The Great Basin Shiga Toxin Direct Test performed on the Portrait™ Analyzer is an automated, in vitro diagnostic assay for the qualitative detection of Shiga toxin 1 (stxl) / Shiga toxin 2 (stx2) genes and specific identification of a conserved genetic region of the E. coli O157 serogroup. Shiga toxin genes are found in Shiga toxin-producing strains of E. coli (STEC) and Shigella dysenteriae. The E. coli 0157 test result is reported only if a Shiga toxin gene is also detected.

The test is performed directly from Cary-Blair or C&S Medium preserved stool specimens from symptomatic patients with suspected acute gastroenteritis, or colitis in hospital laboratories. The assay is intended for use in conjunction with clinical presentation as an aid in the diagnosis of STEC infections. Positive results do not rule out oninfection with other organisms, and may not be the definitive cause of patient illness.

The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Shiga Toxin Direct Test negative results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Device Description

The Portrait System is a fully automated system that includes the Portrait Analyzer, single-use Great Basin Shiga Toxin Direct Test cartridges, and the Portrait System data analysis software. The Portrait System is designed to perform automated sample preparation, PCR, and optical chip-based detection with integrated data analysis in approximately 2 hours.

The single-use Test Cartridge contains blister packs, fluidic channels, processing chambers, a waste chamber, and an assay chip coated with an array of sequence-specific detection probes. All reagents are contained within the integrated blister packs with the exception of the amplification reagents and SPC, which are dried into the Amplification Chamber and SPC Chambers of the Cartridge, respectively.

The appropriate specimen for use in the Test Cartridge is an aliquot of stool from symptomatic patients preserved in Cary-Blair or C&S transport media. A preserved stool specimen is placed into the sample port of the Test Cartridge for processing. Multiple fluidic channels move reagents from integrated blister packs to chambers where reagent mixing and sample processing occur. A waste chamber, self-contained and segregated within the Test Cartridge, collects and stores reagent waste.

AI/ML Overview

Here's a breakdown of the acceptance criteria and study details for the Great Basin Shiga Toxin Direct Test, as extracted from the provided text:

1. Table of Acceptance Criteria and Reported Device Performance:

The document doesn't explicitly state "acceptance criteria" as a separate, pre-defined set of values that the device must meet to be approved. Instead, it presents the clinical performance (sensitivity, specificity, PPA, NPA) as results from the studies, which are then compared to a predicate device (though specific performance metrics for the predicate are noted as "Not Reported").

However, based on the performance data presented, here's a summary of the device's reported performance which implicitly serve as the achieved acceptance level, especially when compared against itself in different study types. The comparison chart with the predicate device also indicates a qualitative "same" for intended use and target sequences which are fundamental acceptance points.

Performance Metric	Acceptance Criteria (Implicitly, Reported Performance)
Shiga Toxin (stx1/stx2)
Prospective Study (Fresh Specimens)
Sensitivity	100.0% (CI: 39.8% - 100.0%)
Specificity	99.3% (CI: 98.5% - 99.7%)
PPV	33.3% (CI: 9.9% - 65.1%)
NPV	100.0% (CI: 99.7% - 100.0%)
Frozen Retrospective Study
PPA	92.7% (CI: 82.4% - 98.0%)
NPA	100.0% (CI: 89.4% - 100.0%)
E. coli O157
Prospective Study (Fresh Specimens)
Specificity	83.3% (CI: 51.6% - 97.9%) (Sensitivity not applicable/reported due to low positives)
PPV	0.0% (CI: 0.0% - 84.2%) (Sensitivity not applicable/reported due to low positives)
NPV	100.0% (CI: 69.2% - 100.0%) (Sensitivity not applicable/reported due to low positives)
Frozen Retrospective Study
PPA	95.7% (CI: 78.1% - 99.9%)
NPA	100.0% (CI: 85.8% - 100.0%)

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

Prospective Clinical Study:
- Sample Size: 1,082 clinical specimens.
- Data Provenance:
  - Origin: Collected prospectively (fresh) at five sites.
  - Retrospective/Prospective: Prospective.
  - Timeframe: June to September 2015 (three-month period).
Frozen Retrospective Clinical Study:
- Sample Size: 88 unique frozen clinical specimens (from an initial panel of 92).
- Data Provenance:
  - Origin: Previously characterized clinical specimens.
  - Retrospective/Prospective: Retrospective.

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

The document does not specify the number of experts used or their qualifications to establish the ground truth for the clinical test sets. It mentions "reference clinical microbiology protocols" for the prospective study and "Clinical Characterization - Molecular and/or Shiga Toxin EIA" for the retrospective study as the basis for ground truth.

4. Adjudication Method for the Test Set:

The document does not detail any specific adjudication method (e.g., 2+1, 3+1). It states that the device's performance was compared to "reference clinical microbiology protocols" and "Molecular and/or Shiga Toxin EIA" results. For discrepant results in the prospective study, it notes:

"Shiga toxin was detected in 8/8 false positive specimens by both bi-directional sequencing and alternate, FDA-cleared comparator NAAT."
"O157 serogroup was detected in 2/2 false positive specimens by alternate, FDA-cleared comparator NAAT."
This describes a characterization process for disagreements rather than a multi-expert adjudication method for the initial ground truth.

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:

This device is an in-vitro diagnostic assay for direct detection of nucleic acids, not an imaging device or an AI-assisted diagnostic tool that involves human readers. Therefore, an MRMC comparative effectiveness study involving human readers and AI assistance is not applicable and was not performed.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done:

Yes, the studies evaluate the Great Basin Shiga Toxin Direct Test (a molecular diagnostic assay run on an automated system) as a standalone device. The device's output (qualitative detection of Shiga toxin genes and E. coli O157) is the primary subject of the performance evaluation. It is an automated, in-vitro diagnostic assay.

7. The Type of Ground Truth Used:

Prospective Clinical Study: "Reference clinical microbiology protocols for the detection of both Shiga Toxin and the E. coli O157 Serotype." This typically involves culture, immunoassay (EIA), and potentially molecular methods. For discrepant results, bi-directional sequencing and FDA-cleared comparator NAAT were used for confirmation.
Frozen Retrospective Clinical Study: "Clinical Characterization - Molecular and/or Shiga Toxin EIA."

8. The Sample Size for the Training Set:

The document does not explicitly mention a "training set" in the context of machine learning or AI algorithm development because this is a molecular diagnostic test. However, the document details various analytical studies which are foundational to the device's design and calibration. These studies use panels of known strains and concentrations:

Analytical Sensitivity (LoD): Involved testing specific E. coli strains (ATCC BAA-2191, ATCC 51434, ATCC BAA-2192, ATCC 43895) at varying concentrations (e.g., 25/25, 21/22, 26/26, 20/20 correct results for LoD determination, indicating numerous replicates).
Analytical Reactivity (Inclusivity): Tested 33 known positive strains (30 STEC, 3 Shigella dysenteriae) with 3 replicates each, totaling 99 tests.
Analytical Specificity (Exclusivity): Tested 118 microorganisms (bacteria, fungi, yeasts, parasites, viruses) and human genomic DNA, each typically with 3 replicates (e.g., 3/3 results mentioned frequently).
Microbial Interference: Tested 42 microorganisms, each typically with 3 or more replicates, against two STEC strains (ATCC 43895 and ATCC 43894) at 2X LoD.
Interfering Substances: Tested 26 different substances, each with 3 replicates, in both positive and negative stool matrices.

9. How the Ground Truth for the Training Set Was Established:

For these analytical studies, the ground truth was established by using well-characterized reference strains (e.g., ATCC strains) with known genetic profiles (presence of stx1, stx2, O157 genes) and established concentrations. This allows for precise control and verification of the device's ability to detect specific targets and its limits of detection, and to ensure it does not cross-react with non-targets. For other studies like specimen stability, known positive samples were contrived by spiking these characterized strains into negative stool matrix.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002 March 22, 2016

GREAT BASIN SCIENTIFIC, INC. CHUCK OWEN DIRECTOR, REGULATORY AFFAIRS & QUALITY ASSURANCE 2441 S. 3850 WEST SALT LAKE CITY, UT 84120

Re: K152955

Trade/Device Name: Great Basin Shiga Toxin Direct Test Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assay Regulatory Class: II Product Code: PCH Dated: March 9, 2016 Received: March 10, 2016

Dear Mr. Owen:

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,

Ribhi Shawar -S

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

Enclosure

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Indications for Use

510(k) Number (if known) K152955

Device Name

Great Basin Shiga Toxin Direct Test

Indications for Use (Describe)

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)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

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510(k) Summary - Shiga Toxin Direct Test

A. Submitted by:

Great Basin Scientific 2441 South 3850 West Salt Lake Citv. Utah 84120 Phone: 801-990-1055 Fax: 801-990-1051

Summary Preparation Date

March 08, 2016

Contact Information

Chuck Owen, Director of Requlatory Affairs Phone: 385-215-3313 801-990-1051 Fax: Email: cowen@gbscience.com

B. Name of Device

Proprietary Name:	Great Basin Shiga Toxin Direct Test
Common or Usual Names:	Shiga Toxin Direct TestShiga Toxin Direct
	Shiga Tox
	STEC Test/Assay

C. Regulatory Information:

a. Requlation Section: 21 CFR 866.3990, Gastrointestinal Microorqanism Multiplex Nucleic Acid-Based Assay
b. Classification: Class II
Classification panel: (83) Microbiology C.
РСН d. Product Code: Gastrointestinal Pathogen Panel Multiplex Nucleic Acid-Based Assay System 001 Real time nucleic acid amplification system

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

The Great Basin Shiqa Toxin Direct Test performed on the Portrait™ Analyzer is an automated, in vitro diagnostic assay for the qualitative detection of Shiga toxin 1 (stxr) / Shiqa toxin 2 (stx2) genes and specific identification of a conserved genetic reqion of the E. col/ 0157 serogroup. Shiga toxin genes are found in Shiga toxin-producing strains of E. col/ (STEC) and Shigella dysenteriae. The E. coli 0157 test result is reported only if a Shiga toxin gene is also detected.

