BioCode Gastrointestinal Pathogen Panel (GPP)

• K Number: K242877
• Device Name: BioCode Gastrointestinal Pathogen Panel (GPP)
• Manufacturer: Applied BioCode, Inc.
• Date Cleared: 2025-01-10 ( 109 days)
• Product Code: PCH
• Regulation Number: 866.3990

Intended Use

The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplexed in vitro diagnostic test intended for use with the BioCode MDx 3000 Instrument. The BioCode GPP is capable of the simultaneous detection of nucleic acids from multiple bacteria, viruses, and parasites extracted directly from unpreserved in Cary-Blair transport medium obtained from individuals with simptoms of gastrointestinal infection. The following bacteria, parasites, and viruses are identified using the BioCode Gastrointestinal Pathogen Panel: - . Campylobacter (C. jejuni/C. coli) - Clostridium difficile (C. difficile) toxin A/B (Fresh samples only) - l 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) lt/st - E. coli 0157 serogroup - Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2 - Shigella/ Enteroinvasive Escherichia coli (EIEC) - Cryptosporidium spp (C. parvum/C. hominis) - 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 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 gastroenteriis 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 Adenovinus 40/41, Campylobacter, E. coli 0157, Shigella(EIEC, Yersinia enterocolitica, and Giardia lamblia were established additionally with retrospective clinical specimens. Performance characteristica, Giardia lamblia, Yersinia enterocolitica and Vibrio (V. parahaemolyticus, V. cholerae) were established primarily using contrived clinical specimens.

Device Description

The BioCode® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid-based test designed to be used with the BioCode MDx-3000 system. The BioCode MDx-3000 is an automated system that integrates PCR amplification, target capture, signal generation and optical detection for multiple gastrointestinal pathogens from a single stool specimen, either unpreserved or in Cary Blair. Stool specimens are processed, and nucleic acids extracted with easyMAG, MagNA Pure 96, KingFisher Flex and KingFisher Apex Dx. Once the PCR plate is set up and sealed, all other operations are automated on MDx-3000. The BioCode MDx-3000 Gastrointestinal Infection 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 (GPP) test are available within less than 4 hours.

Predicate Devices

K190585

Reference Devices

K180041

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a multiplexed nucleic acid-based test and an automated system for PCR amplification, target capture, signal generation, and optical detection. There is no mention of AI or ML in the intended use, device description, or performance studies. The analysis focuses on traditional metrics like percent agreement and CV, not AI/ML-specific metrics or training/test sets for AI/ML models.

Therapeutic

No

The device is an in vitro diagnostic test designed to detect nucleic acids from multiple bacteria, viruses, and parasites for the diagnosis of gastrointestinal illness. It is not intended for treatment or therapeutic purposes.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states, "The BioCode GPP is indicated as an aid in the diagnosis of gastrointestinal illness".

SaMD (Software as a Medical Device)

No

The device description explicitly states it is a "multiplexed nucleic acid-based test designed to be used with the BioCode MDx-3000 system," which is described as an "automated system that integrates PCR amplification, target capture, signal generation and optical detection." This involves significant hardware components and processes beyond just software.

IVD (In Vitro Diagnostic)

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

The "Intended Use / Indications for Use" section explicitly states: "The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplexed in vitro diagnostic test intended for use with the BioCode MDx 3000 Instrument." It also includes the statement "For In Vitro Diagnostic Use Only."

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplexed in vitro diagnostic test intended for use with the BioCode MDx 3000 Instrument. The BioCode GPP is capable of the simultaneous detection of nucleic acids from multiple bacteria, viruses, and parasites extracted directly from unpreserved in Cary-Blair transport medium obtained from individuals with simptoms of gastrointestinal infection. The following bacteria, parasites, and viruses are identified using the BioCode Gastrointestinal Pathogen Panel:

• . Campylobacter (C. jejuni/C. coli)
• Clostridium difficile (C. difficile) toxin A/B (Fresh samples only)
• l 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) lt/st
• E. coli 0157 serogroup
• Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2
• Shigella/ Enteroinvasive Escherichia coli (EIEC)
• Cryptosporidium spp (C. parvum/C. hominis)
• 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 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 gastroenteriis 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 Adenovinus 40/41, Campylobacter, E. coli 0157, Shigella(EIEC, Yersinia enterocolitica, and Giardia lamblia were established additionally with retrospective clinical specimens. Performance characteristica, Giardia lamblia, Yersinia enterocolitica and Vibrio (V. parahaemolyticus, V. cholerae) were established primarily using contrived clinical specimens.

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

PCH, OOI

Device Description

The BioCode® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid-based test designed to be used with the BioCode MDx-3000 system. The BioCode MDx-3000 is an automated system that integrates PCR amplification, target capture, signal generation and optical detection for multiple gastrointestinal pathogens from a single stool specimen, either unpreserved or in Cary Blair. Stool specimens are processed, and nucleic acids extracted with easyMAG, MagNA Pure 96, KingFisher Flex and KingFisher Apex Dx. Once the PCR plate is set up and sealed, all other operations are automated on MDx-3000. The BioCode MDx-3000 Gastrointestinal Infection 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 (GPP) test are available within less than 4 hours.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

Not Found

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

Not Found

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

Reproducibility Study:

• Sample Size: 90 replicates per organism (3 instruments, 3 operators, 2 runs/day, 5 days, 3 replicates per organism per run) for positive agreement; 450 replicates for negative agreement.
• Data Source: Organisms diluted in Cary-Blair and negative stool.
• Annotation Protocol: Not specified, but involved assessing agreement with expected results and calculating CI, Mean MFI, and %CV.

Limit of Detection (LoD) Study:

• Sample Size: Initial screening used 4 replicates; LoD confirmed with 20 replicates for each sample type/extraction method.
• Data Source: Quantified bacteria, virus, or parasite stocks (except norovirus which used clinical samples).
• Annotation Protocol: LoD defined as lowest concentration with >=95% detection of 20 replicates (19 out of 20).

Method Comparison Study (Clinical Investigational Study):

• Sample Size: 468 remnant, de-identified samples (254 frozen unpreserved stool and 214 inoculated Cary-Blair stool) from a previous clinical study (K180041). 54 freshly collected leftover samples for C. difficile testing. 120 contrived samples (15 at 3x LoD and 15 at 6x LoD for each of three targets).
• Data Source: Remnant, de-identified stool samples; freshly collected leftover stool samples; contrived samples.
• Annotation Protocol: Samples were extracted using KingFisher Flex, KingFisher Apex Dx, and easyMAG, and tested on the MDx-3000 system. Positive Percent Agreement (PPA) calculated as TP/(TP+FN). Negative Percent Agreement (NPA) calculated as TN/(TN + FP). Exact binomial two-sided 95% confidence interval calculated. Discordant samples were retested twice with both easyMAG and KingFisher Flex/Apex Dx. Invalid samples were subjected to reflex testing.

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

REPRODUCIBILITY STUDY:

• Study Type: Reproducibility study.
• Sample Size: 90 replicates for positive agreement for each organism/concentration tested (3 instruments, 3 operators, 2 runs/day, 5 days, 3 replicates per organism per run). 450 replicates for negative agreement.
• Key Results (KingFisher Flex - Positive Agreement): All organisms showed 98.9% or 100% agreement with expected results at 1.5X LoD and 3X LoD.
• Key Results (KingFisher Flex - Negative Agreement): All targets showed 100% Negative Agreement (450/450).
• Key Results (KingFisher Apex Dx - Positive Agreement): All organisms showed 98.89% or 100% agreement with expected results at 1.5X LoD and 3X LoD.
• Key Results (KingFisher Apex Dx - Negative Agreement): Clostridium difficile and Yersinia enterocolitica showed 99.56% and 99.33% Negative Agreement respectively, while other targets showed 100%.

LIMIT OF DETECTION (LoD):

• Study Type: Analytical sensitivity study.
• Sample Size: 20 replicates per sample type/extraction method for confirmation.
• Key Results (Unpreserved Stool): LoDs varied by organism and extraction method, generally within 3-fold for each system, with noted exceptions.
• Key Results (Cary-Blair Stool): LoDs varied by organism and extraction method, generally within 3-fold for each system, with noted exceptions.
• Key Results (Norovirus): For unpreserved stool, Norovirus GII LoD was equivalent across systems, while KingFisher Flex was 10-fold more sensitive for Norovirus GI than easyMAG, and 3-fold more sensitive than KingFisher Apex Dx. For Cary-Blair stool, LoD for Norovirus GI was equivalent, and KingFisher Apex Dx was 3-fold more sensitive for Norovirus GII than easyMAG and KingFisher Flex.

