What is the Role of Point-Of-Care Fluorescence Imaging for Bacterial Load Identification in Diabetic Foot Ulcers?

Editorial Summary

We recently published 'Point-Of-Care Fluorescence Imaging Reveals Extent of Bacterial Load in Diabetic Foot Ulcers' in the International Wound Journal, which explores point-of-care fluorescence imaging using a prospective, single-blind, multi-center and cross-sectional clinical trial.1 In this editorial we discuss the relevance of this trial and the impact of new terminology: chronic inhibitory bacterial load (CIBL), and how it intends to help define the presence of bacteria at high loads that is distinct from infection, but may inhibit wound healing.

Introduction

Worldwide, there are almost half a billion people with diabetes. Of these, 1 in 3 will develop diabetic foot ulcer (DFU), which has an associated 1.89 increased mortality risk.1 The most important clinical complication is diabetic foot infection.2 In diabetic patients, 60% of foot ulcers will become infected during clinical management.3 Diabetic foot infections are associated with increased economic costs, in addition to significant morbidities. They are the most frequent diabetes related indication for hospital admission and the most common reason for lower extremity amputation in diabetic patients. Overall, long-term healing rates and outcomes in these patients are poor.2

In addition to infection, other local factors influencing the healing rate of ulcers are pressure at the wound site and adequate blood supply to the site. When treating ulcers, it is often difficult to establish whether the wound is infected or not. High bacterial loads are often found in ulcers that do not appear clinically remarkable.4 Therefore, another important factor to consider in determining the rate of ulcer healing is bacterial infiltration of the wound, with or without overt clinical infection. The presence of a high burden of bacteria at the site of a wound has previously been shown to disrupt wound healing. Left unaddressed, bacterial endotoxins released from Gram-negative bacteria e.g., Pseudomonas aeruginosa, attract immune cells that perpetuate the inflammatory response, stall the healing cascade and force the wound to remain chronically open.5,6 Polymicrobial biofilms, present in 68% - 100% of DFUs, similarly contribute to wound chronicity by stimulating inflammation.8,9 The degree of bacterial infiltration varies and is dependent upon bacterial virulence and species.4,7,8

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The presence of high numbers of bacteria and formation of biofilms at the site of DFUs perpetuates the chronic wound cycle and increases the risk of infection. Earlier and more accurate detection of bacteria, with appropriate management, could reduce the risk of associated morbidity.1

Chronic inhibitory bacterial load (CIBL) is defined as:

“The chronic presence of bacterial microorganisms in a wound or its surrounding tissue at loads which can damage tissues and be inhibitory to healing, as well as require clinical intervention, with or without the presence of clinical symptoms.”

Point-of-Care Fluorescence Imaging

There are few reliable diagnostic tools to directly detect the presence of bacteria in wounds. Of those available, most do not provide immediate results. Point-of-care fluorescence imaging (FL-imaging) of bacteria is a biotechnology well positioned to address this clinical concern.

Under current guidelines, sampling and microbial analysis of a wound is done only if clinical infection is suspected.2 Although this allows treatment with appropriate antibiotics, superficial sampling techniques are suboptimal and are impeded by the presence of bacterial biofilms.1 This also misses the opportunity to treat patients with subclinical infection, where high levels of bacteria are present at the wound site, delaying healing and increasing the risk of future clinical infection.

Point-of-care FL-imaging uses endogenous FL signals, produced by bacterial metabolites and virulence factors to identify bacterial loads in wounds. It is objective, sensitive and noninvasive. FL-imaging is able to identify most bacterial species at clinically relevant levels, considered to be >104 colony-forming unit per gram (CFU/g), and crucially can detect biofilmencased bacteria.1

Since many patients who do not have clinically overt infection do have high levels of bacteria present at the wound site, the term infection is not particularly helpful in characterizing the degree of bacterial infiltration. This value is clinically important, as the presence of high levels of bacteria may inhibit wound healing, even in the absence of infection. Therefore, a new terminology, chronic inhibitory bacterial load (CIBL), helps to make the distinction between infection and high levels of bacterial infiltration requiring intervention.

