(258 days)
DN 140019
Not Found
No
The summary describes a diagnostic test using T2 magnetic resonance technology and nucleic acid amplification. It details the sample processing, detection method, and performance metrics, but there is no mention of AI or ML being used for data analysis, interpretation, or any other part of the process. The results are interpreted by the T2Dx applications software as valid/invalid and positive/negative/indeterminate, which appears to be based on predefined thresholds or algorithms, not AI/ML.
No.
The T2Bacteria Panel is intended for the direct detection of bacterial species as an aid in the diagnosis of bacteremia, not for direct treatment or therapy.
Yes
The device is explicitly stated to be "indicated as an aid in the diagnosis of bacteremia," and its results are intended to be "used in conjunction with other clinical and laboratory data" for this purpose.
No
The device is a system that includes hardware (T2Dx Instrument, Cartridge) and reagents, in addition to software. It is not solely software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use: The intended use explicitly states that the device is for the "direct detection of bacterial species in K2EDTA human whole blood specimens with suspected bacteremia" and is "indicated as an aid in the diagnosis of bacteremia." This clearly indicates that the device is intended to be used in vitro (outside the body) to examine specimens from the human body for diagnostic purposes.
- Specimen Type: The device uses "K2EDTA human whole blood specimens," which are biological specimens taken from the human body.
- Methodology: The device utilizes "nucleic acid amplification and proprietary T2MR® detection technology" to detect and identify bacterial species. These are laboratory-based techniques performed on the collected specimen.
- Device Description: The description details the process of preparing and analyzing the blood specimen within the instrument, which is a typical workflow for an in vitro diagnostic test.
- Results: The results are reported as "Positive or Target not Detected" for specific bacterial species, providing information about the presence or absence of these organisms in the blood specimen, which is used to aid in diagnosis.
The definition of an IVD generally includes devices intended for use in the collection, preparation, and examination of specimens taken from the human body for the purpose of providing information for the diagnosis, prevention, or treatment of a disease or condition. The T2Bacteria Panel fits this definition.
N/A
Intended Use / Indications for Use
The T2Bacteria Panel run on the T2Dx Instrument is a qualitative T2 magnetic resonance (T2MR) test for the direct detection of bacterial species in K2EDTA human whole blood specimens with suspected bacteremia. The T2Bacteria Panel identifies five species of bacteria: Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.
The T2Bacteria Panel is indicated as an aid in the diagnosis of bacteremia and results should be used in conjunction with other clinical and laboratory data. Concomitant blood cultures are necessary to recover organisms for susceptibility testing or further identification, and for organisms not detected by the T2Bacteria Panel.
Results from the T2Bacteria Panel are not intended to be used as the sole basis for diagnosis, treatment management decisions in patients with suspected bacteremia.
Product codes
QBX, NSU
Device Description
The T2Bacteria Panel detects and identifies five bacterial target species directly from whole blood specimens and independent of blood culture using nucleic acid amplification and proprietary T2MR® detection technology. The assay is performed on the proprietary T2Dx platform.
The whole blood specimen, drawn into a blood collection tube containing K₂EDTA is used for the test. The blood collection tube containing a minimum of 3 mL of blood is loaded directly onto the T2Dx instrument as part of the assembled Cartridge, a single use self-contained unit that contains all of the reagents and disposables required to run a single test.
Fully automated on the T2Dx, the blood specimen is mixed with the Lysis Reagent to lyse the red blood cells and the bacterial cells are concentrated by centrifugation. The Internal Control is added to the concentrated bacterial cells, which then undergo a bead beating step to lyse the bacteria cells. The supernatant containing the DNA from the lysed bacterial cells and the Internal Control are amplified with the target and Internal Control specific primers. The generated amplicon is then aliquoted into individual tubes containing targetspecific conjugated particles for Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and the Internal Control. These individual tubes are read in the MR reader and a signal is generated.
Up to seven specimens can be loaded onto the T2Dx Instrument in parallel. When running a single specimen, the first result is reported in 3.5 hours from the time the specimen is loaded onto the instrument. The results are interpreted using the T2Dx applications software as valid or invalid, and if valid, target specific results are reported as Positive or Target not Detected. For one target, the Escherichia coli channel, results are reported as Positive, Indeterminate, or Target not Detected. An Indeterminate result is a valid result, but the presence or absence of Escherichia coli cannot be definitively assessed, and the indeterminate status applies only to the Escherichia coli channel. Results are displayed on the T2Dx touchscreen and can be printed. Raw T2MR data are not available to the end user.
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Not Found
Anatomical Site
Not Found
Indicated Patient Age Range
Not Found
Intended User / Care Setting
Prescription Use
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
Not Found
Summary of Performance Studies
Limit of Detection (LoD):
The LoD for each bacterial species was determined by spiking whole blood specimens with each individual bacteria target species. For each bacterial target, the LoD is defined as the lowest concentration (CFU/mL) of target that can be detected at a rate ≥ 95%. The LoD was established by testing a minimum of twenty replicates each of two strains of each bacteria species at multiple concentrations.
Reproducibility:
To confirm the site-to-site, operator, system-to-system, and lot-to-lot reproducibility of the T2Bacteria Panel, negative blood and blood spiked with Kp and Sa or Efm, Pa, and Eci were tested at 1-2x LoD and 3-4x LoD at three sites, with three to four operators per site, two lots of reagents, and three T2Dx systems. Each sample type in the study was tested over six non-consecutive days. A total of 540 samples comprised of 108 negative samples and 432 positive samples were run with an overall reproducibility of 98.7%.
Analytical Reactivity (Inclusivity):
Analytical reactivity testing was conducted to ensure that the T2Bacteria Panel is capable of detecting multiple strains of the five organisms that constitute the panel. Clinical isolates were chosen based on resistance, phylogenetic, temporal, and geographic diversity and spiked into whole blood at 2-3x the established panel LoDs. Inclusivity panels included: 11 Enterococcus faecium strains, 12 Escherichia coli strains, 13 Klebsiella pneumonia strains, 13 Pseudomonas aeruginosa strains, and 8 Staphylococcus aureus strains. A total of 57 organisms were evaluated. Testing was performed in triplicate. In the event of a false negative result, testing was repeated with 20 replicates and 19/20 replicates had to generate a positive result to be considered passing.
Analytical Exclusivity:
Analytical exclusivity testing was conducted to assess the cross-reactivity of the T2Bacteria Panel to non-panel species at 1,000 units/mL concentrations (CFU, TCID50, or copies /mL where applicable) of pathogenically, phylogenetically, or environmentally relevant organisms in whole blood. Species that were shown to be potentially cross-reactive at the initial test concentration were further evaluated at lower target concentrations using a pre-defined titration scheme (100, 33, and 10 units/mL). Analytical testing included 128 different organisms.
Competitive Inhibition:
A Competitive Inhibition Study was conducted to evaluate assay performance in the presence of two or more Panel bacterial target species at high and low concentrations as well as selected bacterial and fungal non-Panel target organisms. The conditions tested included: co-infection with two Panel target species both at or near the LoD; co-infection with two Panel target species where one species is at high titer (1,000 CFU/mL) and the other is at or near the LoD; and co-infection with one Panel species at or near the LoD and a non-Panel species at high titer (1,000 CFU/mL). Four replicates were tested and if any false negative results were generated, the test was repeated with 20 replicates.
Interfering Substances:
Studies were conducted to evaluate the impact of potential endogenous and exogenous interfering substances on the performance of the T2Bacteria Panel. These substances were added to negative whole blood samples or to whole blood samples multi-spiked with either E. coli, P. aeruginosa, and E. faecium, or K. pneumoniae and S. aureus at 2-3x LoD. Three replicate samples were run for each interfering substance tested.
Clinical Performance Evaluation (Prospective and Contrived Arms):
- Prospective Arm: The performance was evaluated at eleven sites within the US and compared to the reference method of blood culture. Patients were enrolled prospectively and two paired sample collections, one for blood culture and one for testing by the T2Bacteria Panel, were drawn from each subject. A total of 1,427 subjects was tested. Species identification for blood cultures was performed using Gram stain, bioMerieux Vitek® 2, bioMerieux or Bruker MALDI TOF, and PCR. The T2Bacteria Panel result was compared against results from these blood culture systems for Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA).
