title: Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.
abstract: OBJECTIVE
  Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media.
  
  
  METHODS
  The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities.
  
  
  RESULTS
  A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs.
  
  
  CONCLUSIONS
  The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening.
authors.affiliation: Department of Laboratory Medicine, Changi General Hospital, Singapore. Electronic address: thean_yen_tan@cgh.com.sg.
authors.forename: Thean Yen
authors.initials: TY
authors.lastname: Tan
authors.affiliation: Department of Laboratory Medicine, Changi General Hospital, Singapore.
authors.forename: Boran
authors.initials: B
authors.lastname: Jiang
authors.affiliation: Department of Laboratory Medicine, Changi General Hospital, Singapore.
authors.forename: Lily Siew Yong
authors.initials: LSY
authors.lastname: Ng
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D019729:Carbon-Oxygen Ligases / Q000235:genetics; D002863:Chromogenic Compounds / Q000378:metabolism; D003365:Costs and Cost Analysis; D003470:Culture Media / Q000737:chemistry*; D013293:Enterococcus faecalis / Q000235:genetics / Q000302:isolation & purification; D016984:Enterococcus faecium / Q000235:genetics / Q000302:isolation & purification; D016908:Gram-Positive Bacterial Infections / Q000175:diagnosis*; D006801:Humans; D008403:Mass Screening / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D044366:Transition Temperature; D065507:Vancomycin-Resistant Enterococci / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D003470:Culture Media; C071437:VanA ligase, Bacteria; C079913:VanB protein, Enterococcus; D019729:Carbon-Oxygen Ligases
keywords: Enterococcus faecalis; Enterococcus faecium; chromogenic agar; vanA; vanB; vancomycin resistance