The T2Biothreat Panel will not distinguish between detection of Burkholderia mallei and Burkholderia pseudomallei but will present valid detections as a positive detection of Burkholderia species.

The T2Biothreat Panel is intended to test individuals with signs and symptoms of infection from biothreat agents and/or individuals who are at risk for exposure or may have been exposed to these agents. The T2Biothreat Panel is indicated as an aid in the diagnosis of anthrax, tularemia, melioidosis, glanders, typhus fever and plague in response to suspected or confirmed bioterrorism events or outbreaks. Diagnosis of infection must be made in conjunction with clinical, epidemiologic and other laboratory data. Results are for the presumptive identification of Bacillus anthracis, Francisella tularensis, Burkholderia spp. (B. mallei), Yersinia pestis and Rickettsia prowazekii. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidel by the relevant public health authorities. The definitive identification of Bacillus anthracis, Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Yersinia pestis or Rickettsia prowazekii requires additional testing and confirmation procedures in consultation with the appropriate public health authorities for whom reports may be required. Positive results do not rule out co-infections with pathogens not included on the T2Biothreat Panel. 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 T2Biothreat Panel is indicated for use in laboratories that have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities, and personnel trained in the safe handling of clinical specimens potentially containing biothreat organisms.

The T2Biothreat Panel is indicated for use in laboratories that follow public health guidelines 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.

Device Description

The T2Biothreat Panel is a qualitative, multiplexed, nucleic acid-based in vitro diagnostic test intended for use with the T2Dx Instrument. The T2Biothreat Panel detects nucleic acids from the following organisms directly from K2EDTA whole blood samples: Bacillus anthracis (plasmids pXO1 and pXO2), Francisella tularensis, Burkholderia spp. (B. mallei/B. pseudomallei), Yersinia pestis, and Rickettsia prowazekii. The T2Biothreat Panel is run on the T2Dx, a fully automated, benchtop instrument. During processing on the T2Dx, intact pathogen cells are concentrated directly in whole blood, then lysed to release the target DNA. After amplification, target amplicon is hybridized with superparamagnetic particles and then detected by T2MR. The Internal Control on the T2Biothreat Panel monitors performance for each sample. The T2Biothreat Panel is a qualitative molecular diagnostic assay that employs whole blood compatible PCR amplification followed by T2 Magnetic Resonance (T2MR) detection. The T2Biothreat Panel is performed on the T2Dx Instrument, which executes all steps after specimen loading, with the capability of loading up to seven blood specimens at the same time. Individually, a KeEDTA whole blood specimen containing a minimum of 3 mL is loaded directly onto the T2Biothreat Sample Inlet, which is then placed on the T2Biothreat Cartridge along with the T2Biothreat Reagent Tray. The Cartidge and Reagent Tray contain the lysis reagent, internal control, primers, enzyme, buffer and probe-coupled superparamagnetic particles for each detected tarqet. After loading into the T2Dx. the blood specimen is mixed with the red blood cell lysing reagent and the bacterial cells and human cellular debris are concentrated by centrifygation. The internal control is added to the concentrated pellet and a bead-beating process lyses the bacterial cells. The supernatant containing the DNA from the lysed bacterial cells and the internal control is amplified using the target and internal control-specific primers. The generated amplified product is aliquoted into individual tubes containing target-specific probe conjugated particles for each detected target and the internal control. The amplified to target-specific probes attached to superparamagnetic particles causing clustering of the particles. The hybridization occurring in individual tubes is analyzed in the T2MR reader and a signal for each target is generated, which indicates the presence of the target organism(s). This automated process is the same process followed by the FDA cleared T2Candida and T2Bacteria Panels performed on the T2Dx Instrument system. When running a single specimens simultaneously, the first specimen will be reported in approximately 4 hours from the specimen is loaded onto the instrument. The results are interpreted by the device software as valid or invalid (based on the result of the internal control or target detections), and if valid, results are reported as "Positive" or "Target not Detected" for each specific target.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study information for the T2Biothreat Panel, based on the provided text:

Acceptance Criteria and Device Performance

Parameter	Acceptance Criteria (Implicit)	Reported Device Performance
Limit of Detection (LoD)	95% positivity rate at minimum bacterial concentration	Ranges from 2-17 CFU/mL or 9 CAGe/mL
Analytical Reactivity	100% inclusivity for target sequences	All tested strains successfully detected, except Y. pestis strains lacking pPCP plasmid.
Analytical Specificity	No cross-reactivity with common bloodstream infection organisms or genetically similar pathogens	No cross-reactivity observed at 1,000 CFU/mL (or IU/mL) for 30 out of 31 tested strains. One Bacillus cereus strain (G-9241) with a pXO1-like plasmid showed cross-reactivity with the BaPXO1 channel, but the device differentiates this from fully virulent B. anthracis. No cross-reactivity at higher concentrations (1x10^5 copies/mL) for exclusivity strains.
Reproducibility	High agreement with expected positive and negative results across sites, operators, lots, and instruments.	Overall agreement of 98.4% for expected positive results; 100% for expected negative results.
Interfering Substances	No interference with detection of targets or sample validity by specified endogenous and exogenous substances.	None of the tested substances (excluding Feraheme, Magnevist, and Ablavar which are known interferents at high concentrations from previous studies) demonstrated interference.
Competitive Inhibition	No competitive effects impacting positive detection in co-infection scenarios.	No competitive effects observed for any combination of Panel members or non-Panel members.
Clinical Sensitivity (PPA)	High Positive Percent Agreement (PPA) for target analytes.	Ranged from 94.3% to 100% for analyte concentrations at 1-3x LoD.
Clinical Specificity (NPA)	High Negative Percent Agreement (NPA) for all analytes.	100% for all analytes.

