Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

Yes
The description mentions that the quantified transcript values are input to "two fixed classifiers" which output the scores. While not explicitly stating AI/ML, the use of "classifiers" to process biological data and output scores based on learned patterns from training data (described in the "Description of the training set" section) strongly suggests the use of a machine learning model. The tuning of thresholds based on training data further supports this.

Therapeutic

No.
The device is an in vitro diagnostic test that aids in differentiating infections and determining the likelihood of severe illness, rather than directly treating or mitigating a disease.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states, "The TriVerity test is an automated ve in vitro diagnostic test..." and describes its use "as an aid to differentiate bacterial infections, viral infections, and non-infectious illness, as well as to determine the likelihood of 7day need for mechanical ventilation, vasopressors, and/or renal replacement with suspected acute infection or suspected sepsis presenting to the emergency department." This clearly defines its role in diagnosis and prognosis, which are functions of a diagnostic device.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the TriVerity test is an in vitro diagnostic test that uses a physical cartridge ("TriVerity cartridge, a single-use, disposable, multi-chambered fluidic cartridge") and runs on a physical instrument ("Myrna instrument"). While software is involved in processing the data from the instrument, the core of the device includes physical components for sample processing and detection.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Explicitly Stated: The "Intended Use / Indications for Use" section begins with "The TriVerity test is an automated ve in vitro diagnostic test...".
Device Description: The "Device Description" section also states, "The TriVerity test is an in vitro diagnostic test...".
Mechanism of Action: The test measures the relative expression levels of host response genes in RNA isolated from whole blood. This is a biological sample analyzed outside of the body to provide information about a patient's health status.
Intended Use: The test is intended to be used "in conjunction with clinical assessments and other laboratory findings as an aid to differentiate bacterial infections, viral infections, and non-infectious illness, as well as to determine the likelihood of 7day need for mechanical ventilation, vasopressors, and/or renal replacement with suspected acute infection or suspected sepsis". This is a diagnostic purpose.
Sample Type: The test uses whole blood, which is a biological specimen.
Predicate Device: The submission lists a predicate device (K203748; SeptiCyte RAPID) which is also an IVD.

All of these points strongly indicate that the TriVerity test is an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

No
The provided text does not contain any explicit statement that the FDA has reviewed, approved, or cleared a Predetermined Change Control Plan (PCCP) for this specific device. The 'Control Plan Authorized (PCCP) and relevant text' section explicitly states 'Not Found'.

Overview

Intended Use / Indications for Use

The TriVerity test is an automated, semi-quantitative in vitro diagnostic test that measures the relative expression levels of host response genes in RNA isolated from whole blood collected in the PAXgene Blood RNA tube using reverse transcription loop-mediated isothermal amplification (RT-LAMP) on the Myrna instrument.

The TriVerity test is indicated for use in conjunction with clinical assessments and other laboratory findings as an aid to differentiate bacterial infections, viral infections, and non-infectious illness, as well as to determine the likelihood of 7day need for mechanical ventilation, vasopressors, and/or renal replacement therapy in adult patients with suspected acute infection or suspected sepsis presenting to the emergency department.

The test generates three scores that each fall within one of five discrete interpretation bands based on the increasing likelihood of

1. bacterial infection,
1. viral infection, and

severe illness, as defined by the need for mechanical ventilation, vasopressors, and/or renal replacement therapy (RRT) within seven days.

Product codes

PRE, OOI

Device Description

The TriVerity test is an in vitro diagnostic test for simultaneous amplification and detection of 29 informative host response RNA transcripts and 3 housekeeping transcripts (listed in measurands) using total RNA extracted from human blood. The test has been designed, manufactured, and validated for use on the Myrna instrument. The TriVerity test is performed with a TriVerity cartridge, a single-use, disposable, multi-chambered fluidic cartridge that runs on the Myrna instrument. All processing steps are automated and occur within a TriVerity cartridge, including sample extraction/purification and qRT-LAMP for the detection and relative quantification of the 29 informative host response RNA transcripts and 3 housekeeping transcripts (listed in measurands). All cartridge steps in this process, following the addition of the sample, are fully automated and completely integrated. In approximately 30 minutes, the test generates three scores that each fall within one of five discrete interpretation bands based on the increasing likelihood of

1. bacterial infection,
1. viral infection, and
1. severe illness, as defined by the need for mechanical ventilation, vasopressors, and/or renal replacement therapy (RRT) within seven days.

The specimen used for the TriVerity test is a sample of whole blood collected by venipuncture using the PAXgene blood collection tubes within the PAXgene Blood RNA System (K042613). The cartridge contains all the necessary reagents to perform RNA isolation and subsequent amplification from the sample.

The TriVerity test quantifies the amount of each transcript in the sample based on the detection of fluorescence by the Myrna instrument. The cartridge includes the reagents for reverse transcription and LAMP. All 32 transcripts (and two in cartridge controls) are amplified and quantified. These values are input to two fixed classifiers which output the three separate scores, each with five discrete interpretation bands. The bands reflect monotonically increasing likelihood of bacterial infection, viral infection, and severe illness, as defined by the need for mechanical ventilation, vasopressors, and/or renal replacement therapy (RRT) within seven days.

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

adult patients

Intended User / Care Setting

emergency department

Description of the training set, sample size, data source, and annotation protocol

Illness Severity Score thresholds were set (tuned) using predicted probabilities and ground truths for 399 patient samples collected from Emergency Department settings. Bacterial and Viral Score thresholds were tuned using predicted probabilities and ground truths for 472 patient samples collected from Emergency Department and ICU studies. All tuning studies were independent from the final clinical study (INF-04), were collected in PAXgene blood tubes, and were processed on the Myrna instrument using GMP-grade TriVerity cartridges.

Description of the test set, sample size, data source, and annotation protocol

A prospective, multi-center clinical study (SEPSIS-SHIELD; NCT04094818) was conducted to establish the performance characteristics of the TriVerity test on the Myrna instrument. This was a non-interventional, minimal-risk study to analyze gene expression and other laboratory data from biological samples collected from participants with suspected respiratory, urinary, intra-abdominal, skin and soft tissue, meningitis/encephalitis, and at least one vital sign change, and other acute infections, or suspected sepsis of any cause with at least two vital sign changes and a blood culture order.

Study sites included emergency departments (EDs) of community and academic hospitals located in twenty-one geographically diverse locations throughout the United States plus one site in Europe. Enrollment was offered to all patients who met the enrollment criteria at presentation to the ED. Of the 1,441 participants who consented to the study, there were 1,222 evaluable participants.

The primary objective of the study was to evaluate the performance of the TriVerity test for (1) diagnosing bacterial and viral infections using consensus and forced clinical adjudication as the comparator and (2) evaluate the performance of the TriVerity test for predicting severe illness (defined as the need for mechanical ventilation, vasopressors, or renal replacement therapy [RRT] within 7 days) using clinical outcomes as the comparator.

The analysis population consisted of all participants who provided consent, met all inclusion criteria, met no exclusion criteria, and had their PAXgene blood sample collected and successfully processed (resulting in three TriVerity scores). The overall analysis population was further divided into diagnostic and prognostic analysis populations with additional requirements. The diagnostic endpoint analysis population included participants who had a completed adjudication for bacterial and viral infection to determine the ground truth. The diagnostic population was further classified as Consensus (only contains participants with a certain (Yes or No) or Forced adjudication (all patients adjudicated as Yes and/or Probable vs. No and/or Unlikely). The prognostic endpoint analysis population included participants who had information available on the presence of severe illness (acute need for mechanical ventilation, vasopressor and/or renal replacement therapy) within 7 days.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Clinical Studies:
Study Type: Prospective, multi-center clinical study (SEPSIS-SHIELD; NCT04094818).
Sample Size:

1,441 participants consented.
1,222 evaluable participants for the diagnostic endpoint.
1,120 evaluable participants for the prognostic endpoint.
Outcomes:
Bacterial Infection (Forced Adjudication):
- AUROC = 0.76 (0.75-0.78) for N=1222.
- Probability of Bacterial Infection: 89.0% for Very High band, 20.0% for Very Low band.
Viral Infection (Forced Adjudication):
- AUROC = 0.83 (0.81-0.85) for N=1222.
- Probability of Viral Infection: 85.3% for Very High band, 6.8% for Very Low band.
Bacterial Infection (Consensus Adjudication):
- AUROC = 0.83 (0.81-0.85) for N=729.
- Probability of Bacterial Infection: 93.2% for Very High band, 12.1% for Very Low band.
Viral Infection (Consensus Adjudication):
- AUROC = 0.91 (0.89-0.93) for N=729.
- Probability of Viral Infection: 92.9% for Very High band, 2.9% for Very Low band.
Severe Illness (Prognostic Endpoint):
- AUROC = 0.78 (0.75-0.81) for N=1120.
- Probability of Severe Illness: 58.1% for Very High band, 2.7% for Very Low band.

Key Results:

TriVerity demonstrated high AUROCs for bacterial (0.83) and viral (0.91) results using consensus adjudication, and high AUROCs for illness severity (0.78).
Likelihood ratios showed monotonic increase across interpretation bands for bacterial, viral, and severe illness scores, with non-overlapping 80% CIs for non-adjacent bands.
TriVerity bacterial results showed markedly superior AUROCs compared to PCT across different races, indicating better performance, especially in minorities, compared to Procalcitonin.
The diagnostic results for TriVerity were robust across different immune statuses (immunosuppressed vs. immunocompetent).
Sequential use of qSOFA and TriVerity significantly improved risk stratification for severe illness compared to qSOFA alone.

