LIAISON PLEX Gastrointestinal Flex Assay

K253722 · Luminex Corporation · PCH · May 19, 2026 · Microbiology

Device Facts

• Record ID: K253722
• Device Name: LIAISON PLEX Gastrointestinal Flex Assay
• Applicant: Luminex Corporation
• Product Code: PCH · Microbiology
• Decision Date: May 19, 2026
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3990
• Device Class: Class 2
• Attributes: Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K253722 · May 19, 2026	LIAISON PLEX Gastrointestinal Flex Assay	Luminex Corporation	Pre-selected, remnant, de-identified clinical specimens	Retrospective clinical specimens were used to supplement the prospective clinical study to establish performance characteristics for targets with low prevalence in the prospective cohort.	Retrospective clinical specimens; Pre-selected remnant samples; Clinical performance evaluation

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Arm 2: Pre-selected clinical specimens; Retrospective analysis of remnant clinical specimens; Study Period: September 2017 through February 2026	Patients exhibiting clinical signs and symptoms of gastrointestinal infection; Sample Size: 333; Number of Sites: 19 (17 US, 1 France, 1 Germany)	FDA cleared molecular assay, PCR followed by bi-directional sequencing (BDS) assays	Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA)

Indications for Use

The LIAISON PLEX® Gastrointestinal Flex Assay is an automated and qualitative multiplexed test for simultaneous detection and identification of common pathogenic bacteria, viruses, and parasites from liquid or soft stool preserved in Cary-Blair or modified Cary-Blair medium and additionally for parasites, from liquid or soft stool preserved in alcohol-based formalin-free parasite fixative (e.g., EcoFix®, Total-Fix®) collected from individuals with signs and symptoms of gastrointestinal infection. The test is performed on the automated LIAISON PLEX® System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific gastrointestinal microbial nucleic acid gene sequences associated with the pathogenic bacteria, viruses, and parasites: Bacteria: Campylobacter spp. (C. coli/C. jejuni/C. lari/C. upsaliensis) Clostridioides difficile (tcdA/tcdB) Enterotoxigenic Escherichia coli (ETEC) LT/ST Plesiomonas shigelloides Salmonella spp. Shiga-like toxin-producing Escherichia coli (STEC) stx1 Shiga-like toxin-producing Escherichia coli (STEC) stx2 Shigella/Enteroinvasive Escherichia coli (EIEC) Vibrio cholerae Vibrio spp. (V. parahaemolyticus/V. vulnificus) Yersinia enterocolitica Viruses: Adenovirus F40/41 Astrovirus Norovirus GI/GII Rotavirus A Sapovirus I/II/IV/V Parasites: Blastocystis sp. Cryptosporidium spp. Cyclospora cayetanensis Dientamoeba fragilis Entamoeba histolytica Giardia lamblia (also known as G. intestinalis and G. duodenalis) Microsporidia (Encephalitozoon hellem/Encephalitozoon intestinalis/Enterocytozoon bieneusi) Strongyloides stercoralis The LIAISON PLEX Gastrointestinal Flex Assay 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 information; however, it is not to be used to monitor these infections. Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor C. difficile infection. LIAISON PLEX Gastrointestinal Flex Assay results should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Confirmed positive results do not rule out co-infection with other organisms that are not detected by this test and may not be the sole or definitive cause of patient illness. Negative LIAISON PLEX Gastrointestinal Flex Assay results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn’s disease.

Device Story

Automated, bench-top, sample-to-answer system; processes liquid/soft stool samples; performs mechanical/chemical cell lysis; magnetic bead-based nucleic acid extraction; multiplex RT-PCR/PCR amplification; microarray-based hybridization; gold nanoparticle probe binding; silver enhancement; light scatter measurement. Used in clinical labs; operated by trained personnel. Provides qualitative detection of enteric pathogens; results used alongside clinical/epidemiological data to aid diagnosis; does not replace culture for bacterial typing.

Clinical Evidence

Prospective clinical study (n=2233) across 7 US sites, supplemented by pre-selected (n=333) and contrived (n=827) specimens. Comparator: FDA-cleared molecular assays and/or PCR/BDS. Overall success rate 99.5%. PPA/NPA varied by target; most targets showed high agreement. Discordant results resolved via secondary molecular methods.

Technological Characteristics

Bench-top system; uses disposable cartridges with sample processing tubes. Nucleic acid extraction via mechanical/chemical lysis and magnetic beads. Amplification via RT-PCR/PCR. Detection via microarray hybridization with gold nanoparticle probes and silver enhancement. Standalone instrument; barcode-driven workflow. Software-controlled.

Indications for Use

Indicated for individuals with signs/symptoms of GI infection. Detects specific bacterial, viral, and parasitic nucleic acids in liquid/soft stool (Cary-Blair or alcohol-based formalin-free fixative). Not for monitoring C. difficile or other infections. Prescription use only.

Regulatory Classification

Identification

A gastrointestinal microorganism multiplex nucleic acid-based assay is a qualitative in 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.

Special Controls

*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).

Predicate Devices

• Biofire Filmarray Gastrointestinal (GI) Panel (K242367)

Submission Summary (Full Text)

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**FDA** U.S. FOOD & DRUG
ADMINISTRATION

# **510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY AND INSTRUMENT**

# **I Background Information:**

# **A 510(k) Number**

K253722

# **B Applicant**

Luminex Corporation

# **C Proprietary and Established Names**

LIAISON PLEX Gastrointestinal *Flex* Assay

# **D Regulatory Information**

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| PCH | Class II | 21 CFR 866.3990 – Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assay | MI-Microbiology |
| NSU | Class II | 21 CFR 862.2570 – Instrumentation for clinical multiplex test systems | CH - Chemistry |

# **II Submission/Device Overview:**

# **A Purpose for Submission:**

To obtain a substantial equivalence determination for the LIAISON PLEX Gastrointestinal *Flex* Assay for the detection of microbial nucleic acids extracted from human stool specimens.

# **B Measurand:**

Target nucleic acid sequences of the following gastrointestinal microorganisms:

- **Bacteria:** *Campylobacter* spp. (*C. coli*, *C. jejuni*, *C. lari*, *C. upsaliensis*), *Clostridioides difficile* (tcdA/tcdB), Enterotoxigenic *Escherichia coli* (ETEC) LT/ST), *Plesiomonas shigelloides*, *Salmonella* spp., Shiga-like toxin-producing *E. coli* (STEC) stx1, Shiga-like

Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

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toxin-producing E. coli (STEC) stx2, Shigella/Enteroinvasive E. coli (EIEC), Vibrio cholerae, Vibrio spp. (V. parahaemolyticus/V. vulnificus), Yersinia enterocolitica

- Viruses: Adenovirus F40/41, Astrovirus, Norovirus GI/GII, Rotavirus A, Sapovirus I/II/IV/V

- Parasites: Blastocystis sp., Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia, Microsporidia (Encephalitozoon hellem, Encephalitozoon intestinalis, Enterocytozoon bieneusi), Strongyloides stercoralis

# C Type of Test:

Qualitative, multiplex nucleic acid amplification test (NAAT)

# III Intended Use/Indications for Use:

# A Intended Use(s):

See Indications for Use below.

# B Indication(s) for Use:

The LIAISON PLEX Gastrointestinal Flex Assay is an automated and qualitative multiplexed test for simultaneous detection and identification of common pathogenic bacteria, viruses, and parasites from liquid or soft stool preserved in Cary-Blair or modified Cary-Blair medium and additionally for parasites, from liquid or soft stool preserved in alcohol-based formalin-free parasite fixative (e.g., EcoFix, Total-Fix) collected from individuals with signs and symptoms of gastrointestinal infection. The test is performed on the automated LIAISON PLEX System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific gastrointestinal microbial nucleic acid gene sequences associated with the pathogenic bacteria, viruses, and parasites:

# Bacteria:

- Campylobacter spp. (C. coli/C. jejuni/C. lari/C. upsaliensis)
- Clostridioides difficile (tcdA/tcdB)
- Enterotoxigenic Escherichia coli (ETEC) LT/ST
- Plesiomonas shigelloides
- Salmonella spp.
- Shiga-like toxin-producing Escherichia coli (STEC) stx1
- Shiga-like toxin-producing Escherichia coli (STEC) stx2
- Shigella/Enteroinvasive Escherichia coli (EIEC)
- Vibrio cholerae
- Vibrio spp. (V. parahaemolyticus/V. vulnificus)
- Yersinia enterocolitica

# Viruses:

- Adenovirus F40/41
- Astrovirus
- Norovirus GI/GII
- Rotavirus A
- Sapovirus I/II/IV/V

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# **Parasites:**

- *Blastocystis* sp.
- *Cryptosporidium* spp.
- *Cyclospora cayetanensis*
- *Dientamoeba fragilis*
- *Entamoeba histolytica*
- *Giardia lamblia* (also known as *G. intestinalis* and *G. duodenalis*)
- Microsporidia (*Encephalitozoon hellem*/*Encephalitozoon intestinalis*/*Enterocytozoon bieneusi*)
- *Strongyloides stercoralis*

The LIAISON PLEX Gastrointestinal *Flex* Assay 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 information; however, it is not to be used to monitor these infections.

Concomitant culture is necessary for organism recovery and further typing of bacterial agents.

This device is not intended to monitor *C. difficile* infection.

LIAISON PLEX Gastrointestinal *Flex* Assay results should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Confirmed positive results do not rule out co-infection with other organisms that are not detected by this test and may not be the sole or definitive cause of patient illness. Negative LIAISON PLEX Gastrointestinal *Flex* Assay 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.

# **C Special Conditions for Use Statement(s):**

For prescription use only

For *in vitro* diagnostic use only

# **D Special Instrument Requirements:**

LIAISON PLEX Gastrointestinal *Flex* Assay is to be used with the LIAISON PLEX System

# **IV Device/System Characteristics:**

# **A Device Description:**

The LIAISON PLEX Gastrointestinal *Flex* Assay is a test for the detection and identification of nucleic acids from enteric bacteria, viruses, and parasites in stool samples, and the assay is performed on the LIAISON PLEX System. The LIAISON PLEX System is a fully automated, bench-top "sample-to-answer" device that is capable of reverse transcription (RT) and polymerase chain reaction (PCR). The test system performs sample preparation, target amplification, microarray-based hybridization, and signal generation/analysis for the detection of target-specific nucleic acids.

