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Rubitschung, K. Molecular Imaging of DFI. Encyclopedia. Available online: (accessed on 10 December 2023).
Rubitschung K. Molecular Imaging of DFI. Encyclopedia. Available at: Accessed December 10, 2023.
Rubitschung, Katie. "Molecular Imaging of DFI" Encyclopedia, (accessed December 10, 2023).
Rubitschung, K.(2021, December 28). Molecular Imaging of DFI. In Encyclopedia.
Rubitschung, Katie. "Molecular Imaging of DFI." Encyclopedia. Web. 28 December, 2021.
Molecular Imaging of DFI

Advances in imaging have the potential to improve the assessment of diabetic foot infection (DFI), particularly in distinguishing infection of soft tissue alone from osteomyelitis (OM). 

diabetic foot infection (DFI) osteomyelitis Molecular Imaging

1. Introduction

Radiotracers are chemical compounds containing a radioactive atom that may be injected alone or conjugated to a targeting moiety. The total concentration is so low that the underlying physiology is not disturbed by the radioimaging probe. After administration, molecular interactions with the radiotracer at sites of interest allow the metabolism of a tissue to be detected and quantified. Scintigraphy, single-photon emission computed tomography (SPECT), and PET imaging are molecular imaging modalities used for diabetic foot infection (DFI) assessment [1]. They possess higher sensitivity and specificity than anatomical modalities, translating to improved distinction between the infection of soft tissue and bone. Although molecular imaging modalities independently show promise in the evaluation of DFI, the combination of molecular imaging and anatomical studies may possess even greater sensitivity and specificity than either modality alone.

2. Bone Scintigraphy

Planar bone scintigraphy involves intravenous injection of a radiotracer—typically 99mTc-methylene diphosphonate or hydroxymethylene diphosphonate (99mTc-MDP/HDP). Other radiotracers such as 18F-sodium fluoride (18F-NaF) may be used for PET imaging of bone. The diphosphonates are retained in bone by binding to hydroxyapatite crystals. Areas of greater radiotracer incorporation by osteoblasts indicate areas of high bone turnover and are often the focal point of infection. Triple phase bone scintigraphy involves capturing images at three stages: immediately after radiotracer injection, 10 min after radiotracer injection, and 2–4 h after radiotracer injection. Each image is used to assess different aspects of DFI. The first image provides information regarding blood flow to the region of interest. The second image, also called the tissue or blood pool phase, assesses the soft tissues and increased retention is generally associated with inflammation or infection. In the third phase, the amount of radiotracer uptake indicates the rate of new bone formation by osteoblasts.
When assessing diabetic foot infection by three-phase bone scintigraphy, variation in radiotracer uptake is observed between phases. In cases of cellulitis and other soft tissue infections, increased radiotracer uptake is only seen during the first two phases, whereas, in OM, increased radiotracer uptake is seen in all three phases identifying focal hyperperfusion, hyperemia, and bone uptake. Moreover, because the third phase of bone scintigraphy reflects general bone turnover, bone deformities associated with degenerative joint disease, fracture, or orthopedic hardware can significantly reduce specificity [3][4]. A meta-analysis by Llewellyn et al. reported an average sensitivity of 84.2% with a relatively low specificity of 67.7% [5]. Although bone scintigraphy is highly sensitive, arterial occlusion or nonspecific inflammatory conditions such as Charcot joint may render the method susceptible to misdiagnosis in cases of DFI [6][5]. Impaired peripheral perfusion may lead to false negatives when assessing the diabetic foot. Sterile inflammation, fracture healing, or Charcot joint may cause increased radiotracer uptake during the third phase, which could be misidentified as a sign of increased bone turnover associated with OM, resulting in falsely positive interpretations. By utilizing a second scintigraphic modality, such as bone marrow imaging with a 99mTc-labeled colloidal preparation and a tomographic method such as SPECT or SPECT/CT, specificity and anatomic localization are improved. This approach provides improved localization of areas with radiotracer uptake that may have been previously obstructed [3].