METHOD COMPARISON STUDY:

• Study Type: Clinical investigational study comparing KingFisher Flex and KingFisher Apex Dx against easyMAG.
• Sample Size: 468 archived samples (254 frozen unpreserved stool, 214 inoculated Cary-Blair stool). 54 fresh unpreserved stool samples. 120 contrived samples.
• Key Results (KingFisher Flex - Archived Specimens):
  • Positive Agreement (PPA) ranged from 90.9% to 100% for individual targets, and 96.1% to 98.5% for combined targets.
  • Negative Agreement (NPA) ranged from 96.6% to 100% for individual targets, and 99.6% to 99.7% for combined targets.
  • Noted PPA

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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January 10, 2025

Image /page/0/Picture/1 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). The logo consists of two parts: on the left, there is a symbol representing the Department of Health & Human Services - USA, and on the right, there is the text "FDA U.S. FOOD & DRUG ADMINISTRATION" in blue. The text is arranged in three lines, with "FDA" being the largest and boldest, followed by "U.S. FOOD & DRUG" and then "ADMINISTRATION".

Applied BioCode, Inc. Alex Chang Regulatory Consultant 12130 Mora Drive Unit 2 Santa Fe Springs, California 90670

Re: K242877

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 Dated: September 20, 2024 Received: September 23, 2024

Dear Alex Chang:

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 (the 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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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.

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Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30. Design controls; 21 CFR 820.90. Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

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 of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

All medical devices, including Class I and unclassified devices and combination product device constituent parts are required to be in compliance with the final Unique Device Identification System rule ("UDI Rule"). The UDI Rule requires, among other things, that a device bear a unique device identifier (UDI) on its label and package (21 CFR 801.20(a)) unless an exception or alternative applies (21 CFR 801.20(b)) and that the dates on the device label be formatted in accordance with 21 CFR 801.18. The UDI Rule (21 CFR 830.300(a) and 830.320(b)) also requires that certain information be submitted to the Global Unique Device Identification Database (GUDID) (21 CFR Part 830 Subpart E). For additional information on these requirements, please see the UDI System webpage at https://www.fda.gov/medical-device-advicecomprehensive-regulatory-assistance/unique-device-identification-system-udi-system.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatory

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assistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

Image /page/2/Picture/3 description: The image shows a digital signature. The signature is for Bryan M. Grabias. The signature was created on January 10, 2025 at 10:49:43 -05'00'.

Bryan Grabias, Ph. D. Acting Branch Chief Bacterial Respiratory and Medical Countermeasures Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known)

K242877

Device Name

BioCode Gastrointestinal Pathogen Panel (GPP)

Indications for Use (Describe)

The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplexed in vitro diagnostic test intended for use with the BioCode MDx 3000 Instrument. The BioCode GPP is capable of the simultaneous detection of nucleic acids from multiple bacteria, viruses, and parasites extracted directly from unpreserved in Cary-Blair transport medium obtained from individuals with simptoms of gastrointestinal infection. The following bacteria, parasites, and viruses are identified using the BioCode Gastrointestinal Pathogen Panel:

• . Campylobacter (C. jejuni/C. coli)
• Clostridium difficile (C. difficile) toxin A/B (Fresh samples only)
• l 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) lt/st
• E. coli 0157 serogroup
• Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2
• Shigella/ Enteroinvasive Escherichia coli (EIEC)
• Cryptosporidium spp (C. parvum/C. hominis)
• 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 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 gastroenteriis 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 Adenovinus 40/41, Campylobacter, E. coli 0157, Shigella(EIEC, Yersinia enterocolitica, and Giardia lamblia were established additionally with retrospective clinical specimens. Performance characteristica, Giardia lamblia, Yersinia enterocolitica and Vibrio (V. parahaemolyticus, V. cholerae) were established primarily using contrived clinical specimens.

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

X Prescription Use (Part 21 CFR 801 Subpart D)

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510(k) Summary

Prepared in accordance with the requirements of 21 CFR 807.92.

Submitter Information [807.92(a)(1)]

Submitter/	Applied BioCode®, Inc.
Applicant		12130 Mora Drive
		Santa Fe Springs, CA 90670

Submitter Contact: Alex Chang Phone: [phone] Email: [e-mail]

Device Information [807.92(a)(2)]

Trade Name	BioCode Gastrointestinal Pathogen Panel (GPP)
Classification	Gastrointestinal microorganism multiplex nucleic acid-based assay
Name
Regulation	21 CFR § 866.3990

Predicate Device [807.92(a)(3)]

BioCode Gastrointestinal Pathogen Panel (GPP) K190585

Device Description [807.92(a)(4)]

The BioCode® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid-based test designed to be used with the BioCode MDx-3000 system. The BioCode MDx-3000 is an automated system that integrates PCR amplification, target capture, signal generation and optical detection for multiple gastrointestinal pathogens from a single stool specimen, either unpreserved or in Cary Blair. Stool specimens are processed, and nucleic acids extracted with easyMAG, MagNA Pure 96, KingFisher Flex and KingFisher Apex Dx. Once the PCR plate is set up and sealed, all other operations are automated on MDx-3000. The BioCode MDx-3000 Gastrointestinal Infection 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 (GPP) test are available within less than 4 hours.

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Bacteria. Viruses. Diarrheagenic E. coli/Shigella. and Parasites Detected by the BioCode MDx Gastrointestinal Pathogen Panel (GPP)

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

Intended Use/ Indications for Use [807.92(a)(5)]

The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative multiplexed nucleic acidbased 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, parasites, and viruses are identified using the BioCode Gastrointestinal Pathogen Panel:

• Campylobacter (C. jejuni/C. coli) "
• . Clostridium difficile (C. difficile) toxin A/B (Fresh samples only)
• . 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) lt/st
• . E. coli 0157 serogroup
• Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2 "
• Shigella/ Enteroinvasive Escherichia coli (EIEC) .
• " Cryptosporidium spp (C. parvum/C. hominis)
• . 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.

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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 0157, 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.

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Device Comparison [807.92(a)(6)]:

Characteristic	Proposed Device	Predicate
Name	BioCode® Gastrointestinal Pathogen Panel (GPP)	BioCode® Gastrointestinal Pathogen Panel (GPP)
Common Name	Gastrointestinal Microorganism Multiplex Nucleic acid-based assay	Gastrointestinal Microorganism Multiplex Nucleic acid-based assay
510(k) No.	K242877	K190585
Regulation	21CFR 866.3990	21CFR 866.3990
Product Code	PCH, OOI	PCH, OOI
Device Class	II	II
	Similarities
Intended Use	The BioCode Gastrointestinal Pathogen Panel (GPP) is a qualitative, multiplexed 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, parasites, and viruses are identified using the BioCode Gastrointestinal Pathogen Panel:
Campylobacter ( C. jejuni/C. coli ) Clostridium difficile ( C. difficile ) toxin A/B (Fresh samples only) 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) lt/st E. coli O157 serogroup Shiga-like toxin-producing Escherichia coli (STEC) stx1/stx2 Shigella/ Enteroinvasive Escherichia coli (EIEC) Cryptosporidium spp ( C. parvum/C.	Same
Characteristic	Proposed Device	Predicate
	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.
Instrument	Nucleic Acid Purification System
BioCode MDx-3000	Same
Sample Type	Unpreserved stool and stool in Cary Blair
Medium	Same
Controls	Externally Sourced - Zeptometrix	Same
Methodology	Multiplex RT-PCR and probe
hybridization to biotinylated PCR
product(s) followed by fluorescence
detection and decoding of barcoded
magnetic beads (BMB) that are coupled to
target-specific probes	Same
Calibrators	Internal Calibration	Same
Differences
Sample Extraction	easyMAG, Roche MagNA Pure 96,
KingFisher Flex, KingFisher Apex Dx	easyMAG, Roche MagNA Pure
96

Comparison of the Applied BioCode GPP with the Predicate Device

9

10

Summary of Performance Data [807.92(b)]

All necessary testing was conducted on BioCode Gastrointestinal Pathogen Panel (GPP) to support a determination of substantial equivalence to the predicate device.

Nonclinical testing summary [807.92(b)(1)]

REPRODUCIBILITY STUDY

A study was performed to assess the Reproducibility of the BioCode® GPP using samples extracted with the KingFisher Flex and KingFisher Apex Dx. This study was designed to assess intra-assay (within run), Inter-assay (run-to-run), day-to-day and instrument-to-instrument (operator-to-operator) reproducibility. One lot of reagents was assayed at Applied BioCode on 3 instruments by 3 operators, 2 runs per day per operator for 5 days (total of 30 runs).

