The FilmArray Meningitis/Encephalitis (ME) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with FilmArray and FilmArray 2.0 systems. The FilmArray ME Panel is capable of simultaneous detection and identification of multiple bacterial, viral, and yeast nucleic acids directly from cerebrospinal fluid (CSF) specimens obtained via lumbar puncture from individuals with signs and/or symptoms of meningitis and/or encephalitis. The following organisms are identified using the FilmArray ME Panel:

Bacteria:
Escherichia coli K1 Haemophilus influenzae Listeria monocytogenes Neisseria meningitidis (encapsulated) Streptococcus agalactiae Streptococcus pneumoniae

Viruses: Cytomegalovirus Enterovirus Herpes simplex virus 1 Herpes simplex virus 2 Human herpesvirus 6 Human parechovirus Varicella zoster virus
Yeast:

Cryptococcus neoformans/gattii

The FilmArray ME Panel is indicated as an aid in the diagnosis of specific agents of meningitis and/or encephalitis and results are meant to be used in conjunction with other clinical, epidemiological, and laboratory data.

Results from the FilmArray ME Panel are not intended to be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results do not rule out co-infection with organisms not included in the FilmArray ME Panel. The agent detected may not be the definite cause of the disease. Negative results do not preclude central nervous system (CNS) infection. Not all agents of CNS infection are detected by this test and sensitivity in clinical use may differ from that described in the package insert.

The FilmArray ME Panel is not intended for testing of specimens collected from indwelling CNS medical devices.

The FilmArray ME Panel is intended to be used in conjunction with standard of care culture for organism recovery, serotyping, and antimicrobial susceptibility testing.

Device Description

The FilmArray ME Panel is a multiplex nucleic acid-based test designed to be used with FilmArray or FilmArray 2.0 system ("FilmArray systems" or "FilmArray instruments"). The FilmArray ME panel includes a FilmArray ME Panel pouch which contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex polymerase chain reaction (PCR) with DNA melt analysis. The FilmArray ME Panel simultaneously conducts 14 tests for the identification of potential CNS pathogens from CSF specimens obtained via lumbar puncture. Results from the FilmArray ME Panel are available within about one hour.

A test is initiated by loading Hydration Solution into one port of the pouch and a CSF sample mixed with the provided Sample Buffer ampoules into the other port of the pouch and placing it in the FilmArray Instrument. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and the Sample Buffer rehydrates the reagents. After the pouch is prepared, the FilmArray Software on the FilmArray systems guides the user though the steps of placing the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run on the FilmArray systems.

The FilmArray instruments contain a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel.

Nucleic acid extraction occurs within the 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. 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

This document describes the evaluation of the FilmArray Meningitis/Encephalitis (ME) Panel for a De Novo classification. It includes information on analytical and clinical studies to demonstrate the device's performance.

Here's the breakdown of the acceptance criteria and study information:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as strict numerical thresholds in the provided text for all performance metrics. However, they are implied through the detailed reporting of study results and statements like "The overall success rate for initial specimen tests in the prospective study was 98.9%" or "Overall PPA for clinical and contrived specimens combined was 97.5% with the lower bound of the two-sided 95% confidence interval (95% CI) at 92.9%, and overall NPA was 99.7% with the lower bound of the two-sided 95% CI at 99.3%." For analytical studies, acceptance criteria like "a minimum of 9/10 replicates detected" for specimen stability or "Delta Tm values between the two systems were less than or equal to 0.4℃... passed the study acceptance criteria of less than 0.5℃" are explicitly mentioned.

For the purpose of this table, I will use the clinical performance reported as the "device performance" and extract the implied acceptance criteria where possible.

Metric (Implied Acceptance Criteria)	Device Performance (FilmArray ME Panel) - Overall	Comments
Clinical Performance (Prospective Study)
Overall Positive Percent Agreement (PPA) (for all analytes in clinical and contrived specimens combined)	97.5% (95% CI: 92.9-99.2%)	Based on comparison to comparator methods (culture for bacteria, PCR with bi-directional sequencing for viruses/yeast). This combines both clinical and contrived specimens from the comparison studies.
Overall Negative Percent Agreement (NPA) (for all analytes in clinical and contrived specimens combined)	99.7% (95% CI: 99.3-99.9%)	Based on comparison to comparator methods.
Analytical Reproducibility (FilmArray System)
Initially Valid Runs	98.4% (360/366)
Agreement with Expected Results (at 1x LoD for various organisms during reproducibility)	Ranges from 86.7% to 100%	Specific criteria often stated as "minimum 9/10 replicates detected" for analytical studies.
Tm Standard Deviations (for positive results)	≤ 0.5℃ for all analytes	The reported performance of ≤ 0.5℃ for all analytes meets the internal acceptance criterion.
Analytical Reproducibility (FilmArray 2.0 System)
Initially Valid Runs	98.6% (360/365)
Agreement with Expected Results (at 1x LoD for various organisms during reproducibility)	Ranges from 93.3% to 100%	Specific criteria often stated as "minimum 9/10 replicates detected" for analytical studies.
Tm Standard Deviations (for positive results)	≤ 0.5℃ for all analytes	The reported performance of ≤ 0.5℃ for all analytes meets the internal acceptance criterion.
Analytical Limit of Detection (LoD)
Detection at LoD Concentration	Mostly 20/20 (100%), some 19/20 (95%)	The LoD was confirmed for all analytes on both systems, indicating successful detection at the established LoD.
Analytical Inclusivity
Detection of tested isolates at 1x to 3x LoD	Majority positive results at specified concentrations	One HPeV strain detected at 10x LoD. In silico analysis was used for less common strains.
Specimen Stability
Detection for stored samples (e.g., 1 day ambient, 1, 3, 7 days refrigerated)	9/10 or 10/10 replicates detected	Study acceptance criteria: minimum of 9/10 replicates detected.
Mean Cp values (between control and stored samples)	Consistent (within expected system variability)
Clinical Comparison (FilmArray vs. FilmArray 2.0)
Delta Tm values between the two systems	≤ 0.4℃ for all analytes	Passed study acceptance criteria of < 0.5℃.

2. Sample Size Used for the Test Set and Data Provenance

Test Set (Clinical Studies):

Prospective Clinical Study: 1560 specimens.
- Data Provenance: From 11 geographically distinct U.S. study sites. Primarily tested fresh (1015 specimens, 65%) with a portion collected and immediately frozen for later testing (545 specimens, 35%).
Preselected Archived Specimens: 235 specimens (210 positive, 25 negative).
- Data Provenance: Archived clinical specimens.
Contrived Specimens (for clinical performance evaluation): Not a single test set, but for analytes with insufficient prevalence in prospective/archived studies, surrogate CSF specimens were created. At least 25 specimens per analyte, spiked at 2x LoD or other concentrations.
- Data Provenance: Residual CSF specimens that previously tested negative for ME panel analytes. These were prepared, frozen, and distributed to prospective clinical study sites for testing.
Clinical Comparison between FilmArray and FilmArray 2.0 Systems: 149 specimens (21 positive clinical specimens + 128 contrived specimens).
- Data Provenance: Residual, de-identified CSF specimens and contrived CSF specimens. Clinical specimens identified as positive at source laboratories or by culture/PCR comparator methods. Contrived specimens prepared in leftover negative CSF.

Training Set (Analytical Studies):

The document does not explicitly delineate a "training set" in the context of machine learning. However, many of the analytical studies (e.g., LoD, inclusivity, cross-reactivity, interfering substances) involve testing a wide range of strains and conditions to characterize the device's analytical performance, which serves as foundational data for the device's algorithms and performance specifications. The "initial melt ranges" for the assay's algorithm were determined based on a "combination of mathematical modeling using known sequence variations... as well as data from testing of clinical specimens and known isolates." This implies an initial dataset used for establishing the algorithm's parameters, which could be considered an internal "training" or development set.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document focuses on comparator methods rather than expert consensus for establishing ground truth, especially for the clinical studies.

Prospective Clinical Study:
- Comparator Method for Bacteria (E. coli K1, H. influenzae, L. monocytogenes, N. meningitidis, S. agalactiae, S. pneumoniae): CSF bacterial culture (performed at the source laboratory).
- Comparator Method for Viruses/Yeast (CMV, EV, HSV-1, HSV-2, HHV-6, HPeV, VZV, C. neoformans/gattii): Two PCR assays with bi-directional sequencing (performed at BioFire Laboratory).
- Qualifications of Experts for Comparator Methods: Not explicitly stated, but "source laboratories" imply trained laboratory personnel, and "BioFire Laboratory" implies internal molecular diagnostics experts. The comparator methods themselves are "well-accepted comparator methods."
Preselected Archived Specimens: The presence (or absence) of expected analytes was verified in each specimen using a "confirmatory molecular test (e.g., PCR with bi-directional sequencing)." This suggests the ground truth was established by laboratory-based molecular methods.
Contrived Specimens: Ground truth is established by the known composition of the spiked organisms and their concentrations.
For algorithm-specific validation of melt ranges: "expert annotation" was mentioned, but the number and qualifications of these experts are not specified.

4. Adjudication Method for the Test Set

The primary method for establishing ground truth for individual results in the clinical studies was objective laboratory comparator methods (culture or PCR with bi-directional sequencing).

There is no explicit mention of an "adjudication method" involving multiple human readers to resolve discrepancies between the device and comparator results.
However, for certain discrepancies (e.g., false positives for S. pneumoniae, H. influenzae, CMV, EV, HSV-1, HSV-2, HHV-6, HPeV, VZV, and C. neoformans/gattii), further investigations were conducted. These included:
- Re-testing with independent PCR assays.
- Review of de-identified subject medical data (e.g., clinical diagnoses, treatment history, CSF pleocytosis).
- Comparison with other relevant tests (e.g., Cryptococcal Antigen testing).
- These further investigations serve as a form of retrospective adjudication to understand the true clinical status or the reason for the discrepancy, rather than a prospective consensus-based change to the initial ground truth.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done.

This document describes the evaluation of an in vitro diagnostic device (IVD), which is an automated molecular diagnostic test. Such devices are typically evaluated for their standalone performance against established laboratory comparator methods, or for equivalence between different systems/platforms of the same device. MRMC studies are generally relevant for diagnostic imaging interpretation or other tasks where human readers interpret data, often with and without AI assistance, to measure the impact of AI on human performance. The FilmArray ME Panel is an automated assay, not an AI interpretation tool for human readers.

6. If a Standalone (i.e. algorithm only without human-in-the loop performance) was done

Yes, a standalone performance evaluation of the algorithm and device was done.

The entire analytical and clinical study described in the document evaluates the FilmArray ME Panel as an automated, standalone diagnostic device. The device's software automatically interprets the results of the DNA melt curve analysis and generates a test report without physician intervention or interpretation of raw data. The physician then uses this report in conjunction with other clinical data.

The reproducibility studies, limit of detection, inclusivity, cross-reactivity, interfering substances, and specimen stability studies all assess the device's intrinsic analytical performance.
The clinical studies compare the device's outputdirectly to established comparator methods (culture and PCR with bi-directional sequencing).
The comparison study between FilmArray and FilmArray 2.0 systems also evaluates the standalone agreement between the two versions of the automated device.

7. The Type of Ground Truth Used

The ground truth for the device's performance evaluation was established using a combination of methods:

Comparator Laboratory Methods:
- CSF bacterial culture: For bacterial analytes (considered the "gold standard" for these).
- Two PCR assays with bi-directional sequencing: For viral and yeast analytes. These assays targeted different nucleic acid sequences than the FilmArray ME Panel to ensure independent confirmation.
Known Spiked Concentrations: For all contrived specimens used in analytical studies (e.g., LoD, inclusivity, competitive inhibition, interfering substances, specimen stability, matrix equivalence, and a portion of the clinical performance evaluation for rare analytes). Dilutions of known organisms were added to artificial CSF or negative clinical CSF.
Historical/Prior Laboratory Testing: For preselected archived specimens, ground truth was based on their previously confirmed positive status for specific analytes, often verified by confirmatory molecular tests prior to FilmArray testing.

8. The Sample Size for the Training Set

As mentioned in point 2, the document does not refer to explicit "training sets" in the context of typical machine learning. However, the system's "melt ranges" (a core component of its interpretive algorithm) were established using:

"mathematical modeling using known sequence variations of different strains/isolates/variants of targeted organisms"
"data from testing of clinical specimens and known isolates."

The sample size for this initial establishment (which functions like a training phase for the algorithm) is not explicitly quantified as a single number in the document. It's an ongoing process of data collection and refinement based on analytical and early clinical performance data.

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

Similarly, for the purposes of algorithm development and initial melt range establishment:

Known Sequence Variations: For mathematical modeling, genetic sequences of relevant organisms and their variants are used, which are inherently "ground truth" derived from genetic sequencing databases.
Known Isolates: Lab-characterized organisms with confirmed identity and concentration are used.
Early Clinical Specimens: For these, the ground truth would have been established using comparator laboratory methods as described in item 7.

The "final validation of the melt ranges was performed and included review of data from the Inclusivity study and clinical studies," indicating that these studies further refined and confirmed the algorithm's parameters against ground truth established by comparator methods and known spiked samples.

Summary

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR FilmArray® Meningitis/Encephalitis (ME) Panel

DECISION SUMMARY

A. DEN Number:

DEN150013

B. Purpose for Submission:

De Novo request for evaluation of automatic class III designation for the FilmArray Meningitis/Encephalitis (ME) Panel

C. Measurands:

The assay detects and identifies nucleic acids of the following organisms: Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis (encapsulated), Streptococcus agalactiae, Streptococcus pneumoniae, Cytomegalovirus, Enterovirus, Herpes simplex virus 1, Herpes simplex virus 2, Human herpesvirus 6, Human parechovirus, Varicella zoster virus, Cryptococcus neoformans/gattii

D. Type of Test:

The FilmArray Meningitis/Encephalitis (ME) Panel ("FilmArray ME Panel"), performed with FilmArray and FilmArray 2.0 systems, is a nucleic acid-based test for the detection of the above listed bacteria, viruses, and yeast from cerebrospinal fluid (CSF) specimens obtained from patients with signs and symptoms of meningitis or encephalitis.