- Contrived Arm: Due to the low prevalence of target organisms in the prospective arm, an additional 250 contrived specimens were evaluated at three sites. Contrived specimens were prepared by spiking bacteria at defined concentrations (CFU/mL) into healthy donor whole blood. A total of 50 strains of each of the five bacterial species (250 strains total) were used. An additional 100 blood samples not spiked with T2Bacteria Panel members were also evaluated.
- Key Results (Combined Prospective and Contrived): PPA ranged from 81.3% to 100%, and NPA ranged from 95.5% to 100%.
False Positive Results Analysis:
In the prospective study, there were 190 T2 positive results (35 T2+/BC+ concordant, 155 T2+/BC- potential false positives). Of the 155 potential false positives: 39 had an additional positive blood culture within ±14 days, 30 were positive by amplification and gene sequencing from a concurrently drawn blood sample, and 23 had other non-blood specimens positive for the same organism within ±14 days. 63 of the 155 potential false positives (41%) were not associated with evidence of infection. Reagent contamination was identified as a likely source for these false positives. After implementing improved reagent testing methods and release criteria, reagent contamination levels were observed at ≤1% for all targets. Retesting of archived samples suggested that 30 E. coli positive samples were likely false positives, and 11 P. aeruginosa positive samples were likely false positives, while 2 P. aeruginosa samples were likely true positives.
Key Metrics
- Overall Reproducibility: 98.7% (533/540)
- False Positive Rates (Analytical Studies): Efm: 0.1%, Eci: 2.6%, Kp: 0.2%, Pa: 1.5%, Sa: 0.1%.
- Invalid Rate (Analytical Studies, Total): 1.7% (0.3% excluding interfering substances)
- Indeterminate Rate (Analytical Studies, E. coli channel only, Total): 0.9% (0.8% excluding interfering substances)
- PPA (Sensitivity) from Combined Prospective and Contrived Arms:
- E. faecium: 100% (40/40) for Contrived, 100% (1/1) for Prospective
- S. aureus: 92.3% (36/39) for Contrived, 81.3% (13/16) for Prospective
- K. pneumoniae: 100% (40/40) for Contrived, 100% (6/6) for Prospective
- P. aeruginosa: 97.4% (38/39) for Contrived, 100% (5/5) for Prospective
- E. coli: 90.9% (20/22) for Contrived, 90.9% (10/11) for Prospective
- NPA (Specificity) from Combined Prospective and Contrived Arms:
- E. faecium: 100% (300/300) for Contrived, 99.4% (1417/1426) for Prospective
- S. aureus: 100% (300/300) for Contrived, 98.0% (1383/1411) for Prospective
- K. pneumoniae: 99.3% (298/300) for Contrived, 98.5% (1399/1421) for Prospective
- P. aeruginosa: 97.7% (293/300) for Contrived, 97.7% (1389/1422) for Prospective
- E. coli: 97.3% (292/300) for Contrived, 95.0% (1345/1416) for Prospective
- Overall Invalid Rate (Prospective and Contrived Arms): 0.4%
- Overall Indeterminate Rate (Prospective and Contrived Arms, E. coli channel only): 0.6%
Predicate Device(s):
T2Candida Panel (DN 140019)
Reference Device(s):
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
§ 866.3960 Nucleic acid-based device for the amplification, detection, and identification of microbial pathogens directly from whole blood specimens.
(a)
Identification. A nucleic acid-based device for the amplification, detection, and identification of microbial pathogens directly from whole blood specimens is a qualitative in vitro device intended for the amplification, detection, and identification of microbial-associated nucleic acid sequences from patients with suspected bloodstream infections. This device is intended to aid in the diagnosis of bloodstream infection when used in conjunction with clinical signs and symptoms and other laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include detailed device description documentation, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including primer/probe sequence, design, and rationale for sequence selection.
(2) Premarket notification submissions must include detailed documentation from the following analytical and clinical performance studies: Analytical sensitivity (limit of detection), reactivity, inclusivity, precision, reproducibility, interference, cross reactivity, carryover, and cross contamination.
(3) Premarket notification submissions must include detailed documentation from a clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from well-accepted reference methods.
(4) Premarket notification submissions must include detailed documentation for device software, including, but not limited to, software applications and hardware-based devices that incorporate software.
(5) The device labeling must include limitations regarding the need for culture confirmation of negative specimens, as appropriate.
(6) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling.
(7) Premarket notification submissions must include details on an end user device training program that will be offered while marketing the device, as appropriate.
(8) As part of the risk management activities performed as part of your 21 CFR 820.30 design controls, you must document an appropriate end user device training program that will be offered as part of your efforts to mitigate the risk of failure to correctly operate the instrument.
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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.
May 24, 2018
T2 Biosystems, Inc. Tom Lowery Chief Operations Officer 101 Hartwell Avenue Lexington, Massachusetts 02421
Re: K172708
Trade/Device Name: T2Bacteria Panel Regulation Number: 21 CFR 866.3960 Regulation Name: Nucleic acid-based device for the amplification, detection and identification of microbial pathogens directly from whole blood specimens Regulatory Class: Class II Product Code: QBX, NSU Dated: April 30, 2018 Received: May 1, 2018
Dear Tom Lowery:
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. 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 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR
1
Part 820); 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 http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
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/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). 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 (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).
Sincerely,
Ribhi Shawar -S For
Uwe Scherf, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health
Enclosure
2
Indications for Use
510(k) Number (if known) K172708
Device Name T2Bacteria Panel
Indications for Use (Describe)
The T2Bacteria Panel run on the T2Dx Instrument is a qualitative T2 magnetic resonance (T2MR) test for the direct detection of bacterial species in K2EDTA human whole blood specimens with suspected bacteremia. The T2Bacteria Panel identifies five species of bacteria: Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.
The T2Bacteria Panel is indicated as an aid in the diagnosis of bacteremia and results should be used in conjunction with other clinical and laboratory data. Concomitant blood cultures are necessary to recover organisms for susceptibility testing or further identification, and for organisms not detected by the T2Bacteria Panel.
Results from the T2Bacteria Panel are not intended to be used as the sole basis for diagnosis, treatment management decisions in patients with suspected bacteremia.
| Type of Use (Select one or both, as applicable) |
|---|
| Prescription Use (Part 21 CFR 801 Subpart D) |
| Over-The-Counter Use (21 CFR 801 Subpart C) |
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3
| 15. | 510(k) SUMMARY |
|---|---|
| Date of Summary | April 30, 2018 |
| Product Name | T2Bacteria® Panel |
| Sponsor | T2 Biosystems, Inc. |
| 101 Hartwell Avenue | |
| Lexington, MA 02421 | |
| Correspondent | T2 Biosystems, Inc. |
| Thomas J. Lowery, PhD | |
| Chief Scientific Officer | |
| Office Phone : (781) 457-1223 | |
| Mobile Phone : (617) 932-9047 | |
| Fax : (781) 357-3080 | |
| tlowery@t2biosystems.com | |
| Device Trade or Proprietary Name | T2Bacteria® Panel |
| Regulation | 21 CFR 866.3960 |
| Common Name | Nucleic acid-based device for the amplification, |
| detection and identification of microbial pathogens | |
| directly from whole blood specimens | |
| Product Code | QBX, NSU |
| Classification | Class II |
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Substantial Equivalence
COMPARISON OF NEW DEVICE WITH PREDICATE DEVICE
| Characteristic | T2Bacteria Panel
(New Device) | T2Candida Panel (DN 140019)
(Primary Predicate Device) |
|------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | Similarities | |
| FDA Product
Code | QBX, NSU | PII, NSU |
| Regulatory
Classification | Class II | Class II |
| Regulation
Number | 21 CFR 866.3960 | 21 CFR 866.3960 |
| Intended Use | The T2Bacteria Panel run on the T2Dx Instrument
is a qualitative T2 magnetic resonance (T2MR)
test for the direct detection of bacterial species
in K₂EDTA human whole blood specimens from
patients with suspected bacteremia. The
T2Bacteria Panel identifies five species of
bacteria: Enterococcus faecium, Escherichia coli,
Klebsiella pneumoniae, Pseudomonas
aeruginosa, and Staphylococcus aureus.
