The ph of Wound Fluid in Diabetic Foot Ulcers- the Way Forward in Detecting Clinical Infection?

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1 Send Orders for Reprints to Current Diabetes Reviews, 2014, 10, The ph of Wound Fluid in Diabetic Foot Ulcers- the Way Forward in Detecting Clinical Infection? Carla McArdle 1 *, Katie M. Lagan 1 and David A. McDowell 2 1 Centre for Health and Rehabilitation Technologies (CHaRT), School of Health Sciences, University of Ulster, Shore Road, Newtownabbey, Co. Antrim, BT37 0QB, UK; 2 School of Health Sciences, University of Ulster, Shore Road, Newtownabbey, Co. Antrim, BT37 0QB, UK Abstract: Infections within diabetic foot ulcers are often hard to detect and extremely difficult to treat. The normal signs and symptoms of infection including purulence, erythema, pain, tenderness, warmth and induration are frequently absent in such wounds necessitating exploration of other ways of rapidly and accurately detecting infection. This study considers diabetic wound fluid ph as a possible alternative means of monitoring infection status. CINAHL, Ovid SP and MEDLINE were searched for papers in English published between January 2004 to May Key search terms included wound fluid, exudate, wound, ulcer, diabetes, ph, healing, infection, bacteria. This paper considers the potential benefits of augmenting and supporting current clinical practice in the early determination of wound healing trajectory and infection status, by monitoring wound fluid ph. The evidence collected highlights the need for further research and suggests the potential of wound fluid analysis as a possible surrogate marker for detecting infection in diabetic foot ulcers. Keywords: Diabetes, ulcer, wound fluid, ph, infection, bacteria. INTRODUCTION Diabetes is a worldwide chronic disease caused by genetic and environmental factors, which are driving national and international increases in the incidences of this disease, particularly within increasingly obese and/or elderly populations [1]. Currently, 2.9 million people within the UK have been diagnosed with diabetes and a further half a million people have the disease, but remain undiagnosed [2]. Within Northern Ireland alone 3.8% of the population (approximately people) have diabetes [2]. Foot ulceration is one of the most significant complications of diabetes, and will affect 15-20% of people with diabetes at some point in their lives [3]. Diabetic foot ulcers (DFUs) often fail to heal properly, and do not progress through the normal stages of wound healing, i.e. haemostasis, inflammation, proliferation and maturation, and remodelling [4]. The exact mechanisms involved in poor healing of ulcers in patients with diabetes remain unclear, but are known to include both systemic (immune defects, neuropathy, peripheral vascular disease) and local factors (infection, tissue fluid) [4]. Such failing to heal ulcers pose significant risks in relation to the chances of developing localised infection, and a range of longer term, potentially severe sequelae, such as osteomyelitis, systemic infection and/or amputation [1]. DFUs are the main cause of lower extremity amputations, with over 1 million people undergoing an amputation every year [5]. *Address correspondence to this author at the Centre for Health and Rehabilitation Technologies (CHaRT), School of Health Sciences, University of Ulster, Shore Road, Newtownabbey, Co. Antrim, BT37 0QB, UK; Tel: ; mcardle-c8@ .ulster.ac.uk Unfortunately diabetic foot infections (DFI) frequently fail to display overt signs and symptoms including purulence, erythema, pain, tenderness, warmth and induration [6]. This makes it difficult for clinicians to detect infection, and to make timely interventions to limit the highly undesirable consequences of DFIs. Alternative means of diagnosing infection are urgently required. A number of authors have suggested that the analysis of wound fluid ph may provide means of monitoring wound health [7-9]. A number of studies have investigated the relationships between wound ph and healing of chronic wounds, to establish which conditions best promote healing. One relatively early investigation established that weak acidic environments significantly inhibit protease activity and may potentially promote wound healing [7]. A subsequent comprehensive review by Schneider et al. recognised the complexity of the processes involved in healing of chronic wounds and reviewed the available literature on the effects of ph on wound healing [8]. ph has been proven to be an influential factor in the healing process, involving the development of an acidic environment as wounds [8]. A more recent review by Percival et al. has confirmed these processes, and their significance [9]. However, both reviews recognised the dearth of evidence in this area and the need for further research, to inform the development and implementation of treatment strategies incorporating possible shifts in ph to promote healing. There is also a need to establish the differences between chronic non-diabetic wounds (venous and pressure ulcers) and diabetic foot ulcers and explore the possibility of using ph as a chair side measure of not only monitoring wound healing status but also for the detection of diabetic foot infection (DFI) /14 $ Bentham Science Publishers

