Fungal infections caused by members of the Mucorales order are rapidly progressing and fatal. The importance of mucormycosis has grown in recent years as the number of patients with predisposing factors has increased dramatically. Clinical symptoms are elusive and conventional techniques are often insensitive and unspecific; in particular, cultures are often negative even though direct microscopy is positive. For early diagnosis of the causative agent of disease and subsequently guiding therapy to improving patients' outcome, molecular assays are promising add-ons. This article provides an overview on current laboratory methods for diagnosing mucormycosis with a special focus on new molecular-based tools. We aim to highlight the pros and cons of various techniques at hand. Given the increase in number and the severity of these infections, molecular approaches for improved diagnosis are highly warranted.

Mucormycoses are emerging and potentially lethal infections. An increase of breakthrough infections has been found in cohorts receiving short-tailed azoles prophylaxis (e.g. voriconazole (VCZ)). Although VCZ is ineffective in vitro and in vivo, long-tailed triazoles such as posaconazole remain active against mucormycetes. Our goal was to validate the molecular mechanism of resistance to short-tailed triazoles in Mucorales. The paralogous cytochrome P450 genes (CYP51 F1 and CYP51 F5) of Rhizopus arrhizus, Rhizopus microsporus, and Mucor circinelloides were amplified and sequenced. Alignment of the protein sequences of the R. arrhizus, R. microsporus, and M. circinelloides CYP51 F1 and F5 with additional Mucorales species (n = 3) and other fungi (n = 16) confirmed the sequences to be lanosterol 14α-demethylases (LDMs). Sequence alignment identified a pan-Mucorales conservation of a phenylalanine129 substitution in all CYP51 F5s analyzed. A high resolution X-ray crystal structure of Saccharomyces cerevisiae LDM in complex with VCZ was used for generating a homology model of R. arrhizus CYP51 F5. Structural and functional knowledge of S. cerevisiae CYP51 shows that the F129 residue in Mucorales CYP51 F5 is responsible for intrinsic resistance of Mucorales against short-tailed triazoles, with a V to A substitution in Helix I also potentially playing a role.

e Amphotericin B and posaconazole susceptibility patterns were determined for the most prevalent Mucorales, following EUCAST (European Committee on Antimicrobial Susceptibility Testing) broth microdilution guidelines. In parallel, Etest was performed and evaluated against EUCAST. The overall agreement of MICs gained with Etest and EUCAST was 75.1%; therefore, Etest cannot be recommended for antifungal susceptibility testing of Mucorales. Amphotericin B was the most active drug against Mucorales species in vitro, while the activities of posaconazole were more restricted. Mucormycoses are rapidly progressing and frequently lethal infections caused by fungi associated with the order Mucorales (1). Rhizopus is the most commonly found genus, followed by the genera Lichtheimia and Mucor (1). Antifungal treatment options consist of lipid formulations of amphotericin B (AMB) as the first-line therapy (2) and posaconazole (PSC) as salvage therapy (3, 4). The rising number of breakthrough mucormycoses (5-7) has stimulated interest in antifungal susceptibility testing (AST) of Mucorales. Phylogenetic studies found that Mucorales embrace a very heterogeneous group of fungi, with their evolutionary distances mirrored in various levels of intrinsic antifungal susceptibilities (8, 9). The recommended international standard methods of the CLSI (Clinical and Laboratory Standards Institute) (10), and EUCAST (European Committee for Antimicrobial Susceptibility Testing) (11) are based on broth microdilution assays. However, ready-to-use commercial tests, such as the Etest, are popular for routine application, as they are simpler to perform and more time efficient (12). To use and exchange data generated with the Etest in routine laboratories, an evaluation of this commercial test compared to a gold standard method is needed.The aims of the present study were 2-fold: to determine amphotericin B and posaconazole susceptibility patterns for the most frequent agents of mucormycoses and to compare the results obtained with Etest and EUCAST.The strain set comprised 131 Mucorales; the clinical strains were collected between 2008 and 2014 in routine laboratory testing at the University Hospital of Innsbruck (see Table S2 in the supplemental material), and an additional 38 reference strains were gained from the Fungal Biodiversity Centre (CBS, Utrecht, The Netherlands) (see Table S3). The strains were cultured on supplementary minimal medium (SUP) agar (13) at 37°C (except that Mucor spp. were grown at 30°C) for 3 to 5 days and preidentified to genus level by micromorphological (Olympus CX21 microscope; Olympus, USA) and macromorphological (Axioplan microscope; Zeiss, Germany) characteristics (6, 14). Species were determined by internal transcribed spacer (ITS) direct sequencing (15). Mycelium was harvested for genomic DNA extraction as previously described (16). ITS sequences were identified with BLAST comparative nucleic acid sequence analysis of sequences in the NCBI database (www.ncbi.nlm.nih.gov/BLAST).In vitro antifungal susceptibilit...