A multicenter study was conducted to define the most suitable testing conditions for antifungal susceptibility of dermatophytes. Broth microdilution MICs of clotrimazole, itraconazole, and terbinafine were determined in three centers against 60 strains of dermatophytes. The effects of inoculum density (ca. 10 3 and 10 4 CFU/ml), incubation time (3, 7, and 14 days), endpoint criteria for MIC determination (complete [MIC-0] and prominent [MIC-2] growth inhibition), and incubation temperature (28 and 37°C) on intra-and interlaboratory agreement were analyzed. The optimal testing conditions identified were an inoculum of 10 4 CFU/ml, a temperature of incubation of 28°C, an incubation period of 7 days, and MIC-0.Dermatophytes are a group of morphologically and physiologically related molds that cause well-defined infections in vertebrates. The incidence of dermatophytoses has increased over recent years, particularly in immunocompromised patients (29,30,32,33). The choice of the proper treatment is determined by the site and extent of the infection and the species involved, as well as by the efficacy, safety profile, and kinetics of the available drugs. For localized nonextensive lesions, topical therapies with clotrimazole (CLT) are generally used. For tinea unguium, scalp ringworm, extensive infections, or skin lesions with folliculitis, systemic antifungal treatment is necessary (1,4,23,26). Oral drugs such as itraconazole (ITC) and terbinafine (TRB) are the antifungal agents currently most used to treat severe infections (4, 23). Some novel compounds, such as UR-9825, posaconazole, voriconazole, or ravuconazole, also appear to be promising candidates for the treatment of dermatophytosis (2,10,28).In the in vitro method proposed by the National Committee for Clinical Laboratory Standards (NCCLS) for testing molds (25), the dermatophytes were not included. Therefore, it is necessary to develop a reproducible standardized method for these important fungi that would lead to protocols for proper treatment. In recent years, some authors, possibly encouraged by the development of the above-mentioned reference method, have published various articles wherein several species of dermatophytes have been tested (11,27,28,34). In these works, different adaptations or modifications of the NCCLS methods have been assayed, although other techniques have also been used (3,12,15). The results obtained have been clearly contradictory in some aspects, which makes evident the need for standardization and the development of reference methods. Recently, we evaluated the activity of 11 antifungal drugs against an important number of strains of dermatophytes (n ϭ 508) by using a microdilution method (10). In that study the testing conditions adopted were an inoculum size of 10 4 CFU/ml, a temperature and time of incubation of 28°C and 7 days, respectively, and the MIC endpoint determination was 50% growth inhibition for azoles and 100% for the rest. It is unknown whether varying the conditions would have changed the results significantly....

Dectin-1, the major β-glucan receptor in leukocytes, triggers an effective immune response upon fungal recognition. Here we use sortase-mediated transpeptidation, a technique that allows placement of a variety of probes on a polypeptide backbone, to monitor the behavior of labeled functional dectin-1 in live cells with and without fungal challenge. Installation of probes on dectin-1 by sortagging permitted highly specific visualization of functional protein on the cell surface and its subsequent internalization upon ligand presentation. Retrieval of sortagged dectin-1 expressed in macrophages uncovered a unique interaction between dectin-1 and galectin-3 that functions in the proinflammatory response of macrophages to pathogenic fungi. When macrophages expressing dectin-1 are exposed to Candida albicans mutants with increased exposure of β-glucan, the loss of galectin-3 dramatically accentuates the failure to trigger an appropriate TNF-α response.pattern-recognition receptors | sortase A | polysaccharide

No abstract

Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.

No abstract

The use of the agar diffusion Neo-Sensitabs method to determine antifungal susceptibility of 59 isolates of dermatophytes, namely Epidermophyton floccosum, Microsporum canis, M. gypseum, Trichophyton mentagrophytes, T. rubrum and T. tonsurans to Clotrimazole (CLZ), Itraconazole (ITZ) and Terbinafine (TBF) is described. Results obtained are compared to the minimum inhibitory concentrations (MIC) determined by an adaptation of the NCCLS-M38-A procedure. Using the diffusion method, all strains showed a broad zone of inhibition at the first available reading time (3 or 7 days). Using the broth microdilution method, the geometric mean MIC (microg/ml) with regard to all isolates was < or = 0.03 for TBF, < or = 0.069 for CLZ and < or = 0.919 for ITZ. In both methods, TBF was the most active antifungal agent against all isolates tested. The two methods evaluated were able to detect the resistance of the quality control strains of Aspergillus fumigatus to ITZ. Even though a reference method for testing dermatophytes still has not been developed, our data suggest that the Neo-Sensitabs diffusion method could provide a simple procedure for the antifungal susceptibility testing of dermatophytes in the routine clinical laboratory.

No abstract