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.

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.

AI/ML Overview

This detailed document outlines the performance characteristics of the Great Basin Stool Bacterial Pathogens Panel (SBPP). While it does not include AI-specific performance criteria, it provides a comprehensive overview of the device's analytical and clinical validation, which are analogous to acceptance criteria and study data for traditional medical devices. I will extract the relevant information and present it in the requested format, interpreting "acceptance criteria" as the performance benchmarks demonstrated in the studies and "device performance" as the results achieved.

For AI-specific questions (Adjudication method, MRMC, Standalone performance, Training set details), the document does not contain this information as it pertains to a nucleic acid-based assay and not an AI/ML-driven device. I will explicitly state "Not applicable" for these points.

Acceptance Criteria and Device Performance for Great Basin Stool Bacterial Pathogens Panel

1. Table of Acceptance Criteria and the Reported Device Performance

Since this is a diagnostic assay and not an AI-driven image analysis tool, the acceptance criteria are based on analytical and clinical performance metrics. The document describes several studies (Analytical Sensitivity, Analytical Reactivity, Analytical Specificity, Competitive Inhibition, Interfering Substances, Microbial Interference, Carry-over/Cross Contamination, Reproducibility, Specimen Stability, and Clinical Studies).

For simplicity and relevance to a typical "acceptance criteria" table for a diagnostic device, I will focus on the key performance indicators from the reproducibility and clinical studies. The implicit "acceptance criteria" are the demonstrated performance percentages, often with 95% confidence intervals where available.

Performance Metric	Acceptance Criteria (Implicit from Study Results)	Reported Device Performance
Reproducibility (1.5X LoD)	≥ 95% Agreement (Target)	Campylobacter: 100% (90/90) Salmonella: 96.7% (87/90) Shiga toxin 1: 97.8% (90/92) Shiga toxin 2: 95.7% (88/92) E. coli O157: 97.8% (90/92) Shigella: 100% (90/90)
Reproducibility (3X LoD)	100% Agreement (Target)	Campylobacter: 100% (90/90) Salmonella: 100% (90/90) Shiga toxin 1: 100% (91/91) Shiga toxin 2: 100% (91/91) E. coli O157: 100% (91/91) Shigella: 100% (90/90)
Reproducibility (Negative)	100% Agreement (Target)	100% (450/450)
Clinical Performance (Positive Percent Agreement - PPA) - Prospective Study (Fresh Samples)	Point estimate ≥ 80%, lower bound of 95% CI ≥ 70% (General expectation for such assays, not explicitly stated as "acceptance criteria" but implied by regulatory review)	Campylobacter: 96.4% (82.3-99.4) Salmonella: 83.3% (55.2-95.3) Shiga toxin 1: 100.0% (20.7-100.0) Shiga toxin 2: 100.0% (20.7-100.0) E. coli O157: 100% (51.0-100.0) Shigella: 100% (56.6-100.0)
Clinical Performance (Negative Percent Agreement - NPA) - Prospective Study (Fresh Samples)	Point estimate ≥ 95%, lower bound of 95% CI ≥ 90% (General expectation)	Campylobacter: 99.2% (98.6-99.5) Salmonella: 99.6% (99.1-99.8) Shiga toxin 1: 99.5% (99.0-99.8) Shiga toxin 2: 99.4% (98.8-99.7) E. coli O157: 75.0% (46.8-91.1) Shigella: 99.1% (98.4-99.4)
Clinical Performance (PPA) - Frozen Retrospective Study	Point estimate ≥ 90%, lower bound of 95% CI ≥ 80% (General expectation)	Salmonella: 94.4% (81.9-98.5) Shiga toxin 1: 100.0% (88.3-100.0) Shiga toxin 2: 100.0% (89.0-100.0) E. coli O157: 100.0% (81.6-100.0) Shigella: 94.7% (75.4-99.1)
Clinical Performance (NPA) - Frozen Retrospective Study	Point estimate ≥ 95%, lower bound of 95% CI ≥ 90% (General expectation)	Salmonella: 100.0% (97.8-100.0) Shiga toxin 1: 100.0% (97.9-100.0) Shiga toxin 2: 100.0% (97.9-100.0) E. coli O157: 100.0% (89.0-100.0) Shigella: 100.0% (98.0-100.0)
Clinical Performance (PPA) - Selected Fresh Salmonella Study	Point estimate ≥ 90%, lower bound of 95% CI ≥ 80% (General expectation)	Salmonella: 92.9% (77.4-98.0)

