A fluorogenic micrococcal nuclease-based probe for fast detection and optical imaging of Staphylococcus aureus in prosthetic joint and fracture-related infections.

PURPOSE: Staphylococcus aureus is the most common and impactful multi-drug resistant pathogen implicated in (periprosthetic) joint infections (PJI) and fracture-related infections (FRI). Therefore, the present proof-of-principle study was aimed at the rapid detection of S. aureus in synovial fluids and biofilms on extracted osteosynthesis materials through bacteria-targeted fluorescence imaging with the 'smart-activatable' DNA-based AttoPolyT probe. This fluorogenic oligonucleotide probe yields large fluorescence increases upon cleavage by micrococcal nuclease, an enzyme secreted by S. aureus. METHODS: Synovial fluids from patients with suspected PJI and extracted osteosynthesis materials from trauma patients with suspected FRI were inspected for S. aureus nuclease activity with the AttoPolyT probe. Biofilms on osteosynthesis materials were imaged with the AttoPolyT probe and a vancomycin-IRDye800CW conjugate (vanco-800CW) specific for Gram-positive bacteria. RESULTS: 38 synovial fluid samples were collected and analyzed. Significantly higher fluorescence levels were measured for S. aureus-positive samples compared to, respectively, other Gram-positive bacterial pathogens (p < 0.0001), Gram-negative bacterial pathogens (p = 0.0038) and non-infected samples (p = 0.0030), allowing a diagnosis of S. aureus-associated PJI within 2 h. Importantly, S. aureus-associated biofilms on extracted osteosynthesis materials from patients with FRI were accurately imaged with the AttoPolyT probe, allowing their correct distinction from biofilms formed by other Gram-positive bacteria detected with vanco-800CW within 15 min. CONCLUSION: The present study highlights the potential clinical value of the AttoPolyT probe for fast and accurate detection of S. aureus infection in synovial fluids and biofilms on extracted osteosynthesis materials.

Preclinical evaluation of 2-[18F]fluorodeoxysorbitol as a tracer for targeted imaging of Enterobacterales infection.

Fluorine-18-fluorodeoxyglucose ([18F]FDG) positron emission tomography (18F-FDG-PET) is widely used for the detection of inflammatory and infectious diseases. Although this modality has proven to be a useful diagnostic tool, reliable distinction of bacterial infection from sterile inflammation or even from a malignancy remains challenging. Therefore, there is a need for bacteria-specific tracers for PET imaging that facilitate a reliable distinction of bacterial infection from other pathology. The present study was aimed at exploring the potential of 2-[18F]-fluorodeoxysorbitol ([18F]FDS) as a tracer for detection of Enterobacterales infections. Sorbitol is a sugar alcohol that is commonly metabolized by bacteria of the Enterobacterales order, but not by mammalian cells, which makes it an attractive candidate for targeted bacterial imaging. The latter is important in view of the serious clinical implications of infections caused by Enterobacterales. Here we demonstrate that sorbitol-based PET can be applied to detect a broad range of clinical bacterial isolates not only in vitro, but also in blood and ascites samples from patients suffering from Enterobacterales infections. Notably, the possible application of [18F]FDS is not limited to Enterobacterales since Pseudomonas aeruginosa and Corynebacterium jeikeium also showed substantial uptake of this tracer. We conclude that [18F]FDS is a promising tracer for PET-imaging of infections caused by a group of bacteria that can cause serious invasive disease.

Sonication of Vascular Grafts and Endografts to Diagnose Vascular Graft Infection: a Head-To-Head Comparison with Conventional Culture and Its Clinical Impact.

Vascular graft and endograft infection (VGEI) is a severe complication associated with high mortality and is often challenging to diagnose. For the definitive microbiological diagnosis, sonication of vascular grafts may increase the microbiological yield of these biofilm-associated infections. The objective of this study was to determine whether sonication of explanted vascular grafts and endografts results in a higher diagnostic accuracy than conventional culture methods and aids in clinical decision-making. A prospective diagnostic study was performed comparing conventional culture with sonication culture of explanted vascular grafts in patients treated for VGEI. Explanted (endo)grafts were cut in halves and were either subjected to sonication or conventional culture. Criteria based on the Management of Aortic Graft Infection Collaboration (MAGIC) case definition of VGEI were used for definitive diagnosis. The relevance of sonication cultures was assessed by expert opinion to determine the clinical impact on decision-making. Fifty-seven vascular (endo)graft samples from 36 patients (four reoperations; 40 episodes) treated for VGEI were included; 32 episodes were diagnosed with VGEI. Both methods showed a positive culture in 81% of the cases. However, sonication culture detected clinically relevant microorganisms that went unnoticed by conventional culturing in 9 out of 57 samples (16%, 8 episodes) and provided additional relevant information regarding growth densities in another 11 samples (19%, 10 episodes). Sonication of explanted vascular grafts and endografts improves the microbiological yield and aids in the clinical decision-making for patients with a suspected VGEI compared to conventional culture alone. IMPORTANCE Sonication culture of explanted vascular grafts was shown to be a noninferior method compared to conventional culturing in diagnosing vascular graft and endograft infection (VGEI). Moreover, sonication culture has probable additional value in microbiological characterization of VGEI by giving more detailed information on growth densities, especially when the conventional culture shows intermediate growth. In this prospective design, for the first time, a direct comparison is made between sonication culturing and conventional culturing in VGEI, while taking clinical interpretation into account. Therefore, this study is another step toward a more accurate microbiological diagnosis of VGEI, influencing clinical decision-making.

