GB2399881A

GB2399881A - Diagnosing wound infections

Info

Publication number: GB2399881A
Application number: GB0306979A
Authority: GB
Inventors: Breda Mary Cullen
Original assignee: Johnson and Johnson Medical Ltd
Current assignee: Johnson and Johnson Medical Ltd
Priority date: 2003-03-26
Filing date: 2003-03-26
Publication date: 2004-09-29
Anticipated expiration: 2023-03-26
Also published as: GB0306979D0; GB2399881B

Abstract

A method of predicting or diagnosing clinical infection of a wound comprises measuring the concentration of a marker associated with an inflammatory response in wound fluid, where the marker is a proinflammatory cytokine, e.g. TNF- a . Also claimed is use of a wound dressing or biosensor comprising components of an assay system for measuring the concentration of a marker associated with an inflammatory response, wherein the marker is a proinflammatory cytokine, for the manufacture of a medicament for predicting the likelihood of clinical infection of the wound or, for diagnosing clinical infection of a wound Immuno, colorimetric, fluorimetric etc. methods may be used for detecting the marker concentration.

Description

239988 1

PREDICTION AND DETECTION OF WOUND INFECTION

The present invention relates to a method of predicting or diagnosing clinical inflection of a wound comprising measuring the concentration of a marker associated with an inflammatory response in wound fluid, in particular the concentration of a proinflarnmatory cytokine. The present invention also relates to devices and kits for use in such methods.

In mammals, injury triggers an organised complex cascade of cellular and biochemical 1() 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.

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 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 elicits 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. The granulation phase of the healing process will begin only after the infection has subsided.

Chronically contaminated wounds all contain a tissue bacterial flora. These bacteria may be indigenous to the patient or might be exogenous to the wound. Closure, or eventual healing -,f the wound is often based on a physician's ability to control the level of this bacterial flora.

('urrent methods used to identify bacterial infection rely mainly on judgement of the odour and appearance ol'a would. With experience, it is possible to identify an infection in a wountl by certain chemical signs such as redness or pain. Some clinicians take swabs that arc then culturctl in the laboratory to identify specific organisms, but this technique takes time. The prior art also describes the use of certain proteases as an indicator of healing status.

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

There is thus 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.

Tumor Necrosis Factor Alpha {'I'NF-a) is a proinflammatory cytokine that is released by activated macrophages and lymphocytes. Mature human TNF-a is peptide of 157 amino acid residues. It binds to receptors present on the surface of most cells to produce a wide range of effects, due to its ability to activate multiple signal transduction pathways, and to its ability to induce or suppress the expression of a vast number of' genes, including those for growth factors and cytokines.

Over-production of TNt'-a has been implicated in a range of disorders, including cachexia (progressive wasting), septic shock, autoimmune disorders, bacterial toxic shock, graft versus host disease, I llV infection and AIDS.

I'hc present inventors have found that bacterial toxins stimulate the production of TNF-a l'rom macrophages and neutrophils more rapidly than previously understood. 'I'his suggests that the measurement calf TNF-a and/or other proinflammakry cytokinc levels in wound fluid could give early-stage warning ol' wound infection, before obvious clinical signs of int'ection arc prcscnt.

According to the present invention, there is provided a method of predicting or diagnosing clinical infection of a wound comprising measuring the concentration of a marker associated with an inflammatory response in wound fluid, wherein the marker is a proinflammatory cytokine. s

Also provided is a use of a wound dressing or biosensor comprising components of an assay system for measuring the concentration of a marker associated with an inflammatory response, wherein the marker is a proinflammatory cytokine, for use in the manufacture of a medicament for predicting the likelihood of clinical infection of the wound or for diagnosing clinical infection of a wound.

The preferred proinflammatory cytokine is l NF-a. Further examples include: Interleukins such as IL-lbeta, 11-4, IL-6, IL-X, IL-IO, 11,-18, MCP-1, MCP-2, MCP-3 Monocyte chemoattractant proteins, MIP- 1 alpha, MIPI beta, MIP-2 Macrophage inflammatory proteins, Interferons lFN-alpha, IFN-beta, and IFN-gamma, GM-CSF Granulocyte/macrophage colony stimulating factor, PF-4 Platelet factor 4, RANTES a member of the chemokinc family.

By "measuring the concentration of a marker associated with an inflammatory response in wound fluid" we include measuring the activity of a marker associated with an inflammatory response in wound fluid. The term "wound fluid" refers to any wound exudate or other fluid (preferably substantially not including blood) that is present at the surface of the wound, or that is removed from the wound surface by aspiration, absorption or washing. I he term "wound fluid" does not refer to blood or tissue plasma remote from the wound site.

