K Number

Device Name

Manufacturer

BioFire Defense, LLC

Date Cleared

2020-11-20

(147 days)

Product Code

Regulation Number

Type

Panel

Reference & Predicate Devices

N/A

Predicate For

N/A

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 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 for each organism on the panel.

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally demonstrated through the successful performance in the analytical and clinical studies. While explicit "acceptance criteria" are not listed in a separate table, the reported performance metrics (PPA, NPA, agreement rates) indicate the achieved levels that were deemed acceptable.

Metric / Criterion	Acceptance Criteria (Implied by Study Design & FDA Review)	Reported Device Performance (Summary)
Analytical Performance
Reproducibility	High agreement with expected results across sites, operators, and lots; low variability in melt temperature (SD < 0.5°C).	Overall Agreement: 96.0% (94.7-97.1% CI) for all analytes and test levels. Melt temperature variability: 0.2-0.3°C (within expected <0.5°C). Leptospira Low Positive sample had 90% agreement. P. falciparum Low Positive had 70% agreement.
Detection Limit (LoD)	Organism detected in at least a specified percentage of replicates (e.g., 95%) at the estimated LoD.	LoD successfully confirmed for various analytes (specific data is redacted but indicates confirmation).
Analytical Reactivity (Inclusivity)	Detection of representative species, subspecies, or serotypes within >1x LoD.	Most isolates detected within >1x LoD. Limitations noted for P. falciparum (SenTh021.09, 10x LoD), Dengue virus (Serotype 3 BC188/97, Serotype 4 D85-019, ~100x LoD), and Dengue virus (Serotype 2 DKA 811, Not Detected).
Microbial Interference	No interference with pouch controls or specific assay targets by other microorganisms.	No interference observed for 10 tested microorganisms.
Analytical Specificity/Cross-reactivity	No non-specific amplification or detection by off-panel organisms; limited and identified cross-reactivity for on-panel near-neighbors.	Detected cross-reactivity: P. knowlesi with P. vivax/ovale assay (>2.2E+03 copies/mL). P. brasilianum (identical to P. malariae) showed reactivity with P. vivax/ovale assay (undisclosed copies/mL). No other cross-reactivity observed in wet testing. No expected cross-reactivity by in silico analysis.
Interfering Substances	No interference effect from endogenous, exogenous, or technique-specific substances/anticoagulants.	No interference observed for most tested substances. Potential interference from TRIzol (initial, then not reproducible) and Heparin (initial, then improved reproducibility, but still potentially inhibitory near LoD).
Specimen Stability	Consistent detection (e.g., 10/10 replicates positive) across recommended storage conditions (room temp, refrigerated, ultra-low freezer) and consistency between fresh vs. frozen.	All analytes: 10/10 replicates positive for all evaluated storage conditions. Fresh vs. Frozen Contrived Specimens: PPA 100% for all organisms. NPA 100% for all organisms. Fresh vs. Frozen Clinical Specimens: PPA 100% for all organisms. NPA 100% for Leptospira, Dengue, Chikungunya, Plasmodium spp. P. falciparum NPA 97.1%, P. vivax/ovale NPA 97.0%.
Clinical Performance
Positive Percent Agreement (PPA)	High agreement with comparator method (implied by accepted results).	Overall (Fresh & Frozen): Chikungunya 100%, Dengue 94.0%, Leptospira 93.8%, Plasmodium spp. 98.3%, P. falciparum 92.7%, P. vivax/ovale 92.7%.
Negative Percent Agreement (NPA)	High agreement with comparator method (implied by accepted results).	Overall (Fresh & Frozen): Chikungunya 99.9%, Dengue 100%, Leptospira 99.8%, Plasmodium spp. 99.2%, P. falciparum 99.8%, P. vivax/ovale 100%.

Study Design and Proof of Meeting Acceptance Criteria:

The detailed performance characteristics section (L. Performance Characteristics) outlines the studies conducted to demonstrate the device meets these criteria. The approval of the De Novo request signifies that the FDA found these results acceptable and the device adequately characterized for its intended use, with appropriate mitigations for identified risks.

2. Sample Sizes and Data Provenance

Test Set (Clinical Study):
- Total Eligible Specimens: 1875 whole blood specimens.
- Category I (Prospective, Fresh): 1469 (78.3%) specimens.
- Category II (Prospective, Frozen): 406 (21.7%) specimens.
- Data Provenance: Multicenter study conducted at ten geographically distinct study sites, including two in the United States, and sites in Africa, Southeast Asia, and Central & South America. Data was collected prospectively (March 2018 - September 2019).
Analytical Studies (Reproducibility, LoD, etc.): Sample sizes for these studies are distinct and smaller than the clinical study.
- Reproducibility: 270 valid test results (90 replicates per sample, 3 samples, 3 sites, 5 days, 2 operators, 3 instruments, rotating pouch lots).
- Detection Limit: "m replicates" tested for confirmation, and "x replicates" initially. Specific numerical values are redacted.
- Inclusivity/Cross-reactivity/Interference: "replicates" (number often redacted) of spiked samples.

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number or specific qualifications of experts used to establish the ground truth for the clinical test set.

Clinical Study Ground Truth: The ground truth for the clinical samples was established using "well-validated nested PCR assays followed by bi-directional Sanger sequencing."
- "Two comparator assays were utilized for each assay target with positive result from both assays considered positive."
- "If the results of the two comparator assays for a particular analyte disagreed, the samples were subjected to repeat comparator testing with samples determined as positive if at least 2/3 replicates were positive for a single comparator assay."
- This implies a laboratory-based method for ground truth, rather than human expert consensus on clinical presentation.
Assay Cut-off Validation (post-analytical/clinical studies): "a final validation of the melt ranges was performed and included review of data from the Inclusivity study and clinical studies. The observed sensitivity and specificity rates for the individual melt curves and assay calls as compared to expert annotation was greater than 99.8% and 99.9% respectively."
- This indicates that "expert annotation" was used to validate the final melt ranges of the software's interpretation of results, suggesting these experts were likely highly specialized in nucleic acid analysis or molecular diagnostics. Specific number or qualifications are not provided beyond "expert annotation."

4. Adjudication Method for the Test Set

The primary method for establishing ground truth for the clinical test set was through two comparator nested PCR assays followed by Sanger sequencing.
Discrepancy Resolution/Adjudication: "Samples for which false positive and/or false negative results (i.e., discrepant results) were obtained when comparing the FilmArray Global Fever Panel results to the comparator method results were further investigated."
- This investigation typically involved:
  1. Examination to determine if additional testing on Global Fever Panel or with comparator assays could detect the analyte (if near or below detection threshold).
  2. Evaluation by "at least one additional PCR test that used different primers than the Global Fever Panel assay or the comparator assays."
  3. When possible, "unresolved discrepancies were evaluated with additional PCR testing that could be verified by sequence analysis."
- This describes a multi-step, laboratory-based adjudication process for discrepant results, leveraging additional molecular methods. It is not a 2+1 or 3+1 human reader adjudication, but rather a technical/laboratory adjudication.

5. Multi Reader Multi Case (MRMC) Comparative Effectiveness Study

No, an MRMC comparative effectiveness study was not performed.
This device is an in vitro diagnostic (IVD) nucleic acid amplification test, and its performance is evaluated against a molecular gold standard (comparator PCR and sequencing), not through human reader interpretation of images or other data. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" does not apply here.

6. Standalone Performance

Yes, the performance characteristics described in Section L (Performance Characteristics) represent the standalone performance of the FilmArray Global Fever Panel algorithm and system. This includes analytical performance (reproducibility, LoD, specificity, etc.) and the clinical performance (PPA, NPA) which compares the device's output directly to the "ground truth" established by the comparator molecular methods. The system automatically interprets the results of each DNA melt curve analysis and combines data with internal controls to provide a test result.

