XTAG GASTROINTESTINAL PATHOGEN PANEL (GPP)/XTAG DATA ANALYSIS SOFTWARE FOR GPP (TDAS GPP)

K140377 · Luminex Molecular Diagnostics, Inc. · PCH · Sep 16, 2014 · Microbiology

Device Facts

• Record ID: K140377
• Device Name: XTAG GASTROINTESTINAL PATHOGEN PANEL (GPP)/XTAG DATA ANALYSIS SOFTWARE FOR GPP (TDAS GPP)
• Applicant: Luminex Molecular Diagnostics, Inc.
• Product Code: PCH · Microbiology
• Decision Date: Sep 16, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3990
• Device Class: Class 2

Indications for Use

The xTAG® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple viral, bacterial, and parasitic nucleic acids in human stool specimens or human stool in Cary-Blair media from individuals with signs and symptoms of infectious colitis or gastroenteritis. The following pathogen types, subtypes and toxin genes are identified using the xTAG GPP: Viruses - Adenovirus 40/41 - Norovirus GI/GII - Rotavirus A Bacteria - Campylobacter (C. jejuni, C. coli and C. lari only) - Clostridium difficile (C. difficile) toxin A/B - Escherichia coli (E. coli) O157 - Enterotoxigenic Escherichia coli (ETEC) LT/ST - Salmonella - Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2 - Shigella (S. boydii, S. sonnei, S. flexneri and S. dysenteriae) - Vibrio cholerae (V. cholerae) cholera toxin gene (ctx) Parasites - Cryptosporidium (C. parvum and C. hominis only) - Entamoeba histolytica (E. histolytica) - Giardia (G. lamblia only - also known as G. intestinalis and G. duodenalis) The detection and identification of 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, laboratory findings and epidemiological information. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks. xTAG GPP positive results are presumptive and must be confirmed by FDA-cleared tests or other acceptable reference methods. The results of this test 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 xTAG Gastrointestinal Pathogen 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. xTAG GPP is not intended to monitor or guide treatment for C. difficile infections. The xTAG GPP is indicated for use with the Luminex® 100/200™ instrument system with xPONENT® software.

Device Story

The xTAG GPP is a multiplexed RT-PCR assay for qualitative detection of GI pathogens in human stool. Input: 10 µL extracted nucleic acid from stool or Cary-Blair media. Process: Multiplex RT-PCR amplification (58-202 bp amplimers) followed by hybridization to Luminex bead populations coupled with 24-mer anti-tags; signal generated via Streptavidin, R-Phycoerythrin conjugate. Output: Median fluorescence intensity (MFI) values analyzed by TDAS GPP software to generate qualitative results. Used in clinical laboratories by trained personnel. Results aid clinicians in diagnosing GI infections alongside clinical/epidemiological data. Benefits include simultaneous identification of multiple pathogens, facilitating rapid diagnosis and outbreak management.

Clinical Evidence

Clinical performance evaluated in a prospective study (N=1407) across six laboratories. Sensitivity/specificity and positive/negative agreement were calculated against comparator methods (NAAT, sequencing, culture, EIA). For Adenovirus 40/41, sensitivity was 80% (95% CI 37.5-96.4%) and specificity 98.9% (95% CI 98.1-99.3%). E. histolytica and V. cholerae showed high specificity (98.4% and 99.9% respectively). Additional retrospective and contrived sample studies supported performance for these analytes. PCR inhibition rates were monitored.

Technological Characteristics

Multiplex RT-PCR assay using Luminex xTAG Universal Array. Materials: Proprietary 24-mer oligonucleotide tags/anti-tags. Energy: Fluorescence-based detection (Streptavidin, R-Phycoerythrin). Connectivity: Networked via Luminex 100/200 instrument. Software: TDAS GPP (US) for qualitative analysis. Sterilization: N/A (reagents). Form factor: Kit-based reagents for use with Luminex 100/200 platform.

Indications for Use

Indicated for individuals with signs and symptoms of infectious colitis or gastroenteritis. Used for qualitative detection of viral, bacterial, and parasitic nucleic acids in human stool or stool in Cary-Blair media. Aids in diagnosis of gastrointestinal infection and detection of acute gastroenteritis outbreaks.

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

• xTAG Gastrointestinal Pathogen Panel (GPP) (K121454)

Reference Devices

• Luminex 100/200 instrument (K073506)
• xPONENT 4.2 Software for MAGPIX (K121894)
• xPONENT 4.2 software for FLEXMAP 3D (K133302)
• Premier Adenoclone Type 40/41 EIA (K881894)

Submission Summary (Full Text)

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

LUMINEX MOLECULAR DIAGNOSTICS, INC. TINA IP REGULATORY AFFAIRS ASSOCIATE 439 UNIVERSITY AVE. TORONTO, ONTARIO, M5G 1Y8 CANADA

September 16, 2014

#### Re: K140377

Trade/Device Name: XTAG Gastrointestinal Pathogen Panel (GPP) Regulation Number: 21 CFR 866.3990 Regulation Name: Gastrointestinal pathogen panel multiplex nucleic acid-based assay system Regulatory Class: II Product Code: PCH, NSU, JJH Dated: August 13, 2014 Received: August 14, 2014

Dear Ms. Ip:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug. and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA), You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the

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

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

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You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

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Sincerely yours,

Uwe Scherf -S for

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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### Indications for Use

Form Approved: OMB No. 0910-0120

510(k) Number (if known)

#### Device Name

xTAG® Gastrointestinal Pathogen Panel (GPP)

#### Indications for Use (Describe)

The xTAG® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucles squalitative detection and identification of multiple viral, bacterial and parasitic nucleic acids in human stool in Cary-Blair media from individuals with signs and symptoms of infectious of infectious of infectious of infectio or gastroenteritis. The following pathogen types, subtypes and toxin genes are identified using the xTAG GPP.

Viruses

- · Adenovirus 40/41
- · Norovirus GI/GII
- Rotavirus A

#### Bacteria

- · Campylobacter (C. jejuni, C. coli and C. lari only)
- · Clostridium difficile (C. difficile) toxin A/B
- · Escherichia coli (E. coli) O157
- · Enterotoxigenic Escherichia coli (ETEC) LT/ST
- · Salmonella
- · Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2
- · Shigella (S. boydii, S. sonnei, S. flexneri and S. dysenteriae)
- Vibrio cholerae (V. cholerae) cholera toxin gene (ctx)

#### Parasites

- · Cryptosporidium (C. parvum and C. hominis only)
- Entamoeba histolytica (E. histolytica)
- · Giardia (G. lamblia only also known as G. intestinalis and G. duodenalis)

The detection and identification of specific and microbial nucleic acid from individuals exhibiting signs and symptoms of gastrontestinal infection ads in the digencis of gastrontestinal infection when with clinical evaluation, laboratory findings and epidemological information. A gastroinestinal microorganism multiplex nucleic acid-based assay also in the detection and identification of acute gastroenteritis in the context of outbreaks.

