FILMARRAY GASTROINTESTINAL (GI) PANEL

K140407 · Biofire Diagnostics · PCH · May 2, 2014 · Microbiology

Device Facts

• Record ID: K140407
• Device Name: FILMARRAY GASTROINTESTINAL (GI) PANEL
• Applicant: Biofire Diagnostics
• Product Code: PCH · Microbiology
• Decision Date: May 2, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3990
• Device Class: Class 2

Indications for Use

The FilmArray Gastrointestinal (GI) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with the FilmArray Instrument. The FilmArray GI Panel is capable of the simultaneous detection and identification of nucleic acids from multiple bacteria, viruses, and parasites directly from stool samples in Cary Blair transport media obtained from individuals with signs and/or symptoms of gastrointestinal infection. The following bacteria (including several diarrheagenic E. coli/Shigella pathotypes), parasites, and viruses are identified using the FilmArray GI Panel: Campylobacter (C. jejuni/C. coli/C. upsaliensis), Clostridium difficile (C. difficile) toxin A/B, Plesiomonas shigelloides, Salmonella, Vibrio (V. parahaemolyticus/V. vulnificus/V. cholerae) including specific identification of Vibrio cholerae, Yersinia enterocolitica, Enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC) lt/st, Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2 (including specific identification of the E. coli O157 serogroup within STEC), Shigella/Enteroinvasive Escherichia coli (EIEC), Cryptosporidium, Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia (also known as G. intestinalis and G. duodenalis), Adenovirus F 40/41, Astrovirus, Norovirus GI/GII, Rotavirus A, Sapovirus (Genogroups I, II, IV, and V). The FilmArray GI Panel is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness and results are meant to be used in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule out co-infection with organisms not included in the FilmArray GI Panel. The agent detected may not be the definite cause of the disease. Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor or guide treatment for C. difficile infection. Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for E. coli O157, Plesiomonas shigelloides, Yersinia enterocolitica, Astrovirus, and Rotavirus A were established primarily with retrospective clinical specimens. Performance characteristics for Entamoeba histolytica, and Vibrio (V. parahaemolyticus, V. vulnificus, and Vibrio cholerae) were established primarily using contrived clinical specimens. Negative FilmArray GI Panel results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or noninfectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

Device Story

FilmArray GI Panel is a multiplexed nucleic acid-based in vitro diagnostic test for stool samples in Cary Blair media. The device uses a closed pouch system containing freeze-dried reagents for automated nucleic acid purification and nested, multiplex PCR. The FilmArray Instrument controls liquid movement via inflatable bladders and pneumatic pistons; Peltier devices manage thermal cycling. The process involves two-stage PCR: a large-volume multiplexed RT-PCR followed by a nested singleplex PCR in an array. The device uses DNA melt analysis to identify specific amplicons. A digital camera captures fluorescent images of the array, and software interprets the melt curves to provide qualitative results for 22 GI pathogens. The test is performed in a clinical laboratory setting, providing results in approximately one hour. The output aids clinicians in diagnosing GI illness and managing outbreaks when used with other clinical/epidemiological data. It does not replace culture for bacterial typing or C. difficile treatment monitoring.

Clinical Evidence

Clinical performance established via multi-center study (n=1556 prospective specimens). Comparator methods included stool culture and PCR/sequencing. Sensitivity/PPA and specificity/NPA were calculated for 22 analytes. PPA ranged from 94.5% to 100% for most targets; NPA was generally >97%. Retrospective and contrived specimens supplemented data for rare organisms (e.g., E. coli O157, E. histolytica). Study confirmed high concordance with reference methods.

Technological Characteristics

Multiplex nucleic acid-based assay using nested PCR and high-resolution melt analysis. Reagents are freeze-dried in a single-use pouch. Instrument uses pneumatic bladders/pistons for fluidics and Peltier elements for thermal control. Standalone instrument; results interpreted by proprietary software. No specific material standards (e.g., ASTM) cited for pouch construction.

Indications for Use

Indicated for individuals with signs/symptoms of GI infection. Detects nucleic acids from bacteria, viruses, and parasites in stool samples collected in Cary Blair transport media. Not for monitoring/guiding C. difficile treatment.

Regulatory Classification

Identification

A gastrointestinal microorganism multiplex nucleic acid-based assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple gastrointestinal microbial nucleic acids extracted from human stool specimens. The device detects specific nucleic acid sequences for organism identification as well as for determining the presence of toxin genes. The detection and identification of a specific gastrointestinal microbial nucleic acid from individuals exhibiting signs and symptoms of gastrointestinal infection aids in the diagnosis of gastrointestinal infection when used in conjunction with clinical evaluation and other laboratory findings. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

Special Controls

*Classification.* Class II (special controls). The special controls are set forth in FDA's guideline document entitled: “Class II Special Controls Guideline: Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assays for Detection and Identification of Microorganisms and Toxin Genes from Human Stool Specimens.” For availability of the guideline document, see § 866.1(e).

Predicate Devices

• Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) (K121454)

Submission Summary (Full Text)

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K140407

N.J.Y 0 2 2014

## 510(k) Summary BioFire Diagnostics, LLC

## FilmArray Gastrointestinal (GI) 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, LLC 390 Wakara Way Salt Lake City, UT 84108

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

Contact: Beth Lingenfelter, ext. 407

Date Submitted: February 13, 2014

## Device Name and Classification:

Trade Name: FilmArray GI Panel

Regulation Number: 21 CFR 866.3990

Classification Name: Gastrointestinal microorganism multiplex nucleic acid-based assay

#### Predicate Device:

K121454 - Luminex xTAG® Gastrointestinal Pathogen Panel (GPP)

#### Intended Use:

The FilmArray Gastrointestinal (GI) Panel is a qualitative multiplexed nucleic acid-based in vitro diagnostic test intended for use with the FilmArray Instrument. The FilmArray GI Panel is capable of the simultaneous detection and identification of nucleic acids from multiple bacteria, viruses, and parasites directly from stool samples in Cary Blair transport media obtained from individuals with signs and/or symptoms of gastrointestinal infection. The following bacteria (including several diarrheagenic E. coli/Shigella pathotypes), parasites, and viruses are identified using the FilmArray GI Panel:

- Campylobacter (C. jejuni/C. coli/C. upsaliensis) .
- Clostridium difficile (C. difficile) toxin A/B .
- Plesiomonas shigelloides .
- Salmonella .

