Community Research and Development Information Service - CORDIS

Final Report Summary - INFACT (Functional materials for fast diagnosis of wound infection)

Executive Summary:
Surgical site infection is one of the most prevalent healthcare-associated infections and heavy microbial colonization is a key reason for non-healing of chronic wounds. In Germany alone, there are approximately 14 million cases of post-operative wounds and 3 million of chronic wounds. In Europe, this results in estimated treatment cost of > € 5 billion per year while the resulting pain, impairment and social isolation lead to reduced quality of life and, in the worst case, hospitalization, and eventually sepsis and death. Currently, wound infection is not diagnosed until becoming pathologically evident. The InFact project has developed novel diagnostic tools for early detection of infection which can be integrated into wound care devices. Considering the global wound care market of USD 21.6 billion in 2018 together with the social need for early infection detection the economic potential of the InFact devices is clearly evident.
The InFact devices were developed based on the fact that the human inate immune system secretes several enzymes, such as myeloperoxidase, neutrophil elastase, and lysozyme in early and established infection. To allow simple indication of infection status, the InFact partners developed specific substrates for these enzymes which rapidly change colour when contacted with infected wound fluid. An alternative visualization strategy invovled trapping systems for dyes released by the enzyme reaction or which migrate in the wound fluid path as a internal control. Moreover, a pH-sensitive substrate was developed providing additional on infection status. The developed substrates are in-sensitive to wound matrix effects (e.g. influence of heme) and develop strong colours (e.g. dark blue) which are clearly visible in red wound fluid. Apart from the enzyme substrates, internal standards were developed to avoid false negative results. In a next step, immobilization procedures (ultrasound and printing) for these enzyme substrates were developed and optimized. Formulations were designed to allow integration into wound dressings by simple printing procedures. In a second application, these substrates were immobilized onto a cellulose matrix to be used as a standalone test device. A risk assessment was carried out for all chemicals present in the final devices. Substrate production was up-scaled for mass production under required quality standards (clean room etc) with concomittant LCA and LCC analysis. The developed individual substrates and the resulting InFact devices (sensors for integration into dressings, standalone test devices) fulfill essential requirements like storage stability, biocompatibily (e.g. no leaching of chemicals), no cytotoxicity and in-sensitivity sterilization where required. Most importantly, however, several clinical studies conducted throughout the project have demonstrated the functionality of the InFact devices for simple and early detection of wound infection. Interestingly, some of the included enzymes as infection markers show high specificities and lower sensitivities and and others vice versa while the combined result (including pH) clearly has a higher diagnostic value than clinical judgment providing an early signal for wound infection. Based on these successful results a business plan has been developed considering EU regulatory and commercialization aspects, and additional future challenges such as gaining access to the US market.
The collaboration within the InFact project time frame resulted in three joint patents, five publications in peer reviewed journals and a number of conference presentations.

Project Context and Objectives:
The Need: Infection is a global problem of traumatic, post-surgical or chronic wounds.
10% of surgical wounds exhibit bacterial infection within 30 days. Heavy bacterial colonization is the main reason for non-healing of chronic wounds such as decubitus, ulcus cruris and diabetic foot ulcers. The severity and cost of wound infections increase dramatically the longer they remain untreated and reach >5 billion € per year in Europe. The resulting pain, impairment and social isolation lead to reduced quality of life and, in the worst case, hospitalization, and eventually sepsis and death.
Early detection of an incipient wound infection is important for the attending physician since it would allow the timely initiation of treatment, thus reducing the severity of the disease. Currently, however, wound infection is not diagnosed until becoming pathologically evident. As a consequence, the treatment of the patient is further complicated and more likely to have a negative outcome. In addition, wounds are often treated with antibiotics prophylactically, leading to unnecessary selection for bacterial resistance.
Nowadays, a Point of Care Testing (PoCT) device to identify incipient wound infection does not exist. Clinicians rely on a combination of clinical information and long-lasting laboratory tests requiring equipment and experts for diagnosis. However, clinical parameters such as redness, heat, swelling and pain appear only at the lamination of the infection cascade making early clinical judgment so uncertain that 50% of infections are not initially identified. Microbiology laboratory tests used to identify wound infection and the type of pathogen take 3–4 days, and in the meantime, antibiotic treatment is often initiated as a precaution. Furthermore, positive swab findings indicate presence of bacteria only, even, but do not provide objective evidence of a clinically important infection due to variation in host factors, colonization and virulence.
InFact rationale: Consortium partners have patented the know-how to convert wound dressings into a diagnostic tool capable to inform both patient and therapist about the wound status, thus allowing a proactive diagnostic step. The proposed functional materials allow a real time in situ infection diagnostic reaction and, thus, a timely treatment intervention. A next-generation Protective, Predictive and Proactive (triple-P) material for in situ diagnosis of wound infection to be integrated in conventional dressings will be prototyped and advanced to commercialization by InFact.

Under the FP6-LIDWINE project novel active materials for the detection of wound infection were identified and protected by EU patents. The central claims focus on a sensor function that can be embedded into dressing materials to permit simultaneous monitoring of the activities of several enzymes secreted by the human immune system into wound fluids in early stage of infection. In contrast to other off-site methods limited only to detection of microbial colonization of wounds, this novel strategy based on functional materials provides a fast diagnosis (within minutes) at the wound site, without the need of analytic equipment.
While the single use device is convenient for decisions at regular dressing change, it is clear that more and more care is delivered at home by mobile nurses, meaning that wound status is not inspected daily and dressings are often left in place for long periods (up to one week). InFact rationale relies on the anticipation of the next dressing change by identifying early signs of wound infection while the dressing is in place.
The novel InFact material technology will be further translated into a low-cost, real-time diagnostic solution to be applicable to any commercially available primary wound dressing (e.g. hydrocolloids), constituting the "triple-P" materials concept:
✓ Protective - by introducing the concept of surveillance into wound care

✓ Predictive - providing a cumulative wound status signal to predict the infection transition

D1 Ex-vivo study 1 - assessing color reactions by contact of wound fluids and enzyme substrates
D2 Ex-vivo study 2 - assessing color reactions of wound fluids and the ‘in situ wound infection diagnostic material’
D3 In-vivo study - treating patients based on the information derived from the ‘in situ wound infection diagnostic material’ to select therapy in real time with assessment by comparison of clinical outcomes and relative treatment cost

(E) Up-scaling, LCA and LCC studies and process engineering for mass production

E1 Up-scale the dye-labeled enzyme-substrates for production of 100 infection detectors per day
E2 Upscale the process for materials functionalization (reagents immobilization)
E3 Packaging, stability, and storage studies
E3 Life cycle assessment (LCA) and life cycle cost assessments (LCC) of the prototype production - these studies will support the studied processes while supplying recommendations to reduce cost and environmental impact for the up-scaling process
E4 Process engineering for mass production, e.g. 5000 infection detectors per day

(F) Towards CE marking, market survey, and business plan
F1 Requirements for CE Mark
F2 Market estimates, reimbursement prospects and revenue projection by market
F3 Business plan based on the SMEs and industry capabilities in the consortium, and pilot outputs



Project Results:
1.3 Description of the main S&T results/foregrounds
A description of the main S&T results/foregrounds
WP1 Optimization of color reaction between enzyme substrates and the wound infection enzymes (myeloperoxidase, lysozyme and elastase) (Leader: BOKU)
Task 1.1 Phenolic molecules developing colour upon oxidation with myeloperoxidase - Leader UPC
The objective of Task 1.1.1 was to identify sensitive and stable substrates that develop intense colour upon myeloperoxidase (MPO) oxidation, and which will be further integrated in the diagnostic device. The colours of choice (in order to avoid misleading readings of the device) were dark blue, dark green and deep purple or violet. The screening for suitable substrates was carried out based on their structural features and reaction sensitivity to peroxidase oxidation. Several commercially available molecules that contain an aromatic moieties similar to known MPO substrates (e.g. guaiacol) were chosen as possible chromogenic candidates. These compounds were also chosen due to the presence of functional groups that would allow their immobilisation onto a solid surface (diagnostic material). Initially 12 candidates were selected, among these molecules only seven candidates displayed colour after oxidation, out of which 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)diamonium salt (ABTS) and Leukocrystal
violet (LCV) developed more intense colours and represented the fastest response,
However, since only ABTS and LCV displayed one of desired colours (green and blue), although still with insufficient intensity, a new strategy for clear visual perception of the MPO presence was developed, involving combinations of the above phenolic molecules as substrates. Two-component mixtures of all combinations were prepared for reaction with MPO, among different tested combinations the combination composed of ABTS, PHBA and LCV (substrate mix F5) was selected due to its colour change towards blue when oxidized by MPO. (Table 1.1.1).
In order to determine the suitability of the selected substrate mix F5 for its use in a diagnostic device, its immobilization on nitrocellulose strips, mimicking a lateral flow device, under a flow of a MPO reaction mix reaction mix (50 mM PB pH 7.4, 28mU MPO and 2mM H2O2) was tested. Unfortunately the substrate mix F2 did not show immobilisation on the support ,