Analytical Performance Studies:

Reproducibility (Multi-site, Lot-to-lot, Intra-site, Repeatability): Found to be within acceptance criteria for all scores and bands.
Real-time Reagent Stability: Studies ongoing; PAXgene sample stability confirmed for immediate use, 24 hours at room temperature, and 24 hours at -80°C.
Shipping Stability: Cartridge and instrument performance unaffected after simulated shipping tests (ASTM D4169:23, DC-13).
Linearity: Not applicable for scores, but individual markers (32 markers) showed linearity for RNA input amounts from 1x10^6 to 1x10^9 copies/mL with an Allowable Deviation for Linearity (ADL) of 5%.
Analytical Sensitivity: Determined to be 1x10^6 cp/mL (95% amplification).
Detection Limits (LoD and LoQ): Both determined to be 500 cells/µL.
Analytical Specificity: No cross-reactivity with gDNA observed. No interference found for 17 endogenous and exogenous substances tested at high concentrations.
Carryover: No substantial bias or imprecision due to carryover or amplicon contamination observed.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

PPA (Positive Percent Agreement) and NPA (Negative Percent Agreement):
- Bacterial (Forced): Very High Band: PPA 30.5%, NPA 94.2%. Very Low Band: PPA 95.7%, NPA 26.6%.
- Bacterial (Consensus): Very High Band: PPA 35.9%, NPA 95.5%. Very Low Band: PPA 97.2%, NPA 34.9%.
- Viral (Forced): Very High Band: PPA 46.5%, NPA 97.7%. Very Low Band: PPA 88.7%, NPA 45.1%.
- Viral (Consensus): Very High Band: PPA 59.3%, NPA 98.6%. Very Low Band: PPA 95.5%, NPA 48.2%.
- Severity: Very High Band: PPA 14.8%, NPA 98.7%. Very Low Band: PPA 91.8%, NPA 36.8%.
Likelihood Ratio (LR):
- Bacterial (Forced): LR for Very High Band: 5.24. LR for Very Low Band: 0.16.
- Bacterial (Consensus): LR for Very High Band: 8.04. LR for Very Low Band: 0.08.
- Viral (Forced): LR for Very High Band: 20.04. LR for Very Low Band: 0.25.
- Viral (Consensus): LR for Very High Band: 40.93. LR for Very Low Band: 0.09.
- Severity: LR for Very High Band: 11.33. LR for Very Low Band: 0.22.
AUROC (Area Under the Receiver-Operating Characteristics):
- Bacterial Infection (Forced Adjudication): 0.76
- Viral Infection (Forced adjudication): 0.83
- Bacterial Infection (Consensus Adjudication): 0.83
- Viral Infection (Consensus Adjudication): 0.91
- TriVerity Bacterial or Viral vs. Non-Infected (Forced Adjudication): 0.79
- TriVerity Bacterial or Viral vs. Non-Infected (Consensus Adjudication): 0.85
- Severe Infection (Prognostic Endpoint): 0.78
Probabilities of Infection/Severity:
- Bacterial (Forced): Probability of Bacterial Infection in Very High Band: 89.0%. No Bacterial Infection in Very Low Band: 80.0%.
- Viral (Forced): Probability of Viral Infection in Very High Band: 85.3%. No Viral Infection in Very Low Band: 93.2%.
- Severity: Probability of Severe Illness in Very High Band: 58.1%. No Severe Illness in Very Low Band: 97.3%.

Predicate Device(s)

K203748

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3215 Device to detect and measure non-microbial analyte(s) in human clinical specimens to aid in assessment of patients with suspected sepsis.

(a)
Identification. A device to detect and measure non-microbial analyte(s) in human clinical specimens to aid in assessment of patients with suspected sepsis is identified as an in vitro device intended for the detection and qualitative and/or quantitative measurement of one or more non-microbial analytes in human clinical specimens to aid in the assessment of patients with suspected sepsis when used in conjunction with clinical signs and symptoms and other clinical and laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the device's detailed Indications for Use statement describing what the device detects and measures, the results provided to the user, whether the measure is qualitative and/or quantitative, the clinical indications for which the test is to be used, and the specific population(s) for which the device use is intended.
(2) Premarket notification submissions must include detailed documentation of the device description, including (as applicable), all device components, software, ancillary reagents required but not provided, explanation of the device principle and methodology, and for molecular devices include detailed documentation of the primer/probe sequence, design, and rationale for sequence selection.
(3) Premarket notification submissions must include detailed documentation of applicable analytical studies, such as, analytical sensitivity (Limit of Detection, Limit of Blank, and Limit of Quantitation), precision, reproducibility, analytical measuring range, interference, cross-reactivity, and specimen stability.
(4) Premarket notification submissions must include detailed documentation of a prospective clinical study or, if appropriate, results from an equivalent sample set. This detailed documentation must include the following information:
(i) Results must demonstrate adequate device performance relative to a well-accepted comparator.
(ii) Clinical sample results must demonstrate consistency of device output throughout the device measuring range likely to be encountered in the Intended Use population.
(iii) Clinical study documentation must include the original study protocol (including predefined statistical analysis plan), study report documenting support for the Indications for Use(s), and results of all statistical analyses.
(5) Premarket notification submissions must include evaluation of the level of the non-microbial analyte in asymptomatic patients with demographic characteristics (
e.g., age, racial, ethnic, and gender distribution) similar to the Intended Use population.(6) As part of the risk management activities performed under 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.
(7) 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, and a detailed explanation of the interpretation of the limitations of the samples (
e.g., collected on day of diagnosis) must be included in the device's 21 CFR 809.10(b)(10) compliant labeling.

Summary

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January 10, 2025

Inflammatix, Inc. Diana Lane VP, Quality Assurance and Regulatory Affairs 540 Oakmead Pkwy. Sunnyvale, California 94085

Re: K241676

Trade/Device Name: TriVerity Regulation Number: 21 CFR 866.3215 Regulation Name: Device To Detect And Measure Non-Microbial Analyte(s) In Human Clinical Specimens To Aid In Assessment Of Patients With Suspected Sepsis Regulatory Class: Class II Product Code: PRE, OOI Dated: December 9, 2024 Received: December 9, 2024

Dear Diana Lane:

We have reviewed your section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (the Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

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Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30. Design controls; 21 CFR 820.90. Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-device-advicecomprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatory

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assistance/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,

Digitally signed by Bryan M. Bryan M. Grabias -S Grabias -S Date: 2025.01.10 Bryan Grabias, Ph. D. Acting 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

3

Indications for Use

510(k) Number (if known) K241676

Device Name TriVerity

Indications for Use (Describe)

The TriVerity test is an automated ve in vitro diagnostic test that measures the relative expression levels of host response genes in RNA isolated from whole blood collected in the PAXgene Blood RNA tube using reverse transcription loop-mediated isothermal amplification (RT-LAMP) on the Myrna instrument.

The TriVerity test is indicated for use in conjunction with clinical assessments and other laboratory findings as an aid to differentiate bacterial infections, viral infections, and non-infectious illness, as well as to determine the likelihood of 7day need for mechanical ventilation, vasopressors, and/or renal replacement with suspected acute infection or suspected sepsis presenting to the emergency department.

The test generates three scores that each fall within one of five discrete interpretation bands based on the increasing likelihood of

bacterial infection,
viral infection, and
severe illness, as defined by the need for mechanical ventilation, vasopressors, and or renal replacement therapy (RRT) within seven days.

Type of Use (Select one or both, as applicable)
Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)

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510(k) SUMMARY

GENERAL INFORMATION 1.0

Submission Date:	June 10, 2024
Submitter Information
Submitted by:	Inflammatix, Inc.
	540 Oakmead Pkwy.
	Sunnyvale, CA 94085
Contact Person:	Diana Lane
	VP, Quality Assurance and Regulatory Affairs
	Tel: +1.831.820.7560
	Email: dlane@inflammatix.com

2.0 PURPOSE OF SUBMISSION

To obtain a substantial equivalence determination for TriVerity™.

3.0 MEASURAND

Measurement of 29 mRNA host response transcripts and 3 housekeeping transcripts

Host Response Genes
ANKRD22	ARG1	BATF
C3AR1	CD163	CEACAM1
CLEC5A	CTSL1	DEFA4
HERC5	HLA-DMB	IFI27
IFI44	IFI44L	IL18R1
IL1R2	ISG15	JUP
KCNJ2	LY86	OASL
OLFM4	PSMB9	RSAD2
S100A12	TDRD9	TGFBI
XAF1	ZDHHC19
Housekeeping Genes
KPNA6	RREB1	YWHAB

TYPE OF TEST 4.0

Quantitative, Reverse Transcription Loop-mediated Isothermal Amplification (qRT-LAMP)

5.0 APPLICANT

Inflammatix, Inc.

PROPRIETARY AND ESTABLISHED NAMES 6.0

TriVerity™

TriVerity Test

TriVerity Test System

Proprietary Information

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Image /page/5/Picture/0 description: The image contains the logo for "Inflammatix". The logo consists of a shield-shaped emblem on the left, featuring vertical stripes in alternating shades of blue and orange. To the right of the emblem, the word "Inflammatix" is written, with the "Inflammati" portion in orange and the "x" in black.

REGULATORY INFORMATION 7.0

Trade Name:	TriVerity™
Regulation:	866.3215 – Device to detect and measure non-microbial
analyte(s) in human clinical specimens to aid in assessment of
patients with suspected sepsis
Product Code:	PRE – RT-qPCR Assay For mRNA Transcript Immune Biomarkers
Panel:	81 (Microbiology)
Device Class:	Class II

INTENDED USE/INDICATIONS FOR USE 8.0

8.1 Intended Use
See Indications for Use, below.
8.2 Indications for Use
The TriVerity test is an automated, semi-quantitative in vitro diagnostic test that measures the relative expression levels of host response genes in RNA isolated from whole blood collected in the PAXgene Blood RNA tube using reverse transcription loop-mediated isothermal amplification (RT-LAMP) on the Myrna instrument.

The TriVerity test is indicated for use in conjunction with clinical assessments and other laboratory findings as an aid to differentiate bacterial infections, viral infections, and non-infectious illness, as well as to determine the likelihood of 7day need for mechanical ventilation, vasopressors, and/or renal replacement therapy in adult patients with suspected acute infection or suspected sepsis presenting to the emergency department.

The test generates three scores that each fall within one of five discrete interpretation bands based on the increasing likelihood of

1. bacterial infection,
1. viral infection, and

severe illness, as defined by the need for mechanical ventilation, vasopressors, and/or renal replacement therapy (RRT) within seven days.

8.3 Special conditions for use statement(s):
Rx - For Prescription Use Only
8.4 Special instrument requirements:
Myrna instrument

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Image /page/6/Picture/0 description: The image contains the logo for "Inflammatix". The logo consists of a shield-like emblem on the left, with vertical lines in blue and red. To the right of the emblem is the word "Inflammatix", with the first part of the word in orange and the last part in black.

DEVICE DESCRIPTION 9.0

ਰੇ. I Device Description:

The TriVerity test is an in vitro diagnostic test for simultaneous amplification and detection of 29 informative host response RNA transcripts and 3 housekeeping transcripts (listed in measurands) using total RNA extracted from human blood. The test has been designed, manufactured, and validated for use on the Myrna instrument. The TriVerity test is performed with a TriVerity cartridge, a single-use, disposable, multi-chambered fluidic cartridge that runs on the Myrna instrument. All processing steps are automated and occur within a TriVerity cartridge, including sample extraction/purification and qRT-LAMP for the detection and relative quantification of the 29 informative host response RNA transcripts and 3 housekeeping transcripts (listed in measurands). All cartridge steps in this process, following the addition of the sample, are fully automated and completely integrated. In approximately 30 minutes, the test generates three scores that each fall within one of five discrete interpretation bands based on the increasing likelihood of

1. bacterial infection,
1. viral infection, and
1. severe illness, as defined by the need for mechanical ventilation, vasopressors, and/or renal replacement therapy (RRT) within seven days.