E. Applicant:

BioFire Diagnostics, LLC

F. Proprietary and Established Names:

FilmArray Meningitis/Encephalitis (ME) Panel

G. Regulatory Information:

1. Regulation section:

21 CFR 866.3970, Device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid

2. Classification:

Class II (Special Controls)

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1. Product code(s):
  PLO, OOI, NSU
1. Panel:
  83- Microbiology

H. Intended Use:

1. Intended use(s):
  The FilmArray Meningitis/Encephalitis (ME) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with FilmArray and FilmArray 2.0 systems. The FilmArray ME Panel is capable of simultaneous detection and identification of multiple bacterial, viral, and yeast nucleic acids directly from cerebrospinal fluid (CSF) specimens obtained via lumbar puncture from individuals with signs and/or symptoms of meningitis and/or encephalitis. The following organisms are identified using the FilmArray ME Panel:

Bacteria:

Escherichia coli K1 Haemophilus influenzae Listeria monocytogenes Neisseria meningitidis (encapsulated) Streptococcus agalactiae Streptococcus pneumoniae

Viruses: Cytomegalovirus Enterovirus Herpes simplex virus 1 Herpes simplex virus 2 Human herpesvirus 6 Human parechovirus Varicella zoster virus

Yeast:

Cryptococcus neoformans/gattii

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detected may not be the definite cause of the disease. Negative results do not preclude central nervous system (CNS) infection. Not all agents of CNS infection are detected by this test and sensitivity in clinical use may differ from that described in the package insert.

The FilmArray ME Panel is not intended for testing of specimens collected from indwelling CNS medical devices.

The FilmArray ME Panel is intended to be used in conjunction with standard of care culture for organism recovery, serotyping, and antimicrobial susceptibility testing.

1. Indication(s) for use:
  Same as Intended Use
1. Special conditions for use statement(s):
  For prescription use only.
1. Special instrument requirements:
  The FilmArray ME Panel is performed on the FilmArray and FilmArray 2.0 systems.

I. Device Description:

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. 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 21d 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.

Materials provided in each FilmArray ME Panel kit:

Individually packaged FilmArray ME Panel pouches
Single-use (1.0 mL) Sample Buffer ampoules
Single-use pre-filled (1.5 mL) Hydration Injection Vials ●
Single-use Sample Injection Vials ●
Individually packaged Transfer Pipettes

Materials required but not provided: FilmArray system including:

FilmArray or FilmArray 2.0 instrument and software
FilmArray Pouch Loading Station

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

CLSI EP12-A2, User Protocol for Evaluation of Qualitative Test Performance, 2008
CLSI EP07-A2, Interference Testing in Clinical Chemistry, 2005 ●
CLSI MM03-A2, Molecular Diagnostic Methods for Infectious Diseases, 2006 ●
EN ISO 14971:2012, 'Medical devices – Application of risk management to medical devices'
Guidance for Sponsors, Institutional Review Boards, Clinical Investigators and FDA ● Staff - Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable. April 25, 2005
Guidance for Industry and Food and Drug Administration Staff Assay Migration ● Studies for In Vitro Diagnostic Devices, April 25, 2013
Guidance for Industry and Food and Drug Administration Staff Highly Multiplexed ●

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Microbiological/Medical Countermeasure In Vitro Nucleic Acid Based Diagnostic Devices, August 27, 2014

Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests, ● FDA Guidance Document, March 13, 2007
Class II Special Controls Guidance Document: Nucleic Acid Amplification Assay for the ● Detection of Enterovirus RNA, January 2, 2009

K. Test Principle:

The FilmArray ME Panel pouch) is a closed system disposable that houses all the chemistry required to isolate, amplify and detect nucleic acid from multiple meningitis and encephalitis pathogens within a single CSF specimen obtained from a lumbar puncture. 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 ME Panel loads the sample into the pouch, places the pouch into the FilmArray systems, and starts the run. All other operations are automated. Operations and processes that occur during a FilmArray run include the following:

. Nucleic Acid Purification - b(4)
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 approximately ten minutes and the bead-beater apparatus can be heard as a high-pitched whine during the first minute of operation.
Reverse Transcription and 1st Stage Multiplex PCR Some pathogens identified by . the pouch are RNA viruses, and a reverse transcription (RT) step is performed to convert the viral RNA into cDNA prior to amplification. b(4)
for multiplex PCR. The effect of 1st stage PCR is to enrich for the target nucleic acids present in the sample.
. 2nd Stage PCR - The products of 1st stage PCR are diluted and mixed with fresh PCR reagents containing an intercalating fluorescent DNA dye "" Plus, BioFire Defense, LLC). This solution is distributed over the 2nd stage PCR array. The individual wells of the array contain primers for different assays (each present in triplicate) that target specific nucleic acid sequences from each of the pathogens detected, as well as control template material. These primers are 'nested' or internal to the specific products of the 1st stage multiplex reaction, which enhances both the sensitivity and specificity of the reactions.
DNA Melting Analysis After 2nd stage PCR, the temperature is slowly increased and . fluorescence in each well of the array is monitored and analyzed to generate a melt curve. The temperature at which a specific PCR product melts (melting temperature or Tm) is consistent and predictable and the FilmArray software automatically evaluates the data from replicate wells for each assay to report results.

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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.

L. Performance Characteristics:

1. Analytical performance:

a. Reproducibility

Reproducibility studies were performed with the FilmArray ME Panel on both the FilmArray and FilmArray 2.0 systems. Testing for the FilmArray system was performed using multiple instruments at three different testing sites, Biofire Diagnostics and two external laboratories. Testing for the FilmArray 2.0 system was performed internally at BioFire Diagnostics using multiple instruments at three different locations within BioFire Diagnostics.

Assay reproducibility was evaluated for both the FilmArray and FilmArray 2.0 system using a panel of contrived CSF samples prepared in artificial CSF matrix (aCSF) and spiked with combinations of nine different ME Panel analytes, including at least one representative gram-negative bacterium, gram-positive bacterium, yeast, DNA virus and RNA virus. Each spiked analyte was evaluated at three different concentrations: Negative (no analyte), Low Positive (1× the limit of detection (LoD)) and Moderate Positive (3× LoD). Testing on both FilmArray systems incorporated a range of potential testing variables including different operators, three different pouch lots, and different FilmArray Instruments. Samples were tested on five different days with a total of 90 replicates tested per panel member.

For the FilmArray system. 366 runs were initiated and 360 runs were completed (98.4% initially valid results). Of the six initially invalid runs, one invalid run was due to a control failure and five invalid tests were due to instrument or software errors. Retesting of initially invalid specimens gave valid results for all six specimens. There were seven unexpected false positive results observed in the study: six for Streptococcus pneumoniae (6/360 = 1.7%) and one for Human herpesvirus 6 (HHV-6) (1/360 = 0.3%).

For the FilmArray 2.0 system. 365 runs were initiated and 360 runs were completed (98.6% initially valid results). Of the five initially invalid runs, three runs were invalid because they were aborted by the operator due to being run on an incorrect instrument. The two other invalid results were due to a software error (1/365 = 0.3%) and due to an incomplete result caused by a data transfer error (1/365 = 0.3%).

A summary of qualitative results from both reproducibility studies (percent agreement with the expected result) for each analyte and organism concentration is provided in the following tables.

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Reproducibility of the FilmArray ME Panel on FilmArray

Organism/IsolateTested	Concentration	ExpectedTest Result	FilmArray Agreement with Expected Results			All Sites(95%ConfidenceInterval)
			Site A	Site B	Site C
	BACTERIA
E. coli K1b(4)	Moderate Positive3× LoD3×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
E. coli K1b(4)	Low Positive1× LoD1×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/9096.7%(96%-100%)
E. coli K1b(4)	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
H. influenzaeb(4)	Moderate Positive3× LoD3×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
H. influenzaeb(4)	Low Positive1× LoD1×103 CFU/mL	Detected	29/3096.7%	30/30100%	30/30100%	89/9098.9%(94.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
L. monocytogenesb(4)	Moderate Positive3× LoD3×103 CFU/mL	Detected	29/3096.7%	30/30100%	30/30100%	89/9098.9%(94.0%-100%)
	Low Positive1× LoD1×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
N. meningitidis	Negative(No analyte)	Not Detected	120/120100%	120/120100%	120/120100%	360/360100%(99.0%-100%)
S. agalactiaeb(4)	Moderate Positive3× LoD3×103 CFU/mL	Detected	29/3096.7%	30/30100%	27/3090.0%	86/9095.6%(89.0%-98.8%)
	Low Positive1× LoD1×103 CFU/mL	Detected	26/3086.7%	30/30100%	27/3090.0%	83/9092.2%(84.6%-96.8%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
S. pneumoniae	Negative(No analyte)	Not Detected	118/12098.3%	118/12098.3%	118/12098.3%	354/360a98.3%(96.4%-99.4%)
VIRUSES/YEAST
CMV	Negative(No analyte)	Not Detected	120/120100%	120/120100%	120/120100%	360/360100%(99.0%-100%)
	Concentration	ExpectedTest Result	FilmArray Agreement with Expected Results			All Sites(95%ConfidenceInterval)
Organism/IsolateTested			Site A	Site B	Site C
EV	Moderate Positive$3\times LoD$15 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Coxsackievirus A9b(4)	Low Positive$1\times LoD$5 TCID50/mL	Detected	30/30100%	30/30100%	28/3093.3%	88/9097.8%(92.2%-99.7%)
			Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%
HSV-1			Negative(No analyte)	Not Detected	120/120100%	120/120100%	120/120100%
	Moderate Positive$3\times LoD$150 TCID50/mL		Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	HSV-2b(4)	Low Positive$1\times LoD$50 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
		Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
HHV-6		Negative(No analyte)	Not Detected	120/120100%	119/12099.2%	120/120100%	359/36099.7%(98.5%-100%)
	Moderate Positive$3\times LoD$$1.5\times 10^3$TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	HPeVb(4)	Low Positive$1\times LoD$500 TCID50/mL	Detected	29/3096.7%	30/30100%	30/30100%	89/9098.9%(94.0%-100%)
		Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
VZVb(4)		Moderate Positive$3\times LoD$0.3 TCID50/mL	Detected	29/3096.7%	30/30100%	30/30100%	89/9098.9%(94.0%-100%)
	Low Positive$1\times LoD$0.1 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
C. gattiib(4)	Moderate Positive$3\times 10^3$ CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Low Positive$1\times 10^3$ CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Organism/IsolateTested	Concentration	ExpectedTest Result	FilmArray Agreement with Expected Results
Site A				Site B	Site C	All Sites(95%ConfidenceInterval)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)

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4Six false positive S. pneumoniae results were reported. The unexpected results were observed at all three test sites, in different samples, on different days, and with different pouch lots. The overall incidence of false S. pneumoniae results observed in all

reproducibility testing was <1%.

C. gattii was tested at concentrations equivalent to 10× and 30× LoD for the combined Cryptococcus neoformans/gattii test result on the FilmArray.

Reproducibility of the FilmArray ME Panel on FilmArray 2.0

Organism/IsolateTested	Concentration	ExpectedTest Result	FilmArray 2.0 Agreement with Expected Results			All Systems(95% ConfidenceInterval)
			System A	System B	System C
BACTERIA
E. coli K1b(4)	Moderate Positive3× LoD3×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Low Positive1× LoD1×103 CFU/mL	Detected	29/3096.7%	29/3096.7%	29/3096.7%	87/9096.7%(90.6%-99.3%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
H. influenzaeb(4)	Moderate Positive3× LoD3×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Low Positive1× LoD1×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
L. monocytogenesb(4)	Moderate Positive3× LoD3×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Low Positive1× LoD1×103 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
N. meningitidis	Negative(No analyte)	Not Detected	120/120100%	120/120100%	120/120100%	360/360100%(99.0%-100%)
S. agalactiaeb(4)	Moderate Positive3× LoD3×103 CFU/mL	Detected	29/3096.7%	30/30100%	29/3096.7%	88/9097.8%(92.2%-99.7%)
Organism/Isolate	Concentration	ExpectedTest Result	FilmArray 2.0 Agreement with Expected Results
Tested			System A	System B	System C	All Systems(95% ConfidenceInterval)
	Low Positive1× LoD1×103 CFU/mL	Detected	29/3096.7%	29/3096.7%	30/30100%	88/9097.8%(92.2%-99.7%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
S. pneumoniae	Negative(No analyte)	Not Detected	120/120100%	120/120100%	120/120100%	360/360100%(99.0%-100%)
			VIRUSES/YEAST
CMV	Negative(No analyte)	Not Detected	120/120100%	120/120100%	120/120100%	360/360100%(99.0%-100%)
EV	Moderate Positive3× LoD15 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
Coxsackievirus A9b(4)	Low Positive1× LoD5 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
HSV-I	Negative(No analyte)	Not Detected	120/120100%	120/120100%	120/120100%	360/360100%(99.0%-100%)
	Moderate Positive3× LoD150 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
HSV-2b(4)	Low Positive1× LoD50 TCID50/mL	Detected	30/30100%	30/30100%	29/3096.7%	89/9098.9%(94.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
HHV-6	Negative(No analyte)	Not Detected	120/120100%	120/120100%	120/120100%	360/360100%(99.0%-100%)
	Moderate Positive3× LoD1.5×103TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
HPeVb(4)	Low Positive1× LoD500 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
VZVb(4)	Moderate Positive3× LoD0.3 TCID50/mL	Detected	30/30100%	30/30100%	29/3096.7%	89/9098.9%(94.0%-100%)
Organism/IsolateTested	Concentration	ExpectedTest Result	FilmArray 2.0 Agreement with Expected Results
			System A	System B	System C	All Systems(95% ConfidenceInterval)
	Low Positive1× LoD0.1 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100%	180/180100%(98.0%-100%)
C. gattiib(4)	Moderate Positive3× LoD300 CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%(96.0%-100%)
	Low Positive1× LoD100 CFU/mL	Detected	30/30100%	30/30100%	28/3093.3%	88/9097.8%(92.2%-99.7%)
	Negative(No analyte)	Not Detected	60/60100%	60/60100%	60/60100 %	180/180100%(98.0%-100%)

{9}------------------------------------------------

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

The following table includes analyses of assay reproducibility by analyte and panel member based on the Tm for each positive result observed during both the FilmArray system and FilmArray 2.0 system Reproducibility studies. Results demonstrated similar performance for both instruments with Tm standard deviations of ≤ 0.5℃ for all analytes and concentrations evaluated.

