Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The description focuses on automated PCR and optical detection technology, with no mention of AI or ML in the device description, intended use, or performance studies. The data analysis software is described as integrated but not as utilizing AI/ML.

Therapeutic

No
This device is a diagnostic tool that identifies bacterial DNA in stool samples to aid in diagnosing gastrointestinal illness. It does not directly treat or prevent a disease or condition.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states, "The test is intended for use as an aid in the diagnosis of gastrointestinal illness in conjunction with clinical and epidemiological information." Additionally, it performs qualitative detection and identification of DNA targets from enteric bacterial pathogens, which is a diagnostic function.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the system includes hardware components such as the Portrait™ Analyzer, single-use test cartridges, and a Sample Preparation Device, in addition to the software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The device is intended for the "detection and identification of DNA targets of enteric bacterial pathogens" from stool specimens to be used "as an aid in the diagnosis of gastrointestinal illness". This clearly indicates a diagnostic purpose performed in vitro (outside the body) on a biological sample.
Sample Type: It uses "preserved stool specimens", which are biological samples.
Method: It utilizes "automated, hot-start PCR amplification technology" and "hybridization probes immobilized on a modified silicon chip surface" to detect nucleic acids. These are standard techniques used in in vitro diagnostic testing.
Device Description: The description details a system designed to perform automated sample preparation, PCR, and detection on a biological sample to provide a diagnostic result.
Performance Studies: The document includes extensive analytical and clinical performance studies, which are required for IVD devices to demonstrate their accuracy and reliability for diagnostic use.
Predicate Device: The mention of a "Predicate Device(s)" with K numbers (K140083) indicates that this device is being compared to previously cleared IVD devices.

All these factors align with the definition and characteristics of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The Great Basin Stool Bacterial Pathogens Panel is a multiplexed, qualitative test for the detection and identification of DNA targets of enteric bacterial pathogens. The Stool Bacterial Pathogens Panel detects nucleic acids from:

· Campylobacter (C. coli/C. jejuni)
· Salmonella
· Shiga toxin 1 (stx1)
Shiga toxin 2 (stx2)
· Escherichia coli serotype 0157
Shigella

Shiga toxin genes are found in Shiga toxin-producing strains of E. coli (STEC/EHEC/VTEC) and Shigella dysenteriae. The E. coli O157 test result is only reported if a Shiga toxin gene (stx1 and/or stx2) is also detected.

The Stool Bacterial Pathogens Panel is performed directly from Cary Blair or C&S Medium preserved stool specimens from symptomatic patients with suspected acute gastroenteritis, or colitis and is performed on the Portrait™ Analyzer.

The test is intended for use as an aid in the diagnosis of gastrointestinal illness in conjunction with clinical and epidemiological information; however, it is not to be used to monitor these infection . Positive results do not rule out co-infection with other organisms and may not be the definitive cause of patient illness. Negative test 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. Concomitant culture is necessary if organism recovery or further typing of bacterial agents is desired.

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

PCI, PCH, OOI

Device Description

The Great Basin Stool Bacterial Pathogens Panel on the PA500 Portrait™ System utilizes automated, hot-start PCR amplification technology to amplify specific nucleic acid sequences that are then detected using hybridization probes immobilized on a modified silicon chip surface, in a single-use, self-contained test cartridge.
An aliquot of the specimen (stool preserved in stool transport media) is first processed using the Sample Preparation Device (SPD). An aliquot of the eluate obtained from the SPD is loaded into the sample port of the SBPP Test Cartridge.
Genomic DNA is extracted from microbial cells and diluted to reduce potential inhibitors of the PCR. During the PCR process, biotin-labeled primers direct the amplification of specific nucleic acid sequences within a conserved region for identification of: a bacterial sample processing control (SPC), Campylobacter coli/Campylobacter jejuni, Salmonella spp., Shiga toxin 1, Shiqa toxin 2, and E. coli serotype 0157.
Following PCR, biotin-labeled, amplified target DNA sequences are hybridized to sequence specific probes immobilized on the silicon chip surface, and incubated with antibody conjugated to the horseradish peroxidase enzyme (HRP). The unbound conjugate is washed away, and tetramethylbenzidine (TMB) is added to produce a colored precipitate at the location of the probe/target sequence complex. The resulting signal is detected by the automated Portrait™ Optical Reader within the PA500 Portrait™ Analyzer System. The SPC undergoes the same extraction, amplification, and detection steps as the sample in order to inhibitory substances, as well as process inefficiency due to instrument or reagent failure. No operator intervention is required once the sample is loaded into the sample port, and the Stool Bacterial Pathogens Panel cartridge is loaded into the Portrait™ Analyzer.

Test Device:
The PA500 Portrait™ Analyzer System is a fully automated system that includes: the Portrait™ Analyzer, single-use Stool Bacterial Pathogen Panel Cartridges, and the Portrait™ Data Analysis Software Program. The Portrait™ System is designed to perform automated sample preparation, PCR, and optical chip-based detection with integrated data analysis in less than two hours. The Portrait System was granted 510(k) clearance for the Portrait Toxigenic C. difficile Assay (K113358), Portrait GBS Assay (K143312), Staph ID/R Blood Culture Panel (K152470) and the Shiga Toxin Direct Test (K152955).

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

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

Not Found

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

Not Found

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

Analytical Sensitivity (LoD): Assessed LoD for 10 bacterial strains (all target organisms) by serial dilution into negative clinical stool. LoD values range from 1.3 x 10^3 CFU/mL to 1.6 x 10^4 CFU/mL depending on the strain.
Analytical Reactivity (Inclusivity): Tested 91 additional bacterial strains representing SBPP detected organisms (5 Campylobacter coli, 6 Campylobacter jejuni, 16 STEC E. coli, 8 O157+ STEC E. coli, 3 Shigella dysenteriae serotype 1, 33 Salmonella spp., 20 Shigella spp.) at 2X LoD in pooled negative preserved clinical stool. At least 3 replicates per strain were tested. Conclusion: The SBPP correctly identified all 91 organisms tested.
Analytical Specificity (Exclusivity): Evaluated cross-reactivity with 100 non-target organisms (84 bacterial, 3 yeast, 3 parasites, 9 viruses, human genomic DNA) commonly found in stool, prepared in pooled, negative, clinical stool matrix. All bacterial and yeast strains at ≥ 1.0x10^6 CFU/mL, gDNA at ≥1 µg/mL, viral strains at ≥1x10^6 copies/mL, and parasites at ≥1x10^5 TCID50/mL. Minimum 3 replicates. In silico analysis performed for Norovirus. Conclusion: All organisms gave expected "Not Detected" results, except for three Enteroinvasive Escherichia coli (EIEC) strains which were detected due to the shared ipaH gene target with Shigella spp.
Competitive Inhibition: Tested combinations of 8 SBPP target organisms (representing potential dual infections) with one organism at low titer (2X LoD) and a second at high titer (≥10^6 CFU/mL) in pooled, negative, preserved clinical stool. 48 unique combinations, tested in triplicate. Conclusion: Competitive inhibition was only observed for Salmonella when E. coli (stx1+/stx2+/O157+) was present at concentrations ≥ 1 x 10^6 CFU/mL. No other combinations showed competitive inhibition.
Interfering Substances: Evaluated 19 common stool contaminants/substances in positive stool (single SBPP target organism at ≤ 3X LoD) and negative stool. Minimum 3 replicates. Conclusion: No interference observed for substances at tested concentrations, with the exception of S. bongori in presence of 5% Milk of Magnesia (but not at 2.5% Milk of Magnesia).
Microbial Interference: Evaluated potential for cross-reactivity in mixed infections using a panel of 29 non-target GI pathogens (21 bacterial, 2 yeast, 2 parasites, 3 viruses, human genomic DNA) with 8 SBPP target strains at ≤ 3X LoD. Non-target organisms added at ≥10^6 CFU/mL, ≥1 x 10^6 copies/mL, and ≥ 1 µg/mL respectively. Minimum 3 replicates. Conclusion: No interference from non-target organisms was observed.
Carry-over/Cross Contamination: Contrived stool sample with high concentration of analyte (≥ 1 x 10^6 CFU/mL) alternated with clinical negative stool sample in 10 consecutive rounds on 4 different analyzers. 80 SBPP tests (40 positive, 40 negative). Organisms used: C. coli, E. coli (stx1+/stx2+/O157+), S. bongori, S. flexneri. Conclusion: No carry-over or cross contamination was observed.
Reproducibility: Conducted at 3 clinical sites (2 external, 1 internal) with 6 operators, 70 Portrait Analyzers, and 10 cartridge lots. Panel of 7 samples (low positive, moderate positive, negative) tested in triplicate over 5 non-consecutive days. Conclusion: Demonstrated acceptable reproducibility. ≥ 95% agreement for low positives, 100% agreement for moderate positives and negatives.
Specimen Stability and Storage: Tested 7 organisms at 2X LoD after storage at various times (0 hr, 2 hr, 24 hr, 48 hr, 72 hr, 96 hr, 120 hr, 144 hr) and temperatures (room temperature, 2-8°C). Each sample tested in triplicate. Conclusion: 100% agreement with expected results, supporting specimen storage claims.
Clinical Studies:
- Prospective Sample Study: Method comparison with standard stool culture-based methods. 1479 samples from symptomatic individuals with suspected GI infection collected at 4 sites (July-Nov 2016).
- Frozen Retrospective Sample Study: 150 frozen archived de-identified samples previously characterized by standard of care, confirmed by FDA cleared NAAT.
- Selected Sample Study: Additional fresh positive Salmonella samples from Intermountain Healthcare.
- Key Results:
  - Prospective Study (1479 samples):
    - Campylobacter: PPA = 96.4% (82.3-99.4), NPA = 99.2% (98.6-99.5)
    - Salmonella: PPA = 83.3% (55.2-95.3), NPA = 99.6% (99.1-99.8)
    - Shiga Toxin 1: PPA = 100.0% (20.7-100.0), NPA = 99.5% (99.0-99.8)
    - Shiga Toxin 2: PPA = 100.0% (20.7-100.0), NPA = 99.4% (98.8-99.7)
    - E. coli Serotype O157: PPA = 100% (51.0-100.0), NPA = 75.0% (46.8-91.1)
    - Shigella: PPA = 100% (56.6-100.0), NPA = 99.1% (98.4-99.4)
  - Frozen Retrospective Study (206 samples):
    - Salmonella: PPA = 94.4% (81.9-98.5), NPA = 100.0% (97.8-100.0)
    - Shiga Toxin 1: PPA = 100.0% (88.3-100.0), NPA = 100.0% (97.9-100.0)
    - Shiga Toxin 2: PPA = 100.0% (89.0-100.0), NPA = 100.0% (97.9-100.0)
    - E. coli Serotype O157 (48 samples): PPA = 100.0% (81.6-100.0), NPA = 100.0% (89.0-100.0)
    - Shigella: PPA = 94.7% (75.4-99.1), NPA = 100.0% (98.0-100.0)
  - Selected Sample Study (Salmonella, 28 samples): PPA = 92.9% (77.4-98.0)
  - Discrepant Results Resolution: Majority of false positives found concordant with FDA cleared NAAT (BioFire Film Array GI Panel or Nanosphere Verigene® EP test).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Prospective Study:

Campylobacter: Positive Percent Agreement (PPA) = 96.4%, Negative Percent Agreement (NPA) = 99.2%
Salmonella: PPA = 83.3%, NPA = 99.6%
Shiga Toxin 1: PPA = 100.0%, NPA = 99.5%
Shiga Toxin 2: PPA = 100.0%, NPA = 99.4%
E. coli Serotype O157: PPA = 100%, NPA = 75.0%
Shigella: PPA = 100%, NPA = 99.1%

Frozen Retrospective Study:

Salmonella: PPA = 94.4%, NPA = 100.0%
Shiga Toxin 1: PPA = 100.0%, NPA = 100.0%
Shiga Toxin 2: PPA = 100.0%, NPA = 100.0%
E. coli Serotype O157: PPA = 100.0%, NPA = 100.0%
Shigella: PPA = 94.7%, NPA = 100.0%

Selected Sample Study (Salmonella):

PPA = 92.9%

Reproducibility Study (Percent Agreement):

Campylobacter coli/jejuni (1.5X LoD): 100%
Campylobacter coli/jejuni (3X LoD): 100%
Salmonella (1.5X LoD): 96.7%
Salmonella (3X LoD): 100%
Shiga toxin 1 (1.5X LoD): 97.8%
Shiga toxin 1 (3X LoD): 100%
Shiga toxin 2 (1.5X LoD): 95.7%
Shiga toxin 2 (3X LoD): 100%
E. coli Serotype O157 (1.5X LoD): 97.8%
E. coli Serotype O157 (3X LoD): 100%
Shigella (1.5X LoD): 100%
Shigella (3X LoD): 100%
Negative: 100%

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Nanosphere Verigene® Enteric Pathogens Nucleic Acid Test (K140083)

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

BioFire Film Array Gl Panel (K140407), Nanosphere Verigene® EP test (K140083)

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

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).

Summary

0

Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized caduceus symbol, which is a staff with two snakes coiled around it. The symbol is enclosed in a circle with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter of the circle.

July 12, 2017

Public Health Service

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

GREAT BASIN SCIENTIFIC, INC. SUZETTE CHANCE SENIOR DIRECTOR OF CLINICAL AFFAIRS 2441 S. 3850 WEST SALT LAKE CITY UT 84120

Re: K163571

Trade/Device Name: Great Basin Stool Bacterial Pathogens Panel Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assay Regulatory Class: II Product Code: PCI, PCH Dated: December 16, 2016 Received: December 19, 2016

Dear Dr. Chance:

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.

1

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,

Steven R. Gitterman -S for

Uwe Scherf, M.S., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K163571

Device Name Great Basin Stool Bacterial Pathogens Panel

Indications for Use (Describe)

The Great Basin Stool Bacterial Pathogens Panel is a multiplexed, qualitative test for the detection and identification of DNA targets of enteric bacterial pathogens. The Stool Bacterial Pathogens Panel Hetects nucleic acids from:

· Campylobacter (C. coli/C. jejuni)
· Salmonella
· Shiga toxin 1 (stx1)
Shiga toxin 2 (stx2)
· Escherichia coli serotype 0157
Shigella

Shiga toxin genes are found in Shiga toxin-producing strains of E. coli (STEC/EHEC/VTEC) and Shige/la dysenteriae. The E. coli O157 test result is only reported if a Shiga toxin gene (stx1 and/or stx2) is also detected.

The Stool Bacterial Pathogens Panel is performed directly from Cary Blair or C&S Medium preserved stool specimens from symptomatic patients with suspected acute gastroenteritis, or colitis and is performed on the Portrait™ Analyzer.

The test is intended for use as an aid in the diagnosis of gastrointestinal illness in conjunction with clinical and epidemiological information; however, it is not to be used to monitor these infection . Positive results do not rule out co-infection with other organisms and may not be the definitive cause of patient illness. Negative test 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. Concomitant culture is necessary if organism recovery or further typing of bacterial agents is desired.

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

X Prescription Use (Part 21 CFR 801 Subpart D)

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

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5.0 510(k) Summary - Stool Bacterial Pathogens Panel

A. Submitted by:

Great Basin Corporation 2441 South 3850 West Salt Lake City, Utah 84120

Contact Information

Suzette Chance, PhD Senior Director of Clinical Affairs Great Basin Scientific 2441 S. 3850 West Salt Lake City, Utah 84120 Phone: 385-215-3369 Email: schance@@gbscience.com

B. Name of Device

Proprietary Name: Great Basin Stool Bacterial Pathogens Panel Common or Usual Names: Stool Bacteria Pathogens Panel SBPP GI Panel

Regulatory Information: ﻥ

| a. Regulation Section: | 21 CFR 866.3990, Gastrointestinal Microorganism Multiplex Nucleic
Acid-Based Assay
21 CFR 862.2570 - Instrumentation for clinical multiplex test systems |
|-------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| b. Classification: | Class II (Stool Bacterial Pathogen Panel; non-exempt);
Class II (PA500 Portrait Analyzer System) |
| c. Classification panel:
Product Code: | Microbiology Devices, OIVD (83) Microbiology
PCI Gastrointestinal Bacterial Panel Multiplex Nucleic Acid-
Based Assay System
PCH Gastrointestinal Pathogen Panel Multiplex Nucleic
Acid-Based Assay System
OOI Real-Time Nucleic Amplification System |

D. Intended use(s)/Indications for Use:

The Great Basin Stool Bacterial Pathogens Panel is a multiplexed, qualitative test for the detection and identification of DNA targets of enteric bacterial pathogens. The Stool Bacterial Pathogens Panel detects nucleic acids from:

· Campylobacter (C. coli/C. jejuni)
· Salmonella
· Shiga toxin 1 (stx1)
Shiga toxin 2 (stx2)
· Escherichia coli serotype 0157
Shigella

Shiga toxin genes are found in Shiga toxin-producing strains of E. coli (STEC/EHEC/VTEC) and Shigella dysenteriae. The E. coli O157 test result is only reported if a Shiga toxin gene (stx1 and/or stx2) is also detected.

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Image /page/4/Picture/2 description: The image shows a pattern of black ovals arranged in a descending order. The ovals are aligned in rows, with each row containing fewer ovals than the row below it. The ovals are solid black and have a smooth, rounded shape. The pattern creates a visual effect of depth and perspective.

The Stool Bacterial Pathogens Panel is performed directly from Carv Blair or C&S Medi preserved stool specimens from symptomatic patients with suspected acute gastroenteritis. enteritis, or colitis and is performed on the Portrait™ Analyzer.

The test is intended for use as an aid in the diagnosis of specific agents of gastrointestinal illness in conjunction with clinical and epidemiological information: however, it is not to be used to monitor these infections. Positive results do not rule out co-infection with other organisms and may not be the definitive cause of patient illness. Negative test 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. Concomitant culture is necessary if organism recovery or further typing of bacterial agents is desired.