The specimen used for the TriVerity test is a sample of whole blood collected by venipuncture using the PAXgene blood collection tubes within the PAXgene Blood RNA System (K042613). The cartridge contains all the necessary reagents to perform RNA isolation and subsequent amplification from the sample.

The TriVerity test quantifies the amount of each transcript in the sample based on the detection of fluorescence by the Myrna instrument. The cartridge includes the reagents for reverse transcription and LAMP. All 32 transcripts (and two in cartridge controls) are amplified and quantified. These values are input to two fixed classifiers which output the three separate scores, each with five discrete interpretation bands. The bands reflect monotonically increasing likelihood of bacterial infection, viral infection, and severe illness, as defined by the need for mechanical ventilation, vasopressors, and/or renal replacement therapy (RRT) within seven days.

9.2 Principles of Operation
The TriVerity test performs RNA extraction and isolation, followed by Quantitative Reverse Transcription Loop-mediated Isothermal Amplification (qRT-LAMP) to measure the expression levels of 29 hosts response RNA transcripts and 3 housekeeping RNA transcripts isolated from whole blood. Amplicons generated by the qRT-LAMP process are detected and quantitated by an intercalating fluorescent dye. The test will only run on the Myrna instrument. The expression levels of the 32 RNA transcripts measured are input to two fixed classifiers which output three separate scores, each with five discrete interpretation bands. The bands reflect monotonically increasing likelihood of bacterial infection, viral infection, and severe illness, as defined by the need for mechanical ventilation, vasopressors, and/or renal replacement therapy (RRT) within seven days.

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10.0 SUBSTANTIAL EQUIVALENCE

Predicate Device Name:	SeptiCyte RAPID
Predicate 510(k) Number:	K203748
Comparison with Predicate:

Table 1: Comparison with Predicate

| | TriVerity
(Proposed Device) | SeptiCyte RAPID
(K203748) (Predicate) |
|----------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | The TriVerity test is an automated, semi-
quantitative in vitro diagnostic test that
measures the relative expression levels
of host response genes in RNA isolated
from whole blood collected in the
PAXgene Blood RNA tube using reverse
transcription loop-mediated isothermal
amplification (RT-LAMP) on the Myrna
instrument.
The TriVerity test is indicated for use in
conjunction with clinical assessments
and other laboratory findings as an aid
to differentiate bacterial infections, viral
infections, and non-infectious illness, as
well as to determine the likelihood of 7-
day need for mechanical ventilation,
vasopressors, and/or renal replacement
therapy in adult patients with suspected
acute infection or suspected sepsis
presenting to the emergency
department.
The test generates three scores that
each fall within one of five discrete
interpretation bands based on the
increasing likelihood of

bacterial infection,
viral infection, and
severe illness, as defined by the need
for mechanical ventilation, vasopressors,
and/or renal replacement therapy (RRT)
within seven days. | The SeptiCyte RAPID test is a gene
expression assay using reverse
transcription polymerase chain reaction
to quantify the relative expression levels
of host response genes isolated from
whole blood collected in the PAXgene
Blood RNA Tube. The SeptiCyte RAPID
test is used in conjunction with clinical
assessments and other laboratory
findings as an aid to differentiate
infection-positive (sepsis) from infection-
negative systemic inflammation in
patients suspected of sepsis on their first
day of ICU admission. The SeptiCyte
RAPID test generates a score
(SeptiScore) that falls within one of four
discrete Interpretation Bands based on
the increasing likelihood of infection
positive systemic inflammation.
SeptiCyte RAPID is intended for in-vitro
diagnostic use on the Biocartis Idylla
System. |
| Indication for
Use Population | Patients presenting with suspected acute
infection or sepsis in the emergency
department | Patients suspected of sepsis on their first
day of Intensive Care Unit (ICU) admission |
| Classification | II | Same |
| Product Code | PRE | Same |
| Specimen
Type | Whole blood | Same |
| | TriVerity
(Proposed Device) | SeptiCyte RAPID
(K203748) (Predicate) |
| Specimen
Collection
Tube | PAXgene Blood RNA | Same |
| Specimen
Processing | Automated extraction within the
instrument platform | Same |
| Assay
Principle | Quantitative gene expression assay,
based on real-time generation of
fluorescence from intercalating
fluorescent dye during RT-LAMP
amplification of nucleic acid
templates | Quantitative gene expression assay,
based on real-time generation of
fluorescence from hydrolysis of dye-
quencher hydrolysis probes during cycles
of PCR amplification of nucleic acid
templates |
| Detection
Technology | Reverse transcription loop-mediated
isothermal amplification (RT-LAMP) | Reverse transcription polymerase chain
reaction |
| Analytes | 29 mRNA host response genes:
ANKRD22, ARG1, BATF, C3AR1, CD163,
CEACAM1, CLEC5A, CTSL1, DEFA4, HERC5,
HLA-DMB, IFI27, IFI44, IFI44L, IL18R1, IL1R2,
ISG15, JUP, KCNJ2, LY86, OASL, OLFM4,
PSMB9, RSAD2, S100A12, TDRD9, TGFBI,
XAF1, ZDHHC19

3 housekeeping genes:
KPNA6, RREB1, YWHAB | Two mRNA transcript immune
biomarkers: PLA2G7, PLAC8 |
| Instrument | Myrna instrument | Biocartis Idylla system |
| Controls | Two internal controls were developed,
serving as within-cartridge positive
controls. They serve as sample processing
and qRT-LAMP controls. The controls are
RNA spike in controls from the External
RNA Controls Consortium.
External controls are not provided with the
assay and are not required for assay
performance; however, labeling describes
their setup and use. | MS2 bacteriophage particles, serving as
sample processing control (SPC), i.e., as
within-cartridge positive control for both
the sample extraction step and the
coupled RT-qPCR step.
External controls not provided with the
assay but are described in labeling with
protocols available from sponsor. |
| Result Output | TriVerity measures the expression levels of
32 mRNA transcripts which are input into
two fixed classifiers. The result is three
separate scores, each with five discrete
interpretation bands. The bands reflect
monotonically increasing likelihood of
bacterial infection, viral infection, and
severe illness, as defined by the need for
mechanical ventilation, vasopressors,
and/or renal replacement therapy (RRT)
within seven days. | SeptiScore, calculated from the
expression levels of the two mRNA
analytes PLA2G7, PLAC8.

The SeptiScore is placed into four
discrete bands that describe a
monotonically increasing likelihood of
sepsis vs. Systemic Inflammatory
Response Syndrome (SIRS). |

Proprietary Information

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STANDARD/GUIDANCE DOCUMENTS REFERENCED 11.0

CLSI EP05-A3 Evaluation of Precision of Quantitative Measurement Procedures; . Approved Guideline - Third Edition
. CLSI EP06 - Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline - Second Edition
CLSI EP07 – Interference Testing in Clinical Chemistry; Approved Guideline – Third Edition
CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory ● Measurement Procedures; Approved Guideline - Second Edition
CLSI EP25-A – Evaluation of Stability of In Vitro Diagnostic Reagents: Approved Guideline
CLSI EP37 – Supplemental Tables for Interference Testing in Clinical Chemistry (1st Edition)
ASTM D4169-23 – Standard Practice for Performance Testing of Shipping Containers and Systems
ASTM F2825-18 – Standard Practice for Climatic Stressing of Packaging Systems for Single Parcel Delivery
FDA Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices (issued 11 May 2005)

12.0 PERFORMANCE CHARACTERISTICS

12.1 Analytical Performance
- a. Reproducibility

Multi-site Reproducibility

A multi-site reproducibility study was conducted at two external sites and one internal site, each equipped with three instruments and multiple operators. The panel of four contrived samples used in the study spanned the range of scores expected from patient samples. Testing was conducted over five days with two runs per day with each panel in triplicate. Each of the two daily runs was performed by a different operator. The expected sample results are summarized in 2. Standard deviation was calculated for each score (Bacterial/Viral/Severity) and for each band (15 bands). The standard deviation measured in each band, and for each overall score was within acceptance criteria.

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Table 2: Overview of the Contrived Panel Members for Reproducibility and Precision

Sample	Expected Results
Sample A	Bacterial Result: High
Viral Result: Moderate
Severity Result: High
Sample B	Bacterial Result: Very High
Viral Result: Very Low
Severity Result: Very High
Sample C	Bacterial Result: Very Low
Viral Result: Very High
Severity Result: Very Low
Sample F	Bacterial Result: Very Low
Viral Result: Very Low
Severity Result: Very Low

Table 3: Summary of results for multi-site reproducibility

| Sample | Score
Type | Mean
Score | Between
Day SD | Between
Instrument SD | Between
Site SD | Reproducibility
SD |
|--------|---------------|---------------|-------------------|--------------------------|--------------------|-----------------------|
| A | B | 38.17 | 0.00 | 1.25 | 1.82 | 4.91 |
| A | V | 22.33 | 0.00 | 1.52 | 1.77 | 5.21 |
| A | S | 37.48 | 1.35 | 1.63 | 0.00 | 3.65 |
| B | B | 47.42 | 0.00 | 0.14 | 0.00 | 0.75 |
| B | V | 6.24 | 0.59 | 0.35 | 0.00 | 1.91 |
| B | S | 48.20 | 0.00 | 0.23 | 0.00 | 0.84 |
| C | B | 1.13 | 0.05 | 0.16 | 0.07 | 0.51 |
| C | V | 49.10 | 0.18 | 0.18 | 0.00 | 0.40 |
| C | S | 6.86 | 0.00 | 0.65 | 0.00 | 1.56 |
| F | B | 3.49 | 0.23 | 0.23 | 0.19 | 0.84 |
| F | V | 3.17 | 0.00 | 0.44 | 0.00 | 1.01 |
| F | S | 8.89 | 0.00 | 0.00 | 0.00 | 2.55 |

Intra-Site Precision

Precision was assessed using a total of 138 evaluable clinical samples covering all score bands and score types, with at least 15 results in every score band. Each clinical sample was measured consecutively in duplicate within the same instrument and using the same cartridge lot in the same laboratory. The study was performed over 5 days using 3 different cartridge lots and 12 different instruments. The standard deviation (SD) was calculated for each score (Bacterial/Viral/Severity) and for each band (15 bands). The SD measured in each band, and for each overall score was within acceptance criteria. The overall SD for bacterial, viral, and severity scores were 2.71, 2.89, and 2.93, respectively:

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Band	Imprecision SD (N=138)
	Bacterial	Viral	Severity
1	1.59	1.44	1.72
2	3.21	4.32	3.89
3	2.90	3.56	3.61
4	3.69	3.58	2.92
5	1.22	0.85	1.38
Overall	2.71	2.89	2.93

Table 4: Precision measured using replicates of clinical samples

Lot-to-lot Reproducibility

The lot-to-lot reproducibility of the TriVerity test score was assessed using a consistent measurement procedure, instrument, operator, operating conditions, and location to obtain measurements of the same sample using three different cartridge lots (Lot A, Lot B, Lot C) with varied lots of reagent formulation of critical reagents. For each contrived sample level (A, B, C, F), a single Myrna instrument was used to run sample replicates in cartridges with alternating runs for each lot, resulting in a total of six replicates per lot/sample level. This design was applied to all four contrived sample levels to characterize the reproducibility of the different score bands across different cartridge lots. The SDs measured for each score and sample type were within acceptance criteria.

