Current Drug Targets - Volume 6, Issue 8, 2005

The current review issue regarding 'Fungal Infections and Antifungal Strategies' unites reviews regarding (i) the infection process of pathogenic fungi resulting in topical or systemic infections, (ii) fungal biofilms, (iii) currently used antimycotics, (iv) novel antifungal drug targets, (v) antifungal susceptibility testing methods and (vi) the use of radiolabeled antifungal agents for early detection of fungal infections. Fungal infections are categorized in two groups: topical and systemic infections. Topical fungal infections affect body surfaces and can be chronic, as discussed by Borgers and coworkers. Systemic fungal infections can occur in an organ or in the whole body and are transferred via the bloodstream. Compared to other microbial infections, systemic fungal infections are characterized by lower frequencies but generally high mortality rates (40-100%). The most common causes of these infections are Candida spp., as discussed by Mavor and coworkers, and filamentous fungi such as Aspergillus spp., as discussed by Brakhage. Several factors are associated with the increasing incidence of fungal infections, including the larger population of immunocompromized patients and the increasing use of invasive devices and implants that can be colonized by fungal biofilms. Biofilms are crucial for the development of fungal infections as they can serve as a nidus for disease. Moreover, surface attachment causes these fungal cells to enter a special physiological state in which they are highly resistant to most of the currently used antifungal drugs, as discussed by Chandra and coworkers. Since there are no fungal vaccines currently licensed, the only clinical recourse to combat fungal infections is the use of therapeutics. The currently used antifungal therapeutics are prone to resistance and suffer from pharmacological limitations, harmful drug-drug interactions, limited activity spectrum and/or high general cytotoxicity, as discussed by Francois and coworkers. Therefore, the search for new antifungal components with a novel mode of action is imperative. Recent advances in genetics and genome-based technologies will allow for the identification and validation of new antifungal drug targets to design novel target-based screening strategies, as discussed by Tournu and coworkers and Thevissen and coworkers. To drive this antifungal drug discovery process more efficiently, novel in vitro assays that mimic in vivo fungal growth, thereby more accurately predicting in vivo efficacy of antifungal components, are mandatory. Current in vitro susceptibility tests will be discussed by Pfaller. An accurate and rapid diagnosis is a critical factor limiting efficient antifungal therapy in immunocompromized patients. Therefore, fast and localized diagnosis of a broad range of pathogenic fungi is mandatory to efficiently combat fungal infections. Given the limitations of the currently used PCR- and ELISA-based methods, development of newer diagnostic techniques are mandatory. A promising novel diagnostic technique is the use of radiolabeled antifungals to monitor the in vivo distribution of fungal infections in infected hosts by Single Photon Emission Computerized Tomography or Positron Emission Tomography, as discussed by Lupetti and coworkers. In conclusion, it can be stated that the following developments are needed to tackle the increasing problem of systemic fungal infections and the lack of efficient antifungal therapeutics: (i) the identification of novel types of antifungal components with respect to their mode of action and fungal targets, (ii) improved in vitro susceptibility testing methods and animal models to more accurately evaluate in vivo efficacy of these novel antifungals, and (iii) early and reliable detection and localization of fungal infections in order to efficiently target antifungal therapy.

Dermatomycoses are among the most widespread and common superficial and cutaneous fungal infections in humans. These typically nonfatal conditions are difficult to treat, especially infections of the nail. Dermatomycoses are caused by filamentous fungi such as Trichophyton, Microsporum or Epidermophyton species. These filamentous fungi have a high affinity for keratin, an important component of hair, skin and nails, which are the primary areas of infection by dermatophytes. The antifungal agents currently marketed for dermatomycoses are mainly inhibitors of ergosterol biosynthesis, except for griseofulvin, which interferes with the cytoplasmic and nuclear microtubular system. Three different types of inhibitors of the ergosterol biosynthetic pathway have been proven to be effective in clinic: the azoles (e.g. topical miconazole and topical/oral ketoconazole, itraconazole and fluconazole), the allylamines (e.g. terbinafine) and morpholines (amorolfine). Even today more effective antifungal azoles with less adverse effects and short-term therapy are deemed necessary to treat dermatophytosis. A promising novel triazole compound in this respect is R126638, which showed potent in vitro and in vivo activity.

Candida species, in particular C. albicans, represent a major threat to immunocompromised patients. Able to exist as a commensal on mucosal surfaces of healthy individuals, these opportunistic fungi frequently cause superficial infections of mucosae and skin. Furthermore, in hospital settings, Candida species may cause life-threatening invasive infections in a growing population of vulnerable patients. In fact, candidaemia is associated with the highest crude mortality of all bloodstream infections. Candida cells may enter the bloodstream by direct penetration from epithelial tissues, due to damage of barriers in the body caused by surgery, polytrauma or drug treatment, or may spread from biofilms produced on medical devices. From the bloodstream, cells may infect almost all organs but appear to prefer certain organs depending upon the route of infection. The exact mechanisms by which Candida cells survive the challenge of the blood environment and escape from the bloodstream to cause deep-seated infections have not yet been elucidated, but various investigations are reviewed. It is clear, however, that Candida must have particular attributes which enable the organism to survive and grow within the environment of healthy individuals and to invade tissues in the immunocompromised host. Most studies have focussed on C. albicans and this review will therefore summarise work on the various known virulence factors and methods used to identify further virulence attributes of this fungus.

