Development of a Toxin-selective immunotracer for the specific in vivo detection of Clostridioides difficile infection by immunoPET

Development of a Toxin-selective immunotracer for the specific in vivo detection of Clostridioides difficile infection by immunoPET | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of a Toxin-selective immunotracer for the specific in vivo detection of Clostridioides difficile infection by immunoPET Mario González-Arjona, Lorena Cussó, Luis Alcalá, María Isabel González, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6294683/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jun, 2025 Read the published version in EJNMMI Radiopharmacy and Chemistry → Version 1 posted 5 You are reading this latest preprint version Abstract Background: Clostridioides difficile infection (CDI) is a major healthcare challenge, associated with high morbidity and mortality. Current diagnostic methods have limitations in specificity and invasiveness, necessitating the development of novel, non-invasive imaging techniques. This study aims to develop and evaluate an immunoPET radiotracer targeting C. difficile toxin B for in vivo CDI detection in mice model. Results: Monoclonal antibody, Bezlotoxumab, was radiolabeled with [ 125 I]I for in vitro characterization and [ 89 Zr]Zr for in vivo PET imaging, resulting in high radiochemical yields (75.36 ± 4.11 % for [ 125 I]I and 71.58 ± 8.19 % for [ 89 Zr]Zr) and purities (>99.99 % both cases), with stable binding properties. PET/CT imaging 48h post infection in an animal model of CDI (C57BL/6 mice employing ribotype 027 strain) demonstrated specific accumulation of [ 89 Zr]Zr-DFO-Beztxab in the colon and cecum of infected mice, distinguishing CDI from dysbiosis and healthy controls, and confirmed by PET quantification and ex vivo biodistribution. Conclusions: We successfully developed an immunoPET radiotracer targeting toxin B for CDI detection. Its application in a CDI animal model proved its capacity to detect the source of infection with high specificity, avoiding sterile inflammation. Clostridioides difficile ImmunoPET Bacterial toxin targeting Imaging of infection Radiotracer development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Background Clostridioides difficile ( C. difficile ) remains a persistent and formidable challenge in healthcare facilities worldwide, significantly burdening patient health outcomes, healthcare resources and infection control ( 1 ). It is the leading cause of hospital-acquired infections (HAIs) in US hospitals ( 2 ) and has a high prevalence in European hospitals, accounting for 7.3% of all HAI cases ( 3 ). C. difficile infection (CDI) is associated with significant morbidity and mortality, exacerbated by its propensity for recurrent infections and complications such as pseudomembranous colitis and toxic megacolon ( 1 ). Despite advances in infection prevention and control strategies, the incidence and severity of CDI continue to rise, underscoring the urgent need for innovative diagnostic and therapeutic approaches to effectively manage this escalating public health problem. The most commonly used diagnostic strategy for CDI follows an algorithm that begins with initial screening using an enzyme immunoassay (EIA) to detect glutamate dehydrogenase (GDH), an enzyme highly sensitive for identifying CDI ( 4 ). Since both toxigenic and non-toxigenic strains of C. difficile produce GDH, a positive GDH EIA result requires confirmatory testing. This confirmation typically involves real-time PCR targeting toxin B gene, with or without an intermediate EIA for the detection of A and B toxins. The intermediate EIA aims to reduce the overall cost of molecular testing. These diagnostic algorithm has been extensively evaluated in multiple studies, demonstrating a sensitivity of 85–90% and a specificity exceeding 99% compared to toxigenic culture, the gold standard for CDI diagnosis ( 5 – 8 ). Although this approach is widely adopted in clinical practice, it is not without limitations. These challenges are particularly evident in cases where toxin concentrations are low, as patients may not have CDI but instead be asymptomatic carriers of toxigenic C. difficile . Colonoscopy, which would allow direct observation of the damage caused by the microorganism in the colon, is not recommended for severe patients because it significantly increases the risk of perforation. These tests are highly invasive and require patient sedation, which always carries certain risks and side effects ( 9 , 10 ). These challenges highlight the inadequacies of current diagnostic modalities and underscore the need for the development of more accurate and reliable diagnostic tools. Molecular imaging techniques have emerged as promising additions to conventional diagnostic approaches, offering the potential for non-invasive visualization and characterization of infectious diseases. Radiological techniques, including radiography, ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), are frequently used, especially to identify cases of toxic megacolon. However, these purely structural imaging tools rely on anatomic or morphologic changes that often occur after molecular events in the disease process, thereby precluding early detection of the infection. Furthermore, they are nonspecific and may reflect a combination of infection and host inflammatory response ( 10 – 13 ). Nuclear imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) have been investigated for their utility in diagnosing CDI by targeting metabolic processes associated with bacterial infection. Among these, PET imaging with [ 18 F]F-fluorodeoxyglucose ([ 18 F]F-FDG) has attracted considerable interest due to its ability to detect areas of increased glucose metabolism characteristic of infectious foci ( 14 ). Our group has pioneered its application to detect and evaluate CDI in a mouse model ( 15 ). In our study, [ 18 F]F-FDG uptake in the abdominal area was evaluated in two mouse models infected with two C. difficile ribotypes of different virulence. However, [ 18 F]F-FDG can actively incorporate into leukocytes, macrophages, and CD4-positive T cells present at infection sites ( 16 ), making it uncapable to differentiate between infectious, inflammatory, and tumor lesions, or even organs with high basal metabolism. This can lead to confusion when foci are close to organs such as the heart or brain, or to false positives ( 17 , 18 ). These limitations restrict its potential use, highlighting the need for the development of more specific imaging probes. To address these challenges and improve the accuracy of CDI diagnosis, our research efforts aim to develop a novel molecular imaging approach that combines the specificity of targeted molecular therapy with the sensitivity of radiotracer imaging. Our proposed strategy involves radiolabeling a commercially available monoclonal antibody, Bezlotoxumab, with the radionuclide [ 89 Zr]Zr to create a customized radiotracer, tailored for the detection of C. difficile toxins. Bezlotoxumab targets and neutralizes C. difficile toxin B, and thus offers an opportunity for targeted molecular imaging due to its high affinity and selectivity, ( 19 ). By using this radiolabeled antibody in an animal model we aim to explore the possibility of selectively targeting active C. difficile infection within the host, enabling precise localization and visualization of CDI sites. 2. Materials and Methods Unless otherwise specified, all reagents were purchased from Merck (KGaA, Darmstadt, Germany) and used without further purification. 2.1 Radiolabeling of commercial Bezlotoxumab and physico-chemical characterization Commercial Bezlotoxumab (Merck & Co., Inc., Rahway, NJ, USA) was radiolabeled with two different radionuclides; [ 125 I]I for in vitro binding and kinetic studies, and [ 89 Zr]Zr for physico-chemical characterization, in vivo and ex vivo studies. Both radioisotopes were purchased from Revvity, Inc. (Waltham, MA, USA). For both immunoconjugates, radiochemical yield was estimated as the fraction of purified radiotracer activity in comparison to the starting amount of activity (%). Specific activity was calculated as the final radioactivity of radiotracer per milligram of antibody (MBq/mg). 2.1.1 Synthesis and physico-chemical characterization of [ 125 I]I-Beztxab Radioiodination of Bezlotoxumab was performed following traditional direct labeling method ( 20 ). Briefly, 20 µg of Bezlotoxumab (8 µL) were mixed with 95 µL PBS 1x and 2 µL [ 125 I]I (2.00-2.52 MBq). The reaction was started by adding 5 µL of a 1 mg/mL solution of chloramine-T trihydrate and incubated at room temperature for 90 seconds. Then, 10 µL of a 1 mg/mL solution of sodium metabisulfite was added to stop the reaction, and the radiolabeled antibody was isolated using Zeba™ Spin Desalting Columns with a 7K MWCO (Thermo Fisher Scientific, Waltham, MA, USA). The purity of the radiotracer was evaluated by instant thin layer chromatography (iTLC) using silica gel (SG) chromatographic paper (Agilent Technologies, Inc., Santa Clara, CA, USA) as the stationary phase and acetone 70% as the mobile phase. Loss of protein was determined by indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) using an Anti-Human IgG (Fc specific) antibody, labeled and unlabeled Beztxab and calculated by 4PL nonlinear regression model. 2.1.2 Synthesis and physico-chemical characterization of [ 89 Zr]Zr-DFO-Beztxab For the labeling of the mAb with the radiometal [ 89 Zr]Zr, Bezlotoxumab was first conjugated to the isothiocyanatobenzyl-derivative of the chelator desferrioxamine (p-NCS-Bz-DFO, Chematech, Dijon, France ) adapting the labeling approach from previous works ( 21 , 22 ). Briefly, 1 mg of Bezlotoxumab (40 µL) was adjusted to 1 ml with 0.1 M sodium bicarbonate buffer (pH = 9.0). Parallelly, 1 mg of p-NCS-Bz-DFO was diluted in 200 µL of dimethyl sulfoxide and 20 µL were added to the Ab solution in four additions of 5 µL, with gently mixing in-between. Mixture was incubated at 37 ºC, 500 rpm for 30 min in Eppendorf® ThermoMixer® C (Eppendorf, Hamburg, Germany). Conjugated Ab was purified by PD-10 desalting column (GE Healthcare Bio-Science AB, Chicago, IL, USA) and collected in 2 mL of HEPES buffer 0.5 M. Ratio of p-DFO-Bz-NCS to Bezlotoxumab was measured by MALDI-TOF MS/MS (Unidad de Espectrometría de Masas, Universidad Complutense de Madrid, Spain) following previous publications ( 23 ). Briefly, a 1 µL aliquot of unconjugated antibody, p-DFO-Bz-NCS and Bz-DFO-Beztxab samples were combined with an equal volume of sinapic acid, used as the matrix solution (10 mg/mL in 50% acetonitrile: water and 0.1% trifluoroacetic acid). Samples were then deposited onto a stainless-steel target plate and left to dry. Following the determination of the mass (m/z) of both the unaltered antibody and the immunoconjugate, the difference was divided by the chelator's molecular weight, and the ratio p-DFO-Bz-NCS:Bezlotoxumab expressed as number of chelates per antibody unit. For radiolabeling, oxalic acid 1 M was added to 74–111 MBq of [ 89 Zr]Zr-oxalic acid solution to a final volume of 2 mL. Then, 90 µL of sodium carbonate 2 M were added, and mixture was incubated at room temperature (RT), 500 rpm for 3 min in ThermoMixer. Next, 300 µL of HEPES 0.5 M, 710 µL of Bz-DFO-Beztxab and 700 µL of HEPES 0.5 M were added to the mixture and kept on reaction for 60 min at RT and 500 rpm in ThermoMixer. Lastly, radiolabeled Ab was purified using 100 kDa Amicon filters (centrifuged at 4 ºC/21884 rcf/10min, recovered at 4 ªC/5471 rcf/5 min). Purity was evaluated by iTLC using WhatmannTM strips (3 MM CHR; GE Healthcare Bio-Science AB, Chicago, IL, USA) as the stationary phase, and citric acid monohydrate/sodium carbonate (pH 4.9–5.1) as the mobile phase. Radioactivity of TLC plates was read using a miniGita Single system (Elisa-Raytest, Angleur, Belgium). Radiolabeled-antibody mass was determined by Bradford-Coomassie assay, according to the manufacturer instructions and employing a VICTOR Nivo Multimode Microplate Reader (Revvity, Inc., Waltham, MA, USA). 2.2 In vitro characterization 2.2.1 In vitro stability In vitro stability of the radiotracers was evaluated in PBS 1x and mouse serum for [ 125 I]I-Beztxab and in PBS 1x for [ 89 Zr]Zr-DFO-Beztxab. Briefly, aliquots of [ 89 Zr]Zr-DFO-Beztxab (3.70 MBq) and [ 125 I]I-Beztxab (0.74 MBq) were added to 0.5-1 mL of PBS 1x and mouse serum, previously tempered at 37ºC with constant shaking. Samples were then collected at different time-points and analyzed by radio-TLC as described in the synthesis section. [ 89 Zr]Zr-DFO-Beztxab PBS 1x time-points: 0h, 0.5h, 1h, 2h, 4h, 20h, 26.5h, 45h, 51h, 68h, 74h, 95h and 100h. [ 125 I]I-Beztxab PBS 1x and mouse serum time points: 0h, 0.25h, 0.5h, 1h, 2h, 4h, 7h, 24h, 27h, 30h, 48h, 51h, 54h, 72h, 75h, 78h, 96h, 100h, 168h, 192h. 2.2.2 Hydrophobicity assessment of [ 89 Zr]Zr-DFO-Beztxab The hydrophobicity of the radiotracer was assessed using the partitioning method based on the LogP calculation. Briefly, 0.37 MBq of [ 89 Zr]Zr-DFO-Beztxab were added to an immiscible biphasic solution consisting of 500 µL of 1-octanol and 500 µL of PBS 1x (n = 3). This mixture was then incubated at 37ºC for 30 minutes with vigorous shaking. The mixture was then allowed to stand for a further 30 minutes to ensure proper phase separation. Finally, 100 µL samples were taken from each phase and their activity was measured using a Genesys gamma counter (Laboratory Technologies Inc., Elburn, IL, USA). 2.2.3 Binding kinetics for [ 125 I]I-Beztxab Binding kinetics was measured by ELISA and LigandTracer ( 24 ). 1. ELISA: A 96-well half area clear flat bottom polystyrene high bind microplate (Corning Inc, Corning, NY, USA) was coated with 50 µL of 0.5 µg/mL C. diff Toxin B and left at 4 ºC over night (ON). Wells were emptied, filled with 150 µL of ELISA Blocking Buffer (1% BSA, 0.15% Kathon ProClin ™ 150 in PBS 1x, pH 7.4) and shaken at 900 rpm for 1 hour. Then, wells were emptied and washed four times with ELISA Washing Buffer (NaH 2 PO 4 x H 2 O 0.32 mM, Na 2 HPO 4 x 2H 2 O 2.17 mM, NaCl 150 mM, 7.5 x 10 − 3 % Kathon ProClin ™ 150, 0.1% TWEEN ® 20 in MQ water, pH 7.5). Next, 50 µL of unlabeled Bezlotoxumab (n = 5) and [ 125 I]I-Beztxab (n = 3) were added to the plate in serial dilutions from 10 nM to 0.64 pM in ELISA Incubation Buffer (0.1% BSA, 0.15% Kathon ProClin ™ 150, 0.05% TWEEN ® 20 in PBS 1x, pH 7.4), and left at 4 ºC ON. Then, wells were emptied and washed as described above, and incubated with HRP-conjugated polyclonal goat anti-human-IgG-F(ab′) 2 antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), diluted 1:2000, at 900 rpm for 1 hour. Wells were emptied and washed, filled with 50 µL K Blue Aqueous TMB substrate (Neogen Corp., Lexington, KY, USA) and incubated for 5 min. Reaction was stopped by adding 50 µLof H 2 SO 4 1M, and signal absorbance was read with a spectrophotometer at 450 nm. Equilibrium dissociation constant (K D ) was calculated using a One site-Specific binding non-linear regression in Prism 8.3.0 (GraphPad Software, La Jolla, CA, USA). For calculations, concentration of [ 125 I]I-Beztxab was corrected according to the % of loss protein calculated as described above. 