Organisms were diluted in Cary-Blair, and pools were constructed in Cary Blair media at 15X or 30X LoD. The prepared pools were then diluted 1:10 fold in pre-screened negative stool, frozen over night or longer, extracted in triplicate and assayed in singlicate. In total 30 sets of RP pools were run in singlicate on 3 instruments by 3 operators over 5 days.

| Organism | Concentration | % Agreement
with
Expected
Result | 95% CI | Mean
MFI | %CV |
|-----------------------------------|--------------------------------------|-------------------------------------------|----------------|-------------|------|
| Salmonella enterica* | 3X LoD (6.60E+03
CFU/mL) | (90/90) 100% | (95.98,100.0) | 15920 | 10.9 |
| Salmonella enterica* | 1.5X LoD
(3.30E+03
CFU/mL) | (89/90) 98.9% | (93.96, 99.97) | 16923 | 8.0 |
| Clostridium difficile
(tcd A)* | 3X LoD (9.21E+01
CFU/mL) | (90/90) 100% | (95.98,100.0) | 15693 | 19.7 |
| Clostridium difficile
(tcd A)* | 1.5X LoD
(4.61E+01
CFU/mL) | (89/90) 98.9% | (93.96, 99.97) | 10333 | 36.3 |
| Clostridium difficile
(tcd B)* | 3X LoD (9.21E+01
CFU/mL) | (90/90) 100% | (95.98,100.0) | 23783 | 12.0 |
| Clostridium difficile
(tcd B)* | 1.5X LoD
(4.61E+01
CFU/mL) | (89/90) 98.9% | (93.96, 99.97) | 18821 | 17.4 |
| Giardia lamblia | 3X LoD (5.40E+03
cysts/mL) | (89/90) 98.9% | (93.96, 99.97) | 13827 | 28.3 |
| Giardia lamblia | 1.5X LoD
(2.70E+03
cysts/mL) | (90/90) 100% | (95.98,100.0) | 15071 | 27.6 |
| Adenovirus 40* | 3X LoD (9.90E-02
TCID50/mL) | (89/90) 98.9% | (93.96, 99.97) | 13061 | 20.6 |
| Adenovirus 40* | 1.5X LoD (4.95E-02
TCID50/mL) | (90/90) 100% | (95.98,100.0) | 16742 | 19.3 |
| Shigella sonnei | 3X LoD (1.32E+03
CFU/mL) | (90/90) 100% | (95.98,100.0) | 19328 | 13.9 |
| Shigella sonnei | 1.5X LoD
(6.60E+02
CFU/mL) | (90/90) 100% | (95.98,100.0) | 19515 | 14.3 |
| Organism | Concentration | % Agreement
with
Expected
Result | 95% CI | Mean
MFI | %CV |
| ETEC (LT) | 3X LoD (1.68E+03
CFU/mL) | (90/90) 100% | (95.98,100.0) | 13727 | 18.5 |
| ETEC (LT) | 1.5X LoD
(8.42E+02
CFU/mL) | (90/90) 100% | (95.98,100.0) | 13137 | 21.4 |
| ETEC (ST1a) | 3X LoD (1.68E+03
CFU/mL) | (90/90) 100% | (95.98,100.0) | 23135 | 17.2 |
| ETEC (ST1a) | 1.5X LoD
(8.42E+02
CFU/mL) | (90/90) 100% | (95.98,100.0) | 25866 | 11.4 |
| ETEC (ST1b) | 3X LoD (1.68E+03
CFU/mL) | (90/90) 100% | (95.98,100.0) | 26034 | 15.1 |
| ETEC (ST1b) | 1.5X LoD
(8.42E+02
CFU/mL) | (90/90) 100% | (95.98,100.0) | 27576 | 13.0 |
| Vibrio parahaemolyticus
(ToxR) | 3X LoD (3.90E+01
CFU/mL) | (90/90) 100% | (95.98,100.0) | 26590 | 12.1 |
| Vibrio parahaemolyticus
(ToxR) | 1.5X LoD
(1.95E+01
CFU/mL) | (90/90) 100% | (95.98,100.0) | 21967 | 20.0 |
| Vibrio parahaemolyticus
(Vib) | 3X LoD (3.90E+01
CFU/mL) | (90/90) 100% | (95.98,100.0) | 10900 | 12.1 |
| Vibrio parahaemolyticus
(Vib) | 1.5X LoD
(1.95E+01
CFU/mL) | (90/90) 100% | (95.98,100.0) | 9295 | 29.7 |
| Yersinia enterocolitica | 3X LoD (5.01E+02
CFU/mL) | (90/90) 100% | (95.98,100.0) | 14645 | 22.1 |
| Yersinia enterocolitica | 1.5X LoD
(2.51E+02
CFU/mL) | (90/90) 100% | (95.98,100.0) | 13641 | 27.6 |
| STEC | 3X LoD (2.25E+04
CFU/mL) | (90/90) 100% | (95.98,100.0) | 17774 | 15.0 |
| STEC | 1.5X LoD
(1.13E+04
CFU/mL) | (90/90) 100% | (95.98,100.0) | 17965 | 18.1 |
| Campylobacter coli* | 3X LoD (1.68E+02
CFU/mL) | (89/90) 98.9% | (93.96, 99.97) | 22869 | 7.8 |
| Campylobacter coli* | 1.5X LoD
(8.40E+01
CFU/mL) | (90/90) 100% | (95.98,100.0) | 21878 | 12.9 |
| Cryptosporidium*
parvum | 3X LoD (9.30E+03
oocysts/mL) | (90/90) 100% | (95.98,100.0) | 23202 | 16.9 |
| Cryptosporidium*
parvum | 1.5X LoD
(4.65E+03
oocysts/mL) | (89/90) 98.9% | (93.96, 99.97) | 22292 | 11.9 |
| Rotavirus A* | 3X LoD (6.57E+02
TCID50/mL) | (90/90) 100% | (95.98,100.0) | 26666 | 24.4 |
| Rotavirus A* | 1.5X LoD
(3.29E+02
TCID50/mL) | (89/90) 98.9% | (93.96, 99.97) | 22240 | 14.5 |

Table 1. KingFisher Flex - Positive Agreement of Results of Reproducibility Panel.

11

12

*False negatives became positives upon repeat from PCR, using the same extracts. The false negatives were due to sampling errors or PCR amplicon transfer during hybridization of MDx-3000.

| Target | Negative Agreement
(True Negative/True
Negative+ False Positive) | 95% CI |
|--------------------------|------------------------------------------------------------------------|-----------------|
| Salmonella enterica | 450/450 (100.00) | (99.15, 100.00) |
| Clostridium difficile | 450/450 (100.00) | (99.15, 100.00) |
| Giardia lamblia | 450/450 (100.00) | (99.15, 100.00) |
| Adenovirus 40 | 450/450 (100.00) | (99.15, 100.00) |
| Shigella sonnei | 450/450 (100.00) | (99.15, 100.00) |
| Vibrio parahaemolyticus* | 450/450 (100.00) | (99.15, 100.00) |
| ETEC | 450/450 (100.00) | (99.15, 100.00) |
| Yersinia enterocolitica | 450/450 (100.00) | (99.15, 100.00) |
| STEC | 450/450 (100.00) | (99.15, 100.00) |
| Campylobacter coli | 450/450 (100.00) | (99.15, 100.00) |
| Cryptosporidium parvum | 450/450 (100.00) | (99.15, 100.00) |
| Rotavirus A | 450/450 (100.00) | (99.15, 100.00) |

Table 2. KingFisher Flex – Negative Agreement of Results of Reproducibility Panel.

