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
2012; Elsevier BV; Volume: 14; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2012.01.015
ISSN1943-7811
AutoresRodney Arcenas, Stacey Spadoni, Amin A. Mohammad, Frederick L. Kiechle, Kimberly Walker, Robert Fader, Françoise Perdreau‐Remington, J Osiecki, Oliver Liesenfeld, Shelby Hendrickson, Arundhati Rao,
Tópico(s)Mycobacterium research and diagnosis
ResumoRapid 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. 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. Worldwide, the increasing number of health care–associated methicillin-resistant Staphylococcus aureus (MRSA) infections is a focus of medical and public concern. In 2003, nearly 60% of S. aureus isolates recovered from patients in intensive care units in the US were MRSA.1Siegel J.D. Rhinehart E. Jackson M. Chiarello L. Healthcare Infection Control Practices Advisory Committee: 2007 Guidelines for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings [Internet] Centers for Disease Control and Prevention.http://www.cdc.gov/hicpac/pdf/isolation/isolation2007.pdfDate: 2007Google Scholar Similar increases have been observed worldwide, with MRSA accounting for increasing rates of S. aureus bacteremia in areas of Canada, the UK, and Europe.2Laupland K.B. Ross T. Gregson D.B. Staphylococcus aureus bloodstream infections: risk factors, outcomes, and the influence of methicillin resistance in Calgary, Canada, 2000–2006.J Infect Dis. 2008; 198: 336-343Google Scholar, 3Naber C.K. Staphylococcus aureus bacteremia: epidemiology, pathophysiology, and management strategies.Clin Infect Dis. 2009; 48: S231-S237Google Scholar, 4Wyllie D.H. Crook D.W. Peto T.E. Mortality after Staphylococcus aureus bacteraemia in two hospitals in Oxfordshire, 1997–2003: cohort study.Bmj. 2006; 333: 281Google Scholar Since almost 20% of invasive MRSA infections are fatal, the failure to reduce health care–associated MRSA poses a serious threat to patients' health.5Klevens R.M. Morrison M.A. Nadle J. Petit S. Gershman K. Ray S. Harrison L.H. Lynfield R. Dumyati G. Townes J.M. Craig A.S. Zell E.R. Fosheim G.E. McDougal L.K. Carey R.B. Fridkin S.K. Invasive methicillin-resistant Staphylococcus aureus infections in the United States.Jama. 2007; 298: 1763-1771Google Scholar In the US, approximately 126,000 hospitalizations each year are related to MRSA infections and are associated with high morbidity and mortality rates, prolonged hospital stays, increased cost, and greater use of medical and personnel resources.6Francois P. Bento M. Renzi G. Harbarth S. Pittet D. Schrenzel J. Evaluation of three molecular assays for rapid identification of methicillin-resistant Staphylococcus aureus.J Clin Microbiol. 2007; 45: 2011-2013Google Scholar Furthermore, community-acquired MRSA infections have become commonplace and have created a public health crisis in US emergency departments and other clinical settings. A population-based study of MRSA infections in San Francisco, CA, in 2004 and 2005 demonstrated that 90% of MRSA infections had onset in the community, with an incidence rate of 316 cases per 100,000 population; there were many fewer hospital-onset infections, with an incidence of 31 cases per 100,000 population.7David M.Z. Daum R.S. Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic.Clin Microbiol Rev. 2010; 23: 616-687Google Scholar, 8Liu C. Graber C.J. Karr M. Diep B.A. Basuino L. Schwartz B.S. Enright M.C. O'Hanlon S.J. Thomas J.C. Perdreau-Remington F. Gordon S. Gunthorpe H. Jacobs R. Jensen P. Leoung G. Rumack J.S. Chambers H.F. A population-based study of the incidence and molecular epidemiology of methicillin-resistant Staphylococcus aureus disease in San Francisco, 2004–2005.Clin Infect Dis. 2008; 46: 1637-1646Google Scholar Patients who are colonized with MRSA have an increased risk of MRSA disease.9Datta R. Huang S.S. Risk of infection and death due to methicillin-resistant Staphylococcus aureus in long-term carriers.Clin Infect Dis. 2008; 47: 176-181Google Scholar, 10Huang S.S. Platt R. Risk of methicillin-resistant Staphylococcus aureus infection after previous infection or colonization.Clin Infect Dis. 2003; 36: 281-285Google Scholar Thus, minimizing the spread of MRSA to reduce new colonization is one way to lower MRSA infection and its associated morbidity and mortality. Importantly, Hacek and colleagues11Hacek D.M. Paule S.M. Thomson Jr, R.B. Robicsek A. Peterson L.R. Implementation of a universal admission surveillance and decolonization program for methicillin-resistant staphylococcus aureus (MRSA) reduces the number of MRSA and total number of S. aureus isolates reported by the clinical laboratory.J Clin Microbiol. 