Continuing the developing of the task 1.1, new MPO substrates with optimised performance were formulated. The new substrate combination comprises of 1 mM m-phenylenediamine 1 mM 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamonium salt (ABTS) and 10% branched polyethylenimine (PEI) in 50 mM phosphate buffer pH6.5. This combination changed from uncoloured to bright violet upon oxidation by MPO in the presence of H2O2. The striking colour change makes this substrate combination optimum for the detection of MPO with the diagnostic device. Moreover, the substrate mix can be applied and dried onto the surface of a paper strip, mimicking a lateral flow device, where it remains immobilised and changes colour when MPO solution flows through the paper strip.The new substrate mix denominated substrate mix F7 was chosen for its immobilization in medical textiles for the further development of task 3.1 in WP3.
Task 1.2 Optimization of colour reaction with labelled lysozyme substrates - Leader BOKU, participant TNO
Various generations of lysozyme substrates were developed within the project. The first series of substrates was based on dyed peptidoglycanes. The production of this substrate was upscaled, printing techiques developed for integration into bandages and was evaluated clinically. To improve various aspects such as uniformity a second generation of substrates based on chitosans was developed. Different chitosan based substrates were synthesized for the detection of elevated lysozyme activities in human wound fluids. Commerical chitosan with a number average molecular weight of 200 kDa and a degree of N-acetylation (DA) of 13% was further N-acetylated improved the lysozyme responsivity of the materials. A DA of ~ 50% was found best for this purpose. Enabling a colourmetric detection of lysozyme, the N-acetyl chitosan scaffold were stained using different dyes and staining strategies. Co-precipitates with starch and Evens Blue as well as covalent staining with Reactive Black 5 (RB5) resulted in lysozyme substrate of high susceptibility towards lysozyme hydrolysis. The different substrates were successfully tested in with buffered lysozymes solutions and with humand fluids of infected wounds.
The RB5 based substrate was further refined rendering it suitable for the chosen printing technology to produce bandage prototypes. In collaboration with TNO, spray drying technology was used generating uniform, spherical particles of diameter < 1µm. These particles fulfil all requirements for screen printing.
Task 1.3 Synthesis of chromogenic peptides/gelatin with specific elastase cleavage sequences - Leader TEC
For the development of elastase detection sensors, three different approaches were evaluated: (I) coupling of a quenching mechanism into the HNE cleavage sequence (FRET system); (II) association of a switch-on chromogenic recombinant protein, Ultramarine (GFP-like); and (III) based on a dye covalently linked to a specific HNE-peptide obtained from a recombinant protein. Approach (I) was mainly developed in task 1.3 and the others approach (II) and (III) were studied on task 1.4 as alternative HNE substrates. several fluorogenic peptides with a HNE-specific cleavage sequence were incorporated into traditional textile dressings.
The best results were obtained with peptide MeOSuc-AAPV-AFC US-immobilized into a cotton dressing and FRET peptide EDANS-Dabcyl chemically-immobilized into a polyamide dressing. These sensor dressings showed an intense fluorescence emission in the presence of HNE, and its visual assessment was possible using a portable UV light. The FRET peptide showed to be more sensitive and effective strategy than the AFC peptide. .However, its chemical immobilization onto the polyamide dressing greatly decreased its detection. After optimization of the in situ immobilization, it will be possible to use these fluorescence-functionalized dressings for an effective and specific monitoring of chronic wounds by simply using a portable UV light source. Obtained results of this approach were presented in two international conferences (on 251st American Chemical Society National Meeting & Exposition in San Diego and on Designer Biology: From Proteins and Cells to Scaffolds & Materials in Vienna) and published in a peer reviewed journal (Ana V. Ferreira, Ilana Perelshtein, Nina Perkas, Aharon Gedanken, Joana Cunha, and Artur Cavaco-Paulo, Detection of Human Neutrophil Elastase (HNE) on Wound Dressings as Marker of Inflammation, Appl Microbiol Biotechnol, 2016, doi: 10.1007/s00253-016-7889-6).
TEC - Alternative HNE substrates
As described above, other alternatives for HNE substrates were also studied, approach (II) and approach (III).
In approach (II), we genetically modified the ultramarine chromogenic protein to increase the distance between the 10 β-sheets barrel (UM10) and the last 11th β-sheet by introducing a stable surface-exposed α-helix containing the HNE-cleavage sequence (E11). Two strategies were followed, one based in the separated production of recombinant proteins, UM10 and E11, and another, based on the combined recombinant protein, UM10E11. Four recombinant proteins, UM (original ultramarine protein), UM10, E11 and UM10E11 were successfully expressed in E. coli BL21 (DE3) with pET28a. For a better understanding, new UM-based mutants were designed and expressed to explore the chemistry involved in the regeneration of the chromophore of ultramarine protein. In this approach, we sucessfully explored the use of the recombinant protein ultramarine as a switch-off and switch-on sensor for elastase. The promising results here obtained will be a turning point for the development of colour protease-sensors using recombinant chromoprotein since to our knowledge this type of sensors were never reported due to the difficult process of achieving a stable chromophore after cleavage. Obtained results of this approach will result in three publications in a peer reviewed journals, that will be submitted in the following months.

Approach (III) involved the expression of a recombinant protein of repetitive sequences of AAPV in E. coli, and then the covalent linkage of its AAPV-monomers to chromatic labels (dyes). This recombinant “polymer” protein offered a solution for the commonly used method of solid-phase peptide synthesis, which is expensive and time consuming.

Task 1.4 Integration of an internal standard - Leader BOKU
Both simple dye migration as will as a pH senitive material was used as positive control. Therefore Bromocresolpurple was covalently immobilized onto cellulose using a new 3-step strategy developed by QZY. This indicator changes colour when coming into contact with wound fluid from yellow to green. In case of in infection and higher pH values of the woundfluid, the indicator changes its colour into blue.

Task 1.5 Identifying additional enzymes that can be detected in wound infection and their colour reaction - Leader BOKU
The main goal of the task 1.5 is the identification of additional enzymes that can be detected in wound infection. Among a potential pool of enzymes, phospholipase C and phospholipase A2 could be identified showing elevated activities in several wound fluids from infected wounds. Within this task, enzyme assays were additionally developed to reproducibly detect the found enzymes.

Task 1.6 The reporter area: colour trapping strategies - Leader SYN
The objective of Task 1.6 was to develop a method to display the reacted enzyme substrates in the reporter (detection) area through a window in the secondary dressing.
Different options were tested, including trapping based on cation/ anion interaction, filtering effects and material affinity. The investigations resulted in two methods, suitable for the enzyme substrates incorporated into the detection area. For both methods options for production of this reporter area were carried out and tested for their feasibility.
Following methods for visualization of the signals were established. 1. The binding and accumulation of a coloured substance at a trapping area positioned in the detection window and 2. A colour change of a substance (enzyme substrate) directly immobilized in the detection window.
The 1st visualization method based on trapping and accumulation of a coloured dye (either generated by an enzyme reaction or -in case of the liquid control- directly printed on the reaction area). The trap is formulated as a printable mixture and contains functional ammonium groups. After printing, the trap penetrates into the diagnostic material and remains immobile integrated in the fibers. Ionic interaction between the negatively charged dye and positively charged ammonium functions of the trapping zone leads to an accumulation of dye in the reporter area and hence a positive (coloured) signal in the presence of the relevant enzyme. This method is used for the visualization of the lysozyme reaction and the control reaction.
The 2nd visualization method is an “in-place” colour change of an immobile enzyme substrate, directly printed in the window of the reporter area. Immobilization of the substrates is caused by hydrophobic properties of the substance and non-covalent chemical interactions with the carrier material. The elastase reaction as well as the myeloperoxidase reaction are visualized by this “in-place colour change”.
Both methods were extensively tested in prototypes lab scale, consisting of the diagnostic material containing the lead substrates for elastase, MPO and Lysozyme, a pH indicator and a fluid control, placed on hydrofiber dressing and foam.

After the successful upscale of the production of the reporter area the functionality of the semi-automatic produced sensors (CON) was tested in lab experiments (SYN, QZY) and in the in-vitro studies at MST.

The Large scale testing of the semi automatic produced prototypes of the reporter area confirmed that the lead substrates for elastase and MPO can be visualized with the optimized in-place colour change method.
Further the in-place colour change method was transferred to a pH indicator system, leading to a clear colour change from yellow to green at pH values higher pH 7.

The trapping and accumulation method is functional for the liquid control and the dye released after hydrolysis catalysed by lysozyme. The reaction velocity of lysozyme was be improved in the last year resulting in sufficient accumulation of the dye.
1.3.2 WP2. Lab prototype production and characterization of the ‘in situ wound infection diagnostic material (Leader: BIU)
Task 2.1 Continuous production process of the diagnostic material by immobilization based on ink-jet printing Leader BOKU, Participants UPC, TEC, TNO
Based on the enzyme substrates above, formulations were successfully designed to allow simple printing. The printing process is shown in detail in Task 5.2.

