Comparative evaluation of antifungal susceptibility testing methods of invasive Candida species and detection of FKS genes mutations in caspofungin intermediate and resistant isolates

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BACKGROUND: Fungal invasive infections caused by Candida species pose a substantial public health risk with limited therapeutic options. Antifungal susceptibility testing (AFST) is necessary to optimize the therapy. The study aimed to compare different AFST methods of Candida spp. and detect FKS gene mutations among caspofungin-intermediate and resistant isolates. METHODS: A total of 60 non-replicative invasive Candida isolates recovered from various clinical samples were included. In-vitro AFST was carried out using the ATB FUNGUS 3, Vitek-2 AST-YS08, and E-test. Hotspot (HS) regions of FKS genes were sequenced for caspofungin-intermediate and resistant isolates. RESULTS: Candida albicans (58.3%) was the most predominant spp., followed by C. glabrata (28.3%). Based on the clinical breakpoints (CBPs), fluconazole resistance was found in C. albicans (45.7%), C. tropicalis (25%), and the C. parapsilosis isolate, while 35.3% of C. glabrata were susceptible dose-dependent (SDD). None of C. albicans, C. tropicalis, or C. parapsilosis isolates were resistant to voriconazole. Using the epidemiological cut-off values (ECVs) for amphotericin B, 6.7% of isolates were non-wild type (non-WT), including C. guilliermondii (50%), C. tropicalis (25%), and C. glabrata (11.8%), while all C. albicans, C. parapsilosis, and C. kefyr isolates were classified as wild-type (WT). ATB FUNGUS 3 and Vitek-2 had the highest categorical agreement (CA) (83.1%) for amphotericin B, while a lower concordance was detected with voriconazole (23.2%) and fluconazole (52.2%). For caspofungin, Vitek-2 and E-test had a CA of 89.8%. Eleven isolates (10 C. glabrata and one C. parapsilosis) exhibited resistance or intermediate susceptibility to caspofungin (MICs: 0.25‒>32 µg/ml). Molecular characterization of the FKS gene demonstrated that FKS1 mutations V47I, V52K, V56T, D57S, L62F, I71Y, I71Q in the HS1 region, and G7S, P11H mutations in the HS2 region were associated with increased caspofungin MIC values (16 µg/ml). Mutations at the HS1 of the FKS2 gene; K33V, W35K, and W35V; were associated with the highest caspofungin MICs of > 32 µg/ml. CONCLUSIONS: ATB FUNGUS 3 demonstrated acceptable performance for AFST, however, azole activity against Candida spp. should be interpreted carefully. Novel mutations within HS regions of FKS genes elucidated different levels of caspofungin resistance in C. glabrata and C. parapsilosis isolates.

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