Study Information

The provided document describes standalone performance studies for the T2Biothreat Panel. It does not mention any multi-reader multi-case (MRMC) comparative effectiveness study.

2. Sample Sizes and Data Provenance

Test Set (Clinical Performance):
- Negative Arm: Undisclosed number of K2EDTA whole blood samples from healthy donors (no signs/symptoms of infection) and febrile donors (fever ≥ 100.4 °F).
- Positive Arm: Sequence-verified clinical bacterial strains spiked into whole blood collected from febrile donors. The sample sizes for the positive arm are listed as:
  - B. anthracis (pXO1 & pXO2): 6 (< LoD), 32 (1-3x LoD), 12 (3-5x LoD) = 50 samples
  - Burkholderia spp.: 17 (< LoD), 77 (1-3x LoD), 6 (3-5x LoD) = 100 samples
  - F. tularensis: 7 (< LoD), 35 (1-3x LoD), 8 (3-5x LoD) = 50 samples
  - R. prowazekii: Undisclosed (< LoD), 10 (1-3x LoD), 40 (3-5x LoD) = 50 samples
  - Y. pestis: 26 (< LoD), 24 (1-3x LoD), undisclosed (3-5x LoD) = 50 samples
- Data Provenance: Not explicitly stated regarding country of origin, but implies clinical samples from "7 geographically diverse sites" and "healthy and febrile donors." The study appears to be prospective in its design, using collected samples for testing.
Test Set (Analytical Performance - Reproducibility): Sample types consisted of negative K2EDTA-treated whole blood and various positive K2EDTA-treated whole blood samples (single, dual, and triple species spikes) at 1-1.5x LoD or 2-3x LoD. The total number of replicates for each condition varied but was sufficient to achieve the stated agreements (e.g., 128 replicates for B. anthracis at 1-1.5x LoD, 379 for negatives).

3. Number of Experts and Qualifications for Ground Truth

Not Applicable: For this in vitro diagnostic device, ground truth for the clinical performance study was established by spiking sequence-verified clinical bacterial strains into whole blood samples at specific concentrations, meaning the "truth" was known by controlled experimental design rather than expert human interpretation of results. No external "experts" were used to establish ground truth for the test set.

4. Adjudication Method for the Test Set

Not Applicable: As the clinical performance study involved spiking known concentrations of sequence-verified bacterial strains into samples, the ground truth was inherently known. There was no need for an adjudication method as the results are compared directly against the known composition of the spiked samples.

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

No: The document does not mention a multi-reader multi-case (MRMC) comparative effectiveness study or any assessment of how human readers improve with or without AI assistance. This device is an automated in vitro diagnostic panel, not an AI-assisted diagnostic tool for human interpretation.

6. Standalone Performance (Algorithm Only)

Yes: The entire set of analytical and clinical performance studies presented (Limit of Detection, Analytical Reactivity, Analytical Specificity, Reproducibility, Interfering Substances, Competitive Inhibition, Clinical Performance Characteristics) represents the standalone performance of the T2Biothreat Panel without human-in-the-loop for interpretation or decision-making. The device is fully automated, and results are interpreted by the device software.

7. Type of Ground Truth Used

Known Spiked Concentrations / Sequence-Verified Strains: For both analytical and clinical performance, the ground truth was established by carefully spiking known concentrations of sequence-verified clinical bacterial strains into K2EDTA whole blood samples. This provides a definitive "true positive" or "true negative" status against which the device's performance is measured.
For the negative arm of the clinical study, the ground truth was based on samples from "healthy donors" (presumed negative) and "febrile donors" (presumed negative for the target biothreat agents unless proven otherwise through the spiking).

8. Sample Size for the Training Set

Not provided/Applicable in the sense of ML training data: The document does not explicitly mention a "training set" in the context of machine learning model training data. This is an in vitro diagnostic device that uses a pre-defined algorithm (nucleic acid amplification followed by T2 magnetic resonance detection) rather than a machine learning model that requires a distinct training phase with labeled data. The development of the assay (primers, probes, algorithm parameters) would have involved extensive R&D and optimization, but this wouldn't be referred to as a "training set" in the same way as for AI/ML.

9. How Ground Truth for the Training Set Was Established

Not Applicable (for ML training data): As noted above, the concept of a separate "training set" for an AI/ML model with established ground truth is not explicitly addressed or relevant in the provided text for this in vitro diagnostic device. The "ground truth" during device development and optimization would come from standard microbiological and molecular biology techniques to confirm the presence, identity, and concentration of target organisms.

Summary

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September 15, 2023

Image /page/0/Picture/1 description: The image contains the logos of the Department of Health & Human Services and the Food and Drug Administration (FDA). The Department of Health & Human Services logo is on the left, and the FDA logo is on the right. The FDA logo includes the text "U.S. Food & Drug Administration".

T2 Biosystems, Inc. Rachel Gilbert Manager, Regulatory Affairs 101 Hartwell Avenue Lexington, Massachusetts 02421

Re: K231336

Trade/Device Name: T2Biothreat Panel Regulation Number: 21 CFR 866.4000 Regulation Name: Device To Detect And Identify Biothreat Microbial Agents In Human Clinical Specimens Regulatory Class: Class II Product Code: QVR, QBX, NSU Dated: May 8, 2023 Received: May 8, 2023

Dear Rachel Gilbert:

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 (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 located 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.

