The SensiTox B. anthracis Toxin Test

K232545 · First Light Diagnostics, Inc. · QUU · Nov 20, 2023 · Microbiology

Device Facts

• Record ID: K232545
• Device Name: The SensiTox B. anthracis Toxin Test
• Applicant: First Light Diagnostics, Inc.
• Product Code: QUU · Microbiology
• Decision Date: Nov 20, 2023
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3046
• Device Class: Class 2

Indications for Use

The SensiTox B. anthracis Toxin Test for use with the MultiPath Analyzer, is a qualitative immunofluorescence assay to aid in the diagnosis of inhalation anthrax. The test is intended for the rapid, qualitative detection of lethal factor, a biomarker associated with Bacillus anthracis (B. anthracis). The test can be used with whole blood collected with dipotassium EDTA anticoagulant by venipuncture. This test is indicated for testing samples from individuals who have signs and symptoms consistent with inhalation anthrax and a likelihood of exposure to B. anthracis. A positive SensiTox B. anthracis Toxin Test result is presumptively diagnostic for B. anthracis infection. Diagnosis of B. anthracis infection must be made in conjunction with medical history, likelihood of exposure, signs, and symptoms of disease, as well as other laboratory evidence. The definitive identification of B. anthracis from blood samples requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required. Testing should be performed and reported in accordance with current guidelines provided by the appropriate public health authorities. The level of lethal factor present in blood from individuals with early systemic infection is unknown. Negative results do not preclude infection with the biothreat microbial agents targeted by the device and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Laboratories performing the SensiTox B. anthracis Toxin Test must have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities, and personnel trained in the safe handling of clinical specimens potentially containing B. anthracis. The SensiTox B. anthracis Toxin Test is for prescription use only. This assay is not FDA-cleared or approved for testing blood or plasma donors.

Device Story

Device detects lethal factor (LF) biomarker in venous whole blood (K2EDTA) to aid inhalation anthrax diagnosis. Uses MultiPath Analyzer and single-use cartridges. Process: sample added to cartridge; heated to 35°C; split into 3 aliquots; mixed with lyophilized antibody-conjugated fluorescent/magnetic particles; incubated 12 minutes. Magnetic particles (with bound LF) drawn through dye-cushion layer to imaging surface via magnets. Nonmagnified digital imaging quantifies captured fluorescent particles. Operated by trained laboratory personnel in clinical settings. Results displayed on touchscreen; reported as valid/invalid and detected/not detected. Aids clinical decision-making in conjunction with patient history and public health guidelines.

Clinical Evidence

Clinical evaluation included 318 residual blood specimens (NPA) and 105 prospective febrile patient samples (PPA) spiked with native/recombinant LF. Results: 100% NPA (95% CI: 98.8-100.0%) and 96.2% PPA (95% CI: 90.6-98.5%). Bench testing established LoD at 50 pg/mL. Reproducibility was 98.9% across 3 sites.

Technological Characteristics

Immunofluorescence assay; fluidic cartridge with magnetic/fluorescent particle-based detection. Energy source: MultiPath Analyzer (electrical). Connectivity: Standalone/networked (instrument-based). Software: MultiPath applications software for automated interpretation. Materials: Proprietary cartridge with lyophilized beads and dye-cushion reagent.

Indications for Use

Indicated for individuals with signs/symptoms consistent with inhalation anthrax and likelihood of exposure to B. anthracis. For prescription use only. Not for blood/plasma donor screening.

Regulatory Classification

Identification

The Active Anthrax Detect Plus Rapid Test is an in vitro immunochromatographic point-of-care diagnostic device used as an aid in the diagnosis of inhalation anthrax. It provides visual and rapid qualitative detection of lethal factor of Bacillus anthracis (B. anthracis) in serum and venous whole blood (dipotassium EDTA, sodium citrate, and sodium heparin). It is indicated for testing individuals with signs and symptoms consistent with inhalation anthrax and a likelihood of exposure, for use by military personnel, medical, and/or healthcare professionals.

Special Controls

1. The distribution of these devices is limited to laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities. 2. Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of the sample types with which this device is intended to be used; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device. 3. The labeling required under 21 CFR 809.10(b) must include: i. An intended use statement that includes the following: (A) A detailed description of targets the device detects and measures; (B) The results provided to the user (i.e., whether the measurement is qualitative, semi-quantitative, or quantitative); (C) The clinical indications appropriate for test use (e.g., in conjunction with patient history, epidemiological information, clinical observations, and other laboratory evidence to make patient management decisions); (D) Sample types with which it is intended for use; (E) The specific population(s) with which the device is intended to be used; (F) The testing location(s) where the device is to be used (if not intended for all locations); (G) A statement that the device results are for the presumptive identification of Bacillus spp., and definitive identification requires additional testing and confirmation procedures in consultation with the appropriate public health authorities; (H) A statement that negative results do not preclude infection with Bacillus spp. and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions; and (I) A statement that testing is to be performed and reported in accordance with current guidelines provided by the appropriate public health authorities. ii. Detailed instructions for minimizing the risk of user exposure to Bacillus spp. that may be present in test samples and those used as control materials. iii. Detailed instructions for minimizing the risk of generating false positive test results due to contamination from positive test samples and/or positive control materials. iv. A prominent and conspicuous precaution that interpretation of test results is intended to be performed by experienced healthcare professionals who have training in principles and use of infectious disease diagnostics and the expertise to report results. v. A prominent and conspicuous warning statement that the test results alone do not conclusively establish infection and that additional testing and confirmation procedures may be necessary in consultation with the appropriate public health or other authorities to whom reporting is required. vi. A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of samples. vii. Detailed descriptions of the performance characteristics of the device for all claimed sample types as shown by the analytical and clinical studies required under paragraphs (4)(ii) and (4)(iii), except sample stability performance characteristics. viii. For any devices intended for use in a near-patient setting, a brief reference sheet for healthcare professionals that accompanies the device and that includes the name and intended use of the test, step-by-step instructions of all control and sample testing procedures for the claimed sample types, the result(s) interpretation, warning and limitation statements, and information for troubleshooting or technical assistance with the device. ix. A statement that a nationally notifiable disease caused by a biothreat microbial agent must be reported to public health authorities in accordance with local, state, and federal law. x. Limiting statements indicating, as applicable: (A) Situations where the device has been demonstrated to fail or may not perform at its expected performance level (e.g., any disease specific circumstances or circumstances identified by human factors or robustness studies); (B) Any specific circumstances that pose significant risk to public health, and for which the device has not been validated. For example: a. Testing of matrices and patient populations that are not identified in the intended use; or b. Testing individuals without signs and symptoms of infection, including mass infection screening (such as airport or border screening) that is not limited to individuals who have signs and symptoms and a risk of exposure to biothreat microbial agents. 4. Design verification and validation must include: i. A detailed device description, including all device parts, control elements incorporated into the test procedure, reagents required but not provided, the principle of device operation and test methodology, and the computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported result). ii. Detailed documentation of analytical studies, as applicable, including those demonstrating Limit of Detection (LoD), inclusivity, cross-reactivity, microbial interference, interfering substances, carryover/cross contamination, sample stability, within lab precision, hook effect, reproducibility, and other studies relevant to the technology (e.g., linearity), as determined to be appropriate by FDA. iii. Detailed documentation and results from either a clinical study or, when determined to be acceptable by FDA, a study with an equivalent data set. Documentation from this study must include study reports, testing results, and results of all statistical analyses, including line data of all test samples, and an appropriate justification describing how the sample set is representative of the intended use population. This study must compare the device performance to results obtained from a reference or comparator method that FDA has determined to be appropriate. This study must include prospective (sequentially collected) samples for each intended sample type that are representative of the intended use populations and may, when determined to be acceptable by FDA, include additional characterized clinical samples; or, as an alternative, when determined to be acceptable by FDA, an equivalent sample set. This study must include samples spanning all relevant analyte concentrations for all of the indicated sample type(s) and the targeted analyte(s). iv. A detailed description of the impact of any software, including software applications and hardware-based devices that incorporate software, on the device's functions, as applicable. v. For any devices that detect the presence of an analyte directly from sample, detailed documentation and results from a shelf-life assessment that includes samples formulated in the most complex clinical matrix identified in the device's intended use. vi. As part of the risk management activities, if the labeling includes hyperlinks to documents from public health authorities regarding sampling, sample shipment, sample testing, or clinical management of patients suspected of being infected; or if the labeling includes direct contact information for any such public health authority, then the hyperlinks and contact information must be reviewed at least annually and updated to reflect any changes to those hyperlinks or contact information.

