BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel

Q: Who is the manufacturer of BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel?

Biofire Defense, LLC filed the 510(k) submission for BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel (K213362).

Q: What is the FDA product code for BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel?

QVR. It is classified under 21 CFR 866.4000 as a Class 2 device in the Immunology panel.

Q: What is the regulatory pathway for BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel?

510(k) clearance (submission type: Traditional).

Q: What are the predicate devices for BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel?

FilmArray NGDS Warrior Panel (DEN160048); FilmArray Global Fever Panel (DEN200043); BIOFIRE SHIELD Control Kit for the Global Fever Panel (K202382).

Q: Who can help prepare an FDA 510(k) submission like BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel?

Innolitics — a medical-device software consultancy — provides regulatory and engineering support for FDA 510(k) submissions. See https://innolitics.com/services/510ks/ or contact https://innolitics.com/contact.

K213362 · Biofire Defense, LLC · QVR · Mar 22, 2023 · Immunology

Device Facts

Record ID	K213362
Device Name	BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel
Applicant	Biofire Defense, LLC
Product Code	QVR · Immunology
Decision Date	Mar 22, 2023
Decision	SESE
Submission Type	Traditional
Regulation	21 CFR 866.4000
Device Class	Class 2
Attributes	Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K213362 · Mar 22, 2023	BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel	Biofire Defense, LLC	Archived clinical serum, plasma, and whole blood specimens	Archived clinical specimens were used to supplement prospective clinical study data for analytes that were not observed or were encountered in insufficient numbers during the prospective clinical evaluation.	Archived clinical specimens; Clinical validation; Performance supplementation

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Archived Specimen Performance Summary; Retrospective testing of archived clinical specimens; Follow-up/Duration: Not applicable	Patients with signs/symptoms of acute febrile illness; Sample Size: Varies by analyte (e.g., 281 for CCHF, 279 for Ebola, 279 for Marburg, 347 for West Nile)	Comparator testing (well-validated nested PCR assays followed by bi-directional Sanger sequencing)	Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA)

Indications for 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. 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.

Device Story

Multiplexed nucleic acid-based test for BioFire FilmArray 2.0/Torch systems; detects bacterial, viral, and protozoan pathogens from EDTA whole blood. Pouch contains freeze-dried reagents for nucleic acid purification and nested, multiplexed PCR with DNA melt analysis. User loads hydration solution and sample buffer/blood mixture into pouch; instrument automates liquid movement via bladders/pistons and thermal cycling via Peltier devices. Digital camera captures fluorescent images of PCR2 reactions; software interprets melt curves to identify pathogens. Results available in ~1 hour. Used in clinical laboratories by trained personnel. Output provides qualitative identification or presumptive identification of pathogens. Results support clinical decision-making in conjunction with other data; aids in managing patients with acute febrile illness and potential exposure to biothreat/special pathogens.

Clinical Evidence

Prospective clinical study (n=2139) evaluated PPA/NPA against comparator methods. Results showed high sensitivity/specificity for identified pathogens. Archived specimen study (n=416) and contrived specimen study (n=50 replicates per analyte) supplemented performance data for rare pathogens. Performance metrics (PPA/NPA) and 95% CIs reported for all analytes.

Technological Characteristics

Multiplexed nested nucleic acid amplification test; uses magnetic bead-based purification; nested PCR with LCGreen Plus fluorescent dye; DNA melt analysis for detection; closed-system disposable pouch; automated instrument-based processing; compatible with FilmArray 2.0 and Torch systems; room temperature reagent storage.

Indications for Use

Indicated for individuals with signs/symptoms of acute febrile illness and known/suspected exposure to target pathogens. For use in laboratories with appropriate biosafety equipment, PPE, containment, and trained personnel.

Regulatory Classification

Identification

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

Predicate Devices

FilmArray NGDS Warrior Panel (DEN160048)
FilmArray Global Fever Panel (DEN200043)
BIOFIRE SHIELD Control Kit for the Global Fever Panel (K202382)

Submission Summary (Full Text)

