VERIGENE ENTERIC PATHOGEN NUCLEIC ACID TEST ( EP)

K140083 · Nanosphere, Inc. · PCH · Jun 20, 2014 · Microbiology

Device Facts

• Record ID: K140083
• Device Name: VERIGENE ENTERIC PATHOGEN NUCLEIC ACID TEST ( EP)
• Applicant: Nanosphere, Inc.
• Product Code: PCH · Microbiology
• Decision Date: Jun 20, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3990
• Device Class: Class 2

Indications for Use

The Verigene® Enteric Pathogens Nucleic Acid Test (EP) is a multiplexed, qualitative test for simultaneous detection and identification of common pathogenic enteric bacteria and genetic virulence markers from liquid or soft stool preserved in Cary-Blair media, collected from individuals with signs and symptoms of gastrointestinal infection. The test is performed on the automated Nanosphere Verigene System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific gastrointestinal microbial nucleic acid gene sequences associated with the following pathogenic bacteria: - Campylobacter Group (comprised of C. coli, C. jejuni, and C. lari) - Salmonella species - Shigella species (including S. dysenteriae, S. boydii, S. sonnei, and S. flexneri) - Vibrio Group (comprised of V. cholerae and V. parahaemolyticus) - Yersinia enterocolitica In addition, EP detects the Shiga toxin 1 gene and Shiga toxin 2 gene virulence markers. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins 1 and 2. EP is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness, in conjunction with other clinical, laboratory, and epidemiological information; however, is not to be used to monitor these infections. EP also aids in the detection and identification of acute gastroenteritis in the context of outbreaks. Due to the limited number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Yersinia enterocolitica, Vibrio Group and Shigella species were primarily established with contrived specimens. Concomitant culture is necessary for organism recovery and further typing of bacterial agents. EP results should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Confirmed positive results do not rule out co-infection with other organisms that are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative EP 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 non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Device Story

The Verigene System is a bench-top molecular diagnostics workstation for clinical laboratory use. It processes liquid or soft stool samples preserved in Cary-Blair media. The system automates cell lysis, magnetic bead-based DNA isolation, and PCR amplification. Target sequences are identified via microarray hybridization using gold nanoparticle probes; silver enhancement creates gold-silver aggregates that scatter light. The Verigene Reader images the light scatter from capture spots to determine the presence or absence of specific bacterial nucleic acids. The system provides a sample-to-result workflow, reducing manual intervention. Results assist clinicians in diagnosing gastrointestinal illness and managing outbreaks. The device is intended for use in conjunction with other clinical, laboratory, and epidemiological information; it does not replace culture for organism recovery or further typing.

Clinical Evidence

Multi-site prospective clinical study (7 U.S. institutions) evaluated 1,852 evaluable specimens (prospectively collected and simulated). Compared to reference methods (culture, phenotype ID, EIA, PCR/BDS). Results showed high agreement for all targets: Campylobacter (97.0% positive, 99.1% negative), Salmonella (97.2% positive, 99.5% negative), Shigella (98.3% positive, 99.0% negative), Vibrio (91.5% positive, 99.9% negative), Yersinia (100% positive, 100% negative), Stx1 (100% positive, 99.7% negative), and Stx2 (97.3% positive, 99.8% negative).

Technological Characteristics

Molecular assay using microarray format with gold nanoparticle probe-based detection and silver enhancement. Automated bench-top workstation (Verigene Reader and Processor SP). Performs magnetic bead-based DNA isolation, PCR amplification, and hybridization. Connectivity via touchscreen and barcode scanner. Single-use disposable test consumables and cartridges.

Indications for Use

Indicated for individuals with signs and symptoms of gastrointestinal infection to detect and identify pathogenic enteric bacteria (Campylobacter, Salmonella, Shigella, Vibrio, Yersinia enterocolitica) and Shiga toxin 1/2 genes in liquid or soft stool preserved in Cary-Blair media. Aids in diagnosis of gastrointestinal illness and outbreak identification. Not for monitoring infections.

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

• xTAG® Gastrointestinal Pathogen Panel (GPP) (K121894)

Submission Summary (Full Text)

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Image /page/0/Picture/0 description: The image shows the word "Nanosphere" in bold black font, with a stylized logo to the left. The logo is a circle, partially filled with black and partially filled with diagonal lines. The text is simple and clear, making it easily readable.

# 510(K) Summary

JUN 2 0 2014

The Summary for this 510(k) submission is submitted in accordance with the requirements of SMDA 1900 and CFR 807.92

# 510(k) Number:

Verigene® Enteric Pathogens Nucleic Acid Test (EP) K140083:

# Summary Preparation Date:

June 18, 2014

# Submitted by:

Nanosphere, Inc. 4088 Commercial Avenue Northbrook, IL 60062 Phone: [phone] Fax: [phone]

# Contact:

Noah Lermer, Ph.D. Director, Regulatory Affairs

# Proprietary Names:

For the instrument: Verigene® System For the assay: Verigene® Enteric Pathogens Nucleic Acid Test (EP)

# Common Names:

For the instrument:

Bench-top molecular diagnostics workstation

For the assay:

Enteric Pathogens Nucleic Acid Test Enteric Pathogens identification and differentiation system Enteric assay Enteric test

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# Regulatory Information:

Regulation section:

866. 3990 - Gastrointestinal microorganism multiplex nucleic acid-based assay

Classification:

Class II

Panel:

Microbiology (83)

Product Code(s):

Gastrointestinal Pathogen Panel Multiplex Nucleic Acid-Based Assay System PCH

Gastrointestinal Bacterial Panel Multiplex Nucleic Acid-based Assay System PCI

001 Real Time Nucleic Acid Amplification System

Other codes used by predicate device:

Instrumentation for clinical multiplex test systems NSU

TH Clinical Sample Concentrator

## Predicate Devices:

xTAG® Gastrointestinal Pathogen Panel (GPP) (K121894) (Luminex Molecular Diagnostics, Inc.)