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The test reagents are supplied as a single, disposable test cartridge with a sample processing tube and a transfer pipette. The LIAISON PLEX Gastrointestinal Flex Assay Kit includes:

- 12 LIAISON PLEX Gastrointestinal Flex Assay Cartridges
- 15 LIAISON PLEX Gastrointestinal Flex Assay Sample Processing Tubes, 2.0 mL
- 1 bag of LIAISON PLEX Gastrointestinal Flex Assay Transfer Pipettes
- Contains 15 individual Transfer Pipettes

Materials Required But Provided Separately:

- LIAISON PLEX System
- LIAISON PLEX Chassis
- One to six LIAISON PLEX Modules
- LIAISON PLEX Software
- LIAISON PLEX Gastrointestinal Flex Assay Software
- One hand-held barcode reader and stand
- Cartridge Removal Tool

Materials Recommended but Provided Separately:

- Sample Prep Tray

Materials Required but Not Provided:

- Specimen transport system: Cary-Blair, modified Cary-Blair, or alcohol-based formalin-free parasite fixative
- Vortex mixer
- 10% bleach
- Deionized water
- A FAT32-formatted USB drive

B Principle of Operation:

The LIAISON PLEX Gastrointestinal Flex Assay is an automated test for the simultaneous detection and identification of nucleic acids from enteric bacteria, viruses, and parasites in stool samples on the LIAISON PLEX System. The LIAISON PLEX Gastrointestinal Flex Assay is performed on liquid or soft stool samples preserved in Cary-Blair, modified Cary-Blair, and/or alcohol-based formalin-free parasite fixatives (parasite testing only). The user loads the mixed sample from the sample processing tube into the sample port of the LIAISON PLEX Gastrointestinal Flex Assay Cartridge. Next, the user sets up the sample order on the LIAISON PLEX System by entering the sample information or scanning the barcode ID located on the sample tube, then scanning the barcode ID located on the test cartridge. Lastly, the user inserts the test cartridge into the processing module to initiate the test. The LIAISON PLEX System identifies the assay being run and automatically initiates the proper testing protocol to process the sample, analyze the data, and generate test results.

The LIAISON PLEX System automates the LIAISON PLEX Gastrointestinal Flex Assay sample analysis through the following steps:

- Sample Extraction: Nucleic acid extraction via mechanical and chemical cell lysis and magnetic bead-based nucleic acid isolation of prepared stool samples.
- Target Amplification: Multiplex PCR- and RT-PCR-based amplification of the extracted nucleic acids to generate target-specific amplicons.

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- Hybridization: Amplified DNA hybridizes to specific capture DNA arrayed on a glass slide in a microarray format and the bound target DNA, in turn, hybridizes with mediator and gold nanoparticle probes;
- Signal Analysis: Gold nanoparticle probes bound specifically to target-containing spots in the microarray are silver-enhanced, and light scatter from the spots is measured and further analyzed to determine the presence (Detected) or absence (Not Detected) of a target.

# C Instrument Description Information:

1. Instrument Name:

LIAISON PLEX

2. Specimen Identification:

The user creates a new order and scans or manually enters the Sample ID and then scans the assay cartridge ID barcode with the hand-held barcode reader.

3. Specimen Sampling and Handling:

Collect stool samples and place into Cary-Blair, modified Cary-Blair, or alcohol-based formalin-free parasite fixative (parasite testing only), as appropriate.

4. Calibration:

LIAISON PLEX modules are calibrated during the manufacturing process; calibration is not performed by the user.

5. Quality Control:

See Section VII.A.5 below for details.

# V Substantial Equivalence Information:

A Predicate Device Name(s):

Biofire Filmarray Gastrointestinal (GI) Panel

B Predicate 510(k) Number(s):

K242367

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K253722 Subject Device | K242367 Predicate Device |
| --- | --- | --- |
| Device Trade Name | LIAISON PLEX Gastrointestinal *Flex Assay* | Biofire Filmarray Gastrointestinal (GI) Panel |

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| General Device Characteristic Similarities | | |
| --- | --- | --- |
| Intended Use/ Indications For Use | The LIAISON PLEX Gastrointestinal Flex Assay is an automated and qualitative multiplexed test for simultaneous detection and identification of common pathogenic bacteria, viruses, and parasites from liquid or soft stool preserved in Cary-Blair or modified Cary-Blair medium and additionally for parasites, from liquid or soft stool preserved in alcohol-based formalin-free parasite fixative (e.g., EcoFix, Total-Fix) collected from individuals with signs and symptoms of gastrointestinal infection. The test is performed on the automated LIAISON PLEX System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific gastrointestinal microbial nucleic acid gene sequences associated with the pathogenic bacteria, viruses, and parasites: **Bacteria:** - *Campylobacter* spp. (*C. coli/C. jejuni/C. lari/C. upsaliensis*) - *Clostridioides difficile* (*tcdA/tcdB*) - Enterotoxigenic *Escherichia coli* (ETEC) *LT/ST* - *Plesiomonas shigelloides* - *Salmonella* spp. - Shiga-like toxin-producing *Escherichia coli* (STEC) *stx1* - Shiga-like toxin-producing *Escherichia coli* (STEC) *stx2* - *Shigella*/Enteroinvasive *Escherichia coli* (EIEC) - *Vibrio cholerae* - *Vibrio* spp. (*V. parahaemolyticus/V. vulnificus*) - *Yersinia enterocolitica* **Viruses:** - Adenovirus F40/41 - Astrovirus - Norovirus GI/GII - Rotavirus A - Sapovirus I/II/IV/V **Parasites:** - *Blastocystis* sp. - *Cryptosporidium* spp. | The BIOFIRE FILMARRAY Gastrointestinal (GI) Panel is a qualitative multiplexed nucleic acid-based *in vitro* diagnostic test intended for use with BIOFIRE FILMARRAY Systems. The BIOFIRE GI Panel is capable of the simultaneous detection and identification of nucleic acids from multiple bacteria, viruses, and parasites directly from stool samples in Cary- Blair transport media 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 BIOFIRE GI Panel: - *Campylobacter* (*C. jejuni/C. coli/C. upsaliensis*) - *Clostridium difficile* (*C. difficile*) toxin A/B - *Plesiomonas shigelloides* - *Salmonella* - *Vibrio* (*V. parahaemolyticus/V. vulnificus/V. cholerae*), including specific identification of *Vibrio cholerae* - *Yersinia enterocolitica* - Enteroaggregative *Escherichia coli* (EAEC) - Enteropathogenic *Escherichia coli* (EPEC) - Enterotoxigenic *Escherichia coli* (ETEC) *lt/st* - Shiga-like toxin-producing *Escherichia coli* (STEC) *stx1/stx2* (including specific identification of the *E. coli* O157 serogroup within STEC) - *Shigella*/ Enteroinvasive *Escherichia coli* (EIEC) - *Cryptosporidium* - *Cyclospora cayetanensis* - *Entamoeba histolytica* - *Giardia lamblia* (also known as *G. intestinalis* and *G. duodenalis*) - Adenovirus F 40/41 - Astrovirus - Norovirus GI/GII - Rotavirus A - Sapovirus (Genogroups I, II, IV, and V) |

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| | - *Cyclospora cayetanensis* - *Dientamoeba fragilis* - *Entamoeba histolytica* - *Giardia lamblia* (also known as *G. intestinalis* and *G. duodenalis*) - Microsporidia (*Encephalitozoon hellem/Encephalitozoon intestinalis/Enterocytozoon bieneusi*) - *Strongyloides stercoralis* The LIAISON PLEX Gastrointestinal *Flex* Assay 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 information; however, it is not to be used to monitor these infections. Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor *C. difficile* infection. LIAISON PLEX Gastrointestinal *Flex* Assay results should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Confirmed positive results do not rule out co-infection with other organisms that are not detected by this test and may not be the sole or definitive cause of patient illness. Negative LIAISON PLEX Gastrointestinal *Flex* Assay 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. | The BIOFIRE GI Panel 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 BIOFIRE GI Panel. The agent detected may not be the definite cause of the 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 *E. coli* O157, *Plesiomonas shigelloides*, *Yersinia enterocolitica*, *Astrovirus*, and *Rotavirus A* were established primarily with retrospective clinical specimens. Performance characteristics for *Entamoeba histolytica*, and *Vibrio* (*V. parahaemolyticus*, *V. vulnificus*, and *Vibrio cholerae*) were established primarily using contrived clinical specimens. Negative BIOFIRE GI Panel 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. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks. |
| --- | --- | --- |
| Test Interpretation | Automated test interpretation and report generation | Same |
| Measurand | Nucleic acid from organisms detected | Same |
| Cartridge/Pouch | Single use | Same |

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| General Device Characteristic Differences | | |
| --- | --- | --- |
| Analytes | - *Campylobacter* spp. (*C. coli/C. jejuni/C. lari/C. upsaliensis*) - Shiga-like toxin-producing *Escherichia coli* (STEC) stx1 - Shiga-like toxin-producing *Escherichia coli* (STEC) stx2 - *Blastocystis* sp. - Microsporidia (*Encephalitozoon hellem/Encephalitozoon intestinalis/Enterocytozoon bieneusi*) - *Strongyloides stercoralis* | - *Campylobacter* (*C. jejuni/C. coli/C. upsaliensis*) - Enteroaggregative *Escherichia coli* (EAEC) - Enteropathogenic *Escherichia coli* (EPEC) - Shiga-like toxin-producing *Escherichia coli* (STEC) stx1/stx2 (including specific identification of the *E. coli* O157 serogroup within STEC) |
| Specimen Type | Liquid or soft stool in Cary-Blair / modified Cary-Blair; parasite targets also from alcohol-based formalin-free fixative. | Human stool sample collected in Cary Blair transport media |
| Technology | RT-PCR/PCR with microarray hybridization (nanoparticle/silver enhancement). | Nested multiplex PCR followed by high resolution melting analysis to confirm the identity of amplified product. |
| Instrument | LIAISON PLEX | BIOFIRE 2.0 System or BIOFIRE Torch System |
| Time to Result | Average Sample to Result time is 2 hours and 16 minutes | About 1 hour |
| Controls | Automated internal control for extraction/nucleic acid recovery/amplification/detection; synthetic DNA controls; hybridization control; results reported Pass/Fail/N/A. Internal control must pass or entire run is invalid. | Two controls are included in each reagent pouch to control for sample processing and both stages of PCR and melt analysis. |

## VI Standards/Guidance Documents Referenced:

- FDA Guidance – 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
- AAMI. Principles for medical device security – Risk Management. AAMI document TIR57:2016. Association for the Advancement of Medical Instrumentation; 2016.
- AAMI. Principles for medical device security – Postmarket risk management for device manufacturers. AAMI document TIR97:2019. Association for the Advancement of Medical Instrumentation; 2019.
- CLSI. Information Technology Security of In Vitro Diagnostic Instruments and Software Systems; Approved Standard – Second Edition. CLSI document AUTO11-A2. Wayne, PA: Clinical Laboratory Standards Institute; 2014.
- CLSI. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline – Third Edition. CLSI document EP05-A3. Wayne, PA: Clinical Laboratory Standards Institute; 2019.
- CLSI. Interference Testing in Clinical Chemistry. 3rd Ed. CLSI Document EP07. Wayne, PA: Clinical Laboratory Standards Institute; 2018.
- CLSI. Evaluation of Qualitative, Binary Output Examination Performance; Approved Guideline – Third Edition. CLSI document EP12. Wayne, PA: Clinical Laboratory Standards Institute; 2023.

