Frank C. Odds

Antifungal agents: mechanisms of action

Clinical needs for novel antifungal agents have altered steadily with the rise and fall of AIDS-related mycoses, and the change in spectrum of fatal disseminated fungal infections that has accompanied changes in therapeutic immunosuppressive therapies. The search for new molecular targets for antifungals has generated considerable research using modern genomic approaches, so far without generating new agents for clinical use. Meanwhile, six new antifungal agents have just reached, or are approaching, the clinic. Three are new triazoles, with extremely broad antifungal spectra, and three are echinocandins, which inhibit synthesis of fungal cell wall polysaccharides--a new mode of action. In addition, the sordarins represent a novel class of agents that inhibit fungal protein synthesis. This review describes the targets and mechanisms of action of all classes of antifungal agents in clinical use or with clinical potential.

Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences

Candida albicans and related species pathogenic for man become resistant to antifungal agents, in particular triazole compounds, by expression of efflux pumps that reduce drug accumulation, alteration of the structure or concentration of antifungal target proteins, and alteration of membrane sterol composition. The clinical consequences of antifungal resistance can be seen in treatment failures in patients and in changes in the prevalences of Candida species causing disease. These effects were seen unequivocally in HIV-infected patients with oropharyngeal candida infections, but their incidence has decreased dramatically with the introduction of highly active antiretroviral therapy. The evidence for similar emergence of antifungal-resistant yeast strains and species in other types of candida infections is confounded by non-standardised susceptibility testing methods and definitions of a resistant fungal isolate. Recent large-scale surveys of yeasts isolated from blood cultures, based on standardised methodology and resistance definitions, do not support the view that antifungal resistance in pathogenic yeasts constitutes a significant or growing therapeutic problem.

Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors

The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of beta-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of beta-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

Candida orthopsilosis and Candida metapsilosis spp. nov. To Replace Candida parapsilosis Groups II and III

ABSTRACT Two new species, Candida orthopsilosis and C. metapsilosis, are proposed to replace the existing designations of C. parapsilosis groups II and III, respectively. The species C. parapsilosis is retained for group I isolates. Attempts to construct a multilocus sequence typing scheme to differentiate individual strains of C. parapsilosis instead revealed fixed DNA sequence differences between pairs of subgroups in four genes: COX3, L1A1, SADH, and SYA1. PCR amplicons for sequencing were obtained for these four plus a further seven genes from 21 group I isolates. For nine group II isolates, PCR products were obtained from only 5 of the 11 genes, and for two group III isolates PCR products were obtained from a different set of 5 genes. Three of the PCR products from group II and III isolates differed in size from the group I products. Cluster analysis of sequence polymorphisms from COX3, SADH, and SYA1, which were common to the three groups, consistently separated the isolates into three distinct sets. All of these differences, together with DNA sequence similarities <90% in the ITS1 sequence, suggest the subgroups should be afforded species status. The near absence of DNA sequence variability among isolates of C. parapsilosis and relatively high levels of sequence variability among isolates of C. orthopsilosis suggest that the former species may have evolved very recently from the latter.

Antifungal Susceptibility Testing: Practical Aspects and Current Challenges

SUMMARY Development of standardized antifungal susceptibility testing methods has been the focus of intensive research for the last 15 years. Reference methods for yeasts (NCCLS M27-A) and molds (M38-P) are now available. The development of these methods provides researchers not only with standardized methods for testing but also with an understanding of the variables that affect interlaboratory reproducibility. With this knowledge, we have now moved into the phase of (i) demonstrating the clinical value (or lack thereof) of standardized methods, (ii) developing modifications to these reference methods that address specific problems, and (iii) developing reliable commercial test kits. Clinically relevant testing is now available for selected fungi and drugs: Candida spp. against fluconazole, itraconazole, flucytosine, and (perhaps) amphotericin B; Cryptococcus neoformans against (perhaps) fluconazole and amphotericin B; and Aspergillus spp. against (perhaps) itraconazole. Expanding the range of useful testing procedures is the current focus of research in this area.