7. Type of Ground Truth Used

For the clinical study, the primary ground truth was established by molecular comparator testing: "well-validated nested PCR assays followed by bi-directional Sanger sequencing." This is a highly objective, laboratory-based method for detecting and identifying specific nucleic acid sequences.

8. Sample Size for the Training Set

The document does not explicitly state the sample size for a "training set".
This is a molecular diagnostic assay where algorithms (e.g., for melt curve analysis) are likely developed based on known principles of PCR and fluorescence, and validated with a range of characterized samples (analytical studies). There isn't a traditional "training set" in the context of deep learning models for medical image analysis, which require large datasets for supervised learning. Instead, the "initial melt ranges" were determined by "mathematical modeling using known sequence variations... as well as data from testing of clinical specimens and known isolates."

9. How the Ground Truth for the Training Set Was Established

As a molecular diagnostic, the "training" (or development/refinement) data for the device's interpretive algorithm (e.g., Melt Detector software Tm values, fluorescence values, and melt curve analysis) would primarily come from:
- Well-characterized isolates and contrived samples: Used to define expected melt curves, Tm values, and limits of detection. This includes the extensive analytical studies detailed (e.g., inclusivity, cross-reactivity, precision, LoD).
- Mathematical modeling: Based on known sequence variations of target organisms.
- Data from clinical specimens: Likely used to fine-tune and validate the interpretive algorithm (as indicated by the "final validation of the melt ranges" including "review of data from the Inclusivity study and clinical studies" against "expert annotation"). This implies that known positive and negative samples, rigorously characterized by molecular methods (and potentially expert review for complex cases), would inform the algorithm's development.

Summary

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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
1. Classification:
  Class II

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1. Product code(s):
  OMV
1. 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.

1. Indication(s) for use:
  Same as intended use.
1. Special conditions for use statement(s):
  For prescription use only.

For in vitro diagnostic use only.

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

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I. Device Description:

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

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 interrogansserovaricterohaemorrhagiae	Low Positive3.3E+02 copies/mL1 x LoD	Moderate Positive1.0E+03 copies/mL3 x LoD	Negative
Dengue Virus (DENV-2)New Guinea C	Low Positive3.3E+02 copies/mL1 x LoD	Moderate Positive1.0E+03 copies/mL3 x LoD	Negative
Plasmodium falciparumIPC 4884	Moderate Positive5.4E+02 copies/mL1.5 x LoD	Negative	Low Positive9.0E+01copies/mL0.5 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 AnalyteIsolate	Concentration	ExpectedTestResult	% Agreement with Expected Results
					Site 1	Site 2	Site 3	AllSites	95%ConfidenceInterval
BACTERIA		Leptospira interrogans serovaricterohaemorrhagiae	Moderate Positive3x LoD(1.2E+03 copies/mL)	Detected	30/30100%	30/30100%	30/30100%	90/90100%	96.0-100%
			Low Positive1x LoD(3.9E+02 copies/mL)	Detected	27/3090.0%	28/3093.4%	26/3086.7%	81/9090.0%	81.9-95.3%
			Negative(No Analyte)	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%	96.0-100%
VIRUSES		Dengue virusDENV-2New Guinea C	Moderate Positive3x LoD(1.1E+03 copies/mL)	Detected	29/3096.7%	30/30100%	30/30100%	89/9098.9%	94.0-100%
			Low Positive1x LoD(3.6E+02 copies/mL)	Detected	30/30100%	30/30100%	30/30100%	90/90100%	96.0-100%
			Negative(No Analyte)	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%	96.0-100%
PROTOZOA		Plasmodium spp.Detection Results	Moderate Positive1.5x LoD1(2.7E+02 copies/mL)	Detected	30/30100%	30/30100%	30/30100%	90/90100%	96.0-100%
			Low Positive0.5× LOD1(9.0E+01 copies/mL)	Detected	28/3093.3%	30/30100%	29/3096.7%	87/9096.7%	90.7-98.9%
			Negative(No Analyte)	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%	96.0-100%
	IPC 4884	PlasmodiumfalciparumDetection Results	Moderate Positive1.5x LoD1(2.7E+02 copies/mL)	Detected	29/3096.7%	30/30100%	28/3093.4%	87/9096.7%	90.6-99.3%
			Low Positive0.5× LoD1(9.0E+01 copies/mL)	Detected	18/3060%	24/3080%	21/3070%	63/9070.0%	59.4-79.2%
			Negative(No Analyte)	NotDetected	30/30100%	30/30100%	30/30100%	90/90100%	96.0-100%
	Overall Agreement with the Expected Test ResultAll Analytes and All Test Levels (95% Confidence Interval)				1,037/1,08096.0% (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(Isolate tested)	Assay	Observed Tm (°C)
		Site 1Mean	±StDev	Site 2Mean	±StDev	Site 3Mean	±StDev	All SitesMean	±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.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.

{10}------------------------------------------------

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 GlobalFever PanelAnalyte	QuantifiedSpecies	Strain	qPCR KitGeneTarget	FilmArray Global Fever Panel Target
Bacteria
	kirschneri	200701401
		3522 C (Cynopteri)
	interrogans	Serovaricterohaemorrhagiae
		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
ChikungunyaVirus	R80422		(b)(4)
	St. Martin2013			(b)(4)
	DHS4263	(b)(4)
	(b)(4)
Dengue Virus	Serotype 1	Hawaii
		276RK1
		Strain 12150
		BC89/94
		228690
		VN/BID-V1792/2007SL-6-6-04
	Serotype 2	New Guinea CVN/BID-V1002/2006DakArA1247
		BC102/94
		429557	(b)(4)
		1349		(b)(4)
		ArA6894
	Serotype 3	H87VN/BID-V1329/2006
		(b)(4)
		271242C0360/94H241703
	Serotype 4	(b)(4)
		BC13/97BC287/97BC258/97PR 06-65-740

		IPC 4884 PursatCambodia 2011SenTh021.09		(b)(4)

Plasmodium spp.	P.falciparum	St. LuciaTanaznia, 02000708	(b)(4)
	P. vivax	ChessonPanamaWallikeri
	P. ovale	Curtisi
	P. knowlesi	Strain H
	P. malariae	Unknown

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

{12}------------------------------------------------

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.

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Global Fever PanelTest Result	Species/Strain Tested	LoD
		Based on qPCR(copies/mL)	Based on Provided StockConcentration (units/mL)
Leptospirainterrogans	IcterohaemorrhagiaeBudapest		(b)(4)
Chikungunyavirus	R80422 (heatinactivated)
	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 IPC4884Pursat Cambodia 2011
	P. knowlesi H Strain
Plasmodium spp.	P. malariae clinicalspecimen
	P. ovale Wallikeri
	P. vivax Chesson
Plasmodiumfalciparum	IPC4884 PursatCambodia 2011
Plasmodiumvivax /Plasmodiumovale	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.