2009; 47: 3749-3752Google Scholar recently showed that lowering the number of clinical MRSA isolates reduces the overall number of S. aureus clinical isolates recovered from hospitalized patients. Although the effectiveness of MRSA screening is not universally accepted,12McGinigle K.L. Gourlay M.L. Buchanan I.B. The use of active surveillance cultures in adult ICUs to reduce methicillin-resistant Staphylococcus aureus-related morbidity, mortality and costs: a systematic review.Clin Infect Dis. 2008; 46: 1717-1725Google Scholar, 13Tacconelli E. Methicillin-resistant Staphylococcus aureus: source control and surveillance organization.Clin Microbiol Infect. 2009; 15: 31-38Google Scholar the importance of rapid detection of nasal carriers in MRSA control programs has recently been described.14Peterson L.R. Diekema D.J. To screen or not to screen for methicillin-resistant Staphylococcus aureus.J Clin Microbiol. 2010; 48: 683-689Google Scholar Critical features identified in recent successful MRSA control programs include rapid and accurate determination of MRSA colonization.15Hardy K. Price C. Szczepura A. Gossain S. Davies R. Stallard N. Shabir S. McMurray C. Bradbury A. Hawkey P.M. Reduction in the rate of methicillin-resistant Staphylococcus aureus acquisition in surgical wards by rapid screening for colonization: a prospective, cross-over study.Clin Microbiol Infect. 2010; 16: 333-339Google Scholar There are a variety of reports on Food and Drug Administration–cleared nucleic amplification tests for the detection of nasal colonization: the BD GeneOhm (BD Biosciences, Franklin Lakes, NJ), Xpert MRSA (Cepheid, Sunnyvale, CA), and LightCycler MRSA Advanced Test (Roche Molecular Diagnostics, Pleasanton, CA) MRSA assays, with performance compared with that of culture.16Luteijn J.M. Hubben G.A. Pechlivanoglou P. Bonten M.J. Postma M.J. Diagnostic accuracy of culture-based and PCR-based detection tests for methicillin-resistant Staphylococcus aureus: a meta-analysis.Clin Microbiol Infect. 2010; 17: 146-154Google Scholar, 17Rossney A.S. Herra C.M. Brennan G.I. Morgan P.M. O'Connell B. 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.J Clin Microbiol. 2008; 46: 3285-3290Google Scholar, 18Snyder J.W. Munier G.K. Johnson C.L. 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 intensive care unit patients.J Clin Microbiol. 2010; 48: 1305-1309Google Scholar, 19Wolk D.M. Picton E. Johnson D. Davis T. Pancholi P. Ginocchio C.C. Finegold S. Welch D.F. de Boer M. Fuller D. Solomon M.C. Rogers B. Mehta M.S. Peterson L.R. Multicenter evaluation of the Cepheid Xpert methicillin-resistant Staphylococcus aureus (MRSA) test as a rapid screening method for detection of MRSA in nares.J Clin Microbiol. 2009; 47: 758-764Google Scholar Whereas the Xpert MRSA assay is considered an on-demand test, the LightCycler and BD GeneOhm MRSA assays are operated primarily in the laboratory for larger-volume batches. Only limited information is available comparing these molecular assays with each other. Since not all laboratories have the capability of testing for MRSA colonization by molecular methods, chromogenic agar media have been used as a culture method for screening.18Snyder J.W. Munier G.K. Johnson C.L. 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 intensive care unit patients.J Clin Microbiol. 2010; 48: 1305-1309Google Scholar, 20Boyce J.M. Havill N.L. Comparison of BD GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) PCR versus the CHROMagar MRSA assay for screening patients for the presence of MRSA strains.J Clin Microbiol. 2008; 46: 350-351Google Scholar, 21Malhotra-Kumar S. Abrahantes J.C. Sabiiti W. Lammens C. Vercauteren G. Ieven M. Molenberghs G. Aerts M. Goossens H. Evaluation of chromogenic media for detection of methicillin-resistant Staphylococcus aureus.J Clin Microbiol. 2010; 48: 1040-1046Google Scholar, 22Malhotra-Kumar S. Van Heirstraetam L. Lee A. Abrahantes J.C. Lammens C. Vanhommerig E. Molenberghs G. Aerts M. Harbarth S. Goossens H. Evaluation of molecular assays for rapid detection of methicillin-resistant Staphylococcus aureus.J Clin Microbiol. 