BioCode Gastrointestinal Pathogen Panel (GPP)

K242877 · Applied BioCode, Inc. · PCH · Jan 10, 2025 · Microbiology

Device Facts

• Record ID: K242877
• Device Name: BioCode Gastrointestinal Pathogen Panel (GPP)
• Applicant: Applied BioCode, Inc.
• Product Code: PCH · Microbiology
• Decision Date: Jan 10, 2025
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3990
• Device Class: Class 2

Indications for 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 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.

Device Story

Multiplexed in vitro diagnostic test for gastrointestinal pathogens; uses BioCode MDx-3000 system. Input: stool specimens (unpreserved or Cary-Blair). Processing: nucleic acid extraction (via easyMAG, MagNA Pure 96, KingFisher Flex, or KingFisher Apex Dx); PCR amplification; target capture; signal generation; optical detection. Output: qualitative identification of 17 pathogens. Used in clinical laboratories by trained personnel. Results aid diagnosis of gastrointestinal illness; used with other clinical/epidemiological data. Benefits: rapid (under 4 hours) simultaneous detection of multiple pathogens.

Clinical Evidence

Clinical method comparison study using 468 archived (frozen unpreserved/inoculated Cary-Blair) and 54 fresh samples. Compared performance of KingFisher Flex and KingFisher Apex Dx against easyMAG. Results showed high positive and negative percent agreement across all targets. Reproducibility study (30 runs, 3 instruments, 3 operators) confirmed consistent performance. Contrived sample testing (120 samples) supported performance for specific targets.

Technological Characteristics

Multiplex RT-PCR and probe hybridization; fluorescence detection; barcoded magnetic beads (BMB). Automated processing on BioCode MDx-3000. Extraction systems: easyMAG, MagNA Pure 96, KingFisher Flex, KingFisher Apex Dx. Internal calibration. Prescription use.

Indications for Use

Indicated for individuals with signs/symptoms of gastrointestinal infection to detect and identify nucleic acids from bacteria, viruses, and parasites in unpreserved or Cary-Blair preserved stool samples. 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

• BioCode Gastrointestinal Pathogen Panel (GPP) (K190585)

Submission Summary (Full Text)

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FDA U.S. FOOD & DRUG ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K242877

B Applicant

Applied BioCode, Inc.

C Proprietary and Established Names

BioCode Gastrointestinal Pathogen Panel (GPP)

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 |

## II Submission/Device Overview:

### A Purpose for Submission:

To obtain a substantial equivalence determination for the BioCode GPP for the detection of microbial nucleic acids extracted from human stool specimens for use on the Applied BioCode MDx 3000 instrument, using an alternate sample extraction system, the KingFisher Flex.

### B Measurand:

Target nucleic acid sequences of the following gastrointestinal microorganisms: 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. hominis), Entamoeba histolytica,

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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Giardia lamblia (also known as G. intestinalis and G. duodenalis), Adenovirus F 40/41, Norovirus GI/GII and Rotavirus A.

## C Type of Test:
Qualitative multiplexed nucleic acid-based test

## III Intended Use/Indications for Use:

### A Intended Use(s):
See Indications for Use below.

### B Indication(s) for 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. hominis)
- Entamoeba histolytica
- Giardia lamblia (also known as G. intestinalis and G. duodenalis)
- Adenovirus F 40/41

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- 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 this setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease. Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor or guide treatment for *C. difficile* infection.

Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Adenovirus 40/41, *Campylobacter*, *E. coli* O157, *Shigella*/EIEC, *Yersinia enterocolitica*, and *Giardia lamblia* were established additionally with retrospective clinical specimens. Performance characteristics for *Entamoeba histolytica*, *Giardia lamblia*, *Yersinia enterocolitica* and *Vibrio* (*V. parahaemolyticus*, *V. vulnificus*, and *V. cholerae*) were established primarily using contrived clinical specimens.

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

Rx - For Prescription Use Only

## D Special Instrument Requirements:

For use on the MDx 3000 instrument

## IV Device/System Characteristics:

### A 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 BioMérieux NucliSENS easyMAG, Roche MagNA Pure 96, or Thermo Fisher KingFisher Flex. 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 from unpreserved stool specimens or stool collected in Cary-Blair transport medium. Results from the BioCode GPP test are available within less than 4 hours.