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

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801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 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 medical devices and radiation-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

Enclosure

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Indications for Use

510(k) Number (if known) K231336

Device Name T2Biothreat Panel

Indications for Use (Describe)

1. Bacillus anthracis (plasmids pXO1 and pXO2)
1. Francisella tularensis
1. Burkholderia spp. (B. mallei/B. pseudomallei)
1. Yersinia pestis
1. Rickettsia prowazekii

The T2Biothreat Panel will not distinguish between detection of Burkholderia mallei and Burkholderia pseudomallei but will present valid detections as a positive detection of Burkholderia species.

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

Type of Use (Select one or both, as applicable)
-------------------------------------------------

X Prescription Use (Part 21 CFR 801 Subpart D)

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

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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510(k) Summary

Date of Summary	May 8, 2023
Product Name	T2Biothreat Panel
Sponsor	T2Biosystems, Inc.101 Hartwell AvenueLexington, MA 02421
Correspondent	Rachel GilbertManager, Regulatory Affairs781-226-2767, 1970rgilbert@t2biosystems.com
Device Trade or Proprietary Name	T2Biothreat Panel
Regulation	21 CFR 866.4000
Common Name	Multiplex Nucleic Acid Detection System For Biothreat Agents
Product Code	QVR
Classification	Class II

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Intended Use

1. Bacillus anthracis (plasmids pXO1 and pXO2)
1. Francisella tularensis
1. Burkholderia spp. (B. malleilB. pseudomallei)
1. Yersinia pestis
1. Rickettsia prowazekii

The T2Biothreat Panel will not distinguish between detection of Burkholderia mallei and Burkholderia pseudomallei but will present valid detections as a positive detection of Burkholderia species.

The T2Biothreat Panel is intended to test individuals with signs and symptoms of infection from biothreat agents and/or individuals who are at risk for exposure or may have been exposed to these agents. The T2Biothreat Panel is indicated as an aid in the diagnosis of anthrax, tularemia, melioidosis, glanders, typhus fever and plaque in response to suspected or confirmed bioterrorism events or outbreaks. Diagnosis of infection must be made in conjunction with clinical, epidemiologic and other laboratory data. Results are for the presumptive identification of Bacillus anthracis, Francisella tularensis, Burkholderia spp. (B. mallei), Yersinia pestis and Rickettsia prowazekii. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guided by the relevant public health authorities. The definitive identification of Bacillus anthracis, Francisella tularensis, Burkholderia pseudomallei, Yersinia pestis or Rickettsia prowazekii requires additional testing and confirmation with the appropriate public health authorities for whom reports may be required.

Positive results do not rule out co-infections with pathogens not included on the T2Biothreat Panel. 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 not FDA-cleared or approved for testing blood or plasma donors.

Limitations

For prescription use only. Refer to the Biothreat Panel labeling for a more complete list of warnings, precautions, and contraindications.

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Device Technology Overview

The T2Biothreat Panel is run on the T2Dx, a fully automated, benchtop instrument. During processing on the T2Dx, intact pathogen cells are concentrated directly in whole blood, then lysed to release the target DNA. After amplification, target amplicon is hybridized with superparamagnetic particles and then detected by T2MR. The Internal Control on the T2Biothreat Panel monitors performance for each sample.

The T2Biothreat Panel is a qualitative molecular diagnostic assay that employs whole blood compatible PCR amplification followed by T2 Magnetic Resonance (T2MR) detection. The T2Biothreat Panel is performed on the T2Dx Instrument, which executes all steps after specimen loading, with the capability of loading up to seven blood specimens at the same time. Individually, a KeEDTA whole blood specimen containing a minimum of 3 mL is loaded directly onto the T2Biothreat Sample Inlet, which is then placed on the T2Biothreat Cartridge along with the T2Biothreat Reagent Tray. The Cartidge and Reagent Tray contain the lysis reagent, internal control, primers, enzyme, buffer and probe-coupled superparamagnetic particles for each detected tarqet.

After loading into the T2Dx. the blood specimen is mixed with the red blood cell lysing reagent and the bacterial cells and human cellular debris are concentrated by centrifygation. The internal control is added to the concentrated pellet and a bead-beating process lyses the bacterial cells. The supernatant containing the DNA from the lysed bacterial cells and the internal control is amplified using the target and internal control-specific primers. The generated amplified product is aliquoted into individual tubes containing target-specific probe conjugated particles for each detected target and the internal control. The amplified to target-specific probes attached to superparamagnetic particles causing clustering of the particles. The hybridization occurring in individual tubes is analyzed in the T2MR reader and a signal for each target is generated, which indicates the presence of the target organism(s). This automated process is the same process followed by the FDA cleared T2Candida and T2Bacteria Panels performed on the T2Dx Instrument system.

When running a single specimens simultaneously, the first specimen will be reported in approximately 4 hours from the specimen is loaded onto the instrument. The results are interpreted by the device software as valid or invalid (based on the result of the internal control or target detections), and if valid, results are reported as "Positive" or "Target not Detected" for each specific target.