#### xTAG GPP positive results are presumptive and must be confirmed by FDA-cleared tests or other acceptable reference methods.

The results of this test should not be used as 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 of patient illness. Negative xTAG Gastronitestinal Pathogen Panel results in the setting of cliness compatible with gastroenteriis may be due to infection by pathogens that are notinfectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

#### xTAG GPP is not intended to monitor or guide treatment for C. difficile infections.

The xTAG GPP is indicated for use with the Luminex® 100/200™ instrument system with xPONENT® software.

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

X | Prescription Use (Part 21 CFR 801 Subpart D)

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

#### PLEASE DO NOT WRITE BELOW THIS LINE - CONTINUE ON A SEPARATE PAGE IF NEEDED.

#### FOR FDA USE ONLY

Concurrence of Center for Devices and Radiological Health (CDRH) (Signature)

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# 510(k) Summary

This 510(k) Summary is being submitted in accordance with the requirements of 21 CFR 807.92.

510(k) Number: K140377

Submission Type: Traditional 510(k), New Device

Measurand: A panel of viruses, bacteria and parasites and toxins including: Adenovirus 40/41, Norovirus GI/GII, Rotavirus A, Campylobacter (C. jejuni, C. coli and C. lari only), Clostridium difficile toxin A/B, Escherichia coli (E. coli) O157, Enterotoxigenic E. coli (ETEC) LT/ST, Salmonella, Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2, Shigella (S. boydii, S. sonnei, S. flexneri and S. dysenteriae), Vibrio cholerae (V. cholera toxin gene (ctx), Cryptosporidium (C. parvum and C. hominis only), Entamoeba histolytica (E. histolytica), Giardia (G. lamblia only, also known as G. intestinalis and G. duodenalis) and the internal control (bacteriophage MS2).

Type of Test: Qualitative nucleic acid multiplex test

Applicant: Luminex Molecular Diagnostics, Inc., Toronto, Ontario, Canada

Proprietary and Established Names: xTAG Gastrointestinal Pathogen Panel (GPP)

| Product<br>Code | Classification | Regulation Section | Review Panel |
|-----------------|----------------|--------------------------------------------------------------------------------------------|-------------------|
| PCH | II | 21CFR866.3990 Gastrointestinal Pathogen Panel<br>Multiplex Nucleic Acid-Based Assay System | Microbiology (83) |
| NSU | II | 21CFR862.2570 Multiplex Instrument System | Microbiology (83) |

#### Regulatory Information:

#### Device Components

| Product | Description |
|-------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| xTAG GPP Kit | Unchanged from k121454 |
| xTAG GPP TDAS (Software CD) | Revised CD, containing data acquisition protocol and data analysis software (updated to include Adenovirus 40/41, V. cholerae and E. histolytica) |
| Luminex® 100/200TM instrument | Unchanged from k121454 and originally cleared in k073506 |
| xPONENT® Software | xPONENT Software Unchanged from k121454<br>xPONENT 4.2 Software for LX200 (new), very similar to xPONENT 4.2 Software for MAGPIX cleared in k121894 and to xPONENT 4.2 software for FLEXMAP 3D cleared in k133302. |

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#### Intended Use:

The xTAG® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple viral, bacterial, and parasitic nucleic acids in human stool specimens or human stool in Cary-Blair media from individuals with signs and symptoms of infectious colitis or gastroenteritis. The following pathogen types, subtypes and toxin genes are identified using the xTAG GPP:

#### Viruses

- . Adenovirus 40/41
- Norovirus GI/GII ●
- Rotavirus A

#### Bacteria

- Campylobacter (C. jejuni, C. coli and C. lari only) .
- Clostridium difficile (C. difficile) toxin A/B ●
- Escherichia coli (E. coli) 0157
- Enterotoxigenic Escherichia coli (ETEC) LT/ST
- . Salmonella
- Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2 ●
- Shigella (S. boydii, S. sonnei, S. flexneri and S. dysenteriae)
- Vibrio cholerae (V. cholerae) cholera toxin gene (ctx)

#### Parasites

- Cryptosporidium (C. parvum and C. hominis only) .
- Entamoeba histolytica (E. histolytica) ●
- . Giardia (G. lamblia only - also known as G. intestinalis and G. duodenalis)

The detection and identification of 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, laboratory findings and epidemiological information. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

#### xTAG GPP positive results are presumptive and must be confirmed by FDA-cleared tests or other acceptable reference methods.

The results of this test 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 xTAG Gastrointestinal Pathogen 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.

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#### xTAG GPP is not intended to monitor or guide treatment for C. difficile infections.

The xTAG GPP is indicated for use with the Luminex® 100/200™ instrument system with xPONENT® software.

Indication(s) for use: Same as intended use.

Special instrument requirements: Luminex 100/200 instrument with xPONENT software.

#### Device Description:

xTAG GPP incorporates a multiplex reverse-transcription polymerase chain reaction (RT-PCR) with Luminex's proprietary universal sorting system (the xTAG Universal Array) on the Luminex platform. The xTAG Universal Array sorts nucleic acids onto discreet Luminex bead populations by virtue of highly specific "tag/anti-tag" hybridization reactions. The tags and anti-tags comprising the xTAG Universal Array are 24-mer oligonucleotide sequences not found in nature. The assay has been designed to simultaneously detect microbial targets and an internal control (bacteriophage MS2 added to each sample prior to extraction).

For each sample, 10 µL of extracted nucleic acid is amplified in a single multiplex RT-PCR reaction. Amplimers ranging from 58 to 202 bp (not including the 24-mer tag) are generated in this reaction. A five μL aliquot of the RT-PCR product is then subjected to a hybridization/detection reaction that also includes bead populations coupled to 24-mer antitags. Each bead population is coupled to a unique anti-tag which is the exact complement of a 24-mer tag incorporated into a given amplimer. Thus, each Luminex bead population uniquely identifies a microbial target or assay control through a specific tag/anti-tag hybridization reaction. Signal is generated via a Streptavidin, R-Phycoerythrin conjugate.