Sample	Score	Mean Score	Between Lot SD	Overall SD
Sample A	Bacterial	33.56	0.0	4.7
	Viral	27.33	0.0	3.5
	Severity	37.5	1.1	2.7
Sample B	Bacterial	47.61	0.0	0.8
	Viral	6.28	0.0	1.8
	Severity	47.94	0.0	0.6
Sample C	Bacterial	1.00	0.0	0.0
	Viral	49.17	0.0	0.4
	Severity	7.33	0.4	1.2
Sample F	Bacterial	3.33	0.0	0.5
	Viral	3.94	0.0	0.7
	Severity	9.33	1.0	2.5

Table 5: Lot-to-Lot Reproducibility Results

Repeatability

TriVerity test score repeatability was assessed using the same measurement procedure, the same operator, the same measuring system, the same operating conditions, and the same location to obtain replicate measurements of the same object over a period of 12 (nonconsecutive) days. For each contrived sample level (Samples A, B, C, F), a single Myrna instrument was used to measure cartridges from a single cartridge lot per sample level, with duplicate runs in the morning and duplicate runs in the afternoon. This design

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was applied to all 4 contrived sample levels to fully test repeatability of the different score bands. The results obtained across all runs for all sample/score combinations met the acceptance criteria.

| Sample | Score | Mean Score | Repeatability
SD |
|----------|-----------|------------|---------------------|
| Sample A | Bacterial | 24.9 | 4.99 |
| | Viral | 32.0 | 3.97 |
| | Severity | 36.6 | 4.66 |
| Sample B | Bacterial | 45.9 | 1.06 |
| | Viral | 10.4 | 3.23 |
| | Severity | 47.9 | 0.89 |
| Sample C | Bacterial | 0.5 | 0.50 |
| | Viral | 49.8 | 0.38 |
| | Severity | 7.2 | 1.21 |
| Sample F | Bacterial | 3.2 | 0.43 |
| | Viral | 3.3 | 0.88 |
| | Severity | 8.1 | 1.76 |

Table 6: Repeatability Study Results

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

Real-time Reagent Stability

Real-time stability studies are ongoing to support product claims for room temperature storage (15-30°C).

PAXgene Sample Collection/Handling

A specimen stability study was conducted to confirm the equivalence of TriVerity results on the Myrna instrument from paired fresh PAXgene Blood RNA samples (run within 1 hour of collection) compared to the PAXgene Blood RNA samples stored at room temperature for 24 hours and the same blood sample stored frozen at -80 ℃ for 24 hours followed by a thaw cycle. The scores of 60 patient samples were compared when fresh after collection (TO), after 24 hours at room temperature (T1) and when subsequently frozen at -80 °C for 24 hours (T2). The results met acceptance criteria on mean score shift(bias) of T1 and T2 relative to T0.

Additionally, a specimen stability study was conducted to confirm fresh specimen stability between 0 and 12 hours under real-time storage conditions. The study was conducted with fresh PAXgene Blood RNA samples from 16 healthy volunteers. The study compared scores from paired fresh PAXgene Blood RNA samples (run within 1 hour of collection, T0) to the PAXgene Blood RNA samples stored at each of 15 °C and 25 °C for 2 hours (T1), 12 hours (T2), and 14 hours (T3) post-collection. The results met acceptance criteria on mean score shift (bias) of T1, T2, and T3 relative to T0 at both tested temperatures.

These data support TriVerity testing immediately after blood draw, up to 12 hours after blood draw (our recommended testing window with a significant safety factor) or after sample processing according to the manufacturer instructions for the PAXgene sample tube.

Shipping Stability

Shipping studies were conducted for both the TriVerity cartridges and the Myrna instrument. Control tests were run on cartridges prior to conditioning or simulated ship testing. Cartridge shippers were then preconditioned per ASTM F2825-18 prior to ship testing. Cartridge and instrument shippers were subjected to simulated ship testing per ASTM D4169:23, Distribution Cycle 13 (DC-13), with assurance level II. There was no impact on cartridge or instrument performance after testing.

Linearity ﻥ

Linearity is not applicable to scores because scores are determined by logistic regression models. Individual marker linearity was assessed using serial dilutions of pure in vitro transcribed RNA sequences (IVT) corresponding to each of the 32 markers that are measured in the TriVerity test. The TriVerity test was found to be linear for all 32 markers at all RNA

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input amounts tested, from 1x106 copies/mL to 1x109 copies/mL, considering an Allowable Deviation for Linearity (ADL) of 5%. The results validated the reportable dynamic range.

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Table 7: Linearity Study Results

Linearity testing, 1x106 copies/mL to 1x109 copies/mL
Gene	slope	intercept	Pearson	ADL 5%
ANKRD22	-1.65	27.61	0.989	Pass
ARG1	-1.91	31.19	0.985	Pass
BATF	-2.03	35.08	0.988	Pass
C3AR1	-1.88	31.14	0.993	Pass
CD163	-1.61	27.66	0.993	Pass
CEACAM1	-2.10	35.61	0.981	Pass
CLEC5A	-1.78	30.00	0.979	Pass
CTSL1	-2.06	33.66	0.997	Pass
DEFA4	-1.85	30.31	0.996	Pass
HERC5	-1.97	32.63	0.982	Pass
HLA-DMB	-2.36	39.00	0.995	Pass
IFI27	-1.84	32.00	0.980	Pass
IFI44	-1.85	30.30	0.994	Pass
IFI44L	-2.55	41.69	0.996	Pass
IL18R1	-1.85	30.33	0.993	Pass
IL1R2	-2.95	40.95	0.961	Pass
ISG15	-1.74	30.82	0.996	Pass
JUP	-2.03	34.26	0.990	Pass
KCNJ2	-1.84	30.55	0.996	Pass
KPNA6	-1.89	31.37	0.995	Pass
LY86	-1.73	28.66	0.995	Pass
OASL	-1.57	27.59	0.982	Pass
OLFM4	-2.41	37.80	0.991	Pass
PSMB9	-2.06	34.37	0.989	Pass
RREB1	-2.15	34.02	0.991	Pass
RSAD2	-1.84	33.40	0.961	Pass
S100A12	-2.52	40.38	0.995	Pass
TDRD9	-2.06	32.94	0.977	Pass
TGFBI	-2.10	35.75	0.989	Pass
XAF1	-2.20	35.77	0.986	Pass
YWHAB	-2.95	43.84	0.943	Pass
ZDHHC19	-2.06	34.41	0.974	Pass

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d. Analytical Sensitivity

Analytical sensitivity was evaluated using IVT corresponding to each of the 32 markers at concentrations ranging from 1x105 copies/mL to 1x10 copies/mL per concentration and evaluating amplification. The study was executed by a single operator over 3 days with 3 instruments used for testing 20 replicates per concentration. Testing was performed in descending order, from the highest concentration sample to the lowest. Analytical sensitivity was determined to be 1x106 cp/mL which was defined as the lowest concentration at which 95% (19/20) samples for each individual marker tested showed measurable amplification.

	Gene	% Amplification
		T6 1x106	T7 5x105	T8 1x105
1	ANKRD22	100	100	95
2	ARG1	100	100	95
3	BATF	100	100	95
4	C3AR1	100	100	95
5	CD163	100	100	95
6	CEACAM1	100	100	95
7	CLEC5A	100	100	95
8	CTSL1	100	100	100
9	DEFA4	100	100	95
10	ERCC17	100	100	100
11	ERCC59	100	100	100
12	HERC5	100	100	95
13	HLA-DMB	100	100	90
14	IFI27	100	100	90
15	IFI44	100	100	95
16	IFI44L	100	100	90
17	IL18R1	100	100	95
18	IL1R2	100	90	40
19	ISG15	100	100	95
20	JUP	100	100	95
21	KCNJ2	100	100	95
22	KPNA6	100	100	100
23	LY86	100	100	95
24	OASL	100	100	95
25	OLFM4	100	100	95
26	PSMB9	100	100	95
27	RREB1	100	100	95
28	RSAD2	100	100	95
29	S100A12	100	100	90
30	TDRD9	100	100	90
31	TGFBI	100	100	95
32	XAF1	100	100	80
33	YWHAB	100	100	95
34	ZDHHC19	100	100	95
		PASS	FAIL	FAIL

Table 8: Analytical Sensitivity Results

Proprietary Information

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e. Detection limits

Two clinical sample pools were created to give adequate representation of TriVerity scores across the score range. The clinical pools were created by combining individual clinical samples stored in PAXgene tubes, with white blood cell (WBC) counts of 1995 cells/μL for Pool A and 1296 cells/μL for Pool B. The pools were measured undiluted in 10 replicates and then diluted using leukoreduced blood successively down to the equivalent to 125 cells/uL or 108 cells/uL and measured in 20 replicates.

Limit of Blank

Leukoreduced blood was used as a blank. Ten replicates of Leukoreduced blood were tested and none provided any valid result (0/10 valid results).

Limit of Detection

For each pool, the LoD was defined as the lowest WBC concentration for which at least 95% (19/20) replicates generate scores. The final LoD was the higher of the LoDs determined for each individual pool.

Limit of Quantitation

For each pool, the LoQ was defined as the lowest WBC concentration for which at least 95% of WBC replicates generate scores with a standard deviation of 5.50 or less. The final LoQ was the higher of the LoQs determined for each individual pool. As two different clinical pools were used, the highest LoD/LoQ from each pool was chosen as the overall LoQ/LoQ. The LoQ and the LoD are 500 cells/μL (rounded up from 499 cells/uL).

