FilmArray Global Fever Panel

DEN200043 · Biofire Defense, LLC · QMV · Nov 20, 2020 · Microbiology

Device Facts

• Record ID: DEN200043
• Device Name: FilmArray Global Fever Panel
• Applicant: Biofire Defense, LLC
• Product Code: QMV · Microbiology
• Decision Date: Nov 20, 2020
• Decision: DENG
• Submission Type: Direct
• Regulation: 21 CFR 866.3966
• Device Class: Class 2

Intended Use

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 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.

Device Story

The FilmArray Global Fever Panel is a multiplexed, nucleic acid-based diagnostic test used with the FilmArray 2.0 system. It processes EDTA whole blood samples to detect DNA/RNA from Leptospira spp., chikungunya virus, dengue virus (serotypes 1-4), and Plasmodium spp. The device uses a closed-pouch system containing freeze-dried reagents; it performs automated mechanical/chemical lysis, nucleic acid purification via magnetic beads, and nested multiplex PCR. The system uses Peltier devices for thermocycling and a digital camera to capture fluorescent melt curve data. Software automatically interprets melt curves to provide qualitative 'Detected' or 'Not Detected' results for each target. Used in clinical settings, the output aids healthcare providers in diagnosing acute febrile illness in conjunction with clinical/epidemiological data. It benefits patients by enabling rapid identification of specific pathogens, facilitating targeted treatment, and addressing unmet diagnostic needs for these infections.

Clinical Evidence

Clinical study required as a special control; must include prospective (sequentially collected) samples and characterized clinical samples. Performance compared to FDA-accepted comparator methods. Documentation must include study protocol, statistical analysis plan, and results for all claimed specimen types.

Technological Characteristics

The device is a closed-system, single-use pouch containing freeze-dried reagents for nucleic acid purification and nested multiplex PCR. It utilizes magnetic bead technology for purification and LC Green Plus fluorescent dye for melt curve analysis. The system is operated on the FilmArray 2.0 instrument, which uses pneumatic bladders, pistons, and Peltier heating/cooling elements. It is a standalone, non-networked diagnostic system. Software is used for automated data interpretation and report generation.

Indications for Use

Indicated for individuals with signs/symptoms of acute febrile illness or recent acute febrile illness and known or suspected exposure to Leptospira spp., chikungunya virus, dengue virus, or Plasmodium spp. (P. falciparum, P. vivax/ovale).

Regulatory Classification

Identification

A device to detect and identify selected microbial agents that cause acute febrile illness is identified as an in vitro device intended for the detection and identification of microbial agents in human clinical specimens from patients with signs and symptoms of acute febrile illness who are at risk for exposure or who may have been exposed to these agents. It is intended to aid in the diagnosis of acute febrile illness in conjunction with other clinical, epidemiologic, and laboratory data, including patient travel, pathogen endemicity, or other risk factors.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) Any sample collection device used must be FDA-cleared, -approved, or -classified as 510(k) exempt (standalone or as part of a test system) for the collection of specimen types claimed by this device; alternatively, the sample collection device must be cleared in a premarket submission as a part of this device. (2) The labeling required under § 809.10(b) of this chapter must include: (i) An intended use that includes a detailed description of targets the device detects and measures, the results provided to the user, the clinical indications appropriate for test use, and the specific population(s) for which the device is intended. (ii) Limiting statements indicating: (A) Not all pathogens that cause febrile illness are detected by this test and negative results do not rule out the presence of other infections; (B) Evaluation of more common causes of acute febrile illness should be considered prior to evaluation with this test; (C) Test results are to be interpreted in conjunction with other clinical, epidemiologic, and laboratory data available to the clinician; and (D) When using this test, consider patient travel history and exposure risk, as some pathogens are more common in certain geographical locations. (iii) A detailed device description, including reagents, instruments, ancillary materials, all control elements, and a detailed explanation of the methodology, including all pre-analytical methods for processing of specimens. (iv) Detailed discussion of the performance characteristics of the device for all claimed specimen types as shown by the analytical and clinical studies required under paragraphs (b)(3)(ii) and (iii) of this section, except specimen stability performance characteristics. (v) A statement that nationally notifiable results are to be reported to public health authorities in accordance with local, state, and federal law. (3) Design verification and validation must include: (i) A detailed device description ( *e.g.,* all device parts, control elements incorporated into the test procedure, reagents required but not provided, the principle of device operation and test methodology), and the computational path from collected raw data to reported result (*e.g.,* how collected raw signals are converted into a reported result).(ii) Detailed documentation of analytical studies, including those demonstrating Limit of Detection (LoD), inclusivity, cross-reactivity, microbial interference, interfering substances, competitive inhibition, carryover/cross contamination, specimen stability, within lab precision, and reproducibility, as appropriate. (iii) Detailed documentation and performance results from a clinical study that includes prospective (sequentially collected) samples for each claimed specimen type and, when determined to be appropriate by FDA, additional characterized clinical samples. The study must be performed on a study population consistent with the intended use population and compare the device performance to results obtained from FDA-accepted comparator methods. Documentation from the clinical studies must include the clinical study protocol (including a predefined statistical analysis plan), study report, testing results, and results of all statistical analyses. (iv) A detailed description of the impact of any software, including software applications and hardware-based devices that incorporate software, on the device's functions.

