Optimization of a Laboratory-Developed Test Utilizing Roche Analyte-Specific Reagents for Detection of Staphylococcus aureus , Methicillin-Resistant S. aureus , and Vancomycin-Resistant Enterococcus Species

Abstract

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...

Performance Characteristics of the Cepheid Xpert vanA Assay for Rapid Identification of Patients at High Risk for Carriage of Vancomycin-Resistant Enterococci

“…1ϩ, 1 to 9 colonies; 2ϩ, 1 to 49 colonies; 3ϩ, 5 to 300 colonies; 4ϩ, Ͼ300 colonies. ego, CA) and the Roche LightCycler analyte-specific reagents (ASRs) (15,17,21). Different from Bourdon et al, our findings showed a higher PPV (91%, versus 66.7% in the study by Bourdon et al) after resolution of discrepant results for the vanA gene.…”

Identification of Staphylococcus Species Directly from Positive Blood Culture Broth by Use of Molecular and Conventional Methods

“…The mecA PCR results were identical for both the in-house PCR assay and the laboratory-optimized Roche ASR assay, similar to our prior experience (12). Of the 199 specimens tested, 85 (42.7%) were negative and 114 specimens (57.3%) were positive by both assays.…”

Evaluation of GeneXpert vanA/vanB assay for the detection of vancomycin-resistant enterococci in patients newly admitted to intensive care units

“…Despite this, we have discrepant results between PCR and culture. Studies issuing false-positive results due to vanB suggest that follow-up culture be performed on any vanB-positive results (11,25,26). If the presence of vanB is low in the setting, culture backup may not be warranted.…”