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Objectives: Resistance to azole antifungal drugs in Aspergillus fumigatus that is not mediated by target gene mutations is now common in some locations. The aim of this study was to investigate possible new mechanisms of resistance in non-target resistance., Methods: Twelve azole-resistant A. fumigatus isolates previously shown not to carry mutations in cyp51A were tested to determine whether the alternative cyp51B gene was overexpressed., Results: Of 12 isolates one showed overinduction of cyp51B after exposure to itraconazole and another showed high constitutive expression of cyp51B., Conclusions: cyp51B overexpression is a possible azole resistance mechanism in A. fumigatus.

Background: Voriconazole, a broad-spectrum triazole antifungal agent increasingly used to treat aspergillosis, has been linked with acute photosensitivity and skin carcinogenesis. The action spectrum of the photosensitivity is unknown, while an indirect retinol effect secondary to the antifungal's impact on CYP450 enzymes has been proposed to contribute to the underlying mechanism., Objectives: To perform a detailed photobiological assessment of the photosensitivity presenting in a series of 12 patients treated with voriconazole., Methods: Minimal erythemal dose thresholds (MED) to narrow wavebands of ultraviolet (UV) A, UVB and visible light were determined. Provocation testing was performed to broadband UVA (310-400 nm) and to solar-simulated radiation (SSR) (290-400 nm). Patients underwent routine photopatch testing and laboratory investigations including serum vitamin A (retinol)., Results: Patients (eight men, four women; median age 54years, range 40-63) experienced moderate-severe cutaneous erythema (n = 12), burning pain (n=5), itching (n=3), scaling (n=5), vesiculation (n=5) and oedema (n=1) following sunlight exposure; increased lentigines (n=4) and actinic cheilitis (n = 4) were also observed. While the majority (n=8) of patients showed normal MED thresholds to monochromator phototesting to UVB, UVA and visible light, a low MED to UVA was observed in four patients. Repeated provocation testing with broadband UVA and SSR provoked an abnormal erythema in eight and 10 patients, respectively. Serum retinol levels were mildly elevated in two patients but normal in the majority., Conclusion:  UVA sensitivity is the predominant finding in acute voriconazole-induced photosensitivity. We found little evidence of elevated circulating retinol as the causal factor. Patients with voriconazole-induced photosensitivity require education in appropriate UVA protective measures in addition to consideration of skin surveillance for malignant sequelae., (© 2012 The Authors. BJD © 2012 British Association of Dermatologists.)

Aspergillus fumigatus is a clinically important fungus with the ability to cause invasive aspergillosis with high mortality rates in immunocompromised patients and chronic pulmonary aspergillosis in immunocompetent individuals. Virulence of mutants has traditionally been assessed using mammalian hosts such as mice and rats and more recently the fruit fly, Drosophila melanogaster, demonstrated the potential to act as an in vivo host suitable for screening Aspergillus mutants. In this study using a larger thermotolerant invertebrate, Galleria mellonella, the virulence of individual gene deletants of Aspergillus fumigatus (cpcA, sidA, sidC, sidD, sidF and paba,) were compared to the parental and gene-replacement strains, if available. A range of infectious challenges consisting of from 3 × 10(3)-3 × 10(6) spores/larva was followed by observation of larval survival with mean survival time used as a surrogate of microbial pathogenicity. Mutants cpcA, sidA, sidF and paba were avirulent and sidC and sidD showed attenuated virulence. Virulence assessment in G. mellonella correlated closely with the historic data generated using mice and Drosophila. Pre-screening Aspergillus mutants using G. mellonella could significantly reduce the number of mammals required to assess changes in virulence.

Patients with chronic mucocutaneous candidiasis (CMC) have an unknown primary immune defect and are unable to clear infections with the yeast Candida. CMC includes patients with AIRE gene mutations who have autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), and patients without known mutations. CMC patients have dysregulated cytokine production, suggesting that defective expression of pattern recognition receptors (PRRs) may underlie disease pathogenesis. In 29 patients with CMC (13 with APECED) and controls, we assessed dendritic cell (DC) subsets and monocyte Toll-like receptor (TLR) expression in blood. We generated and stimulated monocyte-derived (mo)DCs with Candida albicans, TLR-2/6 ligand and lipopolysaccharide and assessed PRR mRNA expression by polymerase chain reaction [TLR-1-10, Dectin-1 and -2, spleen tyrosine kinase (Syk) and caspase recruitment domain (CARD) 9] in immature and mature moDCs. We demonstrate for the first time that CMC patients, with or without APECED, have normal blood levels of plasmocytoid and myeloid DCs and monocyte TLR-2/TLR-6 expression. We showed that in immature moDCs, expression levels of all PRRs involved in anti-Candida responses (TLR-1, -2, -4, -6, Dectin-1, Syk, CARD9) were comparable to controls, implying that defects in PRR expression are not responsible for the increased susceptibility to Candida infections seen in CMC patients. However, as opposed to healthy controls, both groups of CMC patients failed to down-regulate PRR mRNA expression in response to Candida, consistent with defective DC maturation, as we reported recently. Thus, impaired DC maturation and consequent altered regulation of PRR signalling pathways rather than defects in PRR expression may be responsible for inadequate Candida handling in CMC patients.

