Evaluation of Multiple Real-Time PCR Tests on Nasal Samples in a Large MRSA Surveillance Program

Evaluation of Multiple Real-Time PCR Tests on Nasal Samples in a Large MRSA Surveillance Program Parul A. Patel, MLS(ASCP), CCRP, 1 Ari Robicsek, MD, 1,2 Althea Grayes, MLS(ASCP), 1 Donna M. Schora, MLS(ASCP), 1 Kari E. Peterson, 1 Marc O. Wright, MT(ASCP), MS, CIC, 1 and Lance R. Peterson, MD 1,2 From the 1 NorthShore University HealthSystem, Evanston, IL, and the 2 University of Chicago Pritzker School of Medicine, Chicago, IL. Key Words: Staphylococcus aureus; MRSA surveillance; Real-time PCR Am J Clin Pathol May 2015;143:652-658 ABSTRACT Objectives: We evaluated the LightCycler MRSA Advanced Test (Roche Molecular Diagnostics, Pleasanton, CA), the BD MAX MRSA assay (Becton Dickinson, Franklin Lakes, NJ), and the Xpert MRSA assay (Cepheid, Sunnyvale, CA) on nasal samples using the same population. Methods: Admission and discharge nasal swabs were collected from inpatients using a double-headed swab. One swab was plated onto CHROMagar MRSA (CMA; Becton Dickinson, Sparks, MD) and then broken off into tryptic soy broth (TSB) for enrichment. TSB was incubated for 24 hours and then plated to CMA. The molecular tests were performed on the second swab. We analyzed the cost benefit of testing to evaluate what parameters affect hospital resources. Results: A total of 27,647 specimens were enrolled. The sensitivity/specificity was 98.3%/98.9% for the LightCycler MRSA Advanced Test and 95.7%/98.8% for the Xpert MRSA assay, but the difference was not significant. The positive predictive value was 86.7% for the LightCycler MRSA Advanced Test, 82.7% for the Xpert MRSA assay (P >.1), and 72.2% and for the BD MAX MRSA test (P <.001 compared with the LightCycler MRSA Advanced Test). All three assays were cost-effective, with the LightCycler MRSA Advanced Test having the highest economic return. Conclusions: Our results suggest that the performance of the three commercial assays is similar. When assessing economic cost benefit of methicillin-resistant Staphylococcus aureus screening, the two measures with the most impact are the cost of the test and the specificity of the assay results. Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen frequently causing bacteremia, pneumonia, and skin and soft tissue infections that result in longer hospital stays and significant mortality. 1-4 Importantly, MRSA colonization leads to the greatest risk for MRSA disease, increasing the likelihood of MRSA infection 10-fold for those with nasal carriage, 5 and patients harboring this pathogen serve as a reservoir for transmission to others. 6,7 MRSA surveillance can be part of an effective infection control plan that includes isolation and, perhaps, decolonization of those found harboring MRSA. However, the role of various practices to reduce the risk of MRSA transmission in a hospital setting remains somewhat controversial, 8-11 despite the observation that the Department of Veterans Affairs Healthcare System has now achieved a major reduction in MRSA clinical disease to the degree that 75% of all nursing units nationally have no MRSA nosocomial infections in any given month using universal admission surveillance. 12 It has been reported that there has been a reduction in bloodstream infections nationally by improving basic central venous catheter infection control practices but no reduction in hospital-acquired MRSA colonization or other disease. 2,13,14 Accurate detection of MRSA colonization is needed for those hospitals undertaking active surveillance with subsequent implementation of contact precautions for those found to be MRSA positive to lower MRSA transmission and subsequent infection. 11 Real-time polymerase chain reaction (PCR) is emerging as the current best diagnostic test that is rapid and sensitive for direct detection of MRSA from nasal specimens. 15-17 Worldwide, there are many commercially available PCR assays designed for direct detection of MRSA 652 Am J Clin Pathol 2015;143:652-658

