Cyclodextrin/Adamantane-Mediated Targeting of Inoculated Bacteria in Mice.

Cyclodextrin (CD)-based host-guest interactions with adamantane (Ad) have demonstrated use for functionalizing living cells in vitro. The next step in this supramolecular functionalization approach is to explore the concept to deliver chemical cargo to living cells in vivo, e.g., inoculated bacteria, in order to study their dissemination. We validated this concept in two rodent Staphylococcus aureus models. Bacteria (1 × 108 viable S. aureus) were inoculated by (1) intramuscular injection or (2) intrasplenic injection followed by dissemination throughout the liver. The bacteria were prefunctionalized with 99mTc-UBI29-41-Ad2 (primary vector), which allowed us to both determine the bacterial load and create an in vivo target for the secondary host-vector (24 h post-inoculation). The secondary vector, i.e., chemical cargo delivery system, made use of a 111In-Cy50.5CD9PIBMA39 polymer that was administered intravenously. Bacteria-specific cargo delivery as a result of vector complexation was evaluated by dual-isotope SPECT imaging and biodistribution studies (111In), and by fluorescence (Cy5); these evaluations were performed 4 h post-injection of the secondary vector. Mice inoculated with nonfunctionalized S. aureus and mice without an infection served as controls. Dual-isotope SPECT imaging demonstrated that 111In-Cy50.5CD9PIBMA39 colocalized with 99mTc-UBI29-41-Ad2-labeled bacteria in both muscle and liver. In inoculated muscle, a 2-fold higher uptake level (3.2 ± 1.0%ID/g) was noted compared to inoculation with nonfunctionalized bacteria (1.9 ± 0.4%ID/g), and a 16-fold higher uptake level compared to noninfected muscle (0.2 ± 0.1%ID/g). The hepatic accumulation of the host-vector was nearly 10-fold higher (27.1 ± 11.1%ID/g) compared to the noninfected control (2.7 ± 0.3%ID/g; p < 0.05). Fluorescence imaging of the secondary vector corroborated SPECT-imaging and biodistribution findings. We have demonstrated that supramolecular host-guest complexation can be harnessed to achieve an in vivo cargo delivery strategy, using two different bacterial models in soft tissue and liver. This proof-of-principle study paves a path toward developing innovative drug delivery concepts via cell functionalization techniques.

Tailoring Fluorescent Dyes To Optimize a Hybrid RGD-Tracer.

Quantitative assessment of affinity and kinetics is a critical component in the development of (receptor-targeted) radiotracers. For fluorescent tracers, such an assessment is currently not yet applied, while (small) changes in chemical composition of the fluorescent component might have substantial influence on the overall properties of a fluorescent tracer. Hybrid imaging labels that contain both a radiolabel and a fluorescent dye can be used to evaluate both the affinity (fluorescent label) and the in vivo distribution (radiolabel) of a targeted tracer. We present a hybrid label oriented and matrix-based scoring approach that enabled quantitative assessment of the influence of (overall) charge and lipophilicity of the fluorescent label on the (in vivo) characteristics of αvß3-integrin targeted tracers. Systematic chemical alterations in the fluorescent dye were shown to result in a clear difference in the in vivo distribution of the different hybrid tracers. The applied evaluation technique resulted in an optimized targeted tracer for αvß3-integrin, which combined the highest T/M ratio with the lowest uptake in other organs. Obviously this selection concept would also be applicable during the development of other (receptor-targeted) imaging tracers.

Bis-pyridylethenyl benzene as novel backbone for amyloid-ß binding compounds.

Detection of cerebral ß-amyloid (Aß) by targeted contrast agents is of great interest for in vivo diagnosis of Alzheimer's disease (AD). Partly because of their planar structure several bis-styrylbenzenes have been previously reported as potential Aß imaging agents. However, these compounds are relatively hydrophobic, which likely limits their in vivo potential. Based on their structures, we hypothesized that less hydrophobic bis-pyridylethenylbenzenes may also label amyloid. We synthesized several bis-pyridylethenylbenzenes and tested whether these compounds indeed display improved solubility and lower LogP values, and studied their fluorescent properties and Aß binding characteristics. Bis-pyridylethenylbenzenes showed a clear affinity for Aß plaques on both human and murine AD brain sections. Competitive binding experiments suggested a different binding site than Chrysamine G, a well-known stain for amyloid. With a LogP value between 3 and 5, most bis-pyridylethenylbenzenes were able to enter the brain and label murine amyloid in vivo with the bis(4-pyridylethenyl)benzenes showing the most favorable characteristics. In conclusion, the presented results suggest that bis-pyridylethenylbenzene may serve as a novel backbone for amyloid imaging agents.