Task 2.2 Semi-continuous production process of the diagnostic material by: impregnation, chemical immobilization or ultrasound Leader BIU Participants TEC, BOKU
2.2.1 Impregnation and 2.2.2 Chemical immobilization - Leader BOKU Participants QZY, DAVO, BIU
Simple impregnation of cellulose carriers (e.g. for peptidoglycane) and covalent chemical immobilisation procedures were used. The latter involved the covalent attachment of myeloperoxidase substrates such functionalised Fast Blue and pH indicators via siloxane chemistry. All substrates retained their ability to change colour upon contacting with infected wound fluid after immobilisation (=hydrolysis of siloxane functionalised substrates). For the myeloperoxidase substrates the biochemical mechanism was elucidated to itentify the nature of reaction products (regulatory issues).

Task 2.3 Assessment of the efficiency of the immobilization protocols Leader BIU
BIU has developed a method for sonochemical immobilization of substrates. The substrates for detection of MPO were suggested by UPC, substrates for Lysozyme detection were proposed by BOKU and the deposition of HNE substrtaes and their activity was done in collaboration with TEC.
Generally, the coating requires a dissolution of the desired molecules in an appropriate solvent. Then, the solution is sonicated in the presence of the fabric to be coated and at the end of the reaction the fabric is washed and dried. The quality of the sonochemical coating of organic NPs is influenced by the following parameters:
- Solvent
- Reaction time
- Temperature
- Concentration of the precursor
Therefore, these parameters were varied in order to get the most active coating. The optimization was based on trial and error, by checking the activity of the coated surfaces. The final report summarizes and the most promossing results obtained during the 4 year project.

Detection of MPO
Various phenol substrates were used for coating:
[1] o-Dianisidine – FBB
[2] 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamonium salt – ABTS
[3] 4-Hydroxybenzoic acid - PHBA
[4] Combination of 2 substrates: PHBA+ABTS, FBB+ABTS and etc..
[5] ABTS + PHBA + LeukoCrystal Violet (LCV))
In order to increase the color response signal, it was decided to combine 2 of the above mentioned molecules. The first combination was composed from 4-Hydroxybenzoic acid (PHBA) and ABTS that were sonochemicaly deposited on bandage from their water solution. The coating is clearly seen from the HRSEM images and a strong, blue color was developed upon exposure of the coated bandage to MPO and H2O2.
To further improve the MPO substrate, UPC sugested: ABTS + PHBA + LeukoCrystal Violet (LCV). These 3 compounds were deposited sonochemicaly on bandages.. After the coating reaction, the color of the bandage turned to greenish. As it can be observed from SEM images, a coating composed of nanoparticles was formed. Color response of the coated textiles was tested in the presence of MPO and it was observed that color of the coated bandages changed from greenish to blue. However, during the activity measurements it was observed that the substrates leached to the buffer solution and has changed the color to blue prior the incubation with MPO. This might cause false positive results. The possible reason for changing the color in buffer might be the oxidation of the substrate.
Detection of Lysozyme
During the project various chitosan based substrates were used for coating:
Chitosan-Starch-Evans Blue
N-acetyl chitosan stained with RB5
Succenyl chitosan stained with RB5 – a soluble substrate at pH 8 – a promising candidate
Succenyl chitosan stained with RB5 (CSR) dissolves in neutral or basic pH. The sonochemical deposition of the stained succenyl chitosan was done by dissolving the substrate in water and the sonication of the solution was conducted in the presence of the fabric. During the ultrasound irradiation nanoparticles of succenyl chitosan are formed and simultaneously deposited onto the fabric. Reaction parameters such as: concentration, time were modified. During optimization, initial concentration was increased from 0.001 to 0.004gr/ml
The best candidate for detection of Lysozyme was the bandage coated from the initial concentration of 0.002 gr/ml. However, the coating was not homogeneously distributed along the bandage and further attempts were done in order to improve the quality of the coating. Initial concentration of Succenyl chitosan was increased from 0.001gr/ml up to 0.004 gr/ml, keeping the rest of the reaction parameters unchanged. As it can be seen even by the naked eye, sample 2785 resulted in a homogeneous blue color of the bandages and the homogeneous coating was further approved by HRSEM image.
In terms of color response, sample 2785 presented very good results, in a very short exposure time, in comparison to sample 2738-1 and this sample was chosen as a best candidate for detection of Lysozyme.
A control experiment, in which the coating of succenyl chitosan was done by regular dipping of the bandage into aqueous solution of stained chitosan was conducted. The fabric was washed, dried and analyzed by HRSEM. No coating was formed on the surface of the fibers during the dipping process. However, these samples were also tested or the activity with Lysozyme solution. The signal of the color was intense but a leaching of the substrate was observed, the adsorbed substrate was immediately removed when exposed to Lysozyme solution. This indicates, that when the coating is done by dipping, the molecules adsorbed on the surface are easily removed, while using the sonochemical coating method the substrates are strongly imbedded in the fabric and gave an excellent color response.
An additional approach for the detection of Lysozyme was suggested and it includes formation of microspheres from chitosan and appropriate dye. Generally, the sonochemical technique is known for its ability to form microspheres from proteins, polymers and biological substances by employing short reaction time. The process includes a sonication of a 2 phase solution in which phase 1 composed of a water soluble compound and phase 2 includes an oil soluble substances. After 3 min of sonication an emulsion is formed which is composed of spheres with oil core and a shell made of the protein or the polymer or the biological molecules. In the current case, a solution of succenyl chitosan was used and 2 water soluble dyes were incorporated: Tolui-dine Blue O and Trypan Blue. As a result, spheres were formed with oil core and a shell which composes from chitosan and the dye.

The formed microspheres were analysed by microscope and DLS and the results indicate that most of the spheres have a size of less than 500nm, but there are also spheres of several microns. In terms of color response, these sample are currently under investigation.



In addition, it was tried to coat the mentioned above dye microspheres on bandages using one-step sonochemical reaction. Namely, during the process for formation of the microspheres, the bandage was inserted and coated..

As for the activity of such coated bandages, the color response was weak as compared to activity of succenyl chitosan stained with RB5 which was deposited on bandage. The poor activity of the coated microspheres might be due to their solubility in water and while exposing such substrate to Lysozyme solution it might undergo dissolution, prior to the reaction with Lysozyme. Therefore, in order to improve the colour response, a substrate that is lees soluble in water/plasma should be search for.

Detection of Elastase
For the detection of elastase 3 commercially available substrates were suggested by TEC:
These 3 substrates were deposited sonochemically from water solution.
1. Elastase substrate, fluorogenic
2. SCP0249 Pancreatic Elastase Substrate
3. SCP0170 Human Granulocyte Elastase Substrate

The reaction was done at low temperature, by placing the reaction cell into an ice-water bath, since these substrates are sensitive to heat. As can be seen from the HRSEM images, a coating of nanoparticles was formed. The coated bandages were sent to TEC for efficacy tests. The activity tests have revealed very weak color response. Therefore, the initial amount of the substrates was doubled.. The activity tests have shown a good color response for the fluoregenic substrate that was sonochemically deposited on bandage. Further in-vitro tests were conducted with this substrate as it was chosen as a good candidate for detection of elastase.
Herein are the main mitigated risks/obstacles for sonochemical immobilization:
Risk Risk mitigation plan
Color developed by the substrate interferences with the wound fluid (reddish color) Choosing a substrate which upon a reaction with the enzyme will develop strong color of blue, green, purple....
Leaching of immobilized substrate prior the reaction with wound enzymes Choosing substrates that has no solubility in water / plasma
Low detection signal Increasing amount of coated substrate by changing reaction parameters / combining 2 substrates

To summarize, sonochemical immobilization was proven as an effective method for coating various enzyme’s substrates. By varying reaction parameters such as: solvent, reaction time, concentration and temperature, the quality of the coating can be modified and improved. The sonochemical coating doesn’t damage the molecules and in certain reaction parameters, a good coating, in terms of color response was obtained. Promising results were obtained with substrates for detection of MPO. A good colour response was achieved while combination of 2 different substrates, for example 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamonium salt (ABTS), with 4-Hydroxybenzoic acid (PHBA). Strong blue color was developed upon exposure of the coated surface to solution of MPO and H2O2.
In case, of Lysozyme detection, succinyl chitosan which was stained with RB5 was sonochemically coated on fabric and has shown a high color response upon reaction with Lysozyme solution.
The elastase was detected by Fluorogenic substrate which was sonochemically coated on cotton using optimized reaction parameters. Firthemore, up-scale feassability study for the sonochemical immobilization of MPO substrate was developed and conducted by DAVO.

