ABSTRACT
Yeasts of the Cryptococcus neoformans/gattii species complexes are human pathogens mostly in immune compromised individuals, and can cause infections from dermal lesions to fungal meningitis. Differences in virulence and antifungal drug susceptibility of species in these complexes indicate the value of full differentiation to species level in diagnostic procedures. MALDI-TOF MS has been reported to sufficiently discriminate these species. Here, we sought to re-evaluate sample pre-processing procedures and create a set of publicly available references for use with the MALDI Biotyper system. Peak content using four different pre-processing protocols was assessed, and database entries for 13 reference strains created. These were evaluated against a collection of 153 clinical isolates, typed by conventional means. The use of decapsulating protocols or mechanical disruption did not sufficiently increase the information content to justify the extra hands-on-time. Using the set of 13 reference entries created with the standard formic acid extraction, we were able to correctly classify 143/153 (93.5%) of our test isolates. The majority of the remaining ten isolates still gave correct top matches; only two isolates did not give reproducible identifications. This indicates that the log score cut-off can be lowered also in this context. Ease to identify cryptococcal isolates to the species level is improved by the workflow evaluated here. The database references are freely available from https://github.com/oliverbader/BioTyper-libraries for incorporation into local diagnostic systems.
Subject(s)
Cryptococcosis , Cryptococcus gattii , Cryptococcus neoformans , Antifungal Agents , Humans , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
MALDI-ToF mass spectrometry offers fast and reliable species identification for bacteria and yeasts under clinical routine conditions. Here, we produced mass spectra for identification of clinically important species of the Pseudallescheria/Scedosporium complex using the recently suggested new nomenclature and use this example to discuss to what extent the principle of DNA barcoding might be transferred to mass spectrometry.
Subject(s)
DNA Barcoding, Taxonomic/methods , Mass Spectrometry/methods , Mycoses/microbiology , Proteomics/methods , Pseudallescheria/isolation & purification , Scedosporium/isolation & purification , DNA, Fungal/genetics , Humans , Phylogeny , Pseudallescheria/chemistry , Pseudallescheria/genetics , Pseudallescheria/metabolism , Scedosporium/chemistry , Scedosporium/genetics , Scedosporium/metabolismABSTRACT
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based species identification has become a reliable and fast tool for use in clinical diagnostics, including in mycology. To identify yeasts in the MALDI Biotyper system, a multistep extraction protocol, which is also used to generate the reference spectra, is recommended. Sample preparation by on-target lysis (OTL) requires significantly less hands-on time and is therefore highly desirable, but it results in too-low MALDI Biotyper log score values to allow automated species identification. To overcome this problem, we developed a procedure for generating and validating an OTL spectrum data set for the most relevant and frequently occurring yeast species in clinical specimens. The performance was evaluated against a set of OTL spectra derived during clinical routine procedures and from a set of closely related yeasts. In the diagnostic setting, the OTL procedure significantly decreased the workload but allowed species identification with high specificity and sensitivity. False identifications were not observed. The use of in-house-generated OTL reference spectra can highly accelerate MALDI-TOF MS-based yeast species identification using the MALDI Biotyper.
