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.

Device Description

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.

AI/ML Overview

Here's a summary of the SensiTox B. anthracis Toxin Test's acceptance criteria and the study that proves the device meets them, based on the provided FDA 510(k) summary:

Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as distinct pass/fail thresholds in the provided document, but rather implied by the design and results of the performed studies. The reported performance metrics from the non-clinical and clinical tests are:

Performance Metric	Acceptance Criteria (Implied)	Reported Device Performance
Limit of Detection (LoD)	A defined lowest concentration detectable with ≥95% hit rate.	50 pg/mL for Lethal Factor (achieved ≥95% hit rate).
Reproducibility (Overall Accuracy)	High overall accuracy across multiple sites, operators, and lots.	98.9% (267/270 samples correct).
Reproducibility (Positive Accuracy)	High accuracy for positive samples.	98.3% (177/180 samples correct).
Reproducibility (Negative Accuracy)	High accuracy for negative samples.	100% (90/90 samples correct).
Analytical Reactivity (Inclusivity)	Detection of a diverse panel of B. anthracis strains.	All 29 tested B. anthracis strains (at 3X LoD) were detected.
Analytical Specificity (Cross-Reactivity)	No cross-reactivity with common pathogens (except known LF-producing B. cereus strains).	No cross-reactivity with a wide range of bacteria, viruses, fungi, and protozoa, except for B. cereus G9241 and 03BB102, which possess the LF gene.
Analytical Specificity (Interference)	No interference from common pathogens on LF detection.	None of the tested pathogens interfered with LF detection (at 3X LoD).
Interfering Substances	No interference from common endogenous or exogenous substances.	None of the tested endogenous or exogenous substances interfered.
Hook Effect	No false negatives at high analyte concentrations.	No hook effect observed from 50 pg/mL to 50 µg/mL.
Clinical Performance (NPA - Residual)	High Negative Percent Agreement in residual samples.	100.0% (318/318) NPA.
Clinical Performance (PPA - Prospective)	High Positive Percent Agreement in prospective samples.	96.2% (101/105) PPA.
Clinical Performance (NPA - Prospective)	High Negative Percent Agreement in prospective samples.	100.0% (105/105) NPA.

Study Details

Sample sizes used for the test set and the data provenance:
- Limit of Detection (LoD):
  - Test set: Minimum of 5 serial dilutions, tested in replicates of 20 each.
  - Data provenance: In-house generated data using recombinant Lethal Factor or native Lethal Factor from B. anthracis culture supernatants spiked into whole blood from healthy donors.
- Reproducibility:
  - Test set: 270 samples (90 per site, 3 replicates per sample for each operator). Samples were whole blood spiked with low positive (1.5X LoD), moderate positive (3X LoD) recombinant LF, and negative (unspiked).
  - Data provenance: In-house generated data using spiked whole blood from healthy donors.
- Analytical Reactivity (Inclusivity):
  - Test set: 29 B. anthracis strains. Supernatants spiked into whole blood at 3X LoD, tested in triplicate.
  - Data provenance: In-house generated data using B. anthracis strains.
- Analytical Specificity (Cross-Reactivity):
  - Test set: Over 100 bacterial, viral, protozoan, and fungal pathogens (see Table 5.5 in the document). Each tested in triplicate by addition to pre-screened K₂EDTA human venous whole blood.
  - Data provenance: In-house generated data using specified pathogens.
- Analytical Specificity (Interference):
  - Test set: Over 100 bacterial, viral, fungal, and protozoan pathogens (see Table 5.6 in the document). Each tested in triplicate by addition to pre-screened K₂EDTA human venous whole blood containing 3X LoD recombinant LF.
  - Data provenance: In-house generated data using specified pathogens spiked into blood.
- Interfering Substances:
  - Test set: 8 endogenous and 33 exogenous substances (see Table 5.7 in the document). Each tested in triplicate in unspiked and 3X LoD spiked K₂EDTA whole blood.
  - Data provenance: In-house generated data using specified interfering substances.
- Hook Effect:
  - Test set: 10 dilutions of recombinant LF from 50 pg/mL to 50 µg/mL. Three replicates per dilution.
  - Data provenance: In-house generated data using recombinant LF spiked into whole blood.
- Clinical Performance Evaluation:
  - Negative Percent Agreement (NPA) - Residual specimens: 318 residual whole blood specimens (from a single clinical site).
  - Positive Percent Agreement (PPA) and NPA - Prospective specimens: 105 prospectively collected whole blood specimens from febrile patients (contingent on being eligible after initial 21 withdrawals). These were used for both unspiked and spiked testing. Contrived specimens (prepared from these prospective samples, spiked with native or recombinant LF at 1.5X LoD, 5X LoD, or 5 µg/mL) were also tested blinded at a second site.
  - Data provenance: Clinical sites in the US (residual and prospective).
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- For the non-clinical studies (LoD, Reproducibility, Analytical Reactivity, Specificity, Interference, Hook Effect), the ground truth was based on the precisely controlled spiking of known concentrations of Lethal Factor, specific B. anthracis strains, various pathogens, or interfering substances. No human expert consensus was used for these.
- For the Clinical Performance Evaluation, the ground truth for NPA in residual specimens was that they were presumed negative for B. anthracis Lethal Factor as they were collected for routine hematology analysis. For the prospective study, the ground truth was established by spiking the collected blood with known concentrations of Lethal Factor for PPA calculation. The intrinsic "negative" status of the febrile patients for naturally occurring anthrax Lethal Factor served as the ground truth for NPA. The document does not specify the qualifications of personnel involved in sample collection or initial characterization, but it implies standard clinical laboratory practices.
Adjudication method for the test set: Not applicable for this device as it's an in vitro diagnostic testing for a biomarker, not image-based or human-interpreted data that would require adjudication. The device provides a qualitative "Lethal Factor detected" or "Lethal Factor not detected" result.
If a multi-reader, multi-case (MRMC) comparative effectiveness study was done, if so, what was the effect size of how much human readers improve with AI vs without AI assistance: Not applicable. This is an automated immunofluorescence assay for a biomarker, not an AI-assisted diagnostic device requiring human interpretation of results.
If a standalone (i.e., algorithm only without human-in-the-loop performance) was done: Yes, the SensiTox B. anthracis Toxin Test, run on the MultiPath Analyzer, is a standalone device. The results are "interpreted using the MultiPath applications software as valid or invalid, and if valid, the results are reported as Lethal Factor detected." There is no explicit human-in-the-loop performance described beyond the loading of the cartridge and reading of the digital display.
The type of ground truth used:
- Non-clinical studies: Laboratory-controlled ground truth established by known concentrations of recombinant or native Lethal Factor, quantified culture supernatants, and characterized pathogen strains.
- Clinical Performance Evaluation:
  - Residual specimens (NPA): Presumed negative as they were "residual whole blood specimens collected in K₂EDTA for routine hematology analysis."
  - Prospective specimens (PPA): Artificially created by spiking prospectively collected patient blood with known concentrations of Lethal Factor.
  - Prospective specimens (NPA): Based on the absence of spiked Lethal Factor and the enrollment criteria (febrile patients without confirmed anthrax, ensuring presumed negativity for the target biomarker).
The sample size for the training set: Not explicitly stated as a separate "training set" in the context of machine learning. For this in vitro diagnostic device, the developmental and validation process involves extensive analytical studies (LoD, cross-reactivity, interference, etc.) using numerous spiked samples and characterized strains. The "training" in this context refers to the development and optimization of the assay and software algorithms during product development, which would likely have involved an internal set of samples, but no specific training set size is provided in this summary.
How the ground truth for the training set was established: As above, the ground truth for the development and optimization of the assay and software would have been established by precisely controlling the input (e.g., specific concentrations of LF, specific pathogen strains) in a laboratory setting. This is a common approach for in vitro diagnostic device development.