Device Description

Test Principle:

The Great Basin Stool Bacterial Pathogens Panel on the PA500 Portrait™ System utilizes automated, hot-start PCR amplification technology to amplify specific nucleic acid sequences that are then detected using hybridization probes immobilized on a modified silicon chip surface, in a single-use, self-contained test cartridge.

An aliquot of the specimen (stool preserved in stool transport media) is first processed using the Sample Preparation Device (SPD). An aliquot of the eluate obtained from the SPD is loaded into the sample port of the SBPP Test Cartridge.

Genomic DNA is extracted from microbial cells and diluted to reduce potential inhibitors of the PCR. During the PCR process, biotin-labeled primers direct the amplification of specific nucleic acid sequences within a conserved region for identification of: a bacterial sample processing control (SPC), Campylobacter coli/Campylobacter jejuni, Salmonella spp., Shiga toxin 1, Shiqa toxin 2, and E. coli serotype 0157.

Following PCR, biotin-labeled, amplified target DNA sequences are hybridized to sequence specific probes immobilized on the silicon chip surface, and incubated with antibody conjugated to the horseradish peroxidase enzyme (HRP). The unbound conjugate is washed away, and tetramethylbenzidine (TMB) is added to produce a colored precipitate at the location of the probe/target sequence complex. The resulting signal is detected by the automated Portrait™ Optical Reader within the PA500 Portrait™ Analyzer System. The SPC undergoes the same extraction, amplification, and detection steps as the sample in order to inhibitory substances, as well as process inefficiency due to instrument or reagent failure. No operator intervention is required once the sample is loaded into the sample port, and the Stool Bacterial Pathogens Panel cartridge is loaded into the Portrait™ Analyzer.

Test Device:

The PA500 Portrait™ Analyzer System is a fully automated system that includes: the Portrait™ Analyzer, single-use Stool Bacterial Pathogen Panel Cartridges, and the Portrait™ Data Analysis Software Program. The Portrait™ System is designed to perform automated sample preparation, PCR, and optical chip-based detection with integrated data analysis in less than two hours. The Portrait System was granted 510(k) clearance for the Portrait Toxigenic C. difficile Assay (K113358), Portrait GBS Assay (K143312), Staph ID/R Blood Culture Panel (K152470) and the Shiga Toxin Direct Test (K152955).

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Image /page/5/Picture/2 description: The image shows a series of black ovals arranged in a triangular pattern. The ovals are stacked on top of each other, with the largest ovals at the bottom and the smallest ovals at the top. The ovals are all the same shape, but they vary in size. There are 8 ovals in total.

E. Substantial Equivalence Information:

Predicate Device: Nanosphere Verigene® Enteric Pathogens Nucleic Acid Test (K140083)

The following table provides a comparison of the Stool Bacterial Pathogens Panel and the predicate device:

| Features/Characteristics | Stool Bacterial Pathogens Panel
(SBPP) | Predicate Device
Verigene® EP (K140083) |
|-------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Manufacturer | Great Basin Scientific, Inc. | Nanosphere |
| Trade Name | Great Basin Stool Bacterial Pathogen
Panel | Verigene Enteric Pathogen Nucleic
Acid Test |
| 510(k) Number | K140083 | K140083 |
| Classification | II | II |
| Qualitative/Quantitative | Qualitative | Qualitative |
| Intended Use/Indications for
Use | The Great Basin Stool Bacterial
Pathogens Panel is a multiplexed,
qualitative test for the detection and
identification of DNA targets of enteric
bacterial pathogens. The Stool
Bacterial Pathogens Panel detects
nucleic acids from:
• Campylobacter ( C. coli and C. jejuni )
• Salmonella
• Shiga toxin 1 ( stx1 )
• Shiga toxin 2 ( stx2 )
• Escherichia coli serotype O157
• Shigella

Shiga toxin genes are found in Shiga
toxin-producing strains of E. coli
(STEC/EHEC/VTEC) and Shigella
dysenteriae . The E. coli O157 test
result is only reported if a Shiga toxin
gene ( stx1 and/or stx2 ) is also
detected.

The Stool Bacterial Pathogens Panel is
performed directly from Cary Blair or
C&S Medium preserved stool
specimens from symptomatic patients
with suspected acute gastroenteritis,
enteritis, or colitis and is performed on
the PortraitTM Analyzer.

The test is intended for use as an aid in
the diagnosis of specific agents of
gastrointestinal illness in conjunction
with clinical and epidemiological
information. Positive results do not rule
out co-infection with other organisms
and may not be the definitive cause of
patient illness. Negative test 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. Concomitant culture is
necessary if organism recovery or
further typing of bacterial agents is
desired. | 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.

11

Image /page/11/Picture/2 description: The image shows a collection of black ellipses arranged in a triangular pattern. The ellipses vary in size, with the largest ones at the bottom and the smallest ones at the top. The ellipses are arranged in rows, with each row containing one more ellipse than the row above it. The overall arrangement creates a sense of depth and perspective.

Table 5. Analytical Reactivity: Shiqella

| Shigella spp. | ATCC ID | Concentration
2X LoD
(CFU/mL) | Correct
SBPP
Results |
|--------------------------------------------------------|---------|-------------------------------------|----------------------------|
| Shigella boydii Serotype 2 | 8700 | 2.8 x 104 | 3/3 |
| Shigella boydii Serotype 3 | 8702 | 2.8 x 104 | 3/3 |
| Shigella boydii Serotype 1 | 9207 | 2.8 x 104 | 3/3 |
| Shigella boydii Serotype 8 | 12028 | 2.8 x 104 | 3/3 |
| Shigella boydii | 29928 | 2.8 x 104 | 3/3 |
| Shigella flexneri Serotype 5 | 9204 | 2.8 x 104 | 3/3 |
| Shigella flexneri Serotype 2b | 12022 | 2.8 x 104 | 3/3 |
| Shigella flexneri Serotype 6 | 12025 | 2.8 x 104 | 3/3 |
| Shigella flexneri Serotype 1a | 25929 | 2.8 x 104 | 3/3 |
| Shigella flexneri Serotype 2a | 29903 | 2.8 x 104 | 3/3 |
| Shigella sonnei | 9290 | 2.8 x 104 | 3/3 |
| Shigella sonnei | 11060 | 2.8 x 104 | 3/3 |
| Shigella sonnei | 25931 | 2.8 x 104 | 3/3 |
| Shigella sonnei | 29029 | 2.8 x 104 | 3/3 |
| Shigella sonnei | 29930 | 2.8 x 104 | 3/3 |
| Shigella dysenteriae Serotype 1 | 27345 | 2.8 x 104 | 6/6 |
| Shigella dysenteriae Serotype 2 | 29027 | 2.8 x 104 | 3/3 |
| Shigella dysenteriae Serotype 3 | 29028 | 2.8 x 104 | 5/5* |
| Shigella dysenteriae Serotype 12 | 49551 | 2.8 x 104 | 3/3 |
| Shigella dysenteriae Serotype 13 | 49555 | 2.8 x 104 | 3/3 |
| n= 20 | | | |
| * This set of test runs also contained 1 "Invalid" Run | | | |

Conclusion: The SBPP correctly identified all 91 organisms tested in the Inclusivity Study indicating that the SBPP can detect additional strains of Campylobacter coli, Campylobacter jejuni, Shigella, Salmonella and Shiga toxin producing Escherichia coli.

c. Analytical Specificity (Exclusivity)

The potential for cross-reactivity was evaluated in an Exclusivity Study, by testing non-target organisms commonly found in stool, in the SBPP. The study included 100 organisms phylogenetically related to targeted organisms as well as other bacteria, fungilyeast, parasites, viruses, and human genomic DNA (84 bacterial strains, 3 yeast, 3 parasites, 9 viruses and human genomic DNA). For those isolates that were classified as Biosafety level III, or unable to be cultured via standard clinical microbiology techniques, genomic DNA was tested in place of whole organism.

Each non-target organism or nucleic acid was prepared in pooled, preserved, negative, clinical stool matrix. All bacterial and yeast strains were tested at concentrations ≥ 1.0x106 CFU/mL. Genomic DNA templates, viral strains, and parasites, were tested at ≥1 uq/mL, ≥1x106 copies/mL, or ≥1x105 TCIDso/mL, respectively. A minimum of 3 replicates were tested for each organism evaluated for cross-reactivity.

In addition, in silico analysis was performed on the SBPP primers and probes against the six (6) published complete Norovirus genomes in the NCBI data base (https://www.ncbi.nlm.nih.gov /assembly/?term=norovirus). Based on low % Match Scores, low sequence similarities, and sequence alignments, it is highly unlikely that any of the Noroviruses would be amplified or detected by the SBPP primer-probe set.

The results of the study, including the specific concentrations at which each organism was evaluated are provided in Table 6.

12

Image /page/12/Picture/2 description: The image shows a pattern of black ovals arranged in a triangular shape. The ovals are stacked on top of each other, with the largest ovals at the bottom and the smallest ovals at the top. The ovals are all the same color and shape, and they are evenly spaced apart. There are 8 ovals in total.