2. LigandTracer: In a high bind circular Petri dish (Corning Inc, Corning, NY, USA), 300 µL of a 10 µg/mL solution of C. diff Toxin B were added on a local spot at the edge of the dish. The dish was left tilted at 4 ºC ON. Then, solution was removed, and the surface of the dish was blocked with ELISA Blocking buffer for 1 hour. Blocking buffer was removed, 2 mL of running buffer (0.1% BSA in PBS 1x) were added, and the dish was placed in the Ligand Tracer Grey instrument (Ridgeview Instruments AB, Uppsala, Sweden). Baseline was recorded for 15 min, running buffer removed, and then 2 mL of a 0.14–0.73 nM [ 125 I]I-Beztxab (n = 3) solution were added. Radioactivity at the coated spot and at a non-coated spot (association) was recorded for 3 hours. Then, concentration of [ 125 I]I-Beztxab was increased to 0.47–2.19 nM, and radioactivity further recorder for 3 hours. Lastly, [ 125 I]I-Beztxab solution was removed, dish washed with running buffer, and dissociation recorded in 2 mL of running buffer for 72 hours. Association (k a ), dissociation (k d ) and K D rate constants were calculated with Trace Drawer 1.8.1 software (Ridgeview Instruments AB). 2.3 Toxin A and B quantification assay of Clostridioides difficile strains Toxin quantification of various C. difficile strains was conducted to identify the highest toxin-producing strain for use in our CDI animal model. The study was performed by C. difficile Toxin A/B FIA (SD Biosensor), a fluorescent immunoassay capable of measuring the fluorescence emitted by toxins A and B labeled with a fluorescent antibody. Three strains were included in this assay: ATCC 43255 (ribotype 087), strain 14243227 (ribotype 027), and strain 13061479 (ribotype 001). These strains were previously employed by our team in a study on C. difficile infection using a mouse model ( 15 ). For each strain, two consecutive re-isolations were conducted on Brucella agar at 35°C in anaerobic conditions for a period of 48 hours. A 0.5 McFarland's suspension of the C. difficile strain was prepared from the cultures and 200 µl of the suspension was inoculated into another BHI broth (previously reduced in an anaerobic environment for four days). This was then incubated at 35°C in anaerobiosis for four days. Following incubation, the broth was vortexed and subsequently centrifuged at 1200 rpm for three minutes. The supernatant was then transferred to an immunoassay extraction buffer, vortexed, and 3 µl of the mixture were added to the immunoassay device. After 15 minutes of incubation, the device was read by the F2400 fluorescence reader. This procedure was repeated twice for each of the strains. 2.4 Animal model of CDI CDI was induced in C57BL/6 female mice (n = 16) with a protocol of 10 days of preconditioning antibiotic in drinking water with cefoperazone 0.5 mg/ml followed by a single dose of 10 mg/kg clindamycin intraperitoneally 1 day before orogastric administration of 10 6 colony-forming unit (CFUs) of C. difficile ribotype 027 ( 15 ). As a control, a different group of animals (n = 14) underwent the same antibiotic treatment without infection, to develop an inflammation due to the disruption of the gut microbiota, known as dysbiosis. In addition, wild-type (WT) animals (n = 14) were used as healthy controls. Weight and clinical status of the animals were monitored every 2–3 days following the protocol established by Shelby et al. ( 25 ), starting from the initiation of antibiotic treatment. After infection or administration of the imaging agent, daily monitoring was conducted. We defined clinical CDI if the animals reached a Clinical Sickness Score (CSS) equal to or greater than 6. 2.5 Ex vivo biodistribution of [ 89 Zr]Zr-DFO-Beztxab In order to determine the optimal uptake time for in vivo PET/CT imaging we conducted ex-vivo biodistribution studies at 48 h and 5 days post-infection. Animals were intravenously administered with [ 89 Zr]Zr-DFO-Beztxab (1.11–3.7 MBq in 200 µL of PBS 1x) 24 hours after infection. The animals were then euthanized at their corresponding uptake times, and organs of interest were collected. The experimental groups and sample sizes were as follows: 48 hours post-infection: CDI (n = 4), Dysbiosis (n = 4), WT (n = 4). 5 days post-infection: CDI (n = 3), Dysbiosis (n = 5), WT (n = 5). The harvested organs included blood, heart, lungs, liver, spleen, kidneys, stomach, colon + cecum, skin, bone, intestine, and feces. The activity was measured in a Wallac Wizard 1480-011 Automatic Gamma Counter (Revvity, Inc., Waltham, MA, USA) and biodistribution was expressed as mean % Injected Dose per gram of tissue (%ID/g). 2.6 In vivo PET/CT imaging of [ 89 Zr]Zr-DFO-Beztxab PET/CT studies were conducted using a small-animal PET/CT scanner (PET/CT SuperArgus, SEDECAL Molecular Imaging, Madrid, Spain). 24 h after the infection time-point, the animals (CDI n = 9, Dysbiosis n = 5, WT n = 5) were intravenously administered with [ 89 Zr]Zr-DFO-Beztxab (3.70 MBq in 200 µL of PBS 1x). Image acquisition was performed 24 h after radiotracer administration and 48 h post-infection. Before CT acquisition, 0.3 mL of Iopamiro (Bracco, Milan, Italy) was administered intraperitoneally. During acquisition, animals were anesthetized with 1.5% sevofluorane in oxygen (SevoFlo, Zoetis Belgium SA, Louvain-la-Neuve, Belgium). PET data were collected for 30 min and reconstructed using FORE/2D-OSEM with 16 subsets and 1 iteration (voxel size: 0.388 x 0.388 x 0.775 mm). The CT study was acquired using an X-ray beam current of 340 µA and a tube voltage of 40 kVp, and reconstructed using an FDK algorithm ( 26 , 27 ). PET/CT images were analyzed with Multimodality Workstation software ( 26 ). On each CT image, a region of interest (ROI) was selected in the peritoneal cavity (Fig. 1 ) using the kidneys as reference, and delimited by 1) an axial plane just below the most caudal kidney pole; 2) a coronal plane ventral to the kidneys, always avoiding the bladder. These ROIs were automatically applied to co-registered PET images to measure ROI mean standard uptake values (SUVmean). 2.7 Validation of CDI animal model Model validation was conducted by measuring colon length, culturing C. difficile from feces, and performing Hematoxylin and Eosin (H&E) histology, after in vivo imaging. The length of the colon was determined by extracting the organ and measuring the total length from cecum to rectum with a ruler. Photographs of the colon were taken above the ruler for subsequent analysis. The concentration of toxigenic C. difficile in stool samples was determined as follows: stool samples were weighed and homogenized in vials containing 1 mL of saline solution by using the gentle MACS Dissociator (Miltenyi Biotec) to ensure a uniform mixture. For molecular analysis, 100 µL of the homogenate from each vial were analyzed using the Xpert™ C. difficile assay (GeneXpert, Cepheid, Sunnyvale, California, USA), which detects genes encoding toxin B, binary toxin, and the deletion at position 117 of the tcdC gene. For culture analysis, serial dilutions of 100µL of the homogenate were prepared to achieve 1:1000 and 1:1,000,000 dilutions. A volume of 100 µL from the undiluted homogenate, as well as from the 10 − 3 and 10 − 6 dilutions, was plated in triplicate on brucella agar plates. All plates were incubated anaerobically at 37°C for 48 hours. Following incubation, toxigenic C. difficile (TCD) colonies were counted, and expressed as colony-forming units (CFU) per microgram of stool sample. For histological assessment by H&E, the bowel specimens were fixed for 24 hours in 10% formalin and subsequently dehydrated in 70, 96 and 100% alcohol and xylene for paraffin-embedment. Paraffin blocks were cut in 4 micrometers slides and were stained after rehydration with hematoxylin-eosin. All samples were analyzed by a single pathologist blinded to the kind of intervention performed in each animal. 2.8 Data processing and statistical analysis We used Prism 8.3.0 (GraphPad Software, La Jolla, CA, USA) for data processing and plotting. Values are presented as mean ± standard deviation. For statistical analysis, since some data did not meet the criteria of normality and homoscedasticity, we used the Kruskal-Wallis test followed by the post-hoc Man-Whitney test for all the variables evaluated. In all cases, the significance threshold was set at p < 0.05. 2.9 Ethics C57BL/6 female mice from Charles Rivers were housed in the animal facility of Hospital General Universitario Gregorio Marañón, Madrid, Spain (ES280790000087). All animal procedures conformed to EU Directive 2010/63EU and national regulations (RD 53/2013) and were approved by the local ethics committees and the Animal Protection Board of the Comunidad Autónoma de Madrid (PROEX 244 − 19). 3. Results 3.1 Synthesis and characterization of [ 125 I]I-Beztxab and [ 89 Zr]Zr-DFO-Beztxab radiotracers Radiotracer [ 125 I]I-Beztxab was synthesized with a radiochemical yield of 75.36 ± 4.11%, a specific activity of 167.24 ± 45.14 MBq/mg and a radiochemical purity of higher than 99.99%, as established by TLC (Fig. 2 A). [ 89 Zr]Zr-DFO-Beztxab was synthesized with a radiochemical yield of 71.58 ± 8.19%, a specific activity of 202.76 ± 34.04 MBq/mg and a radiochemical purity of higher than 99.99% (Fig. 3 A). Calculated LogP value using [ 89 Zr]Zr-DFO-Beztxab was − 2.41 ± 0.86, which is in accordance with an hydrophilic behavior. 3.2 In vitro characterization of [ 125 I]I-Beztxab In vitro stability of [ 125 I]I-Beztxab in PBS 1x remained 100% at 24 hours and slowly decreased to 90.7 ± 1.6% at 192 hours. Stability in mouse serum remained 100% after 1 hour and slowly decreased to 86.9 ± 2.6% after 192 hours (Fig. 2 B-C). In the case of [ 89 Zr]Zr-DFO-Beztxab, stability in PBS 1x remained 100% at 26.5 hours and slowly decreased to 95.0 ± 0.6% at 100 hours (Fig. 3 B). The K D of bezlotoxumab and [ 125 I]I-Beztxab measured by ELISA was 24.87 ± 9.04 and 26.03 ± 3.80 pM, respectively (Fig. 4 ). Binding kinetic constants measured by LigandTracer were K D = 5.22 ± 1.66 pM, k a = 3.08 x 10 5 ± 9.76 x 10 4 M − 1 s − 1 , and k d = 1.52 x 10 − 6 ± 2.87 x 10 − 7 s − 1 (Fig. 5 ). 3.4 Quantification of toxin expression in C. difficile strains Table 1 shows the results of the C. difficile toxin A/B FIA immunoassay. Data, expressed in fluorescence units, provides a relative measure of the amount of toxins A and B produced by each strain. Strains ATCC 43255 (ribotype 087), and 14243227 (ribotype 027) showed higher toxin A production capacity, while strains with a higher toxin B production capacity were strain ATCC 43255 (ribotype 087), and, especially, strain 14243227 (ribotype 027). As Bezlotoxumab specifically binds to toxin B, this latter strain was selected for the development of the CDI animal model. Table 1 Toxin A/B FIA immunoassay results Strain Ribotype Mean relative toxin production Toxin A Toxin B ATCC 43255 087 86.52 ± 0.28 10.62 ± 4.76 14243227 027 86.14 ± 3.05 14.87 ± 11.03 13061479 001 63.31 ± 7.30 0.41 ± 0.04 3.5 Ex vivo biodistribution Table 2 and Fig. 6 present the biodistribution values of [ 89 Zr]Zr-DFO-Beztxab, performed in an independent group of animals (48 h and 5 days of infection). Colon + cecum uptake in CDI animals was 1.9-fold higher at 48 h post-infection (7.66 ± 5.02%ID/g) compared to 5 days (4.01 ± 0.88%ID/g). Thus, 48-hour time point was chosen for in vivo studies to optimize PET imaging. Colon + cecum uptake at 48 h was significantly higher in CDI animals (7.66 ± 5.02%ID/g) compared to dysbiosis (3.43 ± 0.43%ID/g) and WT animals (3.21 ± 0.78%ID/g). Table 2 Ex vivo biodistribution results expressed as %ID/g 48 h p. infection CDI Dysbiosis WT Blood 21.46 ± 1.11 27.03 ± 1.47 26.11 ± 2.12 Heart 6.19 ± 0.70 7.43 ± 0.82 8.03 ± 1.75 Lungs 9.25 ± 1.19 12.56 ± 2.15 11.43 ± 1.88 Liver 6.18 ± 0.47 5.93 ± 0.67 6.31 ± 0.63 Spleen 8.53 ± 2.75 6.96 ± 1.53 6.81 ± 1.25 Kidneys 12.23 ± 2.03 11.74 ± 0.47 11.21 ± 1.01 Stomach 3.33 ± 1.38 2.22 ± 0.53 2.76 ± 0.67 Colon + cecum 7.66 ± 5.02 3.43 ± 0.43 3.21 ± 0.78 Skin 3.71 ± 0.78 3.94 ± 1.14 4.99 ± 1.29 Bone 3.50 ± 0.56 3.87 ± 0.64 3.27 ± 0.30 Intestine 3.65 ± 1.68 3.54 ± 0.40 3.76 ± 0.98 Feces 2.71 ± 0.63 1.10 ± 0.52 1.31 ± 0.20 5 d p. infection CDI Dysbiosis WT Blood 11.93 ± 2.08 22.57 ± 8.64 20.03 ± 6.66 Heart 6.61 ± 1.97 7.44 ± 1.94 6.86 ± 1.18 Lungs 9.08 ± 4.16 14.45 ± 1.96 14.27 ± 2.86 Liver 6.31 ± 1.72 8.54 ± 4.67 7.84 ± 1.19 Spleen 7.46 ± 2.07 13.10 ± 8.58 9.92 ± 3.17 Kidneys 7.69 2.39 11.07 ± 1.92 11.49 ± 2.05 Stomach 1.57 ± 0.50 1.78 ± 0.52 2.23 ± 0.18 Colon + cecum 4.01 ± 0.88 2.02 ± 1.68 1.99 ± 0.46 Skin 4.65 ± 1.59 6.18 ± 2.00 5.49 ± 0.90 Bone 3.96 ± 0.53 6.81 ± 0.58 7.43 ± 0.80 Intestine 2.10 ± 0.50 3.50 ± 0.50 3.54 ± 1.07 Feces 1.00 ± 0.15 1.79 ± 1.09 1.95 ± 1.00 3.6 In vivo PET/CT imaging of [ 89 Zr]Zr-DFO-Beztxab Qualitatively in vivo PET/CT images of [ 89 Zr]Zr-DFO-Beztxab confirmed the existence of a specific and localized radiotracer uptake in different segments of the digestive tract. In contrast, the control groups exhibited slight abdominal uptake, basically located in excretion organs and the circulatory system, without no apparent accumulation in the digestive system (Fig. 7 A-B). Quantification of abdominal uptake (SUVmean) showed an increase in [ 89 Zr]Zr-DFO-Beztxab uptake in infected animals (SUVmean 0.85 ± 0.06; p = 0.003) compared with WT animals (SUVmean 0.66 ± 0.07) (Fig. 7 C). 3.7 Validation of CDI animal model The CDI animals exhibited a rapid deterioration within 24 hours following C. difficile administration. At the imaging time point of 48 hours post-infection, all animals in the CDI group exhibit a CSS ≥ 6, whereas in the dysbiosis group only 66% of the animals did. No animal in the WT group showed any symptoms. Ex vivo measurements of colon length demonstrated a significant shortening in the CDI group (5.76 ± 0.40 cm) compared to the dysbiosis (6.80 ± 0.50 cm, p < 0.01) and WT (6.50 ± 0.57 cm, p < 0.05) groups (Fig. 8 ). Toxin quantification from fecal samples was positive for the presence of C. difficile in all CDI animals. Quantification of TCD in stool samples was 177.90 ± 284.81 CFU/µg. The main histopathological lesions were architectural changes in the bowel crypts, increase of lymphocytes and plasma cells in the lamina propria of the bowel, and also presence of polymorphonuclear cells both in the lamina propria and the epithelium. 