# Indications for Use:

The Verigene® Enteric Pathogens Nucleic Acid Test (EP) is a multiplexed. qualitative test for simultaneous detection and identification of common pathogenic enteric bacteria and genetic virulence markers from liquid or soft stool preserved in Cary-Blair media, collected from individuals with signs and symptoms of gastrointestinal infection. The test is performed on the automated Nanosphere Verigene System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific gastrointestinal microbial nucleic acid gene sequences associated with the following pathogenic bacteria:

- Campylobacter Group (comprised of C. coli. C. jejuni. and C. lari) .
- . Salmonella species
- Shigella species (including S. dysenteriae, S. boydii, S. sonnei, and S. flexneri) .
- Vibrio Group (comprised of V. cholerae and V. parahaemolyticus) .
- . Yersinia enterocolitica

In addition, EP detects the Shiga toxin 1 gene and Shiga toxin 2 gene virulence markers. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins l and 2.

EP is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness, in conjunction with other clinical, laboratory, and epidemiological information; however, is not to be used to monitor these infections. EP also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

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Due to the limited number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Yersinia enterocolitica, Vibrio Group and Shigella species were primarily established with contrived specimens.

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

EP results should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Confirmed positive results do not rule out co-infection with other organisms that are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative EP 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 non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

# Technological Characteristics:

The Verigene Enteric Pathogens Nucleic Acid Test (EP) is a molecular assay which relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial nucleic acid sequences detected by EP, unique Capture and Mediator oligonucleotides are utilized, with gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides are covalently bound to the microarray substrate and hybridize to a specific portion of the nucleic acid targets. The Mediator oligonucleotides have a region which bind to a different portion of the same nucleic acid targets and also have a sequence which allows binding of a gold nanoparticle probe. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency and provide accurate detection of target capture.

The EP test is performed on the Verigene System, a "sample-to-result", fully automated, bench-top molecular diagnostics workstation. The System enables automated nucleic acid extraction from unformed stool specimens (liquid or soft) preserved in Cary-Blair media and detection of bacterial-specific target DNA. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP.

The Reader is the Verigene System's user interface, which serves as the central control unit for all aspects of test processing, automated imaging, and result generation using a touchscreen control panel and a barcode scanner. The Verigene Processor SP executes the test procedure, automating the steps of (1) Sample Preparation and Target Amplification – cell lysis and magnetic bead-based bacterial DNA isolation and amplification, and (2) Hybridizationdetection and identification of bacterial-specific DNA in a microarray format by using gold nanoparticle probe-based technology. Once the specimen is loaded by the operator, all other fluid transfer steps are performed by an automated pipette that transfers reagents between wells of the trays and finally loads the specimen into the Test Cartridge for hybridization. Single-use disposable test consumables and a self-contained Verigene Test Cartridge are utilized for each sample tested with the EP assay.

To obtain the test results after test processing is complete, the user removes the Test Cartridge from the Processor SP, and inserts the substrate holder into the Reader for analysis. Light scatter from the capture spots is imaged by the Reader and intensities from the microarray spots are used to make a determination regarding the presence (Detected) or absence (Not Detected) of a bacterial nucleic acid sequence/analyte. This determination is made by means of software-based decision algorithm resident in the Reader.

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# Performance Data - Analytical Testing

# Analytical Sensitivity / Limit of Detection (LoD)

Analytical sensitivity (LoD) of the EP test was determined for 16 strains of enteric pathogens, representing all seven (7) EP test reportable target analytes. The LoD was defined as the concentration at which the test produces a positive result at least 95% of the time. Serial dilutions of the strains were tested and the putative LoD confirmed with 20 replicates. To ensure the accuracy of the LoD determination, if the initial detection rate was 100%, a further 20 replicates were performed at the next lower concentration until <95% was achieved. The LoDs for the 16 strains tested, and the corresponding LoD ranges for the EP test reportable target, are shown in the table below. Overall, the LoDs range from 4.10x10 to 3.33x10 CFU/mL of stool.

| Representative Organism Tested | ATCC<br>Source<br>Number | Organism<br>LoD<br>(CFU/mL) | Reportable<br>Target | EP Test Target<br>LoD<br>(CFU/mL Stool) |
|--------------------------------------------------|--------------------------|-----------------------------|----------------------------|-----------------------------------------|
| Campylobacter jejuni subsp jejuni | 43429 | $3.70x10^4$ | | |
| Campylobacter coli | 43482 | $1.11x10^5$ | Campylobacter | $3.70x10^4$ - $1.11x10^5$ |
| Campylobacter lari | 35222 | $3.70x10^4$ | | |
| Salmonella enterica subsp enterica serovar typhi | 9993 | $3.33x10^5$ | Salmonella | $3.33x10^5$ |
| Salmonella enterica subsp arizonae | 13314 | $3.33x10^5$ | | |
| Shigella dysenteriae / Shiga Toxin 1 | 29026 | $3.70x10^4$ | Shigella, Stx1 | |
| Shigella flexneri | 25929 | $1.11x10^5$ | | $3.70x10^4$ - $1.11x10^5$ |
| Shigella sonnei | 29030 | $3.70x10^4$ | Shigella | |
| Shigella boydii | 12035 | $1.11x10^5$ | | |
| Vibrio cholerae | 39315 | $1.11x10^5$ | | |
| Vibrio parahaemolyticus | 49398 | $3.70x10^4$ | Vibrio | $3.70x10^4$ - $1.11x10^5$ |
| Yersinia enterocolitica | 700822<br>23715 | $3.33x10^5$<br>$1.11x10^5$ | Yersinia<br>enterocolitica | $1.11x10^5$ - $3.33x10^5$ |
| E. coli - Shiga Toxin I | 43890 | $4.10x10^3$ | Stx1 | $4.10x10^3$ - $3.70x10^4$ |
| E. coli - Shiga Toxin 2 | BAA-176 | $1.11x10^5$ | Stx2 | $3.70x10^4$ - $1.11x10^5$ |
| E. coli - Shiga Toxin 1 / Shiga Toxin 2 | 43895 | $3.70x10^4$ | | |

