Departments of 1Medical Microbiology and Infectious Diseases and 2Biostatistics, Erasmus Medical Centre, PO Box 1738, 3000 DR Rotterdam, The Netherlands
Received 15 October 2001; returned 14 December 2001; revised 23 January 2002; accepted 28 January 2002.
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Abstract |
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Introduction |
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In the present study, it was investigated whether the efficacy of AmBisome in our model of IPA in persistently granulocytopenic rats could be improved by the addition of Fungi-zone in the early phase of the disease. Several parameters for assessment of therapeutic response were used.
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Materials and methods |
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Our animal model, first described by Leenders et al.,13 was used with a few modifications. Specified pathogen-free female RP strain albino rats (1825 weeks old, 185225 g) were employed. Profound granulocytopenia was induced by intraperitoneal (ip) administration of 75 mg/kg cyclophosphamide (Sigma-Aldrich Chemie, Steinheim, Germany) 5 days before inoculation, followed by repeated doses of 60 mg/kg 1 day before and 3 and 7 days after fungal inoculation. This protocol resulted in granulocyte counts of <0.1 x 109/L on the day of fungal inoculation. To prevent bacterial superinfections, animals were given daily doses of 40 mg/kg amoxicillin intramuscularly (im) starting 1 day before inoculation and a 6 mg/kg dose of gentamicin im on the day of inoculation. In addition, rats received ciprofloxacin 660 mg/L and polymyxin B 100 mg/L in their drinking water throughout the experiment. Rats were inoculated with a clinical isolate of Aspergillus fumigatus originally isolated from an immunocompromised patient with IPA. In accordance with NCCLS procedures,14 the MIC of amphotericin B for this strain was 0.4 mg/L. Infection was established by intubation of the left main bronchus under general anaesthesia. A cannula was passed through the tube and the left lung was inoculated with 6 x 104 A. fumigatus conidia suspended in 20 µL of phosphate-buffered saline (PBS).
Before experiments in infected rats, treatment regimes were administered to uninfected, granulocytopenic rats in which no noticeable toxicity was observed.
The experimental protocols adhered to the rules laid down in The Dutch Animal Experimentation Act (1977) and the published Guidelines on the Protection of Experimental Animals by the Council of the EC (1986). The present protocols were approved by the Institutional Animal Care and Use Committee of the Erasmus University Rotterdam.
Antifungal treatment
Antifungal agents were administered intravenously via the lateral tail vein to groups of rats daily. Treatment was started at 16 h after fungal inoculation, at which time hyphal growth was established. Fungizone was obtained from Bristol-Myers BV (Woerden, The Netherlands), and was diluted in 5% dextrose. AmBisome was obtained from NeXstar Pharmaceuticals (Wilrijk, Belgium) and diluted in 5% dextrose. The following treatment regimens were investigated: (i) Am-Bisome 10 mg/kg/day for 10 days; and (ii) AmBisome 10 mg/kg/day for 10 days combined with a single dose of Fungizone 1 mg/kg at day 1. When Fungizone and AmBisome were combined on the same day, AmBisome was administered first and Fungizone within 20 min thereafter. Controls received no treatment, since previous studies showed that placebo treatment with either 5% dextrose or empty liposomes did not influence survival, levels of circulating galactomannan (GM) or other parameters of fungal infection (data not shown).
Parameters for efficacy of antifungal treatment
The survival rate of rats was monitored twice daily until day 11 after fungal inoculation. GM was measured on days 3, 5, 7, 9 and 11 after fungal inoculation. At the end of the antifungal treatment rats had died or survived. Rats surviving on day 11 were killed and dissected. In rats that died and killed rats, the size of the pulmonary lesion of the left lung was measured, left lung weight was determined, organs were cultured for fungi, and chitin assays were carried out on the left lung homogenates.
GM detection
Blood for GM detection was sampled by puncture of the orbital venous plexus in the lateral canthus of the orbita. When cyclophosphamide, antifungal agents or antibiotics were administered to the rats on the same day as GM detection was carried out, blood samples for GM detection were taken within 2 h before injection of these agents. Concentrations of circulating GM were measured as described previously.15 Briefly, 300 µL of each serum sample were used in a sandwich ELISA (Platelia Aspergillus; Sanofi Diagnostics Pasteur, Belgium). Each plate contained a calibration curve derived from rat serum samples containing 0, 1, 1.5, 2, 3, 4, 6, 8 and 12 ng/mL GM.
