Predicate Devices

Not Found

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on standard molecular diagnostic techniques (nucleic acid purification, PCR, melt curve analysis) and software interpretation of melt curve data, without mentioning AI or ML.

Therapeutic

No.
The document explicitly states that the FilmArray Meningitis/Encephalitis (ME) Panel is an "in vitro diagnostic test" intended as "an aid in the diagnosis of specific agents of meningitis and/or encephalitis". It is designed to detect and identify nucleic acids from pathogens, not to treat or directly manage a condition, which are characteristic functions of a therapeutic device. Furthermore, it states its results "are not intended to be used as the sole basis for diagnosis, treatment, or other patient management decisions."

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the FilmArray ME Panel is an "in vitro diagnostic test" and "is indicated as an aid in the diagnosis of specific agents of meningitis and/or encephalitis".

SaMD (Software as a Medical Device)

No

The device description clearly states that the FilmArray ME Panel is a multiplex nucleic acid-based test designed to be used with FilmArray or FilmArray 2.0 systems, which are described as "FilmArray instruments." The description details the physical components of the system, including the pouch containing reagents, the instrument with inflatable bladders and seal points, and a digital camera. While software is used to interpret data, the device is fundamentally a hardware-based in vitro diagnostic system that utilizes a physical pouch and instrument to perform the test.

IVD (In Vitro Diagnostic)

Based on the provided text, this device is an IVD (In Vitro Diagnostic).

Here's why:

Explicit Statement: The "Intended Use / Indications for Use" section explicitly states: "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 definition of an in vitro diagnostic device is a medical device that is used to examine specimens, such as blood, tissue, or urine, that have been taken from the human body to detect diseases, conditions, or infections. This device fits that description perfectly as it tests cerebrospinal fluid (CSF) specimens to aid in the diagnosis of meningitis and/or encephalitis.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

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.

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

PLO, OOI, NSU

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 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.

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.

Mentions image processing

Yes

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Cerebrospinal fluid (CSF) specimens obtained via lumbar puncture

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

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

Not Found

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

Description of the training set: Not Found
Description of the test set:

Prospective Clinical Study:
- Sample Size: 1560 prospective CSF specimens (initially 1643, 83 excluded). 545 (35%) were previously frozen, 1015 (65%) were fresh.
- Data Source: CSF specimens collected at 11 geographically distinct U.S. study sites.
- Annotation Protocol: FilmArray ME Panel test results were compared to appropriate comparator/reference methods. For bacterial analytes, CSF bacterial culture was used. For viral and yeast analytes (CMV, EV, HSV-1, HSV-2, HHV-6, HPeV, VZV, C. neoformans/gattii), two PCR assays with bi-directional sequencing were used as comparator methods, targeting different nucleic acid sequences than the FilmArray ME Panel.
Testing of Preselected Archived Specimens:
- Sample Size: 235 archived specimens (25 negative, 210 positive). Of the 210 positives, 150 were confirmed by the comparator method.
- Data Source: Archived clinical specimens previously tested positive or negative for FilmArray ME Panel analytes.
- Annotation Protocol: 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). Only confirmed analytes were used for PPA calculations, but all specimens were used for NPA analyses. Specimens were randomized and blinded to users.
Testing of Contrived Specimens:
- Sample Size: For each analyte, at least 25 specimens were spiked at 2× LoD, and remaining at four additional concentrations.
- Data Source: Residual CSF specimens that had previously tested negative for all ME panel analytes by the FilmArray ME Panel and comparator methods.
- Annotation Protocol: 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. Specimens were frozen and distributed to prospective clinical study sites for testing.