Table 6. Analytical Specificity (Exclusivity) Study Results

| Species NEGATIVE
Result | |---------------------- | Bacteria | Abiotrophia defectiva | Acinetobacter baumannii | Aeromonas hydrophila | Anaerococcus tetradius | Bacillus cereus | Bacteriodes fragilis | Bacteriodes vulgatus | Bifidobacterium adolescentis | Bifidobacterium bifidum | Bifidobacterium longum | Campylobacter curvus (gDNA) | Campylobacter fetus fetus | Campylobacter | Campylobacter hyointestinalis | Campylobacter lari | Campylobacter lari | Campylobacter lari | Campylobacter lari | Campylobacter lari | Campylobacter upsaliensis | Citrobacter amalonaticus | Citrobacter freundii | Clostridium difficle | Clostridium histolyticum | Clostridium perfringens | Clostridium sordellii | Enterobacter aerogenes | Enterobacter cloacae | Enterococcus cecorum | Enterococcus faecalis | Enterococcus faecium | EAEC Escherichia coli | Escherichia coli | EIEC Escherichia coli | EIEC Escherichia coli | EIEC Escherichia coli | ETEC Escherichia coli | Escherichia fergusonii | Escherichia hermannii | Fusobacterium varium | Gardnerella vaginalis | Helicobacter fennelliae | Helicobacter pylori | Klebsiella pneurmoniae | Klebsiella oxytoca | Lactobacillus acidophilus | Lactobacillus casei | Leminorella grimonti | Listeria grayi | Listeria innocua | Listeria monocytogenes | Morganella morganii | Peptostreptococcus anaerobius | Plesiomonas shigelloides | Porphyromonas asaccharolytica | Prevotella melaninogenicus | Strain ID | Input Tested | SBPP
| | | |
----------|-------------------|-------------------|----------------------------|-------------------|-----------------------|-----|-----|
| | | | | | | |
| ATCC 49176 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC19606 | 9.6 x 107 CFU/mL | 3/3 | | | | |
| ATCC 35654 | 8.7 x 108 CFU/mL | 3/3 | | | | |
| ATCC 35098 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 14579 | 1 x 108 CFU/mL | 3/3 | | | | |
| ATCC 23745 | ≥ 1 x 106 CFU/mL# | 3/3* | | | | |
| ATCC 8482 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 15703 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 11863 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 15707 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC BAA-1459D-5 | ≥ 1 µg/mL | 6/6 | | | | |
| ATCC 27374 | 2.65 x 107 CFU/mL | 5/5* | | | | |
fetus venerealis | ATCC 33561 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 35217 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 35222 | 9.0 x 106 CFU/mL | 3/3 | | | | |
| ATCC 35223 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 35221 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 43675 | 1.75 x 107 CFU/mL | 3/3 | | | | |
| ATCC BAA-1060 | 5.95 x 107 CFU/mL | 3/3 | | | | |
| ATCC 49816 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 25406 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 8090 | 8.47 x 107 CFU/mL | 3/3 | | | | |
| ATCC 43594 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 19401 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 13124 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 9714 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 15038 | 8.43 x 107 CFU/mL | 3/3 | | | | |
| ATCC 13047 | 8.33 x 107 CFU/mL | 3/3 | | | | |
| ATCC 43918 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 29212 | 4.8 x 107 CFU/mL | 3/3 | | | | |
| ATCC 19434 | 5.85 x 107 CFU/mL | 3/3 | | | | |
| ATCC 29552 | 8.53 x 107 CFU/mL | 3/3 | | | | |
| ATCC 23544 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 43892 | 2.8 x 104 CFU/mL | 0/3 | | | | |
| ATCC 43893 | 2.8 x 104 CFU/mL | 0/3 | | | | |
| ATCC 12806 | 2.8 x 104 CFU/mL | 0/3 | | | | |
| ATCC 31703 | 3.37 x 107 CFU/mL | 3/3 | | | | |
| ATCC 35469 | 2.47 x 107 CFU/mL | 3/3 | | | | |
| ATCC 33650 | 5.57 x 107 CFU/mL | 3/3 | | | | |
| ATCC 27725 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 14018 | 3.53 x 107 CFU/mL | 3/3 | | | | |
| ATCC 35683 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 49503 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 13883 | 5.7 x 107 CFU/mL | 3/3 | | | | |
| ATCC 49131 | 7.8 x 107 CFU/mL | 3/3 | | | | |
| ATCC 4356 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 393 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 43007 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 19120 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 33090 | 6.6 x 107 CFU/mL | 3/3 | | | | |
| ATCC 15313 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 25829 | 1.72 x 108 CFU/mL | 3/3 | | | | |
| ATCC 27337 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 51903 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| ATCC 27908 | ≥ 1 x 106 CFU/mL# | 3/3 | | | | |
| Proteus mirabilis | ATCC 25845 | ATCC 25933 | ≥ 1 x 106 CFU/mL# | $\ge$ 1 x 106 CFU/mL# | 3/3 | 3/3 |

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Image /page/13/Picture/2 description: The image shows a series of black, oval shapes arranged in a pattern. The ovals are positioned in a way that creates a sense of depth or perspective, with the larger ovals at the bottom and the smaller ovals at the top. The background is plain white, which makes the black ovals stand out.

| Species | Strain ID | Input Tested | SBPP
NEGATIVE
Result |
|-----------------------------------------|----------------|-------------------|----------------------------|
| Proteus penneri | ATCC 33519 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Proteus vulgaris | ATCC 6896 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Providencia alcalifaciens | ATCC 9886 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Providencia rettgeri | ATCC 9250 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Providencia stuartii | ATCC 49762 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Pseudomonas aeruginosa | ATCC 10145 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Pseudomonas putida | ATCC 49128 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Ruminococcus bromii | ATCC 27255 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Serratia liquefaciens | ATCC 27592 | 7.17 x 107CFU/mL | 3/3 |
| Serratia marcescens | ATCC 13880 | 4.27 x 107 CFU/mL | 3/3 |
| Staphylococcus aureus | ATCC 6538 | 3.37 x 107 CFU/mL | 6/7a |
| Staphylococcus epidermidis | ATCC 12228 | 3.7 x 107 CFU/mL | 3/3 |
| Stenotrophomonas maltophilia | ATCC 13637 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Streptococcus agalactiae | ATCC 13813 | 4.43 x 107 CFU/mL | 5/5 |
| Streptococcus dysgalactiae | ATCC 43078 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Streptococcus intermedius | ATCC 27335 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Streptococcus pyogenes | ATCC 4543 | 4.4 x 107 CFU/mL | 5/6b |
| Streptococcus uberis | ATCC 9927 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Trabulsiella guamensis | ATCC 49492 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Veillonella parvula | ATCC 10790 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Vibrio cholera | ATCC 55188 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Vibrio parahaemolyticus | ATCC 17802 | 6.2 x 107 CFU/mL | 3/3 |
| Vibrio vulnificus | ATCC 27562 | 1.48 x 108 CFU/mL | 3/3 |
| Yersinia bercovieri | ATCC 43970 | 2.57 x 108 CFU/mL | 3/3 |
| Yersinia enterocolitica | ATCC 49397 | 1.81 x 108 CFU/mL | 3/3 |
| Yersinia pseudotuberculosis | ATCC 23207 | 4 x 107 CFU/mL | 3/3 |
| Yersinia rohdei | ATCC 43380 | 1.92 x 107 CFU/mL | 3/3 |
| Fungi | | | |
| Candida albicans | ATCC 18804 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Candida catenulata | ATCC 10565 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Saccharomyces boulardii | ATCC MYA-796 | ≥ 1 x 106 CFU/mL# | 3/3 |
| Viruses and Parasites | | | |
| Adenovirus Type 2 (gDNA) | ATCC VR-846D | ≥ 1 µg/mL | 3/3 |
| Adenovirus type 40, strain Dugan (gDNA) | ATCC VR-931D | ≥ 1 µg/mL | 3/3 |
| Adenovirus type 41, strain Tak (gDNA) | ATCC VR-930D | ≥ 1 µg/mL | 3/3 |
| Coxsackie B4 | ATCC VR-184 | 1 x 105 TCID50/mL | 3/3 |
| Cryptosporidium parvum (gDNA) | ATCC PRA-67D | ≥ 1 µg/mL | 3/3 |
| Entamoeba histolytica (gDNA) | ATCC 30459DQ | 1 x 106 copies/mL | 3/3 |
| Enterovirus (RNA) | ATCC VR-1775DQ | 1 x 105 TCID50/mL | 5/5* |
| Giardia intestinalis (gDNA) | ATCC 50803D | ≥ 1 µg/mL | 3/3 |
| Norovirus GI (synthetic RNA) | ATCC VR-3234SD | 1 x 106 copies/mL | 3/3 |
| Norovirus GII (synthetic RNA) | ATCC VR-3235SD | 1 x 106 copies/mL | 3/3 |
| Rotavirus | ATCC VR-1546 | 1 x 105 TCID50/mL | 3/3 |
| Rotavirus A (RNA) | ATCC VR-2018DQ | 1 x 106 copies/mL | 3/3 |

Concentration estimated based on OD600

*This set of test runs also contained 1 'INVALID' run

a 'Salmonella DETECTED' for 1/4 replicates. An additional 3 replicates were run and the expected NEGATIVE result obtained for all replicates.

b One out of 3 replicates gave a 'Salmonella DETECTED' result. An additional 3 replicates were run, and the expected NEGATIVE result obtained for all replicates.

