Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on traditional molecular diagnostic techniques (RT-PCR, hybridization, MFI analysis) and standard statistical analysis of results. There is no mention of AI or ML algorithms being used for data analysis, interpretation, or result generation. The "algorithms" mentioned in the device description refer to standard data processing steps applied to MFI values.

Therapeutic

No.

Explanation: This device is a diagnostic test (in vitro diagnostic device) used to identify pathogens in stool samples, aiding in the diagnosis of gastrointestinal infections. It does not provide any treatment or therapeutic benefit to a patient.

Diagnostic

Yes

Explanation: The "Intended Use/Indications for Use" section explicitly states that the device is "intended for the simultaneous qualitative detection and identification of multiple viral, bacterial, and parasitic nucleic acids in human stool specimens... aids in the diagnosis of gastrointestinal infection." This directly indicates its diagnostic purpose.

SaMD (Software as a Medical Device)

No

The device description clearly states that the xTAG GPP incorporates a multiplex RT-PCR with Luminex's proprietary universal sorting system on the Luminex platform, and is indicated for use with the Luminex® 100/200™ instrument system. This indicates the device relies on specific hardware components for its function, not just software.

IVD (In Vitro Diagnostic)

Based on the provided information, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The intended use explicitly states that the device 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." This describes a test performed in vitro (outside the body) on a human specimen (stool) to provide information for the diagnosis of a disease (gastrointestinal infection).
Device Description: The description details a laboratory-based assay involving nucleic acid extraction, RT-PCR, hybridization with beads, and detection using a Luminex instrument. This is a typical process for an in vitro diagnostic test.
Clinical Performance Studies: The document describes extensive clinical performance studies using human stool specimens from symptomatic and asymptomatic individuals, comparing the device's results to comparator methods. This is a requirement for demonstrating the clinical validity of an IVD.
Predicate Device: The mention of a predicate device (K121454 xTAG GPP) indicates that this device is being compared to a previously cleared IVD, which is a common pathway for regulatory submission of IVDs.

Therefore, the xTAG® Gastrointestinal Pathogen Panel (GPP) clearly fits the definition of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / 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) 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.

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.

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

PCH, NSU, JJH

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).

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

human stool, human stool in Cary-Blair media from individuals with signs and symptoms of infectious colitis or gastroenteritis.

Indicated Patient Age Range

pediatric and adult patients

Intended User / Care Setting

Not Found

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

Not Found

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

Summary of Performance Studies:

Detection in asymptomatic volunteers: 200 clinical stool samples from healthy, asymptomatic donors from various age groups. PCR inhibition was observed in 23 samples (11.5%), and still in 8 samples (4%) after rerunning. Analysis conducted on 192 samples. Comparator method: Sequencing for samples that were positive by xTAG GPP and negative by sequencing.
Detection in symptomatic patients (prospective clinical study in stool specimens): 1407 clinical specimens (stools) prospectively collected between June 2011 and February 2012 from pediatric and adult patients at six (6) independent laboratories (4 in US, 2 in Canada). All specimens were analyzed by reference/comparator at central laboratories independent of xTAG GPP testing sites. Comparator Methods:
- Adenovirus 40/41: Composite comparator consisting of Premier Adenoclone Type 40/41 EIA (Meridian Bioscience, K881894) directly on the stool specimen and Amplification + sequencing directly from clinical specimen using one NAAT+.
- Entamoeba histolytica: Microscopy followed by amplification + sequencing directly from clinical specimens using one NAAT+ (positive specimens by microscopy only).
- Vibrio cholerae: Bacterial culture.
- Other analytes (from K121454): Not explicitly detailed in the provided text but mentioned as applicable from previous submission.
Detection in symptomatic patients (prospective clinical study in Stool in Cary-Blair Media): Remnants of original prospective study samples for which adequate sample was available. Comparator method: Original comparator method test results for all samples in the prospective study.
Pre-selected stool specimens Retrospective Study: 207 archived stool specimens positive by reference/comparator for pathogens of low prevalence in the prospective clinical study, collected at multiple sites in North America, Africa, and Europe. Luminex unable to source Vibrio cholerae positive specimens. Specimens tested at 4 sites (3 external), randomized and blinded. "Negative" designation based on routine algorithms at banking site (e.g., bacterial culture, EIA, microscopy, in-house real time PCR). Confirmatory testing by nucleic acid amplification followed by bi-directional sequencing using analytically validated primers performed on all available specimens, especially those positive by xTAG GPP but not pre-selected at banking site.
Pre-selected stool in Cary-Blair specimens Retrospective Study: Remnants of available pre-selected frozen stool specimens from the original clinical study, mixed proportionally with Cary-Blair medium and tested in a randomized, blinded fashion.
Supplemental Clinical Data (Simulated Stool Specimen Results): Contrived samples made using individual stool matrix spiked with varying levels of pathogen (clinically relevant and LoD challenge). Sample size: 50 for Entamoeba histolytica, 50 for Vibrio cholerae.
Supplemental Clinical Data (Simulated Stool in Cary-Blair Specimen Results): Contrived specimens made from individual negative stool specimens in Cary-Blair prepared at concentrations spanning analytical detection range. Sample size: 50 for Adenovirus 40/41 (25 Adenovirus 40, 25 Adenovirus 41), 50 for Entamoeba histolytica, 50 for Vibrio cholerae.
Supplemental clinical data (Botswana Pediatric Stool Specimens): 313 pediatric stool specimens prospectively collected between February 2011 and January 2012 from symptomatic pediatric patients admitted to two referral hospitals in Botswana, Africa (diarrhea and/or vomiting). Specimens shipped frozen to testing site in Southern Ontario (Canada). Comparator methods:
- Adenovirus 40/41: FDA-cleared EIA (Premier Adenoclone Type 40/41 EIA, Meridian Bioscience, K881894) for all available specimens (N=311).
- Rotavirus, ETEC, Cryptosporidium, Giardia: Nucleic acid amplification followed by bi-directional sequencing for samples positive by xTAG GPP, and a random subset of xTAG GPP negative samples.
- Other analytes (from K121454): Not explicitly detailed in the provided text but mentioned as applicable from previous submission.