4. Discussion Despite ongoing advancements in identifying infection sites, effective non-invasive detection methods remain scarce. A major challenge lies in differentiating between inflammatory and infectious processes, which can lead to false-positive diagnoses ( 18 ). Building on the success of immuno-PET imaging in oncology ( 28 ), and the group’s previous work in targeting bacterial toxins ( 29 ), this study evaluates an immuno-PET tracer using an [ 89 Zr]Zr-radiolabeled antibody targeting C. difficile toxin B. To this end, two different radiolabelings of the monoclonal antibody Bezlotoxumab were performed. The [ 125 I]I-radiolabeling, with a radioisotope half-life of 59.49 days, enabled long-lasting in vitro binding studies up to 2000 min. Additionally, direct radioiodination is a simple, fast and inexpensive reaction, unlike [ 89 Zr]Zr. However, despite its potential for SPECT imaging, its low gamma energy (35 keV) limits its use as an imaging agent ( 30 ). Following in vitro binding validation with [ 125 I]I, we radiolabeled Bezlotoxumab with [ 89 Zr]Zr, a well-established radioisotope used in immunoPET for in vivo PET imaging in humans ( 31 ). Its half-life of 78.42 h allows for longitudinal studies while providing more sensitive PET images. The [ 125 I]I-radiolabeling was performed with a low amount of radioactivity due to the long half-life of the radioisotope and the low activity required for in vitro binding assays. This resulted in a good radiochemical yield of 75.36 ± 4.11% and, consequently, a relatively low specific activity of 167.24 ± 45.14 MBq/mg. The high radio-chemical purity of more than 99.99% and the long in vitro stability in PBS ensure that the results obtained in in vitro binding assays are not caused by the binding of free [ 125 I]I. Radiolabeling with [ 89 Zr]Zr resulted in a good radiochemical yield of 71.58 ± 8.19%, similar to other immunotracers for toxins developed by our group ( 32 ), and a higher specific activity of 202.76 ± 34.04 MBq/mg. As in the previous case, the radio-chemical purity was higher than 99.99%, and in vitro stability in PBS remained above 95.0 ± 0.6% at 100 hours. K D values of [ 125 I]I-Beztxab determined by ELISA showed a high binding affinity of Bezlotoxumab to C. difficile B toxins, in the picomolar range ( 33 ). A slight increase in the K D of [ 125 I]I-Beztxab compared to the unlabeled version may indicate a small loss of affinity. However, the standard deviations of the two values overlap, suggesting that the difference may be due to measurement error. There is a small difference between K D values measured by ELISA (26.03 ± 3.80 pM) and LigandTracer (5.22 ± 1.66 pM). Lower K D values are usually observed with LigandTracer compared to ELISA because it provides a more reliable result by collecting data over several days with different ligand concentrations, allowing for the measurement of k a (3.08 x 10 5 ± 9.76 x 10 4 M − 1 s − 1 ) and k d (1.52 x 10 − 6 ± 2.87 x 10 − 7 s − 1 ) kinetic values. Nevertheless, both K D values are in the picomolar range, demonstrating the high binding affinity of the antibody. The association and dissociation constants also indicate rapid binding and slow release of the antibody to its target, with values approximately ten times higher and lower, respectively, than those of other antibodies ( 34 , 35 ). Among the C. difficile strains evaluated for toxin production, strain 14243227 (ribotype 027) and strain ATCC 43255 (ribotype 087) exhibited the highest relative levels of toxin A production, with strain 14243227 also displaying the highest relative level of toxin B. Bezlotoxumab binds specifically to toxin B; therefore, strain 14243227 was selected for this initial evaluation of a radiotracer based on this antibody, excluding the other strains in order to validate the probe’s performance in an optimal scenario. Furthermore, as this strain also exhibits a relatively high production of toxin A, future studies with different targeting antibodies can be conducted using the same strain. In this study, we used two different animal models, one for C. difficile infection and another for dysbiosis. Dysbiosis, an imbalance in the gut microbiota, is linked to several diseases such as inflammatory bowel disease (IBD) ( 36 ). We induced dysbiosis using a combination of antibiotics disrupting gut microbiota and favoring C. difficile infection ( 37 ). Normally, a healthy gut microbiome protects against pathogens through nutrient competition, antimicrobial production, and maintaining an acidic environment ( 38 ). When disrupted, this barrier is compromised, allowing C. difficile to proliferate. The two models selected allowed us to test the radiotracer’s ability to differentiate between active infection versus inflammatory pathways triggered by dysbiosis. Animal models were validated in three different ways: 1) colon length reduction indicated the existence of inflammation ( 39 ). 2) Bacterial cultures from fecal samples confirmed C. difficile presence in all CDI animals, and not in the rest. 3) H&E histology confirmed colon inflammation, with higher levels in the dysbiosis model. This finding further underscores the capability of our radiotracer to detect B toxins for diagnosing C. difficile infection, while avoiding non-specific uptake by sterile colon inflammation Interestingly, samples from animals with low TCD levels showed similar colonic uptake to those with higher TCD levels. These results suggest that PET/CT imaging with [ 89 Zr]Zr-DFO-Beztxab may be more sensitive and reliable than traditional culture methods at low toxin concentrations in stool samples. Ex vivo biodistribution studies were conducted in the three animal models prior to PET/CT imaging to determine the optimal radiotracer uptake time, aiming to identify the best time-point for early diagnosis. Biodistributions were performed at 48 h and 5 days of infection, with 24 h and 4 days of radiotracer uptake respectively. The uptake in the colon and cecum at 48 h post-infection was found to be 1.9 times higher (7.66 ± 5.02%ID/g) compared to the uptake observed at 5 days post-infection (4.01 ± 0.88%ID/g). Based on this finding, the 48-hour time point was selected for subsequent in vivo PET imaging studies to maximize detection sensitivity. Furthermore, at 48 h post-infection, colon and cecum uptake was significantly increased in CDI animals (7.66 ± 5.02%ID/g) compared to dysbiosis animals (3.43 ± 0.43%ID/g) and wild-type (WT) controls (3.21 ± 0.78%ID/g). These differences suggest that this time frame is optimal for distinguishing active CDI from dysbiosis inflammation and healthy controls. PET/CT imaging analysis of the CDI animal model clearly showed a distinct and precisely localized uptake pattern of [ 89 Zr]Zr-DFO-Beztxab in different segments of the lower digestive tract, typically associated with CDI. Interestingly, dysbiosis and WT models exhibited a markedly different uptake profile, mainly localized in excretory organs and circulatory system, with low accumulation within the gastrointestinal tract. This disparity illustrates the specificity of the radiotracer uptake in delineating the pathophysiological processes associated with bacterial infection. In all animal models, a high bone and joint uptake is observed. Demetallization of [ 89 Zr]Zr in DFO-conjugated 150 kDa IgG isotype antibodies in plasma is a common issue observed in previous studies ( 40 ), often leading to uptake of free [ 89 Zr]Zr in bone tissue, especially in joints ( 41 ). This qualitative analysis of the images is supported by the quantitative analysis, where an increase in the abdominal uptake was observed in the infected group compared to the wild type animals. This study presents certain limitations that should be considered. First, the sample size for the ex vivo biodistribution analysis performed 5 days post-infection was insufficient to obtain statistically significant results, although the trend suggested higher uptake in the colon of CDI animals. Second, only one strain of C. difficile (ribotype 027) was used, the one that demonstrated the highest level of toxin B production among the evaluated strains. While this choice facilitated radiotracer validation in an optimal scenario, future studies should evaluate tracer performance on strains with lower toxin B production to determine a more realistic detection limit. Third, PET image quantification was performed using an abdominal ROI rather than a more specific colonic and cecum ROI due to challenges in differentiating these organs on CT images. This lack of specificity may have affected the accuracy of uptake measurements. In addition, the study was conducted in a mouse model, which, while useful for preclinical evaluation, does not fully replicate the complexity of human CDI. Differences in immune response, gut microbiota composition, and toxin distribution between mice and humans may limit the direct translation of findings to clinical practice ( 42 ). Finally, the study did not evaluate potential cross-reactivity with other gut bacteria or inflammatory conditions, which may affect the specificity of the tracer in clinical settings. Addressing these limitations in future research is warranted to refine the diagnostic accuracy and translational potential of this immunoPET approach. In case of positive results this radiotracer has the potential to enhance the diagnosis of CDI and improve patient prognosis. The ability to non-invasively detect CDI in early stages represents a significant advancement over invasive techniques such as colonoscopy, which carries substantial risks in severely ill patients ( 5 ). Early detection would allow for timely intervention, potentially reducing disease progression and severe complications such as pseudomembranous colitis or toxic megacolon ( 43 ). This radiotracer also enables the specific visualization of toxin-producing bacterial foci in vivo, which may be important for precise differentiation between active infection and colonization. This could be particularly advantageous in complex clinical scenarios, such as patients with coexisting gastrointestinal disorders or recurrent CDI, where misdiagnosis can lead to unnecessary treatment or delayed care. Recurrent CDI remains a major challenge, often requiring repeated and prolonged courses of therapy ( 44 ). The proposed radiotracer could identify residual infection sites and differentiate recurrent infection from post-treatment inflammatory changes, reducing the reliance on empirical therapy and potentially curbing antimicrobial resistance associated with overuse of broad-spectrum antibiotics. Additionally, the ability to monitor the response to therapy in vivo would enable clinicians to adjust treatments dynamically, potentially improving patient outcomes. 5. Conclusions We have synthesized and characterized two C. difficile specific radiotracers based on Bezlotoxumab. Radiolabeling with both [ 125 I]I and [ 89 Zr]Zr was successfully achieved with high radiochemical yield, purity and stability, which supports their use for in vivo assessments. In vitro binding assays using [ 125 I]I-Beztxab confirmed a high and rapid uptake of bezlotoxumab, with slow release even at long time-points. PET/CT imaging using [ 89 Zr]Zr-DFO-Beztxab revealed a distinct and specific uptake in the digestive tract of CDI animals, while this uptake was not observed in the other groups. These findings support the radiotracer's ability to differentiate between active infection and dysbiosis, providing valuable evidence of its diagnostic potential. Abbreviations [ 125 I]I-Beztxab [ 125 I]I radiolabeled bezlotoxumab [ 18 F]F-FDG [ 18 F]F-fluorodeoxyglucose [ 89 Zr]Zr-DFO-Beztxab [ 89 Zr]Zr radiolabeled bezlotoxumab C. difficile Clostridiodes difficile CDI Clostridioides difficile infection CFU Colony-forming units CT Computed Tomography EIA Enzyme immunoassay GDH Glutamate dehydrogenase H&E Hematoxylin and Eosin HAIs Hospital-acquired infections IBD Inflammatory bowel disease MRI Magnetic Resonance Imaging PET Positron Emission Tomography p-NCS-Bz-DFO p-Isothiocyanatobenzyl-deferoxamine ROI Region of interest SPECT Single Photon Emission Computed Tomography TCD Toxigenic C. difficile WT Wild-type Declarations Compliance with Ethical Standards Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work has been funded by Instituto de Salud Carlos III (ISCIII) through the projects "PT23/00027" and “PI23/01405”, and co-funded by the European Union. Work supported by Comunidad de Madrid, project S2022/BMD-7403 (RENIM-CM). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MICIU) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIU/AEI/10.13039/501100011033). Grant PTA2022-021556-I funded by MICIU/AEI /10.13039/501100011033 and by FSE+. Authors also thank Fundación Ramón Areces for their support. Ethics approval and consent to participate C57BL/6 female mice from Charles Rivers were housed in the animal facility of Hospital General Universitario Gregorio Marañón, Madrid, Spain (ES280790000087). All animal procedures conformed to EU Directive 2010/63EU and national regulations (RD 53/2013) and were approved by the local ethics committees and the Animal Protection Board of the Comunidad Autónoma de Madrid (PROEX 244-19). Availability of data and material The datasets supporting the conclusions of this article are included within the article. The data that support the findings of this study are available from the corresponding author upon reasonable request. Authors' contributions MGA and LC contributed equally to this work, performing the main experiments, data analysis and drafting the manuscript. LA performed the C. difficile toxin quantification of different strains and the analysis of stool samples to determine the presence of toxigenic species. MIG assisted in the synthesis of the radiotracer and ex vivo biodistribution studies. MJFA performed the H&E staining of colon tissues and their subsequent analysis. DS and SS provided the 125 I radioisotope and directed its radiolabeling and characterization, including ELISA and ligand tracer assays. EB and PM provided the C. difficile background and opportunity for new diagnostic tools. MD helped with experimental design and interpretation of results. Finally, Dr. BS developed the experimental design and interpretation of results, and supervised the work. All authors read and approved the final manuscript. Consent for publication Not applicable Acknowledgments The authors thank Alexandra de Francisco and Diego Eguibar from the Imaging Laboratory for Small Animal Experimentation of Instituto de Investigación Sanitaria Gregorio Marañón for their excellent work with animal preparation and imaging protocols. References Czepiel J, Dróżdż M, Pituch H, Kuijper EJ, Perucki W, Mielimonka A, Goldman S, Wultańska D, Garlicki A, Biesiada G. Clostridium difficile infection. European Journal of Clinical Microbiology & Infectious Diseases. 