The FLAAG Clinical Trial

In summary, the FL-Imaging Assessment and Guidance (FLAAG) clinical trial was a prospective, single-blind, multi-center, and cross-sectional study. It included 350 adults (>18 years) presenting with wounds of unknown infection status.10 Patients were recruited from 14 outpatient wound care centres across the United States between May 2018 and April 2019. An independent third party (Ironstone Product Development, Toronto, ON, Canada) was used to control for bias and to ensure appropriate blinding. The study aim was to compare the performance of standard clinical signs and symptoms (CSS) assessment using International Wound Infection Institute (IWII) guidelines, to CSS in combination with FLimaging, to detect clinically relevant bacterial loads. The clinicians received relevant training prior to commencing the study. They reviewed the patient’s history and inspected the wounds for all signs and symptoms of covert, overt, and spreading infection, identified by the IWII 2016 guidelines.11 Each sign and symptom was recorded when detected, including delayed healing beyond expectations; this was identified if the wound area had not reduced by at least 30% during the prior 4 weeks of care.

Post-hoc analysis of the FLAAG was conducted. The recorded CSS was reviewed to identify wounds that fulfilled the International Working Group of the Diabetic Foot (IWGDF) criteria for infection. Immediately following clinical assessment, standard and FL images were taken using the FL-imaging device (MolecuLight i:X, Toronto, Canada). This advanced imaging technology creates a map of high bacterial loads in and around wounds, without the use of contrast agents.10,12 Clinicians participating in the trial underwent didactic and hands-on training on use of the device; they were also trained on image interpretation and were required to pass an image interpretation certification test with a score of >80%.

Red FL on images indicates the presence of endogenously produced porphyrins from most common wound pathogens, while cyan FL indicates pyoverdine virulence factors from Pseudomonas aeruginosa specifically, both at loads >104 CFU/g.13,14

Up to 3 punch biopsies were collected from each DFU after cleansing of the wound with saline and gauze. More than 1 biopsy was only taken if an area of interest was identified or red or cyan FL was detected outside the wound centre.

Quantitative culture was subsequently performed, ensuring optimal conditions for difficult-to-culture bacteria were maintained.10 Diluted biopsy samples were cultured on various agars in conditions to support both aerobic and anaerobic growth.10,15 To identify specific bacterial species, matrix assisted laser desorption ionisation-time of flight mass spectrometry (Bruker Daltonics) was used.

Statistical analysis of data calculating both sensitivity and specificity at various bacterial thresholds was performed using MedCalc© Version 19.1.5. This was calculated with 95% confidence intervals (exact Clopper-Pearson). Bacterial loads were compared between the wound centre and periwound biopsies using a 2-sided paired student t-test.1

Results

Data regarding participant characteristics was noted. Importantly, participants with skin tones from across the Fitzpatrick scale (I to VI) were included. Most had a DFU of >3 months duration.1

In this study, 138 DFUs were examined; the majority of these (131) had some bacterial presence. Most ulcers that had some bacterial presence were also found to have high bacterial loads. Of the 131 ulcers where bacteria were found, only 6.1% (8/131) had bacterial loads below 104 CFU/g, while 93.9% (123/131) had bacterial loads exceeding 104 CFU/g, and 83.2% (109/131) had bacterial loads exceeding 105 CFU/g.1 The average bacterial load of DFUs with confirmed bacterial presence was 1.44 x 108 CFU/g. The number of bacterial species varied between wounds, with some ulcers having as many as 8 different species. The most common species found was Staphylococcus aureus (52.4%) and the average number of bacterial species per biopsy was 2.74.1

Although red and/ or cyan FL was detected in the majority of DFUs, as the bacterial load increased so did the proportion of DFUs with FL indicating bacterial loads. Red or cyan FL was identified in 92.3% of DFUs with the highest bacterial loads. In wounds where high volumes of bacteria were identified, IWGDF infection criteria were largely missing.

Five CSS were included in the present study, following those in the IWGDF criteria; these were swelling, erythema, warmth, and purulent discharge. In ulcers where there were very high levels of bacteria (>108 CFU/g), the most common IWGDF criteria detected were swelling (11.5%), erythema (15.4%), pain (11.5%), and local warmth (11.5%). Where bacterial levels were very low, only swelling was detected. Purulent discharge was consistently the least commonly observed. Of the clinically assessed signs and symptoms, none proved better than chance at predicting high bacterial loads of greater than 104 CFU/g.

