CORRECTED ABSTRACT

1. C-025 Screening for Nasal Carriage of Methicillin-Resistant Staphylococcus aureus (MRSA): Reduction of Turnaround Time and Workload by the use of BBLTM CHROMagarTM MRSA. F.E. Hardic, A. W. Pasculle, C.A. Muto. University of Pittsburgh Medical Center, Pittsburgh, PA A William Pasculle, Sc.D. UPMC Presbyterian Rm. A-807, 200 Lothrop St. Pittsburgh, PA 15213 (412) 647-3727 (Ph) (412) 647-3610 (FAX) pasculleaw@upmc.edu CORRECTED ABSTRACT Active surveillance testing for nasal carriage of MRSA is becoming increasingly important and is escalating the service demands on many laboratories. We compared the performance of BBLTM CHROMagarTM MRSA (C-MRSA) (BD Diagnostics, Sparks, MD) to the combination of blood agar and mannitol salt agar with oxacillin (BAP/MSO) which was routinely used in our laboratory. We cultured 1046 nasal swabs from patients in intensive care units on all three media. On BAP/MSO, MRSA were identified by colonial morphology, Gram stain, catalase and/or coagulase tests, and growth on Mueller Hinton agar with oxacillin. On C-MRSA, MRSA were identified after 24 h of incubation by the production of mauve-colored colonies. Mauve-colored colonies at 48 h were confirmed as MRSA by a coagulase test. Of 79 cultures positive for MRSA, BAP/MSO detected 61 (77.2%) while C-MRSA detected 69 (87.3%). Of the 18 cultures that were falsely negative on BAP/MSO, 12 (75%) contained methicillin-susceptible S. aureus that were not differentiated from MRSA by the conventional cultures despite the use of MSO. Positive cultures were also identified faster on C-MRSA. After 24 h of incubation, 81% of all positive cultures were identified by C-MRSA with the remaining 19 % identified after 48 h. In contrast, no cultures were definitively positive on BAP/MSO at 24 h. At 48 h, BAP/MSO identified only 62 % of positive cultures with 33 % identified at 72 h and 5 % identified after 96 h of incubation. The use of C-MRSA also required fewer supplemental tests to identify MRSA. Cultures using BAP/MSO required 300 subcultures, 321 coagulase tests and 180 oxacillin screening tests to identify 61 MRSA isolates while C-MRSA cultures required only 58 coagulase tests to identify 69 MRSA isolates. Less technologist time was required using C-MRSA. Exclusive of specimen planting and result reporting, the average bench time to work up a BAP/MSO culture was 2.64 min while average time spent on each C-MRSA culture was 0.64 min (40 sec). The use of C-MRSA results in more rapid detection of MRSA with a significant reduction in reagents and workload. • Conclusions • C-MRSA performed as well as BAP/MSO. • C-MRSA recovered MRSA from cultures which contained BOTH MRSA and MSSA when MSO did not. • C-MRSA enabled the detection of 80 percent of MRSA after only 24 h of incubation. While BAP/MSO required at least 3 days to recover a similar percentage. • Significantly fewer (93%) supplemental tests were required to identify MRSA on C-MRSA • Adoption of C-MRSA resulted in a reduction in workload of 2 minutes per culture. The savings in salary alone more than offsets the increased cost of C-MRSA. • We believe that the high level of performance of C-MRSA may have been associated with our practice of batch-inoculating the plates so that they were examined after a full 20-24 h of incubation. Results In total, 79 (7.6%) cultures were positive for MRSA. Of the positive cultures, 61 (77.2%) were detected on BAP/MSO while 69 (87.3%) were detected on C-MRSA (Table 1). Among the 18 falsely-negative BAP/MSO cultures, 12 (75%) contained methicillin-sensitive S. aureus that were not differentiated from MRSA despite the use of MSO. Organisms recovered on BAP/MSO were identified using standard methods for our laboratory. Suspicious colonies were identified as Staphylococcus aureus using a latex agglutination reagent (Staphaurex, Remel, Lenexa, KS) according to the manufacturer’s instructions. Isolates which grew initially on BAP were tested for coagulase