The T2Bacteria Panel is indicated as an aid in the
diagnosis of bacteremia and results should be
used in conjunction with other clinical and
laboratory data. Concomitant blood cultures are
necessary to recover organisms for susceptibility
testing or further identification and for organisms
not detected by the T2Bacteria Panel.
Results from the T2Bacteria Panel are not
intended to be used as the sole basis for
diagnosis, treatment, or other patient
management decisions in patients with
suspected bacteremia. | T2Candida Panel and T2Dx Instrument is a
qualitative T2 Magnetic Resonance (T2MR®) assay
for the direct detection of Candida species in
K₂EDTA human whole blood specimens from
patients with symptoms of, or medical conditions
predisposing the patient to, invasive fungal
infections. The T2Candida Panel identifies five
species of Candida and categorizes them into the
following three (3) species groups:
- Candida albicans and/or Candida
tropicalis - Candida parapsilosis
- Candida glabrata and/or Candida krusei
The T2Candida Panel is indicated for the
presumptive diagnosis of candidemia. The
T2Candida Panel is performed independent of
blood culture. Concomitant blood cultures are
necessary to recover organisms for susceptibility
testing or further identification.
The T2Candida positive and negative External
Controls are intended to be used as quality control
samples with the T2Candida Panel when run on
the T2Dx Instrument. These controls are not
intended for use with other assays or systems. |
| Sample Type | 4 ml whole blood collected in a blood collection tube
with K₂EDTA anticoagulant | 4 ml whole blood collected in a blood collection tube
with K₂EDTA anticoagulant |
| Test Platform | T2Dx | T2Dx |
| Test Cartridge
Format | T2Bacteria Test Cartridge and disposables | T2Candida test cartridge and disposables |
| Test Principle | Nucleic acid amplification followed by T2 magnetic
resonance detection | Nucleic acid amplification followed by T2 magnetic
resonance detection |
| Through put | Single cartridge test with random access with
seven (7) draws on T2Dx | Single cartridge test with random access with seven
(7) draws on T2Dx |
| Characteristic | T2Bacteria Panel
(New Device) | T2Candida Panel (DN 140019)
(Primary Predicate Device) |
| Reagent Trays | T2Bacteria Test Reagents for detection of bacteria | T2Candida test reagent for detection of Candida
(difference is specific to primers and probes) |
| Targets | T2Bacteria Panel tests for five (5) different species of
bacteria commonly implicated in bacteremia:
Enterococcus faecium, Escherichia coli, Klebsiella
pneumoniae, Pseudomonas aeruginosa, and
Staphylococcus aureus. | T2Candida Panel tests for five (5) different species
of Candida commonly associated with candidemia:
Candida albicans and/or Candida tropicalis;
Candida parapsilosis; Candida glabrata and/or
Candida krusei |
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Page 3 of 20
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Intended Use
The T2Bacteria Panel run on the T2Dx Instrument is a qualitative T2 magnetic resonance (T2MR) test for the direct detection of bacterial species in K₂EDTA human whole blood specimens from patients with suspected bacteremia. The T2Bacteria Panel identifies five species of bacteria: Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.
The T2Bacteria Panel is indicated as an aid in the diagnosis of bacteremia and results should be used in conjunction with other clinical and laboratory data. Concomitant blood cultures are necessary to recover organisms for susceptibility testing or further identification and for organisms not detected by the T2Bacteria Panel.
Results from the T2Bacteria Panel are not intended to be used as the sole basis for diagnosis, treatment, or other patient management decisions in patients with suspected bacteremia.
Limitations:
For prescription use only.
Please refer to the T2Bacteria Panel labeling for a more complete list of warnings, precautions, and contraindications.
Methodology:
The T2Bacteria Panel detects and identifies five bacterial target species directly from whole blood specimens and independent of blood culture using nucleic acid amplification and proprietary T2MR® detection technology. The assay is performed on the proprietary T2Dx platform.
The whole blood specimen, drawn into a blood collection tube containing K₂EDTA is used for the test. The blood collection tube containing a minimum of 3 mL of blood is loaded directly onto the T2Dx instrument as part of the assembled Cartridge, a single use self-contained unit that contains all of the reagents and disposables required to run a single test.
Fully automated on the T2Dx, the blood specimen is mixed with the Lysis Reagent to lyse the red blood cells and the bacterial cells are concentrated by centrifugation. The Internal Control is added to the concentrated bacterial cells, which then undergo a bead beating step to lyse the bacteria cells. The supernatant containing the DNA from the lysed bacterial cells and the Internal Control are amplified with the target and Internal Control specific primers. The generated amplicon is then aliquoted into individual tubes containing targetspecific conjugated particles for Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and the Internal Control. These individual tubes are read in the MR reader and a signal is generated.
7
Up to seven specimens can be loaded onto the T2Dx Instrument in parallel. When running a single specimen, the first result is reported in 3.5 hours from the time the specimen is loaded onto the instrument. The results are interpreted using the T2Dx applications software as valid or invalid, and if valid, target specific results are reported as Positive or Target not Detected. For one target, the Escherichia coli channel, results are reported as Positive, Indeterminate, or Target not Detected. An Indeterminate result is a valid result, but the presence or absence of Escherichia coli cannot be definitively assessed, and the indeterminate status applies only to the Escherichia coli channel. Results are displayed on the T2Dx touchscreen and can be printed. Raw T2MR data are not available to the end user.
Performance Data
For ease of reference, Table 1 defines the target organisms contained in the T2Bacteria Panel and common acronyms used in the study descriptions and tables.
| Table 1: T2Bacteria Assay Targets | |
|---|---|
| Organism | Acronym |
| Enterococcus faecium | Efm |
| Escherichia coli | Eci |
| Klebsiella pneumoniae | Kp |
| Pseudomonas aeruginosa | Pa |
| Staphylococcus aureus | Sa |
| Internal Control | IC |
i. Limit of Detection (LoD)
The limit of detection (LoD) for each bacterial species was determined by spiking whole blood specimens with each individual bacteria target species. For each bacterial target, the LoD is defined as the lowest concentration (CFU/mL) of target that can be detected at a rate ≥ 95%. The LoD was established by testing a minimum of twenty replicates each of two strains of each bacteria species at multiple concentrations. The LoD established for each bacteria in the T2Bacteria Panel is shown in Table 2a.
| Table 2a: T2Bacteria Panel LoD | ||
|---|---|---|
| Bacteria | LoD (CFU/mL) | |
| Enterococcus faecium | 5 | |
| Escherichia coli | 11 | |
| Klebsiella pneumoniae | 2 | |
| Pseudomonas aeruginosa | 5 | |
| Staphylococcus aureus | 2 |
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ii. Reproducibility
To confirm the site-to-site, operator, system-to-system-to-system, and lot-to-lot reproducibility of the T2Bacteria Panel, negative blood and blood spiked with Kp and Sa or Efm, Pa, and Eci were tested at 1-2x LoD and 3-4x LoD at three sites, with three to four operators per site, two lots of reagents, and three T2Dx systems. Each sample type in the study was tested over six non-consecutive days. A total of 540 samples comprised of 108 negative samples and 432 positive samples were run with an overall reproducibility of 98.7%. The data, summarized in Tables 2b and 2c demonstrate that the T2Bacteria Panel run on the T2Dx across sites, operators, reagent lots, and systems reproducibly.
