WO2009122188A2

WO2009122188A2 - Wound infection monitoring method

Info

Publication number: WO2009122188A2
Application number: PCT/GB2009/000892
Authority: WO
Inventors: David Pritchard; Erling Sundrehagen
Original assignee: Axis-Shield Diagnostics Ltd
Priority date: 2008-04-04
Filing date: 2009-04-03
Publication date: 2009-10-08
Also published as: GB0806186D0; WO2009122188A3

Abstract

The present invention provides a method for indicating the infection-status of a wound by measuring the concentration of at least one component associated with inflammation in a fluid from the wound. Calprotectin is a highly valuable example of an inflammatory component for use in such a method. Chronic wounds such as ulcers are of particularly appropriate for monitoring by the method of the invention. The invention further provides to the use of a component associated with inflammation to monitor the infection status of a wound, and provides devices suitable for carrying out the monitoring. Such devices take the form of, for example, test strips, swabs, sheets or wound dressings.

Description

Wound Infection Monitoring Method

The present application relates to methods for establishing the infection-status of a wound at the skin or mucosal surface of subject. In particular, the invention relates to such methods comprising the measurement of at least one component of a fluid from the wound, and most particularly wherein said component is a component endogenous to the subject.

In mammals, injury triggers an organised complex cascade of cellular and biochemical events that result in a healed wound. Wound healing is a complex dynamic process that results in the restoration of anatomic continuity and function; an ideally healed wound is one that has returned to normal anatomic structure, function and appearance.

A simple, uncontaminated, wound at the skin surface of a human subject heals by a process which can be divided into several stages and sub-stages. These begin at the moment of injury and may continue for years after closure of the wound surface. The typical stages of wound healing are approximately as follows;

I. Inflammatory Phase ( immediate to 2-5 days ) i) Haemostasis

• Vasoconstriction

• Platelet aggregation Coagulation ii) Inflammation

• Vasodilation

• Phagocytosis

• Control of pathogenic organisms

II. Proliferative Phase (2 days to around 3 weeks) iii) Granulation

• Fibroblasts lay bed of collagen • Fills defect and produces new capillaries iv) Contraction

• Wound edges pull together to reduce defect v) Epithelialisation • Formation of new epithelial surface

• Crosses moist surface of wound

Cell travel about 3 cm from point of origin in all directions

III. Remodeling Phase (around 3 weeks to 2 years) vi) New collagen forms which increases tensile strength to wounds vii) Scar tissue typically reaches up to 80 percent the strength of original tissue

Infection of wounds by bacteria delays the healing process, since bacteria compete for nutrients and oxygen with macrophages and fibroblasts, whose activity are essential for the healing of the wound. Infection results when invading organisms (typically bacteria) achieve dominance over the systemic and local factors of host resistance.

Infection is therefore a manifestation of a disturbed host/bacteria equilibrium in favour of the invading bacteria. This may elicit a systemic septic response, and also inhibits the multiple processes involved in.wound healing. Lastly, infection can result in a prolonged inflammatory phase and thus slow healing, or may cause further necrosis of the wound, expanding the wound site and delaying healing still further. The granulation phase of the healing process will begin only after the infection has subsided and thus the dominance of the invading organism has been overcome.

Chronic wounds almost without exception contain bacterial flora. These bacteria may be indigenous to the patient or might be exogenous to the wound. Closure, or eventual healing of the wound is often based on a physician's ability to control the level of this bacterial flora. Whilst bacteria and other organisms may be present within a wound, this does not necessarily mean that the wound is clinically infected. Bacteria and other organisms may colonise the surface of a wound without any negative impact on the host. It is therefore not sufficient to identify whether bacteria or other organisms are present at a wound site in order to assess the infection status of a wound. The information required by a physician in order to determine what if any treatment is required is whether or to what degree bacterial dominance over the host resistance exists. Ideally, an indication of likely bacterial dominance would be provided before extensive bacterial proliferation has occurred, so that treatment may be local and less extensive rather than requiring systemic approaches.

Current methods used to identify bacterial infection rely mainly on judgement of the odour and appearance of a wound. With experience, it is possible to identify an infection in a wound by certain signs, such as redness or pain, particularly once the infection is well established. Some clinicians take swabs that are then cultured in the laboratory to identify specific organisms, but this technique takes time and may simply indicate that the bacteria are present rather than indicating that the equilibrium is shifted in favour of the pathogen. As a result, swabs are primarily of value to identify the invading organism once it is apparent from other signs that infection exists.

If clinicians could respond to wound infection as early as possible, the infection could be treated topically as opposed to having to use systemic antibiotics. This would also lead to less clinical intervention/hospitalisation (e.g. due to sepsis) and would reduce the use of antibiotics and other complications of infection.

There is thus an evident need for improved methods by which the infected or non- infected status of a wound may be assessed. It would be an advantage if such a method was quick, simple to perform and preferably non-invasive, so that a wound could be tested or monitored easily by a clinician or patient, preferably at the "point- of-care" or at home. It would be a further advantage if such a method was able to distinguish between bacterial presence at a wound site and the dominance of bacteria over host factors and would be most advantageous if this could take place before physical signs of infection were evident. It would be a further advantage if such a method could be embodied into a simple device or incorporated into the devices such as dressings brought into contact with a wound during routine treatment. There is furthermore a long felt need for a prognostic aid that would assist in predicting clinical infection of a wound prior to obvious clinical symptoms of infection. Such a prognostic aid would allow early intervention with suitable treatment (e. g. a topical antimicrobial treatment) before wound chronicity sets in. There is also a need for a diagnostic aid that would assist in the early diagnosis of clinical infection, preferably allowing diagnosis prior to obvious clinical symptoms of infection. It would be a further advantage if such a prognostic or diagnostic aid were of greater sensitivity than previous wound infection-status monitoring methods. It would be a still further advantage if such a prognostic or diagnostic aid allowed detection of a forthcoming infection both before clinical signs are manifest and earlier than previous wound infection-status monitoring methods allow.