Submission Summary (Full Text)

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# EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR FILMARRAY GLOBAL FEVER PANEL DECISION SUMMARY

## A. De Novo Number:

DEN200043

## B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the FilmArray Global Fever Panel.

# C. Measurand:

DNA and RNA sequences from the following organisms: Leptospira spp., dengue virus serotypes 1-4, chikungunya virus, and Plasmodium spp.

## D. Type of Test:

Multiplex Nucleic Acid Amplification Test

## E. Applicant:

BioFire Defense, LLC

## F. Proprietary and Established Names:

FilmArray Global Fever Panel

# G. Regulatory Information:

- 1. Regulation section:
21 CFR 866.3966

- 2. Classification:
Class II

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- 3. Product code(s):
OMV

- 4. Panel:
Microbiology (83)

## H. Intended Use:

- 1. Intended use(s):
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 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.

- 2. Indication(s) for use:
Same as intended use.

- 3. Special conditions for use statement(s):
For prescription use only.

For in vitro diagnostic use only.

- 4. Special instrument requirements:
The FilmArray Global Fever Panel is performed on the FilmArray 2.0 system.

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# I. Device Description:

The FilmArrav Global Fever Panel is a multiplex nucleic acid-based test designed to be used with the FilmArray 2.0 system ("FilmArray system" or "FilmArray instrument"). The FilmArray Global Fever Panel includes a FilmArray Global Fever Panel pouch (pouch) which contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex polymerase chain reaction (PCR) with DNA melt analysis. The FilmArray Global Fever Panel simultaneously conducts six tests for the identification of bacterial, viral, and protozoan organisms from whole blood specimens collected in EDTA tubes. Results from the FilmArray Global Fever 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 or positive blood culture specimen mixed with the provided Sample Buffer and protease into the other port of the pouch and placing it in the FilmArray Instrument. 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 rehvdrates 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 liguid 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 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 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 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 2nd stage PCR, or nested PCR, is performed in each well of the array. At the conclusion of the 2nd 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 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

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for each organism on the panel. A description of the individual assays and their result interpretation is included below:

- . Chikungunya Virus: The Global Fever Panel contains two assays for species-level detection of all chikungunya virus strains (CHIKV1 and CHIKV2). The FilmArray software will interpret any single positive chikungunya assay as a Chikungunya Virus Detected result.
- . Dengue Virus: The Global Fever 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 software will interpret any single positive dengue virus assay as a Dengue Virus Detected result.
- . Leptospira: The Global Fever 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.
- Plasmodium: The Global Fever 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 Panel assay.