2019;38:1211-21. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6294683","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":436227274,"identity":"56ad8618-34ac-41a3-a700-5d4944bcdbdc","order_by":0,"name":"Mario González-Arjona","email":"","orcid":"","institution":"Instituto de Investigacion Sanitaria Gregorio Maranon","correspondingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"González-Arjona","suffix":""},{"id":436227275,"identity":"da30ff66-d9ae-471b-8102-b9d16f38c1cf","order_by":1,"name":"Lorena Cussó","email":"","orcid":"","institution":"Instituto de Investigacion Sanitaria Gregorio Maranon","correspondingAuthor":false,"prefix":"","firstName":"Lorena","middleName":"","lastName":"Cussó","suffix":""},{"id":436227276,"identity":"43066da8-fce7-44c7-b8ac-cd2aae4f4124","order_by":2,"name":"Luis Alcalá","email":"","orcid":"","institution":"Instituto de Investigacion Sanitaria Gregorio Maranon","correspondingAuthor":false,"prefix":"","firstName":"Luis","middleName":"","lastName":"Alcalá","suffix":""},{"id":436227277,"identity":"a1860a4f-b0e3-4eca-bb07-0851f4e81631","order_by":3,"name":"María Isabel González","email":"","orcid":"","institution":"CNIC: Centro Nacional de Investigaciones Cardiovasculares Carlos III","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Isabel","lastName":"González","suffix":""},{"id":436227278,"identity":"22d8cc97-bc4f-40d2-8a78-0794ec8d9913","order_by":4,"name":"María Jesús Fernández-Aceñero","email":"","orcid":"","institution":"San Carlos University Hospital Internal Medicine III Service: Hospital Clinico San Carlos Servicio de Medicina Interna","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Jesús","lastName":"Fernández-Aceñero","suffix":""},{"id":436227279,"identity":"b5a9d8b3-d68e-4e1a-b65c-02638b601678","order_by":5,"name":"Dag Sehlin","email":"","orcid":"","institution":"Uppsala University: Uppsala Universitet","correspondingAuthor":false,"prefix":"","firstName":"Dag","middleName":"","lastName":"Sehlin","suffix":""},{"id":436227280,"identity":"1e2e5443-95f7-4829-bc2e-5ebad832f9a5","order_by":6,"name":"Stina Syvänen","email":"","orcid":"","institution":"Uppsala University: Uppsala Universitet","correspondingAuthor":false,"prefix":"","firstName":"Stina","middleName":"","lastName":"Syvänen","suffix":""},{"id":436227281,"identity":"5caaba34-9a38-4ecd-aa8c-1a81c2560107","order_by":7,"name":"Emilio Bouza","email":"","orcid":"","institution":"Instituto de Investigacion Sanitaria Gregorio Maranon","correspondingAuthor":false,"prefix":"","firstName":"Emilio","middleName":"","lastName":"Bouza","suffix":""},{"id":436227282,"identity":"39654d06-05a7-451d-8c03-845e3b132c5e","order_by":8,"name":"Patricia Muñoz","email":"","orcid":"","institution":"Instituto de Investigacion Sanitaria Gregorio Maranon","correspondingAuthor":false,"prefix":"","firstName":"Patricia","middleName":"","lastName":"Muñoz","suffix":""},{"id":436227283,"identity":"c98b2362-324d-473d-a3bc-9599b56f4324","order_by":9,"name":"Manuel Desco","email":"","orcid":"","institution":"Instituto de Investigacion Sanitaria Gregorio Maranon","correspondingAuthor":false,"prefix":"","firstName":"Manuel","middleName":"","lastName":"Desco","suffix":""},{"id":436227284,"identity":"45c2cf22-7432-4214-a4f6-950c455c3869","order_by":10,"name":"Beatriz Salinas","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIie2PsYoCMRRF3zDgNNF6K+cXMiwo+zcZltVGQTsLCRkEt5G1Xf8in5AlkGnyAa8TsbBVBCuRjQ4iFtEtt8gpLrzicO8DCAT+IfSWsVAumwCJeqZEolKii/IKQNgfFKiUc+biWu2jnUw269GAQ/r5U+jBmHdlqT4kjLhXeZuZrLC0BtTmQn8b3Zc2NwhW+4chywpBiRvjFFJTfYnQwWiq/Mpys3PKC6TzlVNOvEsRWhid/MMoknOLexhdS30as0oR8YNfesOFoIxQdC31L50tbK+DzPh/aSel3Ikjb6bzd70nB542ytLgduwfdoXcn+ypEAgEAoFH/AJSqVydcQ8xIAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-9388-5917","institution":"Universidad Carlos III de Madrid","correspondingAuthor":true,"prefix":"","firstName":"Beatriz","middleName":"","lastName":"Salinas","suffix":""}],"badges":[],"createdAt":"2025-03-24 11:09:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6294683/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6294683/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s41181-025-00350-x","type":"published","date":"2025-06-13T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82010149,"identity":"b01c19df-8e35-4d35-b27e-6ce8d6fa1494","added_by":"auto","created_at":"2025-05-06 01:28:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":140429,"visible":true,"origin":"","legend":"\u003cp\u003eAbdominal ROI selected for PET quantification, shown in A) Axial, B) Coronal and C) Sagittal planes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/2bf01afca582fbd4dc5448e1.png"},{"id":82010148,"identity":"aeca24ae-0906-4144-9390-e350114fecb2","added_by":"auto","created_at":"2025-05-06 01:28:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79383,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab. A) TLC chromatogram of purified [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab. B) In vitro stability of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab in PBS 1x. C) In vitro stability of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab in mouse serum.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/c48996a28a40734ba4eb7707.png"},{"id":82010698,"identity":"d4dbdbcf-09b5-42e9-8167-95f053e8eeb7","added_by":"auto","created_at":"2025-05-06 01:36:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":57535,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab. A) TLC chromatogram of purified [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab. B) In vitro stability of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab in PBS.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/49865b3c8b0aa8fbb1e1e57c.png"},{"id":82010154,"identity":"8121a4c1-6758-4fa1-9aa1-c94010eaa7f3","added_by":"auto","created_at":"2025-05-06 01:28:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":81592,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of the dissociation constant (K\u003csub\u003eD\u003c/sub\u003e) of Bezlotoxumab and [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab by ELISA. (Left) Binding curve of Bezlotoxumab to \u003cem\u003eC. difficile\u003c/em\u003e toxin B. (Right) Binding curve of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab to \u003cem\u003eC. difficile\u003c/em\u003e toxin B. Data are presented as the mean ± standard deviation of three independent experiments.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/e980182c3c9969ee27797450.png"},{"id":82010699,"identity":"0ea9f64d-247b-4e09-864d-dd7c58613dfe","added_by":"auto","created_at":"2025-05-06 01:36:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66431,"visible":true,"origin":"","legend":"\u003cp\u003eBinding kinetics assay of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab to \u003cem\u003eC. difficile\u003c/em\u003e toxin B by LigandTracer, showing the ligand association and dissociation (expressed as CPS in the targeted toxin B) over time.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/b60e4580c9596e62ccfbfd6e.png"},{"id":82010151,"identity":"d7fd1b9f-44c6-4fb8-9cef-bbe4059ec317","added_by":"auto","created_at":"2025-05-06 01:28:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":41661,"visible":true,"origin":"","legend":"\u003cp\u003eColon + cecum biodistribution of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab in the three animal models 48 h and 5 d post-infection (48 h post-infection: CDI n = 4, Dysbiosis n = 4, WT n = 4; 5 days post-infection: CDI n = 3, Dysbiosis n = 5, WT n = 5). Statistical analysis was performed by Kruskal Wallis test for independent samples and Mann-Whitney post-hoc test, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/a9a709de381610d15e249aa9.png"},{"id":82010700,"identity":"28b68e69-8896-477e-8d26-b636754fd58e","added_by":"auto","created_at":"2025-05-06 01:36:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":372424,"visible":true,"origin":"","legend":"\u003cp\u003eA) Representative images of in vivo PET/CT imaging of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab in the three animal models 48h post-infection. Red arrow points at colon/cecum uptake. B) Additional images of CDI animal models 48h post-infection. Red arrows point at colon/cecum uptake. C) PET/CT images quantification in the three animal models (CDI n = 9, Dysbiosis n = 5, WT n = 5) expressed as SUVmean. Statistical analysis was performed by Kruskal wallis test for independent samples and Mann-Whitney post-hoc test, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/a48afdb8f8727a191d33136e.png"},{"id":82010164,"identity":"52b725a3-097a-435f-8a21-aaf5fe5946f9","added_by":"auto","created_at":"2025-05-06 01:28:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":267392,"visible":true,"origin":"","legend":"\u003cp\u003eA) Ex vivo measurements of colon length of the three animal models. Statistical analysis was performed by One-way ANOVA, *p\u0026lt;0.05, **p\u0026lt;0.01. B) Representative pictures of the measurement of colon length of the three animal models. C) Representative H\u0026amp;E histology of CDI and Dysbiosis animal models. Statistical analysis was performed by Kruskal Wallis test for independent samples and Mann-Whitney post-hoc test, *p \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/22215e060da30a5d194d74a0.png"},{"id":84726498,"identity":"8e568523-8201-4736-99d9-f72bcd33e806","added_by":"auto","created_at":"2025-06-16 16:06:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2374342,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6294683/v1/0452d6ec-5696-426a-8132-42547addd068.pdf"}],"financialInterests":"","formattedTitle":"Development of a Toxin-selective immunotracer for the specific in vivo detection of Clostridioides difficile infection by immunoPET","fulltext":[{"header":"1. Background","content":"\u003cp\u003e \u003cem\u003eClostridioides difficile\u003c/em\u003e (\u003cem\u003eC. difficile\u003c/em\u003e) remains a persistent and formidable challenge in healthcare facilities worldwide, significantly burdening patient health outcomes, healthcare resources and infection control (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It is the leading cause of hospital-acquired infections (HAIs) in US hospitals (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) and has a high prevalence in European hospitals, accounting for 7.3% of all HAI cases (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). \u003cem\u003eC. difficile\u003c/em\u003e infection (CDI) is associated with significant morbidity and mortality, exacerbated by its propensity for recurrent infections and complications such as pseudomembranous colitis and toxic megacolon (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite advances in infection prevention and control strategies, the incidence and severity of CDI continue to rise, underscoring the urgent need for innovative diagnostic and therapeutic approaches to effectively manage this escalating public health problem.\u003c/p\u003e \u003cp\u003eThe most commonly used diagnostic strategy for CDI follows an algorithm that begins with initial screening using an enzyme immunoassay (EIA) to detect glutamate dehydrogenase (GDH), an enzyme highly sensitive for identifying CDI (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Since both toxigenic and non-toxigenic strains of \u003cem\u003eC. difficile\u003c/em\u003e produce GDH, a positive GDH EIA result requires confirmatory testing. This confirmation typically involves real-time PCR targeting toxin B gene, with or without an intermediate EIA for the detection of A and B toxins. The intermediate EIA aims to reduce the overall cost of molecular testing. These diagnostic algorithm has been extensively evaluated in multiple studies, demonstrating a sensitivity of 85\u0026ndash;90% and a specificity exceeding 99% compared to toxigenic culture, the gold standard for CDI diagnosis (\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough this approach is widely adopted in clinical practice, it is not without limitations. These challenges are particularly evident in cases where toxin concentrations are low, as patients may not have CDI but instead be asymptomatic carriers of toxigenic \u003cem\u003eC. difficile\u003c/em\u003e. Colonoscopy, which would allow direct observation of the damage caused by the microorganism in the colon, is not recommended for severe patients because it significantly increases the risk of perforation. These tests are highly invasive and require patient sedation, which always carries certain risks and side effects (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). These challenges highlight the inadequacies of current diagnostic modalities and underscore the need for the development of more accurate and reliable diagnostic tools.\u003c/p\u003e \u003cp\u003eMolecular imaging techniques have emerged as promising additions to conventional diagnostic approaches, offering the potential for non-invasive visualization and characterization of infectious diseases. Radiological techniques, including radiography, ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), are frequently used, especially to identify cases of toxic megacolon. However, these purely structural imaging tools rely on anatomic or morphologic changes that often occur after molecular events in the disease process, thereby precluding early detection of the infection. Furthermore, they are nonspecific and may reflect a combination of infection and host inflammatory response (\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNuclear imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) have been investigated for their utility in diagnosing CDI by targeting metabolic processes associated with bacterial infection. Among these, PET imaging with [\u003csup\u003e18\u003c/sup\u003eF]F-fluorodeoxyglucose ([\u003csup\u003e18\u003c/sup\u003eF]F-FDG) has attracted considerable interest due to its ability to detect areas of increased glucose metabolism characteristic of infectious foci (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Our group has pioneered its application to detect and evaluate CDI in a mouse model (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In our study, [\u003csup\u003e18\u003c/sup\u003eF]F-FDG uptake in the abdominal area was evaluated in two mouse models infected with two \u003cem\u003eC. difficile\u003c/em\u003e ribotypes of different virulence.