Reproducibility of Tm values for Positive FilmArray ME Assays on FilmArray and FilmArray 2.0 systems

	Assay	Concentration Tested(× LoD)	Mean Tm (°C) (±StDev)
Test Result(Organism/Isolate Tested)			FilmArray			FilmArray 2.0
			Site A	Site B	Site C	All Sites	System A	System B	System C	All Systems
	yeastRNA		82.4	82.5	82.0	82.3	81.7	81.9	81.7	81.8
RNA Process Control			±0.2	±0.3	±0.2	±0.3	±0.3	±0.3	±0.3	±0.3
	PCR2		76.5	76.6	76.1	76.4	75.5	75.7	75.5	75.6
PCR2 Control			±0.1	±0.3	±0.2	±0.3	±0.3	±0.3	±0.3	±0.3
BACTERIA
	Ecoli 3	3× LoD	82.9	82.8	82.6	82.8	82.1	82.3	82.1	82.2
E. coli K1b(4)			±0.1	±0.3	±0.1	±0.3	±0.3	±0.3	±0.3	±0.3
		1× LoD	83.0	83.1	82.6	82.9	82.2	82.4	82.3	82.3
			±0.1	±0.3	±0.2	±0.3	±0.3	±0.3	±0.3	±0.3
	Hinfluenzae 1	3× LoD	78.6	78.6	78.2	78.5	77.9	77.9	77.7	77.8
			±0.1	±0.4	±0.2	±0.4	±0.3	±0.3	±0.2	±0.3
		1× LoD	78.6	78.7	78.3	78.5	78.0	78.0	77.9	78.0
H. influenzaeb(4)b(4)			±0.2	±0.3	±0.2	±0.3	±0.3	±0.3	±0.3	±0.3
	Hinfluenzae 2	3× LoD	82.0	81.9	81.6	81.8	81.0	81.1	81.0	81.0
			±0.1	±0.4	±0.2	±0.3	±0.2	±0.2	±0.2	±0.2
		1× LoD	82.0	82.0	81.7	81.9	81.2	81.3	81.2	81.2
			±0.1	±0.3	±0.2	±0.3	±0.2	±0.2	±0.3	±0.2
	Lmonocytogenes	3× LoD	80.4	80.6	80.2	80.4	80.0	80.1	80.0	80.0
L. monocytogenes (b(4)b(4)			±0.2	±0.3	±0.2	±0.3	±0.4	±0.3	±0.3	±0.3
		1× LoD	80.5	80.6	80.1	80.4	80.0	80.1	79.9	80.0
			±0.2	±0.4	±0.2	±0.4	±0.4	±0.3	±0.2	±0.3
	Sagalactiae	3× LoD	82.0	82.0	81.6	81.9	81.1	81.3	81.2	81.2
S. agalactiaeb(4)b(4)			±0.1	±0.3	±0.2	±0.3	±0.3	±0.3	±0.2	±0.3
		1× LoD	82.1	82.2	81.7	82.0	81.2	81.4	81.2	81.3

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	Assay	Concentration Tested(× LoD)	Mean Tm (°C) (±StDev)
Test Result(Organism/Isolate Tested)			FilmArray			FilmArray 2.0
			Site A	Site B	Site C	All Sites	System A	System B	System C	All Systems
			±0.1	±0.3	±0.2	±0.3	±0.3	±0.2	±0.2	±0.3
VIRUSES
EV(Coxsackievirus A9)b(4)	EV2	3× LoD	89.7	89.7	89.3	89.6	89.1	89.3	89.2	89.2
			±0.1	±0.3	±0.2	±0.3	±0.3	±0.3	±0.3	±0.3
		1× LoD	89.7	89.7	89.3	89.6	89.1	89.3	89.2	89.2
b(4)			±0.1	±0.4	±0.2	±0.3	±0.3	±0.3	±0.2	±0.3
		3× LoD	75.6	75.5	75.1	75.4	74.6	74.8	74.6	74.7
			±0.3	±0.3	±0.3	±0.3	±0.3	±0.3	±0.3	±0.3
	HSV2 1	1× LoD	75.9	76.0	75.4	75.7	74.9	75.1	74.9	75.0
HSV-2			±0.2	±0.2	±0.3	±0.4	±0.3	±0.3	±0.3	±0.3
b(4)	HSV2 2	3× LoD	88.8	88.9	88.4	88.7	88.1	88.3	88.2	88.2
			±0.2	±0.4	±0.2	±0.3	±0.3	±0.3	±0.2	±0.3
		1× LoD	88.9	89.0	88.5	88.8	88.2	88.5	88.3	88.3
			±0.2	±0.3	±0.2	±0.4	±0.3	±0.3	±0.2	±0.3
HPeVb(4)	HPeV	3× LoD	82.8	82.8	82.5	82.7	82.2	82.3	82.1	82.2
			±0.2	±0.4	±0.2	±0.3	±0.3	±0.3	±0.2	±0.3
		1× LoD	82.8	82.9	82.5	82.7	82.3	82.3	82.2	82.3
			±0.2	±0.3	±0.2	±0.3	±0.3	±0.2	±0.3	±0.3
	VZV 1	3× LoD	88.9	89.0	88.5	88.8	88.5	88.5	88.4	88.5
			±0.2	±0.4	±0.2	±0.4	±0.4	±0.2	±0.2	±0.3
		1× LoD	89.0	89.0	88.5	88.8	88.5	88.5	88.3	88.4
VZVb(4)			±0.2	±0.5	±0.2	±0.4	±0.3	±0.3	±0.2	±0.3
	VZV 2	3× LoD	82.0	82.1	81.7	81.9	81.5	81.5	81.4	81.5
			±0.1	±0.3	±0.2	±0.3	±0.3	±0.2	±0.2	±0.2
		1× LoD	82.1	82.1	81.7	82.0	81.5	81.5	81.4	81.5
			±0.1	±0.4	±0.2	±0.3	±0.3	±0.2	±0.2	±0.2
YEAST
C. neoformans/gattii(C. gattii)(b(4)	Cryptococcus	30× LoDa (FilmArray)	82.0	82.0	81.6	81.8	81.2	81.4	81.3	81.3
		3× LoD (FilmArray 2.0)	±0.1	±0.3	±0.2	±0.3	±0.3	±0.3	±0.3	±0.3
		10× LoDa (FilmArray)	82.0	82.1	81.6	81.9	81.3	81.5	81.3	81.4
		1× LoD (FilmArray 2.0)	±0.2	±0.2	±0.2	±0.3	±0.3	±0.4	±0.2	±0.3

9 C. gattii was tested at concentrations equivalent to 10× and 30× LoD for the combined Cryptococcus neoformans/gattii test result on the FilmArray system, while C. gattii was tested at the intended 1× and 3× LoD concentrations on the FilmArray 2.0 system.

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 ME Panel pouch:

RNA Process Control: The RNA Process Control assay targets an RNA transcript ● from the yeast Schizosaccharomyces pombe. b(4)
. 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 ME Panel pouch were successful.

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

. PCR2 Control: The PCR2 Control assay detects a DNA target b(4) . A positive result
indicates that the 2nd stage PCR was successful.

Both internal control assays must be positive for the test run to pass. When 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 user is instructed to repeat the test using a new pouch.

Of the six pouch control failures observed in the prospective clinical study, five were attributed to both RNA Process Control and PCR 2 Control failures and one was attributed to RNA Process Control failure only.

Recommended External Controls:

External controls are not provided with the FilmArray ME Panel, but are recommended in the package insert. Molecular grade water or artificial CSF can be used as an external negative control. Previously characterized CSF specimens or negative matrix spiked with well-characterized organisms can be used as external positive controls. External controls should be used in accordance with the appropriate accrediting organization requirements, as applicable.

d. Detection Limit:

Evaluation of the Limit of Detection:

The limit of detection (LoD) for FilmArray ME Panel analytes was estimated by testing dilutions of contrived samples containing known concentrations of bacteria, viruses or yeast detected by the FilmArray ME Panel. Representative strains were chosen in order to obtain positive results for every assay on the panel and multiple strains were evaluated to cover clinically important species or variants for some analytes.

Samples for LoD testing were prepared in aCSF (artificial CSF obtained from a commercial provider) matrix and contained one or up to five targeted organisms.

b(4)
		. b(4)



		The LoD was confirmed for all
		1

analytes on both FilmArray and FilmArray 2.0 systems. Data presented in the following table are from LoD confirmation testing on the FilmArray system.

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

ME Panel TestResult	Species/Isolate Tested	LoD Concentration	Detection atLoDConcentration
	BACTERIA
E. coli K1	E. coli K1, strain C5 b(4)b(4)	1×103 CFU/mL	20/20100%
H. influenzae	H. influenzae, strain AMC 36-A-1b(4)	1×103 CFU/mL	20/20100%
L. monocytogenes	L. monocytogenes, strain 1071/53,b(4)	1×103 CFU/mL	20/20100%
N. meningitidis	N. meningitidis, strain M-1574b(4)	100 CFU/mL	19/2095%
S. agalactiae	S. agalactiae, type strain, G19,group Bb(4)	1×103 CFU/mL	20/20100%
S. pneumoniae	S. pneumoniae, strain SV 1,serotype 1b(4)	100 cells/mL	19/2095%
	VIRUSES
CMV	CMV, strain AD-169b(4)	100 TCID50/mL(4.30×103 copies/mL)	20/20100%
EV(Species A-D)	Coxsackievirus A6, species A,b(4)	50 TCID50/mL	20/20100%
	Coxsackievirus A9, species Bb(4)	5 TCID50/mL	20/20100%
	Coxsackievirus A17, species C,strain G-12b(4)	5 TCID50/mL	20/20100%
	EV 70, species D, b(4)b(4)	50 TCID50/mL	20/20100%
HSV-1	HSV-1, strain MacIntyreb(4)	250 TCID50/mL(1.51×103 copies/mL)	20/20100%
HSV-2	HSV-2, strain MSb(4)	50 TCID50/mL(1.29×103 copies/mL)	20/20100%
HHV-6	HHV-6A, strain U1102b(4)	1×104 copies/mL	19/2095%
	HHV-6B, strain HSTb(4)	1×104 copies/mL	19/2095%
HPeV	HPeV, type 3b(4)	500 TCID50/mL	19/2095%
VZV	VZV, strain Ellenb(4)	0.10 TCID50/mL(1.66×103 copies/mL)	20/20100%
	YEAST

Limit of Detection Results for FilmArray ME Panel Analytes

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

ME Panel TestResult	Species/Isolate Tested	LoD Concentration	Detection atLoD Concentration
C.neoformans/gattii	C. neoformans var. grubii, typeb(4)	100 CFU/mL	20/20100%
	C. gattii, strain A6MR38,b(4)	100 CFU/mL	20/20100%

Evaluation of Effect on Analyte LoDs in Multi-Spiked Samples:

A study was performed to determine the applicability of evaluating multiple analytes in a single CSF sample during the analytical studies and evaluated for potential loss of detection when multiple targets are present in clinical specimens (co-infections). Samples consisting of three different organism mixes with up to five targeted organisms each were compared to contrived single-spike samples for the following representative panel analytes: bacterium (E. coli K1), yeast (C. neoformans), DNA virus (HSV-1), and RNA virus (HPeV). All samples were prepared in aCSF matrix. Serial dilutions were prepared for both single-spike samples and multiple spiked samples containing the corresponding analyte for comparison.

For E. coli K1 and HSV-1 samples prepared with analyte concentrations at LoD, 4/4 replicates were positive for both single and multi-spiked samples. Dilutions with concentrations below LoD showed a loss of detection between the single-spiked and multi-spiked specimens at the same organism concentration, thus demonstrating no difference in assay detection for E. coli K1 and HSV-1 for single versus multi-spiked samples.

For C. neoformans, 4/4 samples were positive at both 1x LoD and 0.1× LoD, At 0.01× LoD, 4/4 were positive for single-spiked samples and 1/4 replicates were positive for multi-spiked samples. To evaluate whether the observed difference in detection was due to multi-spiking versus single-spiking, mean PCR crossing point (Cp) values were evaluated for each concentration evaluated. The analysis showed that mean Cp values for each concentration tested showed no trend toward lower or higher Cp values between single and multi-spiked samples.

For HPeV at 1× LoD. 3/4 replicates were detected for multi-spiked samples and 4/4 replicates were detected for single-spiked samples. However, at two different dilutions below the LoD, there was no trend in detection differences between single and multispiked samples.

In summary, testing of single versus multi-spiked samples did not show a significant difference in assay performance for the four representative analytes evaluated. The study results supported the use of multi-spiked samples in various analytical studies.

e. Analytical Reactivity (Inclusivity):

Inclusivity testing was performed using contrived samples consisting of 96 isolates

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

spiked into artificial CSF (aCSF) sample matrix at a concentration near the LoD for each analyte (1× to 3× LoD). Strains were selected to represent relevant species, subspecies, or serotypes. For any isolate that was not detected at the initial test concentration, results were reviewed and if reactivity was expected, the isolate was retested at the same concentration up to 5 times. If needed, retesting of the isolate was also performed at a higher concentration (typically 10× LoD) and a detected result was a demonstration of reactivity with the isolate at the elevated concentration.

The FilmArray ME panel gave positive results for the majority of isolates evaluated when tested at sample concentrations near the LoD (1× to 3× LoD). One strain of HpeV (Serotype 5) was detected at 10× LoD as compared to the HpeV strain evaluated in the LoD study.

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 evaluated with empirical testing.

The following tables includes a summary of ME Panel reactivity based on empirical data with footnotes describing predictions of assay reactivity based on in silico analysis.

