FILMARRAY BLOOD CULTURE IDENTIFICATION (BCID) PANEL

K130914 · Biofire Diagnostics, Inc. · PEN · Jun 21, 2013 · Microbiology

Device Facts

• Record ID: K130914
• Device Name: FILMARRAY BLOOD CULTURE IDENTIFICATION (BCID) PANEL
• Applicant: Biofire Diagnostics, Inc.
• Product Code: PEN · Microbiology
• Decision Date: Jun 21, 2013
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3365
• Device Class: Class 2

Intended Use

The FilmArray Blood Culture Identification (BCID) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with the FilmArray Instrument. The FilmArray BCID Panel is capable of simultaneous detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The BCID assay is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrates the presence of organisms as determined by Gram stain. The following gram-positive bacteria, gram-negative bacteria, and yeast are identified using the FilmArray BCID Panel: Enterococci, Listeria monocytogenes, commonly encountered Staphylococci (including specific differentiation of Staphylococcus aureus), commonly encountered Streptococci (with specific differentiation of Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes), Acinetobacter baumannii, commonly encountered Enterobacteriaceae (including specific differentiation of the Enterobacter cloacae complex, Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus, and Serratia marcescens), Haemophilus influenzae, Neisseria meningitidis (encapsulated), Pseudomonas aeruginosa, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis. The FilmArray BCID Panel also contains assays for the detection of genetic determinants of resistance to methicillin (mecA), vancomycin (vanA and vanB), and carbapenems (blakpc) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, and carbapenems exist. FilmArray BCID is indicated as an aid in the diagnosis of specific agents of bacteremia and fungemia and results should be used in conjunction with other clinical and laboratory findings. Positive FilmArray results do not rule out co-infection with organisms not included in the FilmArray BCID Panel. FilmArray BCID is not intended to monitor treatment for bacteremia or fungemia. Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the FilmArray BCID Panel, and for species determination of some Staphylococci, Enterococci, Streptococci, and Enterobacteriaceae that are not specifically identified by the FilmArray BCID Panel assays.

Device Story

FilmArray BCID Panel is a multiplexed nucleic acid test for positive blood culture samples. Input: positive blood culture sample mixed with buffer and hydration solution. Process: automated nucleic acid extraction via mechanical lysis/magnetic beads; nested multiplex PCR; DNA melt curve analysis. Output: identification of 24 organisms/groups and 3 resistance markers. Used in clinical labs; operated by technicians. Results provided to clinicians to aid diagnosis of bacteremia/fungemia. Benefits: rapid identification (under 1 hour) of pathogens and resistance markers to guide clinical decision-making.

Clinical Evidence

Clinical study evaluated 2207 blood culture specimens (1568 prospective, 639 seeded) across 8 U.S. sites. Compared to standard microbiological/biochemical methods and 16S sequencing. Overall sensitivity/PPA and specificity/NPA were high across targets (e.g., Enterococcus 97.7% PPA/99.8% NPA; S. aureus 98.4% PPA/99.8% NPA; Acinetobacter baumannii 100% PPA/99.8% NPA). Resistance gene performance compared to PCR/sequencing.

Technological Characteristics

Multiplex nucleic acid test using nested PCR and DNA melt curve analysis. Reagents are freeze-dried in a single-use pouch. Instrument uses pneumatic bladders/pistons for fluidics and Peltier devices for thermal cycling. Connectivity: standalone instrument. Sterilization: not specified.

Indications for Use

Indicated for use as an aid in the diagnosis of bacteremia and fungemia in patients with positive blood culture samples (identified by continuous monitoring systems and Gram stain). Detects 24 bacterial/yeast targets and 3 antimicrobial resistance genes (mecA, vanA/B, blaKPC). Not for monitoring treatment.

Regulatory Classification

Identification

A multiplex nucleic acid assay for identification of microorganisms and resistance markers from positive blood cultures is a qualitative in vitro device intended to simultaneously detect and identify microorganism nucleic acids from blood cultures that test positive by Gram stain or other microbiological stains. The device detects specific nucleic acid sequences for microorganism identification as well as for antimicrobial resistance. This device aids in the diagnosis of bloodstream infections when used in conjunction with other clinical and laboratory findings. However, the device does not replace traditional methods for culture and susceptibility testing.

Special Controls

In combination with the general controls of the FD&C Act, the Verigene® Gram Positive Blood Culture Nucleic Acid Test is subject to the following special controls: The special controls for the BC-GP Assay are contained in the guideline document entitled "Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures."

*Classification.* Class II (special controls). The special control for this device is FDA's guideline document entitled “Class II Special Controls Guideline: Multiplex Nucleic Acid Assay for Identification of Microorganisms and Resistance Markers from Positive Blood Cultures.” For availability of the guideline document, see § 866.1(e).

Predicate Devices

• Nanosphere Verigene® Gram-Positive Blood Culture Nucleic Acid Test (BC-BG) (K122514)

Submission Summary (Full Text)

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jC130914

## 510(k) Summary BioFire Diagnostics, Inc.

# JUN 2 1 2013

# FilmArray Blood Culture Identification (BCID) Panel Kit

Introduction: According to the requirements of 21 CFR 807.92, the following information provides sufficient detail to understand the basis for a determination of substantial equivalence.

# Submitted by:

BioFire Diagnostics, Inc. 390 Wakara Way Salt Lake City, UT 84108

Telephone: [phone] Facsimile: 801-588-0507

Contact: Beth Lingenfelter, ext. 407

ﻟﻠﺴ

Date Submitted: March 30, 2013

### Device Name and Classification:

Trade Name: FilmArray BCID Panel

Regulation Number: 21 CFR 866.3365

Classification Name: Multiplex devices that use DNA hybridization to detect bacteria and their resistance markers

# Predicate Device:

K122514 - Nanosphere Verigene® Gram-Positive Blood Culture Nucleic Acid Test (BC-BG)

#### Intended Use:

The FilmArray Blood Culture Identification (BCID) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with the FilmArray Instrument. The FilmArray BCID Panel is capable of simultaneous detection and identification of multiple bacterial and yeast nucleic acids and select genetic determinants of antimicrobial resistance. The BCID assay is performed directly on blood culture samples identified as positive by a continuous monitoring blood culture system that demonstrates the presence of organisms as determined by Gram stain.

The following gram-positive bacteria, gram-negative bacteria, and veast are identified using the FilmArray BCID Panel: Enterococci, Listeria monocytogenes, commonly

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encountered Staphvlococci (including specific differentiation of Staphylococcus aureus). commonly encountered Streptococci (with specific differentiation of Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes), Acinetobacter baumannii, commonly encountered Enterobacteriaceae (including specific differentiation of the Enterobacter cloacae complex. Escherichia coli, Klebsiella oxytoca. Klebsiella pneumoniae, Proteus, and Serratia marcescens), Haemophilus influenzae, Neisseria meningitidis (encapsulated), Pseudomonas aeruginosa, Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis.

The FilmArray BCID Panel also contains assays for the detection of genetic determinants of resistance to methicillin (mecA), vancomycin (vanA and vanB), and carbapenems (blaker) to aid in the identification of potentially antimicrobial resistant organisms in positive blood culture samples. The antimicrobial resistance gene detected may or may not be associated with the agent responsible for disease. Negative results for these select antimicrobial resistance gene assays do not indicate susceptibility, as multiple mechanisms of resistance to methicillin, vancomycin, and carbapenems exist.

