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Evaluation of BD Max StaphSR and BD Max MRSAXT Assays Using ESwab-Collected Specimens Suzane Silbert, Carly Kubasek, Faris Galambo, Elaine Vendrone, Raymond Widen Esoteric Testing Laboratory, Pathology Department, Tampa General Hospital, Tampa, Florida, USA

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taphylococcus aureus is a leading cause of health care-associated infections. Infection due to S. aureus imposes a high and increasing burden on health care resources and increases morbidity and mortality. Nasal carriage of S. aureus has been identified as a risk factor for the development of infections in various settings. This has been studied extensively in surgical patients (general, orthopedic, and thoracic surgery), patients on hemodialysis, patients on continuous ambulatory peritoneal dialysis (CAPD), HIV-infected patients, and patients in intensive care units (1, 2). Methicillin-resistant S. aureus (MRSA) is an important cause of community-acquired and hospital-acquired infections (3, 4). MRSA can colonize the nares, skin, and other body sites, serving as a reservoir for infection and transmission in health care environments (5–7). Screening for MRSA colonization in order to help identify patients at greater risk for MRSA infection and to allow for the initiation of isolation precautions has become standard practice in many institutions (5, 6, 8–10). MRSA screening using molecular amplification methods provides faster results than culture and has been demonstrated to be more sensitive (11–15). However, most of the molecular commercially available rapid tests are based on the detection of a sequence indicating the integration of the staphylococcal cassette chromosome mec element (SCCmec) within the chromosome and do not specifically target the methicillin resistance gene mecA (16). S. aureus isolates with an SCCmec lacking the mecA gene have been described and can be incorrectly identified as MRSA by assays that do not specifically target the mecA gene (17). False-positive results can lead to unnecessary and expensive isolation and treatment of patients. In addition, MRSA strains with the newly discovered methicillin resistance gene mecC account for 3 to 4% of all new MRSA cases in Denmark (18) and cannot be detected by assays that do not detect the mecC gene (19). These false-negative results can lead to uncontrolled transmission of undetected MRSA strains. The BD Max StaphSR and the BD Max MRSAXT assays (BD Diagnostics, Québec, Canada) performed on the BD Max system

August 2015 Volume 53 Number 8

(BD Diagnostics, Sparks, MD) are next-generation automated qualitative in vitro diagnostic tests able to detect MRSA strains harboring the mecC gene and decrease the number of false-positive results caused by mecA/mecC dropouts. Both assays utilize real-time PCR and fluorogenic target-specific hybridization probes to detect the SCCmec right-extremity junction (MREJ) and mecA and mecC genes (20–22). The BD Max StaphSR assay also detects the nuc gene encoding a thermostable nuclease of S. aureus, which allows the assay to detect all S. aureus, including MRSA (21). In our laboratory, we previously validated the use of ESwab samples with the BD Max MRSA first-generation assay for MRSA screening (23). The objective of this study was to validate the BD Max StaphSR assay for the detection and differentiation of S. aureus and MRSA and the BD Max MRSAXT assay for the detection of MRSA using ESwab-collected samples and to compare the results to those with the BD Max MRSA first-generation assay and traditional culture. MATERIALS AND METHODS Specimen collection. A total of 255 clinical samples were collected for routine MRSA screening from the nares of patients using anterior nares ESwab (Copan Diagnostics, Murrieta, CA). The use of the ESwab with BD Max assays is outside the claims in the package insert; however, its use with

Received 9 April 2015 Returned for modification 2 May 2015 Accepted 20 May 2015 Accepted manuscript posted online 27 May 2015 Citation Silbert S, Kubasek C, Galambo F, Vendrone E, Widen R. 2015. Evaluation of BD Max StaphSR and BD Max MRSAXT assays using ESwab-collected specimens. J Clin Microbiol 53:2525–2529. doi:10.1128/JCM.00970-15. Editor: S. S. Richter Address correspondence to Suzane Silbert, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.00970-15