| Table 2b: T2Bacteria Panel Reproducibility – Overall Performance | |||
|---|---|---|---|
| Sample | N Accurate / | ||
| N Total | % Accurate | 95% CI | |
| Total | 533/540 | 98.7 | 97.3-99.5 |
| Eci | Efm | Kp | Pa | Sa | ||
|---|---|---|---|---|---|---|
| Table 2c: T2Bacteria Panel Reproducibility | ||||||
| KS 1-2x | ||||||
| LoD | N Pos / N Total | 5/108 | 0/108 | 108/108 | 0/108 | 105/108 |
| % Accurate | 95.4 | 100 | 100 | 100 | 97.2 | |
| 95% Cl | 89.5-98.5 | 96.6-100 | 96.6-100 | 96.6-100 | 92.1-99.4 | |
| KS 3-4x | ||||||
| LoD | N Pos / N Total | 5/108 | 0/108 | 108/108 | 1/108 | 108/108 |
| % Accurate | 95.4 | 100 | 100 | 99.1 | 100 | |
| 95% Cl | 89.5-98.5 | 96.6-100 | 96.6-100 | 94.9-100 | 96.6-100 | |
| EPE 1- | ||||||
| 2x LoD | N Pos / N Total | 108/108 | 107/108 | 0/108 | 108/108 | 0/108 |
| % Accurate | 100 | 99.1 | 100 | 100 | 100 | |
| 95% Cl | 96.6-100 | 94.9-100 | 96.6-100 | 96.6-100 | 96.6-100 | |
| EPE 3- | ||||||
| 4x LoD | N Pos / N Total | 108/108 | 108/108 | 0/108 | 108/108 | 0/108 |
| % Accurate | 100 | 100 | 100 | 100 | 100 | |
| 95% Cl | 96.6-100 | 96.6-100 | 96.6-100 | 96.6-100 | 96.6-100 | |
| Neg | N Neg / N Total | 106 / 108 | 108 / 108 | 108 / 108 | 107/108 | 108 / 108 |
| % Accurate | 98.1 | 100 | 100 | 99.1 | 100 | |
| 95% Cl | 93.5-99.8 | 96.6-100 | 96.6-100 | 94.9-100 | 96.6-100 |
iii. Analytical Reactivity (Inclusivity):
Analytical reactivity testing was conducted to ensure that the T2Bacteria Panel is capable of detecting multiple strains of the five organisms that constitute the panel. Clinical isolates were chosen based on resistance, phylogenetic, temporal, and geographic diversity and spiked into whole blood at 2-3x the established panel LoDs. Inclusivity panels included the following:
- 11 Enterococcus faecium strains
- 12 Escherichia coli strains ●
- 13 Klebsiella pneumonia strains
- 13 Pseudomonas aeruginosa strains ●
- 8 Staphylococcus aureus strains
9
A total of 57 organisms were evaluated for inclusivity in the T2Bacteria Panel. Testing was performed in triplicate. In the event of a false negative result, testing was repeated with 20 replicates and 19/20 replicates had to generate a positive result to be considered passing. Test results summarized in Table 3 demonstrate that the T2Bacteria Panel is able to detect multiple strains of each target species.
| Table 3: T2Bacteria Panel Inclusivity Results | |||
|---|---|---|---|
| Species | Isolate ID | N=3 | N=20 |
| Enterococcus faecium | ATCC BAA-472 | 3/3 | NT |
| Enterococcus faecium | ATCC 51559 | 3/3 | NT |
| Enterococcus faecium | ATCC 49224 | 3/3 | NT |
| Enterococcus faecium | ATCC 700221 | 3/3 | NT |
| Enterococcus faecium | ATCC BAA-2320 | 3/3 | NT |
| Enterococcus faecium | ATCC 51858 | 3/3 | NT |
| Enterococcus faecium | DSM 17050 | 3/3 | NT |
| Enterococcus faecium | LMG 16308 | 3/3 | NT |
| Enterococcus faecium | LMG 20732 | 3/3 | NT |
| Enterococcus faecium | LMG 23226 | 3/3 | NT |
| Enterococcus faecium | LMG 24170 | 3/3 | NT |
| Escherichia coli | ATCC 31705 | 3/3 | NT |
| Escherichia coli | ATCC BAA-2326 | 3/3 | NT |
| Escherichia coli | ATCC 700928 | 3/3 | NT |
| Escherichia coli | ATCC BAA-2452 | 3/3 | NT |
| Escherichia coli | ATCC BAA-1652 | 3/3 | NT |
| Escherichia coli | ATCC BAA-2129 | 3/3 | NT |
| Escherichia coli | ATCC 35150 | 3/3 | NT |
| Escherichia coli | ATCC 23518 | 3/3 | NT |
| Escherichia coli | ATCC 29640 | 3/3 | NT |
| Escherichia coli | ATCC BAA-177 | 3/3 | NT |
| Escherichia coli | ATCC 33780 | 3/3 | NT |
| Escherichia coli | ATCC BAA-179 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC 27736 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC 6908 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC BAA-1144 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC 29013 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC BAA-1898 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC BAA-1903 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC BAA-1904 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC 13884 | 2/3 | 20/20 |
| Klebsiella pneumoniae | ATCC 13886 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC 8045 | 3/3 | NT |
| Klebsiella pneumoniae | CCUG 26108 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC BAA-2578 | 3/3 | NT |
| Klebsiella pneumoniae | ATCC 700603 | 2/3 | 20/20 |
| Pseudomonas aeruginosa | ATCC 47085 | 3/3 | NT |
| Pseudomonas aeruginosa | ATCC 43637 | 3/3 | NT |
| Table 3: T2Bacteria Panel Inclusivity Results | |||
| Species | Isolate ID | N=3 | N=20 |
| Pseudomonas aeruginosa | ATCC 14203 | 3/3 | NT |
| Pseudomonas aeruginosa | ATCC 15692 | 3/3 | NT |
| Pseudomonas aeruginosa | LMG 25200 | 3/3 | NT |
| Pseudomonas aeruginosa | LMG 27623 | 3/3 | NT |
| Pseudomonas aeruginosa | ATCC 27853 | 3/3 | NT |
| Pseudomonas aeruginosa | LMG 24907 | 3/3 | NT |
| Pseudomonas aeruginosa | LMG 24916 | 3/3 | NT |
| Pseudomonas aeruginosa | LMG 24918 | 3/3 | NT |
| Pseudomonas aeruginosa | LMG 24928 | 3/3 | NT |
| Pseudomonas aeruginosa | LMG 25009 | 3/3 | NT |
| Pseudomonas aeruginosa | LMG 25143 | 3/3 | NT |
| Staphylococcus aureus | ATCC 33592 | 2/3 | 19/20 |
| Staphylococcus aureus | ATCC BAA-1556 | 3/3 | NT |
| Staphylococcus aureus | ATCC BAA-42 | 3/3 | NT |
| Staphylococcus aureus | ATCC BAA-1717 | 3/3 | NT |
| Staphylococcus aureus | ATCC 11371 | 3/3 | NT |
| Staphylococcus aureus | ATCC 12598 | 3/3 | NT |
| Staphylococcus aureus | ATCC BAA-44 | 3/3 | NT |
| Staphylococcus aureus | ATCC BAA-1683 | 3/3 | NT |
10
NT: Not tested
Analytical Exclusivity iv.
Analytical exclusivity testing of the T2Bacteria Panel was conducted to assess the cross-reactivity of the T2Bacteria Panel to non-panel species at 1,000 units/mL concentrations (CFU, TCID50, or copies /mL where applicable) of pathogenically, phylogenetically, or environmentally relevant organisms in whole blood. Species that were shown to be potentially cross-reactive at the initial test concentration were further evaluated at lower target concentrations using a pre-defined titration scheme (100, 33, and 10 units/mL).
Analytical testing of the T2Bacteria Panel included 128 different organisms comprised of the T2Bacteria Panel members themselves, viruses, and pathogenically, phylogenetically, or environmentally relevant bacterial and fungal species.
The test results, summarized in Tables 4a and 4b, establish the specificity of the T2Bacteria Panel in the presence of all organisms tested at 1,000 units / mL with the exception of Shigella boydii, Shigella dysenteriae, Shigella sonnei, Enterococcus durans, Escherichia albertii, Escherichia fergusonii, and Klebsiella variicola for which cross-reactivity was established at titer levels ≤ 10 units/mL.
