Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a multiplex nucleic acid test and an automated system for PCR amplification, target capture, signal generation, and optical detection. There is no mention of AI or ML being used for data analysis, interpretation, or any other function of the device. The performance studies focus on standard metrics for molecular diagnostics.

Therapeutic

No.
This device is an in vitro diagnostic (IVD) test that detects nucleic acids from gastrointestinal pathogens in stool samples. It is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness, but it is "not intended to monitor or guide treatment," which means it does not directly provide therapeutic intervention.

Diagnostic

Yes

The product's intended use explicitly states, "The BioCode Gastrointestinal Pathogen Panel (GPP) is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness."

SaMD (Software as a Medical Device)

No

The device description explicitly states that the BioCode Gastrointestinal Pathogen Panel (GPP) is a multiplex nucleic acid test designed to be used with the BioCode MDx 300 system, which is an automated system integrating PCR amplification, target capture, signal generation, and optical detection. This involves significant hardware components and processes beyond just software.

IVD (In Vitro Diagnostic)

Based on the provided information, the BioCode Gastrointestinal Pathogen Panel (GPP) is indeed an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The intended use explicitly states that the device is a "qualitative, gastrointestinal microorganism multiplexed nucleic acid-based assay capable of detecting of nucleic acids from the following organisms in unpreserved stool and Cary-Blair media." It is indicated as "an aid in the diagnosis of specific agents of gastrointestinal illness and results are meant to be used in conjunction with other clinical, laboratory, and epidemiological data." This clearly describes a test performed in vitro (outside the body) on biological specimens (stool) to provide information for diagnostic purposes.
Device Description: The description details a "multiplex nucleic acid test designed to be used with the BioCode MDx 300 system," which is an "automated system that integrates PCR amplification, target capture, signal generation and optical detection." This further confirms that the device performs laboratory testing on biological samples.
Performance Studies: The document describes extensive performance studies including clinical performance testing, reproducibility, limit of detection, interfering substances, and stability studies. These types of studies are standard for demonstrating the analytical and clinical validity of an IVD.
Key Metrics: The document lists key metrics such as Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), Sensitivity, Specificity, etc., which are commonly used to evaluate the performance of IVDs.
Predicate Device: The mention of a predicate device (Luminex xTAG® Gastrointestinal Pathogen Panel (GPP), K121454) is a strong indicator that this device is being compared to an already cleared IVD, which is a common pathway for regulatory submission of new IVDs.

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

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative, gastrointestinal microorganism multiplexed nucleic acid-based assay capable of detecting of nucleic acids from the following organisms in unpreserved stool and Cary-Blair media:

Adenovirus 40/41 ●
Campylobacter (C. jejuni, C. coli) ●
Clostridium difficile (C. difficile) toxin A/B (from fresh specimens only) ●
Cryptosporidium (C. parvum, C. hominis) 0
Entamoeba histolytica ●
Escherichia coli (E. coli) 0157 ●
Enterotoxigenic E. coli (ETEC) LT/ST ●
Enteroaggregative E. coli (EAEC) 0
Giardia lamblia (also known as G. intestinalis and G. duodenalis) ●
Norovirus GI/GII ●
Rotavirus A ●
Salmonella spp. .
Shiga-like Toxin producing E. coli (STEC) stx1/stx2 ●
Shigella (S. boydii, S. sonnei, S. flexneri, S. dysenteriae)/EIEC ●
Vibrio spp. (V. cholerae, V. parahaemolyticus, V. vulnificus), specific identification of V. ● parahaemolyticus
Yersinia enterocolitica

The BioCode Gastrointestinal Pathogen Panel (GPP) is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness and results are meant to be used in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule out co-infection with organisms not included in the BioCode Gastrointestinal Pathogen Panel (GPP). The agent detected may not be the cause of patient illness. 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.

Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor or guide treatment for C. difficile infection.

Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Campylobacter spp., E. coli 0157, Shigella/EIEC, Yersinia enterocolitica, and Adenovirus 40/41 were established primarily with retrospective clinical specimens.

Performance characteristics for Entamoeba histolytica, and Vibrio spp. (V. parahaemolyticus, V. vulnificus, and Vibrio cholerae) were established primarily using contrived clinical specimens.

Product codes

PCH, OOI

Device Description

The BioCode Gastrointestinal Pathogen Panel (GPP) is a multiplex nucleic acid test designed to be used with the BioCode MDx 300 system. The BioCode MDx 3000 is an automated system that integrates PCR amplification, target capture, signal generation and optical detection for nultiple gastrointestinal pathogens from a single stool speciment or in Cary Blair. Stool specimens are processed and nucleic acids extracted with easyMAG, an automated system. Once the PCR plate is set up and sealed, all other operations are automated on MDx 3000. The BioCode Gastrointestinal Pathogen Panel simultaneously tests for 17 pathogens (see table below) from unpreserved stool specimens or stool collected in Cary-Blair transport medium. Results from the BioCode Gastrointestinal Pathogen Panel test are available within less than 5 hours.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Gastrointestinal (via stool specimens)

Indicated Patient Age Range

The clinical study included patients with the following age categories: 99%. Qualitative results were as expected with the exception of one false positive for Giardia lamblia and one false negative for STEC. Quantitative results for mean fluorescence index (MFI) and coefficient of variation (%CV) were provided for each target at Low and Medium positive concentrations, showing instrument precision.

5. Analytical Performance - Limit of Detection (LoD)

Study Type: LoD determination study.
Key results: LoD for each stock was defined as the lowest concentration with ≥95% detection of 20 replicates (19 out of 20), determined separately for unpreserved stool and Cary-Blair preserved stool. Specific LoD values (CFU/mL, oocysts/mL, TCID50/mL, copies/gram) for each organism in both unpreserved and Cary-Blair stool were established.

6. Analytical Performance - Interfering Substances

Study Type: Interference study.
Key results: No interfering substances or microorganisms were identified from a panel of potential microbial interferents and other substances at high concentrations, aside from one false positive for Adenovirus 40/41 with Bacteroides fragilis that was not reproducible.

7. Analytical Performance - Analytical Reactivity/Inclusivity

Study Type: Inclusivity study.
Key results: Demonstrated detection of various strains and serotypes for each analyte at 3X LoD (or higher for some Norovirus strains). All organisms were detected at the indicated concentrations.

8. Analytical Performance - Analytical Specificity/Cross Reactivity

Study Type: Cross-reactivity study (empirical and in silico analysis).
Key results: Cross-reactivity was not observed with most microorganisms tested. Noted potential cross-reactivity:
- Vibrio spp assay may react with V. alginolyticus and V. mimicus.
- Y. enterocolitica assay has potential for cross-reactivity with Y. bercovieri, Y. frederiksenii, Y. intermedia, Y. mollaretii near LoD, and Y. rohdei at high levels.
- Shiga toxin in Shigella dysenteriae may cause STEC and Shigella/EIEC co-detection.
- Assays detect recombinant viruses in Rotavirus vaccines.
- Potential for cross-reactivity with C. meleagridis by Cryptosporidium assay.
- No cross-reactivity predicted by in silico analysis for many other organisms.

9. Analytical Performance - Competitive Inhibition

Study Type: Competitive inhibition study evaluating mixed infections.
Key results: No competitive inhibition was observed when high concentrations of one target were mixed with medium concentrations of two other targets.

10. Analytical Performance - Fresh vs. Frozen Stability

Study Type: Specimen stability study.
Key results: Specimens displayed stability throughout the study length. 95% replicates for positive targets were valid and detected (63/63), and 95% of negative targets were valid and not detected (63/63, with one sample target detected but invalid for RNA IC).

11. Analytical Performance - Specimen Stability

Study Type: Specimen stability study under various storage conditions.
Key results: Samples displayed stability under the various conditions throughout the length of the study (Cary-Blair: 0, 2, 4, 6 days at room temp and 2-8°C; Unpreserved Stool: 0, 2, 4, 6 days at 2-8°C; Fresh vs. Frozen (-90°C to -60°C): 30, 60, 90 days).

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

Clinical sensitivity/positive agreement (PPA) = TP/(TP + FN)
Clinical specificity/negative agreement (NPA) = TN/(TN + FP)
Specific values for PPA and NPA are listed in the "Summary of Performance Studies" for each performance study and target.

Predicate Device(s)

K121454 – Luminex xTAG® Gastrointestinal Pathogen Panel (GPP)

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3990 Gastrointestinal microorganism multiplex nucleic acid-based assay.

(a)
Identification. A gastrointestinal microorganism multiplex nucleic acid-based assay is a qualitativein vitro diagnostic device intended to simultaneously detect and identify multiple gastrointestinal microbial nucleic acids extracted from human stool specimens. The device detects specific nucleic acid sequences for organism identification as well as for determining the presence of toxin genes. The detection and identification of a specific gastrointestinal microbial nucleic acid from individuals exhibiting signs and symptoms of gastrointestinal infection aids in the diagnosis of gastrointestinal infection when used in conjunction with clinical evaluation and other laboratory findings. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.(b)
Classification. Class II (special controls). The special controls are set forth in FDA's guideline document entitled: “Class II Special Controls Guideline: Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assays for Detection and Identification of Microorganisms and Toxin Genes from Human Stool Specimens.” For availability of the guideline document, see § 866.1(e).

Summary

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo features a stylized representation of a human figure in profile, composed of three overlapping shapes. The figure is positioned to the right of a circular seal that contains the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter.

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

September 28, 2018

Applied BioCode Inc.

Robert Di Tullio Regulatory Consultant 10020 Pioneer Blvd. Suite 102 Santa Fe Springs. CA 90670

Re: K180041

Trade/Device Name: BioCode Gastrointestinal Pathogen Panel (GPP) Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal Microorganism Multiplex Nucleic Acid-based Assay Regulatory Class: Class II Product Code: PCH, OOI Dated: January 08, 2018 Received: January 09, 2018

Dear Mr. Di Tullio:

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 Part 801 and Part 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

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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and Part 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/ResourcesforYou/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,

Steven R. Gitterman -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) K180041

Device Name

BioCode Gastrointestinal Pathogen Panel (GPP)

Indications for Use (Describe)

The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative, gastrointestinal microorganism multiplexed nucleic acid-based assay capable of detecting of nucleic acids from the following organisms in unpreserved stool and Cary-Blair media:

Adenovirus 40/41 ●
Campylobacter (C. jejuni, C. coli) ●
Clostridium difficile (C. difficile) toxin A/B (from fresh specimens only) ●
Cryptosporidium (C. parvum, C. hominis) 0
Entamoeba histolytica ●
Escherichia coli (E. coli) 0157 ●
Enterotoxigenic E. coli (ETEC) LT/ST ●
Enteroaggregative E. coli (EAEC) 0
Giardia lamblia (also known as G. intestinalis and G. duodenalis) ●
Norovirus GI/GII ●
Rotavirus A ●
Salmonella spp. .
Shiga-like Toxin producing E. coli (STEC) stx1/stx2 ●
Shigella (S. boydii, S. sonnei, S. flexneri, S. dysenteriae)/EIEC ●
Vibrio spp. (V. cholerae, V. parahaemolyticus, V. vulnificus), specific identification of V. ● parahaemolyticus
Yersinia enterocolitica

The BioCode Gastrointestinal Pathogen Panel (GPP) is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness and results are meant to be used in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule out co-infection with organisms not included in the BioCode Gastrointestinal Pathogen Panel (GPP). The agent detected may not be the cause of patient illness. 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.

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Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor or guide treatment for C. difficile infection.

Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Campylobacter spp., E. coli 0157, Shigella/EIEC, Yersinia enterocolitica, and Adenovirus 40/41 were established primarily with retrospective clinical specimens.

