T2 Biothreat Panel

K231336 · T2biosystems, Inc. · QVR · Sep 15, 2023 · Immunology

Device Facts

• Record ID: K231336
• Device Name: T2 Biothreat Panel
• Applicant: T2biosystems, Inc.
• Product Code: QVR · Immunology
• Decision Date: Sep 15, 2023
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.4000
• Device Class: Class 2

Indications for Use

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: 1. Bacillus anthracis (plasmids pXO1 and pXO2) 2. Francisella tularensis 3. Burkholderia spp. (B. mallei/B. pseudomallei) 4. Yersinia pestis 5. 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 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/B. pseudomallei), Yersinia pestis and Rickettsia prowazekii. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided 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 Story

T2Biothreat Panel is a qualitative, multiplexed, nucleic acid-based in vitro diagnostic test for use on the T2Dx Instrument. Input: K2EDTA whole blood samples. Process: Automated concentration of pathogen cells via centrifugation; lysis to release target DNA; PCR amplification; hybridization with target-specific probe-coupled superparamagnetic particles; T2 Magnetic Resonance (T2MR) detection. Output: Presumptive identification of B. anthracis, F. tularensis, Burkholderia spp., Y. pestis, and R. prowazekii. Used in clinical laboratories by trained personnel. Results are interpreted by device software as 'Positive' or 'Target not Detected'. Clinical utility: Aids in diagnosis of biothreat infections in conjunction with clinical/epidemiologic data; supports rapid identification during outbreaks; requires confirmatory testing by public health authorities.

Clinical Evidence

Clinical performance evaluated via prospective study (265 febrile subjects) and contrived sample arm (300 positive samples). PPA for targets at 1-3x LoD was 100% (except F. tularensis at 94.3%). PPA for samples above LoD ranged 95-100%. NPA was 100% (95% CI 98.34-100%) across healthy and febrile negative cohorts. No clinical data for rare targets in prospective study.

Technological Characteristics

Nucleic acid amplification followed by T2 magnetic resonance (T2MR) detection. Uses superparamagnetic particles functionalized with oligonucleotide capture probes. Fully automated benchtop system (T2Dx). K2EDTA whole blood input. 4.5-hour time-to-result. Internal control included for monitoring lysis, amplification, and detection.

Indications for Use

Indicated for individuals with signs/symptoms of biothreat agent infection or those at risk of exposure. Aids in diagnosis of anthrax, tularemia, melioidosis, glanders, typhus fever, and plague during suspected/confirmed bioterrorism events or outbreaks. For use in qualified laboratories with appropriate biosafety equipment and trained personnel.

Regulatory Classification

Identification

A multiplex nucleic acid detection system for biothreat agents is a qualitative in vitro diagnostic test for detection and identification of nucleic acids from biothreat pathogens in human clinical specimens

Predicate Devices

• FilmArray NGDS Warrior Panel (K170883)

Reference Devices

• T2Candida
• T2Bacteria

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY

## I Background Information:

A 510(k) Number

K231336

B Applicant

T2 Biosystems, Inc.

C Proprietary and Established Names

T2Biothreat Panel

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| QVR | Class II | 21 CFR 866.4000 - Device To Detect And Identify Biothreat Microbial Agents In Human Clinical Specimens | MI - Microbiology |
| QBX | Class II | 21 CFR 866.3960 - Nucleic acid-based device for the amplification, detection, and identification of microbial pathogens directly from whole blood specimens | MI - Microbiology |
| NSU | Class II | 21 CFR 862.2570 - Instrumentation for clinical multiplex test systems | CH - Clinical Chemistry |

## II Submission/Device Overview:

### A Purpose for Submission:

The purpose of this submission is to obtain a substantial equivalence determination for the T2Biothreat Panel on the T2Dx Instrument.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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B Measurand:

The assay amplifies and detects nucleic acids of the following species: Bacillus anthracis, Francisella tularensis, Burkholderia spp. (B. mallei/B. pseudomallei), Yersinia pestis, and Rickettsia prowazekii.

C Type of Test:

Qualitative multiplex nucleic acid amplification test

III Intended Use/Indications for Use:

A Intended Use(s):

See Indications for Use below.

B Indication(s) for Use:

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:

1. Bacillus anthracis (plasmids pXO1 and pXO2)
2. Francisella tularensis
3. Burkholderia spp. (B. mallei/B. pseudomallei)
4. Yersinia pestis
5. 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 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/B. pseudomallei), Yersinia pestis and Rickettsia prowazekii. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided 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.

K231336 - Page 2 of 20

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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.

## C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

## IV Device/System Characteristics:

## A Device Description:

The T2Biothreat Panel is a qualitative nucleic acid-based test for the detection of microorganisms in K₂EDTA whole blood specimens using PCR amplification followed by T2 magnetic resonance (T2MR) detection. The assay detects six species of biothreat pathogens and categorizes the reported results using the following detection channels:

| Targets |
| --- |
| Bacillus anthracis plasmid pXO1 (Ba pXO1)* |
| Bacillus anthracis plasmid pXO2 (Ba pXO2)* |
| Francisella tularensis (Ft) |
| Burkholderia species (B. mallei/B. pseudomallei) (Bu) |
| Yersinia pestis (Yp) |
| Rickettsia prowazekii (Rp) |
| Internal Control |

*The presumptive identification of Bacillus anthracis requires detection of both pXO1 and pXO2 plasmids.

