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Invasive fungal infections are relatively common opportunistic infections in immunocompromised patients and are still associated with a high mortality rate. Furthermore, these infections are often complicated by resistance or refractoriness to current antimicrobial agents. Therefore, an urgent need exists for new therapeutic strategies based on the identification of new microbial targets and novel antimicrobial agents. One such hypothetical therapeutic strategy may involve the use of mycoviruses that are able to selectively infect fungi. Current knowledge of mycoviruses of human pathogenic fungi and the scope for using (recombinant) mycoviruses as future biological control agents are reviewed here.

Recognition of pathogen-associated molecular patterns (PAMPs) of microorganisms by pathogen recognition receptors induces signals responsible for the activation of genes important for an effective host defence, especially those of pro-inflammatory cytokines. Toll-like receptors (TLRs) and lectin-like receptors are the most important classes of pattern-recognition receptors. In addition to their effects on the activation of host defence, recent studies suggest that pathogenic fungi can modulate or interfere with the pattern recognition mechanisms of innate immunity, and can use pattern recognition receptors as mechanisms of escape from host defence. Two major recognition receptor-mediated escape mechanisms have been identified during infection with fungal pathogens: immunosuppression induced by activation of certain pattern recognition receptors, especially induction of IL-10 release through TLR2; and the blockade of TLR recognition by antigen modification during the germination of yeasts into hyphae. Thus, signals mediated by recognition receptors are not only beneficial to the host, but in certain situations can be used by pathogenic fungi to escape immune recognition and promote infection.

Toll-like receptors (TLRs) have been identified as a major class of pattern-recognition receptors. Recognition of pathogen-associated molecular patterns by TLRs, either alone or in heterodimerization with other TLR or non-TLR receptors, induces signals responsible for the activation of the innate immune response. Recent studies have demonstrated a crucial involvement of TLRs in the recognition of fungal pathogens such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans. Through the study of fungal infection in knock-out mice deficient in either TLRs or TLR-associated adaptor molecules, it became apparent that specific TLRs such as TLR2 and TLR4 play differential roles in the activation of the various arms of the innate immune response. Recent data also suggest that TLRs offer escape mechanisms to certain pathogenic microorganisms, especially through TLR2-driven induction of anti-inflammatory cytokines. These new data have substantially increased our knowledge of the recognition of fungal pathogens, and the study of TLRs remains one of the most active areas of research in the field of fungal infections., (Copyright 2004 Springer-Verlag)

For 45 years, amphotericin B has been the drug of choice for the treatment of invasive mycoses. Because of its severe toxicity, lipid-associated formulations of amphotericin B have been developed. Although comparative trials are scarce, there appears to be no convincing advantage of these new formulations in terms of efficacy. The lipid-associated amphotericins are significantly less nephrotoxic than conventional amphotericin B, although there are major differences in the infusion-related toxicity of the various lipid-associated preparations. The current armamentarium of azoles and echinocandins for the treatment of invasive mycoses has only left a very minor role for both conventional and lipid-associated amphotericin B in the treatment of a few specific, rare mycoses.

The azole antifungal voriconazole and the echinocandin caspofungin have recently become available for the treatment of invasive mycoses. Fluconazole remains the drug of choice for candidemia, except for infections with one of the resistent species such as Candida krusei and some strains of Candida glabrata. In these cases, as well as in patients who cannot tolerate azoles in connection with side effects or drug interactions, caspofungin is an attractive alternative. Voriconazole has become the drug of choice for severe invasive aspergillosis. Itraconazole is a good alternative for milder and chronic forms of aspergillosis. The use of conventional amphotericin B will be limited by the availability of the new drugs. In view of their high costs, the lipid-bound forms of amphotericin B will usually be given only as salvage therapy in case of failure, in patients who are unable to tolerate either conventional amphotericin or one of the newer agents, and for the treatment of zygomycosis.