Performance characteristics for Entamoeba histolytica, and Vibrio spp. (V. parahaemolyticus, V. vulnificus, and Vibrio cholerae) were established primarily using contrived clinical specimens.

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

Prescription Use (Part 21 CFR 801 Subpart D)

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

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APPLIED BIOCODE, INC. 10020 PIONEER BLVD. SUITE 102 SANTA FE SPRINGS, CA 90670, USA

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510(k) SUMMARY

Introduction: According to the requirements of 21 CFR 807.92, the following provides sufficient information to understand the basis for a determination of substantial equivalence.

Submitted by:

Applied BioCode, Inc. 10020 Pioneer Blvd. Suite 102 Santa Fe Springs, CA 90670

Contact:

Robert Di Tullio Regulatory Consultant rditullio@apbiocode.com Telephone: 310 801 1235 Fax: 323 372 3816

Date Submitted:

August 22, 2018

Trade Name:

BioCode Gastrointestinal Pathogen Panel

Classification Name and Regulation Number:

Gastrointestinal microorganism multiplex nucleic acid-based assay (21 CFR 866.3990)

Predicate Device:

K121454 – Luminex xTAG® Gastrointestinal Pathogen Panel (GPP)

Intended Use:

The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative, gastrointestinal microorganism multiplexed nucleic acid-based assy capable of detecting of nucleic acids from the following organisms in unpreserved stool and Cary-Blair media:

□ Adenovirus 40/41

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Campylobacter (C. jejuni, C. coli)

Clostridium difficile (C. difficile) toxin A/B (from fresh specimens only)

Cryptosporidium (C. parvum, C. hominis)

Entamoeba histolytica
Escherichia coli (E. coli) 0157
Enterotoxigenic E. coli (ETEC) LT/ST
Enteroaggregative E. coli (EAEC)
Giardia lamblia (also known as G. intestinalis and G. duodenalis)

Norovirus GI/GII

Rotavirus A
Salmonella spp.
Shiga-like Toxin producing E. coli (STEC) stx1/stx2
Shigella (S. boydii, S. sonnei, S. flexneri, S. dysenteriae)/EIEC
Vibrio spp. (V. cholerae, V. parahaemolyticus, V. vulnificus), specific identification of V. parahaemolyticus
Yersinia enterocolitica

The BioCode Gastrointestinal Pathogen Panel in the diagnosis of specific agents of gastrointestinal illness and results are meant to be used in conjunction with other clinical, laboratory, and epidemiological data. Positive results do not rule out co-infection with organisms not included in the BioCode Gastrointestinal Pathogen Panel (GPP). The agent detected may not be the cause of patient illness. Negative results in the setting of clinical illness compatible with gastroenteritis may be due to infection by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor or guide treatment for C. difficile infection.

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Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Compylobacter spp., E. coll O157, Shigella/Elec, Yersinio enterocoltico, and Adenovirus 40/41 were established primarily with retrospective clinical specimens.

Performance characteristics for Entamoebo histolytica, and Vibrio spp. (V. parahaemolyticus, V. vulnificus, and Vibrio cholerae) were established primarily using contrived clinical specimens.

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Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Adenovirus 40/41, Campylobacter, E. coli 0157, Shigello/ElEC, Yersinia enterocoltica, and Giardied additionally with retrospective clinical specimens. Performance characteristics for Entamoeba histolytica, Giardia lamblia, Yersinia enterocolitica and Vibrio (V. parahaemolyticus, V. valificus, and V. cholerae) were established primarily using contrived clinical specimens.

A gastrointestinal microorganism multiplex nucleic acids in the detection and identification of acute gastroenteritis in the context of outbreaks.

Device Description:

The BioCode Gastrointestinal Pathogen Panel (GPP) is a multiplex nucleic acid test designed to be used with the BioCode MDx 300 system. The BioCode MDx 3000 is an automated system that integrates PCR amplification, target capture, signal generation and optical detection for nultiple gastrointestinal pathogens from a single stool speciment or in Cary Blair. Stool specimens are processed and nucleic acids extracted with easyMAG, an automated system. Once the PCR plate is set up and sealed, all other operations are automated on MDx 3000. The BioCode Gastrointestinal Pathogen Panel simultaneously tests for 17 pathogens (see table below) from unpreserved stool specimens or stool collected in Cary-Blair transport medium. Results from the BioCode Gastrointestinal Pathogen Panel test are available within less than 5 hours.

Bacteria	Parasites
▪ Campylobacter spp. (C. jejuni, C. coli)	▪ Cryptosporidium spp.(C. parvum/C. hominis)
▪ Clostridium difficile toxin A/B	▪ Entamoeba histolytica
▪ Enteroaggregative E. coli (EAEC)	▪ Giardia lamblia/intestinalis
▪ Enterotoxigenic E. coli (ETEC): LT/ST	Viruses
▪ Shiga-toxin producing E. coli (STEC): stx1/stx2	▪ Adenovirus 40/41
▪ E.coli O157	▪ Norovirus GI/GII
▪ Shigella spp. /Enteroinvasive E.coli (EIEC)	▪ Rotavirus A
▪ Salmonella spp.	RNA Internal Control
▪ Vibrio parahaemolyticus
▪ Vibrio spp (not parahaemolyticus)
▪ Yersinia enterocolitica

Bacteria, Viruses, and Parasites Detected by the BioCode Gastrointestinal Pathogen Panel

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Device Comparison:

Comparison of the BioCode Gastrointestinal Pathogen Panel with the Predicate Device

Characteristic	Proposed Device	Predicate
Name	Applied BioCode, Inc. BioCode Gastrointestinal Pathogen Panel	xTAG®
Gastrointestinal
Pathogen Panel (GPP)
Common Name	Gastrointestinal Microorganism Multiplex Nucleic acid-based assay	Gastrointestinal
Microorganism
Multiplex Nucleic
acid-based assay
510(k) No.	N/A	K121454
Regulation	21CFR 866.3990	21CFR 866.3990
Product Code	PCH, OOI	PCH, OOI
Device Class	II	II
Similarities
Characteristic	Proposed Device	Predicate
Intended Use	The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplexed	Equivalent
	nucleic acid-based in vitro diagnostic test intended for use with the BioCode MDx
	3000 Instrument. The BioCode GPP is capable of the simultaneous detection and
	identification of nucleic acids from multiple bacteria, viruses, and parasites
	extracted directly from unpreserved stool samples or stool preserved in Cary-Blair
	transport medium obtained from individuals with signs and/or symptoms of
	gastrointestinal infection. The following bacteria (including several diarrheagenic
	E. coli/Shigella pathotypes), parasites, and viruses are identified using the BioCode
	GPP: Campylobacter (C. jejuni/C. coli), Clostridium difficile (C. difficile) toxin A/B,
	Salmonella spp, Vibrio (V. parahaemolyticus/V. vulnificus/ V. cholerae) , including
	specific identification of Vibrio parahaemolyticus, Yersinia enterocolitica ,
	Enteroaggregative Escherichia coli (EAEC), Enterotoxigenic Escherichia coli (ETEC)
	It/st, E. coli O157 serogroup, Shiga-like toxin-producing Escherichia coli (STEC)
	stx1/stx2, Shigella / Enteroinvasive Escherichia coli (EIEC), Cryptosporidium spp,
	Entamoeba histolytica, Giardia lamblia (also known as G. intestinalis and G.
	duodenalis ), Adenovirus F 40/41, Norovirus GI/GII, Rotavirus A. The BioCode GPP is
	indicated as an aid in the diagnosis of specific agents of gastrointestinal illness and
	results are meant to be used in conjunction with other clinical, laboratory, and
	epidemiological data. For In Vitro Diagnostic Use Only. For Prescription Use Only.
	Positive results do not rule out co-infection with organisms not included in the
	BioCode GPP. The agent detected may not be the definite cause of the disease.
	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.
	Concomitant culture is necessary for organism recovery and further typing of
	bacterial agents. This device is not intended to monitor or guide treatment for C.
	difficile infection. Due to the small number of positive specimens collected for
	certain organisms during the prospective clinical study, performance characteristics
	for Adenovirus 40/41, Campylobacter, E. coli O157, Shigella/EIEC, Yersinia
	enterocolitica , and Giardia lamblia were established additionally with retrospective
	clinical specimens. Performance characteristics for Entamoeba histolytica, Giardia
	lamblia, Yersinia enterocolitica and Vibrio (V. parahaemolyticus, V. vulnificus, and V.
	cholerae) were established primarily using contrived clinical specimens. A
	gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the
	detection and identification of acute gastroenteritis in the context of outbreaks.
Characteristic	Proposed Device	Predicate
Instrument	Nucleic Acid Purification System
BioCode MDx 3000	Nucleic Acid
Purification System
PCR Thermocycler
Luminex® 100/200TM
or MAGPIX
instruments
Sample Type	Unpreserved stool and stool in Cary- Blair Media	Same
Controls	Externally Sourced -	Same
Differences
Methodology	Multiplex RT-PCR and probe hybridization followed by fluorescence detection and
decoding of barcoded magnetic beads (BMB) that are coupled to biotinylated
products with streptavidin conjugate	Multiplex RT-PCR and
multiplex TSPE
followed by
fluorescence-
activated sorting of
labeled beads coupled
to streptavidin-
conjugated
biotinylated products.
Calibrators	Internal Calibration	External Calibration
Kit

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Summary of Performance Characteristics of the

BioCode GPP Clinical Performance

Testing of Prospectively collected Specimens (Categories I and II)

A clinical investigational study was performed in which a total of 1558 leftover, de-identified samples were prospectively collected from patients who underwent stool sample collection for clinical indications at four (4) investigational sites, with each collecting approximately 400 samples. The sites were located in the United States, with a broad geographic representation. The sites were located in the Northeast (Baltimore, MD), Southeast (Tampa, FL), Midwest (Memphis, TN), and West (Los Angeles, CA). In addition testing was performed on archived known positives and contrived specimens to further augment the sample numbers. Each raw stool specimen was de-identified at the site and the de-identified leftover sample divided into aliquots and either tested freshly on the ABC IUO assay at the site, or frozen and tested at a later date at the site. Each stool specimen in Cary-Blair was similarly de-identified at the site and the de-identified leftover sample aliquoted into 4 aliquots and tested freshly on the ABC IUO assay at the site. All prospectively collected samples had age, sex, and therapy status collected by the site. Enrollment of samples covered multiple calendar seasons beginning January 2015 and ending in August 2017, within which to enroll the specimens displaying due diligence in attempting to cover all panel targets with native specimens. The results of the clinical study follow.

Prospective Study Specimens
Total Specimens	1558
Gender (n/N(%))
Female	778/1558 (49.9)
Male	780/1558 (51.1)
Age Category (n/N(%))
Shigella/EIEC	ETEC
Total Co-infections
Double Infections
Triple Infections

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Testing of inoculated Cary-Blair specimens from previously frozen prospective specimens

To supplement the number of prospective Cary-Blair specimens. 400 unpreserved stool samples from sites 1 and 2 were thawed and inoculated into Cary-Blair. 3 were removed from the study for improper storage prior to testing. 2 were invalid for RNA IC failure in the unpreserved stool. The following table summarizes the comparison of the results from the initial unpreserved testing and subsequent inoculated results.

Summary of Inoculated Cary-Blair or unpreserved samples Vs. reference method. Agreements were calculated compared to reference method testing of unpreserved stool. Reference testing was not repeated after samples were inoculated to Cary-Blair.

