Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on nucleic acid amplification (PCR) and melt analysis, with software interpreting fluorescent images. There is no mention of AI, ML, or related techniques for data analysis or interpretation.

Therapeutic

No.

This device is an in vitro diagnostic (IVD) test designed for the qualitative detection and identification of multiple bacterial, viral, and protozoan nucleic acids. Its intended use is for diagnostic purposes, providing results that are meant to be used in conjunction with other clinical data for patient management decisions, rather than for direct treatment or therapy.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the device is "for the simultaneous qualitative detection and identification of multiple bacterial, viral, and protozoan nucleic acids directly from EDTA whole collected from individuals with signs and or symptoms of acute febrile illness and known or suspected exposure to the target pathogens". This function of detecting and identifying pathogens to aid in understanding a patient's condition falls directly under the definition of a diagnostic device. Furthermore, the document repeatedly refers to its use in "diagnostic clinical specimens" and "For In Vitro Diagnostic Use."

SaMD (Software as a Medical Device)

No

The device is a nucleic acid-based test that requires specific hardware systems (BioFire® FilmArray® 2.0 and BioFire® FilmArray® Torch Systems) to perform the test. While software is involved in interpreting the data, the core functionality relies on physical reagents and hardware for nucleic acid purification and PCR.

IVD (In Vitro Diagnostic)

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

Here's why:

Explicit Statement: The document explicitly states "For In Vitro Diagnostic Use" in the Intended Use section.
Intended Use: The device is intended for the qualitative detection and identification of nucleic acids from clinical specimens (whole blood) to aid in the diagnosis of acute febrile illness. This is a classic definition of an in vitro diagnostic test.
Device Description: The description details a "multiplexed nucleic acid-based test designed to be used with BioFire® FilmArray® Systems" that performs nucleic acid purification and PCR, all of which are processes performed in vitro (outside the body) on a biological sample.
Performance Studies: The document describes various performance studies (clinical, analytical, reproducibility) conducted to evaluate the device's ability to accurately detect the target pathogens in clinical samples, which is a requirement for IVD devices.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The BioFire® Global Fever Special Pathogens Panel is a qualitative, mucleic acid-based test intended for use with BioFire® FilmArray® 2.0 and BioFire® FilmArray® Torch Systems. The BioFire Global Fever Special Pathogens Panel is for the simultaneous qualitative detection and identification of multiple bacterial, viral, and protozoan nucleic acids directly from EDTA whole collected from individuals with signs and or symptoms of acute febrile illness and known or suspected exposure to the target pathogens described below.

Pathogens identified:
Chikungunya virus Dengue virus (serotypes 1, 2, 3 and 4) Leishmania spp. that cause visceral leishmaniasis (e.g., L. donovani and L. infantum) Leptospira spp. Plasmodium spp. (including species differentiation of Plasmodium falciparum and Plasmodium vivax/ovale) West Nile virus

Pathogens presumptively identified:
Bacillus anthracis Crimean-Congo hemorrhagic fever virus Ebolavirus spp. Francisella tularensis Lassa virus Marburgvirus Yellow fever virus Yersinia pestis

Pathogens for which the panel provides presumptive identification results resting and confirmation procedures in consultation with the appropriate public health authorities for whom reports may be necessary.

Positive results do not rule out co-infections with pathogens not included on the BioFire Global Fever Special Pathogens Panel. Not all pathogens that cause acute febrile illness are detected by this test, and nectude infection with the pathogens targeted by the device and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Evaluation for more common causes of acute illness (e.g., infections of the upper and lower respiratory tract or gastroenteritis, as well as non-infectious causes) should be considered prith this panel. In the United States, patient travel history, exposure risk, and consultation of the CDC Yellow Book should be considered prior to use of the BioFire Global Fever Special Pathogens Panel as some pathogens are more common in certain geographical locations. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guided by the relevant public health authorities.

The BioFire Global Fever Special Pathogens Panel is indicatories having appropriate biosafety equipment, personal protective equipment (PPE), contamment facilities and persomel trained in the safe handling of diagnostic clinical specimens potentially containing any of the pathogens detected by this panel.

The BioFire Global Fever Special Pathogens Panel is indicated for use in laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.

For In Vitro Diagnostic Use.

Product codes (comma separated list FDA assigned to the subject device)

QVR, QMV, PMN

Device Description

The BioFire® Global Fever Special Pathogens Panel is a multiplexed nucleic acid-based test designed to be used with BioFire® FilmArray® Systems (BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch). The BioFire Global Fever Special Pathogens Panel pouch contains freezedried reagents to perform nucleic acid purification and nested, multiplexed polymerase chain reaction (PCR) with DNA melt analysis. The BioFire Global Fever Special Pathogens Panel conducts tests for the identification of bacterial, viral, and protozoan pathogens from whole blood specimens collected in EDTA tubes (Table 1). Results from the BioFire Global Fever Special Pathogens Panel are available in about 1 hour.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

laboratories having appropriate biosafety equipment, personal protective equipment (PPE), containment facilities and personnel trained in the safe handling of diagnostic clinical specimens potentially containing any of the pathogens detected by this panel.

laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.

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

Not Found

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

Prospective Clinical Study
Sample Size: 2139 prospectively collected whole blood specimens
Data Source: 11 sites contributed specimens from individuals who had a recorded or self-reported fever within the past two days.
Annotation Protocol: PPA for each analyte was calculated as 100% × (TP + FN). True positive (TP) indicates that both the BioFire Global Fever Special Pathogens Panel and the comparator method had a positive result for this specific analyte, and false negative (FN) indicates that the BioFire Global Fever Special Pathgogens Panel result was negative while the comparator result was positive. NPA was calculated as 100% × (TN / (TN + FP)). True negative (TN) indicates that both the BioFire Global Fever Special Pathogens Panel and the comparator method had negative results and a false positive (FP) indicates that the BioFire Global Fever Special Pathogens Panel result was positive, but the comparator result was negative.

Archived Specimen Study
Sample Size: 416 archived specimens
Data Source: Archived specimens with known analyte content and/or archived specimens with a high likelihood of containing a given analyte were tested. Wherever possible, archived whole blood specimens were tested; blood plasma and blood serum were tested when whole blood was not available. Specimens were collected from a range of ages and sexes.
Annotation Protocol: Specimens were coded and tested at clinical sites or shipped to BioFire Defense. Nucleic acid was extracted and tested using plate-based PCR comparator methods. Discrepant results (false positive/false negative) were further investigated.

Contrived Specimen Study
Sample Size: For each analyte, fifty (50) replicates were contrived.
Data Source: Residual human whole blood specimens from patients with signs/symptoms of acute febrile illness. Quantified isolates were used to prepare contrived specimens at a range of concentrations relative to the limit of detection (LoD).
Annotation Protocol: The positive percent agreement (PPA) and negative percent agreement (NPA) were defined as agreement between the BioFire Global Fever Special Pathogens Panel result and the known composition of the contrived specimen.

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

Prospective Clinical Study
Study Type: Clinical Performance, Prospective Study
Sample Size: 2139 specimens
Key Metrics: Sensitivity/Positive Percent Agreement (PPA) and Specificity/Negative Percent Agreement (NPA)
Key Results:

Chikungunya virus: PPA 100% (25/25), NPA 99.9% (1848/1850)
Crimean-Congo hemorrhagic fever virus: PPA 100% (1/1), NPA 100% (2138/2138)
Dengue virus: PPA 94.0% (266/283), NPA 100% (1592/1592)
Ebola virus: PPA - (0/0), NPA 100% (2139/2139)
Lassa virus: PPA - (0/0), NPA 100% (2139/2139)
Marbug virus: PPA - (0/0), NPA 100% (2139/2139)
West Nile virus: PPA 100% (1/1), NPA 100% (2138/2138)
Yellow fever virus: PPA - (0/0), NPA 100% (2139/2139)
Bacillus anthracis: PPA - (0/0), NPA 100% (2139/2139)
Francisella tularensis: PPA - (0/0), NPA 100% (2139/2139)
Leptospira spp.: PPA 93.8% (15/16), NPA 99.8% (1855/1859)
Yersinia pestis: PPA - (0/0), NPA 100% (2139/2139)
Leishmania spp.: PPA 100% (10/10), NPA 100% (2129/2129)
Plasmodium spp.: PPA 98.5% (338/343), NPA 99.2% (1519/1532)
Plasmodium falciparum: PPA 92.7% (230/248), NPA 99.8% (1624/1627)
Plasmodium vivax/ovale: PPA 92.7% (115/124), NPA 100% (1751/1751)

Archived Specimen Study
Study Type: Clinical Performance, Archived Specimen Study
Sample Size: 416 specimens
Key Metrics: PPA, NPA
Key Results:

Crimean-Congo hemorrhagic fever virus: PPA - (0/0), NPA 100% (281/281)
Ebolavirus spp.: PPA - (0/0), NPA 100% (279/279)
Lassa virus: PPA 83.3% (10/12), NPA 99.2% (265/267)
Marburgvirus sp.: PPA - (0/0), NPA 100% (279/279)
West Nile virus: PPA 90.8% (59/65), NPA 99.4% (345/347)
Yellow fever virus: PPA - (0/0), NPA 100% (279/279)
Bacillus anthracis: PPA - (0/0), NPA 100% (281/281)
Francisella tularensis: PPA - (0/0), NPA 100% (281/281)
Yersinia pestis: PPA - (0/0), NPA 100% (281/281)
Leishmania spp.: PPA - (0/0), NPA 100% (283/283)

Contrived Specimen Study
Study Type: Clinical Performance, Contrived Specimen Study
Sample Size: 50 replicates per analyte
Key Metrics: PPA, NPA
Key Results:

Bacillus anthracis: PPA 100% (50/50), NPA 100% (332/332)
CCHF virus: PPA 98% (98/100), NPA 100% (282/282)
Ebolavirus spp.: PPA 100% (50/50), NPA 100% (332/332)
Francisella tularensis: PPA 100% (50/50), NPA 100% (332/332)
Lassa virus: PPA 100% (50/50), NPA 100% (332/332)
Leishmania spp.: PPA 100% (50/50), NPA 100% (332/332)
Marburgvirus sp.: PPA 99% (99/100), NPA 100% (282/282)
West Nile virus: PPA 100% (50/50), NPA 99.7% (331/332)
Yellow fever virus: PPA 98% (49/50), NPA 100% (332/332)
Yersinia pestis: PPA 100% (50/50), NPA 100% (332/332)

Analytical Studies - Limit of Detection (LoD)
Study Type: Analytical Study
Key Metrics: LoD
Key Results: Confirmed LoD concentrations for all analytes and detection rate at 1x LoD, ranging from 6.4E+00 copies/mL (Crimean-Congo hemorrhagic fever virus) to 7.0E+04 copies/mL (Ebolavirus spp.). Estimated LoD values for infectious material were also provided.

Analytical Studies - Inclusivity (Reactivity)
Study Type: Analytical Study
Key Metrics: Detection of isolates
Key Results: The BioFire Global Fever Special Pathogens Panel was considered inclusive for most tested isolates (217 isolates of bacteria, viruses, fungi, and protozoa) if detected within 3x LoD. Some isolates showed reduced sensitivity or were not detected (e.g., Dengue virus DKA 811 strain, Zaire ebolavirus Mayinga).

Analytical Studies - Exclusivity (Specificity)
Study Type: Analytical Study
Key Metrics: Cross-reactivity
Key Results: No cross-reactivity observed for 217 on- and off-panel organisms tested at high concentrations. Cross-reactivity was identified for Plasmodium knowlesi and Plasmodium malariae with the Plasmodium vivax/ovale assay; Francisella hispaniensis and Francisella tularensis subsp. mediasiatica with the Francisella tularensis assay; O'nyong-nyong virus with the Chikungunya virus assay; Crithidia fasciculata and Leptomonas seymouri with the Leishmania spp. assay; and various Plasmodium spp. (non-human primates/rodents) with Plasmodium spp. and Plasmodium vivax/ovale assays.

Analytical Studies - Interference
Study Type: Analytical Study
Key Metrics: Interference by substances
Key Results: Most endogenous substances, exogenous substances (drugs), competitive microorganisms, and technique-specific substances showed no interference. Potential interference was observed for heparin and TRIzol when testing analytes at near-LoD concentrations.

Reproducibility (Overall Device)
Study Type: Reproducibility
Sample Size: 270 valid test results from 90 replicates per sample tested over different sites, operators, instruments, and reagent lots.
Key Metrics: Percent agreement with expected results
Key Results: Overall agreement with expected results was 96.8% (1307/1350, 95% CI: 95.7-97.2%) for the BioFire FilmArray 2.0.

Reproducibility Comparison Between BioFire FilmArray 2.0 and BioFire FilmArray Torch (Overall Device)
Study Type: Reproducibility Comparison
Sample Size: 90 replicates for each analyte concentration distributed equally over three BioFire FilmArray 2.0 systems and three BioFire FilmArray Torch systems.
Key Metrics: Overall agreement with expected results
Key Results: Overall agreement was 99.5% (1343/1350, 95% CI: 98.9-99.8%) for FilmArray 2.0 and 99.1% (1338/1350, 95% CI: 98.4-99.5%) for FilmArray Torch, demonstrating similar performance.