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The BD Max MRSAXT and the BD Max StaphSR assays were validated for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in ESwab samples. In addition, the BD Max StaphSR assay was evaluated for its ability to detect and differentiate S. aureus and MRSA in the same sample. A total of 255 ESwab samples collected from the anterior nares of patients were tested by each of three BD Max assays, including the BD Max MRSA first-generation assay. The results were compared to those of direct and enrichment culture. Additionally, a challenge panel comprising 14 control strains was evaluated to determine the ability of these assays to correctly identify MRSA and also appropriately differentiate S. aureus by the BD Max StaphSR assay. Out of 255 clinical samples tested, 161 were negative and 30 were positive for MRSA, and 45 were positive for S. aureus (by BD Max StaphSR) and negative for MRSA by all three PCR assays and culture. Nineteen samples had discrepant results; all of them were retested by additional laboratory testing. All strains from the challenge panel were correctly identified or excluded by the BD Max MRSAXT and BD Max StaphSR assays. The results showed that the BD Max StaphSR and the BD MRSAXT assays have excellent sensitivity (94.3%) and specificity (97.7%) for detecting MRSA. The BD Max StaphSR assay demonstrated excellent sensitivity (96.4%) and specificity (93.6%) for detecting S. aureus.

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er’s instructions and tested by the three BD Max assays, as described above.

RESULTS

A total of 255 ESwab samples were tested by the BD Max MRSA first-generation, BD Max StaphSR, and BD Max MRSAXT assays and by direct and enrichment culture. Out of 255 samples, 161 were negative for MRSA by the three PCR assays and culture, 45 were positive for S. aureus only by the BD Max StaphSR assay and culture, and 30 samples were positive for MRSA by the three PCR assays and culture. The remaining 19 samples had discrepant results when combined culture results were compared to the results from the BD Max PCR assays (Table 1). Retesting of samples with discrepant results was required in 19 samples, all of which were resolved by achieving concordance between at least two of the three additional tests performed (culture, BD Max PCR, and Xpert SA nasal complete PCR). After discrepancies were resolved, samples were assigned as false negative (FN), false positive (FP), true negative (TN), or true positive (TP) according to their initial result. Out of 19 samples for which discrepant results were resolved, 10 were TN MRSA and 4 FP MRSA by the three BD Max PCR assays. The BD Max MRSA first-generation assay also identified one TP MRSA and four FN MRSA samples, while the BD Max MRSAXT and BD Max StaphSR assays identified three TP MRSA and two FN MRSA samples. Additionally, the BD Max StaphSR assay identified nine FP, one TN, six TP, and 3 FN S. aureus samples (Table 1). The sensitivity, specificity, and overall agreement between each one of the BD Max PCR assays tested and culture are presented in Table 2. Challenge panel results. Strains from the challenge panel representing 11 MREJ types, one MSSE, one MSSA, and one mecA dropout MSSA strain were correctly identified by both BD Max MRSAXT and BD Max StaphSR assays. The BD Max MRSA firstgeneration assay was able to correctly identify 8 out of the 14 strains from the challenge panel (Table 3). Analytical studies were performed to test the feasibility of using ESwab collection samples with the BD Max StaphSR and BD Max MRSAXT assays, using two different strains (MRSA ATCC 33591 and MSSA ATCC 25923). An initial suspension of each strain at a 0.5 McFarland standard (1.5 ⫻ 108 CFU/ml) was prepared, followed by eight 10-fold dilutions. The tests were performed in triplicate, and the results revealed a limit of detection (LoD) of 1.5 ⫻ 102 CFU/ml (333 CFU/ml) for the BD Max MRSAXT and BD Max StaphSR assays using ESwab-collected samples. DISCUSSION