Sensitivity did not improve at higher bacterial loads for 3 out of 5 IWGDF criteria. The only criteria which did show a high sensitivity at higher bacterial loads were pain and purulent discharge, however their prevalence was low. The low sensitivity and 1-specificity values indicate a poor predictive value of all 5 IWGDF criteria when assessing high bacterial load.1

In the study, the most commonly observed clinical sign was delayed wound healing beyond expectations. Generally, as bacterial load increased, the time taken for the wound to heal also increased. In over 50% of cases (52%), DFUs with bacterial loads of >104 CFU/g were healing delayed. This increased to 64.7% of wounds with bacterial loads of 107 - 108 CFU/g. Most wounds demonstrating a delay in healing were FL positive (70.0% - 95.5%).1

Sensitivity of delayed healing beyond expectations for predicting high bacterial loads was high across all bacterial thresholds, but specificity was lower than IWGDF criteria.1

In this study, 84.2% of the DFUs that exhibited red or cyan bacterial FL had FL indicating bacteria outside of the wound bed; this was mostly confined to callused tissue in the periwound (2cm radius extending out from the wound edge).16 Red FL signals within this region usually appeared blush pink to yellow due to the bacteria being below the surface. Bright red FL signals observed were attributed to the presence of higher bacterial loads at or near the callus surface.

If an additional biopsy was taken, the number of species and bacterial load at the wound centre was compared to the biopsy from outside the wound bed. The mean bacterial load from regions of red or cyan FL in the periwound region was significantly higher than the mean bacterial load of biopsies collected from the wound centre, implying that higher bacterial loads are present at the edge of the wound.1

Chronic Inhibitory Bacterial Load

The findings of this study led to a proposal of new clinical terminology: chronic inhibitory bacterial load (CIBL). This term refers to the state of bacterial infiltration within a wound that resists healing, but the process is not linked to any known clinically perceptible sign or symptom.1 CIBL is a state that is both CSS independent and is characterized by an inhibition of normal healing processes.

The DFUs in this study experiencing delayed healing were likely to be FL-positive, regardless of the bacterial threshold. Most DFUs that were FL-positive had signals indicating bacteria in the peri-wound region (84.2%). In addition, of the clinically assessed signs and symptoms, none were better than chance at predicting high bacterial loads.1 These findings suggest that a state exists where there are high bacterial loads, resulting in reduced wound healing, without any clinically apparent presentation. Introduction of this new terminology seems to help address this diagnostic gap.

Clinical Implications

In this study, CSS did not correlate with high bacterial loads. In fact, CSS were largely missing, and all 5 IWGDF criteria were infrequent. This suggests that clinical assessment alone is not a suitable method of detecting high bacterial loads, as there is frequently an absence of signs and symptoms even when there are many bacteria present. The term ‘CIBL’ illustrates this important pathogenic state. There is no connection between CIBL and any bacterial threshold, it merely describes the state in which a persistently elevated bacterial load results in pathology, including delayed healing. There is also no link to infection status of the wound.

CIBL may exist in a wound at any point beyond contamination, regardless of clinical markers of infection; it can therefore be addressed with bacteria-based wound care, which may include debridement and cleansing.1,11 This concept acknowledges the role of the biofilm in wound chronicity and is also suited to holistic wound infection prevention and management.

In clinical practice, CIBL can be utilized as an indicator for intervention prior to the onset of infection. Current clinical guidelines do not respond to asymptomatic but potentially pathogenic bacterial loads, despite acknowledging the value of their early and proactive management. This may be due to the inability to clinically detect them. Fl-imaging overcomes this diagnostic limitation, as it can visualize high bacterial loads across the entire wound and periwound.

Previous evidence has demonstrated high positive predictive values (PPV) of FL-imaging, and sensitivity greater than that of the CSS of infection. This study is consistent with previous findings, and has also demonstrated that while the sensitivity of CSS is not proportional to bacterial load, there is a proportional increase in sensitivity of FL-imaging.1 Furthermore, DFUs often present with macerated tissue. This allows futher bacterial colonization,