WOUNDCHEK Bacterial Status (WCBS) is an in vitro diagnostic chromatographic test for the qualitative detection of bacterial protease activity directly from wound fluid samples collected with a swab. The WCBS test is intended for use in adult patients as an aid in assessing the risk for non-healing of chronic venous, diabetic foot, and pressure ulcers associated with wounds where there are no signs of wound infection and where patients are asymptomatic for clinical signs of infection. The test is intended for use with chronic wounds that are between 21 days and < 6 months of age and chronic wounds that are > 6 months of age that are < 1cm² in size.

This test is indicated for use solely by health care professionals whose clinical practice primarily or routinely involves the assessment and treatment of chronic wounds. WCBS results are intended for use in conjunction with the assessment of other known risk factors for wound healing that significantly contribute to the assessment of risk for nonhealing chronic wounds such as wound age, wound size, and vascular status.

Device Description

WOUNDCHEK Bacterial Status (WCBS) is a lateral flow assay that qualitatively detects bacterial protease activity in chronic wound fluid. Wound fluid is collected by rolling a swab over the cleansed wound surface until it is saturated. The swab is then incubated in the assay reagent which contains a substrate that can be cleaved by bacterial proteases and human neutrophil elastase (HNE) and an HNE inhibitor. The WCBS test card is used to detect cleavage products of the substrate. It consists of biotinylated bovine serum albumin (BSA) (Test Line (T)), and a control system protein, (Control line (C)), on a nitrocellulose membrane support. Neutravidin, which is conjugated to visualizing particles, binds the biotinylated end of a synthetic peptide and the biotinylated BSA test line. The test result is based on the presence or absence of a pink-to-purple colored Test Line (T) which means bacterial protease activity was detected. A negative test result is defined by the absence of a Test Line (T) which means bacterial protease activity was not detected. If the control line (C) is not present, then the test result is invalid. The device is intended for use on venous leg ulcers (VLU), diabetic foot ulcers (DFU) and pressure ulcers (PU).

A control kit, consisting of negative and positive swabs, is also available for WOUNDCHEK Bacterial Status.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study that proves the WOUNDCHEK Bacterial Status device meets these criteria, based on the provided text:

Acceptance Criteria and Device Performance

1. Table of Acceptance Criteria and Reported Device Performance

Acceptance Criteria Category	Specific Criteria	Reported Device Performance and Evidence
Precision	>95% agreement with expected results for moderate positive, low positive, and true negative panel members. Acceptable variability for high negative panel member. No significant differences in detection rate by site, operator, or day.	Met.Results from a three-site, three-operator study (total 9 operators) showed: - Moderate Positive: 98.5% (133/135) agreement- Low Positive: 95.6% (129/135) agreement- True Negative: 97.8% (132/135) agreementThe high negative panel member had 93.3% (126/135) agreement, acknowledged as variable but acceptable given its nature. No significant differences by site, operator, or day were reported.
Sample Stability	Testing of all positive and negative swabs for various storage conditions (up to 9 hours at 2-8°C and 30°C) generated expected results.	Met.Ten positive and negative swabs were tested for each storage condition (0.5, 1, 1.5, 2, 4, 6, 9 hours at 2-8°C and 30°C). All generated expected results. Interpretations were made by two blinded operators, with a total of (b)(4) determinations per condition. Daily controls met expectations.
Detection Limit (LOD)	LOD defined as the level of bacterial protease activity generating a positive result ~95% of the time (C95). C5 level also identified.	Met.Preliminary and confirmed LODs (C95, C5, C50) were established for individual proteases (V8, GelE, ZapA, LasB) and a pooled mix of these proteases, consistent with the objective of defining these detection levels. (Specific mU/test values are redacted but are reported as being established).
Cross-reactivity - Human Proteases	Positive swabs must produce a positive result in the presence of any host protease. Negative swabs must produce a negative result in the presence of any host protease.	Met.The study identified specific concentrations of human proteases (e.g., MMP-13, MMP-9, HNE, Plasmin) at which no interference was observed with WCBS performance for both positive and negative samples. If initial levels showed interference, dilutions were tested until criteria were met.
Interference - Microbial	Positive swabs (with analyte) must produce a positive result in the presence of tested microorganisms. Negative swabs must produce a negative result in the presence of tested microorganisms.	Met.The study determined specific levels of various fungi, mold, and viruses (e.g., Candida species, Aspergillus fumigatus, Herpes Simplex viruses) at which no interference was observed. If initial levels showed interference with stock solutions, log dilutions were tested until criteria were met.
Interference - Healthy Skin Flora	Implied: Device should ideally not react to normal skin flora unless these flora are indicative of bacterial protease activity leading to non-healing. However, the study identified that performance is affected by normal skin flora. This led to a labeling mitigation.	Met (with mitigation).Out of (b)(4) apparently healthy skin swab samples, 20 were positive by both operators, and an additional 4 by one operator, totaling 44 false positives out of 102 readings. While the device is affected, this finding is acknowledged as expected due to skin flora secreting proteases. The mitigation is a prominent instruction in the IFU: "Do not swab intact skin."
Interfering Substances	Positive swabs must produce a positive result in the presence of the interfering substance. Negative swabs must produce a negative result in the presence of the interfering substance.	Met.The study identified specific concentrations for a wide range of wound-related substances (e.g., acetic acid, silver dressings, Manuka honey, mupirocin, blood, various antibiotics, skin substitutes, iodine, antifungals) at which no interference was observed. If a substance failed at the initial level, it was diluted twofold until criteria were met.
Clinical Performance (Risk Assessment for Non-Healing)	Provide Positive Likelihood Ratio (PLR) and Negative Likelihood Ratio (NLR) for indication.	Met (with caveats).For the intended use population (wounds < 6 months old or ≥ 6 months old and < 1 cm²): - PLR = 2.06 (95% CI = 1.25 - 3.41)- NLR = 0.69 (95% CI = 0.54 - 0.88)A positive WCBS result resulted in a 16.6% increased risk of non-healing, and a negative result a 9.4% decreased risk. The study acknowledges limitations in establishing a clear correlation with healing due to its observational design and lack of control for numerous clinical factors or interventions.

