The Verigene Enteric Pathogens Nucleic Acid Test (EP) is a multiplexed, qualitative test for simultaneous detection and identification of common pathogenic enteric bacteria, viruses and genetic virulence markers from liquid or soft stool preserved in Cary-Blair medium, 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 and viruses:

· Campylobacter Group (composed of C. coli, C. jejuni, and C. lari)
· Salmonella species
· Shigella species (including S. dysenteriae, S. boydii, S. sonnei, and S. flexneri)
· Vibrio Group (composed of V. cholerae and V. parahaemolyticus)
· Yersinia enterocolitica
Norovirus GI/GII
Rotavirus A

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 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 Description

The Verigene Enteric Pathogens Nucleic Acid Test (EP) is a molecular assay that relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial or viral nucleic acid sequences detected by EP, unique Capture and Mediator oligonucleotides are used, 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 that binds to a different portion of the same nucleic acid targets and also have a sequence that 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, benchtop 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 analyte-specific target nucleic acids. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP.

The Reader is the Verigene System's user interface and serves as the central control unit for all aspects of test processing, automated imaging, and result generation using a touch-screen 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 and viral nucleic acid isolation and amplification, and (2) Hybridization- detection and identification of analyte-specific nucleic acid 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 used 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 targeted nucleic acid sequence/analyte. This determination is made by means of software-based decision algorithm resident in the Reader.

AI/ML Overview

Here's an analysis of the provided text regarding the acceptance criteria and study for the Verigene® Enteric Pathogens Nucleic Acid Test (EP):

1. Table of Acceptance Criteria and Reported Device Performance

The document primarily focuses on overall agreement rates rather than explicitly stated acceptance criteria with specific thresholds for positive and negative agreement. However, we can infer performance targets from the reported results and the nature of medical device studies aiming for high accuracy.

Performance Metric	Acceptance Criteria (Inferred)	Reported Device Performance (Clinical Study - Fresh Specimens)
Norovirus GI/GII	High Positive Agreement	94.9% (37/39)
	High Negative Agreement	99.6% (1250/1255)
Rotavirus A	High Positive Agreement	66.7% (2/3) - Note: Limited positives, primarily established with contrived specimens for certain organisms.
	High Negative Agreement	99.9% (1290/1291)
Campylobacter spp.	High Positive Agreement	90.9% (20/22)
	High Negative Agreement	98.7% (1255/1272)
Salmonella spp.	High Positive Agreement	86.4% (19/22)
	High Negative Agreement	99.4% (1265/1272)
Shigella spp.	High Positive Agreement	66.7% (2/3) - Note: Limited positives, primarily established with contrived specimens for certain organisms.
	High Negative Agreement	98.8% (1275/1291)
Vibrio spp.	High Positive Agreement	100% (1/1) - Note: Limited positives, primarily established with contrived specimens for certain organisms.
	High Negative Agreement	100% (1293/1293)
Y. enterocolitica	High Positive Agreement	No positives reported for fresh specimens in clinical study
	High Negative Agreement	100% (1294/1294)
Stx1	High Positive Agreement	100% (4/4)
	High Negative Agreement	99.8% (1287/1290)
Stx2	High Positive Agreement	100% (6/6)
	High Negative Agreement	99.8% (1286/1288)

Notes on Acceptance Criteria: Based on typical FDA clearance for diagnostic devices, the acceptance criteria would generally be a pre-defined lower bound for the confidence interval of the positive and negative agreement (e.g., >90% or >95% for the lower bound of the 95% CI). While not explicitly stated here, the reported percentages (and confidence intervals where provided) demonstrate that the device aimed for and largely achieved very high agreement rates. The low numbers of positive clinical specimens for some targets (Rotavirus A, Shigella spp., Vibrio spp., Y. enterocolitica) led to the use of contrived specimens to establish performance, which is a common practice when clinical prevalence is low.

