Evaluation of the Bio-Rad MRSASelect agar for Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) from Surveillance Specimens 2005

Transcription

1 Evaluation of the Bio-Rad MRSASelect agar for Detection of Methicillin-Resistant Staphylococcus aureus (MRSA) from Surveillance Specimens 2005 Report requested by: Delphine Cassagne CMD Project Manager Bio-Rad France Study report compiled by: Barbara Willey and Nathan Kreiswirth Infection Control and Methods Development Room 1460 Department of Microbiology Mount Sinai Hospital 600 University Ave Toronto, Ontario Canada M5G 1X5

2 INDEX A. Purpose of Study 4 B. Materials and Methods 4 1. Study setting 4 2. Number of specimens 4 3. Type of specimens 4 4. Specimen collection 4 5. Media types and quality assurance 5 6. numbering and inoculation order 5 7. Specimen and media labeling 6 8. inoculating and streaking 6 9. Incubation temperature conditions Length of incubation and reading time points Blinded plate examination Documentation interpretation Routine Mannitol Salt Cefoxitin Agar protocol Bio-Rad MRSA Select protocol Resolution of discrepancies Use of a relative gold standard Database clean up for this report Presentation dates and secrecy contracts 11 2

3 C. Result tables Table 1. Patient demographics 12 Table 2. Description of specimen type, MRSA yield 13 per medium per specimen type, and number of new versus previously known MRSA cases detected per specimen type per medium Table 3. Lot numbers used in study with corresponding 14 cumulative specimen result distribution per lot Table 4. Specimen plating order and corresponding 15 cumulative specimen result distribution per plate order Table 5. Results of 24h and 48h growth for the specimens in the phase-i temperature study Table 6. Percent sensitivities, specificities, positive and 16 negative predictive values for 24 and 48 hour results obtained in the Phase-I temperature study for the first 900 specimens Table 7. Results of 24 and 48 hour growth for the total specimens incubated at 37 C Table 8. Percent sensitivities, specificities, positive 17 and negative predictive values for the total 2500 specimens incubated at 37 C Table 9. Results of 24h and 48h growth per specimen type incubated at 37 C Table 10. Percent sensitivities, specificities, positive 21 and negative predictive values 24h and 48h per specimen incubated at 37 C Table 11. Summary of presumptive identifications of 22 pink/yellow colonies that were not MRSA 3

4 A. Purpose of Study The objective of this blinded prospective comparison study was to identify the most rapid yet cost effective, sensitive yet specific selective medium for detecting methicillin-resistant Staphylococcus aureus (MRSA) from a wide variety of surveillance specimens. B. Materials and Methods 1. Study setting The study was conducted in a large CAP-accredited clinical microbiology laboratory, the Toronto Medical Laboratories and Mount Sinai Hospital Department of Microbiology, Toronto, Ontario, Canada. The laboratory serves 11 diverse health care facilities in the Greater Toronto Region and processes >75,000 MRSA screens from multiple specimen types annually. All facilities perform screening of patients who are admitted with risk factors for MRSA. Routine surveys of patients residing on medium to high risk units, and monitoring of patients known to have MRSA, are also performed. See Table 1. for summary of patient demographics, specimens per patient and for distribution of specimens and MRSA per facility. 2. Number of specimens Since the number of MRSA-positive screens processed by the laboratory from all specimen types in the months leading up to the study was +/-6%, it was calculated that 2500 specimens would be needed to obtain >100 MRSA. To accommodate staffing requirements, only the specimens received on Mondays and Tuesdays were included in the study. The number of samples plated each day was predetermined according to predicted workloads. This resulted in 150 consecutive samples being enrolled each day for the first three study weeks followed by 200 samples each day for the final four of seven study weeks. 3. Type of specimens The 2500 samples processed included any type received by the laboratory during the study period for MRSA screening purposes. These included 823 nasal swabs, 729 rectal swabs, 42 peri-rectal swabs, 6 stool swab, 222 wound, 20 axilla swabs, 23 axilla-groin swabs, 16 groin swabs, 619 nasalaxilla-groin-perineum swabs. See Table 2 for a summary of specimen type, MRSA yield per plate per specimen type, and whether the MRSA was from a new or previously identified case. This table reflects the total MRSA identified in the study from all four media types tested (Non-Bio-Rad data not shown). 4. Specimen collection Specimens were obtained either by nurses or by infection control practitioners, in accordance with recommended specimen collection 4