S. aureus is still at the top of the list of causative organisms of nosocomial infections and has become even more drug resistant in the last decades (1, 2). MRSA infections are on the rise worldwide, and it appears that these isolates do not replace methicillinsusceptible S. aureus (MSSA) as a cause of disease but rather add to the total S. aureus infections in the population (26, 27). Patients colonized with MRSA have an increased risk of developing MRSA disease. Therefore, routine infection prevention practices, such as active surveillance programs that screen nares for MRSA, have been recommended and established by many accredited health care facilities worldwide. These programs have been showing that minimizing the spread of MRSA to lower new colonization is one way to reduce MRSA infection (3, 5–7, 9, 26, 28).




the BD Max MRSA first-generation assay was validated in previous studies (22, 23). BD Max assays. The BD Max MRSA first-generation, BD Max StaphSR, and BD Max MRSAXT assays were performed according to the manufacturer’s instructions, with a few modifications: (i) ESwabs were used for specimen collection, and (ii) 200 ␮l from the ESwab transport medium was transferred into a BD Max sample buffer tube (SBT). Briefly, for each ESwab sample, three aliquots of 200 ␮l were transferred to three BD Max SBTs, and the tubes were vortexed for 60 s on a multiposition mixer (23). The SBTs were placed in the instrument rack along with the corresponding unitized reagent strip and reagent tubes for each of the 3 PCR assays. The entire assembly was placed in the BD Max instrument, and the runs for each assay were initiated. The total run time for each assay was either 2 h (for up to 12 samples/run) or 2 h and 17 min (for up to 24 samples/run). Bacterial culture. Direct culture was performed from each ESwab specimen by plating 50 ␮l of the ESwab medium to each of the following media: BBL Trypticase soy agar with 5% sheep blood (TSAII) (BD Diagnostics, Sparks, MD), BBL CHROMagar Staph aureus (BD Diagnostics), and CHROMagar MRSA II (BD Diagnostics). Enrichment culture for all samples was performed by adding 50 ␮l of the ESwab medium into BBL Trypticase soy broth with 6.5% sodium chloride (TSB with 6.5% NaCl) (BD Diagnostics) and subculturing 50 ␮l of the TSB with 6.5% NaCl, after overnight incubation onto TSAII, CHROMagar Staph aureus, and CHROMagar MRSA II plates (24). The plates were examined for growth after 20 to 24 h of incubation at 35 to 37°C. The cultures were incubated according to the manufacturer’s instructions. Presumptive S. aureus colonies (mauve-colored colonies) observed on CHROMagar Staph aureus were subcultured on TSAII plates and incubated at 35 ⫾ 2°C for 18 to 24 h. S. aureus identification was confirmed by the Pastorex Staph Plus latex agglutination test (Bio-Rad, Hercules, CA) directly from the TSAII subculture. Presumptive MRSA colonies (mauvecolored colonies) observed on the CHROMagar MRSA II assay were also subcultured on TSAII plates and incubated at 35 ⫾ 2°C for 18 to 24 h. MRSA identification was confirmed by an in-house validated mecA/femA PCR test protocol, using colonies from the TSAII subculture plate and in accordance with a previously published protocol (1). Results and statistical analysis. The results from direct and enrichment cultures were combined, and a final positive or negative culture result was compared to the results from each one of the BD Max PCR assays. The sensitivity and specificity of the three molecular tests were determined with combined direct and enriched culture as the reference method, and the score method was used to calculate confidence intervals. Samples with discrepant results. All samples with discrepant results were retested in the laboratory using the initial TSB-inoculated medium. Additional tests included culture on TSAII plates, Gram stain analysis, Staph Plus latex agglutination, and the Xpert SA nasal complete PCR assay (Cepheid, Sunnyvale, CA) performed on the GeneXpert (Cepheid) platform, according to the manufacturer’s instructions. The Cepheid Xpert SA nasal complete assay is a next-generation assay that detects S. aureus and MRSA DNA using 3 targets (spa, SCCmec, and mecA) (25). This test was used to help with samples with discrepant results, since it is a more accurate assay, which, like the BD Max new-generation assays, has the ability to identify mecA dropouts. After all samples were retested, the results were reanalyzed. Challenge panel. A challenge panel comprising 14 control strains (Microbiologics, Inc., Saint Cloud, MN) was evaluated by the BD Max StaphSR and BD Max MRSAXT assays to verify the ability of these assays to correctly identify and differentiate MRSA and S. aureus. The 14-member challenge panel included 11 MRSA MREJ type strains (10 mecA and 1 mecC), 1 methicillin-susceptible S. aureus strain (MSSA), 1 methicillinsusceptible S. aureus mecA dropout strain (MSSA), and 1 methicillinsusceptible Staphylococcus epidermidis strain (MSSE) (coagulase-negative staphylococci [CoNS]). Suspensions of all 14 lyophilized bacterial organisms constituting the panel were prepared according to the manufactur-