2. Sample sizes used for the test set and the data provenance:

Clinical Test Sets:
- Prospective Study (Fresh Samples): 1479 samples included in the analysis (1506 collected, 24 excluded). Collected from four external, geographically-diverse U.S. clinical study sites (Midwest, Northeast, Southwest and West) from July 2016 to November 2016. These were excess remnants of preserved stool samples. Prospective data.
- Frozen Retrospective Sample Study: 150 frozen archived de-identified specimens initially (for the general panel), with specific numbers for each analyte:
  - Salmonella: 206 samples
  - Shiga Toxin 1: 206 samples
  - Shiga Toxin 2: 206 samples
  - E. coli O157: 48 samples
  - Shigella: 206 samples
    These were de-identified specimens previously characterized (historical result). Retrospective data.
- Selected Fresh Positive Salmonella Samples Study: 28 additional fresh samples. Collected from Intermountain Healthcare (IMC) in Salt Lake City, UT. Prospective data.
Analytical Test Sets:
- Analytical Sensitivity (LoD): 10 bacterial strains, serially diluted.
- Analytical Reactivity (Inclusivity): 91 well-characterized bacterial strains, multiple replicates per strain (at least 3).
- Analytical Specificity (Exclusivity): 100 non-target organisms (84 bacterial, 3 yeast, 3 parasites, 9 viruses) and human genomic DNA, multiple replicates per organism (minimum of 3).
- Competitive Inhibition: 48 unique combinations of pathogens, each tested in triplicate.
- Interfering Substances: 19 different substances, tested with 8 target organisms, minimum of 3 replicates per substance/organism combination.
- Microbial Interference: 29 non-target organisms tested in presence of 8 target analytes, minimum of 3 replicates.
- Carry-over/Cross Contamination: 40 high positive samples and 40 negative samples (total 80 tests).
- Reproducibility: 7 different samples tested in triplicate over 5 non-consecutive days by 6 operators, across 3 sites (90-92 replicates per positive analyte, 450 replicates for negative).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

The document describes the ground truth for clinical samples as "standard stool culture-based methods" and "standard of care method used at the institution (historical result)," further confirmed by "FDA cleared Nucleic Acid Amplification Test (NAAT)" for retrospective samples. The experts are implicitly the microbiologists and clinical laboratory personnel at the clinical sites performing these standard methods. No specific number or explicit qualifications (e.g., years of experience) for these "experts" are provided in the document, which is typical for diagnostic assay submissions relying on established clinical laboratory practices.

4. Adjudication method (e.g., 2+1, 3+1, none) for the test set:

None described. The ground truth for the clinical studies relied on standard microbiological culture methods and FDA-cleared NAATs as reference methods. Discrepant results were investigated by further testing with other FDA-cleared NAATs (BioFire Film Array GI Panel or Nanosphere Verigene® EP test). This is a discrepant analysis approach, not an adjudication process by human experts re-interpreting initial data.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

Not applicable. This device is a molecular diagnostic assay, not an AI-driven imaging or diagnostic tool intended for human-in-the-loop assistance.

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

Yes, the device operates as a standalone automated system. The entire analytical and clinical performance evaluation described in the document represents the standalone performance of the SBPP, as it integrates sample preparation, PCR, and detection with automated software interpreting results. It is an "algorithm only" in the sense of a laboratory assay's automated result interpretation, although not a machine learning algorithm.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc):

The ground truth for the clinical studies was established primarily by:

Standard stool culture-based methods (for prospective samples).
Historical results from clinical sites using their standard of care methods, then confirmed by FDA cleared Nucleic Acid Amplification Tests (NAATs) (for frozen retrospective samples).
Discrepant analysis was performed using other FDA-cleared NAATs (BioFire Film Array GI Panel or Nanosphere Verigene® EP test) for unconcordant results.

Thus, the ground truth is a combination of established microbiological standards and validated molecular diagnostic tests.

8. The sample size for the training set:

Not applicable. This is a nucleic acid-based diagnostic device, not an AI/ML device that requires a "training set" in the conventional sense. The development of the assay (primer/probe design, assay conditions) would be based on scientific knowledge and wet-lab experiments, not a machine learning training data set.