Development of a Hybrid Tracer for SPECT and Optical Imaging of Bacterial Infections.

In trauma and orthopedic surgery, infection of implants has a major impact on the outcome for patients. Infections may develop either during the initial implantation or during the lifetime of an implant. Both infections, as well as aseptic loosening of the implant, are reasons for revision of the implants. Therefore, discrimination between aseptic-mechanical-loosening and septic-bacterial-loosening of implants is critical during selection of a patient-tailored treatment policy. Specific detection and visualization of infections is a challenge because it is difficult to discriminate infections from inflammation. An imaging tracer that facilitates bacterial identification in a pre- and intraoperative setting may aid the workup for patients suspicious of bacterial infections. In this study we evaluated an antimicrobial peptide conjugated to a hybrid label, which contains both a radioisotope and a fluorescent dye. After synthesis of DTPA-Cy5-UBI29-41 and-when necessary-radiolabeling with (111)In (yield 96.3 ± 2.7%), in vitro binding to various bacterial strains was evaluated using radioactivity counting and confocal fluorescence microscopy. Intramuscular bacterial infections (S. aureus or K. pneumoniae) were also visualized in vivo using a combined nuclear and fluorescence imaging system. The indium-111 was chosen as label as it has a well-defined coordination chemistry, and in pilot studies labeling DTPA-Cy5-UBI29-41 with technetium-99m, we encountered damage to the Cy5 dye after the reduction with SnCl2. As a reference, we used the validated tracer (99m)Tc-UBI29-41. Fast renal excretion of (111)In-DTPA-Cy5-UBI29-41 was observed. Target to nontarget (T/NT) ratios were highest at 2 h post injection: radioactivity counting yielded T/NT ratios of 2.82 ± 0.32 for S. aureus and 2.37 ± 0.05 for K. pneumoniae. Comparable T/NT ratios with fluorescence imaging of 2.38 ± 0.09 for S. aureus and 3.55 ± 0.31 for K. pneumoniae were calculated. Ex vivo confocal microscopy of excised infected tissues showed specific binding of the tracer to bacteria. Using a combination of nuclear and fluorescence imaging techniques, the hybrid antimicrobial peptide conjugate DTPA-Cy5-UBI29-41 was shown to specifically accumulate in bacterial infections. This hybrid tracer may facilitate integration of noninvasive identification of infections and their extent as well as real-time fluorescence guidance during surgical resection of infected areas.

First-in-human evaluation of a hybrid modality that allows combined radio- and (near-infrared) fluorescence tracing during surgery.

PURPOSE: The clinical introduction of the hybrid tracer indocyanine green (ICG)-(99m)Tc-nanocolloid, composed of a radioactive and a near-infrared (NIR) fluorescence component, has created the need for surgical (imaging) modalities that allow for simultaneous detection of both signals. This study describes the first-in-human use of a prototype opto-nuclear probe during sentinel node (SN) biopsy using ICG-(99m)Tc-nanocolloid. METHODS: To allow for fluorescence tracing, a derivative of the conventional gamma probe technology was generated in which two optical fibers were integrated to allow for excitation (785 nm) and emission signal collection (> 810 nm). The ability of this opto-nuclear probe to detect the fluorescence signal of the hybrid tracer ICG-(99m)Tc-nanocolloid was firstly determined ex vivo in (non)SNs samples obtained from 41 patients who underwent hybrid tracer-based SN biopsy in the head and neck or urogenital area. In an in vivo proof-of-concept study in nine of these 41 patients, SNs were localized using combined gamma and fluorescence tracing with the opto-nuclear probe. Fluorescence tracing was performed in a similar manner as gamma tracing and under ambient light conditions. RESULTS: Ex vivo, the gamma tracing option of the opto-nuclear probe correctly identified the SN in all 150 evaluated (non)SN samples. Ex vivo fluorescence tracing in the low-sensitivity mode correctly identified 71.7% of the samples. This increased to 98.9% when fluorescence tracing was performed in the high-sensitivity mode. In vivo fluorescence tracing (high-sensitivity mode) accurately identified the SNs in all nine patients (20 SNs evaluated; 100%). CONCLUSION: This study demonstrates the first-in-human evaluation of a hybrid modality capable of detecting both gamma and fluorescence signals during a surgical procedure. Fluorescence tracing could be performed in ambient light.

