title: Complementary use of MALDI-TOF MS and real-time PCR-melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE.
abstract: OBJECTIVE
  To develop a rapid method for routine screening of methicillin-resistant staphylococci and VRE for clinical isolates and positive blood cultures.
  
  
  METHODS
  Our method consisted of two parts: MALDI-TOF MS was used for identification of staphylococci and enterococci, followed by antibiotic resistance detection by real-time PCR-melt curve analysis without DNA extraction. The latter part included a triplex reaction for staphylococcal culture isolates (mecA, mecALGA251 and Panton-Valentine leucocidin genes), dual PCR of mecA/mecALGA251 and nuc genes for staphylococcal blood cultures, and a duplex reaction for enterococci (vanA and vanB genes). A total of 124 clinical isolates and 56 positive blood cultures were tested. MALDI-TOF MS was performed using Microflex LT (Bruker Daltonik, Bremen, Germany) and Rotor-Gene Q (Qiagen, Hilden, Germany) was used for real-time PCR-melt curve analysis. The total assay time was <2.5 h.
  
  
  RESULTS
  The results revealed 100% concordance with antibiotic susceptibility testing or other reference methods for all culture isolates and enterococcal blood cultures. The percentage of concordance for staphylococcal blood cultures was 97.5%.
  
  
  CONCLUSIONS
  The method described herein was fast, economical, reliable and capable of detecting mecALGA251, vanB1 and vanB2 genotypes, which are not included in most commercial assays. Large-scale screening is required to further test the performance of this protocol, especially for genotypes that are infrequently encountered.
authors.affiliation: Department of Pathology, Hong Kong Sanatorium & Hospital, Hong Kong, China.
authors.forename: Wai-Sing
authors.initials: WS
authors.lastname: Chan
authors.affiliation: Department of Pathology, Hong Kong Sanatorium & Hospital, Hong Kong, China.
authors.forename: Tsz-Ming
authors.initials: TM
authors.lastname: Chan
authors.affiliation: Department of Pathology, Hong Kong Sanatorium & Hospital, Hong Kong, China.
authors.forename: Tsz-Wan
authors.initials: TW
authors.lastname: Lai
authors.affiliation: State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong, China.
authors.forename: Jasper Fuk-Woo
authors.initials: JF
authors.lastname: Chan
authors.affiliation: Department of Microbiology, Prince of Wales Hospital, Hong Kong, China.
authors.forename: Raymond Wai-Man
authors.initials: RW
authors.lastname: Lai
authors.affiliation: Department of Pathology, Queen Elizabeth Hospital, Hong Kong, China.
authors.forename: Christopher Koon-Chi
authors.initials: CK
authors.lastname: Lai
authors.affiliation: Department of Pathology, Hong Kong Sanatorium & Hospital, Hong Kong, China bonetang@hksh.com.
authors.forename: Bone Siu-Fai
authors.initials: BS
authors.lastname: Tang
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics; D016983:Enterococcus / Q000145:classification* / Q000187:drug effects / Q000235:genetics*; D005098:Exotoxins / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D007956:Leukocidins / Q000235:genetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000145:classification* / Q000187:drug effects / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D020713:Vancomycin Resistance / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D005098:Exotoxins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin
keywords: MRSA; VRE; antibiotic resistance; mixed blood cultures; multiplex