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Antifungal susceptibility testing (AFST) is necessary to optimize the therapy. The study aimed to compare different AFST methods of <jats:italic>Candida<\/jats:italic> spp. and detect <jats:italic>FKS<\/jats:italic> gene mutations among caspofungin-intermediate and resistant isolates.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>A total of 60 non-replicative invasive <jats:italic>Candida<\/jats:italic> 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 <jats:italic>FKS<\/jats:italic> genes were sequenced for caspofungin-intermediate and resistant isolates.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>\n              <jats:italic>Candida albicans<\/jats:italic> (58.3%) was the most predominant spp., followed by <jats:italic>C. glabrata<\/jats:italic> (28.3%). Based on the clinical breakpoints (CBPs), fluconazole resistance was found in <jats:italic>C. albicans<\/jats:italic> (45.7%), <jats:italic>C. tropicalis<\/jats:italic> (25%), and the <jats:italic>C. parapsilosis<\/jats:italic> isolate, while 35.3% of <jats:italic>C. glabrata<\/jats:italic> were susceptible dose-dependent (SDD). None of <jats:italic>C. albicans<\/jats:italic>, <jats:italic>C. tropicalis<\/jats:italic>, or <jats:italic>C. parapsilosis<\/jats:italic> 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 <jats:italic>C. guilliermondii<\/jats:italic> (50%), <jats:italic>C. tropicalis<\/jats:italic> (25%), and <jats:italic>C. glabrata<\/jats:italic> (11.8%), while all <jats:italic>C. albicans<\/jats:italic>, <jats:italic>C. parapsilosis<\/jats:italic>, and <jats:italic>C. kefyr<\/jats:italic> 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 <jats:italic>C. glabrata<\/jats:italic> and one <jats:italic>C. parapsilosis<\/jats:italic>) exhibited resistance or intermediate susceptibility to caspofungin (MICs: 0.25\u2012&gt;32\u00a0\u00b5g\/ml). Molecular characterization of the <jats:italic>FKS<\/jats:italic> gene demonstrated that <jats:italic>FKS1<\/jats:italic> 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\u00a0\u00b5g\/ml). Mutations at the HS1 of the <jats:italic>FKS2<\/jats:italic> gene; K33V, W35K, and W35V; were associated with the highest caspofungin MICs of &gt;\u200932\u00a0\u00b5g\/ml.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>ATB FUNGUS 3 demonstrated acceptable performance for AFST, however, azole activity against <jats:italic>Candida<\/jats:italic> spp. should be interpreted carefully. Novel mutations within HS regions of <jats:italic>FKS<\/jats:italic> genes elucidated different levels of caspofungin resistance in <jats:italic>C. glabrata<\/jats:italic> and <jats:italic>C. parapsilosis<\/jats:italic> isolates.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/s12879-024-10435-8","type":"journal-article","created":{"date-parts":[[2025,1,24]],"date-time":"2025-01-24T09:54:42Z","timestamp":1737712482000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Comparative evaluation of antifungal susceptibility testing methods of invasive Candida species and detection of FKS genes mutations in caspofungin intermediate and resistant isolates"],"prefix":"10.1186","volume":"25","author":[{"given":"Dalia Saad","family":"ElFeky","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Doaa Mahdy","family":"El-Wakil","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mai M.","family":"Mwafy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohamed M.A.","family":"Atia","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Noha Mahmoud","family":"Gohar","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,1,24]]},"reference":[{"issue":"9","key":"10435_CR1","doi-asserted-by":"publisher","first-page":"1232","DOI":"10.1086\/496922","volume":"41","author":"TE Zaoutis","year":"2005","unstructured":"Zaoutis TE, Argon J, Chu J, Berlin JA, Walsh TJ, Feudtner C. 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