Conclusion: All of the organisms shown in Table 6 gave the expected "Not Detected" result indicating that there was no cross-reactivity with the SBPP. The only exceptions were the three Enteroinvasive Escherichia coli (EIEC) strains which were "Detected" in the SBPP (0/3 SBPP Negative Results for each). This cross-reactivity was expected since the gene target used to detect Shigella spp. (ipaH) is also present EIEC (see Limitations in the Package Insert).

14

Image /page/14/Picture/2 description: The image shows a series of black oval shapes arranged in a step-like pattern. The ovals are oriented horizontally and decrease in size as they ascend. The arrangement creates a visual effect of depth or perspective, with the larger ovals appearing closer and the smaller ones further away.

d. Competitive Inhibition

To evaluate potential for competitive interference in the SBPP, combinations of the 8 SBPP target organisms, representative of potential dual infections, were tested. The orqanisms included: C. coli (ATCC 43486), C. jejuni (ATCC 49943), E. coli (ATCC BAA-2196 stx1+/stx2+), E. coli (ATCC 43895 stx1+/stx2+/0157+), S. bongori (ATCC 43975), S. enterica (ATCC 13311), S. flexneri (ATCC 25929), and S. sonnei (ATCC 29930). The panels were designed such that one organism of each bacterial species was present at a low titer (2X LoD) with a second organism present at a high titer (≥10° CFU/mL). The samples were generated by spiking previously frozen and quantified enriched broth cultures of all bacterial species, into pooled, negative, preserved clinical stool at the required concentration. This resulted in 48 unique combinations, each of which were tested in triplicate in the SBPP. The combinations and concentrations tested along with the study results are shown in Table 7.

	Organisms at High Titer: ≥106 CFU/mL
Organism at Low Titer
(2X) LoD	C. coli
(ATCC

| C. jejuni
(ATCC
| E. coli
(stx1+/stx2+/
non-O157)
(ATCC BAA-
| E. coli
(stx1+/stx2+/
O157+)
(ATCC 43895) | S. bongori
(ATCC
| S. enterica
(ATCC
| S. flexneri
(ATCC
| S. sonnei
(ATCC
|
| Campylobacter coli (ATCC 43486) | -- | -- | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Campylobacter jejuni (ATCC 49943) | -- | -- | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Escherichia coli (stx1+/stx2+/O157-)
(ATCC BAA-2196) | 3/3 | 3/3 | -- | -- | 3/3 | 3/3 | 3/3 | 3/3 |
| Escherichia coli (stx1+/stx2+/O157+)
(ATCC 43895) | 3/3 | 3/3 | -- | -- | 3/3 | 3/3 | 3/3 | 3/3 |
| Salmonella bongori (ATCC 43975) | 3/3 | 3/3 | 3/3 | 3/3 | -- | -- | 3/3 | 5/6a |
| Salmonella enterica (ATCC 13311) | 7/9b | 3/3 | 3/3 | 14/19c
6/6d | -- | -- | 3/3 | 3/3 |
| Shigella flexneri (ATCC 25929) | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | -- | -- |
| Shigella sonnei (ATCC 29930) | 3/3 | 3/3 | 5/6e | 3/3 | 3/3 | 3/3 | -- | -- |
| a In 1/3 replicates, 'high titer' Shigella sonnei was not detected and contamination with a Campylobacter sp. was noted. An
additional 3 replicates were tested and the expected result was obtained for both analytes, in all replicates. | | | | | | | | |
| b For a 'low titer' Salmonella enterica and 'high titer' Campylobacter coli sample, the SBPP did not detect Salmonella in 2/3
replicates, although Campylobacter was correctly identified in all cases. An additional 6 replicates were tested, and the expected
result was obtained for both analytes, in all replicates. | | | | | | | | |
| c For a 'low titer' Salmonella enterica and 'high titer' Escherichia coli (ATCC 43895, ≥106 CFU/mL) sample, the SBPP did not detect | | | | | | | | |

Table 7. Competitive Inhibition Study Results

Salmonella in 1/3 replicates. An additional 16 replicates were tested and 12/16 detected 'low titer' Salmonella.

d We decreased the concentration of the 'E. coli to 1 x 10 CFU/mL in combination with 'low tite' Salmonella and tested 6 replicates. The expected result was obtained for both analytes, in all replicates.

® In 1/3 replicates, low titer' Shigella sonnei was not detected, although Shiga Toxin 1 & 2 was detected in all cases. An additional 3 replicates were tested, and the expected result obtained for both analytes, in all replicates.

Conclusion: Competitive inhibition was only observed for Salmonella when E. coli (stx1+/stx2+/O157+) was present at concentrations ≥ 1 x 10° CFU/mL. No other combinations of organisms showed competitive inhibition.

15

Image /page/15/Picture/2 description: The image shows a series of black oval shapes arranged in a pyramid-like formation. The ovals are stacked on top of each other, with the largest ovals at the bottom and the smallest ovals at the top. The ovals are all the same color and have a smooth, uniform texture. The background is white.

e. Interfering Substances

Potential interference in the SBPP from 19 different substances that are common stool contaminants, or likely to be present in patients with diarrhea were evaluated in an Interfering Substances Study. Each substance was added to a positive stool prepared by adding a single SBPP target organism to pooled, negative, preserved clinical stool at ≤ 3X LoD. The organisms tested represented each analyte detected by the SBPP and included: C. coli (ATCC 43486), C. jejuni (ATCC 49943), E. coli (ATCC BAA-2196 stx1+/stx2+), E. coli (ATCC 43895 stx1+/stx2+/0157+), S. bongori (ATCC 43975), S. enterica (ATCC 13311), S. flexneri (ATCC 25929), and S. sonnei (ATCC 29930).

A clinical, neqative, un-spiked stool matrix was also tested as a control to evaluate potential interference with the internal assay control in the absence of analyte. A minimum of 3 replicates were tested for each substance. Samples for which 1 or more of the replicates gave unexpected results were re-tested. If 1 or more replicates still gave the unexpected result, the substance was considered to demonstrate interference in the SBPP at the concentration tested. The concentration at which each substance was tested along with the SBPP results are summarized in Table 8.

16

Table 8. Interfering Substances Study Results

| Potentially Interfering Substances and
Input Concentration | | Campylobacter
coli
ATCC 43486 | Campylobacter
jejuni
ATCC 49943 | Escherichia coli
(stx1+/stx2+
O157+)
ATCC 43895 | Escherichia coli
(stx1+/stx2+
non-O157)
ATCC 2196 | Salmonella
bongori
ATCC 43975 | Salmonella
enterica
ATCC 13311 | Shigella
flexneri
ATCC 25929 | Shigella
sonnei
ATCC 29930 | Negative
Stool
Samples
Assay
Control
DETECTED |
|---------------------------------------------------------------|------------|---------------------------------------------------|-----------------------------------------------------|-----------------------------------------------------------------|-------------------------------------------------------------------|---------------------------------------------------|----------------------------------------------------|--------------------------------------------------|------------------------------------------------|--------------------------------------------------------------|
| | | ≤3X LoD
(3.6 x 103
CFU/mL) | ≤ 3X LoD
(2.6 x 103
CFU/mL) | ≤ 3X LoD
(3.2 x 104
CFU/mL) | ≤ 3X LoD
(2 x 104
CFU/mL) | ≤ 3X LoD
(7.5 x 103
CFU/mL) | ≤ 3X LoD
(3.8x 104
CFU/mL) | ≤ 3X LoD
(1.6 x 104
CFU/mL) | ≤ 3X LoD
(2.8 x 104
CFU/mL) | |
| | | Ampicillin | 50 mg/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Bacitracin Zinc ointment | 50 mg/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 6/6 | 3/3 | 3/3 | 3/3 |
| Benzalkonium chloride,
ethanol (moist towelettes) | 9.5% v/v | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 4/6 | 3/3 |
| Bovine Mucin | 6.25 mg/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 5/6 | 3/3 |
| Calcium carbonate | 200 mg/mL | 3/3 | 3/3 | 3/3 | 5/6 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Cholesterol | 5% v/v | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Hemoglobin | 10% w/v | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/4 | 3/3 |
| Human Whole Blood | 50% v/v | 3/3 | 3/3 | 3/3 | 3/3 | 5/6 | 3/3 | 3/3 | 3/3 | 3/3 |
| Hydrocortisone | 75 mg/mL | 3/3 | 3/3 | 5/6 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Imodium | 10% v/v | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Kaopectate | 10% v/v | 3/3 | 3/3 | 3/3 | 3/3 | 5/6 | 3/3 | 3/3 | 3/3 | 3/3 |
| Milk of Magnesia | 5% v/v | 3/3 | 3/3 | 2/3 | 3/3 | 2/6b | 3/3c | 3/3 | 3/3 | 3/3 |
| Mineral Oil | 50% v/v | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Naproxen Sodium | 9.5% w/v | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Nystatin | 5% v/v | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Pepto-Bismol | 10% v/v | 3/3 | 3/3 | 3/3 | 5/5*a | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Pork Mucin | 6.25 mg/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Sennosides | 9.7 mg/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Triglycerides | 10% v/v | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| * This set of test runs also contained 1 "Invalid" run. | | | | | | | | | | |

ª One replicate in this set correctly identified Shiga toxin 2, but additionally detected Shigella.