BioFire Diagnostics, LLC 510(k) FilmArray GI Panel

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- Vibrio (V. parahaemolvticus/V. vulnificus/V. cholerae) including specific identification of . Vibrio cholerae
- . Yersinia enterocolitica
- Enteroaggregative Escherichia coli (EAEC) .
- Enteropathogenic Escherichia coli (EPEC) .
- Enterotoxigenic Escherichia coli (ETEC) It/st .
- Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2 (including specific . identification of the E. coli 0157 serogroup within STEC)
- . Shigella/Enteroinvasive Escherichia coli (EIEC)
- . Cryptosporidium
- Cyclospora cayetanensis .
- Entamoeba histolytica .
- Giardia lamblia (also known as G. intestinalis and G. duodenalis) .
- Adenovirus F 40/41 .
- Astrovirus .
- Norovirus GI/GII .
- Rotavirus A .
- Sapovirus (Genogroups I, II, IV, and V) . ●

The FilmArray GI Panel is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness and results are meant to be used in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule out co-infection with organisms not included in the FilmArray GI Panel. The agent detected may not be the definite cause of the disease.

Concomitant culture is necessary for organism recovery and further typing of bacterial agents.

This device is not intended to monitor or guide treatment for C. difficile infection.

Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for E. coli O157, Plesiomonas shigelloides, Yersinia enterocolitica, Astrovirus, and Rotavirus A were established primarily with retrospective clinical specimens.

Performance characteristics for Entamoeba histolytica, and Vibrio (V. parahaemolyticus, V. vulnificus, and Vibrio cholerae) were established primarily using contrived clinical specimens.

Negative FilmArray Gl Panel results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or noninfectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

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# Device Description:

The FilmArray Gastrointestinal (GI) Panel is a multiplex nucleic acid test designed to be used with the FilmArray Instrument. The FilmArray GI pouch contains freeze-dried reagents to perform nucleic acid purification and nested, multiplex PCR with DNA melt analysis. The FilmArray Gastrointestinal (GI) Panel simultaneously conducts 22 tests for the identification of GI pathogens from stool specimens collected in Cary Blair transport medium (Table 1). Results from the FilmArray GI Panel test are available within about one hour.

| Bacteria | Viruses |
|-------------------------------------------------------------------|-----------------------------------------|
| Campylobacter (C. jejuni/C. coli/C. upsaliensis) | Adenovirus F 40/41 |
| Clostridium difficile (toxin A/B) | Astrovirus |
| Plesiomonas shigelloides | Norovirus GI/GII |
| Salmonella | Rotavirus A |
| Vibrio(V. parahaemolyticus/V. vulnificus/V. cholerae) | Sapovirus (Genogroups I, II, IV, and V) |
| Vibrio cholerae | |
| Yersinia enterocolitica | |
| Diarrheagenic E. coli/Shigella | Parasites |
| Enteroaggregative E. coli (EAEC) | Cryptosporidium |
| Enteropathogenic E. coli (EPEC) | Cyclospora cayetanensis |
| Enterotoxigenic E. coli (ETEC) lt/st | Entamoeba histolytica |
| Shiga toxin-producing E. coli (STEC) stx1/stx2 | Giardia lamblia |
| E. coli O157 | |
| Shigella/Enteroinvasive E. coli (EIEC) | |

| | Table 1. Bacteria, Viruses, Diarrheagenic E. coli/Shigella, and Parasites Detected by the FilmArray | |
|----------|-----------------------------------------------------------------------------------------------------|--|
| GI Panel | | |

A test is initiated by loading Hydration Solution into one port of the FilmArray pouch and a stool sample (in Cary Blair transport medium) mixed with the provided Sample Buffer into the other port of the FilmArray GI 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 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

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times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis.

Nucleic acid extraction occurs within the FilmArray pouch using mechanical and chemical Ivsis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the FilmArray performs a nested multiplex PCR that is executed in two stages. During the first stage, the FilmArray performs a single, large volume, highly multiplexed reverse transcription PCR (rt-PCR) reaction. 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). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the arrav 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 200 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 on the panel.

# Substantial Equivalence:

The Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) is a qualitative, multiplexed in vitro diagnostic assay intended to simultaneously detect and identify microorganism nucleic acids from human stool samples. Testing is performed on pre-treated human stool samples. Table 2 outlines the similarities between the two systems and Table 3 outlines the differences.

| Element | FilmArray GI Panel | Luminex xTAG® Gastrointestinal<br>Pathogen Panel (GPP) |
|-----------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------|
| Organisms<br>Detected | Campylobacter, toxigenic Clostridium<br>difficile, Salmonella, Norovirus GI/GII.<br>Rotavirus A, Cryptosporidium, Giardia<br>lamblia, E. coli O157, Shiga toxin-<br>producing E. coli (STEC), Enterotoxigenic<br>E. coli (ETEC), and Shigella/Enteroinvasive<br>E. coli (EIEC). | Same<br>See below for differences |
| Analyte | DNA/RNA | Same |
| Technological<br>Principles | Multiplex nucleic acid | Same<br>See below for differences |

| Table 2. Similarities Between the FilmArray GI Panel and the Luminex xTAC® Gastrointestinal | |
|---------------------------------------------------------------------------------------------|--|
| Pathogen Panel (GPP). | |

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| Element | FilmArray GI Panel | Luminex xTAG® Gastrointestinal<br>Pathogen Panel (GPP) |
|---------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Specimen Types | Human stool sample collected in Cary<br>Blair transport media. | Pre-treated human stool sample. |
| Organisms<br>Detected | Detects the following Campylobacter<br>species: C. jejuni/C. coli/C. upsaliensis.<br>Also detects additional Cryptosporidium<br>species, Plesiomonas shigelloides, Vibrio<br>(V. parahaemolyticus/V. vulnificus/V.<br>cholerae), V. cholerae, Yersinia<br>enterocolitica, Adenovirus F40/41,<br>Astrovirus, Sapovirus (Genogroups I, II,<br>IV, and V), Cyclospora cayetanensis,<br>Entamoeba histolytica, Enteropathogenic<br>E. coli (EPEC), Enteroinvasive E. coli<br>(EIEC), and Enteroaggregative E. coli<br>(EAEC). | Detects the following Campylobacter<br>species: C. jejuni, C. coli, and C. lari. Only<br>detects the following Cryptosporidium<br>species: C. parvum and C. hominis. |
| Technological<br>Principles | Nested multiplex RT-PCR followed by<br>high resolution melting analysis to confirm<br>identity of amplified product. | Multiplex RT-PCR and multiplex TSPE<br>followed by Fluorescence-activated sorting<br>of labeled beads coupled to streptavidin-<br>conjugated biotinylated products. |
| Instrumentation | FilmArray Instrument | Nucleic Acid Purification System<br>PCR Thermocycler<br>Luminex® 100/200™ or MAGPIX<br>instruments |
| Time to result | Less than 1 hour | Approximately 5 hours |
| Reagent Storage | Room temperature | Reagents stored at 4°C and -20°C. |
| Sample<br>Preparation<br>Method | Sample Processing is automated in the<br>FilmArray Instrument. | Up front sample processing is required to<br>extract nucleic acid. |
| Test<br>Interpretation | Automated test interpretation and report<br>generation. User cannot access raw data. | Semi-automated test interpretation. User<br>must review all "no call" results to<br>determine cause and retesting strategy. |
| 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 control added to each sample.<br>External control processed with each batch<br>of samples. |

# Table 3. Differences Between the FilmArray GI Panel Test System and the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP).