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the diagnosis of STEC infections. Positive results do not rule out co-infection with other organisms, and may not be the definitive cause of patient illness.

E. Device Description:

Test Principle:

The 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. The Portrait System was granted 510(k) clearance for the Portrait Toxigenic C. difficile Assay (DEN120013) and the Portrait GBS Assay (K143312).

Target genomic DNA is extracted from microbial cells alongside sample processing control cells (SPC) and diluted to reduce potential inhibitors of the PCR reaction. During the PCR process, double-stranded DNA is separated and target nucleic acid sequences are amplified by thermal cycling. Biotin-labeled primers direct amplification of specific nucleic acid sequences within a conserved region of the stx1, stx2, and 0157 antigen-specific genes for identification of Shiqa toxin producing E. coli.

Following the PCR process, biotin-labeled, amplified target DNA sequences are hybridized to an array of probes immobilized on the silicon chip surface, then incubated with antibody conjugated to the horseradish peroxidase enzyme (HRP). These probes are specific for Shiga toxin 1 (stxr), Shiga toxin 2 (stx2), an O157 antigen marker gene, and SPC. The unbound conjugate is removed by washing 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 Portrait Analyzer. While the Shiqa Toxin Direct Test is designed to detect and distinguish between stx1 and stx2 toxin types, the assay does not report results to the individual toxin level.

Test Device:

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F. Substantial Equivalence Information:

a. Predicate Device: FilmArray® Gastrointestinal (GI) Panel from BioFire Diagnostics, LLC
b. Predicate 510(k) number: K140407
ﻥ Comparison with Predicate, see chart:

Predicate Device Comparison Chart
Item	Shiga Toxin Direct Test	Predicate (K140407)
Manufacturer	Great Basin Scientific, Inc.	BioFire Diagnostics, LLC
Trade Name	Portrait™ Shiga Toxin Direct Test	FilmArray™ Gastrointestinal (GI) Panel
510(k) Number	K141658	K140407
	Similarities
Classification	Class II	same
Intended Use/ Indications for Use	Direct detection of nucleic acids from enteric pathogens and toxin genes directly from transport media preserved clinical stool specimens.	same
Target Sequence Detected	Shiga toxin 1 (stx1) and Shiga toxin 2 (stx2) gene virulence markers for the identification of Shiga toxin-producing Escherichia coli (STEC), including specific identification of the E. coli O157 serotype within STEC.	same
Qualitative/ Quantitative	Qualitative	same
Single-Use Test Cartridge	Disposable, single-use, self-contained fluidic test cartridge	same
Automated	Yes	same
Test Principle	Nucleic Acid Amplification Assay	same
Sample Types	Human stool sample preserved in transport media.	same
Assay Controls	Sample processing controls are incorporated directly into single-use test cartridges in freeze-dried form and are rehydrated into the test prior to sample lysis.	same
Calibration	Not required	same
	Differences
Organisms Detected	Shiga toxin-producing Escherichia coli (STEC), including specific identification of the E. coli O157 serotype within STEC.	· Campylobacter (C. jejuni/C. coli/C. upsaliensis)· Clostridium difficile (C. difficile) toxin A/B· Plesiomonas shigelloides· Salmonella· Vibrio (V. parahaemolyticus/V. vulnificus/ V. cholerae), including specific identification of Vibrio cholerae· Yersinia enterocolitica· Enteroaggregative Escherichia coli (EAEC)· Enteropathogenic Escherichia coli (EPEC)· Enterotoxigenic Escherichia coli (ETEC) lt/st· Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2 (including specific identification of the E. coli 0157 serogroup within STEC)· Shigella/ Enteroinvasive Escherichia coli (EIEC)· Cryptosporidium· Cyclospora cayetanensis· Entamoeba histolytica· Giardia lamblia· Adenovirus F 40/41· Astrovirus· Norovirus GI/GII· Rotavirus A· Sapovirus (Genogroups I, II, IV, and V)
Compatible Media Types	Cary-Blair and C&S stool Preservation and Transport Medias	Cary-Blair Preservation and Transport Media
Analyte	DNA	DNA/RNA
	Shiga Toxin Direct Test	Predicate (K140407)
Item	Differences
Amplification Technology	Multiplex polymerase chain reaction (PCR)	Nested multiplex RT-PCR
Instrument	PA500 Portrait™ Analyzer	FilmArray™ Instrument
Time to Result	Approximately 2 hours	Less than 1 hour
Detection Method	Colorimetric target specific hybridization to probeon a chip surface, optical reader, automatedsoftware with built-in result interpretation.	High resolution melting analysis to confirm identityof amplified product with automated software withbuilt-in result interpretation.
Reagent Storage	Reagents stored at 4°C	Reagents stored at Room Temperature
Clinical Sensitivity Shiga Toxin	100% [95% CI: 39.8% - 100%]	Not Reported
Clinical Specificity Shiga Toxin	99.3% [95% CI: 98.5%- 99.7%]	Not Reported
Clinical Sensitivity 0157Serotype	Not Reported	Not Reported
Clinical Specificity 0157Serotype	83.3% [95% CI: 51.6%- 97.9%]	Not Reported
Positive Percent AgreementShiga Toxin	92.7% [95% CI: 82.4%- 98.0%]	100% [95% CI: 89.4%- 100%]
Negative Percent AgreementShiga Toxin	100% [95% CI: 89.4%- 100%]	99.7% [95% CI: 99.2%- 99.9%]
Positive Percent Agreement0157 Serotype	95.7% [95% CI: 78.1%- 99.9%]	100% [95% CI: 29.2%- 100%]
Positive Percent Agreement0157 Serotype	100% [95% CI: 85.8%- 100%]	97.1% [95% CI: 85.1%- 99.9%]

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Performance Data - Analytical Studies

d. Analytical Sensitivity

The limit of detection (LoD) for four (4) Shiqa toxin-producinq E. coli (STEC) strains was measured for the Shiga Toxin Direct Test. The LoD for each toxin gene, stx1 and stx2, was assessed and measured independently by testing a non-0157 stx1+ Escherichia coli strain (ATCC BAA-2191) and a non-0157 stx2+ Escherichia coli strain (ATCC 51434), respectively. In addition the LoD for a non-0157 Escherichia coli strain containing both toxin genes (stx1+/stx2+/0157-) was measured (ATCC BAA-2192). Finally, the LoD for an 0157 Serotype Escherichia coli strain containing both toxin genes (stx1+/stx2+/0157+) was also measured (ATCC 43895). The LoD for each strain is listed in Table 1.

Shiga toxin-producingE. coli (STEC) Strain	Shiga Toxin(s)Present	Serotype	Expected Shiga Toxin DirectTest Result	CorrectResults	LoD
ATCC BAA-2191	stx1+	045:H2	STEC POSITIVE/Serotype O157 NEGATIVE	25/25	$5.5 x 10^3$ CFU/mL
ATCC 51434	stx2+	091:H21	STEC POSITIVE/Serotype O157 NEGATIVE	21/22	$2.8 x 10^3$ CFU/mL
ATCC BAA-2192	stx1+, stx2+	0145:NM	STEC POSITIVE/Serotype 0157 POSITIVE	26/26	$5.2 x 10^3$ CFU/mL
ATCC 43895	stx1+, stx2+	O157:H7	STEC POSITIVE/Serotype 0157 POSITIVE	20/20	$5.0 x 10^3$ CFU/mL

Table 1. Limit of Detection (LoD) of the Shiga Toxin Direct Test.