FilmArray BCID is indicated as an aid in the diagnosis of specific agents of bacteremia and fungemia and results should be used in conjunction with other clinical and laboratory findings. Positive FilmArray results do not rule out co-infection with organisms not included in the FilmArray BCID Panel. FilmArray BCID is not intended to monitor treatment for bacteremia or fungemia.

Subculturing of positive blood cultures is necessary to recover organisms for susceptibility testing and epidemiological typing, to identify organisms in the blood culture that are not detected by the FilmArray BCID Panel, and for species determination of some Staphylococci, Enterococci, Streptococci, and Enterobacteriaceae that are not specifically identified by the FilmArray BCID Panel assays.

# Device Description:

The FilmArray Blood Culture Identification (BCID) Panel is a multiplex nucleic acid test designed to be used with the FilmArray Instrument. The FilmArray BCID pouch contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex PCR with DNA melt analysis. The FilmArray Blood Culture Identification (BCID) Panel simultaneously tests a single positive blood culture sample to provide results for 24 different organisms and organism groups that cause bloodstream infections and three genetic markers that are known to confer antimicrobial resistance (see Table 1).

| Gram-Positive Bacteria | Gram-Negative Bacteria | Yeast |
|--------------------------|------------------------------|--------------------------------|
| Enterococcus | Acinetobacter baumannii | Candida albicans |
| Listeria monocytogenes | Enterobacteriaceae | Candida glabrata |
| Staphylococcus | Enterobacter cloacae complex | Candida krusei |
| Staphylococcus aureus | Escherichia coli | Candida parapsilosis |
| Streptococcus | Klebsiella oxytoca | Candida tropicalis |
| Streptococcus agalactiae | Klebsiella pneumoniae | Antimicrobial resistance genes |
| Streptococcus pneumoniae | Proteus | mecA – methicillin resistance |

### Table 1, FilmArray RCID Panel Test Results

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| Gram-Positive Bacteria | Gram-Negative Bacteria | Yeast |
|------------------------|---------------------------------------|--------------------------------|
| Streptococcus pyogenes | Serratia marcescens | vanA/B - vancomycin resistance |
| | Haemophilus influenzae | blaKPC - carbapenem resistance |
| | Neisseria meningitidis (encapsulated) | |
| | Pseudomonas aeruginosa | |

A test is initiated by loading Hydration Solution and a positive blood culture sample mixed with the provided Sample Buffer into the FilmArray BCID pouch. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the FilmArray Software guides the user though the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

The FilmArray Instrument contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister. it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical lysis and standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the FilmArray performs a nested multiplex PCR that is exceuted in two stages. During the first stage, the FilmArray performs a single, large volume, highly multiplexed PCR reaction which includes all primers of the outer primer sets. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green Plus-, BioFire Diagnostics). 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 200 stage PCR, or nested PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 2" 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 2nd stage PCR captures fluorescent images of the PCR 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 and antimicrobial resistance gene on the panel.

### Substantial Equivalence:

.

The Nanosphere Verigene® Gram-Positive Blood Culture Nucleic Acid Test is a qualitative, multiplexed in vitro diagnostic test for the simultaneous detection and identification of potentially pathogenic gram-positive bacteria which may cause

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bloodstream infection. Table 2 outlines the similarities between the two systems and Table 3 outlines the differences.

| Element | FilmArray BCID Panel | Nanosphere Verigene® Gram-Positive<br>Blood Culture Nucleic Acid Test |
|------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Organisms<br>Detected | Enterococci, Staphylococci (including<br>specific differentiation of <i>Staphylococcus aureus</i> ), Streptococci (with specific<br>differentiation of <i>Streptococcus agalactiae, Streptococcus pneumoniae</i> ,<br>and <i>Streptococcus pyogenes</i> ) and<br>resistance markers <i>mecA, vanA</i> , and <i>vanB</i> . | Same<br>See below for differences |
| Analyte | DNA | Same |
| Technological<br>Principles | Multiplex nucleic acid | Same<br>See below for differences |
| Sample<br>Processing and<br>Purification | Automated by instrument | Same |
| Controls | Two controls are included in each reagent<br>pouch to control for sample processing and<br>both stages of PCR and melt analysis. | Internal procedural/instrument quality<br>controls; Internal Negative Control,<br>Sample processing control, external<br>positive and negative assay controls. |
| User<br>Complexity | Moderate/Low | Same |

| Table 2. Similarities Between the FilmArray BCID Panel and the Nanosphere Verigene® Gram- | |
|-------------------------------------------------------------------------------------------|--|
| Positive Blood Culture Nucleic Acid Test. | |

| Table 3. Differences Between FilmArray Respiratory Panel Test System and the Nanosphere | |
|-----------------------------------------------------------------------------------------|--|
| Verigene® Gram-Positive Blood Culture Nucleic Acid Test. | |

| Element | FilmArray BCID Panel | Nanosphere Verigene® Gram-Positive<br>Blood Culture Nucleic Acid Test |
|-----------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Specimen<br>Types | Positive blood culture samples containing<br>gram-positive, gram-negative bacteria,<br>and/or yeast. | Positive blood culture bottles<br>(unspecified) which contain gram-<br>positive bacteria. |
| Organisms<br>Detected | Detection of additional targets: Listeria<br>monocytogenes, Acinetobacter<br>baumannii, Enterobacteriaceae<br>(including specific differentiation of<br>Enterobacter cloacae complex species,<br>Escherichia coli, Klebsiella oxytoca,<br>Klebsiella pneumoniae, Proteus, and<br>Serratia marcescens), Haemophilus<br>influenzae, Neisseria meningitidis,<br>Pseudomonas aeruginosa, Candida<br>albicans, Candida glabrata, Candida<br>krusei, Candida parapsilosis, Candida<br>tropicalis, and resistance marker blaKPC | Tests only for gram positive bacteria.<br>Tests for Listeria spp. rather than<br>Listeria monocytogenes. Includes testing<br>for additional Staphylococcus spp.:<br>Staphylococcus epidermidis,<br>Staphylococcus lugdunensis, as well as<br>testing for specific Enterococcus spp.:<br>Enterococcus faecalis,<br>Enterococcus faecium. Includes testing<br>for an additional Streptococcus spp.:<br>Streptococcus anginosus group. Does<br>not include testing for blaKPC |

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| Element | FilmArray BCID Panel | Nanosphere Verigene® Gram-Positive<br>Blood Culture Nucleic Acid Test |
|-----------------------------|-------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Technological<br>Principles | Nested multiplex PCR followed by high<br>resolution melting analysis to confirm<br>identity of amplified product. | Qualitative, multiplexed test for the<br>detection of specific nucleic acid targets<br>in a microarray format using capture and<br>mediator oligonucleotides for gold<br>nanoparticle probe-based endpoint<br>detection. |
| Instrumentation | FilmArray Instrument | Verigene Reader and Processor SP |
| Time to result | Less than 1 hour | 2.5 hours |
| Test<br>Interpretation | Automated test interpretation and report<br>generation. User cannot access raw data. | Diagnostic Software/Decision<br>Algorithm. |