.

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# Summary of Performance Data

## Clinical Performance

The clinical performance of the FilmArray GI Panel was established during a multi-center study conducted at four geographically distinct U.S. study sites (Pacific, North Central, Great Lakes, and Northeast regions) between May and September, 2013. A total of 1578 prospective residual stool specimens in Cary Blair transport media were acquired for the clinical study; 22 of these were excluded. The most common reasons for exclusion were that a valid external control was not completed on the day of testing, that the specimen was not plated to all of the appropriate bacterial culture media required for the reference method, or that the specimen was beyond four days from the date of collection. The final data set consisted of 1556 specimens. Table 4 provides a summary of demographic information for the 1556 specimens included in the prospective study.

| Prospective Study Specimens | |
|-----------------------------|-------------------------|
| Total Specimens | 1556 |
| Sex | Number of Specimens (%) |
| Male | 718 (46%) |
| Female | 838 (54%) |
| Age Group | Number of Specimens (%) |
| <1 year | 121 (8%) |
| 1-5 years | 418 (27%) |
| 6-12 years | 193 (12%) |
| 13-21 years | 240 (15%) |
| 22-64 years | 411 (26%) |
| 65+ years | 173 (11%) |
| Status | Number of Specimens (%) |
| Outpatient | 1350 (87%) |
| Hospitalized | 164 (11%) |
| Emergency | 42 (3%) |

#### Table 4. Demographic Summary for Prospective FilmArray G1 Panel Clinical Evaluation

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

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| FilmArray Test Results | Reference/Comparator Method |
|----------------------------------------|-----------------------------------------------------------------------------------------|
| Campylobacter | Stool cultureb |
| <i>E. coli</i> O157a | (Blood agar, Blood agar with Ampicillin,<br>MacConkey agar, Sorbitol-MacConkey agar, GN |
| <i>Plesiomonas shigelloides</i> | broth + Hektoen enteric agar, Campylobacter agar, |
| <i>Salmonella</i> | Cefsulodin-IrgasanTM-Novobiocin agar, and |
| <i>Vibrio</i> and <i>V. cholerae</i> | Thiosulfate Citrate Bile Salts agar) with standard |
| <i>Yersinia enterocolitica</i> | manual and automated microbiological/biochemical<br>identification methods |
| STEC (stx1/2) | |
| ETEC | |
| EPECc | |
| EIEC/ <i>Shigella</i> d | |
| EAEC | |
| Adenovirus F 40/41 | |
| Astrovirus | |
| Norovirus GI/GIIe | PCR with Bi-directional Sequencingh |
| Rotavirus A | |
| Sapovirusf | |
| <i>Clostridium difficile</i> toxin A/B | |
| <i>Cryptosporidium</i> | |
| <i>Giardia lamblia</i> g | |
| <i>Cyclospora cayetanensis</i> | |
| <i>Entamoeba histolytica</i> | |

#### Mathada for Film Array CI Danal Clinicol Evalu

4 The E. coli O157 comparator method data were only used to determine the accuracy of the FilmArray determination of E. coli 0157 detected or not detected for specimens in which FilmArray detected STEC.

6 Any bacteria isolated from stool culture that could not be identified to the species level by laboratory methods were sequenced using an assay capable of providing species information (c.g., 16S).

& A result for EPEC is only reported in the absence of STEC (same algorithm as FilmArray).

4 Shigella may be identified by routine methods: however. culture detection will be reported for informational purposes only.

CDC Calicinet assays (non-sequenceable) were used for the comparator method for Norovirus.

Sapovirus comparator assays consisted of one well-validated, sequenceable assay and one published assay that was not sequenceable.

8 G. lamblia comparator assays consisted of one well-validated, sequenceable assay and one published assay that was not sequenceable.

" PCR assays were designed to amplify different sequences than those targeted by FilmArray Gl. Positive results for sequenceable assays required a sequencing result of adequate quality to match a sequence of the expected organism/gene deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), with an acceptable E-value.

A total of 1556 specimens were evaluated in this study. Of these specimens, 832 (53.5%) were positive for at least one analyte. Clinical sensitivity or positive percent agreement (PPA) was calculated as 100% x (TP / (TP + FN)). True positive (TP) indicates that both the FilmArray G1 Panel and reference/comparator method had a positive result for this specific analyte, and false negative (FN) indicates that the FilmArray 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 GI Panel and the

BioFire Diagnostics, LLC 510(k) FilmArray GI Panel

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reference/comparator method had negative results, and a false positive (FP) indicates that the FilmArray GI Panel result was positive but the comparator result was negative. The exact binomial two-sided 95% confidence interval was calculated. The results are summarized in Table 6.

| Bacteria | Sensitivity/PPAa | | | Specificity/NPAa | | |
|--------------------------------------------------------|-----------------------------------|------|-----------|-----------------------------------|------|-----------|
| | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Campylobacter (C. jejuni/C. coli/C. upsaliensis) | 34/35b | 97.1 | 85.1-99.9 | 1497/1521b | 98.4 | 97.7-99.0 |
| Clostridium difficile toxin A/Ba | 163/165c | 98.8 | 95.7-99.9 | 1350/1391c | 97.1 | 96.0-97.9 |
| Plesiomonas shigelloides | 3/3 | 100 | 29.2-100 | 1538/1553d | 99.0 | 98.4-99.5 |
| Salmonella | 31/31 | 100 | 88.8-100 | 1519/1525e | 99.6 | 99.1-99.9 |
| Vibrio (V. parahaemolyticus/V. vulnificus/V. cholerae) | 0/0 | - | | 1554/1556f | 99.9 | 99.5-100 |
| Vibrio cholerae | 0/0 | - | | 1555/1556g | 99.9 | 99.6-100 |
| Yersinia enterocolitica | 1/1 | 100 | N/A | 1555/1555 | 100 | 99.8-100 |
| Diarrheagenic E. coli/Shigella | Positive Percent Agreement (PPA)a | | | Negative Percent Agreement (NPA)a | | |
| | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Enteroaqggregative E. coli (EAEC) | 82/83 | 98.8 | 93.5-100 | 1446/1473h | 98.2 | 97.3-98.8 |
| Enteropathogenic E. coli (EPEC) | 314/317 | 99.1 | 97.3-99.8 | 1167/1201i | 97.2 | 96.1-98.0 |
| Enterotoxigenic E. coli (ETEC) lt/st | 22/22 | 100 | 84.6-100 | 1525/1534j | 99.4 | 98.9-99.7 |
| Shiga-like toxin-producing E. coli (STEC) stx1/stx2 | 33/33 | 100 | 89.4-100 | 1518/1523k | 99.7 | 99.2-99.9 |
| E. coli O157a | 3/3 | 100 | 29.2-100 | 34/35l | 97.1 | 85.1-99.9 |
| Shigella/Enteroinvasive E. coli (EIEC) | 47/49 | 95.9 | 86.0-99.5 | 1505/1507 | 99.9 | 99.5-100 |
| Parasites | Positive Percent Agreement (PPA)a | | | Negative Percent Agreement (NPA)a | | |
| | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Cryptosporidium | 18/18 | 100 | 81.5-100 | 1532/1538m | 99.6 | 99.2-99.9 |
| Cyclospora cayetanensis | 19/19 | 100 | 82.4-100 | 1537/1537 | 100 | 99.8-100 |
| Entamoeba histolytica | 0/0 | - | | 1556/1556 | 100 | 99.8-100 |
| Giardia lamblia | 20/20 | 100 | 83.2-100 | 1529/1536n | 99.5 | 99.1-99.8 |
| Viruses | Positive Percent Agreement (PPA)a | | | Negative Percent Agreement (NPA)a | | |
| | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Adenovirus F 40/41 | 42/44o | 95.5 | 84.5-99.4 | 1499/1512o | 99.1 | 98.5-99.5 |
| Astrovirus | 7/7 | 100 | 59.0-100 | 1548/1549p | 99.9 | 99.6-100 |
| Norovirus GI/GII | 52/55q | 94.5 | 84.9-98.9 | 1483/1501q | 98.8 | 98.1-99.3 |
| Rotavirus A | 6/6 | 100 | 54.1-100 | 1538/1550r | 99.2 | 98.7-99.6 |
| Sapovirus (Genogroups I, II, IV, and V) | 46/46 | 100 | 92.3-100 | 1497/1510s | 99.1 | 98.5-99.5 |