FilmArrayGlobal FeverPanel Analyte	#IsolatesDetected/ Tested	Isolates Tested	ConcentrationDetected ¹	x LoD	FilmArray GlobalFever Panel Result(ReplicatesDetected/Tested)
			BACTERIA
		Species	Strain
	b/19	interrogans	Serovar (Budapest)HAI0156(Copenhageni)L495 (Manilae)	(b)(4)		Detected (b)(41)
		alexanderi	L60 (Manhao 3)			Detected (b)
		alstonii	Sichuan 79601			Detected (b)
		borgpetersenii	Castellon 3(Castellonis)Veldrat Bataviae46 (Javanica)
Leptospira spp.		kirschneri	200701401(Bogvere)3522 C(Cynopteri)
		kmetvi	Bejo-Iso9T(Malaysia)			Detected (b)
		mayottensis	200901116
		noguchii	CZ 214T (Panama)
		santarosai	LT 821 (Shermani)6712
		weilii	94-79970/3 TopazA 102 (Mengrun)Celledoni20160426H 27 (Hekoou)LT89-68 (Vughia)
				VIRUSES
			Strain	(b)(4)
ChikungunyaVirus	b/3		R80422DHS4263St. Martin 2013			Detected (b)(41)
						Detected (b)
		Serotype	Strain	(b)(4)
	b/28		HawaiiStrain 12150			Detected (b)(4)
			228690276RK1
		Serotype 1	BC89/94SL-6-6-04			Detected (b)
Dengue virus			UIS 1162
			VN/BID-V1792/2007

		Serotype 2	New Guinea C(DENV2 1)			Detected (b)(41)
FilmArrayGlobal FeverPanel Analyte	# IsolatesDetected / Tested		Isolates Tested	ConcentrationDetected1	x LoD	FilmArray GlobalFever Panel Result(ReplicatesDetected/Tested)
			DakArA1247(DENV2 2)	(b)(4)
			1349
			429557			Detected (b)(4)
			ArA6894
			BC102/94
			DKA 811			Detected (b)(4)
			VN/BID-V1002/2006			Detected (b)
			H87			Detected (b)(4)
			271242
		Serotype 3	BC188/97
			C0360/94			Detected (b)
			VN/BID-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,02000708		Detected (b)
			vivax	Chesson		Detected (b)(4)
				Panama		Detected (b)
			ovale	Wallikeri		Detected (b)(4)
				Curtisi		Detected (b)
			knowlesi	Strain H
				malariae		Clinicalspecimen

Plasmodiumfalciparum	4/4		falciparum	IPC4884	(b)(4)
				SenTh021.09		Detected (b)
				St. Lucia		Detected (b)
				Tanzania,02000708		Detected (b)

Plasmodiumvivax/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}------------------------------------------------

ObservedResult	Detection Level	Analyte	Serotype/Strain/Isolate
Detected(may beunderreported)	10 x LoD	Plasmodiumfalciparum1	SenTh021.09
Detected(may beunderreported)	~100 x LoD	Dengue virus1	Serotype 3 BC188/97Serotype 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(HIV) 1/2	HIV-1: 1.3E+05 U/mLHIV-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 orInactivated		Results
	Live	Inactivated
Leptospira interrogans (Schu S4)	√	-	As expected (only Leptospira spp. Detected)
Chikungunya virus (R80422 culturefluid)	√	-	As expected (only Chikungunya virusDetected)
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)
	DENV-4 (H241)	√	-	As expected (only Dengue virus Detected)
Plasmodium	falciparum(Pursat Cambodia2011)	√	-	As expected (Plasmodium spp. Detected and P.falciparum Detected)
	knowlesi(Strain H)	√	-	Plasmodium spp Detected as expected.
	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)

Although no cross-reactivity with P. malariae was observed in this study, in silico analysis shows be 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 copies/mL.

{18}------------------------------------------------

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 | and PMA (b)(4) 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.

Bacteria		Viruses
Acinetobacter baumannii	Mycoplasma pneumoniae	Adenovirus 1	Omsk hemorrhagic fevervirus
Bacillus anthracis	Neisseria meningitidis	Adenovirus 3	O'nyong-nyong virus1
Bacillus brevis	Proteus mirabilis	Adenovirus 5	Parvo virus
Bacillus cereus	Pseudomonas aeruginosa	Aura virus	Powassan virus
Bacillus circulans	Rickettsia typhi	Barmah Forest virus	Rabies virus
Bacillus coagulans Hammer	Salmonella enterica subsp.arizonae	Bunyamwera virus	Rift Valley Fever Virus
Bacillus halodurans	Salmonella enterica subsp.bongori	Coronavirus NL63	Ross River virus
Bacillus licheniformis	Salmonella enterica subsp.diarizoniae	Crimean-Congo HemorrahagicFever Virus	Human respiratory syncytialvirus
Bacillus megaterium	Salmonella enterica subsp.enterica serovar Dublin	Dugbe virus	Rubella virus
Bacillus mycoides	Salmonella enterica subsp.enterica serovar Enteritidis	Eastern Equine EncephalitisVirus	Saint Louis encephalitis virus
Bacillus pumilus	Salmonella enterica subsp.enterica serovar Javiana	Ebolavirus (Zaire, Sudan,Bundibugyo, Tai Forest,Reston)	Sindbis virus
Bacillus subtilis	Salmonella enterica subsp.enterica serovarManchester	Enterovirus, HEV-71	Spondweni virus
Bacillus thuringiensis	Salmonella enterica subsp.enterica serovarMontevideo	Epstein Barr virus	Tickborne encephalitis virus
Bacteroides fragilis	Salmonella enterica subsp.enterica serovarMuenchen	Flexal virus	Una virus
Bordetella bronchiseptica	Salmonella enterica subsp.	Guanarito virus	Usutu virus
	enterica serovar Newport
Borrelia burgdorferi	Salmonella enterica subsp.enterica serovar Paratyphi	Hantaan virus	Vaccinia virus
Brucella melitensis	Salmonella enterica subsp.enterica serovar Rubislaw	Hazara virus	Varicella zoster virus
Burkholderia cepacia	Salmonella enterica subsp.enterica serovar Saintpaul	Hendra virus	Venezuelan EquineEncephalomyelitis (VEE)virus
Burkholderia mallei	Salmonella enterica subsp.enterica serovar Tennessee	Hepatitis A Virus (HAV)	Western Equine Encephalitis(WEE) virus
Burkholderia pseudomallei	Salmonella enterica subsp.enterica serovarThompson	Hepatitis B Virus (HBV)	West Nile virus Lineage 1
Chlamydophila pneumoniae	Salmonella enterica subsp.enterica serovar Typhi	Hepatitis C Virus (HCV)	West Nile virus Lineage 2
Clostridium bifermentans	Salmonella enterica subsp.enterica serovarTyphimurium	Herpes Simplex Virus 2(HSV-2)	Yellow Fever virus
Clostridium perfringens	Salmonella enterica subsp.houtenae	Human Parainfluenza Virus 1	Zika virus
Clostridum sordelli	Salmonella enterica subsp.indica	Human Parainfluenza Virus 3	Fungi
Clostridium sporogenes	Salmonella enterica subsp.salamae	Hughes virus	Aspergillus fumigatus
Clostridium perfringens	Serratia marcescens	Human herpesvirus 6B	Cryptococcus neoformans
Coxiella burnetii	Staphylococcus aureus	Human ImmunodeficiencyVirus 1	Pathogenic Protozoa
Enterobacter aerogenes	Streptococcus agalactiae	Human ImmunodeficiencyVirus 2	Babesia microti
Enterococcus faecalis	Streptococcus pneumoniae	Human T-lymphotropic virustype 1	Crithidia fasciculata
Enterococcus faecium	Streptococcus pyogenes	Human T-lymphotropic virustype 2	Cyclospora cayetanensis
Francisella hispaniensis	Vibrio cholerae	Influenza A H1N1-2009	Leishmania donovani
Francisella tularensis subsp.tularensis	Yersinia aldovae	Influenza A H3N2	Leptomonas seymouri
Francisella persica	Yersinia bercovieri	Influenza B virus	Schistsoma mansoni
Francisella philomiragia	Yersinia entericolitica	Japanese Encephalitis Virus(JEV)	Toxoplasma gondii
Klebsiella oxytoca	Yersinia fredericksenii	Junin virus	Trypanosoma brucei
Legionella pneumophila	Yersinia intermedia	Lassa virus	Trypanosoma cruzi
Leptospira biflexa	Yersinia kristensenii	Machupo virus	Trypansoma rangeli
Leptospira meyeri	Yersinia mollarettii	Marburg Marburgvirus variantMusoke	Plasmodium berghei2
Leptospira terpstrae	Yersinia pestis	Marburg Marburgvirus(RAVN)	Plasmodium brasilianum3
Leptospira vanthielii	Yersiniapseudotuberculosis	Mayaro virus	Plasmodium cynomolgi4
Leptospira wolbachii	Yersinia rohdei	Measles virus	Plasmodium fieldi5
Leptospira yanagawae		Metapneumovirus	Plasmodium fragile6
Listeria monocytogenes		Mopeia Virus	Plasmodium inui7
Mycobacterium tuberculosis		Mumps Virus	Plasmodium simiovale8