FilmArray MEPanelTest Result	# of Isolates Testedand Detected	ConcentrationDetected	Isolates Tested and Detected
Bacteria
E. coli K1	5	1,000 - 3,000 CFU/mL	E. coli strains of the K1 serotype only
H. influenzae	9	1,000 - 3,000 CFU/mL	Non-typeable and typeable (types a-f) strainsof H. influenzae
L. monocytogenes	6	1,000 - 3,000 CFU/mL	Types 1/2a, 1/2b, and 4b of L. monocytogenes a
N. meningitidis	7	100 - 300 CFU/mL	Encapsulated N. meningitidis (serotypesW135, A, B, C, D, Y and DNA from a strainswith a variant ctrA gene)
S. agalactiae	5	1,000 - 3,000 CFU/mL	Multiple serotypes or isolates of S. agalactiae(Group B Streptococcus )
S. pneumoniae	6	100 - 300 cells/mL	Multiple serotypes of S. pneumoniae
Viruses
CMV	5	100 - 300 TCID50/mL(4.3×103 - 1.3×104 copies/mL)	Multiple strains of Cytomegalovirus (CMV).
EV	18	5 - 50 TCID50/mL	Representative isolates from all species (A-D)and several serotypes of human Enterovirus,Coxsackievirus, and Echovirusb
HSV-1	5	250 - 750 TCID50/mL(1.5×103 - 4.5×103 copies/mL)	Multiple strains of Herpes simplex virus 1(HSV-1)
HSV-2	5	50 - 150 TCID50/mL(1.3×103 - 3.9×103 copies/mL)	Multiple strains of Herpes simplex virus 2(HSV-2)
HHV-6	4	1×104 - 3×104 copies/mL	A and B variants of Human herpesvirus 6(HHV-6)

Summary of FilmArray ME Panel Analytical Reactivity (Inclusivity)

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

FilmArray MEPanelTest Result	# of Isolates Testedand Detected	ConcentrationDetected	Isolates Tested and Detected
HPeV	6	500 - 5,000 TCID50/mL	Serotypes 1-6 of Human parechovirus(HPeV)c
VZV	5	0.1 - 0.3 TCID50/mL(1.7×103-5×103 copies/mL)	Multiple strains of Varicella zoster virus(VZV)
Yeast
C. neoformans/gattii	10(5 per species)	100 - 300 CFU/mL	Multiple strains, serotypes, and genotypes ofCryptococcus neoformans and Cryptococcusgattii

ª In silco analysis of available sequences predicts that the FilmArray ME Panel with all currently characterized strains and

serotypes of L. monocytogenes.
1 1200 condysis of available sequences predicts that the FilmArray ME Panel will react with all currently characterized serotypes (>100) of human enteroviruses (including enteroviruses, coxsackieviruses, and echoviruses).

6 Based on sequence analysis, the FilmArray ME Panel is also predicted to react with HPeV serotypes 7 and 8. No sequence data were available for predicting reactivity with other serotypes.

The following tables include specific strains evaluated with the FilmArray ME Panel.

Results for Escherichia coli K1 Inclusivity Testing

Organism	Isolate ID	Serotype[Strain-Year Isolated]	ConcentrationTested	TestResult
Escherichia coli K1	b(4)	Serotype O18ac:K1:H7[Strain C5 [Bort]-1975]	$1\times10^3$ CFU/mL(1x LoD)	Detected
		Serotype O2:K1:H4[Strain U9-41]	$3\times10^3$ CFU/mL	Detected
		Serotype O16:K1:H-[Strain F11119-41-1952]	$3\times10^3$ CFU/mL	Detected
		Serotype O9:K1:H-[Strain Bi 7509/41-1952]	$3\times10^3$ CFU/mL	Detected
		Serotype O45:K1:H10[Strain H61-1952]	$3\times10^3$ CFU/mL	Detected

Results for Haemophilus influenzae Inclusivity Testing
--------------------------------------------------------	--	--	--

		Type	Concentration	Test
Organism	Isolate ID	[Strain]	Tested	Result
	b(4)	Non-typeable[strain Rd [KW20]]	$3 × 10^3$ CFU/mL	Detected
		Non-typeablebiogroup aegyptius[type strain, 180-a]	$3 × 10^3$ CFU/mL	Detected
Haemophilus		Type a[strain AMC 36-A-3]	$3 × 10^3$ CFU/mL	Detected
influenzae		Type b[strain Rab]	$3 × 10^3$ CFU/mL	Detected
		Type b[biotype 1]	$1 × 10^3$ CFU/mL(1× LoD)	Detected
		Type c[strain C 9007]	$3 × 10^3$ CFU/mL	Detected

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

Isolate ID	Type[Strain]	ConcentrationTested	TestResult
b(4)	Type d[strain AMC 36-A-6]	$3 × 10^3$ CFU/mL	Detected
	Type e[strain AMC 36-A-7]	$3 × 10^3$ CFU/mL	Detected
	Type f[strain GA-1264]	$3 × 10^3$ CFU/mL	Detected

	Results for Listeria monocytogenes Inclusivity Testing

Organism	Source	Type	ConcentrationTested	TestResult
Listeriamonocytogenes	b(4)	Type 1/2a	$3 \times 10^3$ CFU/mL	Detected
		Type 1/2a	$3 \times 10^3$ CFU/mL	Detected
		Type 1/2b	$3 \times 10^3$ CFU/mL	Detected
		Type 1/2b	$3 \times 10^3$ CFU/mL	Detected
		Type 4b	$3 \times 10^3$ CFU/mL	Detected
		Type 4b	$1 \times 10^3$ CFU/mL(1 × LoD)	Detected

Note: At least 12 serotypes of L. monocytogenes have been recognized (i.e., 1/2a, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e and 7) based on somatic (O) and flagellar (H) antigens, but more than 90% of human isolates belong to only three serotypes: 1/2a, 1/2b, and 4b. In silico analysis based on available sequence data suggest that all serotypes are expected to be amplified by the assay and detected by the FilmArray ME Panel.

Organism	Serotype	ConcentrationTested	TestResult
	Serotype W135	100 CFU/mL(1× LoD)[~1.9×103copies/mL]	Detected
	Serotype A	300 CFU/mL	Detected
Neisseria meningitidis(Encapsulated)	Serotype B	300 CFU/mL	Detected
	Serotype C	300 CFU/mL	Detected
	Serotype D	300 CFU/mL	Detected
	Serotype Y	300 CFU/mL	Detected
	DNA with variantctrA gene	5.6×103 copies/mL(~3× LoD)	Detected

Results for Neisseria meningitidis Inclusivity Testing

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Organism	Isolate ID	Serotype	ConcentrationTested	TestResult
Streptococcusagalactiae	b(4)	Serotype I a/cType strain	$1 \times 10^3$ CFU/mL(1× LoD)	Detected
		Serotype III	$3 \times 10^3$ CFU/mL	Detected
		Serotype V	$3 \times 10^3$ CFU/mL	Detected
		Unknown	$3 \times 10^3$ CFU/mL	Detected
		Unknown	$3 \times 10^3$ CFU/mL	Detected

Results for Streptococcus agalactiae Inclusivity Testing

Note: In silico analysis predicts that the FilmArray ME panel should detect S. agalactiae serotypes Ia, Ia/c, Ib, II, III, V and VIII strains.

Organism	Isolate ID	Serotype	ConcentrationTested	Test Result
	b(4)	Serotype 1	100 cells/mL(1× LoD)	Detected
	b(4)	Serotype 4	300 cells/mL	Detected
Streptococcus pneumoniae	b(4)	Serotype 5	300 cells/mL	Detected
	b(4)	Serotype 11A	300 cells/mL	Detected
	b(4)	Serotype 14	300 cells/mL	Detected
	b(4)	Serotype 19A	300 cells/mL	Detected

Results for Streptococcus pneumoniae Inclusivity Testing

Note: Based on the serotype information associated with sequences available in public databases, in silico analysis indicates the assay will react with all serotypes of S. pneumoniae, including those covered by the PPSV23 pneumococcal vaccine (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F).

Organism	Isolate ID	Strain	Location/Year Isolated	ConcentrationTested	TestResult
Cytomegalovirus (CMV)[Human Herpesvirus 5]	b(4)	AD-169	unknown	100 TCID50/mL[ $4.3 \times 10^3$ copies/mL](1 $\times$ LoD)	Detected
		Towne	Unknown	$1.3 \times 10^4$ copies/mL	Detected
		Merlin	Wales, 2003	$1.3 \times 10^4$ copies/mL	Detected

Results for Cytomegalovirus (CMV) Inclusivity Testing

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Organism	Isolate ID	Strain	Location/Year Isolated	ConcentrationTested	TestResult
	b(4)	Davis	1957	$1.3 \times 10^4$ copies/mL	Detected
		Toledo	Virginia, USA2011	$1.3 \times 10^4$ copies/mL	Detected

Results for Enterovirus (EV) Inclusivity Testing

Species	Virus/Serotype	Strain Location/Year	Concentration Tested	Test Result
A	Coxsackievirus A6	Gdulab(4)	50 TCID50/mL(1 × LoD)	Detected
	Coxsackievirus A10	M.K. (Kowalik)b(4)	150 TCID50/mL	Detected
	Coxsackievirus A16	H07314 334	50 TCID50/mL	Detected
	Enterovirus 71	BrCr	150 TCID50/mL	Detected
			50 TCID50/mL	Detected
	Coxsackievirus A9	-	5 TCID50/mL(1 × LoD)	Detected
B	Coxsackievirus B1	H07248 372	15 TCID50/mL	Not Detected
			50 TCID50/mL	Detected
	Coxsackievirus B2	H08084 367	15 TCID50/mL	Not Detected
			50 TCID50/mL	Detected
	Coxsackievirus B3	-	15 TCID50/mL	Detected
	Coxsackievirus B4	-	15 TCID50/mL	Detected
			15 TCID50/mL	Not Detected
	Coxsackievirus B5	-	50 TCID50/mL	Detected
	Echovirus 6	-	15 TCID50/mL	Not Detected
			50 TCID50/mL	Detected
	Echovirus 9	-	15 TCID50/mL	Detected
	Echovirus 18	H07218 472	15 TCID50/mL	Not Detected
			50 TCID50/mL	Detected
C	Coxsackievirus A17	G-12b(4)	5 TCID50/mL(1 × LoD)	Detected
	Coxsackievirus A21	Kuykendallb(4)	15 TCID50/mL	Detected
	Coxsackievirus A24	DN-19b(4)	15 TCID50/mL	Detected
D	Enterovirus 70	J670/71b(4)	50 TCID50/mL(1 × LoD)	Detected
	Enterovirus 68(aka Rhinovirus 87)	-	150 TCID50/mL	Detected
			50 TCID50/mL	Detected

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Note: In silico analysis of available sequences predicts that the FilmArray ME Panel will react with all currently characterized serotypes (>100) of human enteroviruses (including enteroviruses, coxsackieviruses, and echoviruses).

Organism	Strain	ConcentrationTested	TestResult
	MacIntyre	250 TCID50/mL[1.5×105 copies/mL](1× LoD)	Detected
	F	4.5×103 copies/mL	Detected
Herpes simplex virus 1(HSV-1)	HF	4.5×103 copies/mL	Detected
	KOS	4.5×103 copies/mL	Detected
	-2011-1b(4)	4.5×103 copies/mL	Detected

Results for Herpes Simplex Virus 1 (HSV-1) Inclusivity Testing

Results for Herpes Simplex Virus 2 (HSV-2) Inclusivity Testing

Organism	Isolate ID	Strain	ConcentrationTested	TestResult
Herpes simplexvirus 2 (HSV-2)	b(4)	MS	50 TCID50/mL[1.3×10³ copies/mL](1× LoD)	Detected
	b(4)	G	3.9×10³ copies/mL	Detected
	b(4)	-2011-2	3.9×10³ copies/mL	Detected
	131596		3.9×10³ copies/mL	Detected
	HG52		3.9×10³ copies/mL	Detected

Results for Human Herpesvirus 6 (HHV-6) Inclusivity Testing

Organism	Isolate ID	Strain	ConcentrationTested	Test Result
Human Herpesvirus 6A	b(4)	U1102	$1\times10^4$ copies/mL(1× LoD)	Detected
		HST	$1\times10^4$ copies/mL(1× LoD)	Detected
Human Herpesvirus 6B	b(4)	SF	$3\times10^4$ copies/mL	Detected
		Z29	$3\times10^4$ copies/mL	Detected

Results for Human Parechovirus (HPeV) Inclusivity Testing

Organism		Serotype	ConcentrationTested	TestResult
Human Parechovirus(HPeV)	b(4)	Serotype 1	$1.5\times10^3$ TCID50/mL	Detected
		Serotype 2	$1.5\times10^3$ TCID50/mL	Detected

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Isolate ID	Serotype	ConcentrationTested	TestResult
b(4)	Serotype 3	500 TCID50/mL(1× LoD)	Detected
b(4)	Serotype 4	$1.5×10^3$ TCID50/mL	Detected
b(4)	Serotype 5	$1.5×10^3$ TCID50/mL	Not Detected
b(4)	Serotype 5	$5×10^3$ TCID50/mL(10× LoD)	Detected
b(4)	Serotype 6	$1.5×10^3$ TCID50/mL	Detected

Note: In silico analysis predicts detection of all serotypes of HPeV for which there are available sequences in the database (serotypes 1-8).

Results for Varicella Zoster Virus (VZV) Inclusivity Testing

Organism	Isolate ID	Strain	Concentration Tested	Test Result
Varicella Zoster Virus(Human Herpesvirus 3)	b(4)	EllenVR-1367	0.10 TCID50/mL$[1.7\times10^3 copies/mL]$(1× LoD)	Detected
	b(4)	Isolate A	$5\times10^3$ copies/mL	Detected
	b(4)	Isolate B	$5\times10^3$ copies/mL	Detected
	b(4)	Strain 275	$5\times10^3$ copies/mL	Detected
	b(4)	Webster	$5\times10^3$ copies/mL	Detected

Results for Cryptococcus neoformans/gattii Inclusivity Testing

Organism	Isolate ID	Serotype/Strain Info	Isolate Location	ConcentrationTested	Test Result
Cryptococcusneoformans	b(4)	type strain, CBS 132	b(4)	300 CFU/mL	Detected
		Serotype A, strain H99type strain of var. grubii		100 CFU/mL(1 × LoD)	Detected
		Serotype Astrain WM148, type VNI		300 CFU/mL	Detected
		Serotype ADstrain WM628, type VNIII		300 CFU/mL	Detected
		Serotype Dstrain WM629, type VNIV		300 CFU/mL	Detected
Cryptococcusgattii		Serotype Bstrain WM179, type VGI		300 CFU/mL	Detected
		Serotype Bstrain R272, type VGIIb		300 CFU/mL	Detected
		Unknown serotypestrain R38, type VGIIc		100 CFU/mL(1 × LoD)	Detected
		Serotype Bstrain WM161, type VGIII		300 CFU/mL	Detected

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Organism	Isolate ID	Serotype/Strain Info	Isolate Location	ConcentrationTested	Test Result
	b(4)	Serotype C	b(4)	300 CFU/mL	Detected
	b(4)	strain WM779, type VGIV	a

f. Competitive Inhibition/Microbial Interference Studies:

Potentially competing or interfering viruses and other microorganisms were evaluated for their effect on FilmArray ME Panel performance.