9. How the ground truth for the training set was established:

Not applicable. See point 8. The "ground truth" for developing the assay would be pure cultures of target and non-target organisms, along with their known biochemical and genetic characteristics, established through standard microbiology laboratory practices.

Summary

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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.

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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,

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

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Indications for Use

510(k) Number (if known) K163571

Device Name Great Basin Stool Bacterial Pathogens Panel

Indications for Use (Describe)

· 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.

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 NucleicAcid-Based Assay21 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) MicrobiologyPCI Gastrointestinal Bacterial Panel Multiplex Nucleic Acid-Based Assay SystemPCH Gastrointestinal Pathogen Panel Multiplex NucleicAcid-Based Assay SystemOOI Real-Time Nucleic Amplification System

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

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

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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:

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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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 DeviceVerigene® EP (K140083)
Manufacturer	Great Basin Scientific, Inc.	Nanosphere
Trade Name	Great Basin Stool Bacterial PathogenPanel	Verigene Enteric Pathogen NucleicAcid Test
510(k) Number	K140083	K140083
Classification	II	II
Qualitative/Quantitative	Qualitative	Qualitative
Intended Use/Indications forUse	The Great Basin Stool BacterialPathogens Panel is a multiplexed,qualitative test for the detection andidentification of DNA targets of entericbacterial pathogens. The StoolBacterial Pathogens Panel detectsnucleic acids from:• Campylobacter ( C. coli and C. jejuni )• Salmonella• Shiga toxin 1 ( stx1 )• Shiga toxin 2 ( stx2 )• Escherichia coli serotype O157• ShigellaShiga toxin genes are found in Shigatoxin-producing strains of E. coli(STEC/EHEC/VTEC) and Shigelladysenteriae . The E. coli O157 testresult is only reported if a Shiga toxingene ( stx1 and/or stx2 ) is alsodetected.The Stool Bacterial Pathogens Panel isperformed directly from Cary Blair orC&S Medium preserved stoolspecimens from symptomatic patientswith suspected acute gastroenteritis,enteritis, or colitis and is performed onthe PortraitTM Analyzer.The test is intended for use as an aid inthe diagnosis of specific agents ofgastrointestinal illness in conjunctionwith clinical and epidemiologicalinformation. Positive results do not ruleout co-infection with other organismsand may not be the definitive cause ofpatient illness. Negative test results inthe setting of clinical illness compatiblewith gastroenteritis may be due toinfection by pathogens that are notdetected by this test, or non-infectiouscauses such as ulcerative colitis,irritable bowel syndrome, or Crohn'sdisease. Concomitant culture isnecessary if organism recovery orfurther typing of bacterial agents isdesired.	The Verigene® Enteric PathogensNucleic Acid Test (EP) is a multiplexed,qualitative test for simultaneousdetection and identification of commonpathogenic enteric bacteria, viruses,and genetic virulence markers fromliquid or soft stool preserved in Cary-Blair medium, collected from individualswith signs and symptoms ofgastrointestinal infection. The test isperformed on the automatedNanosphere Verigene System utilizingreverse transcription (RT), polymerasechain reaction (PCR), and arrayhybridization to detect specificgastrointestinal microbial nucleic acidgene sequences associated with thefollowing pathogenic bacteria andviruses:• Campylobacter Group (composed ofC. coli, C. jejuni , and C. lari )• Salmonella species• Shigella species (including S.dysenteriae, S. boydii, S. sonnei , andS. flexneri )• Vibrio Group (composed of V.cholerae and V. parahaemolyticus )• Yersinia enterocolitica• Norovirus GI/GII• Rotavirus AIn addition, EP detects the Shiga toxin1 gene and Shiga toxin 2 genevirulence markers. Shiga toxinproducing E. coli (STEC) typicallyharbor one or both genes that encodefor Shiga toxins 1 and 2.EP is indicated as an aid in thediagnosis of specific agents ofgastrointestinal illness, in conjunctionwith other clinical, laboratory, andepidemiological information; however,is not to be used to monitor theseinfections. EP also aids in the detectionand identification of acute