### B Principle of Operation:

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The principle of operation of the BioCode MDx 3000 remains unchanged from the initial clearance in K180041. Refer to the published decision summary for additional information. 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 are extracted with either the BioMérieux NucliSENS easyMAG, Roche MagNA Pure 96, or Thermo Fisher KingFisher Flex automated systems. Once the PCR plate is set up and sealed, all other operations are automated on the MDx 3000.

The BioCode MDx 3000 Software controls the operation of the instrument, collects and analyzes data, and automatically generates interpretation for test reports at the end of the run. Fluorescent signals from BMBs with the same barcode are sorted and calculated to generate median fluorescence index (MFI) for each analyte. The presence or absence of a pathogen is determined relative to the validated assay cutoff by MFI. The software also analyzes the results of external and internal controls to validate the run and individual specimen results for reporting.

V Substantial Equivalence Information:

A Predicate Device Name(s):

BioCode Gastrointestinal Pathogen Panel (GPP), Biocode Gastrointestinal Pathogen Panel (GPP)

B Predicate 510(k) Number(s):

K190585

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K242877 | K190585 |
| --- | --- | --- |
| Device Trade Name | BioCode Gastrointestinal Pathogen Panel (GPP) | BioCode Gastrointestinal Pathogen Panel (GPP) |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For 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 | 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 |

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| | 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. 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. | 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. 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 O157, Shigella/EIEC, Yersinia enterocolitica, and Giardia lamblia were established additionally with retrospective clinical specimens. Performance characteristics for Entamoeba histolytica, Giardia lamblia, Yersinia enterocolitica and Vibrio (V. parahaemolyticus, V. vulnificus, and V. cholerae) were established primarily using contrived clinical specimens. | Positive results do not rule out co-infection with organisms not included in the BioCode GPP. The agent detected may not be the definite cause of the disease. Negative results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn’s disease. Concomitant culture is necessary for organism recovery and further typing of bacterial agents. This device is not intended to monitor or guide treatment for C. difficile infection.

Due to the small number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Adenovirus 40/41, Campylobacter, E. coli O157, Shigella/EIEC, Yersinia enterocolitica, and Giardia lamblia were established additionally with retrospective clinical specimens. Performance characteristics for Entamoeba histolytica, Giardia lamblia, Yersinia enterocolitica and Vibrio (V. parahaemolyticus, V. vulnificus, and V. cholerae) were established primarily using contrived clinical specimens. |
| --- | --- | --- |
| Instrument | 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 | Same |

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| | beads (BMB) that are coupled to target-specific probes | |
| --- | --- | --- |
| Calibrators | Internal Calibration | Same |
| General Device Characteristic Differences | | |
| Sample Extraction | easyMAG, Roche MagNA Pure 96, KingFisher Flex | easyMAG, Roche MagNA Pure 96 |

VI Standards/Guidance Documents Referenced:

CLSI EP09c 3rd Edition, Measurement Procedure Comparison and Bias Estimation Using Patient Samples

CLSI EP12-A2, User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline - Second Edition

CLSI EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition

VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Precision/Reproducibility:

A reproducibility study was conducted to evaluate variability associated with extraction of samples with the KingFisher Flex system. 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 two instruments by three operators. Testing was performed including two runs per day per operator for five days (a total of 30 runs).

Organisms were diluted directly into 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. The reproducibility panel consisted of 7 contrived samples (sample 7 is a negative control) extracted in triplicate and each assayed in singlet. The samples consisted of combinations of 12 representative targets at 1.5x LoD (Low) and 3x LoD (Medium). Results from the study are shown in Table 1 below.

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Table 1. Reproducibility Study Results with KingFisher Flex

