Amphotericin B in combination with 5-fluorocytosine was synergistic against three clinical isolates ofAspergillus fumigatus and one of three clinical isolates of A. flavus. Amphotericin B in combination with rifampin was synergistic against all six clinical isolates ofAspergillus tested. The levels of 5-fluorocytosine and rifampin required for synergism were higher than clinically achievable concentrations when measurements of synergism were based on visual turbidity; but when the effects of the drugs were measured by inhibition of ribonucleic acid synthesis or dry-weight increase, much lower concentrations were effective.The increasing frequency of disseminatedAspergillus infections (7), particularly in the compromised host, has stimulated us to reevaluate present therapeutic measures for this infection and to try to develop newer forms of therapy. 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. In this regard, aspergilli behave in a similar fashion to several other fungi we have tested (6,8,9 Susceptibility tests. All of the susceptibility tests were done in duplicate at least three times, and there was no significant variation within each experimental method. Conidiospores were harvested from 7-day-old cultures by flooding the surface growth with phosphate-buffered saline and shaking the tubes. The conidiospores dislodged from the mycelium were then agitated with glass beads to break down aggregates and yield a uniformly dispersed suspension of single spores. The spores were then separated from the beads and resuspended in 50-ml Erlenmeyer flasks in 2 x Salvin liquid medium at a concentration of 2 x 105 spores per ml. We used Salvin medium in these experiments because of our findings in the accompanying paper (5). One-milliliter samples from each of the flasks were pipetted into capped tubes, the drugs were added at an equivalent volume to each of the flasks or tubes, and the cultures were incubated at 37 C for 24 h. At the end of the 24-h incubation, the tubes were read visually for turbidity and graded 0 to 4+. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of drug or drug combination that caused complete growth inhibition or no detectable visual turbidity. Subcultures of the clear tubes were done to determine the minimal fungicidal concentrations. Synergism was judged to be present when complete growth inhibition occurred with combinations of drugs in concentrations less than half of their respective MICs. This conformed to our previous criterion for synergy (6,8).After the turbidity readings, 0.5 gCi of [3H]uridine per ml was added to each tube and the cultures were reincubated at...
There have been several reported abnormalities in the cellular components of the acute inflammatory response in diabetes mellitus. These studies have dealt primarily with polymorphonuclear leukocytes. In the present investigations, we have examined monocyte metabolic activity in diabetic patients and controls. Following particle ingestion, the microbicidal mechanisms of the monocyte are activated and excited molecular oxygen and carboxyl groups are generated. Upon decay to the ground state, these molecules emit photons, which can be measured as chemiluminescence. Chemiluminescence production was significantly increased in 27 patients with poorly controlled diabetes (blood glucose levels from 208 to 712 mg/dl). The mean peak in the diabetics was 31.3 ± 8.2 (SD) x 10 3 cpm versus 25.8 ± 3.8 x 10 3 cpm in controls. Hexose monophosphate shunt activity as determined by 14 C-l-glucose utilization was also increased (667 ± 284 percent increase in diabetics versus 425 ± 154 in controls). Superoxide dismutase-inhibitable superoxide production was also significantly augmented in diabetic monocytes (9.77 ± 3.06 nmol/10 6 monocytes/20 min versus 6.36 ±1.10 nmol). Addition of glucose to whole blood from a normal individual, or to normal or diabetic monocyte suspensions, resulted in marked enhancement of chemiluminescence production. Increasing blood sugar levels appear, therefore, to be associated with monocyte metabolic activation in diabetic patients. Such activation could conceivably be detrimental to the diabetic host by contributing to cell damage through the release of toxic oxygen products. DIABETES 29:251-256, April 1980.
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 ...
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