Identification of Microorganism in Infected Wounds by Positively Charged Selective Sensor Array and Deep Learning Algorithm.

Microorganism are ubiquitous and intimately connected with human health and disease management. The accurate and fast identification of pathogenic microorganisms is especially important for diagnosing infections. Herein, three tetraphenylethylene derivatives (S-TDs: TBN, TPN, and TPI) featuring different cationic groups, charge numbers, emission wavelengths, and hydrophobicities were successfully synthesized. Benefiting from distinct cell wall binding properties, S-TDs were collectively utilized to create a sensor array capable of imaging various microorganisms through their characteristic fluorescent signatures. Furthermore, the interaction mechanism between S-TDs and different microorganisms was explored by calculating the binding energy between S-TDs and cell membrane/wall constituents, including phospholipid bilayer and peptidoglycan. Using a combination of the fluorescence sensor array and a deep learning model of residual network (ResNet), readily differentiation of Gram-negative bacteria (G-), Gram-positive bacteria (G+), fungi, and their mixtures was achieved. Specifically, by extensive training of two ResNet models with large quantities of images data from 14 kinds of microorganism stained with S-TDs, identification of microorganism was achieved at high-level accuracy: over 92.8% for both Gram species and antibiotic-resistant species, with 90.35% accuracy for the detection of mixed microorganism in infected wound. This novel method provides a rapid and accurate method for microbial classification, potentially aiding in the diagnosis and treatment of infectious diseases.

[Several issues that need attention to improve the diagnosis and treatment level of infectious wounds].

Wound infection is an inflammatory response of the host to pathogenic microorganisms. It is a complex pathological process that may manifest either as a rapid onset acute wound infection, or as a prolonged chronic wound infection. Infectious wounds refer to acute or chronic wounds where infection has occurred, and their diagnosis and treatment involve many links, any loopholes in any aspect can lead to treatment failure. How to improve the diagnosis and treatment level of infectious wounds? This article proposed and discussed several issues, such as biofilm and drug resistance of bacteria, new materials and new-type dressings, special types of infectious wounds, and the combined application of various treatment techniques that need attention, and intended to attract the attention of peers. At the same time, it advocated for full attention to research results in the relevant fields and continuously emerging new technologies, concepts, materials, and methods, and applying them to the diagnosis and treatment of infectious wounds to benefit the majority of patients.

Wound, Ischemia, Foot Infection (Wifi) Classification System And Its Predictive Ability Concerning Amputation-Free Survival, Mortality And Major Limb Amputation In A Portuguese Population: A Single Center Experience.

INTRODUCTION: Commonly used chronic limb-threatening ischemia (CLTI) classifications lack granularity and detail to precisely stratify patients according to risk of limb loss, expected revascularization benefit and mortality. The aim of this study is to evaluate in a Portuguese population the prognostic value of an updated CLTI classification based on Wound, Ischemia, and foot Infection (WIfI) proposed by the Society for Vascular Surgery. MATERIALS AND METHODS: Single-center retrospective evaluation of prospectively collected data of consecutive patients with CLTI submitted to lower limb revascularization from January to December of 2017. All consecutive patients with chronic peripheral artery disease with ischemic rest pain or tissue loss were included. The exclusion criteria were patients with intermittent claudication, vascular trauma, acute ischemia, non-atherosclerotic arterial disease and isolated iliac intervention. The primary end-point was major limb amputation, mortality and amputation-free survival (AFS) at 30 days, 1 year and 2 year follow-up. Secondary end-points were minor amputation, wound healing time (WHT) and rate (WHR). RESULTS: A total of 111 patients with CLTI were submitted to infra-inguinal revascularization: 91 endovascular and 20 open surgery. After categorizing them according to the WIfI: 20 had stage 1 (18.52%), 29 stage 2 (26.85%), 38 stage 3 (35.19%) and 21 stage 4 (19.44%). Overall mortality rate was 1.8%, 17% and 22.3% at 30 days, 1 year and 2 years follow-up. Major amputation rate was 0.9%, 2.7% and 2.7% at 30 days, 1 year and 2 years follow-up. AFS rate was 97.3%, 82.1%, and 76.8% at 30 days, 1 year, 2 years follow-up. In multi-variable analysis, higher WIfI score was the only predictive factor for mortality and AFS. WIfI 3 and 4 were also associated with increased risk of non-healing ulcer. CONCLUSION: This study proved the prognostic value of the WIfI classification in a Portuguese population by showing an association between higher scores and increased mortality, lower AFS and non-healing ulcer.