Objectives: The aim of this study was to assess the dose-response of isavuconazole, voriconazole and fluconazole in disseminated Candida tropicalis and Candida krusei infections., Methods: Mice were immunosuppressed using either one dose [temporarily neutropenic (TN)] or two doses [persistently neutropenic (PN)] of cyclophosphamide. Treatment was started 5 h after infection with oral isavuconazole (6, 15, 30, 60, 90, 120 or 150 mg/kg equivalent active compound), intravenous voriconazole (5, 20 or 40 mg/kg plus grapefruit gavage twice daily) or oral fluconazole (15, 50 or 150 mg/kg) all administered twice daily. Kidney burden was assessed for C. tropicalis, and kidney and brain burden for C. krusei., Results: Vehicle controls developed a non-lethal infection with high burdens in both models. In the TN models, isavuconazole, voriconazole and fluconazole (>50 mg/kg) reduced kidney burden compared with controls; >60 mg/kg isavuconazole and 50 mg/kg fluconazole were superior to alternative treatments (other than voriconazole 40 mg/kg). Isavuconazole (all doses) reduced brain burden (P<0.05) in the C. krusei model; fluconazole (all doses) and voriconazole (5 and 20 mg/kg) did not. In the C. krusei kidney burden model, isavuconazole 120 and 150 mg/kg and voriconazole 40 mg/kg were superior to controls and fluconazole. In the C. tropicalis model, PN isavuconazole (all doses), voriconazole (>5 mg/kg) and fluconazole (all doses) reduced kidney burden (P<0.05). Only isavuconazole (all doses) and 40 mg/kg voriconazole were effective against C. krusei in the brain, isavuconazole and voriconazole reduced tissue burden (P<0.05). Fluconazole had no significant effect on brain burden even at 150 mg/kg., Conclusions: Isavuconazole significantly reduced kidney burden in mice infected with C. tropicalis and both kidney and brain burdens in mice infected with C. krusei. Isavuconazole was as effective as voriconazole and much more effective than fluconazole at reducing brain burden.

Chronic pulmonary aspergillosis is broad term encompassing chronic cavitary, chronic fibrosing pulmonary aspergillosis and aspergilloma. All affect patients with structural lung diseases and many have subtle genetic immune defects. Almost all cases are caused by Aspergillus fumigatus. Here we report a patient with an aspergilloma which had Aspergillus flavus recovered from a surgical specimen and serum containing detectable precipitating antibody.

Toll-like receptors (TLRs) are important components of innate immunity. We investigated the association between polymorphisms in the TLR2, TLR4, and TLR9 genes and susceptibility to noninvasive forms of pulmonary aspergillosis. A significant association was observed between allele G on Asp299Gly (TLR4) and chronic cavitary pulmonary aspergillosis (odds ratio [OR], 3.46; P =.003). Susceptibility to allergic bronchopulmonary aspergillosis was associated with allele C on T-1237C (TLR9) (OR, 2.49; P =. 043). No particular polymorphism was associated with severe asthma with fungal sensitization. These findings reinforce the importance of innate immunity in the pathogenesis of different forms of aspergillosis.

Although several new antifungal drugs have been licensed in the last 5 years, some patients remain difficult to treat. The main reasons for this include intrinsic or acquired antifungal resistance, organ dysfunction preventing the use of some agents and drug interactions. In addition, some drugs penetrate poorly into sanctuary sites including eye and urine, and others are associated with considerable adverse events. Here, we review the preclinical and clinical development progress with four new antifungal agents: isavuconazole, ravuconazole, albaconazole and aminocandin. Isavuconazole and ravuconazole are extremely similar, with a broad spectrum of activity, a very long half-life and large volume of distribution and good in vivo data supporting their efficacy in invasive aspergillosis and candidosis. Both compounds are in early Phase 3 development. Albaconazole has also shown very potent activity against species of Candida, Cryptococcus and Aspergillus. It was well tolerated and effective in women with vaginal candidosis. Aminocandin is an intravenous-only echinocandin with in vivo activity against Candida spp. and Aspergillus spp. Its extended half-life probably permits dosing less frequently than once a day. Overall these new antifungal agents in development offer extended half-lives, possibly reduced drug interaction profiles and good tolerance. Their antifungal spectrum is narrower than posaconazole and probably similar to voriconazole (isavuconazole and ravuconazole) and caspofungin (aminocandin). Licensure and determination of their place in clinical practice requires randomized clinical studies, which are or will be underway.