# Materials provided in each FilmArray Global Fever Panel kit:

- . Individually packaged FilmArray Global Fever Panel pouches (6)
- . Individually-packaged Transfer Pipettes (7)
- . Single-use (1.0 mL) Sample Buffer Tubes (7)
- Single-use pre-filled (1.5 mL) Hydration Injection Vials (Blue) (7) 0
- Single-use Sample Injection Vials (Red) (7) .
- Instructions and Documents .
- FilmArray Global Fever Panel Instructions for Use i
- FilmArray Global Fever Panel Quick Guide i

Materials required but not provided:

- 10% bleach solution .
FilmArray system including:

- FilmArray 2.0 instrument, computer, and software .
- FilmArray Pouch Loading Station .

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## J. Standard/Guidance Document Referenced (if applicable):

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'

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Guidance for Industry and FDA on Alternative to Certain Prescription Device Labeling Requirements

# K. Test Principle:

The FilmArray Global Fever 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 and positive blood culture. 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 FilmArray Global Fever 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:

- (D)(4) . Nucleic Acid Purification- Nucleic acid purification occurs in the 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 highpitched 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 188 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 185 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.

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# L. Performance Characteristics (if/when applicable):

## 1. Analytical performance:

# a. Precision/Reproducibility:

To evaluate the reproducibility of the FilmArray Global Fever 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 instruments; GF Panel pouch lot was rotated daily. In total, 270 valid test results were obtained for the reproducibility evaluation of the FilmArray GF Panel.

| Organism (Isolate) | Sample 1 | Sample 2 | Sample B3 |
|----------------------------------------------------------|-----------------------------------------------------|---------------------------------------------------|---------------------------------------------------|
| Leptospira interrogans<br>serovar<br>icterohaemorrhagiae | Low Positive<br>3.3E+02 copies/mL<br>1 x LoD | Moderate Positive<br>1.0E+03 copies/mL<br>3 x LoD | Negative |
| Dengue Virus (DENV-2)<br>New Guinea C | Low Positive<br>3.3E+02 copies/mL<br>1 x LoD | Moderate Positive<br>1.0E+03 copies/mL<br>3 x LoD | Negative |
| Plasmodium falciparum<br>IPC 4884 | Moderate Positive<br>5.4E+02 copies/mL<br>1.5 x LoD | Negative | Low Positive<br>9.0E+01<br>copies/mL<br>0.5 x LoD |

| Table 1. Contrived Whole Blood Samples for Reproducibility Testing | | |
|--------------------------------------------------------------------|--|--|
| | | |

Combined reproducibility results are shown below in Table 2.

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| | | Test Analyte<br>Isolate | Concentration | Expected<br>Test<br>Result | % Agreement with Expected Results | | | | |
|----------|---------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|-------------------------------------------------------|----------------------------|-----------------------------------|----------------|----------------|----------------|-------------------------------|
| | | | | | Site 1 | Site 2 | Site 3 | All<br>Sites | 95%<br>Confidence<br>Interval |
| BACTERIA | | Leptospira interrogans serovar<br>icterohaemorrhagiae | Moderate Positive<br>3x LoD<br>(1.2E+03 copies/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 30/30<br>100% | 90/90<br>100% | 96.0-100% |
| | | | Low Positive<br>1x LoD<br>(3.9E+02 copies/mL) | Detected | 27/30<br>90.0% | 28/30<br>93.4% | 26/30<br>86.7% | 81/90<br>90.0% | 81.9-95.3% |
| | | | Negative<br>(No Analyte) | Not<br>Detected | 30/30<br>100% | 30/30<br>100% | 30/30<br>100% | 90/90<br>100% | 96.0-100% |
| VIRUSES | | Dengue virus<br>DENV-2<br>New Guinea C | Moderate Positive<br>3x LoD<br>(1.1E+03 copies/mL) | Detected | 29/30<br>96.7% | 30/30<br>100% | 30/30<br>100% | 89/90<br>98.9% | 94.0-100% |
| | | | Low Positive<br>1x LoD<br>(3.6E+02 copies/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 30/30<br>100% | 90/90<br>100% | 96.0-100% |
| | | | Negative<br>(No Analyte) | Not<br>Detected | 30/30<br>100% | 30/30<br>100% | 30/30<br>100% | 90/90<br>100% | 96.0-100% |
| PROTOZOA | | Plasmodium spp.<br>Detection Results | Moderate Positive<br>1.5x LoD1<br>(2.7E+02 copies/mL) | Detected | 30/30<br>100% | 30/30<br>100% | 30/30<br>100% | 90/90<br>100% | 96.0-100% |
| | | | Low Positive<br>0.5× LOD1<br>(9.0E+01 copies/mL) | Detected | 28/30<br>93.3% | 30/30<br>100% | 29/30<br>96.7% | 87/90<br>96.7% | 90.7-98.9% |
| | | | Negative<br>(No Analyte) | Not<br>Detected | 30/30<br>100% | 30/30<br>100% | 30/30<br>100% | 90/90<br>100% | 96.0-100% |
| | IPC 4884 | Plasmodium<br>falciparum<br>Detection Results | Moderate Positive<br>1.5x LoD1<br>(2.7E+02 copies/mL) | Detected | 29/30<br>96.7% | 30/30<br>100% | 28/30<br>93.4% | 87/90<br>96.7% | 90.6-99.3% |
| | | | Low Positive<br>0.5× LoD1<br>(9.0E+01 copies/mL) | Detected | 18/30<br>60% | 24/30<br>80% | 21/30<br>70% | 63/90<br>70.0% | 59.4-79.2% |
| | | | Negative<br>(No Analyte) | Not<br>Detected | 30/30<br>100% | 30/30<br>100% | 30/30<br>100% | 90/90<br>100% | 96.0-100% |
| | Overall Agreement with the Expected Test Result<br>All Analytes and All Test Levels (95% Confidence Interval) | | | | 1,037/1,080<br>96.0% (94.7-97.1%) | | | | |

