a Istituto di Malattie Infettive e Medicina Pubblica, Centro di Gestione Presidenza Medicina e Chirurgia, b Università degli Studi di Ancona, Ancona, Italy
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Abstract |
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Introduction |
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The gold standard therapy for cryptococcosis remains amphotericin B with or without flucytosine.1 For suppression therapy a triazole, such as fluconazole or itraconazole, is the agent of choice.1,3
Recently, the new investigational triazole posaconazole was shown to have potent activity against isolates of C. neoformans in vitro.47 Thus far, few studies have been conducted to correlate the in vitro data on posaconazole MICs for this pathogenic yeast with results in vivo.8
Therefore, in the present study we investigated the efficacy of this new triazole in an experimental model of systemic murine cryptococcosis caused by strains of C. neoformans with variable patterns of posaconazole susceptibilty in vitro.
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Materials and methods |
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Three isolates of C. neoformans, each obtained from an AIDS patient, were used in this study (Table). Two strains were obtained from blood (491 and 2337), while C. neoformans 486 was isolated from CSF. All isolates were identified as C. neoformans var. neoformans on the basis of no colour change on canavanineglycinebromothymol blue agar.9 All the strains were maintained on Sabouraud dextrose agar (SDA; Difco Laboratories, Detroit, MI, USA) slants at 4°C.
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For in vitro studies, a stock solution of posaconazole (Schering Plough Research Institute, Kenilworth, NJ, USA) was prepared in polyethylene glycol (PEG 200; Janssen Chimica, Geel, Belgium), and a stock solution of amphotericin B (Sigma Chemical, Milano, Italy) in dimethyl sulphoxide (Sigma). Further dilutions of both drugs were made in the test medium. The final concentration of the solvent did not exceed 1% in any well.
For in vivo studies, posaconazole was prepared in PEG 200 while amphotericin B was purchased as Fungizone from Bristol-Myers Squibb (S.p.A., Latina, Italy).
Susceptibility testing
Antifungal susceptibility testing was performed using a broth microdilution method, adhering to the recommendations of the NCCLS.10 The test medium was RPMI 1640 (Sigma) buffered to pH 7.0 with 0.165 M MOPS (Gibco Laboratories, Milano, Italy). Final concentrations of both drugs ranged from 0.0078 to 4.0 mg/L. Yeast inocula ranged from 0.5 x 103 to 2.5 x 103 cfu/mL. The microdilution plates were incubated in air at 35°C and read at 72 h. The posaconazole MIC was defined as the lowest concentration of drug at which turbidity in the well was 80% less than that in the control well, while amphotericin B MIC was defined as the lowest concentration of drug at which no fungal growth was detectable.10 Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were routinely tested in parallel with each MIC run.
Animal studies
A murine model of systemic cryptococcosis was established in CD1 male mice (weight 30 g; Charles River Laboratories, Calco, Italy) by injection via the lateral tail vein of viable yeast cells grown overnight in brainheart infusion broth. Animal experiments were conducted with the approval of the University of Ancona ethics committee. Posaconazole was administered by oral gavage at concentrations of 3 and 10 mg/kg/day, while amphotericin B was given ip at 0.3 mg/kg/day. Therapy was started 24 h after the infection and continued for 10 consecutive days. In survival studies the mice were observed through days 40 or 60 and deaths were recorded daily. Moribund mice were killed, and their deaths were recorded as occurring on the next day. In tissue burden studies the mice were killed 24 h after the end of therapy, and the number of viable cfu per gram of brain and lungs of each animal was determined by quantitative plating of organ homogenates onto SDA plates. There were 1013 mice per group in survival studies, and seven mice per group in tissue burden studies. Three additional mice per group were killed 24 h after the end of therapy and were used to study the effects of drugs on the clearance of cryptococcal capsular polysaccharidic antigen (PA) in serum. Testing was performed by a commercially available agglutination assay (Crypto-La Test, Bouty, S.p.A., Milano, Italy).
Statistical analysis
The log rank test was used to determine the difference between survival groups, and the MannWhitney U-test was used to determine the significance in tissue burden studies. The results of PA serum levels were determined by the analysis of variance followed by Bonferroni t-test. Differences were considered significant when P was <0.05.