Panel testing is performed on the T2Dx Instrument (K172708), a benchtop, fully automated sample-to-result system, which performs all assay steps after specimens are loaded into the instrument. To use the T2Biothreat Panel, a 4 mL K₂EDTA tube is first directly loaded onto the Sample Inlet module, which is then placed on the T2Biothreat Cartridge with the T2Biothreat Reagent Tray and loaded into the T2Dx Instrument. An Internal Control is included in each assay cartridge to monitor the integrity of the Panel results.

The T2Biothreat Panel performs sample concentration, lysis, and DNA amplification for direct detection of six target organisms in 4.5 hours. The test incorporates an Internal Control for monitoring test performance in each amplification reaction.

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Device Components

The T2Biothreat Panel is comprised of a cartridge that contains all needed consumables, an inlet module for loading sample into the assay and a reagent tray that contains all reagents needed to run the assay.

The detection particles contained in the T2Biothreat Reagent Tray and used in the T2MR detection system are superparamagnetic particles that have been functionalized with oligonucleotide capture probes for each organism detected.

## Principle of Operation:

Amplicons from target organisms are aliquoted into individual tubes containing target-specific probes conjugated to superparamagnetic particles. The probes hybridize to target sequences causing clustering of the particles. The hybridization occurring in individual tubes is analyzed in the MR reader and a signal for each target is generated and detected by T2 Magnetic Resonance (T2MR) indicating the presence of the target organism.

## V Substantial Equivalence Information:

A Predicate Device Name(s):

FilmArray NGDS Warrior Panel

B Predicate 510(k) Number(s):

K170883

C Comparison with Predicate(s):

| Device & Predicate Device(s): | Device K231336 | Predicate K170883 |
| --- | --- | --- |
| Device Trade Name | T2Biotreat Panel | FilmArray NGDS Warrior Panel |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | 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:
1. Bacillus anthracis (plasmids pXO1 and pXO2)
2. Francisella tularensis
3. Burkholderia spp. (B. mallei/B. | The FilmArray NGDS Warrior Panel is a qualitative, multiplexed, nucleic acid-based in vitro diagnostic test intended for use with the FilmArray 2.0 system. The FilmArray NGDS Warrior Panel detects and identifies Bacillus anthracis, Yersinia pestis, Francisella tularensis, Coxiella burnetii, Ebola virus, and Marburg virus nucleic acids directly from human whole blood (EDTA). The FilmArray NGDS Warrior Panel is also intended to be used to test for Bacillus anthracis or Yersinia pestis nucleic acids in blood cultures that are determined to be |

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K231336 - Page 5 of 20

| | pseudomallei)
4. Yersinia pestis
5. 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 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/B. pseudomallei), Yersinia pestis and Rickettsia prowazekii. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided 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 | positive either by an automated system, by turbidity, or by daily Gram stain even without turbidity, and is indicated to be performed with concomitant Gram stain performed on positive blood culture specimens as per normal laboratory procedure.

The FilmArray NGDS Warrior 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 FilmArray NGDS Warrior Panel is indicated as an aid in the diagnosis of anthrax, plague, tularemia, Q fever, and the hemorrhagic fevers caused by Ebola and Marburg viruses, in response to a suspected or confirmed bioterrorism event or outbreaks. It is for diagnostic use in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the appropriate Department of Defense and public health authorities.

Results are for the presumptive identification of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Coxiella burnetii, Ebola virus, and Marburg virus. The definitive identification of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Coxiella burnetii, Ebola virus, and Marburg virus requires additional testing and confirmation procedures in consultation with the appropriate Department of Defense and public health authorities for whom reports may be necessary. Negative results do not preclude infection with these biothreat agents and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

The FilmArray NGDS Warrior Panel is solely for use by United States Department of Defense laboratories, |
| --- | --- | --- |

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K231336 - Page 6 of 20
| | 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. | and laboratories designated by the
Department of Defense. |
| --- | --- | --- |
| Test Platform Automation | Fully automated | Same |
| Reagent Platform | All reagents contained within a
single-use tray or pouch | Same |
| Sample Type | Whole blood (EDTA) | Whole blood (EDTA), positive blood
culture, and sputum |

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| General Device Characteristic Differences | | |
| --- | --- | --- |
| Instrument Platform | T2Dx Instrument | FilmArray 2.0 System |
| Test Principle | Nucleic acid amplification followed by T2 magnetic resonance detection | Nested multiplex RT-PCR followed by melting analysis |
| Pathogens Detected | Bacillus anthracis, Burkholderia species, Francisella tularensis, Rickettsia prowazekii, and Yersinia pestis. | Bacillus anthracis, Yersinia pestis, Francisella tularensis, Coxiella burnetii, Ebola virus, and Marburg virus. |
| Time to Result | About 4.5 hours | About 1 hour |