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

Analytical Performance: Analytical Reactivity
- Study Type: Empirical testing of GI pathogen strains, genotypes, and isolates.
- Key Results: Reactivity established at 2 to 3 times the limit of detection for new analytes (Adenovirus 40/41, Entamoeba histolytica, Vibrio cholerae).
Analytical Performance: Carry-over Contamination
- Study Type: Testing of simulated samples (High Negative and High Positive) across 6 extractions, assayed in duplicate in a checkerboard manner.
- Sample Size: 144 high negative samples, 144 high positive samples (for Adenovirus 40).
- Key Results: 100% of high negative samples remained negative; 100% of high positive samples remained positive. Lack of carryover contamination demonstrated.
Analytical Performance: Limit of Detection (LoD)
- Study Type: Analysis of serial dilutions of simulated samples in negative clinical matrix (stool). Confirmed with at least 20 replicates for selected dilution.
- Sample Size: At least 20 replicates for each analyte.
- Key Results:
  - Adenovirus 40: LoD 1.45E+01 TCID50/mL.
  - Adenovirus 41: LoD 7.69 TCID50/mL.
  - Entamoeba histolytica: LoD 2.88E+01 cells/mL.
  - Vibrio cholerae: LoD 2.34E+06 CFU/mL.
Analytical Performance: Repeatability
- Study Type: Testing of 20 replicates of two different analyte concentrations (very low positive at LoD, moderate positive 5x-10x above cut-off MFI) starting from extraction.
- Sample Size: 20 replicates per dilution level per analyte.
- Key Results: Correct qualitative result obtained for ≥ 19 of 20 replicates at low positive level and for 20 of 20 replicates at moderate positive level for each analyte.
Analytical Performance: Analytical Specificity and Potential Interfering Agents
- Study Type:
  1. Cross-reactivity: Testing with non-probed GI pathogens, commensal flora, and non-microbial agents at high positive titres.
  2. Interference (false negatives): Testing panel analytes at low positive concentrations in presence of highly concentrated non-panel organisms.
  3. Competitive interference: Testing one microbial target near cut-off with a second at very high concentration.
- Key Results:
  - Entamoeba dispar (ATCC PRA-353) cross-reacted with E. histolytica at 3.0E+05 cells/mL, but not at 4-fold lower titer.
  - No interference seen with Astrovirus (for Adenovirus 40/41).
  - No interference found with common commensal bacteria, yeast, and parasites for Adenovirus, Entamoeba histolytica, and Vibrio cholerae.
  - No competitive interference observed between panel pathogens.
  - In silico analysis: Entamoeba coli not likely to cross-react.
Analytical Performance: Evaluation of Fresh vs. Frozen Stool (Stability)
- Study Type: Assessment of agreement between fresh and frozen samples at different storage conditions and time points (1 month, 3 months).
- Key Results:
  - Adenovirus 40/41 and Vibrio cholerae met 1-month and 3-month stability acceptance criteria for un-extracted, pre-treated, and extracted specimens.
  - Entamoeba histolytica un-extracted specimen stability did not meet 1-month acceptance but met 3-month acceptance criteria; nucleic acid stability met 1-month but not 3-month. Overall, supports 1-month stability for un-extracted and extracted Entamoeba histolytica at -70°C to -80°C.
Analytical Performance: Precision / Reproducibility
- Study Type: Site-to-site reproducibility testing with simulated samples (single and mixed analyte) extracted with NucliSENS easyMAG.
- Sample Size: 90 replicates per sample (3 replicates/run x 5 runs/operator/site x 3 sites).
- Key Results:
  - Single Analyte: For MP samples, 89/90 (99%) to 90/90 (100%) positive calls. For LP samples, 85/90 (94%) to 90/90 (100%) positive calls. For HN samples, 67/90 (74%) to 90/90 (100%) negative calls.
  - Dual Analyte: All HP targets generated positive calls (100%). For LP concentration, 3 of 4 target combinations generated 90/90 (100%) positive calls. 1/90 replicates of C. difficile (HP) / Adenovirus (LP) sample generated a negative call for Adenovirus. Overall, adequate site-to-site reproducibility established.
Analytical Performance: Stool in Cary-Blair Media Limit of Detection Study
- Study Type: Evaluation of LoD equivalency between raw stool and stool in Cary-Blair medium using representative analytes.
- Key Results: Raw stool samples in Cary-Blair media have equivalent limits of detection for C. difficile, Giardia lamblia, and Norovirus GII.