Task 2.4 Assembly and characterization of the diagnostic material Leader SYN, participant CON
To select the diagnostic materials several aspects had to be considered.
- Flexibility and low thickness to preserve the wearing comfort for the patients.
- Stability and integrity to ensure full functionality over the hole application time.
- Liquid handling properties to achieve a sufficient liquid flow for full functionality of the reporter area.
- Compatibility with the substrate chemistry as well as the enzymes released from the wound.
- All materials must be able to be sterilized, preferable by gamma radiation.
- Materials with supporting functions, e.g. the UV ink lines which form the fluid path and the adhesives that stick the reporter area onto the dressing must additionally fullfill their specific requirements

During the process of material selection the diagnostic material was produced and assembled in lab scale. Manual reagent application and assembly was carried out at SYN and enabled production of 30 Prototypes per day. Further all materials were tested in special setups to proof their applicability.
Most important was the selection of a suitable carrier material for the chemistry. At an early timepoint the decision was made to work with a nonwoven material. In the following process more than 30 materials from 3 manufacturers were tested, differing in the material (Polyolefin, PET, PA, wood pulp, viscose, Microfiber or mixtures of these), the weight, tensile, texture and potential surfactant. Further the wicking capazity, wicking time and compatibility with the chemistry had to be tested. This testing procedure clearly showed that a high content of wood pulp (at least 20 %) is a main factor to support the functionality. Further a small fiber size is required and a medium weight.
After selection of the nonwoven carrier material the other materials were selected and tested systematically.
. Following key steps were required to reach functional materia for prototypingl:
• Selection of a set of materials that was compatible with all aspects.
• Testing the reagents for printability by various methods
• Testing of shelf life and storage conditions for all components
• Test of shelf life and storage conditions for the finished prototypes.

Semi-automatic production was done at CON and resulted in the production of >2000 diagnostic material prototypes per week.
Therefore, reagents were incorporated with printing on the carrier material (fluid path material/basic material). Diagnostic fields, as well as top and bottom layers were the cut out automatically with a laser cutter. The following assembly step was done manually.
The produced prototypes were tested in the ex-vivo study at MST.
WP3. In vitro/in vivo assessment of the ‘in situ wound infection diagnostic material’ (Leader: SYN)
Task 3.1 In vitro efficacy: Leader UPC
The aim of Task 3.1 was to assess the stability of sonochemically immobilised MPO substrates on gauzes and textiles formulated in task 1.1 of WP1. Partners BIU and DAVO impregnated sonochemically gauzes and textiles with the MPO substrates. To determine the stability of the sonochemically coated substrates, the gauzes were first incubated in phosphate buffer (PB) pH 7.4 for 30 min, the washing step, before incubation with the MPO reaction mix (50 mM PB pH 7.4, 28mU MPO and 2mM H2O2), the detection step.
Running in parallel with the development of task 1.1 the selected substrate mix F5 was sonochemically immobilised on gauzes by partner BIU. F5 substrate mixture comprises ABTS, PHBA and LCV, called changes from pale green to dark blue colour when is oxidised by MPO in the presence of H2O2. Two types of gauzes with immobilised substrate (2820 and 2821) were provided by BIU and assessed for substrate stability.
The substrate stability assessment revealed that after the wash step, the gauzes changed to pale blue colour) and the washing buffer acquired also a pale blue colour.1) being more accused in the case of gauze type 2820. When the washed gauzes were incubated with the MPO reaction mix the colour solution changed towards a faint blue/green colour a change more accused in the case of gauze type 2820, regarding the gauzes no further colour change was observed. Finally, when MPO and H2O2 were added to the washing solution, a colour change towards green/blue colour was observed thus indicating that the substrates were released from the gauzes during the washing step and, at the same time, explaining the weak and faint colour change induced by the washed gauzes when these were incubated with the MPO reaction mix. The fact that the gauzes changed to pale blue colour during the washing step indicates that the use of substrate F5 can lead to the determination of false positives, therefore is not a suitable substrate for the development of MPO detecting gauzes.
Following the development of task 1.1 the new formulated MPO substrates were immobilised onto gauzes and textiles. Partners BIU and DAVO impregnated sonochemically gauzes and textiles with the new selected MPO substrate mix F7 composed of m-phenylenediamine, ABTS and PEI.
Regarding the gauzes produced by BIU, four types of gauzes with immobilised substrate (3101-1, 3101-2, 3107-1 and 3107-2) were provided by BIU and assessed for substrate stability. After the wash step, the gauzes maintained the initial white colour. When the washed gauzes were incubated with the MPO reaction mix, the reaction mix and the gauzes changed towards a violet colour which is the expected colour change for the substrate mix F7. When MPO and H2O2 were added to the washing solution, a colour change towards violet colour was observed indicating a substrate leak during the washing step, however it didn´t cause loss of the MPO detection properties of the coated gauzes. Further assessment with the gauzes coated with F7 substrate mix was performed testing their ability to detect MPO in wound fluids. Wound fluids were obtained by washing gauzes used in chronic wound treatment with PBS (gauzes were kindly provided by the hospital of Terrassa). In contact with the wound fluid and in the presence of hydrogen peroxide the coated gauzes changed its colour towards violet thus detecting the presence of MPO in the wound fluids moreover the absence of colour change of the gauzes when these are in contact with wound fluid without hydrogen peroxide indicates no cross-reaction with the different elements in the wound fluid other than MPO.
Partner DAVO produced three types of coated textiles (pescarus 100% polyester, 100% polyester and 55% viscose 45% polyester). Despite during the washing step no changes in the textile colour were observed, when textile was incubated with the MPO reaction mix no colour change was observed.. The addition of MPO and H2O2 in the washing solution did not cause colour appearance indicating that the lack of colour change of the coated fabrics in the presence of MPO is not due to the substrate leakage during the washing step. Therefore, the assessment performed reveal that the coated textiles do not show MPO detection properties.
Task 3.2 In vitro biocompatibility Leader SYN
The new dressing had a layered structure consisting of an already existing sodium carboxymethylcellulose fiber nonwoven structure (the wound contact layer); the existing open pore textile/foam structure (the second layer) and a new layer of an additional functionalized nonwoven construction in which the additional chemical excipients are contained; and the existing final outer film layer which allows visualization of the indicator result (color change/development). The additional chemical excipients are identified by CON and SYN. By product design and with intended use, any chemical excipients (released dyes, dyed fragment or other fragments) will be held within the functionalized nonwoven layer of the wound dressing and will not come into contact with the wound contact layer or the wound bed directly. The device is classified as Class IIB medical device (surface device, breached or compromised skin), as the primary purpose of the device is a wound dressing, with an auxiliary function of providing the clinician with an indication of wound infection status.
In advance of testing the in vitro biocompatibility a theoretical toxicological risk assessment of the indicator chemicals was done by the partner CON. This risk assessment, including the chemicals subjected to the risk assessment and the method details, is described in detail in the 2nd periodic report.
A permissible device level (PDL) for each identified chemical was calculated as absolute mass/device and mass/cm2 assuming a device surface area of 600 cm2 (largest device). The values are calculated for a standard reference body weight of 70 kg. These values are at or below the tolerable exposure (as defined individually).
In wound dressing there is the potential that components may come into contact with broken skin and thereby enter the body in a manner distinct from that in the intact skin setting. This quasi parenteral exposure potential is additionally sensitive in that the healing of the wound is potentially complicated if the dressing contains agents that inhibit the action of immune cells, or indeed the cells responsible for healing and re-epithelisation.
It is, therefore, desirable and essential that compounds in a dressing are not specifically toxic to cells.
In our case, we were interested in the toxicity of compounds that generate blue colours which is in the colour range of most viability reagents. Our data indicate that the reagents themselves engender a blue colour that influences the data. The cytotoxicity was tested in an MTT assay, a commonly used colorimetric assay to evaluate cell metabolic activity.
The detailed method is described in deliverable 3.3.
The results of the enzyme substrates and their cleavage products are listed in table 3.2.1.
Table 3.2.1.
CSY2002 ID 6549 CSY 1704 F-moc-
A-A-P-V-OH Indigo F-moc-
A-A-P-OH Life control Dead control
Compound conc. [µM]
100 0,50 1,36 1,37 1,59 1,12 1,58 1,76 0,23
50 1,46 1,66 1,74 1,79 1,00 1,76 1,91 0,14
25 1,89 1,88 2,00 1,87 1,24 1,72 1,95 0,13
12,5 2,00 1,94 2,21 2,07 1,25 1,95 2,00 0,13
6,25 1,96 2,01 1,87 1,99 1,33 2,00 2,02 0,13
3 2,03 2,02 1,90 2,05 1,28 1,98 2,02 0,11
1,5 1,84 2,07 2,03 2,01 1,29 2,01 2,02 0,15
0,75 2,07 2,12 1,95 1,99 1,32 1,79 1,76 0,12
Cut off: OD 1.7
Elastase substrate CSY 1704 as well as its tested cleavage products Fmoc-A-A-P-V-OH and Fmoc-A-A-P-OH do not show changes in cell growth up to concentrations between 50 and 100 µM. For the MPO-substrate CSY2002 and its cleavage product ID 6549) concentrations between 25 and 50 µM are without inhibitory effect.
Generally, indigo appeared to influence MTT assays as no change in visual cell viability was observed under conditions where MTT levels were influenced by Indigo.
The observation that the decyl conjugate of fast blue (CSY2002) is slightly more inhibitory that the free fast blue suggests that the long lipophilic tail is probably providing the molecule with a weak surfactant property. The weak positive charge would make the molecule into a weak cationic lipophile which in a cell culture context is associated with low potency membrane disturbance. These effects are not very significant in the real setting. For example, the far more potent benzalkonium chloride is widely used as a disinfectant and is toxic to cells in the range of 1-3 µM.
Since substrate concentrations on the device will not exceed a maximum of approximately 15 µM for the Elastase substrate and approximately 21 µM for the MPO-substrate the results suggest that CSY2002 and CSY 1704 pose no significant risk. Results in the ink formulations are comparable and within an acceptable range. Furthermore, the dressing construction foresees that the materials are bound to the device, that the back diffusion is not favoured by the construction and that the high levels of protein in the exudate and the low solubility of the materials adds to their overall non-availability in this setting.
Task 3.3 In vivo biocompatibility, leaching and efficacy Leader SYN
The dressing and the reporter area were designed to avoid backflow of the substrate chemistry into the primary dressing material. The purpose of task 3.3 was to test if the materials and reagents used in the sensor could leach back in the primary dressing, leading to the possibility that they could come in contact with the wound bed. As the diagnostic area (containing the materials and reagents for the enzymatic reaction) is connected to the dressing via a small channel for sample supply there is a small possibility that the materials and reagents could leach via back-flow, damage of the sensor cover or simple wearing effects (e.g. movement, hygiene) into dressing. In a wearing experiment and wound simulating tests with differing moisture flow, only in the samples worn 5 days traces of reagents could be detected. Furthermore, full functionality of the indicator reagents after 5 days wearing of the dressing could be confirmed.
Testing to test biocompatibility, leaching and efficiency of the sensor as part of a dressing in in-vivo experiments and wound simulation tests. Effects of e.g. movement, perspiration, hygiene and clothing on the integrity of the sensor materials and reagents should be investigated. Design and materials have been developed to prevent any leaching effects. So an important part of this study was the investigation of possible leaching of the reagents back in the primary dressing to confirm or reject the chosen design.
Sensors, produced semi-automatic at CON were assembled with dressing material (Aquacel foam). These assembled sensors were used for the wearing test and the leaching tests.
a) In the wearing test sensors stuck on Aquacel foam were placed at three different positions on the non-wounded test person`s skin. 1 sensor (on Aquacel foam) was stored as control under dry conditions at room temperature.