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

Total Valid Specimens: 1940
- 1294 prospectively-collected fresh specimens
- 34 prospectively-collected frozen specimens
- 203 selected samples
- 409 simulated specimens
Data Provenance:
- Country of Origin: United States (multi-site prospective investigation study at eight (8) U.S. institutions).
- Retrospective/Prospective: Primarily prospective (1294 fresh, 34 frozen specimens). There were also "selected samples" (203) and "simulated specimens" (409), which would not be considered purely prospective clinical data. The phrase "selected samples" often implies retrospectively identified samples with known disease status.

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

The document does not specify the number of experts used or their qualifications for establishing ground truth.

4. Adjudication Method for the Test Set

The document does not specify an adjudication method. It states that "The viral comparator methods were a composite of a real-time RT-PCR assay and conventional PCR assays with confirmatory bi-directional sequencing." This implies a reference standard rather than an adjudication process between multiple readers.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This study is for a diagnostic test (Verigene EP) and compares its performance against reference laboratory methods, not human readers.

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

Yes, the study described is a standalone performance study. The Verigene EP test is an automated system that produces results directly from the specimen ("sample-to-result") without human interpretation of images or complex data that would necessitate a "human-in-the-loop" assessment. The performance is assessed by comparing the device's output to a reference method.

7. The Type of Ground Truth Used

The ground truth for the clinical study was established using PCR-based viral reference methods, specifically:

A composite of a real-time RT-PCR assay.
Conventional PCR assays with confirmatory bi-directional sequencing.

For bacterial targets and Shiga toxin genes, while not explicitly detailed in the clinical study section, it can be inferred that similar robust molecular or culture-based methods served as the reference standard.

8. The Sample Size for the Training Set

The document does not explicitly state the sample size for a training set. The clinical study described is primarily for performance evaluation (test set). For a molecular diagnostic device like this, the development and training of the underlying detection algorithm would typically occur during the assay development phase, using internal validation samples, and may not have a "training set" in the same way an AI/ML image analysis algorithm would. Analytical performance studies (LoD, inclusivity, cross-reactivity) are part of establishing the robustness of the assay's detection mechanism.

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

As no specific training set is outlined in the context of this 510(k) summary, the method for establishing its ground truth is also not described. For the analytical studies (e.g., LoD, inclusivity, cross-reactivity), the "ground truth" is inherently defined by using characterized microbial strains, known concentrations, and well-established molecular and microbiological techniques to prepare the samples.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

NANOSPHERE, INC. NOAH LERMER, Ph.D DIRECTOR, REGULATORY AFFAIRS 4088 COMMECIAL AVENUE NORTHBROOK IL 60062

October 10, 2014

Re: K142033

Trade/Device Name: Verigene® Enteric Pathogens Nucleic Acid Test (EP) Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal microorganism multiplex nucleic acid-based assay Regulatory Class: II Product Code: PCH, PCI, OOI Dated: July 24, 2014 Received: July 25, 2014

Dear Dr. Lermer:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug. and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21. Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours.

Uwe Scherf -S for

Sally Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K142033

Device Name

Verigene Enteric Pathogens Nucleic Acid Test (EP)

Indications for Use (Describe)

· Campylobacter Group (composed of C. coli, C. jejuni, and C. lari)
· Salmonella species
· Shigella species (including S. dysenteriae, S. boydii, S. sonnei, and S. flexneri)
· Vibrio Group (composed of V. cholerae and V. parahaemolyticus)
· Yersinia enterocolitica
Norovirus GI/GII
Rotavirus A

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.

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

Type of Use (Select one or both, as applicable)

2 Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON A SEPARATE PAGE IF NEEDED.

FOR FDA USE ONLY

Concurrence of Center for Devices and Radiological Health (CDRH) (Signature)

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510(K) Summary

510(k) Number:

Verigene® Enteric Pathogens Nucleic Acid Test (EP) K142033:

Summary Preparation Date:

September 25, 2014

Submitted by:

Nanosphere, Inc. 4088 Commercial Avenue Northbrook, IL 60062 Phone: 847-400-9000 Fax: 847-400-9176

Contact:

Noah Lermer Director, Regulatory Affairs

Proprietary Names:

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

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
OOI Real Time Nucleic Acid Amplification System

Other codes used by predicate devices:

NSU Instrumentation for clinical multiplex test systems

JJH Clinical Sample Concentrator

Predicate Devices:

xTAG Gastrointestinal Pathogen Panel (GPP) (K121894/K121454) (Luminex Molecular Diagnostics, Inc.)