Table 11. Off-Panel Organisms Tested by the FilmArray Global Fever Panel

{19}------------------------------------------------

{20}------------------------------------------------

Image /page/20/Picture/0 description: The image is a gray rectangle with a red border. There is some text in the middle of the rectangle that is difficult to read. The text appears to be a fraction, with the numerator being (6)(4).

There were two instances of cross-reactivity observed during testing:

Image /page/20/Picture/2 description: The image is a gray rectangle with a red border. The numbers 1 and 2 are in the upper left corner of the rectangle. The text (b)(4) is in the upper center of the rectangle.

No other cross-reactivity was observed in wet testing.

In addition to the experimental testing, in silico analyses directed toward the potential to cross-react with specific pathogens that were unavailable for testing was also performed. This included Avalon virus, Bas-Congo virus, Bacillus luciferensis, Chalmydophila psittaci, Francisella mediasiatica, Lymphocytic choriomeningitis virus, Middleburg virus, Murray Valley encephalitis virus, Orientia chuto (tsutsugamushi), Pirital virus, Rickettsia prowazekii, Rickettsia ricketsii, Sabia virus,

{21}------------------------------------------------

Semliki Forest virus, Tonate virus, and Variola major. No expected cross-reactivity with off-panel pathogens were predicted by in silico analysis.

h. Interfering Substances:

Potentially interfering substances that could be present in whole blood or introduced during specimen collection and testing were evaluated for their effect on the FilmArray Global Fever Panel performance. Each substance was added to contrived samples containing representative FilmArray Global Fever Panel organisms (Leptospira interrogans, dengue virus Type 3, and P. falciparum) at concentrations equivalent to approximately 3×LoD. The concentrations of endogenous and exogenous substances tested were based on a reference concentration of "normal" to "high" levels expected to be present in clinical specimens.

The following two tables show the results for tested endogenous and exogenous substances, and for technique specific substances and anticoagulants, respectively.

Endogenous Substances	ConcentrationTested a	Results
Albumin	60.0 mg/mL	No interference observed
Bilirubin (Conjugated)	0.41 mg/mL	No interference observed
Bilirubin (Unconjugated)	0.41 mg/mL	No interference observed
Cholesterol (total)	4.2 mg/mL	No interference observed
Glucose	10.1 mg/mL	No interference observed
Hemoglobin	137.0 mg/mL	No interference observed
Immunoglobulins	60 mg/mL	No interference observed
Triglycerides	15.1 mg/mL	No interference observed
Human cells(K-562 Human Leukemia Cells)	6.1E+06 cells/mL	No interference observed
Exogenous Substances	Concentration Tested a	Results
Artemether-Lumefantrine	0.0004 mg/mL	No interference observed
Atovaquone	0.005 mg/mL	No interference observed
Proguanil	0.001 mg/mL	No interference observed
Mefloquine	0.0017 mg/mL	No interference observed
Amphotericin B	0.002 mg/mL	No interference observed
Pentamidine	0.0015 mg/mL	No interference observed
Fluconazole	0.026 mg/mL	No interference observed
Amoxicillin	0.062 mg/mL	No interference observed
Azithromycin	0.011mg/mL	No interference observed
Cetriazone	1.0 mg/mL	No interference observed
Ciprofloxacin	0.012 mg/mL	No interference observed
Clindamycin	0.055 mg/mL	No interference observed
Doxycycline	0.02 mg/mL	No interference observed

Table 12. Results for Potentially Interfering Substances Tested on the FilmArray
Global Fever Panel (Endogenous and Exogenous Substances)

{22}------------------------------------------------

Gentamicin	0.036 mg/mL	No interference observed
Meropenem	0.39 mg/mL	No interference observed
Sulfamethoxazole	0.38 mg/mL	No interference observed
Vancomycin	0.12 mg/mL	No interference observed
Cycloserine	75.0 mg/mL	No interference observed
Isoniazid	0.06 mg/mL	No interference observed
Oseltamivir	0.0005 mg/mL	No interference observed
Ribavirin	0.011 mg/mL	No interference observed
Tenofovir	0.001 mg/mL	No interference observed
Acetaminophen	0.16 mg/mL	No interference observed
Aspirin	0.03 mg/mL	No interference observed
Ibuprofen	0.22 mg/mL	No interference observed
Prednisone	0.0001 mg/mL	No interference observed
Prednisolone	1.2 mg/mL	No interference observed
Cortisone	0.001 mg/mL	No interference observed
Artesunate	0.1 mg/mL	No interference observed

4Concentrations of interfering substances were based on guidelines contained in CLSI guidelines for interference testing (EP07 and EP37) when available.


	Global Fever Panel (Disinfectants, Solvents, and Anticoagulants)
Technique Specific Substances	Concentration Tested	Results
Bleach	1% (v/v) or 525 ppm NaOCl	No interference observed
Povidone-iodine	1% (v/v)	No interference observed
Ethanol	2% (v/v)	No interference observed
TRIzol	2-3% (v/v)	Potentially Interfering
DMSO1	2% (v/v)	No interference observed
Methanol1	2% (v/v)	No interference observed
Saline1	2% (v/v)	No interference observed
Chloroform1	2% (v/v)	No interference observed
Acetone1	2% (v/v)	No interference observed
Hydrochloric Acid1	0.0005 N	No interference observed
Anticoagulant	Concentration Tested	Results
Citrate (sodium)	~0.32 % (10.9 mmol/L)	No interference observed
K · EDTA in excess (5x) 2	~9 mg/mL	No interference observed
Na · Heparin	~19 USP/mL	Potentially Interfering
Acid-citrate-dextrose (ACD)	~2.2 g/L citrate0.8 g/L citric acid2.5 g/L dextrose	No interference observed
Sodium polyanethenolesulfonate (SPS)	0.72 mg/mL	No interference observed
Serum separation tubes	N/A	No interference observed

1 Testing of these specific substances was performed to account for their use as solvents for resuspension of other potential interferants.

2 Testing was performed to evaluate potential interference caused by a short blood draw and a collection tube that thus contained higher anticoagulant concentration

{23}------------------------------------------------

dengue virus, Leptospira spp., and Plasmodium spp. vaccines were not evaluated in this study but are predicted to be reactive with the corresponding Global Fever Panel assay targets.

All evaluated exogenous substances exhibited no interference effect. Among technique specific substances, initial testing of TRIzol exhibited an interference effect wherein P. falciparum replicates were incorrectly reported as a Not Detected result. Subsequent retesting at the same interferent level and additionally with samples containing 1% TRIzol (v/v) did not produce unexpected assay results but may result in delaved Cp values. Similarly, initial testing with Heparin resulted in two unexpected Not Detected results for a single analyte with only Preplicates positive. However, repeat testing at the same heparin concentration successfully met acceptance criteria with replicates positive. These data demonstrate that Heparin levels near the evaluated concentration may be inhibitory for analytes near the LoD.

i. Assay cut-off:

The FilmArray Global Fever Panel Melt Detector software determines whether a FilmArray Global Fever Panel assay result is positive or negative using a predefined algorithm that includes Tm values, fluorescence values, and analysis of melting curves.