5 guidelines in place in each facility. To reduce specimen numbers, combination nasal-axilla-groin-perineum (NAGP) swabs are collected as per Infection Control Program guidelines and consist of a perineum swab taken simultaneously to a swab that is obtained by first swabbing the patient s anterior nares and then the axillae and groin surface areas. These two swabs are taped together, accessioned as a nasal-axilla-groin-perineum swab and is processed as a pooled specimen on receipt in the laboratory. 5. Media types and quality assurance The study compared the routine MRSA screening medium in use in the clinical laboratory, namely the Oxoid Mannitol Salt Cefoxitin agar (abbreviated as MSF in Tables and MSA in Access raw database), to three other screening media - the Bio-Rad MRSA Select, the BD CHROMagar MRSA and another modified Mannitol Salt Cefoxitin formulation from Oxoid. No results for BD or the Oxoid modified Mannitol Salt Cefoxitin agar will be discussed in this report. Each media company provided a minimum of five separate lots of media that had been checked for performance before shipping. Specifically, Bio-Rad supplied five batches, and Oxoid supplied seven batches. On arrival, the condition of each batch was independently inspected by the routine laboratory quality assurance bench technologist, and representative samples from each batch were processed as per laboratory protocol. This protocol is in accordance with CLSI (formerly NCCLS) standards for sterility, selectivity, specificity and growth rates using appropriate ATCC control organisms. These results were documented directly into the clinical laboratory information system and are available for review if necessary. All batches of media used in the study passed these performance challenges. The lot number of each medium used to process each individual specimen is documented in the raw data within the Access data base against each particular specimen number. The number of specimens and MRSA yield per lot is summarized in Table numbering and inoculation order To prevent bias, the order of inoculation of the various media types was rotated in a predefined way throughout the study so that each medium was inoculated first, second, third etc, no more frequently than any other. No bias was identified and this order is available for detailed review in the raw data and is summarized with MRSA yields in Table 4. In practice, all plates were organized into their respective orders for the following morning. This process included marking each plate with a consecutive Access generated study auto-number from , organizing them according to the predefined rotation, placing the numbered and organized plates into cardboard media boxes to protect them from light, and storing them closed until the following morning in a continuously temperature 5

6 monitored walk-in media storage refrigerator. The numbering and plate organization was performed in a room with reduced light and plates were taken out of the boxes only when inoculation was imminent (to ensure that the chromogenic media were not exposed to light for more than a few hours in total from beginning to end of study). 7. Specimen and media labeling On receipt in the laboratory, specimens were accessioned into the laboratory information system by technicians as per routine practice. Labels were printed during this procedure, one of which was affixed to each specimen and another placed onto a pre-printed worksheet against the Access database generated study auto-number that had been assigned to that specimen. In a darkened room, a final label from each specimen was placed by a study technician onto the routine laboratory plate only. This plate was then replaced into its appropriate position in the box that contained the remaining prenumbered and pre-organized stacks of plates. Once completed, the boxes of plates and their accompanying specimens were taken to the planting area for processing by laboratory technicians. 8. inoculating and streaking Using the frosted marking on the outside of each Petri-dish as a guide, a triangular area of approximately 3x3x3cm was inoculated by applying the swab to the surface and rotating it so that all sides of the swab made contact with the medium. Once inoculated, plates were placed on a Fisher Scientific Iso-r-80 Petri dish streaker, which automatically and consistently spread the specimen inoculums round the remaining three quadrants so as to obtain single colonies. Once streaking was completed, the plates were quickly reorganized into type and numerical study order (except for the routine method which were barcode labeled), in preparation for incubation. 9. Incubation temperature conditions The study was conducted in two phases: the first 900 specimens were planted to two plates of each media type for a total of eight plates per specimen and one of each media type was incubated at 35 o C (as was routine laboratory protocol) and the other at 37 o C (as recommended by manufacturers of chromogenic media). After these 900 specimens were completed, a mid-way analysis was conducted to determine whether there was a difference between these two temperature groups. The outcome showed trending towards improved yield at 37 o C but these differences were not statistically significant. Therefore, the remaining 1600 specimens were planted only to one plate of each media type which was incubated at 37 o C as per Bio-Rad recommendations. All media types from each specimen were always incubated simultaneously. s assigned to 35 o C incubation were placed into large plastic bins (one for each media type) in stacks of 10 in the clinical laboratories continuously 6

