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Rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible Staphylococcus aureus (MSSA) using the KeyPath MRSA/MSSA blood culture test and the Bact/ALERT system in a pediatric population.

Stophylococcus aureus is a concerning etiologic agent in pediatric bacteremia because of its association with elevated mortality and its propensity to cause embolic disease. (1-2) Evidence from the literature suggests that central venous catheter removal, (3) prompt initiation of antimicrobial therapy, (4) and selection of optimal antimicrobial agents can help to mitigate these complications. (5-6)

Although vancomycin is an acceptable therapeutic choice for methicillin-resistant S aureus (MRSA) bacteremia, adult data suggest that treatment failures can be significantly reduced in methicillin-susceptible S aureus (MSSA) bacteremia by initiating narrow-spectrum agents, such as cefazolin or nafcillin, when methicillin-susceptible status is known. (5-6) In 2011, 75% of S aureus bacteremia cases were MSSA at our pediatric hospital, indicating a sizable cohort of patients that could benefit from accelerated detection of MSSA bacteremia and de-escalation of vancomycin to MSSA-focused therapy. Moreover, rapid identification of MRSA bacteremia could potentially decrease in-hospital transmission of MRSA by expediting initiation of MRSA-isolation practices. (7)

The time a laboratory requires to report MSSA/MRSA bacteremia is dependent on the time required to identify the presence of S aureus and to ascertain methicillin susceptibility status from a positive blood culture result. Routine procedures (subculture of positive blood culture tests to bacteriologic media, incubation, assignment of genus and species identification, and susceptibility testing) typically require 24 to 48 hours. Commercially available DNA polymerase chain reaction assays currently dominate the rapid MRSA/MSSA direct-detection market in blood culture tests. These assays were promising initially, but subsequent data revealed a variety of performance-related challenges. (8)

The KeyPath MRSA/MSSA blood culture test (MMBT, MicroPhage, Inc, Longmont, Colorado) is a US Food and Drug Administration (FDA)-approved, nonmolecular, rapid test that detects MSSA and MRSA directly from blood culture tests using bacteriophage-amplification technology. This study examined the diagnostic performance of MMBT in the detection of MSSA and MRSA bacteremia in a tertiary pediatric population.


Positive BacT/ALERT Pediatric FAN (fastidious antibiotic neutralization) blood culture bottles (bioMeerieux, Inc, Durham, North Carolina) were prospectively tested in the microbiology laboratory at the Children's Hospital of Philadelphia (Philadelphia, Pennsylvania) during 3 periods: November to December 2010, April to May 2011, and February to March 2012. Staphylococcus aureus isolates were identified using colonial morphology, catalase reaction, and Staphaurex latex testing (Remel, Lenexa, Kansas). The rapid Alere PBP2a test (Alere Scarborough, Inc, Scarborough, Maine) and the Vitek 2 system (bioMeerieux) were used to determine methicillin-susceptibility status.

The MMBT was performed according to the manufacturer's instructions. The MMBT uses an S aureus-specific bacteriophage cocktail that infects S aureus cells. Within 24 hours of the BacT/ ALERT instrument signaling blood culture positivity, 10 [micro]L of positive blood culture samples were pipetted into tubes containing predispensed "ID" reaction medium (with bacteriophage and culture medium) and "RS" reaction medium (with bacteriophage, culture medium, and cefoxitin). The tubes were incubated at 35[degrees]C in ambient air for 5 hours ([+ or -] 20 minutes), during which, bacteriophage rapidly replicated causing cellular lysis and increasing bacteriophage concentrations. The contents of the ID and RS reaction tubes were then dispensed into the ID and RS wells of the detector device. Detection of phage amplification by phage-specific antibodies in both the ID and RS wells indicated the presence of MRSA. Detection in only the ID well indicated the presence of MSSA.

The sensitivity, specificity, positive predictive value, and negative predictive value of MMBT in detecting S aureus, MSSA, and MRSA were calculated with 95% confidence intervals. Routine pathogen identification and susceptibility testing results were used as the gold standard.


Results are summarized in the Table. Of 188 positive blood cultures tested, 119 (63%) had Gram-positive cocci in clusters. All (119 of 119; 100%) grew Staphylococcus spp. Of these 119 positive results, 46 (39%) grew S aureus (26 MSSA [57%] and 20 MRSA [43%]), and the remainder grew coagulase-negative Staphylococcus spp. In the remaining positive blood cultures (n=69; 37%), the organisms isolated included other Gram-positive pathogens (Streptococcus spp, Corynebacterium spp), Gram-negative pathogens (Enterobacteriaceae, nonfermenting organisms), Candida spp, and Fusarium spp.