| Organism | Target Probe | Concentration | Expected Results | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Instrument 1 | Instrument 2 | Instrument 3 | All Instruments/ Operators |
| Salmonella enterica ATCC 14028 | Salm | 3X LoD (6.60E+03 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (3.30E+03 CFU/mL) | Detected | 30/30 (100%) | 29/30 (96.7%) | 30/30 (100%) | 89/90 (98.9%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| Clostridium difficile Zeptometrix 0801619 | tcdA | 3X LoD (9.21E+01 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (4.61E+01 CFU/mL) | Detected | 30/30 (100%) | 29/30 (96.7%) | 28/30 (93.3%) | 87/90 (96.7%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| Clostridium difficile Zeptometrix 0801619 | tcdB | 3X LoD (9.21E+01 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (4.61E+01 CFU/mL) | Detected | 30/30 (100%) | 29/30 (96.7%) | 30/30 (100%) | 89/90 (98.9%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| Giardia lamblia Waterborne Inc. P101 | G.lam | 3X LoD (5.40E+03 cysts/mL) | Detected | 30/30 (100%) | 29/30 (96.7%) | 30/30 (100%) | 89/90 (98.9%) |
| | | 1.5X LoD (2.70E+03 cysts/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| Adenovirus 40 Zeptometrix 0810084CF | Adeno | 3X LoD (9.90E-02 TCID50/mL) | Detected | 30/30 (100%) | 29/30 (96.7%) | 30/30 (100%) | 89/90 (98.9%) |
| | | 1.5X LoD (4.95E-02 TCID50/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| Shigella sonnei ATCC 29930 | Shig | 3X LoD (1.32E+03 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (6.60E+02 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| ETEC ATCC 35401 | LT | 3X LoD | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |

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| Organism | Target Probe | Concentration | Expected Results | % Agreement with Expected Result | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Instrument 1 | Instrument 2 | Instrument 3 | All Instruments/ Operators |
| | | (1.68E+03 CFU/mL) | | | | | |
| | | 1.5X LoD (8.42E+02 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| ETEC ATCC 35401 | ST1a | 3X LoD (1.68E+03 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (8.42E+02 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| ETEC ATCC 35401 | ST1b | 3X LoD (1.68E+03 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (8.42E+02 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| Vibrio parahaemolyticus ATCC 17802 | V.para | 3X LoD (3.90E+01 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (1.95E+01 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| Vibrio parahaemolyticus ATCC 17802 | Vib | 3X LoD (3.90E+01 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (1.95E+01 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 147/150 (98.0%) | 150/150 (100%) | 150/150 (100%) | 447/450 (99.3%) |
| Yersinia enterocolitica ATCC 23715 | Yent | 3X LoD (5.01E+02 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | 1.5X LoD (2.51E+02 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |
| | | None | Not Detected | 150/150 (100%) | 150/150 (100%) | 150/150 (100%) | 450/450 (100%) |
| STEC ATCC BAA-2217 | Stx2 | 3X LoD (2.25E+04 CFU/mL) | Detected | 30/30 (100%) | 30/30 (100%) | 30/30 (100%) | 90/90 (100%) |

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

| 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) |

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# 2. Linearity:

Not applicable because the BioCode GPP is a qualitative assay.

# 3. Analytical Specificity/Interference:

Analytical specificity of the BioCode GPP was evaluated in the original $510(\mathrm{k})$ Premarket Notification (K180041). No additional testing was conducted.

# 4. Assay Reportable Range:

Not applicable because the BioCode GPP is a qualitative assay.

# 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

No modifications were made to the BioCode GPP assay or MDx 3000 instrument system. Please refer to the published decision summary for the original $510(\mathrm{k})$ submission for additional information (K180041).

# 6. Detection Limit:

The LoD of BioCode GPP assay was evaluated when the KingFisher Flex instrument was used for nucleic acid extraction. Contrived samples were prepared with the 21 quantified bacteria, virus, or parasite stocks (Table 3) as well as 1 high Norovirus GI-positive and 1 high Norovirus GII-positive clinical specimens. Each stock was serially diluted and extracted to challenge analytical sensitivity of the assay. LoD for each stock was the lowest concentration with $\geq 95\%$ detection of 20 replicates (19 out of 20). LoD were verified for both unpreserved stool and Cary-Blair preserved stool. To minimize the required volume of reagents and negative matrix and operator time, the LoD study was performed using dual analyte spiked samples. Side by side dilutions (initial 10-fold dilution series followed by finer dilutions) of the Norovirus GI and Norovirus GII clinical specimens was performed and assayed to demonstrate the equivalence between the cleared extraction platform EasyMag and KingFisher Flex.

