A BLINDED COMPARISON OF T H R E E LABORATORY PROTOCOLS FOR THE IDENTIFICATION OF PATIENTS

OBJECTIVE: To compare three laboratory screening protocols for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from surveillance specimens (mannitol-salt agar containing 2 ug/mL of oxacillin [MSA-2], mannitol-salt agar containing 4 ug/mL of oxacillin [MSA-4], and a broth-containing protocol as recommended by the American Society for Microbiology [M-ASM]). DESIGN: Blinded comparative laboratory study and cost analysis. SETTING: University-affiliated microbiology laboratory. METHODS: Outcome measurements included rate of detection of MRSA-positive specimens and patients, turnaround time, and media and technologist costs. All MRSA culture swabs obtained from any patient site from November 1998 to April 1999 were included. RESULTS: The M-ASM protocol detected between 19.1% and 32.0% more MRSA-positive specimens and between 13.3% and 23.3% more MRSA-positive patients per surveillance event than the MSA-4 and MSA-2 protocols, respectively. There was no difference in positive-culture reporting time between the M-ASM and MSA-4 protocols. The broth-containing protocol was 2to 2.5-fold more expensive than the simpler protocols, taking into account media and laboratory personnel costs. CONCLUSIONS: It remains to be determined whether it is cost beneficial for a hospital to adopt the M-ASM, as the potential cost of MRSA transmission from unidentified MRSA-colonized patients is unknown. A broth-containing protocol should be considered the gold standard in future studies examining newer MRSA screening protocols (Infect Control Hosp Epidemiol 2001;22:152-156). Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most significant nosocomial pathogens of the past 2 decades, being responsible for significant patient morbidity, mortality, and healthcare costs. In an effort to limit the nosocomial spread of MRSA, many centers rely on surveillance programs to identify asymptomatic colonized patients, so that appropriate barrier precautions may be used to interrupt transmission. The laboratory protocol used to isolate MRSA from surveillance sites is a critical component to such programs. Different laboratory protocols have been used for the detection of MRSA-colonized patients. Several small non-blinded studies have examined the use of staphylococcal broth cultures in addition to solid-agar media and have reported that broth culture increases the sensitivity of detecting MRSA from clinical specimens from 11% to 60% over solid-agar media. Based on these studies, the American Society for Microbiology (ASM) recommends as primary culture media a salt-containing, tryptone-based broth in conjunction with a blood agar (BA) plate and a mannitol-salt agar (MSA) plate containing 4 ug/mL of oxacillin. It remains uncertain, however, whether a brothcontaining protocol is truly more sensitive than a solid-agar protocol, as none of the aforementioned studies were blinded. Furthermore, it is unknown whether broth-containing protocols actually detect more MRSA-colonized patients or just a larger percentage of culture sites. Finally, neither the reporting times of broth-containing protocols nor their associated costs have been addressed adequately. We undertook a study of the sensitivity of three surveillance protocols: MSA solid agar containing 2 and 4 ug/mL of oxacillin (MSA-2 and MSA-4 protocols, respectively) and a modified broth-containing ASM protocol (M-ASM protocol) where the subculture MSA plate was replaced by an MSA4 plate to decrease bacterial overgrowth. Reporting times for positive and negative results, as well as laboratory and technologist costs, were determined. From the Infection Prevention and Control Unit (Drs, Gardam, Brunton, and Conly), Department of Microbiology, Toronto Medical Laboratories, University Health Network, University of Toronto; and Department of Microbiology (Drs. McGeer and Low; Ms. Willey), Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada. Address reprint requests to Michael Gardam, MSc, MD, 200 Elizabeth St, NUW13-132, Toronto General Hospital, University Health Network, Toronto, Ontario M5G 2C4, Canada. This research has been presented in part at the 39th ICAAC; September 1999; San Francisco, California. The authors thank the following individuals: Iris Ann Edwards, Helen Didier, Kit Wong, and Dr. Ivii Campbell for laboratory assistance, and Zahir Hirji for assistance in data collection. 00-OA-024. Gardam M, Brunton J, Willey B, McGeer A, Low D, Conly J. A blinded comparison of three laboratory protocols for the identification of patients colonized with methicillin-resistant Staphylococcus aureus. Infect Control Hosp Epidemiol 2001;22:152-156. Vol. 22 No. 3 BROTH CULTURE FOR THE DETECTION OF MRSA 153 METHODS Specimen Source and Processing The microbiology section of the Toronto Medical Laboratories receives 30 to 60 MRSA surveillance specimens daily from six Toronto hospitals (including universityaffiliated teaching hospitals, community hospitals, and chronic-care institutions). Twenty random specimens received by the laboratory each day were processed in triplicate in the following manner: each culture swab was streaked on a sheep BA plate (PML Microbiologicals, Mississauga, Ontario, Canada); an MSA-2 plate (Quelab, Montreal, Quebec, Canada); two MSA-4 plates (Oxoid, Nepean, Ontario, Canada); and finally broken off into a tryptone-based broth containing 7.5% NaCl, 1% mannitol, and 0.25% yeast extract (Difco Laboratories, Detroit, MI). The MSA-2 plate, MSA4 plate, and the M-ASM media (BA plate, MSA-4 plate, and broth culture) were delivered to three different sections of the laboratory. The three protocols were worked on by different experienced technologists who were blinded to each other's work. The technologists were not rotated through the different protocols. Laboratory Protocols All media were incubated at 35°C in 5% C02. The primary agar media of all three protocols were examined at 24 and 48 hours for typical staphylococcal colonies. The broth culture was subcultured after 24 hours to a BA plate and an MSA-4 plate which, in turn, were examined at 24 and 48 hours. Suspicious colonies were identified as S aureus by catalase production, positive slide coagulase (Staphaurex; Murex Biotech Limited, Kent, England), or positive tube coagulase tests. When required, subculture onto colistinnaladixic agar or DNase testing were performed. All presumed MRSA isolates were confirmed for oxacillin resistance by growth on a Mueller-Hinton agar containing 4 ug/mL oxacillin, Vitek gram-positive sensitivity card testing (bioMerieux, Marcy l'Etoile, France), inoculation of a BBL crystal (Becton-Dickinson, Cockeysville, MD), or polymerase chain reaction for the mecA gene. Technologist Times, Reporting Times, and Cost Analysis Technologist times for the M-ASM and MSA-4 protocols were estimated from random time-motion observations. For the M-ASM protocol, reporting times also were calculated separately for each of the primary media. Media costs for the M-ASM protocol were tabulated daily by a technologist, whereas media costs of the MSA-4 protocol were estimated, taking into account the percentage of MRSA-positive specimens reported. Estimated costs relating to MRSA colonization or infection for a Canadian hospital were obtained from the literature. All costs are reported in Canadian dollars. Statistical Analysis Data were entered into SPSS (version 8.0; SPSS Inc, Chicago, IL), and statistical comparisons were tested at the 5% level of significance for a two-tailed test, using either the McNemar Test for correlated proportions or the binomial distribution, where appropriate. The number of MRSAcolonized patients detected by the three protocols was calculated by analyzing whether a patient would have been declared MRSA-positive or -negative by each of the three protocols for each day that surveillance cultures were performed (defined as a "surveillance event"). Review of our laboratory results indicated that approximately 8% of surveillance specimens are positive for MRSA using the MSA-4 protocol. Assuming that the MASM protocol would detect 25% to 35% more positive specimens, as suggested in the literature, the study would have an 80% power of detecting a significant difference between the two protocols if 716 to 2,266 paired specimens were processed.