Clinical Performance: Detection in asymptomatic volunteers
- Sample Size: 192 samples (out of 200 collected from healthy, asymptomatic donors).
- Key Results: ≥97% negative percent agreement across all analytes. 12/192 false positives (xTAG GPP positive, sequencing negative) with MFI values close to cut-offs. 2 samples positive for C. difficile by both xTAG GPP and sequencing.
Clinical Performance: Detection in symptomatic patients (prospective clinical study in stool specimens)
- Sample Size: 1407 clinical specimens.
- Key Results:
  - Adenovirus 40/41: Sensitivity 80% (95% CI: 37.5%-96.4%), Specificity 98.9% (95% CI: 98.1%-99.3%). Invalid Rate 16.7%.
  - Entamoeba histolytica: Sensitivity N/A, Specificity 98.4% (95% CI: 97.5%–99%). Invalid Rate 17.0%.
  - Vibrio cholerae: Sensitivity N/A, Specificity 99.9% (95% CI: 99.5%–100%). Invalid Rate 17.0%.
  - Summary for all analytes: Variable sensitivity (e.g., Campylobacter 100%, ETEC 25%), generally high specificity (>95%). Total 98 mixed infections detected (20.2% of positives).
Clinical Performance: Detection in symptomatic patients (prospective clinical study in Stool in Cary-Blair Media)
- Key Results:
  - Comparable sensitivity and specificity metrics to unpreserved stool for most analytes.
  - Adenovirus 40/41: Positive Agreement 40.0% (95% CI: 11.8%-76.9%), Negative Agreement 99.8% (95% CI: 99.3%-99.9%).
  - C. difficile toxin A/B: Positive Agreement 91.6% (95% CI: 84.8%-95.5%), Negative Agreement 92.0% (95% CI: 90.2%-93.4%).
  - Entamoeba histolytica: Specificity 98.4%.
  - Vibrio cholerae: Specificity 99.9%.
  - Clinical sensitivity for Norovirus G/GII and Clostridium difficile toxin A/B met 90% criterion. Low positivity rate in sample set affected other targets.
Other supportive clinical data: Pre-selected stool specimens Retrospective Study
- Sample Size: 207 archived stool specimens.
- Key Results:
  - Adenovirus 40/41: Positive Agreement 100%.
  - Entamoeba histolytica: Positive Agreement 100%.
  - Generally high positive agreements (e.g., Campylobacter 97.6%, ETEC 97.4%, Giardia 93.7%, Rotavirus A 100%, STEC 100%, Shigella 100%). Salmonella 88.89%.
  - Additional 114 positive calls (by xTAG GPP but not pre-selected at banking site) confirmed by PCR/Bi-directional Sequencing for Adenovirus 40/41 (62.5% confirmed positive) and Entamoeba histolytica (12.5% confirmed positive). No Vibrio cholerae additional positives.
  - Detected 71 mixed infections (29.8% of positives), all mixed infection combinations detected by reference/comparator methods were detected by xTAG GPP.
Other supportive clinical data: Pre-selected stool in Cary-Blair specimens Retrospective Study
- Key Results: Overall high Positive percent agreement (e.g., Campylobacter 100%, E. coli O157 100%, Salmonella 100%, Shigella 100%).
Other supportive clinical data: Supplemental Clinical Data (Simulated Stool Specimen Results)
- Key Results:
  - Entamoeba histolytica: Overall Positive Percent Agreement 100% (50/50). Overall Negative Percent Agreement 100% (100/100).
  - Vibrio cholerae: Overall Positive Percent Agreement 98% (49/50). Overall Negative Percent Agreement 100% (100/100).
Other supportive clinical data: Supplemental Clinical Data (Simulated Stool in Cary-Blair Specimen Results)
- Key Results:
  - Adenovirus 40/41: Overall Positive Agreement 100% (50/50).
  - Entamoeba histolytica: Overall Positive Percent Agreement 96% (47/49).
  - Vibrio cholerae: Overall Positive Percent Agreement 100% (50/50).
Other supportive clinical data: Supplemental clinical data (Botswana Pediatric Stool Specimens)
- Sample Size: 313 pediatric stool specimens.
- Key Results:
  - Adenovirus 40/41: Positive Agreement 65.4% (95% CI: 51.8% - 76.8%), Negative Agreement 100% (95% CI: 98.5% - 100%). High positive calls by xTAG GPP related to higher viral titer.
  - Entamoeba Histolytica: Confirmed Positive Rate N/A (0 positives).
  - Vibrio Cholerae: Confirmed Positive Rate N/A (0 positives).
  - Detected 110 mixed infections (38.9% of positives).
Summary of Sample Failure Rates in Clinical Performance Studies:
- Prospective Study: 17.1% PCR Inhibition re-runs, 3.5% PCR Contamination re-runs.
- Pre-selected Study: 14.2% PCR Inhibition re-runs, 4.4% PCR Contamination re-runs.
- Botswana Study: 1.9% PCR Inhibition re-runs, 1.6% PCR Contamination re-runs.
  Overall Conclusion: The results generated support the intended use of the xTAG GPP in diagnosing gastrointestinal infections.