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

Study Type: Analytical Performance (Reproducibility, Linearity/Assay Reportable Range, Traceability/Stability/Expected Values, Detection Limit, Analytical Reactivity (Inclusivity), Competitive Inhibition/Microbial Interference, Analytical Specificity/Cross-reactivity, Interfering Substances, Assay Cut-off, Specimen Stability, Fresh versus Frozen Study), Clinical Comparison (FilmArray vs. FilmArray 2.0 Systems, Matrix Equivalence Study), Clinical Studies (Prospective Clinical Study, Testing of Preselected Archived Specimens, Testing of Contrived Specimens).
Sample Size:
- Reproducibility: FilmArray System: 366 runs initiated (360 completed). FilmArray 2.0 System: 365 runs initiated (360 completed). 90 replicates per panel member.
- Detection Limit: Not specified for the study as a whole, but individual analyte LoD confirmation in 20 replicates.
- Analytical Reactivity (Inclusivity): 96 isolates tested.
- Competitive Inhibition/Microbial Interference: Not specified for study as a whole, specific numbers for organisms tested.
- Analytical Specificity/Cross-reactivity: 19 on-panel organisms, 107 off-panel organisms.
- Interfering Substances: Not specified for study as a whole.
- Specimen Stability: 10 replicates for each sample mix and storage condition.
- Fresh vs. Frozen Study: 60 contrived specimens, 22 clinical specimens.
- Clinical Performance Comparison (FilmArray vs. FilmArray 2.0): 149 specimens (21 positive clinical, 128 contrived).
- Matrix Equivalence Study: Not specified for study as a whole, 4 replicates for each dilution.
- Prospective Clinical Study: 1560 specimens.
- Preselected Archived Specimens: 235 specimens.
- Contrived Specimens (Clinical): At least 25 specimens per analyte.
AUC: Not Found
MRMC: Not Found
Standalone performance: Not Found
Key results:
- Reproducibility: Overall incidence of false S. pneumoniae results was 15 mg/mL albumin) and bleach (>0.1% v/v). All other substances did not interfere.
- Specimen Stability: Demonstrated 10/10 positive results for most analytes under various storage conditions, supporting stated recommendations (room temperature up to 24 hours, 2-8°C up to 7 days).
- Fresh versus Frozen Study: 100% concordance for fresh and frozen samples for H. influenzae, N. meningitidis, and HPeV. High PPA and NPA for other analytes. Overall Cp values variability within expected range. Supports use of frozen specimens in clinical and reproducibility studies.
- Clinical Comparison (FilmArray vs. FilmArray 2.0): Overall PPA of 97.5% (92.9% 95% CI lower bound) and NPA of 99.7% (99.3% 95% CI lower bound). Delta Tm values

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.

Summary

0

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)

1

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

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

2

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:

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. b(4)

3

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. b(4)

. 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.

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.

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 ●

4

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.

5

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.

