Microbiology and Infectious Disease / Xpert MRSA/SA in Pediatric Blood Cultures Validation and Implementation of the GeneXpert MRSA/SA Blood Culture Assay in a Pediatric Setting David H. Spencer, MD, PhD, 1 Patricia Sellenriek, MBA, 3 and Carey-Ann D. Burnham, PhD 1,2 Key Words: GeneXpert; Staphylococcus aureus; Methicillin resistance; MRSA; Bacteremia DOI: 10.1309/AJCP07UGYOKBVVNC CME/SAM Upon completion of this activity you will be able to: describe the genetic targets that differentiate methicillin-resistant Staphylococcus aureus (MRSA) from methicillin-susceptible S aureus (MSSA). discuss how rapid polymerase chain reaction technology can improve turnaround time in the identification of organisms in positive blood cultures. describe how a genetic test can predict phenotypic susceptibility results for S aureus. Abstract Blood cultures positive for gram-positive cocci in clusters can pose a dilemma for empiric antimicrobial therapy because they could represent coagulasenegative staphylococcus or Staphylococcus aureus bacteremia. The GeneXpert MRSA/SA BC Assay (Cepheid, Sunnyvale, CA) is a polymerase chain reaction based method for identifying S aureus and methicillin resistance that has been approved for use in adults, but data on its use in samples from pediatric patients is limited. We validated the Xpert MRSA/SA BC Assay for use with anaerobic and polymicrobial specimens from pediatric patients and implemented it for routine presumptive identification of S aureus in our pediatric hospital. The assay was 100% sensitive and specific for methicillin-resistant S aureus and 100% sensitive and 99.5% specific for methicillin-susceptible S aureus. Time to presumptive identification of S aureus bacteremia and determination of methicillin susceptibility was reduced by more than 24 hours. We found the Xpert MRSA/SA BC Assay to be a rapid, accurate tool for detecting methicillin-resistant and methicillin-susceptible S aureus in positive pediatric blood cultures, including polymicrobial cultures and those recovered in anaerobic blood culture media. The ASCP is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity for a maximum of 1 AMA PRA Category 1 Credit per article. Physicians should claim only the credit commensurate with the extent of their participation in the activity. This activity qualifies as an American Board of Pathology Maintenance of Certification Part II Self-Assessment Module. The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Questions appear on p 817. Exam is located at www.ascp.org/ajcpcme. A blood culture positive for gram-positive cocci in clusters (GPCCL) poses a dilemma for empiric antimicrobial therapy because this morphologic feature could represent coagulase-negative staphylococcus, which is often a contaminant, or Staphylococcus aureus bacteremia. In the case of a blood culture with GPCCL, patients are often treated with broad-spectrum antimicrobial agents for 24 to 48 hours until phenotypic identification and antimicrobial susceptibility results guide therapy. Molecular methods for distinguishing methicillin-resistant S aureus (MRSA) from methicillinsusceptible S aureus (MSSA) and other organisms have the potential to expedite this process and decrease the time before patients receive optimal antimicrobial therapy. 1-4 The GeneXpert MRSA/SA BC Assay (Cepheid, Sunnyvale, CA) is a real-time polymerase chain reaction (PCR)- based method that can identify S aureus and methicillin resistance from blood cultures with GPCCL within approximately 1 hour of a positive signal from an automated blood culture instrument. 5 This method relies on the detection of the staphylococcal protein A gene (spa) for identification of S aureus and infers methicillin resistance via concomitant detection of the methicillin-resistance gene meca and the junction between the staphylococcal cassette chromosome that harbors meca (SCCmec) and the S aureus genome (orfx). 5,6 All 3 of these targets must amplify for the specimen to be deemed positive for MRSA; thus, the Xpert MRSA/SA BC Assay avoids a pitfall of some other PCRbased assays for MRSA that target only the SCCmec junction, which can falsely identify MSSA strains with cassette chromosomes lacking meca (ie, empty cassettes or meca dropouts ) as MRSA. 5 690 Am J Clin Pathol 2011;136:690-694 690 DOI: 10.1309/AJCP07UGYOKBVVNC