The Luminex instrument sorts the products of these hybridization reactions and generates a signal in the form of a median fluorescence intensity (MFI) value for each bead population. The MFI values are generated by the xPONENT software provided with the instrument using the GPP protocol parameters, and are analyzed by the xTAG Data Analysis Software (TDAS GPP (US)). TDAS GPP (US) applies algorithms to MFI values in order to generate a qualitative result for each microbial target selected for reporting to establish the presence or absence of bacterial, viral or parasitic targets and/or controls in each sample. The data analysis software also generates a qualitative result and compiles a report for patient samples and external controls assayed in a given run. Before data are analyzed, a user has the option to select a subset of the targets from the intended use of the xTAG GPP (for each sample).

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#### Substantial Equivalence Information:

| Item | New Device (k140377) xTAG GPP | Predicate (k121454) xTAG GPP |
|------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Manufacturer (Same) | Luminex Molecular Diagnostics | Luminex Molecular Diagnostics |
| Extraction Method (Same) | bioMérieux NucliSENS® easyMAG® | bioMérieux NucliSENS easyMAG |
| Test Principle and<br>Amplification Method<br>(Same) | Multiplex end point RT-PCR | Multiplex end point RT-PCR |
| Kit Reagents (Same) | xTAG GPP Primer Mix, xTAG OneStep<br>Enzyme Mix, xTAG OneStep Buffer,<br>xTAG RNase-Free Water, xTAG BSA,<br>xTAG MS2, xTAG GPP Bead Mix, xTAG<br>Reporter Buffer, xTAG 0.22 SAPE | xTAG GPP Primer Mix, xTAG OneStep<br>Enzyme Mix, xTAG OneStep Buffer,<br>xTAG RNase-Free Water, xTAG BSA,<br>xTAG MS2, xTAG GPP Bead Mix, xTAG<br>Reporter Buffer, xTAG 0.22 SAPE |
| Test Format (Same) | Multiplex MAGPLEX bead-based<br>universal array | Multiplex MAGPLEX bead-based<br>universal array |
| Detection Method (Same) | Fluorescence based | Fluorescence based |
| Quality Control (Same) | Internal Control (MS2), rotating analyte<br>controls and negative control (RNAse-<br>free water) | Internal Control (MS2), rotating<br>analyte controls and negative control<br>(RNAse-free water) |
| Results (Same) | Qualitative | Qualitative |
| Instrument Software<br>System | Luminex 100/200 with xPONENT<br>Software | Luminex 100/200 with xPONENT<br>Software |

#### Table 2: Differences between New Device and Predicate

| ltem | New Device (k140377) xTAG GPP | Predicate (k121454) xTAG GPP |
|----------|-----------------------------------------------------|----------------------------------------------------------------------------------------------------|
| Specimen | Human stool specimens and human stool in | Human stool specimens |
| Types | Cary-Blair media | |
| Software | Updated assay protocol to acquire and show | Assay protocol file excludes analytes |
| | data for additional 3 analytes: Adenovirus | Adenovirus 40/41, Entamoeba histolytica (E. |
| | 40/41, Entamoeba histolytica (E. histolytica), | histolytica), and Vibrio cholerae (V. cholerae) |
| | and Vibrio cholerae (V. cholerae). | |
| | xPONENT 3.1 software and higher | xPONENT 3.1 software |
| Intended | See above. Addition of sample type human | The xTAG Gastrointestinal Pathogen Panel |
| Use | stool in Cary-Blair media and addition of | (GPP) is a multiplexed nucleic acid test |
| | analytes Adenovirus 40/41, Entamoeba | intended for the simultaneous qualitative |
| | histolytica (E. histolytica), and Vibrio cholerae | detection and identification of multiple viral, |
| | (V. cholerae) cholera toxin gene (ctx). Specified | parasitic, and bacterial nucleic acids in human |
| | software used with Luminex 100/200 | stool specimens from individuals with signs |
| | instrument. Organized analytes listed under | and symptoms of infectious colitis or |
| | sub-heading of viruses, bacteria and parasites. | gastroenteritis. The following pathogen types, |
| | | subtypes and toxin genes are identified using |
| | | the xTAG GPP: |
| | | • Campylobacter (C. jejuni, C. coli and C. Iari<br>only) |
| | | • Clostridium difficile (C. difficile) toxin A/B |
| | | • Cryptosporidium (C. parvum and C. hominis |
| | | only) |
| | | • Escherichia coli (E. coli) O157 |
| | | • Enterotoxigenic Escherichia coli (ETEC) |
| | | LT/ST |
| | | · Giardia (G. lamblia only - also known as G. |
| ltem | New Device (k140377) xTAG GPP | Predicate (k121454) xTAG GPP |
| | | intestinalis and G. duodenalis) |
| | | • Norovirus GI/Gll |
| | | • Rotavirus A |
| | | • Salmonella |
| | | • Shiga-like Toxin producing E. coli (STEC) stx |
| | | 1/stx 2 |
| | | • Shiqella (S. boydii, S. sonnei, S. flexneri and |
| | | S. dysenteriae) |
| | | The detection and identification of 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, laboratory |
| | | findings and epidemiological information. A |
| | | gastrointestinal microorganism multiplex |
| | | nucleic acid-based assay also aids in the |
| | | detection and identification of acute |
| | | gastroenteritis in the context of outbreaks. |
| | | xTAG GPP positive results are presumptive |
| | | and must be confirmed by FDA-cleared tests |
| | | or other acceptable reference methods. |
| | | The results of this test 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<br>or definitive cause of patient illness. Negative |
| | | xTAG Gastrointestinal Pathogen 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. |
| | | xTAG GPP is not intended to monitor or guide |
| | | treatment for C. difficile infections. |
| | | The xTAG GPP is indicated for use with the |
| | | Luminex 100/200 instrument. |
| Targets | Adenovirus 40/41, Campylobacter (C. jejuni, C. | Campylobacter (C. jejuni, C. coli and C. lari |
| Reported | coli and C. lari only), Clostridium difficile (C. | only), Clostridium difficile (C. difficile) toxin |
| | difficile) toxin A/B, Cryptosporidium (C. parvum | A/B, Cryptosporidium (C. parvum and C. |
| | and C. hominis only), Escherichia coli (E. coli) | hominis only), Escherichia coli (E. coli) O157, |
| | O157, Enterotoxigenic Escherichia coli (ETEC) | Enterotoxigenic Escherichia coli (ETEC) LT/ST, |
| | LT/ST, Entamoeba histolytica (E. histolytica), | Giardia (G. lamblia only - also known as G. |
| | Giardia (G. lamblia only - also known as G. | intestinalis and G. duodenalis), Norovirus |
| | intestinalis and G. duodenalis), Norovirus GI/GII, | GI/GII, Rotavirus A, Salmonella, Shiga-like |
| | Rotavirus A, Salmonella, Shiga-like Toxin | Toxin producing E. coli (STEC) stx 1/stx 2, |
| | producing E. coli (STEC) stx 1/stx 2, Shigella (S. | Shigella (S. boydii, S. sonnei, S. flexneri and S. |
| | boydii, S. sonnei, S. flexneri and S. dysenteriae), | dysenteriae) |
| | Vibrio cholerae (V. cholerae) cholera toxin gene | |
| | (ctx) | |