After 5 days the dressings were removed and visually checked for damages e.g. stretching of the sensor, porosity of materials or loss of contact between sensor and Aquacel foam. Afterwards a syringe pump experiment with artificial wound exudate (simulating an infected wound) was performed to test functionality of the indicator substances.
Visual evaluation did not show any influence of the experiment on the physical integrity of the dressing in terms of loss of contact, porosity, stretching or other damages.
Also the functionality testing by application of artificial wound exudate onto the dressing, simulating an infected wound did not show a negative effect of the wearing on the functionality of the dressing. The detailed methods and results are described in deliverable 3.4.
To test if the chemistry is immobile and if the dressing design avoids backflow in a sufficient manner leaching experiments were perfomed. After a 7 day period in which the dressing and sensor material was treated with different liquid flows, the primary dressing was extracted, the extract concentrated and analysed by LC-MS/MS.
The analysis of all test samples revealed no detectable amount of any of the three compounds left in the sensors used in the simulation experiments. Trace amounts (between 1 and 2 ng) were found in the sensors placed in the unwounded skin volunteer.
Wearing of the dressing the in dry state had no effects on biocompatibility or efficiency under the conditions studies in our experiments..
We observed leaching of reagents only in traces in the dressings worn by the volunteer. Detected amounts were less than 0.0025 % of the reagents printed on the sensor.
WP4. In human clinical studies: ex-vivo and in vivo (Leader MST)
Task 4.1 Ex-vivo study 1 - assessing colour reactions by contact of wound samples and enzymes substrates Leader MST Participants QZY, SYN, CON, TNO
The aim of ex-vivo study 1 was to determine the diagnostic accuracy of the newly developed enzyme substrates for myeloperoxidase (MPO), lysozyme and human neutrophil elastase (HNE) for the detection of wound infection. These substrates were developed as first step towards a smart wound dressing that provides a colour change when elevated enzyme levels are present in wound fluid (i.e. presence of infection). MST therefore designed a study in which wound fluid samples were collected from patients with a variety of wounds presenting at the wound care clinics. These wound fluid samples were then tested with the newly developed enzyme substrates at Qualizyme. Enzyme-results were compared to the actual wound infection status as determined by clinical judgment and microbiological culture results. We were able to collect and test a total of 60 wound fluid samples. At Qualizyme, both positive (colour change) and negative signals were observed for each enzyme. Lysozyme and HNE demonstrated both high sensitivity (71 and 86% respectively) and specificity (77 and 68% respectively). MPO seemed less accurate with a low sensitivity of 14%, but a high specificity of 91%. When the three enzyme assays were combined into one result, diagnostic properties were fair with an area under the curve of 0.68. This study has demonstrated that, overall, the enzyme assays have higher diagnostic value than clinical judgment alone, which is the first go-to method in clinical practice. The results from this study have contributed to the further development of enzyme substrates and their incorporation into a smart wound dressing sensor.
Task 4.2 Ex-vivo Study 2 - assessing the colour reactions between wound enzymes and the InFact material Leader MST Participants QZY, SYN, CON, TNO
This study investigated the active part of the wound dressing containing the enzyme substrates (INFACT ID sensor). Patients presenting at the wound care clinic of MST were asked to provide their used wound dressings for testing with the ID sensor. The used dressings were wetted with saline to promote transfer of wound fluid onto the ID sensor, and afterwards the sensors were placed onto the dressing for 24 hours. MST was able to test a total of 72 ID sensors on a variety of wound dressings from different wounds. In 8 dressings, there was no sufficient transfer of fluid from the dressing onto the ID sensor as indicated by the pH indicator (fluid control). Of the remaining 64 dressings, 13 dressings originated from infected wounds, 46 from non-infected wounds and for 5 dressings the wound infection status was dubious. Overall we observed both positive and negative signals for all enzymes. The signal for MPO was somewhat difficult to observe as the developed colour was similar to wound exudate (brownish). However, we did observe fair diagnostic properties for the detection of wound infection (in comparison to clinical judgment), with a sensitivity and specificity of 62% and 65% respectively. Remarkably the InFact ID sensor was more frequently positive than was expected based on clinical judgment. This might indicate that the ID sensors are able to provide an early signal for wound infection, i.e. a signal while wound infection is not (yet) visible. The results from this study have contributed to the further development/improvement of the ID sensor.
Task 4.3 In-vivo Study: compare information from the sensors with clinical judgement and microbiological results of culturing of wound fluids. Leader MST
This study investigated the usability and accuracy of the newly developed INFACT SID test. Market research from ConvaTec pointed out that there was a more urgent need for a point-of-care test, providing results within a few minutes. Smart wound dressings (providing results over the time that the dressing is placed onto the wound), are desired but a point-of-care test could be developed, introduced and probably accepted more rapidly in the market. Therefore, an easy-to-use swab-based device was developed within the INFACT project. This device, the INFACT SID test, contains of a tube in which three indicator strips are incorporated; one strip for pH, one for MPO and one for HNE. After taking a wound swab, the swab should be place into the tube where the swab tip is squeezed to promote transfer of wound fluid onto the indicator strips. In case of elevated enzyme levels in the wound fluid, the indicator strips will show a colour change within 5 minutes. To determine whether this test was able to accurately detect wound infection and to determine user-experiences in clinical, MST designed and conducted this in-vivo study. During this study patients were asked to provide informed consent before a wound swab for the INFACT SID test was taken. Clinical judgment was used to determine wound infection status, with the support of microbiological culture. We were able to include 63 patients with 68 different wounds in this study. Amongst these patients, we were able to perform 91 INFACT SID tests (including 23 follow-up measurements). The test was easy-to-use in clinical practice and colour changes could easily be observed after 5 minutes. We were able to observe both positive and negative signals for all indicators (pH, MPO, HNE). However, design improvements are needed to ensure sufficient transfer of wound fluid from the swab into the device as the indicators were not completely wetted in more than 50% of all measurements. Nonetheless, the test is very promising and received very well in clinical practice. Therefore, ConvaTec aims to improve the device and continue collaboration with MST to further investigate improved versions of the INFACT SID test.
WP5. Up-scaling, LCA and LCC studies and process engineering for mass production (Leader: QZY)
Task 5.1 Upscale the process for enzyme substrates production for 100 InFact materials per day Leader QZY
The minimal amount of substrate to be produced in one process arises from the amount needed to produce 100 devices per day (each with 4 sensors) in 1 month, leading to a minimal amount of 5g substrate per process. For the Infact device, 3 different enzyme substrates for the detection of human myeloperoxidase, elastase and lysozyme are used.
- Ela substrate: CSY 2002, SYN
- MPO substrate CSY 1704 SYN
LYS substrate PG-RB5 QZY
During this project, the synthesis/production of these substrates was optimized and up-scaled. A maximum of 200 mg/day or 5 g/ month of each substrate (including 40% surplus to consider loss during production process) are calculated. All three syntheses were up scaled successfully, in the processes up to 50 g CSY2002, 70 g CSY1704 and 16 g PG-RB5.