First evaluation of a commercial multiplex PCR panel for rapid detection of pathogens associated with acute joint infections.

Background: prompt recognition and identification of the causative microorganism in acute septic arthritis of native and prosthetic joints is vital to increase the chances of successful treatment. The aim of this study was to independently assess the diagnostic accuracy of the multiplex BIOFIRE® Joint Infection (JI) Panel (investigational use only) in synovial fluid for rapid diagnosis. Methods: synovial fluid samples were collected at the University Medical Center Groningen from patients who had a clinical suspicion of a native septic arthritis, early acute (post-operative, within 3 months after arthroplasty) periprosthetic joint infection (PJI) or late acute (hematogenous, ≥ 3 months after arthroplasty) PJI. JI Panel results were compared to infection according to Musculoskeletal Infection Society criteria and culture-based methods as reference standard. Results: a total of 45 samples were analysed. The BIOFIRE JI Panel showed a high specificity (100 %, 95 % confidence interval (CI): 78-100) in all patient categories. Sensitivity was 83 % (95 % CI: 44-97) for patients with a clinical suspicion of native septic arthritis ( n = 12 ), 73 % (95 % CI: 48-89) for patients with a clinical suspicion of a late acute PJI ( n = 14 ), and 30 % (95 % CI: 11-60) for patients with a clinical suspicion of an early acute PJI ( n = 19 ). Conclusion: the results of this study indicate a clear clinical benefit of the BIOFIRE JI Panel in patients with a suspected native septic arthritis and late acute (hematogenous) PJI, but a low clinical benefit in patients with an early acute (post-operative) PJI due to the absence of certain relevant microorganisms, such as Staphylococcus epidermidis, from the panel.

Diagnosis and treatment of vascular graft and endograft infections: a structured clinical approach.

A vascular graft or endograft infection (VGEI) is a severe complication that can occur after vascular graft or endograft surgery and is associated with high morbidity and mortality rates. A multidisciplinary approach, consisting of a team of vascular surgeons, infectious diseases specialists, medical microbiologists, radiologists, nuclear medicine specialists, and hospital pharmacists, is needed to adequately diagnose and treat VGEI. A structured diagnostic, antibiotic, and surgical treatment algorithm helps clinical decision making and ultimately aims to improve the clinical outcome of patients with a VGEI.

Bacteria-targeted fluorescence imaging of extracted osteosynthesis devices for rapid visualization of fracture-related infections.

PURPOSE: Fracture-related infection (FRI) is a serious complication in orthopedic trauma surgery worldwide. Especially, the distinction of infection from sterile inflammation and the detection of low-grade infection are highly challenging. The objective of the present study was to obtain proof-of-principle for the use of bacteria-targeted fluorescence imaging to detect FRI on extracted osteosynthesis devices as a step-up towards real-time image-guided trauma surgery. METHODS: Extracted osteosynthesis devices from 13 patients, who needed revision surgery after fracture treatment, were incubated with a near-infrared fluorescent tracer composed of the antibiotic vancomycin and the fluorophore IRDye800CW (i.e., vanco-800CW). Subsequently, the devices were imaged, and vanco-800CW fluorescence signals were correlated to the results of microbiological culturing and to bacterial growth upon replica plating of the imaged devices on blood agar. RESULTS: Importantly, compared to culturing, the bacteria-targeted fluorescence imaging of extracted osteosynthesis devices with vanco-800CW allows for a prompt diagnosis of FRI, reducing the time-to-result from days to less than 30 min. Moreover, bacteria-targeted imaging can provide surgeons with real-time visual information on the presence and extent of infection. CONCLUSION: Here, we present the first clinical application of fluorescence imaging for the detection of FRI. We conclude that imaging with vanco-800CW can provide early, accurate, and real-time visual diagnostic information on FRI in the clinical setting, even in the case of low-grade infections.