Microbiology and Infectious Disease / Original Article We were interested in using the Xpert MRSA/SA BC Assay to decrease the time to determination of MRSA and MSSA bacteremia in blood cultures from pediatric patients. Although the Xpert MRSA/SA BC Assay has been shown to be highly accurate for the identification of staphylococcal bacteremia in recent studies of blood cultures obtained primarily from adults, 7,8 data on its performance in pediatric populations are lacking. Pediatric blood cultures differ from those of adults because alternative culture media may be used and may have a suboptimal blood-to-broth ratio owing to a smaller volume of inoculated blood. Furthermore, the Xpert MRSA/SA BC Assay is currently cleared by the US Food and Drug Administration only for use with BD BACTEC Plus Aerobic/F blood culture media (BD, Franklin Lakes, NJ), and it is not approved for polymicrobial cultures. To address these issues, we conducted a study to validate the Xpert MRSA/SA BC Assay for use with the pediatric specimens routinely encountered in our clinical microbiology laboratory. The objectives of our study were to validate the assay for polymicrobial specimens and the blood culture media used at our institution (BACTEC Peds Plus/F and BACTEC Lytic Anaerobic/F media, BD) and assess the accuracy and usefulness of the assay when applied to routine testing of GPCCLpositive blood cultures from pediatric patients in our hospital. Materials and Methods Validation Study Validation experiments were conducted using 49 specimens. These included 26 laboratory-derived ( spiked ) blood cultures and 23 clinical blood cultures that contained MRSA, Table 1 Validation of the Xpert MRSA/SA BC Assay With Pediatric Blood Cultures MSSA, coagulase-negative Staphylococcus species (CNS), and nonstaphylococcal organisms in BD BACTEC Peds Plus and BD BACTEC Lytic Anaerobic media Table 1. The spiked cultures were inoculated with known quantities of organisms, as described in Table 2, and then incubated in an automated blood culturing system (BD BACTEC FX) until growth was detected. Polymicrobial cultures were inoculated with 1% to 50% of MRSA or MSSA relative to a second organism (Table 2). The 23 clinical specimens were consecutive blood cultures with GPCCL that signaled positively during the day shift in our laboratory during the validation period and were processed in real time by laboratory technologists. The GeneXpert MRSA/SA BC Assays were performed on all validation cultures according to the manufacturer s package insert, and results were interpreted directly from the report generated by the GeneXpert instrument. Assay Implementation The GeneXpert MRSA/SA BC Assay was implemented in our clinical laboratory for routine testing of GPCCLpositive blood cultures following validation experiments. The procedure involved testing all positive blood cultures exhibiting GPCCL morphologic features on the initial Gram stain, including polymicrobial cultures and those recovered in anaerobic blood culture media. Specimens from patients with a history of MRSA or MSSA-positive blood cultures collected within 3 days of the current positive sample were excluded as a cost-saving measure to avoid unnecessary repeated testing. Testing was performed in real time according to the manufacturer s package insert, and results were interpreted from the GeneXpert software without modification. However, during Xpert MRSA/SA BC Result Validation Sample Aerobic Anaerobic Total MRSA MSSA Negative Spiked cultures MRSA only 1 4 5 5 0 0 MRSA polymicrobial 9 0 9 9 0 0 MSSA only 1 4 5 0 5 0 MSSA polymicrobial 2 0 2 0 2 0 Coagulase-negative Staphylococcus 0 1 1 0 0 1 Coagulase-negative Staphylococcus polymicrobial 3 0 3 0 0 3 Micrococcus 1 0 1 0 0 1 Clinical samples MRSA 3 1 4 4 0 0 MSSA 0 1 1 0 1 0 Coagulase-negative Staphylococcus 13 5 18 0 0 18 Total 33 16 49 MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible S aureus. The Xpert MRSA/SA BC Assay correctly identified all organisms as MRSA, MSSA, or non S aureus organisms. BD BACTEC Peds Plus medium. BD BACTEC Lytic Anaerobic media. Non S aureus organisms in the mixed cultures included coagulase-negative Staphylococcus species, Micrococcus species, Escherichia coli, viridans group Streptococcus species, and Candida albicans. Am J Clin Pathol 2011;136:690-694 691 691 DOI: 10.1309/AJCP07UGYOKBVVNC 691