2 178 Current Diabetes Reviews, 2014, Vol. 10, No. 3 McArdle et al. This paper considers the potential of the analysis of wound fluid ph as an indicator of infection status and a means of wound healing prognosis of DFUs. It is based on a comprehensive search of CINAHL, Ovid SP and MEDLINE, covering articles published between January 2004 and May Searches were performed without restrictions on publication status, but were confined to articles in English. An initial specific search strategy based on combination of appropriate terms including; {ph value} and {wound fluid or wound exudate} and {diabetes or diabetic} and {ulcer or wound} yielded just eight articles of which only one was relevant. An alternative, much broader, multistage approach was therefore applied. The first stage of this more general approach aimed to identify publications dealing with the effects of ph on all wound types. This wider search used the following combination of search terms; {ph value}, {wound or ulcer} and {bacteria or infection or healing} and identified 319 publications. This group was further reduced by the inclusion of an additional wider search term {fluid or exudate}, yielding 118 articles for further analysis. After the removal of duplicates, all remaining publications were screened by two reviewers who separately examined the publication titles and abstracts, and subsequently derived an agreed list of twenty three publications for further consideration. Full text copies of these articles were obtained through the university library and separately examined in their entirety by the above two reviewers in relation to the specific criteria of this study, i.e. diabetes and wound fluid ph. This process identified four articles, which along with two additional articles identified by hand searching of reference list of the above twenty three articles, formed the final core group of articles to be included in the review. The very small number of articles identified as dealing with diabetic wounds, wound fluid ph, and infection/healing status was surprisingly low, bearing in mind the frequency and severe implications of infection in the wounds of those suffering from diabetes, but it does highlight the need for further research in this area. Wound Infection The presence of infection normally results from reductions in host susceptibility and the invasion of microorganisms on the surface of a wound [10, 11]. Successful microorganism invasion of host tissues can lead to a variety of infections. Such infections may involve superficial or deep tissues remaining localised, or spread into soft tissue causing cellulitis, or attacking bone, causing osteomyelitis. The agents of such infections have considerable contact with possible hosts, as they frequently contaminate and colonise almost all surfaces of the human body. However, as noted by Pasteur (1880) the germ is nothing. It is the terrain in which it is found that is everything [12]. Consequently it is usually only when the normal protective skin defences are damaged or pierced, forming a wound, that potential pathogens gain direct access, with the opportunity to establish infection. The most frequent contaminants of DFUs are, in descending order of frequency, Staphylococcus spp, Streptococcus spp, Enterobacter spp, E. coli and Pseudomonas spp [11, 13-16]. In most cases DFI is a consequence rather than a cause of ulceration, but a consequence with very serious sequelae [17-18]. DFIs are usually associated with increased morbidity (and/or mortality) which means that they represent significant clinical events, requiring immediate attention in relation to local (wound dressings) and systemic (antibiotics) issues and well-coordinated management (off-loading) [19]. The detection and elimination of DFIs present major clinical challenges, as a substantial proportion of these infections does not display the normal signs of inflammatory reaction, making them difficult to diagnose, and they are frequently polymicrobial in nature, making them difficult to treat [20-23]. Besides, current methods of clinical assessment of infection fail to support rapid, accurate diagnosis of wound infection in patients with diabetes. McIntosh [24] suggests that individuals suffering from diabetes are frequently hyperglycaemic, which facilitates the development of infection, by simultaneously stimulating the rate of growth of infecting bacteria, and reducing the efficiency with which neutrophils eliminate these bacteria [24]. The severity of the consequences of DFIs, and the absence of rapid accurate strategies to detect the development of infection, underline the need for further investigations into bacterial metabolism and interaction with environmental conditions within the wound fluid of DFUs. Current methods of recovering bacteria from DFUs use needle aspiration, swabbing, tissue biopsy and (more recently) polyvinyl alcohol (PVA) foam discs. However these methods are not very sensitive in terms of determining whether or not the wound is infected, and there is limited correlation between results obtained by these different methods. Perhaps more significantly, sampling techniques cannot reliably differentiate between infecting, contaminating and colonising bacteria. O Meara and colleagues [25] in their systematic review of the detection of infection in DFUs suggested that the definition of infection should include the observation of clinical signs and symptoms, along with the presence of at least 10 5 organisms. However these authors also acknowledged that clinicians are frequently unable to reliably identify infection solely by direct visual clinical assessment. Clinicians may also have difficulty identifying when patients need referral for formal diagnostic testing for infection including x-ray and MRI and are unsure whether empirical treatment with antibiotics leads to better outcomes [25]. Therefore in the absence of normal, observable, timely signs of infection in DFUs, it may be necessary to seek other indicators of the ecology of potentially infective agents in DFUs, to support early and effective interventions to mitigate the very serious and potentially life-threatening effects of such infections. There is a need for additional (ideally objective) parameters and measurements to determine when infection is present. Perhaps, greater certainty could essentially be achieved through analysis of DFU wound fluid, a material which is, after all, the most frequent and accessible first point of contact between bacteria and the wound [26]. Wound Fluid Wound fluid is a complex protein-rich exudate representing the extracellular fluid space within the microenvironment of a wound and thus is suggested to reflect the wound s current clinical condition [26-28].