## Table 6. FilmArray GI Clinical Performance Summary

4 C. difficile performance is reported as positive percent and negative percent agreement, and E. coli O157 performance is reported as sensitivity/specificity, in contrast to the respective sections. The performance mcasures of sensitivity and specificity only refer to those analytes for which the gold-standard bacterial culture was used as the reference method: Campylobacter, E. coli 0157, Plesiomonas shigelloides, Salmonella, Vibrio cholerae, and Yersina

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enterocolitica. Performance measures of positive percent agreement (PPA) and negative percent agreement (NPA) refer to all other analytes, for which PCR/sequencing assays were used as comparator methods.

· Campylobacter jejuni subsp. doyler was identified in the single false negative specimen using bi-directional sequence analysis. Campylobacter was detected in 19/24 false positive specimens using bi-directional sequence analysis.

S C. difficile was detected in 1/2 false negative specimens and 41/41 false positive specimens using bi-directional sequence analysis.

4 P. shigelloides was detected in 15/15 false positive specimens using bi-directional sequence analysis.

Salmonella was detected in 6/6 false positive specimens using bi-directional sequence analysis.

4 Vibrio was detected in 2/2 false positive specimens using bi-directional sequence analysis.

B V. cholerae was detected in the single false positive specimen using bi-directional sequence analysis.

h EAEC was detected in 27/27 false positive specimens using bi-directional sequence analysis.

1 EPEC was detected in 23/34 false positive specimens using bi-directional sequence analysis.

I ETEC was detected in 6/9 false positive specimens using bi-directional sequence analysis. The three remaining false positive results were determined to have been caused by cross-reactivity with Citrobacter koseri (2 instances), and Hafnia alvei (1 instance). These bacteria contain a variant of the firs gene with sequence similarity to assay primers.

* STEC was detected in 5/5 false positive specimens using bi-directional sequence analysis.

E. coli 0157 was detected in the single false positive specimen using bi-directional sequence analysis.

m Cryptosporidium was detected in 6/6 false positive specimens using bi-directional sequence analysis.

9 G. lamblia was detected in 47 false positive specimens using bi-directional sequence analysis. Two false positive results appear to be caused by cross-reactivity with Bifidobacterium longum and Ruminococcus callidus.

9 Adcnovirus was detected in 1/2 false negative specimens and 11/13 false positive specimens using bi-directional sequence analysis

P Astrovirus was detected in the single false positive specimen using bi-directional sequence analysis.

9 The FilmArray G1 system detected Norovirus in 1/3 false negative specimens when retested in 1/2 remaining false negative specimens and 8/18 false positive specimens using bi-directional sequence analysis.

Rotavirus A was detected in 11/12 false positive specimens using bi-directional sequence analysis.

S Sapovirus was detected in 12/13 false positive specimens using bi-directional sequence analysis.

FilmArray GI reports genus level (or multiple species group) results for three bacterial analytes; i.e., Campylobacter (C. jejuni/C. coli/C. upsaliensis), Salmonella, and Vibrio (V. parahaemolyticus/V. vulnificus/V. cholerae). Standard laboratory methods identified various species/serovars within each of these groups during the clinical evaluation. Where standard methods did not provide a species identification, bi-directional sequencing was used to identify the species of the isolate. Stratification of performance by species/serovar is presented below. For Vibrio. no organisms were isolated by the culture methods; however, bi-directional sequencing from the original specimens identified one V. parahaemolyticus and one V. cholerae.

| Campylobacter speciesa | Sensitivity |
|--------------------------------|-------------------------------------|
| <i>C. jejuni</i> b | 31/31 (100%) |
| <i>C. coli</i> | 2/2 (100%) |
| <i>C. jejuni subsp. doylei</i> | 0/1 (0%) |
| <i>C. upsaliensis</i> | 1/1 (100%) |
| Overall Campylobacter | 34/35 (97.1%)<br>95%CI = 81.3-99.3% |

Table 7 Campylabacter Clinical Performance Stratified by Species

a Fifteen (15) Campylobacter were not speciated by the source laboratory and were subject to sequencing of the cadF gene. This method identified 11 C. jejuni, two C. jejuni subsp. doylei, and one C. upsaliensis.

b Two C. jejuni were originally identified by the source lab as "Campylobacter species". Sequencing of the isolates provided by the laboratory identified them as C. jejuni. However, molecular testing of the specimen from which the isolates were obtained also detected the presence of C. upsaliensis. representing co-infection by these two species.

