Abstract
Matrix-assisted laser desorption–ionization/time of flight mass spectrometry (MALDI-TOF MS) has been widely implemented for the rapid identification of microorganisms. Although most bacteria, yeasts and filamentous fungi can be accurately identified with this method, some closely related species still represent a challenge for MALDI-TOF MS. In this study, two MALDI-TOF-based approaches were applied for discrimination at the species-level of isolates belonging to the Cryptococcus neoformans complex, previously characterized by Amplified Fragment Length Polymorphism (AFLP) and sequencing of the ITS1-5.8S-ITS2 region: (i) an expanded database was built with 26 isolates from the main Cryptococcus species found in our setting (C. neoformans, C. deneoformans and AFLP3 interspecies hybrids) and (ii) peak analysis and data modeling were applied to the protein spectra of the analyzed Cryptococcus isolates. The implementation of the in-house database did not allow for the discrimination of the interspecies hybrids. However, the performance of peak analysis with the application of supervised classifiers (partial least squares-discriminant analysis and support vector machine) in a two-step analysis allowed for the 96.95% and 96.55% correct discrimination of C. neoformans from the interspecies hybrids, respectively. In addition, PCA analysis prior to support vector machine (SVM) provided 98.45% correct discrimination of the three analyzed species in a one-step analysis. This novel method is cost-efficient, rapid and user-friendly. The procedure can also be automatized for an optimized implementation in the laboratory routine.
Highlights
The genus Cryptococcus has classically comprised two sibling species with great clinical importance: Cryptococcus neoformans and C. gattii, the causative agents of cryptococcosis
The application of MALDI-TOF MS and the commercial database allowed the correct identification of 18/22 C. neoformans isolates (81.8%) and 1/3 C. deneoformans isolates (33.3%); the remaining
The identification of the interspecies hybrids (n = 19) was not achieved using the commercial database due to the lack of representation of this microorganism. These isolates were identified as C. neoformans complex in nine cases, as C. deneoformans in seven cases and as C. neoformans in three cases—Table 1
Summary
The genus Cryptococcus has classically comprised two sibling species with great clinical importance: Cryptococcus neoformans and C. gattii, the causative agents of cryptococcosis. C. neoformans; AFLP2/VNIV for C. deneoformans, AFLP3/VNIII for the interspecies hybrid C. neoformans x C. deneoformans; and AFLP4/VGI, AFLP5/VGIII, AFLP6/VGII, AFLP7/VGIV and AFLP10/VGIV-VGIIIc for the C. gattii complex [15,16] These molecular techniques have been shown to be accurate and robust, the applied procedure is cumbersome, time consuming and delays the final identification. Non-Candida yeasts still represent a challenge for this technology, especially when applied to the identification of genera poorly represented or lacking in the commercial databases [21]. In this case, expanded in-house databases containing protein spectra from the underrepresented species have been shown to overcome this drawback [17]. A database was built using well characterized isolates for the identification of the Cryptococcus spp. isolates using the Biotyper system developed by Bruker Daltonics (Bremen, Germany) and the identifications obtained were compared with the peak-analysis method
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