Introduction
Invasive fungal diseases are among the leading causes of morbidity and mortality in haematopoietic stem cell and solid organ transplant recipients, as well as in patients with solid tumours and haematological malignancies [
1‐
3]. In recent years, an increase (from 19 % to 25 %) in the incidence of infections caused by opportunistic mould pathogens including
Aspergillus,
Candida, zygomycete,
Fusarium,
Scedosporium and
Acremonium species has been observed [
1,
3,
4], with invasive aspergillosis (IA) being the predominant infection [
5,
6]. The mortality rate associated with IA, which is mainly caused by
Aspergillus fumigatus and primarily affects the lungs, remains unacceptably high (30–95 %) [
2,
3,
7]. Singh et al. have reported that an estimated 9.3–16.9 % of all deaths in transplant recipients in the first year can be attributable to IA [
8,
9].
Early and accurate diagnosis of IA is critical for a favourable outcome, but is difficult to achieve with currently available methods [
10,
11]. Current methods for the diagnosis of IA include prognostic factors, clinical signs, radiology and laboratory tests (e.g. galactomannan antigen, PCR, microscopy and culture) [
10,
11]. However, most of these techniques lack sufficient specificity and/or sensitivity for early detection of IA. Identification of patients at high risk, appropriate prophylaxis, diagnostic surveillance, and early diagnosis remain important for improved patient management [
11], and underline the need for specific and sensitive imaging methods for IA.
Iron is an essential nutrient and is also a key factor in the virulence of pathogenic microorganisms [
12,
13]. In response to low iron availability, iron-dependent microorganisms have evolved different strategies to obtain iron. These strategies include the biosynthesis of low molecular mass iron chelators, termed siderophores, with extremely high affinity for ferric ions, which are employed for iron delivery by almost all bacteria and fungi (including
A. fumigatus) as well as in some plants [
14]. The major siderophore produced by
A. fumigatus for iron acquisition is triacetylfusarinine C (TAFC). The importance of TAFC for
A. fumigatus during virulence is reflected by the transcriptional upregulation of its biosynthesis and uptake during infection as well as the attenuation of virulence by inactivation of TAFC biosynthesis in murine IA models [
15,
16].
Aspergillus recovers iron from iron-siderophore complexes via specific uptake mechanisms involving highly efficient siderophore transporters [
17]. Remarkably, numerous fungi including
Aspergillus species possess specific uptake systems not only for native siderophores, but also for siderophores synthesized exclusively by other fungi [
18].
68Ga is a positron emitter that has recently become the subject of great interest for molecular imaging applications using PET [
19]. It is readily available from a
68Ge/
68Ga generator and has a suitably short half-life of 68 min. In addition, Ga
3+ has comparable complex chemistry to Fe
3+, and binds with high affinity to siderophores [
20].
In a proof of principle study, we recently showed that a
68Ga-labelled TAFC can detect
A. fumigatus infection in a rat animal model using PET imaging [
20]. In a subsequent study, we characterized the in vitro and in vivo behaviour of selected siderophores [
21], showing that besides
68Ga-TAFC,
68Ga-ferrioxamine E (
68Ga-FOXE) also shows high uptake by
A. fumigatus in culture and remains stable in vivo. In this study we compared these most promising candidates including investigations in a rat
A. fumigatus infection model and μPET imaging.
Materials and methods
Chemicals
All commercially available reagents were of analytical grade and used without further purification. Siderophores were obtained from Genaxxon Bioscience (Ulm, Germany). 68Ga was eluted from a 68Ge/68Ga generator (IGG; Eckert & Ziegler, Berlin, Germany).
Radiolabelling and in vitro studies
TAFC and FOXE were labelled with
68Ga using acetate buffer at room temperature for 15 min (TAFC) and at 80 °C for 20 min (FOXE). For all in vitro and in vivo studies, the pH of the final product was adjusted with 1.1 M sodium acetate to pH 6–7. The radiochemical purity, log
P, protein binding and stability of
68Ga-siderophores in various media were determined, as described previously [
20].
Preparation of A. fumigatus cultures for in vitro uptake studies
The
Aspergillus strain used for in vitro studies was
A. fumigatus wild-type ATCC46645 (American Type Culture Collection) cultured at 37 °C in
Aspergillus minimal medium, containing 1 % glucose as the carbon source, 20 mM glutamine as the nitrogen source, salts and trace elements, as described previously [
22]. Iron-sufficient media contained 30 mM FeSO
4. For preparation of iron-deficient media, iron addition was omitted. Iron-deficient conditions were verified by detection of extracellular siderophore production, which is suppressed by iron.
In vitro uptake of 68Ga-siderophores by A. fumigatus
Uptake by A. fumigatus in iron-deficient and iron-sufficient cultures was studied. For the monitoring of uptake over time, 68Ga-siderophores (5 ng) were incubated in microbial media for 10, 20, 30, 45, 60 and 90 min at room temperature in 96-well plates (Millipore, Billerica, MA). For the monitoring of uptake blocking, excess of ferri-siderophore (Fe-TAFC or Fe-FOXE) and/or sodium azide was used. 68Ga-siderophores were incubated in iron-deficient and iron-sufficient media for 45 min at room temperature in 96-well plates. The incubation was interrupted in both cases by filtration of the medium and rapid rinsing with ice-cold TRIS buffer. The filters were collected and counted in a γ-counter.