" Four Salmonella borgori (ATCC 4397, 7.5 x 10° CFUmLine SBP do rot detect Salmonella in 23 replicates. An additional S repicates were tested and similarly, 2/3 replicates did not detect Salmonella.

C The concentration of Milk of Magnesia was decreased to 2.5% v/v. The SBPP detected Salmonella in all replicates tested

Conclusion: No interference in the SBP was observed for the substances tested at the concentrations shown in Table 8, with the exception of S. bongoriin the presence of 5% Milk of Magnesia. However, no interference was observed at 2.5% Milk of Magnesia.

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Image /page/17/Picture/2 description: The image shows a pattern of black ovals arranged in a triangular shape. The ovals are arranged in rows, with each row containing fewer ovals than the row below it. The bottom row contains two ovals, and the top row contains two ovals. The ovals are all the same size and shape.

f. Microbial Interference

The potential for cross-reactivity in a mixed infection was evaluated in a Microbial Interference Study. A panel of non-target gastrointestinal pathogens commonly encountered in stool were tested in the presence of each of the analytes detected in the SBPP. The panel of non-target organisms tested was a subset of 29 of the organisms used in the Exclusivity Study and were commercially purchased at a given concentration, or previously frozen broth cultures, for which concentration was measured at the time of growth.

Similar to the Exclusivity Study, the non-target bacterial and yeast strains were prepared using previously frozen and enumerated aliquots of liguid cultures. Potentially interfering bacterialfungi, viruses, and DNA were added at ≥10° CFU/mL, ≥1 x 10° copies/mL, and ≥ 1 µg/mL, respectively, to pooled, negative, preserved, clinical stool with a single SBPP target analyte added at ≤ 3X LoD. The following 8 strains representing all SBPP targets were tested in the presence of the 29 interfering organisms: C. coli (ATCC 43486), C. jejuni (ATCC 49943), E. coli (ATCC BAA-2196 stx1+/stx2+), E. coli (ATCC 43895 stx1+/stx2+/0157+), S. bongori (ATCC 43975), S. enterica (ATCC 13311), S. flexneri(ATCC 25929), and S. sonnei (ATCC 29930). In total, 21 unique bacterial strains, 2 yeast, 2 parasites, 3 viruses and human genomic DNA were tested for microbial interference with the 8 SBPP target strains.

A minimum of 3 replicates of each sample were tested. The specific concentrations at which each organism was evaluated along with the results are shown in Table 9.

18

| |
|
|--------|------|
| | |
| V
D | |

Table 9. Microbial Interference Study Results

| Species | Strain ID | Input Tested | C. coli
ATCC 43486 | C. jejuni
ATCC 49943 | E. coli
(stx1+/stx2+
non-0157)
ATCC 2196 | E. coli
(stx1+/stx2+
0157+)
ATCC 43895 | Salmonella
bongori
ATCC 43975 | Salmonella
enterica
ATCC 13311 | Shigella
flexneri
ATCC 25929 | Shigella
sonnei
ATCC 29930 |
|--------------------------------------------------------------------------------------------------|------------------------------|-------------------|-----------------------------------|----------------------------------|---------------------------------------------------|-------------------------------------------------|-------------------------------------|--------------------------------------|------------------------------------|-----------------------------------|
| | | | ≤ 3X LoD
(3.6 x 103
CFU/mL) | ≤3X LoD
(2.6 x 103
CFU/mL) | ≤3X LoD
(2 x 104
CFU/mL) | ≤ 3X LoD
(3.2 x 104
CFU/mL) | ≤ 3X LoD
(7.5 x 103
CFU/mL) | ≤ 3X LoD
(3.8 x 104
CFU/mL) | ≤ 3X LoD
(1.6 x 104
CFU/mL) | ≤ 3X LoD
(2.8 x 104
CFU/mL) |
| Bacteria | | | | | | | | | | |
| Aeromonas hydrophilia | ATCC 35654 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Bacteroides fragilis | ATCC 23745 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 5/5* | 3/3 | 3/3 | 3/3 | 3/3 |
| Bacteroides vulgatus | ATCC 8482 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 5/6 | 3/3 | 3/3 | 5/5* |
| Bifidobacterium bifidum | ATCC 11863 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 5/6 | 3/3 |
| Clostridium difficile (toxinA/B) | ATCC 43594 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3a | 3/3 | 3/3 | 3/3 |
| Clostridium perfringens | ATCC 13124 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3* | 3/3 | 3/3 |
| Enterobacter aerogenes | ATCC 15038 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Enterococcus faecalis | ATCC 29212 | ≥106 CFU/mL | 3/3 | 3/3* | 3/3 | 3/3 | 5/6 | 3/3 | 3/3 | 3/3 |
| Escherichia coli (non-STEC O157) | ATCC 700728 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 8/9 | 3/3 | 3/3 |
| Enteroaggregative E. coli (EAEC) | ATCC 29552 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Enterotoxigenic E. coli (ETEC) | ATCC 31703 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Enteropathogenic E. coli (EPEC) | ATCC 49106 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Helicobacter pylori | ATCC 49503 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Klebsiella pneumonia | ATCC 13883 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Lactobacillus acidophilus | ATCC 4356 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 5/5* | 5/6 | 3/3 | 3/3 | 3/3 |
| Listeria monocytogenes | ATCC 15313 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 5/5* | 3/3 | 3/3 | 3/3 | 3/3 |
| Prevotella melaninogenica | ATCC 25845 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3* |
| Pseudomonas aeruginosa | ATCC 10145 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 5/5* | 3/3 | 3/3 |
| Staphylococcus aureus | ATCC 6538 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 5/5* | 3/3 | 3/3 | 3/3 | 3/3 |
| Vibrio cholera | ATCC 55188 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Yersinia enterocolitica | ATCC 49397 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| | | | n=21 | | | | | | | |
| Yeasts, Parasites, Viruses and DNA | | | | | | | | | | |
| Candida albicans | ATCC 18804 | >106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Saccharomyces boulardii | ATCC MYA-796 | ≥106 CFU/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Entamoeba histolytica (gDNA) | ATCC 30459DQ | ≥1 x106 copies/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Giardia intestinalis (gDNA) | ATCC 50803D | ≥1 µg/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Enterovirus 71 (RNA) | ATCC VR-
1775DQ | ≥1 x106 copies/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 5/5 | 3/3 |
| Norovirus G1 (synthetic RNA) | ATCC VR-
3234SD | ≥1 x106 copies/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Rotavirus (RNA) | ATCC VR-
2018DQ | ≥1 x106 copies/mL | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 | 3/3 |
| Human genomic DNA | Roche Cat No.
11691112001 | ≥1 µg/mL | 3/3 | 3/3 | 5/6 | 5/6 | 3/3 | 3/3 | 3/3 | 3/3 |
| | | | n=8 | | | | | | | |
| *This set of tests also contained 1 "Invalid" run | | | | | | | | | | |
| a One replicate in this set correctly identified Salmonella, but additionally detected Shigella. | | | | | | | | | | |

Conclusion: No interference from non-target organisms was observed at the concentrations indicated in Table 9 in the mixed Microbial Interfernes Study.

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Image /page/19/Picture/2 description: The image shows a series of black oval shapes arranged in a pattern. The ovals are organized in rows, with the bottom row containing the largest ovals and the top row containing the smallest ovals. The ovals appear to be arranged in a way that suggests a sense of depth or perspective. The background is plain white.

q. Carry-over/Cross Contamination

To evaluate potential carry-over/cross-contamination of the SBPP a Carry-over Study was conducted. Briefly a contrived stool sample containing a high concentration of an analyte was alternated with a clinical negative stool sample in 10 consecutive testing rounds. The high positive sample was generated by adding previously frozen and quantified enriched broth cultures into pooled negative, preserved, clinical stool at a final concentration ≥ 1 x 10° CFU/mL. The organisms used in this study were C. coli (ATCC 43486), E. coli (ATCC 43895 stx1+/stx2+/0157+), S. bongori (ATCC 43975), and S. flexneri (ATCC 25929). The negative sample was stool from symptomatic patients that previously tested negative for all SBPP targets.