Specimen Storage (Overall Device)
Study Type: Specimen Stability
Key Metrics: Analyte Detections Over Total Number Tested
Key Results: 10/10 detections reported for tested analytes (Leptospira interrogans, Dengue virus, Leishmania donovani, Plasmodium falciparum) under all specified storage conditions: no storage, room temperature (18-21°C and 30°C) for up to 24 hours, refrigerated storage (2-8°C) for Day 1, Day 3, and Day 7, and ultra-low freezing (≤ -70°C).

Reproducibility (Assayed External Controls)
Study Type: Reproducibility
Sample Size: 135 tests for Positive control and 135 for Negative control on BioFire FilmArray 2.0; 135 tests for Positive control and 135 for Negative control on BioFire FilmArray Torch.
Key Metrics: Percent agreement between observed and expected results
Key Results:

BioFire FilmArray 2.0: Overall agreement 98.1% (265/270, 95% CI: 95.7-99.2%).
BioFire FilmArray Torch: Overall agreement 97.4% (263/270, 95% CI: 94.7-98.7%).

Repeatability (Assayed External Controls)
Study Type: Repeatability
Sample Size: 45 Positive and 45 Negative External Controls
Key Metrics: Percent agreement between observed and expected results
Key Results:

Positive Control: 100% (45/45) passed.
Negative Control: 100% (45/45) passed.

Clinical Evaluation (Assayed External Controls)
Study Type: Clinical Evaluation
Sample Size: 160 Positive controls and 159 Negative controls
Key Metrics: Percent Passed (%)
Key Results:

Positive Control: 98.8% (158/160) passed.
Negative Control: 98.7% (157/159) passed.
Overall: 98.7% (315/319) passed.

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

Sensitivity/Positive Percent Agreement (PPA), Specificity/Negative Percent Agreement (NPA), LoD (Limit of Detection), Percent agreement.

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

DEN200043, K170883

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

K143178, K160068

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

N/A

Summary

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March 22, 2023

Image /page/0/Picture/1 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health and Human Services logo. To the right of that is the FDA logo, which consists of a blue square with the letters "FDA" in white, followed by the words "U.S. FOOD & DRUG" in blue, and the word "ADMINISTRATION" in a smaller font size below that.

BioFire Defense, LLC David Rabiger Associate Director of Regulatory and Clinical Affairs 79 W 4500 S. Suite 14 Salt Lake City, Utah 84107

Re: K213362

Trade/Device Name: BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel Regulation Number: 21 CFR 866.4000 Regulation Name: Device To Detect And Identify Biothreat Microbial Agents In Human Clinical Specimens. Regulatory Class: Class II Product Code: QVR, QMV, PMN Dated: October 8, 2021 Received: October 12, 2021

Dear David Rabiger:

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

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's

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requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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

For comprehensive regulatory information about 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-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Noel J. Gerald -S

Noel J. Gerald, Ph.D. Branch Chief Bacterial Respiratory and Medical Countermeasures Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known)

K213362

Device Name

BioFire Global Fever Special Pathogens Panel

Indications for Use (Describe)

The BioFire® Global Fever Special Pathogens Panel is a qualitative, mucleic acid-based test intended for use with BioFire® FilmArray® 2.0 and BioFire® FilmArray® Torch Systems. The BioFire Global Fever Special Pathogens Panel is for the simultaneous qualitative detection and identification of multiple bacterial, viral, and protozoan nucleic acids directly from EDTA whole collected from individuals with signs and or symptoms of acute febrile illness and known or suspected exposure to the target pathogens described below.

Pathogens identified:

Chikungunya virus Dengue virus (serotypes 1, 2, 3 and 4) Leishmania spp. that cause visceral leishmaniasis (e.g., L. donovani and L. infantum) Leptospira spp. Plasmodium spp. (including species differentiation of Plasmodium falciparum and Plasmodium vivax/ovale) West Nile virus

Pathogens presumptively identified:

Bacillus anthracis Crimean-Congo hemorrhagic fever virus Ebolavirus spp. Francisella tularensis Lassa virus Marburgvirus Yellow fever virus Yersinia pestis

Pathogens for which the panel provides presumptive identification results resting and confirmation procedures in consultation with the appropriate public health authorities for whom reports may be necessary.

Positive results do not rule out co-infections with pathogens not included on the BioFire Global Fever Special Pathogens Panel. Not all pathogens that cause acute febrile illness are detected by this test, and nectude infection with the pathogens targeted by the device and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Evaluation for more common causes of acute illness (e.g., infections of the upper and lower respiratory tract or gastroenteritis, as well as non-infectious causes) should be considered prith this panel. In the United States, patient travel history, exposure risk, and consultation of the CDC Yellow Book should be considered prior to use of the BioFire Global Fever Special Pathogens Panel as some pathogens are more common in certain geographical locations. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guided by the relevant public health authorities.

The BioFire Global Fever Special Pathogens Panel is indicatories having appropriate biosafety equipment, personal protective equipment (PPE), contamment facilities and persomel trained in the safe handling of diagnostic clinical specimens potentially containing any of the pathogens detected by this panel.

The BioFire Global Fever Special Pathogens Panel is indicated for use in laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.

For In Vitro Diagnostic Use.

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

X Prescription Use (Part 21 CFR 801 Subpart D)

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

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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510(k) Summary

Submitter I.

BioFire Defense, LLC 79 West 4500 South, Suite 14 Salt Lake City, UT 84107 Phone: (801) 262-3592 Fax: (801) 447-6907

Contact Person: David Rabiger Date Prepared: 2022-Oct-07

II. Device

Name of Device: BioFire® Global Fever Special Pathogens Panel Common or Usual Name: Same Product Code: QMV Regulation: 21 CFR 866.3966 Classification Name: Device to detect and identify selected microbial agents that cause acute febrile illness Regulatory Class: Class II (Special Controls) Panel: Microbiology - 83

Additional Product Code: QVR Regulation: 21 CFR 866.4000 Classification Name: A multiplex nucleic acid detection system for biothreat agents Regulatory Class: Class II (Special Controls) Panel: Microbiology - 83

Predicate Devices III.

BioFire® Global Fever Panel (BioFire Defense, LLC) [DEN200043] This predicate has not been subject to a design-related recall.

FilmArray® NGDS Warrior Panel (BioFire Defense, LLC) [K170883] This predicate has not been subject to a design-related recall.

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IV. Device Description

The BioFire® Global Fever Special Pathogens Panel is a multiplexed nucleic acid-based test designed to be used with BioFire® FilmArray® Systems (BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch). The BioFire Global Fever Special Pathogens Panel pouch contains freezedried reagents to perform nucleic acid purification and nested, multiplexed polymerase chain reaction (PCR) with DNA melt analysis. The BioFire Global Fever Special Pathogens Panel conducts tests for the identification of bacterial, viral, and protozoan pathogens from whole blood specimens collected in EDTA tubes (Table 1). Results from the BioFire Global Fever Special Pathogens Panel are available in about 1 hour.

Type	Disease	Pathogen
Bacterial	Anthrax	Bacillus anthracis 1
	Leptospirosis	Leptospira spp.
	Plague	Yersinia pestis 1
	Tularemia	Francisella tularensis 1
Viral	Crimean-Congo hemorrhagic fever	Crimean-Congo hemorrhagic fever virus 1
	Chikungunya fever	Chikungunya virus
	Dengue fever	Dengue virus (serotypes 1, 2, 3 and 4)
	Lassa fever	Lassa virus 1
	Ebola virus disease	Ebolavirus spp. (Bundibugyo, Reston, Sudan, Taï Forest, Zaire) 1
	Marburg virus disease	Marburgvirus 1
	West Nile fever	West Nile virus
	Yellow fever	Yellow fever virus 2
Protozoan	Malaria	Plasmodium spp.
		Plasmodium falciparum
		Plasmodium vivax/ovale
	Visceral Leishmaniasis	Leishmania spp. including L. donovani and L. infantum

Table 1. Pathogens Detected by the BioFire Global Fever Special Pathogens Panel

1 Select agents are subject to additional requirements. Definitive identification requires confirmatory testing.

2 Definitive identification requires confirmatory testing.

A test is initiated by loading Hydration Solution into one port of the pouch and a whole blood specimen mixed with the provided Sample Buffer into the other port of the pouch. The pouch contains all the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and the Sample Buffer rehydrates the reagents. After the pouch is prepared, the BioFire® FilmArray® Software guides the user through the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, selecting the appropriate protocol, and initiating the run on the BioFire FilmArray system.

The BioFire FilmArray instruments contain a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting

6

channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and subsequent melt.

Nucleic acid extraction occurs within the BioFire FilmArray pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, a nested multiplexed PCR is executed in two stages. During the first stage, a single, large volume, multiplexed reverse transcription PCR (rt-PCR) reaction is performed. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Defense, LLC). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The second stage PCR and melt, or nested singleplex PCR, is performed in each well of the array. At the end of the second stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the array captures fluorescent images of the PCR2 reactions and software interprets the data.

Materials provided in each BioFire Global Fever Special Pathogens Panel Kit:

Individually packaged BioFire Global Fever Special Pathogens Panel Pouches ●
Single-use (1.0 mL) BioFire® FilmArray® Sample Buffer Tubes ●
Single-use pre-filled (1.5 mL) BioFire® FilmArray® Hydration Injection Vials ●
. Individually packaged BioFire® FilmArray® Sample Injection Vials
Individually packaged Transfer Pipettes
. BioFire Global Fever Special Pathogens Panel – Ouick Guide
Instructions available online at: www.biofiredefense.com 0 BioFire Global Fever Special Pathogens Panel – Instructions for Use

Materials required but not provided:

BioFire FilmArray System including: ●
- BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch Instrument System o including accompanying platform-specific core software
- BioFire® FilmArray® Pouch Loading Station O
- BioFire® Global Fever Special Pathogens Pouch Module Software is required to o run the BioFire Global Fever Special Pathogens Panel and is available by request at www.biofiredefense.com if not already installed on the instrument system.
10% bleach solution or a similar disinfectant ●

7

v. Intended Use

The BioFire® Global Fever Special Pathogens Panel is a qualitative, multiplexed, nucleic acid-based test intended for use with BioFire® FilmArray® 2.0 and BioFire® FilmArray® Torch Systems. The BioFire Global Fever Special Pathogens Panel is for the simultaneous qualitative detection and identification of multiple bacterial, viral, and protozoan nucleic acids directly from EDTA whole blood collected from individuals with signs and/or symptoms of acute febrile illness or recent acute febrile illness and known or suspected exposure to the target pathogens described below.

Pathogens identified:

Chikungunya virus Dengue virus (serotypes 1, 2, 3 and 4) Leishmania spp. that cause visceral leishmaniasis (e.g., L. donovani and L. infantum) Leptospira spp. Plasmodium spp. (including species differentiation of Plasmodium falciparum and Plasmodium vivax/ovale) West Nile virus

Pathogens presumptively identified:

Bacillus anthracis Crimean-Congo hemorrhagic fever virus Ebolavirus spp. Francisella tularensis Lassa virus Marburgvirus Yellow fever virus Yersinia pestis

Pathogens for which the panel provides presumptive identification results require additional testing and confirmation procedures in consultation with the appropriate public health authorities for whom reports may be necessary.

Positive results do not rule out co-infections with pathogens not included on the BioFire Global Fever Special Pathogens Panel. Not all pathogens that cause acute febrile illness are detected by this test, and negative results do not preclude infection with the pathogens targeted by the device and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Evaluation for more common causes of acute febrile illness (e.g., infections of the upper and lower respiratory tract or gastroenteritis, as well as non-infectious causes) should be considered prior to evaluation with this panel. In the United States, patient travel history, exposure risk, and consultation of the CDC Yellow Book should be considered prior to use of the BioFire Global Fever Special Pathogens Panel as some pathogens are more common in certain geographical locations. Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities.

8

The BioFire Global Fever Special Pathogens Panel is indicated for use in laboratories having appropriate biosafety equipment, personal protective equipment (PPE), containment facilities and personnel trained in the safe handling of diagnostic clinical specimens potentially containing any of the pathogens detected by this panel.

The BioFire Global Fever Special Pathogens Panel is indicated for use in laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.

For In Vitro Diagnostic Use.

9

Substantial Equivalence VI.

The BioFire Global Fever Special Pathogens Panel is substantially equivalent to the BioFire Global Fever Panel (previously known as the FilmArray Global Fever Panel) and the FilmArray NGDS Warrior Panel. The BioFire Global Fever Panel was granted De Novo classification on November 20, 2020 [DEN200043] and was categorized as a Class II device. The FilmArray NGDS Warrior Panel was cleared on June 22, 2017 and was also categorized as a Class II device [K170883].

In addition, the BioFire Global Fever Special Pathogens Panel is substantially equivalent to the FilmArray NGDS Warrior Panel in that they both have the ability to detect select agents in whole blood specimens. While there are differences between these panels regarding pouch chemistry, pouch protocols, and pouch module software, both panels were developed using the same FilmArray technology and design principles.