3. Radiolabeled WBC and Bone Marrow Planar Scintigraphy, SPECT, and SPECT/CT

While planar bone scintigraphy uses radiotracers to directly detect areas of high bone turnover, leukocyte scans trace localization of radiolabeled white blood cells (WBC) that migrate to the site of infection. In this case, the WBCs act as carriers of the molecular imaging agent. The majority of the WBCs labeled by conventional methods are neutrophils, which are the first and most abundant leukocytes to arrive at the locus of infection [7]. As a result, WBC scans are better for acute infections and can generate highly specific images which can be further improved for spatial localization using SPECT or SPECT/CT.
WBC radiolabeling is performed by in vitro or in vivo methods. In vitro WBC labeling involves the isolation of WBCs from fractionated blood, direct labeling with a radiotracer such as 111In-oxine or 99mTc-HMPAO (exametazine), and reinjection into the subject. In vitro labeling can be limited by the concentration of recovered leukocytes, as optimal labeling requires at least 2000 leukocytes/μL [8]. 111In-oxine displays a normal distribution in the spleen, liver, and bone marrow and because it is highly stable, delayed imaging can be performed to allow labeled cells more time to localize to sites of infection (Figure 1). Importantly, there is no uptake in the uninfected foot. Most importantly, 111In-oxine-labeled cells can be combined with 99mTc radiolabeled agents to target activated bone marrow in dual tracer studies. Such studies are useful in distinguishing OM from Charcot neuropathic changes (Figure 2).
Figure 1. Biodistribution of 111In-oxine WBC in a non-infected individual. Anterior (a) and posterior (b) planar images of a healthy patient 24 h after 111In-oxine-labeled WBC injection. Uptake is seen in the spleen, liver, and bone marrow.
Figure 2. Dual tracer imaging using 111 n-WBC and 99mTc-sulfur colloid. Planar 111In-WBC (a,b) and 99mTc-sulfur colloid (c,d) images from a 62-year-old male with diabetes with a left foot abscess. There is spatial and intensity discordance in activity from the radionuclides. Anterior (a) and lateral (b) 111In-WBC images show focus of increased activity in the left mid foot. Anterior (c) and lateral (d) 99mTc-sulfur colloid images show diffuse activity throughout the mid and hind foot, suggesting the development of Charcot foot. Axial and sagittal 111In-WBC SPECT/CT (e) localized activity to an abscess in the plantar aspect of the left mid foot. Osteomyelitis was excluded.
Unfortunately, 111In-oxine WBC scans produce low-resolution images and require a long interval between patient injection and image acquisition (16–30 h); however, 99mTc-HMPAO (Figure 3) overcomes these problems by producing relatively high-quality images and requiring only a short interval between injection and image acquisition. Previous analyses have found that 99mTc-HMPAO WBC scans are highly sensitive and specific in the detection of OM (91% and 92%, respectively) [9]. At present dual-tracer scans are not performed with 99mTc WBC scans because no suitable marrow-targeting agents are commercially available for simultaneous imaging.
Figure 3. Planar scintigraphic and SPECT/CT 99mTc-WBC images in an individual with DFI. The patient’s presentation was suspicious for OM involving the great toe. Planar images (a,b) demonstrate increased radiolabeled WBCs in the right forefoot, perhaps in the region of the toes. SPECT/CT (ce) allows for precise localization of infection to the proximal phalanx of the right great toe.
WBC scans have also been used to assess the efficacy of antibiotic treatment in patients with DFO. Vouillarmet et al. [10] followed 45 patients with newly diagnosed DFO for either remission or treatment failure at one year. All patients were initially treated with appropriate antibiotics for six weeks, after which WBC-SPECT/CT was performed after infusion of 99mTc-HMPAO-labeled leukocytes. In the 23 with negative scans, antibiotics were discontinued; whereas, in the 22 with positive scans, antibiotic therapy was continued for six more weeks after which a second scan was obtained. At the one-year clinical follow-up, 22 of the 23 with negative scans at 6 weeks remained in remission. The remaining 22 were re-scanned at 12 weeks after completion of the second course of antibiotics. At their one-year follow-up, all 9 with a negative re-scan remained in remission; whereas, of the 13 with positive scans at 12 weeks, 7 were in remission at 1 year, and 6 had failed treatment. Overall, in this two-stage testing and treating study, the sensitivity and specificity of the 99mTc-HMPAO-labeled WBC-SPECT/CT were 82% and 86%, respectively, while the predictive value of a negative scan was 97% and the predictive value of a positive scan was 46%. These results suggest that a 99mTc-HMPAO-labeled leukocyte scan will accurately predict remission near the end of antibiotic treatment. Moreover, based on reports using 111In-labeled WBCs, WBC scans appear well suited for monitoring the response of DFO patients to antibiotic therapy over time because the abnormal scan findings revert to normal within two–eight weeks after the start of successful antibiotic therapy [11].
The dual radiopharmaceutical/radiotracer method is a unique strength of planar and SPECT molecular imaging. In the typical application in DFI, one radiotracer labels WBCs while the other targets bone marrow. This approach is particularly powerful in the evaluation of suspected infection superimposed on confounding pathology such as Charcot neuropathic changes. Sixteen to thirty hours after the initial scan, delayed scans of the bone marrow are often performed using a longer-lived tracer if OM is suspected; however, bone deformities such as fracture or abnormal marrow distribution from atypical hematopoietic activity may lead to false positives [11]. To overcome this, visualization of the bone marrow using a second radiotracer (dual-tracer approach) may be adopted. Depending on the tracers used, some dual-tracer approaches allow for simultaneous visualization, while others such as 99mTc-HMPAO require clearance of the first tracer. 99mTc-labeled sulfur colloid is commonly used for this application because the molecule is phagocytized and sequestered by endothelial cells lining the bone marrow, reticular cells, macrophages, and macrophage precursors [12]. A positive diagnosis is indicated by greater or spatially discordant radiotracer uptake on the leukocyte scan compared with the bone marrow scan. Not only does this combined technique improve sensitivity and specificity, but it is also convenient and can be performed within one scan because of the gamma camera’s ability to distinguish the different energies of 99mTc-sulfur colloid and radionuclides such as 111In.
Although the dual-tracer approach is highly effective, it has limitations; principally, localizing the anatomic compartment of both tracers [3]. To improve this technique, WBC-labeled SPECT may be used in conjunction with CT to better visualize anatomically complex areas of the foot. This combined modality approach has been associated with improved distinction between soft tissue infection and OM as well as fewer false positives, resulting in fewer amputations as well as shorter hospital stays compared to anatomical imaging techniques alone [13]. Still, SPECT/CT in combination with labeled-WBC or labeled-WBC and sulfur colloid scan can be expensive, and in vitro leukocyte labelling is limited by the concentration of leukocytes obtained. Similar to planar imaging, radiotracer uptake—and therefore image quality—depends on the number and type of cells labeled as well as the host response to infection.