Pulmonary macroscopic lesions and left lung weight
Pulmonary macroscopic lesions were observed in both un-treated and treated rats. Angio-invasive lesions, seen as macroscopic dark-red lesions, were histologically characterized by extensive hyphal broncho- and angio-invasion, and haemorrhagic infarction. Responsive lesions, seen as macroscopic light-red coloured lesions, were histologically characterized by the presence of relatively short hyphae and little angio- and bronchio-invasion with resulting less haemorrhagic infarction. The size of the two types of pulmonary lesion was expressed as a percentage of the total lung surface. The pulmonary lesion size was measured from photographs of the anterior of the lungs taken immediately after dissection.
Fungal cultures of organs
Left and right lungs were disrupted in 12 mL and livers in 20 mL of PBS using a tissue homogenizer (The Virtis Co. Inc., Gardiner, NY, USA) for 45 s at 10 000 rpm. Cfu in right lungs and livers were counted in 1:10 and undiluted homogenates. Cfu in left lungs were counted in 1:10 and 1:100 dilutions, and the remaining 10.68 mL of homogenate were used for the chitin assay. Dilutions of homogenates were spread on to Sabouraud agar plates. Undiluted homogenates were cultured according to the pour plate method. After incubation at 37°C for 36 h the cfu were counted.
Chitin assay
A 10.68 mL aliquot of left lung homogenate was used for the chitin assay, as described by Lehmann & White.16 In brief, the homogenate was centrifuged, resuspended in 4 mL of 3% sodium lauryl sulphate (SDS; Sigma Chemical Co., St Louis, MO, USA) and heated at 100°C for 15 min. After cooling, the pellet was washed once with distilled water, resuspended in 3 mL of 120% KOH solution and heated to 130°C for 1 h. Subsequently, 8 mL of ice-cold 75% ethanol were added, tubes were kept at 4°C for 15 min and 0.3 mL of Celite sus-pension (Celite 545; Sigma) were added. After centrifugation, the pellet was washed with cold ethanol (40%) and cold distilled water, and suspended in 0.5 mL of NaNO2 (5%) and 0.5 mL of KHSO4 (95%). After centrifugation, volumes of the supernatant were mixed with 12.5% NH4SO3NH2 followed by 3-methyl-benzo-2-thiazolone hydrazone HCl monohydrate (MBTH; Sigma). After heating for 3 min, the supernatants were cooled, FeCl36H2O (0.83%) was added and they were allowed to stand for 30 min. The optical density at 650 nm was read in a spectrophotometer. The chitin content of the organs was expressed as microgrammes of glucosamine per organ. Final measurements of chitin were corrected for the loss of volume of homogenate.
Toxicity
To check for the presence of toxicity following antifungal treatment, renal and hepatic functions were monitored in treated rats. Renal function was measured by serum creatinine and blood urea nitrogen (BUN) after 10 days of antifungal treatment. Hepatic function was measured by serum aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT).
Influence of treatment start time on survival
In a separate experiment the influence of start time of treatment on therapeutic efficacy was investigated. In this experiment, treatment was started at 16 h after fungal inoculation in one group, and at 24 h in the other, and survival of the rats was compared. All rats received the combination therapy.
Statistical methods
Differences in survival were assessed by the log rank test. Differences in proportions of animals with dissemination to the right lung and liver were examined by Fishers exact test. Differences in parameters of fungal infection were assessed by Students t-test with the exception of differences in chitin amounts, which were calculated using the Mann-Whitney U-test.
To analyse circulating GM concentrations, logarithmically transformed GM values were taken, showing an approximately normal distribution. The mean increase of log10 GM concentration with time was determined for each group of rats by repeated measurements ANOVA (random coefficients regression model) using the SAS Proc Mixed statistical computer package. The correlation between the measured concentrations of GM and mortality was assessed using Cox regression with log10 GM concentration as the time-dependent variable.
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Results |
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Ninety-six per cent of untreated rats died <11 days after fungal inoculation. Both treatment regimens significantly improved survival compared with the untreated controls (P < 0.0001 for both treatment regimens), as shown in Figure 1. The addition of a single dose of Fungizone 1 mg/kg at the start of treatment (day 1) to the 10 day AmBisome regimen significantly improved survival compared with AmBisome monotherapy (P = 0.02).
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In both the untreated and the treated groups, concentrations of circulating GM increased consistently over time (Table 1). The mean daily increases in log10 GM concentrations for controls, AmBisome monotherapy and combination therapy were 0.33 [±0.02 (S.E.M.)], 0.12 (±0.02) and 0.08 (±0.01), respectively. The mean increases in GM for the two treatment regimens were significantly lower than in controls (P < 0.001 for both regimens). The difference in log10 GM increase between AmBisome monotherapy and combination therapy approached significance (P = 0.06).