Predicate Devices

• InBios International, Inc. Active Anthrax Detect Plus Rapid Test (DEN220044)

Submission Summary (Full Text)

{0}------------------------------------------------

November 20, 2023

Image /page/0/Picture/1 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

First Light Diagnostics, Inc. Joanne Spadoro Chief Executive Officer 2 Omni Way Chelmsford. Massachusetts 01824

# Re: K232545

Trade/Device Name: The SensiTox B. anthracis Toxin Test Regulation Number: 21 CFR 866.3046 Regulation Name: Simple In Vitro Diagnostic Device For The Detection Of Secreted Proteins From Bacillus Species (Spp.) In Human Clinical Samples Regulatory Class: Class II Product Code: QUU Dated: August 22, 2023 Received: August 22, 2023

# Dear Joanne Spadoro:

We have reviewed your section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (the Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

{1}------------------------------------------------

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review. the OS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

Noel J. Gerald -S

Noel J. Gerald, Ph.D. Branch Chief Bacterial Respiratory and Medical Countermeasures Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

{2}------------------------------------------------

# Indications for Use

510(k) Number (if known) K232545

Device Name SensiTox B. anthracis Toxin Test

#### Indications for Use (Describe)

The SensiTox B. anthracis Toxin Test for use with the MultiPath Analyzer, is a qualitative immunofluorescence assay to aid in the diagnosis of inhalation anthrax. The test is intended for the rapid, qualitative detection of lethal factor, a biomarker associated with Bacillus anthracis). The test can be used with whole blood collected with dipotassium EDTA anticoagulant by venipuncture. This testing samples from individuals who have signs and symptoms consistent with inhalation anthrax and a likelihood of exposure to B. anthracis. A positive SensiTox B. anthracis Toxin Test result is presumptively diagnostic for B. anthracis infection. Diagnosis of B. anthracis infection must be made in conjunction with medical history, likelihood of exposure, signs, and symptoms of disease, as well as other laboratory evidence. The definitive identification of B. anthracis from blood samples additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required. Testing should be performed and reported in accordance with current guidelines provided by the appropriate public health authorities. The level of lethal factor present in blood from individuals with early systemic infection is unknown. Negative results do not preclude infection with the biothreat microbial agents targeted by the device and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Laboratories performing the SensiTox B. anthracis Toxin Test must have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities, and personnel trained in the safe handling of clinical specimens potentially containing B. anthracis.

The SensiTox B. anthracis Toxin Test is for prescription use only.

This assay is not FDA-cleared or approved for testing blood or plasma donors.

| Type of Use (Select one or both, as applicable) | |
|---------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------|
| <div> <span> Prescription Use (Part 21 CFR 801 Subpart D) </span> </div> | <div> <span> Over-The-Counter Use (21 CFR 801 Subpart C) </span> </div> |

CONTINUE ON A SEPARATE PAGE IF NEEDEDThis section applies only to requirements of the Paperwork Reduction Act of 1995.

#### *DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.*

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

> Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{3}------------------------------------------------

# 510(k) Summary

August 21, 2023

The summary of the 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

## Contact Details

| Sponsor: | First Light<br>Diagnostics, Inc.<br>2 Omni Way<br>Chelmsford, MA 01824 |
|----------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Correspondent: | Joanne Spadoro, Ph.D.<br>President and CEO<br>First Light Diagnostics, Inc.<br>2 Omni Way<br>Chelmsford, MA 01824<br>Phone: [phone], extension 224<br>Email: [e-mail] |

#### Device

| Device Trade Name: | SensiTox® B. anthracis Toxin Test |
|----------------------|---------------------------------------------------------------------------------|
| Common Name: | B. anthracis Toxin Test |
| Classification Name: | Class II |
| Regulation Number: | 866.3046 |
| Product Code: | QUU |
| Predicate Device: | InBios International, Inc. Active Anthrax Detect Plus Rapid Test<br>(DEN220044) |

{4}------------------------------------------------

#### Device Description Summary

The SensiTox B. anthracis Toxin Test, run on the MultiPath Analyzer, detects lethal factor in venous whole blood samples using an immunofluorescence assay and the proprietary MultiPath detection technology.

A whole blood specimen, collected in dipotassium EDTA, from individuals with signs and symptoms consistent with inhalation anthrax and a likelihood of exposure, is used for the test. The blood sample is added directly to the SensiTox B. anthracis Cartridge, a single use consumable that contains all the reagents required to run the test. The Cartridge is loaded onto the MultiPath Analyzer for processing through the steps of the assay.