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

Adenovirus 40/41 (stool - prospective clinical study):
- Sensitivity: 80% (95% CI: 37.5% - 96.4%)
- Specificity: 98.9% (95% CI: 98.1% - 99.3%)
- Invalid Rate: 16.7%
Entamoeba histolytica (stool - prospective clinical study):
- Sensitivity: N/A
- Specificity: 98.4% (95% CI: 97.5% – 99%)
- Invalid Rate: 17.0%
Vibrio cholerae (stool - prospective clinical study):
- Sensitivity: N/A
- Specificity: 99.9% (95% CI: 99.5% – 100%)
- Invalid Rate: 17.0%
Adenovirus 40/41 (stool in Cary-Blair - prospective clinical study):
- Positive Agreement: 40.0% (95% CI: 11.8% - 76.9%)
- Negative Agreement: 99.8% (95% CI: 99.3% - 99.9%)
Clostridium difficile toxin A/B (stool in Cary-Blair - prospective clinical study):
- Positive Agreement: 91.6% (95% CI: 84.8% - 95.5%)
- Negative Agreement: 92.0% (95% CI: 90.2% - 93.4%)
Entamoeba histolytica (stool in Cary-Blair - prospective clinical study):
- Specificity: 98.4%
Vibrio cholerae (stool in Cary-Blair - prospective clinical study):
- Specificity: 99.9%
Adenovirus 40/41 (Botswana Pediatric Stool Specimens):
- Positive Agreement: 65.4% (95% CI: 51.8% - 76.8%)
- Negative Agreement: 100% (95% CI: 98.5% - 100%)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K121454

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

K073506

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.3990 Gastrointestinal microorganism multiplex nucleic acid-based assay.

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

Summary

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Image /page/0/Picture/1 description: The image shows the logo for the Department of Health & Human Services - USA. The logo is a circular seal with the words "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" written around the perimeter. Inside the circle is a stylized image of three human profiles facing to the right, stacked on top of each other. The profiles are rendered in black and white.

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 (800) 638 2041 or (301) 796-7100 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

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely 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

Code	Classification	Regulation Section	Review Panel
PCH	II	21CFR866.3990 Gastrointestinal Pathogen Panel
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
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
Amplification Method
(Same)	Multiplex end point RT-PCR	Multiplex end point RT-PCR
Kit Reagents (Same)	xTAG GPP Primer Mix, xTAG OneStep
Enzyme Mix, xTAG OneStep Buffer,
xTAG RNase-Free Water, xTAG BSA,
xTAG MS2, xTAG GPP Bead Mix, xTAG
Reporter Buffer, xTAG 0.22 SAPE	xTAG GPP Primer Mix, xTAG OneStep
Enzyme Mix, xTAG OneStep Buffer,
xTAG RNase-Free Water, xTAG BSA,
xTAG MS2, xTAG GPP Bead Mix, xTAG
Reporter Buffer, xTAG 0.22 SAPE
Test Format (Same)	Multiplex MAGPLEX bead-based
universal array	Multiplex MAGPLEX bead-based
universal array
Detection Method (Same)	Fluorescence based	Fluorescence based
Quality Control (Same)	Internal Control (MS2), rotating analyte
controls and negative control (RNAse-
free water)	Internal Control (MS2), rotating
analyte controls and negative control
(RNAse-free water)
Results (Same)	Qualitative	Qualitative
Instrument Software
System	Luminex 100/200 with xPONENT
Software	Luminex 100/200 with xPONENT
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
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
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
Devices for the Detection of Clostridium difficile	Nov. 29, 2010
2	Class II Special Controls Guidance Document: Norovirus Serological
Reagents	Mar. 9, 2012
3	Class II Special Controls Guidance Document: Instrumentation for
Clinical Multiplex Test Systems - Guidance for Industry and FDA Staff	Mar. 10, 2005
4	Guidance for the Content of Premarket Submissions for Software
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)
Memorandum #K86-3	June 30, 1986
7	The New 510(k) Paradigm - Alternate Approaches to Demonstrating
Substantial Equivalence in Premarket Notifications - Final Guidance	Mar. 20, 1998
8	The 510(k) Program: Evaluating Substantial Equivalence in Premarket
Notifications [510(k)]	Dec. 27, 2011
9	Guidance for Industry and Food and Drug Administration Staff - eCopy
Program for Medical Device Submissions	Oct. 10, 2013
10	Guidance for Industry and Food and Drug Administration Staff - FDA and
Industry Actions on Premarket Notification (510(k)) Submissions: Effect
on FDA Review Clock and Goals	Oct. 15, 2012

