Radiotracers in Infection Imaging: Comparison
Please note this is a comparison between Version 2 by Katie Lynn Rubitschung and Version 3 by Jason Zhu.

Several developments in the molecular imaging of infections target microorganism-specific metabolism and activity. By targeting substances that are presented by or released from the pathogenic microorganism or microorganism-specific metabolic pathways, more radiotracers can be developed whose localization mechanism is independent of the host immune response.

  • Radiolabeled antibiotics
  • Infection Imaging
  • diabetic foot infections

1. Introduction

Radiolabeled antibiotics are relatively well-studied in the context of imaging diabetic foot infection (DFI). Previously studied radiolabeled antibiotics target crucial features required for bacterial pathogenicity and survival, including the synthesis of folic acid required for nucleic acid synthesis, cell wall construction, cell membrane structure and function, transcription, and translation [1][131]. Antibiotic-resistant bacteria pose a major problem for radiolabeled antibiotics. Depending on the uptake mechanism, some bacteria may demonstrate radiotracer accumulation, while others may not. 99mTc-ciprofloxacin is a well-studied antibiotic imaging agent and, although previous studies originally demonstrated its high specificity, subsequent studies have not been able to reproduce the result [2][3][4][5][132,133,134,135]. In vivo studies of its ability to differentiate gram-positive from gram-negative bacteria have reached contradictory conclusions. The main drawback to 99mTc-ciprofloxacin is its accumulation in dead bacteria [2][132], which leads to false positives, as suggested by a large multicenter clinical trial that reported a sensitivity of 66.7%, a specificity of 85.7%, and an accuracy of 72% for both soft tissue infection and OM [6][136]. Several radiolabeled antibiotics have been studied to date (Table 1); however, the least well-studied are bacterial cell wall synthesis inhibitors such as vancomycin. The SPECT tracer 99 Tc-vancomycin, which was directly labeled, has demonstrated an affinity for S. aureus infectious foci. 99mTc-vancomycin labeled through 99mTc-Tc-HYNIC tetrazine click chemistry corroborated this observation with a three-fold uptake increase in a S. aureus infection site compared to controls [7][137]. Studies using fluorescently labeled vancomycin have also reported promising results [8][138], although isotopic-labeled vancomycin has shown greater uptake [9][139]. Although the 99mTc-vancomycin single-photon emission computed tomography (SPECT) tracer may have a valuable place in the clinical management of DFI, the development of a vancomycin PET tracer may provide better image resolution.
Table 1. Emerging Investigational Radiotracers of Infection Imaging.
  Radiotracer Clinical Trials Parameter Mechanism of Localization Strength/Weakness References
Radiolabeled Antibiotics 99mTc-ciprofloxacin Yes Inhibition of DNA Synthesis Bacterial DNA gyrase High sensitivity (85.4–97.2%), ciprofloxacin already used in DFI treatment [3][4][5][6][133,134,135,136]
Low specificity (66.7–81.7%), antibiotic resistant bacteria
18F-fluoropropyl-trimethoprim No Inhibition of Folic Acid Synthesis Inhibition of thymidine biosynthesis Low background, high uptake in bacteria, detect inflammation from soft tissue infection vs sterile inflammation [10][167]
Antibiotic resistant bacteria
99mTc-sulfonamides (pertechnetate, sulfadiazine) No Inhibition of Folic Acid Synthesis Broad spectrum antibiotics, uptake in bacterial and fungal infections [11][155]
Antibiotic resistant bacteria
99mTc-vancomycin No Inhibition of bacterial cell wall synthesis Binds to D-ala-D-ala lipid moiety Specific for gram positive organisms [7][8][9][137,138,139]
Not specific for gram negative organisms