		(n)	Positive Agreement		Negative Agreement
Target	Specimen Type		PPA (%)	95% CI	NPA (%)	95% CI
Campylobacter spp.a	Unpreserved	394	2/2 (100.0)	(15.81, 100.0)	385/392 (98.21)	(96.36, 99.28)
Campylobacter spp.a	Cary-Blair
(Inoculated)	396	2/2 (100.0)	(15.81, 100.0)	388/394 (98.48)	(96.72, 99.44)
E. coli O157b	Unpreserved	395	1/2 (50.00)	(1.26, 98.74)	389/393 (98.98)	(97.41, 99.72)
E. coli O157b	Cary-Blair
(Inoculated)	397	1/2 (50.00)	(1.26, 98.74)	391/395 (98.99)	(97.43, 99.72)
Enteroaggregative E. coli
(EAEC)c	Unpreserved	394	12/14 (85.71)	(57.19, 98.22)	378/380 (99.47)	(98.11, 99.94)
Enteroaggregative E. coli
(EAEC)c	Cary-Blair
(Inoculated)	396	12/14 (85.71)	(57.19, 98.22)	382/382 (100.0)	(99.04, 100.0)
Enterotoxigenic
E. coli (ETEC)d	Unpreserved	394	4/6 (66.67)	(22.28, 95.67)	387/388 (99.74)	(98.57, 99.99)
Enterotoxigenic
E. coli (ETEC)d	Cary-Blair
(Inoculated)	396	4/6 (66.67)	(22.28, 95.67)	386/390 (98.97)	(97.39, 99.72)
Shiga toxin-producing E.
coli (STEC)	Unpreserved	361	2/2 (100.0)	(15.81, 100.0)	359/359 (100.0)	(98.98, 100.0)
Shiga toxin-producing E.
coli (STEC)	Cary-Blair
(Inoculated)	363	2/2 (100.0)	(15.81, 100.0)	361/361 (100.0)	(98.98, 100.0)
Salmonella spp.e	Unpreserved	395	5/6 (83.33)	(35.88, 99.58)	385/389 (98.97)	(97.39, 99.72)
Salmonella spp.e	Cary-Blair
(Inoculated)	397	6/6 (100.0)	(54.07, 100.0)	389/391 (99.49)	(98.16, 99.94)
Shigella/ EIECf	Unpreserved	395	1/1 (100.0)	(2.50, 100.0)	389/394 (98.73)	(97.06, 99.59)
Shigella/ EIECf	Cary-Blair
(Inoculated)	397	1/1 (100.0)	(2.50, 100.0)	391/396 (98.74)	(97.08, 99.59)
Vibrio parahaemolyticus	Unpreserved	395	N/A	N/A	395/395 (100.0)	(99.07, 100.0)
Vibrio parahaemolyticus	Cary-Blair
(Inoculated)	397	N/A	N/A	397/397 (100.0)	(99.08, 100.0)
Vibrio spp. (not
parahaemolyticus)	Unpreserved	395	N/A	N/A	395/395 (100.0)	(99.07, 100.0)
	Cary-Blair
(Inoculated)	397	N/A	N/A	397/397 (100.0)	(99.08, 100.0)
Yersinia enterocoliticag	Unpreserved	395	N/A	N/A	394/395 (99.75)	(98.60, 99.99)
Yersinia enterocoliticag	Cary-Blair
(Inoculated)	397	N/A	N/A	396/397 (99.75)	(98.60, 99.99)
Cryptosporidium spp	Unpreserved	394	2/2 (100.0)	(15.81, 100.0)	392/392 (100.0)	(99.06, 100.0)
Cryptosporidium spp	Cary-Blair
(Inoculated)	396	2/2 (100.0)	(15.81, 100.0)	394/394 (100.0)	(99.07, 100.0)
Entamoeba histolytica	Unpreserved	394	N/A	N/A	394/394 (100.0)	(99.07, 100.0)
Entamoeba histolytica	Cary-Blair
(Inoculated)	396	N/A	N/A	396/396 (100.0)	(99.07, 100.0)
Target	Specimen Type	(n)	Positive Agreement		Negative Agreement
			PPA (%)	95% CI	NPA (%)	95% CI
Giardia lambliah	Unpreserved	394	N/A	N/A	391/394 (99.24)	(97.79, 99.84)
Giardia lambliah	Cary-Blair
(Inoculated)	396	N/A	N/A	394/396 (99.49)	(98.19, 99.94)
Adenovirus 40/41i	Unpreserved	394	3/6 (50.00)	(11.81, 88.19)	388/388 (100.0)	(99.05, 100.0)
Adenovirus 40/41i	Cary-Blair
(Inoculated)	396	2/6 (33.33)	(4.33, 77.72)	385/390 (98.72)	(97.03, 99.58)
Norovirus
(GI/GII)	Unpreserved	395	28/28 (100.0)	(87.66, 100.0)	364/367 (99.18)	(97.63, 99.83)
Norovirus
(GI/GII)	Cary-Blair
(Inoculated)	397	28/28 (100.0)	(87.66, 100.0)	364/369 (98.64)	(96.87, 99.56)
Rotavirus A	Unpreserved	395	11/12 (91.67)	(61.52, 99.79)	380/383 (99.22)	(97.73, 99.84)
Rotavirus A	Cary-Blair
(Inoculated)	397	11/12 (91.67)	(61.52, 99.79)	380/385 (98.70)	(97.00, 99.58)

22

a – Campylobacter spp. Unpreserved: The 6 false positives compared to reference culture method were tested by bidirectional sequencing and 4 of 6 confirmed as positives. Cary-Blair: The 7 false positives compared to the reference culture method were tested by bidirectional sequencing and 4 of 7 confirmed as positives.

b - E. coli 0157. Unpreserved: The one false negative compared to the reference culture method was tested by bidirectional sequencing and could not be confirmed as positives compared to the reference culture method were tested by bidirectional sequencing and 3 of 4 confirmed as positives. Cary-Blair: The one false negative compared to the reference culture method was tested by bidirectional sequencing and could not be confirmed as positives compared to reference culture method were tested by bidirectional sequencing and 3 of 4 confirmed as positives.

c - EAEC. Unpreserved: The 2 false negatives compared to bidirectional sequencing were tested by 2 additional rounds of sequencing; 1 of the 2 confirmed as positive. Cary-Blair: The 2 false negatives compared to bidirectional sequencing were tested by 2 additional rounds of sequencing; 1 of the 2 confirmed as positives compared to bidirectional sequencing were not confirmed as positive by 2 additional rounds of sequencing.

d - ETEC. Unpreserved: The 2 false negatives compared to bidirectional sequencing were tested by 2 additional rounds of sequencing; none were confirmed as positives were confirmed as positive by 2 additional rounds of sequencing. Cary-Blair: The 2 false negatives compared to bidirectional sequencing were tested by 2 additional rounds of sequencing; none were confirmed as positive compared to bidirectional sequencing was not available for confirmation testing.

e – Salmonella spp. Unpreserved: The 2 false positives compared to the reference culture method were tested by bidirectional sequencing and 1 of 2 confirmed as positives. Cary-Blair: The one false negative compared to the reference culture method was tested by bidirectional sequencing and confirmed as positives compared to the reference culture method were tested by bidirectional sequencing and 2 of 4 confirmed as positives.

f - Shigella/EIEC. Unpreserved: The 5 false positives compared to the reference culture method were tested by bidirectional sequencing and 4 of 5 confirmed as positives. Cary-Blair: The 5 false positives compared to the reference culture method were tested by bidirectional sequencing and 4 of 5 confirmed as positives.

g - Yersinia enterocolitica. Unpreserved: The 1 false positive compared to the reference culture method was tested by bidirectional sequencing and confirmed as positive. Cary-Blair: The 1 false positive compared to the reference culture method were tested by bidirectional sequencing and confirmed as positive.

h - Giardia lamblia. Unpreserved: The 2 false positives compared to bidirectional sequencing were not confirmed as positive by 2 additional rounds of sequencing. Cary-Blair: The 3 false positives compared to bidirectional sequencing were not confirmed as positive by 2 additional rounds of sequencing.

i – Adenovirus 40/41. Unpreserved: The 4 false negatives compared to bidirectional sequencing were tested by 2 additional rounds of sequencing; 1 of 4 was confirmed as positives were not confirmed as positives by an additional round of sequencing. Cary-Blair: The 3 false negatives compared to bidirectional sequencing were tested by 2 additional rounds of sequencing; none confirmed as positive.

23

Testing of Pre-selected Archived Specimens (Category III)

Several analytes were not encountered or had low prevalence in the clinical study. To supplement the results of the prospective clinical study, 260 preselected archived specimens were assayed. These were archived clinical specimens that were previously tested positive by different methods. Prior to testing with the Applied BioCode Gastrointestinal Pathogen Panel, the presence of the expected analyte was verified in each specimen using analyte-specific PCR followed by bi-directional sequencing performed at Applied BioCode, Inc. The specimens were randomized with negative specimens, such that the users performing the BioCode GPP were blinded to the expected test result. A summary of the demographic information of the tested samples and the results of the BioCode GPP testing are presented in Tables below.

Prospective Study Specimens
Total Specimens	260
Gender (n/N(%))
Female	123/260 (47.3)
Male	137/260 (52.7)
Age Category (n/N(%))
60	60

Table. Demographic information for Asymptomatic Volunteers.

Table. Detections in Asymptomatic Volunteers-Stratified by Age

| Analyte | 99%.

	Concentration Level
	Medium Positive		Low Positive		Negative
Target	Detection	95% CI	Detection	95% CI	Agreement	95% CI
	n/N (%)		n/N (%)		n/N (%)
Salmonella bongori	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
Clostridium difficile	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
Giardia lambia	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	449/450
(99.78)	(98.77,
99.99)
Adenovirus 40/41	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
Shigella sonnei	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
Vibrio
parahaemolyticus	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
ETEC	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
Yersinia
enterocolitica	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
STEC	90/90
(100.0)	(95.98,
100.0)	89/90
(98.89)	(93.96,
99.97)	450/450
(100.0)	(99.18,
100.0)
Campylobacter
jejuni	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
Rotavirus A	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)
Cryptosporidium
parvum	90/90
(100.0)	(95.98,
100.0)	90/90
(100.0)	(95.98,
100.0)	450/450
(100.0)	(99.18,
100.0)

Qualitative results from Reproducibility study. All results are as expected with the exception of one false positive for Giardia lamblia and one false negative for STEC.