The alternating pattern of 10 test rounds of high positive and negative samples were performed in direct succession on 4 different analyzers. In total, 80 SBPP tests were performed: 40 tests of a high positive sample and 40 tests of a negative sample. The concentrations tested along with the study results are shown in Table 10.

| Sample Type (Alternating
Positive/Negative) | | Portrait
Analyzer
5.315 | Portrait Analyzer
5.106 | Portrait
Analyzer
5.382 | Portrait
Analyzer
5.072 |
|------------------------------------------------|--------------|-------------------------------|----------------------------------------------------------------------------|-------------------------------|-------------------------------|
| High Positive | Runs 1 - 4 | C. coli/jejuni
DETECTED | Shiga Toxin 1 DETECTED
Shiga Toxin 2 DETECTED
Serotype O157 DETECTED | Salmonella
DETECTED | Shigella
DETECTED |
| Negative | Runs 5 - 8 | Negative | Negative | Negative | Negative |
| High Positive | Runs 9 - 12 | C. coli/jejuni
DETECTED | Shiga Toxin 1 DETECTED
Shiga Toxin 2 DETECTED
Serotype O157 DETECTED | Salmonella
DETECTED | Shigella
DETECTED |
| Negative | Runs 13 - 16 | Negative | Negative | Negative | Negative |
| High Positive | Runs 17 - 20 | C. coli/jejuni
DETECTED | Shiga Toxin 1 DETECTED
Shiga Toxin 2 DETECTED
Serotype O157 DETECTED | Salmonella
DETECTED | Shigella
DETECTED |
| Negative | Runs 21 - 24 | Negative | Shiga Toxin 1 DETECTED | Negative | Negative |
| High Positive | Runs 25 - 28 | C. coli/jejuni
DETECTED | Shiga Toxin 1 DETECTED
Shiga Toxin 2 DETECTED
Serotype O157 DETECTED | Salmonella
DETECTED | Shigella
DETECTED |
| Negative | Runs 29 - 32 | Negative | Negative | Negative | Negative |
| High Positive | Runs 33 - 36 | C. coli/jejuni
DETECTED | Shiga Toxin 1 DETECTED
Shiga Toxin 2 DETECTED
Serotype O157 DETECTED | Salmonella
DETECTED | Shigella
DETECTED |
| Negative | Runs 37 - 40 | Negative | Negative | Negative | Negative |

Table 10. Carry-over/Cross Contamination Study Results

Conclusion: No carry-over or cross contamination was observed in the SBPP.

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Image /page/20/Picture/2 description: The image shows a series of black oval shapes arranged in a pattern. The ovals are organized in rows, with each row containing a different number of ovals. The rows are arranged in a way that creates a sense of progression or sequence. The ovals are all the same color and shape, and they are evenly spaced apart.

h. Reproducibility

A Reproducibility Study was conducted at 3 clinical study sites; 2 external and 1 internal. The study incorporated several variables including 6 different operators (2 per site), 70 different Portrait Analyzers and 10 different cartridge lots.

A panel consisting of 7 different samples was tested in triplicate over 5 non-consecutive days by each operator. Each analyte detected in the SBPP was included as a low positive (1.5X LoD) and a moderate positive (3X LoD) in the reproducibility panel. A single negative was also included.

Analysis of Positive Results

For each analyte, there were 90 possible positive results. However, due to circumstances which required additional runs, some samples have greater than 90 results which is explained below.

For Sample RP-03 two (2) of the three (3) replicates obtained by Operator 4 on Day 3 gave unexpected negative results for all 3 analytes that were supposed to be present in RP-03 (Shiga toxin 1, Shiga toxin 2, and E. coli 0157 at 1.5X LoD). It was suspected that the negative results were due to a mix up with the sample ID. The third replicate was an aborted run ("Test Incomplete") and didn't produce any results. All 3 replicates were repeated. The original results along with the repeat test results were included in the analysis for a total "n" of 92 for each analyte in sample RP-03.

For Sample RP-04 one (1) of the replicates obtained by operator 5 gave positive results for all analytes detected in the SBPP (Sample RP-04 should only contain Shiga toxin 1, Shiga toxin 2, and E. coli O157 at 3X LoD). It was suspected that this sample was contaminated by the operator. Another aliquot of this sample was tested. The original result was included in the analysis along with the repeat test results giving a total "n" of 91 for the analytes in sample RP-04. The total number of replicates for all other samples was 90.

The percent agreement of the positive results is presented separately for each analyte and concentration, by operator, by site and in total, and is shown in Tables 11 through 16.

| Analyte | Conc. | Operator | Correct
Positive
Results | Agreement | Site | Correct
Positive
Results | Agreement | Total
Correct
Positive
Results |
|--------------------|--------------|----------|--------------------------------|-----------|------|--------------------------------|-----------|-----------------------------------------|
| Campylobacter spp. | 1.5 X
LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 90/90
100% |
| | | 2 | 15/15 | 100% | | 30/30 | 100% | |
| | | 3 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 4 | 15/15 | 100% | | 30/30 | 100% | |
| | | 5 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | | 6 | 15/15 | 100% | | 30/30 | 100% | |
| | 3 X LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 90/90
100% |
| | | 2 | 15/15 | 100% | | 30/30 | 100% | |
| | | 3 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 4 | 15/15 | 100% | | 30/30 | 100% | |
| | | 5 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | | 6 | 15/15 | 100% | | 30/30 | 100% | |

Table 11. Campylobacter Reproducibility Results

21

| Analyte | Conc. | Operator | Correct
Positive
Results | Agreement | Site | Correct
Positive
Results | Agreement | Total
Correct
Positive
Results |
|------------|-----------|----------|--------------------------------|-----------|------|--------------------------------|-----------|-----------------------------------------|
| Salmonella | 1.5 X LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 87/90
96.7% |
| | | 2 | 15/15 | 100% | 1 | 30/30 | 100% | |
| | | 3 | 14/15 | 93.3% | 2 | 28/30 | 93.3% | |
| | | 4 | 14/15 | 93.3% | 2 | 28/30 | 93.3% | |
| | | 5 | 14/15 | 93.3% | 5 | 29/30 | 96.7% | |
| | | 6 | 15/15 | 100% | 5 | 29/30 | 96.7% | |
| Salmonella | 3 X LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 90/90
100.0% |
| | | 2 | 15/15 | 100% | 1 | 30/30 | 100% | |
| | | 3 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 4 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 5 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | | 6 | 15/15 | 100% | 5 | 30/30 | 100% | |

Table 12. Salmonella Reproducibility Results

Table 13. Shiga toxin 1 Reproducibility Results

| Analyte | Conc. | Operator | Correct
Positive
Results | Agreement | Site | Correct
Positive
Results | Agreement | Total
Correct
Positive
Results |
|--------------|--------------|----------|--------------------------------|-----------|------|--------------------------------|-----------|-----------------------------------------|
| Shia toxin 1 | 1.5 X
LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 90/92
97.8% |
| | | 2 | 15/15 | 100% | 1 | 30/30 | 100% | |
| | | 3 | 15/15 | 100% | 2 | 30/32 | 93.8% | |
| | | 4 | 15/17 | 88.2% | 2 | 30/32 | 93.8% | |
| | | 5 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | | 6 | 15/15 | 100% | 5 | 30/30 | 100% | |
| Shia toxin 1 | 3 X LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 91/91
100.0% |
| | | 2 | 15/15 | 100% | 1 | 30/30 | 100% | |
| | | 3 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 4 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 5 | 16/16 | 100% | 5 | 31/31 | 100% | |
| | | 6 | 15/15 | 100% | 5 | 31/31 | 100% | |

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| Analyte | Conc. | Operator | Correct
Positive
Results | Agreement | Site | Correct
Positive
Results | Agreement | Total
Correct
Positive
Results |
|---------------|--------------|----------|--------------------------------|-----------|------|--------------------------------|-----------|-----------------------------------------|
| Shiga toxin 2 | 1.5 X
LOD | 1 | 14/15 | 93.3% | 1 | 29/30 | 96.7% | 88/92
95.7% |
| | | 2 | 15/15 | 100% | | 29/32 | 90.6% | |
| | | 3 | 15/15 | 100% | 2 | | | |
| | | 4 | 14/17 | 82.4% | | | | |
| | | 5 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | | 6 | 15/15 | 100% | | | | |
| | 3 X LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 91/91
100.0% |
| | | 2 | 15/15 | 100% | | 30/30 | 100% | |
| | | 3 | 15/15 | 100% | 2 | | | |
| | | 4 | 15/15 | 100% | | | | |
| | | 5 | 16/16 | 100% | 5 | 31/31 | 100% | |
| | | 6 | 15/15 | 100% | | | | |

Table 14. Shiga toxin 2 Reproducibility Results

Table 15. E. coli Serotype 0157 Reproducibility Results

| Analyte | Conc. | Operator | Correct
Positive
Results | Agreement | Site | Correct
Positive
Results | Agreement | Total
Correct
Positive
Results |
|------------------------------|--------------|----------|--------------------------------|-----------|------|--------------------------------|-----------|-----------------------------------------|
| E. coli serotype O157 | 1.5 X
LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 90/92
97.8% |
| | | 2 | 15/15 | 100% | | | | |
| | | 3 | 15/15 | 100% | 2 | 30/32 | 93.8% | |
| | | 4 | 15/17 | 88.2% | | | | |
| | | 5 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | | 6 | 15/15 | 100% | | | | |
| | 3 X LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 91/91
100% |
| | | 2 | 15/15 | 100% | | | | |
| | | 3 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 4 | 15/15 | 100% | | | | |
| | | 5 | 16/16 | 100% | 5 | 31/31 | 100% | |
| | | 6 | 15/15 | 100% | | | | |