## Table 2: Reproducibility of the FilmArray Global Fever Panel Qualitative Results

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 overall. Variability in the melt temperatures for the assays evaluated was within the expected range (<0.5℃) for each assay at each site and overall, with a standard deviation of 0.2-0.3ºC.

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| Analyte<br>(Isolate tested) | Assay | Observed Tm (°C) | | | | | | | |
|-----------------------------|------------------------------|------------------|--------|----------------|--------|----------------|--------|-------------------|--------|
| | | Site 1<br>Mean | ±StDev | Site 2<br>Mean | ±StDev | Site 3<br>Mean | ±StDev | All Sites<br>Mean | ±StDev |
| CONTROLS | | | | | | | | | |
| RNA Process Control | yeast RNA | | | | (b)(4) | | | | |
| PCR2 Control | PCR2 | | | | | | | | |
| BACTERIA | | | | | | | | | |
| Leptospira interrogans | Lepto1 | | | | (b)(4) | | | | |
| VIRUSES | | | | | | | | | |
| Dengue virus Type 2 | DENV2_1 | | | | (b)(4) | | | | |
| PROTOZOA | | | | | | | | | |
| Plasmodium falciparum | Plas spp.<br>Plas falciparum | | | | (b)(4) | | | | |

# Table 3: Summary of Tm (℃) Analyses for FilmArray Global Fever Panel Assays

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 [014) PC.

Cumulatively, the results suggest that there are no significant differences between variables evaluated in the reproducibility study. Therefore, the reproducibility studies for the FilmArray Global Fever Panel are acceptable.

- b. Linearity/assay reportable range:
Not applicable

- c. Traceability, Stability, Expected values (controls, calibrators, or methods):
## Internal Controls:

Two internal controls are included in each FilmArray Global Fever Panel pouch:

- . RNA Process Control: The RNA Process Control assay targets an RNA transcript from the yeast Schizosaccharomyces pombe. The yeast is present in the pouch in a freeze-dried form and becomes rehydrated when sample is loaded. The control material is carried through all stages of the test process, including lysis, nucleic acid purification, reverse transcription, 1st stage PCR, dilution, 2nd stage PCR, and DNA melting. A positive control result indicates that all steps carried out in the FilmArray Global Fever Panel pouch were successful.
- . PCR2 Control: The PCR2 Control assay detects a DNA target that is dried into wells of the array along with the corresponding primers. A positive result indicates that 2nd stage PCR was successful.

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Both control assays must be positive for the test run to pass. If either control fails, the Controls field of the test report will display "Failed" and all results will be listed as "Invalid". If the controls fail, the sample should be retested using a new pouch.

