Apr 21, 1997 - The antimicrobial activities of amphotericin B, itraconazole, and miconazole against 101 filamentous fungi from patients with cystic fibrosis were ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1997, p. 2064–2066 0066-4804/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 41, No. 9
In Vitro Susceptibilities to Amphotericin B, Itraconazole, and Miconazole of Filamentous Fungi Isolated from Patients with Cystic Fibrosis C. HENNEQUIN,1* N. BENAILLY,1 C. SILLY,2 M. SORIN,3 P. SCHEINMANN,3 G. LENOIR,2 J. L. GAILLARD,1 AND P. BERCHE1 Laboratoire de Parasitologie, Service de Microbiologie,1 Service de Pe´diatrie Ge´ne´rale,2 and Service de Pneumologie Pe´diatrique,3 Ho ˆpital NeckerEnfants Malades, 75015 Paris, France Received 9 December 1996/Returned for modification 21 April 1997/Accepted 30 June 1997
The antimicrobial activities of amphotericin B, itraconazole, and miconazole against 101 filamentous fungi from patients with cystic fibrosis were tested by a reproducible microdilution method. Itraconazole was very active against Aspergillus species and Scedosporium species (MIC at which 90% of the isolates were inhibited [MIC90], 0.06 to 0.5 mg/liter), whereas amphotericin B was less effective (MIC90, 0.5 to 8 mg/liter). were chronically colonized (two-thirds of sputum cultures positive for each species). Isolates obtained at intervals of 2 months or more were considered independent. One isolate per positive sample was stored at 220°C in sterile distilled water supplemented with 10% glycerol. The MICs were determined by a microdilution method testing each isolate with an inoculum of 1 3 104 to 5 3 104 CFU/ ml against twofold serial dilutions of antifungal agents (0.03 to 16 mg/liter). This method was adapted from the proposed reference method described by the National Committee for Clinical and Laboratory Standards (NCCLS) for in vitro susceptibility testing of yeasts (18). The reference strains A. fumigatus IP 840, A. flavus IP 954, S. apiospermum IP 1698, and S. prolificans IP 1974 were used as controls (one per microdilution plate). Stock concentrations of antifungal agents (1,280 mg/liter) were prepared by dissolving of powders in dimethyl sulfoxide (DMSO) (Sigma Chemical Company, Saint Louis, Mo.) by incubation for 30 min for amphotericin B (in the dark) and miconazole and for 3 h for itraconazole. Each drug was serially twofold diluted to obtain concentrations from 32 to 6.25 mg/liter. Isolates were incubated on potato dextrose agar plates (Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France) at 37°C for 2 to 3 days for Aspergillus spp. and for 4 to 5 days for Scedosporium spp. The fungal cultures were then covered with 10 ml of 0.05% sterile sodium dodecyl sulfate (Sigma) solution for Aspergillus spp. and 5 ml of 0.09% sterile saline for Scedosporium spp. Colonies were gently probed with the tip of a Pasteur pipette, and the resulting suspension (conidia and hyphae) was transferred to a sterile tube and decanted for 5 min. The supernatant, containing only conidia, was mixed for 15 s with a vortex mixer, and its turbidity at 530 nm was measured with a spectrophotometer (Novaspec II; Pharmacia, Uppsala, Sweden). A. fumigatus, A. flavus, and S. apiospermum suspensions were then adjusted to the ranges of transmission (T) as previously described (7) corresponding to inocula from 1 3 106 to 5 3 106 CFU/ml. We used the same T values for S. apiospermum and S. prolificans. These estimates were confirmed by determination of the numbers of CFU in each suspension by plating on Sabouraud chloramphenicol gentamicin agar (Sanofi-Diagnostics Pasteur) and incubation for 24 h for Aspergillus species and 72 h for Scedosporium species at 37°C. The reliability of this spectrophotometric method for inoculum preparation was evaluated by calculating,
In recent years, filamentous fungi have emerged as important bronchial pathogens in patients with cystic fibrosis (CF). Strains of Aspergillus spp. have been isolated from the sputum of more than 20% and up to 52% of CF patients and are associated with a broad spectrum of clinical diseases (3, 16). Colonization with Aspergillus fumigatus is generally considered asymptomatic, although it may contribute to exacerbations of bronchitis (10). Allergic bronchopulmonary aspergillosis (ABPA) is one of the most common complications of established CF lung disease and can lead to other life-threatening complications, such as pulmonary fibrosis, at the end stage of the disease (11, 16). More rarely, invasive pulmonary aspergillosis may develop most often following lung transplantation (6) or during corticosteroid therapy (2). Scedosporium apiospermum is the second-most-frequent genus of filamentous