13

| Organism | Concentration | % Agreement
with Expected
Result | 95% CI | Mean MFI | %CV |
|------------------------------------|-----------------------------------|----------------------------------------|----------------|----------|------|
| Salmonella enterica | 3X LoD (6.60E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 16189 | 13.2 |
| | 1.5X LoD (3.30E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 17748 | 18.2 |
| Clostridium difficile
(tcd A)* | 3X LoD (9.21E+01
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 18406 | 18.8 |
| | 1.5X LoD (4.61E+01
CFU/mL) | (89/90) 98.89% | (93.96, 99.97) | 12099 | 30.4 |
| Clostridium difficile
(tcd B)* | 3X LoD (9.21E+01
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 26553 | 9.9 |
| | 1.5X LoD (4.61E+01
CFU/mL) | (89/90) 98.89% | (93.96, 99.97) | 20579 | 16.5 |
| Giardia lamblia | 3X LoD (1.80E+03
cysts/mL) | (90/90) 100% | (95.98, 100.0) | 14679 | 26.5 |
| | 1.5X LoD (9.00E+02
cysts/mL) | (90/90) 100% | (95.98, 100.0) | 18652 | 19.5 |
| Adenovirus 40* | 3X LoD (9.90E-02
TCID50/mL) | (89/90) 98.89% | (93.96, 99.97) | 13952 | 21.4 |
| | 1.5X LoD (4.95E-02
TCID50/mL) | (90/90) 100% | (95.98, 100.0) | 19824 | 14.4 |
| Shigella sonnei | 3X LoD (1.32E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 17642 | 17.0 |
| | 1.5X LoD (6.60E+02
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 18937 | 18.5 |
| ETEC (LT)* | 3X LoD (1.68E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 16907 | 11.8 |
| | 1.5X LoD (8.42E+02
CFU/mL) | (89/90) 98.89% | (93.96, 99.97) | 15586 | 15.2 |
| ETEC (ST1a) | 3X LoD (1.68E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 22884 | 17.9 |
| | 1.5X LoD (8.42E+02
CFU/mL) | (89/90) 98.9% | (93.96, 99.97) | 27127 | 10.6 |
| ETEC (ST1b)* | 3X LoD (1.68E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 26896 | 14.3 |
| | 1.5X LoD (8.42E+02
CFU/mL) | (89/90) 98.89% | (93.96, 99.97) | 29785 | 8.6 |
| Vibrio parahaemolyticus
(ToxR)* | 3X LoD (1.30E+01
CFU/mL) | (89/90) 98.89% | (93.96, 99.97) | 24510 | 11.9 |
| | 1.5X LoD (6.50E+00
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 20694 | 16.2 |
| Vibrio parahaemolyticus
(Vib)* | 3X LoD (1.30E+01
CFU/mL) | (89/90) 98.89% | (93.96, 99.97) | 9362 | 16.6 |
| | 1.5X LoD (6.50E+00
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 7110 | 24.2 |
| Yersinia enterocolitica | 3X LoD (1.50E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 7245 | 33.6 |
| | 1.5X LoD (7.52E+02
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 6912 | 34.0 |
| Organism | Concentration | % Agreement
with Expected
Result | 95% CI | Mean MFI | %CV |
| STEC | 3X LoD (2.50E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 19069 | 15.0 |
| | 1.5X LoD (1.25E+03
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 17716 | 20.3 |
| Campylobacter coli | 3X LoD (1.68E+02
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 23830 | 6.2 |
| | 1.5X LoD (8.40E+01
CFU/mL) | (90/90) 100% | (95.98, 100.0) | 23139 | 10.5 |
| Cryptosporidium parvum | 3X LoD (3.09E+03
oocysts/mL) | (90/90) 100% | (95.98, 100.0) | 22639 | 15.5 |
| | 1.5X LoD (1.55E+03
oocysts/mL) | (90/90) 100% | (95.98, 100.0) | 22527 | 12.5 |
| Rotavirus A | 3X LoD (6.57E+02
TCID50/mL) | (90/90) 100% | (95.98, 100.0) | 25647 | 20.8 |
| | 1.5X LoD (3.29E+02
TCID50/mL) | (90/90) 100% | (95.98, 100.0) | 25301 | 18.8 |

Table 3. KingFisher Apex Dx-Positive Agreement of Results of Reproducibility Panel.

14

*False negatives became positives upon repeat from PCR, using the same extracts. The false negatives were due to sampling errors or PCR amplicon transfer during hybridization of MDx-3000.

Table 4. KingFisher Apex Dx-Negative Agreement of Results of Reproducibility Panel.			and the country of the country of the first of the first of the first of the first of the first of the first of the first of the first of the first of the first of the first

| Target | Negative Agreement
(True Negative/True
Negative+ False Positive) | 95% CI |
|-------------------------|------------------------------------------------------------------------|-----------------|
| Salmonella enterica | 450/450 (100.00) | (99.15, 100.00) |
| Clostridium difficile | 448/450 (99.56) | (98.39, 99.88) |
| Giardia lamblia | 450/450 (100.00) | (99.15, 100.00) |
| Adenovirus 40 | 450/450 (100.00) | (99.15, 100.00) |
| Shigella sonnei | 450/450 (100.00) | (99.15, 100.00) |
| Vibrio parahaemolyticus | 450/450 (100.00) | (99.15, 100.00) |
| ETEC | 450/450 (100.00) | (99.15, 100.00) |
| Yersinia enterocolitica | 447/450 (99.33) | (98.06, 99.77) |
| STEC | 450/450 (100.00) | (99.15, 100.00) |
| Campylobacter coli | 450/450 (100.00) | (99.15, 100.00) |
| Cryptosporidium parvum | 450/450 (100.00) | (99.15, 100.00) |
| Rotavirus A | 450/450 (100.00) | (99.15, 100.00) |

15

LIMIT OF DETECTION (LoD)

A study was performed to assess the performance of the BioCode GPP on the BioCode MDx-3000 at the Limit of Detection (LoD) for both unpreserved Stool and Cary-Blair specimens. In this study the GI Panel was tested with quantified bacteria, virus or parasite stocks (except norovirus which used clinical samples). For initial screening, four replicates of each concentration (near LoD for the predicate) in negative stool and Cary-Blair were extracted on the easyMAG, the KingFisher Flex and the KingFisher Apex Dx and tested in singlet with the BioCode® GPP on the BioCode MDx-3000 system. The LoD was confirmed by extracting 20 replicates of each sample type/extraction method 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. The LoDs determined from the quantitated stocks or clinical samples are presented below and in the labeling. The LoDs were the same or within 3-fold for each extraction system with a few notable exceptions identified in the tables with footnotes.

Table 5. Comparison of results for limit of detection testing for Unpreserved Stool extracted
with the easyMAG, the KingFisher Apex Dx, KingFisher Flex and assayed with BioCode® GPP.

		EasyMag		KingFisher Flex		KingFisher Apex Dx
Strain	Source	Unpreserve
d Stool LoD	Detectio
n	Unpreserve
d Stool LoD	Detectio
n	Unpreserve
d Stool LoD	Detectio
n
Campylobacter
coli	ATCC
33559	$5.60 \times 10^1$
CFU/mL	20/20	$5.60 \times 10^1$
CFU/mL	20/20	$5.60 \times 10^1$
CFU/mL	20/20
Campylobacter
jejuni spp. jejuni	ATCC
33292	$2.33 \times 10^2$
CFU/mL	20/20	$2.33 \times 10^2$
CFU/mL	20/20	$2.33 \times 10^2$
CFU/mL	20/20
Clostridium
difficile
(toxinotype 0)	ATCC
9689	$2.11 \times 10^1$
CFU/mL	20/20	$2.11 \times 10^1$
CFU/mL	20/20	$2.11 \times 10^1$
CFU/mL	20/20
Clostridium
difficile
(toxinotype III;
Napl)	Zeptometri
X
0801619cf	$3.07 \times 10^1$
CFU/mL	19/20	$3.07 \times 10^1$
CFU/mL	20/20	$3.07 \times 10^1$
CFU/mL	20/20
Enteroaggregativ
e E. coli
092:H33
(EAEC)	STEC
TW04440	$1.40 \times 10^3$
CFU/mL	20/20	$1.40 \times 10^3$
CFU/mL	20/20	$1.40 \times 10^3$
CFU/mL	20/20
Enteroinvasive
E. coli O29:NM
(EIEC)	ATCC
43892	$3.60 \times 10^2$
CFU/mL	20/20	$3.60 \times 10^2$
CFU/mL	20/20	$3.60 \times 10^2$
CFU/mL	20/20
Enterotoxigenic
E. coli 078:H11
H10407 (ETEC)	ATCC
35401	$1.87 \times 10^2$
CFU/mL	20/20	$5.61 \times 10^2$
CFU/mL	20/20	$5.61 \times 10^2$
CFU/mL	20/20
Salmonella
bongori	SGSC
4900	$1.40 \times 10^3$
CFU/mL	20/20	$4.67 \times 10^2$
CFU/mL	19/20	$1.40 \times 10^3$
CFU/mL	20/20
Salmonella
enterica ssp.
enterica	ATCC
14028	$7.33 \times 10^2$
CFU/mL	20/20	$2.20 \times 10^3$
CFU/mL	19/20	$2.20 \times 10^3$
CFU/mL	20/20