The present inventors have now surprisingly established that rather than looking for the presence or type of bacteria at a wound, the infection-status may be more effectively established by measuring certain endogenous components of fluid present at a wound site. Most particularly, measurement of factors associated with inflammation provides a better indication of the host/pathogen equilibrium than do methods directly assessing the pathogens themselves.

In a first aspect, the present invention thus provides a method for indicating the infection-status of a wound, the method comprising measuring the concentration of at least one component in a fluid from said wound, wherein said component is associated with inflammation.

Any component or combination of components associated with inflammation may usefully be used in all aspects of the present invention, and these components may be elevated upon inflammation or depressed upon inflammation, or the ratio of two components may be changed. Preferred examples of components which are elevated upon inflammation are calprotectin, and/or C-reaCtive protein (CRP). In a preferred embodiment of the invention, the component measured is thus calprotectin and/or CRP and a concentration above at least one threshold level is indicative of wound infection. In the most preferred embodiment, which will be generally illustrated herein, the component measured is calprotectin. Elevated levels of calprotectin are therefore the preferred indicator of inflammation and in the most preferred embodiment, the component measured is calprotectin and a concentration above at least one threshold level is considered indicative of wound infection. It has additionally been established by the present inventors that factors associated with inflammation are altered in concentration in wounds which although not apparently clinically infected will subsequently go on to reveal an infection within a few days. All relevant aspects of the present invention are therefore preferably practiced on wounds which show no clinical signs of infection, or which show only early or ambiguous signs of infection.

It has not previously been proposed to use the concentration of an endogenous component linked to an inflammatory response to determine the infection-status of a wound. In a second aspect, the present invention therefore provides the use of concentration measurement of at least one component in a fluid from a wound for indicating the infection-status of said wound, characterised in that said component is associated with inflammation.

The measurement of components linked to inflammation may be carried out by any suitable methods, including laboratory or "point-of-care" solution, homogeneous, or heterogeneous assays. The present inventors have established, however that use of the method of the present invention as a routine screening method, preferably applied repeatedly at the same wound site potentially allows for detection of wound infection-status at an early stage, as well as monitoring of remedial treatment if wound infection is detected. In a further aspect, the present invention thus provides for a device for indicating the infection-status of a wound, said device comprising; i) at least one specific binder for at least one component in a fluid from said wound; and ii) at least one signal generating moiety, wherein said component is an endogenous component associated with inflammation.

The device of the invention is typically exposed to a fluid from a wound and in use the device generates a signal corresponding to the binding of said component to said specific binder. Suitable signals are described herein, but signals detectable by eye, such as colour-change, opacity change, or fluorescence change are particularly suitable. The device may be for use in a analyser such as those used in a clinical laboratory or at the point of care, or more preferably may be an essentially self- contained analysis device which upon exposure to wound fluid provides a signal change indicating the presence or absence of at least one particular component at above or below a predetermined threshold level. Most suitable devices include "dipstick" type test sticks, test strips, swabs, sheets, liners or dressings.

In a yet further aspect, the present invention provides a method for assessing the progress or course of treatment of a clinically infected wound which is subject to at least one treatment modality, said method comprising measuring the concentration of at least one component in a fluid from said wound, characterised in that said component is associated with inflammation, hi this aspect of the invention, successful treatment of the wound will be associated with a return of the component concentration(s) to or towards their normal values. This may be, for example, a lowering of calprotectin concentrations to lower absolute values, or below one or more threshold values.

In all aspects of the present invention may be practiced on any type of wound, which term is used herein to indicate any pathological or traumatic cut, laceration, graze, burn, lesion, or ulcer of skin or mucous membrane and optionally of underlying tissue. For example, the wound may be an acute wound such as an acute traumatic laceration (e.g. resulting from an intentional elective or emergency operative incision) or the wound may be a chronic wound. In one preferred embodiment, the invention is envisaged is practiced in predicting, diagnosing, or providing an assessment of risk of clinical infection of a chronic wound. Preferably, the chronic wound is selected from the group consisting of venous ulcers, pressure sores, decubitus ulcers, diabetic ulcers and chronic ulcers of unknown aetiology.

The subject having the wound upon which the present invention is practiced or applied is generally a human or non-human mammalian subject and is preferably a human, canine, feline, bovine, porcine or equine subject. Human subjects are more preferred.

Where the subject us human, they may be subjects comprised within groups having an elevated risk of wound infection. Such groups include older (e.g. greater than 60 years of age, such as 70 to 100 years of age), or younger (e.g. less than 12 years, preferably less than 5 years (e.g. 1 day to 5 years), most preferably less than 1 year of age) subjects, subjects with compromised immune activity (such as those taking immunosuppressive drugs or those having immune-compromising infections such as HIV) and subjects with reduced circulation, such as diabetic patients or patients with cardiovascular disease.