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# Analytical Reactivity (Inclusivity)

Analytical reactivity of the EP test was demonstrated with a comprehensive panel of 111 clinically relevant bacterial strains representing temporal, and phylogenic diversity for each claimed target (see table below). For the Stx2 targets, Shiga toxin producing organisms tested included the vast majority of serotypes isolated in the U.S and those that are outbreak-related. All 111 strains generated the expected result when tested in triplicate at a concentration of three times LoD.

| | Total Number of | Species Tested | | | |
|-------------------------|-------------------|-------------------------------------------------------------------------------------|--------|--|--|
| Reportable Target | Organisms/Strains | Name | Total | | |
| | Tested | (No. of Strains) | Number | | |
| Campvlobacter | ો રે | C. coli (5). C. jejuni subsp jejuni (4). C. jejuni<br>subsp dovlei (1), C. lari (5) | 3 | | |
| Salmonella | 31 | S. bongori (1), S. enterica subsp various (5), | 2 | | |
| | | S. enterica subsp enterica serovar various (25) | | | |
| Shigella | 20 | S. bovdii (5), S. dysenteriae (5) , S. flexneri (5), | 4 | | |
| | | S. sonnei (5) | | | |
| Vibrio | 10 | I'. cholerae (5), V. parahaemolvticus (5) | 2 | | |
| Yersinia enterocolitica | 7 | Y, enterocolitica (7) | | | |
| Shiga toxin 1 | 19 | S. dysenteriae (2)a. E. coli (17)0 | 2 | | |
| Shiga toxin 2 | 16 | E. coli (16) " | | | |

Two (2) strains contain Stx I

Five (5) strains contain both Stx1 and Stx2

# Analytical Specificity (Cross-reactivity)

One-hundred and sixty-one (161) organisms, consisting of 135 bacterial organisms, 21 viruses, four (4) parasites and one (1) human cell line were tested with the EP test to determine analytical specificity (see table below). Eight (8) organisms, including Astrovirus and Sapovirus (2 strains). Campylobacter hominis and all four parasites were tested as genomic DNA/RNA. In addition, to rule out cross-reactivity between the analytes detected by the EP test, six organisms representing all of the EP test detected targets, were tested at elevated concentrations of 5 x 10° CFU/mL. The exclusivity of 15 species of Vibrio not associated with human infection, four (4) non-pathogenic strains of Escherichia coli, Yersinia pestis, and Clostridium botulinum were evaluated by in silico analysis alone.

All of the organisms tested yielded the expected "Not Detected" results, indicating that there was no cross-reactivity with the EP test. with the exception of Campylobacter insulaenigrae which yielded a single positive result (1/9) for "Campylobacter". In silico analysis also indicates a potential for low-level cross-reactivity. While Campylobacter insulaenigrae has been isolated primarily from marine mammals, in rare cases it may cause septicemia and gastroenteritis in humans. 11

[1] J Med Microbiol. 2007 Nov;56(Pt 11):1565-7.