#### Recommended External Controls:

External controls are not provided with the FilmArray Global Fever Panel but are recommended in the package insert. External controls should be used in accordance with laboratory protocols and the appropriate accrediting organization requirements. as applicable. Molecular grade water or saline can be used as an external negative control. Previously characterized positive samples or negative samples spiked with well-characterized organisms can be used as external positive controls.

BioFire Defense provides an external positive and negative assayed quality control kit to monitor the performance of in vitro laboratory nucleic acid testing procedures for the qualitative detection of the FilmArray Global Fever Panel performed on FilmArray 2.0 systems. The positive external control is a surrogate control material comprised of dried synthetic DNA (positive only) in buffer and stabilizer, supplied in a FilmArray Injection Vial that is used directly with the FilmArray Global Fever Panel. The FilmArray Global Fever Panel Control Kit is composed of two controls: FilmArray Global Fever Positive External Control Material (Positive ECM) and FilmArray Global Fever Negative External Control Material (Negative ECM). The DNA in the Positive ECM includes DNA segments to assess the presence of each individual assay in the FilmArray Global Fever Panel listed above. There is no DNA in the Negative ECM. The Global Fever Panel Control Kit contains no biological hazards and is 100% non-infectious. To use the product. the operator opens and uses the FilmArray Injection Vial in place of the Sample Injection Vial, and otherwise runs the test according to protocol. This control is shipped and stored at 18-28 ℃.

The external control kit is available for purchase directly from BioFire Defense.

Quantification of nucleic acid derived from live or inactivated viral and bacterial. cultures

Quantification using the genesig qPCR kits was performed at two different locations, 1) [ = live chikungunya virus stocks. 2) BioFire Defense - live and inactivated bacteria, viral, and protozoan stocks.

Image /page/9/Figure/7 description: The image shows text from a document. The text states that organisms evaluated at BioFire Defense were obtained from an unreadable source. Verification of the stocks, whenever available, was provided with a Certificate of Analysis (CofA).

the source. The majority of organisms obtained did not come quantified or came without any enumeration value.

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Image /page/10/Figure/0 description: The image shows a section of a research paper that describes the process of extracting nucleic acids from bacterial and viral stocks. It mentions that all virus stocks were treated with a reagent prior to extraction, and the nucleic acid concentration of each extract was then determined using commercially available genesig qPCR assay kits. The qPCR assays were performed at BioFire Diagnostics/BioFire Defense, and the cycling conditions were as recommended and are shown below in Table 4. Table 4 is titled "Amplification Cycling Conditions for the g qPCR Kits".

15)(4)

Both DNA and RNA target nucleic acid from organism stocks was extracted using the (D(4) The nucleic acid concentration of the extract (Unknown) was then determined using commercially available quantitative real-time PCR assay Standard Kits from TONET

Table 5. Quantified Organisms and the | | | qPCR Assay Kits Targets and the FilmArray Global Fever Panel Targets