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Analytical Performance Characteristics

Limit of Detection (LoD)

LoD was defined as the minimum bacterial concentration at which a 95% positivity rate was achieved. The T2Biothreat Panel has a range of LoDs from 2-17 CFU/mL or 9 Cell-Associated Genomic equivalents (CAGe)/mL for the six target organisms (Table 1), as determined by measuring the percent positive detection for human whole blood spiked samples (№20) at a qiven CFU/mL level, Bacterial concentrations were assigned by quantitative CFUlmL plating of an aliquot of the final spike solution. Testing included two instruments, two reagent lots, and two different strains of each of the Panel members using K2EDTA-treated whole blood. The final LoD for each target was determined as the highest LoD determined across all reagents and strains. The LoD for B. anthracis was determined based on the detection of both pXO1 and pXO2. The data is summarized below:

Species	LoD
Bacillus anthracis	6 CFU/mL
Francisella tularensis	4 CFU/mL
Burkholderia mallei	12 CFU/mL
Burkholderia pseudomallei	17 CFU/mL
Yersinia pestis	2 CFU/mL
Rickettsia prowazekii	9 CAGe/mL

		Table 1: LoD of T2Biothreat Panel
--	--	-----------------------------------	--

Analytical Reactivity (Inclusivity)

To confirm detection of multiple clinically relevant strains of the organisms detected by the T2Biothreat Panel, an in silico analysis of primer and probe designs against the NCBInt data base determined that the assay was 100% inclusive of pathogens containing the target sequence. This was then confirmed with wet testing of B. anthracis, 8 strains of B. mallei, 10 strains of B. pseudomallei, 10 strains of R. prowazekii and 12 strains of Y. pestis on the T2Dx instrument. The strain identities were confirmed by whole genome sequence analysis. All strains were tested at a titer consistent with approximately 2 times the LoD level for each organism. All strains were successfully detected by the T2Biothreat Panel except two Y. pestis strains that lacked the pPCP plasmid that contains the Y. pestis species target sequence. All Y. pestis strains that lack the pPCP plasmid will be outside the inclusivity of the T2Biothreat Panel.

Table 2: Inclusivity Study Results

Organism	Strains	Positivity	RepeatTesting	Result
Bacillusanthracis10 strains	2002013094	3/3	N/A	Inclusive
	A0489	3/3	N/A	Inclusive
	A0707	3/3	N/A	Inclusive
	A0809	3/3	N/A	Inclusive
	Ames	3/3	N/A	Inclusive
	Buffalo	3/3	N/A	Inclusive
	Canadian Bison	3/3	N/A	Inclusive
	G-28	3/3	N/A	Inclusive
	SK-31	3/3	N/A	Inclusive
	Vollum	3/3	N/A	Inclusive
Organism	Strains	Positivity	Repeat Testing	Result
Burkholderiamallei8 strains	AMC Strain China 5	3/3	N/A	Inclusive
	GB8 Horse 4	3/3	N/A	Inclusive
	NCTC 10230	3/3	N/A	Inclusive
	NCTC10245	3/3	N/A	Inclusive
	NCTC 10260	3/3	N/A	Inclusive
	NCTC 120	3/3	N/A	Inclusive
	NCTC 3708	3/3	N/A	Inclusive
	NCTC 3709	3/3	N/A	Inclusive
Burkholderiapseudomallei10 strains	Human/Blood/OH/US/1994 7894	3/3	N/A	Inclusive
	China 3	3/3	N/A	Inclusive
	ATCC 23343	3/3	N/A	Inclusive
	Environment/Thailand/1990	3/3	N/A	Inclusive
	HBPUB10134A	2/3	19/20	Inclusive
	JCU-NCTC 13178	2/3	20/20	Inclusive
	MSHR840	3/3	N/A	Inclusive
	NAU20B16	3/3	N/A	Inclusive
	NCTC 13392	3/3	N/A	Inclusive
	425 100892A	2/3	20/20	Inclusive
Francisellatularensis10 strains	ATCC Vaccine Strain GA99-3549	3/3	N/A	Inclusive
	HN63	3/3	N/A	Inclusive
	JAP (Cincinnati)	3/3	N/A	Inclusive
	KY99	3/3	N/A	Inclusive
	OR96	3/3	N/A	Inclusive
	MO#1	3/3	N/A	Inclusive
	MR#2	3/3	N/A	Inclusive
	103-2P	3/3	N/A	Inclusive
	Addis Abbas	3/3	N/A	Inclusive
	Cairo	3/3	N/A	Inclusive
Rickettsiaprowazekii5 strains	GvF	3/3	N/A	Inclusive
	ZRS	3/3	N/A	Inclusive
	195/P (India)	3/3	N/A	Inclusive
	Angola	3/3	N/A	Inclusive
	AZ94-0666	3/3	N/A	Inclusive
Yersinia pestis12 strains	Bombay	0/3	N/A	Not Detected
	El Dorado 2572-1	3/3	N/A	Inclusive
	Harbin	3/3	N/A	Inclusive
	MAD115	3/3	N/A	Inclusive
	Pestoides G	0/3	0/20	Not Detected
	Shasta	3/3	N/A	Inclusive
	ZE94-2122	3/3	N/A	Inclusive
	PBM19	3/3	N/A	Inclusive
	Nicholick	3/3	N/A	Inclusive

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Analytical Specificity (Exclusivity)

To establish the analytical specificity of the T2Biothreat Panel, 31 bacterial, fungal, and viral strains commonly found to cause blood stream infections or to be genetically similar to pathogens detected by the Panel were tested. All organisms were prepared at 1,000 CFU/mL, or IU/mL K2EDTA-treated whole blood followed by titration to determine if any repeatable reactivity was detected. The results are outlined below:

Non-Reactive Species/Strains
Bacillus cereus NR-608	Burkholderia thailandensis	Human Immunodeficiency Virus3
Bacillus cereus BAG1X1-11	Candida albicans	Klebsiella pneumoniae
Bacillus cereus BAG4X2-12	Citrobacter koseri	Pseudomonas aeruginosa
Bacillus cereus VD1152	Clostridium perfringens	Rickettsia rickettsia
Bacillus tropicus	Enterobacter aerogenes	Rickettsia typhi
Bacillus thuringiensis	Enterococcus faecalis	Staphylococcus epidermidis
Bacillus anthracis A0006(pXO1 plasmid only)	Enterococcus faecium	Staphylococcus lugdunensis
Bacillus anthracis 4229(pXO2 plasmid only)	Escherichia coli	Staphylococcus aureus
Bacteroides fragilis	Francisella hispaniensis	Yersinia pseudotuberculosis
Burkholderia cepacia	Francisella philomiragia	Yersinia enterocolitica

