Methicillin-resistant Staphylococcus Aureus Assay Research Articles

The application of the loop-mediated isothermal amplification method for rapid detection of methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is an important problem associated with significant mortality and morbidity and well known as a predominant bacterial pathogen. The aim of this study was to identify MRSA strains. In this study (June 2018 to June 2019) isolates of S. aureus were obtained from patients referred to teaching hospitals of Ahvaz, Iran. All isolates were confirmed by conventional microbiological methods. In following, antimicrobial susceptibility testing (AST), MRSA screening, PCR detection of MRSA and LAMP assay were performed. Out of a total of 156 staphylococcal isolates, 126 isolates were identified as MRSA. Seventy-two (57.1%) MRSA isolates were recovered from wound. All MRSA isolates were sensitive to vancomycin, linezolid, teicoplanin, quinupristin-dalfopristin, and tigecycline. The results of LAMP showed 100% agreement with PCR. Sensitivity and specificity of the LAMP assays for the mecA genes were 100% and 100%, respectively. The LAMP assay is a rapid and simple method for the identifications of MRSA. Because of its performance without the need for specific instrumentation, this method can be easily employed in medical centers for the detection of mecA.

Methicillin-Resistant Staphylococcus aureus (MRSA) Admission Screening in the Neonatal Intensive Care Unit (NICU): Algorithm for Hospital Transfers

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent source of infection in the neonatal intensive care unit (NICU). Due to the serious consequences associated with MRSA infections in neonates, much effort has been made to prevent and control epidemics in NICUs. Since 2006, our hospital has performed MRSA nasal surveillance screening of all newborns in the NICU in accordance with the recommendations of the Chicago-Area Neonatal MRSA Working Group. In 2017, a MRSA infection was identified in a newborn shortly after transfer from an outside hospital and who had an initial negative MRSA admission screen. As a result, we modified the admission screening process for all transfers from outside NICUs. Methods: The Evanston Hospital Infant Special Care Unit is a level 3 NICU in the northern suburbs of Chicago with 44 NICU beds and 450 admissions per year. Effective July 1, 2017, all NICU transfers have a nasal MRSA screen performed upon admission and after 48 hours. The transferred baby is placed on contact isolation until both screening results return negative. Nasal MRSA testing is performed using both PCR on the BD MAX MRSA Assay platform and is confirmed by culture using MRSA CHROMagar TM. Results: Between July 1, 2017, and October 31, 2019, 112 neonates were transferred from outside NICUs. Moreover, 105 (94%) had at least 1 MRSA screen completed and 99 (88%) had both MRSA screens completed. Of 99 with 2 screens, only 1 neonate had an initial positive nasal MRSA screen. Of the remaining 98 negative babies, none had a repeat positive nasal MRSA screen within 48 hours of admission. of 99 neonates with 2 serial admission MRSA screens, 82 (83%) were transferred within 48 hours of birth. In addition, 17 neonates were transferred >48 hours after birth, including the 1 MRSA-positive baby. Conclusions: In an attempt to identify all potential MRSA-positive neonates transferred to our NICU, we instituted a policy of 2 admission nares swabs. However, our data suggest that a single initial MRSA swab may be sufficient. If continued collection of a second screen is performed, it may be sufficient to screen babies who have been hospitalized for at least 48 hours prior to transfer, which eliminates 83% of admission testing and results in a cost savings.Funding: NoneDisclosures: None