Background: There is uniform consensus that flucytosine blood concentrations should be measured to avoid toxicity and ensure adequate efficacy., Objectives and Methods: The purpose of this study was to evaluate all flucytosine levels performed in a regional centre in the UK from October 1991 to May 2006. Concentrations were measured by bioassay., Results: We reviewed 1071 flucytosine levels in 233 patients, including 33 neonates. Overall, only 20.5% of levels were in the expected therapeutic range. Low levels were observed in 40.5%, of which 5.1% were undetectable levels (<12.5 mg/L). High levels occurred in 38.9%, of which 9.9% were considered potentially toxic (>100 mg/L). High flucytosine levels occurred more frequently amongst neonates, which could be related to an immature renal system resulting in drug accumulation., Conclusions: Our findings reveal that the vast majority of patients were out of range for flucytosine levels. These data emphasize the importance of monitoring flucytosine levels.

The availability of the genome sequences of multiple Aspergillus spp. presents the research community with an unprecedented opportunity for discovery. The genomes of Neosartorya fischeri and Aspergillus clavatus have been sequenced in order to extend our knowledge of Aspergillus fumigatus, the primary cause of invasive aspergillosis. Through comparative genomic analysis, we hope to elucidate both obvious and subtle differences between genomes, developing new hypotheses that can be tested in the laboratory. A preliminary examination of the genomes and their predicted proteomes reveals extensive conservation between protein sequences and significant synteny, or conserved gene order. Comparative genomic analysis at the level of these closely related aspergilli should provide important insight into the evolutionary forces at play and their effect on gene content, regulation and expression.

The availability of new antifungal agents has multiplied the demand for in vitro antifungal susceptibility testing for Aspergillus spp. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has charged its Antifungal Susceptibility Testing Subcommittee (AFST-EUCAST) with the preparation of new guidelines for in vitro susceptibility testing of antifungals against Aspergillus spp (EUCAST-AST-ASPERGILLUS). This committee has modified the reference method for broth dilution antifungal susceptibility testing of filamentous fungi (M38-A) as follows: (i) RPMI 1640 supplemented with 2% glucose (RPMI 2%G) as assay medium, (ii) inoculum preparation by conidium counting in a haemocytometer and (iii) an inoculum size of 2×105-5×105 CFU/ml. The incubation time is about 48 h at 35°C and MIC is read visually. The MIC value is a no-growth visual endpoint. The standard method described herein is intended to provide a valid and economic method for testing the susceptibility to antifungal agents of Aspergillus spp., to identify resistance, and to facilitate an acceptable degree of conformity, e.g. agreement within specified ranges and between laboratories in measuring the susceptibility.

An 83-year-old diabetic man receiving corticosteroids developed a forearm lesion. Histology confirmed the presence of a dematiaceous fungus, with associated granulomatous inflammation. Culture of a biopsy yielded fungal colonies with branching chains of single-celled, melanised, dry, sympodial conidia, which were identified as Cladophialophora devriesii on the basis of morphology and rDNA gene sequencing. To date, C. devriesii has been a relatively rare cause of human disease. To our knowledge, this is only the second case to be described, and the first report of infection in a UK resident.

Aspergillus is a common disease-causing agent, both as an allergen causing ABPA and severe asthma with fungal sensitization (SAFS) and as a pathogen causing invasive aspergillosis in immunocompromised individuals and chronic cavitating disease (CCPA) in apparently immune competent individuals. Currently detection of Aspergillus is problematic and some of the most useful tests rely on detection of antibody response to Aspergillus allergens. Here we examine the IgE antibody response to crude and recombinant allergen tests (Asp f 1, Asp f 2, Asp f 4 and Asp f 6) in individuals with allergic conditions ABPA, SAFS and in individuals with CCPA. Additionally we use recently obtained genomic information to examine the possibility of cross reaction to these allergens and show that possible cross reactive epitopes occur in several species of Aspergillus.

Candida spp. are responsible for most of the fungal infections in humans. Available since 1990, fluconazole is well established as a leading drug in the setting of prevention and treatment of mucosal and invasive candidiasis. Fluconazole displays predictable pharmacokinetics and an excellent tolerance profile in all groups, including the elderly and children. Fluconazole is a fungistatic drug against yeasts and lacks activity against moulds. Candida krusei is intrinsically resistant to fluconazole, and other species, notably Candida glabrata, often manifest reduced susceptibility. Emergence of azole-resistant strains as well as discovery of new antifungal drugs (new triazoles and echinocandins) have raised important questions about its use as a first line drug. The aim of this review is to summarize the main available data on the position of fluconazole in the prophylaxis or curative treatment of invasive Candida spp. infections. Fluconazole is still a major drug for antifungal prophylaxis in the setting of transplantation (solid organ and bone marrow), intensive care unit, and in neutropenic patients. Prophylactic fluconazole still has a place in HIV-positive patients in viro-immunological failure with recurrent mucosal candidiasis. Fluconazole can be used in adult neutropenic patients with systemic candidiasis, as long as the species identified is a priori susceptible. Among non-neutropenic patients with candidaemia fluconazole is one of the first line drugs for susceptible species. Cases reports and uncontrolled studies have also reported its efficacy in the setting of osteoarthritis, endophthalmitis, meningitis, endocarditis and peritonitis caused by Candida spp. among immunocompetent adults. In paediatrics, fluconazole is a well tolerated and major prophylactic drug for high-risk neonates, as well as an alternative treatment for neonatal candidiasis. Importantly 15 years after its introduction in the antifungal armamentarium, fluconazole is still a first line treatment option in several cases of invasive candidiasis. Its prophylactic use should however be limited to selected high-risk patients to limit the risk of emergence of azole-resistant strains.

