400 and detection of mecA and Panton-Valentine leukocidin genes, with discrimination of Staphylococcus aureus from coagulase-negative staphylococci

We developed a novel multiplex PCR assay for rapid identification and discrimination of the USA300 and USA400 strains and concomitant detection of Panton-Valentine leukocidin genes, with simultaneous discrimination of methicillin-resistant Staphylococcus aureus strains from methicillin-susceptible S. aureus strains, S. aureus strains from coagulase-negative staphylococci, and staphylococci from other bacteria.     

Carriage rates of methicillin-resistant Staphylococcus aureus (MRSA) depend on anatomic location, the number of sites cultured, culture methods, and the distribution of clonotypes

The present study was carried out to determine how active surveillance for methicillin-resistant Staphylococcus aureus (MRSA) could be improved by the use of enrichment broth and the inclusion of extra-nasal sites with nares cultures. Molecular typing was also performed to identify colonization by single or multiple strains. Surveillance cultures for MRSA were obtained from 650 patients on admission to a medical and surgical intensive care unit (ICU) in Taiwan. MRSA was detected on directly plated vs. broth-enrichment cultures in any site at 10.0% vs. 24.2%, nares 8.2% vs. 17.5%, throat 4.8% vs. 13.4%, axilla 1.2% vs. 9.1%, and perineum 1.8% vs. 9.5%, respectively. Nares cultures alone detected only 81.5% and 72.5% of all colonized patients by direct and broth-enriched cultures, respectively. The molecular typing of 68 isolates from 17 patients revealed that multisite isolates were largely indistinguishable within each patient, but four patients had multiple subtypes and another three patients had different clonotypes. The detection of MRSA carriers was considerably enhanced by broth-enrichment cultures at multiple anatomic sites and simultaneous colonization by multiple strains at different sites can occur. Epidemiological studies are needed to determine the likelihood of subsequent nosocomial infection among colonized patients detected via direct nasal versus broth-enriched cultures from multiple sites.