Real-Time Fluorescence Wound Imaging for Bacterial Presence

This LCD supplements but does not replace, modify or supersede existing Medicare applicable National Coverage Determinations (NCDs) or payment policy rules and regulations for Real-Time Fluorescence Wound Imaging for Bacterial Presence. Neither Medicare payment policy rules nor this LCD replace, modify or supersede applicable state statutes regarding medical practice or other health practice professions acts, definitions and/or scopes of practice. All providers who report services for Medicare payment must fully understand and follow all existing laws, regulations and rules for Medicare payment for Real-Time Fluorescence Wound Imaging for Bacterial Presence and must properly submit only valid claims for them. Please review and understand them and apply the reasonable and necessary provisions in the policy within the context of the manual rules. Relevant bureaucratic regulations are referenced below.

Social Security Act References:

• Title XVIII of the Social Security Act, Section 1862(a)(1)(A) states that no Medicare payment may be made for items or services which are not reasonable and necessary for the diagnosis or treatment of illness or injury.
• Title XVIII of the Social Security Act, Section 1862(a)(7). This section excludes routine physical examinations.

CMS Internet-Only Manual (IOM) Citations:

• CMS IOM Pub. 100-02, Medicare Benefit Policy Manual,
• Chapter 16, Section 10 General Exclusions from Coverage
• CMS IOM Publication 100-03, Medicare National Coverage Determinations (NCD) Manual,
• Chapter 1, Part 4, Section 270 Wound Treatment
• CMS IOM Pub. 100-04, Medicare Claims Processing Manual,
• Chapter 12, Section 20.3, Bundled Services/Supplies
• Chapter 12, Section 30, Correct Coding Policy
• Chapter 12, Section 30A, Coding Based on Standards of Medical/Surgical Practice
• Chapter 30, Section 50 Advance Beneficiary Notice of Non-coverage (ABN)
• CMS IOM Publication 100-08, Medicare Program Integrity Manual,

Chapter 13, Section 13.5.4 Reasonable and Necessary Provision in an LCD

This contractor has determined Real-Time Fluorescence Wound Imaging (RTFI) for locating bacterial wound pathogens, bacterial load of >10⁴ CFU/g, and measurement of wound size with or without clinical darkness to be noncovered.

RTFI is marketed as a point-of-care (POC) adjunctive technology intended to aid in the visualization of areas of suspected elevated bacterial burden and to assist with wound measurement and documentation. However, the available evidence does not demonstrate that RTFI independently diagnoses wound infection, accurately determines bacterial burden, or results in clinically meaningful improvements in patient health outcomes when compared with standard wound assessment and management.

Additionally, FDA clearance and manufacturer labeling restrict the use of RTFI to adjunctive imaging in combination with clinical signs and symptoms. These regulatory parameters do not support clearance for use as a diagnostic test or as a stand-alone method for assessing bacterial burden or infection.

Accordingly, RTFI for bacterial presence, location, load, and measurement of wound size with or without clinical darkness does not meet Medicare’s statutory requirement of being reasonable and necessary under §1862(a)(1)(A) of the Social Security Act and is therefore noncovered.

BACKGROUND

Generally accepted wound management practices are based on a systematic, multidisciplinary approach aimed at optimizing conditions for healing and preventing complications.¹ Core principles include: comprehensive patient and wound assessment; identification and management of underlying etiologies and comorbid conditions; appropriate wound cleansing and debridement to remove devitalized tissue to reduce biofilm-burden; maintenance of a moist wound environment through appropriate dressing selection; management of exudate and edema; protection of the wound from further trauma or pressure; and ongoing monitoring for clinical signs of infection.^2-5 Treatment decisions are guided by clinical evaluation and standardized care protocols, with therapies tailored to wound type, severity, and patient-specific factors to promote timely, effective healing and functional recovery.¹

Description of the Intervention

Real-time fluorescence wound imaging (RTFI) devices are handheld medical imaging instruments designed for POC use to detect elevated loads of bacteria in wounds based on known intrinsic fluorescence characteristics. The devices are comprised of a high-resolution color LCD display and touch-sensitive screen with integrated optical filters and microelectronic components. These devices use technology to enable real-time digital imaging and fluorescence imaging of wounds and surrounding healthy skin in patients.

The handheld fluorescence imaging device emits a violet light under which bacteria at loads greater than 10⁴ colony-forming units (CFU)/gram (both planktonic and biofilm-encased) produce detectable fluorescence signals in real time.^6,7 When illuminated at 405 nm from violet-blue light-emitting diodes (LEDs), porphyrins produced by 87.5% of chronic wound pathogens (Staphylococcus aureus (S. aureus) and Gram-positive bacteria)⁸ and pyoverdines from Pseudomonas aeruginosa (P. aeruginosa) and other Gram-negative bacteria reportedly cause the wound to fluoresce red or cyan, respectively.⁹ Additionally, fluorophores within the wound can excite up to a depth of 1.5 mm.¹⁰ Overall, the positive predictive value for red and cyan fluorescence signals in detecting bacteria in chronic wounds has been reported as 93–100%.¹¹

Under 405 nm violet light, other tissues can fluoresce due to the presence of red blood cells or extracellular matrix proteins such as collagen, elastin, and fibrin. The intensity and shade of this light differs from that of bacterial fluorescence, which allows it to be distinguished. Collagen-rich tissues such as tendon or bone and high-fibrin slough tissue glow bright green to white. Skin appears as a less intense green (dependent on skin color, as darker skin tones absorb more violet light due to melanin), flaky skin as green with white edges, and blood appearing as a dark maroon color.⁷

RTFI is most effective when used in a dark room for imaging because light contamination can cause false positives or misinterpretation of fluorescence.⁹ If the room itself cannot be adequately darkened, a black drape can be used. The ambient light sensors in some RTFI devices let the operator know if correct light settings are reached. Wounds are imaged at a 90° angle and from 8 to 12 cm. A range finder is included in the device, which confirms the appropriate distance for imaging. After the images are captured on the camera, the fluorescent signals are displayed on a digital touch screen and interpreted by the user. Images and videos can also be browsed in the image library and uploaded to the patient’s electronic records.¹²

In addition to its fluorescence imaging capability, RTFI devices are also able to measure the dimensions of chronic wounds. The MolecuLight DX device has an auto-tracking capability that allows for graphing wound size over time with imaging sessions.¹³

REGULATORY STATUS

The Food and Drug Administration (FDA) initially granted a de Novo classification for MolecuLight i:X™ device (DEN180008) on February 16, 2018,¹⁴ followed by clearance for marketing (K191371) through the FDA Premarket Notification process on December 4, 2019.¹⁵ According to FDA labeling, the device is indicated as a tool for clinicians to view and digitally record images and measurements from the wound and images of fluorescence emitted from a wound when exposed to an excitation light.¹⁵ A newer model, the MolecuLight DX™, received clearance for marketing (K211901) on July 21, 2021, for the same indications.¹⁶

The FDA approved indication for MolecuLight i:X™ states that it is a handheld imaging tool that allows clinicians diagnosing and treating skin wounds, at the POC, to:

1. View and digitally record images of a wound;
2. Measure and digitally record the size of a wound; and
3. View and digitally record images of fluorescence emitted from a wound when exposed to an excitation light.

The fluorescence image, when used in combination with clinical signs and symptoms, has been shown to increase the likelihood that clinicians can identify wounds containing bacterial loads >10⁴ CFU/g as compared to examination of clinical signs and symptoms alone. However, the FDA specifies that “The MolecuLight i:X™ device should not be used to rule out the presence of bacteria in a wound. The MolecuLight i:X™ does not diagnose or treat skin wounds”.¹⁷

A wound autofluorescence imaging (AFI) device is a tool to view autofluorescence images from skin wounds that are exposed to an excitation light. The device is not intended to provide quantitative or diagnostic information (DEN180008). Additionally, the device is a tool-type device intended for capturing and recording both white light and fluorescent images of wounds, and it performs this function without making clinical or diagnostic claims. A potential benefit is the ability to capture and document wound images over time to support monitoring of wound changes. However, according to the FDA, no direct clinical benefit has been demonstrated from the recording of fluorescence images alone; however, a device that records fluorescence images may provide supplementary information to clinicians as part of their overall assessment.¹⁴

TERMINOLOGY

Bacterial load is the quantity of bacteria in a given sample or area, such as a wound.

Biofilms are defined as coherent clusters of bacterial cells embedded in a biopolymer matrix, which, compared with planktonic [freely suspended] cells, show increased tolerance to topical (antiseptics) and systemically (antibiotics) administered antimicrobials and resist the antimicrobial properties of host defenses.¹⁸

Chronic wounds are defined in this policy as wounds that fail to epithelialize due to some physiologic impairment (e.g., disruption of the wound healing cycle from impaired angiogenesis, innervation, or cellular migration among other reasons).¹⁹

Real-time fluorescence is a technique that measures fluorescent signals at the POC to monitor biological or chemical processes as they happen.