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

| Salmonella species/serovar | Sensitivity |
|---------------------------------------------|-----------------------------------|
| S. enterica ser. Enteritidis | 7/7 (100%) |
| S. enterica ser. Typhimurium (i:-) | 7/7 (100%) |
| S. enterica ser. Typhimurium | 3/3 (100%) |
| S. enterica ser. Javiana | 2/2 (100%) |
| S. enterica ser. Newport | 2/2 (100%) |
| S. enterica ser. Agbeni | 1/1 (100%) |
| S. enterica ser. Berta | 1/1 (100%) |
| S. enterica ser. Ealing | 1/1 (100%) |
| S. enterica ser. Gaminara | 1/1 (100%) |
| S. enterica ser. Infantis | 1/1 (100%) |
| S. enterica ser. Mbandaka | 1/1 (100%) |
| S. enterica ser. Miami | 1/1 (100%) |
| S. enterica ser. Muenchen | 1/1 (100%) |
| S. enterica ser. Paratyphi B var L-Tartrate | 1/1 (100%) |
| S. enterica ser. Thompson | 1/1 (100%) |
| Overall Salmonella | 31/31 (100%)<br>95%CI = 88.8-100% |

Table 8. Salmonella Clinical Performance Stratified by Species/Serovan

The FilmArray GI Panel reported multiple organism detections (i.e., mixed infections) for a total of 262 specimens. This represents 31.5% of positive specimens (262/832) and 16.8% of all specimens (262/1556). The majority of multiple detections (199/262; 76.0%) contained two organisms, while 19.1% (50/262) contained three organisms, 3.4% (9/262) contained four organisms. 1.1% (3/262) contained five organisms. and 0.4% (1/262) contained six organisms. The three organisms that were most prevalent in co-infections were also the three most prevalent organisms in the study as a whole (i.e., EPEC, C. difficile, and EAEC). Out of the 262 specimens with multiple detections. 144 specimens (55.0%; 144/262) were concordant with the reference methods. One hundred eighteen specimens (45.0%; 118/262) contained one or more organisms that had not been detected by the reference/comparator methods (i.e., 139 false positive results); however, bi-directional sequence analysis confirmed the presence of the analyte for 88.5% (123/139) of the discrepant results.

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

| Analyte | No. | % |
|-----------------------------------------------------|-----|-------|
| Prevalence in Mixed Infections N = 262 | | |
| <b>Bacteria</b> | | |
| Campylobacter | 30 | 11.5% |
| Clostridium difficile toxin A/B | 109 | 41.6% |
| Plesiomonas shigelloides | 16 | 6.1% |
| Salmonella | 15 | 5.7% |
| Vibrio | 1 | 0.4% |
| Vibrio cholerae | 1 | 0.4% |
| Yersinia enterocolitica | 1 | 0.4% |
| <b>Diarrheagenic E. coli/Shigella</b> | | |
| Enteroaggregative E. coli (EAEC) | 67 | 25.6% |
| Enteropathogenic E. coli (EPEC) | 159 | 60.7% |
| Enterotoxigenic E. coli (ETEC) lt/st | 26 | 9.9% |
| Shiga-like toxin-producing E. coli (STEC) stx1/stx2 | 13 | 5.0% |
| E. coli O157 | 1 | 0.4% |
| Shigella / Enteroinvasive E. coli (EIEC) | 17 | 6.5% |
| <b>Parasites</b> | | |
| Cryptosporidium | 11 | 4.2% |
| Cyclospora cayetanensis | 2 | 0.8% |
| Entamoeba histolytica | 0 | 0% |
| Giardia lamblia | 14 | 5.3% |
| <b>Viruses</b> | | |
| Adenovirus F 40/41 | 34 | 13.0% |
| Astrovirus | 4 | 1.5% |
| Norovirus G1/GII | 43 | 16.4% |
| Rotavirus A | 10 | 3.8% |
| Sapovirus | 33 | 12.6% |

Table 9. Prevalence of Analytes in Mixed Infections as determined by the FilmArray GI Panel

The most prevalent mixed infection was C. difficile with EPEC (2.0% of all specimens; 32/1556) followed by EAEC with EPEC (1% of all specimens; 15/1556); as previously stated these were the most prevalent organisms detected in the study. Mixed infections were observed for all combinations of analyte classes (e.g. bacteria with viruses, diarrheagenic E. coli/Shigella with parasites) and co-infections were observed within classes (e.g. three diarrheagenic E. coli/Shigella combined; ETEC, EAEC, and STEC).

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

| Multiple Detection Combination | Number of Specimens |
|-------------------------------------------|---------------------|
| C. difficile toxin A/B + EPEC | 32 |
| EAEC + EPEC | 15 |
| Campylobacter + EPEC | 11 |
| EPEC + Sapovirus | 10 |
| Adenovirus + EPEC | 9 |
| EPEC + Norovirus GI/GII | 9 |
| C. difficile toxin A/B + EAEC | 7 |
| C. difficile toxin A/B + Norovirus GI/GII | 6 |
| C. difficile toxin A/B + STEC stx1/stx2 | 5 |
| EPEC + ETEC lt/st | 5 |
| EPEC + G. lamblia | 5 |
| EPEC + Shigella/EIEC | 5 |

Table 10. Most Prevalent Multiple Detection Combinations (≥5 instances) as Determined by the FilmArray GI Panel

The overall success rate for initial specimen tests in the prospective study was 99.4% (1544/1557). Four tests were incomplete due to software errors (3) or a user aborted run (1), and nine tests were invalid due to pouch control failures. All specimens but one were retested within four days of specimen collection and were successful after a single retest, for a final success rate of 99.9% (1556/1557).

# Testing of Preselected Archived Specimens

Several analytes were either not encountered or had a low prevalence in the clinical study. To supplement the results of the prospective clinical study, an evaluation of 222 preselected archived specimens was performed. These specimens were archived clinical specimens that were selected because they had previously tested positive for one of the following analytes: E. coli O157, P. shigelloides, Y. enterocolitica, Vibrio, Astrovirus, and E. histolytica, or had been negative in previous laboratory testing. Prior to testing with the FilmArray GI Panel, the presence (or absence for negative specimens) of the expected analytes was verified in each specimen using analyte-specific PCR followed by bi-directional sequencing.

The specimens were organized into "test panels" and randomized such that the users performing the FilmArray GI Panel testing were blinded as to the expected test result. A summary of the available demographic information of the tested samples is provided in Table 11 and the results of the FilmArray GI testing are presented in Table 12.

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

| Preselected Archived Specimens | |
|--------------------------------|-------------------------|
| Total Specimens | 222 |
| Sex | Number of Specimens (%) |
| Male | 57 (25.7%) |
| Female | 48 (21.6%) |
| Unknown | 117 (52.7%) |
| Age Group | Number of Specimens (%) |
| <1 year | 12 (5.4%) |
| 1-5 years | 36 (16.2%) |
| 6-12 years | 15 (6.8%) |
| 13-21 years | 11 (5%) |
| 22-64 years | 18 (8.1%) |
| 65+ years | 4 (1.8%) |
| Unknown | 126 (56.8%) |

Table 11. Demographic Summary for Preselected Archived Specimens

Table 12. FilmArray GI Panel Archived Specimen Performance Data Summary

| Analyte | Positive Percent Agreement (PPA) | | | Negative Percent Agreement (NPA) | | |
|--------------------------------|----------------------------------|------|-----------|----------------------------------|-----|----------|
| | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Bacteria | | | | | | |
| Plesiomonas shigelloides | 12/12 | 100 | 73.5-100 | 107/107 | 100 | 96.6-100 |
| Vibrio | 1/1 | 100 | N/A | 127/127 | 100 | 97.1-100 |
| Yersinia enterocolitica | 8/8 | 100 | 63.1-100 | 117/117 | 100 | 96.9-100 |
| Diarrheagenic E. coli/Shigella | | | | | | |
| (STEC) E. coli 0157ª | 19/19 | 100 | 82.4-100 | 0/0 | - | - |
| Parasites | | | | | | |
| Cryptosporidium | 29/30 | 96.7 | 82.8-99.9 | 66/66 | 100 | 94.6-100 |
| Entamoeba histolytica | 2/2 | 100 | 15.8-100 | 123/123 | 100 | 97.0-100 |
| Giardia lamblia | 26/26 | 100 | 86.8-100 | 66/66 | 100 | 94.6-100 |
| Viruses | | | | | | |
| Astrovirus | 31/32 | 96.9 | 83.8-99.9 | 91/91 | 100 | 96.0-100 |
| Rotavirus A | 29/29 | 100 | 88.1-100 | 65/65 | 100 | 94.5-100 |

4 No non-0157 STEC were included in the data set; therefore, negative percent (NPA) could not be calculated for E. coli 0157.