The method of the present invention provides an indication of the "infection-status" of a wound. This will typically not be an absolute diagnosis of wound condition, but will rather be an indication that the equilibrium of pathogen to host response is upset in favour of the pathogen (generally bacteria). This applies particularly where the wound may show no current signs of clinical infection, and thus may not be

"infected" as such, but may rather have an increased chance of progressing to a clinically infected state Evidently, in such situations, it would be preferable for systemic or more preferably topical treatments to be considered by the clinician immediately such that the progress of the infection is slowed or halted and actual clinical infection of the wound may be completely or partially avoided. Thus, the "infection-status" may be a probability of existing or forthcoming clinical infection, may be an indication of elevated risk or may be a prognostic indicator providing evidence as to how quickly a wound might be expected to heal with and without intervention. Overall, "infection-status" indicates a correlation between an elevated or depressed concentration of at least one analysed component and an increased or decreased likelihood of infection. In the case of the most preferred calprotectin, increasing concentration is correlated with an increasing likelihood of present or future infection, while decreasing concentration is correlated with decreasing likelihood of present or future infection.

In appropriate embodiments, the "infection-status" of a wound undergoing treatment will typically provide an indication of the success or otherwise of an existing treatment modality and might thus be used by a clinician to maintain cease or vary treatment as appropriate.

The various aspects of the present invention, relate to the measurement of a concentration of at least one inflammation-related component in a wound fluid.

By measurement of concentration, as used herein, is indicated the qualitative, semi- quantitative or quantitative assessment of the relevant component or the assessment of a ratio or proportion of two or more components wherein at least one is associated with inflammation.

A qualitative assessment may, for example, be the presence or absence of a relevant component at above a threshold value, and such a measurement of concentration is suitable for all aspects of the invention, and particularly to those relating to a self- contained device such as a dipstick or dressing. In this qualitative embodiment a "positive/negative" result is typically generated indicating the concentration of the marker(s) relative to the threshold.

A semi-quantitative assessment may, for example, be the presence or absence of a relevant component at above, below or between at least two threshold values, and such a measurement of concentration is suitable for all aspects of the invention, and particularly to those relating to a self-contained device such as a dipstick or dressing. In such a case, the type or intensity of signal (e.g. the colour) may provide the indication of the value, or a device may be divided such that a signal at various points is indicative of the relevant concentration or range. Such ranges may be absolute or relative and may be on a clearly defined or arbitrary scale. A typical result in the semi-quantitative embodiment of the invention would be a concentration in a particular range, or a value for the concentration on a short, arbitrary scale (e.g. a value on a 0 to 5 or 0 to 10 arbitrary scale).

A quantitative assessment may provide an absolute value of the concentration of a component on a absolutely-defined or an arbitrary scale. Such a measurement of concentration is suitable for all aspects of the invention, and particularly to those relating to assays for conducting in a analytical device such as a laboratory or point- of-care analyser.

In all cases, the measurement of concentration of a relevant component is compared to a threshold value or scale to allow the subject or clinician to judge what action or further analysis should take place. The scale and/or threshold will be readily determined by one skilled in the art based upon the performance of the particular assay embodying the invention.

Those skilled in the art will readily be able to determine concentration levels of . markers of the inflammatory response, which are indicative of subsequent progression to clinical infection and/or of the presence of clinical infection based upon studies of patients with appropriate wounds. For example, subjects may have appropriate wounds tested at regular intervals and their clinical progress followed over time. This will then reveal the basal concentration level of the appropriate component in the fluid of wounds which heal normally and the degree of variation (typically elevation) of concentration in those wounds which subsequently go on to show clinical infection. Although this may be conducted on an absolute scale (e.g. by the use of standards of known concentration or activity), an absolutely-defined scale is not necessary so long as the assay is reproducible and at least one relevant threshold or scale can be defined.

Typically, a concentration at least twofold greater than the basal level of the inflammation-associated component is considered as being indicative of subsequent progression to clinical infection.

The scale and threshold value(s) associated with the various aspects of the invention, including any thresholds inherent to the construction of an assay device will need to be appropriate to the type of wound analysed. In particular, a generally higher level of background inflammation is associated with chronic wounds than would be observed in an acute wound such as a surgical incision. The "basal" level of the marker-component thus indicates the level of the marker normally associated with a wound of a corresponding type which is not clinically infected and which does not subsequently become clinically infected. It will be appreciated that the basal level of the marker may be much higher for a chronic wound than for an acute wound, but that this inflammation will nonetheless be increased in situations of current or imminent wound infection.

The various aspects of the present invention relate to an analysis conducted on a "wound fluid". This term is used herein to indicate any wound exudate or other fluid that is present at the surface of the wound, that is removed from the wound surface by aspiration, by absorption and/or by washing. The wound fluid should preferably comprise less than 50% blood, and more preferably is substantially free of blood). The term "wound fluid" indicates a fluid secreted or discharged by cells at or in the vicinity of the wound. That is to say, from cells within 2 cm, preferably within 5mm of the wound. For clarity, "wound fluid" does not refer to blood, tissue, plasma or any other sample discharged, secreted or taken remote from the wound site (e.g. originating more than 2 cm, especially more than 5 cm from the wound). The wound fluid should thus not be a systemic fluid or a fluid from a remote site.

