Using routine blood parameters to anticipate clinical outcomes in invasive aspergillosis.

OBJECTIVE: In invasive aspergillosis (IA), monitoring response to antifungal treatment is challenging. We aimed to explore if routine blood parameters help to anticipate outcomes following IA. METHODS: Post hoc secondary analysis of two multicenter randomized trials was performed. The Global Comparative Aspergillosis Study (GCA, n = 123) and the Combination Antifungal Study (CAS, n = 251) constituted the discovery and validation cohorts respectively. The outcome measures were response to treatment and survival to 12 weeks. Interval platelet, galactomannan index (GMI) and C-reactive protein (CRP) levels prior and during antifungal treatment were analysed using logistic regression, Kaplan-Meier survival and receiver operating characteristic (ROC) analyses. RESULTS: The 12-week survival was 70.7% and 63.7% for the GCA and CAS cohorts respectively. In the GCA cohort, every 10 × 109/L platelet count increase at week 2 and 4 improved 12-week survival odds by 6-18% (odds ratio (OR) 1.06-1.18, 95% confidence interval (CI) 1.02-1.33). Survival odds also improved 13% with every 10 mg/dL CRP drop at week 1 and 2 (OR 0.87, 95% CI 0.78-0.97). In the CAS cohort, week 2 platelet count was also associated with 12-week survival with 10% improved odds for every 10 × 109/L platelet increase (OR, 1.10, 95% CI 1.04-1.15). A GMI drop of 0.1 unit was additionally found to increase the odds of treatment response by 3% at the baseline of week 0 (OR 0.97, 95% CI 0.95-0.99). Week 2 platelet and CRP levels performed better than GMI on ROC analyses for survival (area under ROC curve 0.76, 0.87 and 0.67 respectively). A baseline platelet count higher than 30 × 109/L clearly identified patients with >75% survival probability. CONCLUSIONS: Higher serial platelets were associated with overall survival while GMI trends were linked to IA treatment response. Routine and simple laboratory indices may aid follow-up of response in IA patients.

Improvement in the outcome of invasive fusariosis in the last decade.

Invasive fusariosis (IF) has been associated with a poor prognosis. Although recent series have reported improved outcomes, the definition of optimal treatments remains controversial. The objective of this study was to evaluate changes in the outcome of IF. We retrospectively analysed 233 cases of IF from 11 countries, comparing demographics, clinical findings, treatment and outcome in two periods: 1985-2000 (period 1) and 2001-2011 (period 2). Most patients (92%) had haematological disease. Primary treatment with deoxycholate amphotericin B was more frequent in period 1 (63% vs. 30%, p <0.001), whereas voriconazole (32% vs. 2%, p <0.001) and combination therapies (18% vs. 1%, p <0.001) were more frequent in period 2. The 90-day probabilities of survival in periods 1 and 2 were 22% and 43%, respectively (p <0.001). In period 2, the 90-day probabilities of survival were 60% with voriconazole, 53% with a lipid formulation of amphotericin B, and 28% with deoxycholate amphotericin B (p 0.04). Variables associated with poor prognosis (death 90 days after the diagnosis of fusariosis) by multivariable analysis were: receipt of corticosteroids (hazard ratio (HR) 2.11, 95% CI 1.18-3.76, p 0.01), neutropenia at end of treatment (HR 2.70, 95% CI 1.57-4.65, p <0.001), and receipt of deoxycholate amphotericin B (HR 1.83, 95% CI 1.06-3.16, p 0.03). Treatment practices have changed over the last decade, with an increased use of voriconazole and combination therapies. There has been a 21% increase in survival rate in the last decade.

The efficacy of voriconazole in the treatment of 192 fungal central nervous system infections: a retrospective analysis.