Summary

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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).

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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 (DICE@fda.hhs.gov) or phone (1-800-638-2041 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

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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 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)
Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

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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 LightDiagnostics, Inc.2 Omni WayChelmsford, MA 01824
Correspondent:	Joanne Spadoro, Ph.D.President and CEOFirst Light Diagnostics, Inc.2 Omni WayChelmsford, MA 01824Phone: (781) 271-0112, extension 224Email: joanne@firstlightdx.com

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(DEN220044)

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Device Description Summary

Intended Use/Indications for Use

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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.

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.Subject DeviceSensiTox B. anthracis Toxin Test	InBios International, Inc.Predicate DeviceDEN220044Active 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 Testfor use with the MultiPath Analyzer, isa qualitative immunofluorescenceassay to aid in the diagnosis ofinhalation anthrax. The test isintended for the rapid, qualitativedetection of lethal factor, a biomarkerassociated with Bacillus anthracis (B.anthracis). The test can be used withwhole blood collected with	The Active Anthrax Detect Plus RapidTest point-of-care diagnostic test forpulmonary anthrax is an in vitroimmunochromatographic device for useas an aid in the diagnosis of inhalationanthrax. It provides visual and rapidqualitative detection of lethal factor ofBacillus anthracis (B. anthracis). The testcan be used to test serum and venouswhole blood (dipotassium EDTA, sodium

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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.

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.

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This assay is not FDA-cleared orapproved for testing blood or plasmadonors.



Similarities
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
Differences
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.

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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	TotalSamples	#Invalid	FN	TP	TN	% AccuracyPositives	% AccuracyNegatives
Site 1	90	4	0	60	30	100	100
Site 2	90	2	2	58	30	96.7	100
Site 3	90	0	1	59	30	98.3	100
Total	270	6	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.

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B. anthracis 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.

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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, 1/2015isolate 1	8.8E+04
Hepatitis A Virus (HAV), HM 175 (clone 1)**	1.0E+05	Rhinovirus, 16**	1.0E+05
Hepatitis B Virus (HBV), ZeptoMetrix0810031C**	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),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, ZeptoMetrixZ139	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, ZeptoMetrixZ152	1.0E+06
Bacillus thuringiensis, USDA HD522	1.0E+06	Salmonella enterica, Typhimurium,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; HER1292	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, ZeptoMetrixZ022	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.

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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.

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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. anthracisToxin 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. anthracisToxin 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).

Regulation Number and Section

N/A