Aspergillus spp. produce a wide range of invasive and sapropytic syndromes which may involve any tissue. Within a given tissue or organ the pathology and pathogenesis varies enormously, ranging from angioinvasive disease to noninvasive saprophytic disease. The individual invasive and saprophytic syndromes in which a causative role can be attributed to Aspergillus spp. are detailed specifically with reference to the underlying pathology and pathogenesis, the clinical setting and features, and the manner in which a diagnosis can be established.

The sequencing and annotation of the genomes of the first strains of Aspergillus nidulans, Aspergillus oryzae, and Aspergillus fumigatus will be seen in retrospect as a transformational event in Aspergillus biology. With this event the entire genetic composition of A. nidulans, the sexual experimental model organism of the genus Aspergillus, A. oryzae, the food biotechnology organism which is the product of centuries of cultivation, and A. fumigatus, the most common causative agent of invasive aspergillosis is now revealed to the extent that we are at present able to understand. Each genome exhibits a large set of genes common to the three as well as a much smaller set of genes unique to each. Moreover, these sequences serve as resources providing the major tool to expanding our understanding of the biology of each. Transcription profiling of A. fumigatus at high temperatures and comparative genomic hybridization between A. fumigatus and a closely related Aspergillus species provides microarray based examples of the beginning of functional analysis of the genomes of these organisms going forward from the genome sequence.

CADRE is a public resource for housing and analysing genomic data extracted from species of Aspergillus. It arose to enable maintenance of the complete annotated genomic sequence of Aspergillus fumigatus and to provide tools for searching, analysing and visualizing features of fungal genomes. By implementing CADRE using Ensembl, a framework is in place for storing and comparing several genomes: the resource will thus expand by including other Aspergillus genomes (such as Aspergillus nidulans) as they become available. CADRE is accessible at http://www.cadre. man.ac.uk.

We compared the activity of four doses of micafungin (FK463) with that of amphotericin B, liposomal amphotericin B and itraconazole in a murine model of disseminated aspergillosis. Temporarily neutropenic mice were infected with a lethal dose of either an itraconazole-resistant Aspergillus fumigatus isolate or Aspergillus terreus, a species that is less susceptible to amphotericin B. Treatment was started 24 h after infection and lasted for 7 days. Mice were treated with either amphotericin B (0.5 or 5 mg/kg), liposomal amphotericin (5 or 25 mg/kg), itraconazole (25 or 75 mg/kg) or FK463 (either 1, 2, 5 or 10 mg/kg). Treatment of the A. fumigatus model with either amphotericin B, liposomal amphotericin or FK463 prolonged survival. Doses of FK463 5 and 10 mg/kg had a 100% survival. Treatment of A. terreus infection with either itraconazole or FK463, but not amphotericin B, also prolonged survival. Doses of liposomal amphotericin of 5 and 25 mg/kg were ineffective against A. terreus infection. No treatment regime was able to totally clear the liver or kidneys in either model. The data indicate that FK463 may have a clinical role in the treatment of life-threatening invasive aspergillosis.

We investigated the in vitro interaction of terbinafine with itraconazole, fluconazole, amphotericin B and 5-flucytosine, against Aspergillus spp. We tested three isolates of Aspergillus fumigatus (one resistant to itraconazole), and two each of Aspergillus flavus, Aspergillus niger and Aspergillus terreus. We employed a broth microdilution-based method derived from an in vivo validated method capable of detecting itraconazole resistance in A. fumigatus. We studied the effect on the MICs by calculation of the fractional inhibitory concentration (FIC) and fractional fungicidal concentration (FFC) (99.99% kill). Itraconazole and terbinafine were synergic or additive in all strains (FIC = 0.15-1.0). Fluconazole and terbinafine were synergic with A. fumigatus, A. terreus and A. flavus (FIC = 0.3-0.5) and indifferent with A. niger (FIC = 2) isolates. Amphotericin B and terbinafine were mostly indifferent or antagonistic (FIC = 1.0-4.02). Flucytosine and terbinafine were usually indifferent or antagonistic (FIC = 0.63-8.5). FFCs were generally in accord with FICs. The use of terbinafine in combination therapy for Aspergillus infections with azoles seems promising, whereas terbinafine and amphotericin B or flucytosine in combination were less effective.

During the past several decades, there has been a steady increase in the frequency of opportunistic invasive fungal infections (IFIs) in immunocompromised patients. However, there is substantial controversy concerning optimal diagnostic criteria for these IFIs. Therefore, members of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group formed a consensus committee to develop standard definitions for IFIs for clinical research. On the basis of a review of literature and an international consensus, a set of research-oriented definitions for the IFIs most often seen and studied in immunocompromised patients with cancer is proposed. Three levels of probability are proposed: "proven," "probable," and "possible." The definitions are intended for use in the context of clinical and/or epidemiological research, not for clinical decision making.