Table 3: Pathogens for Which no Reactivity was Detected

1 Harbors a pXO-1 like plasmid that was not detected by the Panel;

2 Harbors a pXO-2-like plasmid that was not detected by the Panel;

3 Tested as IU/mL

Table 4: Cross-Reactive Species/Strains at Concentrations from 1,000 to 10 CFU/mL

Cross-reactiveNon-Panel Strain	Panel Channelthat Cross-reacts
Bacillus cereus G-9241	BaPXO1

Five (5) strains of Bacillus cereus were tested, two of which contained a pXO1-like plasmid and two that contained a pXO2-like plasmid. None of these strains were detected except strain G-9241, known to harbor a plasmid similar to pX01 in Bacillus anthracis. The T2Biothreat Panel distinguishes between the detection of a single virulence plasmid in a Bacillus spp. and detection of fully virulent B. anthracis harboring both the pXO1 and pXO2 plasmids and the software identifies results accordingly.

To evaluate higher concentrations of analyte which may be encountered in whole blood samples, 1x105 copies/mL of DNA from the exclusivity strains were spiked individually into whole blood samples and tested in triplicate with the T2Biothreat Panel. No cross-reactivity was observed at this higher analyte concentration.

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Non-Reactive Species/Strains
Bacillus cereus NR-608	Candida albicans	Pseudomonas aeruginosa
Bacillus cereus BAG1X1-1	Citrobacter koseri	Rickettsia rickettsia
Bacillus cereus BAG4X2-1	Clostridium perfringens	Rickettsia typhi
Bacillus cereus VD115	Enterobacter aerogenes	Staphylococcus epidermidis
Bacillus circulans	Enterococcus faecalis	Staphylococcus lugdunensis
Bacteroides fragilis	Enterococcus faecium	Staphylococcus aureus
Bacillus tropicus	Escherichia coli	Yersinia enterocolitica
Bacillus thuringiensis	Francisella hispaniensis	Yersinia pseudotuberculosis
Burkholderia cepacia	Francisella philomiragia
Burkholderia thailandensis	Klebsiella pneumoniae

Table 5: Exclusivity Study Organisms Evaluated at 1x 106 copies/mL

Reproducibility

Results agreement was analyzed at multiple sites over six non-consecutive days for each sample type, minimum of two operators per site, two Reagent Tray Kit lots, and two instruments. Sample types consisted of a negative K2EDTA-treated whole blood sample, and three types of positive K2EDTA-treated whole blood samples (B. anthracis, B. pseudomallei, and Y. pestis triple species spike, B. mallei and R. prowazekii dual species spike, and F. tularensis single species spike) at 2-3x LoD or 1-1.5 LoD. Reproducibility results across instruments, operators, reagent lots, and sample type showed an overall agreement of 98.4% with expected positive results and an overall agreement of 100% for expected negative results.

	Table 6: Summary of Reproducibility Results

Organism	Conc.	ExpectedResult	Agreement with Expected Result [95% CI]
			BaPXO1 Channel	BaPXO2 Channel
B. anthracis	1-1.5x LoD	Detected	128/128100% [97.7-100]	127/12899% [95.7-100]
	2-3x LoD	Detected	104/104100% [97.2-100]	103/10499% [94.8-100]
B. pseudomallei	1-1.5x LoD	Detected	Bu Channel126/12898% [94.5-99.8]	-
	2-3x LoD	Detected	103/10499% [94.8-100]	-
B. mallei	1-1.5x LoD	Detected	96/96100% [96.9-100]	-
	2-3x LoD	Detected	95/9699% [94.3-100]	-
Y. pestis	1-1.5x LoD	Detected	Yp Channel126/12898% [94.5-99.8]	-
	2-3x LoD	Detected	104/104100% [97.2-100]	-

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Organism	Conc.	ExpectedResult	Agreement with Expected Result [95% CI]
R. prowazekii	1-1.5x LoD	Detected	Rp Channel95/9699% [94.3-100]	-
	2-3x LoD	Detected	96/96100% [96.9-100]	-
F. tularensis	1-1.5x LoD	Detected	Ft Channel96/96100% [96.9-100]	-
	2-3x LoD	Detected	95/9699% [94.3-100]	-
Negative	Negative	Not Detected	379/379100% [99.2-100]	-

Interfering Substances

To determine and characterize the effects of potential endogenous interfering substances on the performance of the T2Biothreat Panel, 7 endogenous and 15 exogenous substances were tested with contrived positive and negative samples in KzEDTA-treated whole blood by the T2Dx. Potentially interfering substances were tested at or above clinically relevant concentrations based on the CLSI guidelines. None of the tested substances demonstrated interference with the detection of any T2Biothreat Panel targets nor in the validity of samples tested. Based on previous studies, Feraheme is considered an interferent at ≥21μg/mL, Magnevist is considered an interferent at ≥1.7 mg/mL, and Ablavar is considered an interferent at ≥0.39 mg/mL. No interference was detectable at the specified test concentration for all other substances.