2. Sample Size Used for the Test Set and Data Provenance

Sample Size (Clinical Study - Evaluable): 147 unique wound fluid samples from 139 unique subjects. (Initial enrollment was 350 wounds, reduced due to various exclusions, including 66 wounds from a subpopulation not supported by the claims).
Data Provenance: Prospective observational study conducted in 2016 at seven (7) sites in the U.S.

3. Number of Experts Used to Establish Ground Truth for the Test Set and Qualifications

Number of Experts: The clinical healing status (healed or not healed) was "assessed by the treating clinician." The text does not specify a number of experts or their explicit qualifications beyond being "treating clinicians" at the study sites. This implies that the clinical decision of "healed" or "not healed" served as the primary ground truth, based on their medical judgment and defined criteria.
Qualifications: "Treating clinician" with an implied expertise in wound care, as the studies involved "health care professionals whose clinical practice primarily or routinely involves the assessment and treatment of chronic wounds."

4. Adjudication Method for the Test Set

Clinical Study: Not explicitly stated for the "healing status" ground truth. It was "assessed by the treating clinician." This suggests a single clinician's assessment rather than a multi-reader adjudication process for the final outcome.
Analytical Studies (e.g., Stability, Cross-reactivity, Interference): Results were typically interpreted by "two operators (each blinded to each other's results)" for a total of 10 or (b)(4) determinations. "Any discrepant results between the operators for a given sample type was clarified by a third operator." This indicates a 2+1 adjudication method.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, an MRMC comparative effectiveness study was not done. The clinical study was an observational study comparing the device's results to the clinical healing status over time. It did not involve a direct comparison of human readers with and without AI assistance or a comparison of improvement in human reader performance. Its purpose was to evaluate the device's performance as an aid in assessing risk for non-healing, not to measure human reader effectiveness.

6. Standalone (Algorithm Only) Performance

Yes, a standalone performance was done. The entire clinical study (L.3.a) describes the performance of the WOUNDCHEK Bacterial Status (WCBS) device in isolation against the clinical healing status. The WCBS is a lateral flow assay and does not involve AI or a "human-in-the-loop" for its immediate result generation from the swab. The results (PLR, NLR) directly reflect the device's diagnostic capability.

7. Type of Ground Truth Used (Clinical Study)

Outcomes Data/Expert Clinical Assessment: The ground truth for the clinical study was the "clinical healing status of the wounds," defined as "complete wound closure" (skin re-epithelialization without drainage or dressing requirements) assessed by the treating clinician within a time frame of up to 12 weeks. This is effectively an outcome-based ground truth determined by expert clinical observation.

8. Sample Size for the Training Set

Not Applicable / Not Provided for Device Training: The WOUNDCHEK Bacterial Status is described as a lateral flow chromatographic assay. This type of in vitro diagnostic device does not typically involve "training data" in the sense of machine learning algorithms. Its design and cutoff are established through analytical studies (e.g., LOD, specificity, cross-reactivity) using predefined panels and controls, not a "training set" of patient data. The provided document details analytical verification and clinical validation.

9. How Ground Truth for the Training Set Was Established

Not Applicable: As mentioned above, this device does not use a "training set" in the machine learning context. The "ground truth" for its analytical performance validation was established through controlled laboratory experiments using known concentrations of bacterial proteases, human proteases, microorganisms, and interfering substances. The "ground truth" for the clinical validation was the objective observation of wound healing by treating clinicians.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR WOUNDCHEK Bacterial Status DECISION SUMMARY

A. De Novo Number:

DEN180014

B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the WOUNDCHEK Bacterial Status

C. Measurand:

Bacterial Proteases

D. Type of Test:

Lateral flow chromatographic assay

E. Applicant:

Alere Scarborough, Inc.

F. Proprietary and Established Names:

WOUNDCHEK Bacterial Status WCBS

G. Regulatory Information:

1. Regulation section:
  21 CFR 866.3231
1. Classification:
  Class II

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3. Product code(s):

QFA

1. Panel:
  Microbiology (83)

H. Indications For Use:

1. Indication(s) for use:
  WOUNDCHEK Bacterial Status (WCBS) is an in vitro diagnostic chromatographic test for the qualitative detection of bacterial protease activity directly from wound fluid samples collected with a swab. The WCBS test is intended for use in adult patients as an aid in assessing the risk for non-healing of chronic venous, diabetic foot, and pressure ulcers associated with wounds where there are no signs of wound infection and where patients are asymptomatic for clinical signs of infection. The test is intended for use with chronic wounds that are between 21 days and < 6 months of age and chronic wounds that are > 6 months of age that are < 1cm² in size.

1. Special conditions for use statement(s):
  For prescription use only.

For in vitro diagnostic use only.