\u003c/p\u003e \u003cp\u003eHowever, [\u003csup\u003e18\u003c/sup\u003eF]F-FDG can actively incorporate into leukocytes, macrophages, and CD4-positive T cells present at infection sites (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), making it uncapable to differentiate between infectious, inflammatory, and tumor lesions, or even organs with high basal metabolism. This can lead to confusion when foci are close to organs such as the heart or brain, or to false positives (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). These limitations restrict its potential use, highlighting the need for the development of more specific imaging probes.\u003c/p\u003e \u003cp\u003eTo address these challenges and improve the accuracy of CDI diagnosis, our research efforts aim to develop a novel molecular imaging approach that combines the specificity of targeted molecular therapy with the sensitivity of radiotracer imaging. Our proposed strategy involves radiolabeling a commercially available monoclonal antibody, Bezlotoxumab, with the radionuclide [\u003csup\u003e89\u003c/sup\u003eZr]Zr to create a customized radiotracer, tailored for the detection of \u003cem\u003eC. difficile\u003c/em\u003e toxins. Bezlotoxumab targets and neutralizes \u003cem\u003eC. difficile\u003c/em\u003e toxin B, and thus offers an opportunity for targeted molecular imaging due to its high affinity and selectivity, (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). By using this radiolabeled antibody in an animal model we aim to explore the possibility of selectively targeting active \u003cem\u003eC. difficile\u003c/em\u003e infection within the host, enabling precise localization and visualization of CDI sites.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003eUnless otherwise specified, all reagents were purchased from Merck (KGaA, Darmstadt, Germany) and used without further purification.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Radiolabeling of commercial Bezlotoxumab and physico-chemical characterization\u003c/h2\u003e\n \u003cp\u003eCommercial Bezlotoxumab (Merck \u0026amp; Co., Inc., Rahway, NJ, USA) was radiolabeled with two different radionuclides; [\u003csup\u003e125\u003c/sup\u003eI]I for in vitro binding and kinetic studies, and [\u003csup\u003e89\u003c/sup\u003eZr]Zr for physico-chemical characterization, in vivo and ex vivo studies. Both radioisotopes were purchased from Revvity, Inc. (Waltham, MA, USA). For both immunoconjugates, radiochemical yield was estimated as the fraction of purified radiotracer activity in comparison to the starting amount of activity (%). Specific activity was calculated as the final radioactivity of radiotracer per milligram of antibody (MBq/mg).\u003c/p\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003e2.1.1 Synthesis and physico-chemical characterization of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab\u003c/h2\u003e\n \u003cp\u003eRadioiodination of Bezlotoxumab was performed following traditional direct labeling method (\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e). Briefly, 20 \u0026micro;g of Bezlotoxumab (8 \u0026micro;L) were mixed with 95 \u0026micro;L PBS 1x and 2 \u0026micro;L [\u003csup\u003e125\u003c/sup\u003eI]I (2.00-2.52 MBq). The reaction was started by adding 5 \u0026micro;L of a 1 mg/mL solution of chloramine-T trihydrate and incubated at room temperature for 90 seconds. Then, 10 \u0026micro;L of a 1 mg/mL solution of sodium metabisulfite was added to stop the reaction, and the radiolabeled antibody was isolated using Zeba\u0026trade; Spin Desalting Columns with a 7K MWCO (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e\n \u003cp\u003eThe purity of the radiotracer was evaluated by instant thin layer chromatography (iTLC) using silica gel (SG) chromatographic paper (Agilent Technologies, Inc., Santa Clara, CA, USA) as the stationary phase and acetone 70% as the mobile phase.\u003c/p\u003e\n \u003cp\u003eLoss of protein was determined by indirect Enzyme-Linked ImmunoSorbent Assay (ELISA) using an Anti-Human IgG (Fc specific) antibody, labeled and unlabeled Beztxab and calculated by 4PL nonlinear regression model.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.1.2 Synthesis and physico-chemical characterization of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab\u003c/h2\u003e\n \u003cp\u003eFor the labeling of the mAb with the radiometal [\u003csup\u003e89\u003c/sup\u003eZr]Zr, Bezlotoxumab was first conjugated to the isothiocyanatobenzyl-derivative of the chelator desferrioxamine (p-NCS-Bz-DFO, Chematech, Dijon, France ) adapting the labeling approach from previous works (\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e). Briefly, 1 mg of Bezlotoxumab (40 \u0026micro;L) was adjusted to 1 ml with 0.1 M sodium bicarbonate buffer (pH\u0026thinsp;=\u0026thinsp;9.0). Parallelly, 1 mg of p-NCS-Bz-DFO was diluted in 200 \u0026micro;L of dimethyl sulfoxide and 20 \u0026micro;L were added to the Ab solution in four additions of 5 \u0026micro;L, with gently mixing in-between. Mixture was incubated at 37 \u0026ordm;C, 500 rpm for 30 min in Eppendorf\u0026reg; ThermoMixer\u0026reg; C (Eppendorf, Hamburg, Germany). Conjugated Ab was purified by PD-10 desalting column (GE Healthcare Bio-Science AB, Chicago, IL, USA) and collected in 2 mL of HEPES buffer 0.5 M.\u003c/p\u003e\n \u003cp\u003eRatio of p-DFO-Bz-NCS to Bezlotoxumab was measured by MALDI-TOF MS/MS (Unidad de Espectrometr\u0026iacute;a de Masas, Universidad Complutense de Madrid, Spain) following previous publications (\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e). Briefly, a 1 \u0026micro;L aliquot of unconjugated antibody, p-DFO-Bz-NCS and Bz-DFO-Beztxab samples were combined with an equal volume of sinapic acid, used as the matrix solution (10 mg/mL in 50% acetonitrile: water and 0.1% trifluoroacetic acid). Samples were then deposited onto a stainless-steel target plate and left to dry. Following the determination of the mass (m/z) of both the unaltered antibody and the immunoconjugate, the difference was divided by the chelator\u0026apos;s molecular weight, and the ratio p-DFO-Bz-NCS:Bezlotoxumab expressed as number of chelates per antibody unit.\u003c/p\u003e\n \u003cp\u003eFor radiolabeling, oxalic acid 1 M was added to 74\u0026ndash;111 MBq of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-oxalic acid solution to a final volume of 2 mL. Then, 90 \u0026micro;L of sodium carbonate 2 M were added, and mixture was incubated at room temperature (RT), 500 rpm for 3 min in ThermoMixer. Next, 300 \u0026micro;L of HEPES 0.5 M, 710 \u0026micro;L of Bz-DFO-Beztxab and 700 \u0026micro;L of HEPES 0.5 M were added to the mixture and kept on reaction for 60 min at RT and 500 rpm in ThermoMixer. Lastly, radiolabeled Ab was purified using 100 kDa Amicon filters (centrifuged at 4 \u0026ordm;C/21884 rcf/10min, recovered at 4 \u0026ordf;C/5471 rcf/5 min).\u003c/p\u003e\n \u003cp\u003ePurity was evaluated by iTLC using WhatmannTM strips (3 MM CHR; GE Healthcare Bio-Science AB, Chicago, IL, USA) as the stationary phase, and citric acid monohydrate/sodium carbonate (pH 4.9\u0026ndash;5.1) as the mobile phase. Radioactivity of TLC plates was read using a miniGita Single system (Elisa-Raytest, Angleur, Belgium).\u003c/p\u003e\n \u003cp\u003eRadiolabeled-antibody mass was determined by Bradford-Coomassie assay, according to the manufacturer instructions and employing a VICTOR Nivo Multimode Microplate Reader (Revvity, Inc., Waltham, MA, USA).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 In vitro characterization\u003c/h2\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.1 In vitro stability\u003c/h2\u003e\n \u003cp\u003eIn vitro stability of the radiotracers was evaluated in PBS 1x and mouse serum for [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab and in PBS 1x for [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab. Briefly, aliquots of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab (3.70 MBq) and [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab (0.74 MBq) were added to 0.5-1 mL of PBS 1x and mouse serum, previously tempered at 37\u0026ordm;C with constant shaking. Samples were then collected at different time-points and analyzed by radio-TLC as described in the synthesis section.\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003e[\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab PBS 1x time-points: 0h, 0.5h, 1h, 2h, 4h, 20h, 26.5h, 45h, 51h, 68h, 74h, 95h and 100h.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e[\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab PBS 1x and mouse serum time points: 0h, 0.25h, 0.5h, 1h, 2h, 4h, 7h, 24h, 27h, 30h, 48h, 51h, 54h, 72h, 75h, 78h, 96h, 100h, 168h, 192h.\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cem\u003e2.2.2\u003c/em\u003e Hydrophobicity assessment \u003cem\u003eof [\u003c/em\u003e\u003csup\u003e\u003cem\u003e89\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eZr]Zr-DFO-Beztxab\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eThe hydrophobicity of the radiotracer was assessed using the partitioning method based on the LogP calculation. Briefly, 0.37 MBq of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab were added to an immiscible biphasic solution consisting of 500 \u0026micro;L of 1-octanol and 500 \u0026micro;L of PBS 1x (n\u0026thinsp;=\u0026thinsp;3). This mixture was then incubated at 37\u0026ordm;C for 30 minutes with vigorous shaking. The mixture was then allowed to stand for a further 30 minutes to ensure proper phase separation. Finally, 100 \u0026micro;L samples were taken from each phase and their activity was measured using a Genesys gamma counter (Laboratory Technologies Inc., Elburn, IL, USA).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e2.2.3 Binding kinetics for [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab\u003c/h2\u003e\n \u003cp\u003eBinding kinetics was measured by ELISA and LigandTracer (\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e1. ELISA: A 96-well half area clear flat bottom polystyrene high bind microplate (Corning Inc, Corning, NY, USA) was coated with 50 \u0026micro;L of 0.5 \u0026micro;g/mL \u003cem\u003eC. diff\u003c/em\u003e Toxin B and left at 4 \u0026ordm;C over night (ON). Wells were emptied, filled with 150 \u0026micro;L of ELISA Blocking Buffer (1% BSA, 0.15% Kathon ProClin\u003csup\u003e\u0026trade;\u003c/sup\u003e 150 in PBS 1x, pH 7.4) and shaken at 900 rpm for 1 hour. Then, wells were emptied and washed four times with ELISA Washing Buffer (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e x H\u003csub\u003e2\u003c/sub\u003eO 0.32 mM, Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e x 2H\u003csub\u003e2\u003c/sub\u003eO 2.17 mM, NaCl 150 mM, 7.5 x 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e% Kathon ProClin\u003csup\u003e\u0026trade;\u003c/sup\u003e 150, 0.1% TWEEN\u003csup\u003e\u0026reg;\u003c/sup\u003e 20 in MQ water, pH 7.5). Next, 50 \u0026micro;L of unlabeled Bezlotoxumab (n\u0026thinsp;=\u0026thinsp;5) and [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab (n\u0026thinsp;=\u0026thinsp;3) were added to the plate in serial dilutions from 10 nM to 0.64 pM in ELISA Incubation Buffer (0.1% BSA, 0.15% Kathon ProClin\u003csup\u003e\u0026trade;\u003c/sup\u003e 150, 0.05% TWEEN\u003csup\u003e\u0026reg;\u003c/sup\u003e 20 in PBS 1x, pH 7.4), and left at 4 \u0026ordm;C ON. Then, wells were emptied and washed as described above, and incubated with HRP-conjugated polyclonal goat anti-human-IgG-F(ab\u0026prime;)\u003csub\u003e2\u003c/sub\u003e antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), diluted 1:2000, at 900 rpm for 1 hour. Wells were emptied and washed, filled with 50 \u0026micro;L K Blue Aqueous TMB substrate (Neogen Corp., Lexington, KY, USA) and incubated for 5 min. Reaction was stopped by adding 50 \u0026micro;Lof H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 1M, and signal absorbance was read with a spectrophotometer at 450 nm. Equilibrium dissociation constant (K\u003csub\u003eD\u003c/sub\u003e) was calculated using a One site-Specific binding non-linear regression in Prism 8.3.0 (GraphPad Software, La Jolla, CA, USA). For calculations, concentration of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab was corrected according to the % of loss protein calculated as described above.\u003c/p\u003e\n \u003c/span\u003e \u003cspan\u003e\n \u003cp\u003e2. LigandTracer: In a high bind circular Petri dish (Corning Inc, Corning, NY, USA), 300 \u0026micro;L of a 10 \u0026micro;g/mL solution of \u003cem\u003eC. diff\u003c/em\u003e Toxin B were added on a local spot at the edge of the dish. The dish was left tilted at 4 \u0026ordm;C ON. Then, solution was removed, and the surface of the dish was blocked with ELISA Blocking buffer for 1 hour. Blocking buffer was removed, 2 mL of running buffer (0.1% BSA in PBS 1x) were added, and the dish was placed in the Ligand Tracer Grey instrument (Ridgeview Instruments AB, Uppsala, Sweden). Baseline was recorded for 15 min, running buffer removed, and then 2 mL of a 0.14\u0026ndash;0.73 nM [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab (n\u0026thinsp;=\u0026thinsp;3) solution were added. Radioactivity at the coated spot and at a non-coated spot (association) was recorded for 3 hours. Then, concentration of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab was increased to 0.47\u0026ndash;2.19 nM, and radioactivity further recorder for 3 hours. Lastly, [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab solution was removed, dish washed with running buffer, and dissociation recorded in 2 mL of running buffer for 72 hours. Association (k\u003csub\u003ea\u003c/sub\u003e), dissociation (k\u003csub\u003ed\u003c/sub\u003e) and K\u003csub\u003eD\u003c/sub\u003e rate constants were calculated with Trace Drawer 1.8.1 software (Ridgeview Instruments AB).