2129. When Is Methicillin-resistant Staphylococcus aureus not Methicillin-resistant Staphylococcus aureus?

BackgroundAs part of active surveillance in our NICU for methicillin-resistant Staphylococcus aureus (MRSA), two isolates representing modified S. aureus (MODSA), which are Methicillin resistant but lacking mecA or C were identified. Our current microbiology laboratory workflow for screening for MRSA involves plating isolates on chromoID agar (bioMérieux, Marcy-l’Etoile, France) as well as on sheep blood agar (SBA). Β hemolytic colonies on SBA that are catalase and coagulase positive are set up for confirmation and antimicrobial susceptibility testing on the Vitek 2 (bioMérieux, Marcy-l’Etoile, France).MethodsThese 2 isolates (from Baby 1 and Baby 2) tested positive for green colonies on the chromoagar plates. The Vitek 2 subsequently identified both these isolates as MRSA. However, for research purposes, all positive NICU MRSA isolates are tested via whole-genome sequencing (WGS). Both isolates were identified by WGS as methicillin-susceptible Staphylococcus aureus (MSSA). We subsequently went back and performed additional workup on these isolates. Isolates were plated on SBA and chromagar again and incubated for 24 hours. 2 colonies of different morphologies from the chromagar plates and 3 from the SBA were randomly selected and subcultured to chromagar and SBA for a total of 5 subcultures. Each of the subcultures was tested using staphaurex, mannitol salt agar and the Cepheid Xpert MRSA assay and all were confirmed to be Staphylococcus aureus.ResultsAll 10 isolates tested negative by the Cepheid Xpert MRSA assay for MRSA. Phenotypic testing was set up again for all ten isolates using the vitek GP panel, as well as cefoxitin disk, and oxacillin E test using Mueller–Hinton agar supplemented with 2% NaCl as per CLSI methods. See table attached for results.ConclusionIn conclusion, these two cases highlight the difficulty in identifying non-MecA, non-MecC-mediated MRSA isolates in the clinical microbiology laboratory. This is particularly important as more laboratories rely on testing for MecA by PCR for surveillance testing. These 2 cases were further complicated by heterogeneous sub-populations of Staphylococcus aureus. Failure to recognize these variant forms of MRSA can lead to difficulties in implementing appropriate therapy and infection control measures. Improved methodologies are needed. Disclosures All authors: No reported disclosures.

Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus.

Many countries using sensitive screening methods for detection of carriage of methicillin-resistant Staphylococcus aureus (MRSA) have a sustained low incidence of MRSA infections. For diagnostic laboratories with high sample volumes, MRSA screening requires stability, low maintenance and high performance at a low cost. Herein we designed oligonucleotides for a new nuc targeted hydrolysis probe PCR to replace the standard in-house nuc SybrGreen PCR assay. This new, more time-efficient, PCR assay resulted in a 40% increase in daily sample capacity, with maintained high specificity and sensitivity. The assay was also able to detect Staphylococcus aureus clonal cluster 75 (CC75) lineage strains, recently re-classified as Staphylococcus argenteus, with a sensitivity considerably increased compared to our previous assay. While awaiting consensus if the CC75 lineage of S. aureus should be considered as S. argenteus, and whether methicillin-resistant S. argenteus should be included in the MRSA definition, many diagnostic laboratories need to update their MRSA assay sensitivity/specificity towards this lineage/species. The MRSA screening assay presented in this manuscript is comprised of nuc oligonucleotides separately targeting S. aureus and CC75 lineage strains/S. argenteus, thus providing high user flexibility for the detection of CC75 lineage strains/S. argenteus.

Purified citritin in combination with vancomycin inhibits VRE in vitro and in vivo.

Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) have been frequently associated with bacterial resistance mechanisms. These mechanisms, in turn, restrict a range of therapeutic opportunities for the treatment of infections caused by these micro-organisms. Faced with this problem, the present study aims to isolate and characterize molecules with antimicrobial activity derived from the fungus Penicillium citrinum isolated from Cerrado soil. Furthermore, we also tested possible antibacterial potential alone and in combination with commercial antimicrobial agents. In this context, citrinin was isolated and characterized by nuclear magnetic resonance and electrospray ionization. Functional analyses showed MIC of 128 µg ml-1 against S. aureus ATCC 25923, E. faecalis ATCC 29212 and a clinical isolate of vancomycin-resistant E. faecium (VRE01). However, for a clinical strain of methicillin-resistant S. aureus (MRSA01), the MIC was 256 µg ml-1. In order to avoid such high concentrations and reduce the collateral effects, additive effects were evidenced by combining citrinin with cefoxitin against MRSA01 (FIC index=0.5) and also citrinin with vancomycin toward VRE01 (FIC index=0.5). In vivo studies with BALB/c-tipe mice (MRSA assay) demonstrated a clinical ineffectiveness of cefoxitin associated with citrinin (9.8 mg kg-1 of cefoxitin +0.2 mg kg-1 of citrinin), with this combination being inefficient to increase animal survival. However, the combination used in the treatment of VRE (23.5 mg kg-1 of citrinin +1.5 mg kg-1 of vancomycin) sepsis model was extremely promising, leading to an animal survival rate of 80 percent. In summary, our data show, for the first time, the possible successful use of citrinin associated with vancomycin for pathogenic bacteria control.