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Pulmonary macroscopic lesions and left lung weight
Antifungal treatment had an effect on the weight and macroscopic appearance of the left lung (Table 2). A shift was observed from angio-invasive lesions in untreated animals to responsive lesions in both treatment groups. Histologically, angio-invasive lesions showed extensive fungal broncho- and angio-invasion, and tissue haemorrhagia, all of which in responsive lesions were clearly reduced. Compared with controls, left lung weight was significantly lower in both treatment groups (P < 0.001 for both regimens). Compared with the AmBisome monotherapy group, the number of angio-invasive lesions was lower in the combination therapy group, but this difference was not significant (P = 0.31).
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AmBisome monotherapy did not reduce cfu cultured from the left lung (Table 2). In contrast, the combination therapy significantly reduced the cfu in the left lung compared with both the untreated controls (P < 0.001) and the AmBisome monotherapy group (P = 0.04). Both treatment regimens com-pletely prevented dissemination to the liver.
Chitin content of the infected lung
Both the AmBisome monotherapy and the combination therapy reduced chitin content in the left lung compared with untreated controls (P = 0.03 and <0.01, respectively). Com-pared with AmBisome monotherapy, the combination therapy significantly reduced left-lung chitin content (P = 0.03).
Toxicity
There were no significant differences between the treatment groups in serum creatinine, BUN, ALAT and ASAT con-centrations after 10 days of treatment (data not shown).
Influence of treatment start time on survival
In a separate experiment, it was investigated whether a delay in the start of treatment would have an effect on the therapeutic efficacy in our model. We compared the efficacy of the combination therapy when started at 16 h (n = 10) or at 24 h after fungal inoculation (n = 12) (Figure 2). A significant reduction in survival was seen when start of treatment was delayed from 16 to 24 h (P = 0.02).
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To investigate which parameters of fungal infection were associated with survival in rats receiving antifungal treatment (either AmBisome monotherapy or combination therapy), we compared the results of rats that died during treatment with those of rats that survived until the end of treatment (Table 3). In the surviving animals the size of the responsive lesion was significantly larger, whereas the angio-invasive lesion was smaller. In addition, the parameters of fungal load were lower in surviving animals, with the left lungs containing significantly less cfu and chitin compared with animals that died. The number of cfu in the right lungs was also lower in surviving rats and the difference in the percentage of rats with dissemination approached significance.
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Discussion |
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The aim of our study was to investigate whether the efficacy of high doses of AmBisome could be enhanced by increasing the immediate availability of the biologically active drug in the early phase of treatment. This hypothesis was based on studies carried out in our laboratory by van Etten et al.9,12,18 in a mouse model of invasive candidiasis. In biodistribution studies that measured the levels of amphotericin B in the lung at various time points after a single injection of Fungizone 0.3 mg/kg, they found that amphotericin B could be detected in the lung at all time points from 5 min to 12 h. In contrast, after a single injection with AmBisome 7 mg/kg, concentrations of amphotericin B in the lung could not be detected until 12 h and later. In addition, they reported that the in vitro antifungal activity of AmBisome during short-term exposure (6 h) of Candida albicans was significantly less than that of Fungizone. The latter finding was explained by a relatively slow release of amphotericin B from the liposomes. Furthermore, they found that addition of a single dose of Fungizone to a 5 day regimen of AmBisome at the start of treatment significantly increased survival and decreased fungal load in the kidney of granulocytopenic mice with disseminated candidiasis.
Our findings are in accordance with these studies. In our rat model of IPA we have also observed that rat survival increased significantly when a single dose of Fungizone (on day 1) was added to a 10 day AmBisome regimen. When higher doses of AmBisome (up to 30 mg/kg) were administered on day 1 and AmBisome 10 mg/kg on days 210, no improvement compared with AmBisome monotherapy was seen (data not shown), indicating that changing the absolute amount of AmBisome on day 1 had no significant impact. Multiple doses of Fungizone 1 mg/kg (at day 1, 3 and 5, or at day 1 and 6, or twice at day 1) combined with the 10 day AmBisome regimen did not further enhance survival compared with a single addition of Fungizone 1 mg/kg at day 1 (data not shown). This indicates that only addition of Fungizone at day 1 is relevant for improving survival. In addition, early antifungal activity was shown to be a highly important factor, since an 8 h delay in starting antifungal treatment resulted in significant reduction in survival. Therefore, it is likely that the improved antifungal efficacy resulting from the addition of Fungizone to the AmBisome regimen at day 1 is caused by increased bio-availability of amphotericin B in the lung in the early phase of the disease.