VI Standards/Guidance Documents Referenced:

- CLSI Guideline EP07-A3, Interference Testing in Clinical Chemistry; Approved Guideline, Third Edition. Volume 38, Number 7
- CLSI Guideline EP37, Supplemental Tables for Interference Testing in Clinical Chemistry, First Edition.
- Protection of Laboratory Workers from Occupationally Acquired Infections. Approved Guideline-Third Edition. CLSI Document. Clinical and Laboratory Standards Institute (CLSI). M29-A3 Wayne, PA: CLSI, 2005.
- CLSI EP25-A, Vol 29 No 20. Evaluation of Stability of In Vitro Diagnostics Reagents; Approved Guideline. Wayne, PA: Clinical and Laboratory Standards Institute; 2009
- CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline- 2nd Edition
- FDA Guidance: The 510(k) Program: Evaluating Substantial Equivalence in Premarket Notifications [510(k)] (July 28, 2014)
- FDA Guidance: Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices (May 11, 2005)
- FDA Guidance: Content of Premarket Submissions for Device Software Functions Draft Guidance for Industry and Food and Drug Administration Staff
- FDA Guidance: Recommended Content and Format of Non-Clinical Bench Performance Testing Information in Premarket Submissions (December 20, 2019)
- FDA Guidance: Content of Premarket Submissions for Management of Cybersecurity in Medical Devices (October 2, 2014)
- FDA Guidance: Cybersecurity in Medical Devices: Quality System Considerations and Content of Premarket Submissions
- FDA Guidance: Off-The-Shelf Software Use in Medical Devices
- FDA Guidance: Cybersecurity for Networked Medical Devices Containing Off-The-Shelf (OTS) Software
- FDA Guidance: General Principles of Software Validation
- ISO 15223-1 Fourth edition 2021-07 Medical devices - Symbols to be used with information to be supplied
- ISO 14971 Medical Devices – Application of Risk Management to Medical Devices
- IEC 62304 Edition 1.1 2015-06 CONSOLIDATED VERSION Medical device software - Software life cycle processes

K231336 - Page 7 of 20

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# VII Performance Characteristics (if/when applicable):

# A Analytical Performance:

# 1. Precision/Reproducibility:

A multi-center reproducibility study was performed to determine the run-to-run, reagent lot, day-to-day and site-to-site reproducibility. Result agreement was analyzed across multiple sites over six non-consecutive days, with a minimum of two operators per site, two Reagent Tray Kit lots, and two instruments. Several contrived sample panels were prepared in $\mathrm{K}_2\mathrm{EDTA}$ -treated whole blood (a B. anthracis, B. pseudomallei, and Y. pestis triple-species panel, a B. mallei and R. prowazekii dual-species panel, and an F. tularensis single-species spike) at 2-3x LoD and 1-1.5x LoD. Negative $\mathrm{K}_2\mathrm{EDTA}$ -treated whole blood samples were also prepared. Reproducibility results across instruments, operators, reagent lots, and sample panels were evaluated. A summary of the reproducibility results is shown in Table 1 below. The reproducibility of the T2Biothreat Panel was found to be acceptable.

Table 1. Summary of Reproducibility Study Results

| Organism | Conc. | Expected Result | Agreement with Expected Result [95% CI] | |
| --- | --- | --- | --- | --- |
| B. anthracis | 1-1.5x LoD | Detected | BaPXO1 Channel | BaPXO2 Channel |
| | | | 128/128 100% [97.7-100] | 127/128 99% [95.7-100] |
| | 2-3x LoD | Detected | 104/104 100% [97.2-100] | 103/104 99% [94.8-100] |
| B. pseudomallei | 1-1.5x LoD | Detected | Bu Channel | - |
| | | | 126/128 98% [94.5-99.8] | |
| | 2-3x LoD | Detected | 103/104 99% [94.8-100] | - |
| B. mallei | 1-1.5x LoD | Detected | 96/96 100% [96.9-100] | - |
| | 2-3x LoD | Detected | 95/96 99% [94.3-100] | - |
| Y. pestis | 1-1.5x LoD | Detected | Yp Channel | - |
| | | | 126/128 98% [94.5-99.8] | |
| | 2-3x LoD | Detected | 104/104 100% [97.2-100] | - |
| R. prowazekii | 1-1.5x LoD | Detected | Rp Channel | - |
| | | | 95/96 99% [94.3-100] | |

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| | 2-3x LoD | Detected | 96/96 100% [96.9-100] | - |
| --- | --- | --- | --- | --- |
| F. tularensis | 1-1.5x LoD | Detected | Ft Channel | - |
| | | | 96/96 100% [96.9-100] | |
| | 2-3x LoD | Detected | 95/96 99% [94.3-100] | - |
| Negative | Negative | Not Detected | 379/379 100% [99.2-100] | - |