Once loaded onto the Analyzer, the barcodes on the cartridge that identify the test type and associated test specific information (manufacturer installed barcode) and sample (laboratory affixed barcode) are read. The cartridge is moved to the fluidics station where it is first heated to 35°C. The sample is then split into 3 equal aliquots in 3 distribution wells within the cartridge. The sample aliquots flow from the distribution wells to the reagent wells containing target-specific antibody conjugated fluorescent and magnetic particles in the form of lyophilized beads. Upon contact with the sample, the lyophilized beads rehydrate and the reaction mixtures flow into the imaging wells, the bottoms of which are coated with a dye cushion reagent. Upon contact with the reagents, the dye-cushion dissolves forming a dense opaque aqueous layer that separates the sample and reagents from the bottom optical surface of the Imaging Well. In the upper assay layer, the toxins, if present, bind to the magnetic and fluorescent particles tethering them together. The cartridge is incubated for 12 minutes to allow the reaction to take place and then is moved to the magnetics station. At the magnetics station, the imaging well is placed over permanent magnets that draw the magnetic particles and any fluorescent particles that are tethered to them via the target molecules through the dye-cushion layer, depositing them on the bottom imaging surface. The captured fluorescent particles are imaged and quantified using nonmagnified digital imaging.

The Analyzer can be run in batch mode or by random access. Up to 20 cartridges can be loaded onto the Analyzer in parallel. The first result is reported in approximately 21 minutes of loading the cartridge onto the Analyzer with subsequent results being reported in 2.5-minute increments. The results are interpreted using the MultiPath applications software as valid or invalid, and if valid, the results are reported as Lethal Factor detected. Results are displayed on the instrument touch screen and can be printed.

# Intended Use/Indications for Use

The SensiTox B. anthracis Toxin Test for use with the MultiPath Analyzer, is a qualitative immunofluorescence assay to aid in the diagnosis of inhalation anthrax. The test is intended for the rapid, qualitative detection of lethal factor, a biomarker associated with Bacillus anthracis (B. anthracis). The test can be used with whole blood collected with dipotassium EDTA anticoagulant by venipuncture. This test is indicated for testing samples from individuals who have signs and symptoms consistent with inhalation anthrax and a likelihood of exposure to B. anthracis. A positive SensiTox B. anthracis Toxin Test result is presumptively diagnostic for B. anthracis

{5}------------------------------------------------

infection. Diagnosis of B. anthracis infection must be made in conjunction with medical history, likelihood of exposure, signs, and symptoms of disease, as well as other laboratory evidence. The definitive identification of B. anthracis from blood samples requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required. Testing should be performed and reported in accordance with current guidelines provided by the appropriate public health authorities. The level of lethal factor present in blood from individuals with early systemic infection is unknown. Negative results do not preclude infection with the biothreat microbial agents targeted by the device and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Laboratories performing the SensiTox B. anthracis Toxin Test must have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities, and personnel trained in the safe handling of clinical specimens potentially containing B. anthracis.

The SensiTox B. anthracis Toxin Test is for prescription use only.

This assay is not FDA-cleared or approved for testing blood or plasma donors.

### Special Conditions for Use Statement(s)

For Prescription Use Only. Please refer to the SensiTox B. anthracis Toxin Test labeling for a more complete list of warnings, precautions, and contraindications.

### Special Instrument Requirements

For use with the Multipath Analyzer

# Indications for Use/Technology Comparison

| Description | First Light Diagnostics, Inc.<br>Subject Device<br>SensiTox B. anthracis Toxin Test | InBios International, Inc.<br>Predicate Device<br>DEN220044<br>Active Anthrax Detect Plus Rapid Test |
|--------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Regulation | 866.3046 | Same |
| Product Code | QUU | Same |
| Device Class | Class II | Same |
| Panel | Microbiology | Same |
| Intended Use | The SensiTox B. anthracis Toxin Test<br>for use with the MultiPath Analyzer, is<br>a qualitative immunofluorescence<br>assay to aid in the diagnosis of<br>inhalation anthrax. The test is<br>intended for the rapid, qualitative<br>detection of lethal factor, a biomarker<br>associated with Bacillus anthracis (B.<br>anthracis). The test can be used with<br>whole blood collected with | The Active Anthrax Detect Plus Rapid<br>Test point-of-care diagnostic test for<br>pulmonary anthrax is an in vitro<br>immunochromatographic device for use<br>as an aid in the diagnosis of inhalation<br>anthrax. It provides visual and rapid<br>qualitative detection of lethal factor of<br>Bacillus anthracis (B. anthracis). The test<br>can be used to test serum and venous<br>whole blood (dipotassium EDTA, sodium |

{6}------------------------------------------------

dipotassium EDTA anticoagulant by venipuncture. This test is indicated for testing samples from individuals who have signs and symptoms consistent inhalation anthrax and with a likelihood of exposure to B. anthracis. A positive SensiTox B. anthracis Toxin Test result is presumptively diagnostic for B. anthracis infection. Diagnosis of B. anthracis infection must be made in conjunction with medical history, likelihood of exposure, signs, and symptoms of disease, as well as other laboratory evidence. The definitive identification of B. anthracis from blood samples requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required. Testing should be performed and reported in accordance with current guidelines provided by the appropriate public health authorities. The level of lethal factor present in blood from individuals with early systemic infection is unknown. Negative results do not preclude infection with biothreat microbial agents the targeted by the device and should not be used as the sole basis for diagnosis, or other treatment, patient management decisions.

Laboratories performing the SensiTox B. anthracis Toxin Test must have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities, and personnel trained in the safe handling of clinical specimens potentially containing B. anthracis.

The SensiTox B. anthracis Toxin Test is for prescription use only.

citrate, and sodium heparin). The assay is indicated for testing samples from individuals who have signs and symptoms consistent with inhalation anthrax and a likelihood of exposure. This test is intended for use by military personnel, medical, and/or healthcare professionals only. The diagnosis of B. anthracis infection must be based on history, signs, symptoms, exposure likelihood, and additional laboratory evidence. A positive Active Anthrax Detect Plus Rapid Test result is presumptively diagnostic for B. anthracis infection. The definitive identification of B. anthracis from blood samples requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required. Testing should be performed and reported in accordance with current guidelines provided by the appropriate public health authorities. The level of lethal factor present in blood from individuals with early systemic infection is unknown. Negative results do not preclude infection with the biothreat microbial agents targeted by the device and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. This assay is for prescription use.

The distribution of in vitro diagnostic devices for Bacillus spp. detection must performed in accordance with be guidelines established by the public authorities health that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.

This assay is not FDA-cleared or approved for testing blood or plasma donors.