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#### xTAG® GPP with Luminex® 100/200™ Traditional 510(k) Submission

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#### Standards/Guidance Documents referenced (if applicable):

Table 3: Guidance Documents

| | Title | Date |
|----|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------|
| 1 | Establishing the Performance Characteristics of In Vitro Diagnostic<br>Devices for the Detection of Clostridium difficile | Nov. 29, 2010 |
| 2 | Class II Special Controls Guidance Document: Norovirus Serological<br>Reagents | Mar. 9, 2012 |
| 3 | Class II Special Controls Guidance Document: Instrumentation for<br>Clinical Multiplex Test Systems - Guidance for Industry and FDA Staff | Mar. 10, 2005 |
| 4 | Guidance for the Content of Premarket Submissions for Software<br>Contained in Medical Devices | May 11, 2005 |
| 5 | Guidance document for Format for Traditional and Abbreviated 510(k)s | Aug. 12, 2005 |
| 6 | Guidance on the CDRH Premarket Notification Review Program, 510(k)<br>Memorandum #K86-3 | June 30, 1986 |
| 7 | The New 510(k) Paradigm - Alternate Approaches to Demonstrating<br>Substantial Equivalence in Premarket Notifications - Final Guidance | Mar. 20, 1998 |
| 8 | The 510(k) Program: Evaluating Substantial Equivalence in Premarket<br>Notifications [510(k)] | Dec. 27, 2011 |
| 9 | Guidance for Industry and Food and Drug Administration Staff - eCopy<br>Program for Medical Device Submissions | Oct. 10, 2013 |
| 10 | Guidance for Industry and Food and Drug Administration Staff - FDA and<br>Industry Actions on Premarket Notification (510(k)) Submissions: Effect<br>on FDA Review Clock and Goals | Oct. 15, 2012 |

#### Table 4: Standards

| | Standard<br>No. | Recognition<br>Number<br>(FDA) | Standards Title | Date |
|---|-----------------|--------------------------------|------------------------------------------------------------------------------------------------------------------------------------|------------|
| । | EP05-A2 | 7-110 | Evaluation of Precision Performance of Quantitative<br>measurement Methods (2nd ed.) | 10/31/2005 |
| 2 | EP07-A2 | 7-127 | Interference Testing in Clinical Chemistry (2nd<br>edition) | 05/21/2007 |
| ന | EP12-A2 | 7-152 | User Protocol for Evaluation f Qualitative Test<br>Performance (2nd edition) | 09/09/2008 |
| 4 | EP14-A2 | 7-143 | Evaluation of Matrix Effects (2nd edition) | 03/16/2012 |
| ട | EP15-A2 | 7-153 | User Verification of Performance for Precision and<br>Trueness (2nd edition) | 09/09/2008 |
| ട | EP17-A | 7-194 | Protocol for Determination of Limits of Detection<br>and Limits of Quantitation<br>(NOTE: Original studies included this standard) | 03/28/2009 |
| 7 | EP17-A2 | 7-233 | Evaluation of Detection Capability for Clinical<br>Laboratory Measurement Procedures | 01/15/2013 |
| 8 | ISO 14971 | 5-40 | Application of Risk Management to Medical Devices | 08/20/2012 |
| த | MM03-A2 | 7-132 | Molecular Diagnostic Methods for Infectious<br>Diseases (2nd edition) | 09/09/2008 |

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Image /page/10/Picture/0 description: The image shows the Luminex logo. The word "Luminex" is written in a bold, sans-serif font, with the letters in black. There is a red dot above the "i" in Luminex. The logo is simple and modern.

xTAG® GPP with Luminex® 100/200™ Traditional 510(k) Submission

| Standard<br>No. | Recognition<br>Number<br>(FDA) | Standards Title | Date | |
|-----------------|--------------------------------|-----------------|----------------------------------------------------------------|------------|
| 10 | MM13-A | 7-191 | Collection, Transport, Preparation and Storage of<br>Specimens | 03/18/2009 |

# Analytical Performance:

The reagents tested in submission k121454 remain the same as the reagents used in testing performed towards this submission. Therefore, the study reports and results presented in the submission summary in k121454 for Analytical Reactivity, Carry-Over Contamination, Limit of Detection, Repeatability, Analytical Specificity (including Interference), Evaluation of Fresh vs. Frozen Stool, and Reproducibility / Precision are all still applicable to the new device. Results presented below for each of these studies are additive to results previously presented in k121454 and include results for Adenovirus 40/41, Entamoeba histolytica (E. histolytica), and Vibrio cholerae (V. cholerae) cholera toxin gene (ctx). This section of the summary includes updated results for these three analytes for the following studies:

- 1. Analytical Reactivity
- 2. Carry-over Contamination
- 3. Limit of Detection
- 4. Repeatability
- 5. Analytical Specificity and Interference
- 6. Evaluation of Fresh vs. Frozen Stool
- 7. Reproducibility / Precision

Additionally, a study demonstrating results of testing analytes in stool as compared to stool in Cary-Blair media is presented, at the limit of detection for each analyte to demonstrate that either sample type can be used with xTAG GPP.

Finally, a summary of negative control failures and sample re-run rates for analytical performance studies is provided.

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#### Analytical Reactivity

Analytical reactivity was assessed through empirical testing of a wide range of clinically relevant GI pathogen strains, genotypes and isolates representing temporal and geographical diversity for each analyte. Through testing of unique samples covering the additional intended use pathogens, reactivity was established at concentrations 2 to 3 times the limit of detection.