The fluid should comprise less than 50% blood, and more preferably comprise 10% or less of blood. The various aspects of the present invention relate to the measurement of at least one component of wound fluid "associated with inflammation". The skilled worker will be aware of the use of a number of systemic components which are associated with inflammation. An elevated plasma level of one of these is thought to indicate a major or wide-spread inflammatory response somewhere within the body. Such inflammatory components include C-reactive protein and Interleukin 8 (Saatvedt et al. Scan. J. Thorac. Cardiovasc Surg 1996 30 53-59), amongst others. Calprotectin is also known as a systemic marker of non-specific inflammation and as a faecal marker of inflammatory bowel disease These known markers have been measured systemically to detect wide-spread inflammation, and in the case of major wounds, such as open fractures, systemic inflammation has been proposed a method of diagnosing post-operative infection (Ferard et al. Clin. Chem. Lab. Med 2002 40 1334-1338). Such methods, however, are capable of detection of infection only when inflammation has reached the systemic stage. The present inventors have surprisingly established that inflammatory components are not only raised or altered in concentration systemically after extensive inflammation, but are raised or altered in concentration locally at a much earlier stage in the infection process, typically before any visible or systemic signs of infection are present.

Any component of wound fluid associated with inflammation is suitable for used in any of the major aspects of the present invention, and in particular, any of the components named herein is suitable for use individually or in any combination for use independently in the methods, uses, kits and devices of the invention. There are certain components of wound fluid associated with inflammation, however which are particularly preferred, and these include: Calprotectin, CRP, Procalcitonin, IL-8, IL-6, Lipopolysaccharide binding protein, Protein C, IL- Ira, TNFa, Soluble TNFa receptor, Soluble IL-I receptor, IL-I, IL-10, IL- 18, Transforming growth factor, Macrophage inflammatory protein- 1, High-mobility group box-1 protein, Hepatocyte growth factor, Leptin, Melanocyte-stimulating hormaon, Fibrinogen and al anti-trypsin. The abbreviation "IL" is used herein and in the art to indicate "interleukin" and thus, for example, IL-8 refers to interleukin-8. In one embodiment of the invention, the component of wound fluid associated with inflammation is not and does not include IL-6.

Furthermore, although any of the above may be used individually of in any combination, there are certain components which are believed to change in concentration earlier than many others, and these provide particular benefit in allowing an infected or potentially infected wound to be detected earlier than other markers/components and thus allowing earlier, less extensive, and/or milder treatment. In this respect, the component associated with inflammation is or includes at least one component selected from: Calprotectin, C-reactive protein (CRP), Procalcitonin, IL-8, Lipopolysaccharide binding protein, Protein C, IL- Ira, TNFa, Soluble TNFa receptor and Soluble IL-I receptor. Of these, the most preferred is calprotectin, which may be the sole component associated with inflammation or may be used in combination with at least one other component associated with inflammation (e.g. one, two, three or four other such components).

In addition, certain components are believed to change in concentration more significantly than many others (i.e. the concentration in fluid from an infected wound is a larger multiple of the basal concentration in fluid from a similar uninfected wound). These provide particular benefit in allowing a highly sensitive and/or reliable assay and/or giving a high degree of confidence that a positive result is a true positive indication of infection. Obviously this provides more certainly in the appropriate treatment for a clinician and subject.

The most preferred component for analysis in the various aspects of the present invention is calprotectin. For the avoidance of doubt, the term calprotectin is used herein synonymously with "Ll protein", "MRP8/14", "cystic fibrosis (associated) antigen (CFA)" and "calgranulin". Calprotectin exists in both dimeric and trimeric forms. As a dimer, calprotectin comprises the polypeptide chains SlOOAB and

S100A9. As a trimer, calprotectin is a 36 kD heterotrimeric protein with two heavy chains (14 kD) and one light chain (8kD) non-covalently linked. The concentration of the marker of the inflammatory response may be measured by any method known to those of skill in the art. Suitable methods typically require the binding of a specific binder to the component of interest, the separation or isolation of the bound fraction and the generation of a signal from the bound portion.

Alternatively, the component of the sample may compete for binding to a specific binder with an alternative binding substrate (which is typically labelled) and the inhibition of binding caused by the sample component measured. Such methods may include, for example, the permanent or temporary immobilisation of a specific binder onto an insoluble substrate, the binding of the component, separation of the substrate from the mixture and addition of a labelled reporter which binds to the complex. All such methods are well-known to those of ordinary skill in the relevant art and may readily be adapted for the present invention. A highly effective assay method for calprotectin is described in WO04/057341, which is hereby incorporated herein by reference. This will be readily adaptable to any aspect of the present invention by one of skill in the art.

Generally, all that is required for effective detection of the desired component is a specific binder such as an antibody, antibody- fragment (e.g. FAB or FAB'2 fragments or any suitable fragment preserving the binding characteristics of the complete antibody), antibody construct (e.g. a construct of two or more whole antibodies and/or antibody fragments wherein at least some of the antibodies and/or fragments preserve the binding characteristics of the complete antibody) or single- chain antibody (which is known in the art as a single-chain antigen binding moiety). These can be raised and matured by standard techniques and once available may be used as the specific binding component in many standard and well-known assay formats. The generation of suitable specific binders and various assay methods for relevant components are described in Saatvedt et al. and Ferard et al. above and the references cited therein. These are hereby incorporated herein in their entirety by reference. The signal generated by the assay method may be any conventional signal including those utilising chemical or enzyme-linked reactions, spectrophotometric, scattering/opacity, colorimetric, fluorimetric, or radioactive detection based techniques. Where the assay is embodied in a self-contained assay device, the signal is preferably one which can be assessed by eye, optionally using hand-held visualisation devices such as a visible or UV lamp and/or filters. Such methods include colourimetric, fluorescent and luminescent detection methods. Colourimetric methods are most preferred, in particular enzymatic colourimetric methods.

