a Mycology Reference Laboratory, Public Health Laboratory Service, Bristol BS2 8EL; Departments of b Microbiology, and c Medicine, University of Manchester School of Medicine, Hope Hospital, Salford M6 8HD; d Department of Infectious Diseases and Tropical Medicine (Monsall Unit), Delaunays Road, North Manchester General Hospital, Manchester M8 6RB, UK
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Abstract |
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Introduction |
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Amphotericin B has been used for the treatment of a wide range of fungal infections for four decades.3,4Until recently, amphotericin B was the only effective agent for invasive aspergillosis, despite the fact that its use is seriously limited by nephrotoxicity and other side effects. The introduction of new lipid-associated forms of amphotericin B have reduced nephrotoxicity, hypokalaemia, acute anaphylactic events and infusion-related toxicity.57Overall, the response rate of invasive aspergillosis to conventional amphotericin B treatment is about 34%, with substantial variations according to the degree of immunocompromise in different patient groups.8The efficacy of the lipid-associated amphotericins appears to be similar.9
Although treatment failure in patients with invasive aspergillosis is common, few cases have been attributed to amphotericin B resistance. A number of methods for in vitrosusceptibility testing of Aspergillusspp. to amphotericin B have been described,1013with most strains demonstrating in vitrosusceptibility. However, with few exceptions,14there have been no attempts to correlate the results of these tests with outcome in patients or animal models of infection. In this investigation we used three strains of Aspergillus fumigatusand one of Aspergillus terreusin an attempt to determine the in vitrotest conditions that best correlated with treatment outcome in a murine model of Aspergillusinfection.
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Materials and methods |
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Three clinical strains of A. fumigatus(AF65, AF210 and AF294) and one of A. terreus(AT49) were used in these studies. These strains have been deposited with the United Kingdom National Collection of Pathogenic Fungi, held at the Mycology Reference Laboratory, Bristol, UK as NCPF 7097, 7101, 7102 and 7130, respectively.15Strain AF210 was obtained from a patient who responded to amphotericin B treatment.16Strain AF65 was recovered from the lung of a leukaemic patient who had an intermediate response to amphotericin B but whose aspergillosis later relapsed. Strain AF294 was obtained from a bronchoalveolar lavage from a patient with multiple myeloma and profound neutropenia for 22 days. The patient died despite amphotericin B treatment (1 mg/kg/day for 12 days) and recovery of neutropenia. Strain AT49 was recovered from a bronchoalveolar lavage from a patient who failed to respond to amphotericin B treatment. Further details are unavailable.
The strains were maintained on slopes of Oxoid Sabouraud dextrose agar (Unipath Ltd, Basingstoke, UK) supplemented with 0.5% (w/v) chloramphenicol (SAB agar) at 20°C and in vials of sterile water at room temperature, in Bristol. In Manchester, strains were stored at 70°C in 15% glycerol.
Inoculum preparation for in vitro testing
Strains were retrieved from storage, subcultured on to SAB agar slopes and incubated for 3 days at 28°C. Spores were harvested in 3 mL of sterile water for the agar incorporation method, or in 3 mL of the growth medium under test for the broth microdilution method. The spore suspensions were vortexed for 10 s to break up clumps and counted using a modified Fuchs Rosenthal haemocytometer.
Drug preparation for in vitro testing
Amphotericin B (Sigma Chemical Co., St Louis, MO, USA) was dissolved in dimethylsulphoxide at a concentration of 10,000 mg/L. The drug solution was diluted further with either sterile water or with the growth medium under test for the agar incorporation and broth microdilution methods, respectively.
Agar incorporation MIC method.
Doubling dilutions of amphotericin B from 160 to 0.15 mg/L were prepared in 2 mL volumes of sterile water. Volumes (18 mL) of molten agar (see below) were added, mixed, poured into Petri dishes and allowed to set before drying. Spore suspensions were adjusted to the appropriate density (1 x 1041 x 107spores/mL) with sterile water. Plates were inoculated with the spore suspensions by means of a multipoint inoculator (Denley Instruments Ltd, Bootle, UK) and incubated at 28 or 35°C. Minimum inhibitory concentrations (MICs) were determined after 24, 48 and 72 h incubation. The MIC was defined as the lowest drug concentration at which there was no visible growth. This procedure was repeated three times for each set of growth conditions.
Four media were tested: (a) RPMI-1640 agar (with l- glutamine and without bicarbonate) (Difco Laboratories, Detroit, MI, USA), buffered to pH 7.0 with 0.165 M morpholinopropanesulphonic acid (MOPS) (RPMI agar); (b) Sabouraud dextrose agar (Unipath) (SAB agar); (c) Bacto Antibiotic medium 3 (Difco) with 1.5% (w/v) Oxoid Agar Technical agar No. 3 (Unipath) and 2% (w/v) glucose (M3G agar); (d) Bacto Yeast Nitrogen Base (Difco), with 1.5% (w/v) Oxoid Agar Technical agar No. 3 (Unipath) and 10% (w/v) glucose (YNBG agar).