Wound debridement is the process of removing dead tissue from wounds. The tissue is removed using instruments such as tweezers, a curette, or a scalpel. An enzyme-based gel is sometimes applied too, to help clean the wound.

EVIDENCE CERTAINTY METHODOLOGY

This contractor evaluated peer-reviewed, evidence-based literature reporting the clinical validity and utility of fluorescence imaging using the principles of the GRADE (Grading of Recommendations Assessment, Development and Evaluation) methodology.²⁰ The PICO parameters documented below were prespecified to ensure transparency and reproducibility:

Population: Adults with 1 or more acute or chronic (>4 weeks) wounds of mixed etiologies [e.g., venous leg ulcer (VLU), diabetic foot ulcer (DFU), pressure ulcers (PU), surgical, burns]

Intervention (Index test): Real-time fluorescence wound imaging with clinical darkness (e.g., MolecuLight i:X™ or MolecuLight DX™)

Comparator (Reference Standard): Standard of care (SOC), other POC wound imaging procedures, deep tissue biopsy culture, or wound swab microscopy and culture

Outcomes (critical/important): Diagnostic accuracy, wound healing, adverse effects (e.g., overprescribing of antibiotics), pain, prognostic prediction, healthcare utilization (debridement frequency, length of stay), quality of life (QOL), change in treatment plan (surrogate for Dx accuracy and/or wound healing)

SEARCH PROTOCOL

A literature search was conducted using PubMed or Ovid versions of MEDLINE to identify peer-reviewed, evidence-based studies evaluating fluorescence wound imaging. Additional studies were identified through reference list review. Separate searches were conducted for clinical guidelines. Searches were limited to English language publications from the past 10 years.

Key search terms included:

• Wound*
• Diabetic foot ulcer *
• Venous ulcer*
• Pressure ulcer *
• Surgical wound
• Burn*
• Fluorescence imag*
• Autofluorescence imag*
• Bacterial fluorescence imag*
• Bacterial autofluorescence imag*
• Point-of-care bacterial autofluorescence imag*
• Real-time fluorescence imag*
• MolecuLight

SUMMATION OF DISCOVERED EVIDENCE

The studies included for analysis evaluated diagnostic accuracy or clinical outcomes associated with real-time (POC) fluorescence wound imaging and comprised 28 observational studies (including several post hoc analyses), 2 randomized controlled trials (RCTs), 3 systematic reviews, and 1 scoping review. Additional contextual evidence included several clinical guidelines, including a Delphi-consensus based guideline. Case reports or small case series, commentaries/editorials, narrative reviews, product promotional materials, poster presentations, abstract-only publications, economic studies, pre-clinical studies, white papers, unpublished studies, and protocols were excluded from the review.

Diagnostic Accuracy

RTFI versus Reference Standard

A total of 8 publications were identified that assessed the diagnostic accuracy of RTFI compared to a reference standard.^21-28 Industry funding and/or potential conflicts of interest were disclosed in three studies.^25-27 Sample sizes ranged from 14 to 178, with all but one trial including <50 participants. Wound tissue sampling was also sparse, with most studies reporting <60 swabs. The ages (mean or range) of participants were not routinely reported, compromising judgments about applicability to the U.S. Medicare population. Quantitative tissue biopsy culture, most commonly described as the reference (or “gold”) standard for assessing wound bioburden because it provides an absolute bacterial load (CFU/g), was employed in a single study.²² The remaining studies employed microbial culture methods, with a single trial including quantitative polymerase chain reaction (qPCR).²⁸ Risk of bias (ROB) was assessed using the QUADAS 2 tool.²⁹ Overall, there was very low certainty evidence due to high ROB, and serious indirectness and imprecision across the included studies that RTFI has moderate accuracy for detecting bacterial presence in different types of wounds, with no clear advantage over standard swabbing.

Hurley et al conducted a pilot, prospective, observational trial (N=33, 43 swabs) that sought to establish the accuracy of a wound imaging device in detecting pathogenic bacteria in wounds.²¹ Patients (N=33, mean age 62.2 years) were selected from a plastic surgery wound care outpatient clinic. An RTFI device (MolecuLight i:X) was the index test compared to a reference standard of microbiological swabs. RTFI demonstrated moderate but clinically unreliable accuracy for standalone diagnostic accuracy for detecting the presence of bacteria, with no measurable difference in detection of bacteria. The RTFI device had a sensitivity of 100% and specificity of 78% at identifying pathological bacteria presence in wounds on fluorescent light imaging. The positive predictive value (PPV) was 95.4%. The negative predictive value (NPV) was 100%. It demonstrated a sensitivity and specificity of 100% at detecting the presence of Pseudomonas spp. The authors noted issues with the use of the RTFI device that raised concerns about the feasibility of use in routine clinical practice settings. The imaging device produced black areas on the image in areas of highly vascularized tissue, or in cases of dressings that contained silver. The DarkDrape needed to ensure the production of a quality image was single use only, rendering it impractical for everyday use, according to the authors. Methodological limitations included a high ROB (non-independent sampling, suboptimal reference standard, uncertainty about the flow and timing), indirectness (single population, single center, specialty clinic), and imprecision (small sample size). Additionally, the study design limits the ability to distinguish diagnostic accuracy vs. clinical adjunct utility.

Koo et al investigated the diagnostic accuracy of RTFI (MolecuLight i:X), with a tissue biopsy culture system as the reference standard.²² This was a prospective, observational, single-center (Korea University Guro Hospital) study of 35 patients with 48 diabetic foot ulcers (DFUs). RTFI had an overall sensitivity of 64.1%, specificity of 55.6%, PPV of 86.2%, NPV of 26.3%, and overall accuracy of 62.5%. For P. aeruginosa, the device showed a sensitivity, specificity, PPV, and NPV of 66.7%, 87.2%, 54.6%, and 91.9%, respectively, with an accuracy of 83.3%. For non-Pseudomonas bacteria, the device showed a sensitivity, specificity, PPV, and NPV of 43.8%, 62.5%, 70.0%, and 35.7%, respectively, with an accuracy of 50.0%. While these results indicate a high PPV (i.e., positive results are more likely to reflect true positives) for RTFI, there was limited diagnostic accuracy due to moderate to low sensitivity, poor specificity, and very low NPV (poor rule-out performance). This study was judged to have a high ROB, mainly due to the index-guided biopsy sampling, unclear blinding/independence of image interpretation, and analysis of 48 wounds with repeated measurements in some patients without clear adjustment. There was very serious imprecision as the study included only 35 patients and 48 wounds, and only 9 culture-negative wounds were available for estimating specificity. Additionally, the confidence intervals reported are wide for overall sensitivity and specificity, which is consistent with imprecision. There were some concerns about indirectness, as well. The workflow included manufacturer interpretation and a sampling strategy that may not mirror usual clinical deployment of the device, so applicability to everyday practice is not clear.

Okeahialam et al published a secondary analysis of an observational study.²³ In this analysis, the investigators sought to evaluate the detection of significant bacterial burden using RTFI in 52 women diagnosed with known perineal wound infections following childbirth. Ninety-five percent (95%) of patients in the study were treated with antibiotics prior to perineal clinic review before imaging and targeted sampling. All participants received imaging with the MolecuLight device and wound swabs, with imaging findings compared to culture-confirmed bacterial burden. In this analytic subset, RTFI showed a sensitivity of 83%, specificity of 90%, PPV 92%, and NPV 80%. However, interpretation of these predictive values is limited by the study population. Because all enrolled participants had clinically diagnosed perineal wound infections, the prevalence of the target condition was substantially enriched, which may have artificially inflated the PPV and reduced the informativeness of the NPV. Consequently, the reported predictive values may overestimate rule-in performance and do not permit reliable estimation of the device’s false-positive rate in a broader wound-care population. Therefore, while RTFI performed comparably to the reference standard as a diagnostic test in this highly selected cohort,these findings may not be generalizable to the general wound-care population with a lower base rate of >10⁴ burden. Interpretation of the results is further complicated by concurrent antibiotic treatment, which may have reduced culture sensitivity and increased the likelihood of false-negative culture results. The study was judged to have a high risk of bias (ROB), driven primarily by index-guided sampling, use of an imperfect reference standard, substantial post-enrollment exclusions from the diagnostic accuracy analysis, and the fact that most participants received antibiotics before both imaging and microbiologic testing. There was also serious indirectness because the evidence applies specifically to postpartum perineal wound infections rather than other wound types. In addition, the diagnostic target was moderate-to-heavy bacterial burden identified through semi-quantitative swab and suture analyses, which is related to, but not identical with, the gold-standard construct of quantitative bacterial burden or biopsy-confirmed tissue infection. Although the full cohort included 80 women, the diagnostic accuracy analysis was limited to 52 participants, including 30 positive and 22 negative results according to the study's reference definition. The relatively small sample size contributes to serious imprecision and limits confidence in the reported diagnostic accuracy estimates.