6

Reproducibility of the FilmArray ME Panel on FilmArray

| Organism/Isolate
Tested | Concentration | Expected
Test Result | FilmArray Agreement with Expected Results | | | All Sites
(95%
Confidence
Interval) | |
|----------------------------|---------------------------------------------------------------------|-----------------------------------------------------|-------------------------------------------|-------------------------------------------|------------------|----------------------------------------------|----------------------------------|
| | | | Site A | Site B | Site C | | |
| | BACTERIA | | | | | | |
| E. coli K1
b(4) | Moderate Positive
3× LoD
3×103 CFU/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) | |
| E. coli K1
b(4) | Low Positive
1× LoD
1×103 CFU/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
96.7%
(96%-100%) | |
| E. coli K1
b(4) | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% | 180/180
100%
(98.0%-100%) | |
| H. influenzae
b(4) | Moderate Positive
3× LoD
3×103 CFU/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) | |
| H. influenzae
b(4) | Low Positive
1× LoD
1×103 CFU/mL | Detected | 29/30
96.7% | 30/30
100% | 30/30
100% | 89/90
98.9%
(94.0%-100%) | |
| | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% | 180/180
100%
(98.0%-100%) | |
| L. monocytogenes
b(4) | Moderate Positive
3× LoD
3×103 CFU/mL | Detected | 29/30
96.7% | 30/30
100% | 30/30
100% | 89/90
98.9%
(94.0%-100%) | |
| | Low Positive
1× LoD
1×103 CFU/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) | |
| | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% | 180/180
100%
(98.0%-100%) | |
| N. meningitidis | Negative
(No analyte) | Not Detected | 120/120
100% | 120/120
100% | 120/120
100% | 360/360
100%
(99.0%-100%) | |
| S. agalactiae
b(4) | Moderate Positive
3× LoD
3×103 CFU/mL | Detected | 29/30
96.7% | 30/30
100% | 27/30
90.0% | 86/90
95.6%
(89.0%-98.8%) | |
| | Low Positive
1× LoD
1×103 CFU/mL | Detected | 26/30
86.7% | 30/30
100% | 27/30
90.0% | 83/90
92.2%
(84.6%-96.8%) | |
| | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% | 180/180
100%
(98.0%-100%) | |
| S. pneumoniae | Negative
(No analyte) | Not Detected | 118/120
98.3% | 118/120
98.3% | 118/120
98.3% | 354/360a
98.3%
(96.4%-99.4%) | |
| VIRUSES/YEAST | | | | | | | |
| CMV | Negative
(No analyte) | Not Detected | 120/120
100% | 120/120
100% | 120/120
100% | 360/360
100%
(99.0%-100%) | |
| | Concentration | Expected
Test Result | FilmArray Agreement with Expected Results | | | All Sites
(95%
Confidence
Interval) | |
| Organism/Isolate
Tested | | | Site A | Site B | Site C | | |
| EV | Moderate Positive
$3\times LoD$
15 TCID50/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) | |
| | Coxsackievirus A9
b(4) | Low Positive
$1\times LoD$
5 TCID50/mL | Detected | 30/30
100% | 30/30
100% | 28/30
93.3% | 88/90
97.8%
(92.2%-99.7%) |
| | | | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% |
| HSV-1 | | | Negative
(No analyte) | Not Detected | 120/120
100% | 120/120
100% | 120/120
100% |
| | Moderate Positive
$3\times LoD$
150 TCID50/mL | | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) |
| | HSV-2
b(4) | Low Positive
$1\times LoD$
50 TCID50/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) |
| | | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% | 180/180
100%
(98.0%-100%) |
| HHV-6 | | Negative
(No analyte) | Not Detected | 120/120
100% | 119/120
99.2% | 120/120
100% | 359/360
99.7%
(98.5%-100%) |
| | Moderate Positive
$3\times LoD$
$1.5\times 10^3$
TCID50/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) | |
| | HPeV
b(4) | Low Positive
$1\times LoD$
500 TCID50/mL | Detected | 29/30
96.7% | 30/30
100% | 30/30
100% | 89/90
98.9%
(94.0%-100%) |
| | | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% | 180/180
100%
(98.0%-100%) |
| VZV
b(4) | | Moderate Positive
$3\times LoD$
0.3 TCID50/mL | Detected | 29/30
96.7% | 30/30
100% | 30/30
100% | 89/90
98.9%
(94.0%-100%) |
| | Low Positive
$1\times LoD$
0.1 TCID50/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) | |
| | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% | 180/180
100%
(98.0%-100%) | |
| C. gattii
b(4) | Moderate Positive
$3\times 10^3$ CFU/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) | |
| | Low Positive
$1\times 10^3$ CFU/mL | Detected | 30/30
100% | 30/30
100% | 30/30
100% | 90/90
100%
(96.0%-100%) | |
| | Organism/Isolate
Tested | Concentration | Expected
Test Result | FilmArray Agreement with Expected Results | | | |
| Site A | | | | Site B | Site C | All Sites
(95%
Confidence
Interval) | |
| | Negative
(No analyte) | Not Detected | 60/60
100% | 60/60
100% | 60/60
100% | 180/180
100%
(98.0%-100%) | |

7

8

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 BACTERIA | | | | | | | | | | |
| | Ecoli 3 | 3× LoD | 82.9 | 82.8 | 82.6 | 82.8 | 82.1 | 82.3 | 82.1 | 82.2 |
| E. coli K1
b(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. influenzae
b(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. agalactiae
b(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 |