2. Linearity:

N/A

3. Analytical Sensitivity

To confirm detection of multiple clinically relevant strains of the organisms detected by the T2Biothreat Panel, 10 strains of *B. anthracis*, 8 strains of *B. mallei*, 10 strains of *B. pseudomallei*, 10 strains of *F. tularensis*, 5 strains of *R. prowazekii* and 12 strains of *Y. pestis* were wet tested on the T2Dx instrument. Isolates were selected to represent variations in phylogenic, temporal, and geographic diversity. Isolates were inoculated into human whole blood at a concentration of 2-3X LoD. Testing of each isolate was performed in triplicate. If a set was not positive for 3/3 replicates, a repeat sample set of 20 replicates was prepared and tested. A strain was defined as inclusive if testing resulted in 3/3 positive detections or ≥ 19/20 positive detections. 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 not be detected by the T2Biothreat Panel. Results of the inclusivity study are shown in Table 2. Inclusivity testing demonstrated reactivity against multiple strains of each target organism on the T2Biothreat Panel and the test results are acceptable.

Table 2. Inclusivity Study Results

| Organism | Strains | Positivity | Repeat Testing | Result |
| --- | --- | --- | --- | --- |
| Bacillus anthracis
10 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 |
| Burkholderia mallei | 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 |

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| 8 strains | NCTC 120 | 3/3 | N/A | Inclusive |
| --- | --- | --- | --- | --- |
| | NCTC 3708 | 3/3 | N/A | Inclusive |
| | NCTC 3709 | 3/3 | N/A | Inclusive |
| Burkholderia pseudomallei10 strains | Human/Blood/OH/US/1994 | 3/3 | N/A | Inclusive |
| | 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 |
| Francisella tularensis10 strains | 425 | 3/3 | N/A | Inclusive |
| | 100892A | 2/3 | 20/20 | Inclusive |
| | ATCC Vaccine Strain | 3/3 | N/A | Inclusive |
| | 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 |
| Rickettsia prowazekii5 strains | 103-2P | 3/3 | N/A | Inclusive |
| | Addis Abbas | 3/3 | N/A | Inclusive |
| | Cairo | 3/3 | N/A | Inclusive |
| | GvF | 3/3 | N/A | Inclusive |
| | ZRS | 3/3 | N/A | Inclusive |
| Yersinia pestis12 strains | 195/P (India) | 3/3 | N/A | Inclusive |
| | Angola | 3/3 | N/A | Inclusive |
| | AZ94-0666 | 3/3 | N/A | Inclusive |
| | Bombay¹ | 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 G¹ | 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 |
| | Nicholisk | 3/3 | N/A | Inclusive |

¹Y. pestis strains Bombay and Pestoides G do not harbor the pPCP plasmid which contains the Y. pestis target sequence

## 4. Analytical Specificity:

Analytical specificity testing of the T2Biothreat Panel was conducted to assess the cross reactivity of the panel with non-panel species. 31 bacterial, fungal, and viral organisms commonly found to cause bloodstream infections or to be genetically similar to pathogens detected by the Panel were tested. Organisms were tested at 1000 CFU/mL, 1000 CAGe/mL, or international units (IU)/mL in K₂EDTA-treated human blood on the T2Biothreat Panel in 3 replicates using three production equivalent lots of T2Biothreat reagents. Organisms shown to be potentially cross-reactive at the initial concentration were then titrated to a concentration at which a negative result was achieved. Organisms that exhibited no cross-reactivity with the T2Biothreat Panel target organisms are identified in Table 3 below.

K231336 - Page 10 of 20

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Organisms for which cross-reactivity with the T2Biothreat Panel was observed are included in Table 4 below. B. cereus strain G-9241 harboring a pXO1-like plasmid exhibited cross-reactivity with the Ba pXO1 channel at the initial concentration of $1000\mathrm{CFU / mL}$ and all titrated concentrations. Francisella tularensis subsp. novicida (also referred to as F. novicida) is similar genetically to F. tularensis yet is rarely associated with human disease. This subspecies is not considered a Select Agent and cross-reacts with the Ft channel.

Table 3. Exclusivity Study Organisms with No Cross-Reactivity

| 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 |

1 Harbors a pXO1-like plasmid that was not detected by the Panel
2 Harbors a pXO2-like plasmid that was not detected by the Panel
3 Tested as IU/mL

Table 4. Exclusivity Study Results - Cross-reactive Organisms

| Cross-reactive Non-Panel Strain | Panel Channel that Cross-reacts |
| --- | --- |
| Bacillus cereus G-9241 | BaPXO11 |
| Francisella tularensis subsp. novicida | Ft |

1Panel will detect this strain, but result will report "Equivocal Detection of B. anthracis or detection of B. cereus or B. thuringiensis"

Five 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 pXO1 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. If both pXO1 and pXO2 plasmids are detected, the results reported indicate a positive detection for B. anthracis. If only one of the plasmids is detected, the results report equivocal detection of B. anthracis or detection of B. cereus or B. thuringiensis.