Evaluation of the eazyplex MRSA assay for the rapid detection of Staphylococcus aureus in pleural and synovial fluid

Loop-mediated isothermal amplification (LAMP) is a simple and sensitive technique for rapid microbiological diagnosis. The aim of this study was to evaluate the analytical and diagnostic performance of the LAMP eazyplex MRSA test for the direct detection and differentiation of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) in synovial/pleural fluid. Analytical validation included the determination of the limit of detection (LoD) and analytical specificity of the eazyplex MRSA test. A diagnostic evaluation of the eazyplex test against bacterial culture was performed on routine pleural/synovial samples collected prospectively from patients aged less than 18 years with complicated pneumonia with empyema or arthritis admitted to the Children's Hospital Sant Joan de Déu in Barcelona, Spain, between April 2015 and May 2016. The new system appropriately detected a quality control panel of clinical samples with DNA of MSSA, MRSA, and other pathogens. The LoD was established at 6.4×103 CFU/ml for S. aureus and 1.0×104 CFU/ml for MRSA. Diagnostic validation of the eazyplex MRSA assay was performed on 51 prospective clinical invasive samples, resulting in sensitivity and specificity values of 83.3% and 97.8%, respectively, for S. aureus detection. The mean turnaround time was 70min. The eazyplex MRSA assay was found to be a useful test for the rapid detection of S. aureus in invasive samples such as pleural/synovial fluid.

Characterization of the staphylococcal cassette chromosome mec insertion site in 108 isolates lacking the mecA gene and identified as methicillin-resistant Staphylococcus aureus by the Xpert MRSA assay.

During a 3-year period, 848 patients were detected as carriers of methicillin-resistant Staphylococcus aureus (MRSA) by the Xpert MRSA assay (Cepheid). Among them, 108 patients (12.7 %) were colonized with strains showing methicillin-susceptible phenotypes and absence of the mecA gene, despite being positive with the rapid polymerase chain reaction (PCR) assay. DNA sequences of the staphylococcal cassette chromosome mec (SCCmec) insertion site of these "false-positive" strains was determined by direct sequencing of the genomic DNA. More than half (53.7 %) of the strains had DNA sequences unrelated to either SCC or SCCmec and one-third had DNA sequences related to non-mec SCC. Only 10.2 % of the strains carried sequences related to SCCmec, suggesting that a sequence containing the mecA gene was lost from an SCCmec. These findings differ from the general idea that all methicillin-susceptible S. aureus having positive Xpert MRSA assay results are essentially MRSA that lost the mecA gene.

Isolation of Methicillin-resistant Staphylococcus aureus (MRSA) using a flocked swab elution transport system; with identification by PCR

Admission screening is an essential part of the control of methicillin-resistant Staphylococcus aureus (MRSA). Using collection systems that maintain viability and have high organism recovery, combined with accurate and rapid laboratory testing is critical for the success of a screening program. This review evaluates the performance of PCR testing, particularly the Roche LightCycler MRSA test, in the detection of MRSA. In addition, the clinical performance of the Copan Elution swab (ESwab) is reviewed. Several commercial molecular tests developed for use in detecting MRSA colonisation have been evaluated against culture methods, and all appear to provide high sensitivity, good specificity, and the potential for rapid reporting of MRSA colonisation. The new LightCycler MRSA advanced test is another of these assays with rapid detection time and high sensitivity. Compared with the BD GeneOhm MRSA assay, this new test also appears to have improved test specificity. The ESwab has an equal or even better recovery of bacteria, including MRSA, compared with other swab systems, via culture, and PCR. The advantages of using the ESwab are: the use of a liquid Amies medium allowed multiple uses (both culture, and PCR can be performed with one collected sample); and, specimens from multiple sites can be pooled into the same liquid transport media which limits the test costs and broadens the screening capabilities. However, there are limited publications on the clinical performance of the ESwab and more research is required on this new swab transport system.

