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Original Article
The International Journal of Lower
Extremity Wounds
A Clinical Study to Evaluate 1–8
© The Author(s) 2021
Autofluorescence Imaging of Diabetic Foot Article reuse guidelines:
sagepub.com/journals-permissions
Ulcers Using a Novel Artificial Intelligence DOI: 10.1177/15347346211047098

journals.sagepub.com/home/ijl
Enabled Noninvasive Device
Vijay Viswanathan, MD, PhD, FICP, FRCP(London),

FRCP(Glasgow)1 , Senthil Govindan, MBBS,
MS(General Surgery), MCH(Plastic Surgery)1,
Bamila Selvaraj, MPT(Neurology), MIAP, Dip. in Podiatry1,
Secunda Rupert, MD2, and Raghul Kumar, M Sc3
Abstract
Diabetic foot ulcers, with worldwide prevalence ranging from 12%-25%, are an important cause of nontraumatic lower limb
amputation. Evidence-based assessment of early infection can help the clinician provide the right first line treatment thus
helping improve the wound closure rate. Illuminate®, a novel point of care device working on multispectral autofluores-
cence imaging, helps in the rapid identification and classification of bacteria. This study was aimed to evaluate the diagnostic
accuracy of the device in detecting bacterial gram type against standard culture methods. A total of 178 patients from a
tertiary care center for diabetes was recruited and 203 tissue samples were obtained from the wound base by the plastic
surgeon. The device was handled by the trained investigator to take wound images. The tissue samples were taken from the
color-coded infected region as indicated by the device’s Artificial Intelligence algorithm and sent for microbial assessment.
The results were compared against the Gram type inferred by the device and the device was found to have an accuracy of
89.54%, a positive predictive value of 86.27% for detecting Gram-positive bacteria, 80.77% for Gram-negative bacteria, and
91.67% for no infection. The negative predictive value corresponded to 87.25% for Gram-positive, 92% for Gram-negative,
and 96.12% for no infection. The Results exhibited the accuracy of this novel autofluorescence device in identifying and
classifying the gram type of bacteria and its potential in significantly aiding clinicians towards early infection assessment
and treatment.
Keywords
diabetic foot ulcers, illuminate®, gram-positive bacteria, gram-negative bacteria, multispectral imaging, autofluorescence,
artificial intelligence
Introduction
The International Diabetes Federation estimates that the
expected prevalence of diabetes mellitus will rise to more 1
MV Hospital for Diabetes and Prof M Viswanathan Diabetes Research
than 570 million in 2030.1 Diabetic foot ulcers (DFU), Centre, Chennai, Tamil Nadu
2
and diabetic foot infections are major contributors of world- Stem Cell Research Centre, Government Stanley Medical College &
wide morbidity and mortality as they lead to limb amputa- Hospital, Chennai, Tamil Nadu
3
Adiuvo Diagnostics Private Limited, Chennai, Tamil Nadu
tion in 12%-25% of individuals with diabetes.2 DFU
initially begins with peripheral neuropathy and finally lead Corresponding Author:
to ulcerations. Secondary infections leads to chronically Vijay Viswanathan, M.V. Hospital for Diabetes and Prof. M. Viswanathan
Diabetes Research Centre (WHO Collaborating Centre for Research,
infected wounds over a period of time.3 Prompt diagnosis
Education and Training in Diabetes) (IDF centre for Excellence in Diabetes
of bacteria in these wounds and timely treatment help care), No. 4, West Madha Church Street, Royapuram, Chennai, Tamil
speed up the wound healing process. However, it is clini- Nadu, India.
cally difficult to diagnose the causative pathogens causing Email: [email protected]
2 The International Journal of Lower Extremity Wounds
secondary infections. Routine microbial-based culture T2DM with acute and/or chronic DFU were recruited into
methods, the current standard for identifying and classifying the study. Patients’ peri lesion noninfected site served as
bacteria infecting the wounds, requires taking a swab or their own control.
biopsy from the infected region and take 2 to 3 days for
diagnosis, causing a delay in providing first-line treatment
Illuminate device and Imaging Procedure with
and/or inappropriate usage of antibiotics leading to the
development of bacterial resistance.4 Illuminate®
Autofluorescence-based multispectral imaging has Intrinsic fluorescence exhibited by bacteria and fungi are
recently emerged as a useful technique for the identification attractive methods for the rapid identification of infection
of bacteria and involves shining multiple wavelengths of on wounds without any additional steps. Previous studies
light on wounds and collecting their emission responses have reported that bacteria have characteristic emission fluo-
thus helping understand the pathophysiology of the wounds. rescence when excited in the UV and blue regions of light
Fluorescence methods have been found to be one of the contributed mainly by metabolic and infectious markers
most straightforward, noncontact, and sensitive methods for such as NADH, Flavin and Porphyrin.8 Shelly et al,9 reported
the detection of biomolecules (intrinsic fluorophores) such fluorescence fingerprinting by characterizing each bacteria’s
as tryptophan, nicotinamide adenine dinucleotide hydride excitation and characteristic emission wavelengths for rapid
(NADH), flavins, etc.5 Different bacteria contain varying and accurate identification of infection especially
amounts of these fluorophores in differing microenviron- Pseudomonas species and Staphylococcus aureus. On the
ments which help in distinguishing different types of bacte- other hand, Ammor et al5 developed a method towards
ria.6 This study was carried out to evaluate the accuracy of species level bacterial detection leveraging the intrinsic fluo-
Illuminate®, one such autofluorescence-based point of a rescence of aromatic amino acids and nucleic acids, suggest-
care imaging device that uses a multispectral imaging techni- ing that autofluorescence spectral analysis can be a rapid and
que to shine multiple excitation sources on the region of the inexpensive technique. Based on the above principles of
wound and collects spectral images. An Artificial using autofluorescence imaging of bacteria, a novel
Intelligence (AI) algorithm then runs on the collected spectral imaging device named Illuminate® (Adiuvo Diagnostics
images to compare the biomolecule fluorophore intensity Private Limited, Chennai, India) (Figure 1) was developed,
toward Gram-type bacterial classification. The results combining multiple light sources (370, 395, and 415 nm)
obtained from this device were compared against the current to shine on the wound region and collects multispectral
gold standard method of bacteriological culture. images in approximately 30 sec to detect infection on
wounds. An image processing algorithm then analyses the
Methods
Study Design
An interventional single arm comparative study was con-
ducted at a tertiary care center for diabetes after obtaining
institutional ethics committee approval (IEC/N-009/10/
2017) registered with the Clinical Trial Registry of India
(Reg. No. CTRI/2018/10/016147). Both acute and chronic
wounds were considered for imaging and the total sample
size required for detection of infection using the device
was estimated as 140 (for 99% confidence level, assumed
prevalence of infection as 70% and a precision of 10%.7
Consecutive patients attending the outpatient department
were recruited after screening for inclusion criteria and
obtaining both informed and written consent forms. The
persons with type 2 diabetes (T2DM) and with new and/or
chronic DFU with nonintact skin (stasis ulcers both arterial
and venous) were included in the study. The persons with an
existing autoimmune disease and skin diseases such as
eczema, psoriasis in the area close to the wounds were
excluded. We excluded patients with osteomyelitis, as the
bone region is high autofluorescent in nature and needs
additional algorithm training. A total of 178 persons with Figure 1. Illuminate® imaging device.
Viswanathan et al 3
spectral images, and a pretrained machine learning algorithm Laboratory Standards Institute (CLSI) guidelines.13 The
compares the relative intensity between different autofluores- results of gram-staining, bacterial identification, and its drug
cence biomarkers10 thus enabling classification of bacteria susceptibility profile were obtained from the microbiology
into Gram-positive and Gram-negative in 2 min.10,11 The lab in a report format for each patient sample.
device is noncontact, does not require the addition of any
reagents and removes the burden of interpreting spectral
images. The AI Engine runs on the device and can even be Statistical Analysis
used in remote places with no internet access. The device pro-
The estimates of diagnostic tests such as true positives, true
vides a comprehensive report along with automated wound
negatives, false positives, false negatives were first evalu-
measurement options including a progressive report, which
ated and used for the calculation of sensitivity, specificity,
can be synced with the hospital’s central database. The
accuracy, positive predictive value, and negative predictive
device is easy to use and requires training to image the
value. The results obtained from the imaging device were
wound regions consistently without shaking.
compared with the results of the tissue culture to calculate
Illuminate device was used to collect Multispectral
accuracy, sensitivity, specificity, positive predictive value,
wound images either in a dark room or using a black
and negative predictive value. The statistical analysis was
hood covering the wound region. The wound imaging was
performed using Python software.
done by the trained investigator. Before imaging, wounds
were first cleaned with normal saline solution. The study
participants’ details were entered in the Illuminate
Results
Imaging Application of the device. The user was guided
to image the wound at a distance of 10 to 12 cm from the Clinical Characteristics of the Study Participants
area of interest. A series of 16 multispectral images were
automatically captured by the device in ∼20 sec. The user The imaging of the wound was done for a total of 178
was then prompted to trace the region of the wound post persons with DFU. The tissue sample was taken from 203
image capture. In under 2 min, an algorithm was processed sites and analyzed. Out of 178 study participants, 144 were
in the background and displayed a color-coded wound male and 34 were female with a median age of 59 years.
image along with a Gram type of bacteria (if infected) 13 Patients were on prior antibiotics usage and were included
along with the length and breadth of the wound. in the study (Table 1). About 60% of the participants were
found to have third-grade ulcers based on the Wagner’s
wound classification score, while 34.3% had grade 2 and
Specimen Collection
Tissue samples were taken with Alley’s tissue holding Table 1. Basic and Clinical Profile of the Study Participants.
forceps and scissors by the attending plastic surgeon.
These tissues were collected before and after debridement No. of participants (%)
Variable (n = 178)
from the color-coded region as indicated by the device.
Specimens were collected from all the study participants Age (years)a 59 (12.5)
and placed in a sterile transport container and sent to the Gender (M: F) 144:34
microbiology laboratory in the hospital for routine standard Duration of diabetes
culture and biochemical methods for identifying and classi- <5 years 39 (22.0)
fication of bacteria. 5-10 years 51 (28.6)
11-15 57 (32.0)
>15 31 (17.4)
Culture Wound classification
All tissue samples were cultured in the microbiology lab. All Grade 1 11 (6.2)
tissue samples were analyzed initially by Gram-staining and Grade 2 61 (34.3)
bacterial identification using conventional aerobic phenotypic Grade 3 106 (59.5)
methods by plating into various enriched and differential HbA1c (%)
growth mediums and biochemical assays.12 Bacterial load <7 23 (13.0)
7-8 43 (24.1)
was estimated by plate count semiquantitative analysis
>8 112 (62.9)
method and then graded as scanty, light, moderate, or heavy
Use of antibiotics prior to imaging
(1+, 2+, 3+ or 4+) of which moderate and heavy growth indi-
Yes 13 (7.3)
cated a significant bacterial load (ie, greater than 100 000 per No 165 (92.7)
gram of tissue). The antibiotics susceptibility profile was then
a
obtained using the disk diffusion method as per Clinical Median (IQR).
Figure 2. Percentage of treatment procedures.
6.2% had grade 1 ulcers respectively. A total of 131 patients Organisms Isolated in Culture
underwent debridement, while 20 participants were directed
towards cleaning and dressing, 17 towards sequestrectomy, From the 203 tissue samples collected, 14 clinically
3 underwent exostectomy, 1 escharotomy, 1 bursa excision, relevant pathogens were isolated (Table 2) while 28 cases
and 16 patients had to undergo an amputation. 39 wounds showed no growth which included reference sites as well.
had exudates. Around 63% of the study participants were 63 samples revealed one Gram-negative bacteria, 53 had
found to have an HbA1c level >8%. one Gram-positive bacteria, while 6 tissues had 2-gram-
The percentage of treatment procedures is shown in negatives, 4 samples had 2-gram-positives and 49 patients
Figure 2. had both gram-positive and gram-negative bacteria
(Table 3).
Illuminate Inference
Table 2. Details of Pathogens Isolated From Wound Samples by
Culture Methods. The device color codes Gram-positive bacteria with red
(Figure 3b) and gram-negative with green (Figure 3a, c).
S. No Bacterial Isolates Count
Tissue culture gram type results were compared against
1 Acinetobacter SPP 4
2 Citrobacter Feundii 2
Table 3. Identification of Gram-Positive and Gram-Negative
3 E coli 19
Organisms in Wound Samples by Culture Methods vs Illuminate
4 E coli ESBL 12 Device.
5 Enterococcus SPP 48
6 Enterococcus faecalis 2 Results by illuminate
7 Klebsiella SPP 35 method (Index)
Results by culture
8 No Growth 28
Type of method Correct
9 Proteus mirabilis 25
organism (CLSI, Gram’s stain) diagnosis Misdiagnosis Total
10 Pseudomonas aeruginosa 1
11 Pseudomonas SPP 26 GN 69 63 6 69
12 Staphylococcus aureus 33 GP 57 44 13 57
13 MRSA 33 NG 28 22 6 28
14 Staphylococcus SPP 1 GPGN 49 39 10 49
15 Enterobacter SPP 1 Total 203 168 35 203
MRSA, methicillin-resistant Staphylococcus aureus; ESBL, Extended Spectrum GN, gram-negative; GP, gram-positive; NG, no growth; GPGN,
beta-lactamase. gram-positive and gram-negative.
Viswanathan et al 5
Figure 3. (a) Gram-negative bacteria-infected wound. (b) Gram-positive bacteria-infected wound. (c) Gram-negative bacteria-infected
wound. (d) Gram-positive and gram-negative bacteria-infected wound. Figures 3a-3d are examples of clinical image and Illuminate®
predicted image of Patient’s wound region. Figure 3a and 3c show a Green overlay indicating Gram-Negative infection and the
corresponding culture reports indicated Klebsiella Spp. and Pseudomonas spp. Figure 3b indicated red over overlay predicting
Gram-positive bacteria and the corresponding culture report was Staphylococcus aureus, 3d is an example of a wound infected by both
Gram-positive and negative bacteria.
the device gram type results. Illuminate device displayed The results of the machine learning algorithm are sum-
63-gram-negative bacteria, 44 gram-positive, 22 No marized in the confusion matrix provided in Table 4. The
growths. 49 wounds were found to have a polymicrobial accuracy of the device was found to be 89.54% with a pos-
infection containing both gram-negative and gram-positive itive predictive value of 80.77% for detecting gram-negative
bacteria (Figureure 3d) and the device identified the same bacteria, 86.3% for gram-positive bacteria, and 91.67%
in 39 patients. 6 ulcers were found to have only gram- for no growth. The negative predictive value corresponded
negative colonization and 4, gram-positive colonization. to 92.0% for gram-negative bacteria, 87.2% for gram-
Further statistical inference was made by comparing the positive bacteria, and 96.12% for no infection. The sensitiv-
culture results with Illuminate device results (Table 3). ity for gram-negative bacteria was 91.30%, 77.2% for
Table 4. Confusion Matrix and Results for the Gram Type The device has an accuracy of 89.54%, a positive predictive
Classification Using Illuminate® Device With the Standard value of 86.27% for detecting Gram-positive bacteria,
Culture-Based Test. 80.77% for Gram-negative bacteria, and 91.67% for no
89.54% infection. The negative predictive value corresponded to
Combined Accuracy GN GP NG 87.25% for Gram-positive, 92% for Gram-negative, and
96.12% for no infection.
PPV 80.77 86.27 91.67 The study conducted by DaCosta RS., et al showed that
NPV 92.00 87.25 96.12 only 52.5% of the bacterial load was accurately identified by
Accuracy 86.27 86.93 95.42
clinical assessment whereas 74.6% of the occurrences of
TPR (sensitivity) 91.30 77.19 81.48
wound infection was correctly identified by fluorescent
TNR (specificity) 82.14 92.71 98.41
imaging-based technique.17 Another study has re-confirmed
GN, gram-negative; GP, gram-positive; NG, no growth; GPGN, the high accuracy in the detection of the pathogenic infec-
gram-positive and gram-negative; PPV, positive predictive value; NPV, tion of the wound by an autofluorescence imaging device
negative predictive value; TPR, true positive rate; TNR, true negative rate. compared to the clinical evaluation of signs and symptoms
in detecting the wounds. It also demonstrated an improved
Gram-positive, and 81.5% for No growth. Specificity for sensitivity of the combined use of bacterial fluorescence
Gram-negative was 82.14%, 92.71% for gram-positive, imaging with clinical signs and symptoms (72%) compared
and 98.4% for No Growth. Multiclass area under the to the use of only clinical signs and symptoms (22%) in the
curve (AUC) score calculated using the One-versus-One identification of wounds with moderate to heavy bacterial
method was 0.86. loads (≥104CFU/g).18
Predominantly Gram-positive bacteria such as
methicillin-resistant Staphylococcus aureus19, methicillin
Discussion sensitive S. aureus, Streptococcus species are typically iden-
Understanding the pathophysiology of DFU is essential for tified in the acute stage of wounds while gram-negative bac-
effective treatment and prevention of its downstream com- teria such as Escherichia coli, Pseudomonas aeruginosa,
plications. Clinical Signs and Symptoms Score continue to Klebsiella spp., are detected in chronic wounds.20–22 Our
be the recommended way of detecting wound infection.14 study showed the presence of Enterococcus spp. (17.8%)
However, this assessment can be very subjective as demon- majorly followed with the presence of Klebsiella spp.
strated by various studies. An accurate, objective method (13%) and Staphylococcus aureus (12.2%). A bacterial con-
would be a boon to the doctors to better understand the centration >104 CFU/g of tissue is necessary to cause infec-
infection levels and to provide tailored antibiotic treatments. tion in complex extremity wounds.23,24 Infections may then
Our study findings showed that the sensitivity for gram- lead to a delay in wound healing, thus early detection of bac-
negative and gram-positive bacteria was 91.3% and 77.2% teria above 104 CFU/g of tissue becomes imperative to
respectively. A sensitivity of 81.5% was found for no prevent complications.
growth of any bacteria. The specificity of the test of the The high sensitivity and specificity observed in the
device was 82.1% for gram-negative organisms, 92.7% for present study, along with an exploratory study done
gram-positive, and 98.4% for no growth of the organisms. earlier10 reiterates that this novel device can be used as a
AUC score of 0.86 also indicates a significant discriminating screening device for early infection detection and under-
ability of the device to the presence of pathogens. standing the gram type of bacteria colonizing the wound.
A pilot study conducted in a wound care clinic of Ireland Definitive tests such as culture, polymerase chain reaction
on the efficacy of a bacterial fluorescence imaging device take time are reagent intensive, and are error prone due to
(Moleculight) showed a sensitivity of 100% and specificity reasons such as error during sample transport, proximity
of 78%.15 The same study demonstrated that the device to a diagnostic lab, etc. This device can thus be used as a
has sensitivity and specificity of 100% in identifying the screening tool to overcome these shortcomings. The
Pseudomonas spp. by visually interpreting the cyan fluores- device can also help monitor the treatment efficacy over a
cence from the autofluorescence images. This study also period of wound treatment and identify regions to take a
showed the effect of appropriate selection of antibiotics tissue sample. From our results, we could see a marked
and further recovery of the wound within a 2-week decrease in bacterial fluorescence spatially, before and
period. It is to be noted that these images need to be manu- post debridement and there was a 93% organism correlation
ally interpreted.16 and cannot automatically distinguish from the culture report obtained. Hence it can be used to
Gram-positive and Gram-Negative bacteria. assess wounds qualitatively post debridement.11
Our study of 178 patients using the Illuminate® imaging Currently, wounds are assessed by visual inspection, and it
device can detect infections and differentiate the gram type is difficult to track wound infection and healing post debride-
of bacteria using multispectral image information and AI. ment. More importantly, an instant decision on the gram type
Viswanathan et al 7
of bacteria infecting the wound can aid the doctors towards the Acknowledgments
first line of antibiotic prescription. This device will reduce the We thank the support from Ms Malathi, for her patient assistance,
time and aid the clinicians ineffective treatment by understand- the microbiology team for providing insights from culturing per-
ing the microbial burden on the wound region in real time. spective, Mr Anand Kumar, Dr Satyavani, and Ms Suganya for
Manual interpretation of images is very tough and requires their support during the trial.
trained personnel and possesses some challenges in ensuring
the accuracy of decisions. Incorporation of machine learning Declaration of Conflicting Interests
algorithm aids in easier understanding of infection regions.
The authors declared no potential conflicts of interest with respect
In future, with more images, genus and species level
to the research, authorship, and/or publication of this article.
machine learning training can also be performed which can
guide towards effective antibiotic treatment.
Funding
The authors received no financial support for the research, author-
ship, and/or publication of this article.
Limitations
Bone, tendon, and fat along with betadine and other cleaning Ethical Approval
solutions are autofluorescent in nature. Hence wounds are to Institutional Ethics Committee approval (IEC/N-009/10/2017) was
be cleaned with normal saline before imaging. Also, the obtained before the initiation of the study.
device cannot be used to interpret infections in the bone/fat
region. The light sources used here have a penetration depth ORCID iD
from 0.5 mm to ∼1 mm and hence the device cannot under-
Vijay Viswanathan https://orcid.org/0000-0001-9116-3937
stand the status of infection in closed wounds. In the case of
polymicrobial infection, the device displays the gram type
that is more in number in that particular spatial region. References
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ijdvl.com Case Report
Utility of a real‑time fluorescence imaging Corresponding author:

Dr. Aayush Gupta,
Department of Dermatology,
device in guiding antibiotic treatment in Dr. D. Y. Patil Medical College,
Hospital and Research Centre,
superficial skin infections Pimpri, Pune, Maharashtra, India.
[email protected]
Received: October, 2019

Bhavika M. Shah, Devina Ganvir, Yugal K. Sharma, Shahzad Beg Mirza1, Accepted: February, 2020
Published:
R. N. Misra1, Preeti Kothari, Sweety Darall, Jitendra S. Bhawalkar2,
Aayush Gupta
DOI:
Departments of Dermatology, 1Microbiology and 2Community Medicine, Dr. D. Y. Patil Medical College, Hospital 10.25259/IJDVL_856_19
and Research Centre, Pune, Maharashtra, India
PMID:
***
Abstract
The prescription of antibiotics empirically without confirmation of an infective etiology is on the rise.
Administration of appropriate antibiotics can be guided by real‑time fluorescence imaging using a
point‑of‑care device. These composite images show the presence, type and the burden of infection.
The time saved by this method over microbiological testing, especially in resource‑poor settings, can
lead to a paradigm shift in treatment by facilitating prompt and adequate antimicrobial therapy, surgical
debridement as well as follow‑up. Thumbnail sketches of a series of four cases highlighting different
scenarios in which a fluorescent imaging device utilizing artificial intelligence and machine learning
was found useful is presented in this report.
Key words: Antimicrobial stewardship, fluorescence imaging, Illuminate, infections, wound care
Introduction techniques, all organisms may not be isolated especially in