Broth dilution MIC method.
Doubling dilutions of amphotericin B were prepared in 100 µL volumes of broth in the wells of a microdilution tray, giving final drug concentrations of 160.5 mg/L. Spore suspensions were adjusted to 2 x 105or 2 x 106spores/mL in the growth medium under test. Volumes of 100 µL of the spore suspensions were dispensed into the wells containing the drug dilutions giving final inocula of 1 x 105or 1 x 106spores/mL. The plates were incubated at 28 or 35°C and the MICs were determined after 24 and 48 h incubation. The MIC was taken as the lowest drug concentration at which there was no visible growth.
Three media were tested: (a) RPMI-1640 medium (with l-glutamine and without bicarbonate) (Sigma), buffered to pH 7.0 with 0.165 M MOPS (RPMI broth); (b) Bacto Sabouraud dextrose broth (Difco) (SAB broth); (c) Bacto Antibiotic medium 3 (Difco) with 2% (w/v) d-glucose (M3G broth).
Determination of MICs by Etest.
The inoculum was prepared in water as previously described to produce a final concentration of 1 x 106conidia/mL. Agar plates were prepared by adding 19 mL RPMI-1640 agar, buffered to pH 7.0 with MOPS to produce a depth of 4.0 ± 0.5 mm. The plates were inoculated by dipping a sterile swab into the inoculum suspension making sure any excess moisture was removed by rolling the swab against the side of the tube. The entire agar surface was evenly swabbed in three directions and the plate dried for 1015 min. Etest strips (AB Biodisk, Solna, Sweden) were placed on the agar surface ensuring that the MIC scale was placed facing upwards. Plates were incubated at 37°C and read at 24, 48 and 72 h.
In vivo studies
Animals.
Male CD-1 mice, 45 weeks old and weighing between 20 and 30 g were purchased from Charles River UK Ltd (Margate, UK) The mice were virus free and were allowed free access to food and water. Mice were randomized into groups of 10.
Immunosuppression.
Cyclphosphamide was administered iv via the lateral tail vein to all animals at a dose of 200 mg/kg. A state of profound neutropenia was achieved 3 days after administration and lasted for 4 days.17
Preparation of inoculum.
Each strain was grown in a tissue culture flask containing potato dextrose agar (PDA) (Oxoid, Basingstoke, UK) for c. 10 days. The Aspergillusconidia were harvested in about 30 mL of sterile phosphate buffered saline (PBS) with 0.05% Tween 80 (PBS/Tween) (Fisons, Loughborough, UK). The stock solution was adjusted using serial dilutions to inocula that varied from strain to strain and were known from pilot experiments to produce an LD90. The inoculum was 3.35 x 106conidia/ mL for AF65, 1.72 x 106conidia/mL for AF210, 0.82 x 106conidia/mL for AF294 and 12 x 107conidia/mL for AT49. Inocula were checked by serial dilution in sterile PBS/Tween and inoculated on to horse blood agar [41 g/L Columbia agar base (Lab M, Bury, UK) containing 50 mL sterile horse blood (Botolph Cladon, Buckingham, UK)]. The prepared inoculum was stored at 4°C for up to 7 days until required. Three days after immunosuppression all mice were infected with the prepared inoculum via iv administration into the lateral tail vein.
Antifungal therapy
Desoxycholate amphotericin B (Fungizone, E.R. Squibb, Hounslow, UK) was dissolved in 5% dextrose (BDH, Poole, UK) to a stock concentration of 5 mg/mL. Further dilution in 5% dextrose produced working concentrations of 0.5 mg/kg, 2.0 mg/kg and 5.0 mg/kg. All doses of amphotericin B were given via ip injection 24, 48 and 96 h, and 7 days after infection. On day 11 of the experiment any surviving mice were killed. The lungs and kidneys were removed and transferred to 5 mL of PBS/Tween containing penicillin 1000 IU/mL and streptomycin 1000 mg/L. The organs were homogenized in a tissue grinder (Polytron, Kinematica, Luzern, Switzerland) for c. 1530 s, and 10-1and 10-2dilutions were prepared. A sample of 0.5 mL of each dilution was transferred to SAB agar and the liquid spread over the surface of the plates. Plates were incubated at 37°C, examined daily for 56 days and the number of colonies counted. One colony or less was defined as no growth.
Statistical analysis
Mortality and culture data were analysed using the MannWhitney U-test or the KruskallWallis test if the MannWhitney U-test was not possible (i.e. if all values were identical in one group). Mice that died before day 10 were assumed to have organ quantitative counts at least as high as the highest counts in surviving mice. All data analysis was performed using the computer package Minitab (Minitab Data Analysis Software, Philadelphia, PA, USA).