Voriconazole is a new azole antifungal drug with activity against a wide range of systemic fungal pathogens, including Aspergillus spp. Five patients with chronic invasive aspergillosis were treated for 12-58 weeks with voriconazole, 200 mg twice daily and developed facial erythema and cheilitis. One who received 58 weeks of therapy also developed discoid lupus erythematosus-like lesions on both sides of her neck. Both erythema and cheilitis resolved after discontinuation of voriconazole. Serum retinoids were elevated in the three patients in whom they were measured. Voriconazole has the potential for retinoid-like side-effects and facial erythema.

The activities of amphotericin B and itraconazole were studied in a temporarily neutropenic murine model of disseminated Absidia corymbifera infection, caused by two different strains. Amphotericin B MICs were 0.25 mg/L for both strains and itraconazole MICs were 1 and 2 mg/L. Amphotericin B was effective in vivo with both isolates. Itraconazole was less effective.

It was investigated whether a deficiency of mannose-binding lectin (MBL), which binds Aspergillus species avidly in vitro, could account for chronic necrotizing pulmonary aspergillosis (CNPA), which is seen most commonly in nonimmunocompromised patients. Blood samples were obtained from 11 patients (10 white) with CNPA and were compared with blood samples from 82 white control subjects. MBL haplotype profiles were determined by polymerase chain reaction, using sequence-specific primers and sequence-specific oligonucleotide probing techniques. Seven of the 10 white patients with CNPA had MBL haplotypes that encode for low levels of the protein, compared with 25.6% of the white control subjects (P=.004). Presence of the codon 52 mutation was particularly common in patients with CNPA (P=.015), which suggests a greater involvement of this mutation.

Studies of invasive fungal infections have been and remain difficult to implement. Randomized clinical trials of fungal infections are especially slow and expensive to perform because it is difficult to identify eligible patients in a timely fashion, to prove the presence of the fungal infection in an unequivocal fashion, and to evaluate outcome in a convincing fashion. Because of these challenges, licensing decisions for antifungal agents have to date depended heavily on historical control comparisons and secondary advantages of the new agent. Although the availability of newer and potentially more effective agents makes these approaches less desirable, the fundamental difficulties of trials of invasive fungal infections have not changed. Therefore, there is a need for alternative trial designs and evaluation strategies for therapeutic studies of invasive mycoses, and this article summarizes the possible strategies in this area.

Aspergillosis comprises a variety of manifestations of infection. These guidelines are directed to 3 principal entities: invasive aspergillosis, involving several organ systems (particularly pulmonary disease); pulmonary aspergilloma; and allergic bronchopulmonary aspergillosis. The recommendations are distilled in this summary, but the reader is encouraged to review the more extensive discussions in subsequent sections, which show the strength of the recommendations and the quality of the evidence, and the original publications cited in detail. Invasive aspergillosis. Because it is highly lethal in the immunocompromised host, even in the face of therapy, work-up must be prompt and aggressive, and therapy may need to be initiated upon suspicion of the diagnosis, without definitive proof (BIII). Intravenous therapy should be used initially in rapidly progressing disease (BIII). The largest therapeutic experience is with amphotericin B deoxycholate, which should be given at maximum tolerated doses (e.g., 1-1.5 mg/kg/d) and should be continued, despite modest increases in serum creatinine levels (BIII). Lipid formulations of amphotericin are indicated for the patient who has impaired renal function or who develops nephrotoxicity while receiving deoxycholate amphotericin (AII). Oral itraconazole is an alternative for patients who can take oral medication, are likely to be adherent, can be demonstrated (by serum level monitoring) to absorb the drug, and lack the potential for interaction with other drugs (BII). Oral itraconazole is attractive for continuing therapy in the patient who responds to initial iv therapy (CIII). Therapy should be prolonged beyond resolution of disease and reversible underlying predispositions (BIII). Adjunctive therapy (particularly surgery and combination chemotherapy, also immunotherapy), may be useful in certain situations (CIII). Aspergilloma. The optimal treatment strategy for aspergilloma is unknown. Therapy is predominantly directed at preventing life-threatening hemoptysis. Surgical removal of aspergilloma is definitive treatment, but because of significant morbidity and mortality it should be reserved for high-risk patients such as those with episodes of life-threatening hemoptysis, and considered for patients with underlying sarcoidosis, immunocompromised patients, and those with increasing Aspergillus-specific IgG titers (CIII). Surgical candidates would need to have adequate pulmonary function to undergo the operation. Bronchial artery embolization rarely produces a permanent success, but may be useful as a temporizing procedure in patients with life-threatening hemoptysis. Endobronchial and intracavitary instillation of antifungals or oral itraconazole may be useful for this condition. Since the majority of aspergillomas do not cause life-threatening hemoptysis, the morbidity and cost of treatment must be weighed against the clinical benefit. Allergic bronchopulmonary aspergillosis (APBA). Although no well-designed studies have been carried out, the available data support the use of corticosteroids for acute exacerbations of ABPA (AII). Neither the optimal corticosteroid dose nor the duration of therapy has been standardized, but limited data suggest the starting dose should be approximately 0.5 mg/kg/d of prednisone. The decision to taper corticosteroids should be made on an individual basis, depending on the clinical course (BIII). The available data suggest that clinical symptoms alone are inadequate to make such decisions, since significant lung damage may occur in asymptomatic patients. Increasing serum IgE levels, new or worsening infiltrate on chest radiograph, and worsening spirometry suggest that corticosteroids should be used (BII). Multiple asthmatic exacerbations in a patient with ABPA suggest that chronic corticosteroid therapy should be used (BIII). Itraconazole appears useful as a corticosteroid sparing agent (BII). (ABSTRACT TRUNCATED)