Skin Pigmentation Impacts the Clinical Diagnosis of Wound Infection: Imaging of Bacterial Burden to Overcome Diagnostic Limitations.

Underrepresentation of diverse skin tones in medical education and providers' implicit racial bias drives inequities in wound care, such as disproportionally poor outcomes for Black patients. Diagnostic indicators (e.g., erythema) can present differently depending on skin pigmentation. This post hoc analysis of 350 chronic wounds from a prospective 14-site clinical trial aimed to determine how the perception of clinical signs and symptoms of infection (CSS) differs by patient skin tone and if fluorescence-imaging can offer a more objective diagnostic solution. Participants were grouped by skin tone (low, medium, high) as measured by the Fitzpatrick Skin Phototype Classification (FSPC) scale. CSS and total bacterial load (TBL) were compared across FSPC groups, along with sensitivity to detect TBL >104 CFU/g using CSS alone and combined with fluorescence-imaging. Erythema was reported less often with increasing FSPC score (p = 0.05), from 13.4% (low), to 7.2% (medium), to 2.3% (high), despite comparable bacterial loads (median = 1.8 × 106 CFU/g). CSS sensitivity in the high group (2.9%) was 4.8-fold to 8.4-fold lower than the low (p = 0.003) and medium groups (p = 0.04). Fluorescence-imaging significantly improved the detection of high bacterial load in each group, peaking in the high group at 12-fold over CSS alone. These findings underscore the threat of pervasive racialized health inequities in wound care, where missed diagnosis of pathogenic bacteria and infection could delay treatment, increasing the risk of complications and poor outcomes. Fluorescence-imaging is poised to fill this gap, at least in part, serving as a more objective and equitable indicator of wound bacteria. Clinicaltrials.gov #NCT03540004 registered 16-05-2018.

Intelligent wound dressing for simultaneous in situ detection and elimination of pathogenic bacteria.

Wound infections hinder the healing process and potentially result in life-threatening complications, which urgently require rapid and timely detection and treatment pathogens during the early stages of infection. Here, an intelligent wound dressing was developed to enable in situ detection and elimination of pathogenic bacteria through a combination of point-of-care testing and antibacterial photodynamic therapy technology. The dressing is an injectable hydrogel composed of carboxymethyl chitosan and oxidized sodium alginate, with addition of 4-methylumphulone beta-D-glucoside (MUG) and up-converted nanoparticles coated with titanium dioxide (UCNPs@TiO2). The presence of bacteria can be visually detected by monitoring the blue fluorescence of 4-methylumbellione, generated through the reaction between MUG and the pathogen-associated enzyme. The UCNPs@TiO2 photosensitizers were synthesized and demonstrated high antibacterial activity through the generation of reactive oxygen species when exposed to near-infrared irradiation. Meanwhile, a smartphone-based portable detection system equipped with a self-developed Android app was constructed for in situ detection of pathogens in mere seconds, detecting as few as 103 colony-forming unit. Additionally, the dressing was tested in a rat infected wound model and showed good antibacterial activity and pro-healing ability. These results suggest that the proposed intelligent wound dressing has potential for use in the diagnosis and management of wound infections. STATEMENT OF SIGNIFICANCE: An intelligent wound dressing has been prepared for simultaneous in situ detection and elimination of pathogenic bacteria. The presence of bacteria can be visually detected by tracking the blue fluorescence of the dressing. Moreover, a smartphone-based detection system was constructed to detect and diagnose pathogenic bacteria before reaching the infection limit. Meanwhile, the dressing was able to effectively eliminate key pathogenic bacteria on demand through antibacterial photodynamic therapy under NIR irradiation. The proposed intelligent wound dressing enables timely detection and treatment of infectious pathogens at an early stage, which is beneficial for wound management.