Indications for Use:

. Campylobacter Group (composed of C. coli, C. jejuni, and C. lari)
. Salmonella species
. Shigella species (including S. dysenteriae, S. boydii, S. sonnei, and S. flexneri)
. Vibrio Group (composed of V. cholerae and V. parahaemolyticus)
Yersinia enterocolitica
. Norovirus GI/GII
Rotavirus A

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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.

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:

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Performance Data - Analytical Testing

Note: Please refer to K140083 for information on EP test analytical performance for bacterial and Shiga toxin gene virulence marker targets.

Analytical Sensitivity / Limit of Detection (LoD)

Analytical sensitivity (LoD) of the EP test for Norovirus and Rotavirus was established by testing four (4) representative virus strains. The LoD is 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 in replicates of four and the putative LoD was 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 4 strains tested, and the corresponding LoD ranges for the EP test reportable target, are shown in the table below. Overall, the LoD ranged from 4.12×105 - 1.67×10 copies/mL of stool for Norovirus and 3.70×102 - 1.11×103 TCID50/mL of stool for Rotavirus.

Organism	LoD (/mL Stool)
Norovirus - GI (CDC 2142)	$4.12\times10^5$ copies
Norovirus - GII (D17219)	$1.67\times10^6$ copies
Rotavirus - DS1 (VR-2550)	$1.11\times10^3$ TCID50
Rotavirus - W161 (VR-2551)	$3.70\times10^2$ TCID50

Analytical Reactivity (Inclusivity)

Analytical Reactivity of the EP test for Norovirus and Rotavirus was demonstrated with a comprehensive panel of 41 clinically relevant viral strains representing temporal, geographical, and phylogenic diversity for each claimed target (see table below). All of the Rotavirus and most of the Norovirus strains tested generated the expected result when tested in triplicate at a concentration of 3x LoD. For some of the Norovirus GII strains, slightly higher concentrations were required to demonstrate inclusivity (10x-50x LoD).

Norovirus strains GII.9, GII.14, and GIV.1 and Rotavirus A strains G4, G5, G10, G11, and G15 are predicted to be detected based on in silico analysis. Norovirus GII.11 is not expected to be detected by the EP test. Additionally, based on in silico analysis, rare Norovirus genotypes GII.6 and GII.13 are predicted to either be not detected by EP or to be detected with reduced sensitivity. Inclusivity to Norovirus strains GII.8 and Rotavirus G7, G21, and G24 could not be evaluated due to a lack of available sequences.

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Reportable Target	Number of Organisms/Strains Tested	Genogroups (No. Of Strains)
Norovirus	29	GI (13), GII (16)
Rotavirus	12	Group A (12)

Analytical Specificity (Cross-reactivity)

One-hundred and fifty-eight (158) organisms, consisting of 134 bacterial organisms, 18 viruses, four (4) parasites, one (1) fungal organism, 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 four (4) parasites were evaluated by using genomic DNA/RNA. In addition, the cross-reactivity of 15 species of Vibrio not associated with human infection, four (4) non-pathogenic strains of Escherichia coli, Yersinia pestis, Clostridium botulinum, Rotavirus (Genogroups B, C, D, NADRV), Adenovirus (Species G), and Norovirus (Genotype GIV.2 and Genogroups GIII, GV) were evaluated by in silico analysis. Finally, to rule out cross-reactivity between the analytes detected by the EP test, nine representative in-panel organisms were tested at elevated concentrations.

All of the tests yielded the expected "Not Detected" results, indicating that there was no cross-reactivity with the EP test Norovirus and Rotavirus probes. Additionally, with the exception of porcine strains of Rotavirus C, all of the organisms evaluated for exclusivity through in silico analysis are predicted to not be detected by the EP test.