To evaluate higher concentrations of analyte which may be encountered in whole blood samples, $1 \times 10^{6}$ 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. Results are shown in Table 5. The results of the cross reactivity study are acceptable.

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Table 5. Exclusivity Study Organisms Evaluated at $1 \times 10^{6}$ copies/mL

| 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 | |

# 5. Interfering Substances

An interfering substances study was performed to determine and characterize the effects of potential endogenous and exogenous interfering substances commonly found in blood on the performance of the T2Biothreat Panel. Interferents were screened as pools of interferents in human whole blood sample panels; potential interferents were added in high concentrations to represent worst case scenarios; samples were tested in triplicate. Three contrived sample panels were prepared in $\mathrm{K}_2\mathrm{EDTA}$ -treated whole blood (a B. anthracis, B. pseudomallei, and Y. pestis triple-species panel, a B. mallei and R. prowazekii dual-species panel, and an F. tularensis single-species spike) at 2-3x LoD and panels without interferents were include as controls. Negative blood samples were tested with each potential interferent. Interferents that caused a difference in signal from a panel without interferents were further analyzed by individual organism in blood samples. Five endogenous substances and 15 exogenous substances were screened to assess potential interference to panel performance (Table 6). Based on previous studies, Feraheme is considered an interferent at $\geq 21\mu \mathrm{g / mL}$ , Magnevist is considered an interferent at $\geq 1.7\mathrm{mg / mL}$ , and Ablavar is considered an interferent at $\geq 0.39$ mg/mL. No interference was detectable at the specified test concentration for all other substances and the results of the study are acceptable.

Table 6. Interference Study Results - Substances without Interference Effect

| 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 | Meropenem trihydrate | 884 μmol/mL |
| Creatinine | 150 mg/L | Ciprofloxacin | 36.2 μmol/L | Novobiocin | 450 μmol/mL |
| Hemoglobin | >20 g/dL | Ceftazidime Pentahydrate | 606 μg/mL | Penicillin G sodium salt | 0.777 μg/mL |
| Intralipid (to mimic triglycerides) | 1703 mg/dL | Doxycycline hyclate | 40.5 μmol/L | Streptomycin sulfate | 444 μmol/L |
| Urea | 42.9 mmol/L | Gentamicin sulfate | 62.8 μg/mL | Tetracycline hydrochloride | 54 μmol/L |
| White Blood Cells | ≥16.5x10^6 cells/mL | K2EDTA | 9 mg/mL | Trimethoprim | 145 μmol/L |

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# 6. Competitive Inhibition

A competitive inhibition study was performed to evaluate the ability of the T2Biothreat Panel to detect target bacterial species present at 1-2X LoD in the presence of other clinically relevant organisms (on- and off- panel) that may be present in a co-infection. Three combinations of organisms were tested in human whole blood samples: 1) samples containing two target bacterial species, each in concentrations of 1-2X LoD (low concentrations); 2) samples containing one target bacterial species at a concentration of 1-2X LoD (low concentration) and a second target bacterial species at a concentration of 1000 CFU/mL (high concentration); and 3) samples containing one target bacterial species at a concentration of 1-2 X LoD (low concentration) and a non-target bacterial species, yeast, or virus species at a concentration of 1000 CFU/mL or IU/mL (high concentration). Four replicates of each combination were initially tested; any combination resulting in a false negative was repeated with 20 replicates. If $\leq 95\%$ of the target species were detected in the 20 replicates, the concentration of the competing organisms was decreased to determine the level at which the detection was not inhibited. No competitive effects were observed in any combination of Panel members at $\leq 1,000$ CFU/mL or CAGe/mL or near LoD (Table 7). No competitive effects were observed in any combination of non-Panel members and Panel members at $\leq 1,000$ CFU/mL or IU/mL (Table 8), and the data are acceptable.

Table 7. Competitive Inhibition - High and Low Concentrations of On-Panel Organisms

| Species 1 Low Conc. (1-2x LoD) | Species 2 High and Low Conc. (1000 CFU/mL or CAGe/mL and 1-2x LoD)1 | Positive Detection Rate | | Competitive Inhibition |
| --- | --- | --- | --- | --- |
| | | Species 1 | Species 2 | |
| 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 |
| | R. prowazekii | 100% | 100% | No |
| B. pseudomallei | B. anthracis | 100% | 100% | No |
| | Y. pestis | 100% | 100% | No |
| | F. tularensis | 100% | 100% | No |
| | R. prowazekii | 100% | 100% | No |
| F. tularensis | B. anthracis | 100% | 100% | No |
| | B. mallei | 100% | 100% | No |
| | Y. pestis | 100% | 100% | No |
| | B. pseudomallei | 100% | 100% | No |
| | R. prowazekii | 100% | 100% | No |
| R. prowazekii | B. anthracis | 100% | 100% | No |
| | B. mallei | 100% | 100% | No |