Spencer et al / Xpert MRSA/SA in Pediatric Blood Cultures the course of the study, the procedure was modified to report any positive result from a clinical culture as indeterminate if amplification occurred for any target after 25 cycles. Presumptive identification based on the Xpert MRSA/SA BC Assay results were reported to the clinical team responsible for the patient s care within 1 hour of completion of each test. Data on test accuracy and turnaround time are based on all tests performed on clinical cultures between October 2009 and November 2010. All organisms described in this study were identified by using phenotypic methods, and methicillin resistance was determined by using cefoxitin and the established Clinical and Laboratories Standards Institute guidelines for disk-diffusion susceptibility testing. 9 Results Validation Study The Xpert MRSA/SA BC Assay showed complete concordance with standard phenotypic identification and antimicrobial susceptibility for 49 cultures tested in validation experiments (Table 1). MRSA and MSSA were correctly identified in 10 anaerobic cultures and in 11 polymicrobial spiked derived cultures inoculated with as little as 1% MRSA or MSSA relative to a second organism (Table 2). The Xpert MRSA/SA BC Assay reported negative results for all 23 of the cultures containing non S aureus organisms. Assay Implementation The performance characteristics of the Xpert MRSA/ SA BC Assay in validation experiments led us to implement the test for routine presumptive identification of MRSA and MSSA bacteremia in our clinical laboratory. The assay was performed on 260 GPCCL-positive blood cultures from 223 patients during a 1-year period, and it detected 100% of MRSA and MSSA organisms in the 259 specimens for which the test was successful. Approximately 41% (106/260) of the specimens tested were from anaerobic blood culture media or polymicrobial cultures. The overall sensitivity and specificity for cultures with MRSA was 100% (95% confidence interval [CI] using the binomial test, 75%-100% for sensitivity and 98%-100% for specificity), and the sensitivity and specificity values for MSSA were 100% (95% CI, 84%-100%) and 99.5% (95% CI, 97%-100%), respectively Table 3. One culture of CNS was tested, but results were not obtained because of repeated analytic failures. There was 1 false-positive result among the 208 cultures containing non S aureus organisms: a blood culture that the assay reported as positive for MSSA but was ultimately determined to be a nonpathogenic Neisseria species. Typically, the assay would not have been performed on such a specimen, but the Gram stain of the blood culture broth was initially interpreted as GPCCL. This culture was negative for MSSA in repeated Xpert MRSA/SA testing, and no other false-positives were detected among the 207 cultures containing CNS and 9 nonstaphylococcal organisms. In addition, we observed no cases of MRSA in which the SCCmec target did not amplify, which has been a reported source of false-positives. 6 We conducted additional analysis to determine why the culture containing Neisseria produced a false-positive in testing with the Xpert MRSA/SA BC Assay. Because the assay is intended only for use with GPCCL-positive cultures, the fact that a specimen with Neisseria, a gram-negative organism, was tested at all suggests that the false-positive was Table 2 Culture Composition and Xpert MRSA/SA BC Assay Results for Laboratory-Derived Polymicrobial Validation Samples Xpert MRSA/SA BC Assay Culture Composition Cycle Thresholds (Cycles) Xpert MRSA/SA Organism 1 (CFU) Organism 2 (CFU) spa meca SCCmec BC Result MRSA (90) MSSA (30) 14.7 15.1 16.4 MRSA MRSA (30) MSSA (90) 13.9 13.9 15.3 MRSA MRSA (3) MSSA (300) 13.5 19.1 20.2 MRSA MRSA (30) Coagulase-negative Staphylococcus (90) 13.9 13.9 15.3 MRSA MRSA (30) Escherichia coli (30) 12.2 12.2 13.5 MRSA MRSA (30) Viridans group Streptococcus (30) 14.2 14.3 15.7 MRSA MRSA (30) Viridans group Streptococcus (90) 13.8 13.9 15.3 MRSA MRSA (30) Micrococcus sp (30) 13.1 13.1 14.4 MRSA MRSA (30) Yeast (10) 12.6 12.5 14.1 MRSA MSSA (30) Yeast (30) 12.7 0 0 MSSA MSSA (30) E coli (30) 15.2 0 0 MSSA Coagulase-negative Staphylococcus (30) E coli (30) 38 32.4 0 Negative Coagulase-negative Staphylococcus (30) Viridans group Streptococcus (30) 0 23.2 0 Negative Coagulase-negative Staphylococcus (30) Yeast (10) 0 14.7 0 Negative CFU, colony-forming units; MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible S aureus. This result was considered negative using the manufacturer s specifications for the assay. 692 Am J Clin Pathol 2011;136:690-694 692 DOI: 10.1309/AJCP07UGYOKBVVNC