3 The ph of Wound Fluid in Diabetic Foot Ulcers- the Way Forward in Detecting Clinical Infection? Current Diabetes Reviews, 2014, Vol. 10, No As yet our knowledge of the synergistic and antagonistic processes occurring within chronic DFU related wounds is limited. However, Loffler et al., [26, 29] suggested that analysis of DFU wound fluid, (i.e. the fluid formed during vasodilation [30] containing infecting or contaminating bacteria), may provide a useful window on the wound, predicting local bacterial/host interactions and the future healing or non-healing trajectory of such wounds. Wound fluid within DFU has been defined as being a wounding agent in its own right [30]. It has the capacity to degrade wound repairing growth factors and predispose the wound to further inflammation and delayed healing [31, 32]. Longer term exposure of DFU wound fluid, also means that it is more likely to become contaminated/colonised by microorganisms, which further interfere with normal wound healing processes. Wound fluid diagnostics could emerge as an invaluable tool providing vital information with respect to clinical endpoints such as healing and infection and is suitable for daily clinical routine [26]. Lui and colleagues [33] recognised that in vivo studies carried out to date on DFUs are limited, and suggested that the non-invasive collection of wound fluid might avoid some of the problems associated with other means of identifying infection, and infective agents within DFUs [33]. WOUND FLUID ph ANALYSIS ph is the regulation of hydrogen ions in fluid compartments throughout the body and is of critical importance to the health of all individuals. Changes in ph can have major effect on the chemical reactions occurring within the body, which means that measurement of the ph of wound fluid may provide a useful method of monitoring the condition of the wound bed, and facilitate evaluation of progress in treatment [34]. The nature and content of wound fluid may significantly influence a number of activities and agents within wounds. Investigations of DFU wound fluid have included the effect(s) of ph on the matrix metalloproteinases (MMPs) and the presence of microorganisms in relation to healing and the detection of infection. The Effects of Wound Fluid ph on MMPs Matrix metalloproteinases (MMPs) are enzymes present in the wound bed that cause the breakdown of dead tissue as part of the repair and restructuring activities. They are closely controlled by enzymes inhibitors called tissue inhibitors of matrix metalloproteases (TIMPs) [27, 35]. Widgerow [27] recognised the high proportion of MMPs in wound fluid and their impact in extending inflammatory processes within the on-going breakdown of the wound bed, trapping the wound in the inflammation and proliferation phases of healing. Changes in ph can have a major effect on the chemical reactions occurring within the body, frequently switching on and off the activities of a wide range of enzymes [34]. In particular it has been suggested that the MMPs, like most other enzymes are very sensitive to such changes in their immediate ph environment [35, 36]. Studies of which ph conditions best promote healing in DFUs [36, 37] established that MMPs are inactivated at low ph (ph 4), but are active and capable of degrading granulation tissue, at higher, more alkaline ph values [37]. A clinical study of wounds (including acute wounds and diabetic foot ulcers) compared wound fluid ph using litmus paper strips with clinical classification in terms of being unhealthy, granulating or healing, and the amount of wound fluid present [37]. Observed improvements in wound status were associated with reductions in wound ph, with 34% of the wounds having a ph between and a further 14% having a ph lower than 7.5. Continuing decreases in the wound ph during the study period were associated with wounds progressing from unhealthy status towards a granulating or healing status. Greener and colleagues [36] in their in vitro study suggested that MMPs at the wound bed may be sensitive to changes in ph and that manipulation of wound fluid to lower ph values might improve healing rates in non-healing wounds [36]. Similarly it has been suggested that the wound environment becomes more acidic as healing progresses [36, 37]. However more clinical studies are needed to support this suggestion [9, 38]. The Effects of Wound Fluid ph on the Presence of Microorganisms In terms of the development of DFI, wound fluid ph may also have significant effects on colonising or infecting bacteria [9, 35, 37, 38], influencing rates of survival, metabolism and growth, as well as the expression or action of the virulence factors associated with the establishment and effects of infection. Bearing in mind the very significant impact of infection within wounds, this means that changes (or stasis) of wound fluid ph may considerably modulate the trajectory of DFUs. Such modulation may positively or negatively influence infection and wound healing [8]. The above key roles of wound ph, influencing host and bacterial activities, mean that monitoring wound fluid ph across the various stages of healing/non-healing may enhance clinicians abilities to more rapidly and accurately recognise infection. Such enhancements are important in identifying the need for, and more rapid application of, early intervention(s) to limit the duration and impact of DFIs. Further