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e. Specimen Stability

The recommended storage time and temperature conditions for Cary-Blair or C&S preserved stool specimens prior to testing via the Shiga Toxin Direct Test includes:

Refrigerated storage (2°- 8° C) for up to 120 hours (5 days) .
. Room temperature (RT) storage for up to 4 hours
The combination of up to 4 hours storage at room temperature followed by . refrigerated storage for up to 120 hours.

To assess the stability of the Shiga toxin and serotype 0157 nucleic acid targets of the Shiga Toxin Direct Test under the recommended storage conditions, a Specimen Stability Study was performed to evaluate each recommended time and temperature storage conditions. The study tested two non-O157 STEC strains (ATCC BAA-2191 and ATCC 51434) and one O157 STEC strain (ATCC 43889). Each sample was contrived from freshly cultured STEC cells spiked at 2X LoD into negative stool matrix and stored under the recommended storage conditions. The LoD was approximated for the 0157 STEC strain (ATCC 43889). A second O157 STEC strain (ATCC 43890) was tested but was not included in the final analysis when baseline (Ta) testing results of 80% positivity indicated that the strain was spiked at concentrations below 2X LoD (Tables 2-3).

Total Strains Tested	4 Shiga toxin-producing E.coli (STEC) strains
Strains Tested	1. ATCC BAA-21922. ATCC 514343. ATCC 43889
Replicates	Each strain was formulated into 5 unique stool matrices* at each concentration and tested in a single replicate at each time point
Total Panel Size	30 samples
Panel Runs	6 time points
Time Points	T₀: 0 hr (freshly prepared)T₁: 4 hr Room Temp. storageT₂: 24 hr 2°-8° C storageT₃: 72 hr 2°-8° C storageT₄: 120 hr 2°-8° C storageT₅: 4 hr Room Temp. + 120 hr 2°-8° C storage
Total Runs	30 samples x 6 time points = 180 runs

Table 2. Specimen Stability Study Protocol Overview. A summary of the Specimen Stability panel and testing overview.

Stool matrices for this study were provided by a clinical test site and were

previously characterized as 'Shiga toxin Negative' by Shiga Toxin Immunoassay.

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Table 3. Specimen Stability Study Summary Results. Shiga toxin-producing E. coli samples stored at either RT and/or 2°-8°C and tested by the Shiga Toxin Direct Test at multiple time points.

	% Agreement
Shiga toxin-producingE. coli (STEC) Strain TestedConcentration(2X LoD)Expected Shiga ToxinDirect Test Result	ATCC BAA-2191(stx1+)$1.1 x 10^4$CFU/mLSTEC POSITIVE/Serotype O157 NEGATIVE	ATCC 51434(stx2+)$6.0 x 10^3$CFU/mLSTEC POSITIVE/Serotype O157 NEGATIVE	ATCC 43889^(stx2+/O157)$8.5 x 10^3$ - $1.0 x 10^4$CFU/mLSTEC POSITIVE/Serotype O157 POSITIVE
T₀: 0 hr	100%(5/5)	100%(5/5)	100%(10/10)
T₁: 4 hr Room Temp.	100%(5/5)	100%(5/5)	100%(10/10)
T₂: 24 hr 2°- 8° C storage	100%(5/5)	100%(5/5)	100%(10/10)
T₃: 72 hr 2°- 8° C storage	100%(5/5)	100%(5/5)	100%(10/10)
T₄: 120 hr 2°- 8° C storage	100%(5/5)	100%(5/5)	100%(10/10)
T₅: 4 hr Room Temp. + 120 hr 2°- 8° C storage	100%(5/5)	100%(5/5)	100%(10/10)
Overall	100%(30/30)	100%(30/30)	100%(60/60)

^ Limit of Detection (LoD) was approximated for this strain.

Fresh vs. Frozen f.

A Fresh vs. Frozen Study was performed to support the use of frozen, transport media preserved stool specimens in the Shiga Toxin Direct Test for the Frozen Retrospective and Reproducibility Studies, as well as for follow-up testing of prospective samples (Table 4).

The Fresh vs. Frozen Study tested the performance of the Shiga Toxin Direct Test on contrived positive samples that were subjected to two freeze/thaw cycles. Demonstration of specimen stability after the second freeze/thaw cycle is important because generation of a Frozen Retrospective panel from archived clinical specimens requires a freeze/thaw to prepare panel prior to re-freezing for shipment to clinical sites. Clinical sites performed the second thawing of archived specimens prior to testing. Samples tested in the Reproducibility Study or for follow-up testing at the reference site were subjected to a single freeze/thaw cycle.

The Fresh vs. Frozen was conducted using contrived positive samples that were prepared using fresh (i.e. never frozen) enriched broth cultures. The panel for the Fresh vs. Frozen Study was comprised of 6 STEC strains: ATCC BAA-2191 (stx1+/O157-), ATCC 51434 (stx2+/0157-), ATCC BAA-2192 (stx1+/stx2+/0157-), ATCC 43890 (stx1+/0157+), ATCC 43889 (stx2+/0157+), and ATCC 43895 (stx1+/stx2+/0157+). Each strain was tested in replicate at 4 concentrations: ≤ 0.5X LoD, 1X LoD, 3X LoD, and 10X LoD. The panel was initially tested on the Shiga Toxin Direct Test within 30 minutes of construction to establish the 'fresh' activity prior to freezing (Ta). The entire panel was then placed at ≤-70°C for 1 week at which time it was thawed and re-tested (T1).

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The entire panel was returned to ≤-70°C for a second freezing cycle for an additional 1 week at which time the samples were tested for a second, and final, thaw (T3),

This study demonstrates sufficient integrity of the Shiga Toxin Direct Test targets (nucleic acid from Shiga toxin 1, Shiga toxin 2 gene, and 0157 serotype identification genes) in frozen stool specimens preserved in C&S media for up to 2 freeze/thaw cycles.

Table 4. Fresh vs. Frozen Study. Shiga Toxin Direct Test results of STEC samples evaluated prior to freezing at ≤-70°C and after multiple freeze/thaw cycles.

			% Agreement
Shiga toxin-producing E. coli(STEC) Strain	Expected Shiga ToxinDirect Test Result	Concentration	T₀ = pre-freeze		T₁ = 1X freeze/thaw		T₂ = 2X freeze/thaw
ATCC BAA-2191(stx1+)		10X LoD	2/2	100%	2/2	100%	2/2	100%
		3X LoD	4/4	100%	4/4	100%	4/4	100%
		1X LoD	4/4	100%	4/4	100%	4/4	100%
		≤ 0.5X LoD	3/4	75%	4/4	100%	4/4	100%
ATCC 51434(stx2+)	STEC POSITIVE/Serotype O157 NEGATIVE	10X LoD	2/2	100%	2/2	100%	2/2	100%
		3X LoD	4/4	100%	4/4	100%	4/4	100%
		1X LoD	4/4	100%	4/4	100%	4/4	100%
		≤ 0.5X LoD	3/4	75%	3/4	75%	4/4	100%
ATCC BAA-2192(stx1+/stx2+)		10X LoD	2/2	100%	2/2	100%	2/2	100%
		3X LoD	4/4	100%	4/4	100%	4/4	100%
		1X LoD	4/4	100%	4/4	100%	4/4	100%
		≤ 0.5X LoD	3/4	75%	4/4	100%	4/4	100%
ATCC 43890+(stx1+/0157)		10X LoD	2/2	100%	2/2	100%	2/2	100%
		3X LoD	4/4	100%	4/4	100%	4/4	100%
		1X LoD	4/4	100%	4/4	100%	3/4	75%
		≤ 0.5X LoD	3/4	75%	2/4	50%	3/4	75%
ATCC 43889+(stx2+/0157)	STEC POSITIVE/Serotype 0157 POSITIVE	10X LoD	2/2	100%	2/2	100%	2/2	100%
		3X LoD	4/4	100%	4/4	100%	4/4	100%
		1X LoD	4/4	100%	4/4	100%	4/4	100%
		≤ 0.5X LoD	3/4^	75%	2/4	50%	3/4	75%
ATCC 43895(stx1+/stx2+/0157)		10X LoD	2/2	100%	2/2	100%	2/2	100%
		3X LoD	4/4	100%	4/4	100%	4/4	100%
		1X LoD	4/4	100%	4/4	100%	4/4	100%
		≤ 0.5X LoD	2/5*	40%	2/4*	50%	4/4	100%

Limit of Detection (LoD) was approximated for this strain.

^ Represents each 'INVALID' run in this dataset.

Represents each 'Test Incomplete' run in this dataset.

q. Analytical Reactivity (Inclusivity)

The Analytical Reactivity of the Shiga Toxin Direct Test was tested against 30 wellcharacterized Shiga toxin-producing E. col/ (STEC) strains from ATCC representing the serotypes of E. coli that are most often associated with disease: serotypes 026, 045, 0103, 0111, 0121, 0145, and 0157. The Shiga toxin gene (stx) which is identical in sequence to the STEC stx1 gene is also commonly found in Shigella dysenteriae serotype 1 strains. Therefore in addition to STEC strains, three (3) serotype 1 Shigella dysenteriae strains were tested.