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- • CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition. CLSI document EP17-A2. Wayne, PA: Clinical Laboratory Standards Institute; 2012.
- • CLSI. Assessment of the Diagnostic Accuracy of Laboratory Tests Using Receiver Operating Characteristic Curves; Approved Guideline – Second Edition. CLSI document EP24-A2. Wayne, PA: Clinical Laboratory Standards Institute; 2011.
- • CLSI. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline. CLSI document EP25-A. Wayne, PA: Clinical Laboratory Standards Institute; 2009.
- • CLSI. Collection Transport Preparation and Storage of Specimens for Molecular Methods. 2nd Edition. CLSI Document MM13. Wayne, PA: Clinical Laboratory Standards Institute; 2020.
- • CLSI. Verification and Validation of Multiplex Nucleic Acid Assays. 2nd Edition. CLSI Document MM17. Wayne, PA: Clinical Laboratory Standards Institute; 2018.
- • ISTA. Packaged-Products for Parcel Delivery System Shipment 70 kg (150 lb) or Less. ISTA Document 3A. International Safe Transit Association. 2018.
- • IEC 62366-1 Edition 1.1 2020-06 Consolidated Version; Medical devices – Part 1: Application of usability engineering to medical devices
- • IEC 61010-1 Edition 3.1 2017-01 Consolidated Version; Safety requirements for electrical equipment for measurement, control, and laboratory use – Part 1: General requirements
- • IEC 60601-1-2 Edition 4.1 2020-09 Consolidated Version; Medical electrical equipment – Part 1-2: General requirements for basic safety and essential performance – Collateral Standard: Electromagnetic disturbances – Requirements and tests
- • IEC 61326-1 Edition 3.0 2020-10; Electrical equipment for measurement, control and laboratory use – EMC requirements – Part 1: General requirements IEC 61326-2 Edition 3.0 2020-10; Electrical equipment for measurement, control and laboratory use – EMC requirements – Part 2-6: Particular requirements – In vitro diagnostic (IVD) medical equipment
- • IEC 62304 Edition 1.1 2015-06 Consolidated Version; Medical device software – Software life cycle processes
- • IEC TR 60878 Ed. 4.0 2022-11; Graphical symbols for electrical equipment in medical practice [Including: Corrigendum 1 (2023)]
- • IEC TR 80001-2-2:2012. Application of risk management for IT Networks incorporating medical devices – Part 2-2: Guidance for the disclosure and communication of medical device security needs, risks and controls
- • IEC TR 80001-2-8 Edition 1.0 206-05; Application of risk management for IT – networks incorporating medical devices – Part 2-8: Application guidance – Guidance on standards for establishing the security capabilities identified in IEC TR 80001-2-2
- • ISO 14971:2019 Medical Devices – Application of risk management to medical devices
- • ISO 15223-1: 2021-07 – Medical Devices- Symbols to be used with information to be supplied by the manufacturer – Part 1: General requirements
- • UL ANSI 2900-1 First Edition 2017; Standard for Safety, Standard for Software Cybersecurity Network-Connectable Products, Part 1: General Requirements
- • UL ANSI 2900-2-1 First Edition 2017; Standard for Safety, Software Cybersecurity for Network-Connectable Products, Part 2-1: Particular Requirements for Network Connectable Components of Healthcare and Wellness Systems
- • EN ISO 18113-1 2011; In vitro diagnostic medical devices. Information supplied by the manufacturer (labelling). In vitro diagnostic instruments for professional use
- • EN ISO 18113-2 2011; In vitro diagnostic medical devices — Information supplied by the manufacturer (labelling) Part 2: In vitro diagnostic reagents for professional use
- • ISO 23640 2015; Medical devices. Symbols to be used with medical device labels, labeling and information to be supplied. General requirements
- • CLSI MM13 2nd Edition, MM13Ed2E; Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods
- • CLSI MM17 2nd Edition, MM17Ed2E; Verification and Validation of Multiplex Nucleic Acid Assays

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## VII Performance Characteristics (if/when applicable):

### A Analytical Performance:

#### 1. Precision/Reproducibility:

##### *Within-Laboratory Precision/Repeatability*

Within-laboratory precision and repeatability of the LIAISON PLEX Gastrointestinal *Flex* Assay was evaluated across days, operators, and replicates. A single assay kit lot was tested by two operators at one internal site. Testing included negative, low positive (1.5x LoD), and moderate positive (3.0x LoD) samples prepared in two separate sample matrices (Parasite Fixative Stool Matrix and Cary-Blair Stool Matrix). Negative and positive samples were prepared as described below.

Precision/Repeatability of the LIAISON PLEX Gastrointestinal *Flex* Assay was demonstrated across operators, days, and replicates with a ≥95% agreement with the expected result for stool samples prepared in Cary-Blair and for stool samples prepared in parasite fixative for all analytes, except for Rotavirus (93.3% at 1.5x LoD in Cary-Blair), *Shigella*/*Enteroinvasive E. coli* (EIEC) (90% at 1.5x LoD in Cary Blair), *Entamoeba histolytica* (93.3% at 1.5x LoD in parasite fixative), and Microsporidia (93.3% at 1.5x LoD in parasite fixative) at low positive concentrations. Results of the Precision/Repeatability study are summarized in **Table 1** and **Table 2**. All analytes evaluated were detected within performance expectations for qualitative agreement across the concentrations tested. Precision and Repeatability of the LIAISON PLEX Gastrointestinal Assay was demonstrated across operators, days, and replicates.

**Table 1: Percent Agreement for Cary-Blair Stool Matrix Samples by Target**

| Panel Assay Target | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- |
| | Low Positive | Moderate Positive | Negative | All Concentrations (95% Confidence) |
| **Adenovirus F40/41** | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | 98.9% (89/90) (94.0% - 99.8%) |
| ***Clostridioides difficile* (tcdA/tcdB)** | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| ***Cryptosporidium* spp.** | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| **Norovirus GI/GII** | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| **Rotavirus A** | 93.3% (28/30) | 100% (30/30) | 100% (30/30) | 97.8% (88/90) (92.3% - 99.4%) |
| **Shiga-like toxin-producing *E. coli* (STEC) stx2** | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| ***Shigella*/Enteroinvasive *E. coli* (EIEC)** | 90.0% (27/30) | 100% (30/30) | 100% (30/30) | 96.7% (87/90) (90.7% - 98.9%) |
| **All Other Targets** | 100% (510/510) | 100% (510/510) | 100% (510/510) | 100% (1530/1530) (99.7% - 100%) |

**Table 2: Percent Agreement for Parasite Fixative Stool Matrix Samples by Target**

| Panel Assay Target | % Agreement with Expected result | | | |
| --- | --- | --- | --- | --- |
| | Low Positive | Moderate Positive | Negative | All Concentrations (95% Confidence) |
| ***Blastocystis* sp.** | | | | 98.9% (89/90) |

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| | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | (94.0% - 99.8%) |
| --- | --- | --- | --- | --- |
| *Cryptosporidium* spp. | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | 98.9% (89/90) (94.0% - 99.8%) |
| *Dientamoeba fragilis* | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | 98.9% (89/90) (94.0% - 99.8%) |
| *Entamoeba histolytica* | 93.3% (28/30) | 100% (30/30) | 100% (30/30) | 97.8% (88/90) (92.3% - 99.4%) |
| *Giardia lamblia* | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| *Microsporidia* | 93.3% (28/30) | 100% (30/30) | 100% (30/30) | 97.8% (88/90) (92.3% - 99.4%) |
| All Other Targets | 100% (60/60) | 100% (60/60) | 100% (60/60) | 100% (180/180) (97.9% - 100%) |

### Reproducibility

Site-to-site reproducibility of the LIAISON PLEX Gastrointestinal Flex Assay was evaluated across three sites. A single assay kit lot was tested by two operators at each of the three sites. Samples were randomized for each matrix and blinded to the operators, where each operator conducted nine tests per study day over five non-consecutive days at each site. Testing included negative, low positive (1.5x LoD), and moderate positive (3.0x LoD) samples in two sample matrices (Parasite Fixative Stool Matrix and Cary-Blair Stool Matrix). Negative samples were either negative stool matrix in Cary-Blair media or negative stool matrix in parasite fixative. Negative stool matrices were comprised of samples from multiple donors that had been pooled and confirmed negative for all targets on the LIAISON PLEX Gastrointestinal Flex Assay prior to use in the study. While positive Cary-Blair samples were designed to include analytes representative of each amplification pool and each target type included in the assay design (e.g., an RNA virus, a DNA virus, a gram-negative bacterium, a gram-positive bacterium, a toxin-producing bacterium, and a parasite), the positive parasite fixative samples were designed to include analytes representative of on-panel parasite targets.

Results of the site-to-site reproducibility study are summarized in Table 3 (Cary-Blair) and Table 4 (Parasite Fixative). Except for Shigella/Enteroinvasive E. coli (EIEC) at 1.5x LoD, the percent agreement with expected results was ≥95% for all analytes tested at 1.5x and 3x LoD, meeting the acceptance criteria for the study. The percent agreement for Shigella/Enteroinvasive E. coli (EIEC) at 1.5x LoD was 94.4%. All analytes evaluated were detected within performance expectations for qualitative agreement across all concentrations tested.

Table 3: Percent Agreement for Cary-Blair Stool Matrix Samples by Target (Site-to-Site)

| Panel Assay Target | Sample Type | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | Site 3 | All Sites (95% Confidence) |
| Adenovirus F40/41 | Low Positive | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 96.7% (29/30) | 98.9% (89/90) (94.0% - 99.8%) |
| Clostridioides difficile (tcdA/tcdB) | Low Positive | 100% (30/30) | 96.7% (29/30) | 96.7% (29/30) | 97.8% (88/90) (92.3% - 99.4%) |
| | | 100% | 100% | 96.7% | 98.9% (89/90) |

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| Panel Assay Target | Sample Type | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | Site 3 | All Sites (95% Confidence) |
| | Moderate Positive | (30/30) | (30/30) | (29/30) | (94.0% - 99.8%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| *Cryptosporidium* spp. | Low Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| Norovirus GI/GII | Low Positive | 100% (30/30) | 100% (30/30) | 96.7% (29/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| Rotavirus A | Low Positive | 93.3% (28/30) | 96.7% (29/30) | 96.7% (29/30) | 95.6% (86/90) (89.1% - 98.3%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| Shiga-like toxin-producing *E. coli* (STEC) *stx2* | Low Positive | 100% (30/30) | 96.7% (29/30) | 100% (30/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 96.7% (29/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| *Shigella*/Enteroinvasive *E. coli* (EIEC) | Low Positive | 90.0% (27/30) | 100% (30/30) | 93.3% (28/30) | 94.4% (85/90) (87.6% - 97.6%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| All Other Targets | Negative | 100% (1530/1530) | 100% (1530/1530) | 100% (1530/1530) | 100% (4590/4590) (99.9% - 100%) |