Evaluation of the BD Max StaphSR and MRSAXT Assays

TABLE 1 Samples with discrepant results at initial and additional tests Initial test/repeated test results for culture of a:

Initial performance/resolved assay performance forb:

Xpert SA nasal complete PCR of c:

PCR StaphSR

S. aureus

MRSA

PCR 1st

PCR XT

S. aureus

MRSA

S. aureus

MRSA

Neg/Neg Neg/Neg Neg/Neg Neg/Neg Pos/Pos Pos/Pos Pos/Pos Pos/Pos Pos/Pos Pos/Pos Neg/Pos Neg/Neg Neg/Neg Neg/Neg Neg/Neg Neg/Neg Neg/Neg Pos/Pos Neg/Pos

Neg/Neg Neg/Neg Neg/Neg Neg/Neg Neg/Neg Neg/Pos Pos/Pos Pos/Pos Neg/Neg Pos/Pos Neg/Pos Neg/Neg Neg/Neg Neg/Neg Neg/Neg Neg/Neg Neg/Neg Neg/Neg Neg/Neg

Pos/FP Neg/TN Neg/TN Pos/FP Pos/FP Neg/FN Neg/FN Neg/FN Neg/TN Neg/FN Pos/TP Neg/TN Neg/TN Pos/FP Neg/TN Neg/TN Neg/TN Neg/TN Neg/TN

Pos/FP Neg/TN Neg/TN Neg/TN Pos/FP Neg/FN Neg/FN Pos/TP Neg/TN Pos/TP Pos/TP Neg/TN Pos/FP Neg/TN Neg/TN Neg/TN Neg/TN Pos/FP Neg/TN

Pos/FP Pos/FP Pos/FP Pos/FP Pos/TP Neg/FN Neg/FN Pos/TP Neg/FN Pos/TP Pos/TP Pos/FP Pos/FP Neg/TN Pos/FP Pos/FP Pos/FP Pos/TP Pos/TP

Pos/FP Neg/TN Neg/TN Neg/TN Pos/FP Neg/FN Neg/FN Pos/TP Neg/TN Pos/TP Pos/TP Neg/TN Pos/FP Neg/TN Neg/TN Neg/TN Neg/TN Pos/FP Neg/TN

Neg Neg Neg Neg Pos Neg Pos NA Pos NA Neg Neg Neg NA Neg Neg Neg Neg Pos

Neg Neg Neg Neg Neg Neg Pos NA Neg NA Neg Neg Neg NA Neg Neg Neg Neg Neg

a

Results combined from direct and enrichment culture. Neg, negative; Pos, positive. PCR 1st, BD Max MRSA first-generation assay; PCR XT, PCR performed using BD Max MRSAXT assay; PCR StaphSR, PCR performed using BD Max StaphSR assay; FP, false positive; TN, true negative; TP, true positive; FN, false negative. c PCR performed using the Xpert SA nasal complete assay. This test was performed only when samples with discrepant results were available. NA, not applicable (not enough samples left for retesting). b