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Results |
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Survival results are given in the Table. Mice were infected with 2.0 x 105, 4.5 x 105 and 4.8 x 105 cfu/mouse in experiments with C. neoformans 486, 491 and 2337, respectively. For C. neoformans 486, all treatment regimens were effective in prolonging survival against the controls (P < 0.001). The effectiveness of posaconazole was shown to be dose dependent, with posaconazole at 10 mg/kg/day being more effective than posacoanzole at 3 mg/kg/day (P < 0.05). For C. neoformans 491, neither posaconazole nor amphotericin B was effective in prolonging survival. For C. neoformans 2337, only posaconazole at 10 mg/kg/day (P < 0.01) and amphotericin B (P < 0.001) were effective in prolonging survival. For this strain, amphotericin B was significantly more active than both triazole dosing regimens (P < 0.001).
The second set of experiments consisted of three tissue burden studies. Mice were infected with 1.8 x 105, 2.0 x 105 and 2.1 x 105 cfu/mouse in experiments with C. neoformans 486, 491 and 2337, respectively. Mice were treated for 10 days, and killed 1 day later. In these studies, posaconazole was only given at 10 mg/kg/day. The results are reported in the Table. For C. neoformans 486, both treatments were equally effective at reducing fungal burdens in the brain, whereas only the triazole was effective at reducing fungal burdens in the lung. Posaconazole was also more effective than amphotericin B (P < 0.05). Similar results were obtained for C. neoformans 491. In particular, for this strain posaconazole was more effective than amphotericin B at reducing fungal burdens in both organs (P < 0.05). For C. neoformans 2337, both posaconazole and amphotericin B were effective at reducing fungal burdens in both organs, with the triazole more effective than the polyene in the brain tissue (P < 0.05).
Antigen titres are reported in the Table. Both drugs were significantly effective at reducing PA serum levels for all three strains (P <0.05 to <0.001). In addition, posaconazole was superior to amphotericin B against C. neoformans 486 (P < 0.01).
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Discussion |
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Unlike antibacterial agents, for which standardized susceptibility testing procedures and interpretive breakpoints are well established, reproducible methods and tentative breakpoints for antifungal agents have only recently been introduced.11 So far, most of the studies investigating the relationship between in vitro and in vivo results of antifungal efficacy have involved infections due to Candida spp.11 Only a few reports have tried to correlate the in vitro activity of a given antifungal agent (mainly fluconazole) with the clinical outcome of cryptococcosis.1214 In an early study, Casadevall et al.12 used a broth macrodilution method to analyse fluconazole and amphotericin B MICs for 13 strains of C. neoformans isolated from five AIDS patients, and showed a lack of correlation between in vitro data and clinical outcome. In contrast, Witt et al.14 used a modified broth microdilution method to test fluconazole MICs for clinical isolates of C. neoformans and found a statistically significant correlation between in vitro data and clinical success or failure.
The reason both posaconazole and amphotericin B were ineffective in prolonging the survival of mice infected with C. neoformans 491 is difficult to explain. It can only be hypothesized that failure to prolong survival of mice infected with this isolate might be due to the longer observation time (60 versus 40 days) than that applied in mice infected with the other two isolates of C. neoformans. This situation, owing to a progressive decrease of drug tissue levels over time, would facilitate the replication of the remaining fungi to a critical burden.
Unlike survival data, tissue burden experiments showed that posaconazole given at 10 mg/kg/day was effective at reducing the number of cfu per gram of brain and lung tissues in all isolates of C. neoformans. In particular, posaconazole was more effective than amphotericin B in the brain of mice infected with C. neoformans 491 and 2337 as in the lung of mice infected with C. neoformans 486 and 491. Determination of antigen serum levels of treated mice confirmed the potent in vivo efficacy of this new antifugal molecule.
Overall, our study underlines the excellent activity of posaconazole against this pathogenic yeast and indicates that this new antifungal molecule merits further investigation as a potentially useful agent for the treatment of human cryptococcosis.
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Acknowledgements |
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Notes |
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References |
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Received 28 February 2001; returned 12 June 2001; revised 17 August 2001; accepted 30 August 2001