7 temperature monitored walk-in incubator. For the chromogenic media, these bins were covered with light-weight breathable toweling to protect the plates from light exposure while incubating. s designated to 37 o C incubation, from the beginning to the end of the study, were all treated in the same manner as they were placed in smaller light-tight floor-model incubators. 10. Length of incubation and reading time points Inoculated plates were typically incubated in batches. As processing was completed throughout the day, this generally occurred from within one hour of the laboratory accessioning of the specimen. Planting of study specimens was usually completed by 3pm each day (with a few exceptions). After overnight incubation, all plates were removed simultaneously (as happens in routine practice) for a rapid initial examination to sort positives from negatives. At this point plates would have been incubated for a range of 18h to 24h. After this initial examination, all negative plates were re-incubated immediately. In the early afternoon, the study plates that had been received later in the previous day were again removed from their respective incubators to perform the 24h examination. This was done for the study plates only as it was not part of the routine laboratory procedure to do so. Negative plates were again placed overnight for incubation, and then examined for a final time the following morning. The 18 and 24 hour time points were documented as the 24h time point. 11. Blinded plate examination Each media type was examined by a dedicated study technologist while the routine medium was worked up by various laboratory technologists assigned to the MRSA bench as per normal practice. Both routine and study technologists were specifically instructed to refrain from discussing their results in any specific manner. Prior to discarding any of the study plates on the day of their final examination, another independent technologist was assigned to rapidly check all negative plates to ensure no potential positive had been missed; plates were seldom retained for further workup. In this way, all technologists were blinded to the results obtained by the other members of the study group. 12. Documentation Each study technologist documented their daily work results onto a printed worksheet generated from the Access database. On completion of the daily work, each study technologist entered their results into the specific query set up for results for their assigned medium within the Access database. For the laboratory arm of the study, all work was documented directly into the paperless laboratory information system (LIS). A separate research assistant was assigned the daily task of extracting the real-time information from this system, recording it on the worksheet on which the LIS labels had initially been placed. This extracted information was subsequently entered into the 7