The factors that determine the success or failure of MRSA surveillance programs include the patient population tested, the sensitivity of the laboratory testing used, and the speed of result determination (26, 28, 29). An active MRSA surveillance program that uses a laboratory test with 98% sensitivity and with a reporting time of ⱕ15 h can capture 85% of potential inpatient MRSA isolation days, leading to a significant (⬎70%) reduction in MRSA infection (28). For these reasons, tremendous amounts of effort and resources have been focused on the development of several new molecular-based rapid screening tests for MRSA (12–14, 30–32). In this study, we validated two PCR assays performed on the BD Max system: the BD Max MRSAXT and BD Max StaphSR assays using ESwab-collected samples, and we assessed their ability to correctly identify MRSA and S. aureus (Max StaphSR only). The identification of S. aureus by the BD Max StaphSR assay showed a sensitivity and specificity of 96.4% and 93.6%, respectively. Discrepant results between the BD Max StaphSR PCR assay and culture included 3 PCR-negative and culture-positive samples and 11 BD Max StaphSR PCR-positive and culture-negative samples for S. aureus. The PCR raw analysis of 9 out of the 11 BD

Max StaphSR PCR-positive and culture-negative samples identified higher cycle threshold (CT) values and curves below the limit of detection for S. aureus, suggesting a false-curve/false-positive result detected. Results that were positive by molecular assays but negative by culture may be attributed to the fact that molecular assays pick up nucleic acid from dead organisms as well and thus sometimes give false-positive results. For this reason, additional culture is suggested to confirm the presence or absence of S. aureus on samples with higher CT values. The two remaining samples identified as positive by the BD Max StaphSR assay and negative by culture were assigned as true positives after discordant result analysis was performed. The reasons were that S. aureus colonies grew on repeated cultures from original TSB, and the raw PCR curve analysis showed strong amplification of the three targets tested, all of them around the same CT range. Most commercially available first-generation MRSA molecular tests are based on the detection of a sequence indicating the integration of the SCCmec within the chromosome and do not specifically target the mecA gene. These tests are faster than culture, and most of them are also easy to perform. However, a high proportion of false-positive and false-negative results due to the lack of

TABLE 2 BD Max assays in comparison to combined culture with resolved discrepancies Sensitivity

Specificity

Overall agreement

BD Max assay

% (no./total no.)

95% CI (%)

% (no./total no.)

95% CI (%)

% (no./total no.)

95% CI (%)

MRSA first-generation MRSAXT StaphSR MRSA StaphSR SA

88.6 (31/35) 94.3 (33/35) 94.3 (33/35) 96.4 (81/84)

74.0, 95.5 81.4, 98.4 81.4, 98.4 90.0, 98.8

95.9 (211/220) 97.7 (215/220) 97.7 (215/220) 93.6 (160/171)

92.4, 97.8 94.8, 99.0 94.8, 99.0 88.8, 96.4

94.9 (242/255) 97.3 (248/255) 97.3 (248/255) 94.5 (241/255)

91.5, 97 94.4, 98.7 94.4, 98.7 91, 96.7



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Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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TABLE 3 Challenge panel results Result forb:

Description

MREJ type

1 2800 9 11 16 2937 19 131 2952 797 ATCC 2312 3097 ATCC 29213 ATCC 14990

MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA MRSA (mecC) MSSA (empty cassette) MSSA MSSE

MRSA i MRSA ii MRSA iii MRSA iv MRSA v MRSA vi MRSA vii MRSA ix MRSA xiii MRSA xiv MRSA xxi NAc NA NA