Specimen Type	Human Stool sample preserved in Cary Blair or C&S Preservation and Transport Media	Human Stool sample preserved in Cary-Blair Medium
Sample Lysis and DNA Extraction	Automated sample lysis and DNA extraction in a self-contained cartridge	Same
Amplification Technology	Multiplex polymerase chain reaction (PCR)	Reserve transcription (RT) polymerase change reaction (PCR)
Detection Technology	Colorimetric target specific hybridization to probe on a chip surface, optical reader, automated software with built-in result interpretation.	Gold/Silver nanoparticle probe detection of bacterial-specific DNA on complementary oligo-microarray. Optical light scatter detection of gold-silver aggregates
Controls	One internal processing control (whole organism) - complete assay control	Two internal processing controls (hybridization control and extraction/assay control)
Instrument	PA500 Portrait™ Analyzer	Verigene Reader and Processor SP
Time to Result	<2 hours	~2 hours

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The following summarizes the differences and similarities between the Stool Bacterial Pathogens Pane and the predicate device:

The Stool Bacterial Pathogens Panel (SBPP) is similar to the Verigene® Enteric Pathogens (EP) Nucleic Acid Test in the following ways:

. The SBPP and the EP have similar Intended Uses and both detect the following analytes: Campylobacter coli, jejuni, Salmonella spp., Shiga toxin 1 and Shiga toxin 2
. The SBPP and the EP use the same sample type (preserved human stool)
The SBPP and the EP are both qualitative, automated, multiplex nucleic acid based tests .

The Stool Bacterial Pathogens Panel differs from the Verigene® Enteric Pathogens Nucleic Acid Test in the following:

. The EP detects additional analytes not detected in the SBPP (Campylobater lari, Vibrio Group, Yersinia enterocolitica, Norovirus GI/GII, Rotovirus A)
The SBPP detects E. coli Serotype O157 whereas EP does not .

F. Performance Summary - Analytical Studies

a. Analytical Sensitivity

The limit of detection (LoD) of the SBPP was assessed by testing 10 bacterial strains which included all the target organisms detected in the SBPP. Preparation of E. coli, Salmonella and Shigella included overnight incubations of each bacterial stock in Tryptic Soy Broth. The broths were serially diluted into a pool of preserved negative clinical stool and plated onto Tryptic Soy Agar plates to determine the cell concentrations.

Preparation of Campylobacter spp. included incubation in Campy-Thioglycollate Broth (using cells obtained from a Campy CVA agar plate). The broths were serially diluted into a pool of preserved neqative clinical stool and plated onto blood agar plates to determine the cell concentrations.

The LoDs for each strain tested is shown in Table 1.

Strain	ATCC ID	SBPP TargetGene Present	LoD(CFU/mL)
Campylobacter coli	43486	cadF	$1.8 x 10^3$
Campylobacter jejuni	49943	cadF	$1.3 x 10^3$
Escherichia coli	BAA-2215	stx1	$5.7 x 10^3$
Escherichia coli	51435	stx2	$4.4 x 10^3$
Escherichia coli	BAA-2196	stx1, stx2	$1.1 x 10^4$
Escherichia coli	43895	stx1, stx2, O157	$1.6 x 10^4$
Salmonella bongori	43975	invA	$2.5 x 10^3$
Salmonella enterica	13311	invA	$1.9 x 10^4$
Shigella flexneri	25929	ipaH	$5.2 x 10^3$
Shigella sonnei	29930	ipaH	$1.4 x 10^4$

Table 1. SBPP Limit of Detection (LoD)

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Image /page/8/Picture/2 description: The image shows a pattern of black oval shapes arranged in a triangular formation. The ovals are oriented horizontally and vary in size, with the largest ovals at the bottom and the smallest at the top. The arrangement creates a sense of depth and perspective, as if the ovals are receding into the distance. There are 9 ovals in total.

b. Analytical Reactivity (Inclusivity)

The analytical reactivity of the SBPP was assessed by testing an additional 91 well characterized bacterial strains representing the organisms detected in the SBPP. The strains were obtained from the ATCC and contained the following:

5 Campylobacter coli, ●
6 Campylobacter jejuni, ●
16 Escherichia coli (stx1+ and/or stx2+) ●
8 Escherichia coli (stx1+ and/or stx2+, with serotype 0157+) ●
3 Shigella dysenteriae serotype 1 (stx1+) strains
. 33 Salmonella spp.
20 Shigella spp. ●

Organisms were grown as described in the LoD studies and were diluted to a concentration of 2X LoD into pooled negative preserved clinical stool matrix. At least 3 replicates per strain were tested. The results for are shown in Tables 2 through 5.

Table 2. Analytical Reactivity Campylobacter spp.