The Shiga Toxin Direct Test correctly detected all 21 of the non-O157 Serotype STEC and three (3) Serotype 1 Shigella dysenteriae strains as 'STEC POSITIVE/Serotype 0157

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Negative'. Furthermore, all nine (9) O157 serotype STEC strains were identified as 'STEC POSITIVE/ Serotype 0157 POSITIVE' (Table 5).

Table 5. Analytical Reactivity (Inclusivity) Panel. Shiga toxin-producing E. coli (STEC) and Shigella dysenteriae strains tested for inclusivity by the Shiga Toxin Direct Test.

ATCC Strain	Serotype	Shiga ToxinGene(s) Present	Expected Shiga ToxinDirect Test Result	Concentration	PositiveResults
Shiga toxin-producing Escherichia coli (STEC)
BAA-2181	O26:H11	stx1+		1.0 x 104 CFU/mL	3/3
BAA-2215	O103:H11	stx1+		1.0 x 104 CFU/mL	3/3
BAA-2199	O123:H25	stx1+		1.0 x 104 CFU/mL	3/3
BAA-2210	O103:H2	stx1+		1.0 x 104 CFU/mL	3/3
BAA-2191	O45:H2	stx1+		1.0 x 104 CFU/mL	3/3
BAA-2201	O111:H8	stx1+	STEC POSITIVE/	1.0 x 104 CFU/mL	3/3
51435	O91:H21	stx2+	Serotype O157 NEGATIVE	1.0 x 104 CFU/mL	3/3
51434	O91:H21	stx2+		1.0 x 104 CFU/mL	3/3
BAA-182	O104:H21	stx2+		1.0 x 104 CFU/mL	3/3
BAA-2326	O104:H4	stx2+		1.0 x 104 CFU/mL	3/3
BAA-183	O113: H21	stx2+		1.0 x 104 CFU/mL	3/3
BAA-2220	O121:H19	stx2+		1.0 x 104 CFU/mL	3/3
BAA-2219	O121:H19	stx2+		1.0 x 104 CFU/mL	3/3
BAA-2211	O145: H25	stx2+		1.0 x 104 CFU/mL	3/3
BAA-2129	O145:H28	stx2+		1.0 x 104 CFU/mL	3/3
BAA-2221	O21:H19	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
BAA-2196	O26:H11	stx1+/stx2+	STEC POSITIVE/Serotype 0157 NEGATIVE	1.0 x 104 CFU/mL	3/3
BAA-2193	O45:H2	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
BAA-2440	0111	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
700840	0111:H8	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
BAA-2192	0145	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
		n = 21
43890	0157:H7	stx1+		1.0 x 104 CFU/mL	3/3
700376	0157:NM	stx1+		1.0 x 104 CFU/mL	3/3
43889	0157:H7	stx2+		1.0 x 104 CFU/mL	3/3
700377	0157:NM	stx2+		1.0 x 104 CFU/mL	3/4+
700378	0157:NM	stx1+/stx2+	STEC POSITIVE/Serotype 0157 POSITIVE	1.0 x 104 CFU/mL	3/3
700927	0157:H7:K	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
43894	0157:H7	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
43895	0157:H7	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
35150	O157:H7	stx1+/stx2+		1.0 x 104 CFU/mL	3/3
		n = 9
Shigella dysenteriae
9361	Type 1	^stx+		1.0 x 104 CFU/mL	3/3
27346†	Type 1	^stx+	STEC POSITIVE/	≤1.0 x 104 CFU/mL	3/3
27345†	Type 1	^stx+	Serotype O157 NEGATIVE	≤1.0 x 104 CFU/mL	3/3*
		n = 3
Totaln = 33

Represents each 'Test Incomplete' run in this dataset.

‡ Represents each replicate in this set that resulted in 'STEC NEGATIVE/Serotype 0157 Not Tested'.

† Concentration of broth culture estimated from optical density due to lack of growth on plates for exact colony counting.

^ This Shigella dysenteriae strain contains the Shiga toxin gene (stx) which is identical in sequence to stx1; therefore the Shiga Toxin Direct Test reports this strain as 'STEC POSITIVE/ Serotype 0157 Negative.'

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h. Analytical Specificity (Exclusivity)

Studies were conducted to assess the potential for cross-reactivity of non-target organisms, some of which are commonly found in stool specimens. Included in Analytical Specificity testing were well known enteric pathogens that present clinically with symptoms similar to STEC, such as diarrhea. The study evaluated a total of 118 microorganisms, including: bacteria, fungi/yeasts, parasites, viruses, and human genomic DNA. For organisms that where classified as Biosafety level III or unable to culture via standard clinical microbiology techniques genomic DNA was tested in lieu of whole orqanism. Each non-target organism or nucleic acid was tested in the background of neqative clinical stool matrix consisting of clinical Shiga toxin negative stool preserved in ParaPak® C&S media (Table 6).

Due to the design of the Sample Processing Control (SPC) in the Shiga Toxin Direct Test, very high concentrations of non-STEC O157 E. col/can compete with amplification of the SPC. The Shiga Toxin Direct Test controls are built such that SPC amplification failure in the absence of Shiga Toxin signal results in an 'invalid' test. Therefore during exclusivity testing both non-STEC O157:H7 E. col/strains (ATCC 43888 and ATCC 700728) resulted in 'invalid' test results when tested at concentrations ≥ 1.0x10° CFU/mL. The test concentration for both strains (ATCC 43888 and ATCC 700728) was lowered to approximately 1.0x106 CFU/mL, each strain was re-retested, and resulted in the correct call of 'STEC NEGATIVE/Serotype O157 Not Tested'.

In total 104 unique bacterial strains, three (3) yeast, three (3) parasites, seven (7) viruses, and human genomic DNA were evaluated for cross-reactivity. With the exception of the 2 previously mentioned E. co// strains (43888 and 700728), none of the tested nucleic acids (genomic DNA or viruses) or cultured organisms (bacteria, yeasts, parasites) interfered with the internal controls and all of the calls were 'STEC NEGATIVE/Serotype 0157 Not Tested,' indicating no cross-reactivity (Table 6).

Table 6. Analytical Specificity (Exclusivity) Panel. Non-Shiga toxin-producing enteric flora, including: bacteria, viruses, parasites, fungi and nucleic acids from various pathogens, tested for exclusivity by the Shiga Toxin Direct Test.