11
| Table 4a: T2Bacteria Panel Exclusivity Results | |||||
|---|---|---|---|---|---|
| Organisms With No Reactivity at 1000 units/mL (CFU/mL, TCID50/mL, copies/mL as applicable) | |||||
| Fungi | |||||
| Aspergillus | |||||
| fumigatus | Aspergillus niger | Candida albicans | Candida glabrata | Candida krusei | Candida |
| parapsilosis | |||||
| Candida tropicalis | Cryptococcus | ||||
| albidus | Cryptococcus | ||||
| neoformans | Fusarium | ||||
| moniliforme | Fusarium | ||||
| oxysporum | Rhizomucor | ||||
| meihei | |||||
| Rhizopus microsporus | Rhizopus oryzae | Rhodotorula glutinis | |||
| Viruses | |||||
| Adenovirus | |||||
| type 1 | Cytomegalovirus | Epstein-Barr Virus | Hepatitis A Virus | Hepatitis B Virus | |
| Herpes Simplex Virus 1 | Herpes Simplex Virus 2 | Varicella Zoster Virus | |||
| Gram Positive Bacteria | |||||
| Actinomyces israelii | Clostridium | ||||
| sphenoides | Corynebacterium | ||||
| jeikeium | Enterococcus | ||||
| avium | Enterococcus | ||||
| caccae | Enterococcus | ||||
| casseliflavus | |||||
| Enterococcus | |||||
| cecorum | Enterococcus | ||||
| dispar | Enterococcus | ||||
| faecalis | Enterococcus | ||||
| gallinarum | Enterococcus | ||||
| gilvus | Enterococcus | ||||
| hirae | |||||
| Enterococcus italicus | Enterococcus | ||||
| malodoratus | Enterococcus | ||||
| mundtii | Enterococcus | ||||
| pallens | Enterococcus | ||||
| pseudoavium | Enterococcus | ||||
| raffinosus | |||||
| Finegoldia magna | Lactobacillus | ||||
| acidophilus | Lactococcus lactis | Leuconostoc | |||
| mesenteroides | Listeria | ||||
| monocytogenes | Parvimonas | ||||
| micra | |||||
| Pediococcus | |||||
| pentosaceus | Peptoniphilus harei | Peptostreptococcus | |||
| anaerobius | Propionibacterium | ||||
| acnes | Staphylococcus | ||||
| auricularis | Staphylococcus | ||||
| capitis | |||||
| Staphylococcus | |||||
| epidermidis | Staphylococcus | ||||
| haemolyticus | Staphylococcus | ||||
| hominis | Staphylococcus | ||||
| lugdunensis | Staphylococcus | ||||
| saprophyticus | Staphylococcus | ||||
| warneri | |||||
| Staphylococcus | |||||
| xylosus | Streptococcus | ||||
| agalactiae | Streptococcus | ||||
| anginosus | Streptococcus | ||||
| bovis | Streptococcus | ||||
| constellatus | Streptococcus | ||||
| dysgalactiae | |||||
| Streptococcus | |||||
| mutans | Streptococcus | ||||
| pneumoniae | Streptococcus | ||||
| pyogenes | Streptococcus | ||||
| salivarius | Streptococcus | ||||
| oralis | |||||
| Gram Negative Bacteria | |||||
| Acinetobacter | |||||
| calcoaceticus | Acinetobacter | ||||
| Iwoffi | Acinetobacter | ||||
| nosocomialis | Acinetobacter pittii | Acinetobacter | |||
| radioresistans | Aeromonas | ||||
| hydrophila | |||||
| Bacteroides fragilis | Burkholderia | ||||
| cepacia | Chryseobacterium | ||||
| indologenes | Citrobacter koseri | Cupriavidus pauculus | |||
| Enterobacter | |||||
| aerogenes | Enterobacter | ||||
| cloacae | Enterobacter | ||||
| hormaechei | Fusobacterium | ||||
| necrophorum | Fusobacterium | ||||
| nucleatum | Klebsiella | ||||
| oxytoca | |||||
| Leptotrichia | |||||
| trevisanii | Moraxella | ||||
| catarrhalis | Morganella morganii | Myroides odoratus | Ochrobactrum | ||
| anthropi | Oligella | ||||
| urethralis | |||||
| Pantoea | |||||
| agglomerans | Plesiomonas | ||||
| shigelloides | Proteus mirabilis | Proteus vulgaris | Providencia | ||
| stuartii | Pseudomonas | ||||
| alcaligenes | |||||
| Pseudomonas | |||||
| fluorescens | Pseudomonas | ||||
| luteola | Pseudomonas | ||||
| oryzihabitans | Pseudomonas pseudoalcaligenes | ||||
| (oleovorans) | Pseudomonas | ||||
| putida | |||||
| Pseudomonas | |||||
| stutzeri | Ralstonia pickettii | Raoultella | |||
| ornithinolytica | Raoultella | ||||
| planticola | Salmonella enterica Enteritidis | ||||
| Salmonella enterica | |||||
| Typhimurium | Serratia | ||||
| marcescens | Shewanella | ||||
| putrefaciens | Stenotrophomonas | ||||
| maltophilia | Weeksella virosa | ||||
| Yersinia pseudotuberculosis |
12
| Table 4b: T2Bacteria Panel Exclusivity Results | |
|---|---|
| Organisms That React at ≤ 10 units/mL (CFU/mL) | |
| Cross Reactive Species | Cross Reacts with T2Bacteria Channel |
| Enterococcus durans | E. faecium |
| Escherichia albertii | E. coli |
| Escherichia fergusonii | E. coli |
| Klebsiella variicola | K. pneumoniae |
| Shigella boydii | E. coli |
| Shigella dysenteriae | E. coli |
| Shigella flexneri | E. coli |
| Shigella sonnei | E. coli |
Based on in silico analysis, Yersinia pestis is not expected to cross-react with any T2Bacteria channel. In silico analysis also showed that the new species Klebsiella quasipneumoniae, which was recently reclassified from K. pneumoniae phylogroup Kpll to Klebsiella quasipneumoniae, is expected to cross-react with the K. pneumoniae T2Bacteria channel. Similarly, the new species Staphylococcus argenteus, which was recently reclassified from S. aureus clonal complex 75 to Staphylococcus argenteus, is expected to cross-react with S. aureus T2Bacteria channel. These organisms have not been tested.
For one species, Enterovirus Type 68, 0 of 3 replicates tested at 1,000 TCIDso/mL were positive for E. coli; 1 of 6 replicates tested at 316 TCIDso/mL were positive on the E. coli channel of the Panel. This species was deemed not cross reactive.
Competitive Inhibition V.
T2 Biosystems conducted a Competitive Inhibition Study on the T2Bacteria Panel to evaluate assay performance in the presence of two or more Panel bacterial target species at high and low concentrations as well as selected bacterial and fungal non-Panel target organisms. The conditions tested included: co-infection with two Panel target species both at or near the LoD; co-infection with two Panel target species where one species is at high titer (1,000 CFU/mL) and the other is at or near the LoD; and co-infection with one Panel species at or near the LoD and a non-Panel species at high titer (1,000 CFU/mL). Four replicates were tested and if any false negative results were generated, the test was repeated with 20 replicates. If a ≤95% hit rate in the 20 replicates was generated, the concentration of the competing organism was titrated to determine the level at which the reaction is not inhibited.
Results from combinations of Panel members at or near the LoD tested when co-infected with a competing Panel member at either 1,000 CFU/mL or also at or near the LoD are shown in Table 5a. False negative results were repeated with 20 replicates and all had 100% hit rates with the exception of the co-infection of P. aeruginosa (at or near the LoD) and E. coli (1,000 CFU/mL). For this combination, detection of P. aeruginosa had a hit rate of
13
18/20. When E. coli concentrations were reduced to 100, 33, and 10 CFU/mL, P. aeruginosa at or near the LoD was detected at a rate of 100%.
Results from combinations of Panel members at or near the LoD tested when coinfected with other clinically relevant organisms at 1,000 CFU/mL are shown in Table 5b. False negative results were repeated with 20 replicates and all had 100% hit rate.