# Summary of Performance Data

# Clinical Performance

The clinical performance of the FilmArray BCID Panel was established during a two armed clinical study which was conducted at eight U.S. clinical sites over an eight month time period. The study included a prospective residual blood culture arm and a seeded blood culture arm. In the prospective arm, 1635 prospectively-collected residual blood culture samples (pediatric and adult) were initially included in the study. Sixty-seven (67) specimens were excluded from the study. The most common reasons for exclusion were that the specimens were >8 hours past positivity, incomplete reference/comparator data were provided, or the specimen was from a subject who had a previous specimen included in the study. In the seeded culture arm, analytes proven to be of low prevalence in the prospective arm were evaluated by seeding previously characterized isolates into blood culture bottles and incubating until positivity. A total of 716 seeded cultures were initiated for the study. Seventy-seven (77) cultures were excluded from the study. The most common reasons for exclusion were that the specimens were >8 hours past positivity, the seeded culture was not called positive by the automated blood culture system. or the culture was contaminated or inconsistent with the intended seed organism. The final specimen set consisted of 2207 blood cultures (1568 prospective and 639 seeded). All cultures were grown in Becton Dickinson BACTECTM Plus Aerobic/F Medium. Table 4 provides a summary of demographic information for the 1568 specimens included in the prospective arm of the study.

| | | Table 4. Demographic Summary for Prospective Arm of FilmArray BCID Clinical Evaluation |
|--|--|----------------------------------------------------------------------------------------|
|--|--|----------------------------------------------------------------------------------------|

| Prospective Study Specimens | |
|-----------------------------|---------------------|
| Total Specimens | 1568 |
| Sex | Number of Specimens |
| Male | 917 (58%) |
| Female | 651 (42%) |
| Age Group | Number of Specimens |
| ≤ 1 year | 57 (4%) |

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| Prospective Study Specimens | |
|-----------------------------|-----------|
| Total Specimens | 1568 |
| 1 - 17 years | 92 (6%) |
| 18 - 44 years | 281 (18%) |
| 45 - 64 years | 583 (37%) |
| 65 - 84 years | 442 (28%) |
| ≥ 85 years | 113 (7%) |

Positive blood cultures (prospective and seeded) were tested with the FilmArray BCID Panel. The performance of FilmArray BCID was evaluated by comparing the FilmArray BCID test result for each panel member with the appropriate comparator/reference methods shown in Table 5.

| | Table 5. Reference/Comparator Methods used to Assess FilmArray BCID Performance | | |
|--|---------------------------------------------------------------------------------|--|--|
|--|---------------------------------------------------------------------------------|--|--|

| Test Result | Reference/Comparator Method(s) |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| All organism detections<br>except Acinetobacter baumannii | Standard manual and automated microbiological/biochemical identification<br>methods a |
| Acinetobacter baumannii detection | Standard manual and automated microbiological/biochemical identification<br>methods<br>Plus<br>16S PCR with bi-directional sequencing of all A. calcoaceticus-baumannii<br>complex isolates for characterization as A. baumannii or non-A. baumannii a |
| Antimicrobial resistance gene detections<br>in specimens in which an associated organism was detected<br>( <i>mecA</i> from <i>Staphylococcus</i> ; <i>vanA/B</i> from <i>Enterococcus</i> ,<br><i>KPC</i> from <i>Enterobacteriaceae</i> , <i>Acinetobacter baumannii</i> ,<br>and <i>Pseudomonas aeruginosa</i> ) | Method 1: PCR with bi-directional sequencing for specific resistance gene<br>- direct from blood culture b<br><br>Method 2: PCR with bi-directional sequencing for specific resistance gene from<br>- appropriate cultured isolates b<br><br>Informational: Standard manual and automated phenotypic antimicrobial<br>susceptibility testing of appropriate cultured isolates (methicillin resistance,<br>vancomycin resistance, and carbapenem resistance (and/or carbapenemase<br>production) according to current CLSI criteria) c |

" Performance of FilmArray BCID develing all organisms was compared to standard manual and automated microbiological/biochemical identification methods. Additionally isolatified as being members of the A. calcoaceticus-baunamii vomplex were subjected to 16S PCR and bi-directional sequencing to categorize the is being A. baunamii for final comparison to the FilmArray BCID A. baunamii-specific results required a sequencing result of adequate quality to match sequences of A. baunamii (or negative result if sequences match non-A. baunannii organisms) deposited in the National Center for Biotechnology Information (NCB) GenBank database (www.nebi.nlm.nlh.gov), with an acceptable E-value. This was required due to the inability of phenotypic identification nethous to adequately discriminate between members of the A. calcoaceticus-baumannii complex.

" Performance of FilmArray BCID detecting antimicrobial resistance genes (nec.4, van KPC) was compared to gene-specific PCR tests with bi-directional sequencing. The assays were designed to amplify different sequences than those targeted by FilmArray BCID. Positive results required a sequencing result of adequate of the expect gene deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.goy), with an acceptable E-value,

6 Performance of FilmArray BCID as compared to phenotypic antimicrobial susceptibility testing was performed for informational purposes. The phenotypic methods were performed in accordance with current CLSI criteria.

A total of 2207 blood culture specimens (1568 prospective and 639 seeded) were evaluated in the FilmArray BCID clinical evaluation. Specimens were tested by FilmArray BCID either fresh or from frozen aliquots. A total of 1240 specimens were tested fresh (821 prospective and 419 seeded) and 967 specimens were tested frozen (747 prospective and 220 seeded). Clinical sensitivity or positive percent agreement (PPA) was calculated as 100% x (TP/TP + FN). True positive (TP) indicates that both FilmArray BCID and the reference/comparator method had a positive result for a specific

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analyte, and false negative (FN) indicates that the FilmArray BCID result was negative while the reference/comparator method was positive. Clinical specificity or negative percent agreement (NPA) was calculated as 100% x (TN/TN + FP). True negative (TN) indicates that both FilmArray BCID and the reference/comparator method had a negative result for a specific analyte, and false positive (FP) indicates that the FilmArray BCID result was positive while the reference/comparator method was negative. The exact binomial two-sided 95% confidence interval was calculated. The results are summarized in Tables 6 - 10.