16

		EasyMag		KingFisher Flex		KingFisher Apex Dx
Strain	Source	Unpreserve
d Stool LoD	Detectio
n	Unpreserve
d Stool LoD	Detectio
n	Unpreserve
d Stool LoD	Detectio
n
Shiga-like toxin
producing E. coli
(STEC)*	ATCC
BAA-2217	2.50 x 103
CFU/mL	20/20	7.50 x103
CFU/mL	20/20	8.33 x102
CFU/mL	20/20
E. coli 0157	ATCC
700376	3.30 x 103
CFU/mL	20/20	3.30 x 103
CFU/mL	20/20	3.30 x 103
CFU/mL	20/20
Shigella sonnei	ATCC
29930	4.40 x 102
CFU/mL	20/20	4.40 x 102
CFU/mL	20/20	4.40 x 102
CFU/mL	20/20
Vibrio cholerae	ATCC
25870	1.81 x 101
CFU/mL	19/20	1.81 x 101
CFU/mL	20/20	1.81 x 101
CFU/mL	20/20
Vibrio
parahaemolyticu
S	ATCC
17802	4.33 x100
CFU/mL	19/20	1.31 x101
CFU/mL	20/20	4.33 x100
CFU/mL	20/20
Yersinia
enterocolitica **	ATCC
23715	1.50 x103
CFU/mL	20/20	1.67 x102
CFU/mL	19/20	5.01 x102
CFU/mL	19/20
Cryptosporidium
parvum	waterborne
P102C	3.10 x103
oocysts/mL	20/20	3.10 x103
oocysts/mL	20/20	1.03 x103
oocysts/mL	20/20
Entamoeba
histolytica HB-
301:NIH	BEI NR-
176	3.10 x10-1
cysts/mL	20/20	3.10 x10-1
cysts/mL	20/20	3.10 x10-1
cysts/mL	20/20
Giardia
intestinalis (aka
G. lamblia)	waterborne
P101	6.00 x 102
cysts/mL	20/20	1.80 x 103
cysts/mL	20/20	6.00 x 102
cysts/mL	20/20
Adenovirus 40
(dugan)	Zeptometri
x 0810084	3.33 x10-2
TCID50/mL	20/20	3.33 x10-2
TCID50/mL	19/20	3.33 x10-2
TCID50/mL	20/20
Adenovirus 41
(TAK)	Zeptometri
x 0810085	8.46 x10-1
TCID50/mL	20/20	8.46 x10-1
TCID50/mL	20/20	2.82 x 10-1
TCID50/mL	19/20
Rotavirus A	Zeptometri
X
0810041C
F	2.19 x102
TCID50/mL	20/20	2.19 x102
TCID50/mL	19/20	2.19 x102
TCID50/mL	20/20

*KingFisher Flex 9x less sensitive than KingFisher Apex Dx; **EM 9x less sensitive than KingFisher Flex

Table 6. Comparison of results for limit of detection testing for Cary-Blair Stool extracted with the easyMAG, the KingFisher Flex Apex Dx, and the KingFisher Flex and assayed with BioCode® GPP.

		EasyMag		KingFisher Flex		KingFisher Apex Dx
Strain	Source	Cary-
Blair
Stool LoD	Detection	Cary-Blair
Stool LoD	Detection	Cary-Blair
Stool LoD	Detection
Campylobacter
r coli	ATCC
33559	$1.87 \times 10^1$
CFU/mL	20/20	$1.87 \times 10^1$
CFU/mL	20/20	$1.87 \times 10^1$
CFU/mL	19/20
Campylobacter
r jejuni spp.
jejuni	ATCC
33292	$7.78 \times 10^1$
CFU/mL	19/20	$2.33 \times 10^2$
CFU/mL	20/20	$2.33 \times 10^2$
CFU/mL	20/20
Clostridium
difficile	ATCC 9689	$7.04 \times 10^0$
CFU/mL	20/20	$2.11 \times 10^1$
CFU/mL	20/20	$6.33 \times 10^1$
CFU/mL	20/20
		EasyMag		KingFisher Flex		KingFisher Apex Dx
Strain	Source	Cary-
Blair
Stool LoD	Detectio
n	Cary-Blair
Stool LoD	Detectio
n	Cary-Blair
Stool LoD	Detection
(toxinotype
0)*
Clostridium
difficile
(toxinotype
III; Nap1)	Zeptometrix
0801619cf	$3.07 x10^1$
CFU/mL	20/20	$3.07 x10^1$
CFU/mL	19/20	$3.07 x10^1$
CFU/mL	20/20
Enteroaggrega
tive E. coli
092:H33
(EAEC)**	STEC
TW04440	$1.56 x10^2$
CFU/mL	20/20	$1.40 x10^3$
CFU/mL	20/20	$4.20 x10^3$
CFU/mL	20/20
Enteroinvasive
E. coli
029:NM
(EIEC)	ATCC
43892	$3.60 x10^2$
CFU/mL	20/20	$3.60 x10^2$
CFU/mL	20/20	$1.20 x10^2$
CFU/mL	19/20
Enterotoxigeni
c E. coli
078:H11
H10407
(ETEC)	ATCC
35401	$1.87 x10^2$
CFU/mL	20/20	$5.61 x10^2$
CFU/mL	20/20	$1.87 x10^2$
CFU/mL	20/20
Salmonella
bongori	SGSC 4900	$1.40 x10^3$
CFU/mL	20/20	$1.40 x10^3$
CFU/mL	20/20	$1.40 x10^3$
CFU/mL	20/20
Salmonella
enterica ssp.
enterica	ATCC
14028	$7.33 x10^2$
CFU/mL	20/20	$7.33 x10^2$
CFU/mL	20/20	$2.20 x10^3$
CFU/mL	20/20
Shiga-like
toxin
producing E.
coli (STEC)	ATCC
BAA-2217	$8.33 x10^2$
CFU/mL	19/20	$2.50 x10^3$
CFU/mL	19/20	$2.50 x10^3$
CFU/mL	20/20
E. coli O157	ATCC
700376	$1.10 x10^3$
CFU/mL	19/20	$3.30 x10^3$
CFU/mL	20/20	$3.30 x10^3$
CFU/mL	20/20
Shigella
sonnei	ATCC
29930	$1.47 x10^2$
CFU/mL	19/20	$4.40 x10^2$
CFU/mL	20/20	$1.47 x10^2$
CFU/mL	19/20
Vibrio
cholerae	ATCC
25870	$1.81 x10^1$
CFU/mL	20/20	$1.81 x10^1$
CFU/mL	20/20	$1.81 x10^1$
CFU/mL	20/20
Vibrio
parahaemolyti
cus	ATCC
17802	$1.30 x10^1$
CFU/mL	20/20	$1.30 x10^1$
CFU/mL	20/20	$4.33 x10^0$
CFU/mL	20/20
Yersinia
enterocolitica	ATCC
23715	$1.50 x10^3$
CFU/mL	20/20	$5.01 x10^2$
CFU/mL	20/20	$5.01 x10^2$
CFU/mL	20/20
Cryptosporidi
um parvum	waterborne
P102C	$3.10 x10^3$
oocysts/m
L	20/20	$1.03 x10^3$
oocysts/mL	19/20	$1.03 x10^3$
oocysts/mL	19/20
Entamoeba
histolytica
HB-301:NIH	BEI NR-176	$1.03 x10^{-1}$
cysts/mL	20/20	$1.03 x10^{-1}$
cysts/mL	20/20	$1.03 x10^{-1}$
cysts/mL	20/20
Giardia
intestinalis
(aka G.
lamblia)	waterborne
P101	$6.00 x10^2$
cysts/mL	20/20	$1.80 x10^3$
cysts/mL	20/20	$1.80 x10^3$
cysts/mL	20/20
	Source	EasyMag		KingFisher Flex		KingFisher Apex Dx
Strain		Cary-
Blair
Stool LoD	Detectio
n	Cary-Blair
Stool LoD	Detectio
n	Cary-Blair
Stool LoD	Detection
Adenovirus 40
(dugan)	Zeptometrix
0810084	$3.33 x10^{-2}$
TCID50/m
L	19/20	$3.33 x10^{-2}$
TCID50/mL	20/20	$3.33 x10^{-2}$
TCID50/mL	20/20
Adenovirus 41
(TAK)	Zeptometrix
0810085	$8.46 x10^{-1}$
TCID50/m
L	20/20	$2.54 x10^{0}$
TCID50/mL	20/20	$8.46 x10^{-1}$
TCID50/mL	20/20
Rotavirus A	Zeptometrix
0810041CF	$2.19 x10^{2}$
TCID50/m
L	19/20	$7.29 x10^{1}$
TCID50/mL	20/20	$7.29 x10^{1}$
TCID50/mL	19/20

17

18

• KingFisher Apex Dx 27x less sensitive than EM; ** KingFisher Flex 9x and KingFisher Apex Dx 27x less sensitive than EM

For Norovirus GI and GII targets, positive clinical specimens were used, and serial dilutions (initial 10-fold dilution series followed by finer dilutions) were performed. Four replicates of each dilution in negative unpreserved stool and Cary-Blair stool were extracted with the easyMAG, the KingFisher Flex and KingFisher Apex Dx and tested with the BioCode® GPP on the BioCode MDx-3000 system. The LoD was confirmed by extracting 20 replicates of each sample type with each extraction method and testing at or near presumptive LoD.