Antibiotic resistant bacteria
Radiolabeled Sugars 18F-FDS Yes Bacteria-Specific Glucose Sources for Carbohydrate Metabolism Bacterial Metabolic Substrate Antibiotic treatment monitoring, used in humans [12][13][140,141]
Uptake by Enterobacteriaceae in the human gut
18F-FAG No Sorbitol analogue utilized only by bacteria Selective accumulation in E. coli, rapid accumulation, can differentiate infection from sterile inflammation, shows promise for monitoring response to treatment, small molecule [14][142]
Not applied clinically
18F-maltohexose No Bacterial-specific maltodextrin transporter Can discriminate between live bacteria, metabolically inactive bacteria, and sterile inflammation [15][143]
Poor signal-to-noise ratios, Not applied clinically
6′′-18F-fluoromaltotriose No Bacterial-specific maltodextrin transporter 2nd Gen, improved signal-to-noise ratio, bacterial-selective uptake in vitro and in vivo [16][17][144,145]
Not applied clinically
Amino Acid Uptake D-[methyl-11C] methionine No Bacterial Cell Wall Synthesis Incorporation into the peptidoglycan Distinguish sterile inflammation from infection in both gram—and gram +, broad sensitivity [18][146[19],147]
Not applied clinically
D-5-[11C] glutamine No Incorporation into the peptidoglycan Highly specific, high sensitivity for gram +, no uptake in sterile inflammation, fast clearance [20][148]
Corroborating studies needed, not yet applied clinically
Vitamin Uptake 124I-fialuridine (FIAU) Yes Endogenous TK enzyme of pathogenic bacteria Trapped in the cell after phosphorylation Reduced uptake in the presence of metal artifacts, [21][22][23][154,168,169]
More clinical studies needed to assess clinical efficacy
111In-biotin No Production of Fatty Acid Bacterial growth factor Essential growth factor for S. aureus [24][149]
Corroborating in vivo studies needed to assess clinical relevance
99mTc-PAMA No Vitamin B12 Metabolism Vitamin B12 derivative that accumulates in rapidly proliferating cells High uptake in Gram + and Gram - [25][150]
Not applied clinically
18F or 3H-PABA No Folic Acid Synthesis Inhibition of Thymidine Synthesis Accumulation in MRSA and other resistant organisms [26][151]
In vivo studies needed
Polyclonal Antibodies 64Cu-NODAGA No Membrane protein binding of polyclonal antibody Microbe-specific membrane polyclonal antibody binding Particular to a specific microbe [27][28][164,165]
Slow accumulation time
Siderophores 68Ga-FOXE No Iron Transport Accumulation of Siderophores in the cell High uptake in S. aureus and fungi [29][30][31][160,161,162]
Not used in DFI model
Immunoscintigraphy 99mTc-sulesomab Yes WBC migration to infectious foci Binds to antigen-90 on WBC membranes Ease of preparation? Not sure about this [32][33][63,71]
Dependent upon host response, expensive, limited availability
99mTc-Besilesomab Binds to antigen-95 on granulocytes and their precursors Ease of preparation, good sensitivity and specificity [32][63]
Dependent upon host response, expensive, limited availability
Perhaps the most promising approach is to target the metabolism of the microorganism itself. One approach targets bacteria-specific transport systems for sugars. For example, sorbitol is used by gram-negative bacteria and has been developed as a tracer and been successfully studied in humans. One study, using 18F-fluorodeoxysorbitol (FDS) in humans, reported selective uptake in infected lesions, low background uptake, and fast plasma clearance [12][140]. However, uptake by Enterobacteriaceae of the gut was also seen. A more recent study of 18F-FDS demonstrated the sorbitol-specific pathway by observing uptake in clinically relevant strains of Enterobacterales. Following the in vitro study and a previous in vivo study in murine models, they acquired 18F-FDS PET/CT images in humans, demonstrating the safety and ability of 18F-FDS to differentiate infection from sterile inflammation [13][141]. In addition, it was used to monitor antibiotic treatment [12][140]. Bacterial cell wall amino sugars, present in gram positive as well as gram negative bacteria, have also been targeted for imaging. One study in rats used 2-deoxy-2-[18F]fluoroacetamido-D-glucopyranose (18F-FAG) to successfully identify E. coli infection from sterile inflammation by imaging with confirmation by histology [14][142]. Perhaps the most effective of the previously discussed radiolabeled sugars are maltodextrin-based imaging probes. The maltodextrin transport pathway is bacteria-specific and can discriminate between live bacteria, metabolically inactive bacteria, and inflammation induced by lipopolysaccharides. PET imaging performed with 18F-maltohexose demonstrated uptake in both gram positive and gram-negative bacteria [15][143], although poor signal-to-noise ratios limit this tracer’s clinical application. The second-generation tracer 6′′-18F-fluoromaltotriose has improved signal-to-noise ratios while maintaining bacterial-selective uptake in vitro and in vivo [144,145].
