Decision criteria for MALDI-TOF MS-based identification of filamentous fungi using commercial and in-house reference databases

Abstract

BackgroundSeveral Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry protocols, which differ in identification criteria, have been developed for mold and dermatophyte identification. Currently, the most widely used approach is Bruker technology, although no consensus concerning the log(score) threshold has been established. Furthermore, it remains unknown how far increasing the number of spots to compare results might improve identification performance.In this study, we used in-house and Bruker reference databases as well as a panel of 422 isolates belonging to 126 species to test various thresholds. Ten distinct identification algorithms requiring one to four spots were tested.ResultsOur findings indicate that optimal results were obtained by applying a decisional algorithm in which only the highest score of four spots was taken into account with a 1.7 log(score) threshold. Testing the entire panel enabled identification of 87.41% (in-house database) and 35.15% (Bruker database) of isolates, with a positive predictive value (PPV) of 1 at the genus level for both databases as well as 0.89 PPV (in-house database) and 0.72 PPV (Bruker database) at the species level. Applying the same rules to the isolates for which the species were represented by at least three strains in the database enabled identification of 92.1% (in-house database) and 46.6% (Bruker database) of isolates, with 1 PPV at the genus level for both databases as well as 0.95 PPV (in-house database) and 0.93 PPV (Bruker database) at the species level.ConclusionsDepositing four spots per extract and lowering the threshold to 1.7, a threshold which is notably lower than that recommended for bacterial identification, decreased the number of unidentified specimens without altering the reliability of the accepted results. Nevertheless, regardless of the criteria used for mold and dermatophyte identification, commercial databases require optimization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-0937-2) contains supplementary material, which is available to authorized users.

Fast identification of dermatophytes by MALDI‐TOF/MS using direct transfer of fungal cells on ground steel target plates

“…According to the manufacturer, LS cut‐offs for genus‐level and species‐level identification are 1.7 and 2.0, respectively. However, those rules apply for bacteria and recent studies suggest that a LS threshold of 1.7 is sufficient for a confident identification of filamentous fungi, as long as a robust reference database is available to perform the identifications . For library validation, each strain used to create the library was analysed in duplicate and tested against D‐MSPL and the filamentous fungi library from Bruker (B‐MSPL, version 1.0‐15 dermatophytes species represented by 51 spectra).…”

“…However, those rules apply for bacteria and recent studies suggest that a LS threshold of 1.7 is sufficient for a confident identification of filamentous fungi, as long as a robust reference database is available to perform the identifications. 19 For library validation, each strain used to create the library was analysed in duplicate and tested against D-MSPL and the filamentous fungi library from Bruker (B-MSPL, version 1.0-15 dermatophytes species represented by 51 spectra). Direct transfer of mycelium onto the ground steel target plate performed as previously reported 7 was used at first for library validation.…”

Substandard and falsified antimicrobials: A potential biohazard in disguise?

“…18,19 Identification of the encountered bacteria was performed according to the procedure utilized by Janvier et al 13 18,19 Identification of the encountered bacteria was performed according to the procedure utilized by Janvier et al 13 …”

“…When fungi were detected in the samples, they were analyzed by a validated matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) system, coupled to MALDI Biotyper 3 software (Bruker Daltonics, Bremen, Germany) containing both commercially available spectra and in-house generated spectra. 18,19 Identification of the encountered bacteria was performed according to the procedure utilized by Janvier et al 13…”

Evaluation of a Novel Mitochondrial Pan-Mucorales Marker for the Detection, Identification, Quantification, and Growth Stage Determination of Mucormycetes

“…Recent advances in the molecular diagnostics of mucormycetes have been comprehensively reviewed by Millon et al [5], but key challenges such as standardization remain unmet [8]. The identification of pure cultures using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) [9], plus the development of a novel pan-fungal antigen assay that detects a disaccharide in serum (MS-DS) and covers mucormycetes, are important developments [10]. Recent steps towards a pan-Mucorales marker system have included the use of real-time PCR with partial cytochrome B [11] as a template and a pan-Mucorales PCR that detects the spore coating protein (CotH) gene in urine samples [12,13].…”