Olbrich et al completed a prospective, single-site study, which included 107 examinations in 46 left ventricular assist device (LVAD) patients, in evaluating the performance of RTFI (Moleculight i:X) for diagnosis of driveline infections (DLIs).²⁴ Microbiological wound swabs were the reference standard. Using Moleculight i:X, 19 of 107 examinations (17.76%) were read as positive, whereas microbiological examination was positive for microorganisms in 74 of 107 examinations (69.16%). The most commonly identified organism was Staphylococcus aureus. When fluorescence imaging was compared with microbiology, there were 10 true positives, 9 false positives, 64 false negatives, and 24 true negatives. Reported diagnostic performance was sensitivity 13.51%, specificity 72.73%, PPV 52.63%, and NPV 27.27%. These findings show that RTFI underperforms relative to the reference standard and demonstrates poor diagnostic accuracy, driven primarily by severe under-detection (false negatives). The authors also reported that subgroup analyses by dressing type and prior antibiotic treatment did not show any relevant difference in performance. The conclusion was that Moleculight i:X had low sensitivity and specificity for detecting DLI in the outpatient setting, and that clinical examination and swabs should remain the gold standard in this context. However, the gold standard microbiologic test for diagnosing a wound infection is a quantitative culture of tissue obtained from a deep tissue biopsy (or curetted tissue specimen, rather than a superficial wound swab.³⁰

Study limitations included a high ROB across all domains recorded in QUADAS 2. There was also serious indirectness as the study addresses a specific outpatient population with suspected driveline infection, and the comparator was swabbing microbiology, which may detect colonization or physiological flora rather than definitively establishing clinically important DLI. Serious imprecision was noted. Although there were 107 examinations, these arose from only 46 patients. The extremely poor observed sensitivity with a high number of false negatives suggests unstable and clinically concerning performance in this setting.

An interventional, single-arm comparative study was conducted at a tertiary diabetes center to assess the diagnostic accuracy of the Illuminate® multispectral autofluorescence device for classifying wound infection by Gram type versus standard tissue culture methods.²⁸ The study enrolled 178 persons with type 2 diabetes and DFUs, producing 203 tissue samples from color-coded wound regions selected by the device’s AI output and sampled by the attending plastic surgeon. The device used multispectral excitation (370, 395, and 415 nm) and a machine-learning algorithm to classify wound regions as Gram-negative, Gram-positive, mixed Gram-positive/Gram-negative, or no growth, with image acquisition taking about 20 seconds and the result displayed in under 2 minutes. Tissue samples from the device-indicated regions were analyzed using standard microbiology methods including Gram stain, culture, semiquantitative plate counts, and susceptibility testing. Among the 203 samples, culture identified 69 Gram-negative, 57 Gram-positive, 49 mixed Gram-positive/Gram-negative, and 28 no-growth samples. The device correctly classified 168 of 203 samples and misclassified 35, yielding an overall accuracy of 89.54%. Reported class-specific performance was: Gram-negative sensitivity 91.30%, specificity 82.14%, PPV 80.77%, NPV 92.00%; Gram-positive sensitivity 77.19%, specificity 92.71%, PPV 86.27%, NPV 87.25%; and no growth sensitivity 81.48%, specificity 98.41%, PPV 91.67%, NPV 96.12%. The multiclass area under the curve (AUC) was 0.86. The authors concluded that the device showed good overall accuracy for early infection screening and Gram-type classification and may help clinicians identify tissue sampling regions, assess wounds qualitatively before and after debridement, and support early treatment decisions while awaiting definitive culture results. This study suggests moderate-to-good diagnostic classification accuracy for the studied AI-enabled autofluorescence device, but it does not establish equivalent performance for other RTFI devices e.g., MolecuLight. This study does not show superiority over the microbiologic reference standard. Culture and Gram-based methods remained the comparator used to establish whether the device was correct. The device’s main potential advantages over the reference standard were operational, not evidentiary superiority. This study was judged to have a high ROB. The main driver was index-test–guided reference sampling, which limits confidence that the reported performance reflects independent diagnostic classification. Indirectness was rated as serious. The study evaluates Illuminate®, not other types of RTFI, and focuses on Gram-type classification rather than simple detection of bacterial burden. The study was also confined to a single-center DFU population and excluded osteomyelitis.

Localization of Bacterial Signal

In addition to overall diagnostic performance of RTFI compared to a reference standard, 3 of the 8 studies evaluate whether fluorescence signals are associated with the spatial localization of bacterial burden within wounds.^25-27

Rennie et al reported on a prospective, single-blind, non-randomized clinical trial, evaluating whether red fluorescence detected by RTFI (MolecuLight i:X) predicts the presence of bacteria in chronic wounds.²⁷ Sixty patients with chronic lower limb wounds (predominantly DFUs and VLUs) were included in the study. The reference standards were quantitative PCR (biopsy; gold standard) and semi-quantitative culture. Key findings were: PPV of red fluorescence: 100% (30/30 in biopsy arm; 30/30 in curettage arm); no false positives; detected bacterial loads ranged from 10⁴ to 10⁸ CFU/g, with most samples showing moderate-to-heavy growth; and multiple clinically relevant pathogens were identified, most commonly Staphylococcus aureus. The authors concluded that red fluorescence reliably identifies clinically significant bacterial burden (≥10⁴ CFU/g) and enables real-time localization of bacteria to guide sampling and treatment at the POC. However, overall diagnostic accuracy is indeterminate (i.e., sensitivity, specificity, and NPV cannot be determined). Additionally, no diagnostic superiority was demonstrated. RTFI was not compared independently to biopsy/culture. RTFI could not show higher sensitivity than qPCR or improved classification vs reference. The study was determined to have a high ROB for all QUADAS 2 domains except for the reference standard. Additionally, there was serious indirectness (applies only to red fluorescence-positive wounds) and imprecision (sample size was insufficient for full accuracy assessment).

Raizman et al performed a prospective, single-blind, single-center clinical study evaluating whether cyan fluorescence detected by RTFI (MolecuLight i:X) predicts the presence of Pseudomonas aeruginosa (PA) in chronic wounds.²⁶ The study included 28 patients with cyan-positive wounds; the most common wound type was venous leg ulcer [VLU] (60.7%), followed by DFU (14.3%) and surgical site wounds (7.1%). Culture confirmed PA in 26 of 28 cyan-positive wounds, yielding a positive predictive value (PPV) of 92.9% for cyan fluorescence as an indicator of PA. Most positive cultures showed moderate-to-heavy growth of PA; specifically, 18 wounds (64.3%) had heavy growth, and 6 wounds (21.4%) had moderate growth. The study also reported that less than 20% of wounds positive for PA had classic clinical signs of pseudomonal infection, such as a greenish tinge on dressings, and a sweet smell was not observed. The authors concluded that cyan fluorescence could help rapidly identify and localize PA at the point of care (POC). Therefore, while this study supports a high PPV for a cyan-positive RTFI signal, it does not establish the overall degree of diagnostic accuracy of RTFI because it did not assess sensitivity, specificity, or NPV. This study was judged to have a high ROB for overall diagnostic accuracy assessment. There was also serious indirectness (the evidence applies specifically to cyan-positive chronic wounds) and serious imprecision (small sample).

A prospective, observational, pilot study by Pijpe et al compared the detection of bacteria in burn wounds between RTFI (MolecuLight i:X) and standard microbiological swabs.²⁵ A total of 14 patients with 20 wounds, who were admitted to a hospital burn center, were included in the study. Each wound underwent three swabs at one timepoint: standard (clinician-selected), fluorescence-guided (high-fluorescent), and non-fluorescent. Of the 20 wounds, 9 (45%) had positive swab results in either of the three swabs and 11 (55%) showed a high fluorescent area. Overall, positive and negative proportion agreement between standard swab and high fluorescent swab sample results were 100%. Sensitivity, specificity, PPV, and NPV for presence of high fluorescence were 78%, 64%, 64%, and 78%, respectively. For Pseudomonas aeruginosa detection, these results were 100%, 70%, 44% and 100%, respectively. Overall, there was moderate diagnostic accuracy. Device performance was similar to standard swabbing (no clear added value). There was potential utility in ruling out Pseudomonas (high NPV), but poor rule-in value. ROB assessment was high across all the QUADAS 2 domains. There was wide uncertainty around estimates (serious imprecision), as there were only 20 wounds with 9 positive cases. There was also serious indirectness (burn-only population; the reference standard measures the presence of organisms, not necessarily clinically meaningful infection).