Table 2 outlines the similarities and differences between the BioFire Global Fever Special Pathogens Panel and the predicate devices.

| Element | Subject Device:
BioFire Global Fever Special
Pathogens Panel | Predicate:
BioFire Global Fever Panel
[DEN200043] | Predicate:
FilmArray NGDS Warrior Panel
[K170883] |
|-----------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Specimen Type | Whole Blood (collected in EDTA
tube) | Same as BioFire Global
Fever Special Pathogens
Panel | Whole Blood (collected in EDTA
tube), positive blood culture, or
sputum |
| Intended Use
Setting | Individuals with signs and/or
symptoms of acute febrile
illness or recent acute febrile
illness and known or suspected
exposure to pathogens on the
panel. | Same as BioFire Global
Fever Special Pathogens
Panel | Individuals with signs and
symptoms of infection from
biothreat agents and/or
individuals who are at risk for
exposure or may have been
exposed to pathogens tested by
the panel. |
| Element | Subject Device:
BioFire Global Fever Special
Pathogens Panel | Predicate:
BioFire Global Fever Panel
[DEN200043] | Predicate:
FilmArray NGDS Warrior Panel
[K170883] |
| Pathogens Detected | Identification:
Chikungunya virus, Dengue
virus (serotypes 1, 2, 3 and 4),
Leishmania spp., Leptospira
spp., Plasmodium spp.
(including species
differentiation of Plasmodium
falciparum and Plasmodium
vivax/ovale),
West Nile virus
Presumptive Identification:
Bacillus anthracis, Crimean-
Congo hemorrhagic fever virus,
Ebolavirus spp., Francisella
tularensis, Lassa virus,
Marburgvirus, Yellow fever
virus, Yersinia pestis | Identification:
Chikungunya virus, Dengue
virus (serotypes 1,2,3, and
4), Leptospira spp.,
Plasmodium spp. (including
species differentiation of
Plasmodium falciparum
and Plasmodium
vivax/ovale) | Presumptive Identification in
whole blood EDTA:
Bacillus anthracis, Coxiella
burnetii, Ebolavirus spp.,
Francisella tularensis,
Marburgvirus, Yersinia Pestis
Presumptive Identification in
positive blood culture:
Bacillus anthracis, Yersinia pestis
Presumptive Identification in
sputum:
Francisella tularensis, Yersinia
pestis |
| Analyte | RNA/DNA | Same as BioFire Global
Fever Special Pathogens
Panel | Same as BioFire Global Fever
Special Pathogens Panel |
| Technological
Principles | Highly multiplexed nested
nucleic acid amplification test
with melt analysis | Same as BioFire Global
Fever Special Pathogens
Panel | Same as BioFire Global Fever
Special Pathogens Panel |
| Instrumentation1 | BioFire FilmArray 2.0 and
BioFire FilmArray Torch | BioFire FilmArray 2.0 | BioFire FilmArray 2.0 |
| Time to result | About 1 hour | Same as BioFire Global
Fever Special Pathogens
Panel | Same as BioFire Global Fever
Special Pathogens Panel |
| Reagent Storage | Room temperature | Same as BioFire Global
Fever Special Pathogens
Panel | Same as BioFire Global Fever
Special Pathogens Panel |
| Test Interpretation | Automated test interpretation
and report generation. User
cannot access raw data. | Same as BioFire Global
Fever Special Pathogens
Panel | Same as BioFire Global Fever
Special Pathogens Panel |
| Controls | Two controls are included in
each reagent pouch to control
for sample processing and both
stages of PCR and melt
analysis. | Same as BioFire Global
Fever Special Pathogens
Panel | Same as BioFire Global Fever
Special Pathogens Panel |
| User Complexity | High | Moderate | Moderate |

Table 2. Comparison of the BioFire Global Fever Special Pathogens Panel and Predicate Devices

10

1 The BioFire FilmArray 2.0 [K143178] and BioFire FilmArray Torch [K160068] instruments are based on the same technological principles.

11

VII. Summary of Performance Data

Clinical Performance

Prospective Clinical Study

The prospective clinical study was designed to evaluate the sensitivity/positive percent agreement (PPA) and specificity/negative percent agreement (NPA) for each analyte of the BioFire Global Fever Special Pathogens Panel when testing prospectively collected whole blood specimens in the intended use setting.

Between March 2018 and March 2021, 11 sites contributed 2139 prospectively collected whole blood specimens from individuals who had a recorded or self-reported fever within the past two days. A summary of demographic information for the 2139 specimens included in the prospective study is given in Table 3.

	Demographic	Overall
Sex	Female	1095 (51.2%)
	Male	1044 (48.8%)
Age	50	360 (16.8%)
Total		2139

Table 3. Demographic Summary for Prospective BioFire Global Fever Special Pathogens Panel Clinical Evaluation

PPA for each analyte was calculated as 100% × (TP + FN)). True positive (TP) indicates that both the BioFire Global Fever Special Pathogens Panel and the comparator method had a positive result for this specific analyte, and false negative (FN) indicates that the BioFire Global Fever Special Pathogens Panel result was negative while the comparator result was positive. NPA was calculated as 100% × (TN / (TN + FP)). True negative (TN) indicates that both the BioFire Global Fever Special Pathogens Panel and the comparator method had negative results and a false positive (FP) indicates that the BioFire Global Fever Special Pathogens Panel result was positive, but the comparator result was negative. Results are summarized in Tables 4 (viruses), 5 (bacteria), and 6 (protozoa).

12

BioFire Global Fever		PPA			NPA
Special Pathogens
Panel Detected Result	Number
Tested	TP/(TP +
FN)	%	95% CI	TN/(TN +
FP)	%	95% CI
Chikungunya virusa	1875c	25/25	100%	86.7-
100%	1848/1850	99.9%	99.6-100%
Crimean-Congo
hemorrhagic
fever virus	2139	1/1	100%	20.7-
100%	2138/2138	100%	99.8-100%
Dengue virusb	1875c	266/283	94.0%	90.6-
96.2%	1592/1592	100%	99.8-100%
Ebola virus	2139	0/0	-	-	2139/2139	100%	99.8-100%
Lassa virus	2139	0/0	-	-	2139/2139	100%	99.8-100%
Marburg virus	2139	0/0	-	-	2139/2139	100%	99.8-100%
West Nile virus	2139	1/1	100%	20.7-
100%	2138/2138	100%	99.8-100%
Yellow fever virus	2139	0/0	-	-	2139/2139	100%	99.8-100%

Table 4. BioFire Global Fever Special Pathogens Panel Clinical Performance Summary - Viruses

ª Evidence of Chikungunya virus was found in 2/2 FP specimens by additional PCR.

ֿ Evidence of Dengue virus was found in 15/17 FN specimens were positive upon BioFire Global Fever Special Pathogens Panel retest and by additional PCR, two were positive Global Fever Special Pathogens Panel retest, and eight were detected only by additional PCR.

^ Comparator analysis was not performed for Chikungunya virus on specimens collected after September 2019.

| BioFire Global Fever Special
Pathogens Panel Detected
Result | Number
Tested | TP/(TP + FN) | PPA
% | 95% CI | TN/(TN + FP) | NPA
% | 95% CI |
|--------------------------------------------------------------------|------------------|--------------|----------|----------------|--------------|----------|------------|
| Bacillus anthracis | 2139 | 0/0 | - | - | 2139/2139 | 100% | 99.8-100% |
| Francisella tularensis | 2139 | 0/0 | - | - | 2139/2139 | 100% | 99.8-100% |
| Leptospira spp.a | 1875b | 15/16 | 93.8% | 71.7-
98.9% | 1855/1859 | 99.8% | 99.4-99.9% |
| Yersinia pestis | 2139 | 0/0 | - | - | 2139/2139 | 100% | 99.8-100% |

Table 5. BioFire Global Fever Special Pathogens Panel Clinical Performance Summary - Bacteria

ª Evidence of Leptospira spp. was found in 1/1 FN specimens by BioFire Global Fever Special Pathogens Panel retest and by additional PCR, and in 3/4 FP specimens by additional PCR.

b Comparator analysis was not performed for Leptospira on specimens collected after September 2019.

13

| BioFire Global Fever Special
Pathogens Panel Detected
Result | Number
Tested | TP/(TP + FN) | PPA | | NPA | |
|--------------------------------------------------------------------|------------------|--------------|-------|------------|-------|------------|
| | | | % | 95% CI | % | 95% CI |
| Leishmania spp. | 2139 | 10/10 | 100% | 72.2-100% | 100% | 99.8-100% |
| Plasmodium spp.a,b | 1875e | 338/343 | 98.5% | 96.6-99.4% | 99.2% | 98.6-99.5% |
| Plasmodium falciparumc | 1875e | 230/248 | 92.7% | 88.8-95.4% | 99.8% | 99.5-99.9% |
| Plasmodium vivax/ovaled | 1875e | 115/124 | 92.7% | 86.8-96.1% | 100% | 99.8-100% |
| TN/(TN +
FP) | 2129/2129 | | | | | |
| 1519/1532 | | | | | | |
| 1624/1627 | | | | | | |
| 1751/1751 | | | | | | |

Table 6. BioFire Global Fever Special Pathogens Panel Clinical Performance Summary – Protozoa

® Four (4/5) Plasmodium FN specimens were also P. falciparum FN and one (1/5) was P. vivax/ovale FN. Three (3/13) Plasmodium FP specimens were also P. falciparum FP.

ካ Evidence of Plasmodium spp. was found in 2/5 FN specimen was positive upon BioFire Global Fever Special Pathogens Panel retest and by additional PCR, and one was positive only upon BioFire Global Fever Special Pathogens Panel retest. Evidence of Plasmodium spp. was found in 11/13 FP specimens by additional PCR (10/13) or by species-level comparator assay (1/13).

^ Evidence of P. falciparum was found in 13/18 FN specimens were positive upon BioFire Global Fever Special Pathogens Panel retest and by additional PCR, one was positive only upon BioFire Global Fever Special Pathogens Panel retest, and nine were positive only by additional PCR. Evidence of P. falciparum was found in 2/3 FP specimens by additional PCR. d Evidence of P. vivax/ovale was found in 7/9 FN specimens were positive upon BioFire Global Fever Special Pathogens Panel retest and by additional PCR, two were positive Global Fever Special Pathogens Panel retest, and three were positive only by additional PCR.

e Comparator analysis was not performed for Plasmodium, P. falciparum, or P. vivax/ovale on specimens collected after September 2019.

Archived Specimen Study

Many of the analytes detectable by the BioFire Global Fever Special Pathogens Panel were not observed or were not encountered in large enough numbers in the prospective clinical evaluation to adequately demonstrate system performance. In this study, archived specimens with known analyte content and/or archived specimens with a high likelihood of containing a given analyte were tested with the BioFire Global Fever Special Pathogens Panel to supplement the prospective clinical evaluation data. Wherever possible, archived whole blood specimens were tested. Where no whole blood specimens could be obtained, blood plasma and blood serum were tested instead. Although plasma and serum are not the intended specimen type for the BioFire Global Fever Special Pathogens Panel, these blood components provide similar results to whole blood specimens in a matrix equivalency study. Archived specimens were collected from a range of ages and sexes (Table 7).

14

		Overall	Site 01 a	Site 02	Site 03 a
Sex	Female	160 (38.5%)	79 (39.7%)	49 (59.8%)	32 (23.7%)
	Male	148 (35.6%)	97 (48.7%)	33 (40.2%)	18 (13.3%)
	Unknown	108 (26.0%)	23 (11.6%)	0 (0%)	85 (63%)
Age	50	57 (13.7%)	22 (11.1%)	12 (14.6%)	23 (17%)
	Unknown	108 (26.0%)	23 (11.6%)	0 (0%)	85 (63%)
	Total	416	199	82	135

Table 7. Overall and Per Site Archived Demographic Analysis

a Demographic data was not available for 23 specimens from Site 01 and 85 specimens from Site 03.

Specimens were coded and either tested at the clinical site or shipped to BioFire Defense for testing on the BioFire Global Fever Special Pathogens Panel. Nucleic acid was extracted from each specimen and tested using plate-based PCR comparator methods. Samples for which false positive and/or false negative results (i.e., discrepant results) were obtained when comparing the BioFire Global Fever Special Pathogens Panel results to the comparator method results were further investigated. Results are summarized in Table 8.