14
| Table 5a: T2Bacteria Panel Competitive Inhibition Results (Positivity)
| Co-infection with Two T2Bacteria Panel Members | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Efm | ||||||||||
| (5-10 CFU/mL) | Eci | |||||||||
| (11-22 CFU/mL) | Kp | |||||||||
| (2-4 CFU/mL) | Pa | |||||||||
| (5-10 CFU/mL) | Sa | |||||||||
| (2-4 CFU/mL) | ||||||||||
| Competing | Efm | Comp. | ||||||||
| Org. | Eci | Comp. | ||||||||
| Org. | Kp | Comp. | ||||||||
| Org. | Pa | Comp. | ||||||||
| Org. | Sa | Comp. | ||||||||
| Org. | ||||||||||
| Efm (5-10 CFU/mL) | N/A | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | |
| Efm (1,000 CFU/mL) | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | ||
| Eci (11-22 CFU/mL) | 4/4 | 4/4 | N/A | 4/4 | 4/4 | 2/4* | 4/4 | 4/4 | 4/4 | |
| Eci (1,000 CFU/mL) | 4/4 | 4/4 | N/A | 4/4 | 4/4 | 2/4* | 4/4 | 4/4 | 4/4 | |
| Kp (2-4 CFU/mL) | 4/4 | 4/4 | 4/4 | 4/4 | N/A | 4/4 | 4/4 | 4/4 | 4/4 | |
| Kp (1,000 CFU/mL) | 4/4 | 4/4 | 4/4 | 4/4 | N/A | 4/4 | 4/4 | 4/4 | 4/4 | |
| Pa (5-10 CFU/mL) | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | N/A | 4/4 | 4/4 | |
| Pa (1,000 CFU/mL) | 4/4 | 4/4 | 3/4* | 4/4 | 4/4 | 4/4 | N/A | 4/4 | 4/4 | |
| Sa (2-4 CFU/mL) | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 3/4* | N/A |
| Sa (1,000 CFU/mL) | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 |
*Required repeat testing at N=20; all yielded 20/20 hit rates except Pa (5-10 CFU/mL) with Eci (1,000 CFU/mL),
15
| Table 5b: T2Bacteria Panel Competitive Inhibition Results | ||||||
|---|---|---|---|---|---|---|
| Co-infection with Organisms Other than T2Bacteria Panel Members | ||||||
| Competing | ||||||
| Organism | Test | |||||
| Concentration | ||||||
| (CFU/mL) | T2Bacteria Panel Members (Hit Rate) | |||||
| Efm | ||||||
| (5-10 | ||||||
| CFU/mL) | Eci | |||||
| (11-22 | ||||||
| CFU/mL) | Kp | |||||
| (2-4 | ||||||
| CFU/mL) | Pa | |||||
| (5-10 | ||||||
| CFU/mL) | Sa | |||||
| (2-4 | ||||||
| CFU/mL) | ||||||
| S. epidermidis | 1000 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 |
| S. haemolyticus | 1000 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 |
| S. pneumoniae | 1000 | 4/4 | 4/4 | 4/4 | 3/4* | 4/4 |
| E. cloacae | 1000 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 |
| S. salivarius | 1000 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 |
| C. albicans | 1000 | 4/4 | 4/4 | 4/4 | 4/4 | 4/4 |
- Required repeat testing and yielded 20/20 hit rate.
vi. Interfering Substances
Studies were conducted to evaluate the impact of potential endogenous and exogenous interfering substances on the performance of the T2Bacteria Panel. These substances were added to negative whole blood samples or to whole blood samples multi-spiked with either E. coli, P. aeruginosa, and E. faecium, or K. pneumoniae and S. aureus at 2-3x LoD. Three replicate samples were run for each interfering substance tested.
Tables 6a and 6b summarize the endogenous and exogenous substances and the maximum concentrations at which they were tested. All of the substances were tested in excess of standard reference or physiological levels and did not interfere with the performance of the assay with the exception of Ferumoxytol (Feraheme). Initially, Ferumoxytol was tested at 618 µg/mL, which is three fold higher than its tmax of 206 µg/mL, but was found to be inhibitory to the performance of the T2Bacteria Panel. Dilutions of Ferumoxytol were performed and it was determined that concentrations ≥ 21 µg/mL interfere with the performance of the T2Bacteria Panel.
| Table 6a: Endogenous Substances Tested, No Interference Observed | |
|---|---|
| Substance | Concentration |
| Albumin | 60 g/L |
| ALT | 120 U/liter |
| AST | 144 U/liter |
| Bilirubin (conjugated) | 342 $ μ $ mol/L |
| Bilirubin (unconjugated) | 342 $ μ $ mol/L |
| Creatinine | 50 mg/L |
| Gamma Globulin | 60 g/L |
| Hemoglobin | 22.8 – 23.9 g/dL* |
| Human DNA | 2.2 $ μ $ g/mL |
| Lactoferrin | 7.5 $ μ $ mol/L |
| Urea | 42.9 mmol/L |
| White Blood Cells (buffy coat) | 2.08 x 10 $7$ -2.48 x 10 $7$ WBC/mL* |
| Lipemia (intralipid to mimic triglycerides) | 3270 mg/dL |
*For hemoglobin and white blood cells, the concentration tested varied between sample types.
16
| Table 6b: Exogenous Substances Tested, No Interference Observed | |||
|---|---|---|---|
| Substance | Concentration | Substance | Concentration |
| Azithromycin (Zithromax) | 15.3 µmol/L | Fluconazole | 245 µmol/L |
| Amphotericin B Trihydrate | 240 µg/mL | Gentamicin sulfate | 21 µmol/L |
| Ampicillin | 152 µmol/L | Heparin | 3,000 U/L |
| Caspofungin | 52.8 µg/mL | Isovue 370 | 180 µL per 4 mL |
| vacutainer | |||
| Cefazolin Sodium Salt | 2.643 mmol/L | Linezolid | 55.8 µg/mL |
| Cefepime Hydrochloride | 492 µg/mL | Lisinopril | 0.74 µmol/L |
| Cefoxitin Sodium Salt | 180 µg/mL | Magnevist (gadopentetate dimeglumine) | 1.5 mM |
| Ceftazidime Pentahydrate | 487 µg/mL | Meropenem trihydrate | 186 µg/mL |
| Ciprofloxacin | 30.2 µmol/L | Metronidazole | 701 µmol/L |
| Clindamycin HCI | 89.1 µmol/L | Micafungin | 90 mg/L |
| Cytarabine | 32.4 µg/mL | Piperacillin/Pipril | 117 µg/mL |
| Dexamethasone | 1.53 µg/mL | Primaxin, 50:50 ratio of Imipenem: Cilastatin | 528 µg/mL |
| EDTA | 5.4 mg/mL | Tazobactam (Tazobac) | 18.9 µg/mL |
| Vancomycin | 103 µg/mL |
vii. Summary of False Positive Results
The false positive rates observed during analytical testing for each channel of the T2Bacteria Panel are shown in Table 7. Overall, false positive rates observed during verification testing were similar across all studies.
| Table 7: Summary of False Positive Results in Analytical Studies | ||||||
|---|---|---|---|---|---|---|
| Study | Efm | Eci | Kp | Pa | Sa | |
| LoD | 0.0% | 2.0% | 0.1% | 0.2% | 0.0% | |
| 5x LoD | 0.0% | 0.0% | 0.0% | 0.0% | 0.0% | |
| Single vs. Multi | 0.0% | 1.6% | 0.0% | 0.0% | 0.0% | |
| Interfering Substances | 0.2% | 2.8% | 0.9% | 4.3% | 0.4% | |
| Analytical Reactivity | 0.0% | 3.0% | 0.0% | 0.4% | 0.0% | |
| Competitive Inhibition | 0.4% | 3.2% | 0.0% | 1.7% | 0.2% | |
| Analytical Specificity | 0.0% | 2.9% | 0.0% | 0.3% | 0.0% | |
| Reproducibility | 0.0% | 3.7% | 0.0% | 0.6% | 0.0% | |
| Total | 0.1% | 2.6% | 0.2% | 1.5% | 0.1% |
viii. Summary of Invalid and Indeterminate Results
The invalid and indeterminate rates observed for all samples tested in the verification studies were recorded (Table 8). Excluding interfering substances, invalid rates remained low across all of the studies at an average rate of 0.3%. Interfering substances had an exceptionally high invalid rate of 4.9% due to the nature of the study being the evaluation of substances that interfere with the T2Bacteria Panel. The indeterminate rate for the E. coli channel ranged from 0% to 1.3% across the verification studies, and in total was 0.9% in verification considering all studies or 0.8% after excluding the interfering subtances study.
