Opportunistic fungal pathogens may cause superficial or serious invasive infections, especially in immunocompromised and debilitated patients. Invasive mycoses represent an exponentially growing threat for human health due to a combination of slow diagnosis and the existence of relatively few classes of available and effective antifungal drugs. Therefore systemic fungal infections result in high attributable mortality. There is an urgent need to pursue and deploy novel and effective alternative antifungal countermeasures. Photodynamic therapy (PDT) was established as a successful modality for malignancies and age-related macular degeneration but photodynamic inactivation has only recently been intensively investigated as an alternative antimicrobial discovery and development platform. The concept of photodynamic inactivation requires microbial exposure to either exogenous or endogenous photosensitizer molecules, followed by visible light energy, typically wavelengths in the red/near infrared region that cause the excitation of the photosensitizers resulting in the production of singlet oxygen and other reactive oxygen species that react with intracellular components, and consequently produce cell inactivation and death. Antifungal PDT is an area of increasing interest, as research is advancing (i) to identify the photochemical and photophysical mechanisms involved in photoinactivation;(ii) to develop potent and clinically compatible photosensitizers;(iii) to understand how photoinactivation is affected by key microbial phenotypic elements multidrug resistance and efflux, virulence and pathogenesis determinants, and formation of biofilms;(iv) to explore novel photosensitizer delivery platforms; and (v) to identify photoinactivation applications beyond the clinical setting such as environmental disinfectants.

Currently accepted fungal diagnostic techniques, such as culture, biopsy, and serology, lack rapidity and efficiency. Newer diagnostic methods, such as polymerase chain reaction (PCR)-based assays, have the potential to improve fungal diagnostics in a faster, more sensitive, and specific manner. Preliminary data indicate that, when PCR-based fungal diagnostic assays guide antifungal therapy, they may lower patient mortality and decrease unnecessary antifungal treatment, improving treatment-associated costs and avoiding toxicity. Moreover, newer PCR techniques can identify antifungal resistance DNA loci, but the clinical correlation between those loci and clinical failure has to be studied further. In addition, future studies need to focus on the implementation of PCR techniques in clinical decision making and on combining them with other diagnostic tests. A consensus on the standardization of PCR techniques, along with validation from large prospective studies, is necessary to allow widespread adoption of these assays.

Fusariosis is an emerging infectious complication of immune deficiency, but models to study this infection are lacking. The use of the soil nematode Caenorhabditis elegans as a model host to study the pathogenesis of Fusarium spp. was investigated. We observed that Fusarium conidia consumed by C. elegans can cause a lethal infection and result in more than 90% killing of the host within 120 hours, and the nematode had a significantly longer survival when challenged with Fusarium proliferatum compared to other species. Interestingly, mycelium production appears to be a major contributor in nematode killing in this model system, and C. elegans mutant strains with the immune response genes, tir-1 (encoding a protein containing a TIR domain that functions upstream of PMK-1) and pmk-1 (the homolog of the mammalian p38 MAPK) lived significantly shorter when challenged with Fusarium compared to the wild type strain. Furthermore, we used the C. elegans model to assess the efficacy and toxicity of various compounds against Fusarium. We demonstrated that amphotericin B, voriconazole, mancozeb, and phenyl mercury acetate significantly prolonged the survival of Fusarium-infected C. elegans, although mancozeb was toxic at higher concentrations. In conclusion, we describe a new model system for the study of Fusarium pathogenesis and evolutionarily preserved host responses to this important fungal pathogen.

iNKT cells are innate T lymphocytes recognizing endogenous and foreign lipid antigens presented in the MHC-like molecule CD1d. The semi-invariant iNKT cell TCR can detect certain bacterial and parasitic lipids and drive iNKT cell responses. How iNKT cells respond to fungi, however, is unknown. We found that CD1d-deficient mice, which lack iNKT cells, poorly control infection with the fungal pathogen Aspergillus fumigatus. Furthermore, A. fumigatus rapidly activates iNKT cells in vivo and in vitro in the presence of APCs. Surprisingly, despite a requirement for CD1d recognition, the antifungal iNKT cell response does not require fungal lipids. Instead, Dectin-1- and MyD88-mediated responses to β-1,3 glucans, major fungal cell-wall polysaccharides, trigger IL-12 production by APCs that drives self-reactive iNKT cells to secrete IFN-γ. Innate recognition of β-1,3 glucans also drives iNKT cell responses against Candida, Histoplasma, and Alternaria, suggesting that this mechanism may broadly define the basis for antifungal iNKT cell responses.