In positive blood cultures with Gram-positive cocci in clusters, MMBT had a sensitivity of 87% (95% confidence interval [CI], 74%-94%) for the detection of S aureus, 81% (95% CI, 62%-92%) for MSSA, and 95% (95% CI, 75%100%) for MRSA. Specificity was consistently 100%. Negative predictive value was 92% (95% CI, 84%-96%) for S aureus, 94% (95% CI, 88%-98%) for MSSA, and 99% (95% CI, 94%-100%) for MRSA. Positive predictive value was consistently 100%. Positive blood cultures that did not grow S aureus uniformly tested negative in both the ID and RS wells.

The MMBT failed to detect S aureus in 6 of the 46 blood cultures (13%) with S aureus present (5 MSSA [83%] and 1 MRSA [17%]). In 3 (50%) of these cases (2 MSSA [67%] and 1 MRSA [33%]), the ID well tested negative for S aureus initially but tested positive when MMBT was repeated within the recommended 24-hour testing window. In the 3 remaining cases (all MSSA), MMBT was negative when repeated. We did not observe any cases of MRSA designated as MSSA or vice versa.


Assays that rapidly and accurately detect MSSA and MRSA from positive blood cultures have the potential to improve clinical outcomes in patients with MSSA bacteremia and may help to limit inpatient transmission of MRSA. To date, the FDA-approved BD GeneOhm StaphSR (BD Diagnostics, San Diego, California) and the currently unavailable Cepheid Xpert MRSA/SA BC (Cepheid, Sunnyvale, California) polymerase chain reaction products have dominated the rapid blood culture MSSA/MRSA detection market. The latter has undergone 2 product recalls, most recently, in January 2012, because of high rates of invalid results. (9) In addition, clinical data generated after FDA approval revealed cases of MRSA bacteremia testing falsely as MSSA by both assays. (10) This phenomenon was attributed to failure of the assay primers to detect variants of SCCmec. (11) Misidentification of MRSA as MSSA would be considered a serious hazard to patients because it could prompt inappropriate de-escalation of vancomycin therapy to cefazolin or cloxacillin, leaving the patient on ineffective therapy for a potentially life-threatening condition. Instances of MSSA testing positive for MRSA have also been reported. This observation has been attributed to amplification of the SCCmec target in the absence of the mecA gene (the "empty cassette" phenomenon). (12) Data in the literature suggest that matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) may accurately detect S aureus directly from positive blood cultures. (13) However, MALDI-TOF MS is not FDA-approved nor capable of reliably performing antibiotic susceptibility testing at this time. An alternative approach to rapid diagnosis of MSSA and MRSA bacteremia is clearly desirable.

By using bacteriophage technology, the KeyPath MMBT is able to circumvent the issue of DNA target variation. In this study, however, we observed 6 instances in which blood cultures that were positive for S aureus tested negative. The control lines on the detection device were appropriately reactive in all 6 cases. In 3 cases, repeat testing yielded positive results. We believe that additional incubation of the blood samples allowed bacterial replication to continue, enhancing phage replication to a level that was detectable by the assay. In the 3 remaining cases, we believe that MSSA was present at levels that were too low to generate sufficient phage replication to allow detection. The failure of the ID well to detect S aureus indicated that reporting of negative S aureus results was problematic. An option might be to report S aureus-negative results as "preliminary," awaiting results from traditional culture procedures. These findings were consistent with data presented by Kingery et al. (14)

This study did have limitations. First, specimen accrual was challenging. In an attempt to achieve a larger sample size, the original study protocol was modified to add a third study period. We feel that this was related, in part, to the relatively low levels of central line-associated bloodstream infections at our institution, which were achieved through implementation of aggressive infection-control initiatives during the past decade. Second, the turnaround time of this test is 5 hours. For MMBT to translate successfully to accelerate optimization of antimicrobial therapy, immediate testing of positive blood cultures and the availability of clinicians to modify antimicrobial therapy would be required, ideally, on a 24-hour basis. Many centers may not have such resources available. (15) Third, the use of a commercially available polymerase chain reaction product would have allowed a gold standard definition that included a molecular standard. We opted to rely on conventional identification and susceptibility testing methods because of concerns pertaining to the performance of existing FDA-cleared products.

In summary, at this time, there is considerable need for accurate, reliable, and rapid MSSA/MRSA detection tests that can be performed directly on positive blood cultures.

The KeyPath MMBT provides an option. The positive predictive value of 100% for MSSA and MRSA detection suggested that positive results could be reported immediately to clinicians, but the sensitivity of this assay currently limits the ability to immediately report negative results. Further research aimed at reducing these false-negative S aureus results would be beneficial.

We thank the technologists in the microbiology laboratory at The Children's Hospital of Philadelphia for their assistance with specimen testing. The KeyPath MMBT kits used in this study were donated in kind by MicroPhage, Inc.


(1.) Welsh KJ, Abbott AN, Lewis EM, et al. Clinical characteristics, outcomes, and microbiologic features associated with methicillin-resistant Staphylococcus aureus bacteremia in pediatric patients treated with vancomycin. J Clin Microbiol. 2010;48(3):894-899.