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Exclusivity Testing: Bacterial Non-Test Panel Members		Campylobacter, Vibrio, and Yersinia Species Not Detected by EP
Genus	Species	Genus	Species
Abiotrophia	defectiva	Campylobacter	concisus
Acinetobacter	baumanniilwoffii		curvusfetusgracilishominishyointestinalisinsulaenigraelanienaemucosalisrectusshowaesputorumupsaliensis
Arcobacter	butzlericryaerophilusallosaccharophilabestiarumcaviaeencheleiaenteropelogeneseucrenophilahydrophiliajandaeisalmonicida*	Vibrio	alginolyticuscampbelliicincinnatiensisfluvialisfurnissiiharveyimetschnikoviimimicustubiashiivulnificus (3 strains)
Aeromonas	veronii	Yersinia	aldovaealeksiciaebercovierifrederikseniiintermediakristenseniimollaretiipseudotuberculosisruckerirohdei
Alcaligenes	faecalis
Bacillus	cereuscaccae
Bacteroides	fragilismerdaestercoris
Cedecea	davisae
Citrobacter	amalonaticusfreundiisedlakiibifermentansbolteaebutyricumdifficile (2 strains)difficile, non-tox
Clostridium	haemolyticummethylpentosumnexilenoyviorbiscindensperfringensscindenssepticumsordelliispiroformesporogenes
Collinsella	aerofaciens
Desulfovibrio	piger
Edwardsiella	tarda
Enterobacter	aerogenescloacae
Enterococcus	faecalisfaecium
Escherichia	coli (3 strains)coli (EAEC)coli (EPEC) (2 strains)coli (ETEC) (2 strains)fergusoniihermannii
Fusobacterium	variumhepaticus
Helicobacter	pylori (4 strains)
Klebsiella	oxytocapneumoniae
Lactobacillus	acidophilusreuterirhamnosus
Lactococcus	lactis
Leminorella	grimontii
Listeria	grayimonocytogenes
Morganella	morganii
Peptostreptococcus	anaerobius
Plesiomonas	shigelloides
Porphyromonas	asaccharolytica
Prevotella	melaninogenica
Proteus	mirabilisvulgarispenneri
Providencia	stuartiialcalifaciensrettgeri
Pseudomonas	aeruginosa (2 strains)fluorescensputida
Ruminococcus	bromii
Serratia	liquefaciensmarcescens
Staphylococcus	aureusepidermidis
Streptococcus	agalactiae, O90Rdysgalactiaemutans
Parasites
Blastocystis	hominis
Cryptosporidium	parvum
Entamoeba	histolytica
Giardia	lamblia
Human Cell Line
Colon epithelial cells
Fungal Strain
Candida albicans
Viruses
Name	Serovar / Group
Adenovirus	Type 1/Group CType 2/Group CType 3/Group B1Type 4/Group EType 5/Group CType 14/Group B2Type 26/Group DType 31/Group AType 37/Group DType 40/Group FHuman 4
Astrovirus	-
Coxsackievirus B4	-
Cytomegalovirus	-
Echovirus 11	-
Enterovirus 68Sapovirus (2 strains)	-
* Sub-species masoucida and sub-species salmonicida (2 strains)

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Microbial Interference

Potential interference of 14 microorganisms was evaluated with representative strains of Norovirus and Rotavirus. These microorganisms, Bacteroides fragilis, Prevotella oralis, Prevotella melaninogenica, Bifidobacterium bifidum, Clostridium perfringens, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Lactobacillus acidophilus, Staphylococcus aureus, Blastocystis hominis, Entamoeba histolytica, and Candida albicans, represent the most prevalent organisms in the human colon and therefore are the most likely to be encountered in stool specimens tested with the EP test. The microorganisms were tested at a concentration of 10' CFU/mL, with the exception of the parasites Blastocystis hominis and Entamoeba histolytica, which were tested at 9×10° cells/mL and 7×10° 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. Representative strains of Norovirus and Rotavirus were individually challenged with 22 potentially interfering substances (shown below) at high, medically-relevant concentrations. None of the 22 substances tested showed any inhibitory effect on the EP test detection of the target viruses.