Organism	Strain ID	Input Tested	STEC NEGATIVE/Serotype O157 NotTested Result
Bacteria
Abiotrophia defective	ATCC 49176	≥1.0 x 106 CFU/mL+	3/3
Acinetobacter baumannii	ATCC 19606	1.3 x 108 CFU/mL	3/3
Aeromonas hydrophila	ATCC 35654	4.6 x 108 CFU/mL	3/3
Anaerococcus tetradius	ATCC 35098	1.6 x 107 CFU/mL	3/3
Bacillus cereus	ATCC 14579	7.6 x 107 CFU/mL	3/3
Bacteroides fragilis	ATCC 23745	2.7 x 107 CFU/mL	3/3
Bacteroides vulgatus	ATCC 8482	≥1.0 x 106 CFU/mL+	3/3
Bifidobacterium adolescentis	ATCC 15703	≥1.0 x 106 CFU/mL+	3/3***
Bifidobacterium bifidum	ATCC 11863	≥1.0 x 106 CFU/mL+	3/3
Bifidobacterium longum	ATCC 15707	≥1.0 x 106 CFU/mL+	3/3
Campylobacter coli	ATCC 33559	5.4 x 107 CFU/mL	3/3
Campylobacter fetus	ATCC 15296	≥1.0 x 106 CFU/mL+	3/3^
Campylobacter jejuni	ATCC 49943	4.9 x 107 CFU/mL	3/3
Campylobacter lari	ATCC 35221	4.0 x 106 CFU/mL	3/3
Citrobacter amalonaticus	ATCC 25406	1.3 x 108 CFU/mL	3/3
Citrobacter freundii	ATCC 8090	3.4 x 108 CFU/mL	3/3
Organism	Strain ID	Input Tested	STEC NEGATIVE/Serotype 0157 NotTested Result
Clostridium difficle (A-, B-)	ATCC BAA-1801	≥1.0 x 106 CFU/mL†	3/3
Clostridium difficle (A+, B+) (gDNA)	ATCC BAA-1382D	5.0 x 106 copies/uL	3/3
Clostridium difficle (A+, B+)	ATCC 43255	≥1.0 x 106 CFU/mL†	3/3
Clostridium histolyticum	ATCC 19401	≥1.0 x 106 CFU/mL†	3/3
Clostridium perfringens	ATCC 12915	1.6 x 107 CFU/mL	3/3
Clostridium sordellii	ATCC 9715	≥1.0 x 106 CFU/mL†	3/3
Enterobacter aerogenes	ATCC 15038	2.7 x 108 CFU/mL	3/3
Enterobacter cloacae	ATCC 13047	4.6 x 108 CFU/mL	3/3
Enterococcus cecorum	ATCC 43198	8.2 x 105 CFU/mL	3/3
Enterococcus faecalis	ATCC 29212	1.2 x 108 CFU/mL	3/3
Enterococcus faecium	ATCC 19434	6.4 x 107 CFU/mL	3/3
Enteroaggregative Escherichia coli (EAEC)	ATCC 29552	5.6 x 107 CFU/mL	3/3
Enteroaggregative Escherichia coli (EAEC)	STEC Center JM221	4.0 x 107 CFU/mL	3/3
Enteroinvasive Escherichia coli (EIEC)	STEC Center 1885-77	2.2 x 107 CFU/mL	3/3
Enteroinvasive Escherichia coli (EIEC)	ATCC 43892	2.4 x 108 CFU/mL	3/3
Enteropathogenic Escherichia coli (EPEC)	STEC Center E2348/69	4.4 x 107 CFU/mL	3/3
Enteropathogenic Escherichia coli (EPEC)	STEC Center TW07897	5.1 x 107 CFU/mL	3/3^
Enteropathogenic Escherichia coli (EPEC)	STEC Center TW07886	5.3 x 107 CFU/mL	3/3
Enteropathogenic Escherichia coli (EPEC)	STEC Center E851/71	2.6 x 107 CFU/mL	3/3
Enterotoxigenic Escherichia coli (ETEC)	ATCC 35401	2.5 x 108 CFU/mL	3/3
Escherichia coli (non-STEC O157)	ATCC 700728	3.9 x 108 CFU/mL	0/3^^^
Escherichia coli (non-STEC O157)	ATCC 700728	1.0 x 106 CFU/mL	3/3
Escherichia coli (non-STEC O157)	ATCC 43888	4.9 x 107 CFU/mL	0/3^^^
Escherichia coli (non-STEC O157)	ATCC 43888	1.0 x 106 CFU/mL	3/3
Escherichia fergusonii	ATCC 35469	2.2 x 108 CFU/mL	3/3
Escherichia hermannii	ATCC 33650	3.7 x 108 CFU/mL	3/3^
Fusobacterium varium	ATCC 27725	1.1 x 108 CFU/mL	3/3
Gardnerella vaginalis	ATCC 14018	≥1.0 x 106 CFU/mL†	3/3
Helicobacter fennelliae	ATCC 35683	≥1.0 x 106 CFU/mL†	3/3
Helicobacter pylori	ATCC 49503	≥1.0 x 106 CFU/mL†	3/3
Klebsiella oxytoca	ATCC 13182	2.5 x 108 CFU/mL	3/3
Klebsiella pneumonia	ATCC 13883	3.2 x 108 CFU/mL	3/3*
Lactobacillus acidophilus	ATCC 4356	4.3 x 105 CFU/mL	3/3
Lactobacillus lactis	ATCC 49032	2.6 x 108 CFU/mL	3/3^^
Leminorella grimonti	ATCC 43007	1.2 x 108 CFU/mL	3/3
Listeria grayi	ATCC 19120	4.5 x 108 CFU/mL	3/3^
Listeria innocua	ATCC 33090	1.2 x 108 CFU/mL	3/3
Listeria monocytogenes	ATCC 19115	2.0 x 108 CFU/mL	3/3
Morganella morganii	ATCC 25829	7.7 x 107 CFU/mL	3/3
Peptostreptococcus anaerobius	ATCC 27337	1.0 x 108 CFU/mL	3/3
Plesiomonas shigelloides	ATCC 51903	3.5 x 108 CFU/mL	3/3^
Prevotella melaninogenica	ATCC 25845	1.6 x 107 CFU/mL	3/3
Proteus mirabilis	ATCC 25933	5.4 x 106 CFU/mL	3/3*
Proteus penneri	ATCC 33519	5.5 x 107 CFU/mL	3/3
Proteus vulgaris	ATCC 6896	1.3 x 108 CFU/mL	3/3
Providiencia alcalifaciens	ATCC 9886	4.4 x 107 CFU/mL	3/3
Providencia rettgeri	ATCC 9250	2.4 x 108 CFU/mL	3/3
Providencia stuartii	ATCC 49762	5.7 x 107 CFU/mL	3/3
Pseudomonas aeruginosa	ATCC 10145	2.4 x 108 CFU/mL	3/3
Pseudomonas mosselii	ATCC 49838	5.8 x 106 CFU/mL	3/3
Ruminococcus bromii	ATCC 27255	≥1.0 x 106 CFU/mL†	3/3
Salmonella enterica subp Arizonae	ATCC 13314	3.4 x 108 CFU/mL	3/3
Salmonella enterica subp Cholerasuis	ATCC 13312	5.3 x 108 CFU/mL	3/3^
Salmonella enterica subp Heidelberg	ATCC 8326	4.0 x 108 CFU/mL	3/3
Salmonella enterica subp Newington	ATCC 29628	1.6 x 108 CFU/mL	3/3
Organism	Strain ID	Input Tested	STEC NEGATIVE/Serotype 0157 NotTested Result
Salmonella paratyphi A	ATCC 9150	2.8 x 108 CFU/mL	3/3
Salmonella paratyphi B	ATCC 8759	7.6 x 108 CFU/mL	3/3*
Salmonella typhimurium	ATCC 13311	4.1 x 108 CFU/mL	3/3*
Selenomonas ruminantium	ATCC 35018	≥1.0 x 106 CFU/mL+	3/3
Serratia liquefaciens	ATCC 35551	6.4 x 108 CFU/mL	3/3^
Serratia marcescens	ATCC 13880	9.2 x 108 CFU/mL	3/3
Shigella boydii	ATCC 29928	2.5 x 108 CFU/mL	3/3
Shigella boydii	ATCC 12028	1.8 x 107 CFU/mL	3/3
Shigella dysenteriae (Type 2)	ATCC 29027	7.2 x 107 CFU/mL	3/3
Shigella dysenteriae (Type 3)	ATCC 29028	3.8 x 107 CFU/mL	3/3
Shigella dysenteriae (Type 12)	ATCC 49551	3.4 x 107 CFU/mL	3/3
Shigella dysenteriae (Type 13)	ATCC 49555	4.0 x 107 CFU/mL	3/3
Shigella flexneri	ATCC 25929	3.6 x 108 CFU/mL	3/3
Shigella sonnei	ATCC 25931	2.2 x 108 CFU/mL	3/3
Shigella sonnei	ATCC 29930	6.4 x 107 CFU/mL	3/3
Staphylococcus aureus	ATCC BK23738	4.5 x 108 CFU/mL	3/3
Staphylococcus epidermidis	ATCC 700567	3.5 x 108 CFU/mL	3/3
Stenotrophomonas maltophilia	ATCC 13637	2.4 x 107 CFU/mL	3/3
Streptococcus agalactiae	ATCC BAA-611	4.0 x 107 CFU/mL	3/3
Streptococcus dysgalactiae	ATCC 43078	7.0 x 106 CFU/mL	3/3
Streptococcus intermedius	ATCC 27335	8.0 x 106 CFU/mL	3/3
Streptococcus pyogenes	ATCC 49399	8.0 x 106 CFU/mL	3/3
Streptococus uberis	ATCC 9927	6.0 x 106 CFU/mL	3/3
Trabulsiella guamensis	ATCC 49492	5.5 x 107 CFU/mL	3/3
Veillonella parvula	ATCC 10790	6.6 x 107 CFU/mL	3/3
Vibrio cholera	ATCC 55188	3.8 x 108 CFU/mL	3/3
Vibrio parahaemolyticus	ATCC 17802	3.2 x 106 CFU/mL	3/3
Vibrio vulnificus	ATCC 27562	1.5 x 108 CFU/mL	4/4
Yersinia bercovieri	ATCC 43970	3.4 x 107 CFU/mL	3/3
Yersinia enterocolitica	ATCC 49397	1.6 x 108 CFU/mL	4/4
Yersinia pseudotuberculosis	ATCC 23207	1.3 x 107 CFU/mL	3/3
Yersinia rohdei	ATCC 43380	1.9 x 107 CFU/mL	3/3
Yeasts, Parasites, and Viruses
Candida albicans	ATCC 18804	3.0 x 106 CFU/mL	3/3
Candida catenulata	ATCC 10565	5.0 x 105 CFU/mL	3/3
Cryptosporidium parvum	ATCC PRA-67D	1 ug/mL	3/3
Entamoeba histolytica	ATCC 30459DQ	1.0 x 108 CFU/mL	3/3
Giardia lamblia (G. intestinalis)	ATCC 50803D	1 ug/mL	3/3
Saccharomyces cerevisiae	ATCC MYA-796	≥1.0 x 106 CFU/mL+	3/3^
Human mastadenovirus F	ATCC VR-931D	1 ug/mL	3/3
Adenovirus type 41	ATCC VR-930D	1 ug/mL	3/3
Coxsackie B4	ATCC VR-184	1.0 x 106 TCID50/mL	3/3
Enterovirus 71	ATCC VR-1775DQ	4.8 x 105 copies/uL	3/3
Norovirus G1	ATCC VR-3234SD	4.7 x 105 copies/uL	3/3
Norovirus G2	ATCC VR-3235SD	4.8 x 105 copies/uL	3/3*
Rotavirus	ATCC VR-1546	1.0 x 105 TCID50/mL	3/3
Human genomic DNA (HT-29)	ATCC HTB-38D	1 ug/mL	3/3

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† Actual concentration undetermined, estimate based on optical density measurement.