Microbiology and Infectious Disease / Original Article Table 3 Xpert MRSA/SA BC Assay Results for 259 Clinical Blood Cultures Positive for Gram-Positive Cocci in Clusters Medium/No. of Samples Xpert MRSA/SA BC Results Organism (Culture-Based Identification) Aerobic Anaerobic Total MRSA MSSA Negative MRSA 11 8 19 19 0 0 MSSA 12 17 29 0 29 0 MSSA polymicrobial 3 0 3 0 3 0 Coagulase-negative Staphylococcus 129 64 193 0 0 193 Coagulase-negative Staphylococcus polymicrobial 2 3 5 0 0 5 Nonstaphylococcal organisms 6 5 11 0 1 10 MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible S aureus. Cultures with nonstaphylococcal organisms included 1 of the following: Micrococcus sp (n = 5), Peptostreptococcus species (n = 4), viridans group streptococci (n = 2), Streptococcus pneumoniae (n = 1), Bacillus species (n = 1), Acinetobacter species (n = 1), Pseudomonas aeruginosa (n = 1), Neisseria species (n = 1), and Candida albicans (n = 1). a preanalytic error. Further investigation showed that the amplification curve for this specimen was not in concordance with what we typically observed for this assay: the signal from the spa target reached threshold at 35 cycles, which is significantly higher than other positive results. In fact, all but 1 of the specimens with S aureus demonstrated positive spa signals in fewer than 25 cycles, whereas non S aureus cultures for which target amplification occurred had cycle thresholds greater than 25 cycles Figure 1. The same pattern was apparent for meca and SCCmec amplification in MRSA and non-mrsa organisms, and in cases of MRSA, it is typical for all 3 gene targets to become positive within 2 cycles of one another. Many non S aureus organisms also demonstrated a positive meca signal before 25 cycles, reflecting the fact that CNS frequently carry the meca gene. These observations, along with the fact that the Neisseria-containing specimen Cycle Threshold 60 50 40 30 20 10 Non-S aureus MSSA MRSA Non-S aureus polymicrobial MSSA polymicrobial was negative for S aureus in repeated testing, indicate the initial false-positive signal was likely due to nonspecific amplification or a low amount of environmental contamination. Implementing the Xpert MRSA/SA BC Assay enabled our laboratory to report presumptive identification of MRSA and MSSA significantly faster than when using culture-based identification and susceptibility testing. The time to presumptive identification of MRSA, MSSA, and non S aureus based on the Xpert MRSA/SA BC Assay results was 1 to 5 hours, compared with 30 to 50 hours for the final results based on conventional culture and in vitro susceptibility Figure 2. Discussion In this report, we describe successful use of the Xpert MRSA/SA BC Assay for presumptive identification of Hours 60 50 40 30 20 10 Culture Xpert MRSA/SA BC 0 spa meca Amplification Target SCCmec 0 MRSA MSSA Non-S aureus Figure 1 Cycle thresholds for Xpert MRSA/SA BC targets spa, meca, and SCCmec from pure and polymicrobial cultures with methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S aureus (MSSA), and non S aureus organisms. Only specimens for which amplification was detected are included. The dashed line indicates the maximum cycle threshold for a positive result in our laboratory. Figure 2 Mean interval between culture positivity and final in vitro susceptibility (open bars) and presumptive identification based on Xpert MRSA/SA BC results (filled bars) for methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S aureus (MSSA), and non S aureus organisms. = P < 10 5 ; Student t test. Am J Clin Pathol 2011;136:690-694 693 693 DOI: 10.1309/AJCP07UGYOKBVVNC 693
Spencer et al / Xpert MRSA/SA in Pediatric Blood Cultures MRSA and MSSA from blood cultures obtained from pediatric patients. We performed validation studies so this method could be broadly applied to all GPCCL-positive blood cultures in our laboratory, and we subsequently tested 260 such blood cultures during a 1-year period. In our experience, the Xpert MRSA/SA BC Assay was 100% sensitive and specific for MRSA and 100% sensitive and 99.5% specific for MSSA from aerobic, anaerobic, and polymicrobial cultures. Approximately half of all specimens tested during this study period were from anaerobic or polymicrobial cultures, which highlights the importance of validating the Xpert MRSA/SA BC Assay for these specimen types. False-negatives for MRSA have been reported in isolates with atypical or novel SCCmec types because as a result of these polymorphisms, the SCCmec-orfX PCR target fails to amplify. These spa-positive, meca-positive, SCCmec-negative strains are interpreted as MSSA by the Xpert system, 6 which prompted the manufacturer to issue a voluntary recall for the assay and recommend confirming all samples deemed to be MSSA with standard culture and susceptibility testing methods. 