**Table 4: Percent Agreement for Parasite Fixative Stool Matrix Samples by Target (Site-to-Site)**

| Panel Assay Target | Sample Type | % Agreement with Expected result | | | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | Site 3 | All Sites (95% Confidence) |
| *Blastocystis* sp. | Low Positive | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 96.7% (29/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| *Cryptosporidium* spp. | Low Positive | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | 98.9% (89/90) (94.0% - 99.8%) |

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| Panel Assay Target | Sample Type | % Agreement with Expected result | | | |
| --- | --- | --- | --- | --- | --- |
| | | Site 1 | Site 2 | Site 3 | All Sites (95% Confidence) |
| *Dientamoeba fragilis* | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Low Positive | 96.7% (29/30) | 100% (30/30) | 93.3% (28/30) | 96.7% (87/90) (90.7% - 98.9%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 96.7% (29/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Low Positive | 93.3% (28/30) | 96.7% (29/30) | 93.3% (28/30) | 94.4% (85/90) (87.6% - 97.6%) |
| *Entamoeba histolytica* | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Low Positive | 100% (30/30) | 100% (30/30) | 93.3% (28/30) | 97.8% (88/90) (92.3% - 99.4%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Low Positive | 93.3% (28/30) | 100% (30/30) | 100% (30/30) | 97.8% (88/90) (92.3% - 99.4%) |
| *Giardia lamblia* | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Low Positive | 100% (180/180) | 100% (180/180) | 100% (180/180) | 100% (540/540) (99.3% - 100%) |
| | Moderate Positive | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Low Positive | 93.3% (28/30) | 100% (30/30) | 100% (30/30) | 97.8% (88/90) (92.3% - 99.4%) |
| *Microsporidia* | Moderate Positive | 100% (30/30) | 100% (30/30) | 96.7% (29/30) | 98.9% (89/90) (94.0% - 99.8%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| | Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (90/90) (95.9% - 100%) |
| **All Other Targets** | **Negative** | 100% (180/180) | 100% (180/180) | 100% (180/180) | 100% (540/540) (99.3% - 100%) |

### Lot-to-Lot

Lot-to-Lot reproducibility of the LIAISON PLEX Gastrointestinal *Flex* Assay was evaluated across three lots of assay kits with one testing operator at one internal site. Positive and negative samples were prepared as described above. Lot reproducibility of the LIAISON PLEX Gastrointestinal *Flex* Assay was demonstrated across three lots of assay kits with a percent agreement $\geq 95\%$ for all stool samples prepared in Cary-Blair and for stool samples prepared in parasite fixative, except for *Shigella/Enteroinvasive E. coli* (EIEC) (93.3% at 1.5x LoD in Cary Blair), *Entamoeba histolytica* (91.1% at 1.5x LoD in fixative). Confidence intervals were calculated using the Wilson Score Method. Results of the Lot-to-Lot Reproducibility study are summarized in **Tables 5** and **Table 6** below.

**Table 5. Lot-to-Lot: Percent Agreement for Cary-Blair Stool Matrix Samples by Target**

| Panel Assay Target | Sample Type | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- |
| | | Lot 1 | Lot 2 | Lot 3 | All Lots (95% Confidence) |
| **Adenovirus F40/41** | Low Positive | 93.3% (14/15) | 100% (15/15) | 93.3% (14/15) | 95.6% (43/45) (85.2% - 98.8%) |
| | | | | | 97.8% (44/45) |

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| | Moderate Positive | 93.3% (14/15) | 100% (15/15) | 100% (15/15) | (88.4% - 99.6%) |
| --- | --- | --- | --- | --- | --- |
| | Negative | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Clostridioides difficile (tcdA/tcdB)* | Low Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Moderate Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Negative | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Cryptosporidium spp.* | Low Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Moderate positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Negative | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Norovirus GI/GII* | Low Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Moderate Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Negative | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Rotavirus A* | Low Positive | 93.3% (14/15) | 100% (15/15) | 93.3% (14/15) | 95.6% (43/45) (85.2% - 98.8%) |
| | Moderate Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Negative | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Shiga-like toxin-producing E.coli (STEC) stx2* | Low Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Moderate Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Negative | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Shigella/Enteroinvasive E.coli (EIEC)* | Low Positive | 93.3% (14/15) | 100% (15/15) | 86.7% (13/15) | 93.3% (42/45) (82.1% - 97.7%) |
| | Moderate Positive | 93.3% (14/15) | 100% (15/15) | 100% (15/15) | 97.8% (44/45) (88.4% - 99.6%) |
| | Negative | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| **All Other Targets** | Negative | 99.9% (764/765) | 100% (765/765) | 100% (765/765) | 99.96% (2294/2295) (99.75% - 99.99%) |

**Table 6. Lot-to-Lot: Percent Agreement for Parasite Fixative Stool Matrix Samples**

| Panel Assay Target | Sample Type | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- |
| | | Lot 1 | Lot 2 | Lot 3 | All Lots (95% Confidence) |
| *Blastocystis sp.* | Low Positive | 100% (15/15) | 100% (15/15) | 93.3% (14/15) | 97.8% (44/45) (88.4% - 99.6%) |
| | Moderate Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | Negative | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Cryptosporidium spp.* | Low Positive | 100% (15/15) | 100% (15/15) | 93.3% (14/15) | 97.8% (44/45) (88.4% - 99.6%) |
| | Moderate Positive | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |

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| | **Negative** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| --- | --- | --- | --- | --- | --- |
| *Dientamoeba fragilis* | **Low Positive** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | **Moderate Positive** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | **Negative** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Entamoeba histolytica* | **Low Positive** | 93.3% (14/15) | 93.3% (14/15) | 86.7% (13/15) | 91.1% (41/45) (79.3% - 96.5%) |
| | **Moderate Positive** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | **Negative** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| *Giardia lamblia* | **Low Positive** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | **Moderate positive** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | **Negative** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| **Microsporidia** | **Low Positive** | 100% (15/15) | 100% (15/15) | 93.3% (14/15) | 97.8% (44/45) (88.4% - 99.6%) |
| | **Moderate Positive** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| | **Negative** | 100% (15/15) | 100% (15/15) | 100% (15/15) | 100% (45/45) (92.1% - 100%) |
| **All Other Targets** | **Negative** | 100% (90/90) | 100% (90/90) | 100% (90/90) | 100% (270/270) (98.6% - 100%) |

# 2. Linearity:

Not applicable.

# 3. Analytical Specificity/Interference:

# *Analytical Specificity (Cross-Reactivity)*

A study was performed to verify that the LIAISON PLEX Gastrointestinal *Flex* Assay does not detect nucleic acids off-panel organisms phylogenetically related to the on-panel organisms, as well as organisms typically present in stool samples. Organisms for the study panel were contrived in Cary-Blair Negative Stool Matrix at the highest available concentration and tested in triplicate with the LIAISON PLEX Gastrointestinal *Flex* Assay. When a non-specific positive result was observed, six additional replicates from a new preparation were tested to confirm potential cross-reactivity. Whole organisms and gBlocks with no observed cross-reactivity after wet testing are listed in **Table 7**. Organisms where cross-reactivity was observed through wet testing or predicted by *in silico* analysis (not wet tested) are reported in **Table 8** and **Table 9**, respectively. While *in silico* inclusivity analysis for all targets was performed with sequences available in the GenBank and WGS databases in July 2025, *in silico* exclusivity analysis was performed with sequences available from the GenBank nucleotide (nt) database as of January 10, 2025 for on-panel and off-panel organisms listed in **Table 9** below.

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Table 7: Organisms with No Observed Cross-Reactivity to LIAISON PLEX Gastrointestinal Flex Assay During Wet Testing

| Organism | Concentration Tested | Organism | Concentration Tested |
| --- | --- | --- | --- |
| **Bacteria** | | | |
| *Abiotrophia defectiva* | 5.70E+06 CFU/mL | *Fusobacterium varium* | 1.31E+08 CFU/mL |
| *Acinetobacter baumannii* | 3.16E+08 CFU/mL | *Helicobacter fennelliae* | 5.00E+04 CFU/mL |
| *Acinetobacter lwoffii* | 6.30E+07 CFU/mL | *Helicobacter pylori* | 4.98E+06 CFU/mL |
| *Aeromonas hydrophila* | 1.76E+08 CFU/mL | *Klebsiella oxytoca* | 2.69E+08 CFU/mL |
| *Alcaligenes faecalis* subsp. *faecalis* | 1.09E+09 CFU/mL | *Klebsiella pneumoniae* | 2.66E+08 CFU/mL |
| *Bacillus cereus* | 2.41E+07 CFU/mL | *Lactobacillus acidophilus* | 4.83E+07 CFU/mL |
| *Bacteroides caccae* | 4.47E+08 CFU/mL | *Lactobacillus reuteri* | 1.09E+08 CFU/mL |
| *Bacteroides fragilis* | 7.60+E08 CFU/mL | *Lactococcus lactis* | 3.02E+08 CFU/mL |
| *Bacteroides stercoris* | 2.35E+08 CFU/mL | *Leminorella grimontii* | 2.35E+08 CFU/mL |
| *Bifidobacterium bifidum* | 1.91E+07 CFU/mL | *Listeria grayi* | 2.90E+07 CFU/mL |
| *Campylobacter concisus* | 1.40E+05 CFU/mL | *Listeria monocytogenes* | 3.43E+08 CFU/mL |
| *Campylobacter curvus* | 3.05E+06 CFU/mL | *Morganella morganii* | 2.44E+08 CFU/mL |
| *Campylobacter fetus* | 2.30E+08 CFU/mL | *Peptoniphilus asaccharolyticus* | 8.35E+07 CFU/mL |
| *Campylobacter gracilis* | 2.85E+05 CFU/mL | *Peptostreptococcus anaerobius* | 3.15E+05 CFU/mL |
| *Campylobacter hominis* | 2.25E+05 CFU/mL | *Porphyromonas asaccharolytica* | 2.30E+06 CFU/mL |
| *Campylobacter rectus* | 1.81E+07 CFU/mL | *Prevotella bivia* | 1.14E+08 CFU/mL |
| *Campylobacter showae* | 1.70E+06 CFU/mL | *Prevotella melaninogenica* | 2.32E+07 CFU/mL |
| *Candida albicans* | 1.21E+07 CFU/mL | *Prevotella (Hoylesella) oralis* | 1.70E+07 CFU/mL |
| *Cedecea davisae* | 1.40E+07 CFU/mL | *Proteus mirabilis* | 3.24E+08 CFU/mL |
| *Citrobacter amalonaticus* | 3.57E+08 CFU/mL | *Proteus penneri* | 6.95E+07 CFU/mL |
| *Citrobacter freundii* | 1.30E+08 CFU/mL | *Proteus vulgaris* | 1.69E+08 CFU/mL |
| *Citrobacter sedlakii* | 2.55E+07 CFU/mL | *Providencia alcalifaciens* | 1.74E+08 CFU/mL |
| *Clostridium bifermentans* | 6.05E+06 CFU/mL | *Providencia stuartii* | 3.42E+08 CFU/mL |
| *Clostridium bolteae* | 5.20E+05 CFU/mL | *Providencia rettgeri* | 2.91E+08 CFU/mL |
| *Clostridium butyricum* | 1.14E+07 CFU/mL | *Pseudomonas aeruginosa* | 4.36E+08 CFU/mL |
| *Clostridium difficile* (Non-toxigenic) | 3.19E+06 CFU/mL | *Pseudomonas putida* | 8.45E+06 CFU/mL |
| *Clostridium haemolyticum* | 2.60E+05 CFU/mL | *Ruminococcus bromii* | 5.00E+05 CFU/mL |
| *Clostridium methylpentosum* | 7.90E+06 CFU/mL | *Serratia liquefaciens* | 4.93E+08 CFU/mL |
| *Clostridium nexile* | 1.28E+06 CFU/mL | *Serratia marcescens* | 1.82E+08 CFU/mL |
| *Clostridium novyi* | 9.65E+05 CFU/mL | *Staphylococcus aureus* | 2.71E+08 CFU/mL |
| *Clostridium perfringens* | 1.77E+06 CFU/mL | *Staphylococcus epidermidis* | 4.54E+08 CFU/mL |
| *Clostridium scindens* | 1.20E+07 CFU/mL | *Streptococcus dysgalactiae* | 6.75E+07 CFU/mL |
| *Clostridium septicum* | 2.54E+06 CFU/mL | *Vibrio alginolyticus* | 9.00E+06 CFU/mL |
| *Clostridium sordellii* | 2.86E+06 CFU/mL | *Vibrio fluvialis* | 3.60E+07 CFU/mL |
| *Clostridium sporogenes* | 1.14E+07 CFU/mL | *Vibrio furnissii* | 4.65E+07 CFU/mL |
| *Desulfovibrio piger* | 1.50E+05 CFU/mL | *Vibrio harveyi* | 3.80E+06 CFU/mL |
| *Edwardsiella tarda* | 5.95E+08 CFU/mL | *Vibrio metschnikovii* | 5.60E+06 CFU/mL |
| *Enterobacter (Klebsiella) aerogenes* | 3.95E+07 CFU/mL | *Vibrio mimicus* | 3.40E+07 CFU/mL |
| *Enterobacter cloacae* | 5.70E+08 CFU/mL | *Vibrio natriegens*^{1} | 1.29E+07 CFU/mL |

