Aspergillus terreus is an uncommon but emerging fungal pathogen, which causes lethal infections that are often refractory to amphotericin B (AmB). In comparison to Aspergillus fumigatus, A. terreus was resistant to the in vitro fungicidal effects of safely achievable concentrations of AmB. These in vitro findings correlated directly with resistance of A. terreus to AmB in experimental invasive pulmonary aspergillosis. Residual fungal pulmonary burden and galactomannan antigenemia demonstrated persistent infection, despite therapy with deoxycholate AmB or liposomal AmB. By comparison, posaconazole and itraconazole resolved GM antigenemia, reduced residual fungal burden, and improved survival. There were no differences in phagocytic host response to A. terreus versus A. fumigatus; however, the rate of conidial germination of A. terreus was slower. The strain of A. terreus with the highest minimum inhibitory and minimum lethal concentration of AmB also had the lowest membrane ergosterol content. The hyphae of A. terreus in vivo displayed distinctive aleurioconidia, which may be a practical microscopic feature for rapid preliminary diagnosis.

The purpose of the study was to evaluate the interlaboratory agreement of broth dilution susceptibility methods for five species of conidium-forming (size range, 2 to 7 m) filamentous fungi. The methods used included both macro-and microdilution methods that were adaptations of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (m27-P). The MICs of amphotericin B, fluconazole, itraconazole, miconazole, and ketoconazole were determined in six centers by both macro-and microdilution tests for 25 isolates of Aspergillus flavus, Aspergillus fumigatus, Pseudallescheria boydii, Rhizopus arrhizus, and Sporothrix schenckii. All isolates produced clearly detectable growth within 1 to 4 days at 35؇C in the RPMI 1640 medium. Colony counts of 0.4 ؋ 10 6 to 3.3 ؋ 10 6 CFU/ml (mean, 1.4 ؋ 10 6 CFU/ml) were demonstrated in 90% of the 148 inoculum preparations. Overall, good intralaboratory agreement was demonstrated with amphotericin B, fluconazole, and ketoconazole MICs (90 to 97%). The agreement was lower with itraconazole MICs (59 to 79% median). Interlaboratory reproducibility demonstrated similar results: 90 to 100% agreement with amphotericin B, fluconazole, miconazole, and ketoconazole MICs and 59 to 91% with itraconazole MICs. Among the species tested, the MICs for S. schenckii showed the highest variability. The results of the study imply that it may be possible to develop a reference method for antifungal susceptibility testing of filamentous fungi.Despite the lower volume of serious infections caused by filamentous fungi compared with that of serious infections caused by yeasts, the performance of antifungal susceptibility testing for these opportunistic pathogens is important in the clinical laboratory (8). Progress has been made in developing guidelines for the antifungal susceptibility testing of yeasts to deal with the standardization of different testing parameters such as inoculum preparation, medium composition and pH, length of incubation, and endpoint criteria (3,5,6,(9)(10)(11)(12). The standardization of these antifungal susceptibility testing steps has led to increased interlaboratory reproducibility which has opened the possibility of developing standards by adopting and tailoring these steps for antifungal susceptibility tests for the filamentous fungi. The first priority of the National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Tests was to develop a standard for the preparation of inoculum suspensions. Among four procedures evaluated, a spectrophotometric method was recommended as the procedure that gives the least variable results for the preparation of inoculum suspensions of yeast cells (11). This recommendation has been substantiated by ensuing collaborative studies of the subcommittee and other studies (2,3,5,6,12).The spectrophotometric method has been evaluated further in a single laboratory for the preparation of conidial suspensions of selected medically important filamentous fungi (1). In t...

A model of primary pulmonary aspergillosis in rabbits was developed to reproduce the persistent levels of profound granulocytopenia and the histopathologic features of bronchopneumonia, vascular invasion, and hemorrhagic infarction encountered in humans. D-mannitol was detectable in bronchoalveolar lavage fluid by gas-liquid chromatography/mass spectroscopy, and galactomannan was measurable in serum by latex agglutination immunoassay. A pharmacokinetically distinctive unilamellar vesicle formulation of liposomal amphotericin B, 5 mg/kg/day intravenously, compared with high-dose conventional desoxycholate amphotericin B, 1 mg/kg/day intravenously, was more effective in preventing nephrotoxicity, increasing survival, reducing the number of viable organisms, and decreasing tissue injury due to Aspergillus organisms. Thus, D-mannitol in lavage fluid and galactomannan in serum may be useful markers of pulmonary aspergillosis, and liposomal amphotericin B was significantly more effective and safer than desoxycholate amphotericin B for treatment of pulmonary aspergillosis in profoundly granulocytopenic rabbits.

This model provides a strategy by which relationships among pathogenesis, immunological effectors, and antifungal drug therapy for invasive pulmonary aspergillosis may be further understood.

Standard conditions are not available for evaluating the minimum fungicidal concentrations (MFCs) of antifungal agents. This multicenter collaborative study investigated the reproducibility in three laboratories of itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B MFCs for 15 selected isolates of Aspergillus spp. After MIC determinations for the 15 isolates in each center by the NCCLS M38-A broth microdilution method with four media, standard RPMI 1640 (RPMI), RPMI with 2% dextrose, antibiotic medium 3 (M3), and M3 with 2% dextrose, MFCs were determined for each isolate-medium-drug combination. MFCs were defined as the lowest drug dilutions that yielded <3 colonies (approximately 99 to 99.5% killing activity). The highest reproducibility (96 to 100%) was for amphotericin B MFCs with the four media. Although reproducibility was more variable and medium dependent for the azoles (91 to 98%), agreement was good to excellent for itraconazole, ravuconazole, and voriconazole MFCs with RPMI and M3 (93 to 98%). For posaconazole, the agreement was higher with M3 media (91 to 96%) than with RPMI media (91%). These data extend the refinement of testing guidelines for susceptibility testing of Aspergillus spp. and warrant consideration for introduction into future versions of the M38 document. The role of the MFC under these standardized testing conditions as a predictor of clinical outcome needs to be established in clinical trials.Aspergillus fumigatus and other Aspergillus spp. are responsible for the majority (85 to 90%) of clinical manifestations of severe infections caused by the filamentous fungi (moulds), especially in the immunocompromised host (4). The increased incidence of fungal infections and the development of new antifungal agents have underscored the importance of the laboratory's role in the selection and monitoring of antifungal therapy. The National Committee for Clinical Laboratory Standards (NCCLS) Subcommittee on Antifungal Susceptibility Tests has developed a reproducible reference testing procedure for the antifungal susceptibility testing of moulds (the M38-A document [18]). The recommendations described in the M38-A document for determination of MICs include the use of the standard RPMI 1640 broth (RPMI), which contains 0.2% dextrose (18). However, the document does not describe testing conditions for determination of minimum fungicidal (or lethal) concentrations (MFCs). Whether MICs are the best in vitro predictors of in vivo or clinical response to antifungal therapy is uncertain. Although standard conditions are not available for determination of fungicidal activities for either yeasts or moulds, it has been demonstrated that MFCs may be better predictors than MICs of therapeutic failure of amphotericin B in trichosporonosis (26, 27) and candidemia (20). The fungicidal activities of the new triazoles have also been evaluated during the last few years by nonstandardized methods (3,7,11,13,14,16,17,21,24,25).The purpose of this collaborative study was to investigate the in...