{0} **FDA** U.S. FOOD & DRUG ADMINISTRATION ## 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ### I Background Information: #### A 510(k) Number K213362 #### B Applicant BioFire Defense, LLC #### C Proprietary and Established Names BioFire Global Fever Special Pathogens Panel; BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel #### D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | QVR | Class II | 21 CFR 866.4000 - Device To Detect And Identify Biothreat Microbial Agents In Human Clinical Specimens. | MI - Microbiology | | QMV | Class II | 21 CFR 866.3966 - Device to detect and identify selected microbial agents that cause acute febrile illness | MI - Microbiology | | PMN | Class II | 21 CFR 866.3920 – Assayed quality control material for clinical microbiology assays | MI - Microbiology | ### II Submission/Device Overview: #### A Purpose for Submission: To obtain a substantial equivalence determination for the BioFire Global Fever Special Pathogens Panel and the BioFire SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel. Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} ## **B Measurand:** DNA and RNA sequences from the following organisms: Chikungunya virus, Dengue virus serotypes 1-4, *Leishmania* spp. (*Leishmania donovani* and *Leishmania infantum*), *Leptospira* spp., *Plasmodium falciparum*, *Plasmodium vivax*, *Plasmodium ovale*, *Plasmodium malariae*, and *Plasmodium knowlesi*, West Nile virus, *Bacillus anthracis*, Crimean-Congo hemorrhagic fever virus, *Ebolavirus* spp., *Francisella tularensis*, Lassa virus, *Marburgvirus*, Yellow fever virus, and *Yersinia pestis*. ## **C Type of Test:** Multiplex Nucleic Acid Amplification Test ## **III Intended Use/Indications for Use:** ### **A Intended Use(s):** See Indications for Use below. ### **B Indication(s) for Use:** The 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* K213362 - Page 2 of 56 {2} 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. 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 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 BioFireGlobal Fever K213362 - Page 3 of 56 {3} Special Pathogens Panel. The Negative Control Injection Vial contains no DNA and is non-reactive with the BioFire Global Fever Special Pathogens Panel assays. # **C Special Conditions for Use Statement(s):** Rx - For Prescription Use Only # **D Special Instrument Requirements:** The FilmArray Global Fever Special Pathogens Panel is performed on the FilmArray 2.0 and FilmArray Torch systems. # **IV Device/System Characteristics:** # **A Device Description:** The BioFire Global Fever Special Pathogens Panel is a multiplex nucleic acid-based test designed to be used with the FilmArray 2.0 or the FilmArray Torch instruments. The BioFire Global Fever Special Pathogens Panel includes a pouch which contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex polymerase chain reaction (PCR) with DNA melt analysis. The BioFire Global Fever Special Pathogens Panel simultaneously conducts sixteen tests for the identification of bacterial, viral, and protozoan organisms from whole blood specimens collected in EDTA tubes. Results from the BioFire Global Fever Special Pathogens Panel are available within about one hour. 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 and placing it in the FilmArray Instrument. The pouch contains all of 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 FilmArray Software on the FilmArray system 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 FilmArray system. The 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 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 in order 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 the melt curve analysis. Nucleic acid extraction occurs within the BioFire 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 multiplex PCR is executed in two stages. During the first stage, a single, large volume, highly 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 K213362 - Page 4 of 56 {4} 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 2$^{nd}$ stage PCR, or nested PCR, is performed in each well of the array. At the conclusion of the 2$^{nd}$ 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. The FilmArray instrument software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel. A description of the individual assays and their result interpretation is included below: - • **Chikungunya virus:** The Global Fever Special Pathogens Panel contains two assays for species-level detection of all chikungunya virus strains (CHIKV1 and CHIKV2). The FilmArray instrument software will interpret any single positive chikungunya assay as a Chikungunya Virus Detected result. - • **Dengue virus:** The Global Fever Special Pathogens Panel contains five individual assays for the detection of Dengue virus serotypes 1, 2, 3, and 4 with two assays specifically dedicated to detecting Dengue virus serotype 2 (DENV1, DENV2\_1, DENV2\_2, DENV3, and DENV4). The FilmArray instrument software will interpret any single positive Dengue virus assay as a Dengue Virus Detected result. - • **Leptospira:** The Global Fever Special Pathogens Panel contains a single pan assay for the genus-level detection of all *Leptospira* Group 1 species (LEPTO1). A positive pan-Leptospira assay will result in a *Leptospira* spp. Detected call. - • **Leishmania spp.:** The Global Fever Special Pathogens panel contains a single assay for the genus-level detection of *Leishmania*. The assay detects species that cause visceral leishmaniasis (*L. infantum* and *L. donovani*). Other species of *Leishmania* are expected to be generate a detected result. A positive *Leishmania* assay will result in a Detected *Leishmania* spp. test result. - • **Plasmodium:** The Global Fever Special Pathogens Panel contains three *Plasmodium* assays, one genus-level assay and two species-level assays. The genus-level assay (*Plasmodium* spp.) detects all five *Plasmodium* species known to infect humans (*P. falciparum*, *P. vivax*, *P. ovale*, *P. malariae*, and *P. knowlesi*). One species-level assay detects *Plasmodium falciparum* and a combined species-level assay detects both *Plasmodium vivax* and *Plasmodium ovale*. Each individual assay is reported as a Detected or Not Detected result separately based on the results of the specific Global Fever Special Pathogens Panel assay. - • **Bacillus anthracis:** The Global Fever Special Pathogens Panel contains one assay for the detection of *Bacillus anthracis*. a positive *Bacillus anthracis* assay will result in a *Bacillus anthracis* Detected result. K213362 - Page 5 of 56 {5} - *Francisella tularensis*: The Global Fever Special Pathogens Panel contains two assays for the detection of *Francisella tularensis*. One or more positive *Francisella tularensis* assays will result in a Detected call for *Francisella tularensis*. - *Yersinia pestis*: The Global Fever Special Pathogens Panel contains two assays for the detection of *Yersinia pestis*. One or more positive *Yersinia pestis* assays will result in a Detected call for *Yersinia pestis*. - Crimean-Congo hemorrhagic fever virus: The Global Fever Special Pathogens Panel contains two assays for the detection of Crimean-Congo hemorrhagic fever virus. One or more positive Crimean-Congo hemorrhagic fever virus assays will result in a Detected call for Crimean-Congo hemorrhagic fever virus. - *Ebolavirus* spp.: The Global Fever Special Pathogens Panel contains three assays for the detection of *Ebolavirus* spp. Any positive assay call will result in a Detected call for *Ebolavirus*. - Lassa virus: The Global Fever Special Pathogens Panel contains two assays for the detection of Lassa virus. Any positive assay call will result in a Detected call for Lassa virus. - *Marburgvirus*: The Global Fever Special Pathogens Panel contains one assay for the detection of *Marburgvirus*. A positive assay call will result in a *Marburgvirus* Detected result. - Yellow fever virus: The Global Fever Special Pathogens Panel contains one assay for the detection of Yellow Fever virus. A positive assay call will result in a Yellow Fever virus Detected result. Materials provided in each BioFire Global Fever Special Pathogens Panel kit: - Individually packaged BioFire Global Fever Panel pouches - Individually-packaged Transfer Pipettes - Single-use (1.0 mL) BioFire FilmArray Sample Buffer Tubes - Single-use pre-filled (1.5 mL) BioFire FilmArray Hydration Injection Vials (Blue) - Single-use BioFire FilmArray Sample Injection Vials (Red) - Instructions and Documents - BioFire Global Fever Special Pathogens Panel Instructions for Use - BioFire Global Fever Special Pathogens Panel Quick Guide Materials required but not provided: - 10% bleach solution BioFire FilmArray system including: - BioFire FilmArray 2.0 or FilmArray Torch instrument, computer, and software - FilmArray Pouch Loading Station K213362 - Page 6 of 56 {6} # **B Principle of Operation:** The BioFire Global Fever Special Pathogens Panel pouch