Table 3. Quantitated Stocks for LoD Testing (23 total)

| Species/Strain/Isolate | Source |
| --- | --- |
| Bacteria | |
| E.coli O157:NM CDC 92-3265 | ATCC 700376 |
| Escherichia coli 10C-3114 (STEC) | ATCC BAA-2217 |
| O78:H11 Escherichia coli H10407 (ETEC) | ATCC 35401 |
| O92:H33 Escherichia coli (EAEC) | STEC TW04440 |
| Escherichia coli O29:NM (EIEC) | ATCC 43892 |
| Shigella sonnei | ATCC 29930 |
| Salmonella bongori | SGSC 4900 |
| Salmonella enterica subsp. enterica | ATCC 14028 |
| Campylobacter jejuni subsp. jejuni | ATCC 33292 |
| Campylobacter coli | ATCC 33559 |
| Clostridium difficile (toxinotype 0) | ATCC 9689 |
| Clostridium difficile (toxinotype IIIA+ B+) Nap1 | Zeptometrix 0801619cf |

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Organism stocks were serially diluted in Cary-Blair to 10x final screening concentration and combined with prescreened negative stool or Cary-Blair stool at a 1:10 ratio at the time of extraction. Four (4) replicates of each concentration in unpreserved stool and Cary-Blair stool were extracted on the easyMAG and KingFisher Flex and tested singly with BioCode GPP assay on the BioCode MDx-3000 system to estimate LoD. The LoD was verified by extracting 20 replicates of each sample type with the easyMAG and KingFisher Flex and testing each extracted sample singly for a total of 20 replicates at or near presumptive LoD. Verification testing (20 replicates) was performed with unpreserved stool samples and Cary-Blair samples. Verification testing (20 replicates) was performed with single targets or in combination with other organism(s).

For Norovirus GI and GII targets, high positive clinical specimens were used and side by side dilutions (initial 10-fold dilution series followed by finer dilutions) was performed and assayed. Each dilution was extracted in quadruplicate with the easyMAG and KingFisher Flex and assayed singly. Twenty (20) replicates at the dilution in which all 3 replicates are detected was assayed to confirm the LoD for each of the two systems.

For Norovirus, as shown in Table 6, the LoD of the Norovirus GII in unpreserved stool was equivalent for 3 extraction systems. For Norovirus GI, the KingFisher Flex is 10-fold and 3-fold more sensitive than the easyMAG. For Cary-Blair stool, as shown in Table 7, the LoD of the Norovirus GI was equivalent for the two systems.

Table 4. LoD Study Results Comparing KingFisher Flex and EasyMag Extraction Methods – Unpreserved Stool

| Strain | Source | EasyMag | | KingFisher Flex | |
| --- | --- | --- | --- | --- | --- |
| | | Unpreserved Stool LoD | Detection | Unpreserved Stool LoD | Detection |
| Campylobacter coli | ATCC 33559 | 5.60 x 10^{1} CFU/mL | 20/20 | 5.60 x 10^{1} CFU/mL | 20/20 |
| Campylobacter jejuni spp. jejuni | ATCC 33292 | 2.33 x 10^{2} CFU/mL | 20/20 | 2.33 x 10^{2} CFU/mL | 20/20 |