# Testing of Contrived Specimens

Several analytes, such as Entamoeba histolytica, are so rare that both prospective and archived testing efforts were insufficient to demonstrate system performance. To supplement the prospective and archived data, an evaluation of contrived specimens was performed. Surrogate clinical specimens were prepared using residual specimens from the prospective clinical study that had previously tested negative for all GI panel analytes by FilmArray GI and comparator methods. Specimens were spiked at clinically relevant levels using five different quantified

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

strains for each organism (or unspiked; at least 50 of each). The analyte status of each contrived specimen was blinded to the users analyzing the specimens, and the specimens were randomized before testing. The results of the FilmArray GI testing are presented in the table below:

| | Positive Percent Agreement (PPA) | | | Negative Percent Agreement (NPA) | | |
|--------------------------|----------------------------------|------|-----------|----------------------------------|-----|----------|
| Analyte | TP/(TP + FN) | % | 95% CI | TN/(TN + FP) | % | 95% CI |
| Entamoeba histolytica | 44/50 | 88.0 | 75.7-95.5 | 75/75 | 100 | 95.2-100 |
| Plesiomonas shigelloides | 70/70 | 100 | 94.9-100 | 105/105 | 100 | 96.5-100 |
| Vibrioᵃ | 112/115 | 97.4 | 92.6-99.5 | 60/60 | 100 | 94.0-100 |
| V. choleraeᵇ | 55/65 | 84.6 | 73.5-92.4 | 110/110 | 100 | 96.7-100 |
| Yersinia enterocolitica | 65/65 | 100 | 94.5-100 | 110/110 | 100 | 96.7-100 |

| | Table 13. FilmArray GI Panel Performance using Contrived Specimens | | |
|--------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------|--------------------------------------|-----------------------------------|
| | All Property of Children Comments of Annual Property of Children Comments of Children Comments of Children Comments of Children | | |
| GI Panel Test Result | Species/Isolate Tested | Confirmed LoD<br>Concentration | Detection at LoD<br>Concentration |
| BACTERIA | | | |
| | Campylobacter coli<br>ATCC 33559 | | 20/20<br>100% |
| Campylobacter | Campylobacter jejuni<br>ATCC BAA-1234 | 4 x 104 cells/mL | 20/20<br>100% |
| | Campylobacter upsaliensis<br>ATCC BAA-1059 | | 20/20<br>100% |
| Clostridium difficile (toxin A/B) | Clostridium difficile<br>Toxinotype 0 A+B+<br>ATCC 9689 | 4 x 105 cells/mL | 20/20<br>100% |
| | Clostridium difficile (NAP1)<br>Toxinotype III A+B+<br>Zeptometrix #801619 | 4 x 104 cells/mL | 19/20<br>95% |
| Plesiomonas shigelloides | Plesiomonas shigelloides<br>ATCC 14029 | 1 x 103 CFU/mL | 20/20<br>100% |
| | Salmonella bongori O66:H1z41:H2-<br>SGSC RKS#3041 SarC11 | 1 x 104 CFU/mL | 20/20<br>100% |
| Salmonella | Salmonella enterica ssp. enterica Serovar<br>Typhimurium<br>O1,4,[5].12:Hli:H21.2<br>SGSC RKS#4194 SarC1 | 5 x 103 CFU/mL | 20/20<br>100% |
| Vibrio and<br>Vibrio cholerae | Vibrio cholerae<br>Ogawa serotype O:1<br>ATCC 14035 | 8 x 103 cells/mL | 20/20<br>100% |
| | Vibrio parahaemolyticus<br>ATCC 17802 | 8 x 104 cells/mL | 20/20<br>100% |
| Yersinia enterocolitica | Yersinia enterocolitica<br>ATCC 9610<br>Biovar1 serogroup O:8 | 5 x 104 CFU/mL | 20/20<br>100% |
| DIARRHEAGENIC E. coli/Shigella | | | |
| Enteroaggregative E. coli (EAEC) | Escherichia coli O92:H33<br>STEC Center # JM221 | 1 x 104 CFU/mL | 20/20<br>100% |
| Enteropathogenic E. coli (EPEC) | Escherichia coli E2348/69 O127:H6<br>STEC Center | 1 x 103 CFU/mL | 20/20<br>100% |
| Enterotoxigenic E. coli (ETEC) lt/st | Escherichia coli H10407 O78:H11<br>ATCC 35401 | 1 x 103 CFU/mL | 20/20<br>100% |
| Shiga-like toxin-producing E. coli (STEC)<br>stx1/stx2 | Escherichia coli O25:H11<br>ATCC BAA-2196 | 1 x 103 CFU/mL | 20/20<br>100% |
| E. coli O157 | Escherichia coli O157:H7<br>ATCC 43895 | 1 x 104 CFU/mL | 20/20<br>100% |
| Shigella/Enteroinvasive E. coli (EIEC) | Escherichia coli O29:NM<br>ATCC 43892 | 5 x 103 CFU/mL | 20/20<br>100% |
| | Shigella sonnei<br>ATCC 29930 | 100 CFU/mL | 20/20<br>100% |
| PARASITES | | | |
| Cryptosporidium a | Cryptosporidium parvum<br>Iowa isolate (Harley Moon)<br>Waterborne, Inc. P102C | 5 x 103 oocysts/mL | 20/20<br>100% |
| | Cryptosporidium hominis<br>Clinical Specimen | | 20/20<br>100% |
| Cyclospora cayetanensis | Cyclospora cayetanensis<br>Clinical Specimen | 180 genome<br>equivalents<br>(GE)/mL | 20/20<br>100% |
| GI Panel Test Result | Species/Isolate Tested | Confirmed LoD<br>Concentration | Detection at LoD<br>Concentration |
| Entamoeba histolytica | Entamoeba histolytica HM-1:IMSS<br>ATCC 30459 | $2 x 10^3$ cells/mL | 19/20<br>95% |
| Giardia lamblia | Giardia intestinalis (aka G. lamblia)<br>ATCC 30957 | 50 cells/mL | 20/20<br>100% |
| VIRUSES | | | |
| Adenovirus F 40/41 | Adenovirus F40<br>ATCC VR-931 | 1 TCID50/mL | 20/20<br>100% |
| | Adenovirus F41<br>ATCC VR-930 | 100 TCID50/mL | 20/20<br>100% |
| Astrovirus | Astrovirus - Type 8<br>NCPV#1003071v | 50 FFU/mL | 20/20<br>100% |
| Norovirus GI/GII | Norovirus GI<br>Clinical Specimen | $1 x 10^4$ RNA<br>copies/mL | 19/20<br>95% |
| | Norovirus GII<br>Clinical Specimen | | 20/20<br>100% |
| Rotavirus A | Rotavirus A - Type G4 [P6]<br>NCPV#0904053v | $1 x 10^5$ FFU/mL | 20/20<br>100% |
| Sapovirus | Sapovirus (Genogroup I)<br>Clinical Specimen | $1.1 x 10^7$ RNA<br>copies /mL | 20/20<br>100% |