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| Organisms Tested for Analytical Specificity | | | |
|-----------------------------------------------------------------|-----------------------|----------------------------------------------------|------------------------|
| Bacterial Non-Test Panel Members | | Bacterial EP Test Panel Members | |
| Genus | Species | Genus | Species |
| Abiotrophia | defectiva | Campylobacter | concisus |
| Acinetobacter | baumannii | | curvus |
| | Iwoffli | | fetus |
| Acrobacter | butzleri | | gracilis |
| | cryaerophilus | | hominis |
| | allosaccharophila | | hyointestinalis |
| | bestiarum | | insulaenigrae |
| | caviae | | lanienae |
| | encheleia | | mucosalis |
| | enteropelogenes | | rectus |
| Aeromonas | eucrenophila | | showae |
| | hydrophilia | | sputorum |
| | jandaei | | upsaliensis |
| | salmonicida* | Vibrio | alginolyticus |
| | veronii | | campbellii |
| Alcaligenes | faecalis | | cincinnatiensis |
| Bacillus | cereus | | fluvialis |
| | caccae | | furnissii |
| Bacteroides | fragilis | | harvevi |
| | merdae | | metschnikovii |
| | stercoris | | mimicus |
| Candida | albicans | | tubiashii |
| Cedecea | davisae | | vulnificus (3 strains) |
| | amalonaticus | Yersinia | aldovae |
| Citrobacter | freundii | | aleksiciae |
| | sedlakii | | bercovieri |
| | bifermentans | | frederiksenii |
| | bolteae | | intermedia |
| | butyricum | | kristensenii |
| | difficile (2 strains) | | mollaretii |
| | difficile, non-tox | | pseudotuberculosis |
| | haemolyticum | | ruckeri |
| | methylpentosum | | rohdei |
| Clostridium | nexile | Viruses | |
| | novvi | Name | Serovar / Group |
| | orbiscindens | Adenovirus | Type 1/Group C |
| | perfringens | | Type 2/Group C |
| | scindens | | Type 3/Group B1 |
| | septicum | | Type 4/Group E |
| | sordellii | | Type 5/Group C |
| | spiroforme | | Type 14/Group B2 |
| | sporogenes | | Type 26/Group D |
| Colinsella | aerofaciens | | Type 31/Group A |
| Desulfovibrio | piger | | Type 37/Group D |
| Edwardsiella | tarda | | Type 40/Group F |
| Enterobacter | aerogenes | | Human 4 |
| Enterobacter | cloacae | Human Cell Line | |
| Enterococcus | faecalis | Astrovirus | - |
| | faecium | Coxsackievirus B4 | - |
| * Sub-species masoucida and sub-species salmonicida (2 strains) | | Cytomegalovirus | - |
| | | Echovirus 11 | - |
| | | Enterovirus 68 | - |
| | | Norovirus | Genogroup GI |
| | | | Genogroup GII |
| | | Rotavirus | Genogroup A |
| | | Sapovirus | - |
| Fusobacterium | varium | Lactobacillus | acidophilus |
| Helicobacter | hepaticus | | reuteri |
| | pylori (4 strains) | | rhamnosus |
| Escherichia | coli (3 strains) | Lactococcus | lactis |
| | coli (EAEC) | Leminorela | grimontii |
| | coli (EPEC) (2) | Listeria | gravi |
| | coli (ETEC) (2) | | monocytogenes |
| | fergusonii | Morganella | morganii |
| | hermannii | Peptostreptococcus | anaerobius |
| Klebsiella | oxytoca | Plesiomonas | shigelloides |
| | pneumoniae | Porphyromonas | asaccharoluticus |
| Lactobacillus | acidophilus | Prevotella | melaniogenica |
| | reuteri | Proteus | mirabilis |
| | rhamnosus | | vulgaris |
| Lactococcus | lactis | | penneri |
| Leminorela | grimontii | | stuartii |
| Listeria | gravi | Providencia | alcalifaciens |
| | monocytogenes | | rettgeri |
| Morganella | morganii | Pseudomonas | aeruginosa |
| Peptostreptococcus | anaerobius | | fluroescenes |
| Plesiomonas | shigelloides | | putida |
| Porphyromonas | asaccharoluticus | Ruminococcus | aeruginosa |
| Prevotella | melaniogenica | | bromii |
| Proteus | mirabilis | Serratia | liquefaciens |
| | vulgaris | | marcescens |
| | penneri | Staphylococcus | aureus |
| | stuartii | | epidermidis |
| Providencia | alcalifaciens | | agalactiae, O90R |
| | rettgeri | Streptococcus | dysgalactiae |
| Pseudomonas | aeruginosa | | mutans |
| | fluroescenes | Parasites | |
| | putida | Blastocystis | hominis |
| Ruminococcus | aeruginosa | Cryptosporidium | parvum |
| | bromii | Entamoeba | histolytica |
| Serratia | liquefaciens | Giardia | lamblia |
| | marcescens | Colon epithelial cells (colorectal adenocarcinoma) | |
| Staphylococcus | aureus | | |
| | epidermidis | | |
| | agalactiae, O90R | | |
| Streptococcus | dysgalactiae | | |
| | mutans | | |

・

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## Microbial Interference

Two representative bacterial organisms detected by the EP test, Campylobacter jejuni and Escherichia coli (Shiga toxin 1), were evaluated for potential interference in the presence of 14 potentially interferent microorganisms not detected by the EP test, including Bacteroides fragilis, Prevotella oralis, Prevotella melaninogenicus, Bifidobacterium bifidum, Clostridium perfringens, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Lactobacillus, Staphylococcus aureus, Blastocystis hominis, Entamoeba histolytica, and Candida albicans. These 14 microorganisms represent the most prevalent bacteria known to be present in the human colon and therefore are the most likely to be encountered in stool specimens tested with the EP test. These normal flora bacteria were tested at a concentration of 10' CFU/mL with the exception of the parasites Blastocystis hominis and Entamoeba histolytica which were tested at 9x10 cells/mL and 7x10 cells/mL respectively. No interference was observed with the EP test for any of the samples tested.

## Interference (Exogenous Substances)

A comprehensive interfering substances study was performed to assess the potential inhibitory effect of endogenous and exogenous substances that can commonly be found in clinical stool specimens. Two organisms representative of the target analytes detected by the EP test, i.e., Campvlobacter jejuni and Escherichia coli (Shiga toxin 1), were individually challenged with 22 potentially interfering substances (shown in the following table) at high, medically-relevant concentrations. None of the 22 substances tested showed any inhibitory effect on the detection of target enteric pathogens using the EP test.

| Intralipid | Vaseline Original 100% Pure Petroleum Jelly |
|----------------------------------------------|-------------------------------------------------|
| Cholesterol | Tums Antacid with Calcium Extra Strength 750 |
| Whole Blood | Gaviscon Extra Strength Liquid Antacid |
| Mucus (Nasopharyngeal swab sample in UTM) | Mesalazine |
| Nystatin Suspension | Immodium® AD Anti-Diarrheal |
| Preparation H® Anti-itch Hydrocortisone 1% | Pepto-Bismol Max Strength |
| Desitin Maximum Strength Original Paste | Metronidazole Topical Cream (0.75%) |
| Preparation H® Hemorrhoidal Ointment | Naproxen Sodium |
| Options Conceptrol®Vaginal Contraceptive Gel | Mucin from bovine submaxillary glands, Type I-S |
| Wet Ones® Antibacterial Hand Wipes | Barium Sulfate |
| K-Y®Personal Lubricant Jelly | Amoxicillin (Antibiotic) |