For use with wound fluid swab specimens only.

1. Special instrument requirements:
  Not applicable.

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I. Device Description:

WOUNDCHEK Bacterial Status (WCBS) is a lateral flow assay that qualitatively detects bacterial protease activity in chronic wound fluid. Wound fluid is collected by rolling a swab over the cleansed wound surface until it is saturated. The swab is then incubated in the assay reagent which contains a substrate that can be cleaved by bacterial proteases and human neutrophil elastase (HNE) and an HNE inhibitor. The WCBS test card is used to detect cleavage products of the substrate. It consists of biotinylated4 bovine serum albumin (BSA) (Test Line (T)), and a control system protein, (Control line (C)), on a nitrocellulose membrane support. Neutravidin, which is conjugated to visualizing particles, binds the biotinylated end of a synthetic peptide and the biotinylated BSA test line. The test result is based on the presence or absence of a pink-to-purple colored Test Line (T) which means bacterial protease activity was detected. A negative test result is defined by the absence of a Test Line (T) which means bacterial protease activity was not detected. If the control line (C) is not present, then the test result is invalid. The device is intended for use on venous leg ulcers (VLU), diabetic foot ulcers (DFU) and pressure ulcers (PU).

A control kit, consisting of negative and positive swabs, is also available for WOUNDCHEK Bacterial Status.

J. Standard/Guidance Document Referenced:

De Novo Classification Process (Evaluation of Automatic Class III Designation): Guidance for Industry and Food and Drug Administration Staff. October 2017.

Guidance for Industry Chronic Cutaneous Ulcer and Burn Wounds - Developing Products for Treatment. June 2006.

K. Test Principle:

The production of proteases from bacteria in chronic wounds can delay the healing of these wounds. The WCBS detects the presence of bacterial protease activity in chronic wounds that can indicate a delay in wound healing.

L. Performance Characteristics (if/when applicable):

1. Analytical performance:
- a. Precision/Reproducibility:

Precision

The precision study was performed at three sites, with three operators per site, for a total of nine operators in the study. Positive panel members were made by combining representative bacterial proteases (i.e., V8, GelE, ZapA and LasB) in wound fluid.

1 WCBS has not been evaluated for potential biotin interference. For more information see FDA Biotin Safety Communication

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The concentration of each protease used in the mixture and tested in each panel member is provided in enzyme units (U) in Table1 below.

Protease	Moderate Positive(3x LOD)	Low Positive(2x LOD)	High Negative(C5)
Endoproteinase Glu-C (V8)	(b) (4)
Serralysin (ZapA)
Gelatinase (GelE)
Pseudolysin (LasB)

Table1. Protease Concentrations in Panel Members

Reproducibility

Testing was conducted over five days. Each day operators tested a blinded panel of (b) (4) swabs; three replicates each of a true negative (wound fluid), moderate positive (3x LOD), low positive (2x LOD) and high negative (C5) panel members.

Swabs were prepared fresh daily by pipetting (b) (4) of the bacterial protease mixture in wound fluid or protease-free wound fluid o Once all solution was absorbed, swabs were assembled into blinded panels. Each test operator was blinded to the expected sample result.

One positive and one negative control swab were tested with the WOUNDCHEK Bacterial Status on each day of testing prior to performing study testing. All control swabs produced the expected results.

The results of the reproducibility study are in Table 2 below.

PanelMember	Site 1		Site 2		Site 3		All Sites
	Positive	Negative	Positive	Negative	Positive	Negative	Positive	Negative
ModeratePositive	100%(45/45)	0.0%(0/45)	97.8%(44/45)	2.2%(1/45)	97.8%(44/45)	2.2%(1/45)	98.5%(133/135)	1.5%(2/135)
LowPositive	100%(45/45)	0.0%(0/45)	93.3%(42/45)	6.7%(3/45)	93.3%(42/45)	6.7%(3/45)	95.6%(129/135)	4.4%(6/135)
HighNegative	6.7%(3/45)	93.3%(42/45)	8.9%(4/45)	91.1%(41/45)	4.4%(2/45)	95.6%(43/45)	6.7%(9/135)	93.3%(126/135)
TrueNegative	4.4%(2/45)	95.6%(43/45)	2.2%(1/45)	97.8%(44/45)	0.0%(0/45)	100%(45/45)	2.2%(3/135)	97.8%(132/135)

Table 2. Reproducibility Results by Site

The moderate positive, low positive and true negative panel members had greater than 95% agreement with the expected results; this meets the acceptance criteria.

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The negative results for the high negative panel member was less than 95%. Because of the nature of the high negative panel member, the percent agreement with expected results is more variable; the percent agreement with expected results observed in this study is acceptable.

There were no significant differences in detection rate by site, operator, or day. Acceptance criteria for the study was met.

b. Linearity/assay reportable range:

Not applicable

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

Stability

External Controls

To monitor the assay performance, reagent performance, and procedural errors, positive and negative external controls must be run in accordance with the guidelines or requirements of local, state, and/or federal regulations or accrediting organizations. Control swabs should be tested with each new shipment received and once for each untrained operator.

WOUNDCHEK Bacterial Status comes with a Positive and Negative Control Swab. If additional control swabs are needed, then WOUNDCHEK Bacterial Swab Control Kits can be purchased separately. These swabs will verify the entire assay. If the correct control results are not obtained, do not report patient results.

