(130 days)
Not Found
No
The description focuses on the molecular diagnostic technology (PCR amplification followed by T2 Magnetic Resonance detection) and the automated instrument process. There is no mention of AI or ML in the device description, intended use, or performance studies. The interpretation of results is described as being performed by "the device software" based on internal control and target detections, which is typical for automated IVD systems and does not necessarily imply AI/ML.
No.
The device is an in vitro diagnostic test intended to aid in the diagnosis of certain infections by detecting nucleic acids from specific organisms. It does not provide therapy or treatment.
Yes
The "Intended Use / Indications for Use" section explicitly states that the device is an "in vitro diagnostic testing" and is "indicated as an aid in the diagnosis" of several infections.
No
The device is an in vitro diagnostic test that utilizes a physical instrument (T2Dx Instrument) and physical reagents (T2Biothreat Cartridge, T2Biothreat Reagent Tray) to perform nucleic acid detection. While software is involved in interpreting results, it is an integral part of a larger hardware and reagent system, not a standalone software-only device.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Explicitly Stated in Intended Use and Device Description: Both sections clearly state that the T2Biothreat Panel is an "in vitro diagnostic test."
- Intended Use: The device is intended for use in a laboratory setting to detect nucleic acids from specific organisms in whole blood samples. This is a classic definition of an in vitro diagnostic test, which is performed outside of the body on biological specimens.
- Purpose: The test is intended as an aid in the diagnosis of infections caused by the targeted biothreat agents. This diagnostic purpose is a key characteristic of an IVD.
- Sample Type: The device analyzes K2EDTA whole blood samples, which are biological specimens.
- Methodology: The device uses nucleic acid-based detection (PCR amplification followed by T2MR detection), a common methodology for IVD tests.
- Performance Studies: The document describes analytical and clinical performance studies, which are required for the regulatory evaluation of IVD devices.
- Predicate Device: The mention of a predicate device (FilmArray NGDS Warrior Panel) with a K number (K170883) further indicates that this device is being evaluated as an IVD, likely for regulatory submission.
N/A
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:
-
- Bacillus anthracis (plasmids pXO1 and pXO2)
-
- Francisella tularensis
-
- Burkholderia spp. (B. mallei/B. pseudomallei)
-
- Yersinia pestis
-
- 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), 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.
Product codes
QVR, QBX, NSU
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 specific organisms directly from K2EDTA whole blood samples. The device 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 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 K2EDTA 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 Cartridge and Reagent Tray contain the lysis reagent, internal control, primers, enzyme, buffer and probe-coupled superparamagnetic particles for each detected target.
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 centrifugation. 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 product binds 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 single specimens simultaneously, the first specimen will be reported in approximately 4 hours from when 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.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
K2EDTA whole blood samples
Indicated Patient Age Range
Not Found
Intended User / Care Setting
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.
Laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
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 degrees 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).
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
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. 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.
- 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
- Analytical Reactivity (Inclusivity): 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 confirmed with wet testing of B. anthracis (10 strains), B. mallei (8 strains), B. pseudomallei (10 strains), R. prowazekii (5 strains), and Y. pestis (12 strains) on the T2Dx instrument. 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.
- Analytical Specificity (Exclusivity): 31 bacterial, fungal, and viral strains commonly found to cause blood stream infections or genetically similar to Panel pathogens were tested at 1,000 CFU/mL or IU/mL K2EDTA-treated whole blood. No repeatable reactivity was detected for most species. One strain, Bacillus cereus G-9241, known to harbor a pXO1-like plasmid, showed cross-reactivity with the BaPXO1 channel. The T2Biothreat Panel software distinguishes between single virulence plasmid detection and fully virulent B. anthracis (both pXO1 and pXO2 plasmids). At 1x10^5 copies/mL of DNA from exclusivity strains, no cross-reactivity was observed.
- Reproducibility: Results agreement was analyzed at multiple sites over six non-consecutive days for each sample type, with a minimum of two operators per site, two Reagent Tray Kit lots, and two instruments. Sample types included negative K2EDTA-treated whole blood and positive spiked samples (B. anthracis, B. pseudomallei, Y. pestis triple species spike; B. mallei, R. prowazekii dual species spike; F. tularensis single species spike) at 2-3x LoD or 1-1.5 LoD.
- Overall agreement with expected positive results: 98.4%
- Overall agreement with expected negative results: 100%
- Interfering Substances: 7 endogenous and 15 exogenous substances were tested with contrived positive and negative samples in K2EDTA-treated whole blood by the T2Dx. None of the tested substances demonstrated interference with the detection of any T2Biothreat Panel targets nor in the validity of samples tested, except for Feraheme (>=21μg/mL), Magnevist (>=1.7 mg/mL), and Ablavar (>=0.39 mg/mL).
- Competitive Inhibition: The study assessed the impact on positive detection of Panel members by competitive inhibition from other potential co-infecting species. Conditions included co-infection with two Panel target species (at/near LoD, or one high titer and one at/near LoD) and co-infection with one Panel species (at/near LoD) and a non-Panel species (high titer). No competitive effects were observed in any combination tested.
Clinical Performance Characteristics:
- Study Type: Two-arm study (Negative Arm for specificity, Positive Arm for sensitivity) conducted using samples from healthy donors and febrile donors.
- Sample Size: Not explicitly stated as a single number encompassing all clinical performance, but results show:
- Negative Arm: Healthy and febrile negative blood samples.
- Positive Arm: Contrived positive-spiked whole blood samples from febrile donors.