{7}------------------------------------------------

| This assay is not FDA-cleared or<br>approved for testing blood or plasma<br>donors. | | |
|-------------------------------------------------------------------------------------|----------------------------------------------------------------------------|--------------------------------------------------------------------------------------|
| | | |
| | | |
| | | |
| <b>Similarities</b> | | |
| Sample Type | Whole blood collected with dipotassium EDTA anticoagulant by venipuncture | Serum and venous whole blood (dipotassium EDTA, sodium citrated, and sodium heparin) |
| Analyte | Lethal Factor | Same |
| Prescription Status | Prescription Use Only | Same |
| Antibodies | Anti-Lethal Factor antibodies | Same |
| <b>Differences</b> | | |
| Technology | Immunofluorescent assay | Immunoassay |
| Test Format | Fluidic cartridge with direct digital Imaging and automated interpretation | Lateral flow with visual interpretation |
| Automated | Yes | No |
| Instrument | Multipath Analyzer | None |

#### Non-Clinical and/or Clinical Tests Summary

#### Limit of Detection (LoD)

The limit of detection (LoD) for Lethal Factor was determined by adding recombinant Lethal Factor or native Lethal Factor contained in culture supernatants from the Ames and SK-102 strains of B. anthracis to K₂EDTA venous whole blood collected from healthy donors. A minimum of 5 serial dilutions of known concentrations of Lethal Factor were tested in replicates of 20 at concentrations ranging from 20 to 60 pg/mL.

The data were evaluated by analysis of hit rate (detected results/total number of test) as well as Probit analysis. The LoD was determined by combining the replicate data across the three cartridge lots and identifying the lowest LF concentration (pg/mL) with a ≥95% "Detected" hit rate. The LoD established for Lethal Factor is 50 pg/mL.

#### Reproducibility

The reproducibility of the SensiTox B. anthracis Toxin Test was evaluated at 3 sites over the course of 5 non-consecutive days by 2 operators per site each day. Three lots of cartridges were tested in this study. Randomized and blinded samples comprised of whole blood spiked with two concentrations of recombinant Lethal Factor – low positive (1.5X LoD), moderate positive (3X LoD), and negative (unspiked) were prepared and provided to each participating site. Each operator mixed a blinded sample with whole blood prescreened and known to be negative for Lethal Factor and tested in triplicate using the SensiTox B. anthracis Toxin Test.

As shown in Table 5.2, the overall reproducibility of the SensiTox B. anthracis Toxin Test is 98.9%. Of the 270 samples tested, there were 3 incorrect results reported from 3 samples (Table 5.3). Two low positive samples and 1 moderate positive sample generated negative results.

{8}------------------------------------------------

| Sample | Site 1 | | Site 2 | | Site 3 | | Total | |
|--------------|--------|------------|--------|------------|--------|------------|---------|------------|
| Description | # | % Accuracy | # | % Accuracy | # | % Accuracy | # | % Accuracy |
| Negative | 30/30 | 100 | 30/30 | 100 | 30/30 | 100 | 90/90 | 100 |
| 75 pg/mL LF | 30/30 | 100 | 29/30 | 96.7 | 29/30 | 96.7 | 88/90 | 97.8 |
| 150 pg/mL LF | 30/30 | 100 | 29/30 | 96.7 | 30/30 | 100 | 89/90 | 98.9 |
| Total | 90/90 | 100 | 88/90 | 97.8 | 89/90 | 98.9 | 267/270 | 98.9 |

Table 5.2. Reproducibility study data analyzed by sample type and by site.

Table 5.3. Summary of reproducibility study analyzed by positive and negative samples.

| Site | Total<br>Samples | #<br>Invalid | FP | FN | TP | TN | % Accuracy<br>Positives | % Accuracy<br>Negatives |
|--------|------------------|--------------|----|----|-----|----|-------------------------|-------------------------|
| Site 1 | 90 | 4 | 0 | 0 | 60 | 30 | 100 | 100 |
| Site 2 | 90 | 2 | 0 | 2 | 58 | 30 | 96.7 | 100 |
| Site 3 | 90 | 0 | 0 | 1 | 59 | 30 | 98.3 | 100 |
| Total | 270 | 6 | 0 | 3 | 177 | 90 | 98.3 | 100 |

# Analytical Reactivity (Inclusivity)

To evaluate the inclusivity of the SensiTox B. anthracis Toxin Test, 29 B. anthracis strains containing the wildtype pXO1 plasmid and representing geographical diversity of clinically relevant strains were tested (Table 5.4). Supernatants from mid-log phase cultures were isolated, sterilized, and the LF content quantified by ELISA. Based on this quantitation, supernatants from each strain were spiked into venous K₂EDTA anticoagulated whole blood at 3X LoD (150 pg/mL) and tested in triplicate. All 29 B. anthracis strains were detected.

{9}------------------------------------------------

| <i>B. anthracis</i> Strains | |
|-----------------------------|------------------|
| Vollum 1B | Turkey 32 |
| CDC 684 | Zimbabwe 89 |
| CDC 607 | N-99 |
| K2129 | 108 |
| K3 | 506/ Heroin Ba |
| 2002013094 | SK-31 |
| RA3 | K1811 (Scotland) |
| Vollum | English Vollum |
| Ohio ACB | South African |
| G-28/BA1035 | K1285 |
| SK-128 | K4834 |
| PAK-1 | New Hampshire |
| Buffalo | Bekasi |
| Carbosap | Sterne |
| 205 | |

Table 5.4. Clinically relevant strains of B. anthracis tested and detected.

Additionally, an in silico analysis of Lethal Factor amino sequences found in the National Center for Biotechnology Information (NCBI) database was conducted. Of the 303 unique B. anthracis strains identified, 299 (98.7%) had amino acid sequences identical to the strains tested in the inclusivity study, and 4 (1.3%) had amino acid substitutions not represented in the panel.

# Analytical Specificity – Cross Reactivity

Analytical specificity testing was conducted to assess the potential cross reactivity of the SensiTox B. anthracis Toxin Test to bacterial, viral, protozoan, and fungal pathogens potentially found in human blood. Bacterial, protozoan, and fungal pathogens were typically tested at 1x106 CFU/mL and viral pathogens at 1x105 PFU/mL by the addition to pre-screened K2EDTA human venous whole blood. Each pathogen was tested in triplicate. A list of all pathogens and the concentrations tested is shown in Table 5.5.

With the exception of two B. cereus strains, G9241 and 03BB102, which contain the extrachromosomal plasmid pXO1 that harbors the gene for Lethal Factor, no pathogens crossreacted when tested in the SensiTox B. anthracis Toxin Test. These two cross-reactive strains of B. cereus were detected as expected.