Admission testing (n = 8,066) BD MAX MRSA assay (n = 8,066) Cultured PCR positive only PPV Total enrollment (n = 27,647) LightCycler MRSA Advanced Test All samples cultured (n = 5,854) sensitivity, specificity, PPV, and NPV Figure 1 Flowchart of testing algorithms using the three molecular tests. NPV, negative predictive value; PCR, polymerase chain reaction; PPV, positive predictive value. nasal colonization. 18-20 Peterson and colleagues 16 reported that the LightCycler MRSA Advanced Test (Roche Molecular Diagnostics, Pleasanton, CA) is as sensitive and rapid as the original BD GeneOhm assay (Becton Dickinson, Franklin Lakes, NJ) and had superior specificity during the US Food and Drug Administration (FDA) registration trial. However, this had not been confirmed during actual widespread clinical use of the test. To confirm these findings in a large MRSA control program setting, we evaluated the performance of the LightCycler MRSA Advanced Test on nasal swab samples from the routine NorthShore MRSA Program. To further assess this technology, we evaluated the BD MAX MRSA assay (Becton Dickinson) and the Xpert MRSA assay (Cepheid, Sunnyvale, CA) on nasal samples using the same patient population. Materials and Methods Discharge testing (n = 19,581) Cultured PCR positive only (n = 12,480) PPV Patient Population and Specimen Collection Xpert MRSA assay (n = 1,247) All samples cultured sensitivity, specificity, PPV, and NPV NorthShore University HealthSystem (NorthShore) includes four hospitals in Evanston, Glenview, Highland Park, and Skokie, Illinois, that receive approximately 60,000 admissions per year. This report is based on testing samples from a large quality improvement admission and discharge testing program done at one of our four hospitals where a total of 27,647 specimens were collected for analysis (19,581 from discharge testing and 8,066 from admission). As part of that program, 18,334 tests were done using the LightCycler MRSA Advanced Test (of those, 5,854 samples were cultured regardless of PCR results to determine full test performance, and 12,480 additional tests where only PCR-positive tests were cultured for added assay analysis). Another 1,247 tests were done using the Xpert MRSA assay, in which all samples were cultured to determine full test performance. Finally, the BD MAX MRSA assay was performed on 8,066 samples collected for routine admission MRSA testing, again with culture performed only on PCRpositive samples to compare the positive predictive value (PPV) during routine use with the package insert claim for the new configuration of this next-generation test. The flowchart of testing algorithms is provided in Figure 1. Nasal samples were collected using a double-headed BBL Culture Swab with Liquid Amies (Becton Dickinson, Sparks, MD). During an infection control quality improvement investigation, nasal samples were collected from all inpatients upon their discharge. All patients admitted to the nursing and intensive care units were eligible for inclusion. Only excess deidentified nasal specimens were used for testing after routine laboratory procedures were performed. This study was approved by the Institutional Review Board at NorthShore University HealthSystem. Real-Time PCR Procedures LightCycler MRSA Advanced Test The LightCycler MRSA Advanced Test was performed according to the manufacturer s instructions. One swab was removed from the transport container, broken into a lysis buffer tube, and then heated at 95 C ± 2 C for 2 minutes. Lysis buffer tubes were processed in the MagNA Lyser instrument at 5,000 rpm for 70 seconds and centrifuged at 10,000 rpm for 1 minute. The lysates were placed in a cold block (4 C) until real-time PCR was performed. A working master mix was prepared according to the number of specimens, controls, and dead volume using MRSA reaction mix and detection mix. The working master mix was vortexed for 3 to 5 seconds and centrifuged briefly. A 15-µL aliquot of the master mix was pipetted into the LightCycler capillary, and 5 µl of the processed sample was added. Real-time PCR was performed using the LightCycler 2.0 instrument. Positive and negative controls were included in each run. The positive control, provided in the LightCycler MRSA Advanced Test kit, contains less than 0.001% noninfectious plasmid DNA containing MRSA sequences. A sterile swab was broken into a lysis tube and carried through the whole PCR process for a negative control. BD MAX MRSA Test One swab was removed from the transport container and broken into a sample buffer tube, then vortexed at high Am J Clin Pathol 2015;143:652-658 653