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| Strain | Source | EasyMag | | KingFisher Flex | |
| --- | --- | --- | --- | --- | --- |
| | | Unpreserved Stool LoD | Detection | Unpreserved Stool LoD | Detection |
| Clostridium difficile(toxinotype 0) | ATCC 9689 | 2.11 x 101CFU/mL | 20/20 | 2.11 x 101CFU/mL | 20/20 |
| Clostridium difficile(toxinotype III;Nap1) | Zeptometrix 0801619cf | 3.07 x 101CFU/mL | 19/20 | 3.07 x 101CFU/mL | 20/20 |
| EnteroaggregativeE. coli O92:H33(EAEC) | STEC TW04440 | 1.40 x 103CFU/mL | 20/20 | 1.40 x 103CFU/mL | 20/20 |
| EnteroinvasiveE. coli O29:NM(EIEC) | ATCC 43892 | 3.60 x 102CFU/mL | 20/20 | 3.60 x 102CFU/mL | 20/20 |
| EnterotoxigenicE. coli O78:H11H10407 (ETEC) | ATCC 35401 | 1.87 x 102CFU/mL | 20/20 | 5.61 x102CFU/mL | 20/20 |
| Salmonella bongori | SGSC 4900 | 1.40 x 103CFU/mL | 20/20 | 4.67 x 102CFU/mL | 19/20 |
| Salmonella enterica ssp. enterica | ATCC 14028 | 7.33 x 102CFU/mL | 20/20 | 2.20 x 103CFU/mL | 19/20 |
| Shiga-like toxin producingE. coli(STEC) | ATCC BAA-2217 | 2.50 x 103CFU/mL | 20/20 | 7.50 x103CFU/mL | 20/20 |
| E. coli O157 | ATCC 700376 | 3.30 x 103CFU/mL | 20/20 | 3.30 x 103CFU/mL | 20/20 |
| Shigella sonnei | ATCC 29930 | 4.40 x 102CFU/mL | 20/20 | 4.40 x 102CFU/mL | 20/20 |
| Vibrio cholerae | ATCC 25870 | 1.81 x 101CFU/mL | 19/20 | 1.81 x 101CFU/mL | 20/20 |
| Vibrio parahaemolyticus | ATCC 17802 | 4.33 x100CFU/mL | 19/20 | 1.31 x101CFU/mL | 20/20 |
| Yersinia enterocolitica* | ATCC 23715 | 1.50 x103CFU/mL | 20/20 | 1.67 x102CFU/mL | 19/20 |
| Cryptosporidium parvum | waterborne P102C | 3.10 x103oocysts/mL | 20/20 | 3.10 x103oocysts/mL | 20/20 |
| Entamoeba histolytica HB-301:NIH | BEI NR-176 | 3.10 x10-1cysts/mL | 20/20 | 3.10 x10-1cysts/mL | 20/20 |
| Giardia intestinalis(aka G. lamblia) | waterborne P101 | 6.00 x102cysts/mL | 20/20 | 1.80 x103cysts/mL | 20/20 |
| Adenovirus 40 (dugan) | Zeptometrix 0810084 | 3.33 x10-2TCID50/mL | 20/20 | 3.33 x10-2TCID50/mL | 19/20 |
| Adenovirus 41 (TAK) | Zeptometrix 0810085 | 8.46 x10-1TCID50/mL | 20/20 | 8.46 x10-1TCID50/mL | 20/20 |
| Rotavirus A | Zeptometrix | 2.19 x102TCID50/mL | 20/20 | 2.19 x102TCID50/mL | 19/20 |
| Rotavirus B | Zeptometrix | 1.00 x102TCID50/mL | 20/20 | 1.00 x102TCID50/mL | 19/20 |
| Vibrio cholerae | ATCC 17802 | 1.50 x103CFU/mL | 20/20 | 1.50 x103CFU/mL | 20/20 |
| Vibrio parahaemolyticus | ATCC 23715 | 1.50 x103CFU/mL | 20/20 | 1.50 x103CFU/mL | 20/20 |

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Table 5. LoD Study Results Comparing KingFisher Flex and EasyMag Extraction Methods - Stool in Cary-Blair

| Strain | Source | EasyMag | | KingFisher Flex | |
| --- | --- | --- | --- | --- | --- |
| | | Cary-Blair Stool LoD | Detection | Cary-Blair Stool LoD | Detection |
| Campylobacter coli | ATCC 33559 | 1.87 x101CFU/mL | 20/20 | 1.87 x101CFU/mL | 20/20 |
| Campylobacter jejuni spp. jejuni | ATCC 33292 | 7.78 x101CFU/mL | 19/20 | 2.33 x102CFU/mL | 20/20 |
| Clostridium difficile(toxinotype 0) | ATCC 9689 | 7.04 x100CFU/mL | 20/20 | 2.11 x101CFU/mL | 20/20 |
| Clostridium difficile(toxinotype III;Nap1) | Zeptometrix 0801619cf | 3.07 x101CFU/mL | 20/20 | 3.07 x101CFU/mL | 19/20 |
| EnteroaggregativeE. coli O92:H33(EAEC)* | STEC TW04440 | 1.56 x102CFU/mL | 20/20 | 1.40 x103CFU/mL | 20/20 |
| EnteroinvasiveE. coli O29:NM(EIEC) | ATCC 43892 | 3.60 x102CFU/mL | 20/20 | 3.60 x102CFU/mL | 20/20 |
| EnterotoxigenicE. coli O78:H11H10407 (ETEC) | ATCC 35401 | 1.87 x102CFU/mL | 20/20 | 5.61 x102CFU/mL | 20/20 |
| Salmonella bongori | SGSC 4900 | 1.40 x103CFU/mL | 20/20 | 1.40 x103CFU/mL | 20/20 |
| Salmonella enterica ssp. enterica | ATCC 14028 | 7.33 x102CFU/mL | 20/20 | 7.33 x102CFU/mL | 20/20 |
| Shiga-like toxin producingE. coli(STEC) | ATCC BAA-2217 | 8.33 x102CFU/mL | 19/20 | 2.50 x103CFU/mL | 19/20 |
| E. coli O157 | ATCC 700376 | 1.10 x103CFU/mL | 19/20 | 3.30 x103CFU/mL | 20/20 |
| Shigella sonnei | ATCC 29930 | 1.47 x102CFU/mL | 19/20 | 4.40 x102CFU/mL | 20/20 |
| Vibrio cholerae | ATCC 25870 | 1.81 x101CFU/mL | 20/20 | 1.81 x101CFU/mL | 20/20 |
| Vibrio parahaemolyticus | ATCC 17802 | 1.30 x101CFU/mL | 20/20 | 1.30 x101CFU/mL | 20/20 |
| Yersinia enterocolitica | ATCC 23715 | 1.50 x103CFU/mL | 20/20 | 5.01 x102CFU/mL | 20/20 |