Amino acid uptake by bacteria has also been targeted for radiotracer development. D-stereoisomers of amino acids are found in bacterial cell walls but are not present in humans, making these amino acid isoforms promising pathogen-specific imaging targets. One analog of D-methionine, D-[methyl-11C]methionine, has been used to differentiate sterile inflammation from E. coli and S. aureus infections in a murine model, reporting an SUVmax 8–10 times higher (SUVmax = 0.8% ID/cc) in E.coli and S. aureus compared to [11C]L-methionine (SUVmax = 0.1% ID/cc) [18][146]. An in vitro study demonstrated the broad sensitivity of D-[methyl 11C] methionine across a panel of clinically relevant DFI pathogens, including E. coli, P. aeruginosa, P. mirabilis, S. aureus, S. epidermidis, and E. faecalis, with the greatest uptake occurring in S. aureus and P. aeruginosa [19][147]. A recent study compared the uptake of D-5-[11C]glutamine and L-5-[11C]glutamine isomers in a murine dual pathogen myositis model with localized MRSA and E.coli infections. Uptake of L-5-[11C]glutamine was higher in the heat-killed control site when compared to D-5-[11C]glutamine, indicating that the D-stereoisomer had better pathogen specificity. When D-5-[11C]glutamine was compared to 18F-FDG in a sterile inflammation model, 18F-FDG uptake was significantly higher, further demonstrating the specificity of D-5-[11C]glutamine for bacterial metabolism [20][148]. Although 11C-labeled amino acid-based imaging shows specificity, widespread adoption for clinical use may be limited because of the 20-min half-life of 11C.
Microorganisms may also be targeted based on their vitamin uptake. Since biotin is used in the production of fatty acids and is an essential growth factor for S. aureus and other bacteria, it has been explored for infection imaging. Although little data are currently available, an in vitro study using [111In]-In-DTPA-biotin reported selective uptake in S. aureus cultures [24][149]. In addition to targeting biotin, a radiolabeled vitamin B12 derivative, 99mTc-PAMA has also been used for infection imaging. Since PAMA accumulates in rapidly proliferating cells, the imaging agent has shown high uptake in S. aureus as well as E. coli in vitro and in vivo [25][150]. Microorganisms commonly use a compound called PABA to synthesize folic acid. A study using 3H-PABA reported accumulation in MRSA as well as other therapy resistant microorganisms. Uptake was 100× greater in the microorganisms than in mammalian cells. When labeled with 18F, uptake was seen in S. aureus [26][151].
Other targets for bacterial imaging include nucleoside analogs, peptidoglycan targeting aptamers, siderophores, and radiolabeled antibodies. The nucleoside analog 124I-fialuridine (124I-FIAU) is a bacterial tyrosine kinase substrate trapped in the cell following phosphorylation. Although it has been previously used to detect prosthetic joint infections [34][152], a more recent study found that tracer uptake was reduced in the presence of metal artifacts [35][153]. A clinical study of 16 patients with suspected musculoskeletal infection, who underwent 18F-FDG and 124I-FIAU PET/CT, reported a range of specificity of 51.7–72.7% and a maximum sensitivity of 100%; however, the minimum sensitivity was incalculable. The authors were also unable to come to a definitive conclusion on its diagnostic efficacy because of equivocal clinical findings in a large number of patients [21][154]. Aptamers are oligonucleotides that have been used to target the peptidoglycan cell wall. One group that used 99mTc-Antibac1 demonstrated its ability to distinguish between bacterial and fungal infections in vivo and in vitro [11][155].
Siderophores are small molecules with a high affinity for Fe(III) and are produced primarily by bacteria but are also produced by virtually all microorganisms—including prokaryotes and eukaryotes [36][156]. Mammalian macrophages have been reported as sources of siderophores [37][157]. Organisms use these metal-seeking molecules for extra- or intra-cellular metal transport and storage. Biological processes such as DNA replication, transcription, oxidative stress responses, and respiration all require transition metals. Iron and zinc are among the most abundant in living organisms. Iron is an essential cofactor for respiration and central metabolism. Zinc is the catalytic center for many enzymes and is required for 5–6% of all protein functions [38][158]. Siderophores are often categorized into the following groups according to the chemical moiety that binds iron: catecholates, hydroxamates, and carboxylates. Mixed type siderophores have two or more of these moieties. Siderophores may actively alter their affinity for iron in order to bind other metals by increasing their expression of metal transporters [38][158]. Desferricoprogen—a natural siderophore produced by Penicillium chrysogenum and Neurospora crass—reportedly bound to both trivalent (Al(III) and In(III)) or divalent (Cu(II), Ni(II), Zn(II), and Fe(II) metal ions [39][159]. Transport systems and siderophore production are unregulated during infection and can accumulate in bacteria as well as fungi. An in vitro study which used 68Ga-ferrioxamine (68Ga-FOXE) showed high uptake in S. aureus; however, it was not corroborated in vivo [29][160]. In fungi, Aspergillus fumigatus-specific fluorescent siderophore conjugates were used to assess pulmonary aspergillosis in vitro [30][161]; however, recent in vivo studies have only been performed to assess biodistribution in pulmonary aspergillosis [31][162]. Further research regarding specificity, sensitivity, and use in additional infection models is needed.