Patel et al / Performance of Multiple Real-Time PCR Tests speed for 1 minute with a multitube vortexer. Tubes were loaded into a rack containing the BD MAX MRSA reagent strips, extraction, and master mix reagents. The amplification, detection, and interpretation of the signals are done automatically by the BD MAX system. The BD MAX MRSA assay includes a sample processing control that is present in the extraction tube and undergoes the extraction, concentration, and amplification steps to monitor for inhibitory substances as well as process inefficiency due to instrument or reagent failure. In addition, external specimen processing controls, MRSA (ATCC strain 43300), and methicillin-susceptible S aureus (ATCC strain 25923) were performed according to the Clinical and Laboratory Standards Institute guideline and BD MAX MRSA assay package insert. 21 Xpert MRSA Assay One swab was removed from the transport container and broken into a tube containing the elution reagent and vortexed at high speed for 10 seconds. Next, the cartridge lid was opened, and the entire content of the elution reagent was transferred to the large notched opening in the GeneXpert (Cepheid) cartridge. Real-time PCR was performed by using the GeneXpert Dx instrument (Cepheid) following the Xpert MRSA assay package insert. 22 Each test includes a sample processing control and a probe check control. The sample processing control contains spores of Bacillus globigii that ensures the sample was correctly processed, checks if the PCR reagents are functional, and ensures the PCR reaction conditions are appropriate for the amplification reaction. Probe check control is used to monitor bead rehydration, reaction tube filling, probe integrity, and dye stability. Culture Methods One of the paired swabs from each nasal specimen was directly plated onto CHROMagar MRSA (BBL; Becton Dickinson) agar and incubated at 33 C to 35 C for up to 48 hours. After direct plating, all swabs were inoculated into 5 ml of tryptic soy broth without NaCl (TSB; Remel, Lenexa, KS) for broth enrichment. TSB was incubated overnight at 33 C to 35 C, plated to fresh CHROMagar MRSA, and incubated at 33 C to 35 C for 48 hours before finalizing the negative results. After 24 hours, the plate was examined, and mauve colonies were subcultured to a blood agar plate (BBL; Becton Dickinson). Pure colonies were identified as S aureus by using the Staphaurex agglutination test (Remel). Definition of the Reference Standard Since the purpose of MRSA surveillance testing is to determine those who may need to be placed into contact precautions as a result of being colonized with MRSA, we used the growth of MRSA from the nasal swab combined with any sample giving a positive real-time PCR test in a patient with a prior positive culture for MRSA in the preceding 24 months, even if the concomitant culture for the paired nasal swab was negative, as the reference standard for comparing the PCR assays for the detection of MRSA. Because there is no accepted standard for removing patients from multidrugresistant organism isolation precautions, this is a relevant reference standard for infection control purposes, as previously reported. 23 Economic Cost-Benefit Analysis To evaluate the potential economic impact of test choice, we used our own published data on the overall health care cost of a MRSA infection (adjusted to 2014 dollars), 3 the avoidable expense of isolation driven by test specificity, 24,25 the impact of a universal admission surveillance program on disease cost reduction, 3,26 and the maximum price for the commercial assays (manufacturer s suggested retail price). The data were normalized to the impact per 10,000 patients tested and data converted to 2014 US dollars. Results In total, 27,647 specimens (18,334 specimens tested using the LightCycler MRSA Advanced Test, 8,066 specimens tested with the BD MAX MRSA assay, and 1,247 tested with the Xpert MRSA assay) were enrolled for this study. Results obtained on nasal swabs from eligible samples tested for MRSA using the LightCycler MRSA Advanced Test, the BD MAX