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Journal of Clinical Microbiology, May 2009, p. 1546-1548, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.00114-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Automation of the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay for High-Throughput Screening of Nasal Swab Specimens{triangledown}

Xue-Ping Wang and Christine C. Ginocchio*

Molecular Diagnostics, Department of Laboratory Medicine, North Shore—LIJ Health System Laboratories, 10 Nevada Drive, Lake Success, New York 11042

Received 19 January 2009/ Returned for modification 1 March 2009/ Accepted 9 March 2009


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ABSTRACT
 
This study demonstrated that an automated version of the BD-GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay (BD-MRSA), using achromopeptidase sample lysis and PCR setup performed on the Hamilton MICROLAB STARlet (Auto-MRSA), gave results comparable to those obtained with BD-MRSA. The positive- and negative-result concordance rates and overall concordance of BD-MRSA and Auto-MRSA were 98.2, 97.7, and 97.6%, respectively. Auto-MRSA required 60% less technical time than BD-MRSA, and the time required to obtain results was 50% of that required for BD-MRSA. Auto-MRSA provides a reliable, robust system for high-volume clinical use.


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TEXT
 
The BD-GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay (BD-MRSA; BD GeneOhm, San Diego, CA) is a Food and Drug Administration-cleared multiplex qualitative real-time PCR test for the direct detection of MRSA from nasal swabs (1, 3, 5, 7, 11, 12, 14). Our laboratory uses BD-MRSA to screen admissions and intensive care patients at risk for MRSA nasal colonization to facilitate appropriate infection control procedures (2, 4, 6, 9, 10, 13). Sample processing and PCR setup are labor intensive and delay the time to result reporting, particularly for laboratories that test >100 samples a day. A study by Paule et al. demonstrated that manual achromopeptidase lysis permitted easier sample processing without negatively affecting the performance of BD-MRSA (8). To accommodate high-throughput testing, we developed an automated version of BD-MRSA (Auto-MRSA) using the Hamilton MICROLAB STARlet (HMS; Hamilton Company, Reno, NV). Auto-MRSA replaces manual BD-MRSA mechanical lysis with automated achromopeptidase lysis and PCR setup.

This study compared the performance of Auto-MRSA and that of the FDA-cleared BD-MRSA assay. Assay sensitivity was evaluated by using serial dilutions of MRSA spiked into lysis buffer and onto nasal swabs. The rate of reporting samples as unresolved (internal control failure due to amplification inhibitors or test failure) and the clinical performance of the assays were compared in two separate studies with patient nasal samples. The hands-on time and overall turnaround time required for Auto-MRSA testing of a single-day test volume (188 clinical samples) were evaluated and compared to those required for BD-MRSA.

Spiked samples, for assay validation and controls, and patient nasal swabs were added to 2.0-ml tubes containing 450 µl of Tris-EDTA buffer (Sigma-Aldrich, St. Louis, MO). Tubes were capped with a split-septum cap, vortexed at 2,500 rpm for 30 s, and placed in HMS sample tube carriers. Tube carriers, reagents, pipette tips, SmartCycler reaction tubes (Cepheid, Sunnyvale, CA), and a 96-deep-well plate were loaded on the HMS. HMS dispensed 20 µl of a 1,500-kallikrein-U/ml achromopeptidase solution (Sigma-Aldrich) and 60 µl of Chill-out liquid wax (Bio-Rad, Hercules, CA) into each well and then transferred 90 µl from each sample buffer tube into the plate. Samples and controls were lysed at 37 ± 2°C for 20 min and then heated at 95 ± 2°C for 10 min to inactivate the achromopeptidase. HMS reconstituted and dispensed into each reaction tube 25 µl of BD-MRSA PCR master mix, followed by 2.8 µl of sample or control lysate. Reaction tubes were removed from HMS, spun for 5 s, transferred to SmartCycler instruments (Cepheid), and tested according to the BD-MRSA package insert. Plates were removed from HMS, covered, and stored at –20°C in the event that retesting was indicated due to unresolved results.