| Gram-Positive Bacteria | | Sensitivity/PPA* | | | Specificity/NPA* | | |
|------------------------------------------|--------------------|------------------|------|-----------|------------------|------|-----------|
| | | TP/TP + FN | % | 95% CI | TN/TN + FP | % | 95% CI |
| Enterococcus | Prospective Fresh | 55/55 | 100 | 93.5-100 | 762/766 | 99.5 | 98.7-99.9 |
| | Prospective Frozen | 43/46 | 93.5 | 82.1-98.6 | 701/701 | 100 | 99.5-100 |
| | Seeded Fresh | 12/12 | 100 | 73.5-100 | 407/407 | 100 | 99.1-100 |
| | Seeded Frozen | 17/17 | 100 | 80.5-100 | 203/203 | 100 | 98.2-100 |
| | Overall | 127/130 | 97.7 | 93.4-99.5 | 2073/2077 | 99.8 | 99.5-99.9 |
| Listeria<br>monocytogenes | Prospective Fresh | 0/0 | - | - | 821/821 | 100 | 99.6-100 |
| | Prospective Frozen | 0/0 | - | - | 747/747 | 100 | 99.5-100 |
| | Seeded Fresh | 23/23 | 100 | 85.2-100 | 396/396 | 100 | 99.1-100 |
| | Seeded Frozen | 13/13 | 100 | 75.3-100 | 207/207 | 100 | 98.2-100 |
| | Overall | 36/36 | 100 | 90.3-100 | 2171/2171 | 100 | 99.8-100 |
| Staphylococcus | Prospective Fresh | 405/418 | 96.9 | 94.7-98.3 | 401/403 | 99.5 | 98.2-99.9 |
| | Prospective Frozen | 364/379 | 96.0 | 93.6-97.8 | 359/368 | 97.6 | 95.4-98.9 |
| | Seeded Fresh | 0/0 | - | - | 418/419 | 99.8 | 98.7-100 |
| | Seeded Frozen | 1/1 | 100 | 2.5-100 | 219/219 | 100 | 98.3-100 |
| | Overall | 770/798 | 96.5 | 95.0-97.7 | 1397/1409 | 99.1 | 98.5-99.6 |
| Staphylococcus<br>aureus | Prospective Fresh | 133/136 | 97.8 | 93.7-99.5 | 685/685 | 100 | 99.5-100 |
| | Prospective Frozen | 120/121 | 99.2 | 95.5-100 | 622/626 | 99.4 | 98.4-99.8 |
| | Seeded Fresh | 0/0 | - | - | 419/419 | 100 | 99.1-100 |
| | Seeded Frozen | 0/0 | - | - | 220/220 | 100 | 98.3-100 |
| | Overall | 253/257 | 98.4 | 96.1-99.6 | 1946/1950 | 99.8 | 99.5-99.9 |
| Streptococcus | Prospective Fresh | 73/77 | 94.8 | 87.2-98.6 | 740/744 | 99.5 | 98.6-99.9 |
| | Prospective Frozen | 63/64 | 98.4 | 91.6-100 | 683/683 | 100 | 99.5-100 |
| | Seeded Fresh | 18/18 | 100 | 81.5-100 | 401/401 | 100 | 99.1-100 |
| | Seeded Frozen | 44/44 | 100 | 92.0-100 | 175/176 | 99.4 | 96.9-100 |
| | Overall | 198/203 | 97.5 | 94.3-99.2 | 1999/2004 | 99.8 | 99.4-99.9 |
| Streptococcus<br>agalactiae<br>(Group B) | Prospective Fresh | 8/8 | 100 | 63.1-100 | 813/813 | 100 | 99.5-100 |
| | Prospective Frozen | 10/10 | 100 | 69.2-100 | 737/737 | 100 | 99.5-100 |
| | Seeded Fresh | 3/3 | 100 | 29.2-100 | 416/416 | 100 | 99.1-100 |
| | Seeded Frozen | 15/15 | 100 | 78.2-100 | 205/205 | 100 | 98.2-100 |
| | Overall | 36/36 | 100 | 90.3-100 | 2171/2171 | 100 | 99.8-100 |
| Streptococcus<br>pneumoniae | Prospective Fresh | 15/15 | 100 | 78.2-100 | 805/806 | 99.9 | 99.3-100 |
| | Prospective Frozen | 10/10 | 100 | 69.2-100 | 737/737 | 100 | 99.5-100 |
| | Seeded Fresh | 4/5 | 80.0 | 28.4-99.5 | 413/414 | 99.8 | 98.7-100 |
| | Seeded Frozen | 7/7 | 100 | 59.0-100 | 213/213 | 100 | 98.3-100 |
| | Overall | 36/37 | 97.3 | 85.8-99.9 | 2168/2170 | 99.9 | 99.7-100 |
| Streptococcus<br>pyogenes<br>(Group A) | Prospective Fresh | 5/5 | 100 | 47.8-100 | 815/816 | 99.9 | 99.3-100 |
| | Prospective Frozen | 2/2 | 100 | 15.8-100 | 745/745 | 100 | 99.5-100 |
| | Seeded Fresh | 9/9 | 100 | 66.4-100 | 410/410 | 100 | 99.1-100 |
| | Seeded Frozen | 22/22 | 100 | 84.6-100 | 198/198 | 100 | 98.2-100 |
| | Overall | 38/38 | 100 | 90.7-100 | 2168/2169 | 99.9 | 99.7-100 |

| Table 6. FilmArray BCID Clinical Performance Summary - Gram-Positive Organism Results |
|------------------------------------------------------------------------------------------|
| (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification |

ª Sensitivity and Specificity celer to performate with the prospective speciment (PPA) and Negative Percent Agreement (NPA) refer to performance with the seeded specimens.

b 3/4 false positive Enterococcus specimens contained Staphylococcus; the false positive results may be due to cross-reactivity.

5 Isolaes from 16/28 false negalive Staphylococus species species S, peties S, petterkofer by bi-directional sequencing. Bidirectional sequencing confirmed the presence of Staphylococcus in 10/12 false positive specimens; 2 were S. epidermidis, and 1 was S. haemolyticus.

4 Bidirectional sequencing identified 2 isolates from S. aureus false negative specimens as S. epidernidis; they were not S. aweus. Bidirectional sequencing confirmed the presence of S. aurens in 1/4 false positive and one false negative S. arreas were in sequentially-tested specimens and may be due to sample mix-up.

& Bidirectional sequencing confirmed the presence of S. mitis in 1/5 false positive Streptococcus specimens

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Table 7. FilmArray BCID Clinical Performance Summary - Gram-Negative Organism Results (Comparator Method: Standard Manual/Automated Microbiological/Biochemical Identification plus 16S Sequencing for Speciation for A. baumannii)

