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Previous Article | Next Article 
Antimicrobial Agents and Chemotherapy, January 2006, p. 294-297, Vol. 50, No. 1
0066-4804/06/$08.00+0 doi:10.1128/AAC.50.1.294-297.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Pentamidine Is Active in a Neutropenic Murine Model of Acute Invasive Pulmonary Fusariosis
Michail S. Lionakis,1,2,
Georgios Chamilos,1,
Russell E. Lewis,1,3
Nathan P. Wiederhold,3
Issam I. Raad,1
George Samonis,2 and
Dimitrios P. Kontoyiannis1,3*
Department of Infectious Diseases, Infection Control and Employee Health, The University of Texas M. D. Anderson Cancer Center, Houston, Texas,1
Medical School of the University of Crete, Heraklion, Greece,2
College of Pharmacy, University of Houston, Houston, Texas3
Received 11 August 2005/
Returned for modification 12 September 2005/
Accepted 12 October 2005
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ABSTRACT
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We studied the efficacy of pentamidine (PNT) as prophylaxis or early treatment in acute pulmonary fusariosis in neutropenic mice. PNT-preexposed mice had significantly improved survival and reduced fungal burden compared to amphotericin B-preexposed and untreated mice. PNT-treated mice had increased survival but no difference in fungal burden versus untreated mice.
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INTRODUCTION
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The mortality rate of invasive fusariosis is exceedingly high in immunocompromised hosts (8, 14). Current antifungal drugs have marginal efficacy and the major prognostic determinant in fusariosis is neutrophil recovery (8, 14). Introduction of new, more effective prophylactic and therapeutic approaches is essential for improving the prognosis of fusariosis. We previously showed that pentamidine (PNT), a broad-spectrum antimicrobial (13), has significant activity against a variety of pathogenic Fusarium species in vitro, with preferential activity against Fusarium conidia (10). As described herein, after establishing a reproducible murine model of acute invasive pulmonary fusariosis, we tested the in vivo efficacy of PNT in prophylaxis or early treatment (first 6 h after infection) of infection with Fusarium oxysporum.
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MATERIALS AND METHODS
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Organism and animals.
We used F. oxysporum isolate F15, which was recovered from a patient with acute myelogenous leukemia who died of fusariosis. The MICs of amphotericin B (AMB) and PNT were 2 µg/ml and 4 µg/ml, respectively, as determined by the Clinical and Laboratory Standards Institute M38-A microdilution method (12). Conidia were collected and prepared as described previously (9). Female BALB/c mice (Harlan Sprague-Dawley, Indianapolis, Ind.) weighting 20 to 25 g each were used. Animals were housed (n = 5 per cage) in presterilized, filter-topped cages and provided with sterile food, water, and bedding in the biohazardous isolation suite at The University of Texas M. D. Anderson Cancer Center Animal Care Facilities. Animals had access to food and water ad libitum. All procedures were performed in accordance with the highest standards for humane handling, care, and treatment of research animals and were approved by The University of Texas M. D. Anderson Cancer Center and University of Houston Institutional Animal Care and Use Committees.
Immunosuppression and infection.
Cyclophosphamide (Sigma Chemical Co., St. Louis, Mo.) was administered by intraperitoneal (i.p.) injections (150 mg/kg; 200 to 250 µl of a 15-mg/ml sterile saline solution) 3 days prior to and 1 after infection, rendering the mice neutropenic, as described previously (6). Mice were infected by the sinopulmonary route as reported previously (9) by using 35 µl from different conidial inoculum solutions in independent experiments (range, 107 conidia/ml solution resulting in infection with 
35 x 104 conidia/mouse to 109 conidia/ml solution resulting in infection with 35 x 106 conidia/mouse). The conidial viability was greater than 99%, as determined by quantitative plating of serial dilutions taken from the original inoculum.
All animals were observed for 4 days after infection and weighed daily to monitor for drug toxicity. Animals that appeared moribund before 4 days after infection were euthanized by CO2 asphyxiation, and death was recorded as occurring 12 h later. On day 4 after infection, all of the remaining mice were euthanized.
Prophylaxis and treatment.
Groups of 10 mice each were given (a) PNT prophylaxis (8.5 mg/kg/24 h in distilled water intravenously [i.v.], starting 24 h prior to infection), or (b) AMB prophylaxis (1.5 mg/kg/24 h in distilled water i.p. starting 24 h prior to infection) or (c) early treatment with PNT (8.5 mg/kg/24 h i.v., starting 6 h postinfection), or (d) i.v. saline (control). The experiment was performed in triplicate on different days.
Fungal burden quantification by real-time quantitative PCR (qPCR).