Sample Stability

Positive swab samples were prepared by spiking representative bacterial proteases (V8. GelE, ZapA and LasB) near the assay LOD to wound fluid. Negative swab samples were prepared by adding wound fluid to swabs. Swabs were prepared by ( pipetting (b) (4) f the bacterial protease mix in wound fluid or protease- free wound fluid onto ertical center of the swab head.

The following sample storage conditions were evaluated:

Time 0.0 hours .
After 0.5 hours of storage at 2-8℃ and 30℃ .
After 1 hour of storage at 2-8°C and 30°C .
After 1.5 hours of storage at 2-8℃ and 30℃ .
After 2 hours of storage at 2-8℃ and 30℃ .
After 4 hours of storage at 2-8°C and 30°C .
. After 6 hours of storage at 2-8°C and 30°C
After 9 hours of storage at 2-8°C and 30°C .

Following the WOUNDCHEK Bacterial Status procedure, ten positive and negative swabs for each storage condition were tested. Results were then interpreted by two

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operators (each blinded to each other's results) for a total of the determinations per storage condition

(b) (4) Positive control swab and (b) (4) Negative control swab were tested per the WOUNDCHEK Bacterial Status procedure on each day of the study. All daily control testing generated the expected results.

Testing of all positive and negative swabs generated the expected results, meeting the acceptance criteria of the study.

d. Detection limit:
The objective of this study was to establish the WOUNDCHEK Bacterial Status limit of detection (LOD) for four representative bacterial proteases individually and pooled. The LOD level is defined as the level of bacterial protease activity that generates a positive result approximately 95% of the time, when tested in multiple replicates by multiple operators (i.e., the C95 level). A second objective of this study was to identify the level of protease activity that generates a positive result approximately 5% of the time (i.e., the C5 level).

A range of concentrations for each of the representative bacterial proteases were spiked into protease-free wound fluid, coated onto swabs and tested to identify preliminary levels that generated a positive result 100%, 95%, and 5% of the time. Negative swabs were coated with protease-free wound fluid.

Once the bacterial protease levels producing approximately 95% and 5% positive results were identified, LOD test panels were prepared for each bacterial protease. Each LOD panel was blinded to the operators participating in the study. Each panel for each bacterial protease contained swabs with the preliminary LOD, swabs with dilutions flanking (above and below) the preliminary LoD, a negative and a postive swab. Preliminary LoD and flanking dilutions were tested in duplicate in each panel. Ten panels for each bacterial protease were prepared, 20 replicates of each concentration and tested by participants within 30 minutes of swab sample preparation.

The same approach was used in to identify confirm the LOD for pooled bacterial proteases.

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Preliminary and confirmed LOD for individual and pooled bacterial proteases are presented in the tables below.

	Activity(mU/test)	NumberDetected(+ result / total)	% Detection
Staphylococcus aureus(V8)	TRUE POSITIVE(b) (4)	(b) (4)	100%
			100%
			97.5%(LOD)
			80%
			65%
			27.5%
			50%
			20%
			10%
			0%
			5%
			5%
	TRUE NEG		0%
Enterococcus faecalis(GelE)	TRUE POSITIVE(b) (4)	(b) (4)	100%
			100%
			90% (LOD)
			95%
			95%
			75%
			65%
			65%
			55%
			30%
			5%
			10%
			5%
	TRUE NEG		0%

Table 3. Preliminary LOD -Individual Proteases

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	Activity(mU/test)	NumberDetected(+ result / total)	% Detection
Proteus mirabilis(ZapA)	TRUE POSITIVE	(b)	100%
	(b) (4)	(4)	95% (LOD)
			80%
			65%
			70%
			65%
			40%
			40%
			25%
			10%
			25%
			10%
			5%
	TRUE NEG		0%
Pseudomonas aeruginosa(LasB)	TRUE POSITIVE	(b) (4)	100%
	(b) (4)		95% (LOD)
			95%
			75%
			0%
			5%
			5%
	NEG		0%

Table 4. LOD - Individual Proteases

Protease	C95(mU/test)	C5(mU/test)
Endoproteinase Glu-C (V8)	9.0	(b) (4)
Serralysin (ZapA)	12.6
Gelatinase (GelE)	56.7
Pseudolysin (LasB)	34.5

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Protease	Activity(mU/test)				% Detection
	V8	GelE	ZapA	LasB
Pooled:Staphylococcusaureus (V8)	(b) (4)				100%
Enterococcusfaecalis (GelE)					100%
Proteus mirabilis(ZapA)					100%
Pseudomonasaeruginosa(LasB)					95% (LOD)
					85%
					50%
					60%
					30%
					5%
					35%
					5%
					10%
					5%
					15%
					5%

Table 5. Preliminary LOD - Pooled Proteases

Table 6. LOD - Pooled Proteases (mU/test)

Protease	C95(mU/test)	C5(mU/test)	C50(mU/test)
Endoproteinase Glu-C (V8)	(b) (4)
Serralysin (ZapA)
Gelatinase (GelE)
Pseudolysin (LasB)

e. Analytical specificity:

Cross-reactivity - Human Proteases:

The objective of this study was to determine if the presence of human proteases in wound fluid interfere with the performance of WCBS.

The acceptance criteria for this study was that positive swab samples must produce a positive result in the presence of any host protease. Negative swab samples must produce a negative result in the presence of any host protease.

Positive swab samples were prepared fresh on each study day at the LOD (as defined in by Analytical Sensitivity Study) in wound fluid. Negative swab samples were

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prepared fresh on each study day using protease-free wound fluid. Swabs were prepared by pipetting 30uL of the bacterial protease mixture in protease-free wound fluid or protease-free wound fluid onto the vertical center of the swab head.