| Gram-Negative Bacteria | | Sensitivity/PPA1 | | | Specificity/NPA1 | | |
|---------------------------------|--------------------|------------------|--------|------------------|------------------|--------|-----------|
| | | TP/TP + FN | % | 95% CI | TP/TP + FP | % | 95% CI |
| Acinetobacter<br>baumannii | Prospective Fresh | 7/7 | 100 | 59.0-100 | 813/814 | 99.9 | 99.3-100 |
| | Prospective Frozen | 7/7 | 100 | 59.0-100 | 739/740 | 99.9 | 99.2-100 |
| | Seeded Fresh | 20/20 | 100 | 83.2-100 | 397/399 | 99.5 | 98.2-99.9 |
| | Seeded Frozen | 17/17 | 100 | 80.5-100 | 202/203 | 99.5 | 97.3-100 |
| | Overall | 51/51 | 100 | 93.0-100 | 2151/2156 | 99.8 | 99.5-99.9 |
| Enterobacteriaceae | Prospective Fresh | 153/156 | 98.1 | 94.5-99.6 | 665/665 | 100 | 99.4-100 |
| | Prospective Frozen | 150/154 | 97.4 | 93.5-99.3 | 589/593 | 99.3 | 98.3-99.8 |
| | Seeded Fresh | 93/93 | 100 | 96.1-100 | 326/326 | 100 | 98.9-100 |
| | Seeded Frozen | 94/95 | 98.9 | 94.3-100 | 125/125 | 100 | 97.1-100 |
| | Overall | 490/498c | 98.4 | 96.9-99.3 | 1705/1709c | 99.8 | 99.4-99.9 |
| Enterobacter cloacae<br>complex | Prospective Fresh | 10/11 | 90.9 | 58.7-99.8 | 809/810 | 99.9 | 99.3-100 |
| | Prospective Frozen | 11/11 | 100 | 71.5-100 | 734/736 | 99.7 | 99.0-100 |
| | Seeded Fresh | 8/8 | 100 | 63.1-100 | 411/411 | 100 | 99.1-100 |
| | Seeded Frozen | 9/9 | 100 | 66.4-100 | 211/211 | 100 | 98.3-100 |
| | Overall | 38/39 | 97.4 | 86.5-99.9 | 2165/2168 | 99.9 | 99.6-100 |
| Escherichia coli | Prospective Fresh | 77/79 | 97.5 | 91.2-99.7 | 742/742 | 100 | 99.5-100 |
| | Prospective Frozen | 68/69 | 98.6 | 92.2-100 | 674/678 | 99.4 | 98.5-99.8 |
| | Seeded Fresh | 4/4 | 100 | 39.8-100 | 414/415 | 99.8 | 98.7-100 |
| | Seeded Frozen | 1/1 | 100 | 2.5-100 | 219/219 | 100 | 98.3-100 |
| | Overall | 150/153d | 98 | 94.4-99.6 | 2049/2054d | 99.8 | 99.4-99.9 |
| Klebsiella oxytoca | Prospective Fresh | 4/4 | 100 | 39.8-100 | 817/817 | 100 | 99.5-100 |
| | Prospective Frozen | 1/2 | 50 | 1.3-98.7 | 744/745 | 99.9 | 99.3-100 |
| | Seeded Fresh | 32/36 | 88.9 | 73.9-96.9 | 383/383 | 100 | 99.0-100 |
| | Seeded Frozen | 22/22 | 100 | 84.6-100 | 198/198 | 100 | 98.2-100 |
| | Overall | 59/64e | 92.2 | 82.7-97.4 | 2142/2143 | 99.9 | 99.7-100 |
| Klebsiella<br>pneumoniae | Prospective Fresh | 33/34 | 97.1 | 84.7-99.9 | 786/787 | 99.9 | 99.3-100 |
| | Prospective Frozen | 35/37 | 94.6 | 81.8-99.3 | 705/710 | 99.3 | 98.4-99.8 |
| | Seeded Fresh | 13/13 | 100 | 75.3-100 | 403/406 | 99.3 | 97.9-99.8 |
| | Seeded Frozen | 21/21 | 100 | 83.9-100 | 199/199 | 100 | 98.2-100 |
| | Overall | 102/105f | 97.1 | ≤91.9-99.4 | 2093/2102f | 99.6 | 99.2-99.8 |
| Proteus | Prospective Fresh | 11/11 | 100 | 71.5-100 | 810/810 | 100 | 99.5-100 |
| | Prospective Frozen | 11/11 | 100 | 71.5-100 | 736/736 | 100 | 99.5-100 |
| | Seeded Fresh | 2/2 | 100 | 15.8-100 | 417/417 | 100 | 99.1-100 |
| | Seeded Frozen | 15/15 | 100 | 78.2-100 | 205/205 | 100 | 98.2-100 |
| | Overall | 39/39 | 100 | 91.0-100 | 2168/2168 | 100 | 99.8-100 |
| Serratia marcescens | Prospective Fresh | 14/14 | 100 | 76.8-100 | 807/807 | 100 | 99.5-100 |
| | Prospective Frozen | 8/8 | 100 | 63.1-100 | 739/739 | 100 | 99.5-100 |
| | Seeded Fresh | 28/28 | 100 | 87.7-100 | 390/391 | 99.7 | 98.6-100 |
| | Seeded Frozen | 26/27 | 96.3 | 81.0-99.9 | 193/193 | 100 | 98.1-100 |
| | Overall | 76/77g | 98.7 | 93.0-100 | 2129/2130g | 99.9 | 99.7-100 |
| Haemophilus<br>influenzae | Prospective Fresh | 5/5 | 100 | 47.8-100 | 816/816 | 100 | 99.5-100 |
| | Prospective Frozen | 3/3 | 100 | 29.2-100 | 744/744 | 100 | 99.5-100 |
| | Seeded Fresh | 29/29 | 100 | 88.1-100 | 390/390 | 100 | 99.1-100 |
| | Seeded Frozen | 6/6 | 100 | 54.1-100 | 214/214 | 100 | 98.3-100 |
| | Overall | 43/43 | 100 | 91.8-100 | 2164/2164 | 100 | 99.8-100 |
| Neisseria<br>meningitidis | Prospective Fresh | 1/1 | 100 | 2.5-100 | 820/820 | 100 | 99.6-100 |
| | Prospective Frozen | 0/0 | - | | 747/747 | 100 | 99.5-100 |
| | Seeded Fresh | 30/30 | 100 | 88.4-100 | 389/389 | 100 | 99.1-100 |
| | Seeded Frozen | 5/5 | 100 | 47.8-100 | 215/215 | 100 | 98.3-100 |
| | Overall | 36/36 | 100 | 90.3-100 | 2171/2171 | 100 | 99.8-100 |
| Pseudomonas<br>aeruginosa | Prospective Fresh | 19/19 | 100 | 82.4-100 | 802/802 | 100 | 99.5-100 |
| | Prospective Frozen | 32/33 | 97 | 84.2-99.9 | 713/714 | 99.9 | 99.2-100 |
| | Seeded Fresh | 0/0 | - | | 419/419 | 100 | 99.1-100 |
| | Seeded Frozen | 0/0 | - | | 220/220 | 100 | 98.3-100 |
| | Overall | 51/52h | 98.1 | 89.7-100 | 2154/2155 | 99.9 | 99.7-100 |
| Yeast | Sensitivity/PPA* | | | Specificity/NPA* | | | |
| | TP/TP + FN | % | 95% CI | TN/TN + FP | % | 95% CI | |
| Candida albicans | Prospective Fresh | 12/12 | 100 | 73.5-100 | 808/809 | 99.9 | 99.3-100 |
| | Prospective Frozen | 4/4 | 100 | 39.8-100 | 740/743 | 99.6 | 98.8-99.9 |
| | Seeded Fresh | 47/47 | 100 | 92.5-100 | 372/372 | 100 | 99.0-100 |
| | Seeded Frozen | 1/1 | 100 | 2.5-100 | 219/219 | 100 | 98.3-100 |
| | Overall | 64/64 | 100 | 94.4-100 | 2139/2143 | 99.8 | 99.5-99.9 |
| Candida glabrata | Prospective Fresh | 6/6 | 100 | 54.1-100 | 813/815 | 99.8 | 99.1-100 |
| | Prospective Frozen | 6/6 | 100 | 54.1-100 | 741/741 | 100 | 99.5-100 |
| | Seeded Fresh | 32/32 | 100 | 89.1-100 | 387/387 | 100 | 99.1-100 |
| | Seeded Frozen | 5/5 | 100 | 47.8-100 | 215/215 | 100 | 98.3-100 |
| | Overall | 49/49 | 100 | 92.7-100 | 2156/2158 | 99.9 | 99.7-100 |
| Candida krusei | Prospective Fresh | 2/2 | 100 | 15.8-100 | 819/819 | 100 | 99.6-100 |
| | Prospective Frozen | 2/2 | 100 | 15.8-100 | 745/745 | 100 | 99.5-100 |
| | Seeded Fresh | 28/28 | 100 | 87.7-100 | 391/391 | 100 | 99.1-100 |
| | Seeded Frozen | 5/5 | 100 | 47.8-100 | 215/215 | 100 | 98.3-100 |
| | Overall | 37/37 | 100 | 90.5-100 | 2170/2170 | 100 | 99.8-100 |
| Candida parapsilosis | Prospective Fresh | 3/3 | 100 | 29.2-100 | 818/818 | 100 | 99.6-100 |
| | Prospective Frozen | 4/4 | 100 | 39.8-100 | 742/743 | 99.9 | 99.3-100 |
| | Seeded Fresh | 47/49 | 95.9 | 86.0-99.5 | 370/370 | 100 | 99.0-100 |
| | Seeded Frozen | 5/5 | 100 | 47.8-100 | 214/215 | 99.5 | 97.4-100 |
| | Overall | 59/61 | 96.7 | 88.7-99.6 | 2144/2146 | 99.9 | 99.7-100 |
| Candida tropicalis | Prospective Fresh | 0/0 | - | | 821/821 | 100 | 99.6-100 |
| | Prospective Frozen | 3/3 | 100 | 29.2-100 | 744/744 | 100 | 99.5-100 |
| | Seeded Fresh | 31/31 | 100 | 88.8-100 | 388/388 | 100 | 99.1-100 |
| | Seeded Frozen | 5/5 | 100 | 47.8-100 | 215/215 | 100 | 98.3-100 |
| | Overall | 39/39 | 100 | 91.0-100 | 2168/2168 | 100 | 99.8-100 |