17
| Table 8: Summary of Invalid and Indeterminate Rate Observed in
| Analytical Studies | ||
|---|---|---|
| Study | Invalid Rate | Indeterminate rate |
| ( E. coli channel only) | ||
| LoD | 0.5% | 1.3% |
| LoD subset - E. coli spikes at 11 CFU/mL | 0.0% | 0.8% |
| 5x LoD | 0.0% | 0.0% |
| Single vs multi | 0.0% | 0.0% |
| Interfering Substances | 4.9% | 1.1% |
| Analytical Reactivity | 0.0% | 0.0% |
| Analytical Specificity | 0.2% | 0.7% |
| Reproducibility | 0.0% | 0.0% |
| Total | 1.7% | 0.9% |
| Total without Interfering Substances | 0.3% | 0.8% |
Clinical Performance Evaluation
The performance of the T2Bacteria Panel was evaluated at eleven sites within the US and compared to the reference method of blood culture. Patients were enrolled prospectively and two paired sample collections, one for blood culture and one for testing by the T2Bacteria Panel were drawn from each subject. The blood culture systems used in the study were BD Bactec™ FX, bioMerieux BacT/ALERT™, and Thermo Fisher VersaTREK®. Species identification was performed on all positive bacteria cultures and methods included Gram stain, bioMerieux Vitek® 2, bioMerieux or Bruker MALDI TOF, and PCR. The T2Bacteria Panel result was compared against results from these blood culture systems for Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). A total of 1,427 subjects was tested prospectively.
Due to the low prevalence of the organisms contained in the Panel, an additional 250 contrived specimens were evaluated at three sites. Contrived specimens were prepared by spiking bacteria at defined concentrations (CFU/mL) into healthy donor whole blood. A total of 50 strains of each of the five bacterial species (250 strains total) were used to prepare the contrived specimens. Further, an additional 100 blood samples not spiked with T2Bacteria Panel members were also evaluated as part of the contrived arm of the study.
Table 9 summarizes the overall PPA (sensitivity) and NPA (specificity) from the prospective and contrived arms of the study. The PPA (sensitivity) ranged from 81.3% to 100% and the NPA (specificity) ranged from 95.5% to 100%. Details of the individual arms of the study are summarized in the respective sections below.
18
Table 9. Sensitivity (PPA) and and Specificity (NPA) from Combined Prospective and Contrived Arms of the T2Bacteria Clinical Study
| the T2Bacteria Clinical Study | |||||
|---|---|---|---|---|---|
| Clinical Study | |||||
| Arm | PPA1 | NPA2 | |||
| Target Species | PPA | 95% CI | NPA | 95% CI | |
| E. faecium | Contrived | 100% (40/40) | 91.2 - 100% | 100% (300/300) | 98.7 - 100% |
| Prospective | 100% (1/1) | 20.7 - 100% | 99.4% (1417/1426) | 98.8 - 99.7% | |
| S. aureus | Contrived | 92.3% (36/39) | 79.7 - 97.3% | 100% (300/300) | 98.7 - 100% |
| Prospective | 81.3% (13/16)3 | 57.0 - 93.4% | 98.0% (1383/1411) | 97.1 - 98.6% | |
| K. pneumoniae | Contrived | 100% (40/40) | 91.2 - 100% | 99.3% (298/300) | 97.6 - 99.8% |
| Prospective | 100% (6/6) | 61.0 - 100% | 98.5% (1399/1421) | 97.7 - 99.0% | |
| P. aeruginosa | Contrived | 97.4% (38/39) | 86.8 - 99.5% | 97.7% (293/300) | 95.3 - 98.9% |
| Prospective | 100% (5/5) | 56.6 - 100% | 97.7% (1389/1422) | 96.8 - 98.3% | |
| E. coli | Contrived | 90.9% (20/22) | 72.2 - 97.5% | 97.3% (292/300) | 94.8 - 98.6% |
| Prospective | 90.9% (10/11)4 | 62.3 - 98.4% | 95.0% (1345/1416)5 | 93.7 - 96.0% |
1 Positive Percent Agreement (PPA) or Sensitivity was calculated against samples with titer levels at or above limit of detection (LoD) in Contrived Arm and blood culture positives in Prospective Arm.
²Negative Percent Agreement (NPA) or Specificity was calculated from all samples (including below LoD and unspiked negative samples) as the total number of negative channels divided by total number of non-spiked channels in Contrived Arm and blood culture negatives in Prospective Arm.
3 One subject was blood culture positive for S. aureus but negative in the first blood sample and S. aureus positive in the second concurrently collected blood sample tested by T2Bacteria Panel. Data shown reflects first result, not second result.
4One subject was blood culture positive for E. coli but negative in the first blood sample and E. coli positive in the second concurrently collected blood sample tested by T2Bacteria Panel. Data shown reflects first result, not second result.
5Of the total 1416 blood culture negative subjects, 8 (0.6%) yielded indeterminate results for the E. coli channel.
Prospective Arm: T2Bacteria Panel Performance vs. Blood Culture
In the prospective arm of the study, the overall positivity rate for bacterial blood culture was 6.2%, with 2.7% specific for target organisms contained in the T2Bacteria Panel, whereas the overall positivity rate for the T2Bacteria Panel was 13.3%. The PPA (sensitivity) of the T2Bacteria Panel against blood culture ranged from 81.3% to 100% depending upon bacterial species. The NPA (specificity) of the T2Bacteria Panel against blood culture ranged from 95.5% to 99.4%, where all channels showed an NPA of > 97.5% with exception of E. coli at 95.5% (Figure 1; Table 10).
19
| Eci | ||||
|---|---|---|---|---|
| Blood culture | ||||
| + | - | |||
| T2 | + | 10 | 63 | 73 |
| Ind | 0 | 8 | 8 | |
| - | 1 | 1345 | 1346 | |
| 11 | 1416 | 1427 | ||
| Value | - 95% CI + 95% CI | |||
| PPA | 90.9% | 62.3% | 98.4% | |
| NPA | 95.0% | 93.7% | 96.0% |
| Figure 1: T2Bacteria Panel Performance as Compared to Blood Culture | |||
|---|---|---|---|
| Efm | |||
|---|---|---|---|
| Blood culture | |||
| + | - | ||
| T2 | + | 1 | 9 |
| - | 0 | 1417 | |
| 1 | 1426 | ||
| Value | - 95% CI | + 95% CI | |
| PPA | 100.0% | 20.7% | 100.0% |
| NPA | 99.4% | 98.8% | 99.7% |
| Kp | ||||
|---|---|---|---|---|
| Blood culture | ||||
| + | - | |||
| T2 | + | 6 | 22 | 28 |
| - | 0 | 1399 | 1399 | |
| 6 | 1421 | 1427 | ||
| Value | - 95% CI + 95% CI | |||
| PPA | 100.0% | 61.0% | 100.0% | |
| NPA | 98.5% | 97.7% | 99.0% |
| Pa | ||||
|---|---|---|---|---|
| Blood culture | ||||
| + | - | |||
| T2 | + | 5 | 33 | 38 |
| - | 0 | 1389 | 1389 | |
| 5 | 1422 | 1427 | ||
| Value | - 95% CI | + 95% CI | ||
| PPA | 100.0% | 56.6% | 100.0% | |
| NPA | 97.7% | 96.8% | 98.3% |
| Sa | ||||
|---|---|---|---|---|
| Blood culture | ||||
| + | - | |||
| T2 | + | 13 | 28 | 41 |
| - | 3 | 1383 | 1386 | |
| 16 | 1411 | 1427 | ||
| Value | - 95% CI | + 95% CI | ||
| PPA | 81.3% | 57.0% | 93.4% | |
| NPA | 98.0% | 97.1% | 98.6% |
| Table 10: T2Bacteria Panel Performance as Compared to Blood Culture | ||||
|---|---|---|---|---|
| Species | PPA (Sensitivity) | NPA (Specificity) | ||
| PPA | 95% CI | NPA | 95% CI | |
| E. coli | 90.9% (10/11) | 62.3 - 98.4% | 95.0% (1345/1416)1 | 93.7 - 96.0% |
| E. faecium | 100% (1/1) | 20.7 - 100% | 99.4% (1417/1426) | 98.8 - 99.7% |
| K. pneumoniae | 100% (6/6) | 61.0 - 100% | 98.5% (1399/1421) | 97.7 - 99.0% |
| P. aeruginosa | 100% (5/5) | 56.6 - 100% | 97.7% (1389/1422) | 96.8 - 98.3% |
| S. aureus | 81.3% (13/16) | 57.0 - 93.4% | 98.0% (1383/1411) | 97.1 - 98.6% |
¹Of the total 1416 blood culture negative subjects, 8 (0.6%) yielded indeterminate results for the E. coli channel.