Campylobacter spp.	ATCC ID	Concentration2X LoD(CFU/mL)	CorrectSBPPResults
Campylobacter coli	33559	$3.6 x 10^3$	9/9
Campylobacter coli	43135	$3.6 x 10^3$	3/3
Campylobacter coli	43486	$3.6 x 10^3$	6/6
Campylobacter coli	49941	$3.6 x 10^3$	3/3
Campylobacter coli	51729	$3.6 x 10^3$	3/3
Campylobacter jejuni subsp. jejuni	29428	$2.6 x 10^3$	3/3
Campylobacter jejuni subsp. jejuni	33560	$2.6 x 10^3$	3/3
Campylobacter jejuni subsp. jejuni	43434	$2.6 x 10^3$	3/3
Campylobacter jejuni subsp. doylei	49349	$2.6 x 10^3$	6/6
Campylobacter jejuni	49943	$2.6 x 10^3$	3/3
Campylobacter jejuni subsp. jejuni	33292	$2.6 x 10^3$	3/3
n=11

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Image /page/9/Picture/2 description: The image shows a pattern of black ovals arranged in a triangular shape. The ovals are of varying sizes, with the largest ones at the bottom and the smallest ones at the top. The ovals are arranged in rows, with each row containing fewer ovals than the row below it. The overall effect is one of a pyramid or a stack of objects.

Table 3. Analytical Reactivity Salmonella spp.

Salmonella spp.	ATCC ID	Concentration2X LoD(CFU/mL)	CorrectSBPPResults
Salmonella enterica subsp. enterica Typhi	6539	3.8 x 104	3/3
Salmonella enterica subsp. enterica Newport	6962	3.8 x 104	3/3
Salmonella enterica subsp. enterica Choleraesuis	7001	3.8 x 104	3/3
Salmonella enterica subsp. enterica Stanley	7308	3.8 x 104	3/3
Salmonella enterica subsp. enterica Heidelberg	8326	3.8 x 104	6/6
Salmonella enterica subsp. enterica Muenchen	8388	3.8 x 104	6/6
Salmonella enterica subsp. enterica Paratyphi B	8759	3.8 x 104	6/6
Salmonella enterica subsp. enterica Bareilly	9115	3.8 x 104	6/6*
Salmonella enterica subsp. enterica Kentucky	9263	3.8 x 104	3/3
Salmonella enterica subsp. enterica Saint Paul	9712	3.8 x 104	3/3*
Salmonella enterica subsp. enterica Tennessee	10722	3.8 x 104	6/6
Salmonella enterica subsp. enterica Paratyphi A	12176	3.8 x 104	3/3
Salmonella enterica subsp. enterica Typhimurium	13311	3.8 x 104	3/3
Salmonella enterica subsp. enterica Choleraesuis	13312	3.8 x 104	6/6*
Salmonella enterica subsp. enterica arizonae	13314	3.8 x 104	3/3
Salmonella enterica subsp. enterica Typhimurium	14028	3.8 x 104	3/3
Salmonella enterica subsp. enterica Dublin	15480	3.8 x 104	3/3
Salmonella enterica subsp. enterica houtenae	15788	3.8 x 104	3/3
Salmonella enterica subsp. enterica Newport	27869	3.8 x 104	3/3
Salmonella enterica subsp. enterica diarizonae	29226	3.8 x 104	6/6
Salmonella enterica subsp. enterica Newington	29628	3.8 x 104	3/3
Salmonella enterica subsp. salamae	43972	3.8 x 104	6/6*
Salmonella enterica subsp. diarizonae	43973	3.8 x 104	3/3
Salmonella enterica subsp. houtenae	43974	3.8 x 104	6/6
Salmonella bongori	43975	3.8 x 104	3/3
Salmonella enterica subsp. indica	43976	3.8 x 104	3/3
Salmonella enterica subsp. enterica Virchow	51955	3.8 x 104	5/6
Salmonella enterica subsp. enterica Agona	51957	3.8 x 104	3/3
Salmonella enterica subsp. enterica Bristol	700138	3.8 x 104	3/3
Salmonella enterica subsp. enterica Montevideo	BAA-710	3.8 x 104	3/3
Salmonella enterica subsp. salamae	BAA-1576	3.8 x 104	3/3
Salmonella enterica subsp. enterica Infantis	BAA-1675	3.8 x 104	3/3
Salmonella enterica subsp. enterica Mississippi	BAA-2739	3.8 x 104	3/3
n=33
* This set of test runs also contained 1 "Invalid" Run

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Image /page/10/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 has two ovals, the next row has three ovals, and the top row has two ovals. The ovals are all the same size and shape, and they are all oriented in the same direction.