It will bc appreciated that the concentration of more than one marker may be measured. In certain embodiments, thc concentrations of at Icast two, three or four markers are monitored.

Mcasuring thc concentration ol a marker asscciatcd with an inflammatory response in wound fluid allows the likelihood <-,i (or the presence of) clinical infection to be assessed.

The step of measuring is preferably carried out on wound fluid that has been removed from the body of the patient, but can also be performed on wound fluid in situ.

Any type of wound may be diagnosed for infection according to the method / use of the present invention. For example, the wound may be an acute wound such as an acute traumatic laceration, perhaps resulting from an intentional operative incision, or the wound may be a chronic wound. The method / use of the invention is envisaged as being most useful in predicting or diagnosing clinical infection oi'a chronic wound. Preferably, the chronic wound is selected from the group consisting of venous ulcers, pressure sores, decubitis ulcers, diabetic ulcers and chronic ulcers of unknown aetiology. Chronic wound fluids inherently have levels of markers such as neutrophil elastase that are many times the level found in normal, acute wound fluids. Nevertheless, it has now been found that the levels of such markers are further elevated by a substantial amount when the chronic According to the present invention, the prognostic / diagnostic assay is designed so as to provide a correlation between a given concentration of a marker of an inflammatory response and the likelihood of (or presence ol) clinical inflection.

'1'hosc 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 inl'ection. Preferably, a concentration at least 1.5-, 2-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 5.5-, 6.0-, 7.0-, 8.0- or 9. 0-fold the basal level of the marker is considered as begin indicative of subsequent progression to clinical infection.

By "measuring the concentration of a marker associated with an inflammatory response" we also include techniques that produce a positive or negative signal if the marker is present at one or more of these concentrations.

Preferably, a concentration at least l.S-, 2-. 2.5-, 3.0-, 3.5-, 4.0-, 4. 5-, 5.0-, 5.5-, 6.0-, 7.0-, 8.0-, 9.()-, 10.()-. 1 1.()-, 12.0- 14.0-, 1 6.0-fold the basal Icvcl <> I'the marker is considered as begin indicative of the presence ol'clinical inicclion. By "measuring the concentration ol'a marker associated with an inflammatory response'' we also include techniques that produce a positive <>r negative signal il the marker is present at one or more ol'lhcse concentrations.

By the "basal level of the marker" we include the level of the marker normally associated with a wound 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 a normal, acute wound.

With regard to the concentration of TNF-a in the wound fluid which is associated with subsequent progression to clinical infection, our data indicates that a concentration of at least about 40pg/ml is indicative of the wound subsequently becoming clinically infected.

As used herein, the term wound fluid is meant to refer to the exudate that is secreted or discharged by cells in the environment of the wound. This fluid contains cells, both living and dead, and a variety of inflammatory cytokines.

lS By the "concentration of a marker of an inflammatory response" is meant the free concentration of the marker in the wound fluid. The concentration of the marker may be assessed in situ, or alternatively a sample of wound fluid may be taken as a clinical swab or as a fluid sample.

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 include those utilising chemical or en><yme-linked reactions, or immunological (c.g. ESIISA, western blots), spectrophotomctric, calorimetric, lluorimetric, or radioactive detection based techniques.

In one embodiment the concentration of' the marker is measured by a dipstick type test.

Such a test could be used in the community and by the patient allowing easier and earlier diagnosis.

l'o allow measurement ol' concentration ol 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 Lsiu7 or fluid may be removed from the wound for subsequent analysis.

I'hc decision as to which method is used will depend upon the type of wound in question.

For example, in the case of surface-exposed wounds, a clinical swab, dressing, "dipstick" or other biosensor device may be applied directly to the surface of the wound. The device should contain the components of the assay system for measuring the concentration of the marker so that the assay reaction may itself proceed in situ.

The device can then be removed from the wound and the signal measured by the appropriate means. In many cases, a physician may not actually require an accurate assessment of the precise concentration of the marker, but may just wish to know whether there is a sufficient concentration of the marker to warrant prophylactic or curative action as necessary. In these cases, visible assessment of the dressing may be sufficient to allow identification of the specific areas of infection. Unnecessary treatment of healthy I granulating tissue can then be avoided.

dressing that allows mapping of the infected areas of a wound will be preferable in certain instances. Diagnostic wound mapping sheets that could be adapted to the methods of the present invention are described in GB2323 166 (application no. GB 9705081.9), filed on 1 2th March l 997, the entire content of which is hereby incorporated by reference.

Immobilisation of reaction components onto a dipstick, wound mapping sheet or other solid or gel substrate offers the opportunity of performing a more quantitative measurement. For example, in the case of a reaction linked to the generation of a colour the device may be transferred to a spectrometer. Suitable methods of analysis will be apparent to those of skill in the art.