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Table 8. Competitive Inhibition – High Conc. Off-Panel Organisms

| Species 1 Low Conc. (1-2x LoD) | Species 2 High Conc. (1000 CFU/mL, CAGe/mL, or IU/mL)¹ | Positive Detection Rate | | Competitive Inhibition |
| --- | --- | --- | --- | --- |
| | | Species 1² | Species 2² | |
| B. anthracis | Escherichia coli | 100% | 0% | No |
| | Enterococcus faecium | 75% (3/4 Ba pXO2, 4/4 Ba pXO1) | 0% | No |
| | | 100% (20/20 both channels) | | |
| | Klebsiella pneumoniae | 100% | 0% | No |
| | Pseudomonas aeruginosa | 100% | 0% | No |
| | Staphylococcus aureus | 100% | 0% | No |
| | Candida albicans | 100% | 0% | No |
| | Human Immunodeficiency Virus | 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 |
| | 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

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$^{1}$ Units are CFU/mL for all except Rp, which is in CAGe/mL, and HIV which is in IU/mL
$^{2}$ Unless otherwise listed, the positivity rate applies to the intended biothreat target channel.

7. Assay Reportable Range:

N/A

8. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

**Specimen Stability:**

The stability of K₂EDTA blood specimens when stored at 15-30°C (room temperature) or 2-8°C (refrigerated) was evaluated with the T2Biothreat Panel on the T2Dx Instrument using contrived samples at a final concentration of 25 CFU/mL. Samples were tested at up to 31 hours when stored at room temperature or 8 days when stored at 2-8°C to determine stability. Data are supportive of the specified storage times and temperatures included in the device labeling shown in Table 9.

Table 9. Recommended storage times and temperatures for blood specimens

| Temperature | Storage Time |
| --- | --- |
| 2-8°C | 3 days |
| Room temperature | 8 hr |

**Controls:**

The T2Biothreat Panel utilizes two types of controls, an internal control and two external controls. The Internal Control (IC) is automatically introduced into each specimen during sample processing on the T2Dx instrument and is carried through the lysis, amplification and detection steps of the assay. The IC monitors the amplification and detection process and detects the presence of inhibitors in the specimen.

The external controls include the QCheck Positive Kit and the QCheck Negative Kit. The QCheck Positive Kit is composed of eight positive samples formulated in proteinaceous buffer-based matrix. The samples contain non-pathogenic bacterial strains containing the T2Biothreat Panel target sequences that can interrogate the respective, complementary probe-coated particles in the corresponding T2Biothreat Panel detection reactions. The QCheck Negative Kit contains eight negative samples containing the proteinaceous buffer-based matrix alone, which can be used to detect contamination of the T2Biothreat Panel reagents and/or the T2Dx Instrument by one or more of the target species detected by the T2Biothreat Panel.

9. Detection Limit:

Limit of detection (LoD) determinations were performed using two T2Dx instruments, two reagent lots, and two different strains of each of the T2Biothreat Panel members in K₂EDTA-treated whole blood. Cell concentrations for all species except Rickettsia prowazekii were measured in CFU/mL. R. prowazekii does not grow on solid media because it is an obligate intracellular organism and therefore cannot be measured in CFU/mL. R. prowazekii cultures were enumerated as Cell Associated Genomic equivalents (CAGe). A preliminary LoD determined during test development was used as the starting concentration for confirmation studies. A minimum of 20 replicates per reagent lot and isolate (minimum of 80 samples total

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per species) were tested at increasing concentrations until a positivity rate of $95\%$ was achieved. The LoD value for each target detected by the T2Biothreat Panel was defined as the lowest $\mathrm{CFU / mL}$ or CAGe/mL as appropriate, for which a positive detection rate of $\geq 95\%$ with $n\geq 20$ was obtained. The final LoD for each species will be based on the highest $\mathrm{CFU / mL}$ or CAGe/mL for which a positive detection rate of $\geq 95\%$ was obtained from the different isolates and reagent lots for that species. The LoD for B. anthracis was determined based on the detection of both pXO1 and pXO2. The LoD for each target species is listed in Table 10. The limit of detection study results are acceptable.

Table 10. LoD for Targets Detected by the T2Biothreat Panel

| Organism | Channel | Tray Lot 1 | | | | Tray Lot 2 | | | | Tray Lot 32 | | | | Final LoD |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Strain 1 | | Strain 2 | | Strain 1 | | Strain 2 | | Strain 1 | | Strain 2 | | |
| | | \(LoD^1\) | Hit Rate | \(LoD^1\) | Hit Rate | \(LoD^1\) | Hit Rate | \(LoD^1\) | Hit Rate | \(LoD^1\) | Hit Rate | \(LoD^1\) | Hit Rate | |
| B. anthracis | BaPOX1 | 6 | 95% | 6 | 100% | 6 | 100% | 6 | 100% | N/A | N/A | N/A | N/A | 6 CFU/mL |
| | BaPOX2 | 5 | 100% | 6 | 100% | 6 | 100% | 6 | 100% | N/A | N/A | N/A | N/A | |
| B. pseudomallei | Bu | 10 | 95% | 12 | 95% | 15 | 100% | 17 | 95% | N/A | N/A | N/A | N/A | 17 CFU/mL |
| B. mallei | Bu | 10 | 100% | 10 | 95% | 12 | 95% | 10 | 100% | N/A | N/A | N/A | N/A | 12 CFU/mL |
| F. tularensis | Ft | 1 | 100% | 2 | 100% | 4 | 100% | 4 | 100% | N/A | N/A | N/A | N/A | 4 CFU/mL |
| R. prowazekii | Rp | N/A | N/A | N/A | N/A | 9 | 100% | 9 | 100% | 9 | 100% | 9 | 100% | 9 CAGe/mL |
| Y. pestis | Yp | 1 | 100% | 2 | 100% | 2 | 100% | 2 | 100% | N/A | N/A | N/A | N/A | 2 CFU/mL |