For Norovirus, the LoD of the Norovirus GII in unpreserved stool was equivalent for three extraction systems. For Norovirus GI, the KingFisher Flex is 10-fold and 3-fold more sensitive than the easyMag and the KingFisher Apex Dx, respectively. For Cary-Blair stool, the LoD of the Norovirus GI was equivalent for three systems. For Norovirus GII, the KingFisher Apex Dx was 3-fold more sensitive than the EasyMag and the KingFisher Flex.

Table 7. Norovirus - Comparison of results for limit of detection testing for Unpreserved Stool extracted with easyMAG, KingFisher Flex, or KingFisher Apex Dx, and assayed with BioCode® GPP.

| | Source | Target
Probe | EasyMag | | KingFisher Flex | | KingFisher Apex Dx | |
|-------------------|------------------------------------------------------|-----------------|-----------------------------------|---------------|-----------------------------------|---------------|-----------------------------------|---------------|
| Target | | | Unpreserve
d Stool
Dilution | Detectio
n | Unpreserve
d Stool
Dilution | Detectio
n | Unpreserv
ed Stool
Dilution | Detectio
n |
| Noroviru
s GI* | Clinical
Sample
ID#
XTAG-
24-
0005574 | NoVG
1 | 1:30,000 | 19/20 | 1:3,000* | 20/20 | 1:10,000 | 19/20 |
| Noroviru
s GII | Clinical
Sample
ID#02-
0134 | NoVG
2 | 1:30,000 | 20/20 | 1:30,000 | 19/20 | 1:30,000 | 20/20 |

*KF Flex 10-fold and 3-fold more sensitive than EM and KF Apex Dx.

19

Table 8. Norovirus - Comparison of results for limit of detection testing for Cary-Blair Stool extracted with the easyMAG, KingFisher Flex, or KingFisher Apex Dx, and assayed with BioCode® GPP.

| | Source | Target
Probe | EasyMag | | KingFisher Flex | | KingFisher Apex Dx | |
|------------------|------------------------------------------------------|-----------------|---------------------------------|-----------|---------------------------------|-----------|-------------------------------------|-----------|
| Target | | | Cary-Blair
Stool
Dilution | Detection | Cary-Blair
Stool
Dilution | Detection | Cary-
Blair
Stool
Dilution | Detection |
| Norovirus
GI | Clinical
Sample
ID#
XTAG-
24-
0005574 | NoVG1 | 1:30,000 | 20/20 | 1:30,000 | 20/20 | 1:30,000 | 20/20 |
| Norovirus
GII | Clinical
Sample
ID#02-
0134 | NoVG2 | 1:30,000 | 20/20 | 1:30,000 | 20/20 | 1:10,000* | 20/20 |

*KF Apex Dx 3-fold more sensitive than EM and KF Flex

Clinical testing summary [807.92(b)(2)]

METHOD COMPARISON STUDY

A clinical investigational study was performed in which a total of 468 remnant, de-identified samples (254 frozen unpreserved stool and 214 inoculated Cary-Blair stool) that were prospectively collected for the clinical study that resulted in the K180041 BioCode® GPP FDA clearance were extracted using the KingFisher Flex, KingFisher Apex Dx, and the easyMAG, and tested on the MDx-3000 system. Fifty-four (54) freshly collected leftover samples were used for the C. difficile testing. In addition, a total of 120 samples were contrived at 3x LoD and 6x LoD (15 samples at 3x LoD and 15 samples at 6xLoD for each of three targets) and tested to determine the performance characteristics for Entamoeba histolytica, Yersinia enterocolitica and Vibrio spp. (V. parahaemolyticus, V. vulnificus, and V. cholerae). The demographics of the clinical patients and the results of the clinical investigational study are as follows:

Archived Samples
Total Specimen Count	468
Gender
Male	246/468 (52.6%)
Female	222/468 (47.4%)
Age Category
yrs	87/468 (18.6%)
6-21 yrs	85/468 (18.2%)
22-59 yrs	197/468 (42.1%)
60+yrs	99/468 (21.1%)

Table 9. Demographic data for archived specimens (frozen unpreserved and inoculated Cary-Blair)

20

Fresh Samples
Total Specimen Count	54
Gender
Male	20/54 (37.04%)
Female	34/54 (62.96%)
Age Category
≤ 5 yrs	0/54 (0.00%)
6-21 yrs	2/54 (3.70%)
22-59 yrs	24/54 (44.45%)
60+yrs	28/54 (51.85%)

Table 10. Demographic data for fresh specimens (unpreserved)

The Positive Percent Agreement (PPA) was calculated as TP/(TP+FN). TP = true positive or positive by both the EasyMag and the KingFisher Apex Dx or the KingFisher Flex; FN = false negative or negative by the KingFisher Apex Dx or the KingFisher Flex only. Negative Percent Agreement (NPA) was calculated as TN/(TN + FP). TN = true negative or negative by the EasyMag and the KingFisher Apex Dx or the KingFisher Flex; FP = false positive or positive by the KingFisher Apex Dx or the KingFisher Flex only. The exact binomial two-sided 95% confidence interval was calculated.

A. Method Comparison Study between EasyMag and KingFisher Flex

Table 11. KingFisher Flex: Summary of Clinical Investigational Study Results (Archived
Specimens) Stratified by Sample Type and Storage
			The Market of the country of the country of the county of the county of