\u003c/p\u003e\n \u003c/span\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Toxin A and B quantification assay of Clostridioides difficile strains\u003c/h2\u003e\n \u003cp\u003eToxin quantification of various \u003cem\u003eC. difficile\u003c/em\u003e strains was conducted to identify the highest toxin-producing strain for use in our CDI animal model. The study was performed by \u003cem\u003eC. difficile\u003c/em\u003e Toxin A/B FIA (SD Biosensor), a fluorescent immunoassay capable of measuring the fluorescence emitted by toxins A and B labeled with a fluorescent antibody. Three strains were included in this assay: ATCC 43255 (ribotype 087), strain 14243227 (ribotype 027), and strain 13061479 (ribotype 001). These strains were previously employed by our team in a study on \u003cem\u003eC. difficile\u003c/em\u003e infection using a mouse model (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). For each strain, two consecutive re-isolations were conducted on Brucella agar at 35\u0026deg;C in anaerobic conditions for a period of 48 hours. A 0.5 McFarland\u0026apos;s suspension of the \u003cem\u003eC. difficile\u003c/em\u003e strain was prepared from the cultures and 200 \u0026micro;l of the suspension was inoculated into another BHI broth (previously reduced in an anaerobic environment for four days). This was then incubated at 35\u0026deg;C in anaerobiosis for four days. Following incubation, the broth was vortexed and subsequently centrifuged at 1200 rpm for three minutes. The supernatant was then transferred to an immunoassay extraction buffer, vortexed, and 3 \u0026micro;l of the mixture were added to the immunoassay device. After 15 minutes of incubation, the device was read by the F2400 fluorescence reader. This procedure was repeated twice for each of the strains.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Animal model of CDI\u003c/h2\u003e\n \u003cp\u003eCDI was induced in C57BL/6 female mice (n\u0026thinsp;=\u0026thinsp;16) with a protocol of 10 days of preconditioning antibiotic in drinking water with cefoperazone 0.5 mg/ml followed by a single dose of 10 mg/kg clindamycin intraperitoneally 1 day before orogastric administration of 10\u003csup\u003e6\u003c/sup\u003e colony-forming unit (CFUs) of \u003cem\u003eC. difficile\u003c/em\u003e ribotype 027 (\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). As a control, a different group of animals (n\u0026thinsp;=\u0026thinsp;14) underwent the same antibiotic treatment without infection, to develop an inflammation due to the disruption of the gut microbiota, known as dysbiosis. In addition, wild-type (WT) animals (n\u0026thinsp;=\u0026thinsp;14) were used as healthy controls.\u003c/p\u003e\n \u003cp\u003eWeight and clinical status of the animals were monitored every 2\u0026ndash;3 days following the protocol established by Shelby et al. (\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e), starting from the initiation of antibiotic treatment. After infection or administration of the imaging agent, daily monitoring was conducted. We defined clinical CDI if the animals reached a Clinical Sickness Score (CSS) equal to or greater than 6.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e2.5 Ex vivo biodistribution of\u003c/em\u003e [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab\u003c/h2\u003e\n \u003cp\u003eIn order to determine the optimal uptake time for in vivo PET/CT imaging we conducted ex-vivo biodistribution studies at 48 h and 5 days post-infection.\u003c/p\u003e\n \u003cp\u003eAnimals were intravenously administered with [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab (1.11\u0026ndash;3.7 MBq in 200 \u0026micro;L of PBS 1x) 24 hours after infection. The animals were then euthanized at their corresponding uptake times, and organs of interest were collected. The experimental groups and sample sizes were as follows:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003e48 hours post-infection: CDI (n\u0026thinsp;=\u0026thinsp;4), Dysbiosis (n\u0026thinsp;=\u0026thinsp;4), WT (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003e5 days post-infection: CDI (n\u0026thinsp;=\u0026thinsp;3), Dysbiosis (n\u0026thinsp;=\u0026thinsp;5), WT (n\u0026thinsp;=\u0026thinsp;5).\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eThe harvested organs included blood, heart, lungs, liver, spleen, kidneys, stomach, colon\u0026thinsp;+\u0026thinsp;cecum, skin, bone, intestine, and feces.\u003c/p\u003e\n \u003cp\u003eThe activity was measured in a Wallac Wizard 1480-011 Automatic Gamma Counter (Revvity, Inc., Waltham, MA, USA) and biodistribution was expressed as mean % Injected Dose per gram of tissue (%ID/g).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003e2.6 In vivo PET/CT imaging of\u003c/em\u003e [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab\u003c/h2\u003e\n \u003cp\u003ePET/CT studies were conducted using a small-animal PET/CT scanner (PET/CT SuperArgus, SEDECAL Molecular Imaging, Madrid, Spain). 24 h after the infection time-point, the animals (CDI n\u0026thinsp;=\u0026thinsp;9, Dysbiosis n\u0026thinsp;=\u0026thinsp;5, WT n\u0026thinsp;=\u0026thinsp;5) were intravenously administered with [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab (3.70 MBq in 200 \u0026micro;L of PBS 1x). Image acquisition was performed 24 h after radiotracer administration and 48 h post-infection. Before CT acquisition, 0.3 mL of Iopamiro (Bracco, Milan, Italy) was administered intraperitoneally. During acquisition, animals were anesthetized with 1.5% sevofluorane in oxygen (SevoFlo, Zoetis Belgium SA, Louvain-la-Neuve, Belgium). PET data were collected for 30 min and reconstructed using FORE/2D-OSEM with 16 subsets and 1 iteration (voxel size: 0.388 x 0.388 x 0.775 mm). The CT study was acquired using an X-ray beam current of 340 \u0026micro;A and a tube voltage of 40 kVp, and reconstructed using an FDK algorithm (\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003ePET/CT images were analyzed with Multimodality Workstation software (\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e). On each CT image, a region of interest (ROI) was selected in the peritoneal cavity (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) using the kidneys as reference, and delimited by 1) an axial plane just below the most caudal kidney pole; 2) a coronal plane ventral to the kidneys, always avoiding the bladder. These ROIs were automatically applied to co-registered PET images to measure ROI mean standard uptake values (SUVmean).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Validation of CDI animal model\u003c/h2\u003e\n \u003cp\u003eModel validation was conducted by measuring colon length, culturing \u003cem\u003eC. difficile\u003c/em\u003e from feces, and performing Hematoxylin and Eosin (H\u0026amp;E) histology, after in vivo imaging.\u003c/p\u003e\n \u003cp\u003eThe length of the colon was determined by extracting the organ and measuring the total length from cecum to rectum with a ruler. Photographs of the colon were taken above the ruler for subsequent analysis.\u003c/p\u003e\n \u003cp\u003eThe concentration of toxigenic \u003cem\u003eC. difficile\u003c/em\u003e in stool samples was determined as follows: stool samples were weighed and homogenized in vials containing 1 mL of saline solution by using the gentle MACS Dissociator (Miltenyi Biotec) to ensure a uniform mixture. For molecular analysis, 100 \u0026micro;L of the homogenate from each vial were analyzed using the Xpert\u0026trade; \u003cem\u003eC. difficile\u003c/em\u003e assay (GeneXpert, Cepheid, Sunnyvale, California, USA), which detects genes encoding toxin B, binary toxin, and the deletion at position 117 of the tcdC gene. For culture analysis, serial dilutions of 100\u0026micro;L of the homogenate were prepared to achieve 1:1000 and 1:1,000,000 dilutions. A volume of 100 \u0026micro;L from the undiluted homogenate, as well as from the 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e dilutions, was plated in triplicate on brucella agar plates. All plates were incubated anaerobically at 37\u0026deg;C for 48 hours. Following incubation, toxigenic \u003cem\u003eC. difficile\u003c/em\u003e (TCD) colonies were counted, and expressed as colony-forming units (CFU) per microgram of stool sample.\u003c/p\u003e\n \u003cp\u003eFor histological assessment by H\u0026amp;E, the bowel specimens were fixed for 24 hours in 10% formalin and subsequently dehydrated in 70, 96 and 100% alcohol and xylene for paraffin-embedment. Paraffin blocks were cut in 4 micrometers slides and were stained after rehydration with hematoxylin-eosin. All samples were analyzed by a single pathologist blinded to the kind of intervention performed in each animal.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Data processing and statistical analysis\u003c/h2\u003e\n \u003cp\u003eWe used Prism 8.3.0 (GraphPad Software, La Jolla, CA, USA) for data processing and plotting. Values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\n \u003cp\u003eFor statistical analysis, since some data did not meet the criteria of normality and homoscedasticity, we used the Kruskal-Wallis test followed by the post-hoc Man-Whitney test for all the variables evaluated. In all cases, the significance threshold was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Ethics\u003c/h2\u003e\n \u003cdiv class=\"BlockQuote\"\u003e\n \u003cp\u003eC57BL/6 female mice from Charles Rivers were housed in the animal facility of Hospital General Universitario Gregorio Mara\u0026ntilde;\u0026oacute;n, Madrid, Spain (ES280790000087). All animal procedures conformed to EU Directive 2010/63EU and national regulations (RD 53/2013) and were approved by the local ethics committees and the Animal Protection Board of the Comunidad Aut\u0026oacute;noma de Madrid (PROEX 244\u0026thinsp;\u0026minus;\u0026thinsp;19).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.1 Synthesis and characterization of\u003c/em\u003e [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab and [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab radiotracers\u003c/h2\u003e \u003cp\u003eRadiotracer [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab was synthesized with a radiochemical yield of 75.36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11%, a specific activity of 167.24\u0026thinsp;\u0026plusmn;\u0026thinsp;45.14 MBq/mg and a radiochemical purity of higher than 99.99%, as established by TLC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e[\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab was synthesized with a radiochemical yield of 71.58\u0026thinsp;\u0026plusmn;\u0026thinsp;8.19%, a specific activity of 202.76\u0026thinsp;\u0026plusmn;\u0026thinsp;34.04 MBq/mg and a radiochemical purity of higher than 99.99% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Calculated LogP value using [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab was \u0026minus;\u0026thinsp;2.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86, which is in accordance with an hydrophilic behavior.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.2 In vitro characterization of\u003c/em\u003e [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab\u003c/h2\u003e \u003cp\u003eIn vitro stability of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab in PBS 1x remained 100% at 24 hours and slowly decreased to 90.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6% at 192 hours. Stability in mouse serum remained 100% after 1 hour and slowly decreased to 86.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.6% after 192 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). In the case of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab, stability in PBS 1x remained 100% at 26.5 hours and slowly decreased to 95.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% at 100 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe K\u003csub\u003eD\u003c/sub\u003e of bezlotoxumab and [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab measured by ELISA was 24.87\u0026thinsp;\u0026plusmn;\u0026thinsp;9.04 and 26.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.80 pM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Binding kinetic constants measured by LigandTracer were K\u003csub\u003eD\u003c/sub\u003e = 5.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66 pM, k\u003csub\u003ea\u003c/sub\u003e = 3.08 x 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 9.76 x 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and k\u003csub\u003ed\u003c/sub\u003e = 1.52 x 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e \u0026plusmn; 2.87 x 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Quantification of toxin expression in C. difficile strains\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the results of the \u003cem\u003eC. difficile\u003c/em\u003e toxin A/B FIA immunoassay. Data, expressed in fluorescence units, provides a relative measure of the amount of toxins A and B produced by each strain. Strains ATCC 43255 (ribotype 087), and 14243227 (ribotype 027) showed higher toxin A production capacity, while strains with a higher toxin B production capacity were strain ATCC 43255 (ribotype 087), and, especially, strain 14243227 (ribotype 027). As Bezlotoxumab specifically binds to toxin B, this latter strain was selected for the development of the CDI animal model.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eToxin A/B FIA immunoassay results\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRibotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eMean relative toxin production\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eToxin A\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eToxin B\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATCC 43255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e86.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e10.62\u0026thinsp;\u0026plusmn;\u0026thinsp;4.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14243227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e86.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.87\u0026thinsp;\u0026plusmn;\u0026thinsp;11.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13061479\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e63.31\u0026thinsp;\u0026plusmn;\u0026thinsp;7.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Ex vivo biodistribution\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e present the biodistribution values of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab, performed in an independent group of animals (48 h and 5 days of infection). Colon\u0026thinsp;+\u0026thinsp;cecum uptake in CDI animals was 1.9-fold higher at 48 h post-infection (7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.02%ID/g) compared to 5 days (4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88%ID/g). Thus, 48-hour time point was chosen for in vivo studies to optimize PET imaging. Colon\u0026thinsp;+\u0026thinsp;cecum uptake at 48 h was significantly higher in CDI animals (7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.02%ID/g) compared to dysbiosis (3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43%ID/g) and WT animals (3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78%ID/g).