Pos Pos Pos Pos Pos Neg Pos Neg Neg Neg Neg Pos Neg Neg

Max StaphSR Max MRSAXT

MRSA

SA

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg Neg Neg

Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Neg

a

ID, identification. Pos, positive; Neg, negative. c NA, not applicable. b

mecA in the SCCmec or to the presence of the mecC gene have been reported (16–19). For this reason, next-generation MRSA assays are in high demand. Our study compared the capacity to detect MRSA using two next-generation BD Max PCR assays against the BD Max MRSA first-generation assay. The sensitivity and specificity of the BD Max MRSAXT and BD Max StaphSR assays were higher than the sensitivity and specificity of the BD Max MRSA first-generation assay when the 3 PCR assays were compared to the results from culture. The BD Max MRSA first-generation assay identified six results differently than the two next-generation assays. Out of these six results, four were either false negative or false positive for MRSA on the first-generation assay, showing a better performance of the next-generation BD Max assays when those were compared to the BD Max MRSA first-generation assay. Furthermore, a challenge panel containing 14 control strains representing 11 MREJ types, one MSSE, one MSSA, and one mecA dropout MSSA was also tested by all PCR methods. One of the reasons to test this panel was to identify possible MRSA identification errors caused either by mecA dropout mutants or by the presence of mecC carriers, to test the ability of the assays to identify a broad range of MRSA MREJ types, and to test the ability of the StaphSR assay to correctly identify S. aureus. Excellent results were observed, showing that all strains from the challenge panel were correctly identified or excluded by both the BD Max MRSAXT and BD Max StaphSR assays. In contrast, the first-generation BD Max MRSA assay was not able to correctly identify the mecA empty cassette or the MRSA strains with mecC and missed 3 of the 11 MRSA MREJ type strains. These results corroborate evidence about wrong identification or misidentification of MRSA described by some colleagues (16–20, 33) and demonstrate an improvement of the new-generation BD Max MRSAXT and BD Max StaphSR assays to correctly identify or exclude these strains. A recent study from Europe reports the performance of the Xpert MRSA Gen3 assay, which also detects mecA/mecC and the SCCmec/orfX junction for MRSA (34). In summary, the two next-generation real-time PCR assays evaluated in this study presented high sensitivity and specificity to

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detect and identify MRSA strains in ESwab-collected nares samples, including at least one isolate carrying the mecC gene. One of the limitations of these BD Max next-generation assays, however, was that they could not distinguish strains with mecC and mecA among MRSA-positive isolates, as mecA and mecC amplicons are detected in the same BD Max optical channel (585/630 channel). Importantly, both assays correctly excluded mecA dropout MSSA isolates. The BD Max StaphSR assay was also able to detect and differentiate MSSA and MRSA isolates in nares samples. Additionally, the LoD observed for ESwab-collected samples using 200 ␮l of the ESwab liquid medium (1.5 ⫻102 CFU/ml; 333 CFU/ml) was in line with the analytical sensitivities of the BD Max MRSAXT and BD Max StaphSR assays (64 to 343 CFU/swab) reported previously by the manufacturers (20, 21). To conclude, the data indicate that both the BD Max MRSAXT and BD Max StaphSR assays provide highly accurate and rapid detection of MRSA in nares samples used in screening patients for MRSA colonization, and both assays can potentially assist with isolation steps to help reduce postprocedure dissemination of nosocomial infections. ACKNOWLEDGMENT

This study was supported by BD Diagnostics (Sparks, MD). REFERENCES 1. Kluytmans JA, Wertheim HF. 2005. Nasal carriage of Staphylococcus aureus and prevention of nosocomial infections. Infection 33:3– 8. 2. Levy PY, Ollivier M, Drancourt M, Raoult D, Argenson JN. 2013. Relation between nasal carriage of Staphylococcus aureus and surgical site infection in orthopedic surgery: the role of nasal contamination. A systematic literature review and meta-analysis. Orthop Traumatol Surg Res 99:645– 651. 3. Hardy K, Price C, Szczepura A, Gossain S, Davies R, Stallard N, Shabir S, McMurray C, Bradbury A, Hawkey PM. 2010. 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 16:333–339. http://dx.doi.org/10.1111/j.1469-0691.2009 .02899.x. 4. Drews SJ, Willey BM, Kreiswirth N, Wang M, Ianes T, Mitchell J,




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