4 Sensitivity and Specificity refer to performance with the prospective speciment (PPA) and Negative Percent Agreement (NPA) refer to performance with the seeded specimens

b Bidirectional sequencing identified isolates from 4 false positive species 3); this species appears to cross-rece with the A. baunanii assay. These four ised identified as A. baunamii by phenotypic methods. 6 other isolates originally identified as

{8}------------------------------------------------

A. baunamii by phenotypic methods were identified by bidirectional sequencials (genomospecies 13; 4 isolates), A bereziniae, and A. radioresistens; these 6 isolates did not cross-react with the A. baumannii assay.

5 One false positive and one false negatively in sequentially-tested specimens and may be due to sample mix-up. One isslate from another false negative speciment as E. coll by phenotypic methods, was identified as Pasteurella, and not E. coli, by bidirectional sequencing.

4 One false positive and one false negative E. coli were in sequentially-tested specimens and may be due to sample mix-up.

* Bidirectional sequencing identified 4/5 isolates from false negative K. axyoca species, Rapollella ornithinolvica, and not K. oxyloca. The misidentification is a known limitation of phenotypic testing methods for this species.

The isolate from one false negative K. preunen was identified as the closely related organism, Routella and not K. preunoniae, 69 false positive K. pneumoniae results appear to be due to cross-reactivity with Entercobacter and Roulella ornithinolytica (misidentified as K. oxytoca by phenotypic methods).

8 Bidirectional sequencing identified the isolative S. marcescens specimen as being in the S. proteonucallans(grimesii group and not S. marcescens. The one false S. marcescens result uppears to be due to cross-reactivity with Raoulella ornithinolyica (misidentified as K. oxytoca by phenotypic methods).

" Bidirectional sequencing identified the isolate P. aeraginosa specimen as the closely related species Bendomonas stutzeri and not P. aeruginosu.

| Table 8. FilmArray BCID Clinical Performance Summary - Yeast Organism Results |
|-------------------------------------------------------------------------------------------|
| (Comparator Method: Standard Manual/Automoted Microbiological/Rioghemical Identification) |

" Scusitivity and Specificity refer to performance with the prospective speciment (PPA) and Negative Percent Agreement (NPA) refer to performance with the seeded specimens

b Bidirectional sequencing identified the isolative C. parapsilosis specimens as being the closely related species C. metapsilosis. This misidentification is a known limitation of phenotypic identification methods,

As the antimicrobial resistance gene results are not reported in the absence of a presumptively associated organism, performance was calculated only for samples in which FilmArray BCID detected an appropriate organism. Performance was calculated separately against the two comparator methods; PCR/sequencing direct from the blood culture specimens and PCR/sequencing from organisms isolated from the blood cultures. When comparing to PCR/sequencing from bacterial isolates, performance was only calculated for specimens in which FilmArray BCID detected an appropriate organism and from which an appropriate organism isolate was obtained (i.e., antimicrobial resistance gene results could be obtained for both methods). The NPA for mecA and vanA/B are lower when comparing to PCR/sequencing from bacterial isolates than to

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

PCR/sequencing direct from blood culture primarily due to the reference methods not isolating a resistant clone of an applicable organism. This may be due to heterogeneous resistance within a population of cultured organisms or co-culturing of multiple indistinguishable applicable organisms with different resistance profiles (e.g., culturing a resistant Staphylococcus along with a sensitive Staphylococcus).