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| Strain | Source | EasyMag | | KingFisher Flex | |
| --- | --- | --- | --- | --- | --- |
| | | Cary-Blair Stool LoD | Detection | Cary-Blair Stool LoD | Detection |
| Cryptosporidium parvum | waterborne P102C | 3.10 x103oocysts/mL | 20/20 | 1.03 x103oocysts/mL | 19/20 |
| Entamoeba histolytica HB-301:NIH | BEI NR-176 | 1.03 x10-1cysts/mL | 20/20 | 1.03 x10-1cysts/mL | 20/20 |
| Giardia intestinalis (aka G. lamblia) | waterborne P101 | 6.00 x102cysts/mL | 20/20 | 1.80 x103cysts/mL | 20/20 |
| Adenovirus 40 (dugan) | Zeptometrix 0810084 | 3.33 x10-2TCID50/mL | 19/20 | 3.33 x10-2TCID50/mL | 20/20 |
| Adenovirus 41 (TAK) | Zeptometrix 0810085 | 8.46 x10-1TCID50/mL | 20/20 | 2.54 x100TCID50/mL | 20/20 |
| Rotavirus A | Zeptometrix 0810041CF | 2.19 x102TCID50/mL | 19/20 | 7.29 x101TCID50/mL | 20/20 |

Table 6. Norovirus LoD Study Results Comparing KingFisher Flex and EasyMag Extraction Methods – Unpreserved Clinical Specimen

| Target | Source | Target Probe | EasyMag | | KingFisher Flex | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | Unpreserved Stool Dilution | Detection | Unpreserved Stool Dilution | Detection |
| Norovirus GI | Clinical Sample | NoVG1 | 1:30,000 | 19/20 | 1:3,000 | 20/20 |
| Norovirus GII | Clinical Sample | NoVG2 | 1:30,000 | 20/20 | 1:30,000 | 19/20 |

Table 7. Norovirus LoD Study Results Comparing KingFisher Flex and EasyMag Extraction Methods – Cary-Blair Clinical Specimen

| Target | Source | Target Probe | EasyMag | | KingFisher Flex | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | 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 |
| Norovirus GII | Clinical Sample ID#02-0134 | NoVG2 | 1:30,000 | 20/20 | 1:30,000 | 20/20 |

7. Assay Cut-Off:

Assay cut-off remains unchanged from the previously cleared version of the BioCode GPP panel.

B Comparison Studies:

1. Method Comparison with Predicate Device:

To support the performance of the Biocode GPP when used with the KingFisher Flex extraction system, a total of 468 remnant, de-identified clinical specimens (254 frozen unpreserved stool

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and 214 inoculated Cary-Blair stool) were evaluated with both the KingFisher Flex procedure and previously cleared methods employing the easyMAG extraction platform. These remnant samples were used to support the original $510(\mathrm{k})$ clearance of the BioCode GPP device.

Fifty-four (54) frozen unpreserved 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 enlerocolitica and Vibrio spp. (V. parahaemolyticus, V. vulnificus, and V. cholerae). The demographics of the enrolled subjects in the clinical study are shown in Table 8 below.