Human protease stocks were diluted (D) in wound fluid and tested. Each of the host proteases were tested by pipettin (b) (4) >f each dilution in wound fluid to the top of the swab well through the top hol e card. Five negative swabs and five positive swabs were tested following the WCBS procedure. Each result was interpreted by two operators (each blinded to each other's results) for a total of (b) determinations per host protease. Any discrepant results between the operators for a given sample type was clarified by a third operator.

If any of the host protease replicates did not meet acceptance criteria, the protease stock was diluted in wound fluid in (b) (4) increments and tested as above until a level that met acceptance criteria was identified.

One positive control swab and one negative control swab was tested per the WCBS procedure on each day of the study. All daily and experimental control testing generated the expected results on each day of testing.

Protease	Activity(per test)
MMP-13 Catalytic Domain	16.5 U
MMP-9 Catalytic Domain	10.0 U
MMP-8 Catalytic Domain	127.5 U
MMP-2 Catalytic Domain	3.7 U
Cathepsin	0.5 – 1 mU
Thrombin	10.3 U
Human Neutrophil Elastase (HNE)	82.0 mU
Plasmin	9.2 mU

Table 7. Human Proteases - Lowest level at which interference with WCBS is not observed

Interference - Microbial:

The objective of this study was to determine if fungi, mold and viruses that may be present in chronic wounds interfere with the performance of WCBS.

The acceptance criteria for this study was positive swabs (i.e., swabs with analyte) must produce a positive result in the presence of the tested microorganisms. Negative swabs must produce a negative result in the presence of the tested microorganisms.

Fungus and mold were grown according to their individual requirements. Then harvested, suspended in (b) (4) nd diluted to an appropriate concentration then stored at (b) (4) were provided by the vendor. Viruses were stored at (b) (4) prior to testing. All organisms were tested live.

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Positive swabs were prepared fresh each day with representative bacterial proteases at the LOD in wound fluid. Negative
Swabs were prepared by pipetting (b) (4) were prepared each day using wound fluid. f the bacterial protease dilution in wound fluid or protease-free wound fluid e vertical center of the swab head.

Each microorganism was tested by pipetting 10ul of each stock solution to the top of the swab well through the top hole in the card. Five negative swabs and five positive swabs were tested following the WCBS procedure. Each result was interpreted by two operators (each blinded to each other's results) for a total of 10 determinations per microorganism. Any discrepant results between the operators for a given sample type was clarified by a third operator (i.e. 2/3 operators determined some test results).

If interference was observed with stock solutions, log dilutions of the organism stock solutions were tested until the acceptance criteria were met.

One Positive control swab and one negative control swab were tested on each day of the study. All controls generated expected results.

	Organism	ATCC Identifier	Activity (per test)
FUNGUS/MOLD	Candida parapsilosis	(b) (4)	$2.9 X 10^6$ cells/mL
	Candida albicans		$2.4 X 10^6$ cells/mL
	Candida tropicalis		$9.5 X 10^5$ cells/mL
	Aspergillus fumigatus		$6.4 X 10^5$ cells/mL
	Mucor indicus		$2.9 X 10^6$ cells/mL
	Rhizopus oryzae		$2.0 X 10^6$ cells/mL
	Apophysomyces elegans		$3.2 X 10^5$ cells/mL
VIRUSE	Herpes Simplex 1/Herpesvirus 1		$1.6 X 10^6$ TCID50/mL
	Herpes Simplex 2/Herpesvirus 2		$2.8 X 10^4$ TCID50/mL
	Varicella Zoster/Herpesvirus 3		$8.9 X 10^2$ TCID50/mL
	Cytomegalovirus/Herpesvirus 5		$1.6 X 10^4$ TCID50/mL

Table 8. Microbial Interference - Levels below which microbial interference is not observed

Interference - Healthy Skin Flora

The objective of this study was to determine if the presence of proteases present in normal skin flora interfere with the performance of WCBS.

Using the same swabs provided with WCBS, (b) (4) apparently healthy skin swab samples were collected by study trained personnel. One swab per unique, consented individual was collected from intact skin (i.e., no visible wounds or lesions) on the lower leg of each volunteer. Prior to swabbing, the area was gently cleansed with sterile saline to remove all loose debris and confirmed to be visibly moist without any pooling. The head of the swab was pressed flat against the cleansed area and gently rolled back and forth several times while applying pressure until it was fully coated. Swabs were tested within 30 minutes of collection following the WCBS procedure.

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Each result was interpreted by two operators (each blinded to each other's results) for a total of 100 determinations.

One positive control swab and one negative control swab were tested per the WCBS procedure each day. All daily control testing generated the expected results on each day of testing.

Results from twenty swabs were interpreted as positive by both operators. Operator 1 interpreted an additional four swabs as positive. In the study there were a total of 44 false positives out of 102 readings. See Table 9 below for results.

Sample	Operator 1Result	Operator 2Result	Sample	Operator 1Result	Operator 2Result
(b) (4)

Table 9. Interference - Healthy Skin Flora

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This study demonstrates that the performance of WCBS is affected by normal skin flora. This is not unexpected as skin flora reportedly can secrete exogenous proteases (c.f. Koziel, J., & Potempa, J. (2013). Protease- armed bacteria in the skin. Cell and Tissue Research, 351(2), 325-337). The Sample Collection and Handling section of the Instructions for Use includes instruction that states "Do not swab intact skin."

