Antimicrobial Susceptibility Testing of Six Clinical Isolates of Aspergillus

Abstract

Several different methods of performing susceptibility tests on six clinical isolates of Aspergillus are described. Some of the conditions that affected the level of susceptibility to drugs were: the type of media used, temperature and time of incubation, and the initial inoculum size. For amphotericin B susceptibility testing, the effectiveness of the polyene antibiotic as measured by visual growth was equivalent to the effectiveness as measured by inhibition of ribonucleic acid synthesis and dry-weight increase. For 5-fluorocytosine and rifampin, the visual-turbidity method gave minimum inhibitory concentrations that were much higher than those determined by effects on ribonucleic acid synthesis and dry weight. The reason for these discrepancies in susceptibility testing with 5-fluorocytosine and rifampin are unknown. We conclude that the most relevant test of this fungus to antifungal agents will have to be determined by the correlation of in vitro data with animal experiments and clinical results.Unlike the case with bacteria, there is no standard universally accepted method of antimicrobial susceptibility testing for filamentous fungi. There are several reasons for this, the most important being: fungus infections are uncommon compared with bacteria, and experience with fungal susceptibility testing is limited; the variability in morphology, growth rate, and optimal conditions of growth of fungi have made standardization difficult; and the instability and variable solubility of the most important antifungal agents complicate the tests (8).We have been screening clinical isolates of Aspergillus for their susceptibility to different antimicrobial agents. Besides the usual problems regarding the choice ofmedium, inoculum size, and method of susceptibility testing, the most appropriate temperature and length of incubation for the susceptibility tests are not known. In addition, it is not clear whether the susceptibility tests should be performed on conidiospores, recently germinated spores, or mature mycelium. Although the number ofreports on susceptibility testing ofAspergillus is small, there are contradictory recommendations for each of the above variables (5,6,17).This report describes our efforts to evaluate all of the above alternatives in susceptibility testing of several isolates of Aspergillus. In addition, we have compared the results of the tube dilution turbidity method, which is one of the most accepted methods of determining drug susceptibility, with several other methods we have used.MATERIALS AND METHODS Chemicals. Amphotericin B (AmB), in the form of Fungizone, was purchased from E. R. Squibb & Sons Inc., Princeton, N.J. It was dissolved in sterile water before use. Rifampin was obtained from Dow Chemical Co., Zionsville, Ind. Eight milligrams of rifampin powder was dissolved in 0.5 ml of absolute ethanol plus 7.5 ml of distilled water. 5-Fluorocytosine (5FC) was obtained from Hoffmann-La Roche, Nutley, N.J., and was dissolved in distilled water.[5-3H]uridine (specific activity, 8 Ci/mmol) was ...

Germinated and Nongerminated Conidial Suspensions for Testing of Susceptibilities of Aspergillus spp. to Amphotericin B, Itraconazole, Posaconazole, Ravuconazole, and Voriconazole

“…This study compared the in vitro fungistatic and fungicidal activities of five agents against nongerminated and germinated conidia of Aspergillus isolates. Conidial germination required 7 to 9 h of incubation for five of the six species; germination of A. terreus conidia required 14 to 20 h. Similar results (8 to 10 h) have been reported for A. fumigatus and A. flavus (16,18). Overall, the MICs of the established and investigational agents obtained with both conidial suspensions were the same or within a 2-dilution range (Table 1).…”

“…Prior studies have compared MICs obtained by employing either germinated conidia or hyphal suspensions to those obtained with nongerminated conidia for dematiaceous fungi (13), Aspergillus spp., and other opportunistic moniliaceous molds (2, 5, 13, 16-18, 22, 25). However, findings on the effect of hyphae on MIC determination (2,5,13,17,22,25) have been more contradictory than those on the effect of germinated conidia (16,18).…”

“…Prior studies have compared MICs obtained by employing either germinated conidia or hyphal suspensions to those obtained with nongerminated conidia for dematiaceous fungi (13), Aspergillus spp., and other opportunistic moniliaceous molds (2, 5, 13, 16-18, 22, 25). However, findings on the effect of hyphae on MIC determination (2,5,13,17,22,25) have been more contradictory than those on the effect of germinated conidia (16,18).Although Aspergillus fumigatus is responsible for the majority (85 to 90%) of the different clinical manifestations of Aspergillus infections (4), other Aspergillus spp. also have been associated with severe infection in immunocompromised hosts (4,21,24,25).…”

Pharmacokinetics of aerosol amphotericin B in rats

“…Intravenous amphotericin B (AmB) remains the standard treatment for most serious fungal infections (1,4,(6)(7)(8) and is the only proven treatment for pulmonary aspergillosis. Unfortunately, the drug is toxic, and treatment failures are common.…”

Activity of Amphotericin B, 5-Fluorocytosine, and Rifampin Against Six Clinical Isolates of Aspergillus

“…As a first approach to this problem, we have attempted to define the variables and to standardize susceptibility testing of Aspergillus to amphotericin B (AmB); and we have also tested the responsivenesss of this organism to 5-fluorocytosine (5F0) and rifampin (5). In this report, we show that, with some clinical isolates ofAspergillus, AmB in combination with either 5FC or rifampin is synergistic and leads to an enhanced antifungal effect.…”