Ultrafast Photoclick Reaction for Selective 18F-Positron Emission Tomography Tracer Synthesis in Flow.

The development of very fast, clean, and selective methods for indirect labeling in PET tracer synthesis is an ongoing challenge. Here we present the development of an ultrafast photoclick method for the synthesis of short-lived 18F-PET tracers based on the photocycloaddition reaction of 9,10-phenanthrenequinones with electron-rich alkenes. The respective precursors are synthetically easily accessible and can be functionalized with various target groups. Using a flow photo-microreactor, the photoclick reaction can be performed in 60 s, and clinically relevant tracers for prostate cancer and bacterial infection imaging were prepared to demonstrate practicality of the method.

Comparison of two fluorescent probes in preclinical non-invasive imaging and image-guided debridement surgery of Staphylococcal biofilm implant infections.

Implant-associated infections are challenging to diagnose and treat. Fluorescent probes have been heralded as a technologic advancement that can improve our ability to non-invasively identify infecting organisms, as well as guide the inexact procedure of surgical debridement. This study's purpose was to compare two fluorescent probes for their ability to localize Staphylococcus aureus biofilm infections on spinal implants utilizing noninvasive optical imaging, then assessing the broader applicability of the more successful probe in other infection animal models. This was followed by real-time, fluorescence image-guided surgery to facilitate debridement of infected tissue. The two probe candidates, a labelled antibiotic that targets peptidoglycan (Vanco-800CW), and the other, a labelled antibody targeting the immunodominant Staphylococcal antigen A (1D9-680), were injected into mice with spine implant infections. Mice were then imaged noninvasively with near infrared fluorescent imaging at wavelengths corresponding to the two probe candidates. Both probes localized to the infection, with the 1D9-680 probe showing greater fidelity over time. The 1D9-680 probe was then tested in mouse models of shoulder implant and allograft infection, demonstrating its broader applicability. Finally, an image-guided surgery system which superimposes fluorescent signals over analog, real-time, tissue images was employed to facilitate debridement of fluorescent-labelled bacteria.

Image-guided in situ detection of bacterial biofilms in a human prosthetic knee infection model: a feasibility study for clinical diagnosis of prosthetic joint infections.

PURPOSE: Due to an increased human life expectancy, the need to replace arthritic or dysfunctional joints by prosthetics is higher than ever before. Prosthetic joints are unfortunately inherently susceptible to bacterial infection accompanied by biofilm formation. Accurate and rapid diagnosis is vital to increase therapeutic success. Yet, established diagnostic modalities cannot directly detect bacterial biofilms on prostheses. Therefore, the present study was aimed at investigating whether arthroscopic optical imaging can accurately detect bacterial biofilms on prosthetic joints. METHODS: Here, we applied a conjugate of the antibiotic vancomycin and the near-infrared fluorophore IRDye800CW, in short vanco-800CW, in combination with arthroscopic optical imaging to target and visualize biofilms on infected prostheses. RESULTS: We show in a human post-mortem prosthetic knee infection model that a staphylococcal biofilm is accurately detected in real time and distinguished from sterile sections in high resolution. In addition, we demonstrate that biofilms associated with the clinically most relevant bacterial species can be detected using vanco-800CW. CONCLUSION: The presented image-guided arthroscopic approach provides direct visual diagnostic information and facilitates immediate appropriate treatment selection.

The smart activatable P2&3TT probe allows accurate, fast, and highly sensitive detection of Staphylococcus aureus in clinical blood culture samples.

Staphylococcus aureus bacteraemia (SAB) is associated with high mortality and morbidity rates. Yet, there is currently no adequate diagnostic test for early and rapid diagnosis of SAB. Therefore, this study was aimed at exploring the potential for clinical implementation of a nuclease-activatable fluorescent probe for early diagnosis of SAB. To this end, clinical blood culture samples from patients with bloodstream infections were incubated for 1 h with the "smart" activatable P2&3TT probe, the total assay time being less than 2 h. Cleavage of this probe by the secreted S. aureus enzyme micrococcal nuclease results in emission of a readily detectable fluorescence signal. Incubation of S. aureus-positive blood culture samples with the P2&3TT probe resulted in 50-fold higher fluorescence intensity levels than incubation with culture-negative samples. Moreover, incubation of the probe with non-S. aureus-positive blood cultures yielded essentially background fluorescence intensity levels for cultures with Gram-negative bacteria, and only ~ 3.5-fold increased fluorescence intensity levels over background for cultures with non-S. aureus Gram-positive bacteria. Importantly, the measured fluorescence intensities were dose-dependent, and a positive signal was clearly detectable for S. aureus-positive blood cultures with bacterial loads as low as ~ 7,000 colony-forming units/mL. Thus, the nuclease-activatable P2&3TT probe distinguishes clinical S. aureus-positive blood cultures from non-S. aureus-positive blood cultures and culture-negative blood, accurately, rapidly and with high sensitivity. We conclude that this probe may enhance the diagnosis of SAB.