Adenovirus - The Limit of Detection (LoD) using Adenovirus 40, Zeptometrix 0810084CF (Dugan) and Adenovirus 41, Zeptometrix 0810085CF (Tak) were found to be 1.45E+01 TCID55/mL (or 4.89E+06 Copies/mL) and 7.69E+00 TCIDEg/mL (or 1.48E+07 Copies/mL), respectively (see LoD section below). The following two samples were tested at the Centers for Disease Control and Prevention (CDC) (Atlanta, Georgia, USA). Note: these samples were different isolates of the strains used in the LoD study (See LoD section below). The amount of the viral target DNA for GP-093 and GP-094 was measured by real-time PCR and the Ct values generated were used to calculate the DNA copy number. The lowest reactivity titers for GP-093 and GP-094, were found to be at 3x and 1x multiple of LoD level, respectively.

| Run Batch ID | Target | Source ID | Strain or Serotype | Reactivity<br>Titre<br>(Copies/mL) | Results Summary |
|--------------------------------|------------------|--------------|-----------------------------|------------------------------------|-----------------|
| Analytical reactivity_II_LX200 | Adenovirus<br>40 | CDC – GP-093 | Dugan<br>pCMK2Gr10, 9/23/91 | 1.49E+07 | POS |
| Analytical reactivity_II_LX200 | Adenovirus<br>41 | CDC – GP-094 | Tak<br>HeLa2Gr10, 9/23/91 | 1.43E+07 | POS |

Table 5: Adenovirus Reactivity List

Furthermore, in sequencing analysis of clinical specimens tested as part of the multi-site clinical study of xTAG GPP, 9 Adenovirus 40 and 28 Adenovirus 41 positive samples were detected by the assay and sequencing.

| Target | Clinical Sample ID |
|---------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Adenovirus 40 | GPP03-092B, GPP03-099B, GPP03-101B, GPP03-102B, GPP03-103B, GPP03-106B, GPP03-109B,<br>GPP03-300B, GPP03-240B |
| Adenovirus 41 | GPP03-001B, GPP03-003B, GPP03-007B, GPP03-013B, GPP03-014B, GPP03-019B, GPP03-020B,<br>GPP03-022B, GPP03-025B, GPP03-026B, GPP03-028B, GPP03-029B, GPP03-033B, GPP03-035B,<br>GPP03-036B, GPP03-037B, GPP03-038B, GPP03-039B, GPP03-048B, GPP03-055B, GPP03-060B,<br>GPP03-095B, GPP03-229B, GPP03-313B, GPP04-159, GPP04-174, GPP02-129, GPP02-192 |

Table 6: Adenovirus Clinical Specimen Positive by the xTAG GPP

Entamoeba histolytica - The LoD for Entamoeba histolytica, ATCC 30890 was found to be 2.88E+01 Cells/mL, equivalent to 4.30E+02 Copies/mL (see LoD section below). For E.histolytica, ATCC 50007, 50481, 50738 and 50454, the titer information expressed in Cells/mL could not be obtained. To standardize the quantification units for all E.histolytica strains, in this Analytical Reactivity study the amount of target DNA was measured by real-time PCR and the Ct values generated were used to calculate the DNA copy numbers. The reactivity titers for most of the

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# Luminex.

strains were in the range of 0.4x to 6.7x multiple of LoD level for E.histolytica. The reactivity titer for ATCC 50738 (Rahman) was found to be 0.2x multiple of LoD level.

| | | Table 7: Entamoeba histolytica Reactivity List | | | |
|--|--|------------------------------------------------|--|--|--|
|--|--|------------------------------------------------|--|--|--|

| Run Batch ID | Target | Source | Strain or Serotype | Reactivity Titre<br>(Cells or<br>Copies/mL) | Results<br>Summary |
|-------------------------------|--------------------------|------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------|--------------------|
| 20120216_JF_GPP_Reactivity_LX | Entamoeba<br>histolytica | ATCC 30015 | (HK-9, colonic biopsy<br>from adult human<br>male with amebic<br>dysentery, Korea);<br>frozen | 2.86E+00 Cells/mL,<br>or 1.82E+02<br>Copies/mL | POS |
| 20120216_JF_GPP_Reactivity_LX | Entamoeba<br>histolytica | ATCC 30190 | (HB-301:NIH, feces<br>from adult human<br>male with amebic<br>dysentery, Burma,<br>1960); test tube | 1.07E+03<br>Copies/mL | POS |
| 20120216_JF_GPP_Reactivity_LX | Entamoeba<br>histolytica | ATCC 30457 | (HU-21:AMC, colonic<br>biopsy from male<br>child with amebic<br>dysentery, Little<br>Rock, AR, 1970); test<br>tube | 1.68E+03<br>Copies/mL | POS |
| 20120216_JF_GPP_Reactivity_LX | Entamoeba<br>histolytica | ATCC 30458 | (200:NIH); frozen | 1.83E+02 Cells/mL,<br>or 2.42E+03<br>Copies/mL | POS |
| 20120216_JF_GPP_Reactivity_LX | Entamoeba<br>histolytica | ATCC 30459 | (HM-1:IMSS [ABRM];<br>feces from adult<br>human male,<br>asymptomatic cyst<br>passer, England,<br>1972); test tube | 1.83E+02 Cells/mL,<br>or 1.10E+03<br>Copies/mL | POS |
| 20120314_JF_GPP_React_LX | Entamoeba<br>histolytica | ATCC 30889 | (H-458:CDC<br>[ATCC30217], feces<br>from human adult<br>female with amebic<br>dysentery, Asia (?),<br>(patient in U.S. for<br>treatment), 1971);<br>test tube | 8.78E+02<br>Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba<br>histolytica | ATCC 30923 | (HU-2:MUSC) | 4.98E+02<br>Copies/mL | POS |
| 20120207_JF_GPP_Reactivity | Entamoeba<br>histolytica | ATCC 30925 | (HU-1:CDC, feces of<br>female child,<br>asymptomatic, sero-<br>negative cyst passer,<br>Cherokee, NC, 1978) | 1.89E+02<br>Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba<br>histolytica | ATCC 50007 | DKB | 2.88E+03<br>Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba<br>histolytica | ATCC 50481 | SD157 | 1.36E+03<br>Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba<br>histolytica | ATCC 50738 | Rahman | 8.90E+01<br>Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba<br>histolytica | ATCC 50454 | HB-301:NIH | 1.08E+03<br>Copies/mL | POS |

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Vibrio cholerae - The LoD using Vibrio cholerae Pacini ATCC 14101 (serovar 0:1) was found to be 2.34E+06 CFU/mL. For this Analytical Reactivity study 3xLoD=7.02E+06 CFU/mL, and this was used for initial reactivity testing. In addition to toxinogenic strains, (i.e. 01 and 0139), the xTAG GPP assay also detects any non:01 Vibrio strains that do express cholera toxin gene, ctx (xTAG GPP Vibrio primers target gene), but not the non:01 strains that may cause clinical symptoms such as diarrhea by expressing a different virulence factor, which is likely the case for sample ATCC 14374 and other non:01 strains in this table. Both non-01 ATCC 25872 and non-01 ATCC 25873 strains, were tested in sequencing assays and confirmed to contain the ctx gene with well conserved xTAG GPP Vibrio cholerae primer binding regions.