{10}------------------------------------------------

Table 5.5. Bacteria, viruses, fungi, and protozoa tested for cross-reactivity. The entries marked with (*) indicate that those organisms were tested in % parasitemia. Entries marked with (**) indicate that those organisms were tested in IU/mL. The entries marked with (†) indicate that those organisms were tested in copies/mL

| Bacteria Tested with No Cross-Reactivity | | | |
|----------------------------------------------|---------|----------------------------------------------------|---------|
| Strain | CFU/mL | Strain | CFU/mL |
| Acinetobacter baumannii, 307-0294 | 1.0E+06 | Enterococcus faecium, ZeptoMetrix Z265 | 1.0E+06 |
| Bacillus cereus, ZeptoMetrix Z091 | 1.0E+06 | Escherichia coli, ZeptoMetrix 0801517 | 1.0E+06 |
| Bacillus cereus, 3A (Fri-41) | 1.0E+06 | Francisella philomiragia, O#319-036 | 1.0E+06 |
| Bacillus cereus, D17 | 1.0E+06 | Francisella tularensis, Schu S4 | 7.7E+05 |
| Bacillus cereus, E33L | 7.0E+05 | Haemophilus influenzae, type b; Eagan | 1.0E+06 |
| Bacillus cereus, m1550 | 1.0E+06 | Klebsiella aerogenes, ZeptoMetrix Z052 | 1.0E+06 |
| Bacillus cereus, MSX-A1 | 1.0E+06 | Klebsiella oxytoca, ZeptoMetrix Z115 | 1.0E+06 |
| Bacillus cereus, VD148 | 6.7E+05 | Klebsiella pneumoniae, KPC-2 | 1.0E+06 |
| Bacillus cereus, 03BB108 | 1.0E+06 | Legionella pneumophila, Philadelphia | 1.0E+06 |
| Bacillus circulans Jordan, 26 | 1.0E+06 | Leishmania donovanii, MHOM/IN/80/DD8 | 1.0E+06 |
| Bacillus coagulans Hammer, NRS 609 | 1.0E+06 | Listeria monocytogenes, Serotype 1/2b | 1.0E+06 |
| Bacillus mycoides Flugge, NRS 273 | 1.0E+06 | Mycobacterium tuberculosis (avirulant), H37Ra-1 | 1.0E+06 |
| Bacillus subtilis Marburg | 1.0E+06 | Mycoplasma pneumoniae, M129 | 1.0E+06 |
| Bacillus thuringiensis, Al Hakam | 1.0E+06 | Neisseria gonorrhoeae, ZeptoMetrix Z017 | 1.0E+06 |
| Bacillus thuringiensis, Berliner, CCUG 7429T | 1.0E+06 | Neisseria meningitidis, Serogroup A | 1.0E+06 |
| Bacillus thuringiensis, NRRL B-3792 | 1.0E+06 | Proteus mirabilis, ZeptoMetrix Z050 | 1.0E+06 |
| Bacillus thuringiensis, USDA HD522 | 4.0E+05 | Pseudomonas aeruginosa, ZeptoMetrix Z139 | 1.0E+06 |
| Bacillus thuringiensis, HD1011 | 1.0E+06 | Pseudomonas luteola, JCM 3352 | 1.0E+06 |
| Bacillus thuringiensis, HD974 | 1.0E+06 | Rickettsia prowzekii, Naples-1 | 1.0E+05 |
| Bacillus thuringiensis, HD571 | 1.0E+06 | Rickettsia sibirica, 246 | 1.0E+05 |
| Bacillus thuringiensis, HD682 | 1.0E+06 | Salmonella enterica, Typhi, ZeptoMetrix Z152 | 1.0E+06 |
| Bacillus thuringiensis, ZeptoMetrix Z096 | 1.0E+06 | Salmonella enterica, Typhimurium, ZeptoMetrix Z005 | 1.0E+06 |
| Bacillus thuringiensis, 97-27 | 1.0E+06 | Serratia marcescens, ZeptoMetrix Z053 | 1.0E+06 |
| Bacteroides fragilis, ZeptoMetrix Z029 | 1.0E+06 | Shigella sonnei, ZeptoMetrix Z004 | 1.0E+06 |
| Bordetella bronchiseptica, ZeptoMetrix | 1.0E+06 | Staphylococcus aureus, MSSA; ΔmecA | 1.0E+06 |
| Borrelia burgdorferi, B31 | 1.0E+06 | Staphylococcus epidermidis, MSSE; HER 1292 | 1.0E+06 |
| Brucella melitensis, 16M (NCTC 10094) | 1.0E+06 | Streptococcus agalactiae, ZeptoMetrix Z019 | 1.0E+06 |
| Burkholderia cepacia, ZeptoMetrix Z066 | 1.0E+06 | Streptococcus pneumoniae, ZeptoMetrix Z022 | 1.0E+06 |
| Burkholderia mallei, Ivan (NTCC 10230) | 1.0E+06 | Streptococcus pyogenes, ZeptoMetrix Z018 | 1.0E+06 |
| Burkholderia pseudomallei, NCTC 4845 | 1.0E+06 | Vibrio cholera, ZeptoMetrix Z133 | 1.0E+06 |
| Chlamydophila pneumoniae, ZeptoMetrix Z500 | 1.0E+06 | Yersinia aldovae, CNCTC Y 49/84 | 1.0E+06 |
| Citrobacter koseri, ZeptoMetrix Z039 | 1.0E+06 | Yersinia bercovieri, CDC 2475-87 | 1.0E+06 |
| Clostridium bifermentans, ZeptoMetrix Z176 | 1.0E+06 | Yersinia enterocolitica, ZeptoMetrix Z036 | 1.0E+06 |
| Clostridium botulinum, IBCA 10-7060 | 7.6E+04 | Yersinia fredericksenii, CDC 1462-81 | 1.0E+06 |
| Clostridium perfringens, Type A | 1.0E+06 | Yersinia intermedia, CDC 881-81 | 1.0E+06 |
| Clostridium sordellii, ZeptoMetrix Z077 | 1.0E+06 | Yersinia kristensenii, CDC 1460-81 | 1.0E+06 |
| Clostridium sporogenes, ZeptoMetrix Z355 | 1.0E+06 | Yersinia mollaretti, CDC 2465-87 | 1.0E+06 |
| Corynebacterium diptheriae, ZeptoMetrix Z116 | 1.0E+06 | Yersinia pestis, A1122 | 1.0E+06 |
| Enterococcus faecalis, ZeptoMetrix Z346 | 1.0E+06 | Yersinia pseudotuberculosis, ZeptoMetrix Z222 | 1.0E+06 |
| | | Yersinia ruckeri, CDC 2396-61 | 1.0E+06 |