| | Sensitivity /PPA | | | Specificity /NPA | | | |
|-------------------------------------------------------------------------------------------------------|--------------------|---------|--------|------------------|---------|--------|------------|
| Antimicrobial Resistance Genes | TP/TP + FN | % | 95% CI | TN/TN + FP | % | 95% CI | |
| <i>mecA</i> - Methicillin Resistance Gene | | | | | | | |
| <i>mecA</i><br>All Staphylococcus<br>Detected | Prospective Fresh | 253/257 | 98.4% | 96.1-99.6% | 147/150 | 98.0% | 94.3-99.6% |
| | Prospective Frozen | 233/237 | 98.3% | 95.7-99.5% | 134/136 | 98.5% | 94.8-99.8% |
| | Seeded Fresh | 1/1 | 100% | n/a | 0/0 | | |
| | Seeded Frozen | 1/1 | 100% | n/a | 0/0 | | |
| | Overall | 488/496 | 98.4% | 96.8-99.3% | 281/286 | 98.3% | 96.0-99.4% |
| <i>mecA</i><br>Staphylococcus<br>Detected;<br><i>S. aureus</i> Detected | Prospective Fresh | 67/69 | 97.1% | 89.9-99.6% | 64/64 | 100% | 94.4-100% |
| | Prospective Frozen | 70/70 | 100% | 94.9-100% | 54/54 | 100% | 93.4-100% |
| | Seeded Fresh | 0/0 | | | 0/0 | | |
| | Seeded Frozen | 0/0 | | | 0/0 | | |
| | Overall | 137/139 | 98.6% | 94.9-99.8% | 118/118 | 100% | 96.9-100% |
| <i>mecA</i><br>Staphylococcus<br>Detected;<br><i>S. aureus</i><br>Not Detected | Prospective Fresh | 186/188 | 98.9% | 96.2-99.9% | 83/86 | 96.5% | 90.1-99.3% |
| | Prospective Frozen | 163/167 | 97.6% | 94.0-99.3% | 80/82 | 97.6% | 91.5-99.7% |
| | Seeded Fresh | 1/1 | 100% | n/a | 0/0 | | |
| | Seeded Frozen | 1/1 | 100% | n/a | 0/0 | | |
| | Overall | 351/357 | 98.3% | 96.4-99.4% | 163/168 | 97.0% | 93.2-99.0% |
| <i>vanA/B</i> - Vancomycin Resistance Genes | | | | | | | |
| <i>vanA/B</i><br>Enterococcus<br>Detected | Prospective Fresh | 23/23 | 100% | 85.2-100% | 36/36 | 100% | 90.3-100% |
| | Prospective Frozen | 13/13 | 100% | 75.3-100% | 30/30 | 100% | 88.4-100% |
| | Seeded Fresh | 12/12 | 100% | 73.5-100% | 0/0 | | |
| | Seeded Frozen | 16/16 | 100% | 79.4-100% | 1/1 | 100% | n/a |
| | Overall | 64/64 | 100% | 94.4-100% | 67/67 | 100% | 94.6-100% |
| KPC - Carbapenem Resistance Gene (Carbapenemase) | | | | | | | |
| KPC<br>Enterobacteriaceae<br>and/or<br><i>A. baumannii</i> and/or<br><i>P. aeruginosa</i><br>Detected | Prospective Fresh | 3/3 | 100% | 29.2-100% | 177/177 | 100% | 97.9-100% |
| | Prospective Frozen | 3/3 | 100% | 29.2-100% | 187/187 | 100% | 98.0-100% |
| | Seeded Fresh | 10/10 | 100% | 69.2-100% | 105/105 | 100% | 96.5-100% |
| | Seeded Frozen | 23/23 | 100% | 85.2-100% | 89/89 | 100% | 95.9-100% |
| | Overall | 39/39 | 100% | 91.0-100% | 558/558 | 100% | 99.3-100% |
| KPC<br>Enterobacteriaceae<br>Detected | Prospective Fresh | 3/3 | 100% | 29.2-100% | 150/150 | 100% | 97.6-100% |
| | Prospective Frozen | 3/3 | 100% | 29.2-100% | 151/151 | 100% | 97.6-100% |
| | Seeded Fresh | 10/10 | 100% | 69.2-100% | 83/83 | 100% | 95.7-100% |
| | Seeded Frozen | 23/23 | 100% | 85.2-100% | 71/71 | 100% | 94.9-100% |
| | Overall | 39/39 | 100% | 91.0-100% | 455/455 | 100% | 99.2-100% |
| KPC<br>Enterobacteriaceae<br>Not Detected;<br><i>A. baumannii</i> and/or<br><i>P. aeruginosa</i> | Prospective Fresh | 0/0 | | | 27/27 | 100% | 87.4-100% |
| | Prospective Frozen | 0/0 | | | 36/36 | 100% | 90.3-100% |
| | Seeded Fresh | 0/0 | | | 22/22 | 100% | 84.6-100% |
| | Seeded Frozen | 0/0 | | | 18/18 | 100% | 81.5-100% |
| | Overall | 0/0 | | | 103/103 | 100% | 96.5-100% |

### Table 9. FilmArray BCID Clinical Performance Summary -- Antimicrobial Resistance Genes (Comparator Method: PCR/Sequencing Direct from Blood Culture).

4 Sensitivity and Specificity refer to performance with the prospective speciment (PPA) and Negative Percent Agreement (NPA) refer to performance with the seeded specimens.

### Table 10. FilmArray BCID Clinical Performance Summary - Antimicrobial Resistance Genes (Comparator Method: PCR/Sequencing of Cultured Isolates)

| ANTIMICROBIAL RESISTANCE GENES | | Positive Percent Agreement | | | Negative Percent Agreement * | | |
|-----------------------------------------------|------------------------------------|----------------------------|-------|------------|------------------------------|-------|------------|
| | | TP/TP + FN | % | 95% CI | TN/TN + FP | % | 95% CI |
| | mecA - Methicillin Resistance Gene | | | | | | |
| <i>mecA</i><br>All Staphylococcus<br>Detected | Prospective Fresh | 234/236 | 99.2% | 97.0-99.9% | 149/172 | 86.7% | 80.6-91.3% |
| | Prospective Frozen | 219/222 | 98.6% | 96.1-99.7% | 135/151 | 89.4% | 83.4-93.8% |
| | Seeded Fresh | 0/0 | - | - | 0/0 | - | - |
| | Seeded Frozen | 1/1 | 100% | n/a | 0/0 | - | - |

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

| ANTIMICROBIAL RESISTANCE GENES | | Positive Percent Agreement | | | Negative Percent Agreement * | | |
|--------------------------------------------------------------------------------------------------------------|--------------------|----------------------------|-------|------------|------------------------------|-------|------------|
| | | TP/TP + FN | % | 95% CI | TN/TN + FP | % | 95% CI |
| <i>mecA</i><br>Staphylococcus<br>Detected;<br><i>S. aureus</i> Detected | Overall | 454/459 | 98.9% | 97.5-99.6% | 284/323 | 87.9% | 83.9-91.3% |
| | Prospective Fresh | 64/65 | 98.5% | 91.7-100% | 65/68 | 95.6% | 87.6-99.1% |
| | Prospective Frozen | 66/66 | 100% | 94.6-100% | 54/58 | 93.1% | 83.3-98.1% |
| | Seeded Fresh | 0/0 | - | - | 0/0 | - | - |
| | Seeded Frozen | 0/0 | - | - | 0/0 | - | - |
| <i>mecA</i><br>Staphylococcus<br>Detected;<br><i>S. aureus</i><br>Not Detected | Overall | 130/131 | 99.2% | 95.8-100% | 119/126 | 94.4% | 88.9-97.7% |
| | Prospective Fresh | 170/171 | 99.4% | 96.8-100% | 84/104 | 80.8% | 71.9-87.8% |
| | Prospective Frozen | 153/156 | 98.1% | 94.5-99.6% | 81/93 | 87.1% | 78.6-93.2% |
| | Seeded Fresh | 0/0 | - | - | 0/0 | - | - |
| | Seeded Frozen | 1/1 | 100% | n/a | 0/0 | - | - |
| Overall | | 324/328 | 98.8% | 96.9-99.7% | 165/197 | 83.8% | 77.9-88.6% |
| <i>vanA/B</i> - Vancomycin Resistance Genes | | | | | | | |
| <i>vanA/B</i><br>Enterococcus<br>Detected | Prospective Fresh | 20/20 | 100% | 83.2-100% | 36/39 | 92.3% | 79.1-98.4% |
| | Prospective Frozen | 12/12 | 100% | 73.5-100% | 30/31 | 96.8% | 83.3-99.9% |
| | Seeded Fresh | 12/12 | 100% | 73.5-100% | 0/0 | - | - |
| | Seeded Frozen | 16/16 | 100% | 79.4-100% | 1/1 | 100% | n/a |
| Overall | | 60/60 | 100% | 94.0-100% | 67/71 | 94.4% | 86.2-98.4% |
| KPC - Carbapenem Resistance Gene (Carbapenemase) | | | | | | | |
| KPC<br>Enterobacteriaceae<br>and/or<br><i>A. baumannii</i> and/or<br><i>P. aeruginosa</i><br>Detected | Prospective Fresh | 3/3 | 100% | 29.2-100% | 177/177 | 100% | 97.9-100% |
| | Prospective Frozen | 3/3 | 100% | 29.2-100% | 187/187 | 100% | 98.1-100% |
| | Seeded Fresh | 10/10 | 100% | 69.2-100% | 105/105 | 100% | 96.5-100% |
| | Seeded Frozen | 23/23 | 100% | 85.2-100% | 89/89 | 100% | 95.9-100% |
| Overall | | 39/39 | 100% | 91.0-100% | 558/558 | 100% | 99.3-100% |
| KPC<br>Enterobacteriaceae<br>Detected | Prospective Fresh | 3/3 | 100% | 29.2-100% | 151/151 | 100% | 97.6-100% |
| | Prospective Frozen | 3/3 | 100% | 29.2-100% | 152/152 | 100% | 97.6-100% |
| | Seeded Fresh | 10/10 | 100% | 69.2-100% | 83/83 | 100% | 95.7-100% |
| | Seeded Frozen | 23/23 | 100% | 85.2-100% | 71/71 | 100% | 94.9-100% |
| Overall | | 39/39 | 100% | 91.0-100% | 457/457 | 100% | 99.2-100% |
| KPC<br>Enterobacteriaceae<br>Not Detected:<br><i>A. baumannii</i> and/or<br><i>P. aeruginosa</i><br>Detected | Prospective Fresh | 0/0 | - | - | 26/26 | 100% | 86.8-100% |
| | Prospective Frozen | 0/0 | - | - | 35/35 | 100% | 90.0-100% |
| | Seeded Fresh | 0/0 | - | - | 22/22 | 100% | 84.6-100% |
| | Seeded Frozen | 0/0 | - | - | 18/18 | 100% | 81.5-100% |
| Overall | | 0/0 | - | - | 101/101 | 100% | 96.4-100% |