Fighting Staphylococcus aureus infections with light and photoimmunoconjugates.

Infections caused by multidrug-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared with today's antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of ROS by an appropriate photosensitizer. Here, we conjugated the near-infrared photosensitizer IRDye700DX to a fully human mAb, specific for the invariantly expressed staphylococcal antigen immunodominant staphylococcal antigen A (IsaA). The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human postmortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the nontoxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.

A Facile and Reproducible Synthesis of Near-Infrared Fluorescent Conjugates with Small Targeting Molecules for Microbial Infection Imaging.

Optical imaging of microbial infections, based on the detection of targeted fluorescent probes, offers high sensitivity and resolution with a relatively simple and portable setup. As the absorbance of near-infrared (NIR) light by human tissues is minimal, using respective tracers, such as IRdye800CW, enables imaging deeper target sites in the body. Herein, we present a general strategy for the conjugation of IRdye800CW and IRdye700DX to small molecules (vancomycin and amphotericin B) to provide conjugates targeted toward bacterial and fungal infections for optical imaging and photodynamic therapy. In particular, we present how the use of coupling agents (such as HBTU or HATU) leads to high yields (over 50%) in the reactions of amines and IRDye-NHS esters and how precipitation can be used as a convenient purification strategy to remove excess of the targeting molecule after the reaction. The high selectivity of the synthesized model compound Vanco-800CW has been proven in vitro, and the development of analogous agents opens up new possibilities for diagnostic and theranostic purposes. In times of increasing microbial resistance, this research gives us access to a platform of new fluorescent tracers for the imaging of infections, enabling early diagnosis and respective treatment.

Yeast Infections after Esophagectomy: A Retrospective Analysis.

Esophageal malignancy is a disease with poor prognosis. Curative therapy incorporates surgery and is burdensome with high rates of infection morbidity and mortality. The role of yeast as causative organisms of post-esophagectomy infections is poorly defined. Consequently, the benefits of specific antifungal prophylactic therapy in improving patient outcome are unclear. Therefore, this study aimed at investigating the incidence of yeast infections at the University Medical Center Groningen among 565 post-esophagectomy patients between 1991 and 2017. The results show that 7.3% of the patients developed a yeast infection after esophageal resection with significantly increased incidence among patients suffering from diabetes mellitus. For patients with yeast infections, higher Acute Physiology and Chronic Health Evaluation (APACHE) II scores, more frequent intensive care unit readmissions, prolonged hospital stays and higher mortality rates were observed. One-year survival was significantly lower for patients with a yeast infection, as well as diabetes mellitus and yeast-positive pleural effusion. We conclude that the incidence of yeast infections following esophagectomy is considerable, and that patients with diabetes mellitus are at increased risk. Furthermore, yeast infections are associated with higher complication rates and mortality. These observations encourage further prospective investigations on the possible benefits of antifungal prophylactic therapy for esophagectomy patients.

Publisher Correction: In vitro imaging of bacteria using 18F-fluorodeoxyglucose micro positron emission tomography.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Sonication of heart valves detects more bacteria in infective endocarditis.

Optimal antimicrobial treatment of infective endocarditis requires identification and susceptibility patterns of pathogens. Sonication of explanted heart valves could increase the identification and culture of pathogens, as shown in prosthetic joint and pacemaker/ICD infections. We tested 26 explanted heart valves from 20 patients with active definite endocarditis for added diagnostic value of sonication to the standard microbiological workup in a prospective diagnostic proof of concept study. Two sonication protocols (broth enrichment vs. centrifugation) were compared in an additional 35 negative control valves for contamination rates. We selected sonication/centrifugation based on acceptable false positive rates (11.4%; 4/35). Sonication/enrichment yielded many false positive results in negative controls (28.6%; 10/35), mainly Propionibacterium acnes (next-generation sequencing excluded technical problems). Compared to direct culture only, adding sonication/centrifugation (including molecular testing) significantly increased the diagnostic yield from 6/26 to 17/26 valves (p = 0.003). Most importantly, culture positives almost doubled (from 6 to 10), providing unique quantitative information about antimicrobial susceptibility. Even if direct molecular testing was added to the standard workup, sonication/centrifugation provided additional diagnostic information in a significant number of valves (8/26; 31%; p = 0.013). We concluded that sonication/centrifugation added relevant diagnostic information in the workup of heart valves with infective endocarditis, with acceptable contamination rates.

