Edward R. Block

5-Fluorocytosine Resistance in Cryptococcus neoformans

Isolates of Cryptococcus neoformans from six patients were obtained before and after unsuccessful therapy with 5-fluorocytosine (5-FC). Post-therapy isolates exhibited massive and stable 5-FC resistance. The frequency of drug-resistant mutants in susceptible isolates of C. neoformans was <0.001% (70.4 ± 17.9 per 107 cryptococci), whereas mutant frequencies in resistant isolates approached 100%. Non-drug-induced, spontaneously appearing 5-FC resistant mutants were documented in four susceptible isolates of C. neoformans by use of the statistical method of fluctuation analysis. Mutation rates on these same four isolates ranged from 1.2 × 10−7 to 4.8 × 10−7. Total intracellular uptake and incorporation of cytosine-5-3H (CyH3) and 5-fluorocytosine-2-14C (5-FC14) into a trichloroacetic acid-insoluble fraction were markedly reduced in six isolates with in vivo-acquired resistance when compared with susceptible pretreatment strains from the same patients. Five of these six isolates also had acquired massive resistance to 5-fluorouracil (5-FU), suggesting that a mutation in the uridine-5′-monophosphate pyrophosphorylase was responsible for drug resistance. The sixth isolate, which remained susceptible to 5-FU, appeared to have a defect in a cytosine-specific permease accounting for 5-FC resistance. A single isolate with in vitro-acquired 5-FC and 5-FU resistance had no reduction in uptake or incorporation of CyH3 or 5-FC14. The mechanism of resistance in this isolate is discussed.

Pharmacological Studies with 5-Fluorocytosine

A cylinder plate bioassay for 5-fluorocytosine (5-FC) is described which permits determination of 5-FC concentrations in biological fluids in the presence of amphotericin B. Using this assay, we determined serum concentrations in 12 patients after a single dose of drug and in 8 patients receiving daily 5-FC at 6-hr intervals (4 to 120 days). Drug was detectable in serum as early as 0.5 hr and concentrations were measurable as long as 24 hr after a dose, regardless of renal function. Peak concentrations occurred 6 hr after the initial drug dose, but were seen between 1 and 2 hr after a dose in all patients receiving a minimum of 4 days of therapy. Mild to moderate renal impairment produced marked increases in peak 5-FC concentrations in the serum of a group of eight patients on three different dosage schedules. Five patients were studied before and after amphotericin B-induced renal insufficiency. Peak concentrations increased from 14 to 142% concomitant with the change in renal function. Parallel studies in rabbits confirmed the results in our patients. 5-FC half-life in the serum of 10 rabbits increased from 3.35 ± 0.27 to 24.63 ± 0.70 hr after experimentally induced acute renal failure. Concentrations of 5-FC in the cerebrospinal fluid of five patients ranged from 17 to 62 μg/ml and were 74.4 ± 5.6% of simultaneously determined serum concentrations. The effect of renal function on 5-FC concentrations in cerebrospinal fluid was similar to that seen with serum.

Flucytosine and Amphotericin B: Hemodialysis Effects on the Plasma Concentration and Clearance: Studies in Man

Abstract The efficacy of hemodialysis in removing flucytosine (5-fluorocytosine) and amphotericin B from blood was studied in eight adult patients with renal failure who required chronic hemodialys...

The Combined Effect of 5-Fluorocytosine and Amphotericin B in the Therapy of Murine Cryptococcosis

Summary 5-Fluorocytosine and amphotericin B were demonstrated to be at least additive in the therapy of murine cryptococcosis. The mechanism for this effect is not known. However, AmB seemed to inhibit the in vivo development of 5-FC resistance among C. neoformans in our model. These results raise the possibility that combined-drug therapy might reduce AmB toxicity and/or the appearance of 5-FC resistant cryptococci without compromising therapeutic efficacy in cryptococcal disease. We are greatly indebted to Dr. David W. Alling for constant and invaluable advice on the mathematical problems of experimental design and data analysis.

Variables Influencing Susceptibility Testing of Cryptococcus neoformans to 5-Fluorocytosine

The minimum inhibitory concentration (MIC) of 5-fluorocytosine (5-FC) was determined for 65 isolates of Cryptococcus neoformans by using a twofold serial tube dilution method. The MIC was profoundly influenced by incubation temperature, inoculum size, and duration of incubation. By using a standard set of test conditions, 100% of 49 pretreatment isolates of C. neoformans were susceptible to 10 μg of 5-FC per ml or less, and 9 (56%) of 16 isolates recovered during or after 5-FC therapy were massively drug resistant (MIC > 320 μg/ml). With the standard test conditions recommended here, the tube dilution method was found to be both accurate and reproducible, and the results correlated with the treatment status of patients.

Stability of Amphotericin B in Infusion Bottles

Intravenous solutions of amphotericin B in 5% dextrose water with or without hydrocortisone or heparin demonstrated no appreciable loss of activity when exposed to fluorescent light for up to 24 h at 25 C (room temperature).

Experimental Therapy of Cladosporiosis and Sporotrichosis with 5-Fluorocytosine

Cladosporium trichoides and Sporothrix schenckii are fungi known to be pathogenic for man. No effective chemotherapy is available for cladosporiosis, and systemic sporotrichosis can be very resistant to antifungal chemotherapy. Experimental infections of mice with these fungi resembled their respective infections in man and provided a model for evaluating a new antifungal agent, 5-fluorocytosine (5-FC). Our results with four isolates of C. trichoides demonstrated a statistically significant dose-related therapeutic effect with 5-FC. Mortality was significantly reduced in all treatment groups, and survivors showed no clinical sign of disease despite positive brain cultures. Results with a single isolate of S. schenckii were less encouraging. Fatality rate was significantly decreased in all treatment groups, but no trend was noted with increasing 5-FC dosage. Survivors manifested the signs of active disease, and all liver and spleen cultures were positive for S. schenckii. These results indicated that (i) 5-FC may be the first drug useful in the treatment of cladosporiosis, and (ii) 5-FC may have only limited therapeutic benefit in systemic sporotrichosis. Images

5-Fluorocytosine and Amphotericin B in Bronchial Secretions

The penetration into and clearance from bronchial secretions of 5-fluorocytosine and amphotericin B were studied in a dog model. After a single intravenous dose of 35 mg/kg, 5-fluorocytosine intrabronchial concentrations were greater than the minimal inhibitory concentration for 80 to 90% of Candida species. These inhibitory concentrations persisted up to 3 h. In contrast, amphotericin B in intravenous doses of 0.6 and 1.2 mg/kg penetrated the blood-bronchus barrier poorly.

Effect of Hepatic Insufficiency on 5-Fluorocytosine Concentrations in Serum

Hepatic function failed to influence serum concentrations of the antifungal agent 5-fluorocytosine when the drug was given intravenously to rabbits with CCl4-induced hepatic insufficiency or orally to a patient with postnecrotic cirrhosis.