Initial melt ranges for each analyte-specific FilmArray Global Fever Panel assay were determined based on a combination of mathematical modeling using known sequence variations of different strains/isolates/variants of targeted organisms as well as data from testing of clinical specimens and known isolates.

After completion of the analytical and clinical studies on the FilmArray Global Fever Panel, a final validation of the melt ranges was performed and included review of data from the Inclusivity study and clinical studies. The observed sensitivity and specificity rates for the individual melt curves and assay calls as compared to expert annotation was greater than 99.8% and 99.9% respectively. The sensitivity, specificity, and accuracy for the validation data were determined to be well above the acceptance criteria.

j. Specimen Transport and Storage (Specimen Stability)

Stability for whole blood specimens was evaluated to support labeling recommendations for storage of samples at room temperature for up to 24 hours, or at 2-8°C for up to seven days, or in an ultra-low temperature freezer (<- 70°C). These storage conditions are consistent with or exceed standard storage and transport conditions and times for most laboratory testing of clinical human whole blood specimens. Testing was conducted using samples composed of human WB contrived with representative GF Panel analytes in mixes at a concentration of Their limit of detection (LoD). Samples were tested immediately [DV replicates) after sample preparation as a no storage control or stored at the appropriate condition for

{24}------------------------------------------------

various amounts of time (one day, three days, or seven days) prior to testing of additional @ replicates per sample per storage condition.

As shown in Table 14 below, study results demonstrated 10/10 replicates positive for each evaluated storage condition for all analytes. All P. falciparum contrived specimens were reactive by both the Plasmodium spp. and Plasmodium falciparum assays. Mean Cp values for all analytes were also consistent between the control and stored samples thereby further supporting the storage claims.

Table 14. Summary of Organism Detection in Whole Blood for All Storage Conditions Tested

Organism/Analyte(Strain)	Ambient Storage		Refrigerated Storage
	(b)(4) °Cfor 24 h	(b) °C for24 h	Day 1	Day 3	Day 7
BACTERIA
Leptospirainterrogans	(b)(4)
VIRUSES
Dengue virus	(b)(4)
PROTOZOA
Plasmodiumfalciparum	(b)(4)

Fresh vs. Frozen Study

Frozen specimens were evaluated as part of the clinical validation of the Global Fever panel due to circumstances that did not permit immediate testing. To determine whether specimen freezing adversely affects analyte detection by the Global Fever Panel, a total of contrived specimens and @ previously tested prospective clinical specimens were evaluated both fresh and after freezing at < - 70°C for at least five days. Contrived specimens were prepared with multiple analytes spiked at concentrations near | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | retested after having been frozen for 6-29 days. Clinical specimens were retested after having been frozen for 111-406 days. Overall agreement of the BioFire Global Fever panel results between fresh and frozen specimens were evaluated as PPA and NPA and results are shown in the tables below.

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Organism/Analyte(Strain)	PPA			NPA
	TP / (TP+ FN)	%	95% CI	TN / (TN + FP)	%	95% CI
BACTERIA
Leptospira spp.	(b)(4)	100%	81.6-100%	(b)(4)	100%	92.0-100%
VIRUSES
Dengue virus	(b)(4)	100%	81.6-100%	(b)(4)	100%	91.8-100%
PROTOZOA
Plasmodium falciparum	(b)(4)	100%	51.0-100%	(b)(4)	100%	93.6-100%
P. vivax/ovale	(b)(4)	100%	64.6-100%	(b)(4)	100%	93.2-100%
Plasmodium spp.	(b)(4)	100%	64.6-100%	(b)(4)	100%	91.8-100%

Table 15. Performance Comparison of Fresh vs Frozen Contrived Specimens

Table 16. Performance Comparison of Fresh vs. Frozen Clinical Specimens

Organism/Analyte (Strain)	PPA			NPA
	TP / (TP+ FN)	%	95% CI	TN / (TN + FP)	%	95% CI
BACTERIA
Leptospira spp.	(b)(4)	100%	56.6-100%	(b)(4)	100%	90.8-100%
VIRUSES
Dengue virus (Serotypes 1, 2, 3, 4)	(b)(4)	100%	81.6-100%	(b)(4)	100%	87.1-100%
Chikungunya Virus	(b)(4)	100%	56.6-100%	(b)(4)	100%	90.8-100%
PROTOZOA
Plasmodium falciparum	(b)(4)	100%	70.1-100%	(b)(4)	97.1%	85.1-99.5%
P. vivax/ovale	(b)(4)	100%	72.2-100%	(b)(4)	97.0%	84.7-99.5%
Plasmodium spp.	(b)(4)	100%	80.6-100%	(b)(4)	100%	87.5-100%

"Three of the 16 Plasmodium spp .- positive fresh specimens were positive for both P. falciparum and P. vivax/ovale bone specimen negative for P. falciparum assay when tested fresh was positive for P. falciparum when tested after freezing. *One specimen that was negative for the P. vivax/ovale assay when tested fresh was positive for P. vivax/orde when tested after freczing

2. Comparison studies:

a. Method comparison with predicate device:

Not applicable

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b. Matrix comparison:

Not applicable.

1. Clinical studies:
- a. Clinical Sensitivity:

Prospective Clinical Study

The clinical performance of the Global Fever Panel was established during a multicenter study conducted at ten geographically distinct study sites, including two in the United States.

Prospective whole blood specimens were collected from subjects meeting the following eligibility criteria:

Inclusion criteria

Subject has a recorded or self-reported fever within the past two days .
Subject provides informed consent prior to enrollment and specimen . collection
. Subject has not participated in the study within the last 30 days

Exclusion criteria

. Subject does not have or did not self-report having a fever within the past two days
Subject was unable to provide informed consent .
Subject has participated in the study within the last 30 days. .

As recorded in site-specific study protocols, some sites applied additional eligibility criteria such as age limits, citizenship within the host country, and/or suspicion in the opinion of the physician that the subject's fever could be due to a pathogen on the Global Fever Panel.

A total of 1.971 collected specimens were generated across all study sites. The most common reason for exclusion was difficulty drawing blood (60 (96) [ 0 (9) (96) were excluded due to errors by study personnel. [ 1000 specimens ( 196) were excluded due to inability to obtain a Global Fever Panel or comparator result and (96) were excluded because of the subject withdrawing before the specimen was collected. In total, 1875 eligible whole blood specimens, including 1469 (78,3%) Category I prospective fresh and 406 (21.7%) Category II prospective frozen specimens, were collected over eighteen months (March 2018 - September 2019).

Table 17 below shows the participating study sites and the number of prospective specimens enrolled at each study site.

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Study Site(Location)	Study Site #	EnrolledPopulation	TotalSubjectsEnrolled	Total ValidWBSpecimensEnrolled(excluded)	Study Started –Completed (2016)
USA	Site 7	EmergencyDepartment (ED)	(b)(4)	179(21)	August 2018- November2018
USA	Site 14	Inpatient and ED		9(3)	August 2019- September2019
Africa	Site 1	Inpatient		134(11)	March 2018- May 2019
Africa	Site 2	Inpatient andOutpatient		108(5)	June 2019= September2019
Africa	Site 5	Inpatient andOutpatient		199(4)	January 2019- June 2019
Africa	Site 11	Inpatient andOutpatient		158(5)	July 2019- September2019
Southeast Asia	Site 8	Inpatient andOutpatient		249(31)	November 2018 –September 2019
Southeast Asia	Site 9	Outpatient		406(9)	February 2019 –September 2019
Central & SouthAmerica	Site 12	Outpatient		297(3)	November 2018 – July2019
Central & SouthAmerica	Site 13	Inpatient,Outpatient, and ED		136(4)	August 2019 –September 2019
		Total	1971	1875(96)

Table 17. Prospectively Collected Samples Across Sites in the Global Fever Panel Clinical Study

Table 18 below shows breakdown of gender and age in clinical study.