2. Immunoscintigraphy

In addition to in vitro labeling, WBCs may also be localized in vivo by using radiolabeled antibodies which target WBC antigens—a technique called immunoscintigraphy. For example, 99mTc-besilesomab uses the monoclonal antibody besilesomab to bind to the granulocyte-specific antigen-95. Antigen-95 is present on the membranes of granulocytes and their precursors. Within 45 min of initial injection, 10% of the 99mTc-besilesomab is bound to neutrophils, while 20% is unbound but localizes to the site of infection by nonspecific mechanisms [40][163]. A recent study of 119 patients with suspected OM compared planar 99mTc-HMPAO WBC and 99mTc-besilesomab imaging in the diagnosis of peripheral OM. They found that 99mTc-HMPAO had a lower sensitivity than 99mTc-besilesomab (59.0% and 74.8%, respectively); however, 99mTc-HMPAO had a higher specificity than 99mTc-besilesomab (79.5% and 71.8%, respectively) [32][63]. 99mTc-sulesomab also binds to an antigen found on leukocyte membranes—antigen-90—but only 3–6% of 99mTc-sulesomab is bound to circulating neutrophils, and 35% is found in bone marrow 24 h post-injection [41][111]. The results of a study of combat-related infections suggest that 99mTc-sulesomab may not possess sufficient accuracy (78% accuracy) for the identification of the foci of infections but may have otherwise comparable diagnostic values (93% PPV, 62% NPV, 72% sensitivity, 88% specificity) [33][71]. One study of Yersinia enterocolitica infections used immunoPET with 64Cu-NODAGA-labeled Yersinia-specific polyclonal antibodies to target the membrane protein YadA and reported colocalization in a dose-dependent manner with bacterial lesions in a murine model [27][164]. Another study that used the [64Cu]NODAGA-IgG3 monoclonal antibody from mice to examine C. albicans infections demonstrated its accuracy in diagnosing infection in vitro as well as in vivo [28][165]. A meta-analysis found a sensitivity of 81% and specificity of 86% for anti-granulocyte scintigraphy with monoclonal antibodies [42][166]. The use of intact antibodies takes up to several days to accumulate near infectious foci. Clinically, this could be a limitation because of the need for rapid identification of the infection site. However, the use of antibody fragments may lead to faster accumulation time. Overall, it appears that radiolabeled antibodies do not have the ability to replace existing methods in a clinical setting because of the relatively low diagnostic values reported by the studies discussed above.
One recent therapeutic application of US used in DFI is cavitation, in which ultrasonic waves are used to create microbubbles which form from the dissolved gas that accumulates in the wound. The molecular purpose of this treatment is to induce compression and movement of the wound cells and small ions to increase protein synthesis and the permeability of vascular walls and cell membranes [3][133]. Ultimately, reduced inflammation, angiogenesis, and increased cell proliferation and recruitment are expected at the site of infection [4][134]. Currently, only three studies have used this technique and two of them demonstrated either complete wound closure or a significant reduction in wound area [5][6][7][135,136,137].

3. Conclusion

Although these emerging imaging probes are targeting the microorganism rather than the host response, the quantitative measures of probe retention are generally much lower than the clinically available tracers that target the host response. Further, while most of these have not been used in DFI specifically, this is an opportunity for the expansion of molecular imaging approaches to DFI.