Point-of-care detection devices for wound care and monitoring.

Healthcare resources are heavily burdened by infections that impede the wound-healing process. A wide range of advanced technologies have been developed for detecting and quantifying infection biomarkers. Finding a timely, accurate, non-invasive diagnostic alternative that does not require a high level of training is a critical step toward arresting common clinical patterns of wound health decline. There is growing interest in the development of innovative diagnostics utilizing a variety of emerging technologies, and new biomarkers have been investigated as potential indicators of wound infection. In this review, we summarize diagnostics available for wound infection, including those used in clinics and still under development.

Targeted metagenomic assessment reflects critical colonization in battlefield injuries.

IMPORTANCE: Microbial contamination in combat wounds can lead to opportunistic infections and adverse outcomes. However, current microbiological detection has a limited ability to capture microbial functional genes. This work describes the application of targeted metagenomic sequencing to profile wound bioburden and capture relevant wound-associated signatures for clinical utility. Ultimately, the ability to detect such signatures will help guide clinical decisions regarding wound care and management and aid in the prediction of wound outcomes.

Point-of-care fluorescence imaging reveals extent of bacterial load in diabetic foot ulcers.

Elevated levels of bacteria, including biofilm, increase the risk of chronic wound infection and inhibit healing. Addressing asymptomatic high bacterial loads is challenged by a lack of clinical terminology and diagnostic tools. This post-hoc multicenter clinical trial analysis of 138 diabetic foot ulcers investigates fluorescence (FL)-imaging role in detecting biofilm-encased and planktonic bacteria in wounds at high loads. The sensitivity and specificity of clinical assessment and FL-imaging were compared across bacterial loads of concern (104 -109 CFU/g). Quantitative tissue culture confirmed the total loads. Bacterial presence was confirmed in 131/138 ulcers. Of these, 93.9% had loads >104 CFU/g. In those wounds, symptoms of infection were largely absent and did not correlate with, or increase proportionately with, bacterial loads at any threshold. FL-imaging increased sensitivity for the detection of bacteria across loads 104 -109 (P < .0001), peaking at 92.6% for >108 CFU/g. Imaging further showed that 84.2% of ulcers contained high loads in the periwound region. New terminology, chronic inhibitory bacterial load (CIBL), describes frequently asymptomatic, high bacterial loads in diabetic ulcers and periwound tissues, which require clinical intervention to prevent sequelae of infection. We anticipate this will spark a paradigm shift in assessment and management, enabling earlier intervention along the bacterial-infection continuum and supporting improved wound outcomes.

Bacterial autofluorescence in infected perineal wounds: A prospective cohort study.

Diagnosis of perineal wound infection based solely on clinical signs and symptoms is subjective, and often incorrectly identifies wounds with clinically significant bacterial loads. New advances in wound care such as bacterial fluorescence imaging allow point-of -care assessment of bacterial burden. This single-center, prospective observational study included 80 women with perineal wound infection and aimed to determine the incidence of significant bacterial colonization identified with bacterial fluorescence imaging. Also, to evaluate the diagnostic accuracy of bacterial fluorescence imaging. 30 women (37.5%) had fluorescence in their wounds despite antibiotic therapy. The sensitivity of bacterial fluorescence imaging in the diagnosis of wounds with a clinically significant bacterial burden was 83% and specificity was 90%. The positive predictive value was 92% and negative predictive value was 80%. Overall, diagnostic accuracy was substantial. The results of this study demonstrate that bacterial fluorescence imaging can provide real-time information surrounding the bacterial burden of perineal wounds. ClinicalTrials.gov NCT04480684.

[Diagnostics and treatment of local wound infections]. / Diagnostik und Therapie lokaler Wundinfektionen.