Interfering Substances

The purpose of this study is to determine if substances potentially found in wounds or used for wound treatment interfere with the performance of WCBS.

The acceptance criteria for this study was that positive swabs must produce a positive result in the presence of the interfering substance being tested. Negative swals must produce a negative result in the presence of the interfering substance being tested.

The following approaches were used to test potential interfering substances.

Positive swabs were prepared daily and tested in parallel with pooled representative bacterial proteases (i.e., V8, GelE, ZapA and LasB) near the LOD in wound fluid.

Negative swabs were prepared daily using protease-free wound fluid. Swabs were prepared by pipetting (b) of the pooled bacterial protease in wound fluid or protease-free wound fithd onto the vertical center of the swab head and stored until tested.

Each interfering substance was tested with five positive swabs following the WCBS procedure. Each result was interpreted by two operators (each blinded to each other's results) for a total of 10 determinations per interfering substance.

If a substance failed to meet acceptance criteria at the initial level, it was diluted twofold in wound fluid until acceptance criteria were met.

One positive control swab and one negative control swab were tested according to the WCBS procedure each day of the study. All daily and experimental control testing generated the expected results.

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Substance	Concentration	SubstanceType	TreatmentComponent	Dressing Type*(sub-type(s), ifapplicable)
Acetic acid	0.026%	WoundCleanser	Acid	N/A
Antimicrobial Barrier Dressingwith nanocystalline silver	N/A	Dressing	Silver	Medicated
Activated charcoal dressing withsilver	N/A	Dressing	ActivatedCharcoal- Silver	Medicated
Manuka honey dressing	1.053%	Dressing	Hydrogel, Honey	Modern(Hydrogel)Medicated
Antimicrobian foam dressing withPHMB	N/A	Dressing	PHMB	Modern (Semi-permeable foam)Medicated
Hydrofiber dressing with silver	N/A	Dressing	Hydrogel, Silver	Modern(Hydrogel)Medicated
Mupirocin 2%	1.053%	Dressing	Antibiotic	Medicated
Blood	7.14%	Endogenous	N/A	N/A
Bromelain	0.01 U	Dressing	Enzyme	Medicated
Menthol/zinc oxide ointment	1.053%	Dressing	Zinc oxide.menthol	Medicated
Clindamycin 1%	1.053%	Dressing	Antibiotic	Medicated
Dermal allograft	N/A	Dressing	Skin Substitute	Tissue engineeredskin substitute
Allograft	N/A	Dressing	Skin Substitute	Tissue engineeredskin substitute
Alginate gel with glucoseoxidase/lactoperoxidase	1.053%	Dressing	Hydrogel,Alginate, Enzyme	Modern(Hydrogel,Alginate)Medicated
Antibacterial foam dressingwith methylene blue and gentianviolet	N/A	Dressing	Antimicrobialdyes	Modern (Semi-permeable foam)Medicated
Non-adherent povidone iodinedressing	N/A	Dressing	Iodine	TraditionalMedicated
Bacitracin/Neomycin/Polymixin B	1.053%	Dressing	Antibiotic	Medicated
Hydrogel with alginate	0.527%	Dressing	Hydrogel,Alginate	Modern(Hydrogel,Alginate)
Nystatin	1.053%	Dressing	Antifungal	Medicated
Porcine small intestine submucosa	N/A	Dressing	Skin Substitute	Tissue engineeredskin substitute
Substance	Concentration	SubstanceType	TreatmentComponent	Dressing Type*(sub-type(s), ifapplicable)
Bacitracin zinc/Polymixin Bsulphate	1.053%	Dressing	Antibiotic	Medicated
Promogran Prisma	N/A	Dressing	Collagen, Silver	BioactiveMedicated
Polyaminopropyl Biguanide 0.1%Wound Irrigation Solution	5.26%	WoundCleanser	PHMB, Betaine	N/A
Becaplermin 0.01%	1.053%	Dressing	PDGF	Medicated
Collagenase 250units/g	1.053%	Dressing	Enzyme	Medicated

Table 10. Interfering Substances – Interference not observed below levels listed

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As defined by: Dhivya S, Padma V, Santhini E. Wound dressings - a review. BioMedicine. 2015;5(4):22. doi:10.7603/s40681-015-0022-9.

f. High Dose Hook Effect Study
Not applicable
g. Assay cut-off:
Not applicable
1. Comparison studies:
- a. Method comparison with predicate device:

Not applicable

b. Matrix comparison:
Not applicable
1. Clinical studies:
- a. Clinical Sensitivity:

Prospective Study

Clinical Study - WOUNDCHEK Bacterial Status compared to Clinical Healing Status:

The clinical performance characteristics of WOUNDCHEK Bacterial Status were evaluated in a blinded, prospective observational study conducted in 2016 at seven (7) sites in the U.S. Wound fluid swab samples, collected from adult patients presenting at the study sites with chronic wounds that did not show clinical signs of infection (i.e. had less than three clinical signs of infection) were tested using the

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WOUNDCHEK Bacterial Status, and the results were compared to the clinical healing status of the wounds. The healing status of each wound (healed or not healed) was assessed within a time frame of up to 12 weeks after enrollment in the study. Per the study definition, a healed wound was one that had achieved "complete wound closure", which was defined as "skin re-epithelialization without drainage or dressing requirements" (i.e., 100% of wound is covered and surface is intact), as assessed by the treating clinician. The clinical performance as an aid in the risk assessment for non-healing of chronic venous, diabetic foot, and pressure ulcers associated with wounds where there are no signs of wound infection and where patients are asymptomatic for clinical signs of infection, is stated as:

Positive Likelihood Ratio (PLR) = Sensitivity / 1 - Specificity and;

Negative Likelihood Ratio (NLR) = 1 - Sensitivity / Specificity

Three hundred fifty wounds were enrolled in the study. Of these, one hundred and forty-seven (147) unique wound fluid samples were eligible for inclusion in evaluation of device performance. The table below summarizes the reasons why wounds were excluded from the performance analysis.