11

| (× LoD) | Mean Tm (°C) (±StDev) | | |----------------------------------------------| | Test Result
(Organism/Isolate Tested) | | | | | | | VIRUSES | | EV
(Coxsackievirus A9)
b(4) | EV2 | 3× LoD | | | | | b(4) | | | | | | | HSV-2 | | b(4) | | | | | | | HPeV
b(4) | HPeV | | | | | | | | | | | | VZV
b(4) | | | | | | | | | YEAST | | C. neoformans/gattii
(C. gattii)
(b(4) | Cryptococcus | 30× LoDa (FilmArray) | | | | | | | Assay | Concentration 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 |
| | | | | | | | | |
| 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 |
| | ±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 |
| | ±0.2 | ±0.2 | ±0.3 | ±0.4 | ±0.3 | ±0.3 | ±0.3 | ±0.3 |
| 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 |
| 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 |
| | ±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 |
| | | | | | | | | |
| 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 Test
Result | Species/Isolate Tested | LoD Concentration | Detection at
LoD
Concentration | | |
|-------------------------|-------------------------------------------------------|----------------------------------------|--------------------------------------|--|--|
| | BACTERIA | | | | |
| E. coli K1 | E. coli K1, strain C5 b(4)
b(4) | 1×103 CFU/mL | 20/20
100% | | |
| H. influenzae | H. influenzae, strain AMC 36-A-1
b(4) | 1×103 CFU/mL | 20/20
100% | | |
| L. monocytogenes | L. monocytogenes, strain 1071/53,
b(4) | 1×103 CFU/mL | 20/20
100% | | |
| N. meningitidis | N. meningitidis, strain M-1574
b(4) | 100 CFU/mL | 19/20
95% | | |
| S. agalactiae | S. agalactiae, type strain, G19,
group B
b(4) | 1×103 CFU/mL | 20/20
100% | | |
| S. pneumoniae | S. pneumoniae, strain SV 1,
serotype 1
b(4) | 100 cells/mL | 19/20
95% | | |
| | VIRUSES | | | | |
| CMV | CMV, strain AD-169
b(4) | 100 TCID50/mL
(4.30×103 copies/mL) | 20/20
100% | | |
| EV
(Species A-D) | Coxsackievirus A6, species A,
b(4) | 50 TCID50/mL | 20/20
100% | | |
| | Coxsackievirus A9, species B
b(4) | 5 TCID50/mL | 20/20
100% | | |
| | Coxsackievirus A17, species C,
strain G-12
b(4) | 5 TCID50/mL | 20/20
100% | | |
| | EV 70, species D, b(4)
b(4) | 50 TCID50/mL | 20/20
100% | | |
| HSV-1 | HSV-1, strain MacIntyre
b(4) | 250 TCID50/mL
(1.51×103 copies/mL) | 20/20
100% | | |
| HSV-2 | HSV-2, strain MS
b(4) | 50 TCID50/mL
(1.29×103 copies/mL) | 20/20
100% | | |
| HHV-6 | HHV-6A, strain U1102
b(4) | 1×104 copies/mL | 19/20
95% | | |
| | HHV-6B, strain HST
b(4) | 1×104 copies/mL | 19/20
95% | | |
| HPeV | HPeV, type 3
b(4) | 500 TCID50/mL | 19/20
95% | | |
| VZV | VZV, strain Ellen
b(4) | 0.10 TCID50/mL
(1.66×103 copies/mL) | 20/20
100% | | |
| | YEAST | | | | |

Limit of Detection Results for FilmArray ME Panel Analytes

14

| ME Panel Test
Result | Species/Isolate Tested | LoD Concentration | Detection at
LoD Concentration |
|-------------------------|-----------------------------------------|-------------------|-----------------------------------|
| C.
neoformans/gattii | C. neoformans var. grubii, type
b(4) | 100 CFU/mL | 20/20
100% |
| | C. gattii, strain A6MR38,
b(4) | 100 CFU/mL | 20/20
100% |