| Run Batch ID | Target | Source | Strain or Serotype | Reactivity Titre<br>(CFU/mL) | Results<br>Summary |
|---------------------------------|-----------------------------------------------------|------------|----------------------------------------------------------------------------------------------------------------------------------|------------------------------|--------------------|
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae<br>Pacini | NCTC 30 | Non-O:1, ATCC<br>4735;MARTIN 1 | 6.00E+08 | NEG |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 4714 | Non-O:1, Isolated<br>from pilgrims in El<br>Tor quarantine camp,<br>El Tor 34-D 19 | 6.00E+08 | NEG |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 7260 | O:1, EGYPT 117 | 7.02E+06 | POS |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 11500 | Non-O:1, VL 7050 | 6.00E+08 | NEG |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 11507 | Non-O:1, VL 1941 | 6.00E+08 | NEG |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 11510 | O:1, VL 01211 | 7.02E+06 | POS |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 12945 | 0:139 (Non-O:1<br>(NAG) — reference<br>strain for 0:139<br>serovar | 7.02E+06 | POS |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 12946 | 0:139 (Non-O:1<br>(NAG)) | 7.02E+06 | POS |
| 20120406-JX-AnaReact-Vibrio2-LX | Vibrio cholerae<br>Pacini | ATCC 14033 | O:1, El Tor DO<br>1930;CN 5774;R.<br>Hugh 1092, Serotype<br>Inaba, Non-<br>toxinogenic | 1.50E+08 | NEG |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae<br>asiaticae (Trevisan)<br>Pfeiffer | ATCC 14035 | O:1, Serotype Ogawa<br>[7787] | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae<br>Pacini | ATCC 14101 | O:1, Serotype<br>Ogawa, clinical<br>specimen – human<br>([185754] cholera<br>epidemic circa 1960,<br>Calcutta) Calcutta<br>India | 7.02E+06 | POS |
| 20120406-JX-AnaReact-Vibrio2-LX | Vibrio cholerae<br>Pacini | ATCC 14374 | Non-O:1 (NAG),<br>5035; R. Hugh 1513 | 1.50E+08 | NEG |
| 20120921-MB-VibrioAnalytical-LX | Vibrio cholerae<br>Pacini | ATCC 14730 | Non-O:1 (Serovar<br>O:2), biovar El Tor,<br>Subgroup III of<br>Gardner and<br>Venkatraman, NCTC<br>4711, NANKING | 6.00E+08 | NEG |

| | | Table 8: Vibrio cholerae Reactivity List | | |
|--|--|------------------------------------------|--|--|
|--|--|------------------------------------------|--|--|

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#### xTAG® GPP with Luminex® 100/200™ Traditional 510(k) Submission

| Run Batch ID | Target | Source | Strain or Serotype | Reactivity Titre<br>(CFU/mL) | Results<br>Summary |
|---------------------------------|---------------------------|-------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|--------------------|
| 20120921-MB-VibrioAnalytical-LX | Vibrio cholerae<br>Pacini | ATCC 14731 | Non-O:1, (Serovar O:3), biovar El Tor, Subgroup V of Gardner and Venkatraman, NCTC 4715, El Tor 34-D 23;CN 3426 | 6.00E+08 | NEG |
| 20120921-MB-VibrioAnalytical-LX | Vibrio cholerae<br>Pacini | ATCC 14732 | Non-O:1 (Serovar O:4), biovar El Tor, Subgroup VI of Gardner and Venkatraman, NCTC 4716, KASAULI 73 | 6.00E+08 | NEG |
| 20120921-MB-VibrioAnalytical-LX | Vibrio cholerae<br>Pacini | ATCC 14733 | Non-O:1 (Serovar O:7), biovar El Tor, Subgroup II of Gardner and Venkatraman, NCTC 8042, NANKING 32/124 | 6.00E+08 | NEG |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae<br>Pacini | ATCC 25870 | O:1, Serotype Inaba | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae<br>Pacini | ATCC 25872 | Non-O:1 (NAG), Isolated from a patient with clinical cholera | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae<br>Pacini | ATCC 25873 | Non-O:1 (NAG), Isolated from a patient with clinical cholera | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae<br>Pacini | ATCC 51394 | O:139 (Non-O:1 [NAG]), Cholera patient, Madras, India | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae<br>Pacini | ATCC 51395 | O:139 (non 0:1 [NAG]), clinical specimen - human (cholera patient, Madras, India) | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae | ATCC BAA-<br>2163 | O:1, Isolated from a patient in Artibonite Department, Haiti, October 2010, Serotype Ogawa, Biogroup El Tor cholera toxin positive CDC Isolate 2010 EL-1786 | 7.02E+06 | POS |

Table 9 summarizes the samples reactive with xTAG GPP. Note that in addition to toxinogenic strains, i.e. O1 and O139, xTAG GPP assay detects any non:O1 Vibrio cholerae strains that do express cholera toxin gene (xTAG GPP Vibrio cholerae primers target gene), but not the non:01 strains that may cause clinical symptoms such as diarrhea by expressing a different virulence

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# Luminex.

factor, which is likely the case for ATCC 14374 and other non:01 strains tested in this study. Both non-O1 ATCC 25872 and non-O1 ATCC 25873 strains, were tested in sequencing assays and confirmed to contain the ctx gene with well conserved xTAG GPP Vibrio cholerae primer binding regions. Ten Vibrio cholerae strains that did not react with xTAG GPP assay are listed in Tables 8 and 10.