Admission Screening of Methicillin-Resistant Staphylococcus aureus with Rapid Molecular Detection in Intensive Care Unit: A Three-Year Single-Centre Experience in Hong Kong

Background. The admission screening of methicillin-resistant Staphylococcus aureus (MRSA) by rapid molecular assay is considered to be an effective method in reducing the transmission of MRSA in intensive care unit (ICU). Method. The admission screening on patients from ICU once on their admissions by BD GeneOhm MRSA assay has been introduced to Prince of Wales Hospital, Hong Kong, since 2008. The assay was performed on weekdays and reported on the day of testing. Patients pending for results were under standard precautions until the negative screening results were notified, while contact precautions were implemented for MRSA-positive patients. In this study, we compared the MRSA transmission rate in molecular screening periods (2008 to 2010) with the historical culture periods (2006 to 2007) as control. Results. A total of 4679 samples were tested; the average carriage rate of MRSA on admission was 4.45%. By comparing with the historical culture periods, the mean incidence ICU-acquired MRSA infection was reduced from 3.67 to 1.73 per 1000 patient bed days. Conclusion. The implementation of admission screening of MRSA with molecular method in intensive care unit could reduce the MRSA transmission, especially in the area with high MRSA prevalence situation in Hong Kong.

Analysis of Staphylococcal Cassette Chromosome mec in BD GeneOhm MRSA Assay-Negative Strains

The BD GeneOhm MRSA assay could identify methicillin-resistant Staphylococcus aureus (MRSA) strains at a high ratio (97.8%). Analysis of 11 assay-negative MRSA strains suggested that insertion of non-mec staphylococcal cassette chromosome elements (SCCs) downstream of orfX, and carriage of SCCmecs with a left extremity that cannot be detected by the kit, might lead to their being given an incorrect negative status.

Rapid testing for methicillin-resistant Staphylococcus aureus: Implications for antimicrobial stewardship

Assays for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) infection are reviewed, with a discussion of their potential role in antimicrobial stewardship programs. Relative to standard culture and susceptibility testing methods, rapid MRSA assays developed during the last decade can dramatically shorten laboratory turnaround times (1-5 hours versus 48-96 hours), enabling clinicians to more quickly confirm or rule out MRSA infection and optimize antistaphylococcal therapy. Some rapid MRSA assays are based on polymerase chain reaction techniques while others use bacteriophage technology; four companies offer products approved by the Food and Drug Administration (FDA) for testing certain clinical specimens. In general, the currently available rapid MRSA tests have been demonstrated to have good sensitivity (91-100%) and specificity (95-100%), but one test product with marginally acceptable sensitivity (75%) was withdrawn from the U.S. market after reports of unacceptably high rates of false-positive and false-negative results. There is limited published evidence on the impact of any rapid MRSA assay on patient-level outcome and cost-effectiveness measures. Hospitals evaluating rapid MRSA tests should weigh factors such as their relative costs, reliability, and sample-processing times, as well as the need for policies and processes to ensure the prompt communication of test results to clinicians. Currently available rapid MRSA assays differ in specificity, sensitivity, cost, FDA-approved applications, and laboratory turnaround time, and published data on their comparative merits in terms of patient care and economic outcomes are limited. The optimal role of such tests in antimicrobial stewardship programs remains to be defined.