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| Organism | Concentration Tested | Organism | Concentration Tested |
| --- | --- | --- | --- |
| Enterococcus faecalis | 2.09E+08 CFU/mL | Yersinia bercovieri | 4.70E+07 CFU/mL |
| Enterococcus faecium | 1.43E+08 CFU/mL | Yersinia kristensenii (gBlock) | 1.50E+07 copies/mL |
| Escherichia coli | 1.36E+08 CFU/mL | Yersinia pseudotuberculosis | 2.84E+08 CFU/mL |
| Escherichia coli (EAEC) | 4.47E+08 CFU/mL | Yersinia rochesterensis (gBlock) | 1.50E+07 copies/mL |
| Escherichia coli (EPEC) | 6.10E+08 CFU/mL | Yersinia rohdei | 1.01E+07 CFU/mL |
| Escherichia fergusonii | 2.95E+08 CFU/mL | Yersinia ruckeri | 3.50E+07 CFU/mL |
| Escherichia hermannii | 2.79E+08 CFU/mL | | |
| **Viruses** | | | |
| Human adenovirus 31 (gBlock) | 1.50E+07 copies/mL | Cytomegalovirus, AD-169 | 1.35E+03 TCID50/mL |
| Adenovirus Type 7A, Species B | 9.75E+05 TCID50/mL | Echovirus Type 11 | 2.95E+06 TCID50/mL |
| Human mastadenovirus B (gBlock) | 1.50E+07 copies/mL | Enterovirus Type 68, 09/2014 Isolate 4 | 1.23E+04 TCID50/mL |
| Adenovirus Type 4, Species E | 8.50E+03 TCID50/mL | Hepatitis A virus, HM-175 (clone 1) | 4.45E+05 TCID50/mL |
| Coxsackie virus Type A16 | 2.04E+06 TCID50/mL | Human mastadenovirus C (gBlock) | 1.50E+07 copies/mL |
| Coxsackie virus B3 | 2.04E+06 TCID50/mL | Human mastadenovirus G (gBlock) | 1.50E+07 copies/mL |
| **Parasites** | | | |
| Entamoeba dispar | 1.25E+04 cells/mL | Pentatrichomonas hominis | 1.30E+06 cells/mL |
| Entamoeba dispar (gBlock) | 1.50E+07 copies/mL | Pentatrichomonas hominis (gBlock) | 1.50E+07 copies/mL |
| Entamoeba moshkovskii | 8.50E+04 cells/mL | Trichomonas tenax | 1.10E+06 cells/mL |
| Cyclospora colobi (gBlock) | 1.50E+07 copies/mL | Trichomonas tenax (gBlock) | 1.50E+07 copies/mL |

¹Vibrio cincinnatiensis was unavailable, Vibrio natriegens was used as a replacement.

Table 8: Organisms with Observed Cross-Reactivity to LIAISON PLEX Gastrointestinal Flex Assay in Wet Testing

| Reportable Target | Organism | Lowest Test Concentration with Positivity |
| --- | --- | --- |
| Adenovirus F40/41 | Adenovirus Type 31, Species A¹ | 8.50E+03 TCID50/mL |
| | Adenovirus Type 26, Species D | 5.85E+02 TCID50/mL |
| Blastocystis sp. | Cystoisospora belli (gBlock) | 1.50E+07 copies/mL |
| | Toxoplasma gondii | 7.70E+04 cells/mL |
| Entamoeba histolytica | Entamoeba nuttalli (gBlock)² | 1.50E+07 copies/mL |
| Giardia lamblia | Giardia ardeae (gBlock)³ | 1.50E+07 copies/mL |
| | Giardia cricetidarum (gBlock)⁴ | 1.50E+07 copies/mL |
| | Giardia microti (gBlock)⁴ | 1.50E+07 copies/mL |
| | Giardia muris⁴ | 6.25E+03 cysts/mL |
| | Giardia psittaci (gBlock)³ | 1.50E+07 copies/mL |
| Shiga-like toxin-producing E.coli (STEC) stx1 | Adenovirus Type 1, Species C¹ | 2.09E+05 TCID50/mL |

¹ Organism was wet tested and produced a positive result, however, the organism was not predicted to cross-react by in silico analysis performed January 2025. The associated gBlock did not cross-react, suggesting one of the following: i) the cross-reacting area of the organism genome is not encompassed by the gBlock fragment, ii) the organism was mis-labeled, or iii) there was contamination.

² Primate pathogen which shows homology to Entamoeba histolytica.

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$^{3}$ Avian pathogen which shows homology to *Giardia lamblia*.

$^{4}$ Rodent pathogen which shows homology to *Giardia lamblia*.

**Table 9: Organisms Not Wet Tested but Predicted by *in silico* Analysis (January 2025) to Cross-React with LIAISON PLEX Gastrointestinal *Flex* Assay**

| Reportable Target | Predicted Cross-Reaction |
| --- | --- |
| Astrovirus | Strains of canine, feline, porcine/swine and California sea lion astroviruses |
| Rotavirus A | Strains of porcine rotavirus |
| *Strongyloides stercoralis* | *Strongyloides callosciureus* *Strongyloides cebus* *Strongyloides fuelleborni* *Strongyloides myopotami* *Strongyloides papillosus* *Strongyloides procyonis* *Strongyloides ransomi* *Strongyloides ratti* *Strongyloides robustus* *Strongyloides venezuelensis* |
| *Vibrio cholerae* | *Vibrio tarriae* (accession CP022353.1)^{1} |

$^{1}$According to sponsor, *Vibrio tarriae* was considered a novel member of the *Cholerae clade*.

### *Interfering Substances*

Nineteen endogenous and exogenous substances that may be present in stool specimens were evaluated for potential interference with the LIAISON PLEX Gastrointestinal *Flex* Assay. Various targets were tested at 3x LoD in the presence of negative stool matrix (prepared in either Cary-Blair or Parasite Fixative) and one potentially interfering substance. Also, negative samples with only interfering substances in stool matrix were prepared and tested. In each matrix, at least five replicates of positive and negative samples were tested in the presence of each potentially interfering substance. No interference was observed for the potentially interfering substances listed in **Table 10** at the indicated concentration.

**Table 10: Potentially Interfering Substances Tested with No Observed Interference**

| Non-Microbial Interfering Substance | Concentration Tested |
| --- | --- |
| Triglyceride (Fecal Fat) | 5% v/v |
| Cholesterol (Fecal Fat) | 3% w/v |
| Human Hemoglobin | 5% w/v |
| Human Whole Blood | 25% v/v |
| Nystatin Suspension (Antifungal) | 25% v/v |
| Phenylephrine (Preparation H) | 10% w/v |
| Hydrocortisone (Preparation H) | 12% w/v |
| Aluminum Hydroxide, Magnesium Hydroxide (Mylanta) | 10% v/v |
| Mineral Oil | 25% v/v |
| Sennosides (Ex-lax) | 10% w/v |
| Bismuth subsalicylate (Pepto-Bismol) | 10% v/v |
| Naproxen Sodium | 2.1% w/v |
| Mucin | 0.5% w/v |
| Benzalkonium Chloride | 1% v/v |
| Ethanol | 1% v/v |

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Calcium carbonate, Polymyxin B sulfate/Bacitracin zinc, and Loperamide Hydrochloride were identified as interfering substances at the initial concentration tested. When tested at the next lower input level, no interference was observed for any targets included in the assay. (See results in Table 11 below).

Table 11: Potentially Interfering Substances Tested with Observed Interference

| Product | Active Ingredient | Concentration of Product with Observed Interference | Concentration of Product with No Observed Interference |
| --- | --- | --- | --- |
| Tums Antacid | Calcium carbonate | 10% w/v in Cary-Blair stool | 5% w/v in Cary-Blair stool |
| Polysporin First Aid Antibiotic Ointment | Polymyxin B sulfate/Bacitracin zinc | 10% w/v in Cary-Blair stool | 5% w/v in Cary-Blair stool |
| Imodium AD Anti-Diarrheal Oral Solution | Loperamide Hydrochloride | 10% v/v in Parasite Fixative stool | 5% v/v in Parasite Fixative stool |

### Microbial Inhibition

A Microbial Interference study was designed to evaluate the ability of the LIAISON PLEX Gastrointestinal Flex Assay to detect low positive analytes in the presence of twelve potentially inhibitory non-panel microorganisms that are commonly found in stool specimens. The study was performed utilizing two LIAISON PLEX target groups prepared in negative stool matrix with either Cary-Blair or Parasite Fixative. For the first target panel, positive samples were prepared at 3x LoD with representative organisms (e.g., an RNA virus, a DNA virus, a gram-negative bacterium, a gram-positive bacterium, a toxin-producing bacterium, and a parasite) in Cary-Blair Negative Stool Matrix. The second target panel included analytes representative of parasite targets in Parasite Fixative Negative Stool Matrix. At least five replicates of positive and negative samples were tested in the presence of each potentially inhibitory microorganism at ≥ 10⁶ organisms/mL, or the highest available concentration. No interference was observed for any of the potentially inhibitory microorganisms tested in Table 12 below.