Fungal morphogenesis and host invasion

Many fungal pathogens undergo morphological transformations during host invasion. However, the significance of this for fungal pathogenesis is not clear. Both yeast and hyphal cells have properties well suited to tissue invasion and evasion of the immune system. However, molecular control circuits that regulate morphogenesis also regulate the expression of other virulence traits. To establish the extent to which morphogenesis impacts on pathogenesis, it is necessary to characterise the morphology of the fungus at different stages and locations during the natural history of a disease and to untangle how gene expression is modulated at these stages. This review considers the role of morphogenesis in fungal infection and argues that no simple, universal relationship can be drawn between morphology and the invasive potential of a fungus.

NRG1 represses yeast–hypha morphogenesis and hypha‐specific gene expression in Candida albicans

We have characterized CaNrg1 from Candida albicans, the major fungal pathogen in humans. CaNrg1 contains a zinc finger domain that is conserved in transcriptional regulators from fungi to humans. It is most closely related to ScNrg1, which represses transcription in a Tup1‐dependent fashion in Saccharomyces cerevisiae. Inactivation of CaNrg1 in C.albicans causes filamentous and invasive growth, derepresses hypha‐specific genes, increases sensitivity to some stresses and attenuates virulence. A tup1 mutant displays similar phenotypes. However, unlike tup1 cells, nrg1 cells can form normal hyphae, generate chlamydospores at normal rates and grow at 42°C. Transcript profiling of 2002 C.albicans genes reveals that CaNrg1 represses a subset of CaTup1‐regulated genes, which includes known hypha‐specific genes and other virulence factors. Most of these genes contain an Nrg1 response element (NRE) in their promoter. CaNrg1 interacts specifically with an NRE in vitro. Also, deletion of two NREs from the ALS8 promoter releases it from Nrg1‐mediated repression. Hence, CaNrg1 is a transcriptional repressor that appears to target CaTup1 to a distinct set of virulence‐related functions, including yeast–hypha morphogenesis.

Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans

The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G4655). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints.

ABSTRACT Developing interpretive breakpoints for any given organism-drug combination requires integration of the MIC distribution, pharmacokinetic and pharmacodynamic parameters, and the relationship between the in vitro activity and outcome from both in vivo and clinical studies. Using data generated by standardized broth microdilution and disk diffusion test methods, the Antifungal Susceptibility Subcommittee of the Clinical and Laboratory Standards Institute has now proposed interpretive breakpoints for voriconazole and Candida species. The MIC distribution for voriconazole was determined using a collection of 8,702 clinical isolates. The overall MIC90 was 0.25 μg/ml and 99% of the isolates were inhibited at ≤1 μg/ml of voriconazole. Similar results were obtained for 1,681 Candida isolates (16 species) from the phase III clinical trials. Analysis of the available data for 249 patients from six phase III voriconazole clinical trials demonstrated a statistically significant correlation (P = 0.021) between MIC and investigator end-of-treatment assessment of outcome. Consistent with parallel pharmacodynamic analyses, these data support the following MIC breakpoints for voriconazole and Candida species: susceptible (S), ≤1 μg/ml; susceptible dose dependent (SDD), 2 μg/ml; and resistant (R), ≥4 μg/ml. The corresponding disk test breakpoints are as follows: S, ≥17 mm; SDD, 14 to 16 mm; and R, ≤13 mm.

An insight into the antifungal pipeline: selected new molecules and beyond

Invasive fungal infections are increasing in incidence and are associated with substantial mortality. Improved diagnostics and the availability of new antifungals have revolutionized the field of medical mycology in the past decades. This Review focuses on recent developments in the antifungal pipeline, concentrating on promising candidates such as new azoles, polyenes and echinocandins, as well as agents such as nikkomycin Z and the sordarins. Developments in vaccines and antibody-based immunotherapy are also discussed. Few therapeutic products are currently in active development, and progression of therapeutic agents with fungus-specific mechanisms of action is of key importance.