Additionally, to these 3-enzyme substrate, pH-sensitive material could be produced. The pH indicator paper is pH sensitive in the right range and turns its colour from green to blue in case of infection. To produce this material a novel 3 step immobilization strategy was developed. Using this process, the production of 100.000 spots per day is possible

Task 5.2 Up-scale process for materials functionalization (reagents immobilization) Leader SYN
The production process in terms of reagent functionalisation, including immobilization and impregnation and assembly was established. The substrate chemistry was formulated as printable inks and immobilized on the nonwoven carrier material by screen printing. A bespoke laser-cutting machine was used to cut adhesive films, then a standard lamination process was used to assemble the ID sensors into its novel design that supports stability and immobilisation of the reagents.
Following materials were used to up-scale the process: Nonwoven (PP/PE/cellulose), UV-cured ink, HNE-responsive ink (SYN), MPO-responsive inks (including 2 x co-factor inks) (SYN), LYS-responsive ink (QZY), Control ink (SYN), Trap ink (SYN) and a flat screen printing set-up (CON), enabling the application of the reagents onto the carrier material and supporting the immobilisation.

Nonwoven sheers were printed A4-size so that 20 x ID sensors could be printed per sheet (Fig 2), which could then be laser-cut.
Printed non-woven was then sandwiched between laser cutter-modified adhesive film layers to form the finished ID sensor.
All used materials were selected to be thin, flexible and not interfering with the primary dressing. The materials were specified and all relevant information collected and evaluated by the partners SYN, CON and QZY.
The Up-scale process for the reagent functionalization was possible up to a rate of 1,000 per hour using semi-automated, manual flat screen printing, laser cutting and lamination processes. These processes are already implemented in the wound dressing manufacturing process at CON and do not pose undue technical difficulty.

Task 5.3 Packaging, stability, and storage Leader CON
Primary packing specifications of the SID device have been developed based on mass production of SID InFact devices for the final clinical study at MST (WP4). The packaging for the sensor tube is a light-occlusive material, such as a film laminate containing aluminium foil or metalized film and a desiccant sachet is also added to control moisture. The packaging is also oxygen-occlusive, achieved by a heat seal. The sealed sensor tubes are then packed into cardboard carton secondary packs then into shippers, or in the case of the MST clinical trial, direct to shippers.

Task 5.4 Life cycle assessment (LCA) and life cycle cost assessments (LCC) of the prototype production Leader TNO, participants ALL
A LCA and LCC study was performed for the product developed within this project. The new product replaces traditional, non-diagnostic wound dressings and minimises false diagnosis. The manufacturing of the different substrate inks was included in the last assessment. Data (costs, material, water and energy inputs) were provided by QZY and SYN. The main contributors to environmental impact and costs are outpatient clinic visits and gloves. Travelling also contributed to environmental impact, whereas nurse visits were important for costs. In case of infection, the InFact material showed lower environmental impact and costs due to a shorter healing period of one month.

Task 5.5 Process engineering for high quantities production i.e. 5000 InFact diagnostic materials per day - Leader CON
A semi-automated/semi-manual process was developed to manufacture devices for the InFact clinical study at MST. This process included the automated step of 3D-printing the sensor cap; the semi-automated (bulk) processes of substrate paper slitting, sensor assembly, sensor cutting, and packing; plus the manual processes of tube cutting, sensor and sensor cap insertion, and labelling. Packaging is described above in T5.3. All process were defined in terms of materials and methods with pictorial guidance in report “D5.5 – Design of a mass production line for 5,000 devices per day”. The manufactured devices proved to perform effectively in laboratory and clinical testing. A semi-automated/fully-automated process in development was also summarised.
WP6. Towards CE marking, market survey and business plan (Leader: CON)
Task 6.1 Requirements for CE Mark and regulatory issues Leader CON
The regulatory strategy is to develop this product as an in vitro diagnostic (IVD) device which is consistent with the proposed mode of action and method of application. It will therefore be necessary to comply with the existing directive, In Vitro Diagnostic Medical Devices Directive 98/79/EC (IVDD), and the recently implemented regulation, European In Vitro Diagnostic Regulation (IVDR), which is being phased in over the next 5 years. It is anticipated that existing Design Control procedures will be adequate to document the design of this proposed IVD from a perspective of Code of Federal Regulations Part 820 Quality System Regulation (QSR) Requirements for Medical Device Manufacturers for FDA 21 and ISO 13485 Medical devices – Quality management systems – Requirements for regulatory purposes. CON is working with both BSi and the US FDA to determine regulatory pathways. In Europe, the device is likely to be a ‘general classification IVD’ under the current IVD Directive (98/79/EEC) which does not require notified body (e.g. BSI) involvement (i.e. can be self-certified). An ISO 13485 scope extension to cover the scope of ‘wound IVDs’, ‘infection IVDs’ or similar, would be required and BSI would conduct an on-site inspection to cover control of any subcontractors. The feedback from BSI was also that the project design and development documents (Technical File) would be inspected in this instance. This strategy will need to be reassessed once the impact of the IVDR is fully understood. In the US, regulatory route is likely to be de novo 510(k). In order to seek further clarification, a pre-submission document has been submitted to the FDA and a meeting will be held with the FDA in early March 2018 to present proposals and seek FDA feedback.

Task 6.2 Market estimates, reimbursement prospects and revenue projection by market Leader QZY
One of the key factors for succeeding in the wound care market are the price, reimbursement and having the right launch strategy. Therefore, an independent market research study was carried out including the Standalone ID and Aquacel ID product concepts in the inpatient and outpatient settings. Special focus was on the reimbursement possibilities of the ID products in different countries.

Therefore, a multicentred market research study was carried out to obtain data for the development of reimbursement and launch strategies. 26 detailed interviews were carried out in Germany, UK and US

The respondents had different backgrounds:
- 6 TVNs / WOCNs
- 2 Directors of Nursing Homes
- 9 Vascular Specialists
- 3 orthopaedic / cardiovascular surgeons
- 3 Dermatologists / diabetologists
- 3 Procurement Managers / Medicines Manager


• Price/Price potential
The price of a new product is an important issued to be considered. Pricing is one of the classic 4 Ps of the marketing. Charging too much means that the product won´t sell. Charging too little means loss of revenues and profits. For both applications, Standalone ID and the dressing, pros and cons for the pricing issues were identified. Especially for the Standalone ID, the price potential suitability was high.

• Reimbursement
A special focus was on the reimbursement possibilities of the products in different countries. The price potential of the Standalone ID and the dressing was defined for Germany, the UK and the U. Reimbursement is an important element when developing a new product. Therefore, the reimbursement strategies for outpatient and inpatient settings were developed.

There are different pillars of reimbursement: coverage, coding and payment.

Coverage will only apply to new medical procedures and technologies that are not currently defined in the regulations. This way is challenging since a large amount of clinical data is needed.

Coding is the language of CMS (Centres for Medcare & Medicaid Services), private payers, facilities, and physicians. Without a proper code, procedures and products are not paid for. There are different types of codes, depending on where a procedure is performed (e.g., inpatient, outpatient), who is performing the procedure (physician, nurse, technician), and what equipment is involved.

In this study, reimbursement possibilities were investigated for the inpatient and outpatient clinical settings. Inpatient care generally refers to any medical service that requires admission into a hospital, while outpatient care, on the other hand, is medical service provided that does not require a prolonged stay at a facility. This can include routine services such as check-ups or visits to clinics.

• Launch strategy
Parameters like the price potential, revenue potential, investment needs, time to market were evaluated to generate a launch strategy.
Conclusion:
The plan is to start the product launch with the Standalone ID in inpatient facilities being focused on DFUs/high-risk wounds. In Phase 2 all type of wounds will be included. Aim of both phases is to generate clinical data for reimbursement studies and to gain acceptance for roll-out into the community.

In Phase 3a the Aquacel ID foam dressing will be launched using. In phase 3b more patients will be covered by additional Aquacel ID dressing formats. Launch Standalone ID selectively in inpatient setting to gain momentum and clinical data for mass market, followed by Aquacel ID dressings.

Task 6.3 Business plan based on the SMEs and industry capabilities in the consortium, and pilot outputs Leader CON
A business plan has been developed to document the activities and requirements to achieve commercialisation of the stand-alone infection detection (SID) device. Infection detection is a completely new sector in wound care. Consequently, market research, education and market preparation are critical to the success of this device. It is clear that there is a significant clinical need for devices that can help clinicians determine the infection status of wounds, and the InFact SID device will be amongst the first of its type in this important sector.
Current practice identifying infection / bioburden in wounds (e.g. clinical signs, microbiology) can be subjective and lead to an incorrect diagnosis. A point-of-care diagnostic device that indicates early sign of wound infection is considered a significant advancement over current practice’ leading to faster treatment of infection along with more appropriate use of antibiotics and the associated improved clinical and economic outcomes.
Key challenges include gaining access to the US market via the FDA, and the need to develop a market that does not currently exist. Based on wound prevalence data, and price/volume analysis, peak revenue potentials are estimated to reach ~$50M in year 5 (although this includes/assumes access into the US market).
CON has the R&D, Regulatory and Marketing expertise to successfully commercialise the SID device. QZY & SYN are critical to the supply of chemistry substrates for the SID device. MST is critical to the clinical programme and the generation of both clinical and cost effectiveness data that will be necessary for successful reimbursement of the device.
Scale-up of the chemistry substrates (T5.1 & T5.2) has been established and process scale-up for manufacture of the device is at an advanced stage (T5.5). Clinical proof-of-principle with a prototype device has been demonstrated (T4.3).