To evaluate the potential for competitive inhibition between on-panel analytes (detected by FilmArray ME Panel), two sample mixes were prepared in aCSF matrix using 10 representative FilmArray ME Panel organisms at concentrations equivalent to approximately 3× LoD for each analyte. Samples were co-spiked with high concentrations of four representative ME Panel organisms (E. coli, Coxackievirus A9, HSV-1, and C. neoformans).

To evaluate the potential for interference from off-panel organisms (not detected by the FilmArray ME Panel), the same two sample mixes comprised of 10 FilmArray ME organisms spiked at approximately 3× LoD were co-spiked with high concentrations of off-panel organisms.

All FilmArray ME analytes were detected as expected and therefore the study results demonstrated no competitive inhibition or microbial interference from high concentrations of on-panel or off-panel organisms. The organisms evaluated for potential inhibition/interference are presented in the following table.

Competitive Inhibition	Concentration Tested	Results
Escherichia coli (K1)	$1.02\times10^8$ CFU/mL	No Inhibition/Interference
Coxsackievirus A9 (Enterovirus)	$2.19\times10^5$ TCID50/mL	No Inhibition/Interference
Herpes simplex virus 1	$1.95\times10^6$ TCID50/mL	No Inhibition/Interference
Cryptococcus neoformans	$8.10\times10^5$ CFU/mL	No Inhibition/Interference
Viral or Microbial Interference	Concentration Tested	Results
Epstein-Barr virus	$1.64\times10^9$ TCID50/mL	No Interference
Influenza A H1N1-2009	$2.45\times10^4$ TCID50/mL	No Interference
Proprionibacterium acnes	$1.12\times10^7$ cells/mL	No Interference
Staphylococcus epidermidis	$1.95\times10^7$ CFU/mL	No Interference
Escherichia coli (non-K1)	$1.38\times10^8$ CFU/mL	No Interference
Staphylococcus aureus	$8.55\times10^6$ CFU/mL	No Interference
Candida albicans	$1.01\times10^6$ CFU/mL	No Interference

Organisms Evaluated for Competitive Inhibition/Microbial Interference

g. Analytical Specificity/Cross-reactivity:

Potential cross-reactivity for the FilmArray ME Panel was evaluated by testing high concentrations of organisms in contrived aCSF samples. The strains tested consisted of 19 on-panel (organisms identified by FilmArray ME Panel) and 107 off-panel organisms.

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Samples were prepared in aCSF matrix at the highest concentration possible based on the available material, with most samples containing analyte concentrations of at least 10° CFU/mL for bacteria. 104 units/mL for viruses and 102 CFU/mL for yeast and protists.

No inter-assay cross-reactivity was observed for the 19 on-panel organisms evaluated in the study.

Image /page/23/Figure/2 description: This image shows a table with two columns labeled "Organism" and "Isolate ID". The left side of the table lists several organisms, including Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, Cytomegalovirus, Enterovirus (Coxsackievirus A6), Enterovirus (Coxsackievirus A9), and Enterovirus (Coxsackievirus A17). The right side of the table lists organisms such as Enterovirus (Enterovirus 70), Herpes simplex virus 1, Herpes simplex virus 2, Human herpesvirus 6A, Human herpesvirus 6B, Human parechovirus, Varicella zoster virus, Cryptococcus neoformans, and Cryptococcus gattii.

Image /page/23/Figure/3 description: The image is a title that reads "On Panel Organisms/Viruses Evaluated for Cross-Reactivity". The title is in bold font and is centered on the image. The text is black and the background is white.

Off-panel organisms evaluated in the cross-reactivity study were selected based on their clinical relevance, their likelihood of being present in CSF and/or their genetic similarity to FilmArray ME Panel assay sequences as determined by in silico analysis.

As was also predicted by in silico analysis, study results demonstrated cross-reactivity with H. haemolyticus (positive for H. influenzae) and Rhinoviruses (positive for Enterovirus). Cross-reactivity was also observed with Cryptococcus amylolentus (positive for C. neoformans/gatti), a near-neighbor to Cryptococcus neoformans that does not infect humans. No other cross-reactivity was predicted or observed. The following table lists the off-panel bacteria, viruses, fungi, and protists that were evaluated with the FilmArray ME Panel for this study. Organisms that demonstrated crossreactivity are in bold.

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Gram-positive Bacteria	Gram-negative Bacteria	Viruses	Fungi
Bacillus cereus	Citrobacter freundii	Adenovirus A12	Parainfluenza virus 2
Corynebacterium striatum	Cronobacter sakazakii	Adenovirus C2	Parainfluenza virus 4
Corynebacterium urealyticus	Enterobacter aerogenes	Adenovirus D20	Parvovirus B19
Listeria ivanovii	Enterobacter cloacae	Adenovirus E4	Respiratory SyncytialVirus
Listeria innocua	Escherichia coli (non-K1)	Adenovirus F41	Rotavirus
Mycobacterium tuberculosis	Escherichia fergusonii	BK polyoma virus	Rubella Virus
Proprionibacterium acnes	Escherichia hermanii	Coronavirus 229E	St. Louis EncephalitisVirus
Staphylococcus aureus	Escherichia vulneris	Coronavirus NL63	West Nile Virus
Staphylococcus capitis	Haemophilus ducreyi	Coronavirus OC43	Fungi
Staphylococcus epidermidis	Haemophilushaemolyticusa	Dengue virus (Type 2)	Aspergillus fumigatus
Staphylococcus haemolyticus	Haemophilusparahaemolyticus	Epstein-Barr Virus	Candida albicans
Staphylococcus hominis	Haemophilus parainfluenzae	Hepatitis B virus (HBV)	Candida krusei
Staphylococcus lugdunensis	Klebsiella pneumoniae	Hepatitis C virus (HCV)	Candida parapsilosis
Staphylococcus saprophyticus	Morganella morganii	Human herpesvirus 7	Candida tropicalis
Streptococcus anginosus	Neisseria meningitidis(Unencapsulated)	Human herpesvirus 8	Cryptococcus albidus
Streptococcus bovis	Neisseria gonorrhoeae	HumanImmunodeficiency Virus	Cryptococcusamylolentusc
Streptococcus dysgalactiae	Neisseria lactamica	Human Rhinovirus A1b	Cryptococcus laurentii
Streptococcus intermedius	Neisseria mucosa	Human RhinovirusA16b	Cryptococcusuniguttulatus
Streptococcus mitis (tigurinus)	Neisseria sicca	Human Rhinovirus B3b	Filobasidiumcapsuligenum
Streptococcus mutans	Pantoea agglomerans	Human RhinovirusB83b	Pathogenic Protists
Streptococcus oralis	Proteus mirabilis	Influenza A H1N1	Naeglaria fowleri
Streptococcuspseudopneumoniae	Pseudomonas aeruginosa	Influenza A H1N1-2009	Toxoplasma gondii
Streptococcus pyogenes	Salmonella bongori	Influenza A H3N2
Streptococcus salivarius	Salmonella enterica	Influenza B
Streptococcus sanguinis	Serratia marcescens	JC polyoma virus
	Shigella boydii	La Crosse EncephalitisVirus
	Shigella flexneri	Measles Virus
	Shigella sonnei	Mumps Virus
	Treponema pallidum

Off-Panel Organisms Evaluated for Cross-Reactivity

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4 Detected by the FilmAray ME Panel as Haemophilus influenzae. H. haemolyticus is a commensal bacterium of the upper respiratory tract, rarely isolated from CSF. Cross-reactivity was observed only at concentrations > 1×10 CFUmL. o Detected by the FilmArray ME Panel as Enterovirus. Human Rhinoviruses are respiratory pathogens and rarely isolated from CSF.

^ Detected by the FilmArray ME Panel as Cryptococus neoformans/gattii. C. amylolentus is not isolated from humans (normal habitat is insect frass).

h. Interfering Substances

A study was performed to evaluate the FilmArray ME Panel for potential interference with the assay from substances that could be present in CSF specimens at the time of collection or introduced into CSF specimens during specimen processing. Positive samples contained mixes of 10 different organisms detected by the FilmArray ME Panel with each targeted organism present at concentrations equivalent to approximately 3× LoD. The concentration of each potentially interfering substance added to each sample was equal to or greater than the highest level expected to be present in CSF. Contrived samples without potentially interfering substances added served as positive controls and a potentially interfering substance in negative sample matrix served as a negative or substance-only control. Samples containing each substance were evaluated for effects of the substance on the internal pouch control assays as well as effects on the ability of the FilmArray ME Panel to provide accurate organism test results compared to the control samples.

Interference was observed in the form of false negative results for E. coli and Enterovirus in samples containing high protein concentrations (albumin >15 mg/mL). This observed interference was further supported by overall higher Cp values for the yeast RNA process control (internal control). Additional testing was performed to evaluate samples with lower protein levels and results showed that interference was not observed at concentrations of 15mg/mL and lower.

Interference was also observed in the form of false negative results for several FilmArray ME analytes in samples containing bleach at a concentration > 0.1% (v/v).

All other substances evaluated did not interfere with FilmArray ME Panel results. The substances tested and study results are shown in the following table.

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Endogenous Substances	Reference Concentration		TestedConcentration	Result
	Normal	Meningitis/Encephalitis
Glucose	40-70 mg/dL(0.4-0.7 mg/mL)	≤ 70 mg/dL(≤0.7 mg/mL)	990 mg/dL(9.9 mg/mL)	No Interference
Lactate	10-20 mg/dL(0.1-0.2 mg/mL)	> 30 mg/dL(> 0.3 mg/mL)	220 mg/dL(2.2 mg/mL)	No Interference
Protein[Albumin]	Total Protein45 mg/dL(0.45 mg/mL)	Total Protein50-500 mg/dL(0.5-5.0 mg/mL)	5,000 mg/dL(50 mg/mL)	Interference
			4,000 mg/dL(40 mg/mL)	Interference
			1,500 mg/dL(15 mg/mL)	No Interference
			500 mg/dL(5 mg/mL)	No Interference
			100 mg/dL(1 mg/mL)	No Interference
Immunoglobulin (IgG)	0-8.0 mg/dL(0.0-0.08 mg/mL)	> 8.0 mg/dL(> 0.08 mg/mL)	1000 mg/dL(10 mg/mL)	No Interference
White Blood Cells (WBC)	0-20 cells/µL	5-5,000 cells/µL	10,000 cells/µL	No Interference
Human Genomic DNA	≤ 0.068 ng/µL	≤ 17 ng/µL	20 ng/µL	No Interference
Human Whole Blood	None		10% (v/v)	No Interference
Hemoglobin	None		200mg/dL(2 mg/mL)	No Interference
Transport Media**	Concentration Tested			Result
Trans-Isolate (T-I) Medium	50% (v/v)			No Interference
Viral Transport Medium(VTM)	50% (v/v)			No Interference
Disinfectants	Concentration Tested			Result
Ethanol	7% (v/v)			No Interference
Bleach	1% (v/v)[570 ppm chlorine in sample]			Interference
	0.1% (v/v)[57 ppm chlorine]			No Interference
	0.01% (v/v)[5.7 ppm chlorine]			No Interference

Results for Potentially Interfering Substances Tested on the FilmArray ME Panel

**CSF in transport media were not evaluated in the clinical studies and are not claimed for use with the FilmArray ME Panel.

i. Assay Cut-off:

The FilmArray ME Panel Melt Detector software determines whether a FilmArray ME Panel 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 ME Panel assay were determined based on a combination of mathematical modeling using known sequence variations of

{27}------------------------------------------------

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 ME 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 99.2% and 99.9% respectively. The sensitivity, specificity, and accuracy for the validation data was determined to be well above the acceptance criteria.

j. Specimen Stability

Stability of CSF specimens was evaluated to support labeling recommendations for storage of CSF samples at room temperature for up to 24 hours or at 2 - 8°C for up to seven days prior to testing. b(4)

| 3× LoD for each analyte, with the exception of C. neoformans and C. gattii which were evaluated at 15× and 30× LoD. Ten replicates were tested for each sample mix and storage condition. The analyses performed were both qualitative (percent agreement to the expected result) as well as numerical (change in Cp values). Study acceptance criteria required a minimum of 9/10 replicates detected.

Storage conditions were considered acceptable for use with the FilmArray ME Panel if accurate test results (equivalent to the non-stored samples (Day 0)) were obtained for at least nine of the ten replicates tested after storage.

As shown in the table below, study results demonstrated 10/10 positive results for all storage conditions for nine of ten analytes evaluated. For HSV-1, 9/10 replicates were positive, meeting the study acceptance criteria. Mean Cp values for all analytes were consistent between the control and stored samples, thereby further supporting the storage claims.

Additional testing was performed to evaluate room temperature storage for samples containing C. neoformans at an adjusted concentration of 300 CFU/mL (3× LoD). C. neoformans was detected as expected for 10/10 replicates.

The study data support the specimen handling recommendations provided in the FilmArray ME package insert.