PURPOSE: The efficacy of voriconazole against fungal central nervous system (CNS) infections was examined retrospectively. METHODS: Voriconazole-treated patients with proven (137) or probable (55) CNS infections were identified in the voriconazole database (114) and the literature (78). Investigator-determined success was a complete or partial response. Survival was calculated from the start of voriconazole therapy. RESULTS: The patients' age range was <1-81 years (median 43) and 127 (66%) were male. Aspergillus spp. (63%) and Scedosporium spp. (18%) predominated, but 12 other genera were recorded. Underlying conditions were haematopoietic stem cell transplantation (HSCT, 35), haematologic malignancy (HM, 35), solid-organ transplantation (SOT, 25), chronic immunosuppression (CI, 40) and other conditions (OC, 57). The median voriconazole therapy duration was 93 days (range 1-1,128), with success in 93 patients (48%). Only 35 patients received primary therapy, with success in 63% versus 45% for salvage (p = 0.06 NS). Underlying conditions influenced success; HSCT 14%, HM 54%, SOT 40%, CI 45% and OC 72% (p < 0.001). Additional antifungal combination therapy (37 patients) gave a trend towards an improved response rate (p = 0.09) and superior survival (p = 0.0149), while patients receiving neurosurgical interventions (72) showed superior responses (p = 0.0174) and survival (p = 0.0399). In all, 49% of patients died, 71% (67/94) due to fungal infection. The overall median survival was 297 days (range 3 to >2,000). Paediatric (p = 0.014) and literature patients (p < 0.001) exhibited superior survival compared with adults and voriconazole database patients, respectively. CONCLUSIONS: Voriconazole shows encouraging efficacy against various CNS fungal infections. Combination therapy and/or CNS surgery may improve outcomes.

Large-scale multicentre study of fluconazole in the treatment of hospitalised patients with fungal infections. Multicentre European Study Group.

The purpose of this prospective, open-label, noncomparative, multicentre study was to evaluate the efficacy and safety of fluconazole in the treatment of hospitalised patients with mycoses. A total of 587 patients with diagnosed fungal infections were enrolled. Fluconazole was given orally or intravenously in a 200 or 400 mg loading dose, followed by 100 or 200 mg once daily. The most common candidal infections were fungemia, esophageal candidiasis, bronchopulmonary candidiasis, peritonitis, oropharyngeal candidiasis, urinary tract infection and deep wound infection. Meningitis was the most common cryptococcal infection. Of the 291 evaluable patients with candidiasis, 96% (70/73) of AIDS patients and 79% (171/218) of non-AIDS patients were clinically cured or improved. Of the 36 evaluable patients with cryptococcosis, 69% (20/29) of AIDS patients and 100% (7/7) of non-AIDS patients responded clinically. The overall mycological eradication rate was 85%; eradication was similar in patients with and without AIDS. Most adverse events during fluconazole therapy were mild to moderate in severity. This investigation confirms the results of previous studies demonstrating high response rates to fluconazole therapy in AIDS and non-AIDS patients with fungal infections. Even during long-term therapy treatment-limiting adverse events were uncommon with fluconazole.

Combination therapy of murine invasive candidiasis with fluconazole and amphotericin B.

A study was performed to assess the in vivo relevance of the in vitro antagonism between fluconazole and amphotericin B against Candida albicans. Combinations of fluconazole and amphotericin B were explored for their efficacies against acute (100% mortality in 2 to 5 days) or less acute (100% mortality in 30 days) invasive candidiasis infections in mice with healthy immune systems and immunocompromised mice. Treatment efficacy was assessed by protection from mortality and/or a reduction in the fungal burden in tissue. In models of acute infection in mice with healthy immune systems or less acute infection in immunocompromised mice, combinations of fluconazole and amphotericin B were superior to fluconazole alone, and the effects were at least additive. Combination therapy was at least as efficacious as amphotericin B alone. In a different model of less acute infection in mice with healthy immune systems, combinations of fluconazole and amphotericin B showed no interactions and were no better than either drug alone. We conclude that combination therapy with fluconazole and amphotericin B is not antagonistic in vivo, in contrast to published in vitro studies, and, consequently, suggest that combination therapy should be considered in the management of clinical candidiasis.

Fluconazole resistance in Candida glabrata.

We report a case of infection with Candida glabrata in which the organism became resistant to fluconazole and in which pre- and posttreatment isolates were available for comparison. The organism was cross-resistant to ketoconazole and itraconazole, in common with other azole-resistant yeasts. Fluconazole was a potent inhibitor of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) in lysates of cells from both susceptible and resistant cultures (50% inhibitory concentration, 0.2 microM), indicating that resistance was unrelated to changes in P-450DM. Instead, it appeared to arise from a permeability barrier to fluconazole, since resistant cells were unable to take up radiolabelled drug.

Standardized susceptibility testing of fluconazole: an international collaborative study.