Represents each 'Test Incomplete' run in this dataset.

^ Represents each 'INVALID' run in this dataset.

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Microbial Interference

As a follow up to the previous Exclusivity studies, the Shiga Toxin Direct Test was further evaluated for interference from mixed microbial populations using a subset of 42 of the same microflora (bacterial, yeast, parasite, and viral stock strains). The potential for cross-reactivity in mixed infections was evaluated with a comprehensive panel created with a specific focus on common gastrointestinal pathogens encountered in stool that cause similar disease states to Shiga Toxins, including other common pathogenic, non-STEC E. coli species.

The same high concentrations of potentially interfering DNA and microorganisms were spiked into negative clinical preserved stool matrix containing a stx1+/stx2+/0157+ STEC strain at low positive concentration of 2X LoD. In total two (2) STEC strains (ATCC 43895 and ATCC 43894) containing all assay targets were evaluated.

This Microbial Interference Study assessed potential Shiga Toxin Direct Test interference due to mixed infections by evaluating detection of a STEC strain (ATCC 43895) consisting of all 3 analytes (stx1+/stx2+/0157) at near LoD concentrations in the background of high concentrations of non-Shiga toxin-producing enteric flora, including: bacteria, viruses, parasites, fungi and nucleic acids from various pathogens. A minimum of three replicate Shiga Toxin Direct Tests were performed for each potentially interfering organism or nucleic acid (Table 7).

In total 30 unique bacterial strains, two (2) yeast, four (4) parasites, five (5) viruses, and human genomic DNA were evaluated for microbial interference. All of the valid test runs resulted in the expected 'STEC POSITIVE/Serotype 0157 POSITIVE' call indicating that none of the tested nucleic acids (genomic DNA or viruses) or cultured organisms (bacteria, yeasts, parasites) interfered with the detection of both Shiga Toxin Direct Test analytes (Shiga toxin and the 0157 serotype) at 2X LoD (Table 7).

Table 7. Microbial Interference Panel. A panel of non-Shiga toxin-producing enteric flora, including: bacteria, viruses, parasites, fungi and nucleic acids from various pathogens, tested for microbial interference in detection of Shiga toxins and the 0157 serotype via the Shiga Toxin Direct Test.

			STEC Strain43895	STEC Strain43894
Organism	Strain ID	Input Tested	STEC POSITIVE/Serotype O157 POSITIVETest Result
Bacteria
Aeromonas hydrophila	ATCC 35654	4.6 x 108 CFU/mL	4/4	3/3
Bacteroides fragilis	ATCC 23745	2.7 x 107 CFU/mL	3/3	3/3
Bacteroides vulgatus	ATCC 8482	≥1.0 x 106 CFU/mL+	3/3	3/3
Bifidobacterium bifidum	ATCC 11863	≥1.0 x 106 CFU/mL+	3/3	3/3
Campylobacter jejuni	ATCC 49943	4.9 x 107 CFU/mL	3/3	3/3
Clostridium difficle (A+, B+)	ATCC 43255	≥1.0 x 106 CFU/mL+	3/3	3/3
Clostridium perfringens	ATCC 12915	1.6 x 107 CFU/mL	3/3	3/3
Enterobacter aerogenes	ATCC 15038	2.7 x 108 CFU/mL	3/3	3/3
Enterococcus faecalis	ATCC 29212	1.2 x 108 CFU/mL	3/3	3/3
Escherichia coli (non-STEC O157)	ATCC 700728	3.9 x 108 CFU/mL	3/3	3/3
Enteroaggregative Escherichia coli(EAEC)	ATCC 29552	5.6 x 107 CFU/mL	3/3	3/3
			STEC Strain43895	STEC Strain43894
Organism	Strain ID	Input Tested	STEC POSITIVE/Serotype O157 POSITIVETest Result
Enteroaggregative Escherichia coli(EAEC)	STEC CenterJM221	4.0 x 107 CFU/mL	3/3	3/3
Enteroinvasive Escherichia coli(EIEC)	ATCC 43892	2.4 x 108 CFU/mL	3/3	3/3
Enteroinvasive Escherichia coli(EIEC)	STEC Center1885-77	2.2 x 107 CFU/mL	3/3	3/3
Enteropathogenic Escherichia coli(EPEC)	STEC CenterE2348/69	4.4 x 107 CFU/mL	3/3	3/3
Enteropathogenic Escherichia coli(EPEC)	STEC CenterTW07897	5.1 x 107 CFU/mL	3/3	3/3
Enteropathogenic Escherichia coli(EPEC)	STEC CenterTW07886	5.3 x 107 CFU/mL	3/5§	3/3
Enteropathogenic Escherichia coli(EPEC)	STEC CenterE851/71	2.6 x 107 CFU/mL	3/6‡‡‡	3/3
Enterotoxigenic Escherichia coli(ETEC)	ATCC 35401	2.5 x 108 CFU/mL	3/3	3/3
Helicobacter pylori	ATCC 49503	≥1.0 x 106 CFU/mL†	3/3	3/3
Klebsiella pneumonia	ATCC 13883	3.2 x 108 CFU/mL	3/3	3/4§
Lactobacillus acidophilus	ATCC 4356	4.3 x 105 CFU/mL	3/3	3/3
Listeria monocytogenes	ATCC 19115	2.0 x 108 CFU/mL	3/3	3/3
Prevotella melaninogenicus	ATCC 25845	1.6 x 107 CFU/mL	3/3	3/3
Prevotella oralis	ATCC 33322	≥1.0 x 106 CFU/mL†	3/3	3/3
Salmonella typhimurium	ATCC 13311	4.1 x 108 CFU/mL	3/3	3/4§
Shigella sonnei	ATCC 29930	6.4 x 107 CFU/mL	3/3	3/3
Staphylococcus aureus	ATCC BK-23738	4.5 x 108 CFU/mL	3/3*	3/3
Vibrio cholera	ATCC 55188	3.8 x 108 CFU/mL	3/3	3/3
Yersinia enterocolitica	ATCC 49397	1.6 x 108 CFU/mL	3/3	3/3
Yeasts, Parasites, and Viruses
Blastocystis hominis (gDNA)	ATCC 50177D	1 ug/mL	3/3	6/6
Entamoeba histolytica	ATCC 30459DQ	1.0 x 108 copies/mL	3/3	3/3
Cryptosporidium parvum	ATCC PRA67D	1 ug/mL	3/3	3/3
Giardia lamblia (G. intestinalis)	ATCC 50803D	1 ug/mL	3/3	3/3
Candida albicans	ATCC 18804	3.0 x 106 CFU/mL	3/3	3/3
Saccharomyces cerevisiae	ATCC MYA-796	≥1.0 x 106 CFU/mL†	3/3^	3/4§
Adenovirus 40	ATCC VR-931D	1 ug/mL	3/3	3/3
Adenovirus 41	ATCC VR-930D	1 ug/mL	3/3	3/4§
Norovirus GI	ATCC VR-3234SD	1.0 x 108 copies/mL	3/3	3/4§
Norovirus GII	ATCC VR-3235SD	1.0 x 108 copies/mL	3/3	3/3
Rotavirus	ATCC VR-1546	1.0 x 105 TCID 50/mL	3/3	3/3
Human genomic DNA (HT-29)	ATCC HTB-38D	1 ug/mL	3/3	3/3

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Actual concentration undetermined, estimate based on optical density measurement.

^ Represents each 'INVALID' run in this dataset.

Represents each 'Test Incomplete' run in this dataset.

‡ Represents each replicate in this set that resulted in 'STEC NEGATIVE/ Serotype 0157 Not Tested'.

§ Represents each replicate in this set that resulted in 'STEC POSITIVE/ Serotype O157 NEGATIVE'.

Interfering Substances j.

The Shiga Toxin Direct Test was evaluated for chemical interference by the following panel of 26 different substances that are common stool contaminants or likely present in patients with diarrhea (Table 8). Each substance was tested in the background of a contrived, low positive that was generated by spiking a Shiga toxin-producing E. coli strain (ATCC 43895) containing all 3 Test analytes (stx1+, stx2+, and serotype O157)

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into ParaPak® C&S preserved clinical negative stool matrix at 2X LoD (1x104 CFU/mL). Clinical neqative stool matrix was also tested (i.e. negative stool specimen, non-STEC) to evaluate the potential for chemical substances to interfere with assay controls without analyte present.