Development of a PCR-free electrochemical point of care test for clinical detection of methicillin resistant Staphylococcus aureus (MRSA)

An MRSA assay requiring neither labeling nor amplification of target DNA has been developed. Sequence specific binding of fragments of bacterial genomic DNA is detected at femtomolar concentrations using electrochemical impedance spectroscopy (EIS). This has been achieved using systematic optimisation of probe chemistry (PNA self-assembled monolayer film on gold electrode), electrode film structure (the size and nature of the chemical spacer) and DNA fragmentation, as these are found to play an important role in assay performance. These sensitivity improvements allow the elimination of the PCR step and DNA labeling and facilitate the development of a simple and rapid point of care test for MRSA. Assay performance is then evaluated and specific direct detection of the MRSA diagnostic mecA gene from genomic DNA, extracted directly from bacteria without further treatment is demonstrated for bacteria spiked into saline (10(6) cells per mL) on gold macrodisc electrodes and into human wound fluid (10(4) cells per mL) on screen printed gold electrodes. The latter detection level is particularly relevant to clinical requirements and point of care testing where the general threshold for considering a wound to be infected is 10(5) cells per mL. By eliminating the PCR step typically employed in nucleic acid assays, using screen printed electrodes and achieving sequence specific discrimination under ambient conditions, the test is extremely simple to design and engineer. In combination with a time to result of a few minutes this means the assay is well placed for use in point of care testing.

The Screening of Methicillin-Resistant Staphylococcus aureus in Vascular Surgery Patients: A Comparison of Molecular Testing and Broth-Enriched Culture

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major global health care-associated pathogen. This study sought to examine the prevalence of MRSA in patients who were admitted to a vascular surgery ward during a 3-month period. Methods: MRSA screening was accomplished through the acquisition of nasal, throat and perineal swabs. These swabs were placed in tryptic soy broth that had been supplemented with 6.5% NaCl and incubated for 24 h. The resulting isolates were subcultured on agar plates containing 5% sheep blood. The BD GeneOhm MRSA assay for screening swabs was performed in accordance with the manufacturer’s instructions. Results: A total of 58 patients were included in the study and swabs from 232 sites were obtained during the sampling period. MRSA was detected in 33 samples of 12 patients during the study period; thus, there was a 20.6% prevalence of patients who were recognized as MRSA carriers. There were discrepancies between the results of classical bacteriological screening and molecular MRSA detection methods in 8 of the patients. Conclusions: Nasal, throat and perineal MRSA screening can detect the carriage of this pathogen and allow for the timely use of appropriate infection control measures. The choice of screening techniques poses a challenge; it has been demonstrated that molecular detection methods should be performed with great sensitivity, specificity and, most importantly, speed. The cost of the PCR screening method is the only disadvantage of this approach.

Clinical utility of the Xpert MRSA assay for early detection of methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for many nosocomial and community-acquired infections, resulting in significant morbidity and mortality. A practical way to limit the spread of MRSA is early detection and proper treatment. However, screening culture for MRSA typically requires 2–3 days. The Xpert MRSA assay (Cepheid, Sunnyvale, CA, USA) is a real-time polymerase chain reaction-based assay developed for screening an MRSA-specific DNA sequence within the staphylococcal cassette chromosome in 2 h. Lower respiratory tract specimens, such as transtracheal aspirates (TTAs) and bronchoalveolar lavage fluid (BALF), are commonly obtained from intubated patients. Therefore, using the lower respiratory tract specimens with the Xpert MRSA assay may be a practical tool for patient care. We performed the Xpert MRSA assay on 108 TTA and 21 BALF specimens from 92 patients and compared the results to those obtained by culture. The two assays showed concordant results in 120 (93.0%) cases and discordant results in 9 (7.0%) cases, which were culture-negative and Xpert MRSA-positive. Among the discordant cases, 5 patients developed culture-positive samples 2–15 days after the Xpert MRSA detected MRSA. We conclude that the Xpert MRSA assay is a rapid, sensitive and clinically useful test, particularly for the early detection of MRSA.