Table 12: Potentially Inhibitory Microorganisms Tested with No Observed Interference

| Bacteroides fragilis | Escherichia coli |
| --- | --- |
| Bifidobacterium bifidum | Klebsiella pneumoniae |
| Candida albicans | Lactobacillus acidophilus |
| Clostridium perfringens | Prevotella bivia |
| Klebsiella aerogenes | Prevotella oralis |
| Enterococcus faecalis | Staphylococcus aureus |

### Co-Infection

A Co-Infection study was designed to evaluate the ability of the LIAISON PLEX Gastrointestinal Flex Assay to detect low positive analytes in the presence of other targets at

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high concentrations. Positive samples were prepared in Cary-Blair Negative Stool Matrix with prevalent co-infection targets, where one target was spiked at a low concentration (3x LoD) and the other target was spiked at a high concentration (100x LoD) and vice versa. Each combination was tested in three replicates. All targets in each co-infection combination were detected at 3x LoD and 100x LoD (Table 13).

Table 13: Percent Positivity of Each Target in Prevalent Co-Infection Combinations

| Co-infection Combination | Target at 100x LoD | Target at 3x LoD | % Positivity of Target at 100x LoD | % Positivity of Target at 3x LoD |
| --- | --- | --- | --- | --- |
| 1-A | Adenovirus F40/41 | Rotavirus A | 100.0% (3/3) | 100.0% (3/3) |
| 1-B | Rotavirus A | Adenovirus F40/41 | 100.0% (3/3) | 100.0% (3/3) |
| 2-A | Blastocystis sp. | Salmonella spp. | 100.0% (3/3) | 100.0% (3/3) |
| 2-B | Salmonella spp. | Blastocystis sp. | 100.0% (3/3) | 100.0% (3/3) |
| 3-A | Campylobacter spp. | Shiga-like toxin-producing E. coli (STEC) stx1 | 100.0% (3/3) | 100.0% (3/3) |
| 3-B | Shiga-like toxin-producing E. coli (STEC) stx1 | Campylobacter spp. | 100.0% (3/3) | 100.0% (3/3) |
| 4-A | Clostridioides difficile (tcdA/tcdB) | Blastocystis sp. | 100.0% (3/3) | 100.0% (3/3) |
| 4-B | Blastocystis sp. | Clostridioides difficile (tcdA/tcdB) | 100.0% (3/3) | 100.0% (3/3) |
| 5-A | Clostridioides difficile (tcdA/tcdB) | Campylobacter spp. | 100.0% (3/3) | 100.0% (3/3) |
| 5-B | Campylobacter spp. | Clostridioides difficile (tcdA/tcdB) | 100.0% (3/3) | 100.0% (3/3) |
| 6-A | Clostridioides difficile (tcdA/tcdB) | Enterotoxigenic E. coli (ETEC) LT/ST | 100.0% (3/3) | 100.0% (3/3) |
| 6-B | Enterotoxigenic E. coli (ETEC) LT/ST | Clostridioides difficile (tcdA/tcdB) | 100.0% (3/3) | 100.0% (3/3) |
| 7-A | Clostridioides difficile (tcdA/tcdB) | Norovirus GI/GII | 100.0% (3/3) | 100.0% (3/3) |
| 7-B | Norovirus GI/GII | Clostridioides difficile (tcdA/tcdB) | 100.0% (3/3) | 100.0% (3/3) |
| 8-A | Clostridioides difficile (tcdA/tcdB) | Salmonella spp. | 100.0% (3/3) | 100.0% (3/3) |
| 8-B | Salmonella spp. | Clostridioides difficile (tcdA/tcdB) | 100.0% (3/3) | 100.0% (3/3) |
| 9-A | Clostridioides difficile (tcdA/tcdB) | Sapovirus I/II/IV/V | 100.0% (3/3) | 100.0% (3/3) |
| 9-B | Sapovirus I/II/IV/V | Clostridioides difficile (tcdA/tcdB) | 100.0% (3/3) | 100.0% (3/3) |
| 10-A | Clostridioides difficile (tcdA/tcdB) | Shiga-like toxin-producing E. coli (STEC) stx1 | 100.0% (3/3) | 100.0% (3/3) |

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| Co-infection Combination | Target at 100x LoD | Target at 3x LoD | % Positivity of Target at 100x LoD | % Positivity of Target at 3x LoD |
| --- | --- | --- | --- | --- |
| 10-B | Shiga-like toxin-producing *E. coli* (STEC) *stx1* | *Clostridioides difficile* (tcdA/tcdB) | 100.0% (3/3) | 100.0% (3/3) |
| 11-A | *Clostridioides difficile* (tcdA/tcdB) | *Vibrio cholerae* | 100.0% (3/3) | 100.0% (3/3) |
| 11-B | *Vibrio cholerae* | *Clostridioides difficile* (tcdA/tcdB) | 100.0% (3/3) | 100.0% (3/3) |
| 12-A | *Giardia lamblia* | *Cryptosporidium* spp. | 100.0% (3/3) | 100.0% (3/3) |
| 12-B | *Cryptosporidium* spp. | *Giardia lamblia* | 100.0% (3/3) | 100.0% (3/3) |
| 13-A | Norovirus GI/GII | Adenovirus F40/41 | 100.0% (3/3) | 100.0% (3/3) |
| 13-B | Adenovirus F40/41 | Norovirus GI/GII | 100.0% (3/3) | 100.0% (3/3) |
| 14-A | Norovirus GI/GII | Astrovirus | 100.0% (3/3) | 100.0% (3/3) |
| 14-B | Astrovirus | Norovirus GI/GII | 100.0% (3/3) | 100.0% (3/3) |
| 15-A | Norovirus GI/GII | Rotavirus A | 100.0% (3/3) | 100.0% (3/3) |
| 15-B | Rotavirus A | Norovirus GI/GII | 100.0% (3/3) | 100.0% (3/3) |
| 16-A | Norovirus GI/GII | *Salmonella* spp. | 100.0% (3/3) | 100.0% (3/3) |
| 16-B | *Salmonella* spp. | Norovirus GI/GII | 100.0% (3/3) | 100.0% (3/3) |
| 17-A | Norovirus GI/GII | Sapovirus I/II/IV/V | 100.0% (3/3) | 100.0% (3/3) |
| 17-B | Sapovirus I/II/IV/V | Norovirus GI/GII | 100.0% (3/3) | 100.0% (3/3) |
| 18-A | Norovirus GI/GII | Shiga-like toxin-producing *E. coli* (STEC) *stx1* | 100.0% (3/3) | 100.0% (3/3) |
| 18-B | Shiga-like toxin-producing *E. coli* (STEC) *stx1* | Norovirus GI/GII | 100.0% (3/3) | 100.0% (3/3) |
| 19-A | Rotavirus A | Enterotoxigenic *E. coli* (ETEC) *LT/ST* | 100.0% (3/3) | 100.0% (3/3) |
| 19-B | Enterotoxigenic *E. coli* (ETEC) *LT/ST* | Rotavirus A | 100.0% (3/3) | 100.0% (3/3) |
| 20-A | Rotavirus A | Shiga-like toxin-producing *E. coli* (STEC) *stx1* | 100.0% (3/3) | 100.0% (3/3) |
| 20-B | Shiga-like toxin-producing *E. coli* (STEC) *stx1* | Rotavirus A | 100.0% (3/3) | 100.0% (3/3) |
| 21-A | *Salmonella* spp. | *Campylobacter* spp. | 100.0% (3/3) | 100.0% (3/3) |
| 21-B | *Campylobacter* spp. | *Salmonella* spp. | 100.0% (3/3) | 100.0% (3/3) |

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| Co-infection Combination | Target at 100x LoD | Target at 3x LoD | % Positivity of Target at 100x LoD | % Positivity of Target at 3x LoD |
| --- | --- | --- | --- | --- |
| 22-A | Shiga-like toxin-producing *E. coli* (STEC) *stx1* | *Shigella*/Enteroinvasive *E. coli* (EIEC) | 100.0% (3/3) | 100.0% (3/3) |
| 22-B | *Shigella*/Enteroinvasive *E. coli* (EIEC) | Shiga-like toxin-producing *E. coli* (STEC) *stx1* | 100.0% (3/3) | 100.0% (3/3) |
| 23-A | *Shigella*/Enteroinvasive *E. coli* (EIEC) | *Salmonella* spp. | 100.0% (3/3) | 100.0% (3/3) |
| 23-B | *Salmonella* spp. | *Shigella*/Enteroinvasive *E. coli* (EIEC) | 100.0% (3/3) | 100.0% (3/3) |

#### Carry-Over/Cross Contamination

The potential for carry-over/cross contamination using the LIAISON PLEX Gastrointestinal *Flex* Assay was assessed through testing high positive samples (containing representative assay targets) in an alternating series with negative samples (containing no targets) in a checkerboard design. On each instrument, negative samples were tested immediately following testing of high positive samples within the same module to evaluate possible carry-over. This alternating series was continued across five valid rounds of testing performed by two operators using one of two LIAISON PLEX instruments with six modules. The high positive sample was contrived in Cary-Blair Negative Stool Matrix containing *Salmonella enterica* (representative DNA target) at 10$^{6}$ CFU/mL and Rotavirus A (representative RNA target) at 10$^{6}$ copies/mL. The negative sample was prepared from Cary-Blair Negative Stool Matrix only. All high positive samples and negative samples yielded the expected results. No carry-over or cross contamination was observed.

#### 4. Detection Limit and Assay Reportable Range:

##### Limit of Detection

A Limit of Detection (LoD) study was performed to determine the analytical sensitivity of the LIAISON PLEX Gastrointestinal *Flex* Assay. Testing included an initial estimation of the LoD followed by a final confirmation of each analyte's LoD. The LoD was estimated for each strain/sample using a 3-fold dilution series and four replicates per dilution (concentration). The preliminary LoD was identified as the lowest concentration where a 100% detection rate was observed. The LoD was confirmed for each strain by testing serial concentrations beginning at the preliminary LoD, with subsequent increases or decreases in three-fold increments until the lowest concentration demonstrating ≥95% detection across replicates was observed. During the confirmation phase of testing, twenty replicates were evaluated at each concentration across 3 assay kits. When multiple strains or samples per assay target call were tested, the highest LoD observed was defined as the LoD claim for the target. All assay targets were evaluated in Cary-Blair stool matrix. For parasite targets included in the assay, these were additionally evaluated in two parasite fixative stool matrices. **Table 14** below shows the LoD per strain, as well as the overall LoD reported for each LIAISON PLEX Gastrointestinal *Flex* Assay analyte.