Table 8. Interfering Substances Panel. Shiqa Toxin Direct Test performance evaluation for chemical interference in detecting a Shiga toxin-producing E. col/strain.

		% Agreement
		STEC Strain 43895		Clinical Negative
		Expected Result
Interfering Substance	Concentration Tested	STEC POSITIVE/Serotype 0157 POSITIVE		STEC NEGATIVE/Serotype 0157 Not Tested

	Endogenous Substances
Human Bile	25% v/v	100%	3/3	100%	3/3^^
Human Urine	50% v/v	100%	3/3	100%	3/3
Human Whole Blood	50% v/v	100%	3/3	100%	3/3
Cholesterol	5% w/v (50 mg/mL)	100%	3/3	100%	3/3
Fatty Acids	3.33% w/v (33.3 mg/mL)	100%	3/3	100%	3/3
Mucin	6.25% w/v (6.25 mg/mL)	100%	3/3	100%	3/3
Triglycerides	10% v/v	100%	3/3	100%	3/3
Exogenous Substances
Amoxicillin	5% w/v (50 mg/mL)	100%	3/3	100%	3/3
Baby Wipes	5% v/v	100%	3/3	100%	3/3
Barium Sulfate	9.9% w/v (99 mg/mL)	100%	3/3	100%	3/3
Ciprofloxacin	1.25% w/v (12.5 mg/mL)	100%	3/3	100%	3/3
Fleet Enema	50% v/v	100%	3/3	100%	3/3
Gaviscon Liquid Anacid	10% v/v	100%	3/3	100%	3/3
Glycerin Laxative	50% v/v	100%	3/3	100%	3/3
Hydrocortisone Cream	7.5% w/v (75 mg/mL)	100%	3/3	100%	3/3
Imodium	10% v/v	100%	3/3	100%	3/3
Personal Lubricant (K-Y Jelly)	50% v/v	100%	3/3^	100%	3/3
Laxative Tablet	0.97% w/v (9.7 mg/mL)	100%	3/3	100%	3/3
Metronidazole	5% w/v (50 mg/mL)	100%	3/3	100%	3/3
Milk of Magnesia	10% v/v	100%	3/3	100%	3/3
Mineral Oil	50% v/v	100%	3/3	100%	3/3
Pepto Bismal	10% v/v	100%	3/3	100%	3/3*
Preparation H Cream	9.5% w/v (95 mg/mL)	100%	3/3	100%	3/3
Stool Softener	0.7% w/v (7 mg/mL)	100%	3/3*	100%	3/3**^
Tums	20% (200 mg/mL)	100%	3/3	100%	3/3^
Vaginal Contraceptive Gel	50% v/v	100%	3/3	100%	3/3

Represents each 'Test Incomplete' run in this dataset.

^ Represents each 'INVALID' run in this dataset.

As summarized in Table 8, none of the chemical substances tested interfered with detection of either Shiga toxin or 0157 Serotype gene targets, and each test resulted in 'STEC POSITIVE/Serotype O157 POSITIVE' calls as expected. Additionally, none of the chemical substances interfered with assays controls when negative stool was tested. During evaluation of chemical interference, four (4) runs yielded incomplete testing results and five (5) 'invalid' tests were observed. In each instance the specimen was reretested on a new Shiga Toxin Direct Test cartridge and resulted in the correct call.

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Carry-Over/Cross-Over Contamination

A study was performed to assess the potential of carry-over or cross-contamination of the Shiga Toxin Direct Test by alternatively testing high positive contrived stool samples and clinical negative stool samples in direct succession for six (6) rounds on five (5) Portrait Analyzers. The high positive sample was formulated by spiking previously frozen and quantified enriched broth culture of STEC strain ATCC 43895 (stx1+/stx2+/0157) into negative clinical stool matrix consisting of clinical Shiga toxin neqative stool preserved in ParaPak® C&S media to obtain a final concentration of 1x108 CFU/mL. By running a series of alternating runs of high positive and negative samples on multiple Portrait Analyzers, potential carry-over/ cross-contamination was evaluated. In total, 60 Shiga Toxin Direct Test runs were performed: 30 high positive runs and 30 negative runs.

All of the Shiga Toxin Direct Test results were in concordance with expected test results. Therefore, there was no evidence of carry-over or cross-contamination in any of the tests. During carry-over/cross-contamination assessment, two (2) tests gave 'test incomplete' results and a single 'invalid' test results was observed. All 3 samples were re-tested on new cartridges and all resolved to the expected result.

-Media Equivalency Study (Poolability)

A Media Equivalency (Poolability) Study was conducted to demonstrate equivalent Shiqa Toxin Direct Test performance in six (6) widely used stool preservation media types, including: Thermo Scientific™ Remel™ Cary-Blair Transport Medium, Meridian™ Para-Pak® Enteric Plus Transport System, Thermo Scientific™ Protocol™ Cary-Blair Media, Thermo Scientific™ Protocol™ Culture & Sensitivity (C&S) Medium, Meridian™ Para-Pak® 10% Formalin Stool Transport Vial, Meridian™ Para-Pak® Zn PVA Stool Transport Vial.

Analytical Sensitivity (LoD) was established in Meridian™ Para-Pak® C&S. The Media Equivalency Study was designed to demonstrate equivalent Shiga Toxin Direct Test performance in each test media type by re-evaluating three (3) of the STEC strains for which LoD was initially measured at concentration near LoD (2X LoD), above LoD (5X LoD) and below LoD (0.5X LoD). To generate the unique stool matrices for each media type, raw clinical stool specimens that previously tested negative for Shiga Toxin were preserved in each preservation medium per the Manufacturer's instructions. The resulting stool matrices (6 in total) were evaluated directly as clinical negative samples and as the base for contrived positives of each media type.

For the Thermo Scientific™ Remel™ Cary-Blair Transport Medium, Meridian™ Para-Pak® Enteric Plus Transport System, Thermo Scientific™ Protocol™ Cary-Blair Media, and Thermo Scientific™ Protocol™ Culture & Sensitivity (C&S) Medium, the Shiga Toxin Direct Test performance was as expected and each test media type demonstrated equivalent performance to all other test mediums, as well as equivalent performance to the reference media, Meridian™ Para-Pak® C&S. At 5X LoD, for all strains tested, there was 100% agreement with the expected results in all four (4) media types. Likewise, at 2X LoD there was ≥95% agreement with the expected results for all strains tested across all four (4) media types. Also as expected, the percent agreement for strains below LoD (0.5X LoD) varied from 50% to 100% across these four (4) media types.

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The Meridian™ Para-Pak® 10% Formalin Stool Transport Vial media was initially tested at the highest concentrations, approximately 5X LoD for strain ATCC 51434 and approximately 5X and 2X LoD for ATCC BAA-2191 and ATCC 43895 strains. At these concentrations each strain was expected to return a 'positive' Shiga Toxin Direct Test result in ≥95% of replicates, however all of the test results were either `negative' (12%) or 'invalid' (88%). Clinical neqative stool matrix, formulated with 10% Formalin media, was also evaluated via Shiga Toxin Direct Test and expected to yield 100% 'negative' results, however only 20% (2/10 replicates) resolved as 'negative'. The remaining 80% of neqative stool replicates tested (8/10 replicates) also yielded 'invalid' results. At 85.7%, the overall invalid rate for initial testing (35 samples in total) was abnormally high, suggesting that 10% Formalin transport media inhibits the Shiga Toxin Direct Test. Due to the evident inhibition and hindered performance of the Shiga Toxin Direct Test, no further testing was conducted on this media type.

The Meridian™ Para-Pak® Zn PVA Stool Transport Vial media was initially tested at all three test concentrations for strain ATCC BAA-2191 (approximately 5X LoD, 2XLoD, and 0.5XLoD). At approximately 2X-5X LoD, ≥95% of the Shiga Toxin Direct Test replicates are expected to be 'STEC POSITIVE/Serotype 0157 NEGATIVE'. However in Zn PVA transport media, 100% of the Shiga Toxin Direct Test replicates resulted in 'INVALID' test results. Clinical neqative stool matrix, formulated with Zn PVA transport media, was also evaluated and expected to yield 100% 'negative' results, however 100% of the clinical neqative replicated resulted in 'INVALID' Shiga Toxin Direct Test results. Therefore, the invalid rate for this initial testing (39 samples in total) was 100%, suggesting that Zn PVA transport media completely inhibits the Shiga Toxin Direct Test. No further testing was conducted on this media type.

A summary of all media types tested and their resultant compatibility with the Shiqa Toxin Direct Test is provided in Table 9.