Multicenter Evaluation of the LightCycler MRSA Advanced Test, the Xpert MRSA Assay, and MRSASelect Directly Plated Culture with Simulated Workflow Comparison for the Detection of Methicillin-Resistant Staphylococcus aureus in Nasal Swabs

Rapid detection of nasal colonization with methicillin-resistant Staphylococcus aureus (MRSA) followed by appropriate infection control procedures reduces MRSA infection and transmission. We compared the performance and workflow of two Food and Drug Administration-approved nucleic acid amplification assays, the LightCycler MRSA Advanced Test and the Xpert MRSA test, with those of directly plated culture (MRSASelect) using 1202 nasal swabs collected at three U.S. sites. The sensitivity of the LightCycler test (95.2%; 95% CI, 89.1% to 98.4%) and Xpert assay (99%; 95% CI, 94.8% to 100%) did not differ compared with that of culture; the specificity of the two assays was identical (95.5%; 95% CI, 94.1% to 96.7%) compared with culture. However, sequencing performed on 71 samples with discordant results among the three methods confirmed the presence of MRSA in 40% of samples that were positive by both molecular methods but negative by culture. Workflow analysis from all sites including batch runs revealed average hands-on sample preparation times of 1.40, 2.35, and 1.44 minutes per sample for the LightCycler, Xpert, and MRSASelect methods, respectively. Discrete event simulation analysis of workflow efficiencies revealed that the LightCycler test used less hands-on time for the assay when greater than eight batched samples were run. The high sensitivity and specificity, low hands-on time, and efficiency gains using batching capabilities make the LightCycler test suitable for rapid batch screening of MRSA colonization.

Positive predictive value of the Xpert MRSA assay diagnostic for universal patient screening at hospital admission: influence of the local ecology

The purpose of this study was to assess the accuracy of the Xpert MRSA assay (XP) for the detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage upon hospital admission. Nasal swabs were prospectively collected for MRSA screening from 1,891 patients admitted to a teaching hospital. XP results were compared to chromogenic agar culture results. MRSA was cultured in 61 specimens (3%). Compared with culture, XP had a sensitivity, specificity, positive, and negative predictive value of 60.7, 97.3, 37.8, and 98.9%, respectively. The median turnaround time (TAT) for the results was 3 h. Of 24 MRSA isolated from XP-negative samples, three harbored composite SCCmec. Among 61 samples with culture-negative but XP-positive results, 15 methicillin-susceptible S. aureus (MSSA) isolates tested positive by XP on pure colony lysates. These MSSA included: (i) strains with SCCmec deletion encompassing mecA and (ii) multilocus sequence typing (MLST) clonal complex (CC) 1 strains harboring a chromosomal sequence homologous to one of the orfX-SCCmec junction sequences targeted by XP. On account of the low sensitivity and positive predictive value in a hospital patient population with moderate prevalence of MRSA, culture still appears to be necessary in order to confirm polymerase chain reaction (PCR) results. The emergence of new SCCmec variants and the presence of MSSA harboring cross-reactive SCCmec-like elements may challenge the successful implementation of such detection systems.

Bactericidal Kinetics of Marine-Derived Napyradiomycins against Contemporary Methicillin-Resistant Staphylococcus aureus

There is an urgent need for new antibiotics to treat hospital- and community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections. Previous work has indicated that both terrestrial and marine-derived members of the napyradiomycin class possess potential anti-staphylococcal activities. These compounds are unique meroterpenoids with unusual levels of halogenation. In this paper we report the evaluation of two previously described napyradiomycin derivatives, A80915A (1) and A80915B (2) produced by the marine-derived actinomycete, Streptomyces sp. strain CNQ-525, for their specific activities against contemporary and clinically relevant MRSA. Reported are studies of the in vitro kinetics of these chemical scaffolds in time-kill MRSA assays. Both napyradiomycin derivatives demonstrate potent and rapid bactericidal activity against contemporary MRSA strains. These data may help guide future development and design of analogs of the napyradiomycins that could potentially serve as useful anti-MRSA therapeutics.

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.

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.

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.