(2.) Carillo-Marquez MA, Hulten KG, Mason EO, Kaplan SL. Clinical and molecular epidemiology of Staphylococcus aureus catheter-related bacteremia in children. Pediatr Infect Dis J. 2010;29(5):410-414.

(3.) Fowler VG Jr, Justice A, Moore C, et al. Risk factors for hematogenous complications of intravascular catheter-associated Staphylococcus aureus bacteremia. Clin Infect Dis. 2005;40(5):695-703.

(4.) Lodise TP, McKinnon PS, Swiderski L, Rybak MJ. Outcomes analysis of delayed antibiotic treatment for hospital-acquired Staphylococcus aureus bacteremia. Clin Infect Dis. 2003;36(11):1418-1423.

(5.) Chang FY, Peacock JE Jr, Musher DM, et al. Staphylococcus aureus bacteremia: recurrence and the impact of antibiotic treatment in a prospective multicenter study. Medicine (Baltimore). 2003;82(5):333-339.

(6.) Stryjewski ME, Szczech LA, Benjamin DK Jr, et al. Use of vancomycin or first-generation cephalosporins for the treatment of hemodialysis dependent patients with methicillin-susceptible Staphylococcus aureus bacteremia. Clin Infect Dis. 2007;44(2):190-196.

(7.) Siegel JD, Rhinehart E, Jackson M, Chiarello L; Healthcare Infection Control Practices Advisory Committee. Management of multidrug-resistant organisms in healthcare settings, 2006. MDROGuideline2006.pdf. Accessed June 30, 2012.

(8.) Marlowe EM, Bankowski MJ. Conventional and molecular methods for the detection of methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2011; 49(9):S53-S56.

(9.) US Food and Drug Administration. Class 3 recall;Cepheid Xpert MRSA/SA blood culture. 2012. res.cfm?ID=105528. Accessed June 30, 2012.

(10.) Snyder JL, Munier GK, Heckman SA, Camp P, Overman TL. Failure of the BD GeneOhm StaphSR assay for direct detection of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates in positive blood cultures collected in the United States. J Clin Microbiol. 2009;47(11):3747-4748.

(11.) Huletsky A, Giroux R, Rossbach V, et al. New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci. J Clin Microbiol. 2004;42(5):1875-1884.

(12.) Stamper PD, Louie L, Wong H, Simor AE, Farley JE, Carroll KC. Genotypic and phenotypic characterization of methicillin-susceptible Staphylococcus aureus isolates misidentified as methicillin-resistant Staphylococcus aureus by the BD GeneOhm MRSA Assay. J Clin Microbiol. 2011;49(4):1240-1244.

(13.) Moussaoui W, Jaulhac B, Hoffmann AM, et al. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identifies 90% of bacteria directlyfrom blood culture vials. Clin Microbiol Infect. 2010;16(11):1631-1638.

(14.) Kingery JD, Stamper PD, Peterson LR, et al. A novel phage technology for the detection of S aureus and differentiation of MSSA and MRSA in positive blood culture bottles. In: Proceedings from the 110th General Meeting of the American Society for Microbiology; May 23-27, 2010;San Diego, CA. Abstract C-153.

(15.) Bauer KA, West JE, Balada-Llasat JM, Pancholi P, Stevenson KB, Goff DA. An antimicrobial stewardship program's impact with rapid polymerase chain reaction methicillin-resistant Staphylococcus aureus/S. aureus blood culture test in patients with S. aureus bacteremia. Clin Infect Dis. 2010;51(9):1074-1080.

Kaede V. Sullivan, MD; Nicole N. Turner, MPH; Sylvester S. Roundtree, BS; Karin L. McGowan, PhD

Accepted for publication October 15, 2012.

From the Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania (Dr Sullivan, Ms Turner, and Mr Roundtree);and the Perelman School of Medicine, University of Pennsylvania, Philadelphia (Drs Sullivan and McGowan).

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Kaede V. Sullivan, MD, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Main Building, Room 5112A, 34th Street & Civic Center Blvd, Philadelphia, PA 19104 (e-mail:
Comparison of Culture Versus KeyPath Methicillin-Resistant
Staphylococcus aureus (MRSA) and Methicillin-Susceptible S aureus
(MSSA) Blood Culture Test (MMBT)

              Culture Results, n = 119

MMBT          Positive   Negative

S aureus
  Positive       40         0
  Negative       6          73
  Positive       21         0
  Negative       5          93
  Positive       19         0
  Negative       1          99

                             Performance of MMBT

MMBT          Sensitivity   Specificity   PPV   NPV

S aureus
  Positive        87            100       100   92
  Positive        81            100       100   95
  Positive        95            100       100   99

Abbreviations: NPV, negative predictive value; PPV, positive
predictive value.
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Article Details
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Author:Sullivan, Kaede V.; Turner, Nicole N.; Roundtree, Sylvester S.; McGowan, Karin L.
Publication:Archives of Pathology & Laboratory Medicine
Article Type:Report
Geographic Code:1USA
Date:Aug 1, 2013
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