Local wound infections are a multidisciplinary challenge which should be diagnosed as early as possible and adequately treated. In addition to a stagnation of wound healing, it is in particular the threat of development into systemic infections and even sepsis that represent feared, potentially life-threatening complications. This topic has a particularly high and multidisciplinary significance in the treatment of patients with chronic wounds. Until now, there were no generally accepted criteria for the diagnostics. The newly developed and validated TILI score, as a supplement to vital signs and serological values, enables rapid objectification of local wound infections. In addition, the W.A.R. score can be used to identify patients with an increased risk of infections. With these easy to use tools, the indications for antiseptic wound treatment can be assessed individually, quickly and without problems. For many patients with chronic wounds, polihexanide is then the wound antiseptic of first choice. However, the indications for wound antiseptics should be critically reviewed after a treatment duration of 14 days at the latest.

Diagnosis and treatment of infected wounds: A multi-centre audit of current clinical practice across the UK, Ireland and Scandinavia.

AIMS AND OBJECTIVES: Surveillance of wound infection including signs of infection alongside antimicrobial usage (types, duration, frequency) can highlight knowledge gaps and inconsistencies. This manuscript aims to highlight these, identify and inform opportunities for practice improvement and to show impact of infection management practice may be having on the issue of antimicrobial resistance. BACKGROUND: Infected wounds pose challenges to healthcare professionals. Balancing risk of wound deterioration and progression to systemic infection with appropriate use of antimicrobials is necessary to minimise development of resistance. METHODS: Analysis consisted of a practice survey of 9661 wounds across 70 community sites over a period of one week. Data were collected from projects between 2017 and 2020. The form was available to providers within the UK, Ireland, Norway, Denmark, Sweden and Finland. EQUATOR research guidelines were followed; STROBE checklist for observational research reporting was completed. RESULTS: Infection rates of 8.9% were reported from wounds assessed. These data indicate inconsistencies with diagnosis across practice with non-specialists more likely to be unsure of wound infection. Greater confidence in infection identification was observed as number of signs increased. Inconsistencies were also observed in appropriate treatment; antimicrobials were used in 35% of wounds considered not infected and not used in 41% of wounds that were identified as infected. CONCLUSIONS: This investigation of infection management practice of over 9000 wounds provides an insight into diagnosis and treatment of infection. Inconsistencies in diagnosis and treatment of wound infections reported highlight the need for increased education, awareness of diagnosis and treatment of infection. RELEVANCE TO CLINICAL PRACTICE: Variability in management of infected wounds highlights opportunities to aid more effective diagnosis and treatment of infected wounds. Incorporation of support tools or evidence-based pathways into practice may enhance confidence in management of local infection, balanced with appropriate use, potentially minimising resistance and improving outcomes.

Loop-Mediated Isothermal Amplification of DNA (LAMP) as an Alternative Method for Determining Bacteria in Wound Infections.

The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method.

Establishing a consensus on wound infection definitions.

OBJECTIVES: The aim of this study was to establish an international, interorganisational consensus on wound infection terminology. METHODS: This project consisted of definition scoping and a Delphi process to produce a consensus glossary for 18 wound infection terms. Recent guidelines/consensus documents were reviewed to identify 2-4 definitions for each term. An online consensus process was undertaken using the RAND Appropriateness Method, a consensus method for panels to reach agreement. International wound organisations nominated experts to participate, from whom 21 participants were selected to represent different organisations, geographic regions and disciplines. In the first consensus round, each term was presented alongside 2-3 definitions and participants nominated their preferred definition, with the majority vote used to select a baseline definition. The consensus process then proceeded, with participants using a 9-point Likert scale to score their level of agreement or disagreement with the definition for each term. Participants also provided a justification outlining the reason behind their rating. At the end of each round, an index was calculated to provide a quantitative evaluation indicating whether agreement or disagreement had been reached. RESULTS: Reasoning statements were summarised and the definitions were adjusted to incorporate concepts identified by participants. The adjusted definition was presented in the next consensus round, together with the reasoning statements. Terms for which a final definition was not achieved in three consensus rounds were finalised with preferential voting using 2-3 definitions that had reached consensus. PROJECT PROGRESS AND SIGNIFICANCE: The project generated a glossary of wound infection terms, endorsed through participation of 15 international organisations, for dissemination of guidelines and clinical decision-making/teaching tools.

