J Antimicrob Chemother 2016; 71: 2230 – 2233
doi:10.1093/jac/dkw111 Advance Access publication 26 May 2016
Delay of antifungal therapy influences the outcome of invasive
aspergillosis in experimental models of infection
Francesco Barchiesi1*, Alfredo Santinelli2, Tommasina Biscotti2, Gianfranco Greganti1,
Daniele Giannini3 and Esther Manso4
1
Infectious Diseases Clinic, Polytechnic University of Marche, Ancona, Italy; 2Pathological Anatomy and Histopathology, Department of
Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Italy; 3Management Centre of Medicine and Surgery,
Polytechnic University of Marche, Ancona, Italy; 4Laboratory of Microbiology, Azienda Ospedaliero-Universitaria, Ospedali Riuniti,
Umberto I-Lancisi-Salesi, Ancona, Italy
*Corresponding author. Clinica Malattie Infettive, Università Politecnica delle Marche, Ancona, Italy. Tel: +39-071-5963466;
Fax: +39-071-5963717; E-mail: [email protected]
Received 22 August 2015; returned 23 October 2015; revised 2 March 2016; accepted 7 March 2016
Objectives: The aim of the present study was to evaluate the effects of delayed antifungal therapy on the
outcome of invasive aspergillosis due to Aspergillus fumigatus in experimental models of infection.
Methods: A clinical isolate of A. fumigatus susceptible to amphotericin B (MIC 0.5 mg/L) and micafungin [minimum
effective concentration (MEC) 0.03 mg/L] was used in all experiments. Two models of infection were investigated in
immunosuppressed mice: disseminated infection and pulmonary infection. Twenty-four hours (early therapy) and
48 h (delayed therapy) post-infection, the mice were given vehicle, liposomal amphotericin B, micafungin or liposomal amphotericin B plus micafungin (combination). Drug efficacy was assessed by either survival or tissue burden
experiments.
Results: In disseminated infection, any drug regimen given early significantly prolonged survival. When therapy
was delayed, only micafungin and the combination were effective. In pulmonary infection, although there was a
trend towards a prolongation of survival of mice treated early with liposomal amphotericin B, only the combination was effective. Similarly, when therapy was delayed, only the combination was effective. In disseminated
infection, any drug regimen given early was effective at reducing the cfu in kidney tissue. In pulmonary infection,
only liposomal amphotericin B and the combination given early were effective at reducing the cfu in lung tissue.
Conversely, when therapy was delayed, no regimen was effective at reducing the tissue burden, regardless of the
type of infection.
Conclusions: Our data indicate that delayed initiation of antifungal therapy is deleterious in experimental models
of invasive aspergillosis. A combination regimen seems to have some advantages over a single-drug approach
when the therapy is started late.
Introduction
Invasive aspergillosis (IA) is a life-threatening fungal infection
associated with significant mortality.1,2 Both voriconazole and
liposomal amphotericin B are shown to be effective in the treatment of IA.3,4 To further improve the outcome of IA, several
antifungal combination approaches have been investigated.5,6
For its unique mode of activity, an echinocandin derivative
would represent an interesting drug partner in combination
regimens.
The difficulty in establishing an early diagnosis of IA can delay
antifungal treatment, resulting in an increased risk of mortality.
We hypothesized that a drug combination approach might
be more effective than monotherapy in infections in which
antifungal treatment is started late. Therefore, we investigated
the effects of liposomal amphotericin B and micafungin, administered alone and in combination, in experimental models of IA in
which therapy was initiated either soon or late after the infection.
Materials and methods
Isolate characterization
A clinical isolate of Aspergillus fumigatus (AF 119701) cultured from a
respiratory secretion of a patient diagnosed with proven aspergillosis
was used in all the experiments. Antifungal susceptibility testing, following
the CLSI recommendations, found that the isolate was susceptible in vitro
to both amphotericin B (median MIC 0.5 mg/L) and micafungin [median
minimum effective concentration (MEC) 0.03 mg/L].7
# The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]
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Delayed antifungal therapy in aspergillosis
Drugs
Liposomal amphotericin B (AmBisome, Gilead Sciences) was reconstituted
according to the manufacturer’s instructions and diluted in sterile 5% dextrose solution. Micafungin (Mycamine, Astellas) was rehydrated in sterile
water and diluted in 0.9% sodium chloride solution.
