Rapid Antifungal Susceptibility Testing by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis

Abstract

eThe widespread use of antifungal agents, which is likely to expand with their enhanced availability, has promoted the emergence of drug-resistant strains. Antifungal susceptibility testing (AFST) is now an essential procedure for guiding appropriate antifungal therapy. Recently, we developed a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based method that enables the detection of fungal isolates with reduced echinocandin susceptibility, relying on the proteome changes that are detectable after a 15-h exposure of fungal cells to serial drug concentrations. Here, we describe a simplified version of this approach that facilitates discrimination of the susceptible and resistant isolates of Candida albicans after a 3-h incubation in the presence of "breakpoint" level drug concentrations of the echinocandin caspofungin (CSF). Spectra at concentrations of 0 (null), 0.03 (intermediate), and 32 (maximal) g/ml of CSF were used to create individual composite correlation index (CCI) matrices for 65 C. albicans isolates, including 13 fks1 mutants. Isolates are then classified as susceptible or resistant to CSF if the CCI values of spectra at 0.03 and 32 g/ml are higher or lower, respectively, than the CCI values of spectra at 0.03 and 0 g/ml. In this way, the drug resistance of C. albicans isolates to echinocandin antifungals can be quickly assessed. Furthermore, the isolate categorizations determined using MALDI-TOF MS-based AFST (ms-AFST) were consistent with the wild-type and mutant FKS1 genotypes and the AFST reference methodology. The ms-AFST approach may provide a rapid and reliable means of detecting emerging antifungal resistance and accelerating the initiation of appropriate antifungal treatment.

Intensive care medicine research agenda on invasive fungal infection in critically ill patients

“…Turnaround times are 10-15 min after the growth of a fungal colony or a positive blood culture, and diagnostic accuracy is over 95%. Additional work is required to make MALDI-TOF MS suitable for the detection of antifungal resistance [33].…”

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

“…Notably, earlier MALDI systems used for fungal identification required a protein extraction step prior to the spectrometric analysis, whereas newer methods, such as the Vitek MS system, manage to overcome this by using a single deposit step that avoids formic acid lysis, thus making the technique faster and easier to use (274). Moreover, a MALDI method to rapidly assess the caspofungin susceptibility of isolated yeast and Aspergillus species was recently described (279) and subsequently modified to reduce turnaround times (280). This method, which is based on the identification of changes in the protein composition of fungal cells exposed to caspofungin, was designed as a much faster (3 h versus 24 h) alternative to the traditional CLSI method, with comparable results.…”

Development and Validation of an In-House Database for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Yeast Identification Using a Fast Protein Extraction Procedure

“…The relationships between the replicates of each set of spectra were estimated by the composite correlation index analysis, where values of around 1 represent a high conformance of the spectra and values near 0 indicate clear diversity of the spectra (21). Indeed, each database entry was generated as a composite of 10 spectra, which were imported into the MALDI Biotyper software to produce a list of the most significant peaks in terms of average masses, average intensities, and relative frequencies (1).…”