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

Competitive Inhibition

The potential for competitive inhibition of the EP test for the viral targets was evaluated by testing 26 unique binary combinations of Norovirus and Rotavirus with the EP test panel organisms. Simulated samples were prepared in Negative Stool Matrix (NSM), with one panel organism at a Low Positive titer (3x LoD) and a second organism at a High Positive titer (10-100-fold higher than the organism's LoD). The EP test correctly detected all organisms present in the co-infection combinations tested in replicates of three (3) with one exception: the "Low Titer Rotavirus and High Titer Y. enterocolitica" sample the EP test correctly identified Y. enterocolitica in all cases, but Rotavirus was detected only in two (2) of the three (3) replicates.

K142033.510kSummary.FINAL.docx

CONFIDENTIAL AND PROPRIETARY

Page 8 of 16

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An additional 6 replicates were tested and the expected result was obtained for both analytes in all replicates.

Cut-off Verification

Target mean intensity values were examined from the testing of sixteen (16) bacterial strains and four (4) viral strains used to establish the Limit of Detection of the EP test. In addition, the cutoff dataset included the test results of two (2) negative samples. With replicates of 20 for each sample and fourteen (14) unique target spot groups evaluated per test, a total of 6160 data points (1320 expected positive) were assessed to verify the assay cut-off.

Carryover / Cross-contamination

The potential for carryover and cross-contamination of the EP test on the Verigene system was assessed by alternately testing 3 representative viral samples with negative stool samples across multiple Verigene Processor SPs. No carryover or cross-contamination was observed.

Precision

The EP test precision study for the viral targets was conducted in-house by Nanosphere as part of the full 20-sample precision study. The full panel was composed of three (3) different viral strains and six (6) different EP panel bacterial strains, each at two (2) concentrations, as well as two (2) negative samples (Negative Stool Matrix and Clostridium difficile). In the study, each sample was tested daily in duplicate by two (2) operators for four (4) non-consecutive days for a total of sixteen (16) tests per sample (2 operators / day x 2 replicates / operator x 4 days).

Sample ID	Expected EP Call	Concentration	Agreement withExpected Result(95 % CI)a
Rotavirus	Rotavirus	Moderate	16/16100%(79.4%-100%)
Rotavirus		Low	16/16100%(79.4%-100%)
Norovirus GI	Norovirus	Moderate	16/16100%(79.4%-100%)
Norovirus GI		Low	16/16100%(79.4%-100%)
Norovirus GII	Norovirus	Moderate	16/16100%(79.4%-100%)
Norovirus GII		Low	16/16100%(79.4%-100%)

Results for the viral strains are summarized below.

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

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Performance Data - Clinical Testing

Reproducibility

The inter-laboratory reproducibility of the EP test was determined by conducting a reproducibility study at three external sites. Three (3) different viral strains at two (2) concentrations were tested as part of the full 20-sample EP test reproducibility study, which also included six (6) different EP panel bacterial strains at two concentrations, as well as two (2) negative samples (Negative Stool Matrix and Clostridium difficile). The 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 results of the Reproducibility Study for viral samples are provided in the table below.

Sample	Expected Call	Concentration	Agreement with Expected Result(95 % CI)
			Site 1	Site 2	Site 3	Total
Rotavirus	Rotavirus Detected	Moderate	30/30100%(88.4-100)	30/30100%(88.4-100)	30/30100%(88.4-100)	90/90100%(96.0-100)
		Low	30/30100%(88.4-100)	27/3090.0%(73.5-97.9)	30/30100%(88.4-100)	87/9096.7%(90.6-99.3)
Norovirus GI	Norovirus Detected	Moderate	30/30100%(88.4-100)	30/30100%(88.4-100)	28/3093.3%(77.9-99.2)	86/8897.7%(92.0-99.7)
		Low	30/30100%(88.4-100)	28/3093.3%(77.9-99.2)	28/3093.3%(77.9-99.2)	86/9095.6%(89.0-98.8)
Norovirus GII	Norovirus Detected	Moderate	30/30100%(88.4-100)	30/30100%(88.4-100)	29/3096.7%(82.8-99.9)	86/8798.9%(93.8-100)
		Low	30/30100%(88.4-100)	30/30100%(88.4-100)	30/30100%(88.4-100)	89/89100%(95.9-100)

