Potential Use of MALDI-ToF Mass Spectrometry for Rapid Detection of Antifungal Resistance in the Human Pathogen Candida glabrata

Abstract

The echinocandins are relatively new antifungal drugs that represent, together with the older azoles, the recommended and/or preferred agents to treat candidaemia and other forms of invasive candidiasis in human patients. If “time is of the essence” to reduce the mortality for these infections, the administration of appropriate antifungal therapy could be accelerated by the timely reporting of laboratory test results. In this study, we attempted to validate a MALDI-ToF mass spectrometry-based assay for the antifungal susceptibility testing (AFST) of the potentially multidrug-resistant pathogen Candida glabrata against anidulafungin and fluconazole. The practical applicability of the assay, reported here as MS-AFST, was assessed with a panel of clinical isolates that were selected to represent phenotypically and genotypically/molecularly susceptible or resistant strains. The data show the potential of our assay for rapid detection of antifungal resistance, although the MS-AFST assay performed at 3 h of the in vitro antifungal exposure failed to detect C. glabrata isolates with echinocandin resistance-associated FKS2 mutations. However, cell growth kinetics in the presence of anidulafungin revealed important cues about the in vitro fitness of C. glabrata isolates, which may lead to genotypic or phenotypic antifungal testing in clinical practice.

Proof of Concept for MBT ASTRA, a Rapid Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS)-Based Method To Detect Caspofungin Resistance in Candida albicans and Candida glabrata

“…So far, rapid susceptibility testing by MALDI-TOF MS has mainly been described for bacteria (23,27,(30)(31)(32)(33). One research group has reported on a rapid AFST approach by analyzing the similarities of the MALDI-TOF MS profile spectra derived from different setups containing increasing antibiotic concentrations (34)(35)(36). MBT ASTRA is a semiquantitative method that was first applied for the detection of meropenem susceptibility in bacteria derived from culture plates or from positive blood cultures within 1 to 4 h (32).…”

“…In contrast, the approach of Vella and colleagues (34) analyzed changes in the MS profile spectra induced by antifungals. A recent study of the same group (36) has shown that this approach does not accurately work for C. glabrata strains that are resistant to anidulafungin and that have known FKS2 mutations (36). The complete assay setup of MBT ASTRA is like that of the commonly accepted microdilution method, which is generally accepted as the gold standard.…”

Anidulafungin Susceptibility Testing of Candida glabrata Isolates from Blood Cultures by the MALDI Biotyper Antibiotic (Antifungal) Susceptibility Test Rapid Assay

“…In another study, an AFST-based MALDI-TOF assay was developed to detect C. glabrata isolates resistant to anidulafungin within 3 h. This method was based on the detection of changes in the protein profile spectrum relative to the antifungal concentration by MALDI-TOF MS. The AFST-based MALDI-TOF assay was useful to detect strains with mutations in FKS1; however, it failed to detect C. glabrata resistant isolates carrying known FKS2 mutations (27).…”

New pathogens, new tricks: emerging, drug‐resistant fungal pathogens and future prospects for antifungal therapeutics

“…Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry has been successfully exploited for species‐level fungal pathogen identification based on unique proteomic signatures . This approach has been particularly useful in the identification of rare and commonly misidentified fungal species, including unusual Aspergillus pathogens, and the identification of antifungal drug‐resistant Candida strains . Moreover, quantitative bottom‐up proteomic analysis combined with selected reaction monitoring has discovered secreted cryptococcal proteins in infected tissue samples, suggesting the utility of such techniques for detection of diagnostic biomarkers of fungal infection .…”

“…210,211 This approach has been particularly useful in the identification of rare and commonly misidentified fungal species, including unusual Aspergillus pathogens, 212,213 and the identification of antifungal drug-resistant Candida strains. 214,215 Moreover, quantitative bottom-up proteomic analysis combined with selected reaction monitoring has discovered secreted cryptococcal proteins in infected tissue samples, suggesting the utility of such techniques for detection of diagnostic biomarkers of fungal infection. 216 Finally, compelling recent work has exploited engineered Saccharomyces cerevisiae as a biosensor capable of detecting fungal pathogen-derived peptides in order to develop a low-cost and nontechnical fungal diagnostic.…”