BACKGROUND Candida can cause mucocutaneous and/or systemic infections in hospitalized and immunosuppressed patients. Most individuals are colonized by Candida spp. as part of the oral flora and the intestinal tract. We compared oral and systemic isolates for the capacity to form biofilm in an in vitro biofilm model and pathogenicity in the Galleria mellonella infection model. The oral Candida strains were isolated from the HIV patients and included species of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. norvegensis, and C. dubliniensis. The systemic strains were isolated from patients with invasive candidiasis and included species of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, and C. kefyr. For each of the acquired strains, biofilm formation was evaluated on standardized samples of silicone pads and acrylic resin. We assessed the pathogenicity of the strains by infecting G. mellonella animals with Candida strains and observing survival. RESULTS The biofilm formation and pathogenicity in Galleria was similar between oral and systemic isolates. The quantity of biofilm formed and the virulence in G. mellonella were different for each of the species studied. On silicone pads, C. albicans and C. dubliniensis produced more biofilm (1.12 to 6.61 mg) than the other species (0.25 to 3.66 mg). However, all Candida species produced a similar biofilm on acrylic resin, material used in dental prostheses. C. albicans, C. dubliniensis, C. tropicalis, and C. parapsilosis were the most virulent species in G. mellonella with 100% of mortality, followed by C. lusitaniae (87%), C. novergensis (37%), C. krusei (25%), C. glabrata (20%), and C. kefyr (12%). CONCLUSIONS We found that on silicone pads as well as in the Galleria model, biofilm formation and virulence depends on the Candida species. Importantly, for C. albicans the pathogenicity of oral Candida isolates was similar to systemic Candida isolates, suggesting that Candida isolates have similar biofilm-forming ability and virulence regardless of the infection site from which it was isolated.

The substantial morbidity and mortality associated with invasive fungal infections constitute undisputed tokens of their severity. The continued expansion of susceptible population groups (such as immunocompromised individuals, patients undergoing extensive surgery, and those hospitalized with serious underlying diseases especially in the intensive care unit) and the limitations of current antifungal agents due to toxicity issues or to the development of resistance, mandate the development of novel antifungal drugs. Currently, drug discovery is transitioning from the traditional in vitro large-scale screens of chemical libraries to more complex bioassays, including in vivo studies on whole animals; invertebrates, such as Caenorhabditis elegans, are thus gaining momentum as screening tools. Key pathogenesis features of fungal infections, including filament formation, are expressed in certain invertebrate and mammalian hosts; among the various potential hosts, C. elegans provides an attractive platform both for the study of host-pathogen interactions and the identification of new antifungal agents. Advantages of compound screening in this facile, relatively inexpensive and not as ethically challenged whole-animal context, include the simultaneous assessment of antifungal efficacy and toxicity that could result in the identification of compounds with distinct mechanisms of action, for example by promoting host immune responses or by impeding fungal virulence factors. With the recent advent of using predictive models to screen for compounds with improved chances of bioavailability in the nematode a priori, high-throughput screening of chemical libraries using the C. elegans-C. albicans antifungal discovery assay holds even greater promise for the identification of novel antifungal agents in the near future.

The facile inoculum delivery and handling of the insect Galleria mellonella make it a desirable model for the study of fungal pathogenesis. Here we present methods to study fungal virulence, filamentation and fungal cell associates with insect hemocytes using Candida albicans and Cryptococcus neoformans to illustrate the use of this model. The two types of fungi cause distinct infections thus we compare and contrast the infection characteristics observed in G. mellonella. The protocols presented herein can be adapted to the study of other fungal pathogens using G. mellonella as an infection model.

Candida albicans is an important cause of morbidity in hospitalized and immunosuppressed patients. Virulence factors of C. albicans include: filamentation, proteinases, adherence proteins and biofilm formation. The objective of this work was to use Galleria mellonella as a model to study the roles of C. albicans filamentation in virulence. We focused our study to five genes BCR1, FLO8, KEM1, SUV3 and TEC1 that have been shown to play a role in filamentation. Filaments are necessary for biofilm formation and evading interaction with macrophages in mammalian infections. Among the five mutant strain tested, we found that only the flo8/flo8 mutant strain did not form filaments within G. mellonella. This strain also exhibited reduced virulence in the larvae. Another strain that exhibited reduced pathogenicity in the G. mellonella model was tec1/tec1 but by contrast, the tec1/tec1 strain retained the ability to form filaments. Overexpression of TEC1 in the flo8/flo8 mutant restored filamentation but did not restore virulence in the larvae as well as in a mouse model of C. albicans infection. The filamentation phenotype did not affect the ability of hemocytes, the immune cells of G. mellonella, to associate with the various mutant strains of C. albicans. The capacities of the tec1/tec1 mutant and the flo8/flo8 TDH3-TEC1 strains to form filaments with impaired virulence suggest that filamentation alone is not sufficient to kill G. mellonella and suggest other virulence factors may be associated with genes that regulate filamentation.

Fungal environments range from soil, plants, animal and human hosts....