Search

Background: Neutrophilic polymorphonuclear leukocytes play a crucial role in the host defence against bacterial and fungal infections. They participate in the inflammatory response through the liberation of peptides and enzymes like myeloperoxidase (MPO). Therefore, MPO has a potential as a marker enzyme for the diagnosis of wound infection., Methods: Substrate specificities and reaction pathways of MPO were investigated for new MPO substrates: crystal violet, leuco crystal violet, fast blue RR (4-benzoylamino-2,5-dimethoxybenzenediazonium chloride hemi(zinc chloride) salt) and various systematically substituted model substrates based on 2,7-dihydroxy-1-(4-hydroxyphenylazo)naphtalene-3,6-disulphonic acid. In addition, fast blue RR was covalently bound to siloxanes allowing immobilization of the substrate, while cellobiosedehydrogenase was integrated for generation of hydrogen peroxide required by MPO., Results: Elevated concentrations of MPO were found in infected wounds compared with non-infected wounds (92.2 ± 45.0 versus 1.9 ± 1.8 U/mL). Various soluble and immobilized substrates were oxidized by MPO in wound samples and the influence of substrate structure and reaction pathways were elucidated for selected compounds., Conclusions: Incubation of different MPO substrates with infected wound fluid samples resulted in a clear colour change in the case of elevated MPO concentrations, thus allowing early diagnosis of wound infection.

Detection of wound infection is based on evaluation of the well-known signs of inflammation like rubor (redness), calor (heat), tumor (swelling), and dolor (pain) by medical doctors and/or time-consuming procedures requiring special machinery. There is currently no rapid diagnostic device available for the indication of wound infection, which would especially be helpful in home care of chronic ulcer patients. In this study, a new concept for a fast diagnostic tool for wound infection based on lysozyme and elastase triggered release of dye from a peptidoglycan matrix was investigated. The matrix consisted of alginate/agarose and peptidoglycan covalently labeled with Remazol brilliant blue. Lysozyme activity in postoperative wounds and decubitus wound fluids was significantly elevated upon infection (4830 ± 1848 U mL(-1)) compared to noninfected wounds (376 ± 240 U mL(-1)). Consequently, incubation of 8% (w/v) labeled agarose/peptidoglycan blend layers with infected wound fluid samples for 2 h at 37 °C resulted in a 4-fold higher amount of dye released than measured for noninfected wounds. For alginate/peptidoglycan beads, a 7-fold higher amount of dye was released in case of infected wound fluid samples compared to noninfected ones. Apart from lysozyme, proteases [i.e., gelatinase matrix metalloproteinase MMP-2 and MMP-9 and elastase] were detected in wound fluids (e.g., using Western blotting). When dosed in ratios typical for wounds, a slight synergistic effect was measured for peptidoglycan hydrolysis (i.e., dye release) between lysozyme and these proteases. Incubation of a double-layer system consisting of stained and nonstained peptidoglycan with infected wound fluids resulted in a color change from yellow to blue, thus allowing simple visual detection of wound infection., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non-infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non-infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections., (© 2011 John Wiley & Sons A/S.)