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Results |
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Agar incorporation MIC method.
Initial experiments with the agar incorporation method determined the effect of four different inoculum concentrations (1 x 104, 1 x 105, 1 x 106and 1 x 107spores/mL) on the MICs for the three A. fumigatusstrains. These experiments showed that inocula of <1 x 106conidia/mL and a reading time of 24 h did not give reproducible results (data not shown). For subsequent experiments inocula of 1 x 106and 1 x 107conidia/mL were used and results therefore determined after 48 and 72 h incubation.
Table Isummarizes the MIC ranges for the three strains of A. fumigatusand one of A. terreus(AT49) under different growth conditions on solid agar. Marked differences were seen in the MICs for the different strains on the four media tested, with the highest readings obtained on SAB agar. In general, the MICs for all four strains showed a slight increase with an increase in inoculum concentration, incubation temperature or duration of incubation.
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Broth dilution MIC method.
Initial experiments with the broth dilution method also showed that MICs for the three A. fumigatusstrains were not reproducible with an inoculum concentration of 1 x 104spores/mL (data not shown). Inocula of 1 x 105and 1 x 106spores/mL were therefore used in subsequent tests.
Table IIpresents the variation in MICs of amphotericin B for the three strains of A. fumigatusand one of A. terreusunder different broth dilution growth conditions. Marked differences were seen with the three media tested, the highest readings being obtained in tests with SAB broth. It was not possible to detect differences between the three A. fumigatusstrains. Nor did the A. terreusstrain demonstrate consistently higher MICs under all conditions tested.
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In vivo results
Data from three experiments are the pooled results for AF65, from two for AF210, AF294 and AT49. For all strains control mice showed 90100% mortality (Figure); Table III
). Strains AF65 and AT49 were associated with the poorest survival rates (Table III
) and there was no apparent dose response with these two strains over the 10-fold dose range 0.55 mg/kg. In comparison, strain AF210 had increased survival rates and there was also a clear dose response over the same dose range. Despite the perception from the clinical data that AF294 would be resistant, even the lowest dose of amphotericin B used was 90% effective. There were no deaths with the highest concentration of amphotericin B at (5 mg/kg) for either AF210 or AF294, compared with mortalities of 43% for AF65 and 40% for AT49. Thus, these data are consistent with susceptibility to amphotericin B in Aspergillusranging from susceptible to resistant, although some activity of amphotericin B was discernable at the maximal dose used against both AF65 and AT49.
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Discussion |
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Several testing formats have been used for susceptibility testing of Aspergillusfor amphotericin B.1014Many different media have been employed including yeast nitrogen broth (with 0.5% glucose), RPMI-1640 with and without supplemental glucose, minimal essential medium, antibiotic medium 3 and others. The pH range of the media varied from 4.0 to 7.4, the temperature of incubation from 22 to 37°C and inoculum concentrations from 103to 107conidia/mL. Most tests have been read at 24, 48 or 72 h. End-point determination has usually been visual but specrophotometric reading has also been assessed and either no growth, 75% or 80% endpoints have been quoted.
A broth microdilution method that showed good interlaboratory reproducibility for Aspergillusspp. and other filamentous moulds has recently been proposed as a reference method and this forms the basis of the new NCCLS proposed method 38-P.36However, no information was provided on the therapeutic response of the patients infected with the test strains.
Therapeutic failure is all too frequent with amphotericin B therapy in patients with invasive aspergillosis. This could imply that most strains are resistant as appears to be the case, in vitro, with A. terreus. The publication by Lass-Florl et al. (1998)14would suggest that all A. terreusstrains are resistant to amphotericin B, that 41% of A. flavusand 28% of A. fumigatusare also resistant (MIC > 2 mg/L) and that this correlates with the failure of amphotericin B therapy. However, therapeutic failure is also intimately related to the diagnostic delay and immune status of the patient. It is not clear that amphotericin B dose is important.37,38
In summary, the data presented here are consistent with in vivoresistance of Aspergillusto amphotericin B in some strains. Our data, combined with published data, tend to suggest universal resistance in A. terreusto amphotericin B, but needs in vivoconfirmation in a greater number of strains. The data also indicate that none of the in vitrosusceptibility test formats used in this study are able to identify resistance in A. fumigatus.
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Acknowledgments |
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Notes |
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Present address: Research Institute for the Care of the Elderly, St Martin's Hospital, Bath BA2 5RP, UK;
# Present address: Chief of the Mycotic Diseases Branch, Centers for Disease Control and Prevention, National Center for Infectious Diseases Division, 1600 Clifton Road NE, MES-C09, Atlanta GA 30333, USA
* Corresponding author. Tel: +44-161-720-2734; Fax: +44-161-720-2732.
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References |
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Received 4 May 1999; returned 28 July 1999; revised 6 August 1999; accepted 2 September 1999