2010; 48: 4598-4601Google Scholar, 23Paule S.M. Mehta M. Hacek D.M. Gonzalzles T.M. Robicsek A. Peterson L.R. Chromogenic media vs real-time PCR for nasal surveillance of methicillin-resistant Staphylococcus aureus: impact on detection of MRSA-positive persons.Am J Clin Pathol. 2009; 131: 532-539Google Scholar, 24van Hal S.J. Stark D. Lockwood B. Marriott D. Harkness J. 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.J Clin Microbiol. 2007; 45: 2486-2490Google Scholar, 25Wolk D.M. Marx J.L. Dominguez L. Driscoll D. Schifman R.B. 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.J Clin Microbiol. 2009; 47: 3933-3936Google Scholar Chromogenic agars have antibiotics and chromogenic compounds incorporated into the medium that provide colonies of distinctive colors. This coloring allows for the rapid examination of agar plates without the need for extensive biochemical testing for organism identification. The performance characteristics of the chromogenic agar MRSASelect (Bio-Rad Laboratories, Hercules, CA) compared with those of molecular methods for MRSA screening have not been reported yet. We, therefore, compared the performance of the LightCycler MRSA Advanced Test with that of the Xpert MRSA assay and MRSASelect culture for rapid detection of MRSA in nasal swabs using a collection of representative staphylococcal strains and a large number of consecutively collected patient samples. Hands-on time and total turnaround time were also recorded during predefined routine testing to compare workflow characteristics between the individual assays. To evaluate the inclusivity of the LightCycler MRSA Advanced Test, the Xpert MRSA assay, and MRSASelect medium, a panel of 28 staphylococcal isolates consisting of 25 MRSA and 3 methicillin-susceptible S aureus strains was tested at each site. The MRSA isolates originated from a variety of body sites and sample types, had previously been typed as SSCmec II and IV, and were pulsed-field gel electrophoresis typed as USA 100, 300, 400, 500, 700, 1000, and 1100. Strains were representative of highly prevalent strains currently found in the US and were provided to all sites in a ready-to-use blinded manner. All the strains had been previously characterized based on established molecular epidemiologic techniques (including SCCmec and pulsed-field gel electrophoresis typing, presence or absence of Panton-Valentine leukocidin proteins). Strains were tested singly by all techniques. Individuals included in the study were patients in hospital intensive care units and non–intensive care units, residents in nursing homes/extended care facilities, outpatients undergoing elective surgical procedures, and dialysis patients. All the participants were eligible for routine MRSA screening in the health care facility. All the sites followed the Food and Drug Administration guidance on the use of leftover specimens (with the approval of the respective institutional review boards) or obtained informed consent. Patient samples were evaluated at three sites across the US. The Hines VA Hospital in Chicago, IL (site 1), a 700-bed hospital serving the veteran population, performs universal screening on all patients admitted to the hospital (including the acute and long-term care wards). Nasal swabs were collected by the nursing staff within 12 hours of admission. Specimens were tested without batching every 2 hours from 8 AM to midnight 7 days a week with a goal of reporting out within 4 hours of laboratory receipt for specimens that arrive during testing hours. Site 2, at Memorial Regional Hospital, Hollywood, FL, is a centralized core molecular laboratory that serves five hospitals in the Memorial Healthcare System. Only patients considered high risk [those with a skin and soft-tissue infection, undergoing renal dialysis, with a previous MRSA-positive result (colonization or infection), with presurgical workups, from long-term care facilities, and placed in or transferred to a critical care unit] and who were admitted and patients undergoing certain elective surgical procedures were screened for the presence of MRSA (two nasal swabs used together for both nostrils). Samples were sent to the molecular laboratory for batched analysis and were run two to three times per day. Site 3, at Scott & White