Potential role of antimicrobial peptides in the early onset of Alzheimer's disease.

Cerebral aggregation of amyloid-ß (Aß) is thought to play a major role in the etiology of Alzheimer's disease. Environmental influences, including chronic bacterial or viral infections, are thought to alter the permeability of the blood-brain barrier (BBB) and thereby facilitate cerebral colonization by opportunistic pathogens. This may eventually trigger Aß overproduction and aggregation. Host biomolecules that target and combat these pathogens, for instance, antimicrobial peptides (AMPs) such as Aß itself, are an interesting option for the detection and diagnostic follow-up of such cerebral infections. As part of the innate immune system, AMPs are defensive peptides that efficiently penetrate infected cells and tissues beyond many endothelial barriers, most linings, including the BBB, and overall specifically target pathogens. Based on existing literature, we postulate a role for labeled AMPs as a marker to target pathogens that play a role in the aggregation of amyloid in the brain.

Polyfluorinated bis-styrylbenzenes as amyloid-ß plaque binding ligands.

Detection of cerebral ß-amyloid (Aß) by targeted contrast agents remains of great interest to aid the in vivo diagnosis of Alzheimer's disease (AD). Bis-styrylbenzenes have been previously reported as potential Aß imaging agents. To further explore their potency as (19)F MRI contrast agents we synthetized several novel fluorinated bis-styrylbenzenes and studied their fluorescent properties and amyloid-ß binding characteristics. The compounds showed a high affinity for Aß plaques on murine and human brain sections. Interestingly, competitive binding experiments demonstrated that they bound to a different binding site than chrysamine G. Despite their high logP values, many bis-styrylbenzenes were able to enter the brain and label murine amyloid in vivo. Unfortunately initial post-mortem (19)F NMR studies showed that these compounds as yet do not warrant further MRI studies due to the reduction of the (19)F signal in the environment of the brain.

Screening sensibility and antifungal activity after topical application of a synthetic lactoferrin-derived antimicrobial peptide.

OBJECTIVE: Onychomycosis is the most common disease of the nails and constitutes about half of all nail abnormalities. Onychomycosis is usually caused by dermatophytes and incomparably less frequently by yeast-like fungi and non-dermatophyte molds. Current treatment options for onychomycosis are ineffective. METHODS: This study evaluated the performance of a commercial and CE-registered product containing antimicrobial peptide hLF1-11 in vitro for treating toenail onychomycosis. In a case-control setting, nail samples from 59 volunteers were obtained before and after treatment by a pedicurist and investigated for the presence of fungi by culturing, barcode sequencing, and MALDI-TOF-MS. RESULTS: Of 89 samples, T. rubrum (19%) and C. parapsilosis (17%) were cultured. In total, 47 samples (53%) were positive for culture. MALDI-TOF-MS could identify 28, but 19 remained unidentified; those species were not included in the commercial MALDI-TOF reference database library. A positive effect of treatment by the hLF1-11 product on 41 volunteers (1 placebo, 18 low doses, 22 high doses) was observed. No adverse effects of the peptide were observed or reported by the pedicurist or any of the participants. CONCLUSIONS: This study showed a positive therapeutic effect of a commercial product containing hLF1-11 in the case of 88.9% of the patients with onychomycosis. The present formulation of hLF1-11 into PBS is stable enough to permit storage at room temperature for at least two years.

Insights into Peptidoglycan-Targeting Radiotracers for Imaging Bacterial Infections: Updates, Challenges, and Future Perspectives.