Ex Vivo Tracer Efficacy in Optical Imaging of Staphylococcus Aureus Nuclease Activity.

The key to effective treatment of bacterial infections is a swift and reliable diagnosis. Current clinical standards of bacterial diagnosis are slow and laborious. There are several anatomical imaging modalities that can detect inflammation, but none can distinguish between bacterial and sterile inflammation. Novel tracers such as smart activatable fluorescent probes represent a promising development that allow fast and specific testing without the use of ionizing radiation. Previously, a smart activatable probe was developed that is a substrate for the micrococcal nuclease as produced by Staphylococcus aureus. In the present study, the function of this probe was validated. Practical applicability in terms of sensitivity was assessed by incubation of the probe with 26 clinical S. aureus isolates, and probe specificity was verified by incubation with 30 clinical isolates and laboratory strains of various bacterial pathogens. The results show that the nuclease-specific probe was activated by all tested S. aureus isolates and laboratory strains with a threshold of ~106-107 cells/mL. The probe was also activated by certain opportunistic staphylococci. We therefore propose that the studied nuclease probe represents a significant step forward to address the need for a rapid, practical, and precise method to detect infections caused by S. aureus.

Noninvasive optical and nuclear imaging of Staphylococcus-specific infection with a human monoclonal antibody-based probe.

Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, 89Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.

In vitro imaging of bacteria using 18F-fluorodeoxyglucose micro positron emission tomography.

Positron emission tomography (PET) with fluorine-18-fluorodeoxyglucose (18F-FDG) can be applied to detect infection and inflammation. However, it was so far not known to what extent bacterial pathogens may contribute to the PET signal. Therefore, we investigated whether clinical isolates of frequently encountered bacterial pathogens take up 18F-FDG in vitro, and whether FDG inhibits bacterial growth as previously shown for 2-deoxy-glucose. 22 isolates of Gram-positive and Gram-negative bacterial pathogens implicated in fever and inflammation were incubated with 18F-FDG and uptake of 18F-FDG was assessed by gamma-counting and µPET imaging. Possible growth inhibition by FDG was assayed with Staphylococcus aureus and the Gram-positive model bacterium Bacillus subtilis. The results show that all tested isolates accumulated 18F-FDG actively. Further, 18F-FDG uptake was hampered in B. subtilis pts mutants impaired in glucose uptake. FDG inhibited growth of S. aureus and B. subtilis only to minor extents, and this effect was abrogated by pts mutations in B. subtilis. These observations imply that bacteria may contribute to the signals observed in FDG-PET infection imaging in vivo. Active bacterial FDG uptake is corroborated by the fact that the B. subtilis phosphotransferase system is needed for 18F-FDG uptake, while pts mutations protect against growth inhibition by FDG.

Preclinical Evaluation of Photoacoustic Imaging as a Novel Noninvasive Approach to Detect an Orthopaedic Implant Infection.

INTRODUCTION: Diagnosing prosthetic joint infection (PJI) poses significant challenges, and current modalities are fraught with low sensitivity and/or potential morbidity. Photoacoustic imaging (PAI) is a novel ultrasound-based modality with potential for diagnosing PJI safely and noninvasively. MATERIALS: In an established preclinical mouse model of bioluminescent Staphylococcus aureus PJI, fluorescent indocyanine green (ICG) was conjugated to ß-cyclodextrin (CDX-ICG) or teicoplanin (Teic-ICG) and injected intravenously for 1 week postoperatively. Daily fluorescent imaging and PAI were used to localize and quantify tracer signals. Results were analyzed using 2-way analysis of variance. RESULTS: Fluorescence clearly localized to the site of infection and was significantly higher with Teic-ICG compared with CDX-ICG (P = 0.046) and ICG alone (P = 0.0087). With PAI, the photoacoustic signal per volumetric analysis was substantially higher and better visualized with Teic-ICG compared with CDX-ICG and ICG alone, and colocalized well with bioluminescence and fluorescence imaging. CONCLUSION: Photoacoustic imaging successfully localized PJI in this proof-of-concept study and demonstrates potential for clinical translation in orthopaedics.