| FilmArray Global<br>Fever Panel<br>Analyte | Quantified<br>Species | Strain | qPCR Kit<br>Gene<br>Target | FilmArray Global Fever Panel Target |
|--------------------------------------------|-----------------------|-------------------------------------------------|----------------------------|-------------------------------------|
| Bacteria | | | | |
| | kirschneri | 200701401 | | |
| | | 3522 C (Cynopteri) | | |
| | interrogans | Serovar<br>icterohaemorrhagiae | | |
| | | HAI0156 (Copenhagen) | | |
| | | L495 (Manilae) | | |
| | alexanderi | L 60 (Manhao 3) | | |
| | santarosai | LT 821 (Shermani) | (b)(4) | |
| Leptospira spp. | kmetyi | Bejo-Iso9T (Malaysia) | | (b)(4) |
| | noguchii | CZ 214T (Panama) | | |
| | borgpetersenii | Veldrat Bataviae 46 | | |
| | weilii | Celledoni 20160426 | | |
| | | A 102 (Mengrun) | | |
| | | 6712 (Hainan) | | |
| | | H 27 (Hekou) | | |
| | | LT 89-68 (Vughia) | | |
| | | 94-79970/3 (Topaz) | | |
| Viruses | | | | |
| Chikungunya<br>Virus | R80422 | | (b)(4) | |
| | St. Martin<br>2013 | | | (b)(4) |
| | DHS4263 | (b)(4) | | |
| | (b)(4) | | | |
| Dengue Virus | Serotype 1 | Hawaii | | |
| | | 276RK1 | | |
| | | Strain 12150 | | |
| | | BC89/94 | | |
| | | 228690 | | |
| | | VN/BID-V1792/2007<br>SL-6-6-04 | | |
| | Serotype 2 | New Guinea C<br>VN/BID-V1002/2006<br>DakArA1247 | | |
| | | BC102/94 | | |
| | | 429557 | (b)(4) | |
| | | 1349 | | (b)(4) |
| | | ArA6894 | | |
| | Serotype 3 | H87<br>VN/BID-V1329/2006 | | |
| | | (b)(4) | | |
| | | 271242<br>C0360/94<br>H241<br>703 | | |
| | Serotype 4 | (b)(4) | | |
| | | BC13/97<br>BC287/97<br>BC258/97<br>PR 06-65-740 | | |
| | | | | |
| | | IPC 4884 Pursat<br>Cambodia 2011<br>SenTh021.09 | | (b)(4) |
| | | | | |
| Plasmodium spp. | P.falciparum | St. Lucia<br>Tanaznia, 02000708 | (b)(4) | |
| | P. vivax | Chesson<br>Panama<br>Wallikeri | | |
| | P. ovale | Curtisi | | |
| | P. knowlesi | Strain H | | |
| | P. malariae | Unknown | | |

{11}------------------------------------------------

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# d. Detection limit:

A study to establish the detection limit of the FilmArray Global Fever panel was conducted utilizing whole blood samples contrived with isolates of viruses, bacteria, (6)(4) and protozoa detected by the Global Fever panel.

(1944)

(b)[1] analytical testing was performed simultaneously for all analytes. Specifically, testing utilized a combination of live and inactivated organisms prepared in multi-spiked mixes that contain on individual target organisms as described below:

| Table 6. Composition of Organism Mixes used in GF Panel LoD Testing | | | |
|---------------------------------------------------------------------|--|--|--|
| | | | |

| Mix | Organism | Mix | Organism | |
|-----|----------|-----|----------|--|
| | | 080 | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |
| | | | | |

The LoD was first estimated by testing dilutions of contrived whole blood samples containing known dilutions of organisms bracketing an initial LoD concentration based on early development testing. Confirmation of LoD was achieved by testing m replicates of a contrived sample containing analyte at the estimated LoD. LoD was successfully confirmed when the organism was detected in at least of the of the replicates tested over days using [b] pouch lots. Complete results for individual analytes included on the Global Fever Panel are included in Table 7 below.

{13}------------------------------------------------

| Global Fever Panel<br>Test Result | Species/Strain Tested | LoD | |
|----------------------------------------------|-----------------------------------------------|------------------------------|-----------------------------------------------------|
| | | Based on qPCR<br>(copies/mL) | Based on Provided Stock<br>Concentration (units/mL) |
| Leptospira<br>interrogans | Icterohaemorrhagiae<br>Budapest | | (b)(4) |
| Chikungunya<br>virus | R80422 (heat<br>inactivated) | | |
| | B8635 (live) | | |
| | Indo23574 (live) | | |
| Dengue Virus 1 | Hawaii | | |
| | New Guinea C | | |
| Dengue Virus 2 | DakArA1247 | | |
| Dengue Virus 3 | H87 | | |
| Dengue Virus 4 | H241 | | |
| | P. falciparum IPC4884<br>Pursat Cambodia 2011 | | |
| | P. knowlesi H Strain | | |
| Plasmodium spp. | P. malariae clinical<br>specimen | | |
| | P. ovale Wallikeri | | |
| | P. vivax Chesson | | |
| Plasmodium<br>falciparum | IPC4884 Pursat<br>Cambodia 2011 | | |
| Plasmodium<br>vivax /<br>Plasmodium<br>ovale | P. ovale Wallikeri | | |
| | P. vivax Chesson | | |

## Table 7. Limit of Detection for FilmArray Global Fever Panel Analytes

[ Quantification of the organism stock material was not available.