To allow measurement of concentration of a marker of the inflammatory response in a wound, a sample of wound fluid must be added to the assay system. Measurement may either be made in situ, or fluid may be removed from the wound for subsequent analysis. This provides two particular aspects of the present invention..

In one aspect, the concentration of the inflammation-related component may preferably be measured in an aqueous assay system. Wound fluid may be extracted directly from the environment of the wound or can be washed off the wound using a saline buffer. The resulting solution can then be assayed for the concentration of the marker in, for example, a test tube, a microassay plate or automated analytical systems.

Solution, homogeneous or heterogeneous liquid assays are typically easy to automate and highly accurate. These methods are therefore most suitable for use in clinical laboratories or for specialist analytical equipment at the point of care. These methods typically allow quantitative analysis and so may be more suited for gathering detailed information such as the progress of treatment or where large numbers of samples need to be analysed.

Solution methods will be preferable for use in cases in which the wound is too small or too inaccessible to allow access of a diagnostic device such as a dipstick. This method has the additional advantage that the wound exudate sample may be diluted. In a further embodiment, the concentration of the marker is measured by a dip-stick or other self-contained assay device. Methods for the incorporation of the components of the assay reaction onto a clinical dressing, "dipstick", sheet or other biosensor are routine in the art. See for example Fagerstam and Karlsson (1994) Immunochemistry, 949-970. These are further illustrated in the Examples below. These methods are highly suited to direct in situ sampling, although a sample of wound fluid may be taken by aspiration or washing and applied to the assay device.

Self-contained assay devices, such as dip-stick type tests are highly facile and may be used in the community and by the patient allowing easier and earlier diagnosis and more routine monitoring. Self-contained devices also generate a result very rapidly because there is no delay for a sample to be sent to a laboratory for analysis or the result returned.

If it is required to monitor the progress of treatment using self-contained devices then they can either be made semi-quantitative with a range of reporting modes (e.g. separated spatially or varying by signal generated), or alternatively a set of test devices with a range of thresholds may be provided along with a schedule indicating when the patient or carer should expect to progress from one range to the next.

A highly beneficial use of the present invention is in the generation of "smart" wound dressings. Such dressings may be formed to incorporate the assay system of the "dipstick" type devices described herein, and since they are placed routinely against the wound may give an early warning that healing is not progressing as expected. In addition to dressings, similar devices such as sheets or liners may be made to be placed between the wound and a standard dressing, or swabs may be made which both sample the wound fluid and provide an assay result according to the invention. Sheets or liners will typically be porous to allow the flow of fluids through them and will be non-adherent to the wound surface. Any device of the invention (especially those suitable for or intended for direct contact with a wound) will preferably be sterile or substantially sterile (at least over the area of contact with the wound, where appropriate, and more preferably over the complete device).

It may be the case with large wounds that generation of a dressing or sheet to cover the wound wherein the whole area was treated to act in a method according to the invention would be unacceptably expensive or simply unnecessary. Evidently, such dressings may simply have "reporter spots" incorporated, wherein one or more small portions of the dressing of sheet is formed to generate a signal according to the invention. Such spots may be distributed over the area of the dressing to provide a "map" of possible infection or healing without requiring the entire area of the device to be signal-generating.

In one embodiment, therefore, 0.1 to substantially 100% (e.g. 1 to 99%) of the area of one surface of the device of the invention is signal-generating. In certain devices, such as a small device or an "indicator spot" within or for use with a larger dressing/device, the proportion may be substantially 100% (e.g. 80 to 99%), while for certain other devices, such as a large dressing, only a tiny spot need be reactive, and so in this embodiment typically 0.1 to 10% (e.g. 1 to 10%) of the surface area will generate a signal. Even for a typical smaller dressing, it is likely that only the centre portion need generate a signal since the edges will typically be applied overlapping the wound, and thus, 10 to 80% of the area of one surface may be signal-generating, more preferably 20 to 60% of that area.

The present invention will now be further illustrated by reference to the following non-limiting Examples and the attached Figures, wherein:

Figure 1 shows the concentrations of calprotectin measured in wound exudate samples from patients assessed as having non-infected or infected wounds; and

Figure 2 shows the concentration of calprotectin measured in wound exudate samples from a patient who developed a wound infection which was subsequently successfully treated. EXAMPLE 1

Detection of an calprotectin in wound fluid using Alkaline Phosphatase Test Strip

A test strip for the detection of calprotectin in a wound fluid sample utilising alkaline phosphatase (AP) activity is constructed in accordance with the present invention, as described below. The assay is based on the hydrolysis of the chromogenic phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) in the presence of AP to yield indoxyl and the further reaction of the so produced indoxyl with nitro blue tetrazolium (NBT) to produce indigo and formazan which accumulate at the interface between the two matrices. The receiving matrix is soaked in a solution containing both the chromogenic substrate BCIP and the colour- developing reagent NBT. Various samples are tested with the strip including wound fluid samples for the detection of clinical infection using the calprotectin marker.

A. Preparation of BCIP-NBT-Impregnated Sample Pads

Glass fibre filters (Millipore, GFCPOO 10000, 10 mm x 10 cm) are soaked in BCIP- NBT solution (0.2 mg/ml BCIP+0.3 mg/ml NBT in 0.1 M Tris buffer pH 9.6) for 30 minutes in the dark at room temperature. The glass fibre filters are transferred to drying oven and are dried for 15 minutes at 50 ⁰C. The BCIP-NBT-impregnated glass fibre filters (sample pads) are stored dried and dark in a dry room (RH 5-10%) at room temperature.