- Specific Positive Percent Agreement (PPA) counts:
- B. anthracis: 5/6 (0.1-1x LoD), 32/32 (1-3x LoD), 12/12 (3-5x LoD)
- Burkholderia spp.: 17/17 (0.1-1x LoD), 77/77 (1-3x LoD), 6/6 (3-5x LoD)
- F. tularensis: 6/7 (0.1-1x LoD), 33/35 (1-3x LoD), 8/8 (3-5x LoD)
- R. prowazekii: N/A (0.1-1x LoD), 10/10 (1-3x LoD), 40/40 (3-5x LoD)
- Y. pestis: 25/26 (0.1-1x LoD), 24/24 (1-3x LoD), N/A (3-5x LoD)
- Key Results:
- Analysis of the Panel negative percent agreement (NPA) utilized results from healthy and febrile negative blood samples. The NPA was 100% for all analytes.
- Analysis of the Panel positive percent agreement (PPA) utilized results from the positive samples. The PPA was analyzed for samples above and below LoD as well as all concentrations.
- For analyte concentrations at 1-3x LoD, the PPA ranged from 94.3% (F. tularensis) to 100%.
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
- Positive Percent Agreement (PPA):
- B. anthracis (pXO1 & pXO2): 83.3% (0.1-1x LoD), 100% (1-3x LoD), 100% (3-5x LoD)
- Burkholderia spp. (B. pseudomallei & B. mallei): 100% (0.1-1x LoD), 100% (1-3x LoD), 100% (3-5x LoD)
- F. tularensis: 85.7% (0.1-1x LoD), 94.3% (1-3x LoD), 100% (3-5x LoD)
- R. prowazekii: N/A (0.1-1x LoD), 100% (1-3x LoD), 100% (3-5x LoD)
- Y. pestis: 96.2% (0.1-1x LoD), 100% (1-3x LoD), N/A (3-5x LoD)
- PPA by organism relative to LoD (positive contrived):
- B. anthracis (BaPXO1): 100% (=LoD), 100% (All Concentrations)
- B. anthracis (BaPXO2): 90% (=LoD), 98% (All Concentrations)
- B. pseudomallei (Bu): 100% (=LoD), 100% (All Concentrations)
- F. tularensis (Ft): 90% (=LoD), 94% (All Concentrations)
- R. prowazekii (Rp): 100% (=LoD), 100% (All Concentrations)
- Y. pestis (Yp): 100% (=LoD), 98% (All Concentrations)
- Negative Percent Agreement (NPA): 100% for all analytes.
Predicate Device(s)
Reference Device(s)
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
N/A
0
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
1
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
2
Indications for Use
510(k) Number (if known) K231336
Device Name T2Biothreat Panel
Indications for Use (Describe)
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
-
- 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), 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.
| 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.
3
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4
510(k) Summary
| Date of Summary | May 8, 2023 |
|---|---|
| Product Name | T2Biothreat Panel |
| Sponsor | T2Biosystems, Inc. |
| 101 Hartwell Avenue | |
| Lexington, MA 02421 | |
| Correspondent | Rachel Gilbert |
| Manager, Regulatory Affairs | |
| 781-226-2767, 1970 | |
| rgilbert@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 |
5
Intended 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:
-
- Bacillus anthracis (plasmids pXO1 and pXO2)
-
- Francisella tularensis
-
- Burkholderia spp. (B. malleilB. pseudomallei)
-
- Yersinia pestis
-
- 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.
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.
Limitations
For prescription use only. Refer to the Biothreat Panel labeling for a more complete list of warnings, precautions, and contraindications.
6
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.
7
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 | 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 |
| Organism | Strains | Positivity | Repeat Testing | Result |
| Burkholderia
mallei
8 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 |
| Burkholderia
pseudomallei
10 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 |
| Francisella
tularensis
10 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 |
| Rickettsia
prowazekii
5 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 pestis
12 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 |
8
9
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-reactive
Non-Panel Strain | Panel Channel
that 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.
10
| 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. | Expected
Result | Agreement with Expected Result [95% CI] | |
|------------------------|------------|--------------------|------------------------------------------|---------------------------|
| | | | BaPXO1 Channel | BaPXO2 Channel |
| B. anthracis | 1-1.5x LoD | Detected | 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] | - |
11
| Organism | Conc. | Expected
Result | Agreement with Expected Result [95% CI] | |
|----------------------|------------|--------------------|-----------------------------------------|---|
| R. prowazekii | 1-1.5x LoD | Detected | Rp Channel
95/96
99% [94.3-100] | - |
| | 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] | - |
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 | 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 | ≥16.5x106
cells/mL | K2EDTA | 9 mg/mL | Trimethoprim | 145 μmol/L |
| | | Kanamycin B
sulfate | 186 μmol/L | | |
12
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 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 |
| 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 Low
Conc.
(1-2x LoD) | Species 2 High Conc.
(1000 CFU/mL, CAGe/mL, or IU/mL)1 | Positive Detection Rate
Species 12 | Positive Detection Rate
Species 22 | Competitive
Inhibition |
| B. anthracis | Escherichia coli | 100% | 0% | No |
| | Enterococcus faecium | 75%
(3/4 Ba pXO2,
4/4 Ba pXO1)
100%
(20/20 both
channels) | 0% | No |
| | 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 |
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
13
14
| 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 | |
| | 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.
15
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 | Titer
Bucket | 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 - Burkholderia spp.
( B. pseudomallei &
B. mallei ) | 12 –
17 CFU/mL6 | 0.1-1x LoD | 1.2 - F. tularensis | 4 CFU/mL | 0.1-1x LoD | 0.4 - R. prowazekii | 9 CAGe/mL | 0.1-1x LoD | 0.9 - Y. pestis | 2 CFU/mL | 0.1-1x LoD | 0.2 -