2LoD for live chikungunya virus strains was only estimated by identifying the lowest concentration of analyte for which o replicates were positive.

## e. Analytical Reactivity (Inclusivity):

The analytical reactivity (inclusivity) of the FilmArray Global Fever Panel was evaluated with a collection of isolates to represent relevant species, subspecies, or serotypes. Testing was performed by evaluating that replicates. If there was one undetected result, additional replicates were tested and if lowel replicates were detected the isolate was considered inclusive. Most isolates were detected by the FilmArray Global Fever Panel at spiked concentrations within > 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.

{14}------------------------------------------------

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.

Table 8 includes a summary of FilmArray Global Fever Panel reactivity based on empirical data.

| FilmArray<br>Global Fever<br>Panel Analyte | #<br>Isolates<br>Detected<br>/ Tested | Isolates Tested | Concentration<br>Detected ¹ | x LoD | FilmArray Global<br>Fever Panel Result<br>(Replicates<br>Detected/Tested) | |
|--------------------------------------------|---------------------------------------|-----------------|---------------------------------------------------------------------------------------------------|----------------------------|---------------------------------------------------------------------------|---------------------------------------------------------------------------|
| | | | BACTERIA | | | |
| | | Species | Strain | | | |
| | b/19 | interrogans | Serovar (Budapest)<br>HAI0156<br>(Copenhageni)<br>L495 (Manilae) | (b)(4) | | Detected (b)(41) |
| | | alexanderi | L60 (Manhao 3) | | | Detected (b) |
| | | alstonii | Sichuan 79601 | | | Detected (b) |
| | | borgpetersenii | Castellon 3<br>(Castellonis)<br>Veldrat Bataviae<br>46 (Javanica) | | | |
| Leptospira spp. | | kirschneri | 200701401<br>(Bogvere)<br>3522 C<br>(Cynopteri) | | | |
| | | kmetvi | Bejo-Iso9T<br>(Malaysia) | | | Detected (b) |
| | | mayottensis | 200901116 | | | |
| | | noguchii | CZ 214T (Panama) | | | |
| | | santarosai | LT 821 (Shermani)<br>6712 | | | |
| | | weilii | 94-79970/3 Topaz<br>A 102 (Mengrun)<br>Celledoni<br>20160426<br>H 27 (Hekoou)<br>LT89-68 (Vughia) | | | |
| | | | | VIRUSES | | |
| | | | Strain | (b)(4) | | |
| Chikungunya<br>Virus | b/3 | | R80422<br>DHS4263<br>St. Martin 2013 | | | Detected (b)(41) |
| | | | | | | Detected (b) |
| | | Serotype | Strain | (b)(4) | | |
| | b/28 | | Hawaii<br>Strain 12150 | | | Detected (b)(4) |
| | | | 228690<br>276RK1 | | | |
| | | Serotype 1 | BC89/94<br>SL-6-6-04 | | | Detected (b) |
| Dengue virus | | | UIS 1162 | | | |
| | | | VN/BID-<br>V1792/2007 | | | |
| | | | | | | |
| | | Serotype 2 | New Guinea C<br>(DENV2 1) | | | Detected (b)(41) |
| FilmArray<br>Global Fever<br>Panel Analyte | # Isolates<br>Detected / Tested | | Isolates Tested | Concentration<br>Detected1 | x LoD | FilmArray Global<br>Fever Panel Result<br>(Replicates<br>Detected/Tested) |
| | | | DakArA1247<br>(DENV2 2) | (b)(4) | | |
| | | | 1349 | | | |
| | | | 429557 | | | Detected (b)(4) |
| | | | ArA6894 | | | |
| | | | BC102/94 | | | |
| | | | DKA 811 | | | Detected (b)(4) |
| | | | VN/BID-<br>V1002/2006 | | | Detected (b) |
| | | | H87 | | | Detected (b)(4) |
| | | | 271242 | | | |
| | | Serotype 3 | BC188/97 | | | |
| | | | C0360/94 | | | Detected (b) |
| | | | VN/BID-<br>V1329/2006 | | | |
| | | | H241 | | | Detected (b)(4) |
| | | | 703 | | | |
| | | | BC13/97 | | | |
| | | Serotype 4 | BC287/97 | | | Detected (b) |
| | | | BC258/97 | | | |
| | | | D85-019 | | | |
| | | | PR 06-65-740 | | | |
| | | | | PROTOZOA | | |
| | | Species | Strain | | | |
| | | | | | | |
| Plasmodium spp. | 10/10 | | falciparum | IPC 4884 | (b)(4) | Detected (b)(4) |
| | | | | SenTh021.09 | | Detected (b) |
| | | | falciparum | St. Lucia | | |
| | | | | Tanzania,<br>02000708 | | Detected (b) |
| | | | vivax | Chesson | | Detected (b)(4) |
| | | | | Panama | | Detected (b) |
| | | | ovale | Wallikeri | | Detected (b)(4) |
| | | | | Curtisi | | Detected (b) |
| | | | knowlesi | Strain H | | |
| | | | | malariae | | Clinical<br>specimen |
| | | | | | | |
| Plasmodium<br>falciparum | 4/4 | | falciparum | IPC4884 | (b)(4) | |
| | | | | SenTh021.09 | | Detected (b) |
| | | | | St. Lucia | | Detected (b) |
| | | | | Tanzania,<br>02000708 | | Detected (b) |
| | | | | | | |
| Plasmodium<br>vivax/ovale | 4/4 | | vivax | Chesson | (b)(4) | Detected (b)(4) |
| | | | | Panama | | Detected (b) |
| | | | ovale | Wallikeri | | Detected (b) |

