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06SCIENCE, PRACTICE AND EDUCATION Implications for clinical practice The autofluorescence imaging device can assist doc- tors in evaluating wounds’ bacterial burden level, spatial bioburden extent and the gram type of bac- teria present, thus aiding in effective debridement and proper wound-management protocols. INTRODUCTION Diabetes, one of the most prevalent chronic diseases, is projected to affect more than 700 million peo- ple globally by 2050 according to the International Diabetes Federation (1). People in underdeveloped and low-income countries are more prone to develop diabetes, and about 12–25% of those are at risk of developing diabetic foot ulcers as well, which can further lead to chronic wounds, amputation and even death (2-6). Wound bacterial burdens are known to be initially composed predominantly of gram-posi- tive bacteria (such as Methicillin-susceptible/resist- ant Staphylococcus aureus, Streptococcus), but as the infection progresses, they become colonised by gram- negative bacteria (such as Pseudomonas aeruginosa, Klebsiella species) (7-12). Wounds frequently differ in their clinical character- istics, making the diagnosis of infections through signs and symptoms alone difficult (13). In hospital settings, the current standard method for detecting bacterial burden requires a swab or deep tissue biopsy for culturing microorganisms, followed by biochemi- cal methods for species recognition and antibiotic susceptibility testing, which usually takes 3–5 days. In short, these methods fail to provide the immediate clinical information (such as the presence/absence of bacterial burden and gram type of bacteria) re- quired for first-line treatment (14). In addition, the culture sensitivity methodology has other limitations, including a bias to certain species, depending on the choice of growth medium and incubating conditions; a requirement for special apparatus for anaerobic bac- teria; and an inability to pick up the full diversity of infecting organisms (2). Accurate identification of polymicrobial species infecting the wound can only be achieved using genotypic methods such as16s rRNA sequencing, shotgun sequencing and so on (15-16). However, these methods are cumbersome, costly and have yet to be adopted in mainstream clini- cal practice (14). The ability to offer targeted treatment during a first consultation offers a tremendous advantage for timely wound healing. With proper and timely management of a wound, clinicians can achieve resolution in >90% of mild to moderate soft tissue infections (7); there- fore, there is a significant need for a modality that focuses on the early assessment and classification of pathogenic gram types infecting a wound. Multispectral imaging, which involves shining mul- tiple wavelengths of light and collecting the emission response, has proven to be a useful technique for identifying and classifying bacteria based on their autofluorescence (19). In addition, autofluorescence imaging is label-free, which is advantageous in terms of reduced complexity and cost. Studies have shown that bacteria have characteristic emission fluorescence when excited in the UV and blue regions of light, contributed by metabolic and infectious markers such as NAD(P)H, flavins, porphyrins and pyoverdine (in the case of Pseudomonas) and so on (19-20). Previous studies have also shown that gram-positive bacteria typically have more fluorescence intensity in the red region, due to an increase in the release of porphyrins, compared to gram-negative bacteria. Similarly, some gram-negative bacteria have increased NAD(P)H and flavins, when compared to gram-positive bacteria (21- 22). In addition, Pseudomonas aeruginosa, which is one of the most predominant pathogens present in diabetic foot infections (21), has clearly distinguish- able autofluorescence contributed by pyoverdine. These key differences in the relative concentration of biomarkers and autofluorescence in various spec- tral bands can potentially be used to design a rapid, non-invasive diagnostic technique for the presence/ absence of bacterial burden and the classification of the gram type of the bacteria infecting the wounds. Fluorescence imaging has emerged as a promising point-of-care technique for monitoring bacterial burden in wounds (21,22). Fluorescence imaging, in combination with clinical signs and symptoms, has been used to effectively monitor wounds with moderate-to-heavy bacterial loads (23). Similarly, sur- gical sites with a high prevalence of bacterial burden have been assessed with point-of-care fluorescence imaging devices (24). These results show that when the standard clinical signs and symptoms are assessed in conjunction with the inputs from fluorescence im- aging, bacterial burden diagnostic accuracy improves. A bacterial fluorescence imaging system was also used for effective wound debridement, which results in accelerated wound healing (25). Further studies have demonstrated that the use of fluorescence imaging to monitor wounds results in reduced antibiotic use (>30%), a reduction in antimicrobial wound dress- JOURNAL OF WOUND MANAGEMENT OFFICIAL JOURNAL OF THE EUROPEAN WOUND MANAGEMENT ASSOCIATION  183Page 1Page 3