Most of the skin and soft tissue infections resolve with comparison with that of deep tissue biopsies (current gold
treatment, however, some worsen forming chronic ulcers that standard).4,5
impair not only the patients’ quality of life but strain an already
overburdened healthcare system. An escalating incidence We used a novel handheld imaging device, “Illuminate,”
of secondarily infected chronic wounds has led to increased developed by Adiuvo Diagnostics (Chennai, India), which
hospitalization of patients as well as antibiotic usage.1 aims to accelerate the treatment of skin infections by
rectifying some of the above‑mentioned lacunae. This
The incidence of acute and chronic wounds in India is 10.5 device leverages autofluorescence, artificial intelligence
and 4.5 per 1000 population respectively.2 Secondary bacterial and machine learning to accurately classify/locate the
infections commonly complicate the healing of diabetic microorganisms and provide semiquantitative data regarding
ulcers leading to the loss of a lower limb every 30 seconds.3 the infective burden of the wound. Pointed to the region of
Initial clinical evaluation and microbiological correlation of interest, the device captures 16 serial images at different
an infected wound typically takes up to 10 days. An already excitation emissions and produces a clinical image overlaid
tedious recovery may get prolonged by incorrect therapy with the superimposed infection, if any, marking the
consequent to a cursory examination, pending culture and presence of gram‑positive bacteria in red, gram‑negative in
sensitivity results, or inaccurate reports following improper green, and fungus in blue. Four cases where the device was
swabbing. Moreover, despite proper swabbing/aseptic found invaluable are summarized below.
How to cite this article: Shah BM, Ganvir D, Sharma YK, Mirza SB, Misra RN, Kothari P, et al. Utility of a real‑time fluorescence imaging device in
guiding antibiotic treatment in superficial skin infections. Indian J Dermatol Venereol Leprol 2020;1-6.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, tweak,
and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.
© 2020 Indian Journal of Dermatology, Venereology and Leprology - Published by Scientific Scholar 1
Shah, et al. Fluorescence imaging in antimicrobial stewardship
Case Reports the ulcer did not heal. Cyan and green fluorescence in
Case 1 two different areas of the ulcer examined by Illuminate
A 73‑year‑old female had multiple and painful non‑healing indicated the co‑colonization of Pseudomonas species and
recalcitrant ulcers on both thighs [Figure 1a] for 9 months, Escherichia coli and the same was subsequently confirmed by
despite multiple courses of tablet amoxicillin/clavulanic image‑guided swabbing [Figures 2b and c]. Prompt clinical
acid, tablet azithromycin, and injectable amikacin as per response followed with the use of ciprofloxacin.
sensitivity reports of the culture isolates of Staphylococcus
epidermidis and Klebsiella. The ulcers were well defined, Case 3
punched out, 0.5 to 3 cm in diameter with erythematous A 45‑year‑old HIV‑positive patient presented with a crusted
margins and seropurulent discharge. A punch biopsy revealing ulcer on left shin for 15 days. [Figure 3a] Initial assessment
neutrophilic infiltrate was described as ‘non‑specific’. with Illuminate suggested a high gram‑positive bacterial
Repeated use of Illuminate for many days consistently failed load (subsequently confirmed to be methicillin‑sensitive
to capture the fluorescence indicative of any pathogenic Staphylococcus aureus) and hence cotrimoxazole was given
organisms, although repeated pus swabs showed growth empirically. Seven days later, the sensitivity report confirmed
of coagulase‑negative Staphylococcus [Figure 1b]. Deep it to be the appropriate choice, during which serial assessment
excisional biopsy thence demonstrated a dense band demonstrated reduced fluorescence at every visit indicating
of neutrophilic infiltrate and perivascular lymphocytes a satisfactory response at the end of 10 days of treatment,
suggestive of pyoderma gangrenosum. The patient ultimately obviating the need for repeated wound swabs [Figures 3b‑d].
responded well to corticosteroids (systemic and intralesional)
without any antibiotic coverage. Case 4
A 48‑year‑old male, known diabetic, presented with a
Case 2 malodorous, discharging ulcer over the right toe since 1
A 58‑year‑old female, known diabetic for 10 years developed month. [Figure 4a] Earlier culture of pus swab taken from
an ulcer over her left great toe [Figure 2a] along with difficulty the center of the lesion yielded no growth. Clusters of
in walking for 4 months. Escherichia coli were isolated but methicillin‑resistant Staphylococcus aureus were isolated
despite the complete course of cefoxitin as per sensitivity, from an image‑guided swab taken from the periphery of the
ulcer [Figure 4b] The wound was surgically debrided with
repeated imaging till the device failed to show any fluorescence,
signifying appropriate debridement depth [Figure 4c].
Discussion
Studies have shown that significant bacterial load in wounds is
correctly identified only in 52.5% instances by clinical assessment
as against 74.6% with fluorescent imaging‑based techniques.6
Antibiotic resistance, a growing global concern can be prevented,
at least in part, by antimicrobial stewardship practices.7,8 All of
the key factors listed by the European Wound Management
Association contributing to antimicrobial misuse in wound care,
Figure 1a: Clinical image of recalcitrant thigh ulcers Figure 1b: Lack of fluorescence on imaging with “Illuminate”
2 Indian Journal of Dermatology, Venereology and Leprology | Volume XX | Issue XX | Month 2020
namely— diagnostic uncertainty regarding presence of bacterial Swabbing from inappropriate sites demonstrated false‑negative
infection, clinician ignorance regarding class and duration of culture results in cases 2 and 4. Sapico et al. reported only a
antibiotic to be used, and patient demands for prescription of 63% concordance of isolates between skin biopsy samples
inappropriate antibiotics can be overcome by a point‑of‑care taken from the periphery and in the center of 25 pressure
device providing immediate guidance.9 sores.10
Our case series underscores that real‑time fluorescence
The resistant‑to‑local‑cleaning periphery of wounds was
imaging of microorganisms can greatly facilitate the
appropriate use of antibiotics. The patient with pyoderma suggested to be a better swabbing site for accurate detection
gangrenosum, having received unnecessary and prolonged of isolates in some cases. Also, a mixed infection may go
antibiotic therapy due to a misleading clinical appearance, undetected by a single swab taken even from the area
responded promptly to systemic steroids, due to the instant of maximal clinical involvement. Image‑guided sample
result provided by this device. collection overcomes this problem.
Figure 2a: Ulcer on the great toe with slough Figure 2b: Composite algorithmic image after processing demonstrating
co‑colonization
Figure 2c: Fluorescence highlighting areas of bacterial colonization with Figure 3a: Encrusted ulcer on the left shin
gram‑positive species (red) and Pseudomonas
Indian Journal of Dermatology, Venereology and Leprology | Volume XX | Issue XX | Month 2020 3
Figure 3b: Fluorescence at initial assessment Figure 3c: Day 5 of follow-up
The reduced bioburden following appropriate antibiotics/ The skin itself has a natural fluorescence contributed by
wound debridement seen on serial imaging in cases 3 and 4 collagen/elastin; bacteria interact with the tissue via heme
not only led to rapid healing but also supplanted the subjective pathway and produce porphyrin displayed as red fluorescence
clinical and/or time‑consuming microbiological assessment. while fungi have a characteristic blue fluorescence due to
chitin and dityrosine linkages. Pseudomonas aeruginosa
Illuminate leverages the autofluorescence property produces a greenish‑cyan fluorescence signal due to the
exhibited by pathogens [such as nicotinamide adenine presence of pyoverdine. There are spectral and textural
dinucleotide hydrogen (NADH), flavins] and infectious changes between the green illumination of specific
markers (pyoverdine, porphyrin) present in bacteria and organisms (especially Pseudomonas giving a cyan color) and
fungus. NADH has a strong emission peak at 470 nm surrounding dermis, which enable their identification. The
producing blue fluorescence, while flavin and porphyrin peak device overlays this composite autofluorescence image on
at around 525 nm and 620 nm corresponding to green and red regions of infections. Gram‑positive bacteria are displayed in
fluorescence, respectively. Gram‑negative organisms have red and gram‑negative in green by comparing the difference
a higher intensity of NADH and flavin than gram‑positive in autofluorescence intensity.
organisms thus making it possible to differentiate between
gram types. Pseudomonas possesses pyoverdine, a unique However, limitations of the technology exist in detecting
marker having blue‑green color enabling its detection. deep‑seated bacterial infections and differentiating pathogenic
from commensal bacteria. While an approximation of
An image processing and machine learning algorithm significant bacterial load is possible; the determination of
processes the spectral images to detect and classify pathogens accurate levels thereof still requires tissue culture. The device
based on the intensity of autofluorescence variation amongst also cannot act as a surrogate for culture and sensitivity,
these biomarkers. It can detect bacterial loads as less as which is often required to choose appropriate antibiotics in
103 CFU/gm (colony‑forming unit per gram) allowing the era of drug resistance.
for early intervention (Indian patent No. 323440, 2019).
Contaminants, if present, are usually at a lower density. Point‑of‑care devices may substantially aid the evaluation of
Contamination may also occur at later stages of sample wounds and help institute appropriate treatment promptly by
collection, processing, or in the laboratory.11 instant confirmation of infective etiology thereby reducing
Figure 4a: Ulcer over the right great toe with serous discharge
Figure 3d: Day 10 of follow-up
Figure 4c: Reduction in fluorescence evident after the initial cleaning
Declaration of patient consent

The authors certify that they have obtained all appropriate
patient consent.
Financial support and sponsorship

Nil.
Figure 4b: Highlighted areas showing clusters of bacterial colonization Conflicts of interest
primarily over the periphery of the wound
There are no conflicts of interest.
the investigative/financial burden, especially in resource‑poor References

settings where facilities for microbiological evaluation may 1. Singh B, Singh S, Khichy S, Ghatge A. Clinical presentation of
not be available. soft‑tissue infections and its management: A study of 100 cases. Niger
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J Surg 2017;23:86‑91. First‑in‑human results. PLoS One 2015;10:e0116623.

2. Gupta N, Gupta SK, Shukla VK, Singh SP. An Indian community‑based 7. Shallcross LJ, Howard SJ, Fowler T, Davies SC. Tackling the threat
epidemiological study of wounds. J Wound Care 2004;13:323‑5. of antimicrobial resistance: From policy to sustainable action. Philos
3. Cheng CF, Sahu D, Tsen F, Zhao Z, Fan J, Kim R, et al. A fragment Trans R Soc Lond B Biol Sci 2015;370 (1670):20140082.
of secreted Hsp90α carries properties that enable it to accelerate 8. Bell BG, Schellevis F, Stobberingh E, Goossens H, Pringle M.
effectively both acute and diabetic wound healing in mice. J Clin Invest A systematic review and meta‑analysis of the effects of antibiotic
2011;121:4348‑61. consumption on antibiotic resistance. BMC Infect Dis 2014;14:13.
4. Bowler PG, Duerden BI, Armstrong DG. Wound microbiology and 9. Lipsky BA, Dryden M, Gottrup F, Nathwani D, Seaton RA, Stryja J.
associated approaches to wound management. Clin Microbiol Rev Antimicrobial stewardship in wound care: A Position Paper from the
2001;14:244‑69. British Society for Antimicrobial Chemotherapy and European Wound
5. Spear M. When and how to culture a chronic wound: A culture is a Management Association. J Antimicrob Chemother 2016;71:3026‑35.
valuable tool in wound care if used correctly. Wound Care Advisor 10. Sapico FL, Ginunas VJ, Thornhill‑Joynes M, Canawati HN, Capen DA,
2014;3:23‑5. Klein NE, et al. Quantitative microbiology of pressure sores in different
6. DaCosta RS, Kulbatski I, Lindvere‑Teene L, Starr D, Blackmore K, stages of healing. Diagn Microbiol Infect Dis 1986;5:31‑8.
Silver JI, et al. Point‑of‑care autofluorescence imaging for real‑time 11. Radhakrishnan G, King J, Meenatchi U, Gupta A. Indian Patent No.
sampling and treatment guidance of bioburden in chronic wounds: 323440. IP India; 2019.
Indian Journal of Surgery
https://doi.org/10.1007/s12262-021-03190-6
ORIGINAL ARTICLE
A New Device and Technology for Detecting Bacterial Infection and its

Gram Type in Diabetic Foot Ulcer
Janakrai N. Parekh1 · Payal Soni2 · Mahendra Kumar Meena3 · Chetan Kumar Tandel3 · Geethanjali Radhakrishanan4
Received: 6 January 2021 / Accepted: 24 November 2021

© Association of Surgeons of India 2021
Abstract
The correct diagnosis and classification of bacterial infection are important for initiating appropriate antibiotic therapy
thus leading to a reduction in the incidence of antibiotic-resistant microbial infection. There are many methods to detect
wound bacterial infections. The gold standard method, at present, remains the tissue/swab culture along with a sensitivity
test. However, new fluorescence-based methods of detecting bacterial infection in a POC (point of care) setting have proved
themselves to be simple, prompt, accurate, and affordable. Many commonly infecting bacteria have a characteristic emission
auto fluorescence when excited with ultraviolet and blue light. A newly developed hand-held device captures the emitted
fluorescence and records the spectral signature of different bacteria. The fluorescence is exhibited because of the metabolic
and infectious biomarkers present in a bacterium, e.g., pseudomonas produces pyocyanin, and pyoverdine. Finally, an in-
built image processing and machine learning algorithm detects this native fluorescence produced by the bacteria and helps
classify them according to their Gram type. We studied 100 patients with diabetic foot ulcers, comparing the results of the
routine tissue culture with the Gram type report generated at POC by this novel device and found that this device was able
to detect infection with 99.24% sensitivity and 82.35% specificity with that of the gold standard culture test.
Keywords Auto fluorescence · Non-invasive infection screening · Wound infection · Gram type bacteria classification
Introduction administer the right antibiotics at the right time; in other

words, to develop a POC (point of care) diagnostic test that
The prevalence of antimicrobial resistance remains a major helps identify the presence of bacterial infection [2]. A rapid
problem all over the world. Its potential impact on health identification of bacteria in wounds, blood, or body excre-
care is as significant as that of global warming [1]. Many tions will lead to treatment directed towards the specific
challenges exist in the way of overcoming antimicrobial pathogen.
resistance, with the most important being the ‘creation of At present, the most common method to detect wound
a cost effective, accurate, rapid, and easy to use method for infection is to take a deep tissue biopsy/swab from the
detection of bacterial infections that allows clinicians to ulcer and perform a culture and sensitivity test. For this, a
BSL-2 facility is required in the microbiology laboratory
and it takes about 24 to 48 h to know the status of the infec-
* Chetan Kumar Tandel tion and the type of bacteria. Though many phenotypic and
[email protected] molecular techniques for microbial detection are available,
Janakrai N. Parekh they are time-consuming, reagent intensive, and require
[email protected] trained personnel, not making it suitable for rapid screen-
1 ing. Recent advancements leveraging native fluorescence of
Department of Surgery, GMERS Medical College, Valsad,
Gujarat 396001, India bacterial cellular molecules have been found to be sensitive
2 and effective for the rapid screening of bacterial infection
Department of Microbiology, GMERS Medical College,
Valsad, Gujarat 396001, India and classification [3].
3 A startup based in India has developed and patented
Department of Surgery, GMERS Medical College, Valsad,
Gujarat 396001, India (IN Patent–323,440) a novel handheld POC device, (Illu-
4 minate®), which utilizes auto fluorescence of bacteria for
Adiuvo Diagnostics, Chennai, India
13
Vol.:(0123456789)
Fig. 1 Illuminate: image of the

novel handheld device
its detection [4] (Fig. 1). Each bacterium has characteristic their Gram type and compare the results obtained with the
emission fluorescence when excited with different wave- results of standard tissue culture method.
length of light [5, 6]. The device uses multispectral imaging
combined with an advanced computational algorithm and
proprietary state of the art AI engine for spatial mapping and Patients and Methods
classification of pathogens. The device captures the spectral
signatures of metabolic growth markers along with markers This study was carried out at GMERS Medical College &
released when a micro-organism causes infection, to detect Hospital, Valsad (Gujarat) by the Department of Surgery
and assess the bacterial Gram type. The device leverages the and department of Microbiology, between April 2018 and
auto fluorescence property exhibited by pathogens [such as November 2019. Permission from the Institutional Human
nicotinamide adenine dinucleotide hydrogen (NADH), fla- Ethics Committee was obtained for conducting this study
vins] and infectious markers [Pyoverdine, Porphyrin] present (No. MCV/IHEC/10/18) and the study was registered
in bacteria and fungus [16] Use of such handheld device with CTRI (Clinical Trial Registry of India) (Reg. No
has been done successfully in diagnosing skin and soft tis- CTRI/2018/10/016147).
sue infection—bacterial and fungal infection—and has been Though the device can be used for imaging any wound,
reported in literature also [7, 8]. we decided to include only diabetic foot ulcer patients
This newly developed hand-held device takes non-contact for this study as they are most seen patients in General
images of the ulcer, preferably in a dark room or under a Surgery OPD and wards in any teaching hospital. These
dark hood, from 7 to 10 cm away. The device detects the patients also remain in ward for many days enabling easier
presence or absence of infection in the wound, maps the follow-ups.
exact site of infection spatially and classifies the Gram type Male and female patients, from the OPD or ward, of
of bacteria within 2 min. The device can also measure the all age groups, having ulcer(s) on the foot/leg along with
dimensions of the ulcer, so wound healing can be measured diabetes were included in this study. Patients unwilling to
during follow-up visits. The test report can be immediately give consent or uncooperative in nature were excluded.
generated, and the ‘test’ data can be saved and transferred Pregnant patients and patients on chemotherapy were
either using a USB drive or through the cloud, connecting excluded from the study as follow-up in these patients
with the hospital records. sometimes becomes difficult.
A clinical study on 100 patients of diabetic foot ulcers All foot ulcers were clinically examined, washed with
was conducted using this novel handheld device to detect the saline and a deep tissue biopsy was taken from a loca-
presence of infection, classify infecting bacteria according to tion which was suspected to be infected by the clinician
on visual inspection by either a small scoop or a no. 22
13
blade without the help of the device. After this, the ulcer A total of 100 patients (88 males and 12 females) with
was imaged by this device either in a dark room or under 104 wound sites were recruited for the study and a total of
a black hood and another tissue sample was taken from 163 session images were taken; some ulcers needed to be
the site where the device showed maximum infection. imaged after few days of antibiotics therapy when the treat-
These samples are (1) blindly taken and (2) the device- ing doctor clinically felt that infection was still not under
guided samples were sent to the microbiology laboratory control. Some patients were reimaged after debridement of
for culture and sensitivity testing. It is to be noted that the the ulcer while some patients had multiple ulcers. Thus, all
microbiologist did not know the device result to avoid any ulcers were imaged and tissues were taken separately from
bias. The Gram type displayed immediately by the device each ulcer.
was recorded in a prepared proforma. After 48 h, culture Out of the 163 session images taken, 149 sessions were
reports were compared with the device report (Gram type). considered for study as 14 images had to be removed due
Figs. 2 and 3 show the images taken by the device and to poor imaging (6 sessions), presence of a bony region (3
clearly indicating the site of infection, the Gram type of sessions), and/or interference due to betadine (5 sessions)
bacteria infecting the wound and size of the ulcer. on the wound.
Fig. 2 Example of Gram-nega-
tive bacteria-infected wound
Fig. 3 Example of Gram-posi-
tive bacteria-infected wound
13
Table 1 Comparison of results obtained by standard culture method and 2 show the comparison of Gram type obtained in 149
and by new device samples by culture method and by the device. The device
Gram type Results of Gram type Results of Gram type imaged report displayed Gram-negative bacteria in green
obtained by culture obtained by new overlay and Gram-positive as red overlay. If two types of
method device Gram-negative bacteria were present in the wound, the
Gram positive 11 20 device depicted them as Gram-negative bacteria only as
Gram negative 119 112 it does not differentiate the different types of species of
No growth 17 15 bacteria. This is true for Gram-positive bacteria also. In
Polymicrobial 02 02 case of polymicrobial infection comprising of both Gram-
Total 149 149 positive and Gram-negative bacteria, the device displays
the Gram type of more abundant bacteria in the region.
Sensitivity of the new device is 99.24%, which means
Totally from 104 sites tissues were taken blindly without that the diagnosis of the bacterial wound infection by the
use of the device by the clinician from the area of the ulcer new device will be 99.24% correct.
where he suspected infection. After this, same ulcer was Specificity of the device is 82.35%, which correctly
imaged by the device and tissues were taken from the sites identify those samples without infection.
on ulcer, where the device was indicating infection spatially. Cohen’s Kappa (K) was also tested to determine the
Forty-five more sites were detected by the device where clini- agreement between culture method and new device
cally there was no suspicion of infection. So totally, 149 tis- method. We found K = 0.86 (P < 0.001), which means
sue samples were taken under the device guidance and totally highly significant agreement was seen between the cul-
149 tissue samples were sent to microbiology department for ture method and the new device method. So, new device
culture and sensitivity testing. The results obtained by the perfectly diagnoses the bacterial presents.
device in all 149 cases about the Gram type of infection were Table 3 shows association among Gram-negative,
recorded in proforma. Microbiologist was not aware of the Gram-positive, and no growth by culture and device
Gram type of bacteria identified by the device. method.
(There were 2 polymicrobial infection sites diagnosed
by culture method and by device method; so, for easy cal-
Results culation 1 is added into Total calculation of Gram-positive
and 1 is added into total calculation of Gram-negative
The culture report of 149 tissue samples were obtained bacteria).
from the microbiology department and compared with Cramer’s V is used to measure the strength of the asso-
the Gram type of infection shown by the device. Tables 1 ciation which is 0.765 (P value < 0.01).
Table 2 Screening test culture Screening test culture (standard)/new device Growth according to culture report
(standard)/new device
Growth seen No growth seen Total
Growth according to new device Growth seen 131 (T.P) 3 (F.P) 134
No growth seen 1 (F.N) 14 (T.N) 15
Total 132 17 149
Sensitivity = T.P/(T.P + F.P) 99.24%
Specificity = T.N/(F.N + T.N) 82.35%
Positivity predictive value = T.P/(T.P + F.N) 97.76%
Negative predictive value = T.N/(F.P + T.N) 93.33%
Table 3 Comparison of Culture (standard)/new device Growth according to culture report

detection of Gram-negative,
Gram-positive, and no growth Gram negative Gram positive No growth
by the new device and culture
method Growth according to new device Gram negative 109 (90.8%) 1 (8.3%) 3 (17.6%)
Gram positive 10 (8.3%) 11 (91.7%) 0 (0.0%)
No Growth 1 (0.8%) 0 (0.0%) 14 (82.4%)
P value < 0.001, highly significant association.
13
There is highly significant association between the cul- bacteria on various surfaces like OT table, anesthesia trolley,
ture method and new device method for diagnosing infec- or in ICU like railing of bed, B.P cuff, and nurse’s computer.
tion and Gram type of bacteria. We applied Cramer’s V test This screening device has multiple advantages.
to find out strength of association between culture method
and new device method. (1) The presence of infection in the ulcer and Gram type
of the infecting bacteria can be immediately diagnosed.
So, appropriate antibiotics based on Gram type of bac-
Discussion teria can be started using the hospital antibiogram.
(2) The device can automatically measure the size of the
Bacterial identification is a growing field of interest in wound, helping the clinician during follow up. The
microbiology. Traditional methods of bacterial identifica- treating doctor can immediately know the status of
tion include simple biochemical tests like catalase, oxidase, infection and the reduction in the size of the ulcer, if
and substrate utilization test that mostly rely on phenotypic any.
identification [5]. On the other hand, modern methods for (3) It can detect bacterial loads as less as 103 CFU/gm
microbial identification include PCR, microarray-based (Colony forming unit per gram) allowing for early
identification, immunological methods, chemical analytical intervention [8].
methods etc. [2]. Modern methods usually complement but (4) In our study, when tissues sample were taken under the
may sometimes replace traditional methods [6]. guidance of device, the report showing “no growth”
One promising method for detection of bacterial infec- were significantly less. “No growth” was reported in
tion is by using the inherent fluorescence of bacteria [3]. 28 patients when tissues were taken blindly without
Previous studies have shown fluorescence-based detection using the device and only 15 patients when tissues were
to be sensitive and effective for food born and environmen- taken under guidance of the device. This difference is
tal microorganisms and have even been able to distinguish statistically significant. Overall accuracy of the device
between different cell types. However, this powerful tech- is 93.20% in diagnosing Gram type of infection.
nique is not available for large-scale deployment in clinical (5) The device is useful for getting immediate feedback
use to detect infection [3]. during debridement of an infected wound in OT.
Fluorescence techniques are well developed in flow (6) Before grafting the partial thickness skin graft on recip-
cytometry [9]. Flow cytometry utilizes either the intrinsic ient area, the device can screen the area and show any
fluorescence (auto fluorescence) of cellular molecules or infection pocket on the recipient area, preventing graft
requires the addition of extrinsic fluorescent stains or anti- rejection.
body probes and is being increasingly employed for rou-
tine analysis in environmental and industrial microbiology The device has certain disadvantages.
[10, 11]. Fluorescence techniques can identify potentially
pathogenic or toxic microorganisms in environmental water (1) Dark room or black hoods required for imaging the
and foods [12, 13]. More specifically, within medicine, wound. Wound cannot be imaged in presence of ambi-
fluorescence characterization has provided insights in the ent light.
development and progression of cancerous tissue [14, 15]. (2) Device cannot determine the species of the bacteria at
Giana et al. (2003) [7] successfully discriminated between present, whereas standard culture method can tell us
the clinically important Escherichia coli., Enterococcus the species of the bacteria while the device can only
faecalis, and Staphylococcus aureus using fluorescence differentiate infecting bacteria into Gram-negative or
methods. Gram-positive.
Adiuvo Diagnostics Pvt. Ltd., Chennai, has used the prin- (3) When there is polymicrobial infection the device can
ciples of auto fluorescence of clinically important bacteria not differentiate various species of bacteria and it will
to make a hand-held device (Illuminate) and we at GMERS show the Gram type of abundant bacteria. It becomes
Medical College and Hospital, Valsad, conducted this study difficult to calculate exactly the number of polymicro-
for validation of the said device. The makers of the device bial infections in a particular wound.
first quantified the complete fluorescent response of many
clinically relevant bacteria to identify the most promising
fluorescent features. In the future, more data will lead to Conclusion
the addition of more wavelengths of light and making the
real-time detection of the species of the bacteria possible From the many newly developed techniques for detection of
along with fungus detection as well. More variants of the bacterial infection, techniques using fluorescence character-
hand-held device can be constructed to detect presence of istic of bacteria can be simple, cost-effective, and speedy.
13
This newly developed device is a prime example of the same. 8. Shah BM, Ganvir D, Sharma Misra SB, Misra RN, Kothari P
At present, this device shows only Gram type of bacteria and et al (2020) Utility of a real –time fluorescence imaging device in
guiding antibiotic treatment in superficial skin infections. Indian
not the species of bacteria. In the future with data available, J Dermatol Venereol leprol 2020(1):6
the algorithm in the device will also be capable of differen- 9. Patra D, Mishra AK (2001) Investigation on simultaneous analysis
tiating between the genus/species of infecting bacteria. The of multi component polycyclic aromatic Hydrocarbon mixtures
accuracy of the device is around 93% which is acceptable. in water samples: a simple synchronous fluometric method. Tal-
anta 55:143–153. https://doi.org/10.1016/50039-9140(01)00404.
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5. Pj Modeiros, Weixu Liztein XU, Wilson BC, Rosen C, Linden R 36
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pling and treatment guidance of bio burden in chronic wounds: Publisher's Note Springer Nature remains neutral with regard to
first in human results. PLOS One 10(3):9 jurisdictional claims in published maps and institutional affiliations.
6. Murihead K, Horan G (1985) Flow cytometry: Present and future.
Nat Biotechnol 3:56. https://doi.org/10.1038/nbt0485-337
7. Radhakrishnan Geethajali, Gupta Ayush, King John, Ganvir
Davina (2020) Rapid handheld screening device to detect skin
and soft tissue infection. Proc SPIE 2020:11211. https://doi.org/
10.1117/12.2546404
13
PROCEEDINGS OF SPIE
SPIEDigitalLibrary.org/conference-proceedings-of-spie
Rapid handheld screening device to

detect skin and soft tissue infections
Radhakrishnan, Geethanjali, Gupta, Aayush, King, John,

Ganvir, Devina
Geethanjali Radhakrishnan, Aayush Gupta, John King, Devina Ganvir, "Rapid

handheld screening device to detect skin and soft tissue infections," Proc.
SPIE 11211, Photonics in Dermatology and Plastic Surgery 2020, 112110V
(19 February 2020); doi: 10.1117/12.2546404
Event: SPIE BiOS, 2020, San Francisco, California, United States
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Rapid Handheld Screening Device to Detect Skin and Soft Tissue
Infections
Geethanjali Radhakrishnana, Aayush Guptab, John Kinga, Devina Ganvirb
a
Adiuvo diagnostics private limited, Chennai, India, Chennai, India
b
Department of Dermatology, Dr DY Patil Hospital, Pune
ABSTRACT
Skin and soft tissue infections (SSTIs) are one of the most common infections in India affecting 10-12% of Indian
population. They are caused by a variety of bacteria and fungus, which makes it harder to diagnose and propose an effective
treatment immediately especially in low resource settings due to the lack of access to qualified physicians. Management
of SSTIs requires early expert infection assessment and remains a major challenge for the clinicians. A hand-held device
is developed leveraging the inherent autofluorescence properties of the bacterial and fungal species that can non-invasively
and rapidly identify the pathogens on SSTI using multispectral imaging followed by image processing and machine
learning algorithms. The device can classify the gram type of bacteria with > 85% accuracy.
Keywords: Multispectral imaging, SSTI, Wound, Handheld device, Multispectral imaging, Machine Learning, Gram
type classification, autofluorescence1
1. INTRODUCTION
The principal barrier against microbial invasion is the skin [1]. It provides the first line of defense and interacts with the
external environment constantly and is colonized with a diverse population of microbes. Gram-positive species such as
Staphylococcus epidermidis, Corynebacterium species, Staphylococcus aureus and Streptococcus pyogenes are the typical
species that colonize the skin above the waistline. Both gram positive and gram-negative bacteria colonize the skin below
the waistline.
Skin and soft tissue infections (SSTIs) which includes all pathologic infections of the skin, range from simple impetigo to
rapidly progressive necrotizing fasciitis [2], foot ulcers, surgical infections and so on. Diagnosing the exact extent and
causative organism of the disease is critical for the successful management of a patient through successfully treating the
soft tissue infection [3].
SSTI has an incidence rate of about 24.6 per 1000 person per year along with a prevalence rate in hospital of 7% to 10 %
[4]. It’s important to identify cases which require immediate intervention to the ones that are less severe thus presenting
diverse challenges. The problem is more compounded in patients who have diabetes mellitus and AIDS where a mild
infection can easily progress into a rapidly advancing life-threatening condition. Chronic ulcers, like those associated with
neuropathy are also associated with unique challenges. Hence, infection care remains a major clinical challenge and
presents an enormous burden to health care worldwide. It is important to understand the infection immediately when a
patient is presented with SSTI, identify the causative organism to deal with them effectively.
Currently, assessments of skin and soft tissue infections and determining the underlying pathogens causing infection is
typically visual inspection based followed by swab analysis and culture method. Culture method is considered the gold
standard technique, but the process is laborious, time-consuming, and requires a BSL-2 facility for identification thus
leading to prescription of generic antibiotics resulting in antimicrobial resistance. In case of fungus, the turnaround time
[email protected]; www.adiuvodiagnostics.com, Phone +91-8015316313
Photonics in Dermatology and Plastic Surgery 2020, edited by Bernard Choi,

Haishan Zeng, Proc. of SPIE Vol. 11211, 112110V · © 2020 SPIE
CCC code: 1605-7422/20/$21 · doi: 10.1117/12.2546404
Proc. of SPIE Vol. 11211 112110V-1

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for a culture technique is even longer and requires a dermatologist for assessment. Only 6000 dermatologists cater to 121
crore population [5] making it difficult for diagnosis in low resource setting areas. Recently PCR is used for rapid detection,
but they require trained physicians and cannot be used for continuous monitoring of an SSTI. Commercially available
wound management devices do not provide comprehensive information about specific pathogens infecting the wound and
are expensive. There is a huge unmet need to understand pathophysiology of a wound at an early stage, especially
complications of diabetes foot ulcers leading to increased risk of chronic wounds and amputations. Similarly, hospital
acquired infections at surgical sites are becoming increasingly prevalent and needs preventive measures.
We propose a non-invasive device that can be used in real-time to detect the causative microorganism gram type and
continuously monitor the SSTI along with their corresponding pathogen load to achieve the above-mentioned unmet need
and improve the quality of life of patients.
Previous research findings show that bacteria have characteristic native fluorescence contributed by metabolic markers
such as tryptophan, tyrosine, Nicotinamide Adenine Dinucleotide (NADH), Flavin molecules during its metabolic phase
and siderophore porphyrin during its infectious phase. It is also well known that Pseudomonas aeruginosa in addition
produces siderophore pyoverdine during its heme acquisition pathway [6]. Fungus has chitin and di-tyrosine linkages
which are autofluorescence in nature as well [7]. Metabolic signals, which are indicators of live cells, fluoresce in the blue-
green region. The fluorescence emitted is directly proportional to the concentration of metabolites and thus to the average
number of live cells [8]. Flavins which fluoresce in the green region and protoporphyrin IX, which fluoresce in the red
region are found in both live and dead cells.
Leveraging this, we wanted to further analyze the change in fluorescence intensity contributed by each clinically relevant
bacterium during its growth phase and while acquiring heme from host and if such an information can be used in classifying
the gram type of bacteria.
We have analyzed auto-fluorescence characteristics of clinically relevant bacteria and fungi. From the obtained data,
characteristic excitation and emission wavelengths are chosen to distinguish various bacteria into gram type and identify
few fungi. To aid the ease of use in low resource settings, we have developed an affordable multispectral imaging platform
capable of capturing images of the wound region. A clinical study was conducted using the hand-held device on SSTI
patients at Dr. DY Patil Hospital, Pune, India. The device was able to distinguish gram type of clinically relevant bacteria
and fungus with more than 85% accuracy.
2. FLUORESCENCE SPECTRA OF CLINICALLY RELEVANT PATHOGENS
An Excitation Emission Matrix Spectra (EEMS) is a three-dimensional graph to analyse fluorescence properties exhibited
by an unknown molecule at various excitation wavelengths [9]. Clinically relevant pathogens samples were obtained from
Microbial Type Culture Collection (MTCC), American Type Culture Collection (ATCC) and subcultures taken from
Patients and used for EEMS analysis. Gram staining was done before and after inoculation from the subcultures to ensure
no contamination. Clinically relevant pathogens that are most common in Indian settings were identified as Staphylococcus
aureus, Enterococcus faecalis, Escherichia coli, Klebsiella sp., Proteus sp., and Pseudomonas aeruginosa. In fig.1, we
have shown results obtained from Pseudomonas aeruginosa and Escherichia coli, belonging to the gram-negative type
and Staphylococcus aureus, a gram-positive bacterium and Malassezia sp. of fungal origin for comparison.
Both intracellular and extracellular metabolic biomarkers are known to cause autofluorescence on excitation with
wavelengths between 200 nm to 450 nm with emission wavelengths between 300 nm to 800 nm. The intensity of
fluorescence contributed by each biomarker is inferred from the EEMS recorded. Tryptophan, NADH, and Flavin
molecules contribute to the majority of autofluorescence in specified wavelength region [10].

Pseudomonas aeruginosa Staphylococcus aureus
Malassezia sp.
Figure 1: Excitation Emission Matrix of clinically relevant bacteria and fungus.
Bacterial pathogens require iron-containing heme to cause the disease. Pathogens have elaborate strategies to acquire heme
from host, and this is required to cause an infection. To understand the biomarkers and any change in autofluorescence
intensity during heme acquisition pathways, these clinically relevant pathogens were induced with aminolaevulinic acid
(ALA). From Figure 2, one can understand that while pyoverdine is very evident in Pseudomonas aeruginosa, porphyrin
intensity is higher in Staphylococcus aureus, followed by a double peak between 400-500nm in Escherichia coli and
Malassezia sp. has significant porphyrin secretion between 550 nm-600 nm.

Pseudomonas aeruginosa + ALA Staphylococcus aureus + ALA
Malassezia sp. + ALA
Figure 2: Excitation Emission Matrix of clinically relevant bacteria induced with ALA.
We compared the EEMS before and after ALA induction (see fig. 2) and compared the intensities contributed by
biomarkers: Flavin, NADH, Porphyrin and Pyoverdine. There were significant differences in Porphyrin production for
gram-positive, gram-negative bacteria1 and fungus. We used this information for classification of gram type of bacteria
and identification of fungus.
3. DEVELOPMENT OF MULTISPECTRAL FLUORESCENCE IMAGING DEVICE
Based on the identified excitation and emission wavelengths from EEMS graphs of various Gram positive, Gram negative
bacteria and Fungi, we have developed a portable handheld multispectral imaging device, referred to as “IlluminateR ”,
that illuminates the SSTI region with multiple excitation sources and capturing the emitted auto fluorescence.
3.1 Device Block Diagram:
The handheld device comprises of the following components: Fluorescence imaging module, Image acquisition module,
Image processing module, Display module and Control system.

Fig. 3: Block diagram of handheld device for capturing auto fluorescence from the diabetic wound region
3.1.1 Fluorescence Imaging Module:
Fig. 4: Block diagram of Fluorescence Imaging Module
The components of fluorescence imaging module comprise of multi-wavelength UV LEDs (UV-A, UV-B and visible)
driven by a LED driver board, an optical filter wheel with emission filters over a CMOS imaging sensor forming a
multispectral imaging module. The filter wheel is rotated by a nano servo motor. The whole module is controlled by the
control system which sets excitation/emission wavelength in a sequence and captures multispectral image of the wound.
3.1.2 Control System:
The control system consists of a quad core ARMv8 Cluster and is implemented on a BCM2837 software. The control
system receives commands from the Display Module running on the Android Smart Phone. It controls the fluorescence
imaging module, image acquisition module, image processing module and display module.
3.1.3 Image Acquisition Module:
Image acquisition is done using 13MP CMOS sensor present on the Android smartphone. The frame rate and exposure for
images captured is set automatically by the control system prior to image acquisition.

3.1.4 Display module:
An application runs on the smartphone screen which enables user to enter patients details, set SSTI area on to the capture
window, evoke capture of images, lasso the wound region after capture and finally also to display colour code wound map
with details of infected region. Red to display gram positive bacteria, green to display gram negative bacteria, blue for
fungi. The interface also enables physician to share and print the report, retrieve patient data any time and for adding notes.
3.1.5 Image Processing Module:
The image processing module analyses the auto-fluorescence response collected from the spectral images and analyses
various biomolecular fluorescence intensities contributed by NADH, Flavin, pyoverdine, Chitin, porphyrin and any
unknown molecule. After the spectral images are captured, the image processor implements an image orientation mapper
to correct for the orientation mismatches. Each image is oriented and aligned with each other and the mean intensity in the
region of interest for each filter is calculated and subsequently used for machine learning.
3.1.6 Pathogen Classifier Model:
Fig. 5: Block diagram of the process flow implemented in the handheld device for pathogen classification.
A machine learning approach is followed in order to build the Pathogen classification model as shown in Figure 5. Spectral
images are captured from illuminate device and sent into a decision matrix step where the images are filtered based on hue
and threshold to extract autofluorescence regions from noise and other unwanted signals. The filtered regions are called
ROI, which are sent as a swab or tissue to microbiology lab and a report is obtained with organism name which is used for
labelling. The corresponding ROI spectra in fed into a Machine learning algorithm which predicts the pathogen gram type
and displays the result. In case of fungus a simple thresholding is done, and sent for ML.
Random Forest Classifier is used here with a 70:30 training/test data split. Data is shuffled randomly before applying the
random classifier. During the training and test split, the data is stratified to ensure equal percentage of each classes is
represented in the training and test set to avoid data bias. Five-fold cross validation is also done to calculate the accuracy
and standard deviation values.
4. CLINICAL STUDY TO VALIDATE THE MULTISPECTRAL IMAGING DEVICE
A clinical study was conducted in collaboration with Dr. DY Patil Hospital, Pune, India. The study was approved by
institutional ethics clearance “DYPU/EC/159/2018”and registered under Central Trial Registry of India (CTRI) with
register number “CTRI/2018/10/016147”.

It was a single arm interventional study where the device results were compared against a gold standard culture method in
case of bacteria or sent for KOH staining in case of fungus. A swab/deep tissue or skin scrapping were sent for microbiology
assessment in the region where the device shows presence of bacteria/fungi. The device report was compared against the
microbiology report. Patients with fungal infections, surgical site infections, contact Dermatitis, stasis ulcer both arterial
and venous, diabetic foot ulcer, sterile ulcers, any scars/suture site, sinuses/fistulas were included in the study. Patients
with inability to consent were excluded from the study.
Informed consent was obtained from 182 patients and were imaged using the device. 33 bacterial patients having various
type of SSTI and 149 dermatology related fungal infections as shown in (Table- A) were imaged using the device.
Species Number
CONS 1
Klebsiella sp. 3
MRSA 7
Pseudomonas sp. 11
Staphylococcus aureus 3
Propionibacterium sp. 4
Tinea sp. 81
P.versicolor 68
No infection – Sterile wounds 5
Table A: Summary of bacteria isolated through standard culture method.
The device was mounted on a tripod to avoid any vibration/shaking during the imaging and lights were turned off to
maintain a dark room setting and the device is held 10-15 cm from the wound region while imaging. For smaller diameter
wounds of ~5 cm2, the distance was less than 7 cm. Images were captured automatically in less than 30 seconds and an
additional 1 minute for the machine learning algorithm to display the results. The images were stored in the memory card
within the device. 70% ethanol was used to decontaminate the device before each imaging. Additionally, a black hood was
given if the lights cannot be turned off to minimize noise due to ambient lighting.
Series of images were taken with the “IlluminateR” imaging device on the affected SSTI region. At the end of imaging, a
spatially mapped color-coded image was displayed indicating possible presence of pathogen regions, where a swab/deep
tissue biopsy/scraping were taken and cross-validated against standard visual inspection followed by
microbiology/microscopy wherever applicable (See table 1).
Previously a machine learning model was trained on 121 bacterial patients and this model was ported into the device. This
model was able to predict 14 gram positive sites and 11 gram negatives with greater than 90% sensitivity as per results
shown in Table B. The device also picked up 1 polymicrobial site and showed no fluorescence in 5 patients where there
was no growth of bacteria in culture as well. Further tests were performed on the no infection patients and the physician
identified 2 patients associated with a rare condition called Pyoderma gangrenosum. The device was also used to check
bacterial presence on patients with Acne. The specificity was 100% and accuracy 93% in case of binary classification
between infection and no infection. In case of fungus, the accuracy is greater than 70% towards classification between
Tinea species and Pityriasis versicolor using morphological features alone. In future, accuracy will be improved combining
both spectral and morphological features. In the near future, the device needs will be tested against more reference images
of different skin colors to compensate for background autofluorescence to make the model more robust.

Microbiology result Total no. of sites Imaging device result
Correct classification Incorrect classification
Gram positive bacteria 15 14 1
Gram negative bacteria 12 11 1
Co-colonization 1 1 0
Microbiology result Total no. of sites Imaging device result
Correct classification Incorrect classification
Presence of bacteria 28 28 0
Absence of bacteria 5 5 0
Table B: Results of the wound imaging for gram type classification, presence and absence of bacteria using the portable
handheld imaging device.
5. LIMITATIONS
Imaging needs dark room setup and extra care needs to be taken for no external light to interfere with imaging sequence
else the model predicts random artifacts as infections. Staples, fibers from cloth, sutures, bones and tendons has
autofluorescence on their own and leads to false fluorescence. The device has been fine-tuned with increased sensitivity
to pick up infections in open wounds. The device should only be used for imaging closed skin or aberrations with caution
due to high autofluorescence contributed by collagen and elastin on skin. Sometime edge artifacts contribute to false
overlay, where decision has to be made by comparing the spectral images.
6. CONCLUSION
The portable handheld device, IlluminateR, based on multispectral auto fluorescence imaging can be used as an important
tool in SSTIs for guided swabbing, assessment of an infection, understanding its microbiome pattern and aiding in the right
first line treatment during the golden hour of wound healing. The device helps to identify infected from non-infected
wounds through a color coded over lay image and classifies the infected bacterial wounds broadly according to their gram
type. The device also identifies fungal infection regions in dermatology related skin infections. The results of the study
with >85% accuracy demonstrates attractive potential of the device in any SSTI settings. Since the results of the gram type
classification are instantaneous, the device significantly helps doctors in effective first line antibiotic treatment thus
preventing antibiotic misuse. In future, the device will be tested on more patients and the model will be fine-tuned to
increase the accuracy.

Acknowledgments
We would like to thank Department of Dermatology, Microbiology, Surgery at Dr.DY Patil Hospital, Pune. We also thank
BIRAC, Department of Biotechnology, Government of India for the Grant aid and support to develop and validate the
device. We also thank Villgro Innovation Foundation and Venture Center, Pune for their constant guidance. NCL, Pune
and Golden Jubilee Biotech park for Woman, Chennai for giving access to their Spectrofluorometer.
REFERENCES
[1] Ki, V., & Rotstein, C. (2008). Bacterial Skin and Soft Tissue Infections in Adults: A Review of Their Epidemiology,
Pathogenesis, Diagnosis, Treatment and Site Of Care. Canadian Journal of Infectious Diseases and Medical
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[2] Park, Y. J., & Lee, H. K. (2018). The Role of Skin and Orogenital Microbiota in Protective Immunity and Chronic
Immune-Mediated Inflammatory Disease. Frontiers in Immunology, 8. doi: 10.3389/fimmu.2017.01955
[3] Park, Y. J., & Lee, H. K. (2018). The Role of Skin and Orogenital Microbiota in Protective Immunity and Chronic
Immune-Mediated Inflammatory Disease. Frontiers in Immunology, 8. doi: 10.3389/fimmu.2017.01955
[4] Leong, H. N., Kurup, A., Tan, M. Y., Kwa, A. L. H., Liau, K. H., & Wilcox, M. (2018). Management of complicated
skin and soft tissue infections with a special focus on the role of newer antibiotics. Infection and Drug Resistance, Volume
11, 1959–1974. doi: 10.2147/idr.s172366
[5] Skin diseases to grow in India by 2015: Report. (2014, May 2). BioSpectrum Bureau.
[6] Nitzan, Y., Salmon-Divon, M., Shporen, E., & Malik, Z. (2004). ALA induced photodynamic effects on Gram positive
and negative bacteria. Photochemical & Photobiological Sciences, 3(5), 430. doi: 10.1039/b315633h
[7] Smail, E. H., Briza, P., Panagos, A., & Berenfeld, L. (1995). Candida albicans cell walls contain the fluorescent cross-
linking amino acid dityrosine. Infection and Immunity, 63(10), 4078–4083. doi: 10.1128/iai.63.10.4078-4083.1995
[ 8 ] Kilungo, A. P., Carlton-Carew, N., & Powers, L. S. (2013). Continuous Real-time Detection of Microbial
Contamination in Water using Intrinsic Fluorescence. Journal of Biosensors & Bioelectronics, S12(01). doi: 10.4172/2155-
6210.s12-002
[9] Blough, N. V., & Vecchio, R. D. (2002). Chromophoric DOM in the Coastal Environment. Biogeochemistry of Marine
Dissolved Organic Matter, 509–546. doi: 10.1016/b978-012323841-2/50012-9
[10] Dartnell, L. R., Roberts, T. A., Moore, G., Ward, J. M., & Muller, J.-P. (2013). Fluorescence Characterization of
Clinically-Important Bacteria. PLoS ONE, 8(9). doi: 10.1371/journal.pone.0075270

S C I E N C E , P R A C T I C E A N D E D U C AT I O N
DOI: 10.35279/jowm2022.23.03.06
Clinical significance of a
novel imaging device to
evaluate infection on wounds:
Performance comparison
with culture method
and metagenome sequencing
Rajesh Kesavan1, Changam Sheela Sasikumar2
1 Department of Podiatric Surgery, Hycare Super Specialty Hospital, Chennai, Tamil Nadu, India. Adjunct Faculty, SRM Institute of Science and
Technology, Kattankulathur, Tamil Nadu, India.
2 Department of Clinical Research, Hycare Super Specialty Hospital, Chennai, Tamil Nadu, India. Managing Partner - SS Clini Research LLP, Adjunct Faculty,
Department of Biochemistry, Saveetha Dental College, Saveetha Institute of Medical & Technical Sciences, Saveetha University Chennai, Tamil Nadu, India.
Correspondence: Dr Rajesh Kesavan, [email protected]

Conflict of interest: None
Keyword s:
Autofluorescence, diabetic foot ulcers, machine learning, pathogens, metagenome sequencing,
point-of-care device
ABSTRACT Results
Background A total of 157 patients were imaged, and 177 deep
Diabetic foot and lower limb complications affect tissue biopsies were taken from colour-coded regions
40–60 million people globally. A rapid method is (Gram Positive/Gram Negative infected) identified by
needed to understand the bioburden and type of the machine learning algorithm. The class-averaged
infecting bacteria on diabetic foot ulcers. accuracy of the device was found to be 90.4% for
gram-positive, -negative, polymicrobial and for no
Aim bacterial burden. A total of 26 biopsies were also
To compare the accuracy and efficacy of a novel sent for 16S rRNA sequencing. Of these, cultures
multispectral imaging device, against the standard were positive in 17 and correlated with the species
culture method and correlate bacterial bioburden identified through 16S rRNA results in 16 cases. In
levels with metagenome sequencing. five cases where there was no growth in culture,
the multispectral imaging device could still detect
Method the presence of bacteria as confirmed by 16S rRNA
A clinical study was conducted on diabetic foot ulcer results (using 50 reads as a cut-off) and thus be
patients. Wounds, post-debridement, were imaged used as an adjunct for monitoring wounds’ bacterial
using the multispectral imaging device and the re- burden over time.
port, containing spatially mapped regions of bac-
terial burden along with their bacterial gram type, Conclusions
was compared with the culture sensitivity report. Ad- The novel multispectral imaging device can be used
ditionally, metagenome sequencing was done on a to effectively understand the bioburden in a wound
subset of the patient samples. of clinical significance and the gram type of infect-
ing bacteria.
182 J OU R NAL O F WO UN D M A N AG EM EN T
OFFIC IAL JOURNAL OF THE EU RO PEA N WO U N D M A N AGEM EN T A SSO C IAT IO N
Implications for clinical practice of a wound, clinicians can achieve resolution in >90%
The autofluorescence imaging device can assist doc- of mild to moderate soft tissue infections (7); there-
tors in evaluating wounds’ bacterial burden level, fore, there is a significant need for a modality that
spatial bioburden extent and the gram type of bac- focuses on the early assessment and classification of
teria present, thus aiding in effective debridement pathogenic gram types infecting a wound.
and proper wound-management protocols.
Multispectral imaging, which involves shining mul-
INTRODUCTION tiple wavelengths of light and collecting the emission
Diabetes, one of the most prevalent chronic diseases, response, has proven to be a useful technique for
is projected to affect more than 700 million peo- identifying and classifying bacteria based on their
ple globally by 2050 according to the International autofluorescence (19). In addition, autofluorescence
Diabetes Federation (1). People in underdeveloped imaging is label-free, which is advantageous in terms
and low-income countries are more prone to develop of reduced complexity and cost. Studies have shown
diabetes, and about 12–25% of those are at risk of that bacteria have characteristic emission fluorescence
developing diabetic foot ulcers as well, which can when excited in the UV and blue regions of light,
further lead to chronic wounds, amputation and even contributed by metabolic and infectious markers such
death (2-6). Wound bacterial burdens are known to as NAD(P)H, flavins, porphyrins and pyoverdine (in
be initially composed predominantly of gram-posi- the case of Pseudomonas) and so on (19-20). Previous
tive bacteria (such as Methicillin-susceptible/resist- studies have also shown that gram-positive bacteria
ant Staphylococcus aureus, Streptococcus), but as the typically have more fluorescence intensity in the red
infection progresses, they become colonised by gram- region, due to an increase in the release of porphyrins,
negative bacteria (such as Pseudomonas aeruginosa, compared to gram-negative bacteria. Similarly, some
Klebsiella species) (7-12). gram-negative bacteria have increased NAD(P)H and
flavins, when compared to gram-positive bacteria (21-
Wounds frequently differ in their clinical character- 22). In addition, Pseudomonas aeruginosa, which is
istics, making the diagnosis of infections through one of the most predominant pathogens present in
signs and symptoms alone difficult (13). In hospital diabetic foot infections (21), has clearly distinguish-
settings, the current standard method for detecting able autofluorescence contributed by pyoverdine.
bacterial burden requires a swab or deep tissue biopsy These key differences in the relative concentration
for culturing microorganisms, followed by biochemi- of biomarkers and autofluorescence in various spec-
cal methods for species recognition and antibiotic tral bands can potentially be used to design a rapid,
susceptibility testing, which usually takes 3–5 days. non-invasive diagnostic technique for the presence/
In short, these methods fail to provide the immediate absence of bacterial burden and the classification of
clinical information (such as the presence/absence the gram type of the bacteria infecting the wounds.
of bacterial burden and gram type of bacteria) re- Fluorescence imaging has emerged as a promising
quired for first-line treatment (14). In addition, the point-of-care technique for monitoring bacterial
culture sensitivity methodology has other limitations, burden in wounds (21,22). Fluorescence imaging,
including a bias to certain species, depending on the in combination with clinical signs and symptoms,
choice of growth medium and incubating conditions; has been used to effectively monitor wounds with
a requirement for special apparatus for anaerobic bac- moderate-to-heavy bacterial loads (23). Similarly, sur-
teria; and an inability to pick up the full diversity of gical sites with a high prevalence of bacterial burden
infecting organisms (2). Accurate identification of have been assessed with point-of-care fluorescence
polymicrobial species infecting the wound can only imaging devices (24). These results show that when
be achieved using genotypic methods such as16s the standard clinical signs and symptoms are assessed
rRNA sequencing, shotgun sequencing and so on in conjunction with the inputs from fluorescence im-
(15-16). However, these methods are cumbersome, aging, bacterial burden diagnostic accuracy improves.
costly and have yet to be adopted in mainstream clini- A bacterial fluorescence imaging system was also used
cal practice (14). for effective wound debridement, which results in
accelerated wound healing (25). Further studies have
The ability to offer targeted treatment during a first demonstrated that the use of fluorescence imaging
consultation offers a tremendous advantage for timely to monitor wounds results in reduced antibiotic use
wound healing. With proper and timely management (>30%), a reduction in antimicrobial wound dress- 
J OU R NAL O F WO UN D M A N AG EM EN T
OFFI C IAL JOURNAL OF THE E U RO PEA N WO U N D M A N AG EM EN T A SSO C IAT IO N
183
Figure 1: Overview of Illuminate multi-spectral autofluorescence imaging platform for pathogen

detection and gram-type classification
Multiple Light source Biomarker - Emission Machine Learning Algorithm

illumination Metabolic - NADH, Flavin compares biomarker intensity
Infectious - Porphyrin, Pyoverdine
Gram positive Gram negative

bacteria bacteria
ings (>45%) and improved wound healing rates tive to distinguish pathogenic autofluorescence from
(>20%) over 12 weeks (26). Overall, the improved this background noise. Advanced machine learning
healing rates are projected to reduce annual wound techniques trained on data from skin and wound
care costs by 10% in National Healthcare System, regions can overcome the above limitations by pro-
United Kingdom. Recently, guidelines were evolved viding correction for these interfering factors.
for the detection of bacterial burden in wounds using
fluorescence imaging based on the Delphi consensus Innovation
protocol (27). To the best of our knowledge, lluminate® is the first
device to use multispectral autofluorescence imag-
The current point-of-care devices for fluorescence ing integrated with machine learning to provide spa-
imaging require careful interpretation. In addition to tial–temporal information on the presence/absence
autofluorescence from bacterial bioburden, human of bacterial burden and gram type of bacteria infect-
tissue has its own autofluorescence, contributed to, ing a wound and to provide the ability to monitor
for example, by collagen or elastin, thus it is impera- a wound continuously (Figure 1). We conducted a
Table 1: Age, gender, and ulcer grade distribution
Age No. of participants (%) N = 157

≤ 35 years 4 (2.55 %)
36–45 years 16 (10.19 %)
46–55 years 39 (24.84 %)
56–65 years 44 (28.03 %)
≥66 years 54 (34.39 %)
Median 60
Q1 51
Q3 69
IQR 18
Gender (M: F) 104:53
Wound Classification
Grade 1 5
Grade 2 33
Grade 3 60
Grade 4 59
Clinical Generated Clinical Generated
Figure 2(a): Figure 2(b):

Gram negative bacteria infected wound Gram negative bacteria infected wound
Patient ID: 6_64 Patient ID: 15_24
Highlighted color-coded region is the Illuminate Device Report: Gram Negative Bacteria
prediction of the infection and gram type. Tissue Culture Report: Klebsiella species - Moderate
biopsy region is taken from this highlighted region growth.
and the culture results are compared with the
Illuminate device result.
Device Report: Gram Negative Bacteria
Culture Report: Pseudomonas spp., Heavy
Growth.
Figure 2(c): Figure 2(d):

Gram Negative bacteria infected wound Gram positive bacteria infected wound
Device Report: Gram negative bacteria Device report: Gram positive bacteria
Culture Report: Pseudomonas sp. - Heavy Growth Gram Staining Report: Gram positive cocci in pairs
& short chains
Culture Report: Enterococcus sp. - Scanty Growth
clinical study on diabetic foot ulcer patients to un- or absence of bacterial burden based on visual char-
derstand the clinical significance of this device for acteristics alone is subjective and can be erroneous.
bacterial burden identification and accuracy of the In addition, information on the gram type of bacteria
gram type classification of bacteria in wounds, to aid infecting the wound using standard microscopy and
doctors in administering first-line treatment. culture tests is both time-consuming and reagent-
intensive. Understanding the bacterial burden and
Clinical problem addressed infection status and monitoring these infections in
Currently, the assessment of wounds for the presence a wound over time is of the utmost importance for 
185
Figure 2(e): Figure 2(f ):

Gram positive bacteria infected wound Gram positive bacteria infected wound
Device Report: Gram positive Device Report: Gram positive
Culture Report: Staphylococcus sp. - Moderate Culture Report: Staphylococcus sp.
Growth
enabling wound closure. Further, identifying bacte- ambient light. Patient details were entered into the
rial burdens that are of clinical relevance is equally device, and the wounds were cleaned with normal
important, as wounds have high loads of commen- saline solution before imaging. The device was held
sals present. A bacterial load >104 CFU/g is usually 10–12 cm from the wound region. A guide icon on
considered to be of clinical significance, and earlier top of the application page, which uses information
fluorescence imaging methods have been able to de- from the distance sensor integrated into the device,
tect bacterial burden above this value effectively (23). ensured that the required distance of ~10 cm was
With the imaging device, the algorithm thresholds maintained during imaging. The capture button was
are adjusted to detect a bacterial load >104 CFU/g. then pressed, to record a series of >15 multispectral
autofluorescence images in <20 seconds. Upon suc-
MATERIALS AND METHODS cessful imaging, the user was prompted with a white
Study design light clinical image to ‘lasso’ the region of interest
An interventional single-arm comparative study to assess the bacterial burden. The built-in image
was carried out at a tertiary care centre in Chennai, processing (to compensate for the slight movement
India after obtaining institutional ethics committee of patients during the imaging and identify regions
approval (IEC 031#HYC/IEC/2018). The study was of interest) and an edge-inferencing machine learn-
registered with the Clinical Trial Registry of India ing algorithm (to detect and classify bacteria causing
(Reg. No. CTRI/2018/10/016147). Adults (>18 infections) then displayed a colour-coded overlaid
years) diagnosed with diabetic foot ulcers and who image of the wound, along with the infected region
were willing to give their consent were included in the (if any) and the gram type of bacteria. The device
study and imaged on their first visit, and during any also displayed the length, breadth and area of the
follow-up visits to the hospital. Patients’ diabetic sta- wound based on proprietary wound segmentation
tus was confirmed from various standard biochemical algorithms. A guided swab/tissue sample was taken
tests that measure blood sugar levels. Patients with from the infected region predicted by the device and
infections under intact skin and/or with pre-existing sent for microbial culture, and the presence/absence
skin conditions, such as eczema or psoriasis in areas of the bacterial burden, along with the gram type,
close to the wounds, were excluded from the study. were compared with the report generated from the
device. Patients were imaged progressively, and a pro-
Imaging procedure with the multispectral gressive report tracking/monitoring the wound was
autofluorescence imaging device also displayed to get a quick understanding of the
All multispectral images were collected using the de- wound status during follow-up visits.
vice in either a dark room, or by covering the wound
with a black hood to minimise interference due to
Table 2: Organisms isolated from tissue biopsy samples
Organism type Occurrence in tissue samples

Pseudomonas spp. 40
Enterococcus spp. 22
Klebsiella spp. 20
E. coli 15
Proteus mirabilis 12
Morganella morganii 11
Staphylococcus aureus 13
Non-fermenting gram negative bacilli 6
Streptococcus spp. 7
Enterobacter spp. 5
Acinetobacter spp. 1
Aeromonas spp. 1
Citrobacter koseri 1
MRSA 1
Candida Spp. 1
Image processing and machine area, as indicated by imaging device. Specimens were
learning algorithms placed in sterile transport containers and delivered to
After collecting multispectral autofluorescence im- the certified microbiology laboratory for gram stain-
ages, thresholding was applied to identify the regions ing, culture and antimicrobial susceptibility testing.
exhibiting higher autofluorescence, and spectral The laboratory personnel were blind to the findings
intensity features were extracted from each of the of the device. Some samples were also sent for 16s
threshold images and sent for machine learning. rRNA sequencing, for comparison of the culture re-
The algorithm’s image-processing thresholds were sults for bacterial species identification. Samples were
adjusted to detect bacterial loads, typically >104 also taken from regions of no fluorescence, to serve
CFU/g. The machine learning algorithm based on as controls. No antimicrobial agents were adminis-
random forests (a highly accurate ensemble classi- tered into the wounds before specimen collection.
fier) was trained using an 80:20 data split ratio. The
hyperparameters, such as the number of trees, tree Cultures
depth, split criterion and so on, were optimised for All tissue biopsies obtained post debridement were
maximum accuracy. A five-fold cross-validation was cultured at a National Accreditation Board for Test-
done to assess the accuracy of pathogen detection ing and Calibration Laboratories-accredited labo-
and gram type classification. ratory and analysed initially by gram-staining and
conventional aerobic methods by plating into various
After two minutes, the machine learning algorithm enriched and differential growth medias. The colo-
could detect and classify the gram type of bacteria, nies were then sent for biochemical assay for species
and the built-in application displayed a wound re- recognition. Bacterial load was estimated by a plate
port, as shown in Figure 2a–f. Both clinical and de- count semi-quantitative analysis and then graded as
vice’s post-processed wound images were displayed scanty, light, moderate or heavy (1+, 2+, 3+ or 4+),
for easier comparison. Gram-positive bacteria are wherein moderate and heavy growth indicated a sig-
colour-coded in red, and gram-negative bacteria are nificant bacterial load (i.e., greater than 100,000 per
in green. gram of tissue). An antibiotics susceptibility profile
was then obtained using the disk diffusion method,
Specimen collection as per Clinical and Laboratory Standards Institute
Following debridement, a deep-tissue biopsy was guidelines.
taken from the colour-coded region of the wound 
187
GN Table 3: Confusion matrix for infection and gram type classification
87 2 1 2
GP
3 19 1 8
True label
NG
16 2 24 8
GPGN
5 2 8 13
GN GP NG GPGN
Predicted label
16S rRNA sequencing 53 females) with a median age of 60 years were en-
16S rRNA gene amplicon sequencing was performed rolled in the study, and 177 tissue samples were ob-
on a subset of 26 patient samples at a certified labo- tained from these patients. Nine wounds were present
ratory. DNA was isolated from the tissue samples in the distal phalanges, 34 in the dorsal regions, 8
using the EXpure microbial DNA isolation kit. PCR in the lateral heel region, 28 in the medial aspect of
was used for selective amplification using 27F and the heel, 67 in the plantar region, 10 in the posterior
1492R primers targeting the V3 and V4 regions. regions and 1 in the anterior tibia. In terms of the
Unincorporated PCR primers and dNTPs from the Wagner classification scoring system, 5 patients had a
PCR products were removed using the Montage PCR Grade 1 ulcer, 33 had a Grade 2 ulcer, 60 had Grade
cleanup kit. The quality, quantity (~100 ng/μl) and 3 ulcer and 59 had a Grade 4 ulcer (Table 1).
formulation of the PCR product was checked using
a Qubit Fluorometer 3.0. Nanopore sequencing was Organisms isolated in culture
subsequently used on a 1 µg DNA template, and the Fifteen pathogens were isolated from the 177 tissue
results were analysed using the EPI2ME 16S analysis biopsies collected (Table 2): 112 gram-negative, 43
workflow. Finally, 16S rRNA sequencing results were gram-positive and 1 fungus; 42 reports came back
compared with the results obtained from the culture showing ‘no growth of bacteria’. Pseudomonas species
method and the fluorescence imaging device. has the highest occurrence (40 samples), followed by
Enterococcus (22) and Klebsiella (20) species.
Statistical analysis
The sample size was calculated to be 164 at a 99% Inferences
confidence level, with a 10% margin of error. Cor- The device picked up 111 wounds with gram-nega-
relation between the gram type obtained from fluo- tive bacteria, 25 with gram-positive bacteria and 15
rescence imaging device and the tissue biopsy culture wounds with both gram-positive and -negative bac-
was assessed using Cohen’s Kappa method in IBM’s teria. A further 26 wounds had no bacterial burden.
SPSS software. The results from the device were com-
pared with the culture results for gram type identifica- Statistical data
tion, and the multi-class performance metrics, such Cohen’s kappa was used to determine the probability
as accuracy, sensitivity and specificity were calculated of an agreement between gram type results obtained
using standard formulas (28). from both the semi-quantitative culture test and the
multispectral imaging device in 177 tissue samples.
RESULTS A good agreement was found between both meth-
Clinical characteristics of the subjects ods, ĸ = .774, 95% CI, [-.0359 to .121], p< .001.
A total of 157 diabetic foot ulcer patients (104 males, The results of the machine learning algorithm are
Table 4: Machine learning performance of the Illuminate device
GN GP NG GP/GN
PPV 78.38 76.00 92.31 86.67
NPV 92.42 97.37 88.08 95.68
Accuracy 83.62 94.35 88.70 94.92
TPR (Sensitivity) 94.57 82.61 57.14 65.00
TNR (Specificity) 71.76 96.10 98.52 98.73
GN= Gram Negative; GP= Gram Positive; TP= True Positive; FP= False Positive; TN= True Negative;
FN= False Negative; TPR= True Positive Rate; TNR=True Negative Rate; PPV= Positive Predictive Value;
NPV= Negative Predictive Value
summarised in the confusion matrix provided in sequencing and culture method, the culture method
Table 3. The class-averaged accuracy of the device gave positive results in 17 cases. In these 17 cases,
was found to be 90.4%, with a positive predictive the species identified by the culture method corre-
value of 78.38% for detecting gram-negative bacteria, lated with at least one of the species identified by
76.00% for gram-positive bacteria, 86.67% for pol- the 16S rRNA sequencing results in 16 cases. The
ymicrobial (GP&GN) bacterial burden and 92.31% device also showed significant levels of bacterial bur-
for no significant bacteria burden (no growth). The den, which correlated with both the culture and 16S
negative predictive value was found to be 92.42% rRNA results. However, the culture method showed
for gram-negative bacteria, 97.37% for gram-positive no growth in several cases (9 cases), where the 16S
bacteria, 95.68% for polymicrobial bacterial burden rRNA results showed significant reads (>50 reads for
and 88.08% for no growth. The sensitivity for gram- at least one organism) in five samples. Interestingly,
negative bacteria was 94.57%, 82.61% for gram- in four of the five cases, the fluorescence imaging
positive, 65% for polymicrobial and 57.14% for no device was still able to show the presence of bacterial
growth. Specificity for gram-negative was 71.76%, burden, demonstrating the superior sensitivity of the
96.10% for gram-positive, 98.73% for polymicrobial device, as compared to the culture.
and 98.52% for no growth. It should be emphasised
that, since the accuracy of the gold standard culture DISCUSSION
method is not 100%, the accuracy of the fluorescence Understanding bacterial burden is important for
imaging device could be potentially much higher. the effective management of diabetic foot ulcers
The device’s imaging results and comparisons with and preventing downstream complications. Earlier
the culture report for various wounds with gram-pos- studies have shown that the clinical evaluation of
itive bacteria, gram-negative bacteria and no growth signs and symptoms (CSS) has a low accuracy rate for
are shown in Figure 2. identifying wounds with moderate to heavy bacterial
burdens (23). CSS is typically based on the clinical
16S rRNA sequencing results signs of critical colonisation (NERDS) or infection
To better assess the performance of the fluorescence (STONEES) (29). The accuracy results vary widely
imaging device in the detection of bacterial burden, among the studies, ranging from <30% (23) to <80%
16S rRNA sequencing was conducted to provide (29), hence, a better technique is needed for accu-
more information on the polymicrobial species pre- rate wound bioburden assessment. It is well known
sent in the wound, as compared to a standard cul- that pathogens emit autofluorescence when excited
ture test. The results of 16S rRNA sequencing were with UV and violet/blue regions of light during the
compared with the results obtained by the fluores- phase of growth and infection (21-22). This weak
cence imaging device and the culture method for the autofluorescence is typically studied using standard
presence/absence of significant bacterial bioburden. spectrofluorometers, which are both expensive and
Of the 26 biopsies compared with the 16S rRNA bulky, making them difficult to adopt in a point-of- 
189
care setting (30). A novel portable and handheld mul- rate swabbing by identifying areas with the highest
tispectral imaging device was developed for rapid and levels of bacterial burden, leading to a better under-
non-invasive bacterial burden assessment at the bed- standing of the overall microbial burden, especially
side in under 2 minutes (31-31). The device detects for polymicrobial wounds, for further therapeutic
bacteria that have infected the tissue and are express- interventions. In addition, the device helps doctors
ing significant metabolic activity, as the device mainly to identify bacterial burdens away from the wound,
targets NAD(P)H and flavins. The device integrates which can potentially be missed by visual inspection
multiple wavelengths of light both in the UV-A and alone.
violet regions and captures weak autofluorescence at Despite the above, the manual interpretation of mul-
different spectral emission bands using high optical ti-spectral autofluorescence images is difficult, subjec-
density to minimise background autofluorescence. tive and requires significant training. This cumber-
These spectral images are fed to an image processing some process might pose severe challenges in terms
and machine learning algorithm that compares these of ensuring the accuracy of decisions. This is where
images and identifies significant bacterial burden re- machine learning plays a vital role, as it corrects for
gions based on the differences in autofluorescence the background noise caused by tissue autofluores-
intensities at various spectral bands before classifying cence and automatically classifies infected regions as
them based on the gram type. gram-positive or gram-negative. The colour-coding
aids in effective spatial understanding of pathogen
This study was carried out to detect the accuracy presence in the wound. Typically, it is assumed that
of the device in comparison with the current gold the bacterial burden is present at the centre of the
standard culture method and proved that the device wound, at the site of maximum slough, but the device
is capable of accurately detecting and distinguishing was frequently able to detect the presence of patho-
the gram type of bacteria within 2 minutes. Thus, gens, including pseudomonas, away from wound, at
fluorescence imaging device can be used as a first- the peri wound site, providing hints about the reasons
line screening solution, especially when access to behind delayed wound healing.
microbiology labs is problematic (33). In addition,
the results show that the device has a better sensitiv- Limitations
ity and specificity in identifying bacterial burden, The device has the following limitations. Bone, ten-
compared to the standard CSS technique. The device don and fat regions of wounds have autofluorescence
also enables evidence-based continuous monitoring of their own, primarily contributed by collagen. The
of patients’ wounds during hospitalisation, rather algorithm may misrepresent these regions as infected,
than visual inspection, making wound tracking easier. due to high levels of autofluorescence. Hence, the
At present, it is difficult to accurately assess if infected results from the device must be clinically correlated
tissues have been removed completely post debride- in such scenarios. The presence of betadine and other
ment using CSS alone. Fluorescence imaging can aid cleaning solutions that are fluorescent in nature will
in effective debridement (25), as demonstrated in also interfere with these weak autofluorescence sig-
earlier studies, and the device used in the present nals; hence, the wound must be thoroughly cleaned
study was also able to improve debridement efficiency with saline solution before imaging. In future,
by providing immediate feedback regarding the status through advanced image processing and machine
of bacterial burden after debriding each layer. This learning techniques, it may be possible to compen-
also saves healthy viable tissue from excessive debride- sate for the interference from betadine fluorescence
ment, improving wound healing. Further, the device and extract the bacterial fluorescence signatures to as-
can be used to check whether a wound area is free of sess the bioburden, especially at high bacterial loads.
bacterial burden before tissue grafting. Surgical sutures and cotton dressing remaining on
wounds might also predict a false bacterial burden,
There are several instances when a wound may ap- hence one needs to be cautious while imaging.
pear infected clinically, but the culture report shows
no growth. This could be the result of concurrent CONCLUSION
antibiotic therapy, the ability of culturing to pick only The device is a first-of-its-kind device that uses mul-
out certain types of bacteria or a defective wound tispectral autofluorescence imaging combined with
swabbing/biopsy site (34-37). Devices such as the machine learning for the rapid detection of bacte-
novel multispectral imaging device can guide accu- rial burden and gram type classification on diabetic
foot wounds. The device demonstrated >90% class- Funding

averaged accuracy, compared to the current gold No exclusive funding was provided by Adiuvo Di-
standard culture method in gram type classification. agnostics or any other entity for the clinical studies.
In addition, the device can pick up bacterial bur-
dens, even in cases the culture report has missed, as Ethical statement
confirmed by 16S rRNA sequencing. Thus, the de- The authors are accountable for all aspects of the
vice can guide doctors towards guided debridement, work, including ensuring that questions related to
the right first-line treatment protocol and improved the accuracy or integrity of any part of the work are
wound tracking. appropriately investigated and resolved. The trial
was conducted in accordance with the Declaration
IMPLICATIONS FOR of Helsinki (as revised in 2013). The study was con-
CLINICAL PRACTICE ducted at Hycare Hospital, in Chennai, India, after
n Doctors and clinicians can obtain an instant obtaining institutional ethics committee approval
understanding of the bioburden on the wound (IEC 031#HYC/IEC/2018). All data were collected
and gram type of infecting bacteria. with patient consent prior to their participation in the
study. The study was registered with the Clinical Trial
n The device aids in effective wound debride- Registry of India (Reg. No. CTRI/2018/10/016147).
ment.
Key messages
n Doctors can optimise dosages and prescriptions n A novel multispectral imaging device is a first
based on the bacterial type and bioburden of its kind device which uses multispectral
present on the wound. autofluorescence imaging combined with
machine learning for rapid detection of
Further research bacterial burden and gram type classification
Areas for further research could include: on the diabetic foot ulcers.
n Large-scale studies correlating the performance
of the device with the results from metagenome n The device demonstrated >90% class-averaged
studies. accuracy as compared to the current gold
standard culture method in gram type
n Quantification of the bioburden using a classification and can pick-up bacterial burden
fluorescence imaging device. even in cases where the culture method has
missed as confirmed by 16S rRNA sequencing.
n Longitudinal tracking of bioburden and
correlations with wound healing in ulcer n It can assist doctors towards right first line
patients. treatment protocol, guided debridement, and
effective wound tracking. m
Acknowledgments
The authors thank Vignesh Devaraj, Clinical Re-
search Intern, who was pivotal in data collection and
documentation.
191
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ILLUMINATE ®

Ulcers Using a Novel Artiﬁcial Intelligence DOI: 10.1177/15347346211047098

Enabled Noninvasive Device

Vijay Viswanathan, MD, PhD, FICP, FRCP(London),

Figure 2. Percentage of treatment procedures.

Utility of a real‑time fluorescence imaging Corresponding author:

Received: October, 2019

Introduction techniques, all organisms may not be isolated especially in

Figure 3b: Fluorescence at initial assessment Figure 3c: Day 5 of follow-up

Figure 3d: Day 10 of follow-up

Figure 4c: Reduction in fluorescence evident after the initial cleaning

Declaration of patient consent

Financial support and sponsorship

the investigative/financial burden, especially in resource‑poor References

J Surg 2017;23:86‑91. First‑in‑human results. PLoS One 2015;10:e0116623.

A New Device and Technology for Detecting Bacterial Infection and its

Received: 6 January 2021 / Accepted: 24 November 2021

Introduction administer the right antibiotics at the right time; in other

Fig. 1 Illuminate: image of the

Table 3 Comparison of Culture (standard)/new device Growth according to culture report

P value < 0.001, highly significant association.

Rapid handheld screening device to

Radhakrishnan, Geethanjali, Gupta, Aayush, King, John,

Geethanjali Radhakrishnan, Aayush Gupta, John King, Devina Ganvir, "Rapid

Event: SPIE BiOS, 2020, San Francisco, California, United States

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[email protected]; www.adiuvodiagnostics.com, Phone +91-8015316313

Photonics in Dermatology and Plastic Surgery 2020, edited by Bernard Choi,

Proc. of SPIE Vol. 11211 112110V-1

2. FLUORESCENCE SPECTRA OF CLINICALLY RELEVANT PATHOGENS

Proc. of SPIE Vol. 11211 112110V-2

Figure 1: Excitation Emission Matrix of clinically relevant bacteria and fungus.

Proc. of SPIE Vol. 11211 112110V-3

Malassezia sp. + ALA

3. DEVELOPMENT OF MULTISPECTRAL FLUORESCENCE IMAGING DEVICE

3.1 Device Block Diagram:

Proc. of SPIE Vol. 11211 112110V-4

3.1.1 Fluorescence Imaging Module:

Fig. 4: Block diagram of Fluorescence Imaging Module

3.1.2 Control System:

3.1.3 Image Acquisition Module:

Proc. of SPIE Vol. 11211 112110V-5

3.1.5 Image Processing Module:

3.1.6 Pathogen Classifier Model:

4. CLINICAL STUDY TO VALIDATE THE MULTISPECTRAL IMAGING DEVICE

Proc. of SPIE Vol. 11211 112110V-6

Table A: Summary of bacteria isolated through standard culture method.

Proc. of SPIE Vol. 11211 112110V-7

Correct classification Incorrect classification

Gram positive bacteria 15 14 1

Gram negative bacteria 12 11 1

Microbiology result Total no. of sites Imaging device result

Correct classification Incorrect classification

Proc. of SPIE Vol. 11211 112110V-8

Proc. of SPIE Vol. 11211 112110V-9

Correspondence: Dr Rajesh Kesavan, [email protected]

Figure 1: Overview of Illuminate multi-spectral autofluorescence imaging platform for pathogen

Multiple Light source Biomarker - Emission Machine Learning Algorithm

Gram positive Gram negative

Table 1: Age, gender, and ulcer grade distribution

Fig. 1 Illuminate: image of the

Table 3 Comparison of Culture (standard)/new device Growth according to culture report