Invasive aspergillosis and disseminated candidiasis are the two major manifestations of opportunistic invasive mycoses. Their incidence has risen considerably during the past decades, due to more intensive anticancer chemotherapy, organ transplantations, intensive care, and aggressive surgical interventions. Especially bone marrow transplant recipients are at risk for developing invasive aspergillosis. Whether the infection is acquired through contaminated water or through airborne spores is a matter of much debate. Candidemia and disseminated candidiasis commonly originate from the gastrointestinal tract. Abdominal surgery and mucosal damage due to anticancer chemotherapy are the majors factor through which gut colonization may lead to invasive disease. A shift in the epidemiology of disseminated candidiasis has been noted, with an increasing incidence of Candida glabrata, C. tropicalis and C. krusei strains.

One hundred courses of fluconazole treatment in a university hospital and 81 courses in a non-university teaching hospital have been analysed in a prospective audit to evaluate prescribing practices. The quality of treatments was assessed by an infectious disease specialist and a pharmacist according to standard guidelines. In the non-university hospital, prescribed dosages were lower than in the university hospital, and often below the recommended dose. Mean duration of treatment for oesophageal candidosis and disseminated infections was considerably shorter in the non-university hospital compared with the university hospital, and often judged too short. Microbiological samples were examined in 75% of the cases in both hospitals. The expert reviewers agreed with the indication to use fluconazole in 58-100% of cases in the university hospital and 42-80% in the non-university hospital, depending on the type of infection. There did not appear to be a major problem with inappropriate use of fluconazole. However, important issues for improvement could be identified, such as increasing the dosage and duration of treatment in cases of serious infections, and withholding treatment from patients with colonization rather than infection.

To improve present results with antifungal drugs, modulation of the host immune response is being explored. Human phagocytes of various lineages work cooperatively in vitro with antifungal drugs to inhibit or kill fungal pathogens, and this activity is augmented by several recombinant cytokines. Monoclonal antibodies against the cryptococcal capsule have been shown to act as an adjunct in enhancing the outcome of cryptococcosis in animal models. This approach is now being pursued in systematic clinical trials. In experimental candidiasis, several manipulations of the immune system, via administration of cytokines, gene deletion or antibodies to cytokines, have been shown to significantly affect survival and fungal clearance in vivo. This approach has already been demonstrated to be of benefit with recombinant human granulocyte-colony stimulating factor adjunct therapy of human candidiasis. Combining antifungal drugs of different classes may enhance their therapeutic effect.

In surgical and intensive care units an alarming increase in the number of invasive fungal infections has been observed. This is partly due to temporal transferral of patients from hemato-oncological units or transplant units and partly to the enhanced use of corticosteroids and other immunosuppressants. Candida species have now become a common isolate in ill patients. Amphotericin B with or without flucytosine constituted the standard therapy for candidosis but similar response rates with less toxicity may be obtained with lipid and with fluconazole. Data on an improved outcome of candidemia if a central venous catheter is removed promptly are conflicting. Amphotericin B remains the standard therapy for other invasive mycoses; it is seldom possible to administer adequate doses and therefore the options and limitations of the triazoles and liposomal preparations should be explored.

During the past decades, the incidence and severity of invasive fungal infections has been increasing. These events have lead to the development of new antifungal agents, and amphotericin B no longer is the standard therapy for a variety of invasive mycoses. Fluconazole has become the drug of choice for treatment of C. albicans infections in non-neutropenic as well as neutropenic patients. For treatment of cryptococcal meningitis, amphotericin B with flucytosine, followed by fluconazole consolidation therapy is used. For therapy of invasive aspergillosis, the lipid formulations of amphotericin B may be associated with reduced toxicity and allow for larger doses, although the efficacy of each of the formulations still has to be established in randomized trials. Finally, treatment modalities aimed at improving host defense mechanisms, including adjunctive therapy with rG-CSF for disseminated candidiasis and rG-CSF-elicited granulocyte transfusions, are under way.

Cell-mediated immunity (CMI) has been shown, over many decades of clinical observation and bench research, to be central to the outcome of invasive fungal infections. In recent years, understanding the role of messenger molecules (cytokines), in coordinating and augmenting cellular immunity has been ascendant. These studies have made it possible to consider using cytokines, now available in abundant quantities via recombinant DNA technologies, to treat fungal infections. In this symposium, the most important fungal pathogens that cause infections in humans, particularly in immunocompromised patients, are considered, with emphasis on how recent experimental work may lead to a better understanding of the role of cytokines and their use in therapy.