Table 7: Substances Tested for Interference with the T2Biothreat Panel - No Interference Observed
---------------------------------------------------------------------------------------------------	--

Endogenous Substances &Concentrations		Exogenous Substances &Concentrations
Bilirubin(conjugated)	475 μmol/L	Ampicillin	215 μmol/L	Levofloxacin	99.6 μmol/L
Bilirubin(unconjugated)	684 µmol/L	Chloramphenicol	241 μmol/L	Meropenemtrihydrate	884 µmol/mL
Creatinine	150 mg/L	Ciprofloxacin	36.2 μmol/L	Novobiocin	450 µmol/mL
Hemoglobin	>20 g/dL	Ceftazidimepentahydrate	606 µg/mL	Penicillin Gsodium salt	0.777 µg/mL
Intralipid (tomimictriglycerides)	1703mg/dL	Doxycyclinehyclate	40.5 μmol/L	Streptomycinsulfate	444 μmol/L
Urea	42.9mmol/L	Gentamicin sulfate	62.8 µg/mL	Tetracyclinehydrochloride	54 μmol/L
White Blood Cells	≥16.5x106cells/mL	K2EDTA	9 mg/mL	Trimethoprim	145 μmol/L
		Kanamycin Bsulfate	186 μmol/L

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Competitive Inhibition

Sensitivity of the T2Biothreat Panel was tested in whole blood samples spiked with two organisms to mimic coinfection in a patient sample. The conditions tested included: co-infection with two Panel target species both at, or near, the LoD; co-infection with two Panel target species is at high titer (1,000 CFU/mL or CAGe/mL) and the other is at or near LoD; and co-infection with one Panel species at or near LoD and a non-Panel species at high titer (1,000 CFU/mL or IU/mL). Non-Panel species tested were E. coli, E. faecium, K. pneumoniae, P. aeruginosa, S. aureus, C. albicans, and HV. This study assessed the impact on the positive detection of Panel members by competitive inhibition from other potential co-infecting species. No competitive effects were observed in any combination of Panel members at ≤1,000 CFU/mL or CAGe/mL or near LoD. No competitive effects were observed in any combination of non-Panel members at ≤1,000 CFU/mL or IU/mL.

Species 1 LowConc. (1-2xLoD)	Species 2 High and Low Conc.(1000 CFU/mL or CAGe/mL and 1-2x LoD)1	Positive DetectionRate		CompetitiveInhibition
		Species1	Species2

B. anthracis	B. mallei	100%	100%	No
	Y. pestis	100%	100%	No
	B. pseudomallei	100%	100%	No
	F. tularensis	100%	100%	No
	R. prowazekii	100%	100%	No
B. mallei	Y. pestis	100%	100%	No
	B. pseudomallei	100%	100%	No
	F. tularensis	100%	100%	No
	R. prowazekii	100%	100%	No
Y. pestis	B. mallei	100%	100%	No
	B. anthracis	100%	100%	No
	B. pseudomallei	100%	100%	No
	F. tularensis	100%	100%	No
B. pseudomallei	R. prowazekii	100%	100%	No
	B. anthracis	100%	100%	No
	Y. pestis	100%	100%	No
	F. tularensis	100%	100%	No
F. tularensis	R. prowazekii	100%	100%	No
	B. anthracis	100%	100%	No
	B. mallei	100%	100%	No
	Y. pestis	100%	100%	No
R. prowazekii	B. pseudomallei	100%	100%	No
	R. prowazekii	100%	100%	No
	B. anthracis	100%	100%	No
	B. mallei	100%	100%	No
	Y. pestis	100%	100%	No
	B. pseudomallei	100%	100%	No
	F. tularensis	100%	100%	No
Species 1 LowConc.(1-2x LoD)	Species 2 High Conc.(1000 CFU/mL, CAGe/mL, or IU/mL)1	Positive Detection RateSpecies 12	Positive Detection RateSpecies 22	CompetitiveInhibition
B. anthracis	Escherichia coli	100%	0%	No
	Enterococcus faecium	75%(3/4 Ba pXO2,4/4 Ba pXO1)100%(20/20 bothchannels)	0%	No
	Klebsiella pneumoniae	100%	0%	No
	Pseudomonas aeruginosa	100%	0%	No
	Staphylococcus aureus	100%	0%	No
	Candida albicans	100%	0%	No
	Human ImmunodeficiencyVirus	100%	0%	No
B. mallei	E. coli	100%	0%	No
	E. faecium	100%	0%	No
	K. pneumoniae	100%	0%	No
	P. aeruginosa	100%	0%	No
	S. aureus	100%	0%	No
	C. albicans	100%	0%	No
	HIV	100%	0%	No
Y. pestis	E. coli	100%	0%	No
	E. faecium	100%	0%	No
	K. pneumoniae	100%	0%	No
	P. aeruginosa	100%	0%	No
	S. aureus	100%	0%	No
	C. albicans	100%	0%	No
	HIV	100%	0%	No
B. pseudomallei	E. coli	100%	0%	No
	E. faecium	100%	0%	No
	K. pneumoniae	100%	0%	No
	P. aeruginosa	100%	0%	No
	S. aureus	100%	0%	No
	C. albicans	100%	0%	No
	HIV	100%	0%	No
F. tularensis	E. coli	100%	0%	No
	E. faecium	100%	0%	No
	K. pneumoniae	100%	0%	No
	P. aeruginosa	100%	0%	No
	S. aureus	100%	0%	No
	C. albicans	100%	0%	No
	HIV	100%	0%	No
R. prowazekii	E. coli	100%	0%	No
	E. faecium	100%	0%	No
	K. pneumoniae	100%	0%	No

Table 8. Competitive Inhibition - High and Low Concentrations of On-Panel Organisms

1Units are CFU/mL for all except Rp, which is in CAGe/mL

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Species 2 High Conc.(1000 CFU/mL, CAGe/mL,or IU/mL)1	Positive Detection Rate		CompetitiveInhibition
	Species 12	Species 22
P. aeruginosa	100%	0%	No
S. aureus	100%	0%	No
C. albicans	100%	0%	No
HIV	100%	0%	No

1Units are CFU/mL for all except Rp, which is in CAGe/mL, and HIV which is in IU/mL

²Unless otherwise listed, the positivity rate applies to the intended biothreat target channel.