1 Units are CFU/mL for all except Rp, which is CAGe/mL
2 Insufficient quantities of Lot 2 remained to complete testing of Rp. Testing with Lot 3 was used only for Rp LoD determination.

# 10. Accuracy (Instrument):

N/A

# 11. Carry-Over:

The T2Biothreat Panel is run on the previously cleared T2Dx Instrument (K172708), which demonstrated a lack of cross-contamination between positive and negative samples when co-loaded on the same instrument for the T2Bacteria Panel (K172708) and the T2Candida Panel (K173536). A co-load is defined as an instrument load that had 2 or more of any combination of a positive sample (contrived whole blood or control) and negative sample (whole blood or control) loaded at the same time. The T2Dx can process up to 7 samples simultaneously. A retrospective analysis was conducted to assess potential carry-over/cross-contamination events throughout T2Biothreat verification and validation studies. Results from instrument loads that contained any combination of positive samples and negative were compiled from T2Biothreat verification and validation studies listed in Tables 11 and 12. The purpose of this analysis was to demonstrate a lack of cross-contamination between positive and negative samples when co-loaded on the same instrument.

Table 11. Descriptive information of the co-load analysis for the T2Biothreat Panel

| Study Analyzed | Positive Sample Titer | # Samples in Analysis | Number of Co-Loads |
| --- | --- | --- | --- |

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Table 12. Number of samples in the co-load analysis

| Sample Type | # of Samples Analyzed | Contained B. anthracis | Contained B. mallei | Contained B. pseudomallei | Contained F. tularensis | Contained R. prowazekii | Contained Y. pestis |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Positive Blood | 568 | 167 | 131 | 133 | 155 | 124 | 178 |
| Negative Blood | 430 | | | | | | |
| Positive Control | 182 | | | | | | |
| Negative Control | 132 | | | | | | |
| Total | 1312 | | | | | | |

1 Value does not include combined study loads
2 Data collected as of 10/21/2022

The false positive sample rates from each T2Biothreat verification and validation study were compiled and demonstrated that blood samples had a false positive rate of $0\%$ and Negative QChecks had a false positive rate of $< 0.44\%$ for any one study, supporting a low-risk for carryover contamination. Table 13 lists a summary of the results. The results of the carryover contamination evaluation are acceptable.

Table 13. Summary of co-load analysis results on the T2Biothreat Panel

| Channel | | Ba pXO1 | Ba pXO2 | Bu | Rp | Ft | Yp | Overall |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| False Positives1 (FP) | # FP/total samples | 0/936 | 0/936 | 0/866 | 0/1006 | 0/975 | 0/952 | 0/5671 |
| | FP Rate | 0% | 0% | 0% | 0% | 0% | 0% | 0% |
| | 95% Confidence Interval | 0-0.32% | 0-0.32% | 0-0.35% | 0-0.30% | 0-0.31% | 0-0.30% | 0-0.05% |
| False Negatives2 (FN) | # FN/total samples | 0/583 | 1/583 | 4/486 | 0/626 | 3/595 | 2/572 | 10/3612 |
| | FN Rate | 0% | 0.16% | 0.65% | 0% | 0.50% | 0.35% | 0.28% |
| | 95% Confidence Interval | 0-0.51% | 0.004%-0.95% | 0.22%-2.09% | 0-0.48% | 0.10%-1.47% | 0.04%-1.26% | 0.13%-0.51% |

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1. Negative blood and negative control samples
2. Positive blood and positive control samples