The unique structural architecture of the peptidoglycan allows for the stratification of bacteria as either Gram-negative or Gram-positive, which makes bacterial cells distinguishable from mammalian cells. This classification has received attention as a potential target for diagnostic and therapeutic purposes. Bacteria's ability to metabolically integrate peptidoglycan precursors during cell wall biosynthesis and recycling offers an opportunity to target and image pathogens in their biological state. This Review explores the peptidoglycan biosynthesis for bacteria-specific targeting for infection imaging. Current and potential radiolabeled peptidoglycan precursors for bacterial infection imaging, their development status, and their performance in vitro and/or in vivo are highlighted. We conclude by providing our thoughts on how to shape this area of research for future clinical translation.

Biodistribution of Radioactively Labeled Splice Modulating Antisense Oligonucleotides After Intracerebroventricular and Intrathecal Injection in Mice.

Antisense oligonucleotides (AONs) are promising therapeutic candidates, especially for neurological diseases. Intracerebroventricular (ICV) injection is the predominant route of administration in mouse studies, while in clinical trials, intrathecal (IT) administration is mostly used. There is little knowledge on the differences in distribution of these injection methods within the same species over time. In this study, we compared the distribution of splice-switching AONs targeting exon 15 of amyloid precursor protein pre-mRNA injected via the ICV and IT route in mice. The AON was labeled with radioactive indium-111 and mice were imaged using single-photon emission computed tomography (SPECT) 0, 4, 24, 48, 72, and 96 h after injection. In vivo SPECT imaging showed 111In-AON activity diffused throughout the central nervous system (CNS) in the first hours after injection. The 111In-AON activity in the CNS persisted over the course of 4 days, while signal in the kidneys rapidly decreased. Postmortem counting in different organs and tissues showed very similar distribution of 111In-AON activity throughout the body, while the signal in the different brain regions was higher with ICV injection. Overall, IT and ICV injection have very similar distribution patterns in the mouse, but ICV injection is much more effective in reaching the brain.

Optical imaging as an expansion of nuclear medicine: Cerenkov-based luminescence vs fluorescence-based luminescence.

Integration of optical imaging technologies can further strengthen the field of radioguided surgery. Rather than using two separate chemical entities to achieve this extension, hybrid imaging agents can be used that contain both radionuclear and optical properties. Two types of such hybrid imaging agents are available: (1) hybrid imaging agents generated by Cerenkov luminescence imaging (CLI) of ß-emitters and (2) hybrid imaging agents that contain both a radioactive moiety and a fluorescent dye. One major challenge clinicians are now facing is to determine the potential value of these approaches. With this tutorial review we intend to clarify the differences between the two approaches and highlight the clinical potential of hybrid imaging during image-guided surgery applications.

Microspheres as a Carrier System for Therapeutic Embolization Procedures: Achievements and Advances.

The targeted delivery of anti-cancer drugs and isotopes is one of the most pursued goals in anti-cancer therapy. One of the prime examples of such an application is the intra-arterial injection of microspheres containing cytostatic drugs or radioisotopes during hepatic embolization procedures. Therapy based on the application of microspheres revolves around vascular occlusion, complemented with local therapy in the form of trans-arterial chemoembolization (TACE) or radioembolization (TARE). The broadest implementation of these embolization strategies currently lies within the treatment of untreatable hepatocellular cancer (HCC) and metastatic colorectal cancer. This review aims to describe the state-of-the-art TACE and TARE technologies investigated in the clinical setting for HCC and addresses current trials and new developments. In addition, chemical properties and advancements in microsphere carrier systems are evaluated, and possible improvements in embolization therapy based on the modification of and functionalization with therapeutical loads are explored.

A radio- and fluorescently labelled tracer for imaging and quantification of bacterial infection on orthopaedic prostheses : a proof of principle study.