is a closed system disposable that houses all the chemistry required to isolate, amplify, and detect nucleic acid from multiple biothreat pathogens within whole blood samples. The rigid plastic component (fitment) of the pouch contains reagents in freeze-dried form. The flexible plastic portion of the pouch is divided into discrete segments (blisters) where the required chemical processes are carried out. The user of the BioFire Global Fever Special Pathogens Panel loads the sample into the pouch, places the pouch into the FilmArray instrument, and starts the run. All other operations are automated. Operations and processes that occur during a FilmArray run include the following: - **Nucleic Acid Purification**- Nucleic acid purification occurs in the first three blisters of the pouch. The sample is lysed by agitation (bead beating) and the liberated nucleic acid is captured, washed, and eluted using magnetic bead technology. These steps require about ten minutes and the bead-beater apparatus can be heard as a high-pitched whine during the first minute of operation. - **Reverse Transcription and 1st Stage Multiplex PCR** - Some pathogens identified by the FilmArray Global Fever pouch are RNA viruses, and a reverse transcription (RT) step is performed to convert the viral RNA into cDNA prior to amplification. The purified nucleic acid solution is combined with a preheated master mix to initiate the RT step and subsequent thermocycling for multiplex PCR. The effect of 1st stage PCR is to enrich for the target nucleic acids present in the sample. - **2nd Stage PCR** - The products of 1st stage PCR are diluted and mixed with fresh PCR reagents containing an intercalating fluorescent DNA dye (LCGreen Plus, BioFire Defense, LLC). This solution is distributed over the 2nd stage PCR array. The individual wells of the array contain primers for different assays (each present in triplicate) that target specific nucleic acid sequences from each of the pathogens detected, as well as control template material. These primers are “nested” or internal to the specific products of the 1st stage multiplex reaction, which enhances both the sensitivity and specificity of the reactions. - **DNA Melting Analysis** – After 2nd stage PCR, the temperature is slowly increased and fluorescence in each well of the array is monitored and analyzed to generate a melt curve. The temperature at which a specific PCR product melts (melting temperature or Tm) is consistent and predictable and the FilmArray software automatically evaluates the data from replicate wells for each assay to report results. The FilmArray software controls the operation of the instrument, collects and analyzes data and automatically generates a test report at the end of the run. K213362 - Page 7 of 56 {7} ## V Substantial Equivalence Information: ### A Predicate Device Name(s): FilmArray NGDS Warrior Panel, FilmArray Global Fever Panel, BIOFIRE SHIELD Control Kit for the Global Fever Panel ### B Predicate 510(k) Number(s): DEN160048, DEN200043, K202382 ### C Comparison with Predicate(s): | Device & Predicate Device(s): | K213362 | DEN160048 | DEN200043 | | --- | --- | --- | --- | | Device Trade Name | BioFire Global Fever Special Pathogens Panel | FilmArray NGDS Warrior Panel | FilmArray Global Fever Panel | | **General Device Characteristic Similarities** | | | | | Intended Use/Indications For 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: | The FilmArray NGDS Warrior Panel is a qualitative, multiplexed, nucleic acid-based in vitro diagnostic test intended for use with the FilmArray 2.0 system. The FilmArray NGDS Warrior Panel detects and identifies *Bacillus anthracis*, *Yersinia pestis*, *Francisella tularensis*, *Coxiella burnetii*, Ebola virus, and Marburg virus nucleic acids directly from human whole blood (EDTA). The FilmArray NGDS Warrior Panel is also intended to be used to test for *Bacillus anthracis* or *Yersinia pestis* nucleic acids in blood cultures that are determined to be positive either by an automated system, by turbidity, or by daily Gram stain even | The FilmArray Global Fever Panel is a qualitative, multiplexed, nucleic acid-based in vitro diagnostic test intended for use with the FilmArray 2.0 system. The FilmArray Global Fever Panel detects and identifies selected 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 following target pathogens: *Leptospira* spp., chikungunya virus, dengue virus (serotypes 1, 2, 3 and 4), and *Plasmodium* spp. (including species differentiation of *Plasmodium falciparum* | K213362 - Page 8 of 56 {8} | | 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 | without turbidity, and is indicated to be performed with concomitant Gram stain performed on positive blood culture specimens as per normal laboratory procedure. The FilmArray NGDS Warrior Panel is intended to test individuals with signs and symptoms of infection from biothreat agents and/or individuals who are at risk for exposure or may have been exposed to these agents. The FilmArray NGDS Warrior Panel is indicated as an aid in the diagnosis of anthrax, plague, tularemia, Q fever, and the hemorrhagic fevers caused by Ebola and Marburg viruses, in response to a suspected or confirmed bioterrorism event or outbreaks. It is for diagnostic use in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the appropriate Department of Defense and public health authorities. Results are for the presumptive | and *Plasmodium vivax/ovale*). 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. 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. Positive results do not rule out co-infections with pathogens not included on the FilmArray Global Fever Panel. Not all pathogens that cause acute febrile illness are detected by this test, and negative results do not rule out the presence of other infections. Patient travel history and consultation of the CDC Yellow Book should be considered prior to use of the FilmArray Global Fever Panel as some pathogens are more common in certain geographical locations. | | --- | --- | --- | --- | K213362 - Page 9 of 56 {9} | | 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. | identification of *Bacillus anthracis*, *Yersinia pestis*, *Francisella tularensis*, *Coxiella burnetii*, Ebola virus, and Marburg virus. The definitive identification of *Bacillus anthracis*, *Yersinia pestis*, *Francisella tularensis*, *Coxiella burnetii*, Ebola virus, and Marburg virus requires additional testing and confirmation procedures in consultation with the appropriate Department of Defense and public health authorities for whom reports may be necessary. Negative results do not preclude infection with these biothreat agents and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. The FilmArray NGDS Warrior Panel is solely for use by United States Department of Defense laboratories, and laboratories designated by the Department of Defense. | | | --- | --- | --- | --- | K213362 - Page 10 of 56 {10} | | 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. | | | | --- | --- | --- | --- | | Specimen Type | Whole blood (collected in EDTA tube) | Same | Whole Blood (collected in EDTA tube), positive blood culture, or sputum | | Analyte | RNA/DNA | Same | Same | | Technological Principles | Multiplexed nested nucleic acid amplification test with melt analysis | Same | Same | | Time to result | About 1 hour | Same | Same | | Reagent storage | Room temperature | Same | Same | | Test interpretation | Automated test interpretation and report | Same | Same | K213362 - Page 11 of 56 {11} | | generation. User cannot access raw data. | | | | --- | --- | --- | --- | | Controls | Two controls included in each reagent pouch to control for sample processing and both stages of PCR and melt analysis | Same | Same | | **General Device Characteristic Differences** | | | | | Pathogens detected | *Bacillus anthracis*, Crimean-Congo hemorrhagic fever virus, Ebolavirus spp., *Francisella tularensis*, Lassa virus, *Leishmania* spp. (L. donovani and L. infantum), *Leptospira* spp., Marburgvirus, chikungunya virus, dengue virus (serotypes 1, 2, 3 and 4), and *Plasmodium* spp. (including species differentiation of *Plasmodium falciparum* and *Plasmodium vivax/ovale*), West Nile virus, Yellow fever virus, *Yersinia pestis* | *Bacillus anthracis*, *Yersinia pestis*, *Francisella tularensis*, *Coxiella burnetii*, Ebola virus, and Marburg virus | *Leptospira* spp., chikungunya virus, dengue virus (serotypes 1, 2, 3 and 4), and *Plasmodium* spp. (including species differentiation of *Plasmodium falciparum* and *Plasmodium vivax/ovale*) | | User Complexity | High | Moderate | Moderate | ### Comparison with BIOFIRE SHIELD Control Kit for the Global Fever Panel | Device & Predicate Device(s): | K213362 | K202382 | | --- | --- | --- | | Device Trade Name | BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel | BIOFIRE SHIELD Control Kit for the Global Fever Panel | | **General Device Characteristic Similarities** | | | | Intended Use/Indications For Use | The BIOFIRE SHIELD Control Kit for the BioFire Global Fever Special | The BIOFIRE SHIELD Control Kit for the BioFire Global Fever Panel contains | K213362 - Page 12 of 56 {12} | | 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 | 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 chikungunya virus, dengue virus (serotypes 1, 2, 3, and 4), *Leptospira* spp., and *Plasmodium* spp. (including species differentiation of *Plasmodium falciparum* and *Plasmodium vivax/ovale*) when using the BioFire Global Fever Panel on BioFire FilmArray 2.0 and BioFire FilmArray Torch Systems. The BIOFIRE SHIELD Control Kit for the BioFire Global Fever Panel is designed for and intended to be used solely with the BioFire Global Fever Panel. This product does not replace manufacturer internal controls provided as part of the BioFire Global Fever Panel device. 