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			# Detected/# Tested
	Isolate Tested	Concentration	No	Ambient
FilmArray ME Panel		Tested	Storage	Storage	Refrigerated Storage
Test Result		(3x LoD)	(Control)	1 Day	Day 1	3 Days	7 Days
BACTERIA
Escherichia coli K1	type 018ac:K1:H7b(4)	3.00E+03 CFU/mL	10/10	10/10	10/10	10/10	10/10
Haemophilusinfluenzae	type b. biotype Ib(4)	3.00E+03 CFU/mL	10/10	10/10	10/10	10/10	10/10
Neisseria meningitidis	M-1574 [199/W135]b(4)	3.00E+03 CFU/mL	10/10	10/10	10/10	10/10	10/10
Streptococcuspneumoniae	SV 1/serotype 1b(4)	300 cells/mL	10/10	10/10	10/10	10/10	10/10
VIRUSES
Cytomegalovirus	AD-169b(4)	300 TCID50/mL	10/10	10/10	10/10	10/10	10/10
Enterovirus	Coxsackievirus A6,Gdula, group Ab(4)	150 TCID50/mL	10/10	10/10	10/10	10/10	10/10
Herpes simplex virus1	MacIntyreb(4)	150 TCID50/mL	10/10	9/10	10/10	10/10	10/10
Human parechovirus	type 3b(4)	1.50E+03TCID50/mL	10/10	10/10	10/10	10/10	10/10
YEAST
Cryptococcusneoformans/gattii	C. neoformans; H99,type strainb(4)	1.50E+03 CFU/mL	10/10	10/10	10/10	10/10	10/10
	C. gattii; A6MR38;AFLP6C; VGIIcb(4)	3.00E+03 CFU/mL	10/10	10/10	10/10	10/10	10/10

Results for Specimen Stability Testing

k. Fresh versus Frozen Study

An analytical study was performed to support the use of frozen specimens in the prospective, archived, and contrived specimen arms of the clinical study as well as in the reproducibility study. The study included a panel of 60 contrived specimens comprised of six representative FilmArray ME Panel analytes. Organisms evaluated included three bacteria know to be fastidious and/or known to demonstrate loss of viability upon freezing (S. pneumoniae, N. meningitidis, and H. influenzae), one yeast (C. neoformans), one DNA virus (HHV-6), and one RNA virus (HPeV).

Ten contrived samples were prepared for each analyte in pooled, residual negative CSF matrix. The majority of samples for each analyte were spiked at 2× LoD with the remaining samples having organism concentrations across the clinically relevant range (determined based on Cp values observed from previous FilmArray ME Panel positive test results). In some instances samples were included with concentrations below the

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assay LoD. Each analyte was represented by six different strains with the exception of HHV6 for which two strains were evaluated. Following spiking, specimens were aliquoted and either tested fresh, or immediately frozen at <- 70°C for at least 12 hours before testing.

Study results demonstrated 100% concordance for fresh and frozen samples for H. influenzae, N. meningitidis, and HPeV. S. pneumoniae demonstrated positive percent agreement (PPA) of 88.9% (one missed detection in a frozen specimen spiked at 2× LoD) and negative percent agreement (NPA) of 96% (one missed detection in a fresh specimen spiked at 0.2× LoD and one additional detection in an un-spiked frozen specimen). Two analytes demonstrated NPA of 98%: C. neoformans/gattii (one missed detection in a fresh specimen spiked at 2× LoD) and HHV-6 (one additional detection in an un-spiked frozen specimen). Analysis of overall Cp values between paired fresh and frozen samples demonstrated that differences in mean Cp values were within the expected system variability (typically within two cycles) and no significant trend toward higher or lower Cp values was observed between fresh and frozen samples.

An additional 22 clinical specimens that were tested fresh during the prospective clinical study were also re-tested after storage at -70°C. Retesting showed 100% detection after frozen storage. Similar to what was observed in the contrived specimens, the difference in average and median Cp values between fresh and frozen specimen testing was within the expected variability of the system.

The study results support inclusion of frozen specimens in the clinical and reproducibility studies.

2. Comparison Studies:

Clinical Comparison between FilmArray and FilmArray 2.0 systems: a.

To demonstrate that performance of the FilmArray ME Panel when used with the FilmArray 2.0 system is equivalent to FilmArray system, a combination of residual, deidentified CSF specimens and contrived CSF specimens covering all 14 analytes on the FilmArray ME Panel were evaluated. Specimens were identified as positive for FilmArray ME analytes at source laboratories or by culture (bacterial analytes) or PCR comparator methods (yeast and viruses) during the prospective FilmArray ME panel clinical study. The specimens were not chosen based on analyte levels but were chosen only for their previous analyte-specific positive test results and availability of sufficient volume for testing.

Contrived specimens were prepared as follows: For each analyte, leftover negative CSF specimens (negative as determined by FilmArray and comparator methods) were spiked with isolates of the organisms of interest at low concentrations (~3× LoD or below). Multiple strains for each organism were represented.

A total of 149 specimens were tested; 21 positive clinical specimens and128 contrived specimens. Positive contrived specimens were spiked with FilmArray ME Panel analytes at approximately 3× LoD or below. Specimens were split into two different aliquots for

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testing with the FilmArray and the FilmArray 2.0 systems and all specimen identities were blinded to the operator.

Each analyte was represented a minimum of five times in the specimen set. All specimens were evaluated on both systems and three specimens with invalid runs (one on FilmArray and two on FilmArray 2.0) were excluded due to insufficient volume for retesting.

Test results were analyzed to compare performance between platforms. The FilmArray ME Panel demonstrated 100% concordance for 18 individual clinical specimens. Additionally. 100% concordance was also observed for nine out of 14 analytes for contrived specimens. The agreement for each analyte tested appears in the following table.

Clinical Performance Comparison Summary of FilmArray ME Panel on FilmArray 2.0 and FilmArray systems (Film Array 2.0 results are represented in the numerator and FilmArray results are represented in the denominator)

Analyte		Positive Percent Agreement			Negative Percent Agreement
		FA2.0/FA	%	95% CI	FA2.0/FA	%	95% CI
Bacteria
E. coli K1	Clinical	0/0	-	-	21/21	100%	84.5-100%
	Contrived	5/6	83.3%	43.7-97.0%	122/122	100%	97.0-100%
	Overall	5/6	83.3%	43.7-97.0%	143/143	100%	97.4-100%
H. influenzae	Clinical	0/0	-	-	21/21	100%	84.5-100%
	Contrived	10/10	100%	72.3-100%	118/118	100%	96.9-100%
	Overall	10/10	100%	72.3-100%	139/139	100%	97.3-100%
L. monocytogenes	Clinical	0/0	-	-	21/21	100%	84.5-100%
	Contrived	5/5	100%	56.6-100%	123/123	100%	97.0-100%
	Overall	5/5	100%	56.6-100%	144/144	100%	97.4-100%
N. meningitidis	Clinical	0/0	-	-	21/21	100%	84.5-100%
	Contrived	11/11	100%	74.1-100%	117/117	100%	96.8-100%
	Overall	11/11	100%	74.1-100%	138/138	100%	97.3-100%
S. agalactiae	Clinical	1/1	100%	20.7-100%	20/20	100%	83.9-100%
	Contrived	5/5	100%	56.6-100%	123/123	100%	97.0-100%
	Overall	6/6	100%	61.0-100%	143/143	100%	97.4-100%
S. pneumoniae	Clinical	1/1	100%	20.7-100%	20/20	100%	83.9-100%
	Contrived	6/7	85.7%	48.7-97.4%	120/121	99.2%	95.5-99.9%
	Overall	7/8	87.5%	52.9-97.8%	140/141	99.3%	96.1-99.9%
Viruses
CMV	Clinical	1/1	100%	20.7-100%	20/20	100%	83.9-100%
	Contrived	5/5	100%	56.6-100%	123/123	100%	97.0-100%
	Overall	6/6	100%	61.0-100%	143/143	100%	97.4-100%
EV	Clinical	1/1	100%	20.7-100%	20/20	100%	83.9-100%
	Contrived	11/12	91.7%	64.6-98.5%	114/116	98.3%	93.9-99.5%

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Analyte		Positive Percent Agreement			Negative Percent Agreement
		FA2.0/FA	%	95% CI	FA2.0/FA	%	95% CI
HSV-1	Overall	12/13	92.3%	66.7-98.6%	134/136	98.5%	94.8-99.6%
	Clinical	3/3	100%	43.9-100%	18/18	100%	82.4-100%
	Contrived	3/3	100%	43.9-100%	125/125	100%	97.0-100%
HSV-2	Overall	6/6	100%	61.0-100%	143/143	100%	97.4-100%
	Clinical	2/2	100%	34.2-100%	19/19	100%	83.2-100%
	Contrived	3/3	100%	43.9-100%	125/125	100%	97.0-100%
HHV-6	Overall	5/5	100%	56.6-100%	144/144	100%	97.4-100%
	Clinical	3/3	100%	43.9-100%	18/18	100%	82.4-100%
	Contrived	9/9	100%	70.1-100%	117/119	98.3%	94.1-99.5%
	Overall	12/12	100%	75.8-100%	135/137	98.5%	94.8-99.6%
HPeV	Clinical	0/0	-	-	21/21	100%	84.5-100%
	Contrived	8/8	100%	67.6-100%	120/120	100%	96.9-100%
	Overall	8/8	100%	67.6-100%	141/141	100%	97.4-100%
VZV	Clinical	3/3	100	43.9-100	18/18	100%	82.4-100%
	Contrived	4/4	100%	51.0-100%	124/124	100%	97.0-100%
	Overall	7/7	100%	64.6-100%	142/142	100%	97.4-100%
Yeast
C. neoformans/gattii	Clinical	2/2	100%	34.2-100%	19/19	100%	83.2-100%
	Contrived	15/15	100%	79.6-100%	112/113	99.1%	95.2-99.9%
	Overall	17/17	100%	81.6-100%	131/132	99.2%	95.8-99.9%
Overall Agreement		117/120	97.5%	92.9-99.2%	1960/1966	99.7%	99.3-99.9%

Overall PPA for clinical and contrived specimens combined was 97.5% with the lower bound of the two-sided 95% confidence interval (95% CI) at 92.9%, and overall NPA was 99.7% with the lower bound of the two-sided 95% CI at 99.3%.

An analysis of Tm values was conducted to compare the performance of the FilmArray ME Panel on both FilmArray and FilmArray 2.0 systems. Delta Tm values between the two systems were less than or equal to 0.4℃ for all analytes, which passed the study acceptance criteria of less than 0.5℃.

Overall, the study results establish equivalent performance for clinical specimens when tested with the FilmArray ME Panel on the FilmArray and FilmArray 2.0 systems.

b. Matrix Equivalence Study

An equivalence study was performed to validate artificial CSF (aCSF) as a surrogate CSF sample matrix for contrived samples in analytical performance studies of the FilmArray ME Panel. The aCSF matrix mimics the composition of natural CSF and contains similar electrolyte concentrations. The equivalence study included paired > samples comprised of four organism mixes prepared in natural and aCSF matrices. A total of 19 organisms covering all targeted FilmArray ME panel analytes were evaluated with samples containing mixtures of up to 5 different organisms. A minimum of four 10-fold dilutions

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were tested per organism in each matrix. including concentrations below and above the LoD for each analyte. Four replicates were tested for each dilution. Where needed, testing of additional replicates or dilutions was performed in order to collect the necessary data to evaluate matrix effects for concentrations flanking the estimated LoD.

FilmArray ME panel results were evaluated for each analyte based on differences in qualitative detection as well as numerical analysis of Cp values for samples prepared in natural CSF and aCSF matrices. Study results showed no significant differences in qualitative detection for each organism evaluated (i.e., loss of detection occurred at similar dilutions for each matrix). In addition. analysis of mean Cp values showed no trend toward lower or higher values between samples prepared in natural or aCSF matrices.

Study results established that the FilmArray ME Panel provides equivalent results for contrived samples prepared in either aCSF matrix or a human clinical CSF matrix over a range of concentrations. Therefore aCSF was determined to be an acceptable sample matrix for preparation of samples for analytical studies.

3. Clinical Studies:

Prospective Clinical Study

The clinical performance of the FilmArrav ME Panel was established during a multi-center study conducted at 11 geographically distinct U.S. study sites. A portion of specimens were collected and immediately frozen for later testing at the source laboratory. A total of 1643 prospective CSF specimens were acquired for the clinical study: 83 of these were excluded. The most common reasons for specimen exclusion were that specimens did not meet the inclusion criteria. Other reasons for exclusion included invalid daily external controls, FilmArray ME Panel run failure, testing with the incorrect FilmArray ME Panel pouch version, the specimen was bevond seven days of storage. lack of comparator test result, or internal control failure. The majority of CSF specimens were tested fresh; however a portion of prospective specimens were collected and immediately frozen for later testing. The final data set consisted of 1560 specimens, of which 545 (35%) had been previously frozen before testing. The following table provides a summary of demographic information for the 1560 specimens included in the prospective study.

Prospective Study Specimens (%)
Fresh	1015 (65%)
Frozen	545 (35%)
Total Specimens	1560
Sex	Number of Specimens (%)
Male	797 (51%)
Female	763 (49%)
Age Group	Number of Specimens (%)
< 2 mo.	299 (19%)
2-23 mo.	143 (9%)

Demographic Summary for Prospective Clinical Study

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2-17 years	197 (13%)
18-34 years	224 (14%)
35-64 years	522 (33%)
65+ years	175 (11%)
Status	Number of Specimens (%)
Outpatient	112 (7%)
Hospitalized	920 (59%)

The performance of the FilmArray ME Panel was evaluated by comparing the FilmArray ME Panel test result for each member of the panel with the appropriate comparator/reference methods shown in the following table.

FilmArray Analyte	Comparator Method	Comparator Test Location
E. coli K1
H. influenzae
L. monocytogenes	CSF bacterial culture	Source Laboratory
N. meningitidis
S. agalactiae
S. pneumoniae
CMV
EV
HSV-1	Two PCR assays withbi-directionalsequencinga	BioFire Laboratory
HSV-2
HHV-6
HPeV
VZV
C. neoformans/gattii

arator Methods for FilmArray ME Panel Clinical Evaluation

4 All assays targeted different nucleic acid sequences than those identified by the FilmArray ME Panel.

A total of 1560 specimens were evaluated in this study. Clinical sensitivity or positive percent agreement (PPA) was calculated as 100% x (TP / (TP + FN)). True positive (TP) indicates that both the FilmArray ME Panel and reference/comparator method had a positive result for the specific analyte, and false negative (FN) indicates that the FilmArray ME Panel result was negative while the comparator result was positive. Specificity or negative percent agreement (NPA) was calculated as 100% x (TN / (TN + FP)). True negative (TN) indicates that both the FilmArray ME Panel and the reference/comparator method had negative results, and a false positive (FP) indicates that the FilmArray ME Panel result was positive but the comparator result was negative. The two-sided 95% confidence intervals were calculated.