# Carryover / Cross-contamination

The potential for carryover and cross-contamination of the EP test on the Verigene system was assessed by alternately testing six representative high positive enteric pathogen samples (Yersinia enterocolitica, Shigella dysenteriae / Stx1, Escherichia coli / Stx2, Salmonella enterica enterica, Campylobacter jejuni , and Vibrio cholera) at 5x10° CFU/mL, followed by testing a negative stool sample. The high-titer sample was alternated with the negative sample three times on six unique Verigene SP Processors. No carryover or cross-contamination was observed.

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### Competitive Inhibition

Binary combinations of all six of the EP test panel organisms representing all possible dual infections were evaluated, using simulated samples prepared in Negative Stool Matrix (NSM), with one panel organism present at a Low Positive titer (3x LoD) and a second organism present at a High Positive titer (> 106 CFU/mL stool). The performance of the EP test was evaluated with each of the 30 unique sample combinations tested in replicates of three (3). The EP test correctly detected both bacterial target organisms present in the co-infection combinations tested with one exception. For the Low Titer Campylobacter coli and High Titer E. coli/Stx2 sample, the EP test did not detect Campylobacter in one of the three replicates, although Shiga Toxin 2 was correctly identified in all cases. However, repeat testing indicated that this observation was not indicative of competitive inhibition.

# Cutoff Verification

Target mean intensity values observed with the EP test were examined for the testing of the sixteen bacterial samples used to establish the Limit of Detection of the assay. In addition, the cut-off data set included the test results of three negative control samples. With replicates of 20 for each sample and ten target spot groups evaluated per test, a total of 3800 data points (1120 expected positive) were assessed to verify the assay cut-off.

#### Precision

The precision study was conducted in-house by Nanosphere, during which a fourteen-member simulated sample panel was tested daily in duplicate by two (2) operators for four (4) non-consecutive days for a total of sixteen (16) tests per sample. In total, the study yielded 224 test results. The fourteen (14) sample panel comprised six (6) different strains at two (2) different concentrations (12 positive samples) and two (2) negative samples (Negative Stool Matrix and Clostridium difficile). This panel included for each strain, a "Low Positive" sample (defined as approximately 1-2x LoD), which would be expected to produce a positive result approximately 95% of the time, and a "Moderate Positive" sample (defined as approximately 2-5x LoD). which would be expected to yield a positive result approximately 100% of the time. Results are summarized below.

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| Sample | EP Test Expected<br>Call | Conc. | Agreement<br>w/ Expected<br>Result<br>(95 % CI)ª | Sample | EP Test<br>Expected Call | Conc. | Agreement<br>w/ Expected<br>Result<br>(95 % CI) ª |
|---------------------------------|--------------------------|----------|--------------------------------------------------|----------------------------|-----------------------------|----------|---------------------------------------------------|
| Escherichia<br>coli/Stx2 | E. coli<br>Stx2 | Moderate | 100%<br>16/16<br>(79.4%-100%) | Campvlobacter | Campylobacter | Moderate | 100%<br>16/16<br>(79.4%-100%) |
| | | Low | 100%<br>16/16<br>(79.4%-100%) | jejuni | | Low | l 00%<br>16/16<br>(79.4%-100%) |
| Sulmonella<br>enterica | Salmonella | Moderate | 100%<br>16/16<br>(79.4%-100%) | Vibrio<br>parahaemolyticus | Vibrio | Moderate | 100%<br>16/16<br>(79.4%-100%) |
| | | Low | 93.8%<br>15/16 p<br>(69.8%-99.8%) | | | Low | 100%<br>16/16<br>(79.4%-100%) |
| Shigella<br>dysenteriae<br>Strl | Shigclla<br>Stx I | Moderate | 100%<br>I ୧/ I ୧<br>(79.4%-100%) | Negative Stool<br>Matrix | All Targets Not<br>Detected | NA | 100%<br>16/16<br>(79.4%-100%) |
| | | Low | 100%<br>16/16<br>(79.4%-100%) | Clostridium<br>difficile | All Targets Not<br>Detected | NA | 100%<br>16/16<br>(79.4%-100%) |
| Yersinia<br>enterocolitica | Y. enterocolitica | Moderate | 100%<br>16/16<br>(79.4%-100%) | | | | |
| | | Low | 100%<br>16/16<br>(79.4%-100%) | | | | |

4 95% Two-sided Exact Binomial Confidence Interval calculation using the exact Clopper-Pearson method.

b One sample called "Salmonella" and "Stx2."

# Performance Data - Clinical Testing

#### Reproducibility

The inter-laboratory reproducibility of the EP test was determined by conducting a reproducibility study at three external sites. Fourteen (14) unique samples were tested daily in triplicate by two (2) operators for five (5) non-consecutive days at three (3) sites for a total of ninety (90) tests per sample. The study tested a total of 1260 samples. The fourteen (14) sample panel was the same panel described previously for the precision study comprising six (6) different strains at two (2) different concentrations (12 positive samples) and two (2) negative samples (Negative Stool Matrix and Clostridium difficile). The results of the Reproducibility Study are provided in the table below.