## B Comparison Studies:

1. Method Comparison with Predicate Device: N/A
2. Matrix Comparison: N/A

## C Clinical Studies:

### 1. Clinical Sensitivity:

Clinical performance of the T2Biothreat Panel was evaluated in a prospective study designed as a dual arm investigation that included a “negative arm” and a “positive contrived arm”. The study tested whole blood samples from K₂EDTA tubes from two different subject populations (heathy and febrile). Due to the low prevalence of positive blood cultures for the target bacterial species, evaluation of the positive performance of the T2Biothreat Panel used contrived sample testing. K₂EDTA whole blood from febrile subjects (patients with temperature ≥100.4°F at time of collection) was collected concurrently at 7 geographically diverse sites with samples to be used to evaluate negative performance. Blood samples were collected from 294 febrile subjects, 29 samples were excluded from the study resulting in a total of 265 samples tested and included in the clinical sensitivity analysis. Samples collected from each febrile subject were either contrived for use in the contrived study arm to evaluate positive performance or used in the arm to evaluate negative performance. Contrived samples were prepared in BSL-3 laboratories using 6 strains per target species resulting in a total of 300 tested positive samples (50 samples for each panel species). Contrived sample concentrations were designed to evaluate the T2Biothreat Panel Positive Percent Agreement (PPA) at various levels (10 samples prepared at 0.1-1x LoD, 35 samples prepared at 1-3x LoD, 5 samples prepared at 3-5x LoD). All contrived samples and negative febrile samples were blinded and processed on a T2Dx Instrument. PPA was calculated by comparing T2Biothreat Panel results to the positive contrived clinical samples. Positive and negative controls (QChecks) were tested daily for each instrument used in the study. Controls must pass on each instrument for the clinical samples tested to be considered valid.

When samples were grouped based on concentration all targets had 100% detection at 1-3x LoD except F. tularensis, which was detected at 94.3% at these concentrations (Table 14). In samples above the LoD for each target, the resulting PPA was ≥95% (ranged 95-100%) for each channel (n = 40 per channel except n=80 for Bu channel) (Table 15). These data met the acceptance criteria of ≥90% PPA for each target channel.

Table 14. PPA by organism relative to concentration (positive contrived)

| Organism | LoD | Titer Bucket | Titer Range (CFU/mL)^{1} | PPA^{2,3,4} (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% |

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| 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 |

$^{1}$ Rp is in CAGe/mL
$^{2}$ TP = True Positives
$^{3}$ FN = False Negatives
$^{4}$ PPA = Positive Percent Agreement
The LoD for B. anthracis detection is the concentration needed to detect both pXO1 and 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

Table 15. PPA by organism relative to LoD (positive contrived)

| Organism | LoD | Detection Channel | < LoD | | ≥ LoD | | All Concentrations | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | PPA (TP/TP+FN) | 95% CI | PPA (TP/TP+FN) | 95% CI | PPA (TP/TP+FN) | 95% CI |
| B. anthracis | 6 CFU/mL | Ba pXO1 | 100% (10/10) | 74.11% - 100% | 100% (40/40) | 92.78% - 100% | 100% (50/50) | 94.18% - 100% |
| | | Ba pXO2 | 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% |

2. Clinical Specificity:

Negative performance of the T2Biothreat Panel was evaluated by determining the negative concordance between T2Biothreat Panel results and blood samples that were presumed to be negative for target biothreat pathogens. These studies included K2EDTA whole blood specimens from both febrile (temperature $\geq 100.4^{\circ}\mathrm{F}$ ) and non-febrile donors and patients. Whole blood samples from 362 unique healthy subjects were collected, with 58 samples excluded from the study that did not meet screening criteria, resulting in 304 samples from 304 unique subjects included in the analysis. A total of 194 whole blood samples from febrile patients which were not used to make contrived samples for the sensitivity study were included in the clinical specificity study, with 179 samples producing valid results. The negative percent agreement (NPA) for healthy subjects, febrile subjects, and combined healthy and febrile subjects is shown in Table 16. The NPA of negative healthy subject samples was calculated from 304 valid, unique donor sample results. Each target channel had

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0/304 positive detections giving an NPA of 100% with a 95% CI of 99.02% - 100% for each channel. For febrile negative samples the NPA was calculated from 179 valid, unique subject sample results. Each target channel had 0/179 positive detection giving an NPA of 100% with a 95% CI of 98.34% - 100%. The results of the clinical testing are acceptable.

Table 16. Results from Presumed Negative Specimens (among healthy and febrile subjects)

| Organism | Channel | Healthy Negatives | | Febrile Negatives | |
| --- | --- | --- | --- | --- | --- |
| | | NPA (TN/(FP+TN)) | 95% CI | NPA (TN/(FP+TN)) | 95% CI |
| B. anthracis | Ba pXO1 | 100% (304/304) | 99.02% - 100% | 100% (179/179) | 98.34% - 100% |
| | Ba pXO2 | 100% (304/304) | 99.02% - 100% | 100% (179/179) | 98.34% - 100% |
| Burkholderia spp. | Bu | 100% (304/304) | 99.02% - 100% | 100% (179/179) | 98.34% - 100% |
| F. tularensis | Ft | 100% (304/304) | 99.02% - 100% | 100% (179/179) | 98.34% - 100% |
| R. prowazekii | Rp | 100% (304/304) | 99.02% - 100% | 100% (179/179) | 98.34% - 100% |
| Y. pestis | Yp | 100% (304/304) | 99.02% - 100% | 100% (179/179) | 98.34% - 100% |

D Clinical Cut-Off:
N/A

E Expected Values/Reference Range:
None of the rare T2Biothreat Panel organisms were detected during the prospective clinical evaluation of the T2Biothreat Panel.

F Other Supportive Instrument Performance Characteristics Data:
Not applicable

VIII Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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