			Mean Score			Score SD			% Valid
Sample ID	Cells/μL	N	Bac	Viral	Sev	Bac	Viral	Sev	Runs	LoD/LoQ
Leukoreduced
blood	N/A	10	N/A	N/A	N/A	N/A	N/A	N/A	0%	Below LoD/LoQ
	1995	10	15.30	42.30	12.80	2.8	1.3	3.8	100%	Above LoD/LoQ
	499	20	16.10	38.50	11.29	4.1	4.4	3.3	100%	LoD/LoQ
Pool A	374	20	13.89	38.50	11.50	2.8	3.7	4.9	90%	Below LoD/LoQ
	249	20	12.82	36.59	10.41	3.3	5.1	5.8	89%	Below LoD/LoQ
	125	20	13.08	29.08	10.58	3.0	7.4	4.3	60%	Below LoD/LoQ


Pool B	1296	10	45.70	7.20	42.00	1.2	1.6	3.4	100%	Above LoD/LoQ
	432	20	44.40	8.35	39.80	1.4	1.9	2.7	100%	Above LoD/LoQ
	324	20	44.19	8.95	40.24	1.0	1.8	3.0	100%	Above LoD/LoQ
	216	20	42.53	11.37	36.42	2.3	3.3	4.7	95%	LoD/LoQ
	108	20	38.85	13.54	30.38	4.6	5.5	6.6	65%	Below LoD/LoQ

Table 9: LoD and LoQ results

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f. Analytical Specificity

Cross Reactivity

Cross-reactivity and non-specific amplification of genomic DNA (gDNA) as well as no amplification from no-template controls (NTC) were evaluated for the TriVerity test.

Cross-reactivity was assessed by spiking 10 ng/μL of gDNA into two levels of contrived samples with previously characterized score results and comparing them with unspiked samples, with six test runs and six control runs. The gDNA-spiked sample runs were compared with control runs by calculating the mean result for each of the three scores for spiked and control samples. The standard deviation for both conditions and sample levels were calculated, and no cross-reactivity was observed.

Genomic gDNA non-specific amplification was evaluated by spiking 20 ng/μL of gDNA into RNA stabilization buffer, resulting in a total of 20 runs. None observed.

All 20 test runs (100%) with no template showed no amplification signal from 20 ng/μL human genomic DNA template.

Endogenous and Exogenous Interferents

Based on the CLSI Guidance EP07, "Interference Testing in Clinical Chemistry" the TriVerity test was evaluated in the presence of interfering substances. A specimen panel was prepared by spiking potential interferents into each of two contrived panel members B and C (see Table 2). The potential interferents were dissolved in appropriate solvents, and then spiked into the contrived samples at concentrations higher than the maxima of their normal or expected clinical reference ranges. Each substance was added to a blood sample in a PAXgene Blood RNA tube. Each interfering substance was spiked into four tubes of both panel types and tested with two different cartridge lots, resulting in a total of eight tests per interfering substance. No interference was found for any of the substances in Table 10 at the concentration listed.

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| Interferent | Concentration in PAXgene
Blood RNA Tube
(pre-RNA extraction) | Equivalent
Concentration in Whole
blood (*) |
|---------------------------|--------------------------------------------------------------------|---------------------------------------------------|
| Bilirubin | 400 mg/L | 1504 mg/L |
| Hemoglobin | 10 g/L | 37.6 g/L |
| Rheumatoid Factor | 45 U/mL | 169.2 U/mL |
| Triglycerides | 2000 mg/dL | 7520 mg/dL |
| Albumin | 5 g/dL | 18.8 g/dL |
| Heparin | 10 u/mL | 37.6 U/mL |
| Imipenem/Cilastatin | 100 mg/L | 376 mg/L |
| Vancomycin | 100 mg/L | 376 mg/L |
| Cefotaxime | 400 mg/L | 1504 mg/L |
| Dopamine | 500 mg/dL | 1880 mg/dL |
| CRP (c-reactive protein) | 60 mg/L | 225.6 mg/L |
| Norepinephrine | 670 µmol/L | 2519.2 µmol/L |
| Dobutamine | 11.2 mg/L | 42.1 mg/L |
| Furosemide | 59.9 mg/L | 225.2 mg/L |
| IL-6 (Interleukin-6) | 2000 pg/mL | 7520 pg/mL |
| sCD14 | 5 µg/mL | 18.8 µg/mL |
| LPS (Lipopolysaccharides) | 5 ng/mL | 18.8 ng/mL |

Table 10: Endogenous and Exogenous Interferences Tested

(*) Considering a standard mixture of 2.5mL of whole blood in 6.9mL of PAXgene solution.

Carryover

Carryover contamination was investigated using three contrived Panel members B, C, and F (see Table 2) that were tested with 3 instruments across 2 days with 10 runs per instrument per day. Tests were performed in sequence alternating Panel members and scores were evaluated in terms of bias and imprecision when compared with control conditions. No substantial bias or imprecision were observed and therefore, no carryover or amplicon contamination.

Assay Reportable Range g.

See linearity above.

h. Assay Cut-Off

See clinical cut-off (Section 3b) below.

12.2 Comparison Studies

Method Comparison with Predicate Device: a.
N/A

b. Matrix Comparison

N/A

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Image /page/20/Picture/0 description: The image contains the logo for "Inflammatix" with a shield-like icon to the left of the name. The first part of the name, "Inflamm", is in orange, while the rest of the name is in black. Below the logo is the word "TriVerity™" with a line underneath it. The logo appears to be for a company in the biotechnology or pharmaceutical industry.

12.3 Clinical Studies

Clinical Sensitivity and Clinical Specificity a.

A prospective, multi-center clinical study (SEPSIS-SHIELD; NCT04094818) was conducted to establish the performance characteristics of the TriVerity test on the Myrna instrument. This was a non-interventional, minimal-risk study to analyze gene expression and other laboratory data from biological samples collected from participants with suspected respiratory, urinary, intra-abdominal, skin and soft tissue, meningitis/encephalitis, and at least one vital sign change, and other acute infections, or suspected sepsis of any cause with at least two vital sign changes and a blood culture order.

Study sites included emergency departments (EDs) of community and academic hospitals located in twenty-one geographically diverse locations throughout the United States plus one site in Europe. Enrollment was offered to all patients who met the enrollment criteria at presentation to the ED. Of the 1,441 participants who consented to the study, there were 1,222 evaluable participants.

The primary objective of the study was to evaluate the performance of the TriVerity test for (1) diagnosing bacterial and viral infections using consensus and forced clinical adjudication as the comparator and (2) evaluate the performance of the TriVerity test for predicting severe illness (defined as the need for mechanical ventilation, vasopressors, or renal replacement therapy [RRT] within 7 days) using clinical outcomes as the comparator.

The analysis population consisted of all participants who provided consent, met all inclusion criteria, met no exclusion criteria, and had their PAXgene blood sample collected and successfully processed (resulting in three TriVerity scores). The overall analysis population was further divided into diagnostic and prognostic analysis populations with additional requirements. The diagnostic endpoint analysis population included participants who had a completed adjudication for bacterial and viral infection to determine the ground truth. The diagnostic population was further classified as Consensus (only contains participants with a certain (Yes or No) or Forced adjudication (all patients adjudicated as Yes and/or Probable vs. No and/or Unlikely). The prognostic endpoint analysis population included participants who had information available on the presence of severe illness (acute need for mechanical ventilation, vasopressor and/or renal replacement therapy) within 7 days.

Each of the bands for the TriVerity test were assessed in terms of likelihood ratio, Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and predictive values (post-test probabilities). The calculation of PPA and NPA estimates considered each cutoff independently to assign True Positive, True Negative, False Positive, and False negative status to each assay result.

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Image /page/21/Picture/0 description: The image contains the logo for Inflammatix, a company that specializes in developing diagnostic tests for infectious diseases. The logo consists of a shield with vertical lines in shades of blue and orange, followed by the company name in black and orange text. Below the company name is the text "TriVerity™".

Among 1,441 participants who consented, 188 (13.0%) were excluded due to not meeting the inclusion criteria, meeting exclusion criteria (screen failures), or withdrawals during the study. There were 31 (2.2%) participants without valid TriVerity results. A total of 1,222 participants were evaluable for the diagnostic endpoint (accuracy of the TriVerity test for the identification of bacterial and viral infections) and 1,120 participants were evaluable for the prognostic endpoint (prediction of need for mechanical ventilation, vasopressor use, and/or RRT within 7 days).

Using forced adjudication, 58.1% bacterial infections, 20.0% viral infections, 2.5% co-infections; 19.4% of participants were adjudicated as noninfected. Consensus adjudication yielded 61.5% of bacterial infections compared to 22.6% viral infections and 1.6% bacterial-viral coinfections; 14.3% of participants were adjudicated to not have an infection. In the prognostic population, 10.9% of participants required mechanical ventilation, vasopressor use, and/or RRT within 7 days (prognostic endpoint).

Participant demographics at the time of presentation in the ED are shown in Table 11. These results demonstrate that a representative cohort of U.S. emergency department patients presenting with suspected acute infection and suspected sepsis was enrolled. The results also demonstrate similar age, sex, race, and ethnicity between the forced and consensus diagnostic populations, as well as between the diagnostic and prognostic populations.

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Image /page/22/Picture/0 description: The image contains the logo for "Inflammatix". The logo consists of a shield-like icon on the left, featuring vertical stripes in alternating shades of blue and orange. To the right of the icon is the company name, "Inflammatix", with the "Inflam" portion in orange and the "matix" portion in black.

Populations
	Diagnostic
Population:
Forced
(N=1222)	Diagnostic
Population:
Consensus
(N=729)	Prognostic
Population
(N=1120)
Age
Mean (SD)	50.9 (17.61)	50.6 (17.35)	51.3 (17.62)
Median (Range)	52.0 (18.0, 90.0)	52.0 (18.0, 90.0)	52.0 (18.0, 90.0)
Sex at Birth, n (%)
Female	605 (49.5%)	345 (47.3%)	544 (48.6%)
Male	617 (50.5%)	384 (52.7%)	576 (51.4%)
Race, n (%)*
American Indian or Alaska
Native	2 (0.2%)	0 (0.0%)	1 (0.1%)
Asian*	11 (0.9%)	6 (0.8%)	8 (0.7%)
Black or African American	360 (29.5%)	223 (30.6%)	321 (28.7%)
Native Hawaiian or Other
Pacific Islander	2 (0.2%)	2 (0.3%)	2 (0.2%)
White*	786 (64.3%)	461 (63.2%)	734 (65.5%)
Other	63 (5.2%)	37 (5.1%)	56 (5.0%)
Ethnicity, n (%)
Hispanic or Latino	135 (11.0%)	97 (13.3%)	128 (11.4%)
Not Hispanic or Latino	1064 (87.1%)	623 (85.5%)	972 (86.8%)
Unknown	23 (1.9%)	9 (1.2%)	20 (1.8%)

Table 11: Participant Demographics for the Diagnostic and Prognostic Populations

*Note: Two patients gave their race as biracial (Asian, White), added to forced and prognostic population.