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEx vivo biodistribution results expressed as %ID/g\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e48 h p. infection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDysbiosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e21.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e27.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e26.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.03\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLungs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e9.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.53\u0026thinsp;\u0026plusmn;\u0026thinsp;2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidneys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStomach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColon\u0026thinsp;+\u0026thinsp;cecum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntestine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeces\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5 d p. infection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCDI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDysbiosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e22.57\u0026thinsp;\u0026plusmn;\u0026thinsp;8.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e20.03\u0026thinsp;\u0026plusmn;\u0026thinsp;6.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHeart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e7.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLungs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.08\u0026thinsp;\u0026plusmn;\u0026thinsp;4.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e14.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e8.54\u0026thinsp;\u0026plusmn;\u0026thinsp;4.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpleen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e13.10\u0026thinsp;\u0026plusmn;\u0026thinsp;8.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9.92\u0026thinsp;\u0026plusmn;\u0026thinsp;3.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidneys\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.69 2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e11.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStomach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColon\u0026thinsp;+\u0026thinsp;cecum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e6.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntestine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeces\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.6 In vivo PET/CT imaging of\u003c/em\u003e [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab\u003c/h2\u003e \u003cp\u003eQualitatively in vivo PET/CT images of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab confirmed the existence of a specific and localized radiotracer uptake in different segments of the digestive tract. In contrast, the control groups exhibited slight abdominal uptake, basically located in excretion organs and the circulatory system, without no apparent accumulation in the digestive system (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). Quantification of abdominal uptake (SUVmean) showed an increase in [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab uptake in infected animals (SUVmean 0.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06; p\u0026thinsp;=\u0026thinsp;0.003) compared with WT animals (SUVmean 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Validation of CDI animal model\u003c/h2\u003e \u003cp\u003eThe CDI animals exhibited a rapid deterioration within 24 hours following \u003cem\u003eC. difficile\u003c/em\u003e administration. At the imaging time point of 48 hours post-infection, all animals in the CDI group exhibit a CSS\u0026thinsp;\u0026ge;\u0026thinsp;6, whereas in the dysbiosis group only 66% of the animals did. No animal in the WT group showed any symptoms. Ex vivo measurements of colon length demonstrated a significant shortening in the CDI group (5.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40 cm) compared to the dysbiosis (6.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 cm, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and WT (6.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 cm, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eToxin quantification from fecal samples was positive for the presence of \u003cem\u003eC. difficile\u003c/em\u003e in all CDI animals. Quantification of TCD in stool samples was 177.90\u0026thinsp;\u0026plusmn;\u0026thinsp;284.81 CFU/\u0026micro;g.\u003c/p\u003e \u003cp\u003eThe main histopathological lesions were architectural changes in the bowel crypts, increase of lymphocytes and plasma cells in the lamina propria of the bowel, and also presence of polymorphonuclear cells both in the lamina propria and the epithelium.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDespite ongoing advancements in identifying infection sites, effective non-invasive detection methods remain scarce. A major challenge lies in differentiating between inflammatory and infectious processes, which can lead to false-positive diagnoses (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Building on the success of immuno-PET imaging in oncology (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), and the group\u0026rsquo;s previous work in targeting bacterial toxins (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), this study evaluates an immuno-PET tracer using an [\u003csup\u003e89\u003c/sup\u003eZr]Zr-radiolabeled antibody targeting \u003cem\u003eC. difficile\u003c/em\u003e toxin B.\u003c/p\u003e \u003cp\u003eTo this end, two different radiolabelings of the monoclonal antibody Bezlotoxumab were performed. The [\u003csup\u003e125\u003c/sup\u003eI]I-radiolabeling, with a radioisotope half-life of 59.49 days, enabled long-lasting in vitro binding studies up to 2000 min. Additionally, direct radioiodination is a simple, fast and inexpensive reaction, unlike [\u003csup\u003e89\u003c/sup\u003eZr]Zr. However, despite its potential for SPECT imaging, its low gamma energy (35 keV) limits its use as an imaging agent (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Following in vitro binding validation with [\u003csup\u003e125\u003c/sup\u003eI]I, we radiolabeled Bezlotoxumab with [\u003csup\u003e89\u003c/sup\u003eZr]Zr, a well-established radioisotope used in immunoPET for in vivo PET imaging in humans (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Its half-life of 78.42 h allows for longitudinal studies while providing more sensitive PET images.\u003c/p\u003e \u003cp\u003eThe [\u003csup\u003e125\u003c/sup\u003eI]I-radiolabeling was performed with a low amount of radioactivity due to the long half-life of the radioisotope and the low activity required for in vitro binding assays. This resulted in a good radiochemical yield of 75.36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.11% and, consequently, a relatively low specific activity of 167.24\u0026thinsp;\u0026plusmn;\u0026thinsp;45.14 MBq/mg. The high radio-chemical purity of more than 99.99% and the long in vitro stability in PBS ensure that the results obtained in in vitro binding assays are not caused by the binding of free [\u003csup\u003e125\u003c/sup\u003eI]I.\u003c/p\u003e \u003cp\u003eRadiolabeling with [\u003csup\u003e89\u003c/sup\u003eZr]Zr resulted in a good radiochemical yield of 71.58\u0026thinsp;\u0026plusmn;\u0026thinsp;8.19%, similar to other immunotracers for toxins developed by our group (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), and a higher specific activity of 202.76\u0026thinsp;\u0026plusmn;\u0026thinsp;34.04 MBq/mg. As in the previous case, the radio-chemical purity was higher than 99.99%, and in vitro stability in PBS remained above 95.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6% at 100 hours.\u003c/p\u003e \u003cp\u003eK\u003csub\u003eD\u003c/sub\u003e values of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab determined by ELISA showed a high binding affinity of Bezlotoxumab to \u003cem\u003eC. difficile\u003c/em\u003e B toxins, in the picomolar range (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). A slight increase in the K\u003csub\u003eD\u003c/sub\u003e of [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab compared to the unlabeled version may indicate a small loss of affinity. However, the standard deviations of the two values overlap, suggesting that the difference may be due to measurement error. There is a small difference between K\u003csub\u003eD\u003c/sub\u003e values measured by ELISA (26.03\u0026thinsp;\u0026plusmn;\u0026thinsp;3.80 pM) and LigandTracer (5.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.66 pM). Lower K\u003csub\u003eD\u003c/sub\u003e values are usually observed with LigandTracer compared to ELISA because it provides a more reliable result by collecting data over several days with different ligand concentrations, allowing for the measurement of k\u003csub\u003ea\u003c/sub\u003e (3.08 x 10\u003csup\u003e5\u003c/sup\u003e \u0026plusmn; 9.76 x 10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and k\u003csub\u003ed\u003c/sub\u003e (1.52 x 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e \u0026plusmn; 2.87 x 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) kinetic values. Nevertheless, both K\u003csub\u003eD\u003c/sub\u003e values are in the picomolar range, demonstrating the high binding affinity of the antibody. The association and dissociation constants also indicate rapid binding and slow release of the antibody to its target, with values approximately ten times higher and lower, respectively, than those of other antibodies (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the \u003cem\u003eC. difficile\u003c/em\u003e strains evaluated for toxin production, strain 14243227 (ribotype 027) and strain ATCC 43255 (ribotype 087) exhibited the highest relative levels of toxin A production, with strain 14243227 also displaying the highest relative level of toxin B. Bezlotoxumab binds specifically to toxin B; therefore, strain 14243227 was selected for this initial evaluation of a radiotracer based on this antibody, excluding the other strains in order to validate the probe\u0026rsquo;s performance in an optimal scenario. Furthermore, as this strain also exhibits a relatively high production of toxin A, future studies with different targeting antibodies can be conducted using the same strain.\u003c/p\u003e \u003cp\u003eIn this study, we used two different animal models, one for \u003cem\u003eC. difficile\u003c/em\u003e infection and another for dysbiosis. Dysbiosis, an imbalance in the gut microbiota, is linked to several diseases such as inflammatory bowel disease (IBD) (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). We induced dysbiosis using a combination of antibiotics disrupting gut microbiota and favoring \u003cem\u003eC. difficile\u003c/em\u003e infection (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Normally, a healthy gut microbiome protects against pathogens through nutrient competition, antimicrobial production, and maintaining an acidic environment (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). When disrupted, this barrier is compromised, allowing \u003cem\u003eC. difficile\u003c/em\u003e to proliferate. The two models selected allowed us to test the radiotracer\u0026rsquo;s ability to differentiate between active infection versus inflammatory pathways triggered by dysbiosis.\u003c/p\u003e \u003cp\u003eAnimal models were validated in three different ways: 1) colon length reduction indicated the existence of inflammation (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). 2) Bacterial cultures from fecal samples confirmed \u003cem\u003eC. difficile\u003c/em\u003e presence in all CDI animals, and not in the rest. 3) H\u0026amp;E histology confirmed colon inflammation, with higher levels in the dysbiosis model. This finding further underscores the capability of our radiotracer to detect B toxins for diagnosing \u003cem\u003eC. difficile\u003c/em\u003e infection, while avoiding non-specific uptake by sterile colon inflammation\u003c/p\u003e \u003cp\u003eInterestingly, samples from animals with low TCD levels showed similar colonic uptake to those with higher TCD levels. These results suggest that PET/CT imaging with [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab may be more sensitive and reliable than traditional culture methods at low toxin concentrations in stool samples.\u003c/p\u003e \u003cp\u003eEx vivo biodistribution studies were conducted in the three animal models prior to PET/CT imaging to determine the optimal radiotracer uptake time, aiming to identify the best time-point for early diagnosis. Biodistributions were performed at 48 h and 5 days of infection, with 24 h and 4 days of radiotracer uptake respectively. The uptake in the colon and cecum at 48 h post-infection was found to be 1.9 times higher (7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.02%ID/g) compared to the uptake observed at 5 days post-infection (4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88%ID/g). Based on this finding, the 48-hour time point was selected for subsequent in vivo PET imaging studies to maximize detection sensitivity. Furthermore, at 48 h post-infection, colon and cecum uptake was significantly increased in CDI animals (7.66\u0026thinsp;\u0026plusmn;\u0026thinsp;5.02%ID/g) compared to dysbiosis animals (3.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43%ID/g) and wild-type (WT) controls (3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78%ID/g). These differences suggest that this time frame is optimal for distinguishing active CDI from dysbiosis inflammation and healthy controls.\u003c/p\u003e \u003cp\u003ePET/CT imaging analysis of the CDI animal model clearly showed a distinct and precisely localized uptake pattern of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab in different segments of the lower digestive tract, typically associated with CDI. Interestingly, dysbiosis and WT models exhibited a markedly different uptake profile, mainly localized in excretory organs and circulatory system, with low accumulation within the gastrointestinal tract. This disparity illustrates the specificity of the radiotracer uptake in delineating the pathophysiological processes associated with bacterial infection. In all animal models, a high bone and joint uptake is observed. Demetallization of [\u003csup\u003e89\u003c/sup\u003eZr]Zr in DFO-conjugated 150 kDa IgG isotype antibodies in plasma is a common issue observed in previous studies (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), often leading to uptake of free [\u003csup\u003e89\u003c/sup\u003eZr]Zr in bone tissue, especially in joints (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). This qualitative analysis of the images is supported by the quantitative analysis, where an increase in the abdominal uptake was observed in the infected group compared to the wild type animals.