Healthcare, Temple, TX, used universal screening at four of the system's hospitals with a total bed population of 800. The swabs were run in three to four batches from 7 AM to midnight 7 days a week, with a turnaround time goal of 10 hours from time of receipt in the laboratory. For specimen collection, the double-headed swab (BD CultureSwab liquid stuart; Becton-Dickinson, Sparks, MD) was inserted into the nostril and rotated against the mucosa five times; the same procedure was then repeated for the second nostril. The double-headed swab was separated in the laboratory and rubbed together for consistency. One swab head was processed for directly plated culture on MRSASelect medium, followed by the LightCycler MRSA Advanced Test. The other swab head was directly plated to culture on separate MRSASelect medium and then was processed using the Xpert MRSA assay. Specimens were stored at 2°C to 8°C for up to 24 hours before testing following routine workflow protocols and according to the manufacturers' instructions. Each of the three study sites performed all the testing. The time when PCR was performed and the number of samples processed in batches/groups were not dictated by the study design but rather reflected the daily routine in all the laboratories. The LightCycler MRSA Advanced Test and the Xpert MRSA assay were performed according to the manufacturers' instructions. At the time of this study, the Xpert MRSA assay was routinely used by each of the study sites for MRSA screening and was performed at all the sites using the most recently available approved procedure, including reagents preloaded into cassettes. Both swab heads were directly streaked onto MRSASelect medium before adding the swab head to the sample preparation buffer of the respective molecular assays. MRSASelect medium was incubated in the dark for 18 to 28 hours at 35°C to 37°C. Characteristic pink colonies observed after incubation were considered positive for MRSA. Ambiguous colonies were initially Gram stained to confirm the presence of characteristic staphylococcal morphologic features. Further confirmation as S. aureus was accomplished using Staphaurex latex reagent (Remel Inc., Lenexa, KS), a rapid coagulase test, following the manufacturer's instructions. Light pink but not white breakthrough colonies were subcultured to 5% sheep blood agar (BD Microbiology Systems, Sparks) and were tested by the Staphaurex reagent as described previously herein. Workflow analysis was performed at predefined days and runs to reflect routine laboratory processing procedures for MRSA surveillance testing at each of the three sites and included the following samples: MRSASelect medium (194 samples), Xpert MRSA assay (184 samples), and LightCycler MRSA Advanced Test (335 samples). Time stamps on a workflow log sheet were used to document the time of day and time to completion for the routinely performed Xpert MRSA assay and were compared with the LightCycler MRSA Advanced Test and MRSASelect medium. Workflow calculations were performed to determine the mean sample preparation and turnaround time per specimen for all days and runs documented; we also determined the mean sample preparation and turnaround time per sample when using average batch sizes of 25, 10, and 13 specimens as determined for the LightCycler MRSA Advanced Test, Xpert MRSA assay, and MRSASelect medium, respectively. The GeneXpert system (Cepheid) used in this study is a 1- to 16-site random-access instrument that automates sample preparation through the use of cartridges. For analysis of specimen/cartridge preparation, the start time for the GeneXpert system was initiated when the first cartridge was prepared, and the stop time was documented when the last cartridge was loaded on the GeneXpert instrument. The times required for amplification, detection, viewing, and printing of all the samples were also recorded. Sites 1, 2, and 3 were capable of evaluating 16, 12, and 8 samples per batch, respectively, using the Xpert MRSA assay. The LightCycler real-time PCR instrument is a carousel-based thermal cycler system that has the capability to evaluate specimens in batches of up to 32 (30 samples and 2 controls). Specimen preparation was evaluated by observing the interval between breaking the first swab into the lysis buffer and when the last specimen completed the final centrifugation