"Isolates for 12 Staphylococi, 4 Enterobacteriaceae/A. bannamii/P. aeruginosa did not grow from the subcultured blood culture and could therefore not be tested with the PCR/bi-directional sequencing method. These blood cultures were considered negative for the antimicabial resistance genes by comparator method, and FilmArray performance has been calculated as True Negative (when FilmArray is negative for the analyte) or False Positive for the analyte) for the analyte) for each of these isolates.

Performance of FilmArray BCID as compared to phenotypic antimicrobial susceptibility testing (AST) results was calculated for informational purposes. Results stratified by AST method are presented in Tables 11-13. Some PPA are lower when comparing results from bacterial isolates than to PCR/sequencing direct from blood culture because phenotypic AST testing is capable of detecting antimicrobial resistance due to mechanisms other than acquisition of mecA, vanA/B, or KPC.

# Table 11. mec4 Performance - Comparison to Phenotypic Antimicrobial Susceptibility Testing (AST) Methods

Note: AST results were not provided for several isolates.

| PHENOTYPIC METHODS | | Positive Percent Agreement<br>TP/TP + FN | % (95%CI) | Negative Percent Agreement<br>TN/TN + FP | % (95%CI) |
|--------------------|--------------------------|------------------------------------------|-----------|------------------------------------------|-----------|
| Prospective | Cefoxitin Disc Diffusion | 22/22 | 100% | 15/15 | 100% |

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

| All Staphylococcus | Chromogenic Agar | 42/46 | 91.3% | 25/32 | 78.1% |
|-------------------------------------------------------------|---------------------------------------------------|---------|-------------------------|---------|-------------------------|
| | Automated Antimicrobial<br>Susceptibility Testing | 366/380 | 96.3% | 226/262 | 86.3% |
| | <i>All Methods</i> | 430/448 | 96.0%<br>(93.7 - 97.6%) | 266/309 | 86.1%<br>(81.7 - 89.7%) |
| Prospective<br>Staphylococcus,<br><i>S. aureus Detected</i> | Chromogenic Agar | 10/11 | 90.9% | 8/8 | 100% |
| | Automated Antimicrobial<br>Susceptibility Testing | 117/119 | 98.3% | 108/112 | 96.4% |
| | <i>All Methods</i> | 127/130 | 97.7%<br>(93.4 - 99.5%) | 116/120 | 96.7%<br>(91.7 - 99.1%) |
| Seeded<br>Staphylococcus | Automated Antimicrobial<br>Susceptibility Testing | 1/1 | 100% | 0/0 | - |

### Table 12. vanA/B Performance - Comparison to Phenotypic Vancomycin AST Methods

| PHENOTYPIC METHODS | | | Positive Percent Agreement | Negative Percent Agreement | |
|----------------------------------------------------|---------------------------------------------------|------------|----------------------------|----------------------------|-------------------------|
| | | TP/TP + FN | % (95%CI) | TN/TN + FP | % (95%CI) |
| Prospective<br>Enterococcus | Vancomycin Screen Agar | 3/3 | 100% | 5/5 | 100% |
| | Vancomycin Disc Diffusion | 0/1 | 0.0% | - | - |
| | Automated Antimicrobial<br>Susceptibility Testing | 29/30 | 96.7% | 55/58 | 94.8% |
| | All Methods | 32/34a | 94.1%<br>(80.3 - 99.3%) | 60/63 | 95.2%<br>(86.7 - 99.0%) |
| Seeded<br>Enterococcus | Vancomycin Disc Diffusion | 14/14 | 100% | 1/1 | 100% |
| | Vancomycin Screen Agar | 14/14 | 100% | - | - |
| | All Methods | 28/28 | 100%<br>(87.7 - 100%) | 1/1 | 100% (n/a) |
| Combined Prospective<br>and Seeded<br>Enterococcus | All Methods | 60/62a | 96.8%<br>(88.8 - 99.6%) | 61/64 | 95.3%<br>(86.9 - 99.0%) |

* Two isolates (one E. galinarum and one E. Jaecalis) that were vancomycin resistant by phenotypic AST the van AB genes by bi-directional sequence analysis.

#### Table 13. KPC Performance - Comparison to Phenotypic Carbapenem AST Methods Note: AST results were not provided for several isolates.

Note: Acinetobacter baunannii and Pseudonosa are commonly resistant to carbapencess due to mechanisms other than acquisition of the KPC gene (blakec). These bacteria very rarely carry the KPC gene.

| PHENOTYPIC METHODS | | Positive Percent Agreement<br>TP/<br>TP + FN | % (95%CI) | Negative Percent Agreement<br>TN/<br>TN+ FP | % (95%CI) |
|------------------------------|---------------------------------------------------|----------------------------------------------|-----------|---------------------------------------------|---------------------|
| Prospective<br>A. baumannii | Automated Antimicrobial<br>Susceptibility Testing | 0/10 | 0% | 4/4 | 100% |
| Seeded<br>A. baumannii | Meropenem Disc Diffusion | 0/30 | 0% | 9/9 | 100% |
| A. baumannii - All Methods | | 0/40 | 0% (n/a) | 13/13…