RTFI versus Clinical Signs and Symptoms

There were 10 publications related to 7 primary studies that assessed the diagnostic accuracy of RTFI with or without clinical signs and symptoms (CSS) compared to the assessment of CSS alone.^8,31-39 Studies ranged in size (number of participants or number of wounds) from 19 to 1000, with 70% of publications having sample sizes of <100. None of the studies reported a mean age of the participants as >60 years. Industry funding was disclosed in 60% of the publications. Tissue biopsy and/or microbiological analysis served as reference standards in 6 of 9 studies. Three studies did not include a reference standard. ROB was assessed using the QUADAS 2 tool and C extension, when appropriate.^29,40 Overall, there is moderate to very-low certainty evidence that the use of RTFI in conjunction with CSS may result in an increase in diagnostic accuracy compared to CSS alone.

An industry-sponsored prospective, multi-centered, controlled study, the Fluorescence imaging Assessment and Guidance (FLAAG) study, enrolled 371 and analyzed 350 participants, who were treated at advanced wound care centers for various types of ulcers.³⁶ This trial compared CSS+RTFI (MolecuLight:X) and CSS, with tissue biopsy as the reference standard. Across all wound types, the addition of FL to CSS improved sensitivity (61.0% [95% CI, 55.3–66.6%]) to detect wounds with bacterial loads >10⁴ CFU/g by fourfold compared to CSS alone (15.33% [95% CI, 11.16–19.50]; p < 0.001). The specificity and PPV of CSS+RTFI were comparable. The NPV and accuracy of CSS+RTFI were significantly increased by 64.4% and 2.2-fold, respectively, compared to CSS (p < 0.001). This study was judged to have a high ROB. The reference standard (punch biopsy) was guided by the result of the index test for some of the wounds, which may have led to an incorporation bias. There was no serious indirectness or imprecision.

Three post hoc analyses of the FLAAG trial, which were limited to exploratory hypothesis generation and were not generalizable to clinical practice, were included in this assessment of diagnostic accuracy. Armstrong et al found the sensitivity to detect bacterial loads using CSS ranged from 9.8% to 11.1%, while CSS+RTFI showed a sensitivity of 71.6% to 92.6% as bacterial loads increased from 10⁴ to 10⁸ CFU/g.³¹ Sandy-Hodgetts et al conducted a post-hoc analysis of patients (n=58) with surgical site wounds.³⁸ The CSS assessment had a sensitivity of 6.8% for detecting bacterial loads greater than 10⁴ CFU/g. In contrast, RTFI improved sensitivity to 38.6%. Expert clinicians using RTFI technology achieved a further improved sensitivity of 77.3%, with an overall accuracy of 75.9%. Johnson et al assessed whether RTFI improves detection of clinically significant bacterial burden (>10⁴ CFU/g) compared with standard clinical signs and symptoms (CSS), and whether diagnostic sensitivity varies by skin pigmentation.³⁵ While bacterial burden did not differ by skin tone, clinical detection varied substantially by pigmentation. Sensitivity of CSS alone was low pigmentation14.0%, medium 24.3%, and high 2.9%. The addition of RTFI increased sensitivity 4.4-fold (low), 2.9-fold (medium), and up to 12-fold (high pigmentation). There was a modest loss of specificity. CSS alone had high specificity (93-100%), while RTFI had moderate specificity (78-100%). Overall, these post hoc analyses were further limited by the high ROB associated with the parent trial, concerns about indirectness, and the small number of events resulting in imprecise data.

A small (N=19), industry sponsored, single-center, prospective pilot study evaluated diagnostic accuracy when RTFI was used in combination with CSS for identifying wounds with moderate-to-heavy bacterial load (>10⁴ CFU/g).³⁹ Similar to the overall results of the FLAAG trial, RTFI increased sensitivity threefold (CSS = 22%; RTFI = 72%) and accuracy from 26% to 74% (P=0.002) for detecting moderate-to-heavy bacterial loads compared to CSS alone. This trial does provide useful pilot evidence that adding RTFI to CSS improves sensitivity and accuracy for detecting high bacterial burden, but it was judged, using the QUADAS 2 tool, as having a high ROB (patient selection, index test conduct, and flow/verification domains). Additionally, there were indirectness/applicability concerns (single site, enriched sample i.e., 95% high burden) and serious imprecision (small number of events). Therefore, the diagnostic accuracy estimates should be treated as exploratory, not definitive.

DasGupta et al conducted a prospective trial involving 21 inpatients at 2 sites in Canada.³² RTFI detected an additional 35% additional wounds (9 wounds) that were negative for CSS. This study was judged to have a high ROB and imprecision due to the small number of reported events.

Hill et al reported on a prospective multi-site trial involving participants with a total of 43 chronic wounds that evaluated the utility of incorporating RTFI into the checklists for signs of superficial/local and signs of deep/spreading infection.³³ Based on the findings of microbiology swabs, an 11 additional wounds were identified as positive for bacterial loads greater than 10⁴ CFU/g compared with CSS criteria alone. In addition to the small sample size resulting in imprecise data, the study was determined to have a high ROB.

Jacob et al performed a cross-sectional retrospective analysis of real-world data involving 1000 chronic wounds (e.g., DFU, VLU, PU, surgical, burns).³⁴ RTFI signals indicating elevated bacterial loads were observed in 701 wounds (70.8%), while only 293 (29.6%) showed signs/symptoms of infection. Neither tissue biopsies nor microbiology cultures were acquired to confirm accuracy of RTFI. This study was further limited by the single time-point analysis and a high ROB.

An industry-funded, prospective, single-center, single-blind study of 45 patients enrolled (40 patients evaluated) the diagnostic accuracy of CSS and RTFI against biofilm identification, as validated by gold standard scanning electron microscopy (SEM) imaging and microbiology.³⁷ RTFI demonstrated superior sensitivity (84%) and accuracy (63%) compared to CSS (sensitivity 44% and accuracy 43%). Limitations include concerns about indirectness (single site), imprecision (few events), and a high ROB due to the patient selection method, which used a case-control-like sampling.

A single-center prospective trial, which included 29 adults with non-healing DFUs, evaluated the use of RTFI to visualize bacteria in and around the wound bed and to guide swabbing during the clinical assessment of DFUs, compared with the CSS.⁸ Diagnostic accuracy measures for identifying clinically relevant bacteria found RTFI had a significantly greater specificity compared to CSS (0.78 [0.54, 0.91] vs. 0.38 [0.18, 0.62]; p = 0.0043). RTFI and CSS were comparable for all other diagnostic accuracy measures (sensitivity, PPV, NPV, accuracy, and diagnostic odds ratio). This study was judged to have a high ROB due to concerns about the reference standard (Levine swabbing technique), indirectness (unclear if the patients matched the target population), and imprecise data.

RTFI versus Biofilm Blotting

Two prospective, observational, industry-funded studies compared diagnostic accuracy metrics for RTFI and wound blotting methods.^37,41 Overall, the certainty of the evidence is very low for diagnostic accuracy outcomes.

Mayer et al evaluated the diagnostic accuracy of RTFI (MolecuLight) and wound blotting against biofilm identification as validated by gold standard SEM imaging and microbiology analysis.³⁷ A total of 45 patients were enrolled (40 analyzed). Patient ages ranged from 30-85 years. RTFI demonstrated superior sensitivity (84%) and accuracy (63%) compared to biofilm blotting (sensitivity 24% and accuracy 40%). Biofilm blotting exhibited higher specificity (64%). The certainty of the evidence was downgraded for serious ROB and serious indirectness. ROB concerns arise from non-consecutive patient selection with deliberate enrichment of biofilm-positive and negative cases, partial verification of index tests across participants, and potential dependence between index testing and reference standard sampling, all of which may inflate diagnostic accuracy estimates. Indirectness is present due to the use of a composite reference standard (SEM combined with microbiological thresholds) for which no universally accepted gold standard exists, and due to conduct in a single specialized wound care setting with a predominance of DFUs, limiting generalizability. The evidence was further downgraded for serious imprecision given the small sample size and wide confidence intervals around sensitivity and specificity estimates.

Wu et al compared POC wound blotting with alcian blue grading versus RTFI for biofilm detection in 53 participants (mean age 65.1 years) with chronic wounds.⁴¹ Results were validated using standard wound microbiological culture. The modified wound blotting method showed a strong association with wound culture results (Spearman’s rho = 0.641, p < 0.001), while MolecuLight i:X™ showed a weaker association (Phi (ϕ) = 0.335, p = 0.015). Using QUADAS 2 and C tools, this study was judged to have a high ROB, driven primarily by subjective index‑test interpretation without blinding and use of an imperfect reference standard. Additionally, there was serious indirectness (a single-center Taiwanese cohort) and imprecision (small sample size, with wide uncertainty around predictive estimates).

Studies comparing RTFI to microbiologic reference standards show mixed, inconsistent, and often moderate accuracy, with no clear advantage over standard methods. The overall certainty of evidence is low to very low, driven by small sample sizes, high risk of bias, and differences in how studies were conducted and interpreted. When used with clinical signs and symptoms, RTFI may improve detection of higher bacterial burden; however, these findings are based on studies with important limitations and limited applicability. Comparisons to approaches such as biofilm blotting are similarly based on small, heterogeneous studies, and there is a lack of strong and consistent evidence across settings.