Table 12 shows medical history and immune status of participants. Results obtained are representative of a U.S. emergency department cohort presenting with suspected acute infection and suspected sepsis; we did not observe marked differences in the distribution of underlying diseases and immune status in the different study populations (prognostic vs. forced diagnostic vs. consensus diagnostic populations). Importantly, a total of 219 (17.9%) participants in the forced population were immunosuppressed, among these, 30 fell into more than one of the immunosuppression categories. A total of 206 (18.4%) participants in the prognostic population were immunosuppressed. Among these, 30 participants fell into more than one immunosuppression category.

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Image /page/23/Picture/0 description: The image contains the word "Inflammatix" in a stylized font. The word "Inflammatix" is written in a combination of orange and black colors. To the left of the word is a logo that resembles a shield with vertical lines in blue and orange.

| A

Population
Medical History¹	Diagnostic Population:
Forced
(N=1222)	Diagnostic
Population:
Consensus
(N=729)	Prognostic
Population
(N=1120)
Blood Disorders	86 (7.0%)	51 (7.0%)	80 (7.1%)
Cardiovascular Diseases	519 (42.5%)	303 (41.6%)	485 (43.3%)
Kidney Diseases	224 (18.3%)	140 (19.2%)	213 (19.0%)
Gastrointestinal Tract Diseases	284 (23.2%)	167 (22.9%)	265 (23.7%)
Metabolic/Endocrinological
Diseases	524 (42.9%)	315 (43.2%)	490 (43.8%)
Musculoskeletal Diseases	177 (14.5%)	106 (14.5%)	167 (14.9%)
Neuropsychiatric Diseases	282 (23.1%)	162 (22.2%)	264 (23.6%)
Respiratory Tract Diseases	380 (31.1%)	229 (31.4%)	350 (31.3%)
Skin or Soft Tissue Diseases	61 (5.0%)	36 (4.9%)	58 (5.2%)
Urogenital Diseases	110 (9.0%)	68 (9.3%)	106 (9.5%)
Other	200 (16.4%)	110 (15.1%)	186 (16.6%)
B	Population
Type of Immunosuppression2	Diagnostic Population:
Forced
(N=1222)	Diagnostic
Population:
Consensus
(N=729)	Prognostic
Population
(N=1120)
Bone Marrow, Stem Cell and/or
Other Cell Transplant	3 (0.2%)	2 (0.3%)	3 (0.3%)
Malignancies	125 (10.2%)	73 (10.0%)	121 (10.8%)
HIV/AIDS	26 (2.1%)	16 (2.2%)	22 (2.0%)
Solid Organ Transplant	35 (2.9%)	27 (3.7%)	34 (3.0%)
Steroid Treatment	34 (2.8%)	17 (2.3%)	30 (2.7%)
Other Immunosuppression	30 (2.5%)	17 (2.3%)	29 (2.6%)

Table 12: Medical History (A) and Type of Immunosuppression (B) of Participants

1 Calculated using provider questionnaire information at time of enrollment

2 Note: Participants may have more than a single type of immunosuppression

Using clinical adjudication as the reference standard, the accuracy of the bacterial result readouts of the TriVerity test by interpretation band is shown in Table 13 for the forced adjudication population.

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Image /page/24/Picture/0 description: The image contains the logo for Inflammatix, a company in the biotechnology industry. The logo features a stylized shield with vertical lines in shades of blue and orange on the left. To the right of the shield is the company name, "Inflammatix," with "Inflam" in orange and "matix" in black. Below the Inflammatix logo is the word "TriVerity™" in black.

Table 13: Accuracy of the TriVerity Test for the Diagnosis of Bacterial Infections in the Forced
Population

| TriVerity
Bacterial
Band | Clinically
Adjudicated
Bacterial
Infection
(Forced) | | LR (80% CI) | PPA
(Positive
Percent
Agreement) | NPA
(Negative
Percent
Agreement) | Relative
Frequency of
Result
(% in Band) | Probability
of Bacterial
Infection
(%) | Probability
of No
Bacterial
Infection
(%) |
|--------------------------------|-----------------------------------------------------------------|-----------|---------------------|-------------------------------------------|-------------------------------------------|---------------------------------------------------|-------------------------------------------------|-------------------------------------------------------|
| | Yes
(N) | No
(N) | | | | | | |
| Very High | 226 | 28 | 5.24
(4.19-6.83) | 30.5 | 94.2 | 20.8 | 89.0 | 11.0 |
| High | 184 | 61 | 1.96
(1.65-2.37) | 24.8 | 87.3 | 20 | 75.1 | 24.9 |
| Moderate | 142 | 97 | 0.95
(0.82-1.11) | 19.2 | 79.8 | 19.6 | 59.4 | 40.6 |
| Low | 157 | 167 | 0.61
(0.54-0.69) | 78.8 | 34.7 | 26.5 | 48.5 | 51.5 |
| Very Low | 32 | 128 | 0.16
(0.12-0.2) | 95.7 | 26.6 | 13.1 | 20.0 | 80.0 |
| Prevalence = 60.6% | | | | | | | | |

LR, likelihood ratio.

Compared to the bacterial infection status as determined by clinical adjudication, a high NPA and positive likelihood ratio were observed for the Very High interpretation band. Similarly, the High band showed high NPA and likelihood ratio for the diagnosis of a bacterial infection. In addition, TriVerity demonstrated high PPA and low negative likelihood ratio for the Very Low and Low interpretation bands. Lastly, 80.4% of results fell into the clinically actionable Very High, High, Low or Very Low interpretation. At a prevalence of 60.6% for bacterial infections, probabilities for having or not having a bacterial infection in the outer Very High and Very Low interpretation bands were 89.0% and 80.0%, respectively.

Expectedly, higher accuracy results were observed when applying the consensus adjudication as the reference standard to determine the bacterial infection status (Table 14) due to the exclusion of participants with uncertain (Probable and Unlikely) bacterial infection status.

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Table 14: Accuracy of the TriVerity Test for the Diagnosis of Bacterial Infections in the
Consensus Population

| TriVerity
Bacterial
Band | Clinically
Adjudicated
Bacterial
Infection
(Consensus) | | LR (80% CI) | PPA
(Positive
Percent
Agreement) | NPA
(Negative
Percent
Agreement) | Relative
Frequency
of Result
(% in Band) | Probability
of Bacterial
Infection
(%) | Probability
of No
Bacterial
Infection
(%) |
|--------------------------------|--------------------------------------------------------------------|-----------|----------------------|-------------------------------------------|-------------------------------------------|---------------------------------------------------|-------------------------------------------------|-------------------------------------------------------|
| | Yes
(N) | No
(N) | | | | | | |
| Very High | 165 | 12 | 8.04
(5.66-12.43) | 35.9 | 95.5 | 24.3 | 93.2 | 6.8 |
| High | 107 | 25 | 2.5
(1.97-3.31) | 23.3 | 90.7 | 18.1 | 81.1 | 18.9 |
| Moderate | 90 | 46 | 1.14
(0.94-1.42) | 19.6 | 82.9 | 18.7 | 66.2 | 33.8 |
| Low | 85 | 92 | 0.54
(0.45-0.63) | 81.5 | 34.2 | 24.3 | 48 | 52 |
| Very Low | 13 | 94 | 0.08
(0.05-0.11) | 97.2 | 34.9 | 14.7 | 12.1 | 87.9 |
| Prevalence = 63.1% | | | | | | | | |

LR, likelihood ratio.

The accuracy of the viral result readout of the TriVerity test by interpretation band is shown in Tables 15 and 16 for the forced and consensus populations. Compared to the viral infection status as determined by clinical adjudication, a high NPA (97.7%) and likelihood ratio (20.04) was observed for the Very High interpretation band. Similarly, the High band showed an NPA of 93.8% and a likelihood ratio of 2.04 for the diagnosis of a viral infection. TriVerity demonstrated a high PPA (88.7%) and a low likelihood ratio (0.25) in the Very Low interpretation band. The Low band showed a PPA of 85.1% and a likelihood ratio of 0.48. Likelihood ratios ranged from 0.25 to 20.04 and increased monotonically from the Very Low to the Very High interpretation band. Lastly, 84.9% of TriVerity viral results fell into the clinically actionable Very High, High, Low and Very Low bands; 20.0% of results fell into the clinically actionable Very High and High interpretation bands whereas 65.0% of results fell into the clinically actionable Very Low and Low interpretation bands; only 15.1% of results were found in the moderate interpretation band.

At a prevalence of 22.5% for viral infections, the positive and negative predictive values of the outer Very High and Very Low interpretation bands were 85.3% and 93.2%, respectively.

26

Forced Population
TriVerity
Viral
Band	Clinically
Adjudicated
Viral Infection
(Forced)		LR
(80% Cl)	PPA
(Positive
Percent
Agreement)	NPA
(Negative
Percent
Agreement)	Relative
Frequency of
Result
(% in Band)	Probability of
Viral
Infection (%)	Probability of
No Viral
Infection (%)
	Yes
(N)	No
(N)
Very High	128	22	20.04
(15.45-27.29)	46.5	97.7	12.3	85.3	14.7
High	35	59	2.04
(1.57-2.65)	12.7	93.8	7.7	37.2	62.8
Moderate	40	144	0.96
(0.76-1.18)	14.5	84.8	15.1	21.7	78.3
Low	41	295	0.48
(0.39-0.57)	85.1	31.2	27.5	12.2	87.8
Very Low	31	427	0.25
(0.2-0.31)	88.7	45.1	37.5	6.8	93.2
Prevalence = 22.5%

Table 15: Accuracy of the TriVerity Test for the Diagnosis of Viral Infections in the Forced Population

LR, likelihood ratio.

Expectedly, higher accuracy TriVerity results were observed when applying the consensus adjudication as the reference standard to determine the viral infection status (Table 16) due to the exclusion of participants with uncertain (Probable and Unlikely) viral infection status.

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Image /page/27/Picture/0 description: The image contains the logo for "Inflammatix". The logo consists of a shield-like emblem on the left, featuring vertical stripes in alternating shades of blue and red. To the right of the emblem is the company name, "Inflammatix", with the first part of the name, "Inflamm", in a reddish-orange color, and the rest of the name, "atix", in a dark gray color.