Reason for Exclusion from Initial Performance Analysis	Number ofWound Fluid Samples
Total number of wounds enrolled	350
Eligibility Violation	-85
Enrollment Wound Image Violation	-1
Subject Withdrawal	-6
Surgery - unrelated to wound, but wound removed	-4
Surgery - conducted on the wound to close or treat it,making it unacceptable for further use in the study.	-5
Subject Expired (prior to study endpoint)	-6
Unable to Source Verify Wound	-3
Insufficient numbers of MLU and ALU to demonstrateperformance	-8
Lost to Follow-Up	-19
Total Number of Evaluable Wounds in the InitialAnalysis per Study Protocol	213
Correlation with healing status was not observed in asubpopulation of wounds	-66
Total Number of Evaluable Wounds in the FinalAnalysis	147

Three hundred fifty wounds were enrolled in the study. Two hundred and thirteen (213) wound fluid samples (87 venous leg ulcers, 104 diabetic foot ulcers, and 22 pressure ulcers) were collected from the distinct chronic wounds of 200 adult subjects. Note, thirteen subjects consented to enroll two wounds in the study.

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Sixty-six (66) evaluable subjects enrolled with wounds > 1 cm² and > 6 months of age were excluded from the results reported in the table below. Risk assessment claims for these types of wounds were not supported due to a low correlation of the test result with wound healing in most subjects in this cohort.

The remaining 147 wound samples were collected from 139 unique subjects; four subjects consented to enroll two wounds in the study. Included in the final analysis were 51 venous leg ulcers. 82 diabetic foot ulcers, and 14 pressure ulcers.

The table below summarizes device performance for the remaining 147 wounds.

Performance in the Intended Use Population: Patients with either wounds < 6 months old or wounds ≥ 6 months old that are <1 cm² in area*
		No-healing	Healing
WOUNDCHEK Bacterial Status	Pos	38	15	53
	Neg	43	51	94
		81	66	147
PLR= 2.06, 95% CI =1.25 - 3.41NLR= 0.69, 95% CI =0.54 - 0.88* Excludes 66 evaluable subjects with wounds ≥ 1 cm and ≥ 6 months of age.

Likelihood ratios provide information on the pre- and post-test probability of a disease or condition, in this case healing. In the pivotal study, the pre-test risk of nothealing was 55.1%, the post-test risk of not-healing for wounds with a positive WCBS result was 71.7%, a 16.6% increase risk of not-healing. The post-test risk of not-healing for wounds with a negative WCBS result is 45.7%, a decrease in risk of not healing of 9.4%.

Analysis of the impact of wound treatment was not evaluated in the observational study. An evaluation of the clinical impact of the result was not possible due to the observational design of the study. The observational clinical study did not evaluate correlation of results to a clinical diagnosis of infection or evaluate clinical intervention in response to WOUNDCHEK results. The observational study did not control for numerous clinical factors which impact healing therefore the clinical correlation of the WOUNDCHEK Bacterial Status result with wound healing cannot be clearly established. Differences in clinical practice may affect the numbers of days to wound healing associated with WOUNDCHEK results.

b. Clinical specificity:
See section L.3a above.
Other clinical supportive data (when a. and b. are not applicable): C.
N/A

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4. Clinical cut-off:

Not applicable.

1. Expected values/Reference range:
  Not Applicable

M. Instrument Name:

Not applicable. The device does not utilize an instrument for result generation.

N. System Descriptions:

1. Modes of Operation:
  Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device?

Yes

Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission?

Yes

1. Software:
  FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes __________________________________________________________________________________________________________________________________________________________________________

The device does not contain any software or instrument components.

1. Specimen Identification:
  Not applicable.
1. Specimen Sampling and Handling:
  Prior to swabbing, the wound should be gently rinsed with sterile saline to remove debris, therapeutic agents and necrotic tissue, but pooling of saline in the wound should be avoided. Sharp wound debridement should not be performed, and wound should be free of blood prior to sample collection. Apply gentle pressure as you roll the swab back and

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forth over the wound until saturated. Care should be taken not to swab blood or intact skin as this can cause false results.

1. Calibration:
  Not applicable.
1. Quality Control:
  WCBS contains an internal control which verifies the sample is flowing properly through the test strip. Additionally, each kit comes with a positive and negative external control swab. External controls should be tested, and the expected results obtained, prior to testing patient samples with a new kit and before an untrained user performs the test. Additional external control swabs can be purchased separately.

O. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

None.

P. Proposed Labeling:

The labeling supports the decision to grant the De Novo request for this device.