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| Sample | Expected Call | Conc. | Total Agreement with Expected Result (95 % CI) | | |
|------------------------------|-------------------|----------|------------------------------------------------|-------------------------------|-------------------------------|
| | | | Site 1 | Site 2 | Site 3 |
| Escherichia<br>coli/Stx2 | E. coli<br>Stx2 | Moderate | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| | | Low | 29/30<br>96.7%<br>(82.8-99.9) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| Salmonella<br>enterica | Salmonella | Moderate | 28/30<br>93.3%<br>(77.9-99.2) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| | | Low | 26/30<br>86.7%<br>(69.3-96.2) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| Shigella<br>dysenteriae/Stx1 | Shigella<br>Stx1 | Moderate | 30/30<br>100%<br>(88.4-100) | 28/30<br>93.3%<br>(77.9-99.2) | 30/30<br>100%<br>(88.4-100) |
| | | Low | 29/30<br>96.7%<br>(82.8-99.9) | 29/30<br>96.7%<br>(82.8-99.9) | 28/30<br>93.3%<br>(77.9-99.2) |
| Yersinia<br>enterocolitica | Y. enterocolitica | Moderate | 29/30<br>96.7%<br>(82.8-99.9) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| | | Low | 28/30<br>93.3%<br>(77.9-99.2) | 27/30<br>90.0%<br>(73.5-97.9) | 25/30<br>83.3%<br>(65.3-94.4) |
| Campylobacter<br>jejuni | Campylobacter | Moderate | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| | | Low | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| Vibrio<br>parahaemolyticus | Vibrio | Moderate | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| | | Low | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| Negative Stool<br>Matrix | Negative | NA | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |
| Clostridium<br>difficile | Negative | NA | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) | 30/30<br>100%<br>(88.4-100) |

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#### Clinical Study - Method Comparison

The performance characteristics of the EP test were determined in a multi-site prospective investigation study at seven (7) U.S. institutions by comparing the Verigene EP test results to reference methods, including bacterial culture and automated phenotype identification for the bacterial targets and broth enrichment followed by EIA and PCR amplification/BDS for Stx1/Stx2 typing. The study included the testing of prospectively collected fresh and frozen Cary-Blair specimens and simulated frozen seeded Cary-Blair specimens. Deidentified prospectively-collected specimens were enrolled from individuals receiving routine care requiring enteric pathogens testing. Twelve (12) clinical specimen acquisition sites were used to provide glycerol stocks to seed 408 simulated specimens. These specimens were blinded and shipped to the testing sites and tested alongside prospectively collected specimens.

A total of 1975 specimens were tested with the EP test. Ninety-eight (98) specimens were excluded; 95 prospectively collected and selected specimens and three simulated specimens. Of the remaining 1877 valid specimens, 25 specimens had a final "No Call," resulting in 25 indeterminate specimens. Therefore, a total of 1852 evaluable specimens were used to calculate the performance characteristics for the study. The following table provides a summary of demographic information for 1262 of the 1277 prospectively collected specimens in the valid dataset (age was not recorded for 15 specimens).

| Age Range | No. of Specimens | Percentage |
|-----------|------------------|------------|
| 0-1 | 61 | 4.8% |
| >1-5 | 47 | 3.7% |
| >5-12 | 84 | 6.7% |
| >12-21 | 139 | 11.0% |
| >21-65 | 609 | 48.3% |
| >65 | 322 | 25.5% |
| Total | 1262 | 100% |

The table below provides a summary of the clinical performance, stratified by specimen type, of the EP test for the detection of five (5) bacterial targets and Stx2 (n=1852), compared to the above-described reference methods.