15

Pathogena	PPA			NPA
	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Viruses
Crimean-Congo hemorrhagic fever
virus	0/0	-	-	281/281	100%	98.7-100%
Ebolavirus spp.b	0/0	-	-	279/279	100%	98.6-100%
Lassa virusb,c	10/12	83.3%	55.2-95.3	265/267	99.2%	97.3-99.8%
Marburgvirus sp.b	0/0	-	-	279/279	100%	98.6-100%
West Nile virusb,d,e,f	59/65	90.8%	81.3-95.7%	345/347	99.4%	97.9-99.8%
Yellow fever virusb	0/0	-	-	279/279	100%	98.6-100%
Bacteria
Bacillus anthracis	0/0	-	-	281/281	100%	98.7-100%
Francisella tularensis	0/0	-	-	281/281	100%	98.7-100%
Yersinia pestis	0/0	-	-	281/281	100%	98.7-100%
Protozoa
Leishmania spp.g	0/0	-	-	283/283	283/283	98.7-100%

Table 8. BioFire Global Fever Special Pathogens Panel Archived Performance Summary

ª Results are not shown for assays previously granted in DEN20043 (i.e., chikungunya virus, Leptospira spp., and Plasmodium spp.).

b Due to low specimen volume, comparator results for most pathogens were not obtained for two specimens were only tested for a subset of pathogens including Crimean-Congo hemorrhagic fever virus, Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Leishmania spp.

^ Evidence of Lassa virus was found in ½ FN specimens and ½ FP specimens by additional PCR.

ª A set of 133 specimens tested at Site 03 had been previously characterized and were expected to be negative for all panel targets or positive for West Nile virus. These specimens were only evaluated by comparator methods for West Nile virus. e Archived specimens included blood serum and blood plasma.

f Evidence of West Nile virus was found in 6/6 FN specimens were positive upon Global Fever Special Pathogens Panel retesting and three of these were also positive by additional PCR. The other two FN specimens were positive only by

additional PCR testing. Evidence of West Nile virus was detected in 1/2 FP specimens by additional PCR testing.

ß Two specimens tested at Site 03 had been previously characterized and were only tested on comparator assays for Leishmania spp.

16

Contrived Specimen Study

For analytes for which no archived specimens were available, or for which there were an insufficient number of archived specimens, testing was performed using contrived specimens. This study evaluated BioFire Global Fever Special Pathogens Panel sensitivity and specificity when testing whole-blood specimens contrived with Bacillus anthracis. Crimean-Congo hemorrhagic fever virus, Ebolavirus spp., Francisella tularensis, Lassa fever virus, Leishmania spp., Marburgvirus sp., West Nile virus, Yellow fever virus, and Yersinia pestis.

Contrived specimens were prepared using residual human whole blood specimens from patients with signs/symptoms of acute febrile illness. For each analyte, fifty (50) replicates were contrived using quantified isolates at a range of concentrations relative to the limit of detection (LoD). If known, clinically relevant concentrations were used to adjust testing levels. The contrived specimens also served as a negative replicate for all other analytes evaluated. Specimens were prepared and randomized such that the analyte status of each contrived specimen was blinded to the users performing testing. The positive percent agreement (PPA) and negative percent agreement (NPA) were defined as agreement between the BioFire Global Fever Special Pathogens Panel result and the known composition of the contrived specimen. A summary of the results is shown in Table 9.

	PPA			NPA
Analyte	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Bacillus anthracis	50/50	100	92.9-100	332/332	100	98.9-100
CCHF virusa	98/100b	98	93.0-99.5	282/282	100	98.7-100
Ebolavirus spp.	50/50	100	92.9-100	332/332	100	98.9-100
Francisella tularensis	50/50	100	92.9-100	332/332	100	98.9-100
Lassa virus	50/50	100	92.9-100	332/332	100	98.9-100
Leishmania spp.	50/50	100	92.9-100	332/332	100	98.9-100
Marburgvirus sp.a	99/100c	99	94.6-99.8	282/282	100	98.7-100
West Nile virus	50/50	100	92.9-100	331/332d	99.7	98.3-100
Yellow fever virus	49/50e	98	89.5-99.7	332/332	100	98.9-100
Yersinia pestis	50/50	100	92.9-100	332/332	100	98.9-100

Table 9. Summary of BioFire Global Fever Special Pathogens Panel Contrived Specimen Performance Data

ª Tested at additional concentrations to better represent clinically relevant range.

b Two false negative CCHF virus at 2× LoD.

c Single false negative Marburqvirus sp. at 10× LoD.

d Discrepancy testing showed near LoD levels of WNV in a single whole blood sample.

e Single false negative yellow fever virus at 5× LoD.

17

Select Analytical Studies

Limit of Detection

Table 10. Limit of Detection for BioFire Global Fever Special Pathogens Panel Test Results

1 Stock concentrations determined using commercially available quantitative real-time PCR assay kits.

19

Since decreased sensitivity may be observed due to nucleic acid damage when evaluating inactivated BSL3/4 pathogens, additional testing was performed using live strains within a subcontracted BSL3/4 laboratory.

The concentrations tested were based on the confirmed LoD for inactivated/attenuated material. In general, four test concentrations were tested for each analyte: 10×, 1×, 0.1×, and 0.01× the LoD for inactivated/attenuated stock. Some analytes required additional concentrations. For each concentration, four unique blood draws were individually spiked and tested on the BioFire Global Fever Special Pathogens Panel. The estimated LoD was identified as the lowest concentration at which 4/4 replicates were positive and fewer than four replicates were positive at the next lower concentration. The estimated LoD values for infectious material are provided in Table 11.

	Table 11, BioFire Global Fever Special Pathogens Panel Estimated LoD in Whole Blood for Live BSL3/4
Agents

| BioFire Global Fever
Special Pathogens Panel

Analyte	Species/Strain	Estimated LoD Concentration
		Copies/mL	Units/mL
Bacteria
Bacillus anthracis	Ames	6.4E+01	3.5E+00 cfu/mL
Francisella tularensis	subsp. tularensis Schu	1.2E+01	2.1E+02 cfu/mL
Yersinia pestis	CO92	1.5E+02	3.0E+01 cfu/mL
Viruses
CCHF Virus	IbAr10200	6.4E+02	2.9E+03 pfu/mL
Chikungunya virus	B8635	5.5E+02	4.4E+01 pfu/mL
Chikungunya virus	Indo23574	5.5E+02	9.0E+01 pfu/mL
Ebolavirus spp.	Bundibugyo virus / Uganda (811250)	7.0E+02	5.6E+00 pfu/mL
Ebolavirus spp.	Tai Forest virus / Taï Forest (Ivory Coast)	1.8E+02	N/A
Ebolavirus spp.	Reston virus / H-28	2.7E+03	4.7E+01 pfu/mL
Ebolavirus spp.	Sudan virus / Boniface	1.1E+02	5.3E+00 pfu/mL
Ebolavirus spp.	Zaire ebolavirus / Makona	1.1E+03	1.6E+01 pfu/mL
Lassa Virus	Josiah	5.6E+03	2.6E+01 pfu/mL
Marburg marburgvirus	Ci67	5.0E+02	1.5E+03 pfu/mL
Marburg marburgvirus	Ravn	2.6E+02	4.7E+01 pfu/mL
West Nile virus	Bz NY99 (lin. 1)	1.6E+02	9.5E+00 pfu/mL
Yellow fever virus	Asibi	1.2E+01	9.2E+00 pfu/mL

20

Inclusivity (Reactivity)

The analytical reactivity (inclusivity) of the BioFire Global Fever Special Pathogens Panel was evaluated by testing a diverse collection of strains/species/serotypes representing temporal, geographic, and genetic diversity. Available isolates were prepared as contrived whole blood samples with the isolate at a concentration near the LoD of the analyte. If the isolate was detected within 3× the LoD the BioFire Global Fever Special Pathogens Panel was considered inclusive for that isolate and those with similar sequences. Analytes that were detected at ≥10× the established LoD were considered to have reduced sensitivity.

Table 12 shows a summary of the BioFire Global Fever Special Pathogens Panel reactivity based on empirical data. Isolates used to determine LoD are bolded.