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Table 14: LIAISON PLEX Gastrointestinal Flex Assay

Limit of Detection Summary by Strain/Sample

| LIAISON PLEX GI Assay Target | Organism | Strain / Sample | Media | LoD per Strain | Percent Positivity at LoD | LoD per Target |
| --- | --- | --- | --- | --- | --- | --- |
| **Bacterial Targets** | | | | | | |
| *Campylobacter* spp. | *Campylobacter coli* | ATCC 43482 | Cary-Blair | 5.65E+00 CFU/mL | 95% (19/20) | 4.57E+02 CFU/mL |
| | *Campylobacter jejuni* subsp. *jejuni* | ATCC 29428 | Cary-Blair | 4.57E+02 CFU/mL | 100% (20/20) | |
| | *Campylobacter lari* | ATCC 35222 | Cary-Blair | 1.69E+01 CFU/mL | 100% (20/20) | |
| | *Campylobacter upsaliensis* | ATCC 43953 | Cary-Blair | 5.08E+01 CFU/mL | 100% (20/20) | |
| *Clostridioides difficile* (tcdA/tcdB) | *Clostridioides difficile* | ATCC 43255 | Cary-Blair | 1.88E+00 CFU/mL | 100% (20/20) | 5.65E+00 CFU/mL |
| | *Clostridioides difficile* | ATCC BAA-1805 | Cary-Blair | 5.65E+00 CFU/mL | 95% (19/20) | |
| **Enterotoxigenic *E.coli* (ETEC) *LT/ST*** | *Escherichia coli* (LT+, ST1A+, ST1B+) | ATCC 35401 | Cary-Blair | 4.57E+02 CFU/mL | 100% (20/20) | 4.57E+02 CFU/mL |
| | *Escherichia coli* (ST1B+) | ATCC 43896 | Cary-Blair | 4.57E+02 CFU/mL | 100% (20/20) | |
| *Plesiomonas shigelloides* | *Plesiomonas shigelloides* | ATCC 51903 | Cary-Blair | 1.37E+03 CFU/mL | 100% (20/20) | 4.12E+03 CFU/mL |
| | *Plesiomonas shigelloides* | ATCC 14029 | Cary-Blair | 4.12E+03 CFU/mL | 100% (20/20) | |
| *Salmonella* spp. | *Salmonella enterica* subsp. *enterica* serovar Typhimurium | ATCC 13311 | Cary-Blair | 1.37E+03 CFU/mL | 100% (20/20) | 1.37E+03 CFU/mL |
| | *Salmonella enterica* subsp. *arizonae* | ATCC 13314 | Cary-Blair | 1.37E+03 CFU/mL | 100% (20/20) | |
| **Shiga-like toxin-producing *E.coli* (STEC) *stx1*** | *Escherichia coli* (O157:H7), *STX1* | ATCC 43890 | Cary-Blair | 1.37E+03 CFU/mL | 100% (20/20) | 1.37E+03 CFU/mL |
| **Shiga-like toxin-producing *E.coli* (STEC) *stx2*** | *Escherichia coli* (O104:H4), *STX2* | ATCC BAA-2326 | Cary-Blair | 4.57E+02 CFU/mL | 100% (20/20) | 4.57E+02 CFU/mL |

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| LIAISON PLEX GI Assay Target | Organism | Strain / Sample | Media | LoD per Strain | Percent Positivity at LoD | LoD per Target |
| --- | --- | --- | --- | --- | --- | --- |
| *Shigella/Enteroinvasive E.coli* (EIEC) | *Shigella boydii* | ATCC 25930 | Cary-Blair | 1.52E+02 CFU/mL | 95% (19/20) | 4.57E+02 CFU/mL |
| | *Shigella flexneri* | ATCC 25929 | Cary-Blair | 4.57E+02 CFU/mL | 100% (20/20) | |
| *Vibrio cholerae* | *Vibrio cholerae* (O:1) | ATCC 39315 | Cary-Blair | 4.12E+03 CFU/mL | 100% (20/20) | 4.12E+03 CFU/mL |
| | *Vibrio cholerae* (O:139) | ATCC 51394 | Cary-Blair | 4.12E+03 CFU/mL | 100% (20/20) | |
| *Vibrio* spp. | *Vibrio parahaemolyticus* | ATCC 49398 | Cary-Blair | 1.37E+03 CFU/mL | 100% (20/20) | 1.37E+03 CFU/mL |
| | *Vibrio vulnificus* | ATCC 27562 | Cary-Blair | 4.57E+02 CFU/mL | 100% (20/20) | |
| *Yersinia enterocolitica* | *Yersinia enterocolitica* | ATCC 23715 | Cary-Blair | 1.37E+03 CFU/mL | 95% (19/20) | 1.37E+03 CFU/mL |
| | *Yersinia enterocolitica* | ATCC 700822 | Cary-Blair | 1.37E+03 CFU/mL | 100% (20/20) | |
| **Viral Targets** | | | | | | |
| **Adenovirus F40/41** | Human adenovirus 40 | ATCC VR-931 | Cary-Blair | 4.57E+02 copies/mL | 95% (19/20) | 4.57E+02 copies/mL |
| | Human adenovirus 41 | ATCC VR-930 | Cary-Blair | 4.57E+02 copies/mL | 100% (20/20) | |
| **Astrovirus** | Astrovirus Type 1 | ATCC VR-1936 | Cary-Blair | 1.00E+06 copies/mL | 100% (20/20) | 1.00E+06 copies/mL |
| | Astrovirus Type 2 | ATCC VR-1943 | Cary-Blair | 1.11E+05 copies/mL | 100% (20/20) | |
| **Norovirus GI/GII** | Norovirus GI | Zeptometrix 0830086CFL | Cary-Blair | 2.75E+04 copies/mL | 100% (20/20) | 3.70E+04 copies/mL |
| | Norovirus GII | Zeptometrix 0830087CFL | Cary-Blair | 3.70E+04 copies/mL | 100% (20/20) | |
| **Rotavirus A** | Rotavirus A | ATCC VR-2550 | Cary-Blair | 1.23E+04 copies/mL | 100% (20/20) | 1.23E+04 copies/mL |
| | Rotavirus A | ATCC VR-2551 | Cary-Blair | 4.57E+02 copies/mL | 100% (20/20) | |
| **Sapovirus I/II/IV/V** | Sapovirus GI | Clinical Specimen LMNX-2829 | Cary-Blair | 1.23E+04 copies/mL | 100% (20/20) | 1.23E+04 copies/mL |
| | Sapovirus GII | Clinical Specimen LMNX-2964 | Cary-Blair | 1.23E+04 copies/mL | 95% (19/20) | |
| **Parasitic Targets** | | | | | | |

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| LIAISON PLEX GI Assay Target | Organism | Strain / Sample | Media | LoD per Strain | Percent Positivity at LoD | LoD per Target |
| --- | --- | --- | --- | --- | --- | --- |
| *Blastocystis* sp. | *Blastocystis hominis* | ATCC 50177 | Cary-Blair | 1.69E+01 cells/mL | 100% (20/20) | Cary-Blair: 4.57E+02 cells/mL EcoFix: 1.52E+02 cells/mL Total-Fix: 5.08E+01 cells/mL |
| | | | EcoFix | 1.52E+02 cells/mL | 100% (20/20) | |
| | | | Total-Fix | 5.65E+00 cells/mL | 95% (19/20) | |
| | *Blastocystis hominis* | ATCC 50608 | Cary-Blair | 4.57E+02 cells/mL | 100% (20/20) | |
| | | | EcoFix | 1.52E+02 cells/mL | 100% (20/20) | |
| | | | Total-Fix | 5.08E+01 cells/mL | 100% (20/20) | |
| *Cryptosporidium* spp. | *Cryptosporidium parvum* | Waterborne P102C | Cary-Blair | 1.37E+03 oocysts/mL | 100% (20/20) | Cary-Blair: 4.12E+03 oocysts/mL EcoFix: 4.12E+03 oocysts/mL Total-Fix: 4.12E+03 oocysts/mL |
| | | | EcoFix | 4.12E+03 oocysts/mL | 100% (20/20) | |
| | | | Total-Fix | 1.37E+03 oocysts/mL | 95% (19/20) | |
| | *Cryptosporidium meleagridis* | TU1867 | Cary-Blair | 4.12E+03 oocysts/mL | 100% (20/20) | |
| | | | EcoFix | 1.37E+03 oocysts/mL | 100% (20/20) | |
| | | | Total-Fix | 4.12E+03 oocysts/mL | 100% (20/20) | |
| | *Cryptosporidium muris* | Waterborne P104 | Cary-Blair | 4.12E+03 oocysts/mL | 100% (20/20) | |
| | | | EcoFix | 1.37E+03 oocysts/mL | 100% (20/20) | |
| | | | Total-Fix | 4.12E+03 oocysts/mL | 100% (20/20) | |
| *Cyclospora cayetanensis* | *Cyclospora cayetanensis* | Clinical Specimen LMNX-3415 | Cary-Blair | 4.12E+03 copies/mL | 100% (20/20) | Cary-Blair: 4.12E+03 copies/mL EcoFix: Not Found^{1} Total-Fix: 4.12E+03 copies/mL |
| | *Cyclospora cayetanensis* | Clinical Specimen LMNX-3862 | Cary-Blair | 7.57E+02 copies/mL | 100% (20/20) | |
| | | | EcoFix | Not Found^{1} | N/A | |
| | | | Total-Fix | 4.12E+03 copies/mL | 95% (19/20) | |
| *Dientamoeba fragilis* | *Dientamoeba fragilis* | D.fragilis-1 | Cary-Blair | 1.52E+02 copies/mL | 100% (20/20) | Cary-Blair: 4.57E+02 copies/mL EcoFix: 4.57E+02 copies/mL Total-Fix: 4.57E+02 copies/mL |
| | | | EcoFix | 1.52E+02 copies/mL | 100% (20/20) | |
| | | | Total-Fix | 1.52E+02 copies/mL | 100% (20/20) | |
| | *Dientamoeba fragilis* | D.fragilis-2 | Cary-Blair | 4.57E+02 copies/mL | 100% (20/20) | |