{11}------------------------------------------------

| Bacteria Tested with Cross-Reactivity | | | |
|----------------------------------------------------|---------|---------------------------------------------------------|---------|
| Strain | CFU/mL | Strain | CFU/mL |
| Bacillus cereus, G9242 | 1.0E+06 | Bacillus cereus, 03BB102 | 1.0E+06 |
| Viruses Tested with No Cross-Reactivity | | | |
| Strain | PFU/mL | Strain | PFU/mL |
| Adenovirus (C1), 1 (Species C)** | 1.0E+05 | Influenza A (H1N1), New York/18/09 | 2.9E+04 |
| Chikungunya Virus, S-27 | 1.0E+05 | Influenza A (H3N2), Kumamoto/102/02 | 1.2E+04 |
| Coronavirus 229E, 229E | 9.1E+04 | Influenza B, B/Colorado/06/2017** | 1.0E+05 |
| Cytomegalovirus, AD169 | 1.0E+05 | Japanese Encephalitis Virus (JEV), Nakayama | 1.0E+05 |
| Eastern Equine Encephalitis Virus, FL93-939 | 1.0E+05 | Measles Virus, ZeptoMetrix 0810025CF | 2.9E+04 |
| Enterovirus Type 68, 2014 isolate 1 | 1.0E+05 | Mumps Virus, 1 | 1.7E+04 |
| Epstein-Barr Virus (EBV), B95-8 | 1.0E+05 | Parainfluenza Virus 1, ZeptoMetrix 0810014CF | 8.8E+04 |
| Hantaan Virus, 76-118 | 2.0E+04 | Respiratory Syncytial Virus Type A, 1/2015<br>isolate 1 | 8.8E+04 |
| Hepatitis A Virus (HAV), HM 175 (clone 1)** | 1.0E+05 | Rhinovirus, 16** | 1.0E+05 |
| Hepatitis B Virus (HBV), ZeptoMetrix<br>0810031C** | 1.0E+05 | Rubella Virus, ZeptoMetrix 0810048CF | 2.5E+04 |
| Hepatitis C Virus (HCV), Seracare** | 1.0E+05 | St. Louis Encephalitis Virus, V 07457 | 1.0E+05 |
| Herpes Simplex Virus 1 (HSV-1), MacIntyre | 1.0E+05 | Vaccinia Virus, ZeptoMetrix 0810310CF | 1.0E+05 |
| Herpes Simplex Virus 2 (HSV-2), G Strain** | 1.0E+05 | Varicella Zoster Virus (VZV), A | 1.0E+05 |
| Human Herpesvirus 6A (HHV-6A), GS+ | 1.0E+05 | West Nile Virus, CO 08-13386 | 1.0E+05 |
| Human Immunodeficiency Virus (HIV), BaL** | 1.0E+05 | Western Equine Encephalitis, CO921356 | 1.0E+05 |
| Human Metapneumovirus, IA 10-2003 | 8.8E+04 | Yellow Fever Virus, 17D | 1.7E+04 |
| Fungi and Protozoa Tested with no Cross-Reactivity | | | |
| Strain | CFU/mL | Strain | CFU/mL |
| Aspergillus fumigatus, ZeptoMetrix Z014 | 1.0E+06 | Cryptococcus neoformans, serotype A | 1.0E+06 |
| Candida albicans, ZeptoMetrix Z006 | 1.0E+06 | Trypanosoma brucei, Lister 427 VSG 221 | 1.0E+06 |
| Strain | CFU/mL | Strain | CFU/mL |
| Acinetobacter baumannii, 307-0294 | 1.0E+06 | Enterococcus faecalis, ZeptoMetrix Z346 | 1.0E+06 |
| Bacillus cereus, ZeptoMetrix Z091 | 1.0E+06 | Enterococcus faecium, ZeptoMetrix Z265 | 1.0E+06 |
| Bacillus cereus, 3A (Fri-41) | 1.0E+06 | Escherichia coli, ZeptoMetrix 0801517 | 1.0E+06 |
| Bacillus cereus, D17 | 1.0E+06 | Francisella philomiragia, O#319-036 | 1.0E+06 |
| Bacillus cereus, E33L | 1.0E+06 | Haemophilus influenzae, type b, Eagan | 1.0E+06 |
| Bacillus cereus, m1550 | 1.0E+06 | Klebsiella aerogenes, ZeptoMetrix Z052 | 1.0E+06 |
| Bacillus cereus, MSX-A1 | 1.0E+06 | Klebsiella oxytoca, ZeptoMetrix Z115 | 1.0E+06 |
| Bacillus cereus, VD148 | 7.0E+05 | Klebsiella pneumoniae, KPC-2 | 1.0E+06 |
| Bacillus cereus, 03BB108 | 1.0E+06 | Legionella pneumophila, Philadelphia | 1.0E+06 |
| Bacillus cereus, G9241 | 1.0E+06 | Listeria monocytogenes, Serotype 1/2b | 1.0E+06 |
| Bacillus cereus, 03BB102 | 6.7E+05 | Mycobacterium tuberculosis (avirulant),<br>H37Ra-1 | 1.0E+06 |
| Bacillus circulans Jordan, 26 | 1.0E+06 | Mycoplasma pneumoniae, M129 | 1.0E+06 |
| Bacillus coagulans Hammer, NRS 609 | 1.0E+06 | Neisseria gonorrhoeae, ZeptoMetrix Z017 | 1.0E+06 |
| Bacillus mycoides Flugge, NRS 273 | 1.0E+06 | Neisseria meningitidis, Serogroup A | 1.0E+06 |
| Bacillus subtilis, Marburg | 1.0E+06 | Proteus mirabilis, ZeptoMetrix Z050 | 1.0E+06 |
| Bacillus thuringiensis, Al Hakam | 1.0E+06 | Pseudomonas aeruginosa, ZeptoMetrix<br>Z139 | 1.0E+06 |
| Bacillus thuringiensis, Berliner, CCUG 7429T | 1.0E+06 | Pseudomonas luteola, JCM 3352 | 1.0E+06 |
| Bacillus thuringiensis, NRRL B-3792 | 1.0E+06 | Salmonella enterica, Typhi, ZeptoMetrix<br>Z152 | 1.0E+06 |
| Bacillus thuringiensis, USDA HD522 | 1.0E+06 | Salmonella enterica, Typhimurium,<br>ZeptoMetrix Z005 | 1.0E+06 |
| Bacillus thuringiensis, HD1011 | 1.0E+06 | Serratia marcescens, ZeptoMetrix Z053 | 1.0E+06 |
| Bacillus thuringiensis, HD974 | 4.0E+05 | Shigella sonnei, ZeptoMetrix Z004 | 1.0E+06 |
| Bacillus thuringiensis, HD571 | 1.0E+06 | Staphylococcus aureus, MSSA; $\Delta$ mecA | 1.0E+06 |
| Bacillus thuringiensis, HD682 | 1.0E+06 | Staphylococcus epidermidis, MSSE; HER<br>1292 | 1.0E+06 |
| Bacillus thuringiensis, ZeptoMetrix Z096 | 1.0E+06 | Streptococcus agalactiae, ZeptoMetrix Z019 | 1.0E+06 |
| Bacillus thuringiensis, 97-27 | 1.0E+06 | Streptococcus pneumoniae, ZeptoMetrix<br>Z022 | 1.0E+06 |
| Bacteroides fragilis, ZeptoMetrix Z029 | 1.0E+06 | Streptococcus pyogenes, ZeptoMetrix Z018 | 1.0E+06 |
| Bordetella bronchiseptica, ZeptoMetrix | 1.0E+06 | Vibrio cholera, ZeptoMetrix Z133 | 1.0E+06 |
| Borrelia burgdorferi, B31 | 1.0E+06 | Yersinia aldovae, CNCTC Y 49/84 | 1.0E+06 |
| Burkholderia cepacia, ZeptoMetrix Z066 | 1.0E+06 | Yersinia bercovieri, CDC 2475-87 | 1.0E+06 |
| Chlamydophila pneumoniae, ZeptoMetrix Z500 | 1.0E+06 | Yersinia enterocolitica, ZeptoMetrix Z036 | 1.0E+06 |
| Citrobacter koseri, ZeptoMetrix Z039 | 1.0E+06 | Yersinia fredericksenii, CDC 1462-81 | 1.0E+06 |
| Clostridium bifermentans, Z176 | 1.0E+06 | Yersinia intermedia, CDC 881-81 | 1.0E+06 |
| Clostridium perfringens, Type A | 1.0E+06 | Yersinia kristensenii, CDC 1460-81 | 1.0E+06 |
| Clostridium sordellii, ZeptoMetrix Z077 | 1.0E+06 | Yersinia mollaretti, CDC 2465-87 | 1.0E+06 |
| Bacteria | | | |
| Strain | CFU/mL | Strain | CFU/mL |
| Clostridium sporogenes, ZeptoMetrix Z355 | 1.0E+06 | Yersinia pseudotuberculosis, ZeptoMetrix Z222 | 1.0E+06 |
| Corynebacterium diptheriae, ZeptoMetrix Z116 | 1.0E+06 | Yersinia ruckeri, CDC 2396-61 | 1.0E+06 |
| | | | |
| Viruses | | | |
| Strain | PFU/mL | Strain | PFU/mL |
| Adenovirus (C1), 1 (Species C)* | 1.0E+05 | Influenza A (H1N1), New York/18/09 | 2.9E+04 |
| Coronavirus 229E, 229E | 9.1E+04 | Influenza A (H3N2), Kumamoto/102/02 | 1.2E+04 |
| Cytomegalovirus (CMV), AD169 | 1.0E+05 | Influenza B, B/Colorado/06/2017* | 1.0E+05 |
| Enterovirus Type 68, 2014 isolate 1 | 1.0E+05 | Measles Virus, ZeptoMetrix 0810025CF | 2.9E+04 |
| Epstein-Barr Virus (EBV), B95-8 | 1.0E+05 | Mumps Virus, 1 | 1.7E+04 |
| Hepatitis A Virus (HAV), HM 175 (clone 1)* | 1.0E+05 | Parainfluenza Virus 1, ZeptoMetrix 0810014CF | 8.8E+04 |
| Hepatitis B Virus (HBV), ZeptoMetrix 0810031C* | 1.0E+05 | Respiratory Syncytial Virus Type A, 1/2015 isolate 1 | 8.8E+04 |
| Hepatitis C Virus (HCV), Seracare* | 1.0E+05 | Rhinovirus, 16* | 1.0E+05 |
| Herpes Simplex Virus 1 (HSV-1), MacIntyre | 1.0E+05 | Rubella Virus, ZeptoMetrix 0810048CF | 2.5E+04 |
| Herpes Simplex Virus 2 (HSV-2), G Strain* | 1.0E+05 | Vaccinia Virus, ZeptoMetrix 0810310CF | 1.0E+05 |
| Human Herpesvirus 6A (HHV-6A), GS* | 1.0E+05 | Varicella Zoster Virus (VZV), A | 1.0E+05 |
| Human Immunodeficiency Virus (HIV), BaL* | 1.0E+05 | Yellow Fever Virus, 17D | 1.7E+04 |
| Human Metapneumovirus, IA 10-2003 | 8.8E+04 | | |
| | | | |
| Fungi and Protozoa | | | |
| Strain | CFU/mL | Strain | CFU/mL |
| Aspergillus fumigatus., ZeptoMetrix Z014 | 1.0E+06 | Cryptococcus neoformans, serotype A | 1.0E+06 |