4. 18F-FDG PET and PET/CT

18F-Fluorodeoxyglucose (18F-FDG) is a radiolabeled glucose analogue which accumulates in cells that utilize glucose as an energy source. Facilitative glucose transporter proteins bring 18F-FDG across the cell membrane to the cytoplasm where it is subsequently phosphorylated by hexokinase. 18F-FDG is then trapped in the cell until it is dephosphorylated by glucose-6-phosphatase. The time between retention and clearance reflects the relative activity of glucose transporters, hexokinase, and glucose-6-phosphatase. In comparison to background activity, areas of decreased or absent uptake reflect low or absent glucose metabolism. Regions of increased 18F-FDG uptake reflect greater than normal rates of glucose metabolism [14]. Cells with many glucose transporters and a high metabolic rate, such as active inflammatory cells, display high FDG uptake. In addition, the 18F-FDG molecule is small and can easily penetrate poorly perfused areas, making it useful for imaging patients with vascular insufficiency [4]. When a dual modality approach of 18F-FDG-PET/CT is adopted, simultaneous CT and PET images are acquired, and a hybrid three-dimensional image is generated that aids in the visualization of infection foci, similar to SPECT/CT imaging. PET-CT is a dual modality that uses the strength of CT and the power of PET tracers to quantify infection and inflammatory processes and assess many other features of DFI [15]. From its high image resolution and radiotracer sensitivity and specificity, 18F-FDG PET/CT is useful in evaluating patients with characteristics that might otherwise compromise the accuracy of MRI or CT, including prior surgery, trauma, or orthopedic hardware [16][17][18]. One concern may be that 18F-FDG PET imaging could be compromised in patients with increased blood glucose and concomitant diabetic foot infection. A study by Yang et al. found otherwise. In a clinical evaluation of 21 patients with healthy serum glucose levels (<150 mg/dL) and 27 patients with elevated glucose levels (150–250 mg/dL), 18F-FDG PET demonstrated comparable sensitivity for detecting pedal OM between subjects with healthy glucose levels (sensitivity = 87.5%) and subjects with high serum glucose (sensitivity = 88.9%). Impressive levels of specificity of 96.8%, sensitivity of 88.3%, and accuracy of 93.8% were reported for both groups combined [19]. However, DFI patients in a clinical setting often have poorly regulated insulin levels and may have much higher glucose levels. In a retrospective study by Kagna et al., investigators addressed a potential issue that 18F-FDG activity may be altered in patients who received antibiotic therapy for a previous infection. The study assessed 18F-FDG PET in 176 patients who had received between 1–73 days of antibiotic therapy for an infectious process. They reported no false negative cases, correct identification of infectious foci in 61% of the study population, and true negatives in 29% of the study population; however, the study did not specifically target DFI [20].
Although 18F-FDG imaging boasts an impressive sensitivity and specificity, it is prone to false positives. Patients with a recent surgical history exhibit increased 18F-FDG uptake in the healing tissue because of the increased cellular metabolic rate. Since FDG localizes to sites of increased metabolic activity rather than directly labelling bacteria, 18F-FDG results are skewed by the human immune system and cannot differentiate among infection, neoplasia, and sterile inflammation that is seen in Charcot [1].