Magnification ×20
Magnification ×150
(a) kidney 24 h
(b) kidney 24 h
(c) kidney 48 h
(d) kidney 48 h
(e) lung 24 h
(f) lung 24 h
(g) lung 48 h
(h) lung 48 h
Experimental infections
CD1 female mice (20 g, 8 weeks old; Charles River Laboratories,
Calco, Italy) were used in all studies. Two models of infection were investigated: disseminated infection, performed by giving the conidia intravenously (1.6×104 conidia/mouse in 0.2 mL of saline solution); and
pulmonary infection, performed by giving the conidia intranasally
(1.0×107 conidia/mouse in 0.04 mL of saline solution). The mice were
rendered neutropenic by intraperitoneal administration of cyclophosphamide (200 mg/kg of body weight) on days 24, +1 and +4 and every 3 days
thereafter. Additionally, in the pulmonary infection model, the mice were
given subcutaneously a single 300 mg/kg dose of cortisone acetate suspension on day 21. Experiments were conducted with the approval of
the University of Ancona Ethics Committee, and animals were cared for
in accordance with national regulations.
Twenty-four hours (early therapy) and 48 h (delayed therapy) postinfection, the mice were given vehicle, liposomal amphotericin B, micafungin or liposomal amphotericin B plus micafungin (combination). Drugs
were used at low (3 mg/kg/day) or at high (10 mg/kg/day) dosages.
Liposomal amphotericin B was given intravenously, whereas micafungin
was given intraperitoneally. Both drugs were administered in a final
volume of 0.2 mL. Therapy was given for three consecutive days.
Drug efficacy was assessed either by survival analysis (15 days) or tissue burden experiments (via kidney in disseminated infection; via lung in
pulmonary infection). In the latter experiments, the mice were euthanized
24 h after the last dose of the drug was given, and the quantity of cfu per
organ per mouse was determined. There were 10 to 25 animals for each
treated group in survival studies and seven animals for each treated group
in tissue burden experiments. In order to monitor the progression of infection, additional groups of untreated mice were killed at 24, 48 and 72 h
after the initial infection. Kidneys (disseminated infection) and lungs
(pulmonary infection) were removed, fixed in 10% neutral buffered formalin solution, embedded in paraffin and stained with Grocott – Gomori’s
methenamine silver nitrate for histopathological studies.
Statistical analysis
Survival results were plotted as Kaplan–Meier curves and analysed by log
rank (due to multiple comparisons, a P value ,0.008 was considered statistically significant). The cfu per organ per mouse values were analysed
by one-way analysis of variance followed by the Tukey’s test corrected
for multiple comparison. A P value ,0.05 was considered statistically
significant.
Results
The histopathological appearances showed a disease progression
that slightly differed based on the type of infection (Figure 1).
Particularly, in disseminated infection, renal structure appeared
normal at 24 h post-inoculation, whereas focal areas of hyphal
formation began to appear at 48 h (Figure 1a –d). In pulmonary
infection, lung fungal infiltrates were already evident at 24 h
post-inoculation, and a progressive hyphal extension was seen
at 48 h (Figure 1e–h). At 72 h post-inoculation, a further progression of tissue invasion was seen in both types of infection (data
not shown).
Figure 1. Representative results for histopathological sections of kidney
(disseminated infection; a–d) and lung (pulmonary infection; e–h) tissues
stained with Grocott – Gomori’s methenamine silver nitrate from mice
infected with A. fumigatus. The mice were killed either 24 h (a, b, e and f)
or 48 h (c, d, g and h) post-infection. Arrows indicate focal areas of
hyphal formation. Magnifications×20 (a, c, e and g) and×150 (b, d, f and h).
Survival results of disseminated infection are shown in
Figure 2(a – d). Median survival time in early therapy was 3, 7, 6
and 9 days for mice treated with vehicle, liposomal amphotericin B
at 3 mg/kg, micafungin at 3 mg/kg and the combination, respectively (Figure 2a). Either liposomal amphotericin B (P ¼ 0.0024) or
micafungin (P ¼ 0.0031) were effective at prolonging the survival
against the controls. The combination was more effective than
control (P,0.0001) and more effective than liposomal amphotericin B (P ¼0.0005), but not more effective than micafungin alone
(P ¼ 0.02). Median survival times in delayed therapy were 3, 5, 5
and 7 days for mice treated with vehicle, liposomal amphotericin B
at 3 mg/kg, micafungin at 3 mg/kg and the combination, respectively (Figure 2b). The only regimen which prolonged the survival
was the combination (P ¼ 0.005). When drug doses, singly and
combined, were increased to 10 mg/kg, median survival time in
early therapy was 4, 12, 11.5 and .15 days for mice treated with
vehicle, liposomal amphotericin B, micafungin and the combination, respectively (Figure 2c). All regimens were effective against
the controls (P,0.0001). When therapy was delayed (Figure 2d),
the median survival times were 4, 7, 8.5 and 8 days for mice
treated with vehicle, liposomal amphotericin B, micafungin and
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Barchiesi et al.