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		Overall	Site #1	Site #2	Site #5	Site #7	Site #8	Site #9	Site #11	Site #12	Site #13	Site #14
Sex	Male	895 (52.3%)	58 (43.3%)	67(62.0%)	125(62.8%)	113(63.1%)	107 (43.0%)	206 (50.7%)	87 (55.1%)	132 (44.4%)	80 (58.8%)	5 (55.6%)
	Female	980 (47.7%)	76 (56.7%)	41(38.0%)	74 (37.2%)	66 (36.9%)	142 (57.0%)	200 (49.3%)	71 (44.9%)	165 (55.6%)	56 (41.2%)	4 (44.4%)
	< 5 years	163 (8.7%)	44 (32.8%)	20(18.5%)	0 (0%)	0 (0%)ª	25 (10.0%)	0 (0%)b	66 (41.8%)	3 (1.0%)	5 (3.7%)	0 (0%)ª
	5-21 years	765 (40.8%)	21 (15.7%)	38(35.2%)	128(64.3%)	14 (7.8%)ª	127 (51%)	204 (50.2%)b	70 (44.3%)	102 (34.3%)	61 (44.9%)	0 (0%)ª
Age	22-50years	672 (35.8%)	42 (31.3%)	31(28.7%)	59 (29.6%)	106(59.2%)	63 (25.3%)	139 (34.2%)	21 (13.3%)	146 (49.2%)	61 (44.9%)	4 (44.4%)
	50+ years	275 (14.6%)	27 (20.1%)	19(17.6%)	12 (6%)	59 (33.0%)	34 (13.7%)	63 (15.5%)	1 (0.6%)	46 (15.5%)	9 (6.6%)	5 (55.6%)
	Total	1875	134	108	199	179	249	406	158	297	136	9

Table 18. Overall and Per Site Demographic Analysis for the Global Fever Panel Clinical Study

ª Site was not enrolling subjects <18 years old
b Site was not enrolling subjects <7 years old

Site was not enrolling subjects <7 years old

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In the laboratory, specimens were thoroughly mixed by vortexing or inverting and pipetted into a 700-uL aliquot for FilmArray testing, a 1300-uL aliquot for nucleic acid extraction, and the remaining volume (typically ≥500 uL) into an aliquot for archiving. Extracted nucleic acid was stored at <- 70 °C and shipped on dry ice to BioFire Defense for molecular comparator testing.

0.3% (5/1902) of eligible specimens were ultimately excluded due to inability to obtain a Global Fever Panel test result. (Four specimens were initially eligible and tested on the Global Fever Panel, but were later excluded, for a total of 1898 specimens included in the analysis of System Performance.) The overall success rate for initial specimen tests on the Global Fever Panel was 98.7% (1868/1898); five tests did not complete (two due to loss of power, two instrument errors, and one software error) and 25 had pouch internal control failures. Of the 30 unsuccessful initial tests. all were retested once, and valid results were produced for 25 of the 30 retested specimens.

The incomplete runs and instrument errors accounted for 0.3% (5/1898) of initial. runs, resulting in an instrument success rate of 99.7% (1893/1898). Of the five unsuccessful initial tests due to instrument performance, all were retested once, and three produced valid results. The two specimens that failed after retesting were excluded from further analysis.

Of the 1893 initial tests that completed. 1.3% (25/1893) did not produce valid internal controls, resulting in a pouch control success rate of 98.7% (1868/1893). Of the 25 tests with internal control failures, all were retested once, and 22 produced valid control results. Internal controls failed a second time for three specimens upon retesting, so these three specimens were excluded from further analysis.

All specimens were evaluated with the FilmArray Global Fever Panel at the clinical study sites. Frozen nucleic acid extracts were sent to BioFire Defense for comparator testing with well-validated nested PCR assays followed by bi-directional Sanger sequencing. Two comparator assays were utilized for each assay target with positive result from both assays considered positive. If the results of the two comparator assays for a particular analyte disagreed, the samples were subjected to repeat comparator testing with samples determined as positive if at least 2/3 replicates were positive for a single comparator assay.

PPA for each analyte was calculated as 100% x (TP/(TP + FN). True positive (TP) indicates that both the FilmArray Global Fever Panel and the comparator method had a positive result for this specific analyte, and false negative (FN) indicates that the FilmArray Global Fever Panel result was negative while the comparator result was positive. NPA was calculated as 100% x (TN/(TN + FP)). True negative (TN) indicates that both the FilmArray Global Fever Panel and the comparator method had negative results and a false positive (FP) indicates that the FilmArray Global Fever Panel result was positive, but the comparator result was negative. The exact binomial two-sided 95% confidence interval was calculated. Samples for which false positive

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and/or false negative results (i.e., discrepant results) were obtained when comparing the FilmArray Global Fever Panel results to the comparator method results were further investigated. The discrepancy investigations were typically performed as follows: 1) All FP and FN were examined to determine whether additional testing on Global Fever Panel or with comparator assays could detect the analyte, but initial testing reported as not detected or negative because analyte was near or below the detection threshold: 2) FP and FN were evaluated by at least one additional PCR test that used different primers than the Global Fever Panel assay or the comparator assays: 3) When possible, unresolved discrepancies were evaluated with additional PCR testing that could be verified by sequence analysis. Discrepancy results are footnoted below in Table 19.

Analyte		TP/(TP + FN)	%	95%CI	TN/(TN+FP)	%	95% CI
VIRUSES
Chikungunyavirusa	Fresh	25/25	100%	86.7-100%	1442/1444	99.9%	99.5-100%
	Frozen	0/0	-	-	406/406	100%	99.1-100%
	Overall	25/25	100%	86.7-100%	1848/1850	99.9%	99.6-100%
Dengue Virus(serotypes 1,2, 3, and 4)b	Fresh	249/263	94.7%	91.3-96.8%	1206/1206	100%	99.7-100%
	Frozen	17/20	85.0%	64.0-94.8	386/386	100%	99-100%
	Overall	266/283	94.0%	90.6-96.2%	1592/1592	100%	99.8-100%
BACTERIA
Leptospiraspp.c	Fresh	9/9	100%	70.1-100%	1456/1460	99.7%	99.3-99.9%
	Frozen	6/7	85.7%	48.7-97.4%	399/399	100%	99-100%
	Overall	15/16	93.8%	71.7-98.9%	1855/1859	99.8%	99.4-99.9%
PROTOZOA
Plasmodiumspp.d,e	Fresh	207/210	98.6%	95.99-99.5%	1251/1259	99.4%	98.7-99.7%
	Frozen	132/135	97.8%	93.7-99.2%	267/271	98.5%	96.3-99.4%
	Overall	339/345	98.3%	96.3-99.2%	1518/1530	99.2%	98.6-99.6%
Plasmodiumfalciparumf	Fresh	148/158	93.7%	88.7-96.5%	1309/1311	99.8%	99.4-100%
	Frozen	82/90	91.1%	83.4-95.4%	315/316	99.7%	98.2-99.9%
	Overall	230/248	92.7%	88.8-95.4%	1624/1627	99.8%	99.5-99.9%
Plasmodiumvivax/ovaleg	Fresh	64/69	92.8	84.1-96.9%	1400/1400	100.0%	99.7-100%
	Frozen	51/55	92.7%	92.7-97.1%	351/351	100.0%	98.9-100%
	Overall	115/124	92.7%	86.8-96.1%	1751/1751	100.0%	99.8-100%

Table 19. FilmArray Global Fever Panel Clinical Performance: Summary - Whole Blood (EDTA)

22/2 FP Chikungunya virus specimens were positive by additional PCR

*Evidence of Dengue virus was found in 15/17 FN speciment with Global Fever Panel and additional PCR, two were positive upon Retest with Global Fever Panel, and eight were detected only by additional PCR.