fungi isolated from the sputa of these patients (1), i.e., 3.3% of patients in one CF series (3). It exhibits a pathogenicity similar to that of Aspergillus species (12). Allergic bronchopulmonary manifestations (15) induced by S. apiospermum and invasive infections (12) have previously been described, but not for CF patients. Several preliminary reports have suggested that antifungal therapy may be of benefit for CF patients with ABPA and those colonized with S. apiospermum or Aspergillus species and with worsening respiratory status (2, 5, 9). However, the susceptibility of these pathogens isolated from the sputa of CF patients to antifungal agents is not known. In this work, we studied the antimicrobial activities of amphotericin B, itraconazole, and miconazole against a large group of clinical isolates of filamentous fungi obtained from the sputa of CF patients. A total of 101 isolates of filamentous fungi (73 A. fumigatus isolates, 10 A. flavus isolates, 13 S. apiospermum isolates, and 5 Scedosporium prolificans isolates) isolated from 57 patients (1 to 4 isolates for a given species per patient) were tested. Three patients had been previously treated with itraconazole (two cases) or amphotericin B (aerosols plus intravenous injection) (one case). The isolates were collected between January 1994 and December 1995 from the sputa of CF patients monitored in our hospital (a cohort of approximately 300 patients) who * Corresponding author. Mailing address: Laboratoire de Parasitologie, Service de Microbiologie, Ho ˆpital Necker-Enfants Malades, 149, rue de Se`vres, 75015 Paris, France. Phone: 44 49 49 61. Fax: 44 49 49 60. 2064
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for each species, the coefficients of variation, which were defined as follows: (standard deviation of CFU/mean of CFU) 3 100. Adjusted suspensions were diluted in 1:50 RPMI 1640 medium (ICN Pharmaceuticals, Costa Mesa, Calif.) without sodium bicarbonate, supplemented with 2.5 mM L-glutamine (Sigma), 1.8% glucose (Prolabo, Fontenay sous Bois, France), and 0.165 M morpholinesulfonic propane acid buffer (ICN Pharmaceuticals) and brought to pH 7. Sterile U-shaped 96well microplates (Greiner-Labortechnik, Frickenhausen, Germany) were used. A 100-ml volume of antifungal solution of one concentration was dispensed from row 1 (highest concentration) to row 10 (lowest concentration) of each plate. Positive (row 11) and negative (row 12) controls were included for each strain. The positive control was 100 ml of DMSO (1:20), and the negative control was 50 ml of DMSO (1:20) plus 50 ml of 10% formalin, respectively. The conidial suspensions in RPMI (100 ml) were then dispensed into the lines. The plates were incubated at 37°C for 24 h for Aspergillus spp. and for 72 h for Scedosporium spp. As previously suggested (8), we defined the MICs as the concentrations reducing fungal growth to less than 25% of that of the positive control, as assessed by optical reading of the turbidity. This microdilution method was reproducible and reliable for in vitro determination of the antifungal susceptibilities of filamentous fungi. Previous tests with a smaller number of strains have already demonstrated the good interlaboratory reproducibility of a microdilution method adapted from the NCCLS recommendations (8). This may be due in part to the standardization of numerous parameters, including inoculum size, medium, temperature, and time of incubation, on which reproducibility largely depends. In our experience, the spectrophotometric method appeared to give a reliable estimate of the inoculum size as assessed by the low coefficients of variation, i.e., 33, 25, 24, and 24% for isolates of A. fumigatus, A. flavus, S. apiospermum, and S. prolificans, respectively. Optical determination of the MIC also appeared to be useful for evaluating the growth of filamentous fungi, which is often heterogeneous, and allowed detection of bacterial contamination. Overall, the reproducibility of MIC determinations by this method appeared satisfactory. For all species, none of the MICs of amphotericin B for the strains tested at least twice differed by more than one dilution, i.e., the method was 100% reproducible. For itraconazole, the method was 90, 78, and 100% reproducible for A. fumigatus, A. flavus, and S. apiospermum, respectively. Similar variability has previously been described (8) and probably results from the poor solubility of itraconazole. The results of tests of S. apiospermum isolates against miconazole were 78% reproducible. In contrast, repeated determinations of the MICs of itraconazole and miconazole for S. prolificans gave a large spread of results (18 and 20% reproducible, respectively), possibly