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    • The initial demonstration of the potential

    2023-03-01

    The initial demonstration of the potential of MALDI-TOF MS for testing antifungal susceptibility of pathogenic fungi came from a proof-of-concept study by Marinach et al. [31] who monitored the changes of the proteome of C. albicans 96 4 exposed to different concentrations of fluconazole. Using a rigorous statistical approach, the authors assessed the similarity of the mass spectra at each of the different intermediate concentrations with each of the mass spectra at two extreme concentrations (fluconazole 128 mg/L and fluconazole 0 mg/L), i.e. the ‘extreme’ spectra, to provide a classification of ‘nearer to the 128 mg/L’ or ‘nearer to the 0 mg/L’ spectrum. Hence, the minimal profile change concentration (MPCC) value was proposed as a new AFST end-point, that was defined as the lowest concentration of drug (i.e. fluconazole) at which a mass spectrum profile change is detected. Surprisingly, MPCCs did not differ by more than one dilution step from the MICs determined using the CLSI reference method, so that all the C. albicans isolates tested except one were correctly assigned to the category (resistant or susceptible) to which they were assigned by CLSI [31]. Taking advantage of these observations, we described in 2012 [32] the development of a MALDI-TOF MS-based assay for testing the susceptibility of fungal species to caspofungin. In this assay, we applied the composite correlation index (CCI) analysis to calculate a correlation matrix based on the mass spectra acquired after the fungal cells were exposed to different drug concentrations for 15 h. Following matching of the ‘intermediate’ mass spectra with each of the ‘extreme’ mass spectra, MPCC was assessed as the CCI value at which a spectrum is more similar to the spectrum observed at the maximal caspofungin (32 mg/L) concentration (maximum CCI) than the spectrum observed at the null caspofungin (0 mg/L) concentration (null CCI). Using a panel of WT and FKS1/FKS2 mutant isolates of Candida (34 isolates) and Aspergillus (ten isolates) species, we showed that the MPCC values were fully concordant with the CLSI MIC or the minimum effective concentration values for 100% of the isolates tested [32]. Although the MALDI-TOF MS-based assay may eliminate subjective visual end-point determination, it provides slight time savings compared with the CLSI reference method (15 h versus 24 h). Therefore, in 2013 [33] we simplified our MALDI-TOF MS-based assay to allow discrimination between susceptible and resistant isolates of C. albicans after 3 h of incubation of yeast cells in the presence of three caspofungin levels only: no drug (null concentration), intermediate (‘breakpoint’ concentration) and maximum (maximal concentration). The decision about susceptibility/resistance was achieved by determining the caspofungin concentration at which the CCI values obtained by matching the ‘breakpoint’ spectrum with the ‘maximal’ spectrum were, respectively, higher (for susceptible isolates) or lower (for resistant isolates) than the CCI values obtained when the ‘breakpoint’ spectrum was matched with the spectrum at null concentration. Analysis of WT and FKS1 mutant C. albicans isolates revealed that 100% (51/51) and 90.9% (10/11) of the isolates tested were correctly classified as caspofungin-susceptible and caspofungin-resistant, respectively, in accordance with the FKS1 genotype. The categorical agreement was 98.4%, with only one major error for an isolate harbouring a D648Y genotype—this mutation is known to confer a lower level of echinocandin resistance [33]. Although these data suggest that the MALDI-TOF MS-based assay has the potential to be introduced into clinical microbiology laboratories, the reproducibility and robustness of the assay still have to be definitively proven. Unsurprisingly, in a study performed similarly to that described originally by us [32] but modified to facilitate the discrimination of susceptible and resistant isolates of Candida species (35 C. albicans, 35 C. glabrata and 37 C. tropicalis) to triazoles [34], the overall essential agreement between the MALDI-TOF MS and the CLSI AFST methods ranged from 54% to 97%, with the best essential agreement observed for C. glabrata isolates. The reproducibility of the MALDI-TOF MS-based assay varied between 54% and 83%, and it was higher for C. glabrata isolates than for C. albicans or C. tropicalis isolates. However, as very major and major errors with MALDI-TOF MS-based assay were noticed, applying a 5% tolerance for CCI ratio evaluation did result in error percentages decreased by up to 33.3% [34].