title: Evaluation of three sample preparation methods for the direct identification of bacteria in positive blood cultures by MALDI-TOF.
abstract: BACKGROUND
  Patient mortality is significantly reduced by rapid identification of bacteria from sterile sites. MALDI-TOF can identify bacteria directly from positive blood cultures and multiple sample preparation methods are available. We evaluated three sample preparation methods and two MALDI-TOF score cut-off values. Positive blood culture bottles with organisms present in Gram stains were prospectively analysed by MALDI-TOF. Three lysis reagents (Saponin, SDS, and SepsiTyper lysis bufer) were applied to each positive culture followed by centrifugation, washing and protein extraction steps. Methods were compared using the McNemar test and 16S rDNA sequencing was used to assess discordant results.
  
  
  RESULTS
  In 144 monomicrobial cultures, using ≥2.000 as the cut-off value, species level identifications were obtained from 69/144 (48%) samples using Saponin, 86/144 (60%) using SDS, and 91/144 (63%) using SepsiTyper. The difference between SDS and SepsiTyper was not statistically significant (P = 0.228). Differences between Saponin and the other two reagents were significant (P < 0.01). Using ≥1.700 plus top three results matching as the cut-off value, species level identifications were obtained from 100/144 (69%) samples using Saponin, 103/144 (72%) using SDS, and 106/144 (74%) using SepsiTyper and there was no statistical difference between the methods. No true discordances between culture and direct MALDI-TOF identification were observed in monomicrobial cultures. In 32 polymicrobial cultures, MALDI-TOF identified one organism in 34-75% of samples depending on the method.
  
  
  CONCLUSIONS
  This study demonstrates two inexpensive in-house detergent lysis methods are non-inferior to a commercial kit for analysis of positive blood cultures by direct MALDI-TOF in a clinical diagnostic microbiology laboratory.
authors.affiliation: Public Health England, Public Health Laboratory Birmingham, Heartlands Hospital, Bordesley Green East, Birmingham, B9 5SS, UK. hannah.tanner@heartofengland.nhs.uk.
authors.forename: Hannah
authors.identifier: http://orcid.org/0000-0003-3378-3219
authors.initials: H
authors.lastname: Tanner
authors.affiliation: Public Health England, Public Health Laboratory Birmingham, Heartlands Hospital, Bordesley Green East, Birmingham, B9 5SS, UK.|Wales Centre for Mycobacteria, Public Health Wales, University Hospital Llandough, Penarth, CF64 2XX, UK.
authors.forename: Jason T
authors.initials: JT
authors.lastname: Evans
authors.affiliation: Public Health England, Public Health Laboratory Birmingham, Heartlands Hospital, Bordesley Green East, Birmingham, B9 5SS, UK.
authors.forename: Savita
authors.initials: S
authors.lastname: Gossain
authors.affiliation: Public Health England, Public Health Laboratory Birmingham, Heartlands Hospital, Bordesley Green East, Birmingham, B9 5SS, UK.
authors.forename: Abid
authors.initials: A
authors.lastname: Hussain
mesh_terms: D016470:Bacteremia / Q000382:microbiology; D001419:Bacteria / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000382:microbiology*; D001431:Bacteriological Techniques; D000071997:Blood Culture; D001788:Blood Grouping and Crossmatching; D002498:Centrifugation; D004269:DNA, Bacterial / Q000302:isolation & purification; D003902:Detergents / Q000737:chemistry; D019075:Electrophoresis, Capillary; D006801:Humans; D012336:RNA, Ribosomal, 16S / Q000302:isolation & purification; D012503:Saponins / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D003902:Detergents; D012336:RNA, Ribosomal, 16S; D012503:Saponins; D012967:Sodium Dodecyl Sulfate
keywords: Bacteraemia; Blood culture; Clinical; Identification; MALDI-TOF; Microbiology; Sepsis