ATCC ID	Serotype	Shiga ToxinGene(s)Present	Expected Result	Concentration2X LoD(CFU/mL)	CorrectSBPPResults
Shiga-toxin producing E. coli
700840	0111:H8	stx1+/stx2+		2.2 x 104	3/3
BAA-2196	O26:H11	stx1+/stx2+	Shiga Toxin 1 DETECTED	2.2 x 104	3/3*
BAA-2221	O21:H19	stx1+/stx2+	Shiga Toxin 2 DETECTED	2.2 x 104	3/3
BAA-2440	O111	stx1+/stx2+		2.2 x 104	3/3
BAA-2181	O26:H11	stx1+		1.4 x 104	3/3
BAA-2191	O45:H2	stx1+		1.4 x 104	3/3
BAA-2193	O45:H2	stx1+		1.4 x 104	3/3
BAA-2199	O123:H25	stx1+	Shiga Toxin 1 DETECTED	1.4 x 104	3/3
BAA-2210	O103:H2	stx1+		1.4 x 104	3/3
BAA-2215	O103:H11	stx1+		1.4 x 104	3/3
51435	O91:H21	stx2+		8.8 x 103	3/3
BAA-183	O113:H21	stx2+		8.8 x 103	3/3
BAA-2129	O145:H28	stx2+		8.8 x 103	3/3
BAA-2211	O145:H25	stx2+	Shiga Toxin 2 DETECTED	8.8 x 103	3/3
BAA-2219	O121:H19	stx2+		8.8 x 103	3/3
BAA-2326	O104:H4	stx2+		8.8 x 103	5/6
35150	O157:H7	stx1+/stx2+		3.2 x 104	3/3
43894	O157:H7	stx1+/stx2+	Shiga Toxin 1 DETECTED	3.2 x 104	3/3
700378	O157:NM	stx1+/stx2+	Shiga Toxin 2 DETECTEDSerotype O157 DETECTED	3.2 x 104	5/5*
700927	O157:H7:K	stx1+/stx2+		3.2 x 104	3/3
43890	O157:H7	stx1+	Shiga Toxin 1 DETECTED	3.2 x 104	3/3
700376	O157:NM	stx1+	Serotype O157 DETECTED	3.2 x 104	8/9
43889	O157:H7	stx2+	Shiga Toxin 2 DETECTED	3.2 x 104	3/3
700377	O157:NM	stx2+	Serotype O157 DETECTED	3.2 x 104	3/3
			n=24

Table 4. Analytical Reactivity: Shiga toxin 1, Shiga toxin 2 and E. coli Serotype O157

43889	O157:H7	stx2+	Shiga Toxin 2 DETECTED	$3.2 x 10^4$	3/3
700377	O157:NM	stx2+	Serotype O157 DETECTED	$3.2 x 10^4$	3/3
n=24
Shiga-toxin producing Shigella dysenteriae
9361	Type 1	stx1+		$1.4 x 10^4$	3/3
27345	Type 1	stx1+	Shiga Toxin 1 DETECTED	$1.4 x 10^4$	6/7
27346	Type 1	stx1+		$1.4 x 10^4$	4/6
n=3
* This set of test runs also contained 1 "Invalid" Run

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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	Concentration2X LoD(CFU/mL)	CorrectSBPPResults
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.

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

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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(ATCC43486)	C. jejuni(ATCC49943)	E. coli(stx1+/stx2+/non-O157)(ATCC BAA-2196)	E. coli(stx1+/stx2+/O157+)(ATCC 43895)	S. bongori(ATCC43975)	S. enterica(ATCC13311)	S. flexneri(ATCC25929)	S. sonnei(ATCC29930)
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	14/19c6/6d	--	--	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. Anadditional 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 2/3replicates, although Campylobacter was correctly identified in all cases. An additional 6 replicates were tested, and the expectedresult 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.

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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.