Table 8, Summary of FilmArray Global Fever Panel Analytical Reactivity (Inclusivity)

{15}------------------------------------------------

Organisms which exhibited reduced or no assay reactivity have limitations included in the labeling and are described specifically in the table below.

{16}------------------------------------------------

| Observed<br>Result | Detection Level | Analyte | Serotype/Strain/Isolate |
|---------------------------------------|-----------------|---------------------------|-------------------------------------------|
| Detected<br>(may be<br>underreported) | 10 x LoD | Plasmodium<br>falciparum1 | SenTh021.09 |
| Detected<br>(may be<br>underreported) | ~100 x LoD | Dengue virus1 | Serotype 3 BC188/97<br>Serotype 4 D85-019 |
| Not Detected | - | Dengue virus2 | Serotype 2 DKA 811 |

The reason for the observed reactivity could not be identified based on in silico sequence analysis. Sequences for these strains were not available in public databases.

21n 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.

#### f. Microbial Interference Studies:

Potentially interfering microorganisms were evaluated for their effect on FilmArray Global Fever Panel performance. To evaluate the potential for interference, FilmArray Global Fever 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 FilmArray Global Fever 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.

| 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 TCID50/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<br>(HIV) 1/2 | HIV-1: 1.3E+05 U/mL<br>HIV-2: 2.2E+05 U/mL | No interference observed |
| Plasmodium vivax | 1.5E+06 copies/mL | No interference observed |

Table 9, Organisms Evaluated for Potential Microbial Interference

None of the ten microorganisms tested showed interference with the pouch controls or specific Global Fever Panel assay targets.

{17}------------------------------------------------

## g. 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 (> 106 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.

# Table 10. FilmArray Global Fever Panel Results for On-Panel Organism Testing Assessing Potential Cross-reactivity

| Pathogen | Live or<br>Inactivated | | Results | |
|---------------------------------------------|-----------------------------------------|-------------|--------------------------------------------------|-----------------------------------------------------------------------|
| | Live | Inactivated | | |
| Leptospira interrogans (Schu S4) | √ | - | As expected (only Leptospira spp. Detected) | |
| Chikungunya virus (R80422 culture<br>fluid) | √ | - | As expected (only Chikungunya virus<br>Detected) | |
| Dengue virus | 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)…