B. Card Assembly and Strip Trimming

A test card is assembled according to the following procedure, which specifies the exact longitudinal dimensions and position of each of the card components.

Following preparation, the card is trimmed to form a plurality of strips for AP assay.

1. A clear plastic film with a release liner protected adhesive, namely the back cover, 43 x 250 mm piece (ARcare 8876, Adhesives Research, Limerick, Ireland) is placed on top of a worktable. The release liner is peeled to expose the adhesive side of the tape. 2. The reaction membrane (Nitrocellulose HFl 8004, Millipore, S A3 Jl 54101 , 25 x

300 mm is attached on top of the adhesive side of the back cover, 8 mm from the lower end.

3. The BCIP-NBT impregnated sample pad is attached on top of the lower side of the back cover with 2 mm overlap on top of the reaction membrane. 4. The absorbent pad (Gel blotting paper, S&S, GB003, 21 x 300 mm) is placed on top of the upper side of the back cover with a 12 mm overlap on top of the reaction membrane.

5. The release liner of top laminate film (ARcare 7759, Adhesives Research,

Limerick, Ireland) is peeled to expose the adhesive side and the film is attached, with the adhesive facing done, on top of the reaction membrane, with overlaps on top of the sample pad and absorbent pad.

C. Test for Alkaline Phosphatase (AP) Activity with Anti-calprotectin AP

a. 25 μl samples is prepared by dilutions of anti- calprotectin AP (0.75 unit/μl) in TBS (Tris buffer saline) pH 7.8 to the following levels: 0.0375 units/test, 0.00375 units/test and 0.000375 units/test, b. 25 μl of sample is loaded onto the sample pad of the strip.

Results:

The signal of positive reaction, a brown-purple colour, is accumulated at the interface between the two different matrices (sample pad and the reaction membrane), namely the signal zone. Negative control (where no anti- calprotectin AP is present) will not show any signal. This demonstrates that localisation of the Anti-calprotectin AP within the test device results in a visible reaction and thus may be used to generate a calprotectin specific strip with a standard immobilised- antibody zone using an anti-calprotectin antibody.

D. Test for calprotectin in wound fluid using Alkaline Phosphatase (AP) Activity

A capture zone for calprotectin is constructed by immobilising an antibody to calprotectin on to nitrocellulose membrane, which is placed in contact with the other elements of device described above. Addition of a calprotectin containing sample along with the anti-calprotectin antibody- AP conjugate results in binding of this anti-calprotectin antibody- AP conjugate to the nitrocellulose membrane through antibody-antigen reactions. Excess unbound anti-calprotectin antibody- AP conjugate is washed away, and captured in an adsorbent pad remote from the area where signal is monitored. The signal generated from the signal zone is thus dependent upon the concentration of calprotectin in the sample.

Results

When the wound has a clinical infection a positive signal will appear within 3 minutes.

EXAMPLE 2 Detection of an calprotectin in wound fluid using Peroxidase Test Strip

A test strip for the detection of calprotectin in wound fluid utilising peroxidase (POD) activity in a wound fluid sample is constructed similarly to the manner described above in Example 1. However in this example, the sample receiving matrix is not impregnated with the detection reagents but is assembled into the strip in its clean untreated form. A commercially available solution of chromogenic peroxidase substrate mixture, with tetramethylbenzidine (TMB) as the chromogen, is loaded onto the strip just before loading the sample. In the presence of peroxidase, the TMB substrate mixture yields a coloured product.

A. Running Buffer 0.5% PEG (PolyEthyleneGlycol- 15000, Merck, 819003), 0.5% BSA (01200050, Seracare, Calif, USA), 0.1% Tween 20 (Sigma, P-5927), 0.1% MgC12 (Merck 1200310) in TBS (Tris buffer saline) pH 7.8.

B. Card Assembly and Strip Trimming A test card is assembled according to the following procedure, which specifies the exact longitudinal dimensions and position of each of the card components. Following preparation, the card is trimmed to form a plurality of strips for POD assay.

1. A clear plastic film with a release liner protected adhesive, namely the back cover, 43 x 250 mm piece (ARcare 8876, Adhesives Research, Limerick, Ireland) is placed on top of a worktable. The release liner is peeled to expose the adhesive side of the tape.

2. The reaction membrane (Nitrocellulose HF 18004, Millipore, SA3J154101, 25 x 300 mm is attached on top of the adhesive side of the back cover, 8 mm from the lower end.

3. The sample pad (Glass fibre filter, Millipore, GFCPOOl 0000, 10 mm x 10 cm) is attached on top of the lower side of the back cover with 2 mm overlap on top of the reaction membrane. 4. The absorbent pad (Gel blotting paper, S&S, GB003, 21 x 300 mm) is placed on top of the upper side of the back cover with a 12 mm overlap on top of the reaction membrane.

5. The release liner of top laminate film (ARcare 7759, Adhesives Research, Limerick, Ireland) is peeled to expose the adhesive side and the film was attached, with the adhesive facing done, on top of the reaction membrane, with overlaps on top of the sample pad and absorbent pad. C. Test for Peroxidase (POD) Activity with Anti-calprotectin POD

a. 25 μl samples is prepared by dilutions of anti-Calprotectin POD (0.15 unit/μl) in running buffer to the following levels: 7.5 units/test, 0.75 units/test and 0.075 units/test. b. 5 μl of tetramethylbenzidine (TMB) substrate mixture (Sigma T0565, or Pierce #34028) is placed on the sample pad of the strip. c. The 25 μl of sample is loaded on top of the sample pad.