Preparation of A. fumigatus inoculum for rat infection model
The A. fumigatus (A29) isolate was grown on Sabouraud dextrose agar (BD) for 5 days at 37 °C, and the conidia were harvested in 2 ml of sterile NaCl by gently rubbing with a pipette tip. The conidia suspension was transferred into a sterile 50 ml plastic tube. After homogenization (vortex) and filtration (40 μm nylon cell strainer; BD), the suspension was counted in a Neubauer chamber and adjusted to the volitional concentration in the range 1 × 105 to 1 × 109 conidia per millilitre.
Animal experiments
All animal experiments were conducted in accordance with regulations and guidelines of the Austrian and Dutch animal Protection laws and with the approval of the Austrian Ministry of Science (66011/42-II/10b/2009), and the institutional Animal Welfare Committee of the Radboud University Medical Centre Nijmegen (revised Dutch Act on Animal Experimentation, 1997). Animal studies were performed using Balb/c mice and Lewis rats (both Charles River Laboratories, Wilmington, MA).
Biodistribution in normal mice
Normal noninfected Balb/c mice (female, 6 weeks old) were injected with 68Ga-siderophore (2 MBq and 0.1–0.2 μg of siderophore per mouse) into the tail vein. Animals were killed by cervical dislocation 30 min and 90 min after injection. The organs and tissues (blood, spleen, pancreas, stomach, intestines, kidneys, liver, heart, lungs, muscle and femur) were removed and radioactivity was counted in a γ-counter. The results are expressed as percentage of injected dose per gram of tissue.
Urine, blood, liver and kidneys of normal Balb/c mice injected with
68Ga-siderophores and treated as described previously were collected 30 min after injection. The urine sample was directly injected onto the RP-HPLC column. Blood samples were precipitated with acetonitrile and centrifuged for 2 min, and the supernatant was injected onto the RP-HPLC column. Liver and kidneys were washed in the ice-cold TRIS buffer and liquidized using a mixer in a falcon tube containing 1 ml of TRIS buffer. The liver and kidney homogenates obtained were mixed with acetonitrile and centrifuged for 2 min, and the supernatant was injected onto the RP-HPLC column. In all cases 1 min fractions of the column eluate were collected and measured in a γ-counter. Samples were not collected at 90 min after injection because the measured activity was already low in the samples obtained 30 min after injection. All metabolic studies were performed using a previously described HPLC method [
20].
68Ga-siderophore imaging in the rat infection model
PET images were acquired with an Inveon animal PET/CT scanner (Siemens Preclinical Solutions, Knoxville, TN) with an intrinsic spatial resolution of 1.5 mm [
23]. The animals were placed in a prone position. Static PET images were acquired over 30 min starting 30 min after intravenous injection of
68Ga-siderophore. Dynamic PET imaging was started upon injection and continued up to 60 min after injection. In addition, combined PET/CT scans were performed for anatomical reference. PET emission scans were acquired for 30 min, preceded by CT scans (spatial resolution 113 μm, 80 kV, 500 μA, exposure time 300 ms). After imaging, animals were killed by CO
2/O
2. Scans were reconstructed using Inveon Acquisition Workplace software (version 1.5; Siemens Preclinical Solutions, Knoxville, TN) using a 3-D ordered subset expectation maximization/maximum a posteriori (OSEM3D/MAP) algorithm with the following parameters: matrix 256 × 256 × 159, pixel size 0.43 × 0.43 × 0.8 mm
3 and a MAP prior β-value of 1.5.
In vitro cultures of excised organs
The excised organs were homogenized in a petri dish using a sterile surgical blade and transferred to Sabouraud dextrose agar (BD) plates. The plates were incubated at 37 °C and examined daily for 7 days. Colony-forming counts were recorded from all plates that showed growth. Severe infection was defined as the presence of severe fungal growth 1 day after incubation, mild infection was defined as the presence of minor growth up to 3 days after incubation, and no infection was defined as lack of growth within 1 week of incubation.
Statistical analysis
Student’s t-test (level of significance, P < 0.05) was used to determine the significance of differences in the ex vivo and in vivo data. Analysis was performed using Microsoft Office Excel 2007.