8 Access database. No-one other than the Study coordinator had access to the entire database. 13. interpretation For the routine Mannitol Salt Cefoxitin agar (abbreviated as MSF in Tables and MSA in Access raw data database), positive colonies were considered to be any colony fermenting mannitol (yellow) or any colony specifically resembling S. aureus, even if it had not fermented mannitol. For the Bio-Rad MRSA Select (BR), any pink colony was taken as positive in accordance with the strict manufacturers instructions that were confirmed prior to commencing the study. 14. Routine Mannitol Salt Cefoxitin Agar protocol The MRSA screening algorithm utilized at Mount Sinai Hospital during the study was outlined in the original study proposal, but is repeated below. I. MRSA screen specimens were inoculated onto Oxoid Mannitol Salt agar plate containing cefoxitin (abbreviated as MSF in Tables and MSA in Access raw data database) (Oxoid prepared using BBL Mannitol Salt Agar base) and streaked for single colonies. s were incubated in an aerobic atmosphere at 35 o C for the first 900 specimens and then at 37 o C for the remaining 1600 specimens. (For the temperature study, the first 900 were incubated at 37 o C as discussed previously). II. After 18-24h and 48h of incubation, plates were examined for yellow colonies or mannitol-negative colonies resembling S. aureus. III. Suspect colonies were sub-cultured to 5% sheep blood agar and incubated for 4-24h at 35 o C in a 5% CO 2 atmosphere. IV. Standard tube coagulase tests, performed from the blood agar, were incubated for 4h at 35 o C, examined for clot formation, and if negative, incubated at room temperature overnight. V. Tube coagulase-negative samples were tested with a Pastorex Staph Plus agglutination test (Bio-Rad) that detects the capsular antigens specific to S. aureus. If discrepancies between the tube coagulase and Pastorex tests occurred, these were resolved using a GenProbe Accuprobe test that detects S. aureus species-specific rdna (considered definitive). VI. With newly identified MRSA patients, colonies of S. aureus (tube coagulase-positive or tube coagulase-negative but Pastorex-positive and Accuprobe positive) were tested for the protein product of the meca gene responsible for the altered cell wall that results in resistance to methicillin using the Denka Seiken PBP2a monoclonal antibody agglutination kit. If PBP2a was detected, the sample is reported as positive for MRSA. If 8

9 sufficient pure single colonies were present on the Mannitol Salt Cefoxitin agar, the Pastorex S. aureus agglutination and the Denka Seiken PBP2a agglutination tests would have been performed directly from the selective agar and the blood agar subculture would have been used for the confirmatory tube coagulase and oxacillin screen agar tests. These new MRSA were further processed to determine antimicrobial susceptibility profiles and SmaI pulsed-field gel electrophoresis patterns, and were stored at -80 o C. VII. The 900 Phase 1 temperature study specimens that were incubated at 37 o C were treated as new patients as above. VIII. In the routine laboratory, for cultures from previous MRSA patients (defined as any patient who had a history in our laboratory of a positive culture within the previous 3 months) the tube coagulase and oxacillin screen agar tests were set up simultaneously and the PBP2a result was referred from a previous test. For study purposes, the original PBP2a result was documented in the database. The oxacillin screen was read at 24h as per CLSI (previously NCCLS) guidelines. Samples that were tube coagulase-positive, and that were Pastorex-positive and that grew on the oxacillin screen agar were reported as MRSA without further testing. IX. Results from the routine Mannitol Salt Cefoxitin agar algorithm were recorded into the Laboratory Information System by the regular clinical technologists assigned to the Infection Control MRSA bench. These results were extracted from the LIS to a worksheet and transferred into the Access database daily by an assigned research technician blinded to the results of other media. 15. Bio-Rad MRSA Select protocol All study media, including the two media not shown, were handled in precisely the same manner. I. On day one, MRSA Select plates were examined at ~18h and rechecked again briefly at ~24h for newly visible pink colonies. Results were recorded on worksheets at the bench. Specimens with no growth or no pink colonies at ~ 24h were incubated overnight and again at 48h. II. No patient identifying information or laboratory LIS numbers appeared on the study medium or worksheets but were available for real-time analysis of discrepancies by cross reference to the lab worksheet by the Study coordinator 9