Study results are summarized in the following table.

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Analyte	Sensitivity(compared to culture)			Specificity(compared to culture)
	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Bacteria
E. coli K1	Fresh1/1	100	-	1014/1014	100	99.6-100
	Frozen1/1	100	-	543/544	99.8	99.0-100
	Overall2/2	100	34.2-100	1557/1558b,c	99.9	99.6-100
H. influenzae	Fresh1/1	100	-	1013/1014	99.9	99.4-100
	Frozen0/0	-	-	545/545	100	99.3-100
	Overall1/1	100	-	1558/1559d	99.9	99.6-100
L. monocytogenes	Fresh0/0	-	-	1015/1015	100	99.6-100
	Frozen0/0	-	-	545/545	100	99.3-100
	Overall0/0	-	-	1560/1560	100	99.8-100
N. meningitidis	Fresh0/0	-	-	1015/1015	100	99.6-100
	Frozen0/0	-	-	545/545	100	99.3-100
	Overall0/0	-	-	1560/1560	100	99.8-100
S. agalactiae	Fresh0/1	0.0	-	1013/1014	99.9	99.4-100
	Frozen0/0	-	-	545/545	100	99.3-100
	Overall0/1e	0.0	-	1558/1559e	99.9	99.6-100
S. pneumoniae	Fresh2/2	100	34.2-100	1008/1013	99.5	98.8-99.8
	Frozen2/2	100	34.2-100	536/543	98.7	97.4-99.4
	Overall4/4	100	51.0-100	1544/1556f	99.2	98.7-99.6
Analyte	Positive Percent Agreement(compared to PCR with bi-directionalsequencing)			Negative Percent Agreement(compared to PCR with bi-directionalsequencing)
	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Viruses
CMV	Fresh2/2	100	34.2-100	1010/1013	99.7	99.1-99.9
	Frozen1/1	100	20.7-100	544/544	100	99.3-100
	Overall3/3	100	43.9-100	1554/1557g	99.8	99.4-99.9
EV	Fresh43/44	97.7	88.2-99.6	965/971	99.4	98.7-99.7
	Frozen1/2	50.0	-	542/543	99.8	99.0-100
	Overall44/46h	95.7	85.5-98.8	1507/1514h	99.5	99.0-99.8
HSV-1	Fresh1/1	100	-	1013/1014	99.9	99.4-100
	Frozen1/1	100	-	543/544	99.8	99.0-100
	Overall2/2	100	34.2-100	1556/1558i	99.9	99.5-100
HSV-2	Fresh6/6	100	61.0-100	1008/1009	99.9	99.4-100
	Frozen4/4	100	51.0-100	540/541	99.8	99.0-100
	Overall10/10	100	72.2-100	1548/1550j	99.9	99.5-100
HHV-6	Fresh13/15	86.7	62.1-96.3	997/1000	99.7	99.1-99.9
	Frozen5/6	83.3	43.6-97.0	535/536	99.8	99.0-100
Analyte	Sensitivity(compared to culture)			Specificity(compared to culture)
	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
HPeV	Overall	18/21k	85.7	65.4-95.0	1532/1536k	99.7	99.3-99.9
	Fresh	9/9	100	70.1-100	1003/1006	99.7	99.1-99.9
	Frozen	0/0	-	-	545/545	100	99.3-100
VZV	Overall	9/9	100	70.1-100	1548/1551l	99.8	99.4-99.9
	Fresh	3/3	100	43.9-100	1010/1012	99.8	99.3-99.9
	Frozen	1/1	100	-	543/544	99.8	99.0-100
	Overall	4/4	100	51.0-100	1553/1556m	99.8	99.4-99.9
C. neoformans/gattii	Yeast
	Fresh	0/0	-	-	1015/1015	100	99.6-100
	Frozen	1/1	100	-	540/544	99.3	98.1-99.7
	Overall	1/1	100	-	1555/1559n	99.7	99.3-99.9

FilmArray ME Prospective Clinical Performance Summary4

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a The performance measures of sensitivity and specificity only refer to bacterial analytes for which the gold-standard of CSF bacterial culture was used as the reference method. Performance measures of Positive Percent and Negative Percent Agreement refer to all other analytes, for which PCR/sequencing assays were used as comparator methods.

b The FP specimen was negative for E. coli K1 when tested using an independent PCR assay targeting a nucleic acid region distinct from that identified by the FilmArray ME Panel. Meningitis was clinically excluded in this patient.

C An additional infant presented with CSF pleocytosis (WBC 3738) and E. coli bacteremia. CSF cultures and FilmArray ME Panel were negative, but no information re-treatment with antibiotics was available, and the patient was clinically diagnosed with meningitis.

ਰੋ H. influenzae was detected in the single FP specimen using an independent PCR assay and was also observed via Gram stain; the subject from whom this specimen was collected received a physician diagnosis of gram-negative bacterial meningitis.

The laboratory reported that S. agalactiae was present at a very low level (two colonies) for the FN specimen. The FP specimen was negative for S. agalactiae when tested using an independent PCR assay.
f S. pneumoniae was detected in 5/12 FP specimens using an independent PCR assay; additional information regarding seven unconfirmed FP specimens is detailed below in Table 10.
bi CMV was detected in 1/3 FP specimens using an independent PCR assay.
h EV was detected in 2/2 FN specimens using an independent PCR assay; one specimen was positive upon FilmArray ME retest. EV was detected in 5/7 FP specimens using an independent PCR assay.
i Both FP specimens were negative for HSV-1 when tested using an independent PCR assay.

j HSV-2 was detected in 1/2 FP specimens using an independent PCR assay; the subject from whom this specimen was collected received a physician diagnosis of HSV meningitis.

k HHV-6 was detected in 2/3 FN and 1/4 FP specimens using an independent PCR assay.
1 HPeV was detected in 1/3 FP specimens using an independent PCR assay; the subject from whom this specimen was collected received a physician diagnosis of HPeV meningitis. Both of the subjects from whom the remaining two specimens were collected received a diagnosis of HPeV infection of HPeV in the blood.

" VZV was detected in 1/3 FP specimens using an independent PCR assay; the subject from whom this specimen was collected received a physician diagnosis of herpes zoster. Of the remaining two specimens with FP results, one was collected from a subject who was diagnosed with herpes zoster oticus.

n C. neoformans/gatti was detected in 2/4 FP specimens using a commercially available antigen test. One FP specimen was positive by standard culture. Additional information regarding FilmArray ME Panel performance with respect to cryptococcal antigen testing is detailed below.

Of 12 specimens with false positive results for S. pneumoniae, seven could not be confirmed using an independent PCR assay. A review of de-identified subject medical data was conducted for the subjects from whom these specimens were collected and is summarized in the following table. None of the subjects had evidence of bacterial meningitis/encephalitis. The cause of these false positives was not determined.

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Subjectage	CSFWBC	FilmArrayResult	Comparator Culture/Investigation PCRa	Diagnosis Reported in Medical Record
<2 mo	3	Pos	Neg/Neg	Infection, non-CNS (S. agalactiae urine culture)
65+	2	Pos	Neg/Neg	Unable to obtain
2-17	0	Pos	Neg/Neg	Infection, non-CNS (folliculitis)
<2 mo	3	Pos	Neg/Neg	Infection, non-CNS (Parainfluenza virus)
18-34	1	Pos	Neg/Neg	CNS disease, non-infectious (epilepsy)
35-64	1	Pos	Neg/Neg	Infection, non-CNS (Hep B), multiple myeloma
18-34	1	Pos	Neg/Neg	Infection, non-CNS (Bells' Palsy)

Clinical Characteristics of Subjects with Unconfirmed False Positive S. pneumoniae Results

4 Independent PCR assay is the same as described in footnote f in the table above.

The comparator method used to evaluate FilmArray ME Panel C. neoformans/gattii performance was PCR with bi-directional sequencing. FilmArray ME Panel performance for detection of C. neoformans/gattii was also calculated in comparison to specific testing for Cryptococcus that was performed by the laboratory based upon clinician test requests for a subset of subjects. For data that were available, FilmArray ME Panel performance is shown in the table below relative to cryptococcal antigen (CrAg) testing (N=196), standard culture (N=1560), and fungal culture (N=23). Notably, seven out of eight CrAg-positive specimens were discordant with FilmArray ME Panel results. All seven of these specimens were negative for Cryptococcus when tested with both PCR comparator assays. Medical chart review indicated that each subject was on antifungal therapy for treatment of cryptococcal meningitis or cryptococcosis at the time of specimen collection and/or had prior history of Cryptococcus infection. Therefore, positive antigen results for these patients in the absence of PCR and culture-based organism detection are likely due to antigen persistence rather than the presence of live organism.

Cryptococcus test comparatormethod	Positive Percent Agreement			Negative Percent Agreement
	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Cryptococcal Antigen	1/8a	12.5	2.2-47.1	187/188b	99.5	97.0-99.5
Standard Culture	2/3c	66.7	20.8-93.9	1554/1557d	99.8	99.4-99.9
Fungal Culture	0/0	-	-	22/23e	95.7	79.0-99.2

FilmArray ME Panel C. neoformans/gattii assay performance relative to other comparator methods

Seven specimens were positive by CrAg testing performed at the clinical site, but were negative by standard of care culture, FilmAray ME Panel, and two comparator assays. All seven subjects from whom these specimens were collected received antifungal therapy prior to LP and or history of Cryptococcus infection. The eighth specimen was positive by CrAg. FilmArray ME Panel, and standard culture.

b Cryptococcus was detected in the single FP specimen using a CrAg testing kit at BioFire.

C The single FN specimen was also positive by standard culture, but negative by the FilmArray ME Panel and two comparator assays. The laboratory reported that only one colony was recovered.

ਰ Cryptococus was detected in 1/3 FP specimens using CrAg testing kits at BioFire (this is the same PP described in footnote a).

e The single FP specimen was negative by standard of care culture, CrAg testing performed at the clinical site, and negative by two comparator assays, but was positive by CrAg testing performed at BioFire (this is the same FP described in footnote a).

In the prospective clinical study, the FilmArray ME Panel reported a total of 5 specimens with multiple organism detections (0.3% of all specimens, 5/1560; and 3.7% of positive specimens, 5/136). Each multi-analyte detection contained two organisms, at least one of which was not detected by the reference/comparator method (i.e., each specimen contained at least one false positive result by the FilmArray ME Panel). The organism combinations detected by FilmArray ME Panel are shown in the following table.

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Co-detectionCombination	Number ofSpecimens	False PositiveAnalyte
CMV + S. pneumoniae	1	CMV
EV + HPeV	1	EV
HSV-1 + HHV-6	1	HSV-1
HSV-2 + S. agalactiae	1	S. agalactiae
S. pneumoniae + VZV	1	S. pneumoniae andVZV

Co-detection Combinations as Determined by the FilmArray ME Panel

The overall success rate for initial specimen tests in the prospective study was 98.9% (1560/1577): 17 tests were unsuccessful (11 due to incomplete tests and six due to control failures). Repeat testing was not possible to limited specimen volumes.

Testing of Preselected Archived Specimens

Several analytes were either not encountered or had a low prevalence in the prospective clinical study. To supplement the results of the prospective clinical study, an evaluation of 235 preselected archived specimens (of which 25 were negative) was performed. These specimens were archived clinical specimens that were selected because thev had previously tested positive for one of the following analytes: Cryptococcus, CMV, E. coli, H. influenzae, HSV-1, HSV-2, HHV-6, HPeV, L. monocytogenes, N. meningitidis, S. agalactiae, S. pneumoniae, or VZV; or had been negative in previous laboratory testing. Prior to testing with the FilmArray ME Panel, the presence (or absence) of expected analytes was verified in each specimen using a confirmatory molecular test (e.g. PCR with bi-directional sequencing) Out of the 210 positives, the historical result was confirmed by the comparator method for 150 (150/210; 71.4%); only confirmed analytes were used in calculations of PPA, but all specimens were used for NPA analyses.

The specimens were organized into "test panels" and randomized such that the users performing the FilmArray ME Panel testing were blinded as to the expected test result. A summary of the available demographic information of the tested samples and the results of the FilmArray ME Panel testing are presented in the following tables.

Preselected Archived Specimens
Total Specimens	235
Sex	Number of Specimens (%)
Male	70 (30%)
Female	90 (38%)
Unknown	75 (32%)
Age Group	Number of Specimens (%)
<2 mo	5 (2%)
2-23 mo	19 (8%)
2-17 yrs.	19 (8%)
18-34 yrs.	33 (14%)

Demographic Summary of Archived Specimens

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65+ yrs.	26 (11%)
Unknown	68 (29%)

	Positive Percent Agreement			Negative Percent Agreement
Analyte	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Bacteria
E. coli K1	2/2	100	34.2-100	35/35	100	90.1-100
H. influenzae	3/3	100	43.9-100	39/39	100	91-100
L. monocytogenes	1/1	100	-	41/41	100	91.4-100
N. meningitidis	7/7	100	64.6-100	34/34	100	89.8-100
S. agalactiae	2/2	100	34.2-100	40/40	100	91.2-100
S. pneumoniae	17/17	100	81.6-100	21/21	100	84.5-100
Viruses
CMV	7/8	87.5	52.9-97.8	181/181	100	97.9-100
HSV-1	16/16	100	80.6-100	156/157	99.4	96.5-99.9
HSV-2	33/34	97.1	85.1-99.5	136/136	100	97.3-100
HHV-6	12/16a	75.0	50.5-89.8	168/168	100	97.8-100
HPeV	2/3	66.7	20.8-93.9	187/187	100	98.0-100
VZV	22/22	100	85.1-100	162/164	98.8	95.7-99.7
Yeast
C. neoformans/gattii	19/19b	100	83.2-100	171/171	100	97.8-100

FilmArray ME Panel Archived Specimen Performance Data Summary

4 Two specimens were sequenced and identified as HHV-6A while 14 were HHV-6B. Of the four FilmArray ME Panel FN specimens, one was sequenced and identified as HHV-6A and the remaining three FN specimens were identified as HHV-6B. The resulting PPA was 50% (1/2); 95% CI 9.5-90.5% and 79% (11/14); 95% CI 52.4-92.4% for HHV-6A and HHV-6B, respectively.