23

| Analyte | Conc. | Operator | Correct
Positive
Results | Agreement | Site | Correct
Positive
Results | Agreement | Total
Correct
Positive
Results |
|----------|--------------|----------|--------------------------------|-----------|------|--------------------------------|-----------|-----------------------------------------|
| Shigella | 1.5 X
LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 90/90
100.0% |
| | | 2 | 15/15 | 100% | 1 | 30/30 | 100% | |
| | | 3 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 4 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 5 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | | 6 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | 3 X LOD | 1 | 15/15 | 100% | 1 | 30/30 | 100% | 90/90
100.0% |
| | | 2 | 15/15 | 100% | 1 | 30/30 | 100% | |
| | | 3 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 4 | 15/15 | 100% | 2 | 30/30 | 100% | |
| | | 5 | 15/15 | 100% | 5 | 30/30 | 100% | |
| | | 6 | 15/15 | 100% | 5 | 30/30 | 100% | |

Table 16. Shigella Reproducibility Results

Analysis of Negative Results

To assess negative results a single negative stool sample (RP-05) was tested. All analytes in the SBPP should give negative "Not Detected" results for this sample except for E. coli 0157. Since E. coli 0157 is not evaluated when Shiga toxin 1 and or 2 are "Not Detected" the reported result for 0157 is "Not Tested". Therefore, the SBPP will generate 5 negative results per replicate tested. Each operator tested 15 replicates of RP-05 for a total of 75 negative results per operator. The percent agreement of the negative results is presented by operator, by site and in total, and is shown in Table 17.

			Table 17. Negative Sample Reproducibility Results
--	--	--	----------------------------------------------------	--

| Analyte | Conc. | Operator | Correct
Negative
Results | Agreement | Site | Correct
Negative
Results | Agreement | Total
Correct
Negative
Results |
|----------|-------|----------|--------------------------------|-----------|------|--------------------------------|-----------|-----------------------------------------|
| Negative | N/A | 1 | 75/75 | 100% | 1 | 150/150 | 100% | 450/450
100% |
| | | 2 | 75/75 | 100% | | | | |
| | | 3 | 75/75 | 100% | 2 | 150/150 | 100% | |
| | | 4 | 75/75 | 100% | | | | |
| | | 5 | 75/75 | 100% | 5 | 150/150 | 100% | |
| | | 6 | 75/75 | 100% | | | | |

The overall results of the Reproducibility Study are summarized in Table 18. There was ≥ 95% agreement of positive results for analytes that were present in low concentrations in the samples (1.5X LoD) and 100% agreement of positive results for analytes present at moderate concentration (3X LoD). The was 100% agreement of negative results from the negative sample.

24

Image /page/24/Picture/2 description: The image shows a series of black oval shapes arranged in a triangular pattern. The ovals are stacked on top of each other, with the base of the triangle consisting of two ovals, and the apex consisting of two smaller ovals. The ovals appear to be solid and have a smooth, uniform texture.

Table 18. Summary of Reproducibility Results

Analyte	Concentration	% Agreement
Campylobacter
coli/jejuni	1.5X LoD	100% (90/90)
Campylobacter
coli/jejuni	3X LoD	100% (90/90)
Salmonella	1.5X LoD	96.7% (87/90)
Salmonella	3X LoD	100% (90/90)
Shiga toxin 1	1.5X LoD	97.8% (90/92)
Shiga toxin 1	3X LoD	100% (91/91)
Shiga toxin 2	1.5X LoD	95.7% (88/92)
Shiga toxin 2	3X LoD	100% (91/91)
E. coli
Serotype O157	1.5X LoD	97.8% (90/92)
E. coli
Serotype O157	3X LoD	100% (91/91)
Shigella	1.5X LoD	100% (90/90)
Shigella	3X LoD	100% (90/90)
Negative	N/A	100% (450/450)

Conclusion: The study demonstrated acceptable reproducibility of the SBPP.

Specimen Stability and Storage i.

A sample stability study was conducted to determine the allowable storage conditions (time and temperature) for clinical specimens. The study included the following organisms prepared at a concentration of 2X LoD: C. coli (ATCC 43486), C. jejuni (ATCC 49943), E. coli (BAA-2196), E. coli (ATCC 43895), S. bongori (ATCC 43975), S. enterica (ATCC 13311), and S. sonnei (ATCC 29930), at 2X LoD.

Each sample was tested in triplicate after storage at the times and temperatures shown in Table 19.

Table 19. Specimen Stability Study Time/Temperature Storage Conditions

Time Point	Time tested and Storage Condition
T0	0 hr: Freshly prepared
T1	2 hr: Room temperature storage (20°- 25°C)
T2	24 hr: 2-8°C storage
T3	48 hr: 2-8°C storage
T4	72 hr: 2-8°C storage
T5	96 hr: 2-8°C storage
T6	120 hr: 2-8°C storage
T7	2 hr Room temperature (20°- 25°C) + 120 hr 2-8°C storage
T8	2 hr Room temperature (20°- 25°C) + 144 hr 2-8°C storage

The results demonstrated 100% agreement with the expected results supporting the specimen storage claims in the Product Insert.

25

Image /page/25/Picture/2 description: The image shows a pattern of black ovals arranged in a step-like formation. The ovals are oriented horizontally and decrease in size as they ascend. There are two ovals at each level, creating a symmetrical appearance.

G. Performance Summary - Clinical Studies

The clinical evaluation of the SBPP consisted of both a Prospective Sample and a Frozen Retrospective Sample Study. An additional study using selected fresh positive Salmonella samples was also performed (Selected Sample Study).

A prospective method comparison study was conducted to compare the performance of the SBPP to standard stool culture-based methods for identification of Campylobacter jejuni /Campylobacter coli, Escherichia coli serotype O157 Salmonella spp., Shiga toxin 1 Shiga toxin 2, and Shigella spp. The study was conducted at four external, geographically-diverse U.S. clinical study sites (Midwest, Northeast, Southwest and West) from July, 2016 through November, 2016. The specimens enrolled in the study were excess remnants of preserved stool samples collected from symptomatic individuals suspected of gastrointestinal infection that were processed according to routine standard care. A total of 1506 samples were collected for all four sites combined. Subsequent to enrollment, 24 samples were excluded from the data set leaving 1479 samples included in the analysis.

In addition, frozen archived de-identified specimens that were previously characterized as positive or negative by the standard of care method used at the institution (historical result) were obtained. The historical result for each sample was first confirmed by an FDA cleared Nucleic Acid Amplification Test (NAAT) prior to enrolling the sample in the study. A total of 150 frozen samples were included in the panel. The SBPP results were compared to the historical result.

To further increase the number of positive Salmonella samples evaluated, additional fresh samples selected as positive by the standard of care method used by the clinical study site were collected and tested. Intermountain Healthcare (IMC) in Salt Lake City, UT was also added as a sample collection site and testing samples from IMC was performed internally at Great Basin Scientific.

The positive (PPA) and negative (NPA) percent agreement for each of the three studies (Prospective Study; All sites Combined, Frozen Retrospective Sample study and Selected Sample Study) along with the 95% Confidence Intervals are shown in Table 20.

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Image /page/26/Picture/2 description: The image shows a series of black oval shapes arranged in a pyramid-like formation. The ovals vary in size, with the largest ones at the bottom and the smallest ones at the top. The ovals are aligned in rows, creating a sense of order and structure within the composition. The background is plain white.

	Table 20. Summary of Clinical Study Results
--	---------------------------------------------

Specimen	n	% Agreement (95% CI)
		Positive	Negative
Campylobacter	Fresh	1479	96.4%
(82.3-99.4)
27/28	99.2%
(98.6-99.5)
1439/1451
	Frozen	0	N/A	N/A
Salmonella	Fresh	1479	83.3%
(55.2-95.3)
10/12	99.6%
(99.1-99.8)
1461/1467
	Fresh
Selected	28	92.9%
(77.4-98.0)
26/28	N/A
	Frozen	206	94.4%
(81.9-98.5)
34/36	100.0%
(97.8-100.0)
170/170
Shiga Toxin 1	Fresh	1479	100.0%
(20.7-100.0)
1/1	99.5%
(99.0-99.8)
1471/1478
	Frozen	206	100.0%
(88.3-100.0)
29/29	100.0%
(97.9-100.0)
177/177
Shiga Toxin 2	Fresh	1479	100.0%
(20.7-100.0)
1/1	99.4%
(98.8-99.7)
1469/1478)
	Frozen	206	100.0%
(89.0-100.0)
31/31	100.0%
(97.9-100.0)
175/175
E. coli
Serotype
0157	Fresh	16	100%
(51.0-100.0)
4/4	75.0%
(46.8-91.1)
9/12
	Frozen	48	100.0%
(81.6-100.0)
17/17	100.0%
(89.0-100.0)
31/31
Shigella	Fresh	1479	100%
(56.6-100.0)
5/5	99.1%
(98.4-99.4)
1460/1474
	Frozen	206	94.7%
(75.4-99.1)
18/19	100.0%
(98.0-100.0)
187/187

As shown in Table 20, in the SBPP Prospective Study the point estimate achieved for PPA was ≥ 95% (96.4% -100%) for all analytes, except for Salmonella which was 83.3%. Due to the low number of co-positive samples, the lower bound of the 95% CI was