production directly from the primary plates. Isolates which grew initially on the MSO plate were subcultured to BAP before coagulase testing. Oxacillin resistance was confirmed by inoculating isolates from BAP to Mueller Hinton Agar with 6 µg/mL Oxacillin and 4% NaCl. Organisms producing mauve-colored colonies on C-MRSA after 20-24 h of incubation were presumed to be MRSA with no further testing. Isolates which did not produce mauve colored colonies until 40-48 h were confirmed by performing a coagulase test. Coagulase-positive isolates were presumed to be MRSA1,2. Table 1. Detection of MRSA on C-MRSA and BAP/MSO • References • Flayhart, D, J.F. Hindler, D.A. Bruckner, G. Hall, R.K. Shrestha, S.A. Vogel, S.S. Richter, W. Howard, R. Walther, and K.C. Carroll. 2005. Multicenter evaluation of BBL CHROMagar for direct detection of methicillin-resistant Staphylococcus aureus from surveillance cultures of the anterior nares. J. Clin. Microbiol. 43:5536-5540. • Zuolin, H., E. Lautenbach, N. Fishman, and I. Nachamkin. 2007. Evaluation of mannitol salt agar, CHROMagar Staph aureus and CHROMagar MRSA for detection of methicillin-resistant Staphylococcus aureus from nasal swab specimens. J. Med. Microbiol. 56:43-46. • When we apportioned the supplemental tests across the number of MRSA isolates detected, we documented a significant reduction in work. • BAP/MSO 61 MRSA Isolates. In total 13.6 tests per MRSA identified • 5.25 coagulase tests per MRSA identified • 4.9 Subcultures to BAP per MRSA identified • 3.5 Oxacillin screens per MRSA identified • C-MRSA 69 MRSA isolates • 0.84 coagulase tests per MRSA identified Time studies were performed during one week of the study. We measured the time required for each of the technologists to perform the testing associated with isolation and identification of MRSA in each system. We did not measure time required for media inoculation or for reporting of results. The BAP/MSO system required 2.64 minutes per culture while the C-MRSA system required 0.64 minutes(40 seconds!) per culture. Extrapolated to the 1300 cultures per month processed by our laboratory, this represents a savings of 43 hours per month. Immediately following the completion of this study, C-MRSA became the standard medium for active surveillance cultures in our laboratory. MRSA were detected more quickly on C-MRSA. Eighty-two percent of cultures positive for MRSA were detected after the first day of incubation (Fig. 2). BAP/MSO required four days for the detection of all positive cultures. Materials and Methods Between May 22, and August 8, 2006, 1046 nasal swabs from patients in our intensive care units were cultured using the routine method used in our lab and on C-MRSA (Fig. 1). The routine method consisted of Trypticase Soy Agar with 5% Sheep Blood (BD Diagnostics, Sparks, MD) (BAP) and Mannitol Salt Agar with 4ug/ml oxacillin (MSO) (Hardy Diagnostics, Santa Maria, CA). Since only a single swab was available, each swab was first used to inoculate the BAP and then either the MSO or C-MRSA plate was inoculated next. We alternated inoculating the C-MRSA first on every other culture in order to randomize which selective medium was inoculated. Because color development requires at least 20 h of incubation to develop, we inoculated cultures in batches at 1 PM and 4 PM each day. Inoculated plates were incubated at 35oC for 20-24 h and , because plates were inoculated in batches, they were examined at 11am and 1pm, respectively, the next day. The standard cultures (BAP/MSO) and the C-MRSA cultures were read by separate technologists who were blinded to the results of the other culture method. Supported in part by BD Diagnostics We also tallied the numbers of supplemental tests which were required to identify MRSA by each culture system. For the BAP/MSO system we performed significantly more supplemental tests (coagulase, subculture to BAP and Oxacillin screening) (Fig. 3). As presented at ASM Abstract Poster Session, 2006