Fungal infections are the primary cause of mortality in patients with severely impaired host defense mechanisms, such as neutropenic patients with acute leukemia or those who have undergone bone marrow transplantation. In view of the unacceptably high mortality due to disseminated candidiasis, it is rational to focus on augmentation of host defense mechanisms in addition to conventional antifungal therapy. In vitro, a variety of immunomodulators, including tumor necrosis factor, interferon-g, and the hematopoietic growth factors, enhance the killing of Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans. Various studies have demonstrated beneficial effects of immunomodulatory therapy in animal models of disseminated candidiasis. For further preclinical and clinical studies, recombinant interferon-g, interleukin-1, granulocyte colony-stimulating factor, and the other hematopoietic growth factors are currently the most promising immunomodulators.

Twenty-eight neutropenic (< 500 granulocytes/microliters) adults with microbiologically or histologically proven systemic mycosis were randomly assigned to receive either amphotericin B alone (0.5 mg/kg/day; n = 14) or amphotericin B (0.5 mg/kg/day) plus 5-flucytosine (150 mg/kg/day; n = 14) intravenously. Therapy was given for an average duration of 10 days in both groups, amounting to a total dose of amphotericin B of 338 mg and 308 mg, respectively. The mean duration of granulocytopenia was 18 days in the amphotericin B group and 20 days in the combination group. Only two patients treated with amphotericin B alone and three given the combination survived. Adverse events were similar in both groups with an elevation of the serum creatinine in six cases during the administration of amphotericin B alone and in seven cases treated with the combination. No other serious adverse events were encountered. Treatment with both regimens was disappointing partly because mycosis was too far advanced by the time therapy was begun and neutrophils were recovered in only half the patients.

The incidence of invasive fungal infections in the immunocompromized host has increased during the past decade. Even the recently developed antifungal drugs are unable to cure these infections in patients with severely impaired host defense mechanisms. Cytokines have great potential to augment host resistance and as adjunctive therapy of invasive mycoses. We discuss the mechanisms of host defense against invasive candidiasis, aspergillosis, and cryptococcosis, and review the use of cytokines and growth factors in this setting. Interleukin-1 has been shown effective in an animal model of disseminated candidiasis, even during severe granulocytopenia. Interferon-gamma has been very effective as a modulator of resistance against a variety of fungal infections in vitro. The effect of interferon-gamma against disseminated candidiasis has been demonstrated in a mouse model. Activation of neutrophils is the main mechanism by which interferon-gamma enhances the elimination of Candida, and consequently the agent is not effective in severely granulocytopenic animals. Data on the role of colony-stimulating factors against fungal pathogens are accumulating, and trials with these agents for hematologic patients with invasive fungal infections are now being performed.

Recent studies point to a dual role for galectin-3 as both a circulating damage-associated molecular pattern and a cell membrane-associated pattern recognition receptor. The aim of this study was to assess the potential of circulating galectin-3 for discriminating between infections and non-infectious inflammatory disorders on the one hand, and between fungal and bacterial infections on the other. Galectin-3 and C-reactive protein (CRP) were measured in the plasma of 127 patients with either non-infectious inflammatory disorders (gout, autoinflammatory syndrome or pancreatitis) or an infection (viral lower respiratory tract infection, bacterial sepsis or candidaemia). Circulating galectin-3 concentrations were increased in patients with infections when compared with healthy volunteers or patients with non-infectious inflammatory diseases. At cut-off values with a specificity of 95 %, the sensitivity of galectin-3 (>20.6 ng/ml) to discriminate between an infection and non-infectious inflammation was higher than that of CRP (>156 mg/l): 43 % [95 % confidence interval (CI) 33–53 %] versus 27 % (95 % CI 19–37 %), p = 0.03. After exclusion of patients with CRP <156 mg/l, galectin-3 concentration >20.6 ng/ml could identify 41 % (95 % CI 29–53 %) of the patients with an infection at the cost of one false-positive with non-infectious inflammation. Using this sequential approach, 57 % of the patients with an infection could be selected. Galectin-3 concentrations were similar in patients with bacterial and Candida sepsis, while being lower in viral respiratory infections. Although galectin-3 does not discriminate between bacterial and Candida sepsis, the sequential use of CRP and galectin-3 in distinguishing infectious diseases from non-infectious inflammation may be superior to CRP alone. [ABSTRACT FROM AUTHOR]