spin. Timing was started for the PCR preparation when the master mix was prepared and was stopped when the specimens were started on the LightCycler instrument. Amplification and detection time, including printing, was also documented. The average number of samples processed per run was 25, 28, and 23 by sites 1, 2, and 3, respectively. For directly plated culture on MRSASelect medium, the duration of streaking samples onto culture media was recorded. The start time for the cultures was documented when the plates were struck with the swab, and the end time was documented when all the swabs in a batch were completed. The time it took to read the plates was documented starting when the first plate was observed and ending when the last of plate was read. The average number of samples processed per batch was 13 for site 1, 14 for site 2, and 11 for site 3. In addition to the previous workflow analysis, we simulated the laboratory workflow processes of the two molecular platforms (a random-access instrument using the Xpert MRSA assay and a batching capability instrument using the LightCycler MRSA Advanced Test) using a discrete event simulator to achieve an objective, unbiased appreciation of laboratory efficiency parameters, including test throughput (number of samples that can be tested per day), expected turnaround times, and labor utilization. iGrafx Process 2007 for Six Sigma discrete event simulation software version 1 (Corel Corp., Tualatin, OR) was used to develop simulation models for both molecular processes to allow quantitative determination of the efficiency parameters, described in detail by Mohammad et al.26Mohammad A.A. Elefano E.C. Leigh D. Stredler D. Okorodudu A.O. Petersen J.R. Using computer simulation to study the impact of increasing test volume on turn around times of STAT samples on ci8000intergrated chemistry and immunoassay analyzer.Clin Chem. 2004; 50: 1952-1955Google Scholar, 27Mohammad A.A. Okorodudub A.O. Petersen J.R. Utilization of modeling programs for technology change.in: Lehmann C. Health Technology. AACC Press, Washington, DC2005: 1-20Google Scholar Site 1 most frequently tested samples as they arrived at the laboratory and did not batch as frequently as sites 2 and 3. Sites 2 and 3 used the eight-dock GeneXpert system. The process diagrams for two processes are shown in Figure 1, Figure 2. Time and motion analysis was performed for all the steps in the two processes. The models were validated by comparing the predicted turnaround times and test throughput with the experimental data.Figure 2Simulation logic for analyzing 30 samples using the LightCycler MRSA Advanced Test. MagNA Lyser is manufactured by Roche Applied Science, Indianapolis, IN.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A discrepancy analysis using sequencing was performed by Roche Molecular Diagnostics only on samples that gave discordant results among the LightCycler MRSA Advanced Test, the Xpert MRSA assay, and the MRSASelect medium. Genomic DNA was extracted from frozen lysates obtained from both molecular assays. PCR was performed using primers that flank the LightCycler MRSA Advanced Test target region [the right (attR) and left (attL) extremity attachment site for the SCCmec].28Bode L.G. Kluytmans J.A. Wertheim H.F. Bogaers D. Vandenbroucke-Grauls C.M. Roosendaal R. Troelstra A. Box A.T. Voss A. van der Tweel I. van Belkum A. Verbrugh H.A. Vos M.C. Preventing surgical-site infections in nasal carriers of Staphylococcus aureus.N Engl J Med. 2010; 362: 9-17Google Scholar, 29Creamer E. Dolan A. Sherlock O. Thomas T. Walsh J. Moore J. Smyth E. O'Neill E. Shore A. Sullivan D. Rossney A.S. Cunney R. Coleman D. Humphreys H. The effect of rapid screening for methicillin-resistant Staphylococcus aureus (MRSA) on the identification and earlier isolation of MRSA-positive patients.Infect Control Hosp Epidemiol. 2010; 31: 374-381Google Scholar In addition, the attB, mecA (to detect methicillin resistance), and orfX (to identify S. aureus) genes were amplified to generate amplicons for sequencing.30Katayama Y. Ito T. Hiramatsu K. A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus.Antimicrob Agents Chemother. 2000; 44: 1549-1555Google Scholar, 31Peterson L.R. Liesenfeld O. Woods C.W. Allen S.D. Pombo D. Patel P.A. Mehta M.S. Nicholson B. Fuller D. Onderdonk A. Multicenter evaluation of the LightCycler methicillin-resistant Staphylococcus aureus (MRSA) advanced test as a rapid method for detection of MRSA in nasal surveillance swabs.J Clin Microbiol. 