Immobi]isation of the reaction components to a small biosensor device will also have the advantage that less of the components (such as enzyme and substrate) are needed. 'I'he tievicc will thus be less expensive to manufacture than a dressing that needs to have a large surface area in order to allow the mapping of a large wound area.

Methods t'or the incorporation of the components -'I' the assay reaction onto a clinical dressing, "dipstick" sheer or <ether biosensor arc routine in the art. See for example Fagerstam and Karlsson (1994) Immunochemistry, 949-970, the entire content of which is incorporated herein by reference.

The concentration of the marker may alternatively 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 or in a microassay plate.

Such a method 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.

It will be clear that an aqueous assay system is more applicable to use in a laboratory environment, whereas a wound dressing containing the necessary reaction components will be more suitable for use in a hospital or domestic environment.

Specific embodiments of'the present invention will now be described in more detail, by way of example, with reference to the accompanying drawings, in which:

BRIEF DESCRIPTION OF THE DRAWING

Figure I shows the amounts of TNF-a detected in cell culture supernatants over time for Tl IP- I monocyte/macrophage cells stimulated by the presence of various concentrations of bacterial lipopolysaccharide, at 2X106 cells/ml for 1,4,24 and 48 hours incubated at 37 C F,XAMPLES Unless otherwise stated all chemicals used are l'rom SlGMA-lildrich Company Ltd. Poole,

U K

Example 1

Wound fluid was collcctecl fron1 two patients having, diabetic foot ulcers old least 30 days duration and a surl'acc area of' et least lcrn2. 'I'he first patient did not exhibit any clinical signs of infection at the time of taking of the wound fluid sample, or within 14 days thereafter. Ate second patient showed clinical signs of wound infection at the time that the sample was taken.

Protein Assay Total protein present in each extracted wound fluid sample was determined using the Bradford protein assay. The protein binding solution comprises I ml Coomassie Brillant Blue stock solution 200mgCoomassie Brillant Blue G250, Sigma Chemical Co., dissolved in 50 ml ethanol-90%); 2ml orthophosphoric acid (85% w/v); in a final volume of 20 ml with distilled water. This solution was filtered (Whatman #1 filter paper) and used immediately. The protein level in a sample wound fluid was measured by mixing 10-ill sample or standard with 190-1 of the protein binding solution in a microtitre well and incubating for 30mins at ambient temperature prior to reading absorbance at 595nm. The concentration of protein was estimated from a standard calibration of BSA (bovine serum albumin prepared in distilled water; Sigma Chemical Co.) ranging from 1.0 to 001 mg/ml.

TNF-x us.say I'he levels of TNF-x present in the samples were measured by an ELISA method using a Quantikine (RTM) kit supplied by R&O Systems Europe of 19 Barton Lane, Abingdon I Science Park, OX14 3NB, ILK. Briefly, the assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-a is pre-coated onto a microplatc. Standards and samples are pipetted into the wells of the microplate, and any 'fNF-a is bound by the immobilized antibody. After washing away any unbound substances, an ezyme-linked polyclonal antibody specific for TNI;a is added to the wells.

Folkwing a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and colour develops in proportion to the amount of TNF-a bound in the initial step. The colour development is stopped and the intensity of the colour is measured.

I'he enzyme used is horse radish peroxidase, the colour reagents are stabilised hydrogen peroxide and stahilised tetramethyl benzidcne chromogen,. and the stop solution is 2M sulphuric acid.

Results The non-infected wound fluid contained 22.2 pa/ml, and when adjusted for total protein 6.36 pg/ml/mg of TNF-a. The infected wound fluid contained 135.6 pa/ml, and when adjusted for protein 64.2 pg/ml/mg of TNF-a. It can thus be seen that a very considerable increase of the lF-a levels is seen upon infection of a chronic wounds.

Example 2

The effect of bacterial lipopolysaccharide on production of TNF-a by neutrophil cells isolated from whole blood was studied as follows.

Neutrophil Cell Isolation from Whole Blood Neutrophils were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation7 according to the method of Moseley et al (1997). Briefly, whole blood (40ml) was removed from healthy, human volunteers (age range 20-30 years) into vacutainers containing ED'I'A (Becton Dickinson, Meylan Cedex, France) as an anticoagulant. Blood aliquots (lOml) were layered onto a Ficoll-Hypaque density gradient, consisting ol a dense Ficoll-llypaque layer (lOml), comprised of 9.5% Ficoll 400 (Amersham Pharmacia lliotech, Buckinghamshire, U.K.) and 17% EIypaque solution I (sodium diatrizoate, Intrapharm I aboratories, C;went, U.K.) and a light Ficoll-Hypaque layer (lOml), consisting of comprised ol' 8.17% Ficoll 400 and 10% Hypaque solution.