Invasive fungal infections are relatively common opportunistic infections in immunocompromised patients and are still associated with a high mortality rate. Furthermore, these infections are often complicated by resistance or refractoriness to current antimicrobial agents. Therefore, an urgent need exists for new therapeutic strategies based on the identification of new microbial targets and novel antimicrobial agents. One such hypothetical therapeutic strategy may involve the use of mycoviruses that are able to selectively infect fungi. Current knowledge of mycoviruses of human pathogenic fungi and the scope for using (recombinant) mycoviruses as future biological control agents are reviewed here. [ABSTRACT FROM AUTHOR]

Disseminated fungal infections are increasing. However, the interactions between the body's largest population of tissue macrophages, the Kupffer cells and the fungal pathogens are scarcely understood. The aim of this study was to examine the involvement of Toll-like receptor 4 (TLR4) signalling in cytokine production, using primary cultures of rat and murine Kupffer cells exposed to Aspergillus fumigatus and Candida albicans hyphae and conidia. All fungal components induced the release of tumour necrosis factor-α (TNF-α), but with delayed kinetics compared with lipopolysaccharide (LPS). Candida albicans was the most potent inducer of TNF-α protein and mRNA and the only inducer of interleukin-10 (IL-10) in rat Kupffer cells. All fungal components induced enhanced mRNA levels of macrophage inhibitory protein-2 (MIP-2) in the cells, similar to LPS. Inhibitors of Src tyrosine kinases added to cells prior to stimulation led to attenuation in the release of both TNF-α (60%, P < 0.05) and IL-10 (70%, P < 0.05) induced by C. albicans conidia but did not influence the LPS-mediated cytokine release. Murine Kupffer cells (C57BL/10J) also released TNF-α as well as the chemokines keratinocyte-derived chemokine (KC) and MIP-2 in response to fungal component. Surprisingly, Kupffer cells from TLR4-deficient C57BL/ScCr mice exhibited significantly enhanced production of KC and MIP-2 upon stimulation by fungal components compared with control littermates ( P < 0.05). Our study demonstrates that Aspergillus and Candida components induce cytokine production in rat Kupffer cells and that the response to C. albicans conidia involves Src tyrosine kinases. The experiments with TLR4-deficient Kupffer cells suggest that the cytokine response in these cells to fungal component is not mediated by TLR4. [ABSTRACT FROM AUTHOR]

Data on the salvage treatment of invasive candidiasis with voriconazole in 52 patients intolerant of other antifungal agents or with infection refractory to other antifungal agents were analyzed. Patients had received a mean of two previous antifungal agents (range, 1–4 agents), and 83% had received an azole. Manifestations of invasive candidiasis included candidemia (37%), disseminated disease (25%), and infection of other sites (38%). The median duration of voriconazole therapy was 60 days (range, 1–314 days). The overall rate of response was 56% (95%CI, 41–70), with the following response rates observed for individual Candida species: Candida albicans, 44% (20–70); Candida glabrata, 38% (14–68); Candida krusei, 70% (35–93); Candida tropicalis, 67% (30–93); and other Candida spp., 100% (40–100). The response rate in patients who had failed previous azole therapy was 58% (42–73). Common adverse events (~20%) included nausea and emesis, abnormal liver enzymes, and visual disturbances. Serious adverse events occurred in four patients, and nine patients died. Voriconazole has promise as a salvage agent for the treatment of invasive candidiasis, even in the settings of previous azole therapy and infection due to Candida krusei. [ABSTRACT FROM AUTHOR]