We report the occurrence of invasive aspergillosis (IA) in nonallografted patients with multiple myeloma (MM) who were treated at hematology or oncology centers in Europe during 1984-1996. Thirty-one cases met the criteria for definitive (21 [68%]) or probable (10 [32%]) IA. Of these cases, 23 (74%) were reported during 1992-1996. Twenty-nine cases (94%) occurred in patients with Durie-Salmon stage 3 MM, and 2 (6%) occurred in patients with Durie-Salmon stage 2 MM. The median time between MM and IA diagnoses was 8 months (range, 1-75 months). Sixteen patients (51%) had a neutrophil count

Amphotericin B has been the standard therapy for invasive aspergillosis since its introduction in 1957. It is only moderately effective. Many susceptibility tests have been used but little variation has been noted between strains. We have studied three strains of Aspergillus fumigatusand one of Aspergillus terreusin a neutropenic mouse model of invasive aspergillosis and attempted to correlate the variable efficacy in vivowith MICs generated by over 30 different susceptibility test formats. One strain of A. fumigatus(AF65) and the strain of A. terreus(AT49) were 'resistant' and the remaining two strains of A. fumigatus(AF210 and AF294) were 'susceptible' in vivo. Only AT49 had elevated MICs of amphotericin (MIC 2 mg/L) by 41 of 54 in vitrotesting systems. With each test format, including Etest, there was no distinction between MICs obtained for AF65, AF210 and AF294 (MICs 0.125-64 mg/L depending on the test). Thus despite extensive efforts we have been unable to correlate susceptible test results with in vivooutcome in A. fumigatusbut we have with A. terreus, with some test formats. This suggests that, at present, amphotericin B susceptibility testing of A. fumigatus is of limited clinical value and further work needs to be done to find testing systems that can identify the 'resistance' documented in vivo.

Endemic mycoses remain a major public health problem in several countries and they are becoming increasingly frequent with the spread of HIV infection. Amphotericin B remains the drug of choice during the acute stage of life-threatening endemic mycoses occurring in both immunocompetent and immunocompromised hosts. Ketoconazole is effective in non-AIDS patients with non-life-threatening histoplasmosis, blastomycosis, or paracoccidioidomycosis. Itraconazole is the treatment of choice for non-life-threatening Histoplasma capsulatum or Blastomyces dermatitidis infections occurring in immunocompetent individuals and is the most efficient secondary prophylaxis of histoplasmosis in AIDS patients. Itraconazole is also effective in lymphocutaneous and visceral sporotrichosis, in paracoccidioidomycosis, for Penicillum marneffei infection, and is an alternative to amphotericin B for Histoplasma duboisii infection. Coccidioidomycosis may be effectively treated with prolonged and sometimes life-long itraconazole or fluconazole therapy. Fluconazole has relatively poor efficacy against histoplasmosis, blastomycosis and sporotrichosis. New antifungal agents have been tested in vitro or in animal models and may soon be evaluated in clinical trials.

We developed and compared five scoring systems designed to quantitate therapeutic response in cases of oropharyngeal candidiasis. We utilized prospectively collected data on 114 patients treated with several doses of the azole D0870. Patients were infected with fluconazole-susceptible (n = 49) or -resistant organisms (MIC, > or = 16 mg/mL; n = 61). Patients with fluconazole resistance had lower CD4+ cell counts at baseline; more symptoms (P = .0006); a higher frequency of dysgeusia (P = .004), dysphagia (P = .006), and throat pain (P = .0034); and greater oral coverage by plaques of Candida. There was no difference between the two groups in terms of colony-forming units, and any change did not correlate with response to therapy. Resolution of dysphagia (P < .01) and oral pain (P < .01) correlated well with response to therapy, unlike retrosternal pain and throat pain, which were also less frequent. Xerostomia, a "furry" taste, and dysgeusia were frequent nonspecific symptoms. Scoring system C, weighting resolution of a symptom higher than absence of a symptom at baseline, yielded the best correlation with global outcome (r = 0.86) and allows the quantitation of incomplete but clinically beneficial responses to therapy.