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| Specimen Type | n | % Agreement (95% CI) | | Specimen Type | n | % Agreement (95% CI) | | | | | | | | | | | | | | | |
|--------------------|----------------------------|----------------------|-------------------------|-------------------------------|-----------------------------------|-----------------------------------|-----------------------------------|----------|------|-------------------------------|-----------------------------------|-----------------------------------|-----------------------------------|--|--------------------------|-----|--|--|------|-----------------------------|---------------------------------|
| Campylobacter spp. | | Positive | Negative | Salmonella spp. | | Positive | Negative | | | | | | | | | | | | | | |
| Clinical Specimens | Prospectively<br>Collected | Fresh | 1243 | 90.5%<br>19/21<br>(69.6-98.8) | 98.8%<br>1207/1222<br>(98.0-99.3) | Clinical Specimens | Prospectively<br>Collected | Fresh | 1243 | 85.7%<br>18/21<br>(63.7-97.0) | 99.4%<br>1215/1222<br>(98.8-99.8) | | | | | | | | | | |
| | | Frozen | 34 | 100%<br>2/2<br>(15.8-100) | 100%<br>32/32<br>(89.1-100) | | | Frozen | 34 | 100%<br>1/1<br>(2.5-100) | 97.0%<br>32/33<br>(84.2-99.9) | | | | | | | | | | |
| | | Selected | 166 | 97.5%<br>39/40<br>(86.8-99.9) | 99.2%<br>125/126<br>(95.7-100) | | | Selected | 166 | 98.2%<br>53/54<br>(90.1-100) | 99.1%<br>111/112<br>(95.1-100) | | | | | | | | | | |
| | | All | 1443 | 95.2%<br>60/63<br>(86.7-99.0) | 98.8%<br>1364/1380<br>(98.1-99.3) | | | All | 1443 | 94.7%<br>72/76<br>(87.1-98.6) | 99.3%<br>1358/1367<br>(98.8-99.7) | | | | | | | | | | |
| | Simulated | | 409 | 98.5%<br>67/68<br>(92.1-100) | 100%<br>341/341<br>(98.9-100) | | Simulated | | 409 | 100%<br>67/67<br>(94.6-100) | 100%<br>342/342<br>(98.9-100) | | | | | | | | | | |
| | All | | | 1852 | 97.0%<br>127/131<br>(92.4-99.2) | 99.1%<br>1705/1721<br>(98.5-99.5) | | All | | | 1852 | 97.2%<br>139/143<br>(93.0-99.2) | 99.5%<br>1700/1709<br>(99.0-99.8) | | | | | | | | |
| Shigella spp. | | | | | Vibrio spp. | | | | | | | | | | | | | | | | |
| Clinical Specimens | Prospectively<br>Collected | Fresh | 1243 | 66.7%<br>2/3<br>(9.4-99.2) | 98.7%<br>1224/1240<br>(97.9-99.3) | Clinical Specimens | Prospectively<br>Collected | Fresh | 1242 | 100%<br>1/1<br>(2.5-100) | 100%<br>1242/1242<br>(99.7-100) | | | | | | | | | | |
| | | Frozen | 34 | - | 97.1%<br>33/34<br>(84.7-99.9) | | | Frozen | 34 | 100%<br>1/1<br>(2.5-100) | 100%<br>33/33<br>(89.4-100) | | | | | | | | | | |
| | | Selected | 166 | 100%<br>6/6<br>(54.1-100) | 99.4%<br>159/160<br>(96.6-100) | | | Selected | 166 | 100%<br>1/1<br>(2.5-100) | 100%<br>165/165<br>(97.8-100) | | | | | | | | | | |
| | | All | 1443 | 88.9%<br>8/9<br>(51.8-99.7) | 98.7%<br>1416/1434<br>(98.0-99.3) | | | All | 1443 | 100%<br>3/3<br>(29.2-100) | 100%<br>1440/1440<br>(99.7-100) | | | | | | | | | | |
| | Simulated | | 409 | 100%<br>50/50<br>(92.9-100) | 100%<br>359/359<br>(99.0-100) | | Simulated | | 409 | 91.1%<br>51/56<br>(80.4-97.0) | 99.7%<br>352/353<br>(98.4-100) | | | | | | | | | | |
| | All | | | 1852 | 98.3%<br>58/59<br>(90.9-100) | 99.0%<br>1775/1793<br>(98.4-99.4) | | All | | | 1852 | 91.5%<br>54/59<br>(81.3-97.2) | 99.9%<br>1792/1793<br>(99.7-100) | | | | | | | | |
| Y. enterocolitica | | | | | StxI | | | | | | | | | | | | | | | | |
| Clinical Specimens | Prospectively<br>Collected | Fresh | 1243 | 100%<br>6/6<br>(54.1-100) | 99.8%<br>1235/1237<br>(99.4-100) | Clinical Specimens | Prospectively<br>Collected | Fresh | 1243 | 100%<br>4/4<br>(39.8-100) | 99.7%<br>1236/1239<br>(99.2-99.9) | | | | | | | | | | |
| | | Frozen | 34 | - | 100%<br>34/34<br>(89.7-100) | | | Frozen | 34 | - | 100%<br>34/34<br>(89.7-100) | | | | | | | | | | |
| | | Selected | 166 | 100%<br>9/9<br>(66.4-100) | 100%<br>157/157<br>(97.7-100) | | | Selected | 166 | 100%<br>9/9<br>(66.4-100) | 99.4%<br>156/157<br>(96.5-100) | | | | | | | | | | |
| | | All | 1443 | 100%<br>13/13<br>(75.3-100) | 99.7%<br>1426/1430<br>(99.3-99.9) | | | All | 1443 | 100%<br>1/1<br>(2.5-100) | 100%<br>1442/1442<br>(99.7-100) | | | | | | | | | | |
| | Simulated | | 409 | 100%<br>51/51<br>(93.0-100) | 99.4%<br>356/358<br>(98.0-99.9) | | Simulated | | 409 | 100%<br>59/59<br>(93.9-100) | 100%<br>350/350<br>(99.0-100) | | | | | | | | | | |
| | <i>Y. enterocolitica</i> | All | | | Clinical Specimens | Prospectively Collected | | 1852 | All | 100%<br>64/64<br>(94.4-100) | 1852 | 99.7%<br>1782/1788<br>(99.3-99.9) | 58/59<br>(90.9-100) | | 1775/1793<br>(98.4-99.4) | All | | | 1852 | 100%<br>60/60<br>(94.0-100) | 100%<br>1792/1792<br>(99.8-100) |
| | | Fresh | 1243 | - | | | 100%<br>1243/1243<br>(99.7-100) | | | | | | | | | | | | | | |
| | | Frozen | 34 | - | | | 100%<br>34/34<br>(89.7-100) | | | | | | | | | | | | | | |
| | | Selected | 166 | 100%<br>1/1<br>(2.5-100) | | | 100%<br>165/165<br>(97.8-100) | | | | | | | | | | | | | | |
| | | | All | 1443 | 100%<br>1/1<br>(2.5-100) | 100%<br>1442/1442<br>(99.7-100) | | | | | | | | | | | | | | | |
| | | | Simulated | 409 | 100%<br>59/59<br>(93.9-100) | 100%<br>350/350<br>(99.0-100) | | | | | | | | | | | | | | | |
| | | | All | 1852 | 100%<br>60/60<br>(94.0-100) | 100%<br>1792/1792<br>(99.8-100) | | | | | | | | | | | | | | | |
| <i>Stx2</i> | | Clinical Specimens | Prospectively Collected | | Fresh | 1243 | 100%<br>6/6<br>(54.1-100) | | | | | | | | | | | | | | |
| | | | | | Frozen | 34 | - | | | | | | | | | | | | | | |
| | | | | | Selected | 166 | 100%<br>9/9<br>(66.4-100) | | | | | | | | | | | | | | |
| | | | | | All | 1443 | 100%<br>15/15<br>(78.2-100) | | | | | | | | | | | | | | |
| | | | | Simulated | 409 | 96.7%<br>58/60<br>(88.5-99.6) | 99.7%<br>348/349<br>(98.4-100) | | | | | | | | | | | | | | |
| | | | | All | 1852 | 97.3%<br>73/75<br>(90.7-99.7) | 99.8%<br>1774/1777<br>(99.5-100) | | | | | | | | | | | | | | |
| | <i>Stx1</i> | Clinical Specimens | Prospectively Collected | | Fresh | 1243 | 100%<br>4/4<br>(39.8-100) | | | | | | | | | | | | | | |
| | | | | | | Frozen | 34 | | | | | | | | | | | | | | |
| | | | | | Selected | 166 | 100%<br>9/9<br>(66.4-100) | | | | | | | | | | | | | | |
| | | | | | All | 1443 | 100%<br>13/13<br>(75.3-100) | | | | | | | | | | | | | | |
| | | | | Simulated | 409 | 100%<br>51/51<br>(93.0-100) | 99.4%<br>356/358<br>(98.0-99.9) | | | | | | | | | | | | | | |
| | | | | All | 1852 | 100%<br>64/64<br>(94.4-100) | 99.7%<br>1782/1788<br>(99.3-99.9) | | | | | | | | | | | | | | |

