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.

Molecular and biochemical characterization of Mottled-dappled, an embryonic lethal Menkes disease mouse model.

Mottled-dappled (Mo-dp) is a mouse model of Menkes disease caused by a large, previously uncharacterized deletion in the 5' region of Atp7a, the mouse ortholog of ATP7A. Affected mutants die in utero at embryonic day 17, and show bending and thickening of the ribs and distortion of the pectoral and pelvic girdles and limbs. To characterize this allele, we designed a custom 4x180K microarray on the mouse X chromosome and performed comparative genomic hybridization using extracted DNA from normal and carrier Mo-dp females, and identified an approximately 9 kb deletion. We used PCR to fine-map the breakpoints and amplify a junction fragment of 630 bp. Sequencing of the junction fragment disclosed the exact breakpoint locations and that the Mo-dp deletion is precisely 8990 bp, including approximately 2 kb in the promoter region of Atp7a. Western blot analysis of Mo-dp heterozygous brains showed diminished amounts of Atp7a protein, consistent with reduced expression due to the promoter region deletion on one allele. In heterozygous females, brain copper levels tended to be lower compared to wild type whereas neurochemical analyses revealed higher dihydroxyphenylacetic acid:dihydroxyphenylglycol (DOPAC:DHPG) and dopamine:norepinephrine (DA:NE) ratios compared to normal (P=0.002 and 0.029, respectively), consistent with partial deficiency of dopamine-beta-hydroxylase, a copper-dependent enzyme. Heterozygous females showed no significant differences in body weight compared to wild type females. Our results delineate the molecular details of the Mo-dp mutation for the first time and define novel biochemical findings in heterozygous female carriers of this allele.