MRSA assay, and the Xpert MRSA assay compared with the reference standard are shown in Table 1. There were 120 (0.7%) invalid results by the Light- Cycler MRSA Advanced Test; 51 (0.3%) remained invalid after retesting from the frozen lysate and 17 (1.4%) invalid/ error by the Xpert MRSA assay; these 68 (51 and 17) specimens were negative on CHROMagar MRSA and therefore were not included in the calculation of clinical sensitivity, specificity, PPV, and negative predictive value (NPV) of the respective assay. Culture testing was not performed for all specimens tested with the BD MAX MRSA assay, and therefore, sensitivity, specificity, and NPV results were taken from the current package insert; only the PPV was calculated from this data set. For the LightCycler MRSA Advanced Test and the Xpert MRSA assay, all values were calculated using results of testing in this evaluation. Further testing was performed on all samples that gave discordant results between chromogenic culture and the LightCycler MRSA Advanced Test. The LightCycler MRSA Advanced Test was repeated from frozen lysate, 654 Am J Clin Pathol 2015;143:652-658

Table 1 Sensitivity, Specificity, PPV, NPV, and 95% CI for the LightCycler MRSA Advanced Test, the BD MAX MRSA Assay, and the Xpert MRSA Assay Compared With the Reference Standard Tested on the Same Population Characteristic LightCycler MRSA Advanced Test: Culture All Samples (n = 5,838) LightCycler MRSA Advanced Test: Only Test Culture-Positive Samples (n = 12,445) BD MAX MRSA Assay: Only Test Culture-Positive Samples (n = 8,066) Xpert MRSA Assay: Culture All Samples (n = 1,230) True positive 397 638 345 67 False positive 61 90 133 14 True negative 5,373 1,146 False negative 7 3 Sensitivity (95% CI), % 98.3 a (96.3-99.2) Same data as in the column to the left 96.0 b (88.9-98.6) 95.7 (87.2-98.9) Specificity (95% CI), % 98.9 c (98.6-99.1) Same data as in the column to the left 96.5 b (94.9-97.5) 98.8 (97.9-99.3) PPV (95% CI), % 86.7 d (83.1-89.6) 87.6 d (85.0-89.9) 72.2 d (67.9-76.1) 82.7 (72.4-89.9) NPV (95% CI), % 99.9 (99.7-99.9) Same data as in the column to the left 99.6 b (98.8-99.9) 99.7 (99.2-99.9) CI, confidence interval; NPV, negative predictive value; PPV, positive predictive value;, unable to calculate data. a P >.3 for all sensitivity comparisons. b Based on current package insert. c P >.1 for all specificity comparisons. d P <.001 between the two indicated LightCycler tests and the BD MAX assay. Table 2 Cost Impact of Changes in Test Specificity on Cost Benefit of a Universal Testing Surveillance Program per 10,000 Tests Performed (Assumes Positive Test Prevalence ~6%) Test Absolute Specificity Difference, % Excess Isolation Cost (Assumes 5 Days Mean LOS at a Cost of $300), $ Manufacturer s Suggested Retail Price per Test, $ Total Program Cost, $ Net Program Financial Benefit, $ a LightCycler MRSA Advanced Test [Reference] NA 26.56 265,600 315,400 BD MAX MRSA assay 2.4 72,000 32.00 392,000 189,000 Xpert MRSA assay 0.1 3,000 42.00 423,000 158,500 Simulation b 5 150,000 32.00 470,000 111,000 LOS, length of stay; NA, not applicable. a Assumes $581,000 annual medical cost reduction for every 10,000 admissions. Calculation based on NorthShore program with 120 less clinical methicillin-resistant Staphylococcus aureus cases per year from 60,000 annual admissions. 3,24 Adjusted to 2014 dollars (http://data.bls.gov/cgi-bin/cpicalc.pl). b Simulation based on published performance of a nucleic acid amplification test with lower specificity due to assay design (http://www.accessdata.fda.gov/cdrh_docs/reviews/ K112937.pdf). and culture was repeated from refrigerated enrichment broth samples. We could not repeat PCR testing for the Xpert MRSA assay, but culture was repeated for samples giving discrepant results. The BD MAX MRSA assay also had no remnant samples on which to perform additional tests on discordant samples. For any specimen negative by the culture method but positive by PCR, a medical record review was done to determine if the patient had a documented history of MRSA colonization or infection; if the patient had a recent history of a positive culture for MRSA, the PCR-positive specimen was considered a true positive. Since the assays all performed similarly, the