Doris Schiffer

Analysis of myeloperoxidase activity in wound fluids as a marker of infection

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.

Novel protease-based diagnostic devices for detection of wound infection

A gelatinase‐based device for fast detection of wound infection was developed. Collective gelatinolytic activity in infected wounds was 23 times higher (p ≤ 0.001) than in noninfected wounds and blisters according to the clinical and microbiological description of the wounds. Enzyme activities of critical wounds showed 12‐fold elevated enzyme activities compared with noninfected wounds and blisters. Upon incubation of gelatin‐based devices with infected wound fluids, an incubation time of 30 minutes led to a clearly visible dye release. A 32‐fold color increase was measured after 60 minutes. Both matrix metalloproteinases and elastases contributed to collective gelatinolytic enzyme activity as shown by zymography and inhibition experiments. The metalloproteinase inhibitor 1,10‐phenanthroline (targeting matrix metalloproteinases) and the serine protease inhibitor phenylmethlysulfonyl fluoride (targeting human neutrophil elastase) inhibited gelatinolytic activity in infected wound fluid samples by 11–37% and 60–95%, respectively. Staphylococcus aureus and Pseudomonas aeruginosa, both known for gelatinase production, were isolated in infected wound samples.

Lysozyme-responsive polymer systems for detection of infection

There is a strong need for new point‐of‐care systems for the detection of wound infection. Overseen infections in chronic wounds induce severe complications, such as delayed healing and high risks for the patients, while time‐consuming common gold and silver standard methods for infection assessment cannot be implemented in home care units. This study demonstrates for the first time the between correlation of lysozyme activity and silver‐standard microbiological evaluation of wounds. Significantly higher (eightfold increase; p < 0.001) lysozyme activity in infected wounds was in accordance with increasing bacterial burden of infected wound fluids. Moreover, a two‐layer membrane‐based test system was developed providing visible results on infection in a short time (30 min) while avoiding any intermediate steps such as centrifugation. In the first layer of the system, a size exclusion membrane (1.2–8 μm cut‐off) retained labeled peptidoglycane while allowing only smaller fragments resulting from lysozyme hydrolysis to pass through. These fragments were then captured in a second layer, an anion‐exchanging diethylaminoethyl cellulose membrane, resulting in clearly visible color changes. Colorimetric measurements demonstrated significant differences (p < 0.001) and sixfold higher delta E values between infected and noninfected wound fluids. This system allows a quick and straightforward determination of the status of a wound. The colorimetric readout indicates the increased lysozyme activity in infected wound fluid.

Fast Blue RR—Siloxane Derivatized Materials Indicate Wound Infection Due to a Deep Blue Color Development

There is a strong need for simple and fast methods for wound infection determination. Myeloperoxidase, an immune system-derived enzyme was found to be a suitable biomarker for wound infection. Hence, alkoxysilane-derivatized Fast Blue RR was immobilized via simple hydrolytic polymerization. The resulting enzyme-responsive siloxane layers were incubated with myeloperoxidase, wound fluid or hemoglobin. The reaction was monitored via HPLC measurements and the color development quantified spectrophotometrically. Myeloperoxidase was indeed able to oxidize immobilized Fast Blue RR leading to a blue colored product. No conversion was detected in non-infected wound fluids. The visible color changes of these novel materials towards blue enable an easy distinction between infected and non-infected wound fluids.

Myeloperoxidase-responsive materials for infection detection based on immobilized aminomethoxyphenol.

There is a strong need for simple and fast diagnostic tools for the detection of wound infection. Immune system‐derived enzymes like myeloperoxidase are efficient biomarkers for wound infection that emerge in the early stage infection process. In this study, 5‐amino‐2‐methoxyphenol was functionalized with alkoxysilane to allow visual detection of MPO on carrier materials, for example, in test strips. Indeed, MPO activity was visually detectable in short time in wound background. Oxidation of the substrate was followed spectrophotometrically and proved via HPLC. LC‐ESI TOF and NMR analyses unveiled the reaction mechanism and a dimeric reaction product responsible for the visualization of MPO activity. The substrate specificity and sensitivity toward MPO detection was proved and tests with infected wound fluids were successfully performed. The study demonstrates the suitability of the novel MPO substrate for the detection of wound infection and the covalent immobilization on diagnostic carrier materials. Biotechnol. Bioeng. 2016;113: 2553–2560.