AIMS: Arthroplasty surgery of the knee and hip is performed in two to three million patients annually. Periprosthetic joint infections occur in 4% of these patients. Debridement, antibiotics, and implant retention (DAIR) surgery aimed at cleaning the infected prosthesis often fails, subsequently requiring invasive revision of the complete prosthetic reconstruction. Infection-specific imaging may help to guide DAIR. In this study, we evaluated a bacteria-specific hybrid tracer (99mTc-UBI29-41-Cy5) and its ability to visualize the bacterial load on femoral implants using clinical-grade image guidance methods. METHODS: 99mTc-UBI29-41-Cy5 specificity for Stapylococcus aureus was assessed in vitro using fluorescence confocal imaging. Topical administration was used to highlight the location of S. aureus cultured on femoral prostheses using fluorescence imaging and freehand single photon emission CT (fhSPECT) scans. Gamma counting and fhSPECT were used to quantify the bacterial load and monitor cleaning with chlorhexidine. Microbiological culturing helped to relate the imaging findings with the number of (remaining) bacteria. RESULTS: Bacteria could be effectively stained in vitro and on prostheses, irrespective of the presence of biofilm. Infected prostheses revealed bacterial presence on the transition zone between the head and neck, and in the screw hole. Qualitative 2D fluorescence images could be complemented with quantitative 3D fhSPECT scans. Despite thorough chlorhexidine treatments, 28% to 44% of the signal remained present in the locations of the infection that were identified using imaging, which included 500 to 2,000 viable bacteria. CONCLUSION: The hybrid tracer 99mTc-UBI29-41-Cy5 allowed effective bacterial staining. Qualitative real-time fluorescence guidance could be effectively combined with nuclear imaging that enables quantitative monitoring of the effectiveness of cleaning strategies.Cite this article: Bone Joint Res 2023;12(1):72-79.

The Development and Validation of Radiopharmaceuticals Targeting Bacterial Infection.

The International Atomic Energy Agency organized a technical meeting at its headquarters in Vienna, Austria, in 2022 that included 17 experts representing 12 countries, whose research spanned the development and use of radiolabeled agents for imaging infection. The meeting focused largely on bacterial pathogens. The group discussed and evaluated the advantages and disadvantages of several radiopharmaceuticals, as well as the science driving various imaging approaches. The main objective was to understand why few infection-targeted radiotracers are used in clinical practice despite the urgent need to better characterize bacterial infections. This article summarizes the resulting consensus, at least among the included scientists and countries, on the current status of radiopharmaceutical development for infection imaging. Also included are opinions and recommendations regarding current research standards in this area. This and future International Atomic Energy Agency-sponsored collaborations will advance the goal of providing the medical community with innovative, practical tools for the specific image-based diagnosis of infection.

Pretreatment with interferon-γ enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis.

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohn's disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon-γ (IFN-γ). In this article, we assessed the effects of IFN-γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN-γ and assessed their therapeutic effects in dextran sodium sulfate (DSS)- and trinitrobenzene sulfonate (TNBS)-induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS-induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS-induced colitis. IMSC-treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN-γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN-γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo.

Advancing intraoperative magnetic tracing using 3D freehand magnetic particle imaging.

PURPOSE: Sentinel lymph node biopsy is a routine procedure for nodal staging in penile cancer. Most commonly, this procedure is guided by radioactive tracers, providing various forms of preoperative and intraoperative guidance. This is further extended with fluorescence imaging using hybrid radioactive-fluorescence tracers. Alternatively, a magnetic-based approach has become available using superparamagnetic iron-oxide nanoparticles (SPIONs). This study investigates a novel freehand magnetic particle imaging and navigation modality (fhMPI) for intraoperative localization, along with a hybrid approach, combining magnetic and fluorescence guidance. MATERIALS AND METHODS: The fhMPI set-up was built with a surgical navigation device, optical tracking system and magnetometer probe. A dedicated reconstruction software based on a look-up-table method was used to reconstruct a superficial 3D volume of the SPION distribution in tissue. For fluorescence guidance, indocyanine green (ICG) was added to the SPIONs. The fhMPI modality was characterized in phantoms, ex vivo human skin and in vivo porcine surgery. RESULTS: Phantom and human skin explants illustrated that the current fhMPI modality had a sensitivity of 2.2 × 10-2 mg/mL SPIONs, a resolving power of at least 7 mm and a depth penetration up to 1.5 cm. Evaluation during porcine surgery showed that fhMPI allowed for an augmented reality image overlay of the tracer distribution in tissue, as well as 3D virtual navigation. Besides, using the hybrid approach, fluorescence imaging provided a visual confirmation of localized nodes. CONCLUSION: fhMPI is feasible in vivo, providing 3D imaging and navigation for magnetic nanoparticles in the operating room, expanding the guidance possibilities during magnetic sentinel lymph node procedures. Furthermore, the integration of ICG provides the ability to visually refine and confirm correct localization. Further clinical evaluation should verify these findings in human patients as well.