"Evidence of Leptospira spp. was found in 1/1 FN by GF panel retest and in 3/4 FP specimens by additional PCR. 4Five (5/6) Plasmodium spp. FN specimens were P. falciparum FN and 1/6 was P. vivax/ovale. Three (3/12) Plasmodium spp. FP specimens were also P. falciparum FP

sPlasmodium spp. were detected in 3/6 FN Specimens: two were positive upon GF Panel retest and additional PCR, one was positive only upon GF Panel retest. Plasmodium spp. were also detected in 10/12 FP specimens by additional PCR.

"Evidence of Plasmodium spp. was found in 13/18 FN specimens: three upon GF Panel retest and by additional PCR, nine were only positive by additional PCR. P. falciparum was detected in 2/3 FP specimens by additional PCR.

8P. vivax/ovale was detected in 79 FN specimens: two of which were positive both upon GF Panel retest and by additional PCR, two were positive only by GF Panel retest, and three were positive only by additional PCR.

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Twenty-five specimens had a positive Plasmodium spp. Detected result with a concomitant Not Detected result for either P. falciparum or P. vivax/ovale. For 19 of these specimens, Cp value of the Plasmodium spp. assay was near or later than the LoD range and/or comparator testing identified P. falciparum or P. vivax/ovale infection. A further 3/25 of these specimens had robust Plasmodium spp, amplification but no further evidence of P. falciparum or P. vivax/ovale infection. These cases likely reflect infection with a different malaria species (P. knowlesi or P. malariae) not differentiated by the Global Fever Panel malaria assays.

When possible, performance of malaria testing as part of Standard of Care was compared to the Global Fever Panel result. Because most sites did not attempt to speciate malaria, only the Plasmodium spp. assay result was used in this analysis. Each clinical site followed its own standard procedure for malaria testing with significant differences between sites. Data is listed in Table 20 below as Site-specific PPA and NPA with site-specific microscopic results.

Table 20. Performance Comparison Global Fever Panel and Site-Specific Malaria Testing,

Overall, the Global Fever Panel showed strong correlation with site-specific methods when the site was able to detect malaria true positives versus only false negatives). Importantly, for all of the false negative results, the comparator method agreed with Global Fever Panel. The Global Fever Panel was also able to detect Plasmodium in many specimens that were determined to be negative by site-specific methods ( ( (1) ). For (1) of these Detections, the comparator method agreed with the Global Fever Panel result. While it cannot be ruled out that the Global Fever Panel detected residual nucleic acid following a cleared infection, these results could also reflect that the Global Fever Panel is more sensitive than the other methods utilized.

Assay performance among positive specimens that were quantified by site-specific methods is included in the table below stratified by overall parasitemia.

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Table 21. Global Fever Panel Positive Percent Agreement with Site-Specific Malaria Testing Stratified by Parasitemia

Parasitemia (Parasites/uL)	PPA	95% CI
	05344

The Global Fever Panel reported a total of specimens with multiple analyte detections (1) 1% of all specimens, PM % of all positive specimens) as described in Table 22 below. The majority of co-detections contained Plasmodium spp. ( 1016) ). No co-detections contained more than two discernible analytes.

Distinct Co-detection Combinations			Total Co-detections
Analyte 1	Analyte 2	Analyte 3
Chikungunya virusa	Dengue virus	-	(b)(4)
Dengue virus	Plasmodium spp.	P. falciparum
Dengue virus	Plasmodium spp.	P. vivax/ovale
Leptospira spp.b	Plasmodium spp.b	-
Leptospira spp.	Plasmodium spp.	P. vivax/ovale
Plasmodium spp.	P. falciparum	P. vivax/ovale

Table 22. Global Fever Panel Clinical Study Specimen Co-Detections

"Chikungunya virus was detected by Global Fever Panel but not by comparator testing in one of the two co-detections 6 Comparator testing did not identify Leptospira spp or Plasmodium spp. in this specimen.

b. Clinical specificity:
See section L.3a above.
c. Other clinical supportive data (when a. and b. are not applicable):
See section L.3a above.
1. Clinical cut-off:
  Not applicable.
1. Expected values/Reference range:
  The prevalence of individual analytes detected by the Global Fever Panel as observed during the clinical study is described in Table 23 below.

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	Overall(N=1875)		USASite 07(N=179)		USASite 14(N=9)		AfricaSite 01(N=134)		AfricaSite 02(N=108)		AfricaSite 05(N=199)		AfricaSite 11(N=158)		Southeast AsiaSite 08(N=249)		Southeast AsiaSite 09(N=404)		Central & S. AmericaSite 12(N=297)		Central & S. AmericaSite 13(N=136)
Analyte	#	EV	#	EV	#	EV	#	EV	#	EV	#	EV	#	EV	#	EV	#	EV	#	EV	#	EV
Chikungunya virus	27	1.4%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	0	0.0%	27	6.7%	0	0.0%	0	0.0%
Dengue virus(Serotypes 1, 2,3, and 4)	266	14.2%	0	0.0%	0	0.0%	1	0.7%	0	0.0%	0	0.0%	0	0.0%	90	36.1%	54	13.4%	20	6.7%	101	74.3%
Leptospira spp.	19	1.0%	1	0.6%	0	0.0%	0	0.0%	1	0.9%	0	0.0%	0	0.0%	4	1.6%	4	1.0%	9	3.0%	0	0.0%
Plasmodium spp.	351	18.7%	0	0.0%	0	0.0%	16	11.9%	50	46.3%	141	70.9%	49	31.0%	7	2.8%	4	1.0%	84	28.3%	0	0.0%
P. falciparum	233	12.4%	0	0.0%	0	0.0%	14	10.4%	44	40.7%	125	62.8%	42	26.6%	3	1.2%	0	0.0%	5	1.7%	0	0.0%
P. vivax/ovale	115	6.1%	0	0.0%	0	0.0%	3	2.2%	0	0.0%	12	6.0%	12	7.6%	4	1.6%	4	1.0%	80	26.9%	0	0.0%

Table 23. Prevalence of Detected Analytes Stratified by Region and Clinical Study Site

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The Global Fever Panel detected at least one analyte in 657 of the 1875 included specimens (35.1% positivity rate).

FilmArray Result	Number Detected	% of Total(% of Positives)
Detected (at least one result)	657	35.0%(100%)
One analyte result	629	33.5%(95.7%)
Two analyte resultsa	28	1.5%(4.3%)

Table 24. Expected Values Summary (Detections) as Determined in the Global Fever Panel Clinical Study

"Data for discernible co-detections only. The Plasmodium spp. assay is not considered a unique Global Fever Panel detection when co-detected with a Plasmodium species-level assay. Specimens containing multiple species within a genus are not always discemble (e.g., a specimen containing P. malariae and P. falciparum would not produce a discernible co-detection.)

