In vitro evaluation of combination of fluconazole and flucytosine against Cryptococcus neoformans var. neoformans

Abstract

Cryptococcal meningitis is a life-threatening opportunistic infection in immunocompromised hosts, especially in human immunodeficiency virus-infected patients and transplant recipients. Although amphotericin B and fluconazole are current acceptable therapies for patients with cryptococcal meningitis, the success of these therapeutic modalities remains suboptimal (13). The combination of amphotericin B and flucytosine appeared to provide the highest rate of clinical success in one study (7). This combination, however, is frequently associated with toxic side effects, and the use of amphotericin B requires close laboratory monitoring with long-term catheters for vascular access. In one pilot study, the combination of fluconazole and flucytosine yielded a clinical success rate of 63%, which is higher than that of previously reported experiences with either amphotericin B or fluconazole (6, 13); however, to date, in vitro synergistic testing to examine the biological basis for this empiric finding has not been performed. We therefore investigated the in vitro interaction of fluconazole with flucytosine against Cryptococcus neoformans var. neoformans. MATERIALS AND METHODSFifty clinical isolates of C. neoformans var. neoformans submitted to the Fungus Testing Laboratory, University of Texas Health Science Center at San Antonio, were studied. Forty-four isolates were recovered from cerebrospinal fluid (CSF), and six were recovered from blood of human immunodeficiency virusinfected patients. In addition, two reference C. neoformans strains, ATCC 90112 and 90113, were used as controls.Antifungal agents. Fluconazole (Pfizer Inc., New York, N.Y.) and flucytosine (Hoffman-La Roche Laboratory Inc., Nutley, N.J.) were tested singly and in combination. Stock solutions of fluconazole and flucytosine (each at 2,560 g/ml) were prepared in distilled water. Serial twofold drug dilutions were also prepared in water. Antifungal susceptibility testing. Drug interaction was assessed with a checkerboard titration, adhering to the recommendations of the National Committee for Clinical Laboratory Standards (11). Briefly, susceptibility testing was performed in RPMI 1640 medium (American Biorganics, Inc., Niagara Falls, N.Y.), with L-glutamine, without bicarbonate, and buffered at pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS). Aliquots of 50 l of each drug (and in the case of the single-drug control, 50 l of that drug and 50 l of sterile water) at a concentration of 20 times the targeted final concentration were dispersed in polystyrene plastic tubes (12 by 75 mm; Falcon 2054; Becton Dickonson, Lincoln Park, N.J.). Yeast inocula (0.9 ml), prepared spectrophotometrically and further diluted as described elsewhere (11), were added to the tubes containing drugs. The final drug concentrations ranged from 0.125 to 128 g/ml for both fluconazole and flucytosine. All tubes were incubated without agitation at 35ЊC, and the readings were made at 72 h. Before the readings, each tube was vortexed, and its turbidity was compared with that ...

Combination Antifungal Therapy

“…This interaction has been proposed for combinations of amphotericin B or fluconazole with antibacterials such as rifampin (23,125,127) and quinolones (204,205) and allows these agents to easily penetrate the fungal cell membrane to reach their target of fungal DNA synthesis. Such a mechanism may also explain potential synergism between azoles (3,12,14,101,124,138,223) or amphotericin B (126,156) and flucytosine, in which case the azole or polyene damages the fungal cell membrane, enabling increased uptake of flucytosine. (iii) A transport interaction is proposed for amphotericin B-flucytosine (17,18,20), whereby amphotericin B acts on the fungal cell membrane and inhibits flucytosine transport across the cell membrane and out of the yeast cell.…”

Successful treatment of disseminated cryptococcosis in a liver transplant recipient with fluconazole and flucytosine, an all oral regimen

“…In a pilot study in HIV patients with cryptococcal meningitis, the combination was associated with a clinical success rate (sterilization of the cerebrospinal fluid) in 63 % of the patients [6]; the cerebrospinal fluid sterilization rate with amphotericin B alone is approximately 40% and with fluconazole alone is about 35% [6]. An in vitro combination of fluconazole and flucytosine against 50 clinical strains of Cryptococcus neoforrizans demonstrated synergy in 62 YO of the cases and antagonism in none [9]. In a murine model of cryptococcal meningitis, combined therapy with fluconazole and flucytosine significantly prolonged survival when compared with controls and with groups receiving fluconazole or flucytosine alone [l].…”

“…Flucytosine at currently recommended dosages of 150 mg/kg per day can be associated with serious marrow toxicity. A noteworthy feature of the fluconazole plus flucytosine combination is that the addition of fluconazole greatly reduced the flucytosine minimum inhibitory concentration for the cryptococcal isolates, even for the strains that did not demonstrate frank synergy [9]. Thus, an additional advantage of the combination therapy is the reduction in the amount of flucytosine required and, therefore, the potential to reduce dose-related toxicity.…”

“…Furthermore, the intravenous formulation of amphotericin B necessitates long-term venous access and may prolong hospitalization. Fluconazole can be administered orally; however, the clinical success rate with its use alone in treating HIV-infected patients with cryptococcal meningitis has only reached 34 % It has been shown that the combination of fluconazole and flucytosine is synergistic against Cryptococcus neoformans in vitro [9]. However, the efficacy of this regimen against Cryptococcus has never previously been demonstrated clinically in transplant recipients.…”

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

“…The first approach looks at the results of each of the three replicates separately and determines the interaction. For the interpretation of the FIC index, several different definitions have been described in the literature (1,22,24), but there is no consensus on which definition to use in interaction studies. In most cases, synergy was claimed when the FIC index was lower than or equal to 0.5, and antagonism was claimed when the FIC index was higher than 4.…”