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Table 8. Interfering Substances Study Results

Potentially Interfering Substances andInput Concentration		CampylobactercoliATCC 43486	CampylobacterjejuniATCC 49943	Escherichia coli(stx1+/stx2+O157+)ATCC 43895	Escherichia coli(stx1+/stx2+non-O157)ATCC 2196	SalmonellabongoriATCC 43975	SalmonellaentericaATCC 13311	ShigellaflexneriATCC 25929	ShigellasonneiATCC 29930	NegativeStoolSamplesAssayControlDETECTED
		≤3X LoD(3.6 x 103CFU/mL)	≤ 3X LoD(2.6 x 103CFU/mL)	≤ 3X LoD(3.2 x 104CFU/mL)	≤ 3X LoD(2 x 104CFU/mL)	≤ 3X LoD(7.5 x 103CFU/mL)	≤ 3X LoD(3.8x 104CFU/mL)	≤ 3X LoD(1.6 x 104CFU/mL)	≤ 3X LoD(2.8 x 104CFU/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.

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VD

Table 9. Microbial Interference Study Results

Species	Strain ID	Input Tested	C. coliATCC 43486	C. jejuniATCC 49943	E. coli(stx1+/stx2+non-0157)ATCC 2196	E. coli(stx1+/stx2+0157+)ATCC 43895	SalmonellabongoriATCC 43975	SalmonellaentericaATCC 13311	ShigellaflexneriATCC 25929	ShigellasonneiATCC 29930
			≤ 3X LoD(3.6 x 103CFU/mL)	≤3X LoD(2.6 x 103CFU/mL)	≤3X LoD(2 x 104CFU/mL)	≤ 3X LoD(3.2 x 104CFU/mL)	≤ 3X LoD(7.5 x 103CFU/mL)	≤ 3X LoD(3.8 x 104CFU/mL)	≤ 3X LoD(1.6 x 104CFU/mL)	≤ 3X LoD(2.8 x 104CFU/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 (AlternatingPositive/Negative)		PortraitAnalyzer5.315	Portrait Analyzer5.106	PortraitAnalyzer5.382	PortraitAnalyzer5.072
High Positive	Runs 1 - 4	C. coli/jejuniDETECTED	Shiga Toxin 1 DETECTEDShiga Toxin 2 DETECTEDSerotype O157 DETECTED	SalmonellaDETECTED	ShigellaDETECTED
Negative	Runs 5 - 8	Negative	Negative	Negative	Negative
High Positive	Runs 9 - 12	C. coli/jejuniDETECTED	Shiga Toxin 1 DETECTEDShiga Toxin 2 DETECTEDSerotype O157 DETECTED	SalmonellaDETECTED	ShigellaDETECTED
Negative	Runs 13 - 16	Negative	Negative	Negative	Negative
High Positive	Runs 17 - 20	C. coli/jejuniDETECTED	Shiga Toxin 1 DETECTEDShiga Toxin 2 DETECTEDSerotype O157 DETECTED	SalmonellaDETECTED	ShigellaDETECTED
Negative	Runs 21 - 24	Negative	Shiga Toxin 1 DETECTED	Negative	Negative
High Positive	Runs 25 - 28	C. coli/jejuniDETECTED	Shiga Toxin 1 DETECTEDShiga Toxin 2 DETECTEDSerotype O157 DETECTED	SalmonellaDETECTED	ShigellaDETECTED
Negative	Runs 29 - 32	Negative	Negative	Negative	Negative
High Positive	Runs 33 - 36	C. coli/jejuniDETECTED	Shiga Toxin 1 DETECTEDShiga Toxin 2 DETECTEDSerotype O157 DETECTED	SalmonellaDETECTED	ShigellaDETECTED
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	CorrectPositiveResults	Agreement	Site	CorrectPositiveResults	Agreement	TotalCorrectPositiveResults
Campylobacter spp.	1.5 XLOD	1	15/15	100%	1	30/30	100%	90/90100%
		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%	90/90100%
		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