For each specimen, the results and the validity were determined per the Interpretation Algorithm of the BioCode GPP. The PPA was calculated as $\mathrm{TP} / (\mathrm{TP} + \mathrm{FN})$ . $\mathrm{TP} =$ true positive or positive by both the EasyMag and the Kingfisher Flex; $\mathrm{FN} =$ false negative or negative by the Kingfisher Flex only. The NPA was calculated as $\mathrm{TN} / (\mathrm{TN} + \mathrm{FP})$ . $\mathrm{TN} =$ true negative or negative by the EasyMag and the KingFisher Flex; $\mathrm{FP} =$ false positive or positive by the KingFisher Flex only.

The PPA and NPA of the results between the KingFisher Flex and the easyMAG for individual targets for each sample type (unpreserved stool or Cary-Blair stool) and for the combined sample types plus binomial two-sided $95\%$ confidence interval were calculated. The overall PPA and NPA of the results between the KingFisher Flex and the easyMAG for all targets combined for each sample type (unpreserved stool or Cary-Blair stool) and for the combined sample types plus binomial two-sided $95\%$ confidence interval were calculated. The agreement for samples with co-infections were calculated separately. After discordant analysis, samples that were still invalid were excluded in the agreement calculations. The percentage of invalid samples was calculated for information only.

Table 8. Demographic Information for Clinical Study Subjects

| Archived Samples | |
| --- | --- |
| Total Specimen Count | 468 |
| Gender | |
| Male | 246/468 (52.6%) |
| Female | 222/468 (47.4%) |
| Age Category | |
| ≤ 5 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 Information Subjects with Freshly Collected Specimens (unpreserved)

| 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. | 0/54 (0.00%) |
| 22-59 yrs. | 0/54 (0.00%) |
| 60+yrs | 0/54 (0.00%) |

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Results from testing of clinical samples are summarized in Table 10 below.

Table 10. Summary of Clinical Investigational Study Results (Archived Specimens) Stratified by Sample Type and Storage – Comparison of KingFisher Flex to EasyMAG results

| Target | Specimen Type | (n) | Positive Agreement | | Negative Agreement | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | PPA (%) | 95% CI | NPA (%) | 95% CI |
| Campylobacter spp.a | Inoculated Cary-Blair | 209 | 18/18 (100) | 82.4 – 100 | 191/191 (100) | 98.0 – 100 |
| | Unpreserved (Frozen) | 247 | 25/25 (100) | 86.7 – 100 | 219/222 (98.6) | 96.1 – 99.5 |
| | 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 |
| | Unpreserved (Frozen) | 247 | 31/33 (93.9) | 80.4 – 98.3 | 213/214 (99.5) | 97.4 – 99.9 |
| | 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 |
| | Unpreserved (Frozen) | 246 | 8/8 (100) | 67.6 – 100 | 238/238 (100) | 98.4 – 100 |
| | All Archived | 455 | 13/13 (100) | 77.2 – 100 | 442/442 (100) | 99.1 – 100 |
| 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 |

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| Target | Specimen Type | (n) | Positive Agreement | | Negative Agreement | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | PPA (%) | 95% CI | NPA (%) | 95% CI |
| | 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/ EIECh | 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 parahaemolyticusi | 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 parahaemolyticusi)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 |
| Yersinia enterocoliticak | 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.l | 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 |
| Entamoebahistolyticam | 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 |

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| Target | Specimen Type | (n) | Positive Agreement | | Negative Agreement | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | PPA (%) | 95% CI | NPA (%) | 95% CI |
| Giardia lamblia^{a} | 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 40/41^{e} | 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 |
| 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 |

*Positive agreement <90%.
Thirty-five archived samples with discordant results (between the easyMAG and KingFisher Flex were retested twice with both easyMAG and KingFisher Flex.
$^{a}$ Campylobacter spp. One false positive continued to be positive whereas two false positives became negative by KingFisher Flex upon retesting. Twelve samples were still invalid upon retesting and not used in the agreement calculation.
$^{b}$ Clostridium difficile. Two false positives were still positive upon retesting. Of the four false negatives, three were negative by EasyMag upon retesting whereas one remained positive. Ten samples were invalid, not used in the agreement calculation.
$^{c}$ E. coli O157: Thirteen samples were invalid, not used in the agreement calculation.
$^{d}$ EAEC: Of the two false negatives, one was negative by EasyMag and one remained positive upon retesting. Twelve samples were invalid, not used in the agreement calculation.
$^{e}$ ETEC: Thirteen samples were invalid, not used in the agreement calculation.
$^{f}$ STEC: Two false negatives were Negative by EasyMag upon retesting. One false positive was negative by KingFisher and positive by EasyMag upon retesting. Twelve samples were still invalid, not used in the agreement calculation.
$^{g}$ Salmonella spp. Of the two false positives, one was negative by KingFisher Flex and one remained positive upon retesting. Thirteen samples were invalid, not used in the agreement calculation.
$^{h}$ Shigella/EIEC. The false positive became positive by EasyMag upon retesting. Twelve samples were invalid, not used in the agreement calculation.
$^{i}$ Vibrio parahaemolyticus: Thirteen samples were invalid, not used in the agreement calculation.