investigations are needed to gain more information in this area. Recognition of the key role of wound ph in affecting host tissue repair mechanisms, and in influencing bacterial metabolism, would suggest that the relatively straight forward non-invasive measurement of wound fluid ph may well be a valuable tool in monitoring wound status, healing trajectory and the presence/absence of infection. However, there is a dearth of evidence for the effects of ph on the presence of microorganisms within diabetic foot ulcers, and further research is required in this area to understand the interactions between wound fluid parameters (including ph) in the establishment of infection and the failure to control DFIs. In terms of the specific nature of the range of bacteria involved in DFIs, some information is available. Shukla and colleagues [37] examining patients (n=50) with acute (burns) or chronic wounds (lower leg ulcers and DFUs) observed that 58% of wounds had microorganisms present on the

4 180 Current Diabetes Reviews, 2014, Vol. 10, No. 3 McArdle et al. wound surface and that different microorganisms were present at different wound ph values. Pseudomonas aeruginosa was the most common microorganism recovered from wound fluid samples which had a ph greater than 8.5. Escherichia coli was the most common microorganism recovered from ph8 samples, and Staphylococcus aureus was the most common microorganism recovered from ph 7.5 samples. The major limitations of this study were that the study included, but did not differentiate acute and chronic wounds. Shi et al. [39] investigated wound fluid ph and numbers of microorganisms in induced diabetic wounds in animals (n=5). They observed that although ph decreased as healing progressed, there was no significant change in microbial numbers. While these findings, and the findings of Shukla and colleagues [39] are of interest, it is important to note that these studies used animal models and non-diabetic wounds respectively and further, more robust studies solely on DFU studies remain necessary. A recent in vitro study on bacteria isolated from burns and chronic wounds examined the effect of bacterial phenotype and environmental ph on the efficacy of antibiotics [40]. The study investigated the extent to which antibiotic efficacy at ph 5.5 and ph 7 against gram positive chronic wound isolates (Methicillin resistant Staphylococcus aureus (MRSA), Streptococcus spp. and Methicillin sensitive Staphylococcus aureus (MSSA)) and gram negative rods (included strains of Pseudomonas aeruginosa) were modulated by bacterial phenotype and environmental ph. The authors reported that the activity of ampicillin against MRSA increased as the ph decreased from ph 7 to ph 5.5, suggesting that lower ph values were associated with reduced antibiotic resistance. These observations would suggest that assessing wound fluid ph and being able to select a more wound ph appropriate antibiotic may provide a more effective means of eliminating of resistant bacteria such as MRSA, which pose an increasingly significant and recalcitrant clinical problem in relation to infection in diabetic foot ulcers. A thorough understanding of the extent to which wound ph reflects or modulates the presence and antibiotic sensitivity of microorganisms within DFIs, may inform a number of aspects of clinical practice. It could, for example, support more timely and effective clinical decision making, and interventions, and may in the longer term provide therapeutic strategies in wound ph manipulation to eliminate DFI and ultimately promote healing. Researchers [29, 41] recognised the need for a simple biomarker that enables the detection of infection. If such a biomarker could be monitored through the non-invasive collection of wound fluid, it could be a useful and relatively simple means of establishing wound status. While a number of studies have been carried out in the area to investigate healing and the presence of infection, further work is needed to provide robust evidence to support routine clinical evaluation of wound fluid. CONCLUSION The role of hypoxia, ischaemia, neuropathy, ageing and growth factors in disrupting the healing process of diabetic foot ulcers and chronic wounds has been well documented, but clinicians continue to face challenges in the rapid and accurate estimation of wound condition, infection status and trajectory. Such difficulties are particularly unfortunate as the potential for infection in DFUs is high, many of the normal indicators of infection within acute wounds are not presented by DFUs, and the sequelae of DFU infection can be very severe. This paper considers the potential benefits of augmenting and supporting current clinical practice in the early determination of wound infection status, by monitoring infection related wound fluid parameters [8]. Bearing in mind the immediate and intimate interactions between wound fluid and invading bacteria, and the relative ease with which samples of wound fluid can be collected during routine clinic activities, such analyses may be of considerable benefit. The possible advantages offered by objective, chair side methods for rapid, accurate, and sensitive monitoring of wound infection status, suggest that this approach may become an important tool in wound surveillance and clinical decision making in the resolution of DFIs. 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