title: Identification of Gram-positive cocci by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry: comparison of different preparation methods and implementation of a practical algorithm for routine diagnostics.
abstract: This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.
authors.affiliation: Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland.
authors.forename: Bettina
authors.initials: B
authors.lastname: Schulthess
authors.forename: Katharina
authors.initials: K
authors.lastname: Brodner
authors.forename: Guido V
authors.initials: GV
authors.lastname: Bloemberg
authors.forename: Reinhard
authors.initials: R
authors.lastname: Zbinden
authors.forename: Erik C
authors.initials: EC
authors.lastname: Böttger
authors.forename: Michael
authors.initials: M
authors.lastname: Hombach
mesh_terms: D000465:Algorithms; D001431:Bacteriological Techniques / Q000379:methods*; D016908:Gram-Positive Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D006095:Gram-Positive Cocci / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification*; D006801:Humans; D011446:Prospective Studies; D013048:Specimen Handling / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
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