2010; 48: 1661-1666Google Scholar Sequencing was performed using the ABI BigDye Terminator version 3.1 chemistry and the Applied Biosystems 3730 DNA analyzer (Applied Biosystems, Foster City, CA). The resulting sequences were analyzed to determine any mismatches to the LightCycler MRSA Advanced Test primers and probes that may account for the discrepant results. The presence of MRSA was confirmed if attR- and attL-specific amplicons were detected combined with the presence of the mecA and orfX genes. Absence of the amplicons specific for the previous genes was indicative of the absence of MRSA in the lysate. AttB in the absence of mecA indicated the presence of methicillin-susceptible S aureus. All other results were considered unresolved. The positive and negative percentage agreements of the LightCycler MRSA Advanced Test with respect to the Xpert MRSA assay were calculated overall. The sensitivity and specificity of the two molecular assays compared with MRSASelect medium were calculated overall and by site. Exact binomial 95% CIs were calculated for estimates of positive and negative agreement and sensitivity and specificity. Differences and 95% CIs were used to compare the paired sensitivities and specificities of the two molecular assays with respect to MRSASelect medium. Sequencing was used exclusively to determine the number of true-positive and true-negative samples but was not used to determine the performance criteria (sensitivity and specificity) of the assays. The 28 staphylococcal isolates were correctly identified as either MRSA positive (MRSA) or MRSA negative (methicillin-susceptible S aureus or Staphylococcus epidermidis) at all three sites by the LightCycler MRSA Advanced Test and MRSASelect medium. The Xpert MRSA test did not identify one MRSA strain (SCCmec type IV, USA300, Panton Valentine Leucodidin positive, obtained from a urine culture) at only one of the three sites, possibly due to transport or operator error. In total, 1211 specimens (409, 402, and 400 from sites 1, 2, and 3, respectively) were collected from eligible individuals enrolled at the three clinical study sites. Most specimens were collected from patients in a hospital non–intensive care unit setting (75.8%), followed by specimens from hospital intensive care unit patients (22.5%) and from outpatient facility patients (1.7%). Agreement between the two swab heads collected for this testing was 99.3% (1188 of 1197) among those with valid results. The results obtained using the LightCycler MRSA Advanced Test and the Xpert MRSA assay are compared in Table 1. There was high negative agreement (98.4%; 95% CI, 97.4% to 99.1%) between both assays; positive percentage agreement was 86.2% (95% CI, 79.7% to 91.2%). A total of 131 samples (10.8%) tested positive by both assays.Table 1Comparison of the LightCycler MRSA Advanced Test with the Xpert MRSA TestLightCycler MRSA Advanced TestXpert MRSA testTotalPositiveNegativePositive13117148Negative2110421063Total15210591211Positive percentage agreement (95% exact CI)86.2 (79.7–91.2)Negative percentage agreement (95% exact CI)98.4 (97.4–99.1) Open table in a new tab Table 2 shows a three-way comparison of results obtained in 1202 specimens that yielded valid results using the LightCycler MRSA Advanced Test, the Xpert MRSA assay, and MRSASelect medium. Ninety-nine samples (8.2%) were concordant positive and 1032 (85.9%) were concordant negative by all three techniques. When both PCR assays gave positive results, culture was also positive in 99 of 131 cases (75.6%); in contrast, 32 samples (24.4%) that gave positive results in both PCR assays were culture negative. All of the 17 samples that gave positive results in the LightCycler MRSA Advanced Test but negative results in the Xpert MRSA assay were culture negative; conversely, 17 of 21 samples that gave negative results in the LightCycler MRSA Advanced Test but positive results in the Xpert MRSA assay were culture negative, and 4 were culture positive. The specificity of the two tests was identical at 95.5% (95% CI, 94.1% to 96.7%) compared with culture. The sensitivity of the Xpert MRSA assay was 99.0% (95% CI, 94.8% to
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