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Both freshly collected and frozen blood samples were tested with the T2Bacteria Panel in the Prospective arm. Of the 1,427 patient specimens tested, 672 were fresh and 755 were frozen. An analysis was performed and demonstrated that the fresh and frozen samples did not show a statistically significant difference in PPA or NPA (p > 0.12; Table 11).
| Table 11: T2Bacteria Panel Performance on Fresh v Frozen Blood | |||||
|---|---|---|---|---|---|
| PPA (Sensitivity) | NPA (Specificity) | ||||
| Species | Sample | ||||
| Type | Value | TP / | |||
| (TP + FN) | Value | TN / | |||
| (TN + FP) | |||||
| Eci | Fresh | 100.0% | 4 / 4 | 94.6% | 630 / 666 |
| Frozen | 85.7% | 6 / 7 | 96.4% | 715 / 742 | |
| Efm | Fresh | 100.0% | 1 / 1 | 99.3% | 666 / 671 |
| Frozen | --- | 0 / 0 | 99.5% | 751 / 755 | |
| Kp | Fresh | 100.0% | 4 / 4 | 98.2% | 656 / 668 |
| Frozen | 100.0% | 2 / 2 | 98.7% | 743 / 753 | |
| Pa | Fresh | 100.0% | 2 / 2 | 97.9% | 656 / 670 |
| Frozen | 100.0% | 3 / 3 | 97.5% | 733 / 752 | |
| Sa | Fresh | 80.0% | 8 / 10 | 97.7% | 647 / 662 |
| Frozen | 83.3% | 5 / 6 | 98.3% | 736 / 749 |
Contrived Arm
Results for the Contrived arm were further analyzed based on the spiked pathogen concentration (either above or below the LoD). The positivity rates for contrived clinical testing were consistent with the results determined for the analytical LoD levels, as shown in Table 12.
| Table 12: PPA for Contrived samples above and below the Limit of Detection | |||||
|---|---|---|---|---|---|
| Contrived | |||||
| Sample Type | LoD | ||||
| (CFU/mL) | E. coli | 11 | 67.8% (19/28)1 | 49.3 – 82.1% | 90.9% (20/22)2 |
| E. faecium | 5 | 60.0% (6/10) | 31.3 - 83.2% | 100% (40/40) | 91.2 - 100% |
| K. pneumoniae | 2 | 50.0% (5/10) | 23.7 - 76.3% | 100% (40/40) | 91.2 - 100% |
| P. aeruginosa | 5 | 63.6% (7/11) | 35.4 - 84.8% | 97.4% (38/39) | 86.8 - 99.5% |
| S. aureus | 2 | 18.2% (2/11) | 5.1 - 47.7% | 92.3% (36/39) | 79.7 - 97.3% |
1 Of the total 28 samples spiked at 1 CFU/mL, the positivity rate was 36/40 yielding a PPA of 90.0% (77.0-96.0% 95% CI).
21
Evaluation of False Positive Results
Overall, in the prospective study, there were 190 T2 positive results consisting of 35 T2+/BC+ concordant results and 155 T2+/BC- potential false positive results. As shown in Table 13, of the 155 potential false positive results, 39 represented patients with an additional positive blood culture at a different blood draw within ±14 days of the T2 draw and 30 results for which an additional blood specimen (drawn at the same time as the original positive T2 specimen) was positive by an amplification and gene sequencing method. An additional 23 results were obtained from patients who had other non-blood specimens positive for the same target organism (collected ± 14 days from the positive T2 specimen). Based on this analysis, 63 of the 190 T2 positive results (33%) or 63 of the 155 potential false positive results (41%) were not associated with evidence of infection.
| Table 13: Summary of T2(+) / BC(-) Discordant Analysis in Prospective Arm | ||||||
|---|---|---|---|---|---|---|
| Species | T2(+) / | |||||
| BC(-) | ||||||
| total | Other | |||||
| Blood | ||||||
| Culture | ||||||
| positive1 | Sequencing | |||||
| positive2 | T2(+) / BC(-) | |||||
| associated with | ||||||
| strong | ||||||
| evidence of | ||||||
| infection3 | T2(+) / BC(-) | |||||
| associated with | ||||||
| other evidence of | ||||||
| infection Non- | ||||||
| Blood Matrices | ||||||
| Culture Positive4 | T2(+) / BC(-) | |||||
| associated with | ||||||
| no evidence of | ||||||
| infection | ||||||
| E. faecium | 9 | 2 | 2 | 44.4% (4/9) | 33.3% (3/9) | 22.2% (2/9) |
| E. coli | 63 | 12 | 9 | 33.3% (21/63) | 12.7% (8/63) | 54.0% (34/63) |
| K. pneumoniae | 22 | 6 | 8 | 63.6% (14/22) | 13.6% (3/22) | 22.7% (5/22) |
| P. aeruginosa | 33 | 3 | 8 | 33.3% (11/33) | 12.1% (4/33) | 54.5% (18/33) |
| S. aureus | 28 | 16 | 3 | 67.9% (19/28) | 17.9% (5/28) | 14.3% (4/28) |
| Total | 155 | 39 | 30 | 44.5% (69/155) | 14.8% (23/155) | 40.7% (63/155) |
1 Blood cultures positive for the T2 species identified other than the paired blood culture and processed within ± 14 days of collection of the T2 sample.
2 Sequencing from blood samples drawn at the same time as collection of the T2 sample and positive for the T2 species identified, where this sequencing assay was only run on subjects without positive evidence from other sample sources (footnote 1 and 2).
3 Strong evidence defined as a T2 positive result associated with a blood culture positive from a different draw than T2 draw or a sequencing positive result from a blood sample drawn concurrently with the T2 draw.
4 Other cultures from non-blood sample matrices positive for the T2 species identified within ± 14 days of collection of the T2 sample.
Further investigation identified reagents contamination as the likely source for the false positive results from patients with no evidence of infection. T2 Biosystems implemented improved reagent testing methods and release criteria with the overall goal of reducing the rate of false positive results that can be attributed to reagents contamination. As shown in Table 14, after these improvements reagents contamination was observed at levels of ≤1% for all targets.
22
| Table 14: Positivity of T2Bacteria Panel in QCheck NEG samples before and after | |||||
|---|---|---|---|---|---|
| improvements | |||||
| Species | Efm | Eci | Kp | Sa | Pa |
| Before | |||||
| Improvements | 0.00% | ||||
| (0/875) | 1.94% | ||||
| (17/875) | 0.23% | ||||
| (2/875) | 0.00% | ||||
| (0/875) | 0.91% | ||||
| (8/875) | |||||
| After | |||||
| Improvements | 0.00% | ||||
| (0/286) | 1.05% | ||||
| (3/286) | 0.00% | ||||
| (0/286) | 0.00% | ||||
| (0/286) | 0.35% | ||||
| (1/286) |
Archived samples were tested for 43 of the 52 discordant T2(+) / BC(-) samples that were originally positive for E. coli (30 samples tested) or P. aeruginosa (13 samples tested) but not associated with any other evidence of infection. Upon retesting after improvements, none of the archived samples were positive for E. coli, suggesting that the original 30 tested E. coli positive samples were most likely false positive results. Two of the archived samples were positive for P. aeruginosa from patients who were originally T2 positive for P. aeruginosa, suggesting that these 2 samples were most likely true positive results and the remaining 11 tested samples were most likely false positive results for P. aeruginosa.
After improvements, additional testing was completed on 286 QCheck negative controls and 120 K2EDTA blood samples obtained from healthy donors. False positives were observed only for E. coli (1.7%) and P. aeruginosa (1.7%) in the blood samples and only for E. coli (1.0%) and P. aeruginosa (0.3%) in the QCheck negative controls.
Prospective and Contrived arm, Invalid and Indeterminate rate
The Prospective arm showed an overall invalid rate of 0.5% and an indeterminate rate of 0.6%, where the indeterminate result applies to the E. coli channel only. Similarly, the Contrived arm showed a 0.0% invalid rate and 0.6% indeterminate rate. In aggregate, the combined Prospective and Contrived arms generated a total invalid rate of 0.4% and total indeterminate rate of 0.6%.