Table 4: Standards

| | Standard
No. | Recognition
Number
(FDA) | Standards Title | Date |
|---|-----------------|--------------------------------|------------------------------------------------------------------------------------------------------------------------------------|------------|
| । | EP05-A2 | 7-110 | Evaluation of Precision Performance of Quantitative
measurement Methods (2nd ed.) | 10/31/2005 |
| 2 | EP07-A2 | 7-127 | Interference Testing in Clinical Chemistry (2nd
edition) | 05/21/2007 |
| ന | EP12-A2 | 7-152 | User Protocol for Evaluation f Qualitative Test
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
Trueness (2nd edition) | 09/09/2008 |
| ട | EP17-A | 7-194 | Protocol for Determination of Limits of Detection
and Limits of Quantitation
(NOTE: Original studies included this standard) | 03/28/2009 |
| 7 | EP17-A2 | 7-233 | Evaluation of Detection Capability for Clinical
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
Diseases (2nd edition) | 09/09/2008 |

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

| Standard
No. | Recognition
Number
(FDA) | Standards Title | Date | |
|-----------------|--------------------------------|-----------------|----------------------------------------------------------------|------------|
| 10 | MM13-A | 7-191 | Collection, Transport, Preparation and Storage of
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
1. Carry-over Contamination
1. Limit of Detection
1. Repeatability
1. Analytical Specificity and Interference
1. Evaluation of Fresh vs. Frozen Stool
1. 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
Titre
(Copies/mL) | Results Summary |
|--------------------------------|------------------|--------------|-----------------------------|------------------------------------|-----------------|
| Analytical reactivity_II_LX200 | Adenovirus
40 | CDC – GP-093 | Dugan
pCMK2Gr10, 9/23/91 | 1.49E+07 | POS |
| Analytical reactivity_II_LX200 | Adenovirus
41 | CDC – GP-094 | Tak
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,
GPP03-300B, GPP03-240B
Adenovirus 41	GPP03-001B, GPP03-003B, GPP03-007B, GPP03-013B, GPP03-014B, GPP03-019B, GPP03-020B,
GPP03-022B, GPP03-025B, GPP03-026B, GPP03-028B, GPP03-029B, GPP03-033B, GPP03-035B,
GPP03-036B, GPP03-037B, GPP03-038B, GPP03-039B, GPP03-048B, GPP03-055B, GPP03-060B,
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
(Cells or
Copies/mL) | Results
Summary |
|-------------------------------|--------------------------|------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------|--------------------|
| 20120216_JF_GPP_Reactivity_LX | Entamoeba
histolytica | ATCC 30015 | (HK-9, colonic biopsy
from adult human
male with amebic
dysentery, Korea);
frozen | 2.86E+00 Cells/mL,
or 1.82E+02
Copies/mL | POS |
| 20120216_JF_GPP_Reactivity_LX | Entamoeba
histolytica | ATCC 30190 | (HB-301:NIH, feces
from adult human
male with amebic
dysentery, Burma,
1960); test tube | 1.07E+03
Copies/mL | POS |
| 20120216_JF_GPP_Reactivity_LX | Entamoeba
histolytica | ATCC 30457 | (HU-21:AMC, colonic
biopsy from male
child with amebic
dysentery, Little
Rock, AR, 1970); test
tube | 1.68E+03
Copies/mL | POS |
| 20120216_JF_GPP_Reactivity_LX | Entamoeba
histolytica | ATCC 30458 | (200:NIH); frozen | 1.83E+02 Cells/mL,
or 2.42E+03
Copies/mL | POS |
| 20120216_JF_GPP_Reactivity_LX | Entamoeba
histolytica | ATCC 30459 | (HM-1:IMSS [ABRM];
feces from adult
human male,
asymptomatic cyst
passer, England,
1972); test tube | 1.83E+02 Cells/mL,
or 1.10E+03
Copies/mL | POS |
| 20120314_JF_GPP_React_LX | Entamoeba
histolytica | ATCC 30889 | (H-458:CDC
[ATCC30217], feces
from human adult
female with amebic
dysentery, Asia (?),
(patient in U.S. for
treatment), 1971);
test tube | 8.78E+02
Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba
histolytica | ATCC 30923 | (HU-2:MUSC) | 4.98E+02
Copies/mL | POS |
| 20120207_JF_GPP_Reactivity | Entamoeba
histolytica | ATCC 30925 | (HU-1:CDC, feces of
female child,
asymptomatic, sero-
negative cyst passer,
Cherokee, NC, 1978) | 1.89E+02
Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba
histolytica | ATCC 50007 | DKB | 2.88E+03
Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba
histolytica | ATCC 50481 | SD157 | 1.36E+03
Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba
histolytica | ATCC 50738 | Rahman | 8.90E+01
Copies/mL | POS |
| 20120411_JF_GPP_React_LX | Entamoeba
histolytica | ATCC 50454 | HB-301:NIH | 1.08E+03
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
(CFU/mL) | Results
Summary |
|---------------------------------|-----------------------------------------------------|------------|----------------------------------------------------------------------------------------------------------------------------------|------------------------------|--------------------|
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae
Pacini | NCTC 30 | Non-O:1, ATCC
4735;MARTIN 1 | 6.00E+08 | NEG |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 4714 | Non-O:1, Isolated
from pilgrims in El
Tor quarantine camp,
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
(NAG) — reference
strain for 0:139
serovar | 7.02E+06 | POS |
| 20120827-JX-V cholera-AR-LX | Vibrio cholerae | NCTC 12946 | 0:139 (Non-O:1
(NAG)) | 7.02E+06 | POS |
| 20120406-JX-AnaReact-Vibrio2-LX | Vibrio cholerae
Pacini | ATCC 14033 | O:1, El Tor DO
1930;CN 5774;R.
Hugh 1092, Serotype
Inaba, Non-
toxinogenic | 1.50E+08 | NEG |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae
asiaticae (Trevisan)
Pfeiffer | ATCC 14035 | O:1, Serotype Ogawa
[7787] | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae
Pacini | ATCC 14101 | O:1, Serotype
Ogawa, clinical
specimen – human
([185754] cholera
epidemic circa 1960,
Calcutta) Calcutta
India | 7.02E+06 | POS |
| 20120406-JX-AnaReact-Vibrio2-LX | Vibrio cholerae
Pacini | ATCC 14374 | Non-O:1 (NAG),
5035; R. Hugh 1513 | 1.50E+08 | NEG |
| 20120921-MB-VibrioAnalytical-LX | Vibrio cholerae
Pacini | ATCC 14730 | Non-O:1 (Serovar
O:2), biovar El Tor,
Subgroup III of
Gardner and
Venkatraman, NCTC
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
(CFU/mL) | Results
Summary |
|---------------------------------|---------------------------|-------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------|--------------------|
| 20120921-MB-VibrioAnalytical-LX | Vibrio cholerae
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
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
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
Pacini | ATCC 25870 | O:1, Serotype Inaba | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae
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
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
Pacini | ATCC 51394 | O:139 (Non-O:1 [NAG]), Cholera patient, Madras, India | 7.02E+06 | POS |
| 20120404-JX-AnaReact-Vibrio-LX | Vibrio cholerae
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-
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