Initially, experiments were conducted to determine the sample buffer volume compatible with enzymatic lysis that provided an assay sensitivity equivalent to that of BD-MRSA but did not yield unresolved results. A saline suspension of MRSA reference strain ATCC 43300 (ATCC, Manassas, VA) grown overnight on 5% sheep blood agar (BBL, Becton Dickinson, Sparks, MD) was serially diluted to yield a four-member panel containing 156, 313, 625, and 1,250 CFU/ml as determined by quantitative culture. A 50-µl aliquot of each MRSA dilution was added to nine 300-µl TE (Tris-EDTA) buffer tubes (standard BD-MRSA volume) and to nine 450-µl TE buffer tubes, and three tubes of each dilution/lysis buffer volume were tested over three independent runs with Auto-MRSA. Increasing the sample buffer from 300 to 450 µl reduced the unresolved-result rate from 22.2 to 0% without affecting the sensitivity of the assay (data not shown). Three samples reported as unresolved by Auto-MRSA (300 µl) were reported as positive by Auto-MRSA (450 µl). Therefore, the 450-µl buffer volume was used to compare the analytical sensitivities of Auto-MRSA and BD-MRSA. The MRSA stock (ATCC 43300) was serially diluted to yield a panel containing 313, 625, 1,250, 5,000, and 10,000 CFU/ml. Aliquots (50 µl) of each dilution were added to lysis buffer and tested by Auto-MRSA (n = 30 per dilution) and BD-MRSA (n = 30 per dilution). The lowest dilutions of MRSA detected by Auto-MRSA and BD-MRSA were 31 CFU/tube and 16 CFU/tube, respectively (Table 1). However, the 95% detection rate was between 250 and 500 CFU/tube for Auto-MRSA and >500 CFU/tube for BD-MRSA. Using logistic analysis, the actual Auto-MRSA limit of detection (95% detection rate) was calculated to be 416 to 435 CFU/tube and was consistent with the BD-MRSA package insert, which claims a 92% detection rate at 325 CFU/swab. Auto-MRSA detected more positive samples (93/180, 51.67%) than BD-MRSA (77/180, 42.78%). These data demonstrated that Auto-MRSA had significantly better analytical sensitivity than BD-MRSA (P = 0.0102). Another MRSA panel (ATCC 43300) containing 10-fold serial dilutions (3 to 3 x 105 CFU/ml) was used to evaluate Auto-MRSA sensitivity with spiked swab specimens. A 50-µl aliquot of each dilution was applied to nasal culture swabs (n = 15 per dilution) (CultureSwab; BBL, Becton Dickinson, Sparks, MD). Swabs were replaced into the transport container and tested within 24 h. MRSA was detected in 1/15 swabs (6.67%) at 15 CFU/swab and in 13/15 swabs (86.67%) at 150 CFU/swab (Table 2), yielding results again consistent with the BD-MRSA package insert.


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  		TABLE 1. Limit-of-detection studies for Auto-MRSA and the FDA-cleared BD-MRSA test with spiked sample buffer tubes


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  		TABLE 2. Sensitivity of Auto-MRSA with MRSA-spiked culture swabs

The clinical performance of Auto-MRSA was evaluated by using patient nasal samples collected with dual swabs (BBL) according to hospital surveillance procedures. Routine BD-MRSA was performed according to the manufacturer's instructions with one of the swabs, and the other swab was tested with Auto-MRSA in accordance with an institutional review board-approved protocol. To determine if the clinical comparison could be biased by (i) sampling error and discordant distribution of material on the dual swabs and/or (ii) MRSA false positives due to the detection of methicillin-susceptible S. aureus containing the staphylococcal cassette chromosome mec with a mecA deletion, we performed the following evaluation. One hundred dual swabs were separated and tested independently by BD-MRSA, and another 100 dual swabs were rolled together (both swabs were rolled over each other by touching all of the sides and the tip) to distribute the bacteria as evenly as possible and then tested with BD-MRSA. The concordance of BD-MRSA results was excellent but slightly better with rolling of the swabs (97%) versus not rolling them (95%). Therefore, dual swabs were rolled prior to testing all clinical samples. BD-MRSA results from the dual swabs (n = 200) tested in the comparison study described above were compared to cultures using a 24-h tryptic soy broth (BD) enrichment step, followed by plating on MRSA ChromAgar (BD) and CNA agar (BD). BD-MRSA results were concordant with culture for 371/400 (92.75%) of the swabs. No methicillin-susceptible S. aureus samples were identified that were BD-MRSA positive. Fourteen culture-negative/BD-MRSA-positive swab sets (28 swabs) were probably MRSA positive due to the detection of nonviable or low-level MRSA. One sample was MRSA culture positive/BD-MRSA negative for one of the two swabs.

