(68)Ga-DOTA-Siglec-9 PET/CT imaging of peri-implant tissue responses and staphylococcal infections

Helena Ahtinen, Julia Kulkova, Laura Lindholm, Erkki Eerola, Antti J Hakanen, Niko Moritz, Mirva Söderström, Tiina Saanijoki, Sirpa Jalkanen, Anne Roivainen, Hannu T Aro, Helena Ahtinen, Julia Kulkova, Laura Lindholm, Erkki Eerola, Antti J Hakanen, Niko Moritz, Mirva Söderström, Tiina Saanijoki, Sirpa Jalkanen, Anne Roivainen, Hannu T Aro

Abstract

Background: Staphylococcus epidermidis (S. epidermidis) has emerged as one of the leading pathogens of biomaterial-related infections. Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial molecule controlling extravasation of leukocytes. Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a leukocyte ligand of VAP-1. We hypothesized that (68)Ga-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated Siglec-9 motif containing peptide ((68)Ga-DOTA-Siglec-9) could detect inflammatory response due to S. epidermidis peri-implant infection by positron emission tomography (PET).

Methods: Thirty Sprague-Dawley rats were randomized into three groups. A sterile catheter was implanted into the medullary canal of the left tibia. In groups 1 and 2, the implantation was followed by peri-implant injection of S. epidermidis or Staphylococcus aureus (S. aureus) with adjunct injections of aqueous sodium morrhuate. In group 3, sterile saline was injected instead of bacteria and no aqueous sodium morrhuate was used. At 2 weeks after operation, (68)Ga-DOTA-Siglec-9 PET coupled with computed tomography (CT) was performed with the measurement of the standardized uptake value (SUV). The presence of the implant-related infection was verified by microbiological analysis, imaging with fluorescence microscope, and histology. The in vivo PET results were verified by ex vivo measurements by gamma counter.

Results: In group 3, the tibias with implanted sterile catheters showed an increased local uptake of (68)Ga-DOTA-Siglec-9 compared with the intact contralateral bones (SUVratio +29.5%). (68)Ga-DOTA-Siglec-9 PET detected inflammation induced by S. epidermidis and S. aureus catheter-related bone infections (SUVratio +58.1% and +41.7%, respectively). The tracer uptake was significantly higher in the S. epidermidis group than in group 3 without bacterial inoculation, but the difference between S. epidermidis and S. aureus groups was not statistically significant. The difference between the S. aureus group and group 3 was neither statistically significant.

Conclusion: PET/CT imaging with novel (68)Ga-DOTA-Siglec-9 tracer was able to detect inflammatory tissue response induced by catheter implantation and staphylococcal infections.

Keywords: 68Ga-DOTA-Siglec-9; Implant infection; Osteomyelitis; PET; S. aureus; S. epidermidis; VAP-1.

Figures

Figure 1
Figure 1
Schematic illustration of the operated tibia. (a) The bone was sliced into five sections for further characterization: (1) histological analysis (decalcified sections); (2) histological analysis (non-decalcified sections); (3) microbiological analysis; (4) ex vivo radioactivity measurement; (5) fluorescent microscopy of biofilm formation. (b) Quantification of the in vivo PET/CT data using two regions of interest (ROI).
Figure 2
Figure 2
Histological-analysis. (a)Staphylococcus epidermidis group, van Gieson stain. Inflammatory response is expressed as a sunburst type periosteal reaction (PR) and partial resorption of the cortex (C). Implant is denoted as ‘Impl’. (b)S. epidermidis group, hematoxylin and eosin stain. Increased number of polymorphonuclear leukocytes were observed in the medullary canal in the proximity of the implant (arrow). This layer is surrounded by granulation tissue; (c)Staphylococcus aureus group, van Gieson stain; Inflammatory response is expressed as a circumferential sunburst type periosteal reaction (PR) and an almost complete resorption of the cortex (C). (d)S. aureus group, hematoxylin and eosin stain. Polymorphonuclear leukocytes are seen in the proximity of the implant (arrow). (e) Sterile catheter implant group, van Gieson stain; Periosteal and cortical reactions are absent. Reactive bone formation (RB) is seen around the implant. (f) Sterile catheter implant group, hematoxylin and eosin stain. The implant is surrounded by fibrous capsule and reactive bone (RB).
Figure 3
Figure 3
Fluorescence microscope images of catheters. The catheter surfaces stained with BacLite Kit. Biofilm clusters composed of aggregates of viable coccoid bacterial cells, which were stained with SYTO® 9 (green color) and dead bacteria stained with PI (orange-red). (a)S. epidermidis; (b)S. aureus; (c) sterile catheter.
Figure 4
Figure 4
Representative sagittal and coronal PET/CT images. Representative sagittal and coronal PET/CT images with 68Ga-DOTA-Siglec-9 peptide of the rats with (a) catheter-related S. epidermidis infection of the right tibia, (b) catheter-related S. aureus infection of the right tibia, or (c) catheter implantation in the right tibia without bacterial inoculation. High focal uptake of radioactivity in the infected right tibia is observed (red arrows) compared with the contralateral intact left tibia (white arrows). Excess of radioactivity is excreted through the kidneys (two headed black arrow) to the urinary bladder (black arrow).
Figure 5
Figure 5
Comparison of the uptake of68Ga-DOTA-Siglec-9 in the three groups of animals. The three groups are as follows: S. epidermidis infection, S. aureus infection, and sterile catheter implant. The uptake is shown as the intra-animal SUVratio values of the proximal tibias measured in vivo (ROI1 in Figure 1) and ex vivo (section 4 in Figure 1). Box plots of are showing median, first and third quartiles, minimum and maximum values, and outliers. Comparison between the groups performed with ANOVA with Tukey's post hoc test.

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