Tubes were centrifuged at 2500rpm/45min at room temperature, resulting in the formation of four definite layers. 'I'he two upper layers, consisting of plasma and lymphocytes/monocytes respectively, were removed and discarded. Each third layer, containing the ncutrophils, was removed and pooled into a separate tube, with each remaining erythrocytc layer, also being discarded. ISqual volumes of PBS were added to the pooled ncutropl1il layers and centrifuged at 2000rpm/5min at room temperature. The supernatant layer removed and pellet resuspended in l'BS and centrif'uged at 2000rpm/5mins at room temperature. Any crythrocytc contamination of the PMN pellet 3() was removed by adding cold ().2% sodium chloride (5ml) and the tube ag,itated f'or lmin.

l-he osmolarity within the tube was corrected by the addition of 1.6% sodium chloride, f'ollowed by g,,entlc agitation. An equal volume of P13S (l()ml) was added and the tube centril'uged at 2()()()rpm/5n1in at room temperature. ()nce crythrocyte containination had been removed, the PMN pellet was washed once in PBS (Sml), eentriffiged at 2000rpm/5min, as above, and resuspended in RPMI-1640 medium (Gibco BRL, Paisley, U.K.), supplemented with L-glutamine (2mM) (Gibco BRL), prior to assessment for amount of contamination by other cell types (visual assessment), cell viability determination and cell counting.

Cellular stimulation by bacterial lipopolysaccharide Neutrophils were seeded in the appropriate culture media into 24-well tissue culture plates at a cell density of 2 x 106 cells/0.5ml and/or 5 x lOs cells/0. 5ml. Cells were then stimulated with equal volumes of LPS E.coli 055:B5 was added at a final concentration of 100, 10, 1, 0.1 ng/ml for Ih, 4h, 24h, and 48 h at 37 C in 5% CO2. Media alone and cells incubated with media only served as the negative control. Culture media was removed, aliquoted and immediately frozen at -70 C.

Results The amount of TNF-a detected in the cell culture supernatant at 4 hours had increased to 50pg/ml and at 24 hours had increased to 220pg/ml. '['his illustrates the prompt response of'I'NF-a level to bacterial contamination.

Example 3

The production of TNF-a from a monocyte/macrophage cell line cultured in vitro was studied as follows, in order to determine the ef'f'ect of bacterial lipopolysaccharide on TNF production.

TllP-1 cells monocyte/macrophage cell line were maintained in culture in RPMI-1640 medium containing 2mM L-glutamine (Cilbco BRL, Paisley7 UK) supplemented with 10% foetal call' syndrome. 'l'he cells were then stimulated with dif'i'erent concentrations of bacterial LPS, and measureweuts taken as described f'or Example 2.

I'he results are sh-'wn in Figure 1. It can be seen that substantial stimulation of' TNt'-a production is seen at I hour for the samples treatecl with l()ng/ml or lO()ng/rnl -,l'I PS.

L'urthennore, it can be seen that stimulation with amounts as small as l() ()pg/ml of' bacteria! LPS results in TNF-a production after 4 hours. Thus, it can be seen that measurement of TNF-a is highly promising for earlystage prognostic/diagnostic detection of wound infection, even before any clinical signs are apparent.

The above examples have been described for the purpose of illustration only. Many other embodiments falling within the scope of the accompanying claims will be apparent to the skilled reader.

Claims

1. A method of predicting or diagnosing clinical infection of a wound comprising measuring the concentration of a marker associated with an inflammatory response in wound fluid, wherein the marker is a proinflammatory cytokine

2. Use of a wound dressing or biosensor comprising components of an assay system for measuring the concentration of a marker associated with an inflammatory response, wherein the marker is a proinflammatory cytokine, for the manufacture of a mcdicament for predicting the likelihood of clinical infection of the wound or for diagnosing clinical infection of a wound.

3. The method of claim I or the use of claim 2 wherein the marker is TNFa

4. The method or use according to any one of claims I to 3 wherein the wound is a chronic wound.

5. The method or use according to claim 4 wherein the chronic wound is a chronic ulcer such as a dermal ulcer, venous ulcer, pressure sore or decubitis ulcer.

6. The method or use according to any one of claims I to 5 wherein the concentration of the marker is measured using an immunological, spectrophotometric, calorimetric, fluorimetric, or radioactive detection based technique.

7. The method or use according to any one of claims I to 6 wherein the concentration of the marker is measured by a dip-stick type test.