In clinical study 1, 110 dual-swab sets were rolled and separated and one swab from each set was tested by Auto-MRSA and one swab was tested by BD-MRSA. Positive-, negative-, and unresolved-result rates were not significantly different between the two assays (Table 3). Residual frozen (–20°C) cell lysates from each assay were used for repeat testing of samples with unresolved results and generated valid results for 10/11 (Auto-MRSA) and 5/8 (BD-MRSA) samples. Unresolved-result rates were reduced from 10 to 0.91% and from 7.27 to 2.73% for Auto-MRSA and BD-MRSA, respectively. Samples with discordant results (one Auto-MRSA positive/BD-MRSA negative and two Auto-MRSA negative/BD-MRSA positive) were cultured for MRSA from sample buffer tubes. The Auto-MRSA-positive/BD-MRSA-negative sample was culture negative from both tubes. One Auto-MRSA-negative/BD-MRSA-positive sample was MRSA culture positive only from the BD-MRSA tube, and the other was culture negative from both tubes. In clinical study 2, an additional 219 dual-swab sets, in which one swab in each set was tested by BD-MRSA and one was tested by Auto-MRSA, demonstrated a similar high level of concordant results (Table 3). When the results of the two studies were combined (Table 3), the positive- and negative-result concordance rates and overall concordance of BD-MRSA and Auto-MRSA were 98.18, 97.71, and 97.57%, respectively. The overall unresolved-result rates prior to and after a freeze-thaw step were 8.51 and 2.43%, respectively, for BD-MRSA and 3.34 and 0.31%, respectively, for Auto-MRSA.


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  		TABLE 3. Results from two clinical studies that compared BD-MRSA with Auto-MRSA for the detection of MRSA and the rates of unresolved results

A work flow comparison of BD-MRSA and Auto-MRSA, based upon our normal daily volume of testing, is presented in Table 4. Auto-MRSA (188 results) required one less technologist per day, results were available in half the time (7.5 h versus 15 h), and the hands-on-time was reduced by 60%. The savings in technologist time (at $50/h, including benefits) is approximately $109,200/year based on a volume of 188 tests/day Monday through Friday and 90 tests/day Saturdays, Sundays, and holidays. The cost of the automated platform would be recouped in approximately 1 year, and one technologist would be available to perform other molecular testing. In summary, Auto-MRSA provides a high-throughput, accurate, and rapid format with significant cost savings for the laboratory and a shorter time to the identification of MRSA-colonized patients.


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  		TABLE 4. Daily work flow analysis comparing Auto-MRSA and BD-MRSA


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ACKNOWLEDGMENTS
 
We thank the Departments of Microbiology and Molecular Diagnostics, North Shore—LIJ Health System Laboratories, for their assistance and support.

This study was funded through a research grant from BD GeneOhm Sciences.


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FOOTNOTES
 
* Corresponding author. Mailing address: North Shore—LIJ Health System Laboratories, 10 Nevada Drive, Lake Success, NY 11042. Phone: (516) 719-1079. Fax: (516) 719-1254. E-mail: CGinocch{at}nshs.edu Back

{triangledown} Published ahead of print on 18 March 2009. Back


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Journal of Clinical Microbiology, May 2009, p. 1546-1548, Vol. 47, No. 5
0095-1137/09/$08.00+0     doi:10.1128/JCM.00114-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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