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Clinical Study - Method Comparison

The performance characteristics of the EP test for Norovirus and Rotavirus were determined by comparing the Verigene EP test results to PCR-based viral reference methods in a multi-site prospective investigation study at eight (8) U.S. institutions. A total of 1940 valid specimens were evaluated in the study which included 1294 prospectively-collected fresh specimens, 34 prospectively-collected frozen specimens, 203 selected samples, and 409 simulated specimens.

The following table provides a summary of demographic information for 1313 of the 1328 prospectively-collected specimens in the valid dataset.

Age Range	No. of Specimens	Percentage
0-1	63	4.8%
>1-5	49	3.7%
>5-12	85	6.5%
>12-21	146	11.1%
>21-65	636	48.4%
>65	334	25.4%
Total	1313	100%

The viral comparator methods were a composite of a real-time RT-PCR assay and conventional PCR assays with confirmatory bi-directional sequencing. The PCR assays were designed to amplify different gene regions than those targeted by the EP test. The tables below provide a summary of the clinical performance of the EP test (n=1940), compared to the reference/comparator methods and stratified by specimen type, for the detection of Norovirus and Rotavirus, as well as the five (5) bacterial targets and the Stx1 and Stx2 targets.

		Specimen Type		n	% Agreement (95% CI)				Specimen Type		n	% Agreement (95% CI)
					Positive	Negative						Positive	Negative
Norovirus GI/GII	Clinical SpecimensProspectivelyCollected	Fresh	1294	94.9%37/39(82.7-99.4)	99.6%1250/1255(99.0-99.9)	Rotavirus A	Clinical SpecimensProspectivelyCollected	Fresh	1294	66.7%2/3(9.4-99.2)	99.9%1290/1291(99.6-100)
		Frozen	34	0%0/1(0.0-97.5)	100%33/33(89.7-100)			Frozen	34	-	100%34/34(89.7-100)
		Selected		203	100%18/18(81.5-100)			99.5%184/185(97.0-100)	Selected		203	98.0%50/51(89.6-100)	100%152/152(97.6-100)
	Simulated		409	-	100%409/409(99.1-100)		Simulated		409	-	100%409/409(99.1-100)

K142033.510kSummary.FINAL.docx

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Clinical Specimer

Clinical Specimen

Y. enterocolitica

Shigella spp

Campylobacter spp

	Specimen Type		n	% Agreement (95% CI)			Specimen Type		n	% Agreement (95% CI)
				Positive	Negative					Positive	Negative
Clinical SpecimensProspectivelyCollected	Fresh	1294	90.9%20/22(79.8-98.9)	98.7%1255/1272(97.9-99.2)	Salmonella spp.	Clinical SpecimensProspectivelyCollected	Fresh	1294	86.4%19/22(65.1-97.1)	99.4%1265/1272(98.9-99.8)
	Frozen	34	100%2/2(15.8-100)	100%32/32(89.1-100)			Frozen	34	100%1/1(2.5-100)	97.0%32/33(84.2-99.9)
	Selected	203	97.5%39/40(86.8-99.9)	99.4%162/163(96.6-100)			Selected	203	98.3%58/59(90.9-100)	99.3%143/144((96.2-100)
	Simulated	409	98.5%67/68(92.1-100)	100%341/341(98.9-100)			Simulated	409	100%67/67(94.6-100)	100%342/342(98.9-100)
Clinical SpecimensProspectivelyCollected	Fresh	1294	66.7%2/3(9.4-99.2)	98.8%1275/1291(98.0-99.3)	Vibrio spp.	Clinical SpecimensProspectivelyCollected	Fresh	1294	100%1/1(2.5-100)	100%1293/1293(99.7-100)
	Frozen	34	-	97.1%33/34(84.7-99.9)			Frozen	34	100%1/1(2.5-100)	100%33/33(89.4-100)
	Selected	203	100%8/8(63.1-100)	99.5%194/195(97.2-100)			Selected	203	100%1/1(2.5-100)	100%202/202(98.2-100)
	Simulated	409	100%50/50(92.9-100)	100%359/359(99.0-100)			Simulated	409	91.1%51/56(80.4-97.0)	99.7%352/353(98.4-100)
Clinical SpecimensProspectivelyCollected	Fresh	1294		100%1294/1294(99.7-100)
	Frozen	34		100%34/34(89.7-100)
	Selected	203	100%1/1(2.5-100)	100%202/202(98.2-100)
	Simulated	409	100%59/59(93.9-100)	100%350/350(99.0-100)

