Point-of-care autofluorescence imaging for real-time sampling and treatment guidance of bioburden in chronic wounds: first-in-human results

Abstract

Background: Traditionally, chronic wound infection is diagnosed by visual inspection under white light and microbiological sampling, which are subjective and suboptimal, respectively, thereby delaying diagnosis and treatment. To address this, we developed a novel handheld, fluorescence imaging device (PRODIGI) that enables non-contact, real-time, high-resolution visualization and differentiation of key pathogenic bacteria through their endogenous autofluorescence, as well as connective tissues in wounds.

Methods and findings: This was a two-part Phase I, single center, non-randomized trial of chronic wound patients (male and female, ≥18 years; UHN REB #09-0015-A for part 1; UHN REB #12-5003 for part 2; clinicaltrials.gov Identifier: NCT01378728 for part 1 and NCT01651845 for part 2). Part 1 (28 patients; 54% diabetic foot ulcers, 46% non-diabetic wounds) established the feasibility of autofluorescence imaging to accurately guide wound sampling, validated against blinded, gold standard swab-based microbiology. Part 2 (12 patients; 83.3% diabetic foot ulcers, 16.7% non-diabetic wounds) established the feasibility of autofluorescence imaging to guide wound treatment and quantitatively assess treatment response. We showed that PRODIGI can be used to guide and improve microbiological sampling and debridement of wounds in situ, enabling diagnosis, treatment guidance and response assessment in patients with chronic wounds. PRODIGI is safe, easy to use and integrates into the clinical workflow. Clinically significant bacterial burden can be detected in seconds, quantitatively tracked over days-to-months and their biodistribution mapped within the wound bed, periphery, and other remote areas.

Conclusions: PRODIGI represents a technological advancement in wound sampling and treatment guidance for clinical wound care at the point-of-care.

Trial registration: ClinicalTrials.gov NCT01651845; ClinicalTrials.gov NCT01378728.

Conflict of interest statement

Competing Interests: RSD, BCW and their institution have a pending patent related to this work (“Device and method for fluorescence-based imaging and monitoring” USPTO WO 2009140757 A1), which is being commercialized. RSD an independent investigator with a Scientist appointment at the Princess Margaret Cancer Centre, University Health Network, where he has been the principal investigator on this project. He is also is founder and a shareholder of a spin-off company commercializing the technology. He has Board membership and receives separate compensation as Chief Scientific Officer of the company. LT is a part time employee of the University Health Network (clinical associate); she also receives separate compensation as the clinical trials manager for the company. RSD and LT’s roles in this project were not on behalf of the company but rather in the context of their academic appointments/affiliations. No other authors are associated with or receive compensation or own shares of the company. The authors confirm that the above stated competing interests do not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials. The authors confirm that there are no restrictions on sharing of data and/or materials presented in this work.

Figures

Fig 1. CONSORT Flow diagram for Part 1 of clinical study.

Flow diagram of the progress through the phases of enrollment, allocation, follow-up, and data analysis for Part 1 of the clinical study.

Fig 2. CONSORT Flow diagram for Part 2 of clinical study.

Flow diagram of the progress through the phases of enrollment, allocation, follow-up, and data analysis for Part 2 of the clinical study.

Fig 3. Photographs of the handheld prototype PRODIGI imaging device and its use for real-time autofluorescence imaging of bacterial load invisible by white light examination.

A. Front view of PRODIGI showing wound fluorescence image displayed in real-time on the LCD screen in high definition. B. Back view of PRODIGI showing white light and 405 nm LED arrays providing illumination of the wound, while the fluorescence mission filter is placed in front of the CCD sensor. Inset shows side profile of the device. C-E. Photograph of PRODIGI device used to examine a diabetic foot ulcer with room lights on, in a hard shell carrying case in a typical wound clinic setting, and placed on typical wound care cart, respectively. Room lights are turned off for fluorescence imaging. F. PRODIGI white light image of type II diabetic foot ulcer in a 52 y old male patient. G. Corresponding AF image taken in < 1 sec showing bright red fluorescence of pathogenic bacteria in the wound periphery (yellow arrow) and in ‘off site’ areas (white arrow) away from the primary wound (confirmed by swab microbiology as mainly heavy growth S. aureus). Bacterial fluorescence appears red against a background of green fluorescence from connective tissues of the healthy skin, which provides anatomical context for localizing the bacteria within and around the wound. The bacterial regions were not seen under white-light visualization. H. A magnified view of G. showing S. aureus growing within the fissures of the wound periphery. Bright fluorescent ‘hot spots’ (yellow arrow) illustrate heterogeneity in the distribution of bioburden in the wound periphery. Fluorescence imaging allowed targeted swabbing of bacterial areas not possible by white light visualization. The heavy growth S. aureus growing in the off-site area was invisible by traditional clinical examination. Scale bars: A. 2 cm, B. 2 cm, C. 1 cm.

Fig 4. Autofluorescence detection of clinically-significant bacterial load in wound periphery and off-site areas.

A. White light image of a type II diabetic foot ulcer in a 78 y old female. B. Corresponding AF image showing heavy growth of S. aureus in the wound periphery missed by white light imaging. C. White light shows unremarkable areas between toes, while in D. the corresponding AF imaging detected bacterial biofilm, confirmed by microbiology. E. Schematic illustrates different wound locations where fluorescence imaging detected clinically significant bacterial load. F. Comparison of accuracy for correctly detecting clinically-significant bioburden between standard WL and AF imaging in wound bed, wound periphery and off-site areas. Scale bars: A,B. 1 cm; C. 2 cm, D. 1 cm.

Fig 5. Wound swabs taken under autofluorescence imaging guidance, indicating detected polymicrobial species prevalence.

The percentage of bacteria for each species are plotted for A. all swabs, B. swabs obtained from the wound bed, C. from the wound periphery and D. ‘off-site’ areas away from the primary wound.

Fig 6. Quantitative longitudinal tracking of bacterial load in chronic wounds.

A. Sequential white light (top row) and AF images (middle row) of a non-healing diabetic foot ulcer in a 67 y old female performed over 5.5 months. AF images revealed bluish-green fluorescence within the wound bed and bright red bacteria around the wound periphery. A fluorescence intensity-based segmentation algorithm was used to quantify bacterial load changes over time (bottom row), with the bacteria false-colored and overlaid on original AF images of the middle row. B. Quantitative changes in bacterial load over time measured by relative bacterial fluorescence amount (total red AF area in cm2) indicate clinically-significant microbial load in the wound periphery, both of which are missed during conventional clinical examination. Scale bar: A. ~1 cm.

Fig 7. Fluorescence image-guided treatment accelerates wound closure over time compared with non-guided conventional treatment.

A. Plot showing wound area measurements for twelve individual patients as a function of time from the onset of the study over the first control period (blue circles, the fluorescence image-guided period (red solid circles) and the second control period (green triangles). During the control periods, treatment was administered without real-time fluorescence image guidance. B. Plot showing the rate of change in the average wound area over the course of the study estimated from the regression model. The slopes and their corresponding p-values are shown for each study period. The data indicate that fluorescence image-guided treatment increased the rate of wound closure in a statistically significant manner compared with the control (non-guided) periods indicating that fluorescence image guided wound treatment could be beneficial addition to conventional wound therapy protocols. * p-value tests for change in the growth rate of average wound area in the guided period from the previous period. ** p-value tests for change in the growth rate of average wound area in the 2nd control period from the guided period.