The in-vitro activity of amphotericin B, itraconazole and voriconazole against 130 clinical and 20 environmental Aspergillus fumigatus isolates was tested with an agar dilution method using RPMI 1640 medium. Itraconazole-susceptible (AF71) and -resistant (AF90) isolates were included in each test and all isolates were tested in duplicate. Geometric mean MIC values (ranges) at 48 h and 72 h were, respectively, amphotericin B, 0.91 (0.25-2) and 1.50 (1-4) mg/L; itraconazole, 0.25 (0.06-1) and 0.48 (0.125-8) mg/L; and voriconazole, 0.56 (0.25-8) and 0.78 (0.25-8) mg/L. The reproducibility of the results was high for all drugs. In-vitro resistance of A. fumigatus to the tested antifungal agents was uncommon and the MICs of itraconazole were half those of voriconazole (P < 0.01).

The in-vitro activity of the triazole antifungal agent voriconazole was compared with itraconazole and amphotericin B against 60 aspergillus isolates. Susceptibility tests were performed using two methods: a macrodilution and a microdulution method. For macrodilution methodology, geometric mean (GM) MIC values and ranges were 0.4 and 0.125-4 mg/L for voriconazole, 0.24 and 0.06-->16 mg/L for itraconazole, and 1.04 and 0.5-4 mg/L for amphotericin B. For the microdilution method, GM MICs and ranges were 0.66 and 0.125-2 mg/L for voriconazole, 0.27 and 0.06-->16 mg/L for itraconazole, and 1.62 and 0.25-32 mg/L for amphotericin B. In conclusion, voriconazole is active against Aspergillus spp. in vitro, and at similar concentrations to itraconazole.

Little is known of the phospholipid composition of Aspergillus species. The aim of this study was to determine individual phospholipid analogues in Aspergillus. Twenty-nine clinical and environmental isolates from five Aspergillus species were analysed. Fast atom bombardment mass spectrometric data were considered in two ranges, m/z 190-500 and m/z 500-1000, to facilitate the recognition of major fatty acyl groups and phospholipids. Quantitative comparison of major anions in both m/z ranges was undertaken. Confirmation of major phospholipid anions from eight representative isolates was achieved by tandem mass spectrometry. The major phospholipid families were phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). Anions were detected consistent with the presence of specific phospholipid moieties, such as palmitoyl-linolenoyl phosphatidic acid, palmitoyl-oleoylphosphatidylethenolamine, oleoyl-linoleoyl-phophatidylserine and palmitoyl-linoleoyl-phosphatidylinositol. It appears that there is some commonality of sn1 or sn2 fatty acyl substituents, frequently C18:2 at sn1 accompanied by C16:0 at sn2, with differing molecular weights being attributable to analogues with differing head groups. Differences in certain phospholipids (e.g. minor peak with m/z 933) were detected between A. fumigatus, A. nidulans and other species examined which could have diagnostic value.

There have been several recent developments in the diagnosis of fungal infections, including the production of novel antigen detection tests, the G-test, and assays for the detection of fungal metabolites. In this article we present an overview of these developments and examine the value of including these tests in clinical trials.

Given the increased choice of therapeutic agents and the rising incidence of serious invasive disease, it is important that reliable in-vitro methods for detecting antifungal drug resistance in Aspergillus spp. are developed. Six clinical isolates of Aspergillus fumigatus, obtained from patients in whom the clinical outcome was known, were selected for study. Each was used to examine a range of parameters affecting agar dilution and broth microdilution susceptibility test results. The in-vitro results were compared with outcome in a neutropenic mouse model of invasive aspergillosis. Groups of animals were treated with itraconazole at 25 mg/kg and 75 mg/kg and survival rates and organ burdens were determined. Itraconazole was efficacious against four isolates (susceptible) but failed for two (resistant) in the animal model of infection. Both the resistant isolates had been obtained from patients receiving itraconazole treatment with good serum concentrations of the drug. Conditions for the agar dilution test which produced results that correlated best with our in-vivo observations included the use of RPMI agar with L-glutamine buffered to pH 7 with MOPS, inoculated with 10(6)-10(7) conidia/mL and incubated for 48-72 h at 28 or 35 degrees C with a no-growth endpoint. Optimal conditions for the broth microdilution method included the use of RPMI medium with L-glutamine and 2% glucose buffered to pH 7 with MOPS, an inoculum of 2 x 10(5) conidia in 200 microL incubated for 48 h at 35 degrees C with a growth (or trace) endpoint. The MICs for the susceptible isolates were 0.12-1.0 mg/L and > or = 16 mg/L for the resistant isolates. With careful selection and standardization of test conditions it is possible to generate reproducible in-vitro susceptibility data for Aspergillus spp. that will predict clinical outcome.

Several methods of susceptibility testing were compared for their ability to detect amphotericin B resistance among yeast isolates, including isolates known to be amphotericin-B-resistant in an animal model. A microtitre MIC method using antibiotic medium no. 3 and Etest strips were able to discriminate susceptible and resistant isolates. A disc susceptibility test was also able to identify resistant isolates and this method may be suitable for routine use as a screening test. A survey of resistance in 644 clinical isolates showed that the incidence of elevated MICs (> or = 0.25 mg/L) was only 1.2%.