Results:

The signal of positive reaction, a blue-purple colour, is accumulated at the sample pad and at the interface between the two different matrices (sample pad and the reaction membrane), namely the signal zone. Negative control (where no anti- Calprotectin POD is present) didn't show any signal. This demonstrates that localisation of the Anti-calprotectin POD within the test device results in a visible reaction and thus may be used to generate a calprotectin specific strip with a standard immobilised-antibody zone using an anti-calprotectin antibody.

D. Test for calprotectin in wound fluid using Peroxidase (POD) Activity Activity

A capture zone for calprotectin is constructed by immobilising an antibody to calprotectin on to nitrocellulose membrane, which is placed in contact with the other elements of device described above. Addition of a calprotectin containing sample along with the anti-calprotectin antibody-POD conjugate results in binding of this anti-calprotectin antibody-POD conjugate to the nitrocellulose membrane through antibody-antigen reactions. Excess unbound anti-calprotectin antibody-POD conjugate is washed away, and captured in an adsorbent pad remote from the area where signal is monitored. The signal generated from the signal zone is thus dependent upon the concentration of calprotectin in the sample. Results

EXAMPLE 3

Calprotectin in infected and non-infected wounds.

Samples of wound exudate were collected by swab from 10 wounds, 5 of which were judged to be infected by assessment of wound appearance by Wound Care Specialist collecting samples (and confirmed by microbiological culture) and 5 of which were judged to be non-infected by wound appearance.

After collection, swabs were placed in 1 ml of sterile phosphate buffered saline (pH 7.4), to allow material collected on the swab to elute into the saline. Dilution series of the saline eluates were prepared and assayed using a commercially available Calprotectin ELISA assay (Buhlman Laboratories AG, Baselstrasse 55, CH-4124 Schonenbuch/Basel, Switzerland). Assays were performed by prewashing the precoated microtitre plate with the supplied wash buffer, then adding 100 ul of calibrator or sample (diluted saline eluate) to wells of the plate and incubating for 30 minutes at 22⁰C. Well contents were then discarded and wells washed three times with the supplied wash buffer. 100 ul of enzyme labelled antibody conjugate was added to each well, and incubated for 30 minutes at 22⁰C. Well contents were then discarded and wells washed five times with the supplied wash buffer. 100 ul of TMB (3,3',5,5'-tetramethylbenzidine) substrate was added to each well and incubated for 15 minutes at 22⁰C prior to addition of 100 ul of stop solution and recording of absorbance at 450 nm.

Calprotectin concentrations in the saline eluates were calculated selecting the dilutions which fell within the calibration curve range, reading the concentration of the sample from this curve and then correcting for the dilution factor. Results obtained are summarised in Figure 1, which demonstrates clear distinction between calprotectin levels in the infected and non-infected samples.

EXAMPLE 4 Changes in calprotectin associated with changes in wound infection status.

A sample of wound exudate were collected by swab from an individual who at the time did not exhibit physical signs of a wound infection (upon assessment by wound care specialist). The individual subsequently developed a wound infection, and a further swab was taken at the time of this overt infection. The infection was treated by topical anti-microbials and systemic antibiotic therapy, and a further sample of wound exudate was collected 2 weeks post cessation of this therapy.

After collection, swabs were placed in 1 ml of sterile phosphate buffered saline (pH 7.4), to allow material collected on the swab to elute into the saline. Dilution series of the saline eluates were prepared and assayed using a commercially available Calprotectin ELISA assay (Buhlman Laboratories AG, Baselstrasse 55, CH-4124 Schonenbuch/Basel, Switzerland). Assays were performed by prewashing the precoated microtitre plate with the supplied wash buffer, then adding 100 ul of calibrator or sample (diluted saline eluate) to wells of the plate and incubating for 30 minutes at 22⁰C. Well contents were then discarded and wells washed three times with the supplied wash buffer. 100 ul of enzyme labelled antibody conjugate was added to each well, and incubated for 30 minutes at 22⁰C. Well contents were then discarded and wells washed five times with the supplied wash buffer. 100 ul of TMB (3,3 ',5,5'-tetramethylbenzidine) substrate was added to each well and incubated for 15 minutes at 22⁰C prior to addition of 100 ul of stop solution and recording of absorbance at 450 nm.

Calprotectin concentrations in the saline eluates were calculated selecting the dilutions which fell within the calibration curve range, reading the concentration of the sample from this curve and then correcting for the dilution factor. Results obtained are summarised in Figure 2. It can be seen from these data that the initial sample provides a higher calprotectin concentration than the negative samples in Figure 1 , indicating that calprotectin may be used to identify wound infection prior to it being apparent by physical examination. Calprotectin levels are greatly increased once the wound infection becomes overt (physical signs of infection apparent), but are much reduced following successful therapy. These data indicate that calprotectin can be used for the early detection of wound infection, and for following the progression of infection and therapeutic efficacy.

Claims

Claims:

1 ) A method for indicating the infection-status of a wound comprising measuring the concentration of at least one component in a fluid from said wound, characterised in that said component is associated with inflammation.

2) A method as claimed in claim 1 wherein said wound does not show symptoms of clinical infection.

3) A method for assessing the progress or course of treatment of a clinically infected wound which is subject to at least one treatment modality, said method comprising measuring the concentration of at least one component in a fluid from said wound, characterised in that said component is associated with inflammation.