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 ME
Panel
Test Result | # of Isolates Tested
and Detected | Concentration
Detected | 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) strains
of 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 (serotypes
W135, A, B, C, D, Y and DNA from a strains
with 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 ME
Panel
Test Result | # of Isolates Tested
and Detected | Concentration
Detected | 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 of
Cryptococcus neoformans and Cryptococcus
gattii |

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] | Concentration
Tested | Test
Result |
|---------------------|------------|-------------------------------------------------|----------------------------------|----------------|
| 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-typeable
biogroup 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

| Organism | Isolate ID | Type
[Strain] | Concentration
Tested | Test
Result |
|----------|------------|-------------------------------|-------------------------|----------------|
| | 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 | Isolate ID | Type | Concentration
Tested | Test
Result |
|-----------------------------------|--------|------------|-----------|-------------------------------------|----------------|
| Listeria
monocytogenes | 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 | Isolate ID | Serotype | Concentration
Tested | Test
Result |
|------------------------------------------|------------|-------------------------------|---------------------------------------------------|----------------|
| | | Serotype W135 | 100 CFU/mL
(1× LoD)
[~1.9×103
copies/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 variant
ctrA gene | 5.6×103 copies/mL
(~3× LoD) | Detected |

Results for Neisseria meningitidis Inclusivity Testing

18

| Organism | Isolate ID | Serotype | Concentration
Tested | Test
Result |
|-----------------------------|------------|-------------------------------|------------------------------------|----------------|
| Streptococcus
agalactiae | b(4) | Serotype I a/c
Type 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 | Concentration
Tested | 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 | Concentration
Tested | Test
Result |
|------------------------------------------------|------------|--------|----------------------------|---------------------------------------------------------------------|----------------|
| 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

19

| Organism | Isolate ID | Strain | Location/
Year Isolated | Concentration
Tested | Test
Result |
|----------|------------|--------|----------------------------|-----------------------------|----------------|
| | b(4) | Davis | 1957 | $1.3 \times 10^4$ copies/mL | Detected |
| | | Toledo | Virginia, USA
2011 | $1.3 \times 10^4$ copies/mL | Detected |

Results for Enterovirus (EV) Inclusivity Testing

Species	Virus/Serotype	Strain Location/Year	Concentration Tested	Test Result
A	Coxsackievirus A6	Gdula
b(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-12
b(4)	5 TCID50/mL
(1 × LoD)	Detected
	Coxsackievirus A21	Kuykendall
b(4)	15 TCID50/mL	Detected
	Coxsackievirus A24	DN-19
b(4)	15 TCID50/mL	Detected
D	Enterovirus 70	J670/71
b(4)	50 TCID50/mL
(1 × LoD)	Detected
	Enterovirus 68
(aka Rhinovirus 87)	-	150 TCID50/mL	Detected
			50 TCID50/mL	Detected

20

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 | Isolate ID | Strain | Concentration
Tested | Test
Result |
|-----------------------------------|------------|-----------------|--------------------------------------------------|----------------|
| | | 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 |
| | | -2011-1
b(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 | Concentration
Tested | Test
Result |
|-----------------------------------|------------|---------|-------------------------------------------------|----------------|
| Herpes simplex
virus 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 | Concentration
Tested | 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 | Concentration
Tested | Test
Result |
|------------------------------|------|------------|---------------------------|----------------|
| 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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| Organism | Isolate ID | Serotype | Concentration
Tested | Test
Result |
|----------|------------|------------|---------------------------------|----------------|
| | 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)	Ellen
VR-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 | Concentration
Tested | Test Result |
|------------------------------------|------------|------------------------------------------------------|------------------|-------------------------|-------------|
| Cryptococcus
neoformans | b(4) | type strain, CBS 132 | b(4) | 300 CFU/mL | Detected |
| | | Serotype A, strain H99
type strain of var. grubii | | 100 CFU/mL
(1 × LoD) | Detected |
| | | Serotype A
strain WM148, type VNI | | 300 CFU/mL | Detected |
| | | Serotype AD
strain WM628, type VNIII | | 300 CFU/mL | Detected |
| | | Serotype D
strain WM629, type VNIV | | 300 CFU/mL | Detected |
| Cryptococcus
gattii | | Serotype B
strain WM179, type VGI | | 300 CFU/mL | Detected |
| | | Serotype B
strain R272, type VGIIb | | 300 CFU/mL | Detected |
| | | Unknown serotype
strain R38, type VGIIc | | 100 CFU/mL
(1 × LoD) | Detected |
| | | Serotype B
strain WM161, type VGIII | | 300 CFU/mL | Detected |

22

| Organism | Isolate ID | Serotype/Strain Info | Isolate Location | Concentration
Tested | 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.