| Analyte Detected /
Tested | Isolates Tested | Source / ID Detected
Tested up to
100x LoD¹ | Limitations | |-------------------------------------------------------------------| | BACTERIA | | Bacillus
anthracis | 4/4 | | | | | | | Francisella
tularensis | 5/5 | | | | | | | | KM14S | BEI BEI / NR-573 | | Leptospira spp. | 19/19 | | HAI0156 (Copenhageni)
L495 (Manilae) | ATCC / 23581
BEI / NR-19891
BEI / NR-19816 | 3.4E+02 copies/mL
1.2E+03 copies/mL
| | | | | | | Veldrat Bataviae 46 (Javanica) | ATCC / 23580
ATCC / 43292 | 1.2E+03 copies/mL
1.0E+03 copies/mL | | | | | | | | | | | | | | Analyte Detected /
Tested | Isolates Tested | Source / ID Detected
Tested up to
100x LoD¹ | Limitations | | | | | | | | | | | | | | Yersinia pestis | 3/3 | | | | | | | | | Chikungunya
virus | 3/3 0810105CFHI | 5.5E+02 copies/mL | | (formerly NR-
50055) | 1.7E+03 copies/mL | | (formerly NR-
49901) | 1.4E+03 copies/mL | Crimean–
Congo
hemorrhagic
fever virus | 1/1 | | | Dengue virus | | | | | | | | | | | | | | V1792/2007 | BEI / NR-44083 | | (DENV 2_1) | ZeptoMetrix /
0810089CF | 3.4E+02 copies/mL | | (DENV 2_2) | BEI / NR-12221 | | of strain
DKA 811 |
| | | | | | | | | | V1002/2006 | BEI / NR-44085 | | 0810090CF | 1.3E+02 copies/mL sensitivity for | | | Analyte Detected /
Tested | Isolates Tested | Isolates Tested Detected
Tested up to
100x LoD¹ | Limitations | Ebolavirus | 6/6 BC188/97 | | | | V1329/2006 | BEI / NR-44087 | | 0810091CF | 6.4E+01 copies/mL | | sensitivity for
strain
D85-019 |
| | | | | | | | | | | Ebolavirus | 6/6 sensitivity for
inactivated
Zaire
ebolavirus
Mayinga |
| | | | | | | | | | | Lassa Virus | 1/1 | Marburgvirus | 3/3 | | | | | West Nile virus | 5/5 0810081cfhi | 2.3E+04 copies/mL | | | | 0810033cfhi | 1.1E+03 copies/mL | | 0810082cfhi | 2.2E+03 copies/mL | | | Yellow fever
virus | 1/1 | PROTOZOA | | Leishmania
spp. | 12/12 | | | | | | sensitivity for
braziliensis
and infantum
strains |
| | | | | | | | | | | | | | | | | Plasmodium
spp. | 10/10 | | | Analyte Detected /
Tested | Isolates Tested | Source / ID Detected
Tested up to
100x LoD¹ | Limitations | | (Plasmodium
spp. assay) | | | | | | | | | | | | | | | 026015 | 1.9E+02 copies/mL | Plasmodium
falciparum
(Plasmodium
falciparum
assay) | 4/4 | falciparum | | SenTh021.09 |
| | | | | Plasmodium
vivax/ovale
(Plasmodium
vivax/ovale
assay) | 4/4 | vivax | | | | | | | Analyte Detected/Tested | Isolates Tested | UCC1 ID Detected | Limitations | Bacillus
anthracis | 10/12 | | | | | | | | | | | | | | | | | | | | | | | | | Francisella
tularensis | 9/9 | | | | | | | | | | | | | | DB082106G | FRAN035 | | | | | GA993550 | FRAN003 | Yersinia
Pestis | 12/12 | | | | | | | | | | | | | | | | | | | | | | | | | | | Zaire
ebolavirus | 4/4 | | | | | | | | | | | Lassa virus | 2/2 | | | | | Analyte Detected/Tested | Isolates Tested | Detected | Limitations | Marburg
Marburgvirus | 2/2 | | | | | | | | | West Nile
virus
(Lineage 1) | 1/1 Virus |
| | | Yellow fever
virus | 4/4 | | | | | | | | | # Isolates
| Concentration
|
------------------------------------|------------------------|---------------------------------------------------------------|--------------------------------------------------------|--------------------------------------------------------|-----------------------------------------------------------------------------|
| | | | | |
| Ames35 | BEI / NR-10355 | 4.2E+01 copies/mL | None | |
| Sterne 34Fs | BEI / NR-1400 | | | |
| UM23 | BEI / NR-10351 | 1.3E+02 copies/mL | | |
| Weybridge | BEI / NR-10350 | | | |
| Subspecies | | | | |
| tularensis | SCHU S4 | BEI / NR-15753 | 1.2E+03 copies/mL | |
| holarctica | LVSR | BEI / NR-597 | | |
| | Type B LVS (CDC) | BEI / NR-646 | 3.6E+03 copies/mL | |
| novicida | CG62
/ NR-580
| |
| Species | | | | |
| interrogans | Serovar (Budapest)
|
| alexanderi | L60 (Manhao 3) | ATCC / 700520 | 1.0E+03 copies/mL | |
| alstonii | Sichuan 79601 | ATCC / BAA-2439 | 1.2E+03 copies/mL | |
| borgpetersenii | Castellon 3 (Castellonis)
|
| kirschneri | 200701401 (Bogvere) | BEI / NR-19942 | 1.2E+03 copies/mL | |
| | 3522 C (Cynopteri) | ATCC / 49945 | 5.3E+02 copies/mL | |
| kmetyi | Bejo-Iso9T (Malaysia) | BEI / NR-22254 | | |
| mayottensis | 200901116 (undesignated) | KIT / 0254 | 1.2E+03 copies/mL | |
| noguchii | CZ 214T (Panama) | BEI / NR-22283 | | |
| santarosai | LT 821 (Shermani) | ATCC / 43286 | 8.7E+02 copies/mL | |
| # Isolates
| Concentration
|
| 6712 | KIT / 0220 | 1.2E+03 copies/mL | | |
| 94-79970/3 (Topaz) | KIT / 0237 | | | |
| A 102 (Mengrun) | KIT / 0023 | | | |
| weilii | Celledoni 20160426 | | | ATCC / 43285 |
| H 27 (Hekou) | KIT / 0074 | | | |
| LT 89-68 (Vughia) | KIT / 0127 | | | |
| Biovar | Strain | | None | |
| Orientalis | A1122 | BEI / NR-636 | | 1.3E+02 copies/mL |
| Orientalis | PY-013 | BEI / NR-51666 | | 3.9E+02 copies/mL |
| Antiqua | PH 80/63 | BEI / NR-51667 | | |
| VIRUSES | | | | |
| R80422 | ZeptoMetrix /
| None | |
| DHS4263 | BEI / NR-50884
| | |
| St. Martin 2013 | BEI / NR-50883
| | |
| Nigeria / IbAr10200 | | BEI / NR-37383 | 6.4E+00 copies/mL | None |
| Serotype | Strain | ZeptoMetrix / | | |
| 27/28 | Serotype 1 | Hawaii | 0810088CF | 2.2E+02 copies/mL | None |
| | Strain 12150 | BEI / NR-3785 | | |
| | 228690 | BEI / NR-3786 | | |
| | 276RK1 | BEI / NR-3782 | | |
| | BC89/94 | BEI / NR-3787 | 6.6E+02 copies/mL | |
| | SL-6-6-04 | BEI / NR-49744 | | |
| | UIS 1162 | BEI / NR-49707 | | |
| | VN/BID-
| | |
| | New Guinea C
| |
| | DakArA1247
| 2.7E+03 copies/mL | |
| Serotype 2 | 1349 | BEI / NR-12219 | | Not inclusive
| | 429557 | BEI / NR-12216 | 1.1E+03 copies/mL | |
| | ArA6894 | BEI / NR-12220 | | |
| | BC102/94 | BEI / NR-3789 | 9.4E+02 copies/mL | |
| | DKA 811 | BEI / NR-49747 | Not Detected | |
| | VN/BID-
| 1.1E+03 copies/mL | |
| Serotype 3 | H87 | ZeptoMetrix /
| Reduced
|
| | 271242 | BEI / NR-3802 | 3.9E+02 copies/mL | |
| # Isolates
| Source / ID | Concentration
|
| | BC188/97 | BEI / NR-3801 | 1.6E+04 copies/mL | strain
|
| | C0360/94 | BEI / NR-48800 | | BC188/97 |
| | VN/BID-
| 3.9E+02 copies/mL | |
| | H241 | ZeptoMetrix /
| |
| Serotype 4 | 703 | BEI / NR-48801 | | Reduced
| | BC13/97 | BEI / NR-3805 | | |
| | BC287/97 | BEI / NR-3806 | 1.9E+02 copies/mL | |
| | BC258/97 | BEI / NR-3807 | | |
| | D85-019 | BEI / NR-3804 | 7.6E+03 copies/mL | |
| | PR 06-65-740 | BEI / NR-49757 | 2.2E+02 copies/mL | |
| | Bundibugyo Uganda | BEI / NR-31813 | 7.0E+04 copies/mL | Reduced
| | Reston MKY 53 | BEI / NR-44238 | 2.7E+04 copies/mL | |
| | Sudan | BEI / NR-31810 | 1.1E+04 copies/mL | |
| | Taï Forest | BEI / NR-44241 | 8.3E+03 copies/mL | |
| Zaire | Guéckédou | BEI / NR-49462 | 1.0E+02 copies/mL | |
| Zaire | Mayinga | BEI / NR-31807 | 1.1E+04 copies/mL | |
| | Josiah | BEI / NR-31822 | 5.6E+04 copies/mL | None |
| | Marburg virus Musoke | BEI / NR-48951 | 5.0E+02 copies/mL | |
| | Marburg virus Voege | BEI / NR-31816 | 1.5E+03 copies/mL | None |
| | Ravn virus Kenya | BEI / NR-31819 | 2.6E+02 copies/mL | |
| | B-956 Uganda (lin. 2) | Zeptometrix/
| |
| | B-956 (lin. 2) | BEI / NR-50885 | 6.9E+04 copies/mL | |
| | NY 2001-6263 (lin. 1) | Zeptometrix/
| None |
| | 1986 (lin. 1) | Zeptometrix/
| |
| Bird 114 (lin. 1) | BEI / NR-50886 | 3.3E+03 copies/mL | | |
| | 17D | BEI / NR-116 | 1.2E+02 copies/mL | None |
| | | | | |
| | donovani 9515 | BEI / NR-48822 | 1.0E+01 copies/mL | |
| | amazonensis | BEI / NR-49247 | 3.0E+01 copies/mL | |
| | braziliensis Vianna | ATCC / 30879 | 1.0E+02 copies/mL | |
| | donovani, 1S | BEI / NR-48821 | 3.4E+01 copies/mL | Reduced
| | donovani chagasi | ATCC / 50133 | 3.0E+01 copies/mL | |
| | infantum | ATCC / 50134 | 1.0E+02 copies/mL | |
| | major IR173 | BEI / NR-48816 | 3.0E+01 copies/mL | |
| | mexicana MHOM/BZ/82/BEL21 | ATCC / 50157 | 2.9E+01 copies/mL | |
| | panamensis PSC-1 | BEI / NR-50162 | | |
| | tropica (MHOM/AF/87/RUP) | BEI / NR-48820 | 3.0E+01 copies/mL | |
| | tropica (MHOM/SU/58/strain-OD) | ATCC / 50130 | | |
| | venezuelensis MHOM-VE/80/H-16 | BEI / NR-29184 | 3.4E+01 copies/mL | |
| | falciparum | BEI / MRA-1238 | 1.8E+02 copies/mL | None |
| | | BEI / MRA-1182 | 3.0E+02 copies/mL | |
| # Isolates
| Concentration
|
| St. Lucia | BEI / MRA-331 | | | |
| Tanzania, 02000708 | BEI / MRA-1169 | | | |
| Chesson | BEI / MRA-383 | 1.5E+02 copies/mL | | |
| vivax | Panama | ATCC / 30138 | 3.1E+02 copies/mL | |
| ovale | Wallikeri | CDC / N8K9QK19 | 2.4E+02 copies/mL | |
| Curtisi | CDC / N8K9QL0S | 7.2E+02 copies/mL | | |
| knowlesi | Strain H | BEI / MRA-456G | 2.4E+01 copies/mL | |
| malariae | Clinical Sample | DLS / DLS17-
| |
| IPC 4884 | BEI / MRA-1238 | 1.8E+02 copies/mL | Reduced |
| | SenTh021.09 | BEI / MRA-1182 | 1.8E+03 copies/mL | sensitivity for
| | St. Lucia | BEI / MRA-331 | 3.0E+02 copies/mL | |
| | Tanzania, 02000708 | BEI / MRA-1169 | | |
| Chesson | BEI / MRA-383 | 1.5E+02 copies/mL | |
| | Panama | ATCC / 30138 | 3.1E+02 copies/mL | |
| ovale | Wallikeri | CDC / N8K9QK19 | 2.4E+02 copies/mL | None |
| | Curtisi | CDC / N8K9QL0S | 7.2E+02 copies/mL | |
| # Isolates
| Concentration
| |
| Ames | BACI008 | 6.4E+01 copies/mL | Reduced sensitivity for strains SK-102 and Vollum 1B | |
| 108 | BACI226 | 1.9E+02 copies/mL | | |
| 2002013094 | BACI293 | 1.9E+02 copies/mL | | |
| Canadian Bison | BACI153 | 1.9E+02 copies/mL | | |
| K3 | BACI261 | 1.9E+02 copies/mL | | |
| Ohio ACB | BACI259 | 1.9E+02 copies/mL | | |
| PAK-1 | BACI309 | 1.9E+02 copies/mL | | |
| RA3 | BACI225 | 1.9E+02 copies/mL | | |
| SK-102 (Pakistan) | BACI126 | 6.4E+02 copies/mL | | |
| South Africa (BA 1035) | BACI207 | 1.9E+02 copies/mL | | |
| Sterne | BACI012 | 1.9E+02 copies/mL | | |
| Turkey #32 | BACI260 | 1.9E+02 copies/mL | | |
| Vollum 1B | BACI124 | 6.4E+02 copies/mL | | |
| Subspecies | Strain | | None | |
| | Schu4 | FRAN016 | | 1.2E+01 copies/mL |
| tularensis | Scherm | FRAN031 | | 3.6E+01 copies/mL |
| | WY96 | FRAN072 | | 3.6E+01 copies/mL |
| | Holarctica, LVS | FRAN004 | | 3.6E+01 copies/mL |
| | Holarctica | FRAN012 | | 3.6E+01 copies/mL |
| holarctica | Holarctica, 425 | FRAN029 | | 3.6E+01 copies/mL |
| | HD,
| | 2.3E+01 copies/mL |
| | VT68 | FRAN025 | | 3.6E+01 copies/mL |
| | F6168 | FRAN134 | | 3.6E+01 copies/mL |
| novicida | U112,
| 3.6E+01 copies/mL | | |
| Biovar | Strain | | None | |
| | CO92 | YERS023 | | 1.5E+02 copies/mL |
| | A1122 | YERS078 | | 4.5E+02 copies/mL |
| | Dodson 2 | YERS073 | | 3.6E+02 CFU/mL |
| Orientalis | Java 9 | YERS022 | | 4.5E+02 copies/mL |
| | PBM19 | YERS018 | | 4.5E+02 copies/mL |
| | Shasta | YERS074 | | 4.5E+02 copies/mL |
| | Angola | YERS080 | | 4.5E+02 copies/mL |
| | Antigua | YERS016 | | 4.5E+02 copies/mL |
| Antiqua | Nairobi | YERS017 | | 4.5E+02 copies/mL |
| | Pestoides F 2 | YERS020 | | 3.6E+02 CFU/mL |
| | Harbin 35 | YERS021 | | 5.3E+02 copies/mL 3 |
| Medievalis | KIM5 | YERS082 | | 6.4E+02 copies/mL 3 |
| | Nicholisk 41 | YERS083 | | 4.5E+02 copies/mL |
| Strain/Isolate | | | None | |
| Makona – SL3864.1 | | Ebola027 | | 1.1E+03 copies/mL |
| Mayinga (Zaire 76) | | Ebola001 | | 2.1E+03 copies/mL |
| Gabon | | Ebola034 | | 3.2E+03 copies/mL |
| Kikwit '95 | | Ebola007 | | 3.1E+03 copies/mL |
| Luebo | | Ebola035 | 3.1E+03 copies/mL | |
| Josiah | | Arena002 | 2.4E+01 PFU/mL | None |
| Macenta 4 | | Arena009 | 7.2E+01 PFU/mL | |
| | | Arena009 | 7.2E+01 PFU/mL | |
| # Isolates
| UCC1 ID | Concentration
|
| Lineage | Strain | | | None |
| Ravn virus | RAVN | Marb002 | 2.6E+02 copies/mL | |
| Marburg virus | Ci67 | Marb003 | 5.0E+02 copies/mL | |
| | Angola | Marb005 | 1.5E+03 copies/mL | |
| | Musoke | Marb001 | 7.8E+02 copies/mL | |
| Bz NY99 | | Flavi022 | 1.6E+02 copies/mL | None for West Nile
| Eg101 | | Flavi016 | 4.8E+02 copies/mL | Lineage 1 |
| Asibi | | FLAVI005 | 1.2E+01 copies/mL | None |
| SVM 3-18-09 | | BEI NR-49799 | 1.2E+04 copies/mL | Reduced sensitivity5 |
| CAREC M2-09 | | BEI NR-50062 | 1.2E+03 copies/mL | |
| INHRR 7a-05 | | BEI NR-50071 | 1.2E+03 copies/mL | |
| INHRR 10a-10 | | BEI NR-50063 | 1.2E+03 copies/mL | |

Table 12. BioFire Global Fever Special Pathogens Panel Analytical Reactivity (Inclusivity)

21

BioFire® Global Fever Special Pathogens Panel 510(k) Summary
BioFire Defense, LLC

22

23

¹ Test concentrations were based on the LoD for the analyte in copies/mL with stock concentrations determined using a commercial qPCR assay.

Additionally, detection of BSL 3/4 infectious strains/isolates/serovars/species of Bacillus anthracis, Francisella tularensis, Yersinia pestis, West Nile virus, Zaire ebolavirus, Lassa virus, Marburgvirus, Yellow fever virus were evaluated by spiking whole blood at near infectious estimated LoD levels. Analytes detected only at ≥10-fold the estimated LoD were considered to have reduced sensitivity. Results are summarized in Table 13. Isolates used to evaluate the infectious estimated LoD are bolded.