One specimen was sequenced and identified as C. gattii and 18 were C. neoformans.

Testing of Contrived Specimens

For some rare FilmArray ME Panel analytes, the prospective and archived clinical study arms included insufficient positive specimens to establish system performance. To supplement the prospective and archived data, an evaluation of contrived specimens was performed. Surrogate specimens were prepared using residual CSF specimens that had previously tested negative for all ME panel analytes by the FilmArray ME Panel and comparator methods. For each analyte, at least 25 specimens were spiked at 2× LoD and the remaining were spiked at four additional concentrations spanning the clinically relevant range using at least five different quantified strains for each organism. Specimens were prepared and randomized along with negative (unspiked) CSF specimens such that the analyte status of each contrived specimen was blinded to the users analyzing the specimens. Contrived specimens were frozen and then distributed to prospective clinical study sites for testing. The results of contrived specimens testing are presented in the following table.

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	PPA			NPA
Analyte	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
E. coli K1	47/49a	95.9	86.3-98.9	245/245	100	98.5-100
H. influenzae	50/50	100	92.9-100	243/244	99.5	97.7-99.9
L. monocytogenes	50/50	100	92.9-100	244/244	100	98.5-100
N. meningitidis	75/75	100	95.1-100	219/219	100	98.3-100
S. agalactiae	48/50b	96.0	86.5-98.9	244/244	100	98.5-100
CMV	47/49c	95.9	86.3-98.9	245/245	100	98.5-100
HHV-6	50/50	100	92.9-100	243/244	99.5	97.7-99.9
HPeV	50/50	100	92.9-100	244/244	100	98.5-100

FilmArray ME Panel Performance Using Contrived Specimens

4 One E coli K1 false negative was observed at 2 × LoD and one E coli K1 false negative was observed at 0.2 × LoD.

b Both S. agalactiae false negatives were observed at 0.2 × LoD.

& Both CMV false negatives were observed at 0.2 × LoD.

4. Clinical cut-off:

Not applicable

5. Expected values/Reference range:

The number and percentage of positive results as determined by the FilmArray ME Panel in the prospective clinical study are presented in the following table stratified by age group. Overall, the FilmArray ME Panel detected at least one organism or virus in a total of 136 prospective specimens (8.7% positivity rate) with a total of 141 analyte detections (codetections were observed in five specimens).

Expected Values (as determined by the FilmArray ME Panel) Summary by Age Group for the Prospective Clinical Evaluation (February through September 2014)

FilmArray ME PanelResult	Overall(n=1560)	< 2 mo.(n=299)	2-23 mo.(n=143)	2-17 years(n=197)	18-34 years(n=224)	35-64 years(n=522)	65+ years(n=175)
Bacteria
E. coli K1	3 (0.2%)	0 (0%)	1 (0.7%)	0 (0%)	0 (0%)	2 (0.4%)	0 (0%)
H. influenzae	2 (0.1%)	0 (0%)	1 (0.7%)	0 (0%)	0 (0%)	1 (0.2%)	0 (0%)
L. monocytogenes	0 (0.0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
N. meningitidis	0 (0.0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
S. agalactiae	1 (0.1%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	1 (0.6%)
S. pneumoniae	16 (1.0%)	2 (0.7%)	2 (1.4%)	2 (1%)	3 (1.3%)	4 (0.8%)	3 (1.7%)
Viruses
CMV	6 (0.4%)	4 (1.3%)	0 (0%)	1 (0.5%)	1 (0.4%)	0 (0%)	0 (0%)
EV	51 (3.3%)	31 (10.4%)	5 (3.5%)	11 (5.6%)	4 (1.8%)	0 (0%)	0 (0%)
HSV-1	4 (0.3%)	0 (0%)	2 (1.4%)	0 (0%)	0 (0%)	2 (0.4%)	0 (0%)
HSV-2	12 (0.8%)	0 (0%)	0 (0%)	0 (0%)	1 (0.4%)	8 (1.5%)	3 (1.7%)
HHV-6	22 (1.4%)	9 (3%)	7 (4.9%)	2 (1%)	3 (1.3%)	1 (0.2%)	0 (0%)

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FilmArray ME PanelResult	Overall(n=1560)	< 2 mo.(n=299)	2-23 mo.(n=143)	2-17 years(n=197)	18-34 years(n=224)	35-64 years(n=522)	65+ years(n=175)
HPeV	12 (0.8%)	12 (4%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
VZV	7 (0.4%)	0 (0%)	0 (0%)	0 (0%)	3 (1.3%)	3 (0.6%)	1 (0.6%)
Yeast
C. neoformans/gattii	5 (0.3%)	1 (0.3%)	0 (0%)	0 (0%)	1 (0.4%)	2 (0.4%)	1 (0.6%)

FilmArray ME Panel Positivity Rate In the Prospective Clinical Evaluation; Overall and By Age Group

Overall (n=1560)
Negatives	1424 (91.3%)
Positives	136 (8.7%)
Single Detections	131 (8.4%)
Co-Detections	5 (0.3%)
Positivity by Age Group
< 2 mo. (n=299)	58 (19.4%)
2-23 mo. (n=143)	17 (11.9%)
2-17 years (n=197)	15 (7.6%)
18-34 years (n=224)	15 (6.7%)
35-64 years (n=522)	23 (4.4%)
65+ years (n=175)	8 (4.6%)

M. Instrument Name

FilmArray and FilmArray 2.0

N. System Descriptions:

1. Modes of Operation:
  After samples and hydration reagent have been placed in the reagent pouch, the remaining processing steps are executed under control of the instrument.
1. Software:
  FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes X

1. Specimen Identification:
  Specimen identification can be entered manually or via barcode.
1. Specimen Sampling and Handling:
  The FilmArray ME Panel is intended for use with CSF specimens that have not been

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centrifuged. The operator places a Hydration Vial and a Sample Injection Vial into the FilmArray Pouch Loading Station. The operator hydrates the test pouch with the Hydration Injection Vial and then using a transfer pipette, adds~200 ul of CSF into the Sample Injection Vial. The operator removes the Sample Injection Vial containing the CSF from the Loading Station, inverts the vial at least three times to mix, and then inserts it into the Loading Station port where the proper amount of specimen is pulled into the FilmArray ME Panel pouch by vacuum. The FilmArray ME Panel is then placed onto the FilmArray instrument for testing.

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

O. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

Not Applicable

P. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809 and the specials controls for this device type.

O. Identified Risks and Required Mitigations:

Identified Risks to Health	Required Mitigations (See Section S below for Special Controls)
Incorrect identification or lack of identification of a pathogenic microorganism by the device can lead to improper patient management	Special Controls (1), (2), (3), (4), and (5)
Failure to correctly interpret test results	Special Controls (6), (7), (8), and (9)
Failure to correctly operate the instrument	Special Control (10)

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R. Benefit/Risk Analysis:

Summary
Summary ofthe Benefit(s)	The morbidity and mortality associated with some forms of meningitis and encephalitis can be substantial, therefore it is desirable to the patient to identify different causes of meningitis and/or encephalitis quickly. The primary benefit provided by the FilmArray ME Panel is the swift diagnosis of meningitis and/or encephalitis when compared to traditional culture.
	Clinicians cannot distinguish between the various bacterial, viral and fungal causes of meningitis and/or encephalitis without additional laboratory testing. Also, cerebrospinal fluid must be obtained through an invasive procedure, and specimen volume may limit the number of tests that can be performed. The FilmArray ME Panel simultaneously tests for 14 analytes using only a small amount of CSF fluid, therefore additional fluid may be more readily available for additional testing.
	Negative test results may provide additional clinician confidence when delaying empiric therapy or discontinuing empiric therapy. In these cases, patients may experience fewer side effects from antimicrobial therapy, and there may be additional benefits to antimicrobial stewardship programs. Such decisions will be at the discretion of the managing physician, who will have supplemental lab testing to guide the ultimate therapeutic decisions.
Summary ofthe Risk(s)	False positive and false negative results, inaccurate identification of a pathogenic microorganism by the device, failure to correctly interpret test results, or failure to correctly operate the instrument are the primary risks associated with use of the FilmArray ME Panel.
	A false positive result may lead to unnecessary antimicrobial therapy, with associated toxicities and side effects, including potential allergic reaction. These side effects from antimicrobial therapy are generally reversible, and limited to the duration of the antimicrobial agent. Certain viral analytes are known to reactivate in association with physiological stress or other infections. Positive results with the herpesviruses in particular may not reflect true causative infection, and could result in incorrect diagnosis. As a result, the true diagnosis could be delayed or missed, including non- infectious diseases or other infectious pathogens. The risk of a false-positive result is mitigated by device use in association with traditional culture as noted in the device's labeling.
	False negative results could result in delayed diagnosis of meningitis and/or encephalitis, or delayed initiation of appropriate antimicrobial therapy. An incorrect identification could result in inappropriate antimicrobial therapy. As a result, the patient could experience a delay in effective antimicrobial therapy. The risk of a false- negative result is mitigated by device use in association with traditional culture as noted in the device's labeling.

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Summary ofOtherFactors

None.

ConclusionsDo theprobablebenefitsoutweigh theprobablerisks?

The probable benefits of the FilmArray ME Panel likely outweigh the potential risks inlight of the listed special controls and applicable general controls, including designcontrols. The FilmArray ME Panel is the first of its kind, and represents a potential forpatient benefit through more rapid diagnosis of meningitis and/or encephalitis. Potentialrisks associated with false positive or false negative results may be mitigated by use oftraditional bacterial and fungal cultures, which would be necessary to recover the organismfor further identification and susceptibility testing even without the FilmArray ME Panel.Data obtained from additional studies with contrived specimens demonstrated highsensitivity. The prospective clinical study found that the sensitivity for some analytes islow but given the small number of positive samples, it is difficult to establish withstatistical significance how the FilmArray ME Panel will perform in real-world use.When used in conjunction with traditional cultures and clinical evaluation, much of therisk presented by this device can be mitigated. End user education will also allowclinicians to make informed judgements about how they will implement the FilmArray MEPanel in their clinical practice. Ultimately, the majority of risks associated with theFilmArray ME Panel may be minimized with appropriate precautions and the FilmArrayME Panel may provide substantial benefit to patients.

S. Conclusion:

The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 866.3970. FDA believes that the stated special controls, and applicable general controls, including design controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:

Product Code:	PLO, OOI, NSU
Device Type:	Device to detect and identify microbial pathogen nucleic acids incerebrospinal fluid.
Class:	II (special controls)
Regulation:	21 CFR 866.3970

(a) Identification. A device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid is identified as a qualitative in vitro device intended for the detection and identification of microbial-associated nucleic acid sequences from patients suspected of meningitis or encephalitis. A device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid is intended to aid in the diagnosis of meningitis or

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encephalitis when used in conjunction with clinical signs and symptoms and other clinical and laboratory findings.

(b) Classification. Class II (special controls). A device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid must comply with the following special controls:
- 1. Premarket notification submissions must include detailed device description documentation, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology including primer/probe sequence, design, and rationale for sequence selection.
- 1. Premarket notification submissions must include detailed documentation from the following analytical studies: Analytical sensitivity (Limit of Detection), inclusivity, reproducibility, interference, cross reactivity, and specimen stability.
- 1. Premarket notification submissions must include detailed documentation from a clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from well-accepted comparator methods.
- 1. Premarket notification submissions must include detailed documentation for device software, including, but not limited to, software applications and hardware-based devices that incorporate software.
- 1. The Intended Use statement in the device labeling must include a statement that the device is intended to be used in conjunction with standard of care culture.
- 1. A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling.
- 1. The device labeling must include a limitation that negative results do not preclude the possibility of central nervous system infection.
- 1. The device labeling must include a limitation that device results are not intended to be used as the sole basis for diagnosis, treatment, or other patient management decisions.
- 1. The device labeling must include a limitation stating that positive results do not mean that the organism detected is infectious or is the causative agent for clinical symptoms.
- 1. As part of the risk management activities performed as part of your 21 CFR 820.30 design controls, you must document an appropriate end user device training program that will be offered as part of your efforts to mitigate the risk of failure to correctly operate the instrument.

Regulation Number and Section

§ 866.3970 Device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid.

(a)
Identification. A device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid is a qualitative in vitro device intended for the detection and identification of microbial-associated nucleic acid sequences from patients suspected of meningitis or encephalitis. A device to detect and identify microbial pathogen nucleic acids in cerebrospinal fluid is intended to aid in the diagnosis of meningitis or encephalitis when used in conjunction with clinical signs and symptoms and other clinical and laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include detailed device description documentation, including the device components, ancillary reagents required but not provided, and a detailed explanation of the methodology, including primer/probe sequence, design, and rationale for sequence selection.
(2) Premarket notification submissions must include detailed documentation from the following analytical studies: Analytical sensitivity (limit of detection), inclusivity, reproducibility, interference, cross reactivity, and specimen stability.
(3) Premarket notification submissions must include detailed documentation from a clinical study. The study, performed on a study population consistent with the intended use population, must compare the device performance to results obtained from well-accepted comparator methods.
(4) Premarket notification submissions must include detailed documentation for device software, including, but not limited to, software applications and hardware-based devices that incorporate software.
(5) The Intended Use statement in the device labeling must include a statement that the device is intended to be used in conjunction with standard of care culture.
(6) A detailed explanation of the interpretation of results and acceptance criteria must be included in the device's 21 CFR 809.10(b)(9) compliant labeling.
(7) The device labeling must include a limitation stating that the negative results do not preclude the possibility of central nervous system infection.
(8) The device labeling must include a limitation stating that device results are not intended to be used as the sole basis for diagnosis, treatment, or other patient management decisions.
(9) The device labeling must include a limitation stating that positive results do not mean that the organism detected is infectious or is the causative agent for clinical symptoms.
(10) As part of the risk management activities performed as part of your 21 CFR 820.30 design controls, you must document an appropriate end user device training program that will be offered as part of your efforts to mitigate the risk of failure to correctly operate the instrument.