| Target | Specimen
Type | (n) | Positive Agreement | | Negative Agreement | |
|-------------------------------------------|--------------------------|------|--------------------|-------------|---------------------|-------------|
| Campylobacter spp.a | Inoculated
Cary-Blair | 209 | 18/18 (100) | 82.4 - 100 | 191/191 (100) | 98.0 - 100 |
| Campylobacter spp.a | Unpreserved
(Frozen) | 247 | 25/25 (100) | 86.7 - 100 | 219/222 (98.6) | 96.1 - 99.5 |
| Campylobacter spp.a | All Archived | 456 | 43/43 (100) | 91.8 - 100 | 410/413 (99.3) | 97.9 - 99.8 |
| Clostridium difficileb | Inoculated
Cary-Blair | 211 | 35/37 (94.6) | 82.3 - 98.5 | 173/174 (99.4) | 96.8 - 99.9 |
| Clostridium difficileb | Unpreserved
(Frozen) | 247 | 31/33 (93.9) | 80.4 - 98.3 | 213/214 (99.5) | 97.4 - 99.9 |
| Clostridium difficileb | All Archived | 458 | 66/70 (94.3) | 86.2 - 97.8 | 386/388 (99.5) | 98.1 - 99.9 |
| E. coli O157c | Inoculated
Cary-Blair | 209 | 5/5 (100) | 56.6 - 100 | 204/204 (100) | 98.2 - 100 |
| E. coli O157c | Unpreserved
(Frozen) | 246 | 8/8 (100) | 67.6 - 100 | 238/238 (100) | 98.4 - 100 |
| E. coli O157c | All Archived | 455 | 13/13 (100) | 77.2 - 100 | 442/442 (100) | 99.1 - 100 |
| Target | Specimen Type | (n) | Positive Agreement | | Negative Agreement | |
| | | | PPA (%) | 95% CI | NPA (%) | 95% CI |
| Enteroaggregative E. coli (EAEC) d | Inoculated
Cary-Blair | 210 | 30/30 (100) | 88.6-100 | 180/180 (100) | 97.9-100 |
| | Unpreserved
(Frozen) | 246 | 20/22 (90.9) | 77.2-97.5 | 224/224 (100) | 98.3-100 |
| | All Archived | 456 | 50/52 (96.2) | 87.0-98.9 | 404/404 (100) | 99.1-100 |
| Enterotoxigenic
E. coli (ETEC) e | Inoculated
Cary-Blair | 209 | 14/14 (100) | 78.5-100 | 195/195 (100) | 98.1-100 |
| | Unpreserved
(Frozen) | 246 | 11/11 (100) | 74.1-100 | 235/235 (100) | 98.4-100 |
| | All Archived | 455 | 25/25 (100) | 86.7-100 | 430/430 (100) | 99.1-100 |
| Shiga toxin-producing
E. coli (STEC) f | Inoculated
Cary-Blair | 209 | 12/12 (100) | 75.8-100 | 197/197 (100) | 98.1-100 |
| | Unpreserved
(Frozen) | 247 | 20/22 (90.9) | 72.2-97.5 | 224/225 (99.6) | 97.5-99.9 |
| | All Archived | 456 | 32/34 (94.1) | 80.9-98.4 | 421/422 (99.80) | 98.7-100 |
| Salmonella spp.g | Inoculated
Cary-Blair | 209 | 20/20 (100) | 83.9-100 | 188/189 (99.4) | 97.1-99.9 |
| | Unpreserved
(Frozen) | 246 | 21/21 (100) | 84.5-100 | 224/225 (99.6) | 97.5-99.9 |
| | All Archived | 455 | 41/41 (100) | 91.4-100 | 412/414 (99.5) | 98.3-99.9 |
| Shigella/ EIEC h | Inoculated
Cary-Blair | 210 | 13/13 (100) | 77.2-100 | 196/197 (99.5) | 97.2-99.9 |
| | Unpreserved
(Frozen) | 246 | 18/18 (100) | 82.4-100 | 228/228 (100) | 98.3-100 |
| | All Archived | 456 | 31/31 (100) | 89.0-100 | 424/425 (99.8) | 98.7-100 |
| Vibrio
parahaemolyticus i | Inoculated
Cary-Blair | 209 | 1/1 (100) | 20.7-100 | 208/208 (100) | 98.2-100 |
| | Unpreserved
(Frozen) | 246 | 1/1 (100) | 20.7-100 | 245/245 (100) | 98.5-100 |
| | All Archived | 455 | 2/2 (100) | 34.2-100 | 453/453 (100) | 99.2-100 |
| Vibrio spp. (not
parahaemolyticus) j | Inoculated
Cary-Blair | 209 | N/A | N/A | 208/209 (99.5) | 97.3-99.9 |
| | Unpreserved
(Frozen) | 246 | 1/2 (50%)* | 9.5–90.5 | 244/244 (100) | 98.5-100 |
| | All Archived | 455 | 1/2 (50%) | 9.5-90.5 | 452/453 (99.8) | 98.8-100 |
| Target | Specimen
Type | (n) | Positive Agreement | | Negative Agreement | |
| | | | PPA (%) | 95% CI | NPA (%) | 95% CI |
| Yersinia
enterocolitica k | Inoculated
Cary-Blair | 209 | 3/3 (100) | 43.9 - 100 | 204/206 (99.0) | 96.5 - 99.7 |
| | Unpreserved
(Frozen) | 246 | 3/3 (100) | 43.9 - 100 | 243/243 (100) | 98.4 - 100 |
| | All Archived | 455 | 6/6 (100) | 61.0 - 100 | 447/449 (99.6) | 98.4 - 99.9 |
| Cryptosporidium spp. 1 | Inoculated
Cary-Blair | 209 | 9/9 (100) | 70.1 - 100 | 200/200 (100) | 98.1 - 100 |
| | Unpreserved
(Frozen) | 248 | 18/18 (100) | 82.4 - 100 | 230/230 (100) | 98.4 - 100 |
| | All Archived | 457 | 27/27 (100) | 87.5 - 100 | 430/430 (100) | 99.1 - 100 |
| Entamoeba
histolytica m | Inoculated
Cary-Blair | 209 | N/A | N/A | 209/209 (100) | 98.2 - 100 |
| | Unpreserved
(Frozen) | 246 | N/A | N/A | 246/246 (100) | 98.5 - 100 |
| | All Archived | 455 | N/A | N/A | 455/455 (100) | 99.2 - 100 |
| Giardia lamblia n | Inoculated
Cary-Blair | 209 | 4/4 (100) | 51.0 - 100 | 205/205 (100) | 98.2 - 100 |
| | Unpreserved
(Frozen) | 246 | 11/11 (100) | 74.1 - 100 | 235/235 (100) | 98.4 - 100 |
| | All Archived | 455 | 15/15 (100) | 79.6 - 100 | 440/440 (100) | 99.1 - 100 |
| Adenovirus 40/41o | Inoculated
Cary-Blair | 209 | 5/5 (100) | 56.6 - 100 | 202/204 (99.0) | 96.5 - 99.7 |
| | Unpreserved
(Frozen) | 246 | 7/8 (87.5)* | 52.9 - 97.8 | 238/238 (100) | 98.4 - 100 |
| | All Archived | 455 | 12/13 (92.3) | 66.7 - 98.6 | 440/442 (99.5) | 98.4 - 99.9 |
| Norovirus
(GI/GII) p | Inoculated
Cary-Blair | 210 | 22/23 (95.7) | 79.0 - 99.2 | 187/187 (100) | 98.0 - 100 |
| | Unpreserved
(Frozen) | 247 | 19/20 (95.0) | 76.4 - 99.1 | 226/227 (99.6) | 97.5 - 99.9 |
| | All Archived | 457 | 41/43 (95.3) | 84.5 - 98.7 | 413/414 (99.8) | 98.6 - 100 |
| Rotavirus A q | Inoculated
Cary-Blair | 210 | 10/10 (100) | 72.2 - 100 | 198/200 (99.0) | 96.4 - 99.7 |
| | Unpreserved
(Frozen) | 246 | 9/9 (100) | 70.1 - 100 | 229/237 (96.6) | 93.5 - 98.3 |
| | All Archived | 456 | 19/19 (100) | 83.2 - 100 | 427/437 (97.7) | 95.8 - 98.8 |
| | Specimen
Type | (n) | Positive Agreement | | Negative Agreement | |
| Target | | | PPA (%) | 95% CI | NPA (%) | 95% CI |
| Combined Targets | Inoculated
Cary-Blair | 3559 | 201/204
(98.5) | 95.8-99.5 | 3345/3355
(99.7) | 99.5-99.8 |
| | Unpreserved
(Frozen) | 4188 | 223/232
(96.1) | 92.8 - 97.9 | 3941/3956
(99.6) | 99.4-99.8 |

21

22

23

*Positive agreement 500 cutoff) for Campylobacter spp. The sample was Campvlobacter spp negative after being retested twice with each extraction system. b. Sample TRI 056A resulted in a false positive agreement and remained false positive after discordant analysis.

Table 15. KingFisher Flex: Summary of Contrived Specimen Results

Note. One hundred twenty (120) contrived samples were tested. Five (5) samples were invalid with the easyMAG on initial testing. When the invalid samples were tested again with PCR, the samples remained invalid.

a- Of the five (5) invalids, one (1) of the E. histolytica positives was tested E. his positive but RNA IC was invalid.

b- Of the five (5) invalids, one (1) of the Yersinia enterocolitica positives was tested Yersinia enterocolitica, but RNA IC was invalid.

c - One (1) of the negative stools being used as matrix for contriving samples was a low-positive C. difficile and erroneously enrolled as a negative sample. Because the C. difficile was not intended as part of the study, the positive agreement calculation for this target was not included.