An international collaborative study of broth dilution (MIC) and disk diffusion susceptibility testing of fluconazole was conducted by using a chemically defined medium (High-Resolution Antifungal Assay Medium; Oxoid Ltd., Basingstoke, United Kingdom) and standard test methods performed in eight reference laboratories. Ten yeast isolates were tested by each test method in duplicate on each of 3 separate days. The intralaboratory reproducibility of the MIC test was excellent; 95.7% of the replicate tests (n = 220) were within 2 doubling dilutions of the other values in the set for the eight laboratories. The intralaboratory reproducibility of the disk test was also good, with 91% of the replicate tests (n = 234) agreeing with each other within an arbitrarily chosen value of 4 mm. Interlaboratory agreement of MIC test results was acceptable, with 84% of the MICs agreeing within 2 doubling dilutions. In contrast, the interlaboratory agreement of the disk test was not good, with only 59% of test results agreeing within 4 mm. Comparison of the rank order of MICs obtained in each laboratory with the reference rank order gave an agreement of 70 to 80% (median, 80%) with the MIC test and 70 to 90% (median, 80%) with the disk test. These preliminary results are encouraging for the development of standardized testing methods for testing fluconazole.

Fluconazole and other azoles: translation of in vitro activity to in vivo and clinical efficacy.

Fluconazole is polar, soluble in water, and metabolically stable and exhibits low protein binding. In contrast, other systemic azoles are lipophilic, metabolically vulnerable compounds with high protein binding and negligible solubility in water. The physical and pharmacokinetic properties of these drugs plus their inherent antifungal potency determine their efficacy. Although fluconazole is less active than ketoconazole in vitro, its distribution throughout the body and the high levels of free drug reached in the blood contribute to and are of value in predicting its efficacy. Even for ketoconazole the levels of free drug in blood may be indicative of efficacy. For very lipophilic agents (itraconazole), blood levels of drug are very low, and organ levels may correlate better with efficacy, although tissue binding will be high and total drug levels in an organ may be misleading indicators of efficacy. The excellent efficacy of fluconazole in vivo despite its low activity in vitro has caused confusion. Consequently, a disk test is being developed to assess whether fungal isolates are sensitive to therapeutically achievable levels of drug.

Discovery of fluconazole, a novel antifungal agent.

Fluconazole, 2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)-2-propanol, is the result of a research program aimed at the development of a broad-spectrum antifungal agent active by both the oral and the intravenous routes for the treatment of superficial and systemic infections. The program began in 1978, and azole derivatives were chosen as a starting point because they were generally well tolerated and offered the advantage of a selective mode of action--inhibition of fungal sterol C-14 demethylation. Initial investigations focused on imidazole derivatives, but attention soon switched to triazole analogues because of their reduced susceptibility to metabolic activity. Polar derivatives were emphasized in an effort to facilitate the attainment of high levels in the blood and to reduce metabolic breakdown still further. This strategy resulted in a number of novel 1,3-bis-triazole-2-arylpropan-2-ol derivatives with good activity in a range of models of fungal infection. These models included systemic fungal infections in both normal and immunosuppressed animals and superficial infections in animals with normal immune function. The 2,4-difluorophenyl analogue, fluconazole, was chosen for development on the basis of an optimal combination of antifungal efficacy, pharmacokinetic characteristics, aqueous solubility, and safety profile.

Interaction of microsomal cytochrome P-450 isolated from Aspergillus fumigatus with fluconazole and itraconazole.

A simple procedure has been developed for isolating a microsomal fraction from Aspergillus fumigatus which contains cytochrome P-450. Maximum absorbance of the carbon monoxide/ferrous cytochrome P-450 difference spectrum was at a wavelength of 451 nm. The triazole antifungal agents, fluconazole and itraconazole, produced type II binding difference spectra with ferric cytochrome P-450 in A. fumigatus microsomes and an azole concentration of 0.5 microM was sufficient to saturate the spectroscopic response when the cytochrome P-450 concentration was 0.07 microM. This extremely high affinity binding precluded the determination of apparent dissociation constants for the cytochrome P-450-azole complexes. Itraconazole was found to have a marginally greater affinity for cytochrome P-450 than fluconazole, as determined from a comparison of their potential to reduce the rate of binding of carbon monoxide to the ferrous haemoprotein. The high affinity binding of both compounds to the cytochrome P-450 from A. fumigatus is consistent with their proposed antifungal mode of action through inhibition of the sterol 14 alpha-demethylase cytochrome P-450 required for ergosterol biosynthesis.

A novel method for studying ergosterol biosynthesis by a cell-free preparation of Aspergillus fumigatus and its inhibition by azole antifungal agents.

