The SPaCE diagnostic: a pilot study to test the accuracy of a novel point of care sensor for point of care detection of burn wound infection.

BACKGROUND: Wound infection in burn patients is common and has an impact on outcomes. There is no objective method to diagnose infection at point of care (PoC). Early diagnosis prevents progression to sepsis. Diagnostic subjectivity supports over-diagnosis, unnecessary hospitalization, and antibiotic overuse. AIM: This pilot study aimed to investigate the accuracy of a novel PoC wound infection diagnostic in burn patients. METHODS: We produced, and in vitro tested, a PoC diagnostic for early wound infection diagnosis. The prototype SPaCE diagnostic uses a patented lipid vesicle suspension into which a clinical swab is placed. The diagnostic delivers a colour-response to Staphylococcus aureus, Pseudomonas aeruginosa, Candida species and Enterococcus faecalis at toxin release. A pilot clinical diagnostic accuracy study was undertaken. The reference standard was a retrospective decision made by an expert clinical panel using routinely available data. FINDINGS: Data was available from 33 of 34 patients. Of these, 52% were considered to have a wound infection, 42% not, and two (6%) were equivocal. The diagnostic results showed 24% were infected, 42% were not and 33% produced intermediate results. Agreement between clinical judgement and diagnostic result, assessed using a weighted Kappa, was 0.591 suggesting moderate agreement. If the intermediate results were excluded, 22 sets of data with definitive results achieved a Kappa statistic of 0.81 suggesting 'almost perfect' agreement. Sensitivity and specificity were 57% (8/14) and 71% (12/17), respectively. CONCLUSION: This pilot study provided evidence that the SPaCE diagnostic could provide valuable and timely data to support clinical decision-making at PoC for wound infection.

Prototype Development of the Intelligent Hydrogel Wound Dressing and Its Efficacy in the Detection of Model Pathogenic Wound Biofilms.

The early detection of wound infection in situ can dramatically improve patient care pathways and clinical outcomes. There is increasing evidence that within an infected wound the main bacterial mode of living is a biofilm: a confluent community of adherent bacteria encased in an extracellular polymeric matrix. Here we have reported the development of a prototype wound dressing, which switches on a fluorescent color when in contact with pathogenic wound biofilms. The dressing is made of a hydrated agarose film in which the fluorescent dye containing vesicles were mixed with agarose and dispersed within the hydrogel matrix. The static and dynamic models of wound biofilms, from clinical strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis, were established on nanoporous polycarbonate membrane for 24, 48, and 72 h, and the dressing response to the biofilms on the prototype dressing evaluated. The dressing indicated a clear fluorescent/color response within 4 h, only observed when in contact with biofilms produced by a pathogenic strain. The sensitivity of the dressing to biofilms was dependent on the species and strain types of the bacterial pathogens involved, but a relatively higher response was observed in strains considered good biofilm formers. There was a clear difference in the levels of dressing response, when dressings were tested on bacteria grown in biofilm or in planktonic cultures, suggesting that the level of expression of virulence factors is different depending of the growth mode. Colorimetric detection on wound biofilms of prevalent pathogens (S. aureus, P. aeruginosa, and E. faecalis) is also demonstrated using an ex vivo porcine skin model of burn wound infection.

Effect of lipid and fatty acid composition of phospholipid vesicles on long-term stability and their response to Staphylococcus aureus and Pseudomonas aeruginosa supernatants.

Phospholipid vesicles have been the focus of attention as potential vehicles for drug delivery, as they are biomimetic, easy to produce, and contain an aqueous compartment which can be used to carry hydrophilic material, such as drugs or dyes. Lipid vesicles used for this purpose present a particular challenge, as they are not especially stable and can rapidly break down and release their contents away from the target area, especially at physiological temperatures/environments. This study aims to investigate optimum methods for vesicle stabilization where the vesicles are employed as part of a system or technology that signals the presence of pathogenic bacteria via the effect of secreted cytolytic virulence factors on a sensor interface. A number of approaches have been investigated and are presented here as a systematic study of the long-term (14 day) stability at 37 °C, and at various pHs. The response of vesicles, both in suspension and within hydrogels, to Staphylococcus aureus (RN 4282) and Pseudomonas aeruginosa (PAO1) whole bacteria, and supernatants from overnight cultures of both (containing secreted proteins but free of cells), was measured via a sensitive encapsulated carboxyfluorescein release assay. The results showed that lipid chain length, cholesterol concentration, and stabilization via photopolymer stable components were critical in achieving stability. Finally, dispersion of the optimum vesicle formulation in hydrogel matrixes was investigated, culminating in the in vivo demonstration of a simple prototype wound dressing.

Visible, colorimetric dissemination between pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent dye containing lipid vesicles.

This paper describes a biosensing concept for exotoxins secreted by Staphylococcus aureus and Pseudomonas aeruginosa based on the toxin mediated breakdown and subsequent fluorescent dye release from phospholipid vesicles (liposomes). The sensitivity of vesicles to toxins was tuned by altering the lipid and fatty acid composition of the membranes such that vesicles could be engineered to respond to toxins/enzymes from S. aureus only; P. aeruginosa only; and both S. aureus and P. aeruginosa. Nineteen types of vesicle were made with varying compositions of phosphocholine (PC), phosphoethanolamine (PE), cholesterol and the photo-polymerizable ampiphile 10,12-tricosadiynoic acid (TCDA). The selectivity of the vesicles was measured via a simple fluorescence "switch on" assay. Sensitivity of the vesicles to 40 clinically derived strains of S. aureus and P. aeruginosa was also demonstrated. This work suggests that this technology could be utilised in a diagnostic tool to discriminate between the species of S. aureus and P. aeruginosa in wound dressings.