10 III. The lot numbers and performance parameters for all media were recorded throughout the study and entered into a Quality Control query within the Access database IV. Identification of MRSA was performed as defined by the routine laboratory algorithm: each suspect isolate from a study medium was subjected to either direct or indirect testing (5% sheep blood agar subculture only when necessary this was documented in the database as a Y per subculture) using the Pastorex Staph Plus and tube coagulase tests. V. If both the Pastorex and tube coagulase tests were negative, no further work was performed. VI. If the Pastorex was positive directly from the study medium, a Denka Seiken PBP2a agglutination test was performed, also directly from the BioRad medium if the organism was present in sufficient numbers and in sufficient purity. VII. When the PBP2a test was positive, the study technologist notified the Study coordinator of the presumptive MRSA; this date was documented as turn-around-time to notification. VIII. All positive PBP2a agglutinations were confirmed by oxacillin screen agar. No discrepancies between PBP2a and oxcacillin occurred during the study and no susceptible S. aureus were identified. IX. The times to detection of positives were taken as the documented time to notification following receipt of the specimens in the laboratory, and not the final report date nor the date of the specimen order X. All supplemental testing required, including the number of subcultures, for final identification of both positives and negatives from each media, was recorded and entered into the study database. XI. Discrepancies were investigated as soon as results from each arm of the study were completed and had been entered into the study database. 16. Resolution of discrepancies If MRSA was identified from one medium only, it was re-identified from the original plate, and the isolate was typed by pulsed field gel electrophoresis to determine if it belonged to one of the circulating MRSA clones. As well, the original swab was enriched using a broth protocol and reprocessed. In addition, results obtained from previous (and sometimes subsequent) specimens from the same patient were reviewed by the Study coordinator when monitoring the discrepancies. This review is a common and accepted laboratory practice, and is routinely done to determine whether a patient was 10

11 a known (previous) or a newly identified case. This was documented in the Access database for all MRSA as N indicating a new case or P indicating that MRSA had previously been identified and fully confirmed from this patient. This added information lent weight to the correctness of a single positive isolate obtained during the study. 17. Use of a relative gold standard When evaluating a selective medium using specimens from contaminated sources, direct comparison to a non-selective medium is not an appropriate gold standard. This is because the non-selective media are vulnerable to overgrowth of the target organism by competing commensal flora. With MRSA, higher yields are frequently achieved from selective media. Comparison of yield from a selective medium to that from broth enrichment methodology (which is known to increase yield) was not used in this study. This was because in practice enrichment protocols delay turn-around-times, are too costly and laborious, and no perfect selective medium for sub-culture post-enrichment has been described. PCR performed directly from the original specimen may in the future resolve this issue. However, since at present, no Gold Standard exists in the conventional sense, a relative gold standard was applied in this study. In practice, this meant that if a confirmed MRSA was identified from a specimen from any one (or more) of the four study media, then that specimen was considered to be a true positive. The relative Gold Standard in this study was total cumulative number of MRSA identified from all media combined (see Tables 5, 7 and 9). 18. Database clean up for this report The raw data pertaining to the BD and Oxoid study media is not contained in this report as it is proprietary to each of the aforementioned companies. However, as results obtained from these media impact the final study results (see Tables 5-11), the raw data includes a composite variable to indicate which MRSA was identified from one of these media but not detected by either the routine method or the MRSA Select plate. As such, even though no substantiating test results for these MRSA are presented, all statistics have been based on this number. To obtain access to this missing information, specific written permission would have to be obtained from each respective media company. 19. Presentation dates and secrecy contracts Complete data from this study will be delivered in an oral presentation at the ECCMID conference in Nice, France in April In accordance with the Secrecy Agreement, the data detailed in this report shall not be divulged before this presentation has taken place. 11

12 C. Result tables The results of the study are summarized in the various tables below: Table 1. Patient demographics Patient demographics Gender Number Female 1314 Male 1147 Not specified 39 Age distribution Range Year Youngest 0yrs 2005 Oldest 105yrs 1900 Mean 69yrs 1936 No. of specimens MRSA pos MRSA neg No. of patients screened MRSA pos MRSA neg Specimens per patient Range Mean during study period 1 to 13 2 No. of Facilities Tn specimens Tn MRSA % MRSA A B C G H L M P Q R T W Z FACILITIES