Stool Preservation Medias that are Compatible with the Shiga Toxin Direct Test
Meridian™ Para-Pak® C&S
Thermo Scientific™ Remel™ Cary-Blair Transport Medium
Meridian™ Enteric Plus Transport System
Thermo Scientific™ Protocol™ Cary-Blair Media
Thermo Scientific™ Protocol™ Culture & Sensitivity (C&S) Medium
Fixative-containing Medias that are not Compatible with Shiga Toxin Direct Test(Interference Observed)
Meridian™ Para-Pak® 10% Formalin Stool Transport Vial
Meridian™ Para-Pak® Zn PVA Stool Transport Vial

Table 9. Summary of Media Equivalency for Shiga Toxin Direct Test.

m. Reproducibility

Reproducibility testing of the Shiga Toxin Direct Test was conducted using a panel of five (5) prepared samples consisting of four 'positive' samples and one 'neqative'. The 'positive' panel constituents comprised two Shiga toxin-producing E. coli (STEC) strains: ATCC BAA-2192 (0145:NM) and ATCC strain 43895 (0157:H7) each at a 'Moderate Positive' concentration (~3X LoD) and a 'Low Positive' concentration (~1.5X LoD). The contrived positive samples were made by spiking the respective enriched broth cultures

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of known concentration into negative clinical stool matrix consisting of clinical Shiga toxin negative stool preserved in ParaPak® C&S media. The 'negative' samples consisted of only clinical negative stool matrix.

The Reproducibility studies were performed at three external clinical sites using randomized, blind-coded panels and two (2) different Shiga Toxin Direct Test cartridge lots. At each site, these studies were performed over the course of five (5), nonconsecutive days. For each day of testing, two (2) panel runs were performed with three (3) replicates of each sample per run (Table 10) on each day. A minimum of two (2) operators was required to perform Reproducibility testing at each site. Results of the Reproducibility studies are summarized in Table 11.

Test Sites	3 external sites
Panel Size	5 samples
Panel Constituents	1. Moderate Positive ATCC BAA-2192
	2. Low Positive ATCC BAA-2192
	3. Moderate Positive ATCC 43895
	4. Low Positive ATCC 43895
	5. Clinical Negative
Runs per Sample	3 replicates
Runs per Day (2 operators)	5 samples x 3 replicates x 2 operators = 30
Total Runs per Site (5 days)	30 runs/day x 5 days = 150
Total Runs	150 runs/site x 3 sites = 450

Table 10. Reproducibility Study Testing Protocol Overview.

Table 11. Overall Results of the Reproducibility Studies.

Sample Type(Panel Constituents)	Expected Result	% Agreement
		Site 1	Site 2	Site 5	All Sites
1. Moderate PositiveATCC BAA-2192	STEC POSITIVE/Serotype O157 NEGATIVE	30/30	100%	30/30	100%	30/30	100%	90/90	100%
2. Low PositiveATCC BAA-2192	STEC POSITIVE/Serotype O157 NEGATIVE	30/30	100%	30/30	100%	29/30	97%	89/90	99%
3. Moderate PositiveATCC 43895	STEC POSITIVE/Serotype O157 POSITIVE	30/30	100%	30/30	100%	30/30	100%	90/90	100%
4. Low PositiveATCC 43895	STEC POSITIVE/Serotype O157 POSITIVE	30/30	100%	30/30	100%	30/30	100%	90/90	100%
5. Clinical Negative	STEC NEGATIVE/Serotype O157 Not Tested	30/30	100%	30/30	100%	30/30	100%	90/90	100%

The cumulative data for Reproducibility testing of the Shiga Toxin Direct Test across all three sites is summarized in Table 11. The Shiga Toxin Direct Test results agreed with the expected results 100% across all three sites, with the exception of a single Low Positive replicate for ATCC BAA-2192 that produced a 'STEC POSITIVE/Serotype 0157 POSITIVE' test result instead of the expected result of 'STEC POSITIVE/Serotype 0157 NEGATIVE'.

The invalid and incomplete test rates for the reproducibility study were 1.1% (5 invalid runs/ 458 total runs) and 0.7% (3 test incomplete runs/ 458 total runs), respectively. In all eight (8) instances, the sample was re-tested on a new cartridge according to the

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package insert and each resolved to the expected result. All panel members produced acceptable performance results.

G. Performance Data - Prospective Clinical Studies

Specimens for the clinical study were collected prospectively (fresh) at five sites during a threemonth period from June to September 2015. A combined total of 1,116 stool samples were enrolled and evaluated. Of these, 1,082 clinical specimens met the inclusion criteria and were used in the prospective study to evaluate the performance of the Shiga Toxin Direct Test. Of these 1,082 specimens, 1,047 used C&S preservation medium, 34 used Cary-Blair preservation medium, and the preservation medium type was not specified for one specimen. Prospective evaluation was conducted by comparing the performance of the Portrait Shiga Toxin Direct Test to the reference clinical microbiology protocols for the detection of both Shiga Toxin and the E. coli 0157 Serotype. Results from these studies from all five sites are combined and summarized in Table 12.

Shiga toxin ( stx1/stx2 )
Reference Clinical Microbiology -Shiga Toxin EIA
	Positive	Negative	Total
Shiga ToxinDirect Test	Positive	4	8‡	12
	Negative	0	1,070	1,070
	Total	4	1,078	1,082
			LowerCl95	UpperCl95
Sensitivity		100.0%	39.8%	100.0%
Specificity		99.3%	98.5%	99.7%
PPV		33.3%	9.9%	65.1%
NPV		100.0%	99.7%	100.0%
ShigaDirect		Negative
	Total	0	12	12
			LowerCI95	UpperCI95
	Sensitivity	N/A	N/A	N/A
	Specificity	83.3%	51.6%	97.9%
	PPV	0.0%	0.0%	84.2%
	NPV	100.0%	69.2%	100.0%

Table 12. Overall Shiga Toxin Direct Test Performance from Prospective Testing at all Clinical Evaluation Sites.

Tes oxi

0157

Positive

Reference Clinical Microbiology -0157 Culture

Negative

Total

‡ Shiga toxin was detected in 8/8 false positive specimens by both bi-directional sequencing and alternate, FDA-cleared comparator NAAT.

‡ O157 serogroup was detected in 2/2 false positive specimens by alternate, FDA-cleared comparator NAAT.

Due to the low clinical prevalence of Shiga-toxin producing E. col/ (STEC), especially of the O157 serotype, a Frozen Retrospective panel was constructed and tested at three (3) clinical test sites in order to enrich the sample set for positives. This panel consisted of 92 unique clinical specimens previously characterized as positive or negative for STEC and the 0157 serotype. Of these, 88 frozen clinical specimens met the inclusion criteria and were used in the Frozen Retrospective study to evaluate the performance of the Shiga Toxin Direct Test. Of these 88 specimens, 44 used C&S preservation medium, 40 used Cary-Blair preservation medium, and 4 used Enteric Transport medium. Results from this study is summarized in Table The performance of the Shiga Toxin Direct Test, both in prospective and frozen 13. retrospective specimen testing, is summarized in comparison in Table 14.

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Table 13. Overall Shiga Toxin Direct Test Performance Results from Frozen Retrospective Specimen Testing.

Shiga toxin (stx1/stx2)
Clinical Characterization -
Molecular and/or Shiga Toxin EIA
	Positive	Negative	Total
Shiga ToxinDirect Test	Positive	51	0	51
	Negative	4	33	37
	Total	55	33	88
		LowerCI95	UpperCI95
PPA		92.7%	82.4%	98.0%
NPA		100.0%	89.4%	100.0%

O157
Clinical Characterization -
Molecular and/or O157 Culture
	Positive	Negative	Total
Shiga ToxinDirect Test	Positive	22	0	22
	Negative	1+	24+	25
	Total	23	24	47
		LowerCI95	UpperCI95
PPA		95.7%	78.1%	99.9%
NPA		100.0%	85.8%	100.0%

‡ The Shiga Toxin Direct Test result was 'STEC NEGATIVE/Serotype O157 Not Tested' in 1/1 false negative and 2/24 true negative specimens.

Table 14. Combined Shiga Toxin Direct Test Clinical Performance Results including Prospective and Frozen Retrospective Studies.

Specimen Type		n	% Agreement (95% CI)
			Positive	Negative
Shiga toxin(sx1/stx2)	Clinical Specimens	Fresh	1,082	100%4/4(39.8-100)	99.3%1,070/1,078(98.5-99.7)
		Frozen	88	92.7%51/55(82.4-98.0)	100%33/33(89.4-100)
E.coli O157	Clinical Specimens	Fresh	12	-	83.3%10/12(51.6-97.9)
		Frozen	47	95.7%22/23(78.1-99.9)	100%24/24(85.8-100)

H. Conclusion

The submitted information in this product notification is complete and supports a substantial equivalence decision.

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).