Table 16: Accuracy of the TriVerity Test for the Diagnosis of Viral Infections in the Consensus Population

| TriVerity
Viral
Band | Clinically
Adjudicated
Viral
Infection
(Consensus) | | LR (80% CI) | PPA
(Positive
Percent
Agreement) | NPA
(Negative
Percent
Agreement) | Relative
Frequency
of Result
(% in Band) | Probability
of Viral
Infection (%) | Probability
of No Viral
Infection (%) | |
|----------------------------|----------------------------------------------------------------|-----------|------------------------|-------------------------------------------|-------------------------------------------|---------------------------------------------------|------------------------------------------|---------------------------------------------|--|
| | Yes
(N) | No
(N) | | | | | | | |
| Very High | 105 | 8 | 40.93
(27.73-72.16) | 59.3 | 98.6 | 15.5 | 92.9 | 7.1 | |
| High | 25 | 33 | 2.36
(1.68-3.25) | 14.1 | 94.0 | 8 | 43.1 | 56.9 | |
| Moderate | 22 | 79 | 0.87
(0.64-1.13) | 12.4 | 85.7 | 13.9 | 21.8 | 78.2 | |
| Low | 17 | 166 | 0.32
(0.22-0.41) | 90.4 | 30.1 | 25.1 | 9.3 | 90.7 | |
| Very Low | 8 | 266 | 0.09
(0.05-0.14) | 95.5 | 48.2 | 37.6 | 2.9 | 97.1 | |
| Prevalence = 24.3% | | | | | | | | | |

LR, likelihood ratio

Taken together, the results shown above demonstrate that the TriVerity test has very high accuracy for the diagnosis of bacterial and viral infections.

Table 17 shows the accuracy of the TriVerity illness severity results for the prediction of the need for mechanical ventilation, vasopressors, and/or RRT within 7 days. The data demonstrate that the TriVerity test was able to predict the need for mechanical ventilation, vasopressors, and/or RRT within 7 days (the prognostic severity endpoint of the study). The NPA of the Very High and High illness severity interpretation bands for the prediction of severe disease was 98.7% and 86.1%, with likelihood ratios of 11.33 and 2.41, respectively. Conversely, the PPA of the Very Low and Low illness severity bands were 91.8% and 87.7% with likelihood ratios of 0.22 and 0.43, respectively. 79.6% of results were observed in the clinically actionable Very High, High, Very Low, and Low interpretation bands; 18.8% of these in the Very High and High bands, 60.7% in the Low and Very Low bands. At a prevalence of 10.9% severe illness, probabilities of having or not having severe illness were 58.1% and 97.3% for the Very High and Very Low interpretation bands, respectively.

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Image /page/28/Picture/0 description: The image contains the logo for "Inflammatix". The logo consists of a shield-shaped emblem on the left and the company name on the right. The shield is divided into two sections with the top section colored in blue and the bottom section colored in red. The company name is written in a combination of orange and black, with "Inflammati" in orange and "x" in black.

Table 17: Accuracy of the Severity Result Readout of the TriVerity Test for the Prediction of Mechanical Ventilation, Vasopressor Use, and/or Renal Replacement Therapy within 7 days

| TriVerity
Illness
Severity
Band | Need for
Mechanical
Ventilation,
Vasopressors,
and/or RRT
Within 7 Days | | LR
(80% CI) | PPA
(Positive
Percent
Agreement) | NPA
(Negative
Percent
Agreement) | Relative
Frequency
of Result
(% in Band) | Probability
of Severe
Illness (%) | Probability
of
No Severe
Illness (%) |
|------------------------------------------|----------------------------------------------------------------------------------------|-----|-----------------------|-------------------------------------------|-------------------------------------------|---------------------------------------------------|-----------------------------------------|-----------------------------------------------|
| | Yes | No | | | | | | |
| Very High | 18 | 13 | 11.33
(7.07-17.75) | 14.8 | 98.7 | 2.8 | 58.1 | 41.9 |
| High | 41 | 139 | 2.41
(1.96-2.91) | 33.6 | 86.1 | 16.1 | 22.8 | 77.2 |
| Moderate | 38 | 191 | 1.63
(1.35-1.97) | 31.1 | 80.9 | 20.4 | 16.6 | 83.4 |
| Low | 15 | 288 | 0.43
(0.3-0.57) | 87.7 | 28.9 | 27.1 | 5 | 95 |
| Very Low | 10 | 367 | 0.22
(0.14-0.31) | 91.8 | 36.8 | 33.7 | 2.7 | 97.3 |
| Prevalence = 10.9% | | | | | | | | |

LR, likelihood ratio.

We also determined the accuracy of the three TriVerity test results readouts using AUROCs in Table 18.

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Image /page/29/Picture/0 description: The image contains the logo for Inflammatix, which includes a shield icon with vertical lines in shades of blue and orange. The word "Inflammatix" is written in a combination of orange and black text next to the shield. Below the Inflammatix logo, the word "TriVerity" is written in black text with a trademark symbol.

Table 18: Area Under the Receiver-Operator Characteristics for the TriVerity Bacterial, Viral, and Severe Illness Results

Population	N	AUROC	Lower 80% CI	Upper 80% CI
Bacterial Infection
(Forced Adjudication)	1222	0.76	0.75	0.78
Viral Infection
(Forced adjudication)	1222	0.83	0.81	0.85
Bacterial Infection
(Consensus Adjudication)	729	0.83	0.81	0.85
Viral Infection
(Consensus Adjudication)	729	0.91	0.89	0.93
TriVerity Bacterial or Viral
vs. Non-Infected
(Forced Adjudication)	1222	0.79	0.78	0.81
TriVerity Bacterial or Viral
vs. Non-Infected
(Consensus Adjudication)	729	0.85	0.83	0.86
Severe Infection
(Prognostic Endpoint)	1120	0.78	0.75	0.81

These results demonstrate the high AUROCs of the TriVerity bacterial (0.83) and viral (0.91) results when using the consensus adjudication. Expectedly, slightly lower results were achieved using the forced adjudication. Similarly, AUROCs for the illness severity results were high (0.78) for the prognostic endpoint.

30

Image /page/30/Picture/1 description: The image contains the logo for Inflammatix, which includes a shield-like emblem with vertical lines in shades of orange and blue. To the right of the emblem is the word "Inflammatix," with "Inflamm" in orange and "atix" in black. Below the logo is the word "TriVerity™" in black.

b. Clinical Cut-Off

TriVerity cut-off values were established prior to the registrational clinical trial (INF-04). PPA and NPA of the outside bands were the primary performance targets with a target goal of:

. "Very Low" Band PPA ≥95%: Minimize the number of false negative patients (patients who would go undetected, hence untreated)
. "Very High" Band NPA ≥95%: Minimize the number of false positive patients (minimize overdiagnosis, leading to unnecessary treatment)

Threshold setting across all bands also considered other clinical performance metrics such as likelihood ratios, moderate band coverage, outer band coverage, and monotonicity and was executed in an iterative fashion.

Illness Severity Score thresholds were set (tuned) using predicted probabilities and ground truths for 399 patient samples collected from Emergency Department settings. Bacterial and Viral Score thresholds were tuned using predicted probabilities and ground truths for 472 patient samples collected from Emergency Department and ICU studies. All tuning studies were independent from the final clinical study (INF-04), were collected in PAXgene blood tubes, and were processed on the Myrna instrument using GMP-grade TriVerity cartridges. See Table 19 for recommended interpretation of results.

Table 19: Recommendations for Interpretation of TriVerity Results: (A) Bacterial Score, (B) Viral Score, (C) Severity Score

A)

Bacterial Score	Result	Interpretation
0-10	Very Low Score	Very low bacterial infection likelihood
11-20	Low Score	Low bacterial infection likelihood
21-30	Moderate Score	Moderate bacterial infection likelihood
31-40	High Score	High bacterial infection likelihood
41-50	Very High Score	Very high bacterial infection likelihood

B)

Viral Score	Result	Interpretation
0-10	Very Low Score	Very low viral infection likelihood
11-20	Low Score	Low viral infection likelihood
21-30	Moderate Score	Moderate viral infection likelihood
31-40	High Score	High viral infection likelihood
41-50	Very High Score	Very high viral infection likelihood

C)

Severity Score	Result	Interpretation
0-10	Very Low Score	Very low 7-day severe illness likelihood
11-20	Low Score	Low 7-day severe illness likelihood
21-30	Moderate Score	Moderate 7-day severe illness likelihood
31-40	High Score	High 7-day severe illness likelihood
41-50	Very High Score	Very High 7-day severe illness likelihood

Proprietary Information

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Image /page/31/Picture/1 description: The image contains the logo for Inflammatix, which is written in black text except for the "Inflamm" portion, which is written in orange. To the left of the word Inflammatix is a shield-shaped logo with red and blue vertical lines. Below the Inflammatix logo is the word "TriVerity" with a trademark symbol.

Other Clinical Supporting Data ﻦ

Secondary and subgroup analyses were performed to demonstrate that accuracy results for the TriVerity test presented above are representative across the entire clinical performance study population and defined patient.

Probabilities of bacterial infection by TriVerity test interpretation band are shown below. Results across the three TriVerity (Bacterial, Viral, and Severity) Score readouts are consistent, indicating a clear relationship between TriVerity scores and increasing likelihoods of bacterial infection, viral infection, and illness severity endpoints across TriVerity interpretation bands. The 80% Cls for nonadjacent bands do not overlap in any of the analyses.

Figure 1: Probabilities of Bacterial Infection by TriVerity Interpretation Band: (A) Forced Population, (B) Consensus Population

Image /page/31/Figure/6 description: The image contains two plots, A and B, comparing the fraction of positive samples in different bands for 'Forced Bacterial' and 'Consensus Bacterial' respectively. Plot A, 'Forced Bacterial (N=1,222)', shows the fraction of positive samples for bands 1 to 5, with values ranging from 20.0% for Band 1 (N=160) to 89.0% for Band 5 (N=254). Plot B, 'Consensus Bacterial (N=729)', also shows the fraction of positive samples for bands 1 to 5, with values ranging from 12.1% for Band 1 (N=107) to 93.2% for Band 5 (N=177). Both plots display an increasing trend in the fraction of positive samples from Band 1 to Band 5.

Note: Band 1=Very Low, Band 2=Low, Band 3=Moderate, Band4=High, Band 5=Very High.

32

Image /page/32/Picture/0 description: The image shows the logo for Inflammatix, a company that specializes in developing diagnostic tests for infectious diseases. The logo features a shield with red and blue flames, followed by the company name in orange and black text. Below the company name is the word "TriVerity" with the trademark symbol.