	Table 25. Expected Values Summary (Analytes) as Determined in the Global Fever
Panel Clinical Study
FilmArray Result	Number Detected	% of Positives
Chikungunya virus	27	4.1%
Dengue virus (serotypes 1,2, 3, and 4)	266	40.5%
Leptospira spp.	19	2.9%
Plasmodium spp.	351	53.4%
Plasmodium falciparum	233	35.5%
Plasmodium vivax/ovale	115	17.5%

The most prevalent analyte result was Plasmodium spp. (351/657; 53.4%), of which, 233/351 (66.4%) also had Plasmodium falciparum identified, 115/351 (32.8%) also had Plasmodium vivax/ovale identified, 25/351 (7.1%) had no species-level identification, and 22/351 (6.3%) had a combination of P. falciparum and P. vivax/ovale. The second most prevalent analyte was dengue virus (266/657, 40.5%). Chikungunya virus was detected in 4.1% (27/657) of specimens and Leptospira spp. was detected in 2.9% (19/657).

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M. Instrument Name:

Not applicable. The device does not utilize an instrument for result generation.

N. System Descriptions:

1. Modes of Operation:

Does the applicant's device contain the ability to transmit data to a computer, webserver, or mobile device?

Yes __________________________________________________________________________________________________________________________________________________________________________

Does the applicant's device transmit data to a computer, webserver, or mobile device using wireless transmission?

Yes X - or No - - - -

1. Software:
  FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes X or No or No

The device does not contain any software or instrument components.

3. Specimen Identification:

The Sample ID can be entered manually or scanned in by using the FilmArray barcode scanner

1. Specimen Sampling and Handling:
  Not applicable.
1. Calibration:
  Not applicable.
1. Quality Control:
  See Quality Control Section above (L.1.c "Traceability, Stability, Expected Values (controls, calibrators, or methods)")

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O. Other Supportive Instrument Performance Characteristics Data Not Covered in the "Performance Characteristics" Section above:

None.

P. Proposed Labeling:

The labeling supports the decision to grant the De Novo request for this device.

Q. Identified Risks to Health and Identified Mitigations

Identified Risks to Health	Mitigation Measures
Risk of an inaccurate test result (falsepositive or false negative result) leadingto improper patient management	Certain labeling information, including certainlimiting statements and performanceinformation.Certain design verification and validation,including certain analytical studies and clinicalstudies.Use of certain specimen collection devices.
Misinterpretation of test results leading tomisdiagnosis and associated risk of falsetest results	Certain labeling information, including certainlimiting statements and performanceinformation.Certain design verification and validation,including certain analytical studies and clinicalstudies.
Failure to correctly operate the deviceleading to inaccurate test results	Certain labeling information, including certainlimiting statements and performanceinformation.Certain design verification and validation,including certain analytical studies and clinicalstudies.Use of certain specimen collection devices.

R. Benefit/Risk Analysis:

Patient Perspectives

This submission did not include specific information on patient perspectives for this device.

Summary of the Assessment of Benefit

Fever is a host response to infection that is not pathognomonic of a specific disease. The benefit of the assay is aiding the accurate diagnosis of infections that cause acute febrile illness in the specific population of patients who have been potentially exposed to pathogens detected by the Global Fever Panel. When guided by appropriate risk factors and epidemiological information, the Global Fever Panel can be helpful to identify

{37}------------------------------------------------

the specific cause of fever and initiate appropriate treatment, including, but not limited to. antimicrobial therapy. Earlier identification of fever causing pathogens and an appropriate course of treatment may improve patient outcomes. Additionally, the Global Fever Panel fills an unmet need for in vitro diagnostics as no currently approved or cleared tests exist for many of the pathogens on the Global Fever Panel.

While the performance of the device in the clinical and analytical studies suggests that patients will benefit from the assay, expected and acceptable sources of uncertainty are the wide confidence intervals around point estimates of performance. The special controls, including the interpretation of results and the limiting statements in device labeling will help to ensure that errors will be uncommon and will facilitate accurate assay implementation and interpretation of results.

Summary of the Assessment of Risk

The risks associated with the device, when used as intended, are those related to the risk of inaccurate test results, the failure to correctly interpret test results, and failure to correctly operate the device.

The risk of a false positive test result is improper patient management, including potentially inappropriate administration of unnecessary antibiotics or anti-malarial medications. Inappropriate administration of prolonged courses of antibiotics is associated with toxicity, allergic reactions, and other adverse outcomes, including secondary infections such as C. difficile colitis. Inappropriate administration of antimalarial medications may also have adverse effects that depend upon the drug administered. No specific treatments exist for dengue and chikungunya virus infections, although a vaccine was recently developed for dengue virus. This vaccine is only recommended in individuals who had previously had confirmed dengue infection and individuals who have not been infected are at increased risk of severe dengue if they are infected after being vaccinated. In the broader population, individual false positive results could lead to increased burden on the CDC to perform confirmatory testing since all targets detected by the Global Fever Panel are nationally notifiable.

During the analytical evaluation of exclusivity, some pathogen cross-reactivity was identified that could pose risks of patient harm. Specifically, P. knowlesi and P. malariae may cross-react with the P. vivax/ovale assay on the Global Fever Panel. P. vivax/ovale infections require a specific course of treatment to clear hypnozoite liver stage of infection and prevent recrudescence. A false positive P. vivax/ovale result could therefore lead to patients receiving unnecessary and potentially toxic treatment.

The risk of a false negative test result is delayed identification of the cause of the disease in the patient, which could lead to improper patient management, including administration of unnecessary treatment and/or discontinuation of appropriate treatment. An undiagnosed infection or delaved diagnosis, particularly with P, falciparum or Leptospira can result in increased morbidity and mortality.

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Failure to correctly operate the device can lead to false test results. Failure to correctly interpret test results can lead to treatment of a clinically positive patient in the same manner as a false negative test result and a clinically negative patient in the same manner as a false positive test result, with the corresponding implications discussed above.

Summary of the Assessment of Benefit-Risk

General controls are insufficient to mitigate the risks associated with the device. However, the probable clinical benefits outweigh the probable risks for the proposed assay, considering the mitigations of the risks provided for in the listed special controls established for this device, as well as general controls. The required special controls will help ensure that errors will be uncommon and will facilitate accurate assay implementation and interpretation of results.

The risk of inaccurate test results (both positive and negative) is mitigated by the intended use clearly stating that the assay results are intended to be used with other clinical, epidemiologic, and laboratory data. The risk of false results is also mitigated by the inclusion of performance characteristics from analytical and clinical studies in the labeling. Risks from cross-reactivity are mitigated by appropriate limitations in the labeling that indicate potential cross-reactivity of these malaria strains and further state that a P. vivax/ovale result should be further confirmed. Additionally, a separate limitation states that all Plasmodium spp. detected results from patients exposed in Southeast Asia should be further investigated for possible P. knowlesi infection.

Risks of failure to correctly interpret the test results are mitigated through the inclusion in the labeling of a detailed description of what the device detects, the specimen type for which testing is indicated, the type of results provided to the user in the intended use statement, as well as a detailed explanation of the interpretation of results. Finally, the risk of failure to correctly operate the device is mitigated by the inclusion of detailed directions for use in the package insert, such that the operator can successfully use the instrument.

The clinical performance observed in the clinical trial suggests that errors will be uncommon and that the assay will provide substantial benefits to patients in the diagnosis of acute febrile illness and when used in conjunction with other clinical and diagnostic findings.

Given the combination of the device's indications for use, labeling, the required general controls, and the special controls established for this device, the probable benefits would outweigh the probable risks.

S. Conclusion

The De Novo request is granted and the device is classified under the following and subject to the special controls identified in the letter granting the De Novo request:

Product Code: OMV

{39}------------------------------------------------

Device Type: Device to detect and identify selected microbial agents that cause acute febrile illness
Class: II Regulation: 21 CFR 866.3966

Regulation Number and Section

§ 866.3966 Device to detect and identify selected microbial agents that cause acute febrile illness.

(a)
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.(b)
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.