Potential Impact:
The Need: Infection is a global problem of traumatic, post-surgical or chronic wounds.
10% of surgical wounds exhibit bacterial infection within 30 days. Heavy bacterial colonization is the main reason for non-healing of chronic wounds such as decubitus, ulcus cruris and diabetic foot ulcers. The severity and cost of wound infections increase dramatically the longer they remain untreated and reach >5 billion € per year in Europe. The resulting pain, impairment and social isolation lead to reduced quality of life and, in the worst case, hospitalization, and eventually sepsis and death.

Exploitation strategy

i) The markets medical dressings and specialized wound care materials
According to Transparency Market Research (www.transparencymarketresearch.com) “Wound Dressings Market – Global Industry Analysis, Size, Share, Trends, and Forecast, 2012 – 2018,” the global wound dressings market was ca. USD 12.8 billion in 2011 and is expected to reach USD 21.6 billion by 2018 at a CAGR of 7.8%.
The growth in the market will center on advanced wound dressings and will be driven by the diabetes epidemic and demographic change.
Given the size of the market, any technical change with significant impact can be expected to have significant revenue.

European market penetration study (performed by partners QZY, SYN and CON)
The market in Europe can be grouped into 1) post-surgical wound infections and 2) chronic wounds. In Europe there are annually about 5 million cases for each of the two groups.(see figure)
A total predicted revenue for the diagnostic material is €46.8M, in the 5th marketing year. The market penetration is estimated at 30% in post-operative wounds and 50% in chronic wounds.
The calculation for Europe is based on a population of 350 million and a study performed for Germany.
The study performed for Germany was extrapolated to 100 million inhabitants to include the German speaking countries (DE, AT and CH) and is detailed below.


CON, the large industry has expertise in wound dressing design and development and associated materials.

In the agreement between CON and the SME partners QZY and SYN is that the SMEs are suppliers of enzyme-substrates and CON is final manufacturer and marketing.
Other opportunity will be
• The SMEs will license their IP to CON, which will manufacture the entire diagnostic device.
• The SMEs will produce the new device and CON will market it.
Foreseen sales of the new product in German speaking European states
⇒ For post-surgical wounds are estimated at 10M Euros in the fifth year based on the following data:
- 13.84 million cases of post-operative wounds in Germany (80 million inhabitants)
- Application of the new diagnostic material on every 5th wound
- End user price is estimated at € 10
- Penetration of the market of 30%

⇒ For chronic wounds are estimated at € 9.3 million in the fifth year based on the following data:
- 3 million chronic wounds
- application for each second wound
- end user price of € 10
- market penetration of 50% at 5 years
The sales of the new product in German speaking European states
⇒ For post-surgical wounds are estimated at 10M Euros in the fifth year based on the following data:
- 13.84 million cases of post-operative wounds in Germany (80 million inhabitants)
- Application of the new diagnostic material on every 5th wound
- End user price is estimated at € 10
- Penetration of the market of 30%

⇒ For chronic wounds are estimated at € 9.3 million in the fifth year based on the following data:
- 3 million chronic wounds
- application for each second wound
- end user price of € 10
- market penetration of 50% at 5 years

ii) Exploitation by the SMEs
The direct output of this project is protected by three patents (see above) and will be further protected by granting licenses to partner CON via QZY..
As part of the previous project that developed the patents which are the basis of this application, The Technical University of Graz formed a spin-out (now QZY) in collaboration with SYN. QZY is the owner of the basic IP for the materials to be developed here. QZY has reached a general agreement with CON with the support of SYN to develop aspects of the technology.
In this regard, it can be seen that the R&D centers in the previous project were able to convert IP into a viable business.
It is therefore clear that this process can be maintained by the consortium R&D centers

iii) Exploitation by the R&D centers
The research organizations and universities involved in the project that will produce scientific and technological results of commercial value will be involved in their exploitation both directly and indirectly.

Furthermore as part of the exploitation there are plans for product-line extension into other critical care applications (i.e. drainage lines). Similarly, the detection materials can be refined to be applicable to larger area wounds to identify "hotspots" of infection (wound map) as opposed to generalized signs of infection in the exudate. In this way, the developed platform of responsive materials can be used to build a new range of products to support and encourage various users to adopt the technology. These plans are entirely in line with the orientation of the manufacturing and marketing project concepts.

Early detection of an incipient wound infection is important for the attending physician since it would allow the timely initiation of treatment, thus reducing the severity of the disease. Currently, however, wound infection is not diagnosed until becoming pathologically evident. As a consequence, the treatment of the patient is further complicated and more likely to have a negative outcome. In addition, wounds are often treated with antibiotics prophylactically, leading to unnecessary selection for bacterial resistance.
Nowadays, a Point of Care Testing (PoCT) device to identify incipient wound infection does not exist. Clinicians rely on a combination of clinical information and long-lasting laboratory tests requiring equipment and experts for diagnosis. However, clinical parameters such as redness, heat, swelling and pain appear only at the lamination of the infection cascade making early clinical judgment so uncertain that 50% of infections are not initially identified. Microbiology laboratory tests used to identify wound infection and the type of pathogen take 3–4 days, and in the meantime, antibiotic treatment is often initiated as a precaution. Furthermore, positive swab findings indicate presence of bacteria only, even, but do not provide objective evidence of a clinically important infection due to variation in host factors, colonization and virulence.
InFact rationale: Consortium partners have patented the know-how to convert wound dressings into a diagnostic tool capable to inform both patient and therapist about the wound status, thus allowing a proactive diagnostic step. The proposed functional materials allow a real time in situ infection diagnostic reaction and, thus, a timely treatment intervention. A next-generation Protective, Predictive and Proactive (triple-P) material for in situ diagnosis of wound infection to be integrated in conventional dressings will be prototyped and advanced to commercialization by InFact.

Under the FP6-LIDWINE project novel active materials for the detection of wound infection were identified and protected by EU patents. The central claims focus on a sensor function that can be embedded into dressing materials to permit simultaneous monitoring of the activities of several enzymes secreted by the human immune system into wound fluids in early stage of infection. In contrast to other off-site methods limited only to detection of microbial colonization of wounds, this novel strategy based on functional materials provides a fast diagnosis (within minutes) at the wound site, without the need of analytic equipment.
While the single use device is convenient for decisions at regular dressing change, it is clear that more and more care is delivered at home by mobile nurses, meaning that wound status is not inspected daily and dressings are often left in place for long periods (up to one week). InFact rationale relies on the anticipation of the next dressing change by identifying early signs of wound infection while the dressing is in place.
The novel InFact material technology will be further translated into a low-cost, real-time diagnostic solution to be applicable to any commercially available primary wound dressing (e.g. hydrocolloids), constituting the "triple-P" materials concept:
✓ Protective - by introducing the concept of surveillance into wound care

✓ Predictive - providing a cumulative wound status signal to predict the infection transition

✓ Proactive - changing the dressing according to a signal, rather than on a schedule base avoiding unnecessary or late therapeutic response.
More specifically, the new functional diagnostic material will incorporate substrates for three enzymes that are markers for infection: myeloperoxidase, lysozyme and elastase .
Upon infection, elevated activity of these enzymes is encountered in wound fluids, detected by color change of the functional dressing material. InFact will use the know-how developed and patented in the LIDWINE project to further prototype these functional materials into a marketable, simple to use, universal and robust diagnostic device for wound infection.

The final product can be designed in two versions:
1) standalone diagnostic insert and
2) incorporated diagnostic material. The incorporated diagnostic material will be attached to a commercially available wound dressing (e.g. a hydrocolloid) with a defined ‘window’ for color visualization (Fig.1.3).

The aim is sell the product to the end user at a price lower than €10.
Consortium partners (QZY, SYN and CON) performed a preliminary market study for the new product and estimated the sales at €46.8M in the 5th marketing year.