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Figure 2. Survival curves of mice infected intravenously (a –d) or intranasally (e and f) with 1.6×104 (a –d) or 1.0×107 (e and f) A. fumigatus conidia
per mouse and treated with vehicle (circles), liposomal amphotericin B (squares), micafungin (triangles) or the combination of liposomal amphotericin B
plus micafungin (upside-down triangles). Both drugs, alone or in combination, were given at 3 mg/kg/day (a, b, e and f) or 10 mg/kg/day (c and d) for
three consecutive days. Therapy was started 24 h (early therapy; a, c and e) or 48 h (delayed therapy; b, d and f) post-infection.
the combination, respectively, and only micafungin (P¼0.001) and
the combination (P¼0.0026) were effective.
Survival results of pulmonary infection are shown in Figure 2(e
and f). Pulmonary infection experiments were conducted with
drugs given at 3 mg/kg. The median survival times in early therapy
were 7, .15, 10 and .15 days for mice treated with vehicle, liposomal amphotericin B, micafungin and the combination, respectively
(Figure 2e). Although there was a trend towards a prolongation of
survival of mice treated with liposomal amphotericin B, the results
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of this regimen did not reach statistical significance (P¼0.025). The
only regimen that significantly prolonged the survival compared
with the control was the combination (P¼0.007). Median survival
time in delayed therapy was 7, 9, 9 and .15 days for mice treated
with vehicle, liposomal amphotericin B, micafungin and the combination, respectively (Figure 2f). Only the combination approach was
effective (P¼0.004).
Tissue burden experiments were performed by giving the drugs
at 3 mg/kg. In early therapy of disseminated infection, all regimens
JAC
Delayed antifungal therapy in aspergillosis
were effective at reducing the cfu/kidney with respect to controls
being the mean cfu/organ+standard deviation (SD) of 398+154,
110+60, 196+115 and 25+15 for mice treated with vehicle,
liposomal amphotericin B, micafungin and the combination,
respectively. Additionally, the combination was more effective
than micafungin but not more effective than liposomal amphotericin B. In early therapy of pulmonary infection, only liposomal
amphotericin B and the combination were effective at reducing
the cfu/lung with respect to controls; the mean cfu/organ+SD
were 8057+1201, 498+380, 4429+2719 and 488+303 for
mice treated with vehicle, liposomal amphotericin B, micafungin
and the combination, respectively. In delayed therapy, no regimen was effective, regardless of the type of infection (data
not shown).
Discussion
Our data showed that the delayed onset of antifungal therapy is
deleterious to the treatment of experimental IA. An earlier study
conducted in a neutropenic rabbit model of IA showed that
amphotericin B deoxycholate therapy given at 1 mg/kg and
initiated ≥48 h post-infection was associated with suboptimal
therapeutic outcome.8 Here, we showed that liposomal amphotericin B given at doses as high as 10 mg/kg was not effective
when initiated 48 h post-infection. Although one can hypothesize
that the highest polyene dose used in this study can be toxic in
mice (and therefore not effective), its effectiveness when the
same dose is initiated earlier eliminates this possibility. Doses of
both drugs selected in this study were either based on previously
published murine aspergillosis models showing a clear dosedependent effectiveness or based on their clinical relevance.3,9 – 13
Here, we showed that an extensive hyphal invasion, as seen in
both models at 48 h post-infection, makes delayed therapy ineffective. It is interesting to note that the combination approach
was often the only effective regimen when therapy was delayed.
This phenomenon was observed at both low and high doses of
both drugs. It is likely that a mutual potentiation of drugs acting
against different fungal targets still persists at a later stage of
hyphal growth when any monotherapy lacks efficacy. Data from
the literature investigating the effects of liposomal amphotericin B
plus an echinocandin in experimental models of infections have
produced varying results.10,11 In A. fumigatus infections, the combination was generally as effective as the polyene alone either in terms
of survival or tissue burden results.10 In Aspergillus flavus infections,
concomitant therapies produced significantly enhanced survival and
reduction in fungal burden, while antagonism was seen in some
sequential regimens.11 Although it is difficult to generalize our
results into clinical practice, our data suggest that a combination
regimen may have an advantage over monotherapy when treatment is started late. Further studies involving other drug combinations, multiple strains and species of Aspergillus, and additional
schemes of experimental treatment initiation are warranted to
corroborate these findings.
Funding
This project was supported by internal funding from Ricerca di Ateneo
to F. B.
Transparency declarations
None to declare.
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