33

Quantitative results from Reproducibility study. Fluorescent signals from BMBs with the same barcode and calculated to generate median fluorescence index (MFI) for each analyte (shown below). The presence or absence of a pathogen is determined relative to the validated assay utoff by MFI.

| Target
Analyte/ Probe | Analyte
Concentration | Analyte
Mean | Repeatability
SD | Repeatability
%CV | Between Runs
SD | Between Runs
%CV | Between Days
SD | Between Days
%CV | Between Sites
SD | Between Sites
%CV | Total
SD | Total
%CV |
|--------------------------|---- | Salmonella | Low | Salmonella | Med | tcdA (C. difficile) | Low | tcdA (C. difficile) | Med | tcdB (C. difficile) | Low | tcdB (C. difficile) | Med | Giardia lamblia | Low | Giardia lamblia | Med | Adenovirus 40 | Low | Adenovirus 40 | Med | Shigella sonnei | Low | Shigella sonnei | Med | Vibrio parahaemolyticus | Low | Vibrio parahaemolyticus | Med | ST-a (ETEC) | Low | ST-a (ETEC) | Med | ST-b (ETEC) | Low | ST-b (ETEC) | Med | Yersinia enterocolitica | Low | Yersinia enterocolitica | Med | stx2 | Low | stx2 | Med | Campylobacter jejuni | Low | Campylobacter jejuni | Med | | | Rotavirus A | Low | Rotavirus A | Med | Cryptosporidium parvum | Low | Cryptosporidium parvum | Med ----------------------|-----------------|---------------------|----------------------|--------------------|---------------------|--------------------|---------------------|---------------------|----------------------|-------------|--------------|
| 9174 | 1853 | 20.2 | 622 | 6.78 | 1464 | 15.95 | 0 | 0 | 2442 | 26.62 |
| 11572 | 1204 | 10.41 | 469 | 4.05 | 1980 | 17.11 | 1190 | 10.28 | 2647 | 22.87 |
| 5144 | 1407 | 27.35 | 1306 | 25.4 | 689 | 13.39 | 882 | 17.15 | 2222 | 43.2 |
| 8802 | 2381 | 27.05 | 1444 | 16.4 | 1645 | 18.69 | 1337 | 15.19 | 3499 | 39.76 |
| 11595 | 2004 | 17.29 | 847 | 7.31 | 2293 | 19.78 | 0 | 0 | 3161 | 27.27 |
| 16441 | 1878 | 11.42 | 2219 | 13.49 | 2804 | 17.05 | 682 | 4.15 | 4096 | 24.91 |
| 30206 | 2436 | 8.06 | 2164 | 7.16 | 2516 | 8.33 | 3342 | 11.07 | 5302 | 17.55 |
| 20485 | 1024 | 5 | 3824 | 18.67 | 0 | 0 | 2839 | 13.86 | 4871 | 23.78 |
| 17155 | 2043 | 11.91 | 2850 | 16.62 | 1987 | 11.58 | 1030 | 6.01 | 4160 | 24.25 |
| 20850 | 1699 | 8.15 | 2666 | 12.79 | 1949 | 9.35 | 2447 | 11.74 | 4448 | 21.33 |
| 6115 | 1654 | 27.05 | 1487 | 24.31 | 1337 | 21.87 | 1043 | 17.06 | 2797 | 45.74 |
| 9707 | 2492 | 25.68 | 1968 | 20.28 | 0 | 0 | 1669 | 17.19 | 3588 | 36.96 |
| 9927 | 2690 | 27.1 | 792 | 7.98 | 2956 | 29.78 | 966 | 9.73 | 4188 | 42.18 |
| 12421 | 1540 | 12.39 | 2018 | 16.25 | 3126 | 25.16 | 0 | 0 | 4026 | 32.42 |
| 14272 | 3469 | 24.31 | 3562 | 24.96 | 3910 | 27.4 | 0 | 0 | 6326 | 44.32 |
| 21453 | 4151 | 19.35 | 2943 | 13.72 | 4869 | 22.7 | 2322 | 10.83 | 7416 | 34.57 |
| 13193 | 3246 | 24.6 | 1992 | 15.1 | 3719 | 28.19 | 733 | 5.56 | 5373 | 40.73 |
| 19807 | 3205 | 16.18 | 2141 | 10.81 | 4756 | 24.01 | 1829 | 9.23 | 6389 | 32.26 |
| 19049 | 1970 | 10.34 | 1729 | 9.08 | 1647 | 8.65 | 2326 | 12.21 | 3872 | 20.33 |
| 20020 | 1246 | 6.22 | 1474 | 7.36 | 2036 | 10.17 | 2156 | 10.77 | 3538 | 17.67 |
| 16291 | 3347 | 20.54 | 5757 | 35.34 | 2353 | 14.44 | 5161 | 31.68 | 8747 | 53.69 |
| 21117 | 2977 | 14.1 | 6931 | 32.82 | 0 | 0 | 5593 | 26.49 | 9390 | 44.47 |
| 21865 | 2384 | 10.9 | 3282 | 15.01 | 2727 | 12.47 | 2302 | 10.53 | 5402 | 24.71 |
| 26561 | 2992 | 11.26 | 3871 | 14.58 | 2549 | 9.6 | 2903 | 10.93 | 6234 | 23.47 |
| | | | | | | | | | | |
| 45792 | 4269 | 9.32 | 3274 | 7.15 | 2960 | 6.46 | 5063 | 11.06 | 7959 | 17.38 |
| 45552 | 4673 | 10.26 | 0 | 0 | 4124 | 9.05 | 7622 | 16.73 | 9846 | 21.61 |
| 25298 | 3754 | 14.84 | 2212 | 8.74 | 3314 | 13.1 | 3639 | 14.38 | 6573 | 25.98 |
| 27966 | 2649 | 9.47 | 3123 | 11.17 | 2672 | 9.56 | 3226 | 11.54 | 5859 | 20.95 |

34

Premarket Notification 510(k)

35

LIMIT OF DETECTION (LoD)

A study was performed to assess the performance of the BioCode Gastrointestinal Pathogen Panel on the BioCode MDx 3000 at the Limit of Detection (LoD) for both unpreserved Stool and Cary- Blair specimens. In this study the BioCode GPP was tested with quantified bacteria, virus or parasite stocks (note Norovirus GI and Norovirus GII were tested by CDC). For initial screening, four replicates of each concentration in negative stool and Cary-Blair were extracted on the easyMAG System and tested in singlet with the BioCode GPP on the BioCode MDx 3000 system to estimate LoD. The LoD was confirmed by extracting 20 replicates of each sample type and testing each in singlet for a total of 20 replicates at or near presumptive LoD. LoD for each stock was defined as the lowest concentration with ≥95% detection of 20 replicates (19 out of 20), and was determined separately unpreserved stool and Cary-Blair preserved stool.

| Strain | Source | Unpreserved Stool LoD | Unpreserved
Stool
Detection | Cary-Blair Stool LoD | Cary-Blair
Stool
Detection |
|---------------------------------------------------|--------------------------|-----------------------|-----------------------------------|----------------------|----------------------------------|
| Campylobacter coli | ATCC 33559 | 5.6 x 101 CFU/mL | 20/20 | 5.6 x 101 CFU/mL | 20/20 |
| Campylobacter jejuni
subsp. jejuni | ATCC 33292 | 7.0 x 102 CFU/mL | 20/20 | 7.0 x 102 CFU/mL | 20/20 |
| Clostridium difficile
(toxinotype 0) | ATCC 9689 | 1.9 x 102 CFU/mL | 20/20 | 1.9 x 102 CFU/mL | 20/20 |
| Clostridium difficile
(toxinotype III; Nap1) | Zeptometrix
0801619cf | 8.3 x 102 CFU/mL | 20/20 | 3.3 x 103 CFU/mL | 20/20 |
| Enteroaggregative E. coli
092:H33 (EAEC) | STEC TW04440 | 1.4 x 103 CFU/mL | 20/20 | 1.4 x 103 CFU/mL | 20/20 |
| Enteroinvasive E. coli
029:NM (EIEC) | ATCC 43892 | 3.6 x 102 CFU/mL | 20/20 | 7.5 x 102 CFU/mL | 20/20 |
| Enterotoxigenic E. coli
078:H11 H10407 (ETEC) | ATCC 35401 | 5.6 x 102 CFU/mL | 20/20 | 5.6 x 102 CFU/mL | 20/20 |
| Salmonella bongori | SGSC 4900 | 1.4 x 103 CFU/mL | 20/20 | 5.5 x 103 CFU/mL | 20/20 |
| Salmonella enterica ssp.
enterica | ATCC 14028 | 2.2 x 103 CFU/mL | 20/20 | 1.1 x 103 CFU/mL | 19/20 |
| Shiga-like toxin producing
E. coli (STEC) | ATCC BAA-2217 | 2.5 x 103 CFU/mL | 20/20 | 2.5 x 103 CFU/mL | 20/20 |
| E. coli 0157 | ATCC 700376 | 3.3 x 103 CFU/mL | 20/20 | 3.3 x 103 CFU/mL | 20/20 |
| Shigella sonnei | ATCC 29930 | 4.4 x 102 CFU/mL | 20/20 | 1.7 x 103 CFU/mL | 20/20 |
| Vibrio cholerae | ATCC 25870 | 4.9 x 102 CFU/mL | 20/20 | 4.9 x 102 CFU/mL | 20/20 |
| Vibrio parahaemolyticus | ATCC 17802 | 1.3 x 101 CFU/mL | 20/20 | 5.0 x 101 CFU/mL | 20/20 |

Limit of Detection (LoD) for BioCode GPP.

36

| Strain | Source | Unpreserved Stool LoD | Unpreserved
Stool
Detection | Cary-Blair Stool LoD | Cary-Blair
Stool
Detection |
|------------------------------------------|------------------------|---------------------------|-----------------------------------|---------------------------|----------------------------------|
| Yersinia enterocolitica | ATCC 23715 | 1.5 × 103 CFU/mL | 20/20 | 1.5 × 103 CFU/mL | 20/20 |
| Cryptosporidium hominis | UKRC | 1.3 × 104
oocysts/mL | 20/20 | 1.3 × 104
oocysts/mL | 19/20 |
| Cryptosporidium parvum | waterborne
P102C | 3.1 × 103
oocysts/mL | 20/20 | 3.1 × 103
oocysts/mL | 20/20 |
| Entamoeba histolytica
HB-301:NIH | BEI NR-178 | 3.1 × $10-1$ cysts/mL | 20/20 | 3.1 × $10-1$
cysts/mL | 20/20 |
| Giardia intestinalis
(aka G. lamblia) | waterborne
P101 | 1.8 × 103 cysts/mL | 20/20 | 1.8 × 103 cysts/mL | 20/20 |
| Adenovirus 40 (dugan) | Zeptometrix
0810084 | 1.0 × $10-1$
TCID50/mL | 20/20 | 1.0 × $10-1$
TCID50/mL | 20/20 |
| Adenovirus 41 (TAK) | Zeptometrix
0810085 | 9.4 × $10-2$
TCID50/mL | 20/20 | 7.5 × $10-1$
TCID50/mL | 20/20 |
| Rotavirus A | ATCC VR-2018 | 2.5 × 103 TCID50/mL | 20/20 | 6.2 × 102
TCID50/mL | 20/20 |
| Norovirus Gla | CDC | 6.4 × 105
copies/gram | 20/20 | 6.5 × 105
copies/gram | 20/20 |
| Norovirus Glla | CDC | 5.2 × 104
copies/gram | 20/20 | 9.96 × 104
copies/gram | 20/20 |

37

INTERFERING SUBSTANCES

A study was performed to demonstrate the accuracy of the BioCode GPP on the BioCode MDx 3000 in the presence of potentially inhibiting substances or microorganisms. Each member of the interfering substance panel (ISP) was added to prescreened negative stool sample spiked with representative members of the BioCode GPP at 3X LoD and a negative matrix only pre-screened negative stool. One parasite, one virus, one gram-positive bacterium, and one gram-negative bacterium were used as representatives for this study. Each sample was tested with and without potentially interfering substances. Each sample was extracted in triplicate on the easyMag System and tested in singlet with the BioCode GPP on the BioCode MDx 3000 system. Concentrations were determined by reviewing 510k summary results of previous GI panel clinical trials and FDA Guidance. No interfering substances or microorganisms were identified.

Interference Test Panel.