15

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

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
to the estimated | Average
MFI | |
|--------------------------|-----------------------------------------|------------------------------------------|----------------|--------|
| Analyte | Strain ID | LoD) | Value | %CV |
| Adenovirus
40/41 | Adenovirus 40, 0810084CF (Dugan) | $1.45E+01$ TCID50/mL | 548 | 34.09% |
| Adenovirus
40/41 | Adenovirus 41, 0810085CF (Tak) | $7.69$ TCID50/mL | 360 | 22.04% |
| Entamoeba
histolytica | Entamoeba histolytica, 30890 | $2.88E+01$ cells/mL | 883 | 16.96% |
| Vibrio
cholerae | Vibrio cholerae, 14101 (Serovar
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
Calls | Mean MFI
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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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.
1. 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.
1. 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
(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 and yeast were tested at a concentration of 6E+08 cfu/mL except for Blastocystis hominis (ATCC 50587 concentration ≥ 1E+06 cells/mL and ATCC 50608 - concentration 2.00E+07 cells/mL). No interference was found with the xTAG GPP analytes Adenovirus, Entamoeba histolytica and Vibrio cholerae.

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| xTAG GPP Analyte (concentration) | Source | Potentially Interfering
Organism (concentration) | Source | Interference
Yes (Y) /No (N) |
|------------------------------------------------------|--------|-----------------------------------------------------|--------|---------------------------------|
| Adenovirus serotypes 40 (LP)
(1.49E+07 copies/mL) | CDC | None | | N |
| | | Astrovirus (HP)
(6.00E+10 copies/mL) | CDC | N |
| Adenovirus serotypes 41 (LP)
(1.43E+07 copies/mL) | CDC | None | | N |
| | | Astrovirus (HP)
(6.00E+10 copies/mL) | CDC | N |