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Specimen Type		n	% Agreement (95% CI)		Specimen Type		n	% Agreement (95% CI)
			Positive	Negative				Positive	Negative
Stx1	Clinical SpecimensProspectivelyCollected	Fresh	1294	100%4/4(39.8-100)	99.8%1287/1290(99.3-100)	Stx2	Clinical SpecimensProspectivelyCollected	Fresh	1294	100%6/6(54.1-100)	99.8%1286/1288(99.4-100)
		Frozen	34	-	100%34/34(89.7-100)			Frozen	34	-	100%34/34(89.7-100)
		Selected	203	100%9/9(66.4-100)	99.5%193/194(97.2-100)			Selected	203	100%10/10(69.2-100)	100%193/193(98.1-100)
		Simulated	409	100%50/50(92.9-100)	99.2%356/359(97.6-99.8)			Simulated	409	96.6%57/59(88.3-99.6)	99.4%348/350(98.0-99.9)

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Substantial Equivalence

The Verigene Enteric Pathogen Nucleic Acid Test (EP test) for Norovirus GI/GII and Rotavirus A targets 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 device 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
	New Device:Enteric Pathogens Nucleic Acid Test (EP)K142033	Predicate:xTAG Gastrointestinal Pathogen Panel (GPP)K121894
Element
Intended Use	The Verigene Enteric Pathogens Nucleic AcidTest (EP) is a multiplexed, qualitative test forsimultaneous detection and identification ofcommon pathogenic enteric bacteria, virusesand genetic virulence markers from liquid orsoft stool preserved in Cary-Blair medium,collected from individuals with signs andsymptoms of gastrointestinal infection. Thetest is performed on the automated NanosphereVerigene System utilizing reverse transcription(RT), polymerase chain reaction (PCR), andarray hybridization to detect specificgastrointestinal microbial nucleic acid genesequences associated with the followingpathogenic bacteria and viruses:Campylobacter Group (composed of C. coli,C. jejuni, and C. lari) Salmonella species Shigella species (including S. dysenteriae, S.boydii, S. sonnei, and S. flexneri) Vibrio Group (composed of V. cholerae andV. parahaemolyticus) Yersinia enterocolitica Norovirus GI/GII Rotavirus A In addition, EP detects the Shiga toxin 1 geneand Shiga toxin 2 gene virulencemarkers. Shiga toxin producing E. coli (STEC)typically harbor one or both genes that encodefor Shiga Toxins 1 and 2.EP is indicated as an aid in the diagnosis ofspecific agents of gastrointestinal illness, inconjunction with other clinical, laboratory, andepidemiological information; however, is not tobe used to monitor these infections. EP alsoaids in the detection and identification of acute	The xTAG Gastrointestinal Pathogen Panel(GPP) is a multiplexed nucleic acid test intendedfor the simultaneous qualitative detection andidentification of multiple viral, parasitic, andbacterial nucleic acids in human stool specimensfrom individuals with signs and symptoms ofinfectious colitis or gastroenteritis. Thefollowing pathogen types, subtypes and toxingenes are identified using the xTAG GPP:Campylobacter (C. jejuni, C. coli and C. larionly) Clostridium difficile (C. difficile) toxin A/B Cryptosporidium (C. parvum and C. hominisonly) Escherichia coli (E. coli) 0157 Enterotoxigenic Escherichia coli (ETEC)LT/ST Giardia (G. lamblia only - also known as G.intestinalis and G. duodenalis) Norovirus GI/GII Rotavirus A Salmonella Shiga-like Toxin producing E. coli (STEC) stx1/stx 2 Shigella (S. boydii, S. sonnei, S. flexneri andS. dysenteriae) The detection and identification of specificgastrointestinal microbial nucleic acid fromindividuals exhibiting signs and symptoms ofgastrointestinal infection aids in the diagnosis ofgastrointestinal infection when used inconjunction with clinical evaluation, laboratoryfindings and epidemiological information. Agastrointestinal microorganism multiplexnucleic acid-based assay also aids in the