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 Panel. The Negative Control Injection Vial contains no DNA and is non-reactive with the BioFire Global Fever Panel assays. | | --- | --- | --- | K213362 - Page 13 of 56 {13} | | presence of each individual assay on the BioFire Global Fever Special Pathogens Panel. The Negative Control Injection Vial contains no DNA and is non-reactive with the BioFire Global Fever Special Pathogens Panel assays. For In Vitro Diagnostic Use. | | | --- | --- | --- | | Device Description | Positive and negative external assayed quality controls to monitor assay performance | Same | | Physical Format | External control material dried on Control Injection Filter | Same | | Composititon | Tm-shifted synthetic DNA (positive control only) | Same | | Time to result | About 1 hour | Same | | Reagent storage | Room temperature | Same | | Test interpretation | Automated test interpretation and report generation. User cannot access raw data. | Same | | Instrumentation | BioFire FilmArray 2.0 or BioFire FilmArray Torch systems | Same | | **General Device Characteristic Differences** | | | | Targets Monitored | *Bacillus anthracis*, Crimean-Congo hemorrhagic fever virus, *Ebolavirus* spp., *Francisella tularensis*, Lassa virus, *Leishmania* spp. (L. donovani and L. infantum), *Leptospira* spp., Marburgvirus, chikungunya virus, dengue virus (serotypes 1, 2, 3 and 4), and *Plasmodium* spp. (including species differentiation of *Plasmodium falciparum* and *Plasmodium vivax/ovale*), West Nile virus, Yellow fever virus, *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*) | K213362 - Page 14 of 56 {14} ## **VI Standards/Guidance Documents Referenced:** 14971:2007/(R)2010 (Corrected 4 October 2007), ‘Medical Devices – Applications of Risk Management to Medical Devices’ Guidance for Industry and Food and Drug Administration Staff – De Novo Classification Process (Evaluation of Automatic Class III Designation) (October 30, 2017) Guidance for Industry and Food and Drug Administration Staff – Highly Multiplexed Microbiological/Medical Countermeasure In Vitro Nucleic Acid Based Diagnostic Devices (August 27, 2014) Guideline for Industry and Food and Drug Administration Staff – Class II Special Controls Guideline: Dengue Virus Nucleic Acid Amplification Test Reagents Guideline for Industry and Food and Drug Administration Staff – Class II Special Controls Guidance Document: Plasmodium Species Antigen Detection Assays Guidance for Industry and FDA Staff – Assayed and Unassayed Quality Control Material Guidance for Industry and FDA Staff – Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests (March 13, 2007) MM03-Ed3, Molecular Diagnostic Methods for Infectious Diseases, CLSI Approved Guideline—Third Edition EP07-Ed3, Interference Testing in Clinical Chemistry; Approved Guideline—Third Edition Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices, FDA Guidance Document Guidance for Industry, FDA Reviewers and Compliance on Off-The-Shelf Software Use in Medical Devices Guidance for Industry, FDA Reviewers and Compliance on Off-The-Shelf Software Use in Medical Devices ISO 62304:2006, ‘Medical Device Software – Software Life Cycle Processes’ – IEC 62304:2006, November 27, 2008 ISO 62304:2006, ‘Medical Device Software – Software Life Cycle Processes’ – IEC 62304:2006, November 27, 2008 ISO 15223-1:2012, ‘Medical Devices – Symbols to be used with medical device labels, labeling and information to be supplied – Part 1: General requirements’ K213362 - Page 15 of 56 {15} Guidance for Industry and FDA on Alternative to Certain Prescription Device Labeling Requirements # **VII Performance Characteristics (if/when applicable):** # **A Analytical Performance:** # 1. Precision/Reproducibility: To evaluate the reproducibility of the BioFire Global Fever Special Pathogens Panel, three contrived whole blood samples were prepared with different mixtures of representative panel analytes. For each analyte, one sample was spiked at a Moderate Positive concentration (3 x LoD), another sample at a Low Positive (1 x LoD) and a third sample that was negative (unspiked) for the given analyte. For *P. falciparum*, a dilution error occurred that resulted in lower than expected analyte concentration being evaluated for all replicates. Six replicates of each sample were tested across 3 different sites 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 FilmArray 2.0 instruments; GF 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. **Table 1. Contrived Whole Blood Samples for Reproducibility Testing** | Organism (Isolate) | Sample 1 | Sample 2 | Sample 3 | | --- | --- | --- | --- | | *Leptospira interrogans* serovar icterohaemorrhagiae | Low Positive 3.3E+02 copies/mL 1 x LoD | Moderate Positive 1.0E+03 copies/mL 3 x LoD | Negative | | **Dengue Virus (DENV-2)** New Guinea C | Low Positive 3.3E+02 copies/mL 1 x LoD | Moderate Positive 1.0E+03 copies/mL 3 x LoD | Negative | | *Plasmodium falciparum* IPC 4884 | Moderate Positive 5.4E+02 copies/mL 1.5 x LoD | Negative | Low Positive 9.0E+01 copies/mL 0.5 x LoD | | *Leishmania donovani* | None | Low Positive 1.1E+01 copies/mL 1.1 x LoD | Moderate Positive 3.4E+01 copies/mL 3.4 x LoD | Combined reproducibility results are shown below in **Table 2**. K213362 - Page 16 of 56 {16} **Table 2: Reproducibility of the FilmArray Global Fever Special Pathogens Panel Qualitative Results** | | Test Analyte Isolate | Concentration | Expected Test Result | % Agreement with Expected Results | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | | Site 1 | Site 2 | Site 3 | All Sites | 95% Confidence Interval | | **BACTERIA** | *Leptospira interrogans* serovar *icterohaemorrhagiae* | Moderate Positive 3× LoD (1.2E+03 copies/mL) | Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | | | Low Positive 1× LoD (3.9E+02 copies/mL) | Detected | 27/30 90.0% | 28/30 93.4% | 26/30 86.7% | **81/90 90.0%** | 81.9-95.3% | | | | Negative (No Analyte) | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | **VIRUSES** | **Dengue virus** **DENV-2** New Guinea C | Moderate Positive 3× LoD (1.1E+03 copies/mL) | Detected | 29/30 96.7% | 30/30 100% | 30/30 100% | **89/90 98.9%** | 94.0-100% | | | | Low Positive 1× LoD (3.6E+02 copies/mL) | Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | | | Negative (No Analyte) | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | **PROTOZOA** | *Plasmodium falciparum* IPC 4884 | Moderate Positive 1.5× LoD^{1} (2.7E+02 copies/mL) | Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | | | Low Positive 0.5× LoD^{1} (9.0E+01 copies/mL) | 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%** | 96.0-100% | | | *Plasmodium falciparum* Detection Results | Moderate Positive 1.5× LoD^{1} (2.7E+02 copies/mL) | Detected | 29/30 96.7% | 30/30 100% | 28/30 93.4% | **87/90 96.7%** | 90.6-99.3% | | | | Low Positive 0.5× LoD^{1} (9.0E+01 copies/mL) | Detected | 18/30 60% | 24/30 80% | 21/30 70% | **63/90 70.0%** | 59.4-79.2% | | | | Negative (No Analyte) | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | | *Leishmania donovani* 1S (MHOM/SD/62/1S) | Moderate Positive 3.4× LoD (3.4E+01 copies/mL) | Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | | | Low Positive 1.1× LoD^{1} (1.1E+01 copies/mL) | Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | | | Negative (No Analyte) | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 96.0-100% | | **Overall Agreement with the Expected Test Result** **All Analytes and All Test Levels (95% Confidence Interval)** | | | | **1,307/1,350** **96.8% (95.7-97.6%)** | | | | | $^{1}$Due to a correction in the stock concentration, *P. falciparum* was evaluated at 1.5x LoD and 0.5x LoD. Detected results were as expected for all analytes except for the Low Positive *Leptospira interrogans* sample which exhibited 90% agreement. The observed negative specimens were distributed across all runs, sites, testing days, and reagent lots and likely reflect underspiking with *Leptospira interrogans*. A secondary assessment of reproducibility is based on variability in the melt temperature (Tm) of the amplification products (measured as standard deviation). Melt temperature mean and standard deviations are shown in **Table 3.