Q. Identified Risks to Health and Mitigation Measures

Identified Risks to Health	Mitigation Measures
Risk of false test results	Use of certain specimen collection and transportdevices identified in special control (1)Certain labeling information identified in specialcontrol (2)Certain design verification and validationactivities identified in special control (3)
Failure to correctly interpret test results	Certain labeling information identified in specialcontrol (2)
Failure to correctly operate the device	Certain labeling information identified in specialcontrol (2)

R. Benefit/Risk Analysis:

Summary of the Assessment of Benefit

The primary benefit associated with use of the WOUNDCHEK Bacterial Status Assay is identification of a risk factor for wound non-healing. The WOUNDCHEK Bacterial Status Assay can be used in chronic venous, diabetic foot, and pressure ulcers between 21 days and six months of age, or wounds more than six months of age that are less than 1 cm2 in which there are no

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signs of wound infection and where patients are asymptomatic for clinical signs of infection. Clinicians may change their management of chronic wounds based upon the results of the WOUNDCHEK Bacterial Status assay with subsequent decrease in unnecessary therapy for wounds likely to heal and increased therapeutic interventions for wounds not likely to heal.

Summary of the Assessment of Risk

The primary risk associated with use of the WOUNDCHEK Bacterial Status assay is erroneous results in which wounds that are identified as being at risk for non-healing may heal and wounds identified as being likely to heal may not heal. Clinicians may change their clinical management of chronic wounds due to the presumption that a wound will not heal based upon their perception of increased or decreased risk due to the WOUNDCHEK Bacterial Status assay result, with subsequent increases in patient morbidity.

Patient Perspectives

This submission did not include specific information on patient perspectives for this device.

Summary of the Assessment of Benefit-Risk

The probable benefits of the WoundChek Bacterial Status Assay as an assay that identifies a risk factor for wound non-healing outweigh the potential risks in light of the listed special controls and applicable general controls. The proposed special controls, including the description of the recommended training (e.g., knowledge and experience) for safe and effective use of the device, are necessary to ensure safe use of the assay and mitigate the risks associated with use of the device. The proposed labeling and quick reference guide will communicate the limitations of the device to users. Overall, the probable benefits of the WOUNDCHEK Bacterial Status outweigh the probable risks for the proposed indications for use in light of the special controls for this type of device and in combination with the general controls.

S. Conclusion

The De Novo request is granted and the device is classified under the following and subject to the special controls identified in the letter granting the De Novo request:

Product Code(s): OFA Device Type: Device to detect bacterial protease activity in chronic wound fluid Class: II (special controls) Regulation: 21 CFR 866.3231

Regulation Number and Section

§ 866.3231 Device to detect bacterial protease activity in chronic wound fluid.

(a)
Identification. A device to detect bacterial protease activity in chronic wound fluid is a lateral flow prescription in vitro diagnostic device that may include a sterile swab. The device is intended for use in patients as an aid in assessing the risk for non-healing of chronic venous, diabetic foot, and pressure ulcers associated with wounds where there are no signs of wound infection and where patients are asymptomatic for clinical signs of infection.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Any swab used to collect a patient specimen must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of wound fluid specimens; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device.
(2) The labeling required under § 809.10(b) of this chapter must include:
(i) An intended use that includes the following statements:
(A) A statement that the device detects and measures bacterial proteases from a swab saturated with wound fluid.
(B) A statement that the device provides a qualitative output to aid the user in assessing the risk for non-healing of wounds (
e.g., chronic venous, diabetic foot and pressure ulcers).(C) A description of the clinical indications for test use.
(D) The specific population(s) for which the device is intended.
(E) A description of the recommended training (
e.g., knowledge and experience) for safe and effective use of the device and to minimize the risks of incorrect results and misinterpretation.(ii) A detailed description of the performance characteristics of the device from the analytical and clinical studies required under paragraphs (b)(3)(ii) and (iii) of this section.
(iii) A detailed explanation of the interpretation of results.
(iv) A warning statement describing situations where the device has not been validated or may not perform as identified in the labeling (
e.g., not for use with wounds which are ≥6 months of age and ≥1 cm2 in size).(v) The following limiting statements:
(A) That the device is not intended to provide a risk assessment of chronic wound infection status or aid in the diagnosis of infection in chronic wounds, nor is the device intended for monitoring the effectiveness of anti-infective therapy.
(B) That a negative result does not exclude the presence of bacterial proteases. Therefore, the results should be used in conjunction with clinical findings to make an accurate assessment of risk of nonhealing. The test result should be interpreted in conjunction with other risk factors, along with clinical and laboratory data available to the clinician.
(C) That the device has been validated using wound fluid samples only. Other sample types (
e.g., whole blood from venous or capillary draws, other body fluids) have not been evaluated.(D) That skin flora may secrete bacterial proteases therefore, swab contact with intact skin should be avoided as this may yield false positive results.
(vi) Labeling must include a brief reference sheet for healthcare professionals that includes the intended use, summary of clinical performance, results from analytical testing on normal skin and human proteases, and warning and limiting statements.
(3) Design verification and validation must include the following:
(i) A detailed device description (
e.g., all device parts, control elements incorporated into the test procedure, reagents required but not provided, and the principle of device operation and test methodology).(ii) Detailed documentation and results from analytical studies, including the limit of detection, inclusivity, cross-reactivity, microbial interference, analytical sensitivity for normal skin flora and human proteases, interfering substances, specimen stability, within-lab precision, and reproducibility.
(iii) Detailed documentation and results from a clinical study that includes prospective (sequentially collected) samples for the intended specimen type that are representative of the intended use population(s). The clinical study must compare the device performance to results obtained from a reference or comparator method that FDA has determined is appropriate.