Image /page/32/Figure/2 description: The image contains two plots, A and B, comparing the fraction of positive samples in different bands for forced viral and consensus viral populations. Plot A, titled "Forced Viral (N=1,222)," shows the fraction of positive samples in bands 1 to 5, with corresponding sample sizes (N) of 458, 336, 184, 94, and 150, respectively. Plot B, titled "Consensus Viral (N=729)," displays the same metric for bands 1 to 5, with sample sizes (N) of 274, 183, 101, 58, and 113, respectively. The y-axis in both plots represents the fraction of positive samples in each band, ranging from 0.0 to 0.9.

Figure 2: Probabilities of Viral Infection by TriVerity Interpretation Band: (A) Forced Population, (B) Consensus Population

Note: Band 1=Very Low, Band 2=Low, Band 3=Moderate, Band4=High, Band 5=Very High.

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Image /page/33/Picture/1 description: The image contains the logo for "Inflammatix" with a shield icon to the left of the name. The word "Inflam" is in orange, while "matix" is in black. Below the logo is the word "TriVerity™" in black. The shield icon is divided into vertical sections with alternating blue and orange colors.

Figure 3: Probabilities of Need of Mechanical Ventilation, Vasopressors, and Renal Replacement Therapy Within 7 Days

Image /page/33/Figure/3 description: The image is a plot titled "Illness Severity (N=1120)". The y-axis is labeled "Fraction of positive samples in band", and the x-axis shows the bands 1 through 5. The plot shows the fraction of positive samples in each band, with band 1 at 2.7%, band 2 at 5.0%, band 3 at 16.6%, band 4 at 22.8%, and band 5 at 58.1%.

Note: Band 1=Very Low, Band 2=Low, Band 3=Moderate, Band4=High, Band 5=Very High.

Next, we plotted the likelihood ratios and 80% Cls for each interpretation band for the bacterial, viral, and severity result readouts of the TriVerity test. Results across the three TriVerity (bacterial, viral, and illness severity) result readouts are consistent, indicating a clear relationship between TriVerity scores and increasing likelihoods of bacterial infection, viral infection, and illness severity across TriVerity interpretation bands. CIs for non-adjacent bands do not overlap in any of the analyses. Of importance, the point estimates of the band likelihood ratios are monotonically increasing, and the 80% Cls of the likelihood ratios in non-adjacent bands are non-overlapping, for all scores.

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Image /page/34/Picture/0 description: The image contains the logo for Inflammatix, a company that specializes in developing diagnostic tests for infectious diseases. The logo features a shield with red and blue vertical lines on the left, followed by the company name in orange and black text. Below the company name is the word "TriVerity" with the trademark symbol.

A)

Figure 4: Likelihood Ratios for Bacterial Infection by TriVerity Interpretation Band: (A) Forced Cohort, (B) Consensus Cohort

Image /page/34/Figure/3 description: The image contains two plots comparing LR values across five bands for "Forced Bacterial" and "Consensus Bacterial" conditions. The "Forced Bacterial" plot (N=1222) shows LR values of 0.16, 0.61, 0.95, 1.96, and 5.24 for bands 1 through 5, respectively. The "Consensus Bacterial" plot (N=729) shows LR values of 0.08, 0.54, 1.14, 2.50, and 8.04 for the same bands, with the y-axis representing the LR value on a logarithmic scale.

B)

LR, likelihood ratio; LR is calculated as nominal likelihood on a log scale Band 1=Very Low, Band 2=Low, Band 3=Moderate, Band 4=High, Band 5=Very High.

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Image /page/35/Picture/0 description: The image contains the logo for "Inflammatix" in a stylized font, with the first part of the word in orange and the rest in black. To the left of the word is a shield-like design with vertical lines in blue and orange. Below the logo is the word "TriVerity" with the trademark symbol.

Image /page/35/Figure/2 description: The image shows two plots, (A) Forced Cohort and (B) Consensus Cohort, displaying LR values for Forced Viral (N=1222) and Consensus Viral (N=729) respectively. Both plots have a y-axis labeled "LR value" on a logarithmic scale and an x-axis representing different bands with corresponding N values. In plot A, the LR values range from 0.25 to 20.04, while in plot B, they range from 0.09 to 40.93, indicating varying levels of viral presence across the bands in each cohort.

Figure 5: Likelihood Ratios for Viral Infection by TriVerity Interpretation Band: (A) Forced Cohort, (B) Consensus Cohort

LR, likelihood ratio; LR is calculated as nominal likelihood on a log scale Band 1=Very Low, Band 2=Low, Band 3=Moderate, Band 4=High, Band 5=Very High.

Image /page/35/Figure/5 description: The image is titled "Figure 6: Likelihood Ratios for Need of Mechanical Ventilation, Vasopressors and Renal Replacement Therapy Within 7 Days by TriVerity Interpretation Band". The title describes the figure as showing likelihood ratios for the need of mechanical ventilation, vasopressors, and renal replacement therapy within 7 days. The data is categorized by TriVerity Interpretation Band.

Image /page/35/Figure/6 description: The image shows a plot titled "Illness Severity (N=1120)". The y-axis is labeled "LR value" and has a logarithmic scale. There are 5 data points, labeled Band 1 through Band 5, with corresponding sample sizes N = 377, N = 303, N = 229, N = 180, and N = 31, respectively. The LR values for each band are approximately 0.22, 0.43, 1.63, 2.41, and 11.33.

LR, likelihood ratio; LR is calculated as nominal likelihood on a log scale Band 1=Very Low, Band 2=Low, Band 3=Moderate, Band 4=High, Band 5=Very High.

Proprietary Information Information herein is not to be disclosed without permission from Inflammatix, Inc.

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Image /page/36/Picture/1 description: The image shows the logo for Inflammatix, a company that specializes in diagnostic testing. The logo features a shield with red and blue vertical lines on the left, followed by the company name in orange and black text. Below the company name is the word "TriVerity" with the trademark symbol.

Of importance, the point estimates of the band likelihood ratios are monotonically increasing, and the 80% Cls of the likelihood ratios in nonadjacent bands are non-overlapping, for all scores.

The diagnostic accuracy of TriVerity appears superior to that of commonly used diagnostic biomarkers. The AUROC gap between the TriVerity bacterial score and PCT is of major clinical importance and statistically significant. In addition, PCT does not detect non-infectious disease nor severe illness in the ED, whereas TriVerity does.

Table 20: Accuracy of TriVerity Test Results Compared to Biomarkers Expressed as
Area Under the Receiver-Operating Characteristics

| Result Biomarker | N | AUROC | 80% CI
(lower - upper) |
|------------------------|--- | Diagnostic Results | | Forced Adjudication Protein | CRP | Forced Adjudication Protein | TriVerity Bacterial | | Forced Adjudication | Forced Adjudication | Forced Adjudication Cells | WBC | Forced Adjudication Cells | TriVerity Bacterial | | Consensus Adjudication Protein | CRP | Consensus Adjudication Protein | TriVerity Bacterial | | Consensus Adjudication | Procalcitonin | Consensus Adjudication | Procalcitonin | Consensus Adjudication Cells | WBC | Consensus Adjudication Cells | TriVerity Bacterial | | Population/Endpoint | Prognostic Population | Lactate | Prognostic Population | Lactate | Prognostic Population | qSOFA | Prognostic Population | qSOFA | Population/Endpoint | Comparator
--------------------|-------------------------|------|-------|---------------------------|
| | | | |
| C-Reactive
| 1200 | 0.73 | 0.71-0.75 |
| C-Reactive
| 0.77 | 0.75-0.78 |
| Procalcitonin | PCT | 1186 | 0.69 | 0.67-0.71 |
| Procalcitonin | TriVerity Bacterial | | 0.77 | 0.75-0.79 |
| White Blood
| 1179 | 0.70 | 0.68-0.72 |
| White Blood
| 0.76 | 0.75-0.78 |
| C-Reactive
| 714 | 0.74 | 0.72-0.77 |
| C-Reactive
| 0.83 | 0.81-0.85 |
| PCT | 711 | 0.71 | 0.68-0.73 |
| TriVerity Bacterial | | 0.83 | 0.81-0.85 |
| White Blood
| 709 | 0.76 | 0.73-0.78 |
| White Blood
| 0.83 | 0.81-0.85 |
| Prognostic Results | | | | |
| Lactate | 1058 | 0.76 | 0.73-0.80 |
| TriVerity Severity | 1058 | 0.78 | 0.75-0.80 |
| qSOFA | 1042 | 0.78 | 0.75-0.81 |
| TriVerity Severity | 1042 | 0.78 | 0.76-0.81 |

Table 21 shows the accuracy of TriVerity bacterial results vs. PCT results stratified by race. TriVerity bacterial results demonstrated markedly superior AUROCs compared to PCT across Whites, African American, and other races using forced and consensus adjudication. AUROCs ranged from 0.76 - 0.86 (forced adjudication) and from 0.82 - 0.91 (consensus adjudication) for the TriVerity bacterial result; for PCT they

Proprietary Information

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ranged from 0.55 - 0.73 (forced adjudication) and from 0.62 - 0.74 (consensus adjudication) across different races; ranges were narrow (max. 0.10) for the TriVerity bacterial results whereas they were markedly wider (max. 0.18) for PCT potentially indicating an impact of race on accuracy of PCT.

Table 21: Accuracy of TriVerity Bacterial Scores Compared to Procalcitonin by Race

| Population | Test | Race | N | AUROC | Lower
80% CI | Upper
80% CI |
|---------------------------|---------------------|------------------------------|-----|-------|-----------------|-----------------|
| Forced
Adjudication | TriVerity Bacterial | Black or African
American | 352 | 0.76 | 0.72 | 0.79 |
| | TriVerity Bacterial | Other¹ | 76 | 0.86 | 0.81 | 0.91 |
| | TriVerity Bacterial | White | 758 | 0.76 | 0.74 | 0.78 |
| Forced
Adjudication | Procalcitonin | Black or African
American | 352 | 0.63 | 0.59 | 0.67 |
| | Procalcitonin | Other¹ | 76 | 0.55 | 0.47 | 0.64 |
| | Procalcitonin | White | 758 | 0.73 | 0.70 | 0.75 |
| Consensus
Adjudication | TriVerity Bacterial | Black or African
American | 218 | 0.83 | 0.80 | 0.87 |
| | TriVerity Bacterial | Other¹ | 44 | 0.91 | 0.86 | 0.96 |
| | TriVerity Bacterial | White | 449 | 0.82 | 0.79 | 0.84 |
| Consensus
Adjudication | Procalcitonin | Black or African
American | 218 | 0.66 | 0.62 | 0.71 |
| | Procalcitonin | Other¹ | 44 | 0.62 | 0.50 | 0.73 |
| | Procalcitonin | White | 449 | 0.74 | 0.71 | 0.77 |

4 for the calculation of AUROCs, racial groups with