** for control and organism assays for the three test sites and K213362 - Page 17 of 56 {17} overall. Variability in the melt temperatures for the assays evaluated was within the expected range (≤0.5°C) for each assay at each site and overall, with a standard deviation of 0.2-0.3°C. Table 3: Summary of Tm (°C) Analyses for FilmArray Global Fever Special Pathogens Panel Assays | Analyte (Isolate tested) | Assay | Observed Tm (°C) | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | Site 1 | | Site 2 | | Site 3 | | All Sites | | | | | Mean | ±StDev | Mean | ±StDev | Mean | ±StDev | Mean | ±StDev | | **CONTROLS** | | | | | | | | | | | RNA Process Control | yeast RNA | 81.7 | ±0.2 | 81.6 | ±0.3 | 81.7 | ±0.2 | 81.6 | ±0.2 | | PCR2 Control | PCR2 | 75.5 | ±0.3 | 75.4 | ±0.3 | 75.6 | ±0.2 | 75.5 | ±0.3 | | **BACTERIA** | | | | | | | | | | | *Leptospira interrogans* | Lepto1 | 81.4 | ±0.2 | 81.3 | ±0.3 | 81.5 | ±0.2 | 81.4 | ±0.2 | | **VIRUSES** | | | | | | | | | | | Dengue virus Type 2 | DENV2_1 | 80.1 | ±0.2 | 80.0 | ±03 | 80.1 | ±0.2 | 80.1 | ±0.2 | | **PROTOZOA** | | | | | | | | | | | *Plasmodium falciparum* | Plas spp. | 79.0 | ±0.2 | 78.9 | ±0.2 | 79.0 | ±0.2 | 78.9 | ±0.2 | | | Plas falciparum | 74.0 | ±0.2 | 74.0 | ±0.2 | 74.1 | ±0.2 | 74.0 | ±0.2 | | *Leishmania donovani* | Leish | 85.4 | ±0.2 | 85.3 | ±0.2 | 85.4 | ±0.2 | 85.4 | ±0.2 | Variability in the melt temperatures for the assays evaluated was within the expected range (≤0.5°C) for each assay at each site and overall, with a standard deviation of 0.2-0.3°C. Cumulatively, the results suggest that there are no significant differences between variables evaluated in the reproducibility study. Therefore, the reproducibility studies conducted for the BioFire Global Fever Special Pathogens Panel are acceptable. Additional testing was also performed to demonstrate equivalent performance of the Global Fever Special Pathogens Panel on both the FilmArray 2.0 and FilmArray Torch instruments. Instead of performing the evaluation at three different sites, individual sites were simulated using different FilmArray Torch and different FilmArray 2.0 systems and two different operators for each of the simulated sites. Three contrived whole blood samples were prepared with different mixtures of six representative panel analytes, two bacteria, two viruses, 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 each simulated site on five different days, providing a total of 90 replicate test results per sample per FilmArray system. On each test day, two operators per simulated site used three FilmArray 2.0 instruments and three FilmArray Torch modules; three Global Fever Special Pathogens Panel pouch reagent lots were rotated daily. Results of this study are summarized in Table 4 and Table 5 below. K213362 - Page 18 of 56 {18} **Table 4. Reproducibility of the Global Fever Special Pathogens Panel on FilmArray Torch and FilmArray 2.0 Instruments.** | Analyte (Source / ID) | Concentration Tested (copies/mL) | Expected Result | Detection Rate (n/N) % Agreement with Expected Result | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | FilmArray 2.0 Platform | | | | 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] | | | *Leptospira interrogans* serovar *icterohaemorrhagiae* | | 3×LoD (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%] | | | | 1×LoD (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%** [87.6-97.6%] | | | | Negative | 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 | | 3×LoD (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%] | | | | 1×LoD (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 | 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^{1} 96.7% | **89/90 98.9%** [94.0-99.8%] | | *Leishmania donovani* 15 (MHOM/SD/62/1S) | | 3×LoD (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%] | | | | 1×LoD (3.4E+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%] | | | | Negative | 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* IPC 4884 (BEI / MRA-1238) | *Plasmodium* spp. **Detection Results** | 3×LoD (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%] | | | | 1×LoD (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%] | | | | Negative | Not Detected | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | 30/30 100% | 30/30 100% | 30/30 100% | **90/90 100%** | K213362 - Page 19 of 56 {19} | Analyte (Source / ID) | Concentration Tested (copies/mL) | Expected Result | Detection Rate (n/N) % Agreement with Expected Result | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | FilmArray 2.0 Platform | | | | 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] | | | | | | | | | | [95.9-100%] | | | | [95.9-100%] | | | *Plasmodium falciparum* Detection Results | 3×LoD (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%] | | | | 1×LoD (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%] | | | | Negative | 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%] | | Overall Agreement with Expected Result | | | | **1343/1350 99.5%** [98.9-99.8%] | | | | **1338/1350 99.1%** [98.4-99.5%] | | | | Table 5. Summary of Tm (°C) Analyses for FilmArray Global Fever Special Pathogens Panel Assays on FilmArray 2.0 and FilmArray Torch instruments | Analyte | Assay | FA Platform | Observed Tm (°C) | | | | | | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | | | Simulated Site 1 | | | Simulated Site 2 | | | Simulated Site 3 | | | Overall | | | | | | | Mean | ±StDev | CV | Mean | ±StDev | CV | Mean | ±StDev | CV | Mean | ±StDev | CV | | **CONTROLS** | | | | | | | | | | | | | | | | **RNA Process Control** | Yeast RNA | FA 2.0 | 82.0 | 0.3 | 0.4% | 82.1 | 0.3 | 0.4% | 82.1 | 0.3 | 0.4% | 82.1 | 0.3 | 0.4% | | | | Torch | 82.4 | 0.2 | 0.2% | 82.3 | 0.2 | 0.2% | 82.3 | 0.2 | 0.2% | 82.3 | 0.2 | 0.2% | | **PCR2 Control** | PCR2 | FA 2.0 | 76.0 | 0.3 | 0.4% | 76.0 | 0.2 | 0.3% | 76.1 | 0.2 | 0.3% | 76.0 | 0.2 | 0.3% | | | | Torch | 76.3 | 0.2 | 0.3% | 76.2 | 0.2 | 0.3% | 76.2 | 0.2 | 0.3% | 76.2 | 0.2 | 0.3% | | **BACTERIA** | | | | | | | | | | | | | | | | *Leptospira interrogans* | Lepto 1 | FA 2.0 | 81.7 | 0.3 | 0.4% | 81.7 | 0.3 | 0.4% | 81.8 | 0.2 | 0.2% | 81.8 | 0.3 | 0.4% | | | | Torch | 82.0 | 0.2 | 0.2% | 81.9 | 0.2 | 0.2% | 81.9 | 0.2 | 0.2% | 82.0 | 0.2 | 0.2% | | **VIRUSES** | | | | | | | | | | | | | | | | **Dengue virus Type 2** | DENV 2_1 | FA 2.0 | 80.3 | 0.3 | 0.4% | 80.3 | 0.2 | 0.2% | 80.4 | 0.2 | 0.2% | 80.3 | 0.2 | 0.2% | | | | Torch | 80.6 | 0.2 | 0.2% | 80.5 | 0.2 | 0.2% | 80.5 | 0.2 | 0.2% | 80.5 | 0.3 | 0.4% | | **PROTOZOA** | | | | | | | | | | | | | | | | | Leish | FA 2.0 | 85.6 | 0.3 | 0.4% | 85.6 | 0.2 | 0.2% | 85.7 | 0.2 | 0.2% | 85.6 | 0.2 | 0.2% | K213362 - Page 20 of 56 {20} | *Leishmania donovani* | | Torch | 86.1 | 0.2 | 0.2% | 85.9 | 0.2 | 0.2% | 85.9 | 0.2 | 0.2% | 86.0 | 0.2 | 0.2% | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | *Plasmodium falciparum* | Plas spp. | FA 2.0 | 79.1 | 0.3 | 0.4% | 79.2 | 0.3 | 0.4% | 79.3 | 0.2 | 0.3% | 79.2 | 0.2 | 0.3% | | | | Torch | 79.5 | 0.2 | 0.3% | 79.4 | 0.2 | 0.3% | 79.4 | 0.2 | 0.3% | 79.4 | 0.2 | 0.3% | | | Plas falciparum | FA 2.0 | 74.2 | 0.3 | 0.4% | 74.3 | 0.2 | 0.3% | 74.3 | 0.2 | 0.3% | 74.3 | 0.2 | 0.3% | | | | Torch | 74.6 | 0.2 | 0.3% | 74.5 | 0.2 | 0.3% | 74.5 | 0.2 | 0.3% | 74.5 | 0.2 | 0.3% | Cumulatively, these data demonstrate that no performance difference is expected for the Global Fever Special Pathogens Panel between the FilmArray 2.0 and FilmArray Torch instrument systems. Reproducibility of the BioFire SHIELD Control Kit for the BioFire Global Fever Special Pathogens Panel was also evaluated on both the FilmArray 2.0 and FilmArray Torch Systems. Briefly, the control material was assessed for reproducibility over three different instrument systems and three independent reagent lots. Testing took place over five days with evaluation of the FilmArray 2.0 instrument runs occurring across three separate sites and evaluation of FilmArray Torch instrument runs occurring at BioFire across three simulated sites. Results from these evaluations are summarized below in Table 6 and Table 7. **Table 6. Reproducibility Test Results of the BioFire SHIELD Control Kit for the Global Fever Special Pathogens Panel on the FilmArray 2.0 System** | External Control Type | Expected Result | Observed/Expected (Percent Agreement) | | | | | --- | --- | --- | --- | --- | --- | | | | Site 1 | Site 2 | Site 3 | All Sites [95% Confidence Interval] | | Positive | Passed^{1} | 42/45 (93.3%) | 45/45 (100%) | 45/45 (100%) | 132/135 (97.8%) [93.7-100%] | | 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%] | | | | $^{1}$All GF Special Pathogens Panel assays have a positive amplicon melt in the ECM melt range. $^{2}$All GF special Pathogens Panel assays have no melt in both the ECM melt range and in the organism/virus melt range. $^{3}$Unexpected detection of pathogen amplicon for Leish assay. $^{4}$Unexpected detection of pathogen amplicon