Sample Name	Organism	Source
Sample A	Clostridium difficile (NAP1)
Cryptosporidium parvum	Zeptometrix 0801619cf
Waterborne P102C
Sample B	Human Rotavirus A
Escherichia coli 10C-3114 (STEC)	ATCC VR-2018
ATCC BAA-2217
Sample C	Negative Matrix Only	N/A

Potential Microbial Interferents. No inhibition or unexpected results were observed in the presence of high titer for the organisms in the table below.

| Microbial Interferent | Source | Concentration
(CFU/mL) | Interference Yes
(Y) or No (N) |
|-------------------------------------|---------------------|---------------------------|-----------------------------------|
| No Interferent (Control) | N/A | N/A | N |
| Bacteroides fragilisa | Zeptometrix 0801583 | 1 × 106 | N |
| Blastocystis hominisb | ATCC 50752 | 1 × 105 | N |
| Candida albicans | ATCC 14053 | 1 × 105 | N |
| Clostridium difficile non-toxigenic | ATCC 700057 | 1 × 106 | N |
| Enterococcus faecalis | ATCC 51299 | 1 × 106 | N |
| Escherichia coli nonpathogenic | ATCC BAA-97 | 1 × 106 | N |
| Pseudomonas aeruginosa | ATCC 39324 | 1 × 106 | N |
| Saccharomyces boulardii | ATCC MYA-796 | 1 × 105 | N |

a – 1/3 wells of B. fragilis in the negative stool only sample produced a false positive result for Adenovirus 40/41. An additional 5 extractions were then repeated with no false positives.

b - Blastocystis hominis is titered in cells/mL.

38

Potential Interfering Substances.

| Substance Interferent | Brand/Source | Concentration | Interference Yes
(Y) or No (N) |
|------------------------------------------------|----------------------|---------------|-----------------------------------|
| No Interferent | N/A | N/A | N |
| Blood (EDTA) | Clinical Sample | 40% w/v | N |
| Ampicillin | Ampicillin | 152 umol/L | N |
| Sodium hypochlorite | Bleach (10%) | 50% w/v | N |
| Cholesterol | Cholesterol | 5% w/v | N |
| Mineral Oil | Mineral oil, USP | 50% w/v | N |
| Hydrocortisone | Hydrocortisone cream | 50% w/v | N |
| Loperamide hydrochloride | Imodium | 5% v/v | N |
| Sennosides | Senokot | 5% w/v | N |
| Magnesium Hydroxide, Aluminum Hydroxide | Maalox | 5% w/v | N |
| Metronidazole | Metronidazole | 14 mg/mL | N |
| Benzalkonium chloride, Ethanol | Moist towelettes | 50% w/v | N |
| Mono, di, triglycerides mix | Supelco | 5% w/v | N |
| Mucin | Mucin | 3 mg/ml | N |
| Naproxen sodium | Naproxen sodium | 14mg/ml | N |
| Polymyxin B sulfate, bacitracin zinc, Neomycin | Neosporin | 50% w/v | N |
| Nystatin | Nystatin | 1000 U/mL | N |
| Bismuth subsalicylate | Pepto-Bismol | 5% v/v | N |
| Petrolatum | Preparation H | 5% v/v | N |
| Calcium carbonate | Tums | 5% w/v | N |
| Vancomycin | Vancomycin | 12.5 mg/mL | N |

ANALYTICAL REACTIVITY/INCLUSIVITY

A study was performed to verify Analytical Reactivity/Inclusivity of the BioCode Gastrointestinal Pathogen Panel. Different strains were selected that represent various temporal, geographic, and genetic diversity for each analyte. This study tested a panel of titered stocks for relevant organisms diluted in Pre-Screened Negative Stools at 3X LoD. Stocks not detected at 3X LoD, if applicable, were tested at higher concentrations. Due to a lack of titered specimens, Adenovirus 40/41 clinical samples and Cryptosporidium DNA from the Cryptosporidium reference unit were used (Public Health England). Norovirus Gl and GII genotypes and the Rotavirus vaccine strain were tested by the CDC. All of the organisms were detected at the concentrations indicated.

Shiga-like toxin producing E. coli (STEC) stx1/stx2 and E. coli O157 Inclusivity results

| Organism | Serotype | Source | Concentration
Detected | Multiples
of LoD
Detected |
|---------------------|-------------------------|----------------------|---------------------------|---------------------------------|
| E. coli 0157 | E. coli 0157:H45 | STEC SC373/2 TW07922 | $9.9 x 10^3$ CFU/mL | 3x |
| | E. coli 0157:HNM | STEC DA-26 TW07952 | $9.9 x 10^3$ CFU/mL | 3x |
| | E. coli 0157:H7 | STEC 93-111 TW04863 | $9.9 x 10^3$ CFU/mL | 3x |
| | E. coli 0157: H7 | STEC MI06-19 TW14301 | $9.9 x 10^3$ CFU/mL | 3x |
| | E. coli 0157:HNT | STEC DA-27 TW07953 | $9.9 x 10^3$ CFU/mL | 3x |

39

| Organism | Serotype | Source | Concentration
Detected | Multiples
of LoD
Detected |
|--------------------------------------------|-------------------------------|----------------------|---------------------------|---------------------------------|
| Shiga toxin
producing E. coli
(STEC) | E. coli O157:H7 Strain EDL933 | BEI NR-11 | $9.9 x 10^3$ CFU/mL | 3x |
| | E. coli O26:H11 | STEC 2332/00 TW08998 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O113:H21 | STEC CL-15 TW02318 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O45:H2 | STEC DEC11C DEC11c | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O103:H2 | STEC 107-226 TW07881 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O104:H21 | STEC G5506 TW04909 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O111:H2 | STEC RD8 TW06296 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O111:H8 | STEC DEC8B | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O26:NM | STEC DA-22 TW07948 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O145:NM | ATCC BAA-2192 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O146:21 | STEC DEC16E TW01383 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O45:H2 | STEC MI05-14 TW14003 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O121:19 | STEC MDCH-4 TW07614 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O121:NM | STEC DA-37 TW07972 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli *O104:H4 | ATCC BAA-2326 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O45:H2 | ATCC BAA-2193 | $7.5 x 10^3$ CFU/mL | 3x |
| | E. coli O26: H11 | BAA-2196 | $7.5 x 10^3$ CFU/mL | 3x |
| E. coli O121:H19 | BAA-2219 | $7.5 x 10^3$ CFU/mL | 3x | |

Enterotoxigenic E. coli (ETEC), Enteroaggregative E. coli (EAEC) Inclusivity results

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|--------------------------------------------|---------------------------------------|---------------------|---------------------------|-----------------------------|
| Enteroaggregative
E. coli (EAEC) | E. coli O44:H18 | STEC 042
TW04393 | 4.08 x 103 CFU/mL | 3x |
| | E. coli O111a,
111b:K58:H21 | ATCC 29552 | 4.08 x 103 CFU/mL | 3x |
| | E. coli O104:H4 | ATCC BAA-2326 | 4.08 x 103 CFU/mL | 3x |
| | E. coli O3:K2a | BEI NR-102 | 4.08 x 103 CFU/mL | 3x |

Shigella/Enteroinvasive E. coli (EIEC) Inclusivity results

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|----------------------------------|-------------------------------|------------------------|---------------------------|-----------------------------|
| Enteroinvasive
E. coli (EIEC) | E. coli O121 | ATCC BAA-2190 | $1.08 x 10^3$ CFU/mL | 3x |
| | E. coli O124:HNM | STEC 929-78
TW16574 | $1.08 x 10^3$ CFU/mL | 3x |
| | E. coli O136:H | STEC LT-41
TW06139 | $1.08 x 10^3$ CFU/mL | 3x |
| | E. coli O285A:HNM | BEI NR-101 | $1.08 x 10^3$ CFU/mL | 3x |
| | E. coli O15 | ATCC 49105 | $1.08 x 10^3$ CFU/mL | 3x |
| Shigella boydii | Shigella boydii (Type 1) | ATCC 9207 | $1.31 x 10^3$ CFU/mL | 3x |
| | Shigella boydii (Type 2) | BEI NR-521 | $1.31 x 10^3$ CFU/mL | 3x |
| | Shigella boydii (Type 7) | ATCC 9905 | $1.31 x 10^3$ CFU/mL | 3x |
| | Shigella boydii (Type 20) | ATCC BAA-1247 | $1.31 x 10^3$ CFU/mL | 3x |
| | Shigella boydii (Type 3) | ATCC 8702 | $1.31 x 10^3$ CFU/mL | 3x |
| | Shigella dysenteriae (Type 1) | BEI NR-520 | $1.31 x 10^3$ CFU/mL | 3x |

40

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|-------------------------|----------------------------------------------|------------|---------------------------|-----------------------------|
| Shigella
dysenteriae | Shigella dysenteriae (Type 3) | ATCC 9751 | 1.31 x 103 CFU/mL | 3x |
| | Shigella dysenteriae (Type 2) | ATCC 9750 | 1.31 x 103 CFU/mL | 3x |
| | Shigella dysenteriae (Type 5) | ATCC 9764 | 1.31 x 103 CFU/mL | 3x |
| | Shigella dysenteriae (Type 12) | ATCC 49552 | 1.31 x 103 CFU/mL | 3x |
| Shigella flexneri | Shigella flexneri, strain 24570
(Type 2a) | BEI NR-517 | 1.31 x 103 CFU/mL | 3x |
| | Shigella flexneri, strain 2457T
(Type 2a) | BEI NR-518 | 1.31 x 103 CFU/mL | 3x |
| | Shigella flexneri (Type 2b) | ATCC 12022 | 1.31 x 103 CFU/mL | 3x |
| | Shigella flexneri (Type 6) | ATCC 12025 | 1.31 x 103 CFU/mL | 3x |
| | Shigella flexneri (Type 1b) | ATCC 9380 | 1.31 x 103 CFU/mL | 3x |
| Shigella sonnei | Shigella sonnei | ATCC 25931 | 1.31 x 103 CFU/mL | 3x |
| | Shigella sonnei | ATCC 11060 | 1.31 x 103 CFU/mL | 3x |
| | Shigella sonnei | ATCC 9290 | 1.31 x 103 CFU/mL | 3x |
| | Shigella sonnei | ATCC 29029 | 1.31 x 103 CFU/mL | 3x |

Salmonella Inclusivity results

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|----------------------------------------|-------------------------------------------|----------------------|---------------------------|-----------------------------|
| Salmonella enterica
subsp. arizonae | Salmonella enterica subsp.
Enterica | ATCC 13314 | 6.45 × 103 CFU/mL | 3× |
| Salmonella enterica
subsp. salamae | serovar Tranoroa | ATCC 700148 | 6.45 × 103 CFU/mL | 3× |
| Salmonella enterica
subsp. enterica | serovar Montevideo | ATCC 7001
BAA-710 | 6.45 × 103 CFU/mL | 3× |
| | serovar Enteritidis | SGSC4901 | 6.45 × 103 CFU/mL | 3× |
| | serovar Enteritidis | ATCC 4931 | 6.45 × 103 CFU/mL | 3× |
| | serovar Oranienburg | SGSC4079 | 6.45 × 103 CFU/mL | 3× |
| | serovar Paratyphi B var.L(+)
tartrate+ | SGSC4150 | 6.45 × 103 CFU/mL | 3× |
| | serovar Typhimurium | SGSC1412 | 6.45 × 103 CFU/mL | 3× |
| | serovar Saintpaul | SGSC2512 | 6.45 × 103 CFU/mL | 3× |
| | serovar S. typhimurium LT2 | SGSC2666 | 6.45 × 103 CFU/mL | 3× |
| | serovar Newport | SGSC2493 | 6.45 × 103 CFU/mL | 3× |
| | serovar Newport | ATCC 6962 | 6.45 × 103 CFU/mL | 3× |
| | serovar Muenchen | SGSC2490 | 6.45 × 103 CFU/mL | 3× |
| | serovar Agona | SGSC2458 | 6.45 × 103 CFU/mL | 3× |
| | serovar Javiana | SGSC4917 | 6.45 × 103 CFU/mL | 3× |
| | serovar Schwarzengrund | SGSC2514 | 6.45 × 103 CFU/mL | 3× |
| | serovar Heidelberg | SGSC2480 | 6.45 × 103 CFU/mL | 3× |
| | serovar Infantis | SGSC2484 | 6.45 × 103 CFU/mL | 3× |
| | serovar Montevideo | SGSC2487 | 6.45 × 103 CFU/mL | 3× |
| | serovar Thompson | SGSC 2519 | 6.45 × 103 CFU/mL | 3× |
| | serovar Hadar | SGSC4965 | 6.45 × 103 CFU/mL | 3× |
| | serovar Mississippi | SGSC4078 | 6.45 × 103 CFU/mL | 3× |
| | serovar Paratyphi A | SGSC2499 | 6.45 × 103 CFU/mL | 3× |
| | serovar Choleraesuis | SGSC4770 | 6.45 × 103 CFU/mL | 3× |
| | serovar Choleraesuis | ATCC 13312 | 6.45 × 103 CFU/mL | 3× |

