WOUNDCHEK Bacterial Status

• K Number: DEN180014
• Device Name: WOUNDCHEK Bacterial Status
• Manufacturer: Alere Scarborough, Inc.
• Date Cleared: 2019-12-02 ( 619 days)
• Product Code: QFA
• Regulation Number: 866.3231

Intended 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. 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.

Predicate Devices

Not Found

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description details a lateral flow assay that provides a qualitative result based on the presence or absence of a colored line. There is no mention of any computational analysis, algorithms, or learning processes that would indicate the use of AI or ML. The performance study focuses on traditional statistical metrics like likelihood ratios.

Therapeutic

No
This device is an in vitro diagnostic test for detecting bacterial protease activity in wound fluid and is intended as an aid in assessing the risk for non-healing wounds. It does not provide therapy or treatment.

Diagnostic

Yes

The device is explicitly described as an "in vitro diagnostic chromatographic test" used for the qualitative detection of bacterial protease activity directly from wound fluid samples. Its purpose is to aid in assessing the risk for non-healing of chronic wounds, clearly indicating its role in diagnosis or risk assessment.

SaMD (Software as a Medical Device)

No

The device description clearly states it is a "lateral flow assay" and involves a "test card" with a "nitrocellulose membrane support," indicating it is a physical, hardware-based diagnostic test.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

• Intended Use: The "Intended Use / Indications for Use" section explicitly states that WOUNDCHEK Bacterial Status (WCBS) is an "in vitro diagnostic chromatographic test".
• Sample Type: It analyzes "wound fluid samples collected with a swab". This is a biological sample taken from the human body.
• Purpose: The test is used to detect "bacterial protease activity" in these samples, which is a biological marker.
• Diagnostic Aid: The intended use clearly states it is an "aid in assessing the risk for non-healing of chronic venous, diabetic foot, and pressure ulcers". This indicates it provides information used in the diagnostic process.
• In Vitro: The test is performed outside of the living organism, using the collected wound fluid sample.

All of these points align with the definition of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications 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 that are

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.

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
• 2. Classification:
     Class II

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3. Product code(s):

QFA

• 4. 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 that are 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.

| Panel

Member	Site 1		Site 2		Site 3		All Sites
	Positive	Negative	Positive	Negative	Positive	Negative	Positive	Negative
Moderate
Positive	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)
Low
Positive	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)
High
Negative	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)
True
Negative	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) | Number
Detected
(+ 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) | Number
Detected
(+ 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) | C50
(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) | | | | Number
Detected | % Detection |
|------------------------------------------|-----------------------|------|------|------|--------------------|-------------|
| | V8 | GelE | ZapA | LasB | | |
| Pooled:
Staphylococcus
aureus (V8) | (b) (4) | | | | | 100% |
| Enterococcus
faecalis (GelE) | | | | | | 100% |
| Proteus mirabilis
(ZapA) | | | | | | 100% |
| Pseudomonas
aeruginosa
(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

9

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 1
Result | Operator 2
Result | Sample | Operator 1
Result | Operator 2
Result |
|---------|----------------------|----------------------|--------|----------------------|----------------------|
| (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 | Substance
Type | Treatment
Component | Dressing Type*
(sub-type(s), if
applicable) |
|--------------------------------------------------------------------------|---------------|-------------------|-------------------------------|---------------------------------------------------|
| Acetic acid | 0.026% | Wound
Cleanser | Acid | N/A |
| Antimicrobial Barrier Dressing
with nanocystalline silver | N/A | Dressing | Silver | Medicated |
| Activated charcoal dressing with
silver | N/A | Dressing | Activated
Charcoal- Silver | Medicated |
| Manuka honey dressing | 1.053% | Dressing | Hydrogel, Honey | Modern
(Hydrogel)
Medicated |
| Antimicrobian foam dressing with
PHMB | 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 engineered
skin substitute |
| Allograft | N/A | Dressing | Skin Substitute | Tissue engineered
skin substitute |
| Alginate gel with glucose
oxidase/lactoperoxidase | 1.053% | Dressing | Hydrogel,
Alginate, Enzyme | Modern
(Hydrogel,
Alginate)
Medicated |
| Antibacterial foam dressing
with methylene blue and gentian
violet | N/A | Dressing | Antimicrobial
dyes | Modern (Semi-
permeable foam)
Medicated |
| Non-adherent povidone iodine
dressing | N/A | Dressing | Iodine | Traditional
Medicated |
| 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 engineered
skin substitute |
| Substance | Concentration | Substance
Type | Treatment
Component | Dressing Type*
(sub-type(s), if
applicable) |
| Bacitracin zinc/Polymixin B
sulphate | 1.053% | Dressing | Antibiotic | Medicated |
| Promogran Prisma | N/A | Dressing | Collagen, Silver | Bioactive
Medicated |
| Polyaminopropyl Biguanide 0.1%
Wound Irrigation Solution | 5.26% | Wound
Cleanser | 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

• 2. Comparison studies:
  • a. Method comparison with predicate device:

Not applicable

• b. Matrix comparison:
  Not applicable

• 3. 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 of
Wound 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 demonstrate
performance | -8 |
| Lost to Follow-Up | -19 |
| Total Number of Evaluable Wounds in the Initial
Analysis per Study Protocol | 213 |
| Correlation with healing status was not observed in a
subpopulation of wounds | -66 |
| Total Number of Evaluable Wounds in the Final
Analysis | 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