23

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.

24

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 Syncytial
Virus
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 Encephalitis
Virus
Staphylococcus aureus	Escherichia vulneris	Coronavirus NL63	West Nile Virus
Staphylococcus capitis	Haemophilus ducreyi	Coronavirus OC43	Fungi
Staphylococcus epidermidis	Haemophilus
haemolyticusa	Dengue virus (Type 2)	Aspergillus fumigatus
Staphylococcus haemolyticus	Haemophilus
parahaemolyticus	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	Human
Immunodeficiency Virus	Cryptococcus
amylolentusc
Streptococcus dysgalactiae	Neisseria lactamica	Human Rhinovirus A1b	Cryptococcus laurentii
Streptococcus intermedius	Neisseria mucosa	Human Rhinovirus
A16b	Cryptococcus
uniguttulatus
Streptococcus mitis (tigurinus)	Neisseria sicca	Human Rhinovirus B3b	Filobasidium
capsuligenum
Streptococcus mutans	Pantoea agglomerans	Human Rhinovirus
B83b	Pathogenic Protists
Streptococcus oralis	Proteus mirabilis	Influenza A H1N1	Naeglaria fowleri
Streptococcus
pseudopneumoniae	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 Encephalitis
Virus
	Shigella flexneri	Measles Virus
	Shigella sonnei	Mumps Virus
	Treponema pallidum

Off-Panel Organisms Evaluated for Cross-Reactivity

25

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.

26

| Endogenous Substances | Reference Concentration | | Tested
Concentration | 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 Protein
45 mg/dL
(0.45 mg/mL) | Total Protein
50-500 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.

28

			# 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:H7
b(4)	3.00E+03 CFU/mL	10/10	10/10	10/10	10/10	10/10
Haemophilus
influenzae	type b. biotype I
b(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
Streptococcus
pneumoniae	SV 1/serotype 1
b(4)	300 cells/mL	10/10	10/10	10/10	10/10	10/10
VIRUSES
Cytomegalovirus	AD-169
b(4)	300 TCID50/mL	10/10	10/10	10/10	10/10	10/10
Enterovirus	Coxsackievirus A6,
Gdula, group A
b(4)	150 TCID50/mL	10/10	10/10	10/10	10/10	10/10
Herpes simplex virus
1	MacIntyre
b(4)	150 TCID50/mL	10/10	9/10	10/10	10/10	10/10
Human parechovirus	type 3
b(4)	1.50E+03
TCID50/mL	10/10	10/10	10/10	10/10	10/10
YEAST
Cryptococcus
neoformans/gattii	C. neoformans; H99,
type strain
b(4)	1.50E+03 CFU/mL	10/10	10/10	10/10	10/10	10/10
	C. gattii; A6MR38;
AFLP6C; VGIIc
b(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

29

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 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

32

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 (%)
E. coli K1
H. influenzae
L. monocytogenes	CSF bacterial culture
N. meningitidis
S. agalactiae
S. pneumoniae
CMV
EV
HSV-1	Two PCR assays with
bi-directional
sequencinga	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.