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| LIAISON PLEX GI Assay Target | Organism | Strain / Sample | Media | LoD per Strain | Percent Positivity at LoD | LoD per Target |
| --- | --- | --- | --- | --- | --- | --- |
| | | | EcoFix | 4.57E+02 copies/mL | 100% (20/20) | |
| | | | Total-Fix | 4.57E+02 copies/mL | 100% (20/20) | |
| *Entamoeba histolytica* | *Entamoeba histolytica* | ATCC 30459 | Cary-Blair | 6.97E-02 cells/mL | 100% (20/20) | Cary-Blair: 1.88E+00 cells/mL EcoFix: 1.88E+00 cells/mL Total-Fix: 1.88E+00 cells/mL |
| | | | EcoFix | 6.97E-02 cells/mL | 100% (20/20) | |
| | | | Total-Fix | 6.97E-02 cells/mL | 95% (19/20) | |
| | *Entamoeba histolytica* | ATCC 50524 | Cary-Blair | 1.88E+00 cells/mL | 100% (20/20) | |
| | | | EcoFix | 1.88E+00 cells/mL | 100% (20/20) | |
| | | | Total-Fix | 1.88E+00 cells/mL | 100% (20/20) | |
| *Giardia lamblia* | *Giardia intestinalis* | ATCC 30888 | Cary-Blair | 5.08E+01 cells/mL | 95% (19/20) | Cary-Blair: 1.52E+02 cells/mL EcoFix: 1.52E+02 cells/mL Total-Fix: 1.52E+02 cells/mL |
| | | | EcoFix | 1.52E+02 cells/mL | 95% (19/20) | |
| | | | Total-Fix | 5.08E+01 cells/mL | 95% (19/20) | |
| | *Giardia lamblia* | ATCC PRA-254 | Cary-Blair | 1.52E+02 cells/mL | 100% (20/20) | |
| | | | EcoFix | 1.52E+02 cells/mL | 100% (20/20) | |
| | | | Total-Fix | 1.52E+02 cells/mL | 100% (20/20) | |
| **Microsporidia** | *Encephalitozoon hellem* | ATCC 50451 | Cary-Blair | 2.08E+03 copies/mL | 95% (19/20) | Cary-Blair: 2.08E+03 copies/mL EcoFix: 1.87E+04 copies/mL Total-Fix: 6.25E+03 copies/mL |
| | | | EcoFix | 1.87E+04 copies/mL | 100% (20/20) | |
| | | | Total-Fix | 6.25E+03 copies/mL | 100% (20/20) | |
| | *Encephalitozoon intestinalis* | ATCC 50651 | Cary-Blair | 3.97E+02 copies/mL | 95% (19/20) | |
| | | | EcoFix | 3.57E+03 copies/mL | 100% (20/20) | |
| | | | Total-Fix | 1.19E+03 copies/mL | 100% (20/20) | |
| | *Enterocytozoon bieneusi* | Clinical Specimen LMNX-2885 | Cary-Blair | 1.37E+03 copies/mL | 100% (20/20) | |
| | *Enterocytozoon bieneusi* | Clinical Specimen LMNX-4140 | Cary-Blair | 1.37E+03 copies/mL | 100% (20/20) | |
| | | | EcoFix | 4.12E+03 copies/mL | 100% (20/20) | |

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| LIAISON PLEX GI Assay Target | Organism | Strain / Sample | Media | LoD per Strain | Percent Positivity at LoD | LoD per Target |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Total-Fix | 1.37E+03 copies/mL | 100% (20/20) | |
| *Strongyloides stercoralis* | *Strongyloides stercoralis* | Clinical Specimen LMNX-3433^{2} | Cary-Blair | 2.03E+04 copies/mL | 95% (19/20) | Cary-Blair: 2.03E+04 copies/mL EcoFix: 2.72E+04 copies/mL Total-Fix: 2.16E+04 copies/mL |
| | | | EcoFix | 2.72E+04 copies/mL | 95% (19/20) | |
| | | | Total Fix | 2.16E+04 copies/mL | 100% (20/20) | |

$^{1}$ To further assess analytical sensitivity in EcoFix relative to Total-Fix, an additional clinical raw stool specimen (LMNX-4486) containing *Cyclospora cayetanensis* was identified and used for additional evaluation. At neat (undiluted) concentration, detection was 19/20 (95%) in Eco-Fix and 20/20 (100%) in Total-Fix. The LoD per target for both EcoFix and Total-Fix was reported as 9.95E+02 copies/mL. At a three-fold dilution in Negative Stool Matrix, the detection rate of the same specimen was 15/19 (79%) in EcoFix and 18/20 (90%) in Total-Fix. The analytical data from parallel matrix testing and the contrived clinical study results provide supportive evidence that detection of *Cyclospora cayetanensis* in EcoFix is comparable to that observed in Total-Fix at concentrations near the limit of detection.

$^{2}$ In the original LoD study, testing with this clinical specimen yielded substantial variability in the stock concentration across matrices. Results demonstrated that the variability in measured copies/mL in the original study was present even during the quantification stage pre-LoD. Therefore, testing was repeated with independently prepared material. Results of new LoD testing showed detection at similar copies/mL, which are included in the table above.

### Inclusivity

Analytical Reactivity (Inclusivity) of the LIAISON PLEX Gastrointestinal *Flex* Assay was assessed with a collection of 243 strains and clinical samples, representing the genetic diversity of the analytes targeted by the assay. While fifty of the strains/clinical samples were used to determine the LoD for each assay target, the remaining 193 strains/clinical samples were diluted in Cary-Blair Negative Stool Matrix to a final concentration of 3x LoD or the highest available concentration where stock concentrations were limiting. Each diluted strain/clinical sample was tested in triplicate. In cases where 100% positivity was not achieved at 3x LoD, higher concentrations of each analyte (up to 1000x LoD) were tested until 100% positivity was achieved.

Of the 243 strains/clinical samples tested, four had unknown concentrations. All four of the samples with unknown concentrations demonstrated 100% positivity in wet testing. Of the 239 strains/clinical samples tested at known concentrations, fifty demonstrated ≥95% positivity at 1x LoD (strains used previously to determine LoD). Of the remaining 189 strains/clinical samples tested at known concentrations: 168 demonstrated 100% positivity at ≤3x LoD, five (5) demonstrated 100% positivity at 10x LoD, thirteen (13) demonstrated 100% positivity at 100x LoD, and three (3) demonstrated 100% positivity at 1000x LoD. Results of the Analytical Reactivity study are shown in **Table 15** through **Table 18** below.

**Table 15: Summary of Analytical Reactivity Study Results**

| Organism | Number of Strains/Clinical Samples | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Total Tested | ≥95% Positivity at 1x LoD | 100% Positivity at ≤3x LoD | 100% Positivity at 10x LoD | 100% Positivity at 100x LoD | 100% Positivity at 1000x LoD | 100% Positivity at Unknown Concentration |
| **Bacteria** | 147 | 22 | 111 | 3 | 11 | --- | --- |
| **Viruses** | 55 | 10 | 39 | 2 | 2 | 1 | 1^{1,2} |
| **Parasites** | 41 | 18 | 18 | --- | --- | 2 | 3^{2} |
| **Total** | 243 | 50 | 168 | 5 | 13 | 3 | 4 |

$^{1}$Sample tested neat

$^{2}$Sample concentration unknown

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**Table 16: Analytical Reactivity (Inclusivity) Bacterial Strains**

| Target Call | Species/ Species Type | Serotype/ Toxin | Source/ | Strain/ Clinical Sample (LoD strains are in bold font) | Test Level (xLoD) | Test Concentration (CFU/mL) | % Positivity |
| --- | --- | --- | --- | --- | --- | --- | --- |
| *Campylobacter* spp. | *Campylobacter coli* | N/A | N/A | **ATCC 43482** | 1x | 5.65E+00 | 95% (19/20) |
| | *Campylobacter coli* | | | ATCC 43488 | 100x | 5.65E+02 | 100% (3/3) |
| | *Campylobacter coli* | | | ATCC 43483 | 100x | 5.65E+02 | 100% (3/3) |
| | *Campylobacter coli* | | | ATCC BAA-371 | 100x | 5.65E+02 | 100% (3/3) |
| | *Campylobacter coli* | | | ATCC 43489 | 100x | 5.65E+02 | 100% (3/3) |
| | *Campylobacter jejuni* (Including subsp.jejuni and subsp.doylei) | | | ATCC 49349 | 3x | 1.37E+03 | 100% (3/3) |
| | *Campylobacter jejuni* (Including subsp.jejuni and subsp.doylei) | | | **ATCC 29428** | 1x | 4.57E+02 | 100% (20/20) |
| | *Campylobacter jejuni* (Including subsp.jejuni and subsp.doylei) | | | ATCC 43479 | 3x | 1.37E+03 | 100% (3/3) |
| | *Campylobacter jejuni* (Including subsp.jejuni and subsp.doylei) | | | ATCC 43474 | 3x | 1.37E+03 | 100% (3/3) |
| | *Campylobacter jejuni* (Including subsp.jejuni and subsp.doylei) | | | ATCC 43465 | 3x | 1.37E+03 | 100% (3/3) |
| | *Campylobacter upsaliensis* | | | ATCC 43954 | 100x | 5.08E+03 | 100% (3/3) |
| | *Campylobacter upsaliensis* | | | ATCC BAA-1059 | 3x | 1.52E+02 | 100% (3/3) |
| | *Campylobacter upsaliensis* | | | **ATCC 43953** | 1x | 5.08E+01 | 100% (20/20) |
| | *Campylobacter upsaliensis* | | | ATCC 49816 | 3x | 1.52E+02 | 100% (3/3) |
| | *Campylobacter upsaliensis* | | | ATCC 49815 | 3x | 1.52E+02 | 100% (3/3) |
| | *Campylobacter lari* | | | **ATCC 35222** | 1x | 1.69E+01 | 100% (20/20) |
| | *Campylobacter lari* | | | ATCC 43675 | 100x | 1.69E+03 | 100% (3/3) |
| | *Campylobacter lari* | | | ATCC 35223 | 100x | 1.69E+03 | 100% (3/3) |
| | *Campylobacter lari* | | | ATCC BAA-1060 | 100x | 1.69E+03 | 100% (3/3) |
| | *Campylobacter lari* | | | ATCC 35221 | 100x | 1.69E+03 | 100% (3/3) |
| *Clostridioides difficile* (*tcdA/tcdB*) | *Clostridioides difficile* | 0, A+, B+ | | **ATCC 43255 (CCUG19126, VPI 10463)** | 1x | 1.88E+00 | 100% (20/20) |
| | *Clostridioides difficile* | 0, A+, B+ | | ATCC 9689 (90556-M6S) | 3x | 1.70E+01 | 100% (3/3) |
| | *Clostridioides difficile* | 0, A+, B+ | | ATCC 700792 (14797-2) | 3x | 1.70E+01 | 100% (3/3) |
| | *Clostridioides difficile* | 0, A+, B+ | | ATCC 17858 (1253) | 3x | 1.70E+01 | 100% (3/3) |
| | *Clostridioides difficile* | IIIb, A+, B+ | | **ATCC BAA-1805 (NAP I strain)** | 1x | 5.65E+00 | 95% (19/20) |

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| Target Call | Species/ Species Type | Serotype/ Toxin | Source/ | Strain/ Clinical Sample (LoD strains are in bold font) | Test Level (xLoD) | Test Concentration (CFU/mL) | % Positivity |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | *Clostridioides difficile* | 0, A+, B+ | | ATCC BAA-1382 (630) | 3x | 1.70E+01 | 100% (3/3) |
| | *Clostridioides difficile* | 0, A+, B+ | | ATCC 51695 (BDMS 18 AN) |…