In addition to survival, we investigated the value of several parameters of fungal infection as markers for treatment response. Antifungal treatment appeared to have an effect on most of these parameters. Concentrations of serially sampled serum GM were significantly lower in both treatment groups compared with untreated controls, whereas the difference between treatment groups approached significance. Other investigators have also described a suppressive effect of antifungal treatment on circulating GM concentrations in both animal models and patients with IPA.19,20 In addition, increases in GM concentration in our model were associated with a significantly increased mortality. This indicates that serum GM concentrations are a relevant parameter for measuring treatment response.
Antifungal treatment resulted in a reduction in lung weight and size of the dark-red coloured haemorrhagic angio-invasive lesion, and an increase in the size of the light-red coloured responsive lesion. The size of this responsive lesion was also associated with increased survival, as opposed to the angio-invasive lesion. Others have also described decreased haemorrhagic lesions in a rabbit model of IPA and increased lighter coloured resolving lesions under antifungal treatment.21 These findings indicate that these lesions reflect an important aspect of the pathogenesis of IPA. Since responsive lesions represented areas with reduced broncho-angio invasion and haemorrhagia, inhibition of the invasive spread of the fungus is probably important for successful antifungal treatment.
The numbers of cfu cultured from the left lungs of rats receiving combination therapy were lower than those of rats receiving AmBisome monotherapy. However, compared with untreated controls the cfu of the AmBisome monotherapy group were not significantly decreased. This may be explained by the fact that the number of cfu does not represent the real viable fungal load: larger hyphae represent a larger fungal load but not necessarily a larger number of cfu.22 In accordance with this, several studies reported that the numbers of cfu cultured from the organs in their animal models of IPA did not significantly increase over time,16,23,24 in contrast with quantitatively measured fungal DNA in one of these studies.24 Chitin may be a more reliable indicator for fungal load than cfu, since it is a constituent from the fungal cell wall and the amount of chitin increases with the growth of the hyphae.16 Indeed, we found a significant decrease in chitin amount in the left lung in both treatment groups compared with untreated rats, as well as significant differences between the two treatment groups.
In conclusion, we have demonstrated that the addition of a single dose of Fungizone at the start of AmBisome treatment enhanced the therapeutic efficacy of AmBisome monotherapy. Apart from survival, parameters of fungal disease that best represented increased treatment response were left-lung chitin content and serum GM concentration.
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Acknowledgements |
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Footnotes |
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References |
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2 . Warnock, D. W. (1991). Amphotericin B: an introduction. Journal of Antimicrobial Chemotherapy 28, Suppl. B, 2738.[ISI][Medline]
3 . Meyer, R. D., Young, L. S., Armstrong, D. & Yu, B. (1973). Aspergillosis complicating neoplastic disease. American Journal of Medicine 54, 615.[ISI][Medline]
4 . Branch, R. A. (1988). Prevention of amphotericin B-induced renal impairment. A review on the use of sodium supplementation. Archives of Internal Medicine 148, 238994.[Abstract]
5 . Bodey, G. P., Anaissie, E. J., Elting, L. S., Estey, E., OBrien, S. & Kantarjian, H. (1994). Antifungal prophylaxis during remission induction therapy for acute leukemia: fluconazole versus intravenous amphotericin B. Cancer 73, 2099106.[ISI][Medline]
6 . Leenders, A. C. & de Marie, S. (1996). The use of lipid formulations of amphotericin B for systemic fungal infections. Leukemia 10, 15705.[ISI][Medline]
7 . Leenders, A. C., Daenen, S., Jansen, R. L., Hop, W. C., Lowenberg, B., Wijermans, P. W. et al. (1998). Liposomal amphotericin B compared with amphotericin B deoxycholate in the treatment of documented and suspected neutropenia-associated invasive fungal infections. British Journal of Haematology 103, 20512.[ISI][Medline]
8 . Blau, I. W. & Fauser, A. A. (2000). Review of comparative stu-dies between conventional and liposomal amphotericin B (Am-Bisome) in neutropenic patients with fever of unknown origin and patients with systemic mycosis. Mycoses 43, 32532.[ISI][Medline]
9 . van Etten, E. W., ten Kate, M. T., Stearne, L. E. & Bakker-Woudenberg, I. A. (1995). Amphotericin B liposomes with prolonged circulation in blood: in vitro antifungal activity, toxicity, and efficacy in systemic candidiasis in leukopenic mice. Antimicrobial Agents and Chemotherapy 39, 19548.[Abstract]
10 . Barnes, A. J., Oppenheim, B. A., Chang, J., Morgenstern, G. R. & Scarffe, J. H. (1999). Early investigation and initiation of therapy for invasive pulmonary aspergillosis in leukaemic and bone marrow transplant patients. Mycoses 42, 4038.[ISI][Medline]
11 . Caillot, D., Casasnovas, O., Bernard, A., Couaillier, J. F., Durand, C., Cuisenier, B. et al. (1997). Improved management of invasive pulmonary aspergillosis in neutropenic patients using early thoracic computed tomographic scan and surgery. Journal of Clinical Oncology 15, 13947.[Abstract]
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.