In two of the three above experiments, lungs of AMB-preexposed, PNT-preexposed, PNT-treated, and control untreated animals were removed after euthanization and stored at –80°C until analysis of pulmonary fungal burden by qPCR. DNA was extracted from lung homogenates by using the DNeasy tissue kit (QIAGEN, Valencia, CA.), and DNA samples were analyzed in duplicate by using the ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA). Primers and dual-labeled fluorescent hybridization probes specific for the F. oxysporum 18S rRNA were designed using the ABI PRISM SeqScape software program (version 2; Applied Biosystems). The primers and probe used were as follows: forward primer, 5'-TGGTGCATGGCCGTTCTTA-3'; reverse primer, 5'-GGTCTCGTTCGTTATCGCAATT-3'; probe, 5'-6-carboxyfluorescein-TTGGTGGAGTGATTTGTCTGCT-6-carboxytetramethylrhodamine-3'. The threshold cycle (Ct) of each sample was interpolated from a six-point standard curve of Ct values prepared by spiking uninfected mouse lungs with 102 to 107 F. oxysporum conidia. Results were reported as conidial equivalents of F. oxysporum DNA.
Histopathology.
In other experiments, whole-lung tissue samples obtained from three AMB-preexposed, three PNT-preexposed, three PNT-treated, and three control untreated animals euthanized 48 h after infection were submitted to compare disease severity by histopathology and Grocott-Gomori methenamine-silver nitrate staining.
Statistical analysis.
Survival curves were plotted by Kaplan-Meier analysis and differences in survival between the groups of mice were analyzed by the log-rank test. For all comparisons, P values of 
0.05 were considered statistically significant. Analysis of variance was performed to assess differences in fungal burden among the different mouse groups.
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RESULTS
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Establishment of a neutropenic murine model of acute invasive pulmonary fusariosis.
Sinopulmonary inoculation of mice with F. oxysporum conidia led to the development of acute pneumonia with mortality rates that were reproducible and inoculum-dependent. Inoculation using a 107-conidia/ml solution resulted in mortality of 30% at day 4 after infection, whereas inoculation using a 109-conidia/ml solution resulted in hyperacute infection with mortality of 90% at day 1 after infection. For our drug protection experiments, we infected mice using a 2 x 108-conidia/ml solution that resulted in infection with 70 x 105-conidia/mouse (Fig. 1A). The possibility that the acute mortality observed 24 h postinfection (50%) was due to a bacterial infection was excluded by performing plating of lung homogenates from representative mice that died 24 h post-Fusarium inoculation on routine culture media. No bacterial growth was appreciated. In addition, in other experiments, histopathology sections with GMS staining of lung tissue from mice that died 24 h postinfection demonstrated the presence of extensive hyphal invasion by Fusarium and hemorrhage consistent with fatal pulmonary fusariosis but no evidence of bacterial infection.
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FIG. 1. (A) Survival of neutropenic BALB/c mice infected with F. oxysporum and administered PNT prophylaxis, AMB prophylaxis, early PNT treatment, or saline (untreated control). Survival was significantly improved in mice given PNT prophylaxis vs. control mice (P = 0.0003), mice given PNT versus AMB prophylaxis (P = 0.01), and mice given early PNT treatment (or AMB prophylaxis) vs. control mice (P = 0.02). Results are the means from three independent experiments. (B) Difference in lung fungal burden (conidial equivalents of F. oxysporum DNA) by real-time qPCR between control mice and mice given PNT or AMB prophylaxis or early PNT treatment. Fungal burden was significantly decreased in mice given PNT prophylaxis versus control mice and mice given PNT treatment or AMB prophylaxis (P < 0.05). Results are the means from two independent experiments. AMB, AMB prophylaxis; PNT TX, PNT treatment; PNT P, PNT prophylaxis.
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Prophylaxis with PNT.
Infected mice preexposed to PNT had significantly improved survival at day 4 after infection when compared with untreated mice (77% and 10%, respectively; P < 0.001) (Fig. 1A). Also, PNT prophylaxis was more effective than AMB prophylaxis (survival at day 4 postinfection, 27%; P = 0.01). In addition to having better survival, PNT-preexposed mice also had substantially lower lung fungal burden than AMB-preexposed mice (P < 0.05) and control mice (P < 0.05) by both qPCR (Fig. 1B) and histopathology (Fig. 2). AMB preexposure improved survival when compared with no treatment (P = 0.02) (Fig. 1A) but did not markedly decrease the lung fungal burden according to qPCR (Fig. 1B) and histopathology (Fig. 2).
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FIG. 2. Representative histopathological sections (stained with Grocott-Gomori methenamine-silver nitrate) of lung tissue recovered from control mice (A and B), mice given AMB prophylaxis (C and D), and mice given PNT prophylaxis (E and F). The arrows show F. oxysporum branching hyphae, which are seen as dark staining. Of note is the significant reduction in the lung fungal burden in PNT-preexposed mice when compared with that in control mice. Magnifications: panels A, C, and D, x196; panels B, D, and E, x980.