for DENV 2_1 assay. Three Positive External Controls failed; one failed due a single assay being called negative, and the other two showed no amplification of ECM for any of the Global Fever Special Pathogens Panel assays. K213362 - Page 21 of 56 {21} **Table 7. Reproducibility Test Results of the BioFire SHIELD Control Kit for the Global Fever Special Pathogens Panel on the FilmArray Torch System** | External Control Type | Expected Result | Observed/Expected (Percent Agreement) | | | | | --- | --- | --- | --- | --- | --- | | | | System 1 | System 2 | System 3 | Overall [95% Confidence Interval] | | Positive | Passed^{1} | 43/45 (95.6%) | 43/45 (95.6%) | 44/45 (100%) | 130/135 (96.3%) [91.6-98.4%] | | Negative | Passed^{2} | 43/45^{3} (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 GF Special Pathogens Panel assays have a positive amplicon melt in the ECM melt range. $^{2}$All GF Special Pathogens Panel assays have no melt in both the ECM melt range and in the organism/virus melt range. $^{3}$One run had unexpected positive results for Ebolavirus and West Nile virus assays and another run had unexpected positive results for Dengue, Ebolavirus, and *P. falciparum* assays ## 2. Linearity: Not applicable. ## 3. Analytical Specificity/Interference: ### *Inclusivity* The analytical reactivity (inclusivity) of the BioFire Global Fever Special Pathogens Panel was evaluated with a collection of isolates to represent relevant species, subspecies, or serotypes. Testing was performed by evaluating three replicates. If there was one undetected result, two additional replicates were tested and if both replicates were detected the isolate was considered inclusive. Most isolates were detected by the FilmArray Global Fever Panel at spiked concentrations within 3 × LoD of testing either inactivated or live organisms (based on molecular quantification of nucleic acids for each isolate) in whole blood. Several isolates with reduced assay reactivity are described in more detail below. When possible, in silico analysis of sequence data was used to make predictions of assay reactivity for less common strains or serotypes that were not tested. Additional inclusivity testing of live organisms for BSL3/4 organisms was also performed the results from this testing have been incorporated into the table below. Table 8 includes a summary of FilmArray Global Fever Panel reactivity based on empirical data. K213362 - Page 22 of 56 {22} **Table 8. Summary of FilmArray Global Fever Panel Analytical Reactivity (Inclusivity)** | FilmArray Global Fever Panel Analyte | # Isolates Detected / Tested | Isolates Tested | Concentration Detected ^{1} | x LoD | FilmArray Global Fever Panel Result (Replicates Detected/Tested) | | --- | --- | --- | --- | --- | --- | | **BACTERIA** | | *Bacillus anthracis* | 4/4 | Ames35 | 4.2E+01 copies/mL | 1 x LoD | Detected (19/20) | | Sterne 34Fs | 1.3E+02 copies/mL | 2.5 x LoD | Detected (3/3) | | UM23 | | Weybridge | | 10/12 | Ames (Live) | 6.4E+01 copies/mL | 1 x LoD | Detected (4/4) | | 108 (Live) | 1.9E+02 copies/mL | 3 x LoD | Detected (3/3) | | 2002013094 (Live) | | Canadian Bison (Live) | | K3 (Live) | | Ohio ACB (Live) | | PAK-1 (Live) | | RA3 (Live) | 6.4E+02 copies/mL | 10 x LoD | Detected (4/5) | | SK-102 (Live) | | South Africa (Live) | 1.9E+02 copies/mL | 3 x LoD | Detected (3/3) | | Sterne (Live) | | Turkey #32 (Live) | | Vollum 1B (Live) | 6.4E+02 copies/mL | 10 x LoD | Detected (3/3) | | *Francisella tularensis* | 5/5 | **Subspecies** | **Strain** | | | | *F. tularensis* | SCHU S4 | 1.2E+03 copies/mL | 1 x LoD | Detected (20/20) | | *F. holarctica* | LVSR | 3.6E+03 copies/mL | 3 x LoD | Detected (3/3) | | Type B LVS (CDC) | 3.6E+03 copies/mL | 3 x LoD | Detected (3/3) | | *F. novicida* | KM14S | 3.6E+03 copies/mL | 3 x LoD | Detected (3/3) | | CG62 | 3.6E+03 copies/mL | 3 x LoD | | | 9/9 | *F. tularensis* | Schu4 (Live) | 1.2E+01 copies/mL | 1 x LoD | Detected (4/4) | | Scherm (Live) | 3.6E+01 copies/mL | 3 x LoD | Detected (3/3) | | WY96 (Live) | | *F. holarctica* | Holarctica LVS (Live) | | Holarctica (Live) | | Holarctica 425 (live) | 2.3E+01 copies/mL | 1.9 x LoD | | | HD, DB082106G (Live) | | *F. novicida* | VT68 (Live) | 3.6E+01 copies/mL | 3 x LoD | | | F6168 (Live) | | U112, GA993550 (Live) | | *Leptospira* spp. | 19/19 | **Species** | **Strain** | | | | *L.interrogans* | Serovar (Budapest) | 3.4E+02 copies/mL | 1 x LoD | Detected (20/20) | | HAI0156 (Copenhageni) | 1.2E+03 copies/mL | 3.5 x LoD | Detected (3/3) | | L495 (Manilae) | | *L. alexanderi* | L60 (Manhao 3) | 1.0E+03 copies/mL | 3 x LoD | | | *L. alstonii* | Sichuan 79601 | 1.2E+03 copies/mL | 3.5 x LoD | Detected (4/5) | | *L. borgpetersenii* | Castellon 3 (Castellonis) | Detected (3/3) | | Veldrat Bataviae 46 (Javanica) | 1.0E+03 copies/mL | 3 x LoD | | *L. kirschneri* | 200701401 (Bogvere) | 1.2E+03 copies/mL | 3.5 x LoD | | K213362 - Page 23 of 56 {23} | FilmArray Global Fever Panel Analyte | # Isolates Detected / Tested | Isolates Tested | Concentration Detected ^{1} | x LoD | FilmArray Global Fever Panel Result (Replicates Detected/Tested) | | --- | --- | --- | --- | --- | --- | | | | | 3522 C (Cynopteri) | 5.3E+02 copies/mL | 1.6 x LoD | | | *L. kmetyi* | Bejo-Iso9T (Malaysia) | 1.2E+03 copies/mL | 3.5 x LoD | | *L. mayottensis* | 200901116 | | *L. noguchii* | CZ 214T (Panama) | | *L. santarosai* | LT 821 (Shermani) | 8.7E+02 copies/mL | 2.6 x LoD | | *L. weilii* | 6712 | 1.2E+03 copies/mL | 3.5 x LoD | | 94-79970/3 Topaz | | A 102 (Mengrun) | | Celledoni 20160426 | | H 27 (Hekoou) LT89-68 (Vughia) | | *Yersinia pestis* | 3/3 | A1122 (biovar Orientalis) | 1.3E+02 copies/mL | 1 x LoD | Detected (20/20) | | PY-013 (biovar Orientalis) | 3.9E+02 copies/mL | 3 x LoD | Detected (3/3) | | PH 80/63 (biovar Antiqua) | 3.9E+02 copies/mL | 3 x LoD | Detected (3/3) | | 12/12 | **Biovar** | **Strain** | | | | | Orientalis | CO92 (Live) | 1.5E+02 copies/mL | 1 x LoD | Detected (8/8) | | A1122 (Live) | 4.5E+02 copies/mL | 3 x LoD | Detected (3/3) | | Dodson (Live) | 3.6E+02 CFU/mL | 3 x LoD | Detected (3/3) | | Java 9 (Live) | 4.5E+02 copies/mL | 3 x LoD | Detected (3/3) | | PBM19 (Live) | | Shasta (Live) | | Antigua | Angola (Live) | 3.6E+02 copies/mL | 3 x LoD | Detected (3/3) | | Antigua (Live) | | Nairobi (Live) | | Medievalis | Pestoides F (Live) | Pestoides F (Live) | 3.6E+02 CFU/mL | 3 x LoD | Detected (3/3) | | Harbin 35 (Live) | Harbin 35 (Live) | 6.4E+02 copies/mL | 4.2 x LoD | | KIM5 (Live) | KIM5 (Live) | | | | Nicholisk 41 (Live) | 4.5E+02 copies/mL | 3 x LoD | Detected (3/3) | | **VIRUSES** | | Chikungunya Virus | 3/3 | **Strain** | | | | | R80422 | 5.5E+02 copies/mL | 1 x LoD | Detected (20/20) | | DHS4263 | 1.7E+03 copies/mL | 3 x LoD | Detected (3/3) | | St. Martin 2013 | 1.4E+03 copies/mL | 2.5 x LoD | | Crimean-Congo Hemorrhagic Virus | 1/1 | Nigeria/IbAr10200 | 6.4E+00 copies/mL | 1 x LoD | Detected (20/20) | | Dengue virus | 27/28 | **Serotype** | **Strain** | | | | | Serotype 1 | Hawaii | 2.2E+02 copies/mL | 1 x LoD | Detected (19/20) | | Strain 12150 | 6.6E+02 copies/mL | 3 x LoD | Detected (3/3) | | 228690 | | 276RK1 | | BC89/94 | | SL-6-6-04 | | UIS 1162 | | Serotype 2 | VN/BID-V1792/2007 | 3.4E+02 copies/mL | 1 x LoD | Detected (20/20) | | New Guinea C (DENV2 1) | | DakArA1247 (DENV2 2) | 2.7E+03 copies/mL | 1 x LoD | | 1349 | 1.1E+03 copies/mL | 3.2 x LoD | Detected (3/3) | | | | 429557 | | | | K213362 - Page 24 of 56 {24} | FilmArray Global Fever Panel Analyte | # Isolates Detected / Tested | Isolates Tested | | Concentration Detected ^{1} | x LoD | FilmArray Global Fever Panel Result (Replicates Detected/Tested) | | --- | --- | --- | --- | --- | --- | --- | | | | | ArA6894 | 1.1E+03 copies/mL | 0.4 x LoD | Detected (1/1) | | | | | BC102/94 | 9.4E+02 copies/mL | 2.8 x LoD | | | | | | DKA 811 | 2.4E+06 copies/mL | ~10,000 x LoD | | | | | | VN/BID-V1002/2006 | 1.1E+03 copies/mL | 3.2 x LoD | | | | | Serotype 3 | H87 | 1.3E+02 copies/mL | 1 x LoD | Detected (20/20) | | | | | 271242 | 3.9E+02 copies/mL | 3 x LoD | | | | | | BC188/97 | 1.6E+04 copies/mL | 123 x LoD | | | | | | C0360/94 | 3.9E+02 copies/mL | 3 x LoD | | | | | | VN/BID-V1329/2006 | | | | | | | Serotype 4 | H241 | 6.4E+01 copies/mL | 1 x LoD | Detected (20/20) | | | | | 703 | 1.9E+02 copies/mL | 3 x LoD | | | | | | BC13/97 | | | | | | | | BC287/97 | | | | | | | | BC258/97 | | | | | | | | D85-019 | 7.6E+03 copies/mL | 119 x LoD | | | | | | PR 06-65-740 | 2.2E+02 copies/mL | 3.4 x LoD | | | Ebolavirus spp. | 6/6 | Bundibugyo virus Uganada | | 7.0E+04 copies/mL | 1 x LoD | Detected (20/20) | | | | Tai Forest virus | | 8.3E+03 copies/mL | 1 x LoD | | | | | Reston virus | | 2.7E+04 copies/mL | 1 x LoD | | | | | Sudan virus | | 1.1E+04 copies/mL | 1 x LoD | | | | | Zaire | Gueckedou | 1.1E+02 copies/mL | 1 x LoD | | | | | | Mayinga | 1.1E+04 copies/mL | 110 x LoD | | | | 4/4 | Zaire | Makona (Live) | 1.1E+03 copies/mL | 1 x LoD | Detected (3/3) | | | | | Mayinga '76 (Live) | 2.1E+03 copies/mL | 2 x LoD | | | | | | Gabon (Live) | 3.2E+03 copies/mL | 2.9 x LoD | | | | | | Kikwit '95 (Live) | 3.1E+03 copies/mL | 2.8 x LoD | | | | | | Luebo (Live) | 3.1E+03 copies/mL | 2.8 x LoD | | | | | | | | | | | Lassa virus | 1/1 | Josiah | | 5.6E+04 copies/mL | 1 x LoD | Detected (20/20) | | | 2/2 | Josiah (Live) | | 2.4E+01 PFU/mL | 1 x LoD | Detected (4/4) | | | | Macenta (Live) | | 7.2E+01 PFU/mL | 3 x LoD | Detected (3/3) | | | | Pinneo (Live) | | 7.2E+01 PFU/mL | 3 x LoD | | | Marburgvirus | 3/3 | Musoke | | 5.0E+02 copies/mL | 1 x LoD | Detected (20/20) | | | | Voege | | 1.5E+03 copies/mL | 3 x LoD | Detected (4/5) | | | | Ravn virus, Kenya | | 2.6E+02 copies/mL | 1 x LoD | Detected (20/20) | | | 2/2 | Ravn (Live) | | 2.6E+02 copies/mL | 1 x LoD | Detected (4/4) | | | | Ci67 (Live) | | 5.0E+02 copies/mL | 1 x LoD | Detected (4/4) | | | | Angola (Live) | | 1.5E+03 copies/mL | 3 x LoD | Detected (3/3) | | | | Musoke (Live) | | 7.8E+02 copies/mL | 1.5 x LoD | | | West Nile virus | 5/5 | B-956 Uganda | | 2.3E+04 copies/mL | 1 x LoD | Detected (20/20) | | | | B-956 (lineage 2) | | 6.9E+04 copies/mL | 3 x LoD | Detected (3/3) | | | | NY 2001-6263 (lineage 1) | | 1.1E+03 copies/mL | 1 x LoD | Detected (20/20) | | | | Bird 114 (lineage 1) | | 3.3E+03 copies/mL | 3 x LoD | Detected (3/3) | | | | 1986 (lineage 1) | | 2.2E+03 copies/mL | 2 x LoD | | | | 1/1 | Bz NY99 (Live) | | 1.6E+02 copies/mL | 1 x LoD | Detected (4/4) | | | | Eg101 (Live) | | 4.8E+02 copies/mL | 3 x LoD | Detected (3/3) | | Yellow Fever virus | 1/1 | 17D | | 1.2E+02 copies/mL | 1 x LoD | Detected (20/20) | | | 4/4 | Asibi (Live) | | 1.2E+01 copies/mL | 1 x LoD | Detected (3/3) | | | | SVM 3-18-09 (Live) | | 1.2E+04 copies/mL | 1000 x LoD | | | | | CAREC M2-09 (Live) | | 1.2E+03 copies/mL | 100 x LoD | | | | | INHRR 7a-05 (Live) | | 1.2E+03 copies/mL | 100 x LoD | | | | | INHRR 10a-10 (Live) | | 1.2E+03 copies/mL | 100 x LoD | | | **PROTOZOA** | | | | | | | | Leishmania | 4/4 | L. donovani 9515 | | 1.0E+01 copies/mL | 1 x LoD | Detected (20/20) | K213362 - Page 25 of 56 {25} | FilmArray Global Fever Panel Analyte | # Isolates Detected / Tested | Isolates Tested | | Concentration Detected^{1} | x LoD | FilmArray Global Fever Panel Result (Replicates Detected/Tested) | | --- | --- | --- | --- | --- | --- | --- | | spp. | | *L. donovani*, 1S | | 3.4E+01 copies/mL | 3.4 x LoD | Detected (3/3) | | | | *L. donovani* chagasi | | 3.0E+01 copies/mL | 3 x LoD | Detected (4/5) | | | | *L. infantum* | | 1.0E+02 copies/mL | 10 x LoD | Detected (3/3) | | Plasmodium spp. | 10/10 | **Species** | **Strain** | | | | | | | *falciparum* | IPC 4884 | 1.8E02 copies/mL | 1 x LoD | Detected (20/20) | | | | | SenTh021.09 | 3.0E+02 copies/mL | 1.7 x LoD | Detected (5/5) | | | | | St. Lucia | | | Detected (3/3) | | | | | Tanzania, 02000708 | | | | | | | *vivax* | Chesson | 1.5E+02 copies/mL | 1 x LOD | Detected (20/20) | | | | | Panama | 3.1E+02 copies/mL | 2.1 x LOD | Detected (3/3) | | | | *ovale* | Wallikeri | 2.4E+02 copies/mL | 1 x LOD | Detected (20/20) | | | | | Curtisi | 7.2E+02 copies/mL | 3 x LoD | Detected (3/3) | | | | *knowlesi* | Strain H | 2.4E+01 copies/mL | 1 x LOD | Detected (20/20) | | | | *malariae* | Clinical specimen | 1.9E+02 copies/mL | 1 x LOD | | | *Plasmodium falciparum* | 4/4 | *falciparum* | IPC4884 | 1.8E+02 copies/mL | 1 x LoD | Detected (3/3) | | | | | SenTh021.09 | 1.8E+03 copies/mL | 10 x LoD | | | | | | St. Lucia | 3.0E+02 copies/mL | 1.7 x LoD | Detected (4/5) | | | | | Tanzania, 02000708 | | | Detected (3/3) | | *Plasmodium vivax/ovale* | 4/4 | *vivax* | Chesson | 1.5E+02 copies/mL | 1 x LoD | Detected (20/20) | | | | | Panama | 3.1E+02 copies/mL | 2 x LoD | Detected (3/3) | | | | *ovale* | Wallikeri | 2.4E+02 copies/mL | 1 x LoD | Detected (20/20) | | | | | Curtisi | 7.2E+02 copies/mL | 3 x LoD | Detected (3/3) | Organisms which exhibited reduced or no assay reactivity are described specifically in the table below. | Observed Result | Detection Level | Analyte | Serotype/Strain/Isolate | | --- | --- | --- | --- | | Detected (may be underreported) | 10 x LoD | *Bacillus anthracis* | SK-102 (Live) | | | 10 x LoD | *Bacillus anthracis* | Vollum 1B (Live) | | | 10 x LoD | *Leishmania infantum* | | | | 10 x LoD | *Leishmania braziliensis* | Vianna | | | 10 x LoD | *Plasmodium falciparum*^{1} | SenTh021.09 | | | ~100 x LoD | Dengue virus^{1} | Serotype 3 BC188/97 | | | | | Serotype 4 D85-019 | | | 110 x LoD | Ebolavirus | Zaire, Mayinga (Inactivated)^{3} | | | 100 x LoD | Yellow Fever Virus | CAREC M2-09^{4} | | | | | INHRR 7a-05^{4} | | | | | INHRR 10a-10^{4} | | 1000 x LoD | | SVM 3-18-09^{4} | | | Not Detected | - | Dengue virus^{2} | Serotype 2 DKA 811 | $^{1}$The reason for the observed reduced reactivity could not be identified based on in silico sequence analysis. Sequences for these strains were not available in public databases. $^{2}$In silico analysis predicted reduced sensitivity or missed detection of this isolate due to sequence variation. Wet testing of this rare sylvatic strain at 10,000 x LoD confirmed detection was significantly impaired. K213362 - Page 26 of 56 {26} $^{3}$Results of live testing did not show reduced sensitivity for the detection of *Zaire evolvavirus* Mayinga, and therefore the observed reduced sensitivity for inactivated *Zaire evolvavirus* Mayinga may be the result of damage to nucleic acid due to inactivation process. $^{4}$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. Available sequences for these strains do not contain the full target region of the assay, therefore the reduced sensitivity could not be fully investigated. ### Microbial Interference Studies Potentially interfering microorganisms were evaluated for their effect on FilmArray Global Fever Special Pathogens Panel performance. To evaluate the potential for interference, FilmArray Global Fever Special Pathogens Panel test results from a control blood sample containing representative panel analytes (*Leptospira interrogans*, *P. falciparum*, dengue virus type 3) at concentrations near 3×LoD were compared to results from a sample with the same composition plus the potentially interfering microorganism, as well as a negative sample (no analytes) containing only the potentially interfering microorganism. Each potentially competing microorganism was tested at the highest concentration possible (1:10 dilution of the stock). The samples containing the potentially interfering microorganism were evaluated for their effects on the BioFire Global Fever Special Pathogens Panel internal control assays and analyte detection. Reproducible internal control failures or loss of analyte detection associated with the presence of a particular potentially interfering microorganism would be recognized as interference by that microorganism. **Table 9. Organisms Evaluated for Potential Microbial Interference** | Microorganisms | Concentration Tested | Results | | --- | --- | --- | | *Corynebacterium diphtheriae* | 1:10 dilution of stock | No interference observed | | *Staphylococcus epidermidis* | 3.8E+06 CFU/mL | No interference observed | | *Escherichia coli* | 1:10 dilution of stock | No interference observed | | *Klebsiella pneumoniae* | 5.5E+04 CFU/mL | No interference observed | | *Haemophilus influenzae* | 1.0E+08 CFU/mL | No interference observed | | Herpes Simplex virus | 1.2E+05 TCID_{50}/mL | No interference observed | | Epstein-Barr virus | 3.3E+07 copies/mL | No interference observed | | Cytomegalovirus | 1:10 dilution of stock | No interference observed | | Human Immunodeficiency virus (HIV) 1/2 | HIV-1: 1.3E+05 U/mL HIV-2: 2.2E+05 U/mL | No interference observed | | *Plasmodium vivax* | 1.5E+06 copies/mL | No interference observed | None of the ten microorganisms tested showed interference with the pouch controls or specific Global Fever Panel assay targets. ### Analytical specificity/Cross-reactivity The potential for non-specific amplification and detection by the FilmArray Global Fever Panel assays (cross-reactivity) was evaluated by testing high concentrations of on-panel (identified by the FilmArray Global Fever Panel assays) and off-panel (not intended to be identified by the FilmArray Global Fever Panel assays) organisms or purified nucleic acids, as well as by *in silico* analysis. All on-panel organisms were tested live at high concentration (> 10$^{6}$ copies/mL). As can be seen in Table 10 below, testing of *P. knowlesi* demonstrated cross-reactivity with the *P. vivax/ovale* assay at concentrations above 2.2E+03 copies/mL. K213362 - Page 27 of 56 {27} **Table 10. BioFireGlobal Fever Special Pathogens Panel Results for On-Panel Organism Testing Assessing Potential Cross-reactivity** | Pathogen | | Results | | --- | --- | --- | | *Bacillus anthracis* | | As expected (only *B. anthracis* Detected) | | *Francisella tularensis* subsp. *tularensis* | | As expected (only *F. tularensis* Detected) | | *Leptospira interrogans* (Schu S4) | | As expected (only *Leptospira* spp. Detected) | | *Yersinia Pestis* | | Yersinia pestis