ID-CARD: A clade-specific molecular assay for the detection of 25 Candida spp. causing candidemia based on antifungal susceptibility patterns and in vitro testing of the antifungal activity of a synthetic antimicrobial peptide derived from human lactoferrin (hLF1-11).

Fungal infections are a serious health concern affecting over 1.5 million individuals annually. ID-CARD aims to improve diagnostics taking into account phylogeny and antifungal susceptibility patterns of Candida spp. involved in candidemia.Twenty-five Candida spp. were chosen. Based on ribosomal DNA sequences, clade-specific primers/Taqman probes were designed. Different multiplex panels consisting of four clades that exhibited similar antifungal susceptibility profiles were created. To create the groups, we tested fluconazole and anidulafungin with broth microdilution according to EUCAST against 3-5 isolates/species (n=121), which were also used for specificity testing of the molecular assay. Furthermore, we tested the in vitro activity of hLF(1-11) peptide against isolates that exhibited elevated minimum inhibitory concentrations (MICs) for one or both of the drugs. The groups created are : i. Lodderomyces, Kluyveromyces, Metschnikowiaceae Sensitive, Internal control, (all with low MICs) ii. Pichiaceae, Nakaseomyces, Wickerhamomycetaceae, Debaryomyces & Diutina, (all with high MICs to azoles) and iii. Yarrowia, Wickerhamiella & Meyerozyma, Candida auris, Candida haemulonii complex (all with high MICs to both azoles & echinocandins). The primers/probes showed 100% specificity and capacity for multiplexing. In vitro experiments indicated that hLF(1-11) is fungicidal against various Candidaspp. A synergistic effect of antifungal and hLF(1-11) against various Candida species was shown as combinations of the peptide with antifungals were more effective than these alone ID-CARD will contribute to a fast and reliable molecular detection of yeasts involved in candidiasis. AMPs is a novel way to treat Candida spp. exhibiting high MICs to commonly used antifungal drugs.