# Analytical Specificity – Microbial Interference

Babesia microti, R1*

Testing was conducted to assess the interference of bacterial, viral, fungal, and protozoan pathogens potentially found in human blood on the detection of B. anthracis Lethal Factor with the SensiTox B. anthracis Toxin Test. Bacterial, fungal, and protozoan pathogens were typically tested at 1x106 CFU/mL and viral pathogens at 1x105 PFU/mL by addition to pre-screened K₂EDTA human venous whole blood containing 150 pg/mL of recombinant Lethal Factor (3X LoD). Each pathogen was tested in triplicate.

3.9 %

None of the organisms shown in Table 5.6 interfered with the SensiTox B. anthracis Toxin Test at the concentrations shown and generated detected results for all replicates containing 150 pg/mL of Lethal Factor.

{12}------------------------------------------------

Table 5.6. Bacteria, viruses, fungi, and protozoa tested for interference. Entries marked with (*) indicate that those organisms were tested in IU/mL. The entries marked with (†) indicate that those organisms were tested in copies/mL.

{13}------------------------------------------------

# Interfering Substances

Candida albicans, ZeptoMetrix Z006

Endogenous and exogenous substances commonly found in blood were evaluated for their potential effect on the performance of the SensiTox B. anthracis Toxin Test. Drugs and their metabolites were tested at approximately 3 times the highest concentration reported following therapeutic dosage. Endogenous substances were tested at the highest physiological concentration expected in the population. The solvents used to dissolve exogenous substances used in the study were tested independently.

Trypanosoma brucei, Lister 427 VSG 221

1.0E+06

Each compound was tested in K₂EDTA anticoagulated whole blood unspiked and spiked with 150 pg/mL (3X LoD) of recombinant Lethal Factor and run in triplicate. The list of endogenous and exogenous substances evaluated and the concentrations at which they were tested is shown in Table 5.7. None of the substances tested interfered with the SensiTox B. anthracis Toxin Test.