41

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|------------------------------------|--------------------------------|------------------------|---------------------------|-----------------------------|
| | serovar Dublin | SGSC4157 | 6.45 × 103 CFU/mL | 3x |
| | serovar Braenderup | ATCC® 700136 | 6.45 × 103 CFU/mL | 3x |
| | serovar Bareilly | ATCC® 9115 | 6.45 × 103 CFU/mL | 3x |
| | serovar Typhi | Zeptometrix
0801933 | 6.45 × 103 CFU/mL | 3x |
| Salmonella enterica
subsp. II | Salmonella enterica subs. II | SGSC3039 | 6.45 × 103 CFU/mL | 3x |
| Salmonella enterica
subsp. IIIa | Salmonella enterica subs. IIIa | SGSC3061 | 6.45 × 103 CFU/mL | 3x |
| Salmonella enterica
subsp. IIIb | Salmonella enterica subs. IIIb | SGSC3068 | 6.45 × 103 CFU/mL | 3x |
| Salmonella enterica
subsp. IV | Salmonella enterica subs. IV | SGSC3074 | 6.45 × 103 CFU/mL | 3x |
| Salmonella enterica
subsp. VI | Salmonella enterica subs VI | SGSC3116 | 6.45 × 103 CFU/mL | 3x |

Campylobacter inclusivity results.

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD
Detected |
|--------------------|------------------------------------|------------|---------------------------|--------------------------------|
| Campylobacter spp. | Campylobacter jejuni subsp. jejuni | BEI NR-399 | 2.10 x 103 CFU/mL | 3x |
| | Campylobacter jejuni subsp. jejuni | BEI NR-400 | 2.10 x 103 CFU/mL | 3x |
| | Campylobacter jejuni subsp. doylei | ATCC 49350 | 2.10 x 103 CFU/mL | 3x |
| | Campylobacter jejuni subsp. doylei | ATCC 49349 | 2.10 x 103 CFU/mL | 3x |
| | Campylobacter coli | ATCC 43478 | 1.68 x 102 CFU/mL | 3x |
| | Campylobacter coli | ATCC 43485 | 1.68 x 102 CFU/mL | 3x |
| | Campylobacter coli | BEI HM-296 | 1.68 x 102 CFU/mL | 3x |
| | Campylobacter coli | ATCC 43484 | 1.68 x 102 CFU/mL | 3x |

Vibrio spp inclusivity results.

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD
Detected |
|---------------------------------------|------------------------------------------------|--------------|---------------------------|--------------------------------|
| Vibrio spp. (not
parahaemolyticus) | Vibrio cholerae (O:1 Inaba,
Biotype E1 Tor) | BEI NR-147 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio cholerae (O:1 Inaba,
Biotype E1 Tor) | BEI NR-146 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio cholerae(O:2) | BEI NR-149 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio cholerae (O:4) | BEI NR-151 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio cholerae (O:139) | BEI NR-144 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio cholerae (O:1 Ogawa) | ATCC 14035 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio vulnificus | ATCC 27562 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio vulnificus | ATCC BAA-88 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio vulnificus | ATCC 43382 | $4.9 x 10^3$ CFU/mL | 10X |
| | Vibrio vulnificus | ATCC 29306 | $1.47 x 10^3$ CFU/mL | 3x |
| | Vibrio vulnificus | ATCC 29307 | $1.47 x 10^3$ CFU/mL | 3x |
| Vibrio
parahaemolyticus | Vibrio parahaemolyticus | BEI NR-21990 | $3.75 x 10^1$ CFU/mL | 3x |
| | Vibrio parahaemolyticus | BEI NR-21991 | $3.75 x 10^1$ CFU/mL | 3x |

42

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD
Detected |
|----------|--------------------------------|---------------|---------------------------|--------------------------------|
| | Vibrio parahaemolyticus | BEI NR-22002 | 3.75 x 101 CFU/mL | 3x |
| | Vibrio parahaemolyticus | Zepto 0801903 | 3.75 x 101 CFU/mL | 3x |
| | Vibrio parahaemolyticus | BEI NR-22013 | 3.75 x 101 CFU/mL | 3x |

Yersinia enterocolitica inclusivity results.

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|-------------------------|-------------------------------|------------|---------------------------|-----------------------------|
| Yersinia enterocolitica | Yersinia enterocolitica (O:8) | ATCC 9610 | 4.43 x 103 CFU/mL | 3x |
| | Yersinia enterocolitica (O:3) | BEI NR-212 | 4.43 x 103 CFU/mL | 3x |
| | Yersinia enterocolitica (O:8) | BEI NR-206 | 4.43 x 103 CFU/mL | 3x |
| | Yersinia enterocolitica | ATCC 29913 | 4.43 x 103 CFU/mL | 3x |
| | Yersinia enterocolitica | BEI NR-213 | 4.43 x 103 CFU/mL | 3x |

Clostridium difficile inclusivity results.

| Organism | TOXINOTYPE | Source | Concentration
Detected | Multiple of
LoD
Detected |
|-----------------------|-----------------|------------------|---------------------------|--------------------------------|
| Clostridium difficile | 0 A+B+ | ATCC 43255-FZ | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC 700792-FZ | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC BAA-1382-fz | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC 51695-fz | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC 43599-fz | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC 43596-fz | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC 17858-fz | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC 43594 | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC 43600 | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC 17857 | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC BAA-1871 | 2.48 x 103 CFU/mL | 3x |
| | 0 A+B+ | ATCC BAA-1872 | 2.48 x 103 CFU/mL | 3x |
| | VIII A-B+ | ATCC 43598 | 2.48 x 103 CFU/mL | 3x |
| | III A+B+ (Nap1) | ATCC BAA-1805 | 2.48 x 103 CFU/mL | 3x |
| | XXII A+B+ | ATCC BAA-1814 | 2.48 x 103 CFU/mL | 3x |
| | III A+B+ | ATCC BAA-1870 | 2.48 x 103 CFU/mL | 3x |
| | V A+B+ | ATCC BAA-1875 | 2.48 x 103 CFU/mL | 3x |

43

| | Organism | Serotype | Source | Concentration Detected | Multiple of
LoD
Detected |
|---------------|---------------|-----------------------------|--------------------|-------------------------|--------------------------------|
| | Adenovirus 40 | Human adenovirus 40 (dugan) | lot #K1220A | 3.0E-01 TCID50/mL | 3 |
| Adenovirus 40 | | n/a | sample ID # SP143 | Clinical Sample unknown | 3 |
| | | n/a | sample ID # SP258 | Clinical Sample unknown | 3 |
| Adenovirus 41 | | n/a | sample ID # SP170 | Clinical Sample unknown | 3 |
| | | n/a | sample ID # SP174 | Clinical Sample unknown | 3 |
| | | n/a | sample ID # SP 276 | Clinical Sample unknown | 3 |
| | | n/a | sample ID # SP288 | Clinical Sample unknown | 3 |
| | | n/a | sample ID # SP309 | Clinical Sample unknown | 3 |
| | | n/a | sample ID # SP329 | Clinical Sample unknown | 3 |
| | | n/a | sample ID # SP446 | Clinical Sample unknown | 3 |

Adenovirus 40/41 inclusivity results.

Norovirus GI/GII inclusivity results.

| Organism | Genotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|----------------|-------------------|-------------------|---------------------------|-----------------------------|
| *Norovirus GI | GI.1 | Clinical Specimen | $1.93 x 10^6$ copies/gram | 3x |
| | GI.2 | Clinical Specimen | $1.93 x 10^6$ copies/gram | 3x |
| | GI.3 | Clinical Specimen | $1.93 x 10^8$ copies/gram | 300x |
| | GI.4 | Clinical Specimen | $1.93 x 10^6$ copies/gram | 3x |
| | GI.5 | Clinical Specimen | $1.93 x 10^6$ copies/gram | 3x |
| | GI.6 | Clinical Specimen | $1.93 x 10^6$ copies/gram | 3x |
| | GI.7 | Clinical Specimen | $1.93 x 10^6$ copies/gram | 3x |
| | GI.8 | Clinical Specimen | $1.93 x 10^6$ copies/gram | 3x |
| aNorovirus GII | GII.1 | Clinical Specimen | $1.57 x 10^5$ copies/gram | 3x |
| | GII.2 | Clinical Specimen | $1.57 x 10^6$ copies/gram | 30x |
| | GII.3 | Clinical Specimen | $1.57 x 10^6$ copies/gram | 30x |
| | GII.4 New Orleans | Clinical Specimen | $1.57 x 10^6$ copies/gram | 30x |
| | GII.4 Sydney | Clinical Specimen | $1.57 x 10^5$ copies/gram | 3x |
| | GII.5 | Clinical Specimen | $1.57 x 10^5$ copies/gram | 3x |
| | GII.6 | Clinical Specimen | $1.57 x 10^5$ copies/gram | 3x |
| | GII.7 | Clinical Specimen | $1.57 x 10^5$ copies/gram | 3x |
| | GII.8 | Clinical Specimen | $1.57 x 10^6$ copies/gram | 30x |
| | GII.12 | Clinical Specimen | $1.57 x 10^5$ copies/gram | 3x |
| | GII.13 | Clinical Specimen | $1.57 x 10^5$ copies/gram | 3x |
| | GII.14 | Clinical Specimen | $1.57 x 10^6$ copies/gram | 30x |
| | GII.16 | Clinical Specimen | $1.57 x 10^6$ copies/gram | 30x |
| | GII.17 | Clinical Specimen | $1.57 x 10^5$ copies/gram | 3x |

a — Assayed at CDC.

44

Norovirus GI/GII inclusivity results.

| Organism | Serotype | Source | Concentration
Detected | Multiples of
LoD
Detected |
|-------------|---------------------------|-----------------|---------------------------|---------------------------------|
| Rotavirus A | Rotavirus A (DS-1) | ATCC VR-2550 | $7.44 x 10^3$ IU/mL | 3x |
| | Rotavirus A (HRV 89-12C2) | ATCC VR-2272 | $7.44 x 10^3$ IU/mL | 3x |
| | Rotavirus A (WISC2) | ATCC VR-2417 | $7.44 x 10^3$ IU/mL | 3x |
| | Rotavirus A (HRV 408) | ATCC VR-2273 | $7.44 x 10^3$ IU/mL | 3x |
| | Rotavirus A (HRV 248) | ATCC VR-2274 | $7.44 x 10^3$ IU/mL | 3x |
| | Rotavirus A (HU) | ATCC VR-1546 | $7.44 x 10^3$ IU/mL | 3x |
| | Rotavirus A (HRV CJN) | ATCC VR-2275 | $7.44 x 10^3$ IU/mL | 3x |
| Rotavirus A | Rotavirus A G1 | Clinical Sample | Ct= 20 | N/Aa |
| Rotavirus A | Rotavirus A G2 | Clinical Sample | Ct= 22 | |
| Rotavirus A | Rotavirus A G3 | Clinical Sample | Ct= 26 | |
| Rotavirus A | Rotavirus A G4 | Clinical Sample | Ct= 21 | |
| Rotavirus A | Rotavirus A G9 | Clinical Sample | Ct= 20 | |
| Rotavirus A | Rota vaccine strain | RotaTeq vaccine | $1.00 x 10^8$ pfu/mL | |
| | Rota vaccine strain | Rotarix vaccine | $1.00 x 10^6$ CCID50/mL | |

a – Tested at CDC at indicated concentration at CDC, concentration cannot be related to LoD.