Rapid Colorimetric Detection of Wound Infection with a Fluidic Paper Device.

Current procedures for the assessment of chronic wound infection are time-consuming and require complex instruments and trained personnel. The incidence of chronic wounds worldwide, and the associated economic burden, urge for simple and cheap point-of-care testing (PoCT) devices for fast on-site diagnosis to enable appropriate early treatment. The enzyme myeloperoxidase (MPO), whose activity in infected wounds is about ten times higher than in non-infected wounds, appears to be a suitable biomarker for wound infection diagnosis. Herein, we develop a single-component foldable paper-based device for the detection of MPO in wound fluids. The analyte detection is achieved in two steps: (i) selective immunocapture of MPO, and (ii) reaction of a specific dye with the captured MPO, yielding a purple color with increasing intensity as a function of the MPO activity in infected wounds in the range of 20-85 U/mL. Ex vivo experiments with wound fluids validated the analytic efficiency of the paper-based device, and the results strongly correlate with a spectrophotometric assay.

Logic-Based Diagnostic and Therapeutic Nanoplatform with Infection and Inflammation Monitoring and Microenvironmental Regulation Accelerating Wound Repair.

Infectious cutaneous wounds are a thorny clinical problem. The microenvironment of the infectious wound is complicated and changes at different healing stages. Traditional treatments either have a single effect such as anti-inflammation, antibacteria, or angiogenesis or a simple mixture of several functions. They fail to deal with the change of the physiological healing process, leading to unsatisfactory outcomes. Herein, we have designed a logic-based smart nanoplatform (named as ZEM), aiming to self-monitor the wound microenvironment and accordingly react to the changes of the healing process, fitting multiple needs of physiological repair at different stages. ZEM was synthesized using zeolitic imidazolate framework-8 (ZIF-8) coated with an epigallocatechin gallate (EGCG)/Mg2+ complex. We characterized ZEM in the aspects of morphology, physical and chemical properties, and ion release pattern. At the initial stage, ZEM sensed the weakly acidic environment and responsively released a large number of zinc ions to eliminate bacterial infection. Then came the second inflammation stage, where ZEM responded to the oxidative stress of the local wound area with EGCG absorbing excessive reactive oxygen species (ROS), contributing to the downregulation of intracellular ROS. Meanwhile, local inflammation was alleviated by reducing the expression of proinflammatory M1 phenotype factors (IL-6, TNF-α, and IL-1ß). Since the balance of local ROS had been achieved, the resulting disintegration of the EGCG/Mg2+ complex gave rise to the sustainable release of Mg2+ at the proliferation stage, promoting vascularized healing. In vivo animal experiments further proved the diagnostic and therapeutic functions of ZEM. All these results demonstrated that ZEM was a promising treatment strategy in soft tissue engineering.

Rapid, label-free pathogen identification system for multidrug-resistant bacterial wound infection detection on military members in the battlefield.

US military service members experiencing combat-related wounds have higher risk of infection by multidrug-resistant bacteria. The gold standard culture-based antimicrobial susceptibility testing (AST) is not feasible in the battlefield environment. Thus, a rapid deployable system for bacteria identification and AST directly from wound sample is urgently needed. We report the potential of a Rapid, Label-free Pathogen Identification (RAPID) diagnostic system based on ATR-FTIR method to detect and distinguish multi-drug resistant strains for six different species in the ESKAPEE group. Our RAPID system combines sample processing on-broad to isolate and enrich bacteria cells from wound sample, ATR-FTIR measurement to detect antimicrobial-induced bacterial cell spectral changes, and machine learning model for automated, objective, and quantitative spectral analysis and unknown sample classification. Based on experimental results, our RAPID system is a promising technology for label-free, sensitive (104 cfu/mL from mixture), species-specific (> 95% accuracy), rapid (< 10 min for identification, ~ 4 hours for AST) bacteria detection directly from wound samples.