5. Gallium Scan

Two isotopes of gallium are in clinical use, namely 67Ga and 68Ga. Gallium, as an iron mimetic, may be recruited into several processes that localize the isotope to sites of infection. First, gallium creates a complex with the ferric ion delivering glycoprotein transferrin. During uptake of 67/68Ga, much of the tracer is bound to circulating plasma transferrin. The increased blood flow and vascular permeability to the site of infection then facilitate the delivery and accumulation of 67/68Ga to infectious foci. Any 67/68Ga which is not bound to transferrin can also be bound to lactoferrin and circulating leukocytes, be directly taken up by bacteria, or form a complex with bacterial-produced siderophores, which are then transported into the bacterium and phagocytized by macrophages [4][21][22].
Historically, 67Ga scans were widely used in institutions with limited access to ex vivo WBC labeling or MRI [3]; however, as the availability of these methods has improved over time, 67Ga scans are now mainly used for the imaging of spinal infections. Studies have found other imaging modalities such as Sulesomab-labeled WBC scans (sensitivity = 67%, specificity = 85%) superior to 67Ga bone scintigraphy (sensitivity = 44%, specificity = 77%) [23]. 68Ga is more commonplace now compared to 67Ga. One study assessing the diagnostic potential of 68Ga-citrate PET/CT for osteomyelitis reported a sensitivity of 100%, a specificity of 76%, PPV of 85%, NPV 100%, and overall accuracy of 90%; however, this was not categorically evaluated in a DFI model [24]. Another study compared the kinetics of 18F-FDG, 68Ga-citrate, 11C-methionine, and 11C-donepezil in a porcine OM model. They found that 68Ga-citrate uptake was slow, irreversible, and limited by diffusion whereas the 18F-FDG uptake rate is determined by perfusion [25].
Because 68Ga possesses a shorter half-life and is a positron emitter, it yields higher quality images than those acquired with 67Ga. In addition, 68Ga may be produced by a generator or cyclotron, while 67Ga production requires the use of a cyclotron. As a result, 68Ga has displaced 67Ga as the gallium isotope of choice in some molecular imaging applications, such as neuroendocrine tumor imaging [26]. Although 68Ga retention in infectious sites is, in part, a bacteria-specific localization mechanism (siderophore binding), 18F-FDG PET/CT may still have superior sensitivity and specificity due to favorable emission features of 18F and glucose utilization by WBCs. However, a recent single institution study found that, for identifying infectious foci in patients with Staphylococcus aureus bacteremia, 68Ga citrate was comparable to 18F-FDG in the detection of osteomyelitis, whereas 18F-FDG resulted in a higher signal for detection of soft tissue infection [27].


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