1.1.2 Main technological and scientific objectives

(A) Optimization of color reaction between enzyme substrates and the wound infection enzymes (myeloperoxidase, lysozyme and elastase)

A1 Myeloperoxidase (MPO) substrates - phenolic molecules developing color upon oxidation with MPO (non-interacting with heme)

A2 Lysozyme (LYS) substrates - optimization of color reaction with dye-labeled Micrococcus luteus (M. luteus) peptidoglycan fragments or specific oligosaccharides

A3 Elastase (HNE) substrates - synthesis of chromogenic peptides/gelatin with specific cleavage sequences

A4 Internal standard - Integration of an internal standard using the displacement concept e.g. to measure protein content in wound fluid

A5 Development of color trapping strategies to generate a signal readable via an external window in the dressing


(B) Lab prototype: production and characterization of the ‘in situ infection diagnostic material’

B1 Continuous process for production of the ‘in situ wound infection diagnostic material’ by immobilization of the detector and capturing reagents using ink-jet printing
B2 Semi-continuous process for production of the in situ wound infection diagnostic material’ by immobilization of the detector and capturing reagents based on:
• Impregnation: spraying/dispensing of the enzyme-substrates and capturing compounds using contact and non-contact tips
• Chemical immobilization
• Ultrasound assisted immobilization – simultaneous sonochemical production of enzyme substrate nanoparticles, and their embedding on the diagnostic material
B3 Assessment of the efficiency for developed immobilization protocols
B4 Assembling of a lab prototype and characterization

(C) In vitro/in vivo assessment of the ‘in situ wound infection diagnostic material’

C1 In vitro efficacy
C2 In vitro biocompatibility - the diagnostic material will be assessed for biocompatibility based on the response of fibroblasts, immune cells and epidermal cells
C3 In vivo biocompatibility, leaching and efficacy - individual components will be assessed for stand-alone toxicity as required by the regulators after consultation and demonstration of the prototype


(D) In human clinical studies: ex vivo/in vivo
D1 Ex-vivo study 1 - assessing color reactions by contact of wound fluids and enzyme substrates
D2 Ex-vivo study 2 - assessing color reactions of wound fluids and the ‘in situ wound infection diagnostic material’
D3 In-vivo study - treating patients based on the information derived from the ‘in situ wound infection diagnostic material’ to select therapy in real time with assessment by comparison of clinical outcomes and relative treatment cost

(E) Up-scaling, LCA and LCC studies and process engineering for mass production

E1 Up-scale the dye-labeled enzyme-substrates for production of 100 infection detectors per day
E2 Upscale the process for materials functionalization (reagents immobilization)
E3 Packaging, stability, and storage studies
E3 Life cycle assessment (LCA) and life cycle cost assessments (LCC) of the prototype production - these studies will support the studied processes while supplying recommendations to reduce cost and environmental impact for the up-scaling process
E4 Process engineering for mass production, e.g. 5000 infection detectors per day

(F) Towards CE marking, market survey, and business plan
F1 Requirements for CE Mark
F2 Market estimates, reimbursement prospects and revenue projection by market
F3 Business plan based on the SMEs and industry capabilities in the consortium, and pilot outputs


Impact:
The advantages of the InFact technology, compared to existing methods such as microbiological analysis after taking swab samples, is a much faster diagnostic response (only minutes compared to days) and the simplicity of the technology (simple colour reaction of materials compared to instrumental analysis). For the first time functional materials for wound infection detection will become available at affordable cost for a wide use both in home care and in hospitals. This will be feasible based on the integration of the diagnostic function in materials already commonly used in wound dressings.

1. Scientific impact
Tangible
• A novel ‘in situ’ wound infection diagnostic material validated via clinical study
• Synthesis of enzyme substrates and process scale-up
• Production processes for coating
Non tangible
• Know-how on enzymes substrates and their reaction with the wound fluid enzymes
• Know-how on coating of bio-reagents and nanoparticles on different materials: cellulose-based and synthetic
• Know-how on synthesis of enzyme substrates

2. Expected impact for SMEs
In this project there are five SMEs out of 11 partners, representing 45% of the consortium and 40.2% of the funds. The InFact will be led by research intensive SMEs, that participate as leaders in 2 WPs out of 5 scientific WPs (partners TEC and SYN). SMEs will have the majority in the Governing Board, of the consortium thus leading on vote decisions to be taken by vote.

The project efforts will facilitate the high-risk stage R&D activities of the SMEs with a potential of developing beyond-the-art technology and intellectual property, thus providing the added value for the SMEs’ stockholders. Project outputs could be implemented by these partners for their further growth and enhanced competitiveness in biotechnology and medicine.

Reinforcing the competitiveness of European industry
The InFact project is based on strong intellectual property which has attracted the interest of the wound therapy industry. Three different large organisations have bid for the general IPR on which the project is based.
Novel approaches to detect or prevent infection are one of the most important medical issues. InFact proposes capitalising on this leading IP position to deliver a revolutionary product to wound care.
The realization of this product will effectively contribute, to the transformation of the European biotechnology and medicine as well as the relevant industries from a resource-intensive to a knowledge-intensive phase, which will further contribute to the EU WW position in these fields. This project is part of the chain in this transformation, introducing high added value technologies in a sustainable manner.
This project is at the crossroads between different disciplines and technologies (chemistry, biology nanotechnology, engineering and wound biology). The outputs of the project will increase European scientific and technological qualities, thus preventing relocation of European science to other areas worldwide, and at the same time creating industrial and employment growth within Europe.

Being at the crossroads between different disciplines and technologies, InFact will enhance the European medical industry competitiveness by providing a unique means to prevent/detect infections early in wounds and thus save dramatically on hospital care.

Socio-economic impact
The diagnostic materials developed in InFact have a large socio economic impact and market potential related to wound management, both in home care and hospitals.
In the western industrialized countries, about 2% of the population suffers from chronic wounds. Decubitus wounds, ulcus cruris and diabetic feet are the most abundant types of chronic wounds, mostly associated with age.

Demographic trends show a significantly increased life expectancy, combined with strong growth in chronically ill and dependent people. 25% of these chronic wounds become infected once a year. In addition, 10% of postsurgical wounds are infected within 30 days, leading to additional costs of 23 Mrd. € per year in Europe. Patients with wound infection require an additional median duration of 6.5 days in hospital resulting in doubling the hospital costs22. The annual total costs for treating infected wounds exceed 5 billion € per year in Europe placing a substantial financial burden on the health care system. The magnitude is expected to increase as the population ages. Consequently, early diagnosis of wound infection is of tremendous importance to improve both the current societal impacts and economic consequences.

The wound care market has two main components: Dressings and associated materials, and the hospital and personal care aspect. These have a cost ratio in the range of about 1:10-100, depending on the case. In other words, the vast majority of cost in wound care is the delivery of care by medical staff.
The goal of the novel diagnostic materials is to save spending on care, by providing more intelligent dressings that reduce the need for intensive efforts.

• Employment:
While the economy is adding jobs at lower levels than workers would like, during the last two years, analysts expect buds of growth in a wide range of service jobs this year-retail, information technology, professional, scientific and technical jobs, as well as continuing growth in the health-care industry.
With the aging population, health care remains the go-to field for job growth. "Health care is always adding jobs. That will clearly continue," says Dean Baker, co-director of the Center for Economic and Policy Research, a Washington think tank.
Among companies that expect to increase full-time, permanent workers in 2011, the top areas, by function, according to the survey are: sales, information technology, customer service, engineering, technology, administrative, business development, marketing, research/development and accounting/finance . InFact is at the intersection of the above mentioned discipline thus having a positive impacting the employment in these fields.

Quality of life
Battling serious wound infection becomes a dominant factor in the lives of affected patients. Wound infection is painful, socially limiting and potentially lethal - thus it imposes dramatic psychological as well as physical burdens on patients. The prospect of wound infection is also a significant disincentive to undertake treatment of chronic injuries. Thus, a person who may ordinarily benefit from, say, a knee re-construction, may forego the treatment for fear (not-unreasonably) that a deep bone infection could render them less mobile than their pre-operative status.
Thus, steps to reduce rates of infection or otherwise more effectively deal with incipient infections will have a dramatic effect on both the lives of existing patients as well as increasing the confidence with which the general public undertakes medical treatment

European added value
The challenging objectives of this project demand a multidisciplinary, international collaboration of cell biology, biotechnology, chemistry, physics, engineering, and medicine as well as training, exploitation, dissemination and project management. These disciplines were chosen from different countries to establish a consortium of great expertise. The participants will benefit from different approaches in Europe. The consortium is formed form 11 organizations from 8 different countries: 7 member states: DE, AT, ES, PT, RO, UK, NL, and one associated state: IL. It is obvious that the research work cannot be achieved at a national level, as research expertise and the IP gathered in this European project is critical for its success. The success of the project is highly dependent on this expert consortium. This challenging project objectives needs European cooperation of the SMEs and the academies since the specific knowledge and IP of these is essential for promoting the know-how to a commercial product.

Europe is well positioned to spearhead the development of a bio-based economy but must invest in demonstration activities to gain a competitive edge, says new study. In this context the ”European paradox" or the “valley of death” refers to the perceived failure of European countries to translate scientific advances into marketable innovations. (The term was coined in a European Commission Green Paper in 1995).

Thus, in accordance with the 7th Framework Programme’s goals and topic NMP.2013.4.0-3, InFact will develop new products, with optimal intrinsic properties, patented and suitable for integration into production, thus diminishing the ‘valley of death’.



List of Websites:
http://in-fact.eu/

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UNIVERSITAET FUER BODENKULTUR WIEN
Austria
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