ª Includes 64/65 V. cholerae (five different strains were used in spiked near the assay limit of detection was not detected) and 48/50 non-V. cholerae (four V. parahaemolyticus strain were used in spiking; two specimens spiked with V. parahaemolyticus near the assay limit of detection were not detected).

b Ten (10) of these specimens were spiked with an isolate which was found to have a highly divergent toxR gene that was not present in the NCBI database and non-reactive with the FilmArray G1 Panel V, cholerae assay, The FilmArray G1 Panel Vibrio assay was positive for nine of these specimens.

# Selected Analytic Studies

## Limit of Detection

A study was performed to determine the analytical sensitivity, or limit of detection (LoD), of the FilmArray GI Panel for each test result included in the panel. LoD (or LoD95) is defined as the lowest concentration of organism that can be consistently detected (≥ 95% of samples test positive) in the defined sample type (stool in Cary Blair transport medium).

The LoD for each organism was estimated with limiting dilutions as single-spiked and multispiked samples (up to four organisms per mix), to provide an estimated LoD concentration, and to determine whether assay sensitivity is affected by the presence of multiple panel organisms in a single sample.

Confirmation of LoDs was performed by spiking organism (single or multi-spike) at the LoD estimate determined by the dilutions series, into 20 independent stool samples. LoD was confirmed when the correct organism/assay results were obtained from at least 19 of the 20 samples (19/20 = 95%) tested.

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

Table 14. Table of Confirmed Limit of Detection (LoD) for GI Panel Analytes

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

ª Limited testing with a clinical specimen containing Cryptosporidium meleagridis indicates that the LoD for this species is similar to that of C. parvum and C. hominis.

# Inclusivity

The analytical reactivity (inclusivity) of the FilmArray GI Panel was evaluated with a collection of 270 isolates that represent the diversity of the FilmArray GI Panel analytes. Isolates were selected to represent relevant subspecies or serotypes and selection was biased toward more common species and known human pathogens. When possible, in silico analysis of sequence data was used to make predictions of assay reactivity for less common species, strains, serovars, or serotypes that were not tested.

Organisms were tested at concentrations near the limit of detection (LoD). If a sample containing a particular strain was positive (detected) at the initial test level, no further testing was required. If a strain was not detected, the strain was retested at the same level (up to five additional times) and if necessary, additional testing was performed at 10- and 100-fold higher concentrations to determine if the strain can be detected by the GI Panel. Based upon predicted assay reactivity, a few select isolates were initially tested at a high concentration, followed by evaluation at lower concentrations if detection was observed. Results are provided below for each FilmArray GI Panel test result.

| Organism | Isolate ID | Concentration<br>Detected<br>(cells/mL) | Multiple of LoD<br>Detected |
|---------------------|---------------|-----------------------------------------|-----------------------------|
| Campylobacter colia | ATCC BAA-1061 | 1.2 x 105 | 3×LoD |
| | BEI HM-296 | 1.2 x 105 | 3×LoD |
| | ATCC43485 | 1.2 x 105 | 3×LoD |
| | ATCC 43478 | 1.2 x 105 | 3×LoD |

Table 15. FilmArray Campylobacter Inclusivity Results (C. coli/C. jejuni/C. upsaliensis)

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

| Organism | Isolate ID | Concentration<br>Detected<br>(cells/mL) | Multiple of LoD<br>Detected |
|------------------------------------|----------------|-----------------------------------------|-----------------------------|
| Campylobacter jejuni subsp. doylei | ATCC 33559b | 4.0 x 104 | 1×LoD |
| Campylobacter jejuni subsp. doylei | ATCC 49349 | 4.0 x 106 | Not Detectedc |
| Campylobacter jejuni subsp. doylei | ATCC 49351 | 4.0 x 106 | 100×LoDc |
| Campylobacter jejuni subsp. doylei | ATCC 49350 | 4.0 x 106 | Not Detectedc |
| Campylobacter jejuni subsp. jejuni | ATCC 43430 | 1.2 x 105 | 3×LoD |
| Campylobacter jejuni subsp. jejuni | ATCC BAA-1062 | 1.2 x 105 | 3×LoD |
| Campylobacter jejuni subsp. jejuni | ATCC BAA-1234b | 4.0 x 104 | 1×LoD |
| Campylobacter jejuni subsp. jejuni | BEI NR-128 | 1.2 x 105 | 3×LoD |
| Campylobacter upsaliensis | ATCC BAA-1059 | 4.0 x 104 | 1×LoD |
| Campylobacter upsaliensis | CCUG 24191 | 1.2 x 105 | 3×LoD |
| Campylobacter upsaliensis | ATCC 43953 | 1.2 x 105 | 3×LoD |
| Campylobacter upsaliensis | ATCC 43954d | 4.0 x 106 | Not Detectedd |
| Campylobacter upsaliensis | ATCC 49815 | 1.2 x 105 | 3×LoD |
| Campylobacter upsaliensis | BEI HM-297 | 1.2 x 105 | 3×LoD |

4 In silico analysis indicates primer mismatches that might lead to reduced assay sensitivity or lack of reactivity with 11/138 C. coli sequences.

b Isolate was used to establish the LoD for this assay.

& In silico analysis indicates primer mismatches that might lead to reduced assay sensitivity for this subspecies.