Table 15: Common Commensal Bacteria, Yeast and Parasites Tested for Interference

Pathogen
Bacteroides thetaiotaomicron (ATCC 29148)
Citrobacter koseri (ATCC 27028)
Clostridium sporogenes (ATCC 3584)
E. coli strain ECOR2 (ATCC 35321)
Enterobacter cloacae (ATCC 13047)
Klebsiella pneumoniae subsp. pneumoniae (ATCC 13883)
Pseudomonas putida (ATCC 47054)
Proteus penneri (ATCC 35198)
Candida albicans (ATCC 10231)
Blastocystis hominis (ATCC 50587 or 50608)

Potential interference with GI pathogens that are a part of the assay (competitive interference) was evaluated with one target prepared at a concentration near the assay cut-off (LP) and the other target prepared at a very high concentration (HP) and vice versa. In each case, xTAG GPP Analyte 1 was also run without a second analyte present. Results (interference in making the appropriate calls) are shown in Table 16. There was no competitive interference observed between pathogens probed by xTAG GPP when testing was carried out with the mixed analyte samples described below.

xTAG GPP Analyte #1	xTAG GPP Analyte #2
Adenovirus serotype 40
(HP)
(3.80E+06 TCID50/mL)	No Analyte #2
Norovirus (LP) (160x dilution of stock)
Salmonella enterica subsp. enterica serovar Typhimurium (LP)
(8.78E+04 cfu/mL)
Campylobacter jejuni (LP) (2.93E+05 cfu/mL)
Adenovirus serotype 40
(LP)
(5.25E+01 TCID50/mL)	No Analyte #2
Norovirus (HP)
Salmonella enterica subsp. enterica serovar Typhimurium (HP)
(6.00E+08 cfu/mL)
Campylobacter jejuni (HP)
(6.00E+08 cfu/mL)

Table 16: Competitive Interference with Panel Pathogens

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The pathogens listed in Table 17 were not attainable. However, an in silico analysis was performed to assess the potential for non-specific cross-reactivity of these microbial pathogens with the primers used in xTAG GPP. These pathogens do not exhibit sufficient sequence homology against the xTAG GPP primer sequences, and therefore would not be expected to cross-react with the exception of Entamoeba coli and Taenia saginata.

Pathogen
Ascaris lumbricoides (roundworm)
Chilomastix mesnili
Cryptosporidium canis
Cryptosporidium felis
Cyclospora cayetanensis
DF-3 – Dysgonomonas capnocytophagoides
Dientamoeba fragilis
Diphyllobothrium species
Endolimax nana
Entamoeba coli
Entamoeba hartmanni
Entamoeba polecki
Enterobius vermicularis (pinworm)
Enteromonas hominis
Hymenolepis nana (the dwarf tapeworm)
Idamoeba buetschlii
Isospora belli
Strongyloides stercoralis
Taenia sp.
Trichuris trichiura

Table 17: In silico Evaluation of Pathogens for Potential Cross-reactivity

From the in silico analysis, Entamoeba coli may cross-react with xTAG GPP primers based on the strong forward primer alignment of E histolytica-FR RVM77 (16 bp contig. on the 3' end) and reverse primer E coli stx1-Rev Biosg 2 (10 bp contig. on the 3' end), as well as an amplimer size (138 bp) which is well within the design of the kit. To further elucidate, a thermal melting temperature (Tm) analysis was performed using the DINAMelt (Di-Nucleic Acid hybridization and melting prediction) program available at http://mfold.rna.albany.edu/?q=DINAMelt. Sequences of Entamoeba coli that aligned to the xTAG primers were analyzed to see if they would form a stable interaction with the xTAG primers which could possibly result in cross reactivity with the

22

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xTAG GPP kit. Mismatches would negatively impact the Tm of the primers and Entamoeba coli. At the xTAG GPP reaction temperature of 58°C, the Entamoeba coli sequences would bind to the E. histolytica forward primer with approximately 64.4% of the Entamoeba coli sequences bound to the primer sequence, compared to binding of the forward primer to its target sequence without any mismatches (98.3%). However, binding of the reverse E. coli stx1 primer to Entamoeba coli would be reduced to 0.1% compared to this primer binding to its target sequence without any mismatches (81.8%). Therefore, Entamoeba coli is not likely to cross-react with the analytes in the xTAG GPP assay.

Fresh vs. Frozen

As in the original study results presented in K121454, results from the Fresh versus Frozen study using samples for the additional analytes are presented here. This evaluation generated data to demonstrate that there is no significant difference in the performance of xTAG GPP between specimens tested from the "fresh" state (i.e. unfrozen) and specimens that were tested after being stored frozen at -70°C to -80°C. Each of the three additional analytes, Adenovirus 40/41, Entamoeba histolytica and Vibrio cholerae were assessed in a set of simulated specimens prepared in negative clinical matrix at a concentration close to the assay cut-off MFI (Low Positive), 5-10x the assay cut-off MFI (Moderate Positive) and, where possible, more than 10x the assay cut-off MFI (High Positive), where MFI is median fluorescent intensity value. Stability of un-extracted specimens, as well as pre-treated specimens, and finally, pre-treated and extracted nucleic acids were evaluated.