Infections with mould pathogens have emerged as an increasing risk faced by patients under sustained immunosuppression. Species of the Aspergillus family account for most of these infections and in particular Aspergillus fumigatus can be regarded as the most important airborne-pathogenic fungus. The improvement in transplant medicine and the therapy of hematological malignancies is often complicated by the threat of invasive aspergillosis. Specific diagnostics are still limited, as are the possibilities of therapeutic intervention. Hence, invasive aspergillosis is still associated with a high mortality rate that ranges from 30 % to 90 %. In recent years, considerable progress has been made in understanding the genetics of A. fumigatus and molecular techniques for the manipulation of the fungus have been developed. Molecular genetics offers not only approaches for the detailed characterization of gene products that appear to be key components of the infection process but also selection strategies that combine classical genetics and molecular biology to identify virulence determinants of A. fumigatus. The review discusses aspects of the current knowledge of the infection process, mechanisms of protection of the fungus against immune effector cells, and virulence determinants of A. fumigatus.

Device-related infections in most nosocomial diseases can be traced to the formation of biofilms (microbial communities encased within a polysaccharide-rich extracellular matrix) by pathogens on surfaces of these devices. Candida species are the most common causative agents of these infections, and biofilms formed by these fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents. This enhanced resistance contributes to the persistence of this fungus despite antifungal therapy. Recent studies showed that Candida biofilms exhibit antifungal resistance against most antifungal agents with the exception of echinocandins and lipid formulations of AMB. This review discusses methods used to evaluate biofilm resistance and provide information on susceptibility pattern of candidal biofilm as well as studies investigating the mechanisms underlying biofilm resistance.

The increasing incidence of fungal infections combined with the emerging problem of antifungal drug resistance have prompted investigations of the mode of action of the currently used antifungal therapeutics (antimycotics). The routinely used antimycotics can be grouped into six different classes based on their mode of action. In this review, the mode of action and antifungal spectrum of these classes are discussed, together with possible resistance development against them.

With the rise of fungal infection incidence amongst the patient population, the importance of developing new antifungal drug targets is higher than ever. This review mainly focuses on the three most prevalent fungal pathogens, Candida, Aspergillus and Cryptococcus, and on the most recent progresses in molecular research that contribute to a better understanding of the pathogen itself, but also its host and the interaction with its host. We consider the progress made in comparative genomics following the huge effort of fungal genome sequence projects undertaken in the last few years. We focus not only on currently used mammalian animal models such as mice, but also on novel non-mammalian models, such as the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, which offer useful tools in the area of the innate immune response to fungal infections. In addition we relate to the recent genomic and proteomic studies and focus on the use of these approaches in in vivo experiments in the pathogen itself as well as in the host. Finally, we describe the latest targeted mutagenesis strategy available in C. albicans and the use of RNA interference in both Cryptococcus neoformans and A. fumigatus. Our aim is not to give an exhaustive list of all new strategies but rather to give an overview of what will contribute most to the identification of new antifungal drug targets and the establishment of novel antifungal strategies.

Sphingolipids are essential membrane components, present in all eukaryotic cells, but structurally distinct in mammalian and fungal cells. Therefore, they represent an attractive new target for the development of novel antimycotics. This review will briefly highlight sphingolipid biosynthesis and functions in the yeast Saccharomyces cerevisiae. In addition, naturally occurring antifungal compounds that interact with fungal-specific sphingolipids, resulting in fungal growth arrest, will be discussed regarding their mode of action, and therapeutic value. These compounds include plant and insect defensins, syringomycin E and antifungal antibodies to sphingolipids.

The number of systemically active antifungal agents has increased dramatically in recent years in response to the challenge of invasive mycoses. Additional work is needed to better understand the mechanisms of action of these agents as well as the mechanisms of resistance expressed by the fungal pathogens. Antifungal susceptibility testing has been standardized and refined and now may be considered to play an important role in the management of invasive mycoses. More work is needed to optimize the methods for testing new antifungal agents and for testing pathogens other than Candida. The ongoing efforts and international collaborations designed to address these issues will provide important information that will improve the management of serious fungal infections.

The outcome of antifungal therapy depends on the progression of the infection at the start of therapy. Unfortunately, most patients are diagnosed once the fungal infection has progressed considerably as a result of the non-specific clinical signs of fungal infections in immunocompromised patients and the poor sensitivity of current mycological diagnostic tests. This review will highlight current fungal diagnostic techniques and will focus on scintigraphic methods for the specific detection of fungal infections in mice. For this purpose, antifungal components (e.g. fluconazole and antifungal peptides) are radiolabeled e.g. with technetium-99m (99mTc) and their in vivo distribution is monitored in infected mice. It has been demonstrated that 99mTc-fluconazole is an excellent tracer to detect Candida albicans infections in mice as it distinguishes these infections from bacterial infections and sterile inflammations. However, this radiopharmaceutical only poorly detects infections with Aspergillus fumigatus in mice. 99mTc-peptides derived from antifungal peptides/ proteins, such as human ubiquicidin and lactoferrin, can distinguish C. albicans and A. fumigatus infections from sterile inflammations, but not from bacterial infections, in mice. Furthermore, the efficacy of fluconazole in C. albicansinfected mice could be successfully monitored using 99mTc-ubiquicidin. In conclusion, neither 99mTc-fluconazole nor the 99mTc-peptides tested are optimal tracers for fungal infections. Nonetheless, since early initiation of antifungal therapy for candidemia reduces its high mortality rate, a positive result with 99mTc-fluconazole scintigraphy is of clinical relevance. Finally, the possibility that other (radiolabeled) antifungal agents, e.g. voriconazole, caspofungin, antifungal plant or insect defensins, can be useful for detection of fungal infections should be considered.