34

| Analyte | Sensitivity
(compared to culture) | | | Specificity
(compared to culture) | | | |
|----------------------|-----------------------------------------------------------------------------------|------------|------------|-----------------------------------------------------------------------------------|-------------------|-------------|------------------|
| | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI | |
| Bacteria | | | | | | | |
| E. coli K1 | Fresh
1/1 | 100 | - | 1014/1014 | 100 | 99.6-100 | |
| | Frozen
1/1 | 100 | - | 543/544 | 99.8 | 99.0-100 | |
| | Overall
2/2 | 100 | 34.2-100 | 1557/1558b,c | 99.9 | 99.6-100 | |
| H. influenzae | Fresh
1/1 | 100 | - | 1013/1014 | 99.9 | 99.4-100 | |
| | Frozen
0/0 | - | - | 545/545 | 100 | 99.3-100 | |
| | Overall
1/1 | 100 | - | 1558/1559d | 99.9 | 99.6-100 | |
| L. monocytogenes | Fresh
0/0 | - | - | 1015/1015 | 100 | 99.6-100 | |
| | Frozen
0/0 | - | - | 545/545 | 100 | 99.3-100 | |
| | Overall
0/0 | - | - | 1560/1560 | 100 | 99.8-100 | |
| N. meningitidis | Fresh
0/0 | - | - | 1015/1015 | 100 | 99.6-100 | |
| | Frozen
0/0 | - | - | 545/545 | 100 | 99.3-100 | |
| | Overall
0/0 | - | - | 1560/1560 | 100 | 99.8-100 | |
| S. agalactiae | Fresh
0/1 | 0.0 | - | 1013/1014 | 99.9 | 99.4-100 | |
| | Frozen
0/0 | - | - | 545/545 | 100 | 99.3-100 | |
| | Overall
0/1e | 0.0 | - | 1558/1559e | 99.9 | 99.6-100 | |
| S. pneumoniae | Fresh
2/2 | 100 | 34.2-100 | 1008/1013 | 99.5 | 98.8-99.8 | |
| | Frozen
2/2 | 100 | 34.2-100 | 536/543 | 98.7 | 97.4-99.4 | |
| | Overall
4/4 | 100 | 51.0-100 | 1544/1556f | 99.2 | 98.7-99.6 | |
| Analyte | Positive Percent Agreement
(compared to PCR with bi-directional
sequencing) | | | Negative Percent Agreement
(compared to PCR with bi-directional
sequencing) | | | |
| | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI | |
| Viruses | | | | | | | |
| CMV | Fresh
2/2 | 100 | 34.2-100 | 1010/1013 | 99.7 | 99.1-99.9 | |
| | Frozen
1/1 | 100 | 20.7-100 | 544/544 | 100 | 99.3-100 | |
| | Overall
3/3 | 100 | 43.9-100 | 1554/1557g | 99.8 | 99.4-99.9 | |
| EV | Fresh
43/44 | 97.7 | 88.2-99.6 | 965/971 | 99.4 | 98.7-99.7 | |
| | Frozen
1/2 | 50.0 | - | 542/543 | 99.8 | 99.0-100 | |
| | Overall
44/46h | 95.7 | 85.5-98.8 | 1507/1514h | 99.5 | 99.0-99.8 | |
| HSV-1 | Fresh
1/1 | 100 | - | 1013/1014 | 99.9 | 99.4-100 | |
| | Frozen
1/1 | 100 | - | 543/544 | 99.8 | 99.0-100 | |
| | Overall
2/2 | 100 | 34.2-100 | 1556/1558i | 99.9 | 99.5-100 | |
| HSV-2 | Fresh
6/6 | 100 | 61.0-100 | 1008/1009 | 99.9 | 99.4-100 | |
| | Frozen
4/4 | 100 | 51.0-100 | 540/541 | 99.8 | 99.0-100 | |
| | Overall
10/10 | 100 | 72.2-100 | 1548/1550j | 99.9 | 99.5-100 | |
| HHV-6 | Fresh
13/15 | 86.7 | 62.1-96.3 | 997/1000 | 99.7 | 99.1-99.9 | |
| | Frozen
5/6 | 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

35

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.

36

| Subject
age | CSF
WBC | FilmArray
Result | Comparator Culture/
Investigation PCRa | Diagnosis Reported in Medical Record |
|----------------|------------|---------------------|-------------------------------------------|--------------------------------------------------|
|