Cryptosporidium spp inclusivity results.

| Organism | Serotype | Source | Concentration Detected | Multiple of
LoD Detected |
|------------|-------------------------|------------|-------------------------|-----------------------------|
| C. parvum | DNA subtype /IIaA17G1R1 | UKCR UK28 | Clinical Sample unknown | 3 |
| | DNA subtype /IIaA15G2R1 | UKCR UK29 | Clinical Sample unknown | 3 |
| | DNA subtype /IIaA19G1R1 | UKCR UK30 | Clinical Sample unknown | 3 |
| | DNA subtype /IIdA22G1 | UKCR UK31 | Clinical Sample unknown | 3 |
| | DNA subtype /IIdA15G1 | UKCR UK32 | Clinical Sample unknown | 3 |
| C. hominis | DNA subtype IaA14R3 | UKCR UKH14 | Clinical Sample unknown | 3 |
| | DNA subtype IdA18 | UKH12 | Clinical Sample unknown | 3 |
| | DNA subtype IbA10G2 | UKH13 | Clinical Sample unknown | 3 |
| | DNA E. coli O103:H2 | NR2520 | Clinical Sample unknown | 3 |
| | | | | |

Entamoeba histolytica inclusivity results.

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|-----------------------|------------|------------|---------------------------|-----------------------------|
| Entamoeba histolytica | 200:NIH | BEI NR-177 | 9.36 x 10-1 cysts/mL | 3x |
| Entamoeba histolytica | HB-301:NIH | BEI NR-176 | 9.36 x 10-1 cysts/mL | 3x |
| Entamoeba histolytica | Rahman | BEI NR-179 | 9.36 x 10-1 cysts/mL | 3x |
| Entamoeba histolytica | H-303:NIH | BEI NR-180 | 9.36 x 10-1 cysts/mL | 3x |

45

| Organism | Serotype | Source | Concentration
Detected | Multiple of
LoD Detected |
|----------------------------------|----------|-------------|---------------------------|-----------------------------|
| Giardia lamblia/
intestinalis | Egypt-4 | BEI NR-9231 | 5.42 x 103 cysts/mL | 3x |
| | Mario | BEI NR-9232 | 5.42 x 103 cysts/mL | 3x |
| | D.Hall | BEI NR-9234 | 5.42 x 103 cysts/mL | 3x |
| | DAN | BEI NR-9235 | 5.42 x 103 cysts/mL | 3x |

Giardia lamblia inclusivity results.

ANALYTICAL SPECIFICITY/CROSS REACTIVITY

A study was performed to verify that the BioCode GPP does not detect DNA or RNA from organisms commonly found in stool specimens or from organisms that can cause similar clinical symptoms. In addition, on-panel organisms were tested at high concentrations to insure there is no cross-reactivity with other panel targets. This study tested a panel of titered stocks and genomic DNA extracts for relevant organisms. Organisms that were not available for were analyzed in silico comparing the whole organism sequence against all primers to assess potential for cross reactivity. Analysis was conducted using BlastN and Primer Blast programs.

Cross-reactivity was not observed with microorganisms tested in this study except for the following.

Empirical testing and in silico sequence analysis indicate that the Vibrio spp assay may also react with some less common Vibrio species (i.e., V. alqinolyticus, and V. mimicus).

Empirical testing and in silico sequence analysis indicate a potential for cross-reactivity with Y. bercovieri, Y. frederiksenii, Y. intermedia and Y. mollaretii near the established LoD for Y. entericolitica (~1.5 x 10³ CFU/mL). Y. rohdei was also detected when present at high levels (>6.8 x 10° CFU/mL). These species are in the Y. enterocolitica group and are suspected human pathogens.

Shiga toxin (stx; identical to stx1 of STEC) is found in Shigella dysenteriae; therefore, a BioCode GPP report with positive test results for Shiga-like toxin-producing E. coli (STEC) and Shigella/Enteroinvasive E. coli (EIEC) in the same sample may indicate the presence of S. dysenteriae.

Empirical testing with a gene fragment construct and in silico sequence analysis do not predict cross reactivity with the closely related E. dispar.

Empirical testing has demonstrated that these assays will detect recombinant viruses included in Rotavirus vaccines.

Empirical testing indicates potential for cross reactivity with C. meleagridis with the Cryptosporidium assay.

46

No Cross reactivity was observed for the organisms tested below.

Bacteria
Aeromonas jandaei	Egglerthella lenta	Proteus penneri
Aeromonas media	Enterobacter aerogenes	Proteus vulgaris
Aeromonas trota	Enterobacter cloacae	Providencia alcalifaciens
Aeromonas caviae	Enterococcus faecalis	Providencia stuartii
Aeromonas hydrophila	Enterococcus faecium	Pseudomonas aeruginosa
Acinetobacter baumannii	Enteropathogenic Escherichia coli	Pseudomonas fluorescens
Acinetobacter lwoffii	Escherichia coli Non pathogenic	Pseudomonas putida
Alcaligenes faecalis	Escherichia coli Non pathogenic	Saccharomyces boulardii
Bacillus cereus	Escherichia coli Non pathogenic	Serratia liquefaciens
Bacteroides fragilis	Escherichia hermannii	Serratia marcescens
Bacteroides thetaiotaomicron	Escherichia vulneris	Shewanella algae
Bifidobacterium breve	Fusobacterium varium	Staphylococcus aureus
Campylobacter fetus	Gardnerella vaginalis	Staphylococcus epidermidis
Campylobacter hyointestinalis	Gemella morbillorum	Stenotrophomonas maltophilia
Campylobacter lari	Grimontia hollisae (formerly vibrio)	Streptococcus agalactiae
Campylobacter upsaliensis	Haemophilus influenzae	Streptococcus intermedius
Candida albicans	Hafnia alvei	Streptococcus pyogenes
Cedecea davisae	Helicobacter pylori	Streptococcus salivarius
Chlamydia trachomatis	Klebsiella oxytoca	Streptococcus suis
Citrobacter amalonaticus	Klebsiella pneumoniae	Trabulsiella guamensis
Citrobacter freundii	Lactobacillus acidophilus	Veillonella parvula
Clostridium difficile non-toxigenic	Lactobacillus reuteri	Vibrio alginolyticusa
Clostridium difficile non-toxigenic	Lactococcus lactis	Vibrio fluvialis
Clostridium difficile non-toxigenic	Leminorella grimontii	Vibrio mimicusa
Clostridium histolyticum	Listeria monocytogenes	Yersinia bercovierib
Clostridium perfringens	Morganella morganii	Yersinia frederikseniib
Clostridium septicum	Peptoniphilus asaccharolyticus	Yersinia intermediab
Clostridium sordellii	Plesiomonas shigelloides	Yersinia mollaretiib
Clostridium sporogenes	Porphyromonas asaccharolytica	Yersinia pseudotuberculosis
Clostridium tetani	Prevotella melaninogenica	Yersinia rohdeiᵇ
Edwardsiella tarda	Proteus mirabilis

a – Detected as Vibrio spp at high titers, see full submission for details.

b – Detected as Yersinia enterocolitica at high titers, see full submission for details.

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No Cross reactivity was observed for the viruses and parasites tested below.

Viruses		Parasites
Adenovirus 3	Enterovirus 68	Cryptosporidium cuniculus (DNA)b
Adenovirus 4	Enterovirusa	Cryptosporidium felis (DNA)
Adenovirus 7a	Echovirus 11a	Cryptosporidium meleagridis b
Adenovirus 8	HSV Type 2	Cryptosporidium meleagridis (DNA)b
Adenovirus 14	Norovirus GIV	Cryptosporidium muris
Adenovirus 37	Rhinovirus 1A	Cryptosporidium ubiquitum (DNA)
Astrovirus type 1a	Sapovirus Gla	Encephalitozoon cuniculi
Astrovirus type 4a	Sapovirus GIVa	Encephalitozoon hellem
Coronavirus 229E	Sapovirus GVa	Encephalitozoon intestinalis
Coronavirus NL63		Giardia muris
Coxsackie virus A16a	Blastocystis hominisa	Pentatrichomonas hominis
Coxsackievirus B3	Blastocystis hominis	Toxoplasma gondii
Cytomegalovirus (CMV)	Cryptosporidium canis (DNA)

a — tested at CDC

b – Detected as Cryptosporidium spp.

No Cross reactivity was observed for BioCode GPP targets.

BioCode GPP Targets
Campylobacter coli	Enterotoxigenic E. coli O78:H11
H10407 (ETEC)	Vibrio parahaemolyticus
Campylobacter jejuni spp. jejuni	Shiga toxin producing E. coli (STEC)	Yersinia enterocolitica
Clostridium difficile (toxinotype 0 )	E. coli O157	Cryptosporidium parvum
Clostridium difficile (toxinotype III;
Nap1)	Salmonella bongori	Entamoeba histolytica HB-301:NIH
Enteroaggregative E. coli O92:H33
(EAEC)	Salmonella enterica ssp. enterica	Giardia intestinalis (aka G. lamblia)
Enteroinvasive E. coli O29:NM (EIEC)	Shigella sonnei	Adenovirus 40 (dugan)
		Rotavirus A

No Cross reactivity was predicted by in silico analysis.

Bacteria	Bacteria	Parasites
Anaerococcus tetradius	Eubacterium cylindroides	Ancylostoma duodenale
Bifidobacterium adolescentis	Eubacterium rectale	Ascaris lumbricoides
Bifidobacterium longum	Megamonas hypermegale	Balantidium coli
Campylobacter concisus	Methanobrevibacter smithii	Chilomastix mesnili
Campylobacter curvus	Peptoniphilus asaccharolyticus	Cryptosporidium bovis
Campylobacter gracilis	Ruminococcus bromii	Cryptosporidium canis
Campylobacter helveticus	Ruminococcus flavefaciens	Cryptosporidium cuniculusb
Campylobacter hominis	Ruminococcus obeum	Cryptosporidium felis
Campylobacter lari	Selenomonas ruminantium	Cryptosporidium fetus
Campylobacter mucosalis	Vibrio cincinnatiensis	Cryptosporidium meleagridisb

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Campylobacter rectus	Vibrio furnissii	Cryptosporidium muris
Campylobacter showae	Vibrio metschnikovii	Cryptosporidium ryanae
Campylobacter sputorum	Yersinia kristensenii	Cryptosporidium xiaoi
Campylobacter upsaliensis	Viruses	Dientamoeba fragilis
Campylobacter ureolyticus	Norovirus GIV	Endolimax nana
Clostridium acetobutylicum	Rotavirus B	Entamoeba coli
Clostridium methylpentosum	Rotavirus Ca	Entamoeba dispar
Clostridium novyi	Rotavirus D	Entamoeba hartmanni
Clostridium ramosum	Rotavirus E	Entamoeba moshkovskii
Collinsella aerofaciens	Rotavirus F	Entamoeba polecki
Desulfovibrio piger	Sapovirus

a – Cross-reactivity predicted with Porcine Rotavirus C strains only, no cross-reactivity with human Rotavirus C.

b - C. Cuniculus and C. meleagridis cross reactivity for Cryptosporidium spp assay observed in lab testing was not predicted by in silico analysis (Primer Blast).