One Month Stability Results

Positive agreement between fresh and frozen un-extracted specimens was ≥ 95% with a lower bound of the 95% (two-sided) confidence interval exceeding 85% for Adenovirus 40/41 and Vibrio cholerae.

Positive agreement between fresh and frozen pre-treated specimens was ≥ 95% with a lower bound of the 95% (two-sided) confidence interval exceeding 85% for Adenovirus 40/41, Entamoeba histolytica and Vibrio cholerae.

Positive agreement between fresh and frozen extracted specimens was ≥ 95% with a lower bound of the 95% (two-sided) confidence interval exceeding 85% for Adenovirus 40/41, Entamoeba histolytica and Vibrio cholerae.

Adenovirus 40/41 and Vibrio cholerae met the 1-month stability acceptance criteria, and the MFIs generated on HP, MP and LP replicates of frozen un-extracted, extracted and extracted specimens were generally close to those generated at baseline. However, the un-extracted specimen stability of Entamoeba histolytica did not meet the acceptance criteria.

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Three Month Stability Results

Positive agreement between fresh and frozen un-extracted specimens was ≥ 95% with a lower bound of the 95% (two-sided) confidence interval exceeding 85% for Adenovirus 40/41, Entamoeba histolytica and Vibrio cholerae.

Positive agreement between fresh and frozen extracted specimens was ≥ 95% with a lower bound of the 95% (two-sided) confidence interval exceeding 85% for Adenovirus 40/41 and Vibrio cholerae.

The 3-month stability results for Entamoeba histolytica are of particular interest as they do not reflect the 1-month stability results. That is study criteria were met for the un-extracted specimen at 3-month stability time point but not at the 1-month time point. The 3-month stability data supports the stability of un-extracted Entamoeba histolytica frozen at -70°C to -80°C for 1 month. Study criteria for Entamoeba histolytica nucleic acid stability were met at the 1-month time point but not at the 3-month time point. Overall, the data supports the stability of un-extracted and extracted Entamoeba histolytica specimens frozen at -70°C to -80°C for 1 month.

Supplemental Stability Results - Entamoeba histolytica (un-extracted)

Additional data to support the stability of un-extracted Entamoeba histolytica specimens was also generated by analyzing LP and MP results obtained at site 1 (LMD) during the multi-site reproducibility study as well as testing LP and MP remnants at a later date. These results also suggest that un-extracted Entamoeba histolytica specimens are stable for at least 1-month when stored frozen at -70°C to -80°C.

Results are summarized for the un-extracted, pre-treated and extracted sample stability for the additional analytes in the following table.

| Analyte Target | Un-extracted
1 month | Un-extracted
3 months | Pre-Treated
1 month | Extracted
1 month | Extracted
3 months |
|-----------------------|-------------------------|--------------------------|------------------------|----------------------|-----------------------|
| Adenovirus 40/41 | V | V | V | V | V |
| Entamoeba histolytica | V^ | V | V | V | X |
| Vibrio cholerae | V | V | V | V | V |

Table 18: Summary of Stability Results Additional Analytes xTAG GPP (also see K121454)

^Based on supplemental testing results, possible titer or extraction issue with sample rather than stability failure

The results generated support the inclusion of frozen clinical specimens positive for all three targets, Adenovirus 40/41, Entamoeba histolytica and Vibrio cholerae, in the multi-site clinical evaluation of the xTAG GPP. Results generated also indicate that pre-treated material and nucleic acid extracts of all three targets evaluated are stable for at least 1 month post freezing.

24

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Precision / Reproducibility

Site-to-site reproducibility was assessed for each of the additional targets and for mixed analyte samples (representing co-infected samples). Original study results for the other analytes were presented in submission K121454. Replicates of simulated samples were tested across 3 sites by 2 operators at each site. One exception was made for testing of the Vibrio cholerae samples at Site 3, where due to operator illness the runs for the second operator were performed by two individuals. All sample replicates tested were prepared through serial dilutions of stock material (pre-treated negative stool spiked with a pathogen or positive stool) containing a microbial target from the intended use. Each sample replicate assayed in the study contained either a single microbial target or 2 microbial targets detected by xTAG GPP in addition to the internal control (bacteriophage MS2). For single analyte samples, dilutions tested fell into 1 of the following 3 categories:

1. High Negative (HN): microbial target concentrations which generate MFI values not lower than 20-30% below the cut-off MFI for the indicated analyte
1. Low Positive (LP): microbial target concentrations which generated MFI values that were 1-5X the cut-off MFI for the indicated analyte
1. Moderate Positive (MP): microbial target concentrations which generated MFI values 7-10X the cut-off MFI for the indicated analyte

For those samples prepared to simulate co-infections, one microbial target was present at the LP level defined above and the other at a High Positive (HP) level. HP levels were