Bacterial protease activity: a prognostic biomarker of early wound infection.

OBJECTIVE: High bacterial burden is one of several reasons that wounds fail to heal. At present, clinicians rely primarily on clinical signs and symptoms (CSS) to diagnose infection in hard-to-heal wounds; however, studies have demonstrated that CSS can be unreliable. This is especially true in the early stages of bacterial infection. Bacteria release proteases, virulence factors that promote invasive infection. This clinical trial evaluated the use of bacterial protease activity (BPA) as a biomarker to detect whether a wound was in the period of pathogenicity, prior to overt clinical signs. METHOD: Participants were drawn from six US wound centres and had their wounds assessed clinically for infection. In addition, wound fluid swabs were collected and analysed for BPA, inflammatory cytokines (interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α)), and cultured for quantitative microbiology. RESULTS: A total of 366 patients were recruited. The median BPA level increased with the increasing number of signs of infection. The majority of wounds tested positive for elevated BPA prior to exhibiting at least three CSS of infection, the level at which the criteria for infection are met. BPA tended to increase with the bioburden (colony forming unit (CFU)/ml) although some wounds with high bioburden were negative for BPA, and others with low bioburden were positive for BPA. The mean levels of IL-1ß and TNF-α were significantly higher in BPA-positive wounds (p<0.0001 and p=0.0002, respectively). CONCLUSION: The results of this clinical trial suggest that measuring BPA can lead to the early detection of pathogenic bacteria in the wound that impede wound healing and may progress to invasive infection. In a large percentage of cases, BPA detected virulent bacteria in the absence of CSS of infection. As a biomarker, BPA has an advantage over measuring bacterial load-hard-to-heal wounds are often colonised with non-pathogenic bacteria that do not inhibit wound healing and, conversely, a low number of highly virulent species could disrupt the healing process.

Bacterial protease activity as a biomarker to assess the risk of non-healing in chronic wounds: Results from a multicentre randomised controlled clinical trial.

Millions worldwide suffer from chronic wounds challenging clinicians and burdening healthcare systems. Bacteria impede wound healing; however, the diagnosis of excessive bacterial burden or infection is elusive. Clinical signs and symptoms of infection are inaccurate and unreliable. This trial evaluated a novel, point-of-care, lateral flow diagnostic designed to detect virulence factors released by the most common bacteria found in chronic wounds. A multicentre prospective cohort clinical trial examined the efficacy of a diagnostic test in detecting bacterial proteases taken from swab samples of chronic venous, arterial, pressure and mixed aetiology chronic wounds. Two hundred and sixty six wounds were included in the analysis of the study. The wounds were tested at the start of the study after which investigators were permitted to use whatever dressings they desired for the next 12 weeks. Healing status at 12 weeks was assessed. The presence of elevated bacterial protease activity decreased the probability of wound healing at 12 weeks. In contrast, a greater proportion of wounds were healed at 12 weeks if they had little or no bacterial protease activity at study start. In addition, the presence of elevated bacterial protease activity increased the time it takes for a wound to heal and increased the risk that a wound would not heal, when compared to the absence of bacterial protease activity. The results of this clinical trial indicate that bacterial protease activity, as detected by this novel diagnostic test, is a valid clinical marker for chronicity in wounds. The diagnostic test offers a tool for clinicians to detect clinically significant bacteria in real time and manage bacteria load before the clinical signs and symptoms of infection are evident.

Defining a new diagnostic assessment parameter for wound care: Elevated protease activity, an indicator of nonhealing, for targeted protease-modulating treatment.

It is widely accepted that elevated protease activity (EPA) in chronic wounds impedes healing. However, little progress has occurred in quantifying the level of protease activity that is detrimental for healing. The aim of this study was to determine the relationship between inflammatory protease activity and wound healing status, and to establish the level of EPA above which human neutrophil-derived elastase (HNE) and matrix metalloproteases (MMP) activities correlate with nonhealing wounds. Chronic wound swab samples (n = 290) were collected from four wound centers across the USA to measure HNE and MMP activity. Healing status was determined according to percentage reduction in wound area over the previous 2-4 weeks; this was available for 211 wounds. Association between protease activity and nonhealing wounds was determined by receiver operating characteristic analysis (ROC), a statistical technique used for visualizing and analyzing the performance of diagnostic tests. ROC analysis showed that area under the curve (AUC) for HNE were 0.69 for all wounds and 0.78 for wounds with the most reliable wound trajectory information, respectively. For MMP, the corresponding AUC values were 0.70 and 0.82. Analysis suggested that chronic wounds having values of HNE >5 and/or MMP ≥13, should be considered wound healing impaired. EPA is indicative of nonhealing wounds. Use of a diagnostic test to detect EPA in clinical practice could enable clinicians to identify wounds that are nonhealing, thus enabling targeted treatment with protease modulating therapies.

A commercial whole blood glucose biosensor with a low sensitivity to hematocrit based on an impregnated porous carbon electrode.

Erythrocytes (red blood cells) are a major source of response variation in biosensor electrodes expected to operate in whole blood. Such a blood-to-plasma difference (hematocrit effect) must be minimized for those sensors directed towards the hospital market where wide variations in hematocrit can be seen. Typically, many current glucose sensors demonstrate a decreasing response to the analyte in the presence of increasing hematocrit levels. A sensor electrode for glucose is described which displays a reduced sensitivity to changes in hematocrit. The working electrode comprises a base porous conducting carbon layer, which is impregnated with a mixture including glucose oxidase and a ferrocene redox mediator. The base carbon layer has a void volume of 50%, an average pore diameter of less than 0.1 microm and a thickness of about 20 microm. The interior void volume of the base carbon layer is filled entirely with a substantial proportion of the impregnating mixture such that very little remains on the exterior. The resulting impregnated porous electrode excludes erythrocytes and is consequently capable of operating acceptably in venous, capillary, arterial and neonatal blood over a wide hematocrit range of 20-70%.