due to the texture of the colonies which are often moist and yeasty. The results of MIC determinations are reported in Table 1. The amphotericin B MICs for all S. apiospermum isolates were .1 mg/liter, which is probably indicative of resistance, although breakpoints for filamentous fungi have not been defined. In contrast, the growth of 92.3 and 100% of these isolates was inhibited by 0.5 mg of itraconazole and 0.5 mg of miconazole per liter. This suggests sensitivity, and, indeed, a recent report proposed a MIC breakpoint for itraconazole versus Candida at 1 mg/liter (19). As far as could be assessed in view of the poor reproducibility of results, S. prolificans isolates were resistant to all three antifungal agents tested (Table 1). Aspergillus isolates were highly susceptible, with the amphotericin B MIC for 98.7% of the isolates being #1 mg/ liter and the itraconazole MIC for 100% of the isolates was
NOTES
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TABLE 1. Antifungal activities of amphotericin B, itraconazole, and miconazole against 101 filamentous fungi isolated from patients Species (no. of isolates tested)
A. fumigatus (73) A. flavus (10) S. apiospermum (13)
S. prolificans (5)
MIC (mg/ml)a
Antifungal agent
Amphotericin Itraconazole Amphotericin Itraconazole Amphotericin Itraconazole Miconazole Amphotericin Itraconazole Miconazole
B B B
B
50%
90%
Range
0.25 0.03 0.5 0.06 4 0.5 0.12 NC NC NC
0.5 0.06 0.5 0.12 8 0.5 0.12 NC NC NC
0.125–1 0.03–0.12 0.25–1 0.06–0.125 1–8 0.125–8 0.06–1 2–16 2–16 0.5–.16
a MIC was defined as 75% inhibition with an optical reading after 24 h of incubation for Aspergillus spp. and after 72 h for Scedosporium spp. MIC 50 and 90%, MICs at which 50 and 90% of the isolates studied, respectively, were inhibited. NC, not calculated (considering the small number of isolates tested).
#0.25 mg/liter. Until now, very few clinical isolates of A. fumigatus have been reported to be resistant to itraconazole (MIC, $6.3 mg/liter) (4). More recently, resistant mutants were obtained by culturing a strain on peptone yeast extract glucose agar containing 8 mg of itraconazole per liter at a frequency of 3.6 3 1027 (13). This is significant for CF patients who are in some cases colonized with a high burden of Aspergillus and may be treated empirically with itraconazole. However, none of the Aspergillus isolates tested in our study could be suspected of resistance, even for those obtained from patients previously treated with antifungal agents. Moreover, no changes in susceptibility were observed when serial isolates from the same patient were tested. In CF patients, antifungal treatment is not usually recommended for fungus-associated complications except invasive infections. However, the good pharmacokinetics and the safety profile of itraconazole have prompted some physicians to use itraconazole in CF patients. Its potential value has been underlined in the treatment of ABPA, in which itraconazole mostly gave clinical and biological improvement or cure (5, 9, 14, 17). Our results encourage further experimental work and controlled clinical trials with itraconazole for CF patients with associated Aspergillus diseases. We thank E. Gueho for confirmation of identification of Scedosporium isolates and C. de Bievre for providing reference strains. This work was supported by the University Paris V and INSERM. REFERENCES 1. Chabasse, D., J. P. Bouchara, J. P. Chazalette, J. Carre`re, J. L. Ginies, L. De Gentille, and B. Cimon. 1991. Mucoviscidose et colonisation fongique `a Scedosporium apiospermum. J. Mycol. Me´d. 1:152–155. 2. Chung, Y., J. R. Kraut, A. M. Stone, and J. Valaitis. 1994. Disseminated aspergillosis in a patient with cystic fibrosis and allergic bronchopulmonary aspergillosis. Pediatr. Pulmonol. 17:131–134. 3. Cimon, B., J. Carre`re, J. P. Chazalette, J. L. Ginies, P. Six, J. F. Vinatier, D. Chabasse, and J. P. Bouchara. 1995. Fungal colonisation and immune response to fungi in cystic fibrosis. J. Mycol. Me´d. 5:211–216. 4. Denning, D. W., R. M. Tucker, L. H. Hanson, and D. A. Stevens. 1989. Treatment of invasive aspergillosis with itraconazole. Am. J. Med. 86:791– 800. 5. Denning, D. W., J. Van Wye, N. J. Lewinston, and D. A. Stevens. 1991. Adjunctive therapy of allergic bronchopulmonary aspergillosis with itraconazole. Chest 100:813–819. 6. Drent, M., M. T. Van Rens, S. S. Wagenaar, B. M. De Jongh, H. Van Velzen Blad, and J. M. Van Den Bosch. 1995. Invasive aspergillosis after bilateral lung transplantation in cystic fibrosis. Respir. Med. 89:449–451. 7. Espinel-Ingroff, A., and T. M. Kerkering. 1991. Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous
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