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# Substantial Equivalence

The Verigene® Enteric Pathogen Nucleic Acid Test (EP test) has been shown to be substantially equivalent to the xTAG Gastrointestinal Pathogen Panel (GPP). The EP test has similar intended use and indications, technological characteristics, and performance characteristics. The minor differences between the EP test and its predicate devices raise no new issues of safety or effectiveness. Performance data demonstrate that the EP test is as safe and effective as the predicate device. Thus, the EP test is substantially equivalent to the predicate device.

| Similarities | | | |
|----------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--|
| Element | New Device:<br>Enteric Pathogens Nucleic Acid Test<br>(EP)<br>K140083 | Predicate:<br>xTAG® Gastrointestinal Pathogen<br>Panel (GPP)<br>K121894 | |
| Intended Use | The Verigene Enteric Pathogens Nucleic<br>Acid Test (EP) is a multiplexed,<br>qualitative test for simultaneous detection<br>and identification of common pathogenic<br>enteric bacteria and genetic virulence<br>markers from liquid or soft stool<br>preserved in Cary-Blair media, collected<br>from individuals with signs and<br>symptoms of gastrointestinal infection.<br>The test is performed on the automated<br>Nanosphere Verigene System utilizing<br>reverse transcription (RT), polymerase<br>chain reaction (PCR), and array<br>hybridization to detect specific<br>gastrointestinal microbial nucleic acid<br>gene sequences associated with the<br>following pathogenic bacteria:<br>• Campylobacter Group (comprised of C.<br>coli, C. jejuni, and C. lari)<br>• Salmonella species<br>• Shigella species (including S.<br>dysenteriae, S. boydii, S. sonnei, and S.<br>flexneri)<br>• Vibrio Group (comprised of V. cholerae<br>and V. parahaemolyticus)<br>• Yersinia enterocolitica<br>In addition, EP detects the Shiga toxin 1<br>gene and Shiga toxin 2 gene virulence<br>markers. Shiga toxin producing E. coli<br>(STEC) typically harbor one or both<br>genes that encode for Shiga Toxins 1 and<br>2.<br>EP is indicated as an aid in the diagnosis<br>of specific agents of gastrointestinal<br>illness, in conjunction with other clinical,<br>laboratory, and epidemiological<br>information; however, is not to be used to<br>monitor these infections. EP also aids in | The xTAG® Gastrointestinal<br>Pathogen Panel (GPP) is a multiplexed<br>nucleic acid test intended for the<br>simultaneous qualitative detection and<br>identification of multiple viral,<br>parasitic, and bacterial nucleic acids in<br>human stool specimens from<br>individuals with signs and symptoms<br>of infectious colitis or gastroenteritis.<br>The following pathogen types,<br>subtypes and toxin genes are identified<br>using the xTAG® GPP:<br>• Campylobacter (C. jejuni, C. coli<br>and C. lari only)<br>• Clostridium difficile (C. difficile)<br>toxin A/B<br>• Cryptosporidium (C. parvum and C.<br>hominis only)<br>• Escherichia coli (E. coli) 0157<br>• Enterotoxigenic Escherichia coli<br>(ETEC) LT/ST<br>• Giardia (G. lamblia only - also<br>known as G. intestinalis and G.<br>duodenalis)<br>• Norovirus GI/GII<br>• Rotavirus A<br>• Salmonella<br>• Shiga-like Toxin producing E. coli<br>(STEC) stx 1/stx 2<br>• Shigella (S. boydii, S. sonnei, S.<br>flexneri and S. dysenteriae)<br>The detection and identification of<br>specific gastrointestinal microbial<br>nucleic acid from individuals<br>exhibiting signs and symptoms of<br>gastrointestinal infection aids in the<br>diagnosis of gastrointestinal infection<br>when used in conjunction with clinical<br>evaluation, laboratory findings and | |
| | Similarities…