| Pathogen | ATCC / Other<br>Reference | Pathogen | ATCC / Other<br>Reference |
|---------------|---------------------------|----------------------------------------------------------------------------------------|---------------------------|
| Adenovirus 40 | CDC - GP-093 | Adenovirus 41 | GPP03-095B |
| Adenovirus 40 | GPP03-092B | Adenovirus 41 | GPP03-229B |
| Adenovirus 40 | GPP03-099B | Adenovirus 41 | GPP03-313B |
| Adenovirus 40 | GPP03-101B | Adenovirus 41 | GPP04-159 |
| Adenovirus 40 | GPP03-102B | Adenovirus 41 | GPP04-174 |
| Adenovirus 40 | GPP03-103B | Adenovirus 41 | GPP02-129 |
| Adenovirus 40 | GPP03-106B | Adenovirus 41 | GPP02-192 |
| Adenovirus 40 | GPP03-109B | Entamoeba histolytica | ATCC 30015 |
| Adenovirus 40 | GPP03-240B | Entamoeba histolytica | ATCC 30190 |
| Adenovirus 40 | GPP03-300B | Entamoeba histolytica | ATCC 30457 |
| Adenovirus 41 | CDC - GP-094 | Entamoeba histolytica | ATCC 30458 |
| Adenovirus 41 | GPP03-001B | Entamoeba histolytica | ATCC 30459 |
| Adenovirus 41 | GPP03-003B | Entamoeba histolytica | ATCC 30889 |
| Adenovirus 41 | GPP03-007B | Entamoeba histolytica | ATCC 30923 |
| Adenovirus 41 | GPP03-013B | Entamoeba histolytica | ATCC 30925 |
| Adenovirus 41 | GPP03-014B | Entamoeba histolytica | ATCC 50007 |
| Adenovirus 41 | GPP03-019B | Entamoeba histolytica | ATCC 50481 |
| Adenovirus 41 | GPP03-020B | Entamoeba histolytica | ATCC 50738 |
| Adenovirus 41 | GPP03-022B | Entamoeba histolytica | ATCC 50454 |
| Adenovirus 41 | GPP03-025B | Vibrio cholerae, serovar 0:1 | NCTC 7260 |
| Adenovirus 41 | GPP03-026B | Vibrio cholerae, serovar 0:1 | NCTC 11510 |
| Adenovirus 41 | GPP03-028B | Vibrio cholerae, serovar O:139 (Non-O:1<br>(NAG)) - reference strain for O:139 serovar | NCTC 12945 |
| Adenovirus 41 | GPP03-029B | Vibrio cholerae, serovar O:139 (Non-O:1<br>(NAG)) | NCTC 12946 |
| Adenovirus 41 | GPP03-033B | Vibrio cholerae asiaticae (Trevisan) Pfeiffer,<br>serovar 0:1, serotype Ogawa | ATCC 14035 |
| Adenovirus 41 | GPP03-035B | Vibrio cholerae Pacini, serovar 0:1, Serotype<br>Ogawa | ATCC 14101 |
| Adenovirus 41 | GPP03-036B | Vibrio cholerae Pacini, serovar 0:1, Serotype<br>Inaba | ATCC 25870 |
| Adenovirus 41 | GPP03-037B | Vibrio cholerae Pacini, serovar Non-O:1<br>(NAG) | ATCC 25872 |
| Adenovirus 41 | GPP03-038B | Vibrio cholerae Pacini, serovar Non-O:1<br>(NAG) | ATCC 25873 |
| Adenovirus 41 | GPP03-039B | Vibrio cholerae Pacini, serovar O:139 (Non-<br>O:1 [NAG]) | ATCC 51394 |
| Adenovirus 41 | GPP03-048B | Vibrio cholerae Pacini, serovar O:139 (Non-<br>0:1 [NAG]) | ATCC 51395 |
| Adenovirus 41 | GPP03-055B | Vibrio cholerae, serovar 0:1, serotype<br>Ogawa, biovar El Tor, cholera toxin positive | ATCC BAA-2163 |
| Adenovirus 41 | GPP03-060B | | |

Table 9: Reactivity of Adenovirus 40/41, Entamoeba histolytica and Vibrio cholerae

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| Table 10: Vibrio cholerae strains that did not react with xTAG GPP | | | | |
|--------------------------------------------------------------------|--|--|--|--|
|--------------------------------------------------------------------|--|--|--|--|

| Pathogen | ATCC / Other Reference | Pathogen | ATCC / Other Reference |
|-------------------------------------------------------------------------------------|------------------------|---------------------------------------------------------------------------------------------|------------------------|
| Vibrio cholerae Pacini, Serovar Non-O:1 (NAG) | NCTC 30 | Vibrio cholerae Pacini, Serovar Non-O:1 (NAG) | ATCC 14374 |
| Vibrio cholerae, Serovar Non-O:1 | NCTC 4714 | Vibrio cholerae Pacini, Serovar O:2, biovar El Tor, Subgroup III of Gardner and Venkatraman | ATCC 14730 |
| Vibrio cholerae, Serovar Non-O:1 | NCTC 11500 | Vibrio cholerae Pacini, Serovar O:3, biovar ElTor, Subgroup V of Gardner and Venkatraman | ATCC 14731 |
| Vibrio cholerae, Serovar Non-O:1 | NCTC 11507 | Vibrio cholerae Pacini, Serovar O:4, biovar El Tor; Subgroup VI of Gardner and Venkatraman | ATCC 14732 |
| Vibrio cholerae Pacini, Serovar O1, biotype El Tor, serotype Inaba, non-toxinogenic | ATCC 14033 | Vibrio cholerae Pacini, Serovar O:7, biovar El Tor; Subgroup II of Gardner and Venkatraman | ATCC 14733 |

#### Carry-over Contamination

The likelihood of carry-over contamination events was initially assessed and presented in k121454 by testing 2 representative pathogens (a bacteria and a parasite): C. difficile, and Giardia respectively. In this study, a representative virus (Adenovirus 40) was tested. This analyte was examined in the form of simulated samples prepared at concentrations just below the assay cut-off (High Negative, HN) and well above the assay cut-off (High Positive, HP). The target was examined in a set of 6 independent extractions. Each extraction was assayed in duplicate arranged in a checkerboard manner on a 96-well plate using xTAG GPP. As with the results in k121454 for the representative bacteria (C. difficile) and parasite (Giardia), results with the virus (Adenovirus 40) showed that all 144 high negative samples remained negative when run on the Luminex 100/200 instrument for all three targets (100% HN). In addition, results for Adenovirus 40 showed that all 144 high positive samples remained positive when run on the Luminex 100/200 instrument (100% HP), as with the targets previously tested. Therefore a lack of carryover contamination has been demonstrated.