The activity of the new triazole antifungal D0870 was compared with those of itraconazole and fluconazole against Candida krusei and Aspergillus fumigatus, two fungi showing inherent tolerance of fluconazole. The activity of D0870 resembled that of itraconazole against whole cells of C. krusei, but it was less effective against A. fumigatus. However, the effect on sterol biosynthesis, in terms of the sterol type accumulating and IC50 for in-vitro biosynthesis, appeared similar in both species. The superior antifungal effect of D0870 over fluconazole appeared related to better inhibition of ergosterol biosynthesis in A. fumigatus, but in C. krusei this did not account for the entire difference which may result mainly from reduced efflux of drug.

Phialophora species are occasional pathogens causing subcutaneous and invasive disease. We report the first case of eumycetoma caused by P. parasitica in an otherwise healthy U.K. resident who visited India. She failed to respond to surgical excision and itraconazole, 400 mg daily, but responded to itraconazole, 400 mg daily, and flucytosine, 1 g three times daily, for 12 months. In vitro susceptibility testing predicted a response.

A review of series of > or = 4 cases of invasive aspergillosis (total, 1,223 cases) was undertaken to establish the crude mortality and rate of response to therapy with amphotericin B in the major at-risk host groups. In association with pulmonary, sinus, and cerebral aspergillosis in immunocompromised patients, the crude mortality rates were 86%, 66%, and 99%, respectively. No untreated patient survived. Among 84 patients treated for 1-13 days, only one survived. Among those with invasive pulmonary aspergillosis treated for > or = 14 days, the response rates to amphotericin B deoxycholate were 83% (in cases of heart and renal transplantation), 54% (leukemia), 33% (bone marrow transplantation) and 20% (liver transplantation). Patients with AIDS mostly received both amphotericin B and itraconazole, and 37% of those treated for > or = 14 days responded to therapy. Substantial variation in outcome from series to series was related to underlying disease status, site of disease, and management. Invasive aspergillosis remains a devastating opportunistic infection despite current treatment.

Fungal infections affect virtually all patients with the Acquired Immunodeficiency Syndrome (AIDS). Superficial infection (seborrheic dermatitis, tinea capitis, tinea corporis and tinea cruris) is more common than in the general population and can be difficult to eradicate. Mucosal disease (oropharyngeal, oesophageal and vaginal candidosis) is very common and often recurs. In advanced AIDS, patients usually fail to respond to topical therapy and often to systemic therapy and isolates of Candida spp. from these patients are frequently resistant in vitro to fluconazole and other azoles. Systemic fungal infection is less common but life threatening. The commonest such infection is Pneumocystis carinii pneumonia (PCP) although prophylaxis is usually successful in preventing either the first episode or recurrent episodes. Histoplasmosis, coccidioidomycosis and Penicillium marneffei infections are common in endemic areas. Cryptococcal meningitis and invasive aspergillosis occur worldwide. The prophylaxis and treatment of all these except PCP are discussed and reviewed.

This article describes the current situation with regard to intrinsic and acquired resistance to antifungal compounds and progress with the development of standardized methods of susceptibility testing for amphotericin B, flucytosine, and the azoles. Recommendations for testing of isolates from patients destined to receive antifungal drug treatment, or in whom therapeutic failure or relapse is suspected, are presented.

Mucosal candidosis is an almost inevitable consequence of AIDS. Resistance to fluconazole therapy associated with enhanced tolerance, detectable in microbiological estimation of sensitivity, occurs in up to 10% of cases with late-stage AIDS. We report here our biochemical analysis of the basis of resistance in a study of two susceptible and two resistant isolates. Resistance was not associated with a change in the target enzyme sterol 14 alpha-demethylase, as indicated by equivalent levels of fluconazole inhibition of activity in extracts from all four isolates, or by mutations in sterol delta desaturase as previously observed in Saccharomyces cerevisiae and Ustilago maydis. Reduced cellular content of fluconazole in the resistant isolates of between six to ten-fold was observed which could account for their resistant phenotype.

We present two cases of aspergillus infection confirmed by blood culture and review 30 other cases of genuine aspergillus fungemia and 34 cases of aspergillus pseudofungemia. Multiple different media and blood culture systems were used to isolated Aspergillus. The median time to positive blood culture was 8.5 days (range, 1-27 days) in the genuine cases. Genuine aspergillus fungemia was observed more often after cardiac surgery (n = 11 [34%]) or during neutropenia (n = 9 [28%]) than in other settings. In a recent series of fungemia during neutropenia, 7.6% of all episodes were due to Aspergillus. Other patients at risk for aspergillus fungemia were similar to those at risk for invasive aspergillosis, including patients with AIDS. Seven (44%) of 19 patients who were treated survived. In the group of patients with aspergillus pseudofungemia, there were no deaths, and cultures of additional specimens from the same patient were not positive. Criteria that may be applied to ascertain whether the isolation of Aspergillus from blood cultures is clinically significant are put forward.