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.

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.

Test-retest reliability and concurrent validity of the Adapted Short QUestionnaire to ASsess Health-enhancing physical activity (Adapted-SQUASH) in adults with disabilities.

The current study determined the test-retest reliability and concurrent validity of the Adapted Short QUestionnaire to ASsess Health-enhancing physical activity (Adapted-SQUASH) in adults with disabilities. Before filling in the Adapted-SQUASH twice with a recall period of 2 weeks, participants wore the Actiheart activity monitor up to 1 week. For the test-retest reliability (N = 68), Intraclass correlation coefficients (ICCs) were 0.67 (p < 0.001) for the total activity score (min x intensity/week) and 0.76 (p < 0.001) for the total minutes of activity (min/week). For the concurrent validity (N = 58), the Spearman correlation coefficient was 0.40 (p = 0.002) between the total activity score of the first administration of the Adapted-SQUASH and activity energy expenditure from the Actiheart (kcals kg-1 min-1). The ICC was 0.22 (p = 0.027) between the total minutes of activity assessed with the first administration of the Adapted-SQUASH and Actiheart. The Adapted-SQUASH is an acceptable measure to assess self-reported physical activity in large populations of adults with disabilities but is not applicable at the individual level due to wide limits of agreement. Self-reported physical activity assessed with the Adapted-SQUASH does not accurately represent physical activity assessed with the Actiheart in adults with disabilities, as indicated with a systematic bias between both instruments in the Bland-Altman analysis.

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.