Discussion
The need for novel approaches to the imaging of IA is reflected by the number of radiopharmaceuticals that have been described and proposed for this application [
24,
25]. Radiopharmaceuticals for which clinical applications have been proposed include
67Ga-citrate [
26], but it has known limitations in terms of pharmacokinetics, sensitivity and specificity, as it is a general marker for imaging malignancies and inflammatory processes. Even though
18F-FDG has also recently been proposed as an imaging agent for IA [
27], it has comparable limitations, in particular related to the low specificity for imaging glucose metabolism. Various attempts have been made to develop more specific radiopharmaceuticals for this application, including
99mTc-labelled polyethyleneglycol liposomes [
28],
99mTc-interleukin-8 [
29],
99mTc-fluconazole [
30] and
99mTc-antimicrobial peptides (e.g. ubiquicidin) [
30,
31]. None of these agents has proven to show specific uptake mechanisms in
Aspergillus species and none has entered clinical trials. Recently a hypha-binding peptide (c(CGGRLGPFC)-NH
2) labelled with
111In has been described [
32] potentially having higher specificity, but further evaluation towards clinical application has not been reported.
The use of a
68Ga-labelled siderophore that is actively taken up via specific iron transporters by the microorganism acquiring iron during the course of infection holds the potential of a unique and specific way to image IA. In a proof of principle study [
20], we have shown that siderophores can be labelled with
68Ga with high affinity and stability in biological systems. In vitro energy-dependent uptake of
68Ga-siderophores in
A. fumigatus was observed and preliminary in vivo studies were performed, proving the potential of
68Ga-labelled siderophores for infection imaging. After the promising study with
68Ga-TAFC in the rat infection model, we focused on the selection, characterization and optimization of the most promising candidates for diagnostic applications as a basis for clinical implementation of PET (PET/CT) in imaging of fungal infections [
21].
In this study we compared two
68Ga-labelled siderophores as the most promising candidates from previous studies [
20,
21] and evaluated their potential as radiopharmaceuticals for IA imaging. Both
68Ga-FOXE and
68Ga-TAFC showed hydrophilic properties, low protein binding and high in vitro stability. In vitro studies showed rapid and high uptake by
A. fumigatus in iron-deficient media, which could be blocked with excess of ferri-siderophore or sodium azide. Both compounds showed excellent pharmacokinetic properties with high metabolic stability. Nevertheless,
68Ga-FOXE showed significantly lower metabolic stability in the liver than
68Ga-TAFC, which could explain the higher accumulation of radioactivity in the liver and intestinal tissue observed in biodistribution studies of Balb/c mice. This was confirmed in μPET imaging of rats. Both
68Ga-siderophores showed highly selective accumulation in infected lung which was shown to be correlated with severity of disease in the rat infection model using μPET or μPET/CT. Even though
68Ga-FOXE had higher uptake values in biodistribution studies indicating a potentially higher sensitivity, PET/CT imaging did not show significant differences either in uptake or in target/non-target ratios.
Today CT is the standard imaging technique for the detection of pulmonary infections [
33,
34], and previous studies have shown that the halo sign is indicative of pulmonary aspergillosis in neutropenic patients [
35]. However, CT scans may not allow differentiation between
Aspergillus and other pathogenic fungi [
36] and CT has limited specificity and predictive value, especially in non-neutropenic stem-cell transplant recipients [
36,
37]. The combination with PET could provide additional functional information on the lesion detected, thereby increasing sensitivity and specificity of patient imaging within one diagnostic procedure. The effectiveness of combined PET and CT imaging is illustrated in Fig.
6. The PET images of both
68Ga-siderophores in rats infected with
A. fumigatus show abnormal uptake of radiotracer in the thoracic area. The fused PET/CT image permits precise localization of the lung tissue affected by
A. fumigatus infection. We found matching uptake in pathological areas on CT images with the accumulation of our investigated
68Ga-siderophores in the infected lung areas. The combination of PET and
68Ga-labelled siderophores (TAFC or FOXE) and CT therefore holds potential for early detection of invasive fungal infections.
In the clinical setting, one of the risk factors for IA in immunocompromised patients is a high iron load [
38], which is frequently occurs due to blood transfusions or iron supplementation during the course of their underlying disease. As
68Ga-siderophores mimic iron-transporting mechanisms in microorganisms, we wanted to see whether an iron preload would have an effect on the uptake of
68Ga-labelled siderophores in the IA rat model. In the small series of animals tested, no significant decrease in uptake of either
68Ga-TAFC or
68Ga-FOXE in fungal infection could be observed indicating that iron supply does not influence uptake of the tracers. Another important factor in judging the suitability of this imaging approach is the selectivity of
68Ga-siderophores for fungal infections. We are currently investigating this issue in an ongoing study to determine the uptake of these
68Ga-siderophores by a variety of microorganisms, which will help in choosing the optimal candidate for noninvasive detection of fungal infections by PET in a clinical setting.
Conclusion
Our study showed that both 68Ga-labelled TAFC and FOXE are very promising agents for detection of IA with high sensitivity. The high metabolic stability, favourable pharmacokinetics with rapid renal excretion and high specific uptake in A. fumigatus cultures were confirmed in imaging studies in a rat IA model that showed high focal uptake in infected lung tissue corresponding to pathological findings seen on CT. 68Ga-TAFC showed advantages in terms of radiolabelling and a somewhat higher metabolic stability, and 68Ga-FOXE showed a trend towards higher uptake in infected tissue. Only currently ongoing selectivity studies will enable selection of the optimal candidate for potential clinical applications.