A Rapid Point of Care Test for Bacterial Protease in Chronic Wounds is Predictive Amputation Risk

Introduction
Non-traumatic lower limb amputations (LLAs) cause significant morbidity and mortality and are costly to health care systems. The in-patient care cost of a LLA for a diabetic in the UK is estimated to be £9,546 (95%CI: £6,416-£13,463)1. In the UK alone, approximately 20 LLAs occur daily2, with one year mortality of 13-40% and five year mortality of 39-80%3. A patient with a chronic wound is more likely to receive a non-traumatic LLA due partly to increased risk of infection4. An indication of pathogenic behavior of bacteria is the production of enzymatic virulence factors or bacterial proteases5. The detection of bacterial protease activity (BPA) in a chronic wound is indicative of a period of pathogenesis which is a precursor to observable clinical signs and symptoms of infection. WOUNDCHEKTM Bacterial Status is a rapid, qualitative, point of care test using a wound fluid swab to detect BPA6. In this study, patients asymptomatic for infection were tested for BPA and then monitored for 12 weeks.

Method
314 chronic wounds (128 venous leg ulcers, 144 diabetic foot ulcers, 31 pressure ulcers, 6 mixed etiology leg ulcers and 5 arterial ulcers) on patients attending 7 US wound clinics, without signs of infection and not currently being treated with topical antimicrobials, were enrolled after informed consent. At Time 0 the wound was swabbed and tested using the WOUNDCHEKTM Bacterial Status device. The test result was blinded to the clinician. The patients were followed for 12 weeks to determine if the wound healed or not and whether an amputation occurred due to the wound.

Results
The analysis combines data from a prospective clinical trial at seven US wound clinics (n=266 wounds) and unaudited data from a preceding pilot cohort (n=48 wounds). 43% (135/314) of the wounds were positive for BPA of which 99 were non-healing at 12 weeks yielding a positive predictive value of 73% (99/135, 95% CI=66.9-78.9%). 124 wounds healed within 12 weeks giving a healing rate of 39% (124/314). Of the 10 patients that received amputations due to their wounds (8 DFU’s, 2 VLU’s), 8 were positive for BPA. The relative risk for amputation in patients positive for BPA was 5.3 (p=0.03).

Discussion
Presence of pathogenic bacteria in a wound can cause a period of pathogenesis that leads to local infection, impairs healing and can result in the need for LLA. Clinical examination can wrongly diagnose infection with some chronic wounds failing to exhibit the classic signs of infection and chronic inflammation being misinterpreted as infection4. Testing wound fluid for BPA using a rapid point of care test may be useful for detecting the presence of pathogenic bacteria, at a clinically significant stage in the infection continuum, even before the signs of infection are apparent. Integrating a point of care test for BPA as part of routine wound assessment could be a valuable tool in treatment pathways to inform clinicians that the wound is in a period of pathogenesis which could lead to overt infection and be a possible contributor to wound chronicity and increase the risk of amputation6.

Conclusion
The study demonstrates there is a statistically significant increased risk of amputation when bacterial proteases are detected in a wound asymptomatic for infection. Prospective studies are required to confirm that using the WOUNDCHEKTM Bacterial Status test to aid diagnosis and assist in directing appropriate therapy can improve healing outcomes and reduce amputation risk.
References
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2. Public Health England (2016). Diabetes Footcare Activity Profiles. Calculated from the average annual number of amputations per year from 2012-15.
3. Singh N, Armstrong D, Lipsky B. Preventing foot ulcers in patients with diabetes. Jama 2005 293:217-28
4. Gardner S, Frantz R.Wound Bioburden and Infection-Related Complications in Diabetic Foot Ulcers. Biol Res Nurs, 2008;10 (1):44-53
5. Lebrun I, Marques-Porto R, Pereira AS, Pereira A, Perpetuo EA. Bacterial toxins: an overview on bacterial proteases and their action as virulence factors. Mini-reviews in medicinal chemistry 2009; 9 (7): 820-8.
6. Serena T, Bayliff S, Brosnan P. Bacterial Proteases: A Marker for a ‘State of Pathogenesis’ in Chronic Wounds. Poster presentation SAWC Fall 2015.