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    • In Pham et al employed the

    2023-12-19

    In 2014, Pham et al. [22] employed the microsphere-based technology to create a Luminex probe-based assay for the rapid identification of single nucleotide polymorphisms that are known to confer resistance to one or more echinocandins in C. glabrata. The targets for the FKS probes were generated using 94 8 PCRs—where single-stranded PCR products allow the probes to anneal; hence, the discriminatory affinity of FKS probes for their targets was determined by using a panel of reference isolates with known FKS mutations. The MagPix assay was first validated with a set of 102 isolates obtaining 100% of results concordant with the isolates' DNA sequencing profiles; after that, the assay was used for high-throughput screening of 1032 C. glabrata surveillance isolates, enabling the detection of 16 new isolates with mutations, the presence of which was confirmed by DNA sequencing of the corresponding region [22]. Of the isolates displaying an irregular FKS2 HS1 probe signal pattern, three had the F659del mutation—not included in the original assay but known to confer echinocandin resistance. The remaining two isolates had the S663P mutation in addition to a silent mutation at G1986A. As a rapid and highly versatile format—FKS1 HS1 and FKS2 HS1 profiles of up to 95 isolates can be determined in as little as 5 h—the multiplex FKS MagPix might be used in place of DNA sequencing, particularly for those laboratories that are already equipped with the Luminex technology [22]. In the meanwhile, Dudiuk et al. [23] developed a set of classical PCRs able to detect ten of the most frequent mutations associated with clinical echinocandin resistance in C. glabrata within 4 h. Using a blind collection of 50 C. glabrata strains, including 16 FKS1 and/or FKS2 mutants, the assay allowed that 98% (49/50) of the strains were correctly identified as echinocandin susceptible or resistant compared with the echinocandin AFST results. The molecular diagnostic results from the PCR assay were 98% concordant with those obtained from DNA sequencing. The one false result regarded an FKS2 mutant, in which Fks2p carried the aforementioned deletion at the 659 residue (F659del). In this case, the deletion could not be detected because a few nucleotides were deleted and the nucleotide sequence where the primer was aligned did not change [23]. Very recently, Zhao et al. [24] proposed a novel diagnostic assay platform for rapid FKS genotyping of C. glabrata isolates, to identify echinocandin resistance-associated mutations without the assay design and set-up complications of previously described assays [22], [23]. Using asymmetrical PCR in conjunction with molecular beacon probe-based melting curve analysis, a dual assay for FKS1 and FKS2 was developed to accurately discriminate WT from mutated FKS genes within 3 h. In this assay, signature melting profiles and corresponding T values were generated, through direct colony PCRs from reference strains, for eight FKS1 HS1 and seven FKS2 HS1 genotypes. Hence, the FKS genotype of the testing isolate could be easily identified by comparing the T value with those of the reference strains representing all the mutations included in the dual assay. In the subsequent proof-of-concept clinical validation study, using a blinded panel of 188 C. glabrata isolates, both FKS1 HS1 and FKS2 HS1 assays showed 100% accuracy for WT/NWT genotype discrimination compared with DNA sequencing results [24]. Collectively, these studies show that the molecular testing can be ideal for detecting echinocandin resistance in Candida species, whereas this is not the same for azole resistance because of the complexity of underlying resistance mechanisms in these yeasts [2].
    MALDI-TOF MS-based assays for detection of echinocandin or azole resistance MALDI-TOF MS has been adopted as a rapid and robust tool for the accurate identification of microorganisms with medical importance [25], [26], including yeasts and filamentous fungi [27], [28]. Recently, MALDI-TOF MS profiling of microbial isolates has taken exciting new trajectories beyond microbe identification, offering opportunities for detection of antimicrobial susceptibility/resistance in fungal organisms [29]. So, as with bacteria [30], promising approaches have been developed for the detection of antifungal resistance in Candida and Aspergillus species [29]. Similar to MALDI-TOF MS-based species identification, these approaches may considerably decrease the time to result associated with the current AFST assays, so facilitating the implementation of targeted antifungal therapy.