Nasal and perianal swab specimens were tested for detection of Staphylococcus aureus and vancomycinresistant Enterococcus species (VRE) using a laboratory-developed real-time PCR test and microbiological cultures. The real-time PCR and culture results for S. aureus were similar. PCR had adequate sensitivity, but culture was more specific for the detection of VRE.Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), and vancomycin-resistant Enterococcus species (VRE) are important pathogens associated with health care facility outbreaks worldwide (1,2,8,21). It has been reported that identifying asymptomatically colonized individuals and placing them into contact isolation within a short time frame are a good management tool for reducing the spread of these pathogens and for lowering health care-associated infections (5,11,16,18). Furthermore, there is rising interest in identifying all S. aureus carriers prior to surgery to decolonize them to reduce postoperative S. aureus surgical-site infections (4, 10).Reliable surveillance requires accurate testing (17), and cultures can take from 2 to 5 days to obtain the final results (3a). PCR offers detection of S. aureus and VRE directly from swab specimens within a few hours (4,12,14) and can help with the rapid deployment of infection control and prevention measures (16,18).The purpose of our study was to develop an optimized test using Roche analyte-specific reagents (ASRs) for detection of S. aureus and VRE by real-time PCR with a single set of amplification conditions and to compare the PCR results to the results of conventional culture.Inpatients at Evanston Northwestern Healthcare during August 2004 made up the patient population. Premoistened, double-headed swabs (culture swab; BBL, Becton Dickinson, Franklin Lakes, NJ) were used to collect paired anterior nasal specimens and paired perianal specimens as part of an infection control activity to determine colonization prevalence. There were 387 nasal specimens (for S. aureus) and 309 perianal specimens (for VRE). This investigation was approved by the Institutional Review Board of Evanston Northwestern Healthcare.For S. aureus, one of the paired nasal swabs was plated onto Columbia colistin-nalidixic agar with 5% sheep blood (Remel, Inc., Lenexa, KS) (3a) and incubated at 35°C for 48 h. S. aureus was identified by colony morphology and Staphaurex latex agglutination testing (Remel, Inc.). Methicillin resistance was determined on colonies by using PCR as described below. For any swabs that were PCR positive only for S. aureus (n ϭ 13), the original cultures were reexamined beyond their initial 48-hour incubation, with five additional swab samples yielding S. aureus after reexamination (two yielded MRSA).VRE was cultured by plating one of the paired perianal swabs on bile esculin azide agar containing 6 g of vancomycin/ml agar (Remel, Inc.) and by incubation at 35°C for 72 h. Colonies black in color (bile esculin positive) were confirmed to be Enterococcus faecium or Enterococcus faecalis by usi...