10 Despite these caveats, the rapid turnaround and high overall accuracy make the Xpert MRSA/SA BC test an attractive option for clinical laboratories. Although in our experience the Xpert MRSA/SA BC Assay is highly accurate, the voluntary product recall attributed to false-negative results for MRSA and the false-positive result we encountered in this study led us to reevaluate our procedure in detail. We made 2 modifications to the interpretation of the assay to ensure that erroneous results are not reported. First, specimens that are spa-positive, meca-positive, SCCmec-negative are reported as methicillin-indeterminate to avoid potential false-negative results because of strains with a novel SCCmec variant. This testing pattern occurred once in this study, and it was produced by a polymicrobial culture containing MSSA and a coagulase-negative Staphylococcus species. Second, specimens that are called positive for MSSA or MRSA but have a cycle threshold greater than 25 for any of the targets would be reported as indeterminate and reviewed by the laboratory director. This step is meant to prevent reporting false-positive results due to spurious or nonspecific amplification, which resulted in 1 false-positive result in this study. An important aspect of our implementation of the Xpert MRSA/SA BC Assay was to coordinate with clinical pharmacists and infectious disease physicians to capitalize on the rapid turnaround time provided by this test. Presumptive results from the Xpert MRSA/SA BC Assay were reported as soon as they were available to the patient s provider and the clinical pharmacist associated with the patient. This information has the potential to reduce length of stay, unnecessary hospital admissions, unnecessary antimicrobial therapy, and/ or the time to optimization of therapy. While additional studies are needed to quantify cost reduction and whether the decreased time to optimization of therapy provided by this test affects patient outcomes, we believe this study shows that it is a rapid, accurate tool for detecting MRSA and MSSA in pediatric blood cultures. From the 1 Departments of Pathology & Immunology and 2 Pediatrics, Washington University School of Medicine, St Louis, MO; and 3 St Louis Children s Hospital, St Louis. Presented in part at the 110th American Society for Microbiology General Meeting; May 23-27, 2010; San Diego, CA. Address reprint requests to Dr Burnham: 660 S Euclid Ave, Campus Box 8118, St Louis, MO 63110. Acknowledgments : We thank the staff of the St Louis Children s Hospital Microbiology Laboratory for technical expertise and assistance; and David Hunstad, MD, and Gregory Storch, MD, for critical review of the manuscript. References 1. Bauer KA, West JE, Balada-Llasat JM, et al. 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;51:1074-1080. 2. Chambers HF, Deleo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol. 2009;7:629-641. 3. Chambers HF. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev. 1997;10:781-791. 4. Parta M, Goebel M, Thomas J, et al. Impact of an assay that enables rapid determination of Staphylococcus species and their drug susceptibility on the treatment of patients with positive blood culture results. Infect Control Hosp Epidemiol. 2010;31:1043-1048. 5. Xpert MRSA-SA BC G2 [package insert]. Sunnyvale, CA: Cepheid; 2008. 6. Laurent C, Bogaerts P, Schoevaerdts D, et al. Evaluation of the Xpert MRSA assay for rapid detection of methicillin-resistant Staphylococcus aureus from nares swabs of geriatric hospitalized patients and failure to detect a specific SCCmec type IV variant. Eur J Clin Microbiol Infect Dis. 2010;29:995-1002. 7. Parta M, Goebel M, Matloobi M, et al. Identification of methicillin-resistant or methicillin-susceptible Staphylococcus aureus in blood cultures and wound swabs by GeneXpert. J Clin Microbiol. 2009;47:1609-1610. 8. Wolk DM, Struelens MJ, Pancholi P, et al. Rapid detection of Staphylococcus aureus and methicillin-resistant S aureus (MRSA) in wound specimens and blood cultures: multicenter preclinical evaluation of the Cepheid Xpert MRSA/SA skin and soft tissue and blood culture assays. J Clin Microbiol. 2009;47:823-826. 9. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement. Wayne, PA: Clinical and Laboratory Standards Institute; 2010. CLSI document M100-S20. 10. US Food and Drug Administration. Cepheid Xpert MRSA/ SA Blood Culture Assay for Use with the GeneXpert Dx System. 2010. http://www.fda.gov/medicaldevices/safety/ RecallsCorrectionsRemovals/ListofRecalls/ucm218002.htm. Accessed January 2011. 694 Am J Clin Pathol 2011;136:690-694 694 DOI: 10.1309/AJCP07UGYOKBVVNC