4 Sequencing under the primers identified an insertion/deletion in the primer binding region of the target gene.

| Organism | Toxinotype | Isolate ID | Concentration<br>Detected<br>(cells/mL) | Multiple of LoD<br>Detected |
|-----------------------|------------|-------------------------|-----------------------------------------|-----------------------------|
| Clostridium difficile | 0 A+B+ | ATCC 9689ª | $4.0 x 10^5$ | 1xLoD |
| | | ATCC BAA-1382 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 17857 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 17858 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 43255 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 43594 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 43596 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 43599 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 43600 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 51695 | $1.2 x 10^6$ | $3×LoD$ |
| | | ATCC 700792 | $1.2 x 10^6$ | $3×LoD$ |
| | III A+B+ | ATCC BAA-1805<br>(NAPI) | $1.2 × 10^6$ | $3×LoD$ |

Table 16. FilmArray Clostridium difficile toxin A/B Inclusivity Results

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

| Organism | Toxinotype | Isolate ID | Concentration<br>Detected<br>(cells/mL) | Multiple of LoD<br>Detected |
|----------|--------------------|---------------------------------|-----------------------------------------|-----------------------------|
| | | Zeptometrix<br>#0801619 (NAP1)a | $4.0 x 10^4$ | 1×LoD |
| | V A+B+ | ATCC BAA-1875 | $1.2 x 10^6$ | 3×LoD |
| | VIII A-B+ | ATCC 43598 | $1.2 x 10^6$ | 3×LoD |
| | X A-B+ | CCUG 8864 | $1.2 x 10^6$ | 3×LoD |
| | XII A+B+ | ATCC BAA-1812 | $1.2 x 10^6$ | 3×LoD |
| | XXII A+B (unknown) | ATCC BAA-1814 | $1.2 x 10^6$ | 3×LoD |

ª This isolate was used to establish the LoD for this assay.

# Table 17. FilmArray Plesiomonas shigelloides Inclusivity Results

| Organism | Geographic<br>Isolation | Isolate ID | Concentration<br>Detected (cells/mL) | Multiple of LoD<br>Detected |
|--------------------------|-------------------------|--------------------------|--------------------------------------|-----------------------------|
| Plesiomonas shigelloides | CDC 3085-55 | ATCC 14029ª | 1.0×103 | lxLoD |
| | CDC 16408 | ATCC 14030 | 3.0 x 103 | 3×LoD |
| | Dakar. Senegal | ATCC 51572 | 3.0 x 103 | 3×LoD |
| | Unknown | ATCC 51903 | 3.0 x 103 | 3×LoD |
| | Colorado | CDPH HUM-2011019465 | 3.0 x 103 | 3×LoD |
| | Czech Republic | NIPH-Czech Republic 6300 | 3.0 x 103 | 3×LoD |

ª This isolate was used to establish the LoD for this assay. The organism was quantified in CFU/mL by plate enumeration.

| | Table 18. FilmArrav Salmonella Inclusivitv Results | |
|--|----------------------------------------------------|--|
| | | |

| Organism<br>(species, subspecies, and serovar) | Isolate ID | Concentration<br>Detected<br>(cells/mL) | Multiple of<br>LoD Detected |
|----------------------------------------------------------|-------------------|-----------------------------------------|-----------------------------|
| <i>Salmonella bongori</i> | SGSC<br>RKS 3041a | $1.0 \times 10^4$ | 1xLoD |
| <i>Salmonella bongori</i> | NCTC 10946 | $3.0 \times 10^4$ | 3xLoD |
| <i>Salmonella enterica</i> subsp. <i>salamae II</i> | SGSC<br>RKS 3044 | $3.0 \times 10^4$ | 3×LoD |
| <i>Salmonella enterica</i> subsp. <i>arizonae IIIa</i> | SGSC<br>RKS 2985 | $1.5 \times 10^4$ | 3×LoD |
| <i>Salmonella enterica</i> subsp. <i>diarizonae IIIb</i> | SGSC<br>RKS 2980 | $1.5 \times 10^4$ | 3xLoD |
| <i>Salmonella enterica</i> subsp. <i>houtenae IV</i> | SGSC<br>RKS 2978 | $1.5 \times 10^4$ | 3xLoD |
| <i>Salmonella enterica</i> subsp. <i>indica VI</i> | SGSC<br>RKS 3027 | $1.5 \times 10^4$ | 3xLoD |
| <i>Salmonella enterica</i> Typhimurium | SGSC<br>RKS 2995 | $1.5 \times 10^4$ | 3xLoD |
| <i>Salmonella enterica</i> Typhimurium | SGSC<br>RKS 4194a | $5.0 \times 10^3$ | IxLoD |

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

| Organism<br>(species, subspecies, and serovar) | Isolate ID | Concentration<br>Detected<br>(cells/mL) | Multiple of<br>LoD Detected | |
|------------------------------------------------|-------------------------------------------------------|-----------------------------------------|-----------------------------|-------|
| subsp. entericab<br> | Enteritidis | ATCC BAA-708 | 1.5 x 104 | 3×LoD |
| | Newport | ATCC 27869 | 1.5 x 104 | 3×LoD |
| | Javiana | ATCC 10721 | 1.5 x 104 | 3×LoD |
| | Heidelberg | ATCC 8326 | 1.5 x 104 | 3×LoD |
| | Montevideo | ATCC BAA-710 | 1.5 x 104 | 3×LoD |
| | 4,[5],12:i:- | Cornell CU0580 | 1.5 x 104 | 3×LoD |
| | Oranienburg | ATCC 9239 | 1.5 x 104 | 3×LoD |
| | Saintpaul | ATCC 9712 | 1.5 x 104 | 3×LoD |
| | Muenchen | ATCC 8388 | 1.5 x 104 | 3×LoD |
| | Braenderup | ATCC 700136 | 1.5 x 104 | 3×LoD |
| | Infantis | ATCC<br>BAA-1675 | 1.5 x 104 | 3×LoD |
| | Thompson | ATCC 8391 | 1.5 x 104 | 3×LoD |
| | Mississippi | Cornell CU0633 | 1.5 x 104 | 3×LoD |
| | Paratyphi B var. L(+)<br>tartrate+<br>(formerly java) | CCUG 9561 | 1.5 x 104 | 3×LoD |
| | Typhi (Purified DNA)b | ATCC<br>700931D-5 | 1.5 x 104 | 3×LoD |
| | Agona | ATCC 51957 | 1.5 x 104 | 3×LoD |
| | Schwarzengrund | CCUG 21280 | 1.5 x 104 | 3×LoD |
| | Bareilly | ATCC 9115 | 1.5 x 104 | 3×LoD |
| | Hadar | ATCC 51956 | 1.5 x 104 | 3×LoD |

4 This isolate was used to establish the LolD for this assay. The organism was quantified in CFUmL by plate cnumeration.

b Purified DNA was quantified in GE/mL by spectrophotometer.

Note: In addition to those evaluated in this study, in silico sequence analysis indicates the FilmArray Salmonella assay should react with all species and subspecies of Salmonella, including all serotypes of S. enterica subsp. enterica.

| Table 19. FilmArray Vibrio ( <i>V. parahaemolyticus/V. vulnificus/V. cholerae</i> ) and Vibrio cholerae |
|---------------------------------------------------------------------------------------------------------|
| Inclusivity Results |

| Organism<br>(species, biotype and serotype) | Source/Isolate ID | Concentration<br>Detect…