major economic cost-benefit results were driven by the simple measures of test price and specificity, as shown in Table 2. While actual test cost is driven by negotiation between the user and provider and based on test volume, this table provides an example of the impact that test price and specificity have on hospital resource utilization. Specificity was important because even small changes in this test performance parameter had a considerable impact on unnecessary isolation that changed the resources needed for the MRSA program. Discussion Several critical issues can affect the outcome of a program undertaken to control MRSA infection, 11 one of them being the performance of the chosen assay. MRSA surveillance using molecular assays increases the sensitivity of detection that can be an important component for decreasing the prevalence of MRSA infection and bacteremia. 17,26,27 Currently, many realtime PCR assays have been developed for rapid detection of MRSA colonization using a nasal sample. 15-19,23,26,28-30 Regarding specificity, our results support the report of the FDA clearance trial by Peterson and colleagues, 16 in which using the LightCycler MRSA Advanced Test had improved test specificity. A high specificity minimizes the false-positive rate, which can be important not only to avoid placing patients unnecessarily into contact isolation but also to minimize the cost of the control program. For example, our prior experience conservatively found that isolation costs approximate $300 ($302-$323 per hospital stay) for a patient with an average hospital length of stay. 24,25 Thus, for every 10,000 patients admitted where MRSA test screening has an absolute specificity difference of even 1%, in a setting where Am J Clin Pathol 2015;143:652-658 655

Patel et al / Performance of Multiple Real-Time PCR Tests the average length of stay is 5 days (mean US length of stay is 4.8 days), 31 an annual excess isolation expense of $30,000 is generated (Table 2). We performed testing to document the PPV of tests where not all specimens were cultured to validate available data for specificity analysis. In our large assessment of the LightCycler assay culturing only positive specimens, we found the PPV virtually identical to the samples where we cultured all specimens (87.6% vs 86.7%); comparing our test results with that of the current BD MAX test package insert similarly found concurrent results for PPV (72.2% vs 71.3%), and thus we believe it is reasonable to use the specificity data presented in Table 2. For our organization with an average of 60,000 annual admissions, this would mean a tangible excess isolation cost of nearly $900,000 between using the highest and lowest specificity test in Table 2. Sensitivity is also a crucial component for MRSA screening and has a major role in the success or failure of any type of control program, 11,32 since the major cost of any program is one that does not have the outcome as planned. In our study, the LightCycler MRSA Advanced Test also had the best sensitivity and therefore would be expected to miss the fewest of the MRSA isolation opportunity days, 25 although the differences between the tests evaluated were not significant. An interesting recent report from Hong Kong suggested a lower sensitivity for the LightCycler MRSA Advanced Test, but this was due to strains most consistent with animal origin being found in human populations. 33 In societies where crowded populations of humans and animals live closely together, this may be important, and when this occurs, one needs to carefully assess the results and report as positive those results with a low melting point, as suggested by the authors. 33 Another report from Singapore found that some MRSA strains may not be detectable by the available commercial assays due to disruption of the target area(s). 