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

Abstract

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...

Combination Antifungal Therapy

“…(46,125) and flucytosine (127). The results of in vitro studies of amphotericin B combined with rifampin (46,87,95,127) (58,87,104), synergy (58,95,140), and antagonism (210). These effects did not differ significantly according to the species of Aspergillus tested or according to levels of baseline resistance to flucytosine (58), but in some cases results differed according to the testing methodology employed by investigators.…”

“…We concur that single-agent therapy in most cases of invasive candidiasis and candidemia will most likely be the treatment of choice. Combination therapy may be (58) Results differ between studies and are variable amongst strains in same study (58,95), different methodologies and doses employed Mice (6, 157) and rats (187) Improved survival (6,157) Improved survival with 5FC ϩ AmB in mouse model (6). No survival benefit with 5FC ϩ AmB vs. AmB alone in steroid-suppressed rats (187).…”

In Vitro Interactions between Amphotericin B, Itraconazole, and Flucytosine against 21 Clinical Aspergillus Isolates Determined by Two Drug Interaction Models

“…Previous in vitro studies showed that this combination was mostly additive or synergistic against Aspergillus species (7,13,14,16,25). However, in one study antagonism was observed (5).…”

Aspergillosis of the CNS in a Pediatric Liver Transplant Recipient: Case Report and Review

“…Initial support for the use of a combination of chemotherapeutic agents for the treatment of fungi originated from an in vitro evaluation of the synergistic action of an1photericin Band 5-fluorocytosine, which showed a tenfold increase in the killing activity when the efficacy of the combination of both drugs was compared to that of either drug alone [26]. Further evaluation of the in vitro activity of amphotericin B with 5-fluorocytosine and with rifampin against isolates of both A. jumigatus and Aspergillus fiavus also demonstrated a synergy in inhibition but not killing [27]. It is unfortunate that results of antifungal susceptibility testing may be erratic [28], thereby raising doubts about the significance of the previously mentioned findings in regard to efficacy.…”