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Similarities
Element	New Device:Enteric Pathogens Nucleic Acid Test (EP)K142033	Predicate:xTAG Gastrointestinal Pathogen Panel (GPP)K121894
	gastroenteritis in the context of outbreaks.Due to the limited number of positivespecimens collected for certain organismsduring the prospective clinical study,performance characteristics for Yersinia enterocolitica , Vibrio Group and Shigellaspecies were primarily established withcontrived specimens.Concomitant culture is necessary for organismrecovery and further typing of bacterial agents.EP results should not be used as the sole basisfor diagnosis, treatment, or other patientmanagement decisions. Confirmed positiveresults do not rule out co-infection with otherorganisms that are not detected by this test, andmay not be the sole or definitive cause ofpatient illness. Negative EP results in thesetting of clinical illness compatible withgastroenteritis may be due to infection bypathogens that are not detected by this test ornon-infectious causes such as ulcerative colitis,irritable bowel syndrome, or Crohn's disease.	detection and identification of acutegastroenteritis in the context of outbreaks.xTAG GPP positive results are presumptive andmust be confirmed by FDA cleared tests or otheracceptable reference methods.The results of this test should not be used as thesole basis for diagnosis, treatment, or otherpatient management decisions. Confirmedpositive results do not rule out coinfection withother organisms that are not detected by this test,and may not be the sole or definitive cause ofpatient illness. Negative xTAG GastrointestinalPathogen Panel results in the setting of clinicalillness compatible with gastroenteritis may bedue to infection by pathogens that are notdetected by this test or non-infectious causessuch as ulcerative colitis, irritable bowelsyndrome, or Crohn's disease. xTAG GPP is notintended to monitor or guide treatment for C. difficile infections.The xTAG GPP is indicated for use with theLuminex MAGPIX instrument.
Specimen Type	Human Stool sample in Cary-Blair Media	Same
DNAAmplification	PCR	Same
Organisms/NATargetsDetected	Campylobacter Group( C. coli, C. jejuni, and C. lari )Salmonella speciesShigella species( S. dysenteriae, S. boydii, S. sonnei, and S. flexneri )Vibrio Group (comprised of V. cholerae and V. parahaemolyticus )Yersinia enterocoliticaNorovirus GI/GIIRotavirus AShiga toxin 1 gene and Shiga toxin 2 gene	Same with additional analytes (excluding VibrioGroup and Yersinia enterocolitica ).

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Differences
Element	New Device:Enteric Pathogens Nucleic Acid Test (EP)K142033	Predicate:xTAG Gastrointestinal Pathogen Panel (GPP)K121894
Time to Result	~ 2 hours	5 hours
Sample prep	On-board, automated NA extraction andamplification	Off-line NA Extraction and amplification
DetectionMethod	Gold/Silver nanoparticle probe detection ofmicrobial-specific DNA on complementaryoligo- microarray	Specific microbial target or control beadpopulations coupled to sequences fromUniversal Array streptavidin, R-phycoerythrinconjugate
OpticalDetection	Light scatter	Multi-color fluorescence

Regulation Number and Section

§ 866.3990 Gastrointestinal microorganism multiplex nucleic acid-based assay.

(a)
Identification. A gastrointestinal microorganism multiplex nucleic acid-based assay is a qualitativein 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.(b)
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).