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Introduction There is no definitive method to determine infection status in non-healing wounds. Measurement of wound pH might be a promising indicator of infection as it is relatively easy to perform, provides objective results within a few seconds, and is inexpensive. The aim of this investigation was to determine if wound pH could be a potential indicator of early or established infection in non-healing wounds. Methods We explored the relationship between wound pH and two indicators of wound infection: expert clinical judgement and elevated neutrophil-derived enzyme activity. Data was used from 120 wound samples previously collected at Medisch Spectrum Twente hospital. Results With increasing wound pH, there was also an increase in the proportion of infected wounds as determined by expert clinical judgement. In addition, increases in the activities of myeloperoxidase, elastase and lysozyme were also associated with elevated pH. Conclusions The strength of the relationship between wound pH and clinical judgement or enzyme activities observed in this study is not sufficient to promote the use of elevated pH alone as an indicator for wound infection. However, the use of pH in combination with other indicators for wound infection, such as elevated neutrophil enzyme activity, warrants further research.

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. © 2016 Wiley Periodicals, Inc.

The question remains whether wound swabs yield similar culture results to the traditional gold standard, biopsies. Swabs are not invasive and easy to perform. However, they are believed to capture microorganisms from the surface rather than microorganisms that have invaded tissue. Several studies compared swabs and biopsies using different populations and sampling methods, complicating the ability to draw conclusions for clinical practice. This study aimed to compare swab and biopsy in clinical practice, by including a variety of wounds and using standard sampling and culture procedures. Swabs (Levine technique) and biopsies were taken for microbiological culture in a standardized manner from the same location of one wound for each patient. Statistical analyses were performed to determine overall agreement, and observed agreement and kappa for specific microorganisms. A variety of wounds of 180 patients from different healthcare facilities in The Netherlands were included. Skin flora was more frequently cultured from swabs, resulting in similar recovery rates when excluding skin flora (1.34 vs 1.35). Swabs were able to identify all microorganisms cultured from biopsies in 131 wounds (72.8%) wounds. Most frequently identified organisms were Staphylococcus aureus, Pseudomonas aeruginosa, and beta-haemolytic streptococci species. Observed agreement and kappa for these organisms varied between 87.2 and 97.8% and 0.73 and 0.85, respectively. This study demonstrates that swabs and biopsies tend to yield the same culture results when taken from the same location. For frequently occurring microorganisms, agreement between the two methods was even higher. Therefore, there seems to be no direct need for invasive biopsy in clinical practice.

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.

Wound infection is a severe complication causing delayed healing and risks for patients. Conventional methods of diagnosis for infection involve error-prone clinical description of the wound and time-consuming microbiological tests. More reliable alternatives are still rare, except for invasive and unaffordable gold standard methods. This review discusses the diversity of new approaches for wound infection determination. There has been progress in the detection methods of microorganisms, including the assessment of the diversity of the bacterial community present in a wound, as well as in the elaboration of specific markers. Another interesting strategy involves the quantification of enzyme activities in the wound fluid secreted by the immune system as response to infection. Color-changing substrates for these enzymes consequently have been shown to allow detection of an infection in wounds in a fast and easy way. Promising results were also delivered in measuring pH changes or detecting enhanced amounts of volatile molecules in case of infection. A simple and effective infection detection tool is not yet on the market, but innovative ideas pave the way for the investigation of fast and easy point-of-care devices.

In clinical practice, diagnosis of wound infection is based on the classical clinical signs of infection. When infection is suspected, wounds are often swabbed for microbiological culturing. These methods are not accurate (clinical judgment in chronic wounds) or provide results after several days (wound swab). Therefore, there is an urgent need for an easy-to-use diagnostic tool for fast detection of wound infection, especially in chronic wounds. This study determined the diagnostic properties of the enzymes myeloperoxidase, human neutrophil elastase (HNE), lysozyme and cathepsin-G in detecting wound infection when compared to wound swabs. Both chronic and acute wounds of 81 patients were assessed through clinical judgment, enzyme analysis and wound swab. Three promising enzyme models for detecting wound infection were identified. A positive test was defined as: at least one enzyme positive after 30 minutes (model 1), lysozyme and HNE positive after 30 minutes (model 2), myeloperoxidase positive after 5 minutes, and HNE or lysozyme positive after 30 minutes (model 3). All models were significant (p≤0.001). There was no correlation between clinical judgment and wound swab, indicating the need for novel diagnostic systems. Enzyme analysis is fast, easy to use and superior to clinical judgment when compared to wound swabs.

A high risk of infection is also reported for chronic wounds, an issue predominantly affecting immune-suppressed people. As a consequence of manifestation of wound infection, wounds fail to heal. This greatly complicates the treatment modalities and life of the patients. The infection progress strongly correlates with the interaction of invading bacteria with the wound environment. This process is divided into four sections, namely, contamination, colonization, critical colonization and infection, whereby impaired wound healing is considered to start at the stage of critical bacterial colonization. This chapter discusses the promising methods available for microbial infection detection divided in direct bacterial detection and indirect infection detection. It describes the advantages and disadvantages of the available detection systems are elucidated and future perspectives in early detection of bacterial wound infection. The need of point-of-care (PoC) diagnostics for infection detection drives research to innovation in online measurement tools that provide fast information on the infection status.

Infection in wounds affects about 2% of the population in developed countires at least once in their lifetime, and the lack of tools for its rapid diagnosis is still a problem 1. Standard procedures of infection detection include the judgement of the classical clinical signs, the detection of signals specific to secondary wounds, or the quantification of the microbial load 2–5. The determination of the microbial load is a time-consuming standard procedure, although the presence of microbes per se is not indicative of infection 2.

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. © 2016 Wiley Periodicals, Inc.

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.

In clinical practice, diagnosis of wound infection is based on the classical clinical signs of infection. When infection is suspected, wounds are often swabbed for microbiological culturing. These methods are not accurate (clinical judgment in chronic wounds) or provide results after several days (wound swab). Therefore, there is an urgent need for an easy-to-use diagnostic tool for fast detection of wound infection, especially in chronic wounds. This study determined the diagnostic properties of the enzymes myeloperoxidase, human neutrophil elastase (HNE), lysozyme and cathepsin-G in detecting wound infection when compared to wound swabs. Both chronic and acute wounds of 81 patients were assessed through clinical judgment, enzyme analysis and wound swab. Three promising enzyme models for detecting wound infection were identified. A positive test was defined as: at least one enzyme positive after 30 minutes (model 1), lysozyme and HNE positive after 30 minutes (model 2), myeloperoxidase positive after 5 minutes, and HNE or lysozyme positive after 30 minutes (model 3). All models were significant (p≤0.001). There was no correlation between clinical judgment and wound swab, indicating the need for novel diagnostic systems. Enzyme analysis is fast, easy to use and superior to clinical judgment when compared to wound swabs.

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.

De abstractprijs is tijdens het 6e Jaarcongres V&VN VS gewonnen door Miriam Blokhuis en haar team en werd uitgereikt door dr. Erik de Laat, voorzitter van de Stichting Ondersteuning Wetenschap Verpleegkundig Specialisten (OWVS).