Feasibility of fluorescence imaging at microdosing using a hybrid PSMA tracer during robot-assisted radical prostatectomy in a large animal model.

BACKGROUND: With the rise of prostate-specific membrane antigen (PSMA) radioguided surgery, which is performed using a microdosing regime, demand for visual target confirmation via fluorescence guidance is growing. While proven very effective for radiotracers, microdosing approaches the detection limit for fluorescence imaging. Thus, utility will be highly dependent on the tracer performance, the sensitivity of the fluorescence camera used, and the degree of background signal. Using a porcine model the ability to perform robot-assisted radical prostatectomy under fluorescence guidance using the bimodal or rather hybrid PSMA tracer (99mTc-EuK-(SO3)Cy5-mas3) was studied, while employing the tracer in a microdosing regime. This was followed by ex vivo evaluation in surgical specimens obtained from prostate cancer patients. RESULTS: T50% blood and T50% urine were reached at 85 min and 390 min, in, respectively, blood and urine. Surgical fluorescence imaging allowed visualization of the prostate gland based on the basal PSMA-expression in porcine prostate. Together, in vivo visualization of the prostate and urinary excretion suggests at least an interval of > 7 h between tracer administration and surgery. Confocal microscopy of excised tissues confirmed tracer uptake in kidney and prostate, which was confirmed with PSMA IHC. No fluorescence was detected in other excised tissues. Tumor identification based on ex vivo fluorescence imaging of human prostate cancer specimens correlated with PSMA IHC. CONCLUSION: Intraoperative PSMA-mediated fluorescence imaging with a microdosing approach was shown to be feasible. Furthermore, EuK-(SO3)Cy5-mas3 allowed tumor identification in human prostate samples, underlining the translational potential of this novel tracer. Trial registration Approval for use of biological material for research purposes was provided by the Translational Research Board of the Netherlands Cancer Institute-Antoni van Leeuwenhoek hospital (NKI-AvL) under reference IRBm19-273 (22/10/2019).

In vivo biodistribution of stem cells using molecular nuclear medicine imaging.

Studies on stem cell are rapidly developing since these cells have great therapeutic potential for numerous diseases and has generated much promise as well as confusion due to contradictory results. Major questions in this research field have been raised as to how and in which numbers stem cells home to target tissues after administration, whether the cells engraft and differentiate, and what their long-term fate is. To answer these questions, reliable in vivo tracking techniques are essential. In vivo molecular imaging techniques using magnetic resonance imaging, bioluminescence, and scintigraphy have been applied for this purpose in experimental studies. The aim of this review is to discuss various radiolabeling techniques for early stem cell tracking, the need for validation of viability and performance of the cells after labeling, and the routes of administration in experimental animal models. In addition, we evaluate current problems and directions related to stem cell tracking using radiolabels, including a possible role for their clinical implementation.

Imaging Bacteria with Radiolabelled Probes: Is It Feasible?

Bacterial infections are the main cause of patient morbidity and mortality worldwide. Diagnosis can be difficult and delayed as well as the identification of the etiological pathogen, necessary for a tailored antibiotic therapy. Several non-invasive diagnostic procedures are available, all with pros and cons. Molecular nuclear medicine has highly contributed in this field by proposing several different radiopharmaceuticals (antimicrobial peptides, leukocytes, cytokines, antibiotics, sugars, etc.) but none proved to be highly specific for bacteria, although many agents in development look promising. Indeed, factors including the number and strain of bacteria, the infection site, and the host condition, may affect the specificity of the tested radiopharmaceuticals. At the Third European Congress on Infection/Inflammation Imaging, a round table discussion was dedicated to debate the pros and cons of different radiopharmaceuticals for imaging bacteria with the final goal to find a consensus on the most relevant research steps that should be fulfilled when testing a new probe, based on experience and cumulative published evidence.