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Clinical Performance Characteristics

The clinical performance of the T2Biothreat Panel was established through a two-arm study conducted using samples from healthy donors and febrile donors collected from 7 geographically diverse sites. One arm comprised the Negative Arm, which tested negative whole blood samples from healthy and febrile donors and was used to assess the Panel specificity. KzEDTA-treated whole blood samples from healthy donors were collected from patients presenting with no signs or symptoms of infection and were tested at a clinical site. Whole blood samples from febrile donors were collected from patients presenting with a fever of ≥ 100.4 °F and were tested at two (2) laboratory sites. The Positive Arm, which tested contrived positive-spiked whole blood samples from febrile donors, was designed to assess the Panel sensitivity. This arm of the study included the testing of sequence-verified clinical bacterial strains spiked at specific concentrations into whole blood collected from febrile donors. Contrived positive samples were produced to contain a single strain for each species being tested, (spiked at concentrations less than the LoD and greater than or equal to the LoD). Analysis of the Panel negative percent agreement (NPA) utilized results from healthy and febrile negative blood samples of the Panel positive percent agreement (PPA) utilized results from the positived samples. The PPA was analyzed for samples above and below LoD as well as all concentrations. The PPA and NPA were assessed for each analyte at a range of concentrations. The PPA ranged from 95% to 100% for analyte concentrations at 1-3x LoD except for F. tularensis which had a PPA of 94.3% and the NPA was 100% for all analytes.

Organism	LoD	TiterBucket	Titer Range1(CFU/mL)	PPA2,3,4(TP/TP+FN)	95% CI
B. anthracis(pXO1 & pXO2)5	6 CFU/mL	0.1-1x LoD	0.6 - <6.0	83.3% (5/6)	35.9-99.6%
		1-3x LoD	6.0 - <18.0	100% (32/32)	91.1-100%
		3-5x LoD	18.0 - 30.0	100% (12/12)	77.9-100%
Burkholderia spp.( B. pseudomallei &B. mallei )	12 –17 CFU/mL6	0.1-1x LoD	1.2 - <17.0	100% (17/17)	83.8-100%
		1-3x LoD	12.0 - < 51.0	100% (77/77)	96.2-100%
		3-5x LoD	36.0 - 85.0	100% (6/6)	60.7-100%
F. tularensis	4 CFU/mL	0.1-1x LoD	0.4 - <4.0	85.7% (6/7)	42.1-99.6%
		1-3x LoD	4.0 - <12.0	94.3% (33/35)	80.8-99.3%
		3-5x LoD	12.0 - 20.0	100% (8/8)	68.8-100%
R. prowazekii	9 CAGe/mL	0.1-1x LoD	0.9 - <9.0	N/A	N/A
		1-3x LoD	9.0 - < 27.0	100% (10/10)	74.1-100%
		3-5x LoD	27.0 - 45.0	100% (40/40)	92.8-100%
Y. pestis	2 CFU/mL	0.1-1x LoD	0.2 - <2.0	96.2% (25/26)	80.4-99.9%
		1-3x LoD	2.0 - <6.0	100% (24/24)	88.3-100%
		3-5x LoD	6.0 - 10.0	N/A	N/A

1Rp is in CAGe/mL

2TP = True Positives

3FN = False Negatives

4PPA = Positive Percent Agreement

5The LoD for B. anthracis detection is the concentration needed to detect both pXO2 plasmids (the higher of the two LoD)

6 Burkholderia species are not differentiated in the results reporting, the LoD for B. pseudomallei is 17 CFU/mL, the LoD for B. mallei is 12 CFU/mL

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Target Species	LoD	DetectionChannel	< LoD		≥ LoD		All Concentrations
			PPA1	95% CI	PPA1	95% CI	PPA1	95% CI
B. anthracis	6 CFU/mL	BaPXO1	100%(10/10)	74.11% -100%	100%(40/40)	92.78% -100%	100%(50/50)	94.18%- 100%
B. anthracis	6 CFU/mL	BaPXO2	90%(9/10)	55.50% -99.75%	100%(40/40)	92.78% -100%	98%(49/50)	89.35%- 99.95%
B. pseudomallei	17 CFU/mL	Bu	100%(20/20)	86.09% -100%	100%(80/80)	96.32% -100%	100%(100/100)	97.05% -100%
B. mallei	12 CFU/mL
F. tularensis	4 CFU/mL	Ft	90%(9/10)	55.50% -99.75%	95%(38/40)	83.08% -99.39%	94%47/50	83.45%- 97.75%
R. prowazekii	9 CAGe/mL	Rp	100%(10/10)	74.11% -100%	100%(40/40)	92.78% -100%	100%(50/50)	94.18%- 100%
Y. pestis	2 CFU/mL	Yp	100%(10/10)	74.11% -100%	98%(39/40)	86.84% -99.94%	98%(49/50)	89.35%- 99.95%

Table 11: PPA by organism relative to LoD (positive contrived)

The analytical and clinical performance data support the T2Biothreat Panel is safe and effective for its intended use and is substantially equivalent to the predicate device.