13 Table 2. Description of specimen type, MRSA yield per medium per specimen type and number of new versus previously known MRSA cases detected per specimen type per medium Total number specimens in study: 2500 Total number of: Specimen type No. MRSA from MSF MRSA from Bio-Rad MRSA in study New MRSA cases Previous MRSA cases WOUND Swabs Stool Rectal swabs Perineum swabs FAECAL Specimens NASAL Swabs Groin swabs Axilla and Groin combination swabs Axilla AXILLA/GROIN Swabs NASAL-AXILLA-GROIN-PERINEUM (NAGP) Total

14 Table 3. Lot numbers used in study with the corresponding cumulative specimen result distribution per lot LOT NUMBERS MSF 37 C = MSF 35 C = MRSA MRSA False Positives False Positives No growth or No Yellow colonies No growth or No Yellow colonies No MRSA No MRSA Total Total BR 37 C = BR 35 C = MRSA MRSA False Positives False Positives No growth or No Pink colonies No growth or No Pink colonies No MRSA No MRSA Total Total MSF 37 C = MRSA False Positives No growth or No Yellow colonies No MRSA Total BR 37 C = /2041 MRSA False Positives No growth or No Pink colonies No MRSA Total

15 Table 4. Specimen plating order and corresponding cumulative specimen result distribution per plate order PLATING ORDER Mannitol Salt Cefoxitin 37 o C n= MRSA False positives No growth or No yellow colonies No MRSA Total Bio-Rad MRSA Select 37 o C n= MRSA False positives No growth or No pink colonies No MRSA Total plated Mannitol Salt Cefoxitin 35 o C n= MRSA False positives No growth or No yellow colonies No MRSA Total plated Bio-Rad MRSA Select 35 o C n= MRSA False positives No growth or No pink colonies No MRSA Total plated Mannitol Salt Cefoxitin 37 o C n= MRSA False positives No growth or No yellow colonies No MRSA Total plated Bio-Rad MRSA Select 37 o C n= MRSA False positives No growth or No pink colonies No MRSA Total plated

16 Table 5. Results of 24h and 48h growth for the 900 specimens in the Phase-I temperature study MSF 900 MSF Media, hrs, temp MRSA NO MRSA Total Yellow colonies MSF 24h 37 C MSF 48h 37 C MSF 24h 35 C MSF 48h 35 C No growth or No yellow colonies MSF 24h 37 C MSF 48h 37 C MSF 24h 35 C MSF 48h 35 C Gold Standard Bio-Rad 900 BR Media, hrs, temp MRSA NO MRSA Total Pink colonies BIO 24h 37 C BIO 48h 37 C BIO 24h 35 C BIO 48h 35 C No growth or No pink colonies BIO 24h 37 C BIO 48h 37 C BIO 24h 35 C BIO 48h 35 C Gold Standard Table 6. Percent sensitivities, specificities, positive and negative predictive values for 24 hour and 48 hour results obtained in the Phase-I temperature study for the 900 specimens MSF 900 Sens Spec PPV NPV MSF C 66.7% 87.2% 23.8% 97.8% MSF C 82.4% 66.5% 13.0% 98.4% MSF C 52.9% 94.1% 35.1% 97.1% MSF C 84.3% 67.4% 13.4% 98.6% Bio-Rad 900 Sens Spec PPV NPV BR C 92.2% 96.8% 63.5% 99.5% BR C 92.2% 42.0% 8.7% 98.9% BR C 88.2% 98.8% 81.8% 99.3% BR C 88.2% 47.0% 9.1% 98.5% 16

17 Table 7. Results of 24h and 48h growth for the total 2500 specimens incubated at 37 C MSF 2500 Media, inc, temp MRSA NO MRSA Total Yellow colonies MSF C MSF C No growth or No yellow colonies MSF C MSF C Gold Standard Bio-Rad 2500 Media, inc, temp MRSA NO MRSA Total Pink colonies BIO C BIO C No growth or No pink colonies BIO C BIO C Gold Standard Table 8. Percent sensitivities, specificities, positive and negative predictive values for the total 2500 specimens incubated at 37 C MSF 2500 Sens Spec PPV NPV PDLR NDLR MSF C 51.7% 91.3% 27.0% 96.8% MSF C 82.3% 68.1% 14.0% 98.4% Bio-Rad 2500 Sens Spec PPV NPV PDLR NDLR BR C 91.2% 97.9% 73.2% 99.4% BR C 91.2% 48.2% 9.9% 98.9%