1.0E+06

{14}------------------------------------------------

| Endogenous Substances | Concentration | | |
|-----------------------------|---------------------|------------------------------|---------------------|
| Glucose | 10 mg/mL | | |
| Hemoglobin | 2 mg/mL | | |
| Human Immunoglobulins (IgG) | 19.7 mg/mL | | |
| Triglycerides | 22 mg/mL | | |
| Cholesterol | 2.5 mg/mL | | |
| Human Serum Albumin | 50 mg/mL | | |
| Bilirubin | 200 µg/mL | | |
| Human Anti-Mouse Antibodies | 1 µg/mL | | |
| Exogenous Substances | Concentration | Exogenous Substances | Concentration |
| Acetaminophen | 200 µg/mL | Heparin | 90.3 U/mL |
| Acid-citrate-dextrose 3x | 300 µL/mL | Ibuprofen | 500 µg/mL |
| Albuterol (Salbutamol) | 400 ng/mL | Malarone (Atovaquone) | 180 µg/mL |
| Amoxicillin | 75.2 µg/mL | Mefloquine | 18 µg/mL |
| Artemisinin | 9 µg/mL | N-acetylcysteine | 1.7 mg/mL |
| Ascorbic acid | 60 µg/mL | Naproxen sodium | 500 µg/mL |
| Aspirin | 652 µg/mL | Oseltamivir (Tamiflu) | 4.95 µg/mL |
| Biothrax | 0.9 µL/mL | Ribavirin | 51.3 µg/mL |
| Cefotaxime | 306 µg/mL | Rifampin | 66 µg/mL |
| Chloroquine | 36 µg/mL | Sodium citrate | 9.6 mg/mL |
| Ciprofloxacin | 10 µg/mL | Sodium polyanetholesulfonate | 1.5 mg/mL |
| Cromolyn sodium | 825 ng/mL | Streptomycin | 450 µg/mL |
| Doxycycline | 30 µg/mL | Sulfamethoxazole | 400 µg/mL |
| EDTA | 5.4 mg/mL | Tetracycline | 15 µg/mL |
| Erythromycin | 60 µg/mL | Tobramycin | 24 µg/mL |
| Flunisolide (Flovent) | 90 ng/mL | Trimethoprim | 40 µg/mL |
| Gentamicin sulfate | 10 µg/mL | | |

Table 5.7 Endogenous and exogenous interferents tested.

#### Hook Effect

This study was conducted to determine if a false negative result could be produced by high concentrations of analyte. Recombinant LF was diluted to concentrations ranging from 50 pg/mL to 50 µg/mL in K₂EDTA anticoagulated venous whole blood and three replicates of each dilution were tested.

Table 5.8 summarizes the qualitative results obtained in the study. All concentrations of LF greater than 50 pg/mL yielded 3/3 detected results. At 50 pg/mL, 1/3 replicates yielded a result of not detected. This result was not unexpected as 50 pg/mL is the limit of detection (LoD) of the SensiTox B. anthracis Toxin Test. No hook effect was observed in the reporting of the qualitative data between 50 pg/mL to 50 µg/mL of LF in K₂EDTA anticoagulated venous whole blood.

{15}------------------------------------------------

| Dilution Name | Lethal Factor (pg/mL) | # Detected / # Tested | # Invalid |
|---------------|-----------------------|-----------------------|-----------|
| S1 | 50,000,000 | 3 / 3 | 0 |
| S2 | 10,000,000 | 3 / 3 | 0 |
| S3 | 2,000,000 | 3 / 3 | 0 |
| S4 | 400,000 | 3 / 3 | 0 |
| S5 | 80,000 | 3 / 3 | 0 |
| S6 | 16,000 | 3 / 3 | 1 |
| S7 | 3,200 | 3 / 3 | 0 |
| S8 | 640 | 3 / 3 | 0 |
| S9 | 128 | 3 / 3 | 0 |
| S10 | 50 | 2 / 3 | 0 |

| Table 5.8. Qualitative results with LF concentrations ranging from 50 pg/mL – 50 µg/mL | | |
|----------------------------------------------------------------------------------------|--|--|
| | | |

### Clinical Performance Evaluation

The performance of the SensiTox B. anthracis Toxin Test was evaluated at three sites across the US. The evaluation consisted of two study arms, one with residual whole blood specimens collected in K₂EDTA for routine hematology analysis to determine negative percent agreement (NPA), and one with prospectively collected whole blood specimens from consented febrile patients that were spiked with recombinant or native Lethal Factor to determine positive percent agreement (PPA). All specimens were tested by the SensiTox B. anthracis Toxin Test within 72 hours of draw.

A total of 325 residual blood specimens were collected at a single clinical site. Seven specimens were withdrawn from this arm of the study, resulting in 318 specimens that were tested at two sites and included in the data analysis.

For the prospective study, a total of 126 subjects presenting with fever and flu-like symptoms were enrolled for the collection of fresh venous whole blood specimens. Twenty-one (21) specimens were withdrawn from the study resulting in 105 eligible samples.

For the prospective arm of the study, patient specimens were tested at a single site. Additionally, contrived specimens prepared from the prospectively collected blood specimens and containing either native Lethal Factor (LF) contained in cell supernatants at 75 pg/mL (1.5X LoD) and 250 pg/mL (5X LoD) or recombinant LF at concentrations of 75 pg/mL, and 5 μg/mL were prepared and tested blinded at a second site.

{16}------------------------------------------------

Table 5.9 summarizes the data from the testing of residual specimens at two sites. All specimens tested reported not detected results for B. anthracis Lethal Factor for a 100% NPA.

| All Sites | | Expected | | |
|--------------------------------------|-----------------------|-------------------------|-----|-------|
| | | Pos | Neg | Total |
| Residual whole blood specimens | | | | |
| SensiTox B. anthracis<br>Toxin Test | Pos (LF detected) | 0 | 0 | 0 |
| | Neg (LF Not detected) | 0 | 318 | 318 |
| | Total | 0 | 318 | 318 |
| All Sites | | (95% C.I.) | | |
| Negative Percent Agreement (NPA) (%) | | 100.0% (98.8% - 100.0%) | | |

| Table 5.9. Performance summary for residual whole blood specimens tested |
|--------------------------------------------------------------------------|
| |

In the prospective study, two vials of blood drawn from 105 subjects presenting with fever and flu-like symptoms were tested at two sites. Table 5.10 shows the overall data summary with a performance of 96.2% PPA and 100% NPA. Four false negative results, generated at a concentration of 75 pg/mL LF (1.5X LoD), were reported in the contrived study.

| All Sites | | Expected | | |
|--------------------------------------|-----------------------|-------------------------|-----|-------|
| | | Pos | Neg | Total |
| Specimens from the Prospective Arm | | | | |
| SensiTox B. anthracis<br>Toxin Test | Pos (LF detected) | 101 | 0 | 101 |
| | Neg (LF not detected) | 4 | 105 | 109 |
| | Total | 105 | 105 | 210 |
| All Sites | | (95% C.I.) | | |
| Positive Percent Agreement (PPA) (%) | | 96.2%* (90.6% - 98.5%) | | |
| Negative Percent Agreement (NPA) (%) | | 100.0% (96.5% - 100.0%) | | |

* All samples were confirmed positive when retested.

{17}------------------------------------------------

# Conclusion

The conclusions drawn from the nonclinical and clinical tests demonstrate that the device is as safe, as effective, and performs as well as or better than the legally marketed device. 807.92(b)(3).