van Etten, E. W., Snijders, S. V., van Vianen, W. & Bakker-Woudenberg, I. A. (1998). Superior efficacy of liposomal amphotericin B with prolonged circulation in blood in the treatment of severe candidiasis in leukopenic mice. Antimicrobial Agents and Chemotherapy 42, 24313.
13 . Leenders, A. C., de Marie, S., ten Kate, M. T., Bakker-Woudenberg, I. A. & Verbrugh, H. A. (1996). Liposomal amphotericin B (AmBisome) reduces dissemination of infection as compared with amphotericin B deoxycholate (Fungizone) in a rate model of pulmonary aspergillosis. Journal of Antimicrobial Chemotherapy 38, 21525.[Abstract]
14 . Espinel-Ingroff, A., Dawson, K., Pfaller, M., Anaissie, E., Breslin, B., Dixon, D. et al. (1995). Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi. Antimicrobial Agents and Chemotherapy 39, 3149.[Abstract]
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.
Becker, M. J., de Marie, S., Willemse, D., Verbrugh, H. A. & Bakker-Woudenberg, I. A. (2000). Quantitative galactomannan detection is superior to PCR in diagnosing and monitoring invasive pulmonary aspergillosis in an experimental rat model. Journal of Clinical Microbiology 38, 14348.
16 . Lehmann, P. F. & White, L. O. (1975). Chitin assay used to demonstrate renal localization and cortisone-enhanced growth of Aspergillus fumigatus mycelium in mice. Infection and Immunity 12, 98792.[ISI][Medline]
17 . Rex, J. H., Walsh, T. J., Nettleman, M., Anaissie, E. J., Bennett, J. E., Bow, E. J. et al. (2001). Need for alternative trial designs and evaluation strategies for therapeutic studies of invasive mycoses. Clinical Infectious Diseases 33, 95106.[ISI][Medline]
18 . van Etten, E. W., Otte-Lambillion, M., van Vianen, W., ten Kate, M. T. & Bakker-Woudenberg, A. J. (1995). Biodistribution of liposomal amphotericin B (AmBisome) and amphotericin B-desoxycholate (Fungizone) in uninfected immunocompetent mice and leucopenic mice infected with Candida albicans. Journal of Antimicrobial Chemotherapy 35, 50919.[Abstract]
19 . Patterson, T. F., Miniter, P., Ryan, J. L. & Andriole, V. T. (1988). Effect of immunosuppression and amphotericin B on Aspergillus antigenemia in an experimental model. Journal of Infectious Diseases 158, 41522.[ISI][Medline]
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.
Roberts, J., Schock, K., Marino, S. & Andriole, V. T. (2000). Efficacies of two new antifungal agents, the triazole ravuconazole and the echinocandin LY-303366, in an experimental model of invasive aspergillosis. Antimicrobial Agents and Chemotherapy 44, 33818.
21 . Allende, M. C., Lee, J. W., Francis, P., Garrett, K., Dollenberg, H., Berenguer, J. et al. (1994). Dose-dependent antifungal activity and nephrotoxicity of amphotericin B colloidal dispersion in experimental pulmonary aspergillosis. Antimicrobial Agents and Chemotherapy 38, 51822.[Abstract]
22
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Manavathu, E. K., Cutright, J. L. & Chandrasekar, P. H. (1998). Organism-dependent fungicidal activities of azoles. Antimicrobial Agents and Chemotherapy 42, 301821.
23 . Duong, M., Ouellet, N., Simard, M., Bergeron, Y., Olivier, M. & Bergeron, M. G. (1998). Kinetic study of host defense and inflammatory response to Aspergillus fumigatus in steroid-induced immunosuppressed mice. Journal of Infectious Diseases 178, 147282.[ISI][Medline]
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Bowman, J. C., Abruzzo, G. K., Anderson, J. W., Flattery, A. M., Gill, C. J., Pikounis, V. B. et al. (2001). Quantitative PCR assay to measure Aspergillus fumigatus burden in a murine model of disseminated aspergillosis: demonstration of efficacy of caspofungin acetate. Antimicrobial Agents and Chemotherapy 45, 347481.