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Analyte	Conc.	Operator	CorrectPositiveResults	Agreement	Site	CorrectPositiveResults	Agreement	TotalCorrectPositiveResults
Salmonella	1.5 X LOD	1	15/15	100%	1	30/30	100%	87/9096.7%
		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%	90/90100.0%
		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	CorrectPositiveResults	Agreement	Site	CorrectPositiveResults	Agreement	TotalCorrectPositiveResults
Shia toxin 1	1.5 XLOD	1	15/15	100%	1	30/30	100%	90/9297.8%
		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%	91/91100.0%
		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	CorrectPositiveResults	Agreement	Site	CorrectPositiveResults	Agreement	TotalCorrectPositiveResults
Shiga toxin 2	1.5 XLOD	1	14/15	93.3%	1	29/30	96.7%	88/9295.7%
		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%	91/91100.0%
		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	CorrectPositiveResults	Agreement	Site	CorrectPositiveResults	Agreement	TotalCorrectPositiveResults
E. coli serotype O157	1.5 XLOD	1	15/15	100%	1	30/30	100%	90/9297.8%
		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%	91/91100%
		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%

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Analyte	Conc.	Operator	CorrectPositiveResults	Agreement	Site	CorrectPositiveResults	Agreement	TotalCorrectPositiveResults
Shigella	1.5 XLOD	1	15/15	100%	1	30/30	100%	90/90100.0%
		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%	90/90100.0%
		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	CorrectNegativeResults	Agreement	Site	CorrectNegativeResults	Agreement	TotalCorrectNegativeResults
Negative	N/A	1	75/75	100%	1	150/150	100%	450/450100%
		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.

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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
Campylobactercoli/jejuni	1.5X LoD	100% (90/90)
Campylobactercoli/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. coliSerotype O157	1.5X LoD	97.8% (90/92)
E. coliSerotype 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.

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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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	Table 20. Summary of Clinical Study Results
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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
	FreshSelected	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. coliSerotype0157	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 < 80% for all analytes, except for Campylobacter which was 82.3%. The point estimate for NPA was still ≥ 95% (99.1% - 99.6%) for all analytes except E. coli O157 which was 75.0%.

Samples with discrepant results for the Campylobacter, Salmonella, Shigella and E. coli Serotype O157 analytes were investigated by further testing in the BioFire Film Array Gl Panel (K140407). Samples with discrepant results for Shiga toxin 1 and/or 2 were investigated by further testing in the Nanosphere Verigene® EP test (K140083). The results are shown in Table 21.

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Analyte	False NegativesResolved byNAAT ReferenceMethod	False PositivesResolved byNAAT ReferenceMethod
Campylobacter	1/1	10/12
Salmonella	0/2	6/6
stx1	N/A	6/7a
stx2	N/A	8/9b
E. coli O157	N/A	3/3
Shigella	N/A	14/14

			Table 21. Summary Table - Prospective Study Discrepant Results Resolution
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a The one false positive not concordant with Verigene® EP (negative for stx1 in the Verigene® EP) was positive for stx1/2 in the BioFire GI Panel.

b The one false positive not concordant with Verigene® EP (negative for stx2 in the Verigene® EP) was positive for stx1/2 in the BioFire GI Panel.

As shown in Table 21, the majority of the false positive results were found to be concordant (resolved) with the FDA cleared NAAT that was used in the investigation of the discrepant results (BioFire GI Panel or Verigene® EP).

The results from the Frozen Retrospective Study which utilized frozen archived samples further support the acceptable performance of the SBPP. As shown in Table 20, in the SBPP Frozen Retrospective Study the point estimate achieved for PPA was ≥ 90% (94.4% - 100.0%) for all analytes detected in the SBPP. The lower bound 95% Cl for the PPA was ≥ 80% for all analytes except Shigella which was 75.4%. The point estimate for NPA was 100.0% for all analytes evaluated. The lower bound of the 95% Cl was ≥ 95% for all analytes except for E. coli O157 which was 89.0% (due to the low sample size of the negatives). In the Frozen Retrospective study, there was one (1) false negative result for Shigella. This sample was further investigated and was also negative in the Verigene® EP test. There were two (2) false negative results for Salmonella which were further investigated. One (1) sample was positive and one (1) sample was negative in the BioFire GI Panel.

The results from the Selected Sample Study are also summarized in Table 20 and provide further support of the performance for the detection of Salmonella using fresh samples. The point estimate was obtained for PPA was 92.9% (77.4% - 98.0%). There were two (2) false negative results which were further investigated by testing in the Verigene® EP. One (1) of these was also negative in the Verigene® EP and the other was positive for Salmonella in the Verigene® EP.

Conclusion: The method comparison studies conducted as part of the clinical studies demonstrated acceptable performance of the SBPP and support the Intended Use of this product.

H. Overall Conclusion

The submitted information in this 510(k) pre-market notification is complete and supports substantial equivalence.

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).