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Page 14 of 16

Predicate Comparison

Device & PredicateDevice(s):	Device	Predicate
	K231336	K170883
Device Trade Name	T2Biotreat Panel	FilmArray NGDS Warrior Panel
General DeviceCharacteristic Similarities	The T2Biothreat Panel is aqualitative, multiplexed, nucleic acid-based in vitro diagnostic testintended for use with the T2DxInstrument. The T2Biothreat Paneldetects nucleic acids from thefollowing organisms directly fromK2EDTA whole blood samples:1. Bacillus anthracis (plasmidspXO1 and pXO2)2. Francisella tularensis3. Burkholderia spp. (B.mallei/B. pseudomallei)4. Yersinia pestis5. Rickettsia prowazekii	The FilmArray NGDS Warrior Panel is aqualitative, multiplexed, nucleic acid-basedin vitro diagnostic test intended for usewith the FilmArray 2.0 system. TheFilmArray NGDS Warrior Panel detectsand identifies Bacillus anthracis, Yersiniapestis, Francisella tularensis, Coxiellaburnetii, Ebola virus, and Marburg virusnucleic acids directly from human wholeblood (EDTA). The FilmArray NGDSWarrior Panel is also intended to be usedto test for Bacillus anthracis or Yersiniapestis nucleic acids in blood cultures thatare determined to be positive either by anautomated system, by turbidity, or by dailyGram stain even without turbidity, and isindicated to be performed withconcomitant Gram stain performed onpositive blood culture specimens as pernormal laboratory procedure.
Intended Use/Indications ForUse	The T2Biothreat Panel will notdistinguish between detection ofBurkholderia mallei and Burkholderiapseudomallei but will present validdetections as a positive detection ofBurkholderia species.The T2Biothreat Panel is intended totest individuals with signs andsymptoms of infection from biothreatagents and/or individuals who are atrisk for exposure or may have beenexposed to these agents. TheT2Biothreat Panel is indicated as anaid in the diagnosis of anthrax,tularemia, melioidosis, glanders,typhus fever and plague in responseto suspected or confirmedbioterrorism events or outbreaks.Diagnosis of infection must be madein conjunction with clinical,epidemiologic and other laboratorydata. Results are for thepresumptive identification of Bacillusanthracis, Francisella tularensis,Burkholderia spp. (B. mallei/B.pseudomallei)	The FilmArray NGDS Warrior Panel isintended to test individuals with signs andsymptoms of infection from biothreatagents and/or individuals who are at riskfor exposure or may have been exposed tothese agents.The FilmArray NGDS Warrior Panel isindicated as an aid in the diagnosis ofanthrax, plague, tularemia, Q fever, andthe hemorrhagic fevers caused by Ebolaand Marburg viruses, in response to asuspected or confirmed bioterrorism eventor outbreaks. It is for diagnostic use inconjunction with other clinical,epidemiologic, and laboratory data, inaccordance with the guidelines providedby the appropriate Department of Defenseand public health authorities.Results are for the presumptiveidentification of Bacillus anthracis, Yersiniapestis, Francisella tularensis, Coxiellaburnetii, Ebola virus, and Marburg virus.

	Rickettsia prowazekii. Results aremeant to be used in conjunction withother clinical, epidemiologic, andlaboratory data, in accordance withthe guidelines provided by therelevant public health authorities.The definitive identification ofBacillus anthracis, Francisellatularensis, Burkholderia mallei,Burkholderia pseudomallei, Yersiniapestis or Rickettsia prowazekiirequires additional testing andconfirmation procedures inconsultation with the appropriatepublic health authorities for whom	The definitive identification of Bacillusanthracis, Yersinia pestis, Francisellatularensis, Coxiella burnetii, Ebola virus,and Marburg virus requires additionaltesting and confirmation procedures inconsultation with the appropriateDepartment of Defense and public healthauthorities for whom reports may benecessary. Negative results do notpreclude infection with these biothreatagents and should not be used as the solebasis for diagnosis, treatment, or otherpatient management decisions.The FilmArray NGDS Warrior Panel is
	reports may be required.Positive results do not rule out co-infections with pathogens notincluded on the T2Biothreat Panel.Negative results do not precludeinfection with the biothreat microbialagents targeted by the device andshould not be used as the sole basisfor diagnosis, treatment, or otherpatient management decisions.The T2Biothreat Panel is indicatedfor use in laboratories that have theappropriate biosafety equipment,personal protective equipment(PPE), containment facilities, andpersonnel trained in the safehandling of clinical specimenspotentially containing biothreatorganisms.The T2Biothreat Panel is indicatedfor use in laboratories that followpublic health guidelines that addressappropriate biosafety conditions,interpretation of test results, andcoordination of findings with publichealth authorities.This assay is not FDA-cleared orapproved for testing blood or plasmadonors.	solely for use by United States Departmentof Defense laboratories, and laboratoriesdesignated by the Department of Defense.
Test Platform Automation	Fully automated	Same
Reagent Platform	All reagents contained within asingle-use tray or pouch	Same
Sample Type	Whole blood (EDTA)	Whole blood (EDTA), positive bloodculture, and sputum
General DeviceCharacteristic Differences
	T2Dx Instrument	FilmArray 2.0 System
Test Principle	Nucleic acid amplification followedby T2 magnetic resonance detection	Nested multiplex RT-PCR followed bymelting analysis
Pathogens Detected	Bacillus anthracis, Burkholderiaspecies, Francisella tularensis,Rickettsia prowazekii, and Yersiniapestis.	Bacillus anthracis, Yersinia pestis,Francisella tularensis, Coxiella burnetii,Ebola virus, and Marburg virus.
Time to Result	About 4.5 hours	About 1 hour

Table 12: Comparison between T2Biothreat Panel and Predicate Device

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Regulation Number and Section

N/A