24

Table 13. Summary of BioFire Global Fever Special Pathogens Panel Reactivity (Inclusivity) for Infectious BSL3/4 Analytes

BioFire® Global Fever Special Pathogens Panel 510(k) Summary BioFire Defense, LLC

25

4 U.S. Department of Defense Unified Culture Collection

2 The Y. pestis bv. Orientalis Dodson and Y. pestis bv. Antiqua Pestoides F strains lack the pPCP plasmid targeted by the qPCR assay, and as a result the nucleic acid concentration could not be quantified. Inclusivity for these two strains was therefore based on concentrations obtained for these two strains, and the CO92 reference strain, by enumeration in Colony Forming Units (CFU)/mL.

3 After testing was completed, it was determined that Yersinia pestis bv. Medievalis strains Harbin35 and KIM5 were evaluated at higher than 3× the estimated LoD, at 3.5× and 4.2×, respectively. Based on amplification data it is expected that these two Y. pestis strains will be detected at 3×LoD.

4 The Lassa virus Macenta and Pinneo strains were not detected by the Josiah strain evaluated in the Estimated LoD study was. Therefore, inclusivity testing for Lassa virus was performed based on concentrations obtained by enumeration in Plaque Forming Units (PFU)/mL.

5 YFV strains SVM 3-18-09, CAREC M2-09, NHRR 7a-05, and INHRR 10a-10 were isolated from neighboring regions (Trinidad and Venezuela). In silico analyses indicate these strains are closely related and do not represent a broad diversity of sequences.

Exclusivity (Specificity)

Analytical specificity (exclusivity) of the BioFire Global Fever Special Pathogens Panel assays was evaluated by challenging the system with a large collection of organisms and viruses prepared at high concentrations. On-panel organisms and viruses were tested to assess the potential for intra-panel cross-reactivity. Off-panel organisms and viruses (those not intended to be detected by the panel) were tested to assess the potential for non-specific amplification of other pathogens or potential contaminants. Organisms and viruses for off-panel testing were selected based on a combination of several factors including: relatedness to specific species detected by the BioFire Global Fever Special Pathogens Panel (near-neighbors); clinical relevance (cause illness or symptoms similar to the panel pathogens); likelihood of being present in blood as a co-infection based on a geographical region or specific population to which a panel pathogen is endemic; and genetic similarity to BioFire Global Fever Special Pathogens Panel assay primers, as determined by in silico analyses.

On-panel and off-panel testing included 217 isolates of bacteria, viruses, fungi, and protozoa tested at high concentration (typically >106 genomic copies/mL). Table 14 shows a complete list of the on- and off-panel bacteria, viruses, protozoa, and fungi that were tested and received the expected BioFire Global Fever Panel Special Pathogens test result (negative for all assays; no cross-reactivity) or for which in silico analysis does not predict cross-reactivity.

26

Table 14. On and Off Panel Organisms and Viruses with No Cross-Reactivity with BioFire Global Fever Special Pathogens Panel Assays

ON-PANEL
Bacteria
Bacillus anthracis	Leptospira interrogans
Francisella tularensis subsp.
tularensis	Yersinia pestis (2 strains: A1122 and CO92)
Viruses
Crimean-Congo hemorrhagic
fever virus	Zaire ebolavirus	Marburg Marburgvirus variant
Musoke
Chikungunya virus	Sudan ebolavirus	Marburg marburgvirus (RAVN)
Dengue virus Serotype 1	Bundibugyo ebolavirus	West Nile virus Lineage 1
Dengue virus Serotype 2	Taï Forest ebolavirus	West Nile virus Lineage 2
Dengue virus Serotype 3	Reston ebolavirus	Yellow fever virus
Dengue virus Serotype 4	Lassa virus
Protozoa
Leishmania donovani	Plasmodium vivax
Plasmodium falciparum		Plasmodium ovale
OFF-PANEL
Bacteria
Tested
Acinetobacter baumannii	Enterococcus faecium	Salmonella enterica subs. enterica
serovar Muenchen
Bacillus brevis	Francisella persica (formerly
Wolbachia persica)	Salmonella enterica subs. enterica
serovar Newport
Bacillus cereus	Francisella philomiragia (formerly
Yersinia)	Salmonella enterica subs. enterica
serovar Rubislaw
Bacillus circulans	Klebsiella oxytoca	Salmonella enterica susb. enterica
serovar Saintpaul
Bacillus coagulans	Legionella pneumophila	Salmonella enterica subs. enterica
serovar Tennessee
Bacillus halodurans	Leptospira biflexa	Salmonella enterica subs. enterica
serovar Thompson
Bacillus licheniformis	Leptospira meyeri	Salmonella enterica subs. enterica
serovar Typhimurium
Bacillus megaterium	Leptospira terpstrae genomospecies 4	Salmonella enterica subs. houtenae
Bacillus mycoides	Leptospira vanthielii genomospecies 3	Salmonella enterica subs. indica
Bacillus pumilus	Leptospira wolbachii	Salmonella enterica subs. salamae
Bacillus subtilis	Leptospira yanagawae
genomospecies 5	Salmonella enterica subsp. enterica
Paratyphi A
Bacillus thuringiensis	Listeria monocytogenes	Salmonella enterica subsp. enterica
Typhi
Bacteroides fragilis	Mycobacterium tuberculosis	Serratia marcescens
Bordetella bronchiseptica	Mycoplasma pneumoniae	Staphylococcus aureus
Borrelia burgdorferi	Neisseria meningitidis	Streptococcus agalactiae
Brucella melitensis	Orientia chuto (tsutsugamushi)	Streptococcus pneumoniae
Burkholderia cepacia	Proteus mirabilis	Streptococcus pyogenes

Burkholderia mallei	Pseudomonas aeruginosa	Treponema pallidum pallidum
Burkholderia pseudomallei	Rickettsia prowazekii	Vibrio cholerae
Chlamydophila pneumoniae	Rickettsia ricketsii	Yersinia aldovae
Chlamydophila psittaci	Rickettsia typhi	Yersinia bercovieri
Citrobacter koseri	Salmonella enterica subs.bongori	Yersinia entericolitica
Clostridium bifermentans	Salmonella enterica subs. arizonae	Yersinia fredericksenii
Clostridium
botulinum/sporogenes	Salmonella enterica subs. diarizoniae	Yersinia intermedia
Clostridium perfringens	Salmonella enterica subs. enterica
serovar Enteritidis	Yersinia kristensenii
Clostridium sordellii	Salmonella enterica subs. enterica
serovar Heidelberg	Yersinia mollaretii
Coxiella burnetii	Salmonella enterica subs. enterica
serovar Javiana	Yersinia pseudotuberculosis
Enterobacter aerogenes	Salmonella enterica subs. enterica
serovar Montevideo	Yersinia rohdei
Enterococcus faecalis	Yersinia similis	Yersinia ruckeri
	In silico Analysis Only
Bacillus luciferensis
	Viruses
	Tested
Adenovirus 1	HPIV-1	Omsk hemorrhagic fever
Adenovirus 3	HPIV-3	Parvovirus
Adenovirus 5	Hughes virus	Powassan virus
Aura virus	Human herpesvirus 6B	Rabies virus
Barmah Forest virus	Human immunodeficiency virus, type
1	Rift Valley fever virus
Bunyamwera virus	Human immunodeficiency virus, type
2	Zika virus
Coronavirus NL63	Human T-lymphotropic virus, type 1	Ross River virus
Cytomegalovirus	Human T-lymphotropic virus, type 2	Human respiratory syncytial virus
Dugbe virus	Influenza A H1N1-2009	Rubella virus
Eastern equine encephalitis virus	Influenza A H3N2	SARS-CoV-2
Enterovirus, HEV-71	Influenza B virus	Saint Louis encephalitis virus
Epstein Barr virus	Japanese encephalitis virus	Semliki Forest virus
Flexal virus	Junin virus (2 strains: XJ and Candid)	Sindbis virus
Guanarito virus	Machupo virus	Spondweni virus
Hantaan virus	Mayaro virus	Tickborne encephalitis virus
Hazara virus	Measles virus	Tonate virus
Hendra virus	Metapneumovirus	Una virus
Hepatitis A virus	Middelburg virus	Usutu virus
Hepatitis B virus	Mopeia virus	Vaccinia virus
Hepatitis C virus	Mumps virus	Varicella zoster virus
Herpes simplex virus type 2	Murray Valley encephalitis virus	Venezuelan equine
encephalomyelitis virus
In silico Analysis Only
Avalon virus	Lymphocytic choriomeningitis virus	Sabia virus (Brazilian hemorrhagic fever)
Bas-Congo virus	Pirital virus	Variola major
Protozoa
Tested
Babesia microti	Toxoplasma gondii	Trypanosoma cruzi
Cyclospora cayetanensis	Trypanosoma brucei	Trypanosoma cruzi
Fungus
Tested
Aspergillus fumigatus	Cryptococcus neoformans var. grubii
Helminths
Tested
Schistosoma mansoni

27

28

Table 15 lists the BioFire Global Fever Special Pathogens Panel assays and corresponding organisms and viruses for which some cross-reactivity was identified (either empirically observed in testing or predicted by in silico analyses).

| CROSS-REACTIVE ORGANISM | BIOFIRE GLOBAL FEVER SPECIAL PATHOGENS
PANEL TEST RESULT |
|----------------------------------------------|-------------------------------------------------------------|
| On-Panel | |
| Plasmodium knowlesi 1 | Plasmodium vivax/ovale Detected |
| Plasmodium malariae 2 | |
| Off-Panel | |
| Francisella hispaniensis 3 | Francisella tularensis Detected |
| Francisella tularensis subsp. mediasiatica 4 | |
| O'nyong-nyong virus | Chikungunya virus Detected |
| Crithidia fasciculata 5 | Leishmania spp. Detected |
| Leptomonas seymouri 6 | |
| Plasmodium berghei7 | |
| Plasmodium brasilianum 7 | Plasmodium spp. and
Plasmodium vivax/ovale Detected |
| Plasmodium cynomolgi 7 | |
| Plasmodium fieldi 7 | |
| Plasmodium fragile 7 | |
| Plasmodium inui 7 | |
| Plasmodium simiovale 7 | |

Table 15. Observed or Predicted Cross-Reactivity of the BioFire Global Fever Special Pathogens Panel

1 Cross-reactive with the Plasmodium vivax/ovale assay at concentrations ≥2.2E+04 copies/mL (*100×LoD).

2 In silico analysis predicts potential cross-reactivity with the Plasmodium vivax assay; cross-reactivity was not observed at the concentration evaluated, 1.9E+05 copies/mL.

3 Cross-reactivity was predicted by in silico analysis and observed during wet testing; Francisello hispaniensis is a pathogenic Francisella species.

4 In silico analysis predicts cross-reactivity; the potential for Francisella tularensis subsp. mediasatica to cause disease in humans is unknown.

5 Crithidia fasciculota is a non-human infective trypanosomatid, however there have been very rare cases where a closely related Crithidia strain was found to be infective and act much like Leishmania spp.

5 Leatomonos seymouri is an opportunistic parasite individuals, particularly those infected with visceral leishmaniasis.

7 Plasmodium spp. that typically infect non-human primates and rodents but are rarely found in humans.

BioFire® Global Fever Special Pathogens Panel 510(k) Summary BioFire Defense, LLC

29

Interference

Potentially interfering substances were selected for evaluation based upon whether the substance may normally be found in blood or may be introduced into blood specimens during collection, handling, or testing. These substances included endogenous substances (e.g., albumin, immunoglobulin, etc.), exogenous substances (e.g., antibiotic and antiviral drugs), microorganisms such as viral and bacterial species that may be present as co-infections, and technique-specific substances that may be introduced into a sample during routine laboratory handling including collection tubes.

The concentrations of endogenous and exogenous substances tested on the BioFire Global Fever Special Pathogens Panel were selected to represent a worst-case scenario based on a reference concentration of normal to high levels expected to be present in clinical specimens. Potentially competing microorganisms were tested at the highest levels possible based on stock concentrations. Technique-specific substances were tested at levels exceeding what would be expected to be found due to incidental contact, accidental addition, or during their use as solvents. Results are shown in Table 16.

Potential interference was only observed for heparin and TRIzol when testing analytes at near-LoD concentrations.