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Treatment with PNT.
PNT-treated mice had better survival (33%) when compared with control mice (10%; P = 0.02) (Fig. 1A) but had comparable lung fungal burden by qPCR (Fig. 1B) and histopathology (data not shown).
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DISCUSSION
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Fusariosis is an emerging opportunistic mycosis against which antifungals have mediocre activity, especially in the setting of neutropenia (8, 14). Current in vivo models used to study the activity of antifungals against fusariosis are established by intravenous inoculation of Fusarium conidia (1, 5). Herein, we developed a reproducible murine model of acute invasive pulmonary fusariosis that simulates the pathophysiology of the human infection (3, 14) through the introduction of Fusarium conidia via the sinopulmonary route.
We tested PNT to determine its efficacy in prophylaxis for and early treatment of F. oxysporum infection. PNT prophylaxis substantially improved survival and significantly reduced the lung fungal burden when compared with AMB prophylaxis and no prophylaxis. Although AMB prophylaxis improved survival, its effect was less pronounced than that of PNT prophylaxis, and it did not decrease the lung fungal burden. The marginal efficacy of AMB as prophylaxis is in part explained by, and in agreement with, the high AMB MIC of the tested Fusarium isolate. The poor in vivo efficacy of AMB in the setting of neutropenia has been well-established both in animal models (1) and in humans with fusariosis (8).
Similarly, early PNT treatment increased survival. However, it did not affect the lung fungal burden when compared with no treatment. The greater activity of PNT in prophylaxis than in treatment is in agreement with our previous work, in which we found that PNT had preferentially increased activity against Fusarium conidia versus hyphae in vitro (10). This relative resistance of hyphae versus conidia to antifungals (i.e., AMB, azoles) has been shown in several filamentous fungi (i.e., Aspergillus, Cladosporium, Paecilomyces, Scopulariopsis, Cladophialophora species) (4, 7).
Our initial plan was to demonstrate a dose-response effect of PNT against Fusarium and also to use different routes of PNT administration. Unfortunately, drug toxicity limited our efforts. Hence, when i.v. PNT was administered at 17 mg/kg/24 h, death of a substantial number of animals within few minutes after i.v. administration was seen (data not shown). This advent outcome was likely due to electrolytic disturbances and/or cardiac arrhythmias, which are known adverse effects of PNT use in humans (13). Moreover, in other experiments using i.p. PNT injections (dose, 8.5 or 17 mg/kg/24 h), significant toxicity was also observed resulting in intra-abdominal necrosis (as determined at necropsy) in a substantial proportion of animals (data not shown). We believe that the reason for this outcome is probably necrotizing pancreatitis, which can occasionally occur with PNT use in humans (13).
Our study has certain limitations. First, we used a PNT dosage that was shown to result in lung concentrations of PNT in mice similar to lung levels achieved in humans with conventional PNT dosages (2, 15). However, we did not confirm the lung or serum concentrations of PNT in pharmacokinetic studies. Additionally, the hyperacute nature of the murine model of fusariosis keeps it from entirely mimicking the course of fusariosis in humans, which tends to have a more subacute tempo of progression (14). Therefore, our model might not be suitable for demonstrating the therapeutic potential of PNT, as shown by our early infection survival data.
Despite these limitations, our study expands upon our previous findings showing that PNT has anti-Fusarium activity in vitro (10) and demonstrates that PNT, at pharmacologically relevant concentrations, has significant in vivo efficacy against fusariosis, particularly when administered as prophylaxis. The use of PNT, despite its toxicity, in prophylaxis against Pneumocystis jiroveci pneumonia in recipients of allogeneic bone marrow transplants and patients with AIDS has been well studied (11). Thus, speculating that such patients may also benefit from protection against fusariosis while receiving prophylaxis with PNT is appealing. However, considering the low incidence of fusariosis, studying this hypothesis may be difficult.
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ACKNOWLEDGMENTS
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This work was supported in part by The University of Texas M. D. Anderson Faculty E. N. Cobb Scholar Award Research Endowment and the M. D. Anderson Cancer Center Core Grant (CA 16672) from The University of Texas (to D.P.K.). M.S.L. is supported in part by the Agricultural Bank of Greece Fellowship.
We thank Nathaniel D. Albert for excellent technical assistance.
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FOOTNOTES
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* Corresponding author. Mailing address: Department of Infectious Diseases, Infection Control and Employee Health, Unit 402, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 792-6237. Fax: (713) 745-6839. E-mail: dkontoyi{at}mdanderson.org. 
These authors contributed equally to this work.
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Antimicrobial Agents and Chemotherapy, January 2006, p. 294-297, Vol. 50, No. 1
0066-4804/06/$08.00+0 doi:10.1128/AAC.50.1.294-297.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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Abstract
Introduction