34 These reports highlight the need to understand the local MRSA epidemiology to properly apply the best test(s) for local surveillance. In support of our choice of a reference standard, Sanford and colleagues 35 reported that MRSA-colonized people may remain carriers for years, and thus a transient negative culture, particularly when due to recent decolonization, is important to consider when evaluating a molecular test. Patients with positive results likely carried the microbe even though the nasal culture was negative at the time of sample collection, which increases the sensitivity of PCR and decreases the performance of culture. Mehta and colleagues 36 have documented that a single course of mupirocin decolonization keeps the nares culture free of S aureus, but this is no assurance that the effect is maintained beyond 30 days since that was the longest period they studied. However, decolonization can affect laboratory testing, making it reasonable to assume a positive PCR assay is accurate in the setting where MRSA-colonized patients are routinely treated with mupirocin. Our results have limitations in that some of the testing was done only on PCR-positive samples. However, this provides valuable information on PPV, and the distinction between culturing all samples vs testing in which we cultured only PCR-positive tests is clearly demarcated in the report. Also, we used both a positive nasal culture as well as a history of culture-positive MRSA colonization and/or disease for the reference standard. This was appropriate due to our aggressive decolonization program at the time of this study and is very consistent with the current isolation practice of maintaining contact precautions for MRSA-positive persons until at least three cultures or a negative PCR test are obtained. 37 In conclusion, real-time PCR assays provide accurate and rapid results for MRSA detection of nasal colonization. Our data show that the three commercial assays all performed well in this high-volume comparison. When choosing a laboratory test for infection control surveillance, it appears useful to not only consider the testing cost in the laboratory but also the impact on isolation practices throughout the hospital since both can have a significant impact on the utilization of health care resources. Address reprint requests to Dr Peterson: NorthShore University HealthSystem, 2650 Ridge Ave, Walgreen Building, SB Rm 525, Evanston, IL 60201; lance1@uchicago.edu. This work was primarily funded by an investigator-initiated grant from Roche Molecular Diagnostics and partially funded by the Agency for Healthcare Research and Quality (grant 1R18HS019968). Disclosure: The authors are solely responsible for this report. During the past 5 years, Dr Peterson has received research grants from BD-GeneOhm, Cepheid, MicroPhage, Nanosphere, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Roche, 3M, Agency for Healthcare Research and Quality, GeneWeave, IMDx, CareFusion, and the Washington Square Health Foundation for work in molecular diagnostics. Dr Peterson has performed consultation for BD-GeneOhm, Cepheid, MicroPhage, Nanosphere, and Roche in this area of research. Dr Robicsek has received research grants from BD-GeneOhm and the Centers for Disease Control and Prevention, as well as performed consultation for BD-GeneOhm and Roche. No other authors have any conflicts to report. References 1. Delorme T, Rose S, Senita J, et al. Epidemiology and susceptibilities of methicillin-resistant Staphylococcus aureus in northeastern Ohio. Am J Clin Pathol. 2009;132:668-677. 2. Mertz D, Dafoe N, Walter SD, et al. Effect of a multifaceted intervention on adherence to hand hygiene among healthcare workers: a cluster-randomized trial. Infect Contr Hosp Epidemiol. 2010;31:1170-1176. 656 Am J Clin Pathol 2015;143:652-658

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Patel et al / Performance of Multiple Real-Time PCR Tests 35. Sanford MD, Widmer AF, Bale MJ, et al. Efficient detection and long-term persistence of the carriage of methicillin-resistant Staphylococcus aureus. Clin Infect Dis. 1994;19:1123-1128. 36. Mehta MS, Hacek DM, Kufner BA, et al. Dose-ranging study to assess the application of intranasal 2% mupirocin calcium ointment to eradicate Staphylococcus aureus nasal colonization. Surg Infect. 2013;14:69-72. 37. Shenoy ES, Kim J, Rosenberg ES, et al. Discontinuation of contact precautions for methicillin-resistant Staphylococcus aureus: a randomized controlled trial comparing passive and active screening with culture and polymerase chain reaction. Clin Infect Dis. 2013;57:176-184. 658 Am J Clin Pathol 2015;143:652-658