Potentially Interfering Substance	Concentration Tested	Results
Endogenous Substances
Albumin	60.0 mg/mL	No interference
Bilirubin (Conjugated)	0.41 mg/mL	No interference
Bilirubin (Unconjugated)	0.41 mg/mL	No interference
Cholesterol (total)	4.2 mg/mL	No interference
Glucose	10.1 mg/mL	No interference
Hemoglobin	137.0 mg/mL	No interference
Immunoglobulins	60.0 mg/mL	No interference
Triglycerides	15.1 mg/mL	No interference
White Blood Cells	6.1E+06 cells/mL	No interference
Exogenous Substances
Artemether-Lumefantrine	0.0004 mg/mL	No interference
Atovaquone	0.005 mg/mL	No interference
Proguanil	0.001 mg/mL	No interference
Mefloquine	0.0017 mg/mL	No interference
Amphotericin B	0.002 mg/mL	No interference
Pentamidine	0.0015 mg/mL	No interference
Fluconazole	0.026 mg/mL	No interference
Amoxicillin	0.062 mg/mL	No interference
Azithromycin	0.011 mg/mL	No interference
Ceftriaxone	1.0 mg/mL	No interference
Ciprofloxacin	0.012 mg/mL	No interference
Clindamycin	0.055 mg/mL	No interference
Doxycycline	0.02 mg/mL	No interference

Table 16. Results for Potentially Interfering Substances Tested on the BioFire Global Fever Special Pathogens Panel

30

Potentially Interfering Substance	Concentration Tested	Results
Gentamicin	0.036 mg/mL	No interference
Meropenem	0.39 mg/mL	No interference
Sulfamethoxazole	0.38 mg/mL	No interference
Vancomycin	0.12 mg/mL	No interference
Cycloserine	75.0 mg/mL	No interference
Isoniazid	0.06 mg/mL	No interference
Oseltamivir	0.0005 mg/mL	No interference
Ribavirin	0.011 mg/mL	No interference
Tenofovir	0.001 mg/mL	No interference
Acetaminophen	0.16 mg/mL	No interference
Aspirin (Acetylsalicylic Acid)	0.03 mg/mL	No interference
Ibuprofen	0.22 mg/mL	No interference
Prednisone	0.0001 mg/mL	No interference
Prednisolone	1.2 mg/mL	No interference
Cortisone	0.001 mg/mL	No interference
Artesunate	0.1 mg/mL	No interference
Competitive Microorganisms			Corynebacterium diphtheriae	1:10 of Stock	No interference	Staphylococcus epidermidis	3.8E+06 CFU/mL	No interference	Escherichia coli	1:10 of Stock	No interference	Klebsiella pneumoniae	5.5E+04 CFU/mL	No interference	Haemophilus influenzae	1.0E+08 CFU/mL	No interference	Herpes Simplex virus	1.2E+05 PFU/mL	No interference	Epstein-Barr virus	3.3E+07 copies/mL	No interference	Cytomegalovirus (CMV) AD-169	1:10 of Stock	No interference	Human Immunodeficiency virus
(HIV-1 and HIV-2)	1:10 of Stocks	No interference	Plasmodium vivax	1.5E+06 copies/mL	No interference	Technique Specific Substances			Bleach	1% v/v	No interference	Povidone-iodine	1% v/v	No interference	Ethanol	2% v/v	No interference	TRIzol	2-3% v/v	Potentially Interfering	DMSO	2% v/v	No interference	Methanol	2% v/v	No interference	Saline	2% v/v	No interference	Chloroform	2% v/v	No interference	Acetone	2% v/v	No interference	Hydrochloric Acid (HCl)	0.0005N	No interference	Blood Collection Tubes	Citrate (sodium)	~0.32%	No interference	EDTA in excess (5x)	~9.0 mg/mL	No interference	Heparin	~19.0 USP/mL	Potentially Interfering	Acid-citrate-dextrose (ACD)	2.2mg/mL (trisodium citrate)
0.8 mg/mL (citric acid)
2.5 mg/mL (dextrose)	No interference	Sodium polyanethenole sulfonate
(SPS)	0.72 mg/mL	No interference	Serum Separation Tubes	N/A	No interference
Competitive Microorganisms
Corynebacterium diphtheriae	1:10 of Stock	No interference
Staphylococcus epidermidis	3.8E+06 CFU/mL	No interference
Escherichia coli	1:10 of Stock	No interference
Klebsiella pneumoniae	5.5E+04 CFU/mL	No interference
Haemophilus influenzae	1.0E+08 CFU/mL	No interference
Herpes Simplex virus	1.2E+05 PFU/mL	No interference
Epstein-Barr virus	3.3E+07 copies/mL	No interference
Cytomegalovirus (CMV) AD-169	1:10 of Stock	No interference
Human Immunodeficiency virus
(HIV-1 and HIV-2)	1:10 of Stocks	No interference
Plasmodium vivax	1.5E+06 copies/mL	No interference
Technique Specific Substances
Bleach	1% v/v	No interference
Povidone-iodine	1% v/v	No interference
Ethanol	2% v/v	No interference
TRIzol	2-3% v/v	Potentially Interfering
DMSO	2% v/v	No interference
Methanol	2% v/v	No interference
Saline	2% v/v	No interference
Chloroform	2% v/v	No interference
Acetone	2% v/v	No interference
Hydrochloric Acid (HCl)	0.0005N	No interference
Blood Collection Tubes
Citrate (sodium)	~0.32%	No interference
EDTA in excess (5x)	~9.0 mg/mL	No interference
Heparin	~19.0 USP/mL	Potentially Interfering
Acid-citrate-dextrose (ACD)	2.2mg/mL (trisodium citrate)
0.8 mg/mL (citric acid)
2.5 mg/mL (dextrose)	No interference
Sodium polyanethenole sulfonate
(SPS)	0.72 mg/mL	No interference
Serum Separation Tubes	N/A	No interference

31

Reproducibility

Assay reproducibility was evaluated using three contrived whole blood samples with a mixture of four representative panel analytes: one bacterium, one virus, and two protozoa. For each analyte, one sample was spiked at a moderate positive (3×LoD) level, another sample at a low positive (1×LoD) level, and the third sample was not spiked (negative). Six replicates of each sample were tested at three locations on five different days, providing a total of 90 replicate test results per sample. On each test day at each site, two different operators used three BioFire FilmArray 2.0 instruments; the BioFire Global Fever Special Pathogens Panel pouch lot was rotated daily. In total, 270 valid test results were obtained for the reproducibility evaluation of the BioFire Global Fever Special Pathogens Panel.

The primary assessment of reproducibility is based on a comparison of the observed test results (Detected)Not Detected) to the expected test results (Detected for spiked samples, Not Detected for un-spiked samples). The detection rate and percent agreement between observed and expected test results are shown in Table 17. The expected percent agreement was >95%. Based on the percent agreement between observed and expected test results, the reproducibility evaluation demonstrates that the BioFire Global Fever Special Pathogens Panel can provide accurate and highly reproducible test results in the context of multiple variables that may be expected in a clinical testing environment, including analyte concentration, location, test day, operator, instruments, and reagent lot.

32

| Analyte
(Source / ID) | Concentration Tested
(copies/mL) | Expected Result | Detection Rate (n/N)
% Agreement with Expected Result
[95% Confidence Interval] | | | | Analyte
(Source / ID) | | Concentration
Tested
(copies/mL) | Expected
Result | BioFire FilmArray 2.0 Platform | | | All FA 2.0
Systems
[95% CI] | BioFire FilmArray Torch Platform | | | All FA Torch
Systems
[95% CI] | |
|-------------------------------------------------------------------------|--------------------------------------------------|-----------------------------------------------------|---------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------|----------------|------------------------------------|----------------------------------------------------------------------------|--------------------------------------------|----------------------------------------------------------------------------|-----------------------------------------|--------------------------------|----------------|----------------|-----------------------------------|----------------------------------|----------------|----------------|-------------------------------------|--------------------------------|
| | | | Site 1 | Site 2 | Site 3 | All Sites | | | | | System
1 | System
2 | System
3 | | System
1 | System
2 | System
3 | | |
| Leptospira interrogans
serovar icterohaemorrhagiae
(ATTC / 23581) | Moderate Positive
3x LoD
(1.0E+03) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | Leptospira interrogans
serovar
icterohaemorrhagiae
(ATTC / 23581) | | Moderate Positive
3xLoD
(1.0E+03) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | |
| | Low Positive
1x LoD
(3.4E+02) | Detected | 27/30
90.0% | 28/30
93.3% | 26/30
86.7% | 81/90
90.0%
[82.1-
94.6%] | | | Low Positive
1xLoD
(3.4E+02) | Detected | 29/30
96.7% | 29/30
96.7% | 28/30
93.3% | 86/90
95.6%
[89.1-98.3%] | 29/30
96.7% | 28/30
93.3% | 28/30
93.3% | 85/90
94.4%
[87.6-97.61] | |
| | Negative
(No Analyte) | Not Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | | | Negative
(No Analyte) | Not
Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | |
| Dengue virus
DENV-2
New Guinea C
(Zeptometrix / 0810089CF) | Moderate Positive
3x LoD
(1.0E+03) | Detected | 29/30
96.7% | 30/30
100% | 30/30
100% | 89/90
98.9%
[94.0-
99.8%] | Dengue virus
DENV-2
New Guinea C
(Zeptometrix / 0810089CF) | | Moderate Positive
3xLoD
(1.0E+03) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | |
| | Low Positive
1x LoD
(3.4E+02) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | | | Low Positive
1xLoD
(3.4E+02) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 29/30
96.7% | 30/30
100% | 89/90
98.9%
[94.0-99.8%] | |
| | Negative
(No Analyte) | Not Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | | | Negative
(No Analyte) | Not
Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 29/30
96.7% | 89/90
98.9%
[94.0-99.8%] | |
| Leishmania donovani
1S (MHOM/SD/62/1S)
(BEI / NR-48821) | Moderate Positive
3.4x LoD 1
(3.4E+01) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | Leishmania donovani
1S (MHOM/SD/62/1S)
(BEI / NR-48821) | | Moderate Positive
3xLoD
(3.0E+01) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | |
| | Low Positive
1.1x LoD 1
(1.1E+01) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | | | Low Positive
1xLoD
(1.0E+01) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | |
| | Negative
(No Analyte) | Not Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | | | Negative
(No Analyte) | Not
Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | |
| Plasmodium
falciparum | Plasmodium
spp. | Moderate Positive
1.5x LoD 2
(2.7E+02) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | Plasmodium
spp.
Detection
Results | Plasmodium
falciparum
IPC 4884
(BEI / MRA-
1238)
falciparum | Moderate Positive
3xLoD
(5.4E+02) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] |
| Analyte
(Source / ID) | | Concentration Tested
(copies/mL) | Expected Result | Detection Rate (n/N)
% Agreement with Expected Result
[95% Confidence Interval] | | | | | | Low Positive
1xLoD
(1.8E+02) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] |
| IPC 4884
(BEI / MRA-
1238) | Detection Results | Low Positive
$0.5\times LoD^2$
(9.0E+01) | Detected | 28/30
93.3% | 30/30
100% | 29/30
96.7% | 87/90
96.7%
[90.7-
98.9%] | | | Negative
(No Analyte) | Not
Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] |
| | | Negative
(No Analyte) | Not Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | | | Moderate Positive
3xLoD
(5.4E+02) | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 29/30
96.7% | 30/30
100% | 89/90
98.9%
[94.0-99.8%] |
| | Plasmodium
falciparum
Detection
Results | Moderate Positive
$1.5\times LoD^2$
(2.7E+02) | Detected | 29/30
96.7% | 30/30
100% | 28/30
93.3% | 87/90
96.7%
[90.7-
98.9%] | | | Low Positive
1xLoD
(1.8E+02) | Detected | 30/30
100% | 28/30
93.3% | 29/30
96.7% | 87/90
96.7%
[90.7-98.9%] | 30/30
100% | 28/30
93.3% | 28/30
93.3% | 86/90
95.6%
[89.1-98.3%] |
| | | Low Positive
$0.5\times LoD^2$
(9.0E+01) | Detected | 18/30
60.0% | 24/30
80.0% | 21/30
70.0% | 63/90
70.0%
[59.9-
78.5%] | | | Negative
(No Analyte) | Not
Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] |
| | | Negative
(No Analyte) | Not Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
[95.9-100%] | | | | | | | | | | | | |
| Overall Agreement with
Expected Result | | All Concentrations | All Results | 1307/1350
96.8%
[95.7-97.2%] | | | | | | | | | | | | | | | |

Table 17. Reproducibility of the BioFire Global Fever Special Pathogens Panel

33

1 Due to a correction in the stock concentration, L. donovani was evaluated at 3.4×LoD and 1.1×LoD.

2 Due to a correction in the stock concentration, P. falciparum was evaluated at 1.5×LoD and 0.5×LoD.

Reproducibility Comparison Between BioFire FilmArray 2.0 and BioFire FilmArray Torch

Performance of the BioFire Global Fever Special Pathogens Panel was compared between the BioFire FilmArray 2.0 and BioFire FilmArray Torch systems by examining analyte detection of contrived whole blood samples. The testing incorporated a range of potential variation introduced by operator, instrument system, analyte concentration, and reagent lot for a total of 90 replicates for each analyte concentration distributed equally over three BioFire FilmArray 2.0 systems and three BioFire FilmArray Torch systems.

A summary of results (percent (%) agreement with the expected Detected or Not Detected result) for the analytes by BioFire FilmArray system is provided in Table 18. Overall agreement between observed and expected results was 99.5% for the BioFire FilmArray 2.0 system and 99.1% for the BioFire FilmArray Torch system demonstrating similar performance between both instrument systems.