#### Limit of Detection

As in the original study results presented for k121454, the LoD was assessed by analyzing serial dilutions of simulated samples made from high-titre stocks of commercial strains or high-titre clinical specimens (when commercial strains were not available). All simulated specimens were prepared in negative clinical matrix (stool). The data from serial dilutions were confirmed in at least 20 replicates of the selected dilution for each analyte target. Results of testing for the three additional analytes were as follows:

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| | | Titre (corresponding<br>to the estimated | Average<br>MFI | |
|--------------------------|-----------------------------------------|------------------------------------------|----------------|--------|
| Analyte | Strain ID | LoD) | Value | %CV |
| Adenovirus<br>40/41 | Adenovirus 40, 0810084CF (Dugan) | $1.45E+01$ TCID50/mL | 548 | 34.09% |
| Adenovirus<br>40/41 | Adenovirus 41, 0810085CF (Tak) | $7.69$ TCID50/mL | 360 | 22.04% |
| Entamoeba<br>histolytica | Entamoeba histolytica, 30890 | $2.88E+01$ cells/mL | 883 | 16.96% |
| Vibrio<br>cholerae | Vibrio cholerae, 14101 (Serovar<br>O:1) | $2.34E+06$ CFU/mL | 255 | 23.62% |

#### Table 11: Summary of Limit of Detection (LoD) for Additional Analytes

#### Repeatability

As in the original study results presented for k121454, repeatability was assessed for each target by testing 20 replicates of each of two different analyte concentrations: a very low positive sample (at the LoD) and a moderate positive dilution level (5x-10x above the cut-off MFI). All replicates for each dilution level were examined starting from sample extraction with the bioMérieux NucliSENS easyMAG system followed by xTAG GPP in a single run. For each set of 20 replicates, the same operator performed the testing on the same instrument system, using the same lot of extraction kit and xTAG GPP reagents. Results of testing were as follows:

| Analyte | Dilution Level | Concentration | xTAG GPP<br>Calls | Mean MFI<br>Value | %CV |
|-------------|-------------------|--------------------|-------------------|-------------------|--------|
| Adenovirus | Moderate Positive | 5.80E+01 TCID50/mL | 20 of 20 POS | 1355 | 9.22% |
| 40/41 | Low Positive/LoD | 1.45E+01 TCID50/mL | 20 of 20 POS | 548 | 34.09% |
| Entamoeba | Moderate Positive | 5.76E+01 cells/mL | 20 of 20 POS | 889 | 7.83% |
| histolytica | Low Positive/LoD | 2.88E+01 cells/mL | 20 of 20 POS | 883 | 16.96% |
| Vibrio | Moderate Positive | 4.68E+06 CFU/mL | 20 of 20 POS | 450 | 15.91% |
| cholerae | Low Positive/LoD | 2.34E+06 CFU/mL | 19 of 20 POS | 255 | 23.62% |

Table 12: Assay Repeatability Assessed by Confirmation of Calls

The correct qualitative result was obtained for ≥ 19 of 20 replicates at the low positive level and for 20 of 20 replicates at the moderate positive level for each analyte tested at these concentrations.

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# Lumines

#### Analytical Specificity and Potential Interfering Agents

Analytical specificity was assessed with respect to the following:

- 1. Propensity for cross-reactivity leading to false positive results: Potential cross-reactivity with pathogens (viruses, bacteria and parasites) associated with gastrointestinal (GI) infections that are not probed by the assay. Potential cross-reactivity was also assessed for commensal flora and non-microbial agents. Organisms were tested at high positive titres.
- 2. Propensity for interference leading to false negative results: Potential interference by pathogens (viruses, bacteria and parasites) associated with gastrointestinal (GI) infections that are not probed by the assay. Potential interference by commensal flora was also assessed. Panel analytes were tested at low positive concentrations in the presence of highly concentrated non-panel organisms.
- 3. Propensity for competitive interference leading to false negative results: Potential interference by GI pathogens that are detected by the assay was evaluated by testing one microbial target prepared at a concentration near the assay cut-off (LP) in the presence of a second microbial target prepared at a very high concentration (HP), and vice-versa. The combinations of analytes tested were selected based on the frequency of co-infections reported in the literature.

Results for the 3 categories of testing outlined above were detailed in the decision summary presented for submission k12454.

The following additions relevant to results for the additional 3 analytes are included here:

Two strains of Entamoeba dispar, ATCC PRA-353 and PRA-368, were tested as commensal flora for potential cross-reactivity with xTAG GPP Assay (Table 13), in addition to Entamoeba dispar PRA-260 included in k121454. One of the three E.dispar strains, ATCC PRA-353, tested at 3.0E+05 cells/mL (or over 10" times LoD for E. histolytica) cross-reacted with E.histolytica. Testing at 4fold lower titre (equivalent to 2.6E+03 multiples of E. histolytica LoD) did not produce a falsepositive call. E. histolytica xTAG GPP kit primers were analyzed in silico for cross-reactivity with E.dispar. Two E. dispar sequences were available in Genbank, Z49256 (unknown strain) and AB282661 (strain SAW1734Rc1AR). In addition, three ATCC strains, PRA-260, PRA-353 and PRA-368, were sequenced at Luminex with primers flanking the xTAG GPP kit E. histolytica primer binding region. All five E. dispar sequences were identical in the E. histolytica GPP kit amplicon region. The forward primer was a perfect match to the E. dispor sequences, whereas the reverse primer had multiple mismatches, most notably, a 2-nt contiguous mismatch on the 3' end. These mismatches in the reverse primer would cause a significant decrease in amplification efficiency, and, therefore, result in a negligible risk of obtaining a false-positive xTAG GPP result for E. histolytica.

As the xTAG GPP testing demonstrated, a false-positive call is only possible when E. dispar is present at a very high concentration, 3.0E+05 cells/mL (or over 10* times LoD for E. histolytica) or higher. Testing at 4-fold lower titer (equivalent to 2.6E+03 multiples of E. histolytica LoD) does not produce a false-positive call.

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Table 13: Cross-reactivity of xTAG GPP Assay with non-Panel Organisms (Commensal Flora)

| Commensal Flora | ATCC/Other Reference | Titer Tested | Cross-Reactive Yes<br>(Y) / No (N) |
|------------------|----------------------|--------------------|------------------------------------|
| Entamoeba dispar | ATCC PRA-260 | 6.80E+06 copies/mL | N |
| Entamoeba dispar | ATCC PRA-353 | 3.00E+05 cells/mL | Y |
| Entamoeba dispar | ATCC PRA-353 | 7.50E+04 cells/mL | N |
| Entamoeba dispar | ATCC PRA-368 | 7.00E+04 cells/mL | N |

Astrovirus was used as a representative interfering pathogen associated with gastrointestinal (GI) infections that are not probed by the assay (See Table 14). The xTAG GPP analyte, in this case Adenovirus 40/41, was also run without a second analyte present. No interference was seen.

Non-panel interference with common commensal bacteria, yeast and parasites was evaluated for each target in the xTAG GPP assay. Organisms tested are presented in Table 15 below. Low positive samples of each analyte target in the assay were tested in the presence of a high positive sample of the potential interfering microorganism. All non-panel bacteria…