30

B. Method Comparison Study between EasyMag and KingFisher Apex Dx

| Target | Specimen
Type | (n) | Positive Agreement
PPA (%) | 95% CI | Negative Agreement
NPA (%) | 95% CI | |
|-----------------------------------------------|--------------------------|------|-------------------------------|-------------|-------------------------------|-------------|--|
| Campylobacter spp.a | Inoculated
Cary-Blair | 210 | 18/18 (100) | 82.4 – 100 | 192/192 (100) | 98.0 – 100 | |
| | Unpreserved
(Frozen) | 249 | 24/25 (96.0) | 80.5 – 99.3 | 221/224 (98.7) | 96.1 – 99.5 | |
| | All Archived | 459 | 42/43 (97.7) | 87.9 – 99.6 | 413/416 (99.3) | 97.9 – 99.8 | |
| Clostridium difficile b | Inoculated
Cary-Blair | 211 | 34/37 (91.9) | 78.7 – 97.2 | 173/174 (99.4) | 96.8 – 99.9 | |
| | Unpreserved
(Frozen) | 249 | 17/19 (89.5)* | 68.6 – 97.1 | 229/230 (99.6) | 97.6 – 99.9 | |
| | All Archived | 460 | 51/56 (91.1) | 80.7 – 96.1 | 402/404 (99.5) | 98.2 – 99.9 | |
| E. coli O157 c | Inoculated
Cary-Blair | 210 | 4/5 (80.0)* | 37.6 – 96.4 | 205/205 (100) | 98.2 – 100 | |
| | Unpreserved
(Frozen) | 249 | 8/8 (100) | 67.6 – 100 | 240/241 (99.6) | 97.7 – 99.9 | |
| | All Archived | 459 | 12/13 (92.3) | 66.7 – 98.6 | 445/446 (99.8) | 98.7 – 100 | |
| Enteroaggregative E.
coli (EAEC) d | Inoculated
Cary-Blair | 211 | 30/30 (100) | 88.6 – 100 | 181/181 (100) | 97.9 – 100 | |
| | Unpreserved
(Frozen) | 249 | 22/22 (100) | 85.1 – 100 | 226/227 (99.6) | 97.5 – 99.9 | |
| | All Archived | 460 | 52/52 (100) | 93.1 – 100 | 407/408 (99.8) | 98.6 – 100 | |
| Enterotoxigenic
E. coli (ETEC) e | Inoculated
Cary-Blair | 210 | 14/14 (100) | 78.5 – 100 | 194/196 (99.0) | 96.4 – 99.7 | |
| | Unpreserved
(Frozen) | 249 | 11/11 (100) | 74.1 – 100 | 237/238 (99.6) | 97.7 – 99.9 | |
| | All Archived | 459 | 25/25 (100) | 86.7 – 100 | 431/434 (99.3) | 98.0 – 99.8 | |
| Shiga toxin-producing
E. coli (STEC) f | Inoculated
Cary-Blair | 210 | 12/12 (100) | 75.8 – 100 | 198/198 (100) | 98.1 – 100 | |
| | Unpreserved
(Frozen) | 249 | 20/22 (90.9) | 72.2 – 97.5 | 226/227 (99.6) | 97.5 – 99.9 | |
| | All Archived | 459 | 32/34 (94.1) | 80.9 – 98.4 | 424/425 (99.8) | 98.7 – 100 | |
| Salmonella spp. g | Inoculated
Cary-Blair | 210 | 20/20 (100) | 83.9 – 100 | 187/190 (98.4) | 95.5 – 99.5 | |
| | Specimen | (n) | Positive Agreement | | Negative Agreement | | |
| Target | Type | | PPA (%) | 95% CI | NPA (%) | 95% CI | |
| | Unpreserved
(Frozen) | 249 | 21/21 (100) | 84.5 – 100 | 228/228 (100) | 98.3 – 100 | |
| | All Archived | 459 | 41/41 (100) | 91.4 – 100 | 415/418 (99.3) | 97.9 – 99.8 | |
| | Inoculated
Cary-Blair | 211 | 13/13 (100) | 77.2 – 100 | 194/198 (98.0) | 94.9 – 99.2 | |
| Shigella/ EIECh | Unpreserved
(Frozen) | 249 | 17/18 (94.4) | 74.2 – 99.0 | 231/231 (100) | 98.4 – 100 | |
| | All Archived | 460 | 30/31 (96.8) | 83.8 – 99.4 | 425/429 (99.1) | 97.6 – 99.6 | |
| | Inoculated
Cary-Blair | 210 | 1/1 (100) | 20.7 – 100 | 208/209 (99.5) | 97.3 – 99.9 | |
| Vibrio parahaemolyticus i | Unpreserved
(Frozen) | 249 | 1/1 (100) | 20.7 – 100 | 248/248 (100) | 98.5 – 100 | |
| | All Archived | 459 | 2/2 (100) | 34.2 – 100 | 456/457 (99.8) | 98.8 – 100 | |
| | Inoculated
Cary-Blair | 210 | N/A | N/A | 209/210 (99.5) | 97.4 – 99.9 | |
| Vibrio spp.j
(not parahaemolyticus) | Unpreserved
(Frozen) | 249 | 1/2 (50%)* | 9.5 – 90.5 | 247/247 (100) | 98.4 – 100 | |
| | All Archived | 459 | 1/2 (50%)* | 9.5 – 90.5 | 456/457 (99.8) | 98.8 – 100 | |
| | Inoculated
Cary-Blair | 210 | 3/3 (100) | 43.9 – 100 | 206/207 (99.5) | 97.3 – 99.9 | |
| Yersinia enterocolitica k | Unpreserved
(Frozen) | 249 | 3/3 (100) | 43.9 – 100 | 245/246 (99.6) | 97.7 – 99.9 | |
| | All Archived | 459 | 6/6 (100) | 61.0 – 100 | 451/453 (99.6) | 98.4 – 99.9 | |
| | Inoculated
Cary-Blair | 210 | 9/9 (100) | 70.1 – 100 | 201/201 (100) | 98.1 – 100 | |
| Cryptosporidium spp.l | Unpreserved
(Frozen) | 250 | 17/18 (94.4) | 74.2 – 99.0 | 231/232 (99.6) | 97.6 – 99.9 | |
| | All Archived | 460 | 26/27 (96.3) | 81.7 – 99.3 | 432/433 (99.8) | 98.7 – 100 | |
| | Inoculated
Cary-Blair | 210 | N/A | N/A | 210/210 (100) | 98.2 – 100 | |
| Entamoeba histolytica m | Unpreserved
(Frozen) | 249 | N/A | N/A | 249/249 (100) | 98.5 – 100 | |
| | All Archived | 459 | N/A | N/A | 459/459 (100) | 99.1 – 100 | |
| Giardia lamblia n | Inoculated
Cary-Blair | 210 | 4/4 (100) | 51.0 – 100 | 206/206 (100) | 98.2 – 100 | |
| | Specimen | | Positive Agreement | | Negative Agreement | | |
| Target | Type | (n) | PPA (%) | 95% CI | NPA (%) | 95% CI | |
| | Unpreserved
(Frozen) | 249 | 10/11 (90.9) | 62.3 - 98.4 | 238/238 (100) | 98.4 - 100 | |
| | All Archived | 459 | 14/15 (93.3) | 70.2 - 98.8 | 444/444 (100) | 99.1 - 100 | |
| | Inoculated
Cary-Blair | 210 | 5/5 (100) | 56.6 - 100 | 204/205 (99.5) | 97.3 – 99.9 | |
| Adenovirus 40/41 ° | Unpreserved
(Frozen) | 249 | 7/8 (87.5)* | 52.9 - 97.8 | 240/241 (99.6) | 97.7 - 99.9 | |
| | All Archived | 459 | 12/13 (92.3) | 66.7 - 98.6 | 444/446 (99.6) | 98.4 - 99.9 | |
| Norovirus | Inoculated
Cary-Blair | 210 | 22/23 (95.7) | 79.0 - 99.2 | 187/187 (100) | 98.0 - 100 | |
| (GI/GII) p | Unpreserved
(Frozen) | 250 | 20/20 (100) | 83.9 - 100 | 230/230 (100) | 98.4 - 100 | |
| | All Archived | 460 | 42/43 (97.7) | 87.9 – 99.6 | 417/417 (100) | 99.1 - 100 | |
| | Inoculated
Cary-Blair | 211 | 10/10 (100) | 72.2 - 100 | 201/201 (100) | 98.1 - 100 | |
| Rotavirus A 9 | Unpreserved
(Frozen) | 249 | 9/9 (100) | 70.1 - 100 | 240/240 (100) | 98.4 - 100 | |
| | All Archived | 460 | 19/19 (100) | 83.2 - 100 | 441/441 (100) | 99.1 - 100 | |
| Combined Targets | Inoculated
Cary-Blair | 3512 | 216/218
(99.1) | 96.7 — 99.7 | 3280/3294
(99.6) | 99.3 — 99.7 | |
| | Unpreserved
(Frozen) | 4235 | 208/218
(95.4) | 91.8 – 97.5 | 4006/4017
(99.7) | 99.5 - 99.8 | |

Table 16. KingFisher Apex Dx: Summary of Clinical Investigational Study Results (Archived Specimens) Stratified by Sample Type and Storage

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*Positive agreement 500 cutoff) for Campylobacter spp. The sample was Campylobacter spp negative after being retested twice with each extraction system.

b. C. difficile positive sample TRI 011A initially tested as false negative resulted in a true positive after retesting. The Xpert C. difficile-Epi test results of TRI- 011A had Ct 34.8 which indicates the sample was low positive.

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Table 20. KingFisher Apex Dx: Summarv of Contrived Specimen Results (Contrived at 3x LoD and 6x LoD)

Note. One hundred twenty (120) contrived samples were tested. Five (5) samples were invalid with the easyMAG on initial testing. When the invalid samples were tested again with PCR, the samples remained invalid.

a- Of the five (5) invalids, one of the E. histolytica positives was tested E. his positive but RNA IC was invalid.

b- Of the five (5) invalids, one of the Yersinia enterocolitica positives was tested Yersinia enterocolitica, but RNA IC was invalid.

c - One (1) of negative stools being used as matrix for contriving samples was a low-positive C. difficile and erroneously enrolled as a negative sample. Because the C. difficile was not intended as part of the study, the positive agreement calculation for this target was not included.

Co-infection summary
+1 Target	246/466 (52.8%)
+2 Targets	78/466 (16.7%)
+3 Targets	13/466 (2.8%)

Table 21. Clinical Co-infection Summary of Archived Samples

Conclusions [807.92(b)(3):

The intended use and fundamental scientific technology of the BioCode GPP using the KingFisher Flex and KingFisher Apex Dx is substantially equivalent to the predicate device. Non-clinical studies and clinical studies have established that the BioCode GPP with the KingFisher Flex and KingFisher Apex Dx is substantially equivalent to the predicate device.