4) A method as claimed in any of claims 1 to 3 wherein at least on of said components associated with inflammation is selected from the group of: Calprotectin, CRP, Procalcitonin, IL-8, Lipopolysaccharide binding protein, Protein C, IL- Ira, TNFa, Soluble TNFa receptor, Soluble IL-I receptor, IL-I, IL-IO, IL- 18, Transforming growth factor, Macrophage inflammatory protein- 1, High-mobility group box-1 protein, Hepatocyte growth factor, Leptin, Melanocyte-stimulating hormone, Fibrinogen and al anti-trypsin.

5) A method as claimed in any of claims 1 to 4 wherein one of said components associated with inflammation is calprotectin, and wherein a concentration of calprotectin above at least one threshold level is indicative of wound infection.

6) A method as claimed in claim 5 wherein one of said components associated with inflammation is calprotectin, said method additionally comprising measuring the concentration of at least one additional component in said fluid from said wound, wherein said additional component is selected from the group of:

CRP, Procalcitonin, IL-8, Lipopolysaccharide binding protein, Protein C, IL- Ira, TNFa, Soluble TNFa receptor, Soluble IL-I receptor, IL-I, IL-IO, IL- 18, Transforming growth factor, Macrophage inflammatory protein- 1, High-mobility group box-1 protein, Hepatocyte growth factor, Leptin, Melanocyte-stimulating hormaon, Fibrinogen and al anti-trypsin.

7) A method as claimed in any of claims 1 to 6 wherein said wound is a pathological or traumatic cut, laceration, graze, burn, lesion, or ulcer of skin or mucous membrane and optionally of underlying tissue.

8) A method as claimed in claim and of claims 1 to 7 wherein said wound is a chronic wound.

9) A method as claimed in claim 8 wherein said chronic wound is a chronic ulcer.

10) A method as claimed in claim 9 wherein said chronic ulcer is selected from a dermal ulcer, a venous ulcer, a diabetic ulcer, a pressure sore or a decubitis ulcer.

11 ) The use of concentration measurement of at least one component in a fluid from a wound for indicating the infection-status of said wound characterised in that said component is associated with inflammation.

12) The use as claimed in claim 11 wherein said wound does not show symptoms of clinical infection.

13) The use as claimed in any of claims 11 to 12 wherein at least one of said components associated with inflammation is selected from the group of: Calprotectin, C-reactive protein, Procalcitonin, IL-8, Lipopolysaccharide binding protein, Protein C, IL- Ira, TNFa, Soluble TNFa receptor, Soluble IL-I receptor, IL- 1, IL-10, IL- 18, Transforming growth factor, Macrophage inflammatory protein- 1, High-mobility group box-1 protein, Hepatocyte growth factor, Leptin, Melanocyte- stimulating hormaon, Fibrinogen and al anti-trypsin. 14) The use as claimed in any of claims 11 to 13 wherein one of said components associated with inflammation is calprotectin, and wherein a concentration of calprotectin above at least one threshold level is indicative of wound infection.

15) The use as claimed in claim 14 additionally comprising use of a concentration measurement of at least one additional component in said fluid from said wound, wherein said additional component is selected from the group of: CRP, Procalcitonin, IL-8, Lipopolysaccharide binding protein, Protein C, IL- Ira, TNFa, Soluble TNFa receptor, Soluble IL- 1 receptor, IL- 1 , IL- 10, IL- 18,

Transforming growth factor, Macrophage inflammatory protein- 1, High-mobility group box-1 protein, Hepatocyte growth factor, Leptin, Melanocyte-stimulating hormone, Fibrinogen and al anti-trypsin.

16) A device for indicating the infection-status of a wound, said device comprising;

i) at least one specific binder for at least one component in a fluid from said wound; and

ii) at least one signal generating moiety,

wherein said component is associated with inflammation.

17) A device as claimed in claim 16 wherein at least one of said component or components associated with inflammation is selected from the group of: Calprotectin, C-reactive protein , Procalcitonin, IL-8, Lipopolysaccharide binding protein, Protein C, IL- Ira, TNFa, Soluble TNFa receptor, Soluble IL-I receptor, IL- 1, IL-10, IL-18, Transforming growth factor, Macrophage inflammatory protein- 1, High-mobility group box-1 protein, Hepatocyte growth factor, Leptin, Melanocyte- stimulating hormone, Fibrinogen and al anti-trypsin. 18) A device as claimed in claim 16 or claim 17 wherein said component associated with inflammation is calprotectin and wherein in use said device generates a signal when the concentration of said component is above at least one threshold.

19) A device as claimed in any of claims 16 to 18 wherein said component associated with inflammation is C-reactive protein and wherein in use said device generates a signal when the concentration of said component is above at least one threshold.

20) A device as claimed in any of claims 16 to 19 wherein in use said device generates a signal corresponding to the binding of said component to said specific binder.

21) A device as claimed in claim 20 wherein said signal is generated over 1 to 95% of the area of said device.

22) A device as claimed in any of claims 16 to 21 wherein said specific binder is an antibody, an antibody fragment, an antibody construct, a single-chain antibody or an aptamer.

23) A device as claimed in any of claims 16 to 22 in the form of a dip-stick.

24) A device as claimed in any of claims 16 to 22 in the form of a swab, liner, sheet or dressing.

25) A device as claimed in claim 24 wherein a signal is generated over 1 to 50% of the area of one face of the device.

26) A kit for use in a method as claimed in any of claims 1 to 10, said kit comprising i) at least one specific binder for at least one component in a fluid from said wound; and ii) at least one signal generating moiety, wherein said component is associated with inflammation.