18 Table 9. Results of 24h and 48h growth per specimen type incubated at 37 C WOUND SWABS (Total number = 222) MSF 24h MRSA NO MRSA Total Yellow colonies No growth or No yellow colonies Gold Standard BR 24h Pink colonies No growth or No pink colonies Gold Standard MSF 48h Yellow colonies No growth or No yellow colonies Gold Standard BR 48h Pink colonies No growth or No pink colonies Gold Standard FAECAL SPECIMENS (Total number = 777) MSF 24h MRSA NO MRSA Total Yellow colonies No growth or No yellow colonies Gold Standard BR 24h Pink colonies No growth or No pink colonies Gold Standard MSF 48h Yellow colonies No growth or No yellow colonies Gold Standard BR 48h Pink colonies No growth or No pink colonies Gold Standard

19 Table 9. cont d NASAL SWABS (Total number = 823) MSF 24h MRSA NO MRSA Total Yellow colonies No growth or No yellow colonies Gold Standard BR 24h Pink colonies No growth or No pink colonies Gold Standard MSF 48h Yellow colonies No growth or No yellow colonies Gold Standard BR 48h Pink colonies No growth or No pink colonies Gold Standard AXILLA/GROIN SWABS (Total number = 59) MSF 24h MRSA NO MRSA Total Yellow colonies No growth or No yellow colonies Gold Standard BR 24h Pink colonies No growth or No pink colonies Gold Standard MSF 48h Yellow colonies No growth or No yellow colonies Gold Standard BR 48h Pink colonies No growth or No pink colonies Gold Standard

20 Table 9. cont d NASAL-AXILLA-GROIN-PERINEUM SWABS (Total number = 619) MSF 24h MRSA NO MRSA Total Yellow colonies No growth or No yellow cols Gold Standard BR 24h Pink colonies No growth or No pink cols Gold Standard MSF 48h Yellow colonies No growth or No yellow cols Gold Standard BR 48h Pink colonies No growth or No pink cols Gold Standard

21 Table 10. Percent sensitivities, specificities, positive and negative predictive values obtained at 24h and 48h per specimen type incubated at 37 C WOUND SWABS (Tn=222) % SENS % SPEC % PPV % NPV PDLR NDLR MSF 24h MSF 48h BR 24h BR 48h FAECAL SPECIMENS (Tn=777) MSF 24h MSF 48h BR 24h BR 48h NASAL SWABS (Tn=823) MSF 24h MSF 48h BR 24h BR 48h AXILLA/GROIN SWABS (Tn=59) MSF 24h MSF 48h Minute BR 24h Infinite 0.1 BR 48h NASAL-AXILLA-GROIN-PERINEUM SWABS (Tn=619) MSF 24h MSF 48h BR 24h BR 48h

22 Table 11. Summary of presumptive identifications of pink/yellow colonies that were not MRSA Bio-Rad MRSA Select agar 37 C n= C n= C n=900 Organism or ID 24h 48h Total 24h 48h Total 24h 48h Total Not Staphylococcus spp. (catalase-neg) Enterococcus species Gram positive bacilli Enterobacteriacae 1 1 Coagulase-negative staphylococci Diphtheroids Staphylococcus lugdenensis 1 Yeast Bacillus species Auto agglutinations/tc-negative Mannitol Salt Cefoxitin agar 37 C n= C n= C n=900 Organism or ID 24h 48h Total 24h 48h Total 24h 48h Total Enterococcus species Enterobacteriacae 1 1 Yeast 1 1 Auto agglutinations/tc-negative 2 2 Coagulase-negative staphylococci