COMPETITIVE INHIBITION

A study was performed to evaluate the potential for inhibition in samples with mixed infections. Prescreened negative stool was spiked with one target at high concentration (≥10°CFU/mL for bacteria and ≥105 units/mL for viruses or parasites) and two targets at medium concentration (≤3x LoD). Common co-infections were determined by reviewing results of previous Gl Panel clinical trials from 510(k) summaries, publications/posters and internal clinical sample testing. Each sample was extracted in triplicate on the easyMag and each extraction tested in singlet with the Gastrointestinal Pathogen Panel on the BioCode MDx 3000 system. No competitive inhibition was observed.

Competitive inhibition testing results.

| Panel
Designation | Viral/Bacteria Strain | Source | Level | Screening Titer | Target
Probe | Average
MFI | Screening
Result
(n of 3) |
|----------------------|-----------------------------------------|-----------------------|--------|------------------------|-----------------|----------------|---------------------------------|
| CI-1 | Clostridium difficile | Zepto 801619 | High | 3.0x $10^6$ CFU/mL | tcdB | 23969 | 3/3 |
| | Rotavirus A | ATCC VR-2018 | Medium | 7.44X $10^3$ TCID50/mL | Rota | 33899 | 3/3 |
| | Escherichia coli
E2348/69 (EPEC) | STEC TW06375 | Medium | 7.02x $10^3$ CFU/mL | EPEC | 11585 | 3/3 |
| CI-2 | 092:H33 Escherichia
coli (EAEC) | STEC JM221
TW04440 | High | 3.0x $10^6$ CFU/mL | EAEC | 26578 | 3/3 |
| | Escherichia coli
E2348/69 (EPEC) | STEC TW06375 | Medium | 7.02X $10^3$ CFU/mL | EPEC | 10680 | 3/3 |
| | Clostridium difficile | Zepto 801619 | Medium | 5.7x $10^2$ CFU/mL | tcdB | 8775 | 3/3 |
| CI-3 | Escherichia coli
E2348/69 (EPEC) | STEC TW06375 | High | 3.0x $10^6$ CFU/mL | EPEC | 27671 | 3/3 |
| | Clostridium difficile | Zepto 801619 | Medium | 5.7x $10^2$ CFU/mL | tcdB | 8876 | 3/3 |
| Panel
Designation | Viral/Bacteria Strain | Source | Level | Screening Titer | Target
Probe | Average
MFI | Screening
Result
(n of 3) |
| | Rotavirus A | ATCC VR-2018 | Medium | $7.44X10^3$ TCID50/mL | Rota | 31617 | 3/3 |
| CI-4 | Escherichia coli
E2348/69 (EPEC) | STEC TW06375 | High | $3x10^6$ CFU/mL | EPEC | 26059 | 3/3 |
| | 092:H33 Escherichia
coli (EAEC) | STEC JM221
TW04440 | Medium | $4.08X10^3$ CFU/mL | EAEC | 22175 | 3/3 |
| | Campylobacter jejuni
subsp .jejuni | ATCC 33292 | Medium | $2.1X10^3$ CFU/mL | Campy | 32286 | 3/3 |
| | Campylobacter jejuni
subsp. Doylei | ATCC 49349 | High | $3.0x10^6$ CFU/mL | Campy | 24738 | 3/3 |
| CI-5 | Escherichia coli
E2348/69 (EPEC) | STEC TW06375 | Medium | $7.02X10^3$ CFU/mL | EPEC | 7382 | 3/3 |
| | 092:H33 Escherichia
coli (EAEC) | STEC JM221
TW04440 | Medium | $4.08X10^3$ CFU/mL | EAEC | 22488 | 3/3 |
| CI-6 | 092:H33 Escherichia
coli (EAEC) | STEC JM221
TW04440 | High | $3x10^6$ CFU/mL | EAEC | 24968 | 3/3 |
| | Campylobacter jejuni
sub sp .jejuni | ATCC 33292 | Medium | $2.1X10^3$ CFU/mL | Campy | 33202 | 3/3 |
| | Escherichia coli
E2348/69 (EPEC) | STEC TW06375 | Medium | $7.02X10^3$ CFU/mL | EPEC | 10342 | 3/3 |
| | Shiga-toxin producing
E. coli (STEC) | ATCC BAA-2217 | High | $3.0x10^6$ CFU/mL | stx2 | 34913 | 3/3 |
| CI-7 | Giardia intestinalis | waterborne P101 | Medium | $5.42X10^3$ cysts/mL | G.lam | 37282 | 3/3 |
| | Shigella sonnei | ATCC 29930 | Medium | $1.31X10^3$ CFU/mL | Shig | 10518 | 3/3 |
| Cl-8 | Giardia intestinalis | waterborne P101 | High | $3.0x10^5$ cysts/mL | G.lam | 12832 | 3/3 |
| | Shigella sonnei | ATCC 29930 | Medium | $1.31X10^3$ CFU/mL | Shig | 12530 | 3/3 |
| | Shiga-toxin producing
E. coli (STEC) | ATCC BAA-2217 | Medium | $7.5X10^3$ CFU/mL | stx2 | 29785 | 3/3 |
| CI-9 | Shigella sonnei | ATCC 29930 | High | $3.0x10^6$ CFU/mL | Shig | 12483 | 3/3 |
| | Shiga-toxin producing
E. coli (STEC) | ATCC BAA-2217 | Medium | $7.5X10^3$ CFU/mL | stx2 | 31890 | 3/3 |
| | Giardia intestinalis | waterborne P101 | Medium | $5.42X10^3$ cysts/mL | G.lam | 31227 | 3/3 |

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CROSS CONTAMINATION/SAMPLE CARRYOVER

A study was performed to demonstrate the absence of carryover or cross-contamination when using the BioCode Gastrointestinal Pathogen Panel in conjunction with the easyMag. High-positive samples (EAEC at 10° CFU/mL) were tested alternating with no-template control samples in a

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"checkerboard" pattern. Samples were extracted checkerboard and assayed in singlet. The study consisted of five complete runs from extraction to BioCode MDx 3000 results on one instrument. No cross contamination was observed.

FRESH VS. FROZEN STABILITY

Since many of the unpreserved samples in three of the four sites using 60 spiked unpreserved stool specimens to assess fresh vs frozen specimen stability. Specimens were contrived and tested at time 0 and after ≥2 freeze thaw cycles. Samples were tested at Low positive (1X-1.5X LoD) spiked into 7 negative stool samples. Cary-Blair specimens were not subjected to frozen storage during the clinical study, therefore there were not tested. Frozen samples displayed stability throughout the length of the study.

Fresh Vs. Frozen results.

Acceptance Criteria	Results
95% replicates for all positive targets in the contrived sample should be
Valid and Detected (≥60/63)	63/63
95% of negative targets should be Valid and not detected (≥60/63)	63a/63

a - Sample Target was detected, but was invalid for RNA IC.

Analysis of results from Fresh Vs Frozen study.

| Analysis | | | Delta Fresh -
Frozen | Percent
Decrease |
|---------------------------------------------------|------------------|------|-------------------------|---------------------|
| Campylobacter jejuni subsp. jejuni | ATCC 33292 | FF1 | 3835 | 14% |
| Escherichia coli 10C-3114 (STEC) | ATCC BAA-2217 | FF2 | 3019 | 12% |
| Human rotavirus A | ATCC VR-2018 | FF3 | 4437 | 13% |
| Vibrio parahaemolyticus | ATCC 17802 | FF4 | 1260 | 7% |
| O78:H11 Escherichia coli strain H10407 (ETEC) STa | ATCC 35401 | FF5 | 2503 | 8% |
| Shigella sonnei | ATCC 29930 | FF6 | 2365 | 20% |
| Salmonella enterica subsp. Enterica | ATCC 14028 | FF7 | -148 | -2% |
| Human adenovirus 40 (dugan) | Zeptometrix | FF8 | 610 | 3% |
| Cryptosporidium parvum | waterborne P102M | FF9 | 3134 | 14% |
| Negative | Negative | FF10 | 1719 | 6% |

SPECIMEN STABILITY

A study was performed to assess the specimen stability limitations for the optimal performance of the BioCode Gastrointestinal Pathogen Panel on the BioCode MDx 3000. This study employed the use of spiked specimens to assess the following storage conditions:

Samples in Cary-Blair - 0, 2, 4 and 6 days at room temperature (20-25°C); 2, 4 and 6 days at 2-8°C Unpreserved Stool - 0, 2, 4 and 6 days at 2-8°C Fresh vs. Frozen (-90°C to -60°C) (2x freeze thaws) - 30, 60 and 90 days at -90°C to -60°C S.T.A.R. buffer after pretreatment (SK38 Tubes prior to extraction) - 24 hours at 2-8°C Fresh vs. Frozen (-90°C to -60°C) (2x freeze thaws) - 30, 60 and 90 days at -90°C to -60°C Extracted Nucleic Acid - 24 hours at 2-8°C Fresh vs. Frozen (-90°C to -60°C) (2x freeze thaws) - 30, 60 and 90 days at -90°C to -60°C

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One parasite, one virus, one-gram positive bacterium, and one-gram negative bacterium was used for testing. Spiked and clinical samples were tested at ~3X LoD with two organisms in each sample. Concentrated organism stocks were serially diluted in Cary-Blair and combined with prescreened negative stool and each condition was assayed with 3 replicates at each time point. Samples displayed stability under the various conditions throughout the length of the study.

Image /page/51/Figure/2 description: The image contains four line graphs that show the MFI (Mean Fluorescence Intensity) values over time for different stool samples under different preservation conditions. The top two graphs show "Upreserved stool at 4°C" over 6 days, with lines representing SSA-Un tcdB, SSA-Un Crypto, SSB-Un Stx2, and SSB-Un Rota. The bottom left graph shows "Upreserved stool at -80°C" over 90 days, with lines for SSA-Un Crypto, SSB-Un Stx2, and SSB-Un Rota. The bottom right graph shows "SK38 tubes at 4°C" over 24 hours, with lines for SSA SK38 tcdB, SSA-SK38 Crypto, SSB-SK38 Stx2, and SSB-SK38 Rota.

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Image /page/52/Figure/1 description: The image contains four line graphs that show the MFI (Mean Fluorescence Intensity) values over time in days for different specimens. The top two graphs show data for Nucleic Acid and SK38 tubes stored at -80°C, while the bottom two graphs show data for Cary-Blair Specimens stored at 4°C and at Room Temperature. Each graph plots four different lines representing SSA-Un tcdB, SSA-Un Crypto, SSB-Un Stx2, and SSB-Un Rota, with MFI values ranging from 0 to 50000 on the y-axis and time in days on the x-axis. The Cary-Blair Specimens graphs show data over a shorter time period of 0 to 6 days, while the Nucleic Acid and SK38 tubes graphs show data over a period of 0 to 90 days.

Image /page/52/Figure/2 description: The image is a title for a figure. The title states that the figure is a graphic display of MFI results at indicated storage temperatures and timepoints. The title is written in a clear, easy-to-read font.

Figure. Graphic display of MFI results at indicated storage temperatures and timepoints.

CONCLUSION

The intended use and fundamental scientific technology of the BioCode Gastrointestinal Pathogen Panel Assay is substantially equivalent to the predicate device. Clinical and non-clinical studies have established that the BioCode Gastrointestinal Pathogen Panel is substantially equivalent to the predicate device.