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$^{1}$ Vibrio spp. One false positive was negative by KingFisher Flex upon retesting. For the false negative retested, the easyMAG had an invalid result with the 2nd run and a negative result with the 3rd run, whereas the KingFisher Flex had negative results with both runs. Thirteen samples were invalid, not used in the agreement calculation.

$^{k}$ Yersinia enterocolitica: Of the false positives, one was negative by EasyMag upon retesting, and one remained false positive upon retesting. Thirteen samples were invalid, not used in the agreement calculation.

$^{l}$ Cryptosporidium spp: Eleven samples were invalid, not used in the agreement calculation.

$^{m}$ Entamoeba histolytica: Thirteen samples were invalid, not used in the agreement calculation.

$^{n}$ Giardia lamblia: Thirteen samples were invalid, not used in the agreement calculation.

$^{o}$ Adenovirus 40/41: The false negative was negative by EasyMag after retesting and the false positive was positive by EasyMag upon retesting, respectively. Thirteen samples were invalid, not used in the agreement calculation.

$^{p}$ Norovirus GI/GII: The false positive was negative on the KingFisher Flex upon retesting and the false negative was positive by the KingFisher Flex upon retesting. Eleven samples were invalid, not used in the agreement calculation.

$^{q}$ Rotavirus: Of the ten false positives, six were negatives by the KingFisher Flex upon retesting and four remained false positives upon retesting. Twelve samples were invalid, not used in the agreement calculation.

To supplement testing of archived samples, additional testing of contrived specimens were evaluated for select analytes. Results of this additional testing are shown in Table 12 below.

Table 11. Summary of Contrived Specimen Results with the KingFisher Flex

| Target | Specimen Type | (n) | Positive Agreement | | Negative Agreement | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | PPA (%) | 95% CI | NPA (%) | 95% CI |
| Vibrio parahaemolyticus | Unpreserved (Frozen) | 115 | 26/28 (92.9%) | 77.4-98.0% | 86/87 (98.9%) | 93.8-99.8% |
| Vibrio spp. (not parahaemolyticus) | Unpreserved (Frozen) | 115 | 29/30 (96.7%) | 83.3-99.4% | 82/85 (96.5%) | 90.1-98.8% |
| Yersinia enterocolitica a | Unpreserved (Frozen) | 116 | 28/28 (100%) | 87.9-100.0% | 88/88 (100%) | 95.8%-100.0% |
| Entamoeba histolytica b | Unpreserved (Frozen) | 116 | 28/28 (100%) | 87.9-100.0% | 86/88 (97.7%) | 92.1-99.4% |
| All other targets c | Unpreserved (Frozen) | 1491 | N/A | N/A | 1491/1491 (100%) | 99.7-100.0% |
| Combined targets | Unpreserved (Frozen) | 1953 | 111/114 (97.4%) | 92.5 - 99.1% | 1833/1839 (99.7%) | 99.3 - 99.9% |

Note. One hundred twenty contrived samples were tested. Five 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 invalids, one of the E. histolytica positives was had a positive assay result for E. histolytica but the RNA internal control was invalid.
$^{b}$ Of the five invalids, one had a positive assay result for Yersinia enterocolitica, but the RNA internal control was invalid.
One of the negative stools being used as matrix for contriving samples exhibited weak positivity for 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.

2. Matrix Comparison: Not applicable.

C Clinical Studies:

1. Clinical Sensitivity:

Not applicable.

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{20}

2. Clinical Specificity:
Not applicable.

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable): None

D. Clinical Cut-Off: Not applicable

E. Expected Values/Reference Range:
Refer to the published decision summary for the original clearance in K180041.

VIII. Proposed Labeling:
The labeling supports the finding of substantial equivalence for this device.

IX. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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