34

Table 18. Reproducibility of the BioFire Global Fever Special Pathogens Panel Test Results on BioFire FilmArray 2.0 and BioFire FilmArray Torch

35

| Analyte
(Source / ID) | Concentration
Tested
(copies/mL) | Expected
Result | BioFire FilmArray 2.0 Platform | | | | BioFire FilmArray Torch Platform | | | |
|-------------------------------------------|----------------------------------------|--------------------|--------------------------------|----------|----------|------------------------------------|----------------------------------|----------|----------|------------------------------------|
| | | | System 1 | System 2 | System 3 | All FA 2.0 Systems
[95% CI] | System 1 | System 2 | System 3 | All FA Torch Systems
[95% CI] |
| Overall Agreement with
Expected Result | All Concentrations | All Results | | | | 1343/1350
99.5%
[98.9-99.8%] | | | | 1338/1350
99.1%
[98.4-99.5%] |

Abbreviations: FA – FilmArray; 95% CI – 95% Confidence Interval

Specimen Storage

Stability of whole blood specimens was evaluated to support labeling recommendations for storage of samples at room temperature for up to 24 hours or at 2-8°C for up to seven days. These conditions were selected to be consistent with, or exceed, standard storage and transport conditions for most laboratory testing of clinical human whole blood specimens. Evaluation of frozen samples at ultra-low temperatures prior to testing was performed to support other analytical and clinical testing.

Testing was conducted using contrived samples composed of human whole blood and four representative BioFire Global Fever Special Pathogens Panel analytes in two mixes at a concentration of 3× their respective LoD or less. Organisms were selected to be representative of the analytes detected by the BioFire Global Fever Special Pathogens Panel, including bacteria, viruses, and protozoa. A time point zero was tested immediately after sample preparation as a no-storage control. Ten replicates were tested at each of the storage conditions. Two different room temperature conditions (18-21℃ and 30℃) were also tested. The results are summarized in Table 19.

| | | Room Temperature | | Refrigerated Storage
2-8°C | | | |
|-----------------------|-------------------------|------------------------------------|--------------------------------------|-------------------------------|----------------|----------------|----------------------------------|
| Analyte | No Storage
(Control) | Ambient
18-21°C
for 24 Hours | Upper Limit
30°C
for 24 Hours1 | Day 1 | Day 3 | Day 7 | Ultra-low
Freezing
≤ -70°C |
| | Leptospira interrogans | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 |
| Dengue virus | | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 |
| | Leishmania donovani | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 |
| Plasmodium falciparum | | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 | 10/10
10/10 |

Table 19. Summary of Analyte Detections Over Total Number Tested by Storage Condition

4 Testing of the upper limit of room temperature storage, 30°C, was performed separately and required a separate no storage control.

36

Assayed External Controls VIII.

Name of Device: BIOFIRE® SHIELD™ Control Kit for the BioFire Global Fever Special Pathogens Panel Common or Usual Name: Same Product Code: PMN Regulation: 21 CFR 866.3920 Classification Name: Assayed quality control material for clinical microbiology assays Regulatory Class: Class II (Special Controls) Panel: Microbiology - 83 Predicate Device: BIOFIRE® SHIELD™ Control Kit for the Global Fever Panel (BioFire Defense, LLC) [K202382] This predicate has not been subject to a design-related recall.

Device Description

The BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens (GF SP) Panel is a surrogate control to monitor performance of the BioFire GF SP Panel assays. The BIOFIRE SHIELD Control Kit for the BioFire GF SP Panel is designed to mitigate the risk of control contamination or misuse when evaluating clinical specimens on BioFire FilmArray Systems. Good laboratory practice recommends running positive and negative external controls regularly. Evaluation of external controls is recommended prior to using a new shipment or new lot of BioFire GF SP Panel kits, when there is a new operator, and following replacement or repair of a BioFire FilmArray System. It is the responsibility of each laboratory to determine the frequency of external control testing with the BioFire GF SP Panel as part of the laboratory's Quality Control program. Quality control materials should be used in accordance with local, state, federal regulations and accreditation requirements.

Materials provided in each BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel:

Six individually packaged Positive External Control Injection Vials ●
Six individually packaged Negative External Control Injection Vials ●
Instructions available online at www.biofiredefense.com: ●
- 0 BIOFIRE® SHIELD™ Control Kit for the BioFire Global Fever Special Pathogens Panel - Instructions for Use
- BIOFIRE® SHIELD™ Control Kit for the BioFire Global Fever Special o Pathogens Panel - Quick Guide

Materials required but not provided:

BioFire® FilmArray® System including: ●
- 0 BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch Instrument System including accompanying platform-specific core software
- 0 BioFire® FilmArray® Pouch Loading Station

37

BioFire® Global Fever Special Pathogens Panel (Part No. DFA2-ASY-0018) and accompanying pouch module software
10% bleach solution or a similar disinfectant

Intended Use

The BIOFIRE® SHIELD™ Control Kit for the BioFire® Global Fever Special Pathogens Panel contains Positive and Negative External Controls intended for use as assayed quality controls to monitor the performance of in vitro diagnostic laboratory nucleic acid testing procedures for the qualitative detection of Bacillus anthracis, chikungunya virus, Crimean-Congo hemorrhagic fever virus, dengue virus (serotypes 1, 2, 3, and 4), Ebolavirus spp., Francisella tularensis, Lassa virus, Leishmania spp., Leptospira spp., Marburgvirus, Plasmodium spp. (including species differentiation of Plasmodium falciparum and Plasmodium vivax/ovale), West Nile virus, yellow fever virus, and Yersinia pestis when using the BioFire® Global Fever Special Pathogens Panel on BioFire® FilmArray® 2.0 and BioFire® FilmArray® Torch Systems. The BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel is designed for and intended to be used solely with the BioFire Global Fever Special Pathogens Panel. This product does not replace manufacturer internal controls provided as part of the BioFire Global Fever Special Pathogens Panel.

Both the Positive and Negative External Controls are provided in a FilmArray Control Injection Vial format. The Positive Control Injection Vial contains dried synthetic DNA segments in buffer and stabilizer to assess the presence of each individual assay on the BioFire Global Fever Special Pathogens Panel. The Negative Control Injection Vial contains no DNA and is nonreactive with the BioFire Global Fever Special Pathogens Panel assays.

For In Vitro Diagnostic Use.

38

Substantial Equivalence

| Element | Subject Device:
BIOFIRE SHIELD Control Kit for
the BioFire Global Fever
Special Pathogens Panel | Predicate:
BIOFIRE SHIELD Control Kit for
the BioFire Global Fever Panel
[K202382] |
|---------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Device Description | Positive and negative external
assayed quality controls to
monitor assay performance in the
BioFire Global Fever Special
Pathogens Panel | Positive and negative external
assayed quality controls to
monitor assay performance in the
BioFire Global Fever Panel |
| Physical Format | External control material dried on
Control Injection Vial filter | Same as subject device |
| Composition | Tm-shifted synthetic DNA
(positive control only) | Same as subject device |
| Targets Monitored | Bacillus anthracis, chikungunya
virus, Crimean-Congo
hemorrhagic fever virus, dengue
virus (serotypes 1, 2, 3, and 4),
Ebolavirus spp., Francisella
tularensis, Lassa virus, Leishmania
spp., Leptospira spp.,
Marburgvirus, Plasmodium spp.
(including species differentiation
of P. falciparum and P.
vivax/ovale), West Nile virus,
yellow fever virus, and Yersinia
pestis | Chikungunya virus, dengue virus
(serotypes 1, 2, 3, and 4),
Leptospira spp., Plasmodium spp.
(including species differentiation
of P. falciparum and P.
vivax/ovale) |
| Instrumentation | BioFire Global Fever Special
Pathogens Panel run on BioFire
FilmArray 2.0 or BioFire FilmArray
Torch systems | BioFire Global Fever Panel run on
BioFire FilmArray 2.0 |
| Test Interpretation | Automated test interpretation
and report generation; user
cannot access raw data | Same as subject device |
| Reagent Storage | Room temperature | Same as subject device |

39

Summary of Performance Data

Reproducibility

Reproducibility for the BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel was evaluated both for the BioFire FilmArray 2.0 System and the BioFire FilmArray Torch System. Reproducibility on the BioFire FilmArray 2.0 System was evaluated using SHIELD lots from three different manufacturing events at three test sites by two operators and three instruments per test site using three different BioFire GF SP Panel reagent lots. Testing took place over 5 days at each site. Reproducibility on the BioFire FilmArray Torch was performed in a similar manner with the exception that sites were simulated using three different BioFire FilmArray Torch Systems. Reproducibility was evaluated by calculating the percent agreement between observed test results (Passed or Failed) and expected test results (Passed) for both negative and positive SHIELD controls. Table 21 show summarized results for BioFire FilmArray 2.0 and BioFire FilmArray Torch Systems, respectively.

| SHIELD Control Type | Expected Result | Observed/Expected
(Percent Agreement)
[95% Confidence Interval] | | | |
|----------------------------------------|-----------------|-----------------------------------------------------------------------|--------------------|--------------------|------------------------------------|
| | | Site 1 | Site 2 | Site 3 | All Sites |
| Positive | Passed 1 | 42/45
(93.3%) | 45/45
(100%) | 45/45
(100%) | 132/135
(97.8%)
[93.7-99.2%] |
| Negative | Passed 2 | 45/45
(100%) | 44/45 3
(97.8%) | 44/45 4
(97.8%) | 133/135
(98.5%)
[94.8-99.6%] |
| Overall Agreement with Expected Result | | | | | 265/270
(98.1%)
[95.7-99.2%] |

Table 20. Multi-Site Reproducibility Test Results of the BIOFIRE SHIELD Control Kit for the BioFire GF SP Panel on BioFire FilmArrav 2.0 Systems

1 All BioFire GF SP Panel assays have a positive amplicon melt in the SHIELD control melt range.

2 All BioFire GF SP Panel assays have no melt in both the SHIELD control melt range and in the pathogen melt range.

3 Unexpected detection of pathogen amplicon for Leishmania spp.

4 Unexpected detection of pathogen amplicon for dengue virus.

40

Table 21. Reproducibility Test Results of the BIOFIRE SHIELD Control Kit for the BioFire GF SP Panel on BioFire FilmArray Torch Systems

| SHIELD Control Type | Expected Result | Observed/Expected
(Percent Agreement) | | | Overall
[95% Confidence
Interval] |
|-------------------------------------------|-----------------|------------------------------------------|----------------------|----------------------|-----------------------------------------|
| | | FA Torch
System 1 | FA Torch
System 2 | FA Torch
System 3 | |
| Positive | Passed 1 | 43/45
(95.6%) | 43/45
(95.6%) | 44/45
(97.8%) | 130/135
(96.3%)
[91.6-98.4%] |
| Negative | Passed 2 | 43/45
(95.6%) | 45/45
(100%) | 45/45
(100%) | 133/135
(98.5%)
[94.8-99.6%] |
| Overall Agreement with Expected
Result | | | | | 263/270
(97.4%)
[94.7-98.7%] |

1 All BioFire GF SP Panel assays have a positive amplicon melt in the positive SHIELD control melt range. 2 All BioFire GF SP Panel assays have no melt in either the positive SHIELD control melt range or the pathogen melt range.

Repeatability

Repeatability was performed by a single operator testing 45 Positive and 45 Negative External Controls from a single BIOFIRE SHIELD Control Kit, using a single BioFire GF SP Panel reagent lot on one BioFire FilmArray 2.0 instrument over a period of 14 days. The primary assessment is based on a comparison of the observed External Control test results (Passed/Failed) to the expected test results (Passed). Table 22 shows the results for the Repeatability evaluation.

Table 22. Summary of BIOFIRE SHIELD Control Kit for the BioFire GF SP Panel Repeatability Test
Results

| SHIELD Control Type | Expected Result | Observed/Expected
(Percent Agreement) |
|---------------------|-----------------|------------------------------------------|
| Positive | Passed 1 | 45/45
(100%) |
| Negative | Passed 2 | 45/45
(100%) |

1 All BioFire GF SP Panel assays have a positive amplicon melt in the positive SHIELD control melt range.

2 All BioFire GF SP Panel assays have no melt in either the positive SHIELD control melt range.

41

Clinical Evaluation

Six clinical sites evaluated the BIOFIRE SHIELD Control Kit by testing a Positive or Negative External Control each day prior to testing clinical specimens. Results for the BioFire GF SP Panel on BioFire FilmArray 2.0 Systems are shown in Table 23.

| SHIELD Control Type | Completed with Passed
Result | Total Completed | Percent Passed (%) |
|---------------------|---------------------------------|-----------------|--------------------|
| Positive | 158a | 160 | 98.8% |
| Negative | 157a | 159 | 98.7% |
| Overall | 315 | 319 | 98.7% |

Table 23. Summary of BIOFIRE SHIELD Control Kit for the BioFire GF SP Panel Clinical Evaluation Results

? Site tested a Positive and a Negative External Controls were most likely swapped as the Negative External Control failed because all External Control targets were Detected, and the Positive External Control failed because all External Control targets were Not Detected.