\u003c/p\u003e \u003cp\u003eThis study presents certain limitations that should be considered. First, the sample size for the ex vivo biodistribution analysis performed 5 days post-infection was insufficient to obtain statistically significant results, although the trend suggested higher uptake in the colon of CDI animals. Second, only one strain of \u003cem\u003eC. difficile\u003c/em\u003e (ribotype 027) was used, the one that demonstrated the highest level of toxin B production among the evaluated strains. While this choice facilitated radiotracer validation in an optimal scenario, future studies should evaluate tracer performance on strains with lower toxin B production to determine a more realistic detection limit. Third, PET image quantification was performed using an abdominal ROI rather than a more specific colonic and cecum ROI due to challenges in differentiating these organs on CT images. This lack of specificity may have affected the accuracy of uptake measurements. In addition, the study was conducted in a mouse model, which, while useful for preclinical evaluation, does not fully replicate the complexity of human CDI. Differences in immune response, gut microbiota composition, and toxin distribution between mice and humans may limit the direct translation of findings to clinical practice (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Finally, the study did not evaluate potential cross-reactivity with other gut bacteria or inflammatory conditions, which may affect the specificity of the tracer in clinical settings. Addressing these limitations in future research is warranted to refine the diagnostic accuracy and translational potential of this immunoPET approach.\u003c/p\u003e \u003cp\u003eIn case of positive results this radiotracer has the potential to enhance the diagnosis of CDI and improve patient prognosis. The ability to non-invasively detect CDI in early stages represents a significant advancement over invasive techniques such as colonoscopy, which carries substantial risks in severely ill patients (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Early detection would allow for timely intervention, potentially reducing disease progression and severe complications such as pseudomembranous colitis or toxic megacolon (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). This radiotracer also enables the specific visualization of toxin-producing bacterial foci in vivo, which may be important for precise differentiation between active infection and colonization. This could be particularly advantageous in complex clinical scenarios, such as patients with coexisting gastrointestinal disorders or recurrent CDI, where misdiagnosis can lead to unnecessary treatment or delayed care. Recurrent CDI remains a major challenge, often requiring repeated and prolonged courses of therapy (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). The proposed radiotracer could identify residual infection sites and differentiate recurrent infection from post-treatment inflammatory changes, reducing the reliance on empirical therapy and potentially curbing antimicrobial resistance associated with overuse of broad-spectrum antibiotics. Additionally, the ability to monitor the response to therapy in vivo would enable clinicians to adjust treatments dynamically, potentially improving patient outcomes.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWe have synthesized and characterized two \u003cem\u003eC. difficile\u003c/em\u003e specific radiotracers based on Bezlotoxumab. Radiolabeling with both [\u003csup\u003e125\u003c/sup\u003eI]I and [\u003csup\u003e89\u003c/sup\u003eZr]Zr was successfully achieved with high radiochemical yield, purity and stability, which supports their use for in vivo assessments. In vitro binding assays using [\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab confirmed a high and rapid uptake of bezlotoxumab, with slow release even at long time-points. PET/CT imaging using [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab revealed a distinct and specific uptake in the digestive tract of CDI animals, while this uptake was not observed in the other groups. These findings support the radiotracer's ability to differentiate between active infection and dysbiosis, providing valuable evidence of its diagnostic potential.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e[\u003csup\u003e125\u003c/sup\u003eI]I-Beztxab\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;[\u003csup\u003e125\u003c/sup\u003eI]I radiolabeled bezlotoxumab\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]F-FDG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;[\u003csup\u003e18\u003c/sup\u003eF]F-fluorodeoxyglucose\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;[\u003csup\u003e89\u003c/sup\u003eZr]Zr radiolabeled bezlotoxumab\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. difficile\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Clostridiodes difficile\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCDI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Clostridioides difficile infection\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCFU\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Colony-forming units\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Computed Tomography\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEIA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Enzyme immunoassay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGDH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Glutamate dehydrogenase\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hematoxylin and Eosin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHAIs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hospital-acquired infections\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIBD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Inflammatory bowel disease\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMRI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Magnetic Resonance Imaging\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePET\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Positron Emission Tomography\u003c/p\u003e\n\u003cp\u003ep-NCS-Bz-DFO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;p-Isothiocyanatobenzyl-deferoxamine\u003c/p\u003e\n\u003cp\u003eROI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Region of interest\u003c/p\u003e\n\u003cp\u003eSPECT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Single Photon Emission Computed Tomography\u003c/p\u003e\n\u003cp\u003eTCD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Toxigenic \u003cem\u003eC. difficile\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Wild-type\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCompeting interests\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been funded by Instituto de Salud Carlos III (ISCIII) through the projects \"PT23/00027\" and “PI23/01405”, and co-funded by the European Union. Work supported by Comunidad de Madrid, project S2022/BMD-7403 (RENIM-CM). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MICIU) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIU/AEI/10.13039/501100011033). Grant PTA2022-021556-I funded by MICIU/AEI /10.13039/501100011033 and by FSE+. Authors also thank Fundación Ramón Areces for their support.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eEthics approval and consent to participate\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6 female mice from Charles Rivers were housed in the animal facility of Hospital General Universitario Gregorio Marañón, Madrid, Spain (ES280790000087). All animal procedures conformed to EU Directive 2010/63EU and national regulations (RD 53/2013) and were approved by the local ethics committees and the Animal Protection Board of the Comunidad Autónoma de Madrid (PROEX 244-19).\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAvailability of data and material\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are included within the article. The data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAuthors' contributions\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eMGA and LC contributed equally to this work, performing the main experiments, data analysis and drafting the manuscript. LA performed the \u003cem\u003eC. difficile\u003c/em\u003e toxin quantification of different strains and the analysis of stool samples to determine the presence of toxigenic species. MIG assisted in the synthesis of the radiotracer and ex vivo biodistribution studies. MJFA performed the H\u0026amp;E staining of colon tissues and their subsequent analysis. DS and SS provided the \u003csup\u003e125\u003c/sup\u003eI radioisotope and directed its radiolabeling and characterization, including ELISA and ligand tracer assays. EB and PM provided the \u003cem\u003eC. difficile\u003c/em\u003e background and opportunity for new diagnostic tools. MD helped with experimental design and interpretation of results. Finally, Dr. BS developed the experimental design and interpretation of results, and supervised the work. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eConsent for publication\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAcknowledgments\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Alexandra de Francisco and Diego Eguibar from the Imaging Laboratory for Small Animal Experimentation of Instituto de Investigación Sanitaria Gregorio Marañón for their excellent work with animal preparation and imaging protocols.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCzepiel J, Dr\u0026oacute;żdż M, Pituch H, Kuijper EJ, Perucki W, Mielimonka A, Goldman S, Wultańska D, Garlicki A, Biesiada G. Clostridium difficile infection. European Journal of Clinical Microbiology \u0026amp; Infectious Diseases. 2019;38:1211-21.\u003c/li\u003e\n\u003cli\u003eRose AN, Baggs J, Kazakova SV, Guh AY, Sarah HY, McCarthy NL, Jernigan JA, Reddy SC. 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European Journal of Nuclear Medicine and Molecular Imaging. 2019;46:1966-77.\u003c/li\u003e\n\u003cli\u003eAbou DS, Ku T, Smith-Jones PM. In vivo biodistribution and accumulation of 89Zr in mice. Nuclear medicine and biology. 2011;38(5):675-81.\u003c/li\u003e\n\u003cli\u003eBest EL, Freeman J, Wilcox MH. Models for the study of Clostridium difficile infection. Gut microbes. 2012;3(2):145-67.\u003c/li\u003e\n\u003cli\u003eRajack F, Medford S, Naab T. Clostridioides difficile infection leading to fulminant colitis with toxic megacolon. Autopsy and Case Reports. 2023;13:e2023457.\u003c/li\u003e\n\u003cli\u003eFinn E, Andersson FL, Madin-Warburton M. Burden of Clostridioides difficile infection (CDI)-a systematic review of the epidemiology of primary and recurrent CDI. BMC infectious diseases. 2021;21(1):456.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Clostridioides difficile, ImmunoPET, Bacterial toxin targeting, Imaging of infection, Radiotracer development","lastPublishedDoi":"10.21203/rs.3.rs-6294683/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6294683/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e \u003cem\u003eClostridioides difficile\u003c/em\u003e infection (CDI) is a major healthcare challenge, associated with high morbidity and mortality. Current diagnostic methods have limitations in specificity and invasiveness, necessitating the development of novel, non-invasive imaging techniques. This study aims to develop and evaluate an immunoPET radiotracer targeting \u003cem\u003eC. difficile\u003c/em\u003e toxin B for in vivo CDI detection in mice model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Monoclonal antibody, Bezlotoxumab, was radiolabeled with [\u003csup\u003e125\u003c/sup\u003eI]I for in vitro characterization and [\u003csup\u003e89\u003c/sup\u003eZr]Zr for in vivo PET imaging, resulting in high radiochemical yields (75.36 ± 4.11 % for [\u003csup\u003e125\u003c/sup\u003eI]I and 71.58 ± 8.19 % for [\u003csup\u003e89\u003c/sup\u003eZr]Zr) and purities (\u0026gt;99.99 % both cases), with stable binding properties. PET/CT imaging 48h post infection in an animal model of CDI (C57BL/6 mice employing ribotype 027 strain) demonstrated specific accumulation of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Beztxab in the colon and cecum of infected mice, distinguishing CDI from dysbiosis and healthy controls, and confirmed by PET quantification and ex vivo biodistribution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e We successfully developed an immunoPET radiotracer targeting toxin B for CDI detection. Its application in a CDI animal model proved its capacity to detect the source of infection with high specificity, avoiding sterile inflammation.\u003c/p\u003e","manuscriptTitle":"Development of a Toxin-selective immunotracer for the specific in vivo detection of Clostridioides difficile infection by immunoPET","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 01:28:29","doi":"10.21203/rs.3.rs-6294683/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-04-05T11:35:03+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-31T09:43:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-31T09:12:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-31T08:21:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2025-03-27T04:57:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b2eaf6b-1fa1-4bf6-80b5-896c9c644547","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-16T16:00:59+00:00","versionOfRecord":{"articleIdentity":"rs-6294683","link":"https://doi.org/10.1186/s41181-025-00350-x","journal":{"identity":"ejnmmi-radiopharmacy-and-chemistry","isVorOnly":false,"title":"EJNMMI Radiopharmacy and Chemistry"},"publishedOn":"2025-06-13 15:57:34","publishedOnDateReadable":"June 13th, 2025"},"versionCreatedAt":"2025-05-06 01:28:29","video":"","vorDoi":"10.1186/s41181-025-00350-x","vorDoiUrl":"https://doi.org/10.1186/s41181-025-00350-x","workflowStages":[]},"version":"v1","identity":"rs-6294683","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6294683","identity":"rs-6294683","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}