A novel procedure has been developed for measuring ergosterol biosynthesis from [14C]mevalonate in a cell-free extract prepared from Aspergillus fumigatus. Ergosterol accounted for approximately 60% of the 4, 14-desmethylated sterol fraction which in turn totalled 13.2% of the non-saponifiable lipid produced. The other major sterol fractions were 4, 4-dimethylated sterols and 4-monomethylated sterols which accounted for 30.8% and 20.1% respectively of non-saponifiable lipid. The cell-free system had a narrow pH optimum of 7.2-7.4 for desmethylated sterol biosynthesis. Activity decreased by 94% at pH 6.5. Fluconazole (10(-4) M), ketoconazole (10(-6) M) and itraconazole (10(-6) M) inhibited formation of desmethylated sterols by greater than 85%, while 4-monomethylated sterols and 4, 4-dimethylated sterols were increased. The IC50s for inhibition of desmethylated sterol biosynthesis were 1.4 x 10(-6) M for fluconazole, 4.0 x 10(-8) M for ketoconazole, and 3.3 x 10(-8) M for itraconazole. The difference in intrinsic potency between fluconazole and ketoconazole is particularly interesting in view of the fact that fluconazole is a more effective agent than ketoconazole in an animal infection model of systemic aspergillosis.

Efficacy of fluconazole (UK-49,858) against experimental aspergillosis and cryptococcosis in mice.

The efficacy of fluconazole, a new bis-triazole antifungal agent, was compared with that of orally administered ketoconazole and parenterally administered amphotericin B against aspergillus and cryptococcus infections in mice. Fluconazole was 5-20-fold more active than ketoconazole against systemic aspergillosis and against systemic, intracranial and pulmonary cryptococcosis but was less active than amphotericin B.

Improved method for estimation of azole antifungal inhibitory concentrations against Candida species, based on azole/antibiotic interactions.

Low, reproducible minimal inhibitory concentrations against Candida species, with sharp, precise end points in complex media were achieved for imidazoles (clotrimazole, econazole, miconazole, tioconazole and ketoconazole) and triazoles (fluconazole, itraconazole, vibunazole, ICI 153066) by including in the test medium antibacterial antibiotics that bind to the 80S eukaryotic ribosome and inhibit protein synthesis, i.e. blasticidin, cycloheximide, doxycycline, neomycin and gentamicin. The presence of these antibiotics reduced MICs, on average, by 50- to 250-fold. Other protein synthesis inhibitors (rifampicin, erythromycin, lincomycin, clindamycin, chloramphenicol and fusidic acid) were not effective, and the antibiotics did not affect MICs for Aspergillus species. The low azole MICs were in close agreement with MICs obtained in a defined, tissue culture-based medium lacking added antibiotics.

Efficacy of UK-49,858 (fluconazole) against Candida albicans experimental infections in mice.

UK-49,858 (fluconazole), a new, orally absorbed bis-triazole derivative, has been evaluated against systemic infections with Candida albicans in normal and immunosuppressed mice and against an intestinal infection with C. albicans in immunosuppressed mice. Orally administered ketoconazole was used as a comparison agent throughout, and orally administered amphotericin B was included for comparative in the experimental intestinal infection. In a 10-day dosage regimen, UK-49,858 was far more active than ketoconazole against systemic infections with C. albicans in normal and immunosuppressed mice. In normal mice, extension of UK-49,858 dosing to 30 days resulted in prolongation of survival to over 90 days, and up to 60% of treated animals had no detectable C. albicans in their kidneys. In addition, over 90% of mice with intestinal candidiasis had culture-negative feces after a 3-day treatment with UK-49,858, but only 62 and 23% of mice gave this response after amphotericin B and ketoconazole therapy, respectively. These data suggest that UK-49,858 may be of value in the treatment of systemic and gastrointestinal infections due to C. albicans in humans.

Activity of UK-49,858, a bis-triazole derivative, against experimental infections with Candida albicans and Trichophyton mentagrophytes.

The therapeutic potential of UK-49,858, a difluorophenyl bis-triazole derivative, has been assessed by evaluating its activity against systemic infections with Candida albicans in normal mice and rats and in mice with impaired defence mechanisms, against vaginal infections with C. albicans in mice, and against dermal infections with Trichophyton mentagrophytes in guinea pigs. Orally administered ketoconazole was used as a comparative agent throughout, and parenterally administered amphotericin B was included in the study of C. albicans systemic infection in normal mice. The activity of UK-49,858 given orally to mice or rats infected systemically with C. albicans was far superior to that of ketoconazole. In addition, UK-49,858 showed activity comparable to that of amphotericin B when given parenterally, although the latter gave more prolonged protection. UK-49,858 was also effective orally in curing experimental candidal vaginitis in mice and trichophytosis in guinea pigs, against which it was approximately 10 times more active than ketoconazole. These data suggest that UK-49,858 may be of value in the treatment of both C. albicans and dermatophyte fungal infections in man.