Detected Unexpected Detected Results for *F. tularensis*^{1} | | Chikungunya virus (R80422 culture fluid) | | As expected (only Chikungunya virus Detected) | | Crimean-Congo Hemorrhagic fever virus | | As expected (only CCHF Detected) | | Dengue | DENV-1 (Hawaii) | As expected (only Dengue virus Detected) | | | DENV-2 ( New Guinea C) | As expected (only Dengue virus Detected) | | | DENV-3 (H87) | As expected (only Dengue virus Detected) | | | DENV-4 (H241) | As expected (only Dengue virus Detected) | | *Zaire ebolavirus* | | As expected (only Ebolavirus spp. Detected) | | *Sudan ebolavirus* | | Ebolavirus spp. Detected Unexpected Detected Result for *F. tularensis*^{1} | | *Bundibugyo ebolavirus* | | As expected (only Ebolavirus spp. Detected) | | *Tai Forest ebolavirus* | | As expected (only Ebolavirus spp. Detected) | | *Reston ebolavirus* | | As expected (only Ebolavirus spp. Detected) | | Lassa virus | | As expected (only Lassa virus Detected) | | Marburg Marburgvirus variant Musoke | | As expected (only Marburgvirus Detected) | | Marburg marburgvirus (RAVN) | | As expected (only Marburgvirus Detected) | | West Nile virus lineage 1 | | As expected (only West Nile Virus Detected) | | West Nile virus lineage 2 | | As expected (only West Nile Virus Detected) | | Yellow Fever virus | | As expected (only Yellow Fever Virus Detected) | K213362 - Page 28 of 56 {28} | Pathogen | | Results | | --- | --- | --- | | *Leishmania donovani* | | As expected (only *Leishmania* spp. Detected) | | *Plasmodium* | *falciparum* (Pursat Cambodia 2011) | As expected (*Plasmodium* spp. Detected and *P. falciparum* Detected) | | | *knowlesi* (Strain H) | *Plasmodium* spp Detected as expected. *Plasmodium vivax/ovale* detected at concentrations above 2.2E+03 copies/mL. | | | *malariae* (DLS17-026015) | As expected (only *Plasmodium* spp. Detected) | | | *vivax* (11 Chesson) | As expected (*Plasmodium* spp. Detected and *P. vivax/ovale* Detected) | | | *ovale* (Wallikeri) | As expected (*Plasmodium* spp. Detected and *P. vivax/ovale* Detected) | ¹Due to potential contamination event Although no cross-reactivity with *P. malariae* was observed in this study, in silico analysis shows 1-3 mismatches between *P. malariae* and the *P. vivax/ovale* assay primers. Wet testing with *P. brasilianum* (a monkey *Plasmodium* thought to have descended from *P. malariae* and which has identical sequence as *P. malariae*) showed reactivity for the *P. vivax/ovale* assay at 1.6E+06 copies/mL. Off-panel organisms were selected for testing based on a combination of several factors including: - Relatedness to the species detected by the Global Fever Panel (near-neighbors) - Clinical relevance (causing 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 - genetic similarity to Global Fever Panel assay primers as determined by in silico analysis BSL3/4 organisms and viruses that could not be obtained as inactivated stocks or purified nucleic acids were tested live in an appropriate facility (USAMRIID and The Center) to assess potential cross-reactivity. Table 11 below lists the off-panel organisms that were tested at high concentrations (> 10⁶ copies/mL) for potential cross-reactivity with the FilmArray Global Fever Panel. Table 11. Off-Panel Organisms Tested by the BioFire Global Fever Special Pathogens Panel | Bacteria | | Viruses | | | --- | --- | --- | --- | | *Acinetobacter baumannii* | *Leptospira wolbachii* | Adenovirus 1 | Omsk hemorrhagic fever virus | | *Bacillus brevis* | *Leptospira yanagawae* | Adenovirus 3 | **O'nyong-nyong virus**² | | *Bacillus cereus* | *Listeria monocytogenes* | Adenovirus 5 | Parvo virus | K213362 - Page 29 of 56 {29} | Bacillus circulans | Mycobacterium tuberculosis (avirulent) | Aura virus | Powassan virus | | --- | --- | --- | --- | | Bacillus coagulans Hammer | Mycoplasma pneumoniae | Barmah Forest virus | Rabies virus | | Bacillus halodurans | Neisseria meningitidis | Bunyamwera virus | Rift Valley Fever Virus | | Bacillus licheniformis | Proteus mirabilis | Coronavirus NL63 | Ross River virus | | Bacillus megaterium | Pseudomonas aeruginosa | Crimean-Congo Hemorrhagic Fever Virus | Human respiratory syncytial virus | | Bacillus mycoides | Rickettsia typhi | Dugbe virus | Rubella virus | | Bacillus pumilus | Salmonella enterica subsp. arizonae | Eastern Equine Encephalitis Virus | Saint Louis encephalitis virus | | Bacillus subtilis | Salmonella enterica subsp. bongori | Ebolavirus (Zaire, Sudan, Bundibugyo, Tai Forest, Reston) | Sindbis virus | | Bacillus thuringiensis | Salmonella enterica subsp. diarizoniae | Enterovirus, HEV-71 | Spondweni virus | | Bacteroides fragilis | Salmonella enterica subsp. enterica serovar Dublin | Epstein Barr virus | Tickborne encephalitis virus | | Bordetella bronchiseptica | Salmonella enterica subsp. enterica serovar Enteritidis | Flexal virus | Una virus | | Borrelia burgdorferi | Salmonella enterica subsp. enterica serovar Javiana | Guanarito virus | Usutu virus | | Brucella melitensis | Salmonella enterica subsp. enterica serovar Manchester | Hantaan virus | Vaccinia virus | | Burkholderia cepacia | Salmonella enterica subsp. enterica serovar Montevideo | Hazara virus | Varicella zoster virus | | Burkholderia mallei | Salmonella enterica subsp. enterica serovar Muenchen | Hendra virus | Venezuelan Equine Encephalomyelitis (VEE) virus | | Burkholderia pseudomallei | Salmonella enterica subsp. enterica serovar Newport | Hepatitis A Virus (HAV) | Western Equine Encephalitis (WEE) virus | | Chlamydophila pneumoniae | Salmonella enterica subsp. enterica serovar Paratyphi | Hepatitis B Virus (HBV) | West Nile virus Lineage 1 | | Clostridium bifermentans | Salmonella enterica subsp. enterica serovar Rubislaw | Hepatitis C Virus (HCV) | West Nile virus Lineage 2 | | Clostridium perfringens | Salmonella enterica subsp. enterica serovar Saintpaul | Herpes Simplex Virus 2 (HSV-2) | Yellow Fever virus | | Clostridium sordelli | Salmonella enterica subsp. enterica serovar Tennessee | Human Parainfluenza Virus 1 | Zika virus | | Clostridium sporogenes | Salmonella enterica subsp. enterica serovar Thompson | Human Parainfluenza Virus 3 | Fungi | | Clostridium perfringens | Salmonella enterica subsp. enterica serovar Typhi | Hughes virus | Aspergillus fumigatus | | Coxiella burnetii | Salmonella enterica subsp. enterica serovar Typhimurium | Human herpesvirus 6B | Cryptococcus neoformans | | Enterobacter aerogenes | Salmonella enterica subsp. houtenae | Human Immunodeficiency Virus 1 | Pathogenic Protozoa | | Enterococcus faecalis | Salmonella enterica subsp. indica | Human Immunodeficiency Virus 2 | Babesia microti | | Enterococcus faecium | Salmonella enterica subsp. salamae | Human T-lymphotropic virus type 1 | Crithidia fasciculata² | | Francisella hispaniensis | Serratia marcescens | Human T-lymphotropic virus type 2 | Cyclospora cayetanensis | K213362 - Page 30 of 56 {30} | Francisella persica | Staphylococcus aureus | Influenza A H1N1-2009 | Leishmania donovani | | --- | --- | --- | --- | | Francisella philomiragia | Streptococcus agalactiae | Influenza A H3N2 | Leptomonas seymouri^{1} | | Klebsiella oxytoca | Streptococcus pneumoniae | Influenza B virus | Schistosoma mansoni | | Legionella pneumophila | Streptococcus pyogenes | Japanese Encephalitis Virus (JEV) | Toxoplasma gondii | | L. amazonensis^{1} | Vibrio cholerae | Junin virus | Trypanosoma brucei | | L. braziliensis Vianna^{1} | Yersinia aldovae | Lassa virus | Trypanosoma cruzi | | L. major IR173^{1} | Yersinia bercovieri | Machupo virus | Trypanosoma rangeli | | L. Mexicana^{1} | Yersinia entericolitica | Marburg Marburgvirus variant Musoke | Plasmodium berghei^{5} | | L. panamensis^{1} | Yersinia fredericksenii | Marburg Marburgvirus (RAVN) | Plasmodium brasilianum^{6} | | L. tropica (MHOM/AF/87/RUP)^{1} | Yersinia intermedia | Mayaro virus | Plasmodium cynomolgi^{7} | | L. tropica (MHOM SU/58/strain-OD)^{1} | Yersinia kristensenii | Measles virus | Plasmodium fieldi^{8} | | Leptospira biflexa | Yersinia mollarettii | Metapneumovirus | Plasmodium fragile^{9} | | Leptospira meyeri | Yersinia pestis | Mopeia Virus | Plasmodium inui^{10} | | Leptospira terpstrae | Yersinia pseudotuberculosis | Mumps Virus | Plasmodium simiovale^{11} | | Leptospira vanthielii | Yersinia rohdei | | | $^{1}$Assay reactivity is expected for these Leishmania species which may be present in the blood due to atypical visceral leishmaniasis. $^{2}$At high concentrations (≤ 1:1000 dilution of stock), the o'nyong-nyong virus triggered a detected result for chikungunya virus. O'nyong-nyong and chikungunya virus infections can occur in the same geographic locations and present with similar symptoms. Appropriate warnings will be included in product literature. $^{3}$Crithidia fasciculata generated Leishmania spp. Detected result. It is a non-human infective trypanosomatid, however there have been very rare cases where a closely related Crithidia strain was found to be…

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