Challenges in the diagnosis and management of wound infection.

Human epithelia are constantly exposed to microorganisms present in the environment or residing as part of commensal flora. Despite this exposure, infections involving the skin and subcutaneous tissue in healthy individuals are, fortunately, quite rare. Many of the wounds that afflict the human body occur in individuals of ill health and/or where the mechanism of wounding is impeded by host immunological, physiological or regenerative dysfunction. The interplay between microorganisms and host immunity is complex and remains ill defined; however, the interpretation of downstream manifestations of the host response to invading microorganisms is still based largely on the clinical signs and symptoms of an active infectious process. In this review article we will provide a brief overview of the current challenges clinicians face in diagnosing wound infections, how chronic infections caused by biofilms are a major challenge, and how there have been minimal advancements in developing new diagnostics or therapeutics in the identification and management of wound infections.

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.

Detecting bacterial infections in wounds: a review of biosensors and wearable sensors in comparison with conventional laboratory methods.

Bacterial infection is a common impediment towards wound healing. Detecting bacterial infections is important to promote wound healing and curb chronic non-healing wounds. In this review, we firstly discuss bacterial communities, including aerobic and anaerobic bacteria in various types of wounds. Following the discussion of wound sampling methods (swab, biopsy) for different wounds, we then discuss laboratory based conventional methods (bacteria cultures, Gram staining, analytical profile index systems, polymerase chain reaction, and gas chromatography coupled with mass spectrometry), focusing on their recent improvement. After that we discussed the contemporary biosensor methods, including e-Nose, electrochemical sensors, surface enhanced Raman spectroscopy, and nucleic acid lateral flow immunoassay. Biosensors embedded into wound dressing, termed wearable sensors or smart wound dressing, are also discussed for their ability of enabling bacteria detection directly from wound sites without the need for obtaining swab/biopsy samples. We have compared all the detection methods for their performance according to their respective targets (either bacteria cells or volatile/non-volatile metabolites); after that we evaluate the suitability of various methods in providing timely and accurate diagnostic results towards real-time, point-of-care testing of bacterial infections.

Electrical monitoring of infection biomarkers in chronic wounds using nanochannels.

Chronic wounds represent an important healthcare challenge in developed countries, being wound infection a serious complication with significant impact on patients' life conditions. However, there is a lack of methods allowing an early diagnosis of infection and a right decision making for a correct treatment. In this context, we propose a novel methodology for the electrical monitoring of infection biomarkers in chronic wound exudates, using nanoporous alumina membranes. Lysozyme, an enzyme produced by the human immune system indicating wound infection, is selected as a model compound to prove the concept. Peptidoglycan, a component of the bacterial layer and the native substrate of lysozyme, is immobilized on the inner walls of the nanochannels, blocking them both sterically and electrostatically. The steric blocking is dependent on the pore size (20-100 nm) and the peptidoglycan concentration, whereas the electrostatic blocking depends on the pH. The proposed analytical method is based on the electrical monitoring of the steric/electrostatic nanochannels unblocking upon the specific degradation of peptidoglycan by lysozyme, allowing to detect the infection biomarker at 280 ng/mL levels, which are below those expected in wounds. The low protein adsorption rate and thus outstanding filtering properties of the nanoporous alumina membranes allowed us to discriminate wound exudates from patients with both sterile and infected ulcers without any sample pre-treatment usually indispensable in most diagnostic devices for analysis of physiological fluids. Although size and charge effects in nanochannels have been previously approached for biosensing purposes, as far as we know, the use of nanoporous membranes for monitoring enzymatic cleavage processes, leading to analytical systems for the specific detection of the enzymes has not been deeply explored so far. Compared with previously reported methods, our methodology presents the advantages of no need of neither bioreceptors (antibodies or aptamers) nor competitive assays, low matrix effects and quantitative and rapid analysis at the point-of-care, being also of potential application for the determination of other protease biomarkers.