While these studies report diagnostic accuracy metrics for detecting bacterial presence, RTFI detects fluorescence signals rather than directly identifying bacteria or infection. These signals are used to infer bacterial burden, and this relationship is indirect and variable across studies. In addition, evidence evaluating whether fluorescence signals accurately identify the spatial location of bacterial pathogens within wounds is limited and uncertain.

The device is limited to detecting signals from superficial tissue depths (approximately 1.5 mm), meaning deeper or undermined bacteria may not be identified, and it does not provide information about specific bacterial species or nonbacterial pathogens.^33,42,43 In addition, RTFI may not detect all bacteria, and results require correlation with clinical assessment.^42,43 Technical and environmental factors, such as lighting conditions and image capture variability, may also affect interpretation.^21,25,26

Taken together, these limitations and evidence gaps make it unclear how reliably the device can identify the location and extent of bacterial burden, and its clinical validity remains uncertain.

Wound Measurement Validation

Raizman et al⁴³ evaluated the MolecuLight i:X as a digital wound assessment tool capable of measuring wound dimensions. The wound measurement component was validated using 17 wound models of known dimensions and repeated measurements by 5 trained clinicians, followed by testing on 17 clinical wound images. Wound area was determined using calibrated digital planimetry, whereby 2 reference stickers were placed adjacent to the wound, a standardized image was captured, and the software automatically or manually traced the wound perimeter to calculate area, maximum length, and maximum width. The study demonstrated high measurement performance, with wound area accuracy exceeding 94%, length and width accuracy exceeding 95%, and low intra- and inter-user variability. In clinical use, digital planimetry was successfully performed in 96% of wounds and was found to be substantially more accurate than conventional ruler-based length × width calculations, which overestimated wound area by approximately 31% on average and by as much as 52% in some cases. The authors concluded that the device could be readily incorporated into routine wound care to provide objective, reproducible wound measurement and documentation.⁴³

Using QUADAS-2, this study would be considered at high ROB, primarily due to manufacturer sponsorship, involvement of investigators employed by the device manufacturer, and limitations in patient selection and reference standard methodology. Validation was conducted largely using wound models and stored clinical images rather than a prospective consecutive clinical cohort, and wound measurements were not compared against an independent gold-standard planimetric measurement system. Applicability concerns are considered moderate because the measurement technique is directly relevant to clinical practice, although the validation environment may not fully reflect routine clinical use.⁴³

Consistent with these QUADAS-2 findings, the overall certainty of evidence would be rated as low. Although the study demonstrated high technical accuracy and reproducibility, confidence in the estimates is limited by serious ROB, potential publication bias, and serious indirectness arising from validation against wound models and stored images rather than prospective clinical outcome standards. Consequently, the available evidence suggests that the MolecuLight i:X provides accurate and reproducible wound measurements and is likely superior to conventional ruler-based assessment; however, independent external validation studies using recognized reference standards in prospective multicenter clinical settings are needed to increase confidence in these findings and establish their generalizability.⁴³

A prospective observational study (n=27 wounds, 2-week follow-up) compared manual measurement (ruler: length × width) and digital measurement (MolecuLight i:X).⁴⁴ Measurement outcomes were absolute wound area at each visit and wound area reduction (WAR) over time. For the longitudinal monitoring of healing (WAR), there was no meaningful difference between manual WAR: −3.80 cm² (−46.88%) versus digital WAR: −2.62 cm² (−46.05%) [% change: p = 0.9057; absolute change: p = 0.2017]. Manual ruler-based measurement demonstrated systematic overestimation (~23%) of wound area compared with digital measurement via MolecuLight, which was statistically significant at single timepoints. However, the relative change in wound area over time (WAR) did not significantly differ between methods, showing that despite absolute differences, relative healing trajectories (WAR) were essentially equivalent. This suggests that consistent measurement bias may not materially affect longitudinal healing assessment. The certainty of evidence was rated as very low for serious ROB (no independent reference standard), serious indirectness (single examiner), and serious imprecision (small sample, feasibility study). However, this study compares measurement methods rather than true accuracy against a reference standard. There is a lack of studies evaluating whether RTFI accurately measures wound dimensions relative to known standards. As such, there is insufficient evidence to determine with confidence the analytic validity of the wound measurement function.

Clinical Outcomes and Utility

Wound Healing

RTFI versus Standard of Care

A total of 5 studies were identified, which assessed healing-related outcomes for various types of wounds, comparing RTFI to a standard of care (SOC).^6,42,45-47 There were no significant industry funding or potential conflicts of interest disclosed in the included studies. Sample sizes ranged from 35 to 229, with 3 of the 5 trials including >100 participants. The mean/median ages of participants ranged from 42 to 78 years. ROB for RCTs was assessed using the ROB 2 and ROBUST-RCT tools.^48,49 Overall, there is low to very low certainty evidence that RTFI may result in clinically meaningful improvement in wound healing.

Randomized Controlled Trials (RCTs)

A single-center, prospective pilot RCT involving 56 patients with active DFUs and no suspected clinical infection, compared standard care informed by RTFI (MolecuLight i:X) versus standard care alone over 12 weeks.⁴⁷ The primary outcome was the proportion of ulcers healed at 12 weeks, assessed by a blinded outcome assessor. In the intention-to-treat analysis, 45% (13/29) of ulcers healed by 12 weeks in the RTFI arm versus 22.2% (6/27) in the control arm. Median wound-area reduction at 12 weeks was 91.3% in the RTFI group versus 72.8% in the control group. At 4 weeks, wound-area reduction was similar (40.7% vs 38.6%). While these findings indicated that adding RTFI to standard DFU care was associated with a numerically higher 12-week healing proportion and greater 12-week wound-area reduction, the authors explicitly stated that the study was a pilot and was not powered to show a difference in the primary outcome. This study was judged to have a high ROB due to the open label design (possible differences in care and concordance between groups related to knowledge of treatment assignment). Serious imprecision was present due to the pilot design, which was not adequately powered to provide definitive evidence. Overall, this pilot RCT provides low-certainty evidence that RTFI-guided management may improve healing outcomes in diabetic foot ulcers, but interpretation is limited by small sample size, pilot design, and risk of bias related to lack of clinician/patient blinding and potential differences in co-interventions.

A single-center, patient‑blinded RCT (N=200) compared RTFI (MolecuLight i:X)–assisted debridement vs standard surgical debridement in adults with chronic wounds.⁴² RTFI was used intraoperatively to guide repeated debridement until the residual bacterial area was <10%. Standard single-pass surgical debridement was guided by surgeon judgment. RTFI-assisted debridement significantly reduced bacterial burden (RTFI = 6.8%, SOC = 30%, p<0.001) and was associated with shorter healing time (49.2 vs 63.0 days; p<0.001), complete healing at 12 weeks (87% vs 80%), These findings are suggestive of clinically meaningful benefit in time-to-healing (accelerated by ~14 days; ~22%) and the likelihood of closure at 12 weeks (+7%).

However, certainty is downgraded for risk of bias. Treating surgeons could not be blinded following randomization, creating the potential for performance bias. Importantly, the intervention protocol required repeated fluorescence-guided debridement until a predefined bacterial threshold (<10% residual bacterial area) was achieved, whereas the control group underwent a single-pass debridement based on surgeon judgment. Consequently, the observed benefit may reflect not only the diagnostic value of RTFI but also differences in treatment intensity, procedural thoroughness, and clinician behavior that resulted from knowledge of treatment allocation. Thus, the trial evaluates an RTFI-guided debridement strategy rather than the imaging modality in isolation, making it difficult to fully separate the effect of fluorescence imaging from the effect of more intensive, target-driven debridement.

Certainty is also downgraded for indirectness/generalizability concerns, as the study was conducted at a single center and may not fully represent outcomes achievable across broader wound care settings with different patient populations, clinical workflows, and provider experience. Although the treatment effects were statistically significant and clinically relevant, the evidence base currently consists of a single study, leaving some residual uncertainty regarding the magnitude and reproducibility of benefit.

This RCT provides low-certainty evidence that RTFI-guided debridement may improve wound healing outcomes compared with standard debridement; however, confidence in the magnitude of benefit is limited by potential performance bias arising from differential treatment intensity between study groups, imprecision due to the absence of confidence interval reporting for key clinical outcomes, and uncertainty regarding the generalizability of findings from a single-center study.

Observational Studies

A retrospective pre/post interventional cohort study of 167 pressure injuries (PIs) in 100 Medicare beneficiaries in long-term care (LTC)/skilled nursing facilities, compared standard of care (SOC) vs RTFI–guided care (MolecuLight i:X).⁶ At 12-weeks follow-up, the comparative healing rates were 38.5% (RTFI) vs 22.5% (SOC); a 16% absolute difference (p=0.007) favoring RTFI. The time to healing also favored RTFI vs SOC (12.4 vs 17.2 weeks; 4.8 weeks faster; ~27.7% reduction; p=0.043). There was a trend toward improved healing favoring RTFI over SOC. The likelihood of healing (HR) was 1.40 overall, with an adjusted HR of 1.80. Low-certainty evidence suggests RTFI-guided care may improve healing outcomes. Confidence is limited by the nonrandomized design, and potential temporal and practice-pattern confounding.

A retrospective pre/post observational study of 229 lower-extremity (primarily diabetic foot) wounds treated over 2 sequential 12-month periods before and after implementation of RTFI (MolecuLight i:X) reported on wound healing rates.⁴⁶ The 12-week healing rate during the RTFI period was 48% and was 39% during the pre-implementation (SOC) phase. This represents a modest 9% absolute difference in favor of RTFI. The certainty of evidence was rated as low due to the observational design.

Thirty-eight adult burn patients undergoing split‑thickness skin grafting (STSG) after clinically and microbiologically “clean” wound bed preparation were included in a single-center prospective observational study that compared graft outcomes associated with intraoperative RTFI and swab microbiology.⁴⁵ RTFI-positive areas resulted in 27.9% graft success per cm². RTFI-negative areas had a 99.2% graft success per cm² rate. RTFI -positive was predictive in 100% of graft failure cases, while swab microbiology predicted 31% of failures. The certainty of evidence was very low, down rated due to serious indirectness (graft integration was viewed as a surrogate for wound healing) and serious imprecision (small number of events).

Clinical Management Outcomes

RTFI versus Standard of Care/Clinical Signs and Symptoms

A total of 10 studies were identified, which quantitively assessed outcomes representing changes in clinical management for various types of wounds, comparing RTFI to a SOC, including CSS.^{32-34,36,39,43,47,50-52} Three studies reported industry funding or potential conflicts of interest. Sample sizes ranged from 19 to 1000. ROB was not formally assessed; however, key methodological and interpretive limitations were described. Overall, this analysis found RTFI is associated with evidence of a consistent impact on clinical decision-making in the management of various types of wounds. However, the evidence is limited to an intermediate outcome pathway (diagnosis → management change) rather than final health outcomes. The evidence is, therefore, indirect with respect to net health outcomes, as no controlled data link these changes to improved healing or reduced complications.

Randomized Controlled Trials (RCTs)

Rahma et al employed a single-center, pilot RCT (N=56) comparing standard care plus RTF (MolecuLight) vs SOC alone in patients with non‑infected DFUs.⁴⁷ Change in management was reported in 56% of patients with positive imaging at baseline. Ongoing changes with repeat imaging were reported as 50% at week 4 and 27% at week 8. The most common change reported was additional/targeted debridement. The more important limitations were imprecision (small pilot study), potential confounding (e.g., higher off-loading in intervention arm), no consistent use of adjunct therapies following imaging, and there was no formal statistical significance testing for the outcome.

Observational Studies

DasGupta et al conducted a prospective implementation study conducted at 2 inpatient hospital sites including 21 patients (26 wound assessments) with complex wounds of mixed etiology.³² The addition of RTFI (MolecuLight) to CSS was compared to CSS alone. Treatment plans changed in 35% of assessments (9/26 wounds) after RTFI. This result exceeds the predefined threshold for meaningful impact (10%), representing a moderate effect. Types of changes included additional microbiologic testing (targeted sampling), initiation of systemic or topical antibiotics, and expanded debridement/cleaning. The main interpretive limitation was the minimal direct evidence of improved patient-important health outcomes.

Hill et al performed a prospective, multisite observational study including 43 chronic wounds of mixed etiology assessed using clinical infection checklists (UPPER/LOWER) with and without RTFI (MolecuLight).³³ The integration of RTFI into structured clinical assessment increased detection of clinically relevant bacterial burden and influenced treatment selection (care was escalated in 13.9% of cases), including debridement, antimicrobial use, and escalation of care; however, direct patient outcome measures (e.g., healing) were not systematically assessed.

Jacob et al described a retrospective, single time‑point observational analysis of ~1000 chronic wounds evaluated across 211 wound care facilities in the U.S., comparing treatment plans before and after RTFI (MolecuLight).³⁴ Overall, 53.3% of wounds had ≥1 management change after RTFI. Types of management changes included wound bed preparation (more extensive debridement [18.7%], targeted (RTFI-guided) debridement [17.2%], more extensive cleansing/hygiene [17.2%]), antimicrobial and therapeutic decisions (new topical therapies [10.1%], new systemic antibiotics [9.0%]), diagnostic refinement (RTFI-guided microbiology sampling [6.2%]), and dressing changes [3.2%]. Quantitatively, the impact on antimicrobial prescribing showed an increase in topical therapy use by 37.7% (p < 0.0001) and systemic antibiotic use by 47.3% (p < 0.0001). The lack of assessment of associated clinical outcomes was a major limitation of this study.

Le et al conducted a multicenter, prospective diagnostic accuracy study (N=350) that evaluated RTFI (MolecuLight) versus standard clinical assessment (CSS for detecting clinically significant bacterial burden (>10⁴ CFU/g), using quantitative biopsy culture as the reference standard.³⁶ The use of RTFI at the POC altered treatment plans in ~69% of wounds, changed diagnosis of bacterial burden in ~52%, influenced wound bed preparation in ~85%, affected downstream care decisions (e.g., debridement 48%, antimicrobials 53%, sampling location 45%), and reported improvement in overall patient care in ~90% of wounds. These management changes and “improvements in patient care” were based on anecdotal information i.e., derived from post-assessment clinician surveys, not objective outcome measures. Additionally, the study does not demonstrate improved patient-centered outcomes (e.g., healing, infection rates, amputation), which is a key limitation.

A post hoc analysis of 350 chronic wounds sought to evaluate whether reliance on CSS to guide treatment decisions leads to inappropriate antimicrobial prescribing patterns in chronic wound care.⁵⁰ Antimicrobials were prescribed in 73.1% of all wounds. Prescribing patterns were similar regardless of clinical indication (CSS+ wounds: 75.0%, CSS– wounds: 72.8%, p=0.75). There was poor alignment with clinical indications. One third of patients receiving systemic antibiotics had no CSS. Antibiotic prescribing did not correlate with bacterial load. Only 66.7% of high-burden wounds (>10⁸ CFU/g) received antimicrobials. A primary limitation of this analysis was the lack of a comparator (e.g., RTFI).

A post hoc, multicenter comparison analyzed subset consisted of 78 hard-to-heal wounds drawn from the 350-wound FLAAG cohort.⁵¹ The study aimed to compare the microbiology of tissue incisional biopsies taken from a SOC wound center site versus a RTFI–guided site in hard-to-heal wounds. The aim was to determine whether RTFI helps identify a more informative biopsy location. RTFI-guided biopsy had a reported sensitivity of 98.7%, compared with 87.2% for SOC biopsy (p=0.0059 by McNemar test) for detecting any bacterial loads ≥10⁴ CFU/g. RTFI-guided biopsies also detected more bacterial species on average than SOC biopsies (3.03 species versus 2.2 species, respectively, p<0.001). Additionally, RTFI-guided biopsies identified a higher average number of antibiotic resistance pathogens of concern than SOC biopsies (1.7 versus 1.4, p=0.002). The authors concluded that RTFI provides a more accurate and relevant microbiological profile for guiding tissue sampling than clinical judgment alone, especially in larger and more complex wounds. In addition to limitations associated with the parent study, this post hoc analysis had a high risk of selection bias. The cohort subset was not randomly or consecutively selected. Further, the additional sampling process increased the risk that the analyzed subset overrepresented wounds where SOC sampling was less suitable.

Serena et al performed a prospective, single-center pilot study (N=19 wounds; primarily VLUs) evaluating RTFI in addition to CSS for detecting moderate-to-heavy bacterial burden (≥10⁴ CFU/g), confirmed by molecular microbiology (qPCR/16S sequencing).³⁹ Clinician-reported impact on clinical management included treatment plan modification (73% of cases), influences on wound care (debridement, antimicrobial selection, sampling location, and cleansing), stewardship of antimicrobial decisions was altered in ~47% of cases, and 95% of overall patient care was judged to have been improved. In addition to the anecdotal nature of the reported results, key limitations affecting interpretation include the very small sample size, single-center design, and single-visit study without follow-up to evaluate whether management changes improved outcomes.

Raizman et al⁴³ conducted a prospective observational study evaluating the use of a fluorescence imaging device (MolecuLight i:X) in 50 wounds from 39 patients with various chronic wound types. Fluorescence signals were observed in 72% of wounds, and fluorescence-guided curettage sampling identified higher bacterial loads compared with standard Levine swab sampling. In a subgroup of diabetic foot ulcers, use of the device resulted in additional fluorescence-targeted debridement following standard care, with 17 of 20 wounds requiring further debridement and all demonstrating residual fluorescence after initial debridement. The study was limited by small sample size, industry sponsorship, and indirectness due to the absence of diagnostic accuracy metrics and lack of clinical outcome assessment.

Trafalet et al conducted a prospective, multicenter audit of antimicrobial prescribing practices across outpatient wound centers (N=1438 wounds), comparing SOC versus SOC plus RTFI.⁵² Data were collected at a single initial visit. Compared with SOC alone, the use of RTFI reduced systemic antibiotic use substantially per-patient (SOC = 47.6% vs RTFI = 8.2%), shifted prescribing toward topical/local therapies (e.g., antimicrobial dressings) by 92% with RTFI versus 64% with SOC, and reduced the overall intensity of prescribing per patient (mean RTFI = 1.0 vs SOC = 1.4). Key limitations affecting confidence in the reported results include the single-visit snapshot that did not permit an assessment of treatment effectiveness or outcomes, the absence of patient-centered endpoints (healing, infection resolution, complications), and the lack of adjustment for wound severity or case-mix differences.

Healthcare Utilization Outcomes

RTFI versus Standard of Care/Clinical Signs and Symptoms

Three studies were identified that quantitively reported outcomes representing changes in healthcare utilization (hospitalization, antibiotic use) for various types of wounds compared RTFI to a SOC, including CSS.^6,42,46 There was no reported industry funding or potential conflicts of interest by any author group. Sample sizes ranged from 100 to 229. ROB was not formally assessed; however, key methodological and interpretive limitations were described. Overall, this analysis found RTFI is associated with evidence of a consistent favorable impact on healthcare utilization with the management of various types of wounds. However, the evidence is limited to low/very low certainty due to the preponderance of observational designs, indirectness, and the risk of confounding.

Randomized Controlled Trials (RCTs)

A single-center, patient-blinded RCT (N=200) compared perioperative RTFI (MolecuLight)–assisted debridement versus standard surgical debridement in adults with chronic wounds.⁴² RTFI-assisted debridement was associated with statistically significant reductions in multiple utilization metrics relative to standard care. The mean length of hospital stay was reduced by ~4 days (17.5 ± 9.3 vs 21.5 ± 12.5 days; p < 0.01). Fewer overall operations took place during hospitalization (2.3 vs 2.7; p ~ 0.03). There was a shorter duration of antibiotic use of 3.2 days in the RTFI-assisted group (15.5 ± 8.7 vs 18.7 ± 6.7 days; p < 0.01). While the randomized design supports causal interpretation, key limitations include the single-center/single-operator setting, partial lack of blinding intraoperatively, and the potential imbalance in wound etiology (e.g., more DFUs in the control group).

Observational Studies

Healthcare utilization outcomes were reported in a retrospective pre/post cohort study of 167 pressure injuries from 100 Medicare beneficiaries in long-term care/skilled nursing facilities, comparing SOC versus RTFI–guided management.⁶ Infection-related, wound-associated hospitalization was reduced by an absolute difference of 12.7% in the RTFI cohort (4.2% vs 16.9%; p=0.007). There was a shift in the use of local therapy, with nearly half of RTFI-treated wounds receiving topical-only antibiotics (46.4%). Overall antibiotic prescribing differed significantly between groups (p<0.0001), reflecting a change in resource utilization rather than simple volume reduction. The strength of inference is limited by study design (retrospective, non-randomized), potential confounding (temporal trends, practice changes, patient differences), and multiple wounds per patient.

A retrospective pre/post observational analysis of 229 lower extremity wounds treated in a UK podiatry clinic before and after implementation of routine POC RTFI (MolecuLight i:X) described healthcare utilization outcomes.⁴⁶ There was a 33% (67% to 45%) decrease in patients requiring antibiotics following implementation of RTFI. Antimicrobial dressing use (a resource utilization proxy) was reduced almost by half (85% to 44%). Study limitations included the observational design (susceptibility to confounding), the single-center setting (indirectness), and the lack of direct measurement of hospitalization.

Patient-Reported Outcomes

RTFI versus Any or No Comparator

Two studies were identified that provided data regarding patient-reported outcomes for lower extremity wounds that describe the use of RTFI.^47,53 ROB was not formally assessed; however, key methodological and interpretive limitations were described. Overall, this analysis found there is sparse evidence that RTFI, when used in the management of DFUs and VLUs, is associated with a favorable-neutral impact on patient-reported outcomes. The evidence is judged to be limited to low/very low certainty due to the preliminary nature of the studies and associated methodological concerns.

Pain Intensity

Pain intensity, using the 0-10 numeric rating scale (greater is worse pain), was reported in a prospective, single-center observational study of 46 adults with VLUs (baseline pain ≥4/10).⁵³ The authors evaluated the relationship between bacterial burden (via RTFI) and patient-reported pain, and assessed changes following RTFI-informed treatment. Pain immediately following treatment was reduced by 1.3 points (p < 0.0001). The next day following RTFI-informed intervention, the mean pain score had reduced by 3.4 points compared to baseline. Pain and bacterial fluorescence overlapped in 87% of patients, suggesting a mechanistic relationship. There was significant heterogeneity of response. While pain decreased in 52% of patients, almost half the cohort reported no change (~39%) or increased pain (~9%). Methodological limitations that reduced certainty in the reported results included the absence of a control group, small sample size, short follow-up, and unblinded subjective outcome reporting. Interpretively, absolute benefit was observed, but comparative effectiveness vs. SOC cannot be determined.

Quality of Life

Rahma et al reported quality of life (QOL) outcomes in a pilot RCT (N=56) that compared SOC versus SOC plus RTFI (MolecuLight) in patients with DFUs.⁴⁷ QOL was assessed using the EQ-5D-5L (generic health-related QOL) and Diabetic Foot Ulcer Scale–Short Form (DFS-SF) (disease-specific QOL). There were modest changes in disease-specific QOL over time. The RTFI group showed a small improvement that was most pronounced at 4 weeks, while the SOC group showed a decline that was most pronounced at 12 weeks. For generic QOL, there were minimal changes in both groups, with no clear between-group effect. Study strengths include the randomized design and use of validated QOL instruments. Limitations were the small (pilot) sample size that was underpowered for QOL outcomes and the short-term follow-up.

Systematic Reviews

Three systematic reviews and a single scoping review were identified as relevant for this analysis. The quality of the systematic reviews was evaluated utilizing the AMSTAR 2 tool.⁵⁴ The scoping review, however, was not appropriate for a formal critical appraisal.

Badrie et al narratively performed a systematic review of 17 observational studies, including 3 case reports, involving 834 patients in any healthcare setting with an infected or non-healing wound (DFU, VLU, burn, surgical).⁵⁵ RTFI was compared to CSS and white light inspection in 5 studies. Reference standards included microbiology swabs (9 studies), tissue biopsies (3 studies), modified wound blotting (1 study), and 4 studies did not specify a test. The reviewers reported on diagnostic accuracy. Across all studies, RTFI exhibited sensitivity ranging from 45% to 100% and specificity between 55.6% and 92.3% for detecting bacterial burden in wounds. PPV varied from 64.0% to 95.4%, while NPV ranged from 35.3% to 100%. Four studies found RTFI to be significantly more accurate than CSS and CSS and white light in detecting bacterial loads, while one study did not report statistical significance. RTFI showed high accuracy in detecting Pseudomonas aeruginosa, with a sensitivity of 66.7% and specificity of 87.2% (p < 0.001). Five studies investigated the impact of RTFI on treatment. The studies consistently report RTFI's capacity to inform clinicians about bacterial burden in the wound, leading to refinements in treatment plans that promote effective wound closure. Limitations included the observational designs of all studies, the quality appraisal tool (Evidence-Based Literature [EBL] checklist) was not appropriate for the analysis, significant methodologic heterogeneity, and the lack of standardized diagnostic thresholds for bacterial fluorescence positivity. The overall quality rating was judged to be critically low (ROB was not assessed and the quality of the individual studies was not considered in the review).

Berenguer-Pérez et al systematically reviewed a total of 19 studies (6 observational studies included RTFI) that compared arrange of non-culture-based methods for microbial detection and CSS.⁵⁶ Microbiological culture was the reference standard. The reviewers concluded that RTFI combined with CSS showed improved diagnostic accuracy, particularly in differentiating bacterial loads. However, species distinction required supplementary microbiological analysis. The authors further determined that RTFI combined with CSS showed improved guidance with treatment modifications. These conclusions should be taken in the context of the limitations of the review. Four of the 6 studies were small, with <30 wounds sampled. Two studies reported on the same data set of 350 wound samples. Five of 6 studies were rated as having a high ROB. Bias assessment using QUADAS-2 identified the main sources of bias as the use of microbiological cultures or CSS as gold standards, which may skew the accuracy of index tests. Additional biases arose from retrospective study designs, antibiotic treatments at sampling, and lack of control in test interpretation that could lead to potential misinterpretations. The heterogeneity of wound types, patient selection, sample collection and processing methodologies introduced significant variability across studies, complicating the synthesis of results. Further complicating the analysis, conflicts of interest and reliance on variable ‘gold standards’ add layers of complexity to understanding diagnostic accuracy. Publication bias may further skew the data landscape. This systematic review was rated as critically low quality. The reviewers stated there wasn’t an antecedent protocol. The likely impact of ROB in individual studies was not discussed in the review.

Edwards et al performed a systematic review that aimed to determine the diagnostic accuracy of different methods currently available to identify infection in chronic wounds applicable to adult patients in a community setting.⁵⁷ Two (n=309) of the 4 included studies assessed the diagnostic accuracy of RTFI (Moleculight i:X). Additionally, enzymatic methods and staining and microscopy were other non-culture-based methods for microbial detection included in the review. Wound swab microscopy/culture and punch biopsies were reference standards. The review did not identify any methods for diagnosing infection in chronic wounds with a sufficient quality of evidence to currently recommend their use in community settings. This review was limited by the small number of studies included and a lack of generalizability. The review was judged to be of low quality as it did not assess or discuss reasons for heterogeneity observed in the results of the review.

In a scoping review, Jeffery et al summarized the current evidence on RTFI detection of elevated bacterial loads in burn wounds and explored its potential applications across all stages of burn wound management.⁵⁸ The review included 6 observational studies (N=148, 185 wounds). Populations included adult and pediatric patients with burn injuries, and the sample sizes ranged from 3 to 63 patients (14 - 77 wounds imaged). The diagnostic accuracy reported for RTFI of burn wounds ranged from 70% to 87% relative to microbial swabbing. Sensitivities ranged from 78% to 100% and specificities ranged from 64% to 98%. PPV for fluorescence imaging in burns ranged from 26% to 93%. There were several limitations associated with this non-systematic review. Sample sizes were limited, some included children resulting in a lack of generalizability. There was variation in swabbing procedures among studies. The wide range of PPV may be a result of the limited data points available. One study set the threshold for a positive microbiological sample at 10² CFU/g, which is below the threshold of detection using fluorescence imaging (10⁴ CFU/g), possibly inflating the number of false negatives.

Clinical Guidelines and Positions of National and Specialty Organizations

The available guidance supporting RTFI is limited to expert opinion and Delphi‑based consensus statements, which rank low on the evidence hierarchy and do not constitute formal clinical practice guideline development. Delphi consensus reflects the views of selected contributors and does not represent a broad consensus of the medical community.

A Delphi consensus-based statement was developed by a multidisciplinary panel of 32 wound experts across multiple clinical settings.⁵⁹ Two rounds of online questionnaires were conducted to evaluate clinical experiences with fluorescence (FL) imaging. Over 80% of panelists reported treatment plan changes, 96% reported improved wound healing due to imaging informed treatment plans, 78% reported reduced amputation rates, and 83% reported reduced rates of microbiological sampling. Based on the consensus, the guideline recommends training to perform imaging, applicability across multiple wound types (DFUs, VLUs, PUs, surgical site infections, post-operative wounds, traumatic wounds), and imaging being complimentary to clinical wound assessment and treatment. The recommendations also emphasize reliance on clinical judgement when incorporating imaging information into treatment decision making. Imaging may be done before, during, or following common wound care procedures and therapies, with suggested frequency of no more than weekly unless indicated by medical necessity. However, the consensus statement is limited by its consensus-based design, reliance on expert opinion, small panel size, potential selection bias, and inclusion of an incomplete RCT referenced within the document.

The IWII is a multidisciplinary expert consensus group focused on wound infection education and best‑practice principles and does not represent a physician specialty society or a general medical community consensus. The utility of microbiological fluorescence examination was summarized in their updated guidelines.⁶⁰ Considerations for use included: a) species can be identified and their relative locations mapped with fluorescent dyes/labels; b) only fluorescent structures can be observed; c) use is limited to microbial cell suspensions and thin tissue sections; and d) cost of dyes and probes is a limitation. Additionally, the guidelines note an alternative approach that yields rapid results, “Direct microscopy examination (Gram stain) can be performed quickly by the laboratory to assess the number and type of microorganisms present in the wound sample. This allows the clinician to commence antibiotics without delay while waiting for the culture results (identification of the specific species), which can take 24 to 48 hours.”

The National Institute for Health and Care Excellence (NICE) assessed the MolecuLight i:X™ device, reporting on the limitations of evidence and key uncertainties.⁶¹ The remaining 2 clinical guidelines did not explicitly comment on or include wound imaging technologies (e.g., MolecuLight).^3,62

Importantly, no clinical practice guidelines issued by major specialty societies, including the Infectious Diseases Society of America (IDSA), Wound, Ostomy and Continence Nurses Society (WOCN), International Working Group on the Diabetic Foot (IWGDF), Society for Vascular Surgery (SVS), or American Academy of Family Physicians (AAFP) recommend routine use of RTFI.

The available evidence evaluating real-time fluorescence imaging (RTFI; MolecuLight) for wound assessment demonstrates that, while the technology may increase detection of elevated bacterial burden compared with clinical signs and symptoms (CSS) alone, the overall certainty of evidence is low to very low and insufficient to establish clinical benefit. Diagnostic accuracy studies are limited by small sample sizes, high risk of bias, heterogeneity in reference standards, and inconsistent performance relative to microbiologic testing. Although the addition of RTFI to CSS increases sensitivity for detecting bacterial loads >10⁴ CFU/g, including in subgroups where clinical assessment may perform poorly (e.g., highly pigmented skin), this represents improvement in an intermediate diagnostic endpoint rather than demonstration of improved clinical outcomes. Evidence evaluating patient-centered outcomes, including wound healing, infection resolution, and complications, remains limited to small randomized and observational studies with methodological limitations and does not demonstrate consistent or definitive benefit.

In addition, while certain RTFI devices include functionality to measure wound dimensions, wound measurement is a routine component of comprehensive wound assessment and evaluation. The work associated with wound measurement is inherently included in the evaluation and management of the wound and is not separately payable under Medicare. Therefore, even if the wound measurement capability of an RTFI device is considered accurate, use of the device solely for wound sizing does not represent a distinct reasonable and necessary service and would be considered part of the underlying wound assessment. Evidence related to downstream clinical impact is similarly limited. While observational and pilot studies suggest that use of RTFI may influence clinical decision-making, including debridement, antimicrobial prescribing, and sampling strategies, these findings represent changes in management rather than demonstrated improvements in net health outcomes. The available data fail to establish a causal relationship between RTFI-informed care and improved wound healing, reduced infection rates, or other patient-centered endpoints.

Consideration of clinical practice guidelines and position statements further limits support for routine use of RTFI. Available guidance is primarily derived from expert opinion and Delphi-based consensus statements, which represent a low level of evidence and do not constitute formal clinical practice guidelines. While these consensus statements propose that fluorescence imaging may be used as an adjunct to clinical judgment and standard wound assessment, such recommendations are based primarily on expert opinion and low- to very low-certainty evidence and are subject to important limitations, including small panel size, potential selection bias, and reliance on subjective reporting.

Importantly, no major clinical practice guidelines issued by national or specialty organizations (i.e., including the Infectious Diseases Society of America (IDSA), Wound, Ostomy and Continence Nurses Society (WOCN), International Working Group on the Diabetic Foot (IWGDF), Society for Vascular Surgery (SVS), or American Academy of Family Physicians (AAFP)) recommend the routine use of RTFI for wound assessment or infection diagnosis. Published guidelines and specialty society recommendations instead continue to emphasize established diagnostic approaches, including clinical assessment and microbiologic testing, and note limitations and uncertainties in the evidence supporting fluorescence-based techniques.

In accordance with §1862(a)(1)(A) of the Social Security Act, items and services must be reasonable and necessary for the diagnosis or treatment of illness or injury. While RTFI demonstrates biologic plausibility and may enhance detection of bacterial wound pathogens as a diagnostic adjunct, there is insufficient evidence that its use results in improved patient-centered outcomes, including wound healing, infection resolution, or reduced complications. The evidence base is characterized by low to very low certainty, important methodological limitations, and imprecise and unreliable estimates of effect. Furthermore, there is no consistent evidence that RTFI-guided management improves net health outcomes compared with standard wound assessment and care. In the absence of high-quality evidence demonstrating clinical benefit and given the lack of endorsement from major evidence-based clinical practice guidelines, RTFI has not been established as reasonable and necessary for the Medicare population. Accordingly, Medicare coverage is not supported. Accordingly, Medicare coverage is not supported.

Please refer to the related Draft Local Coverage Article: Billing and Coding: Real-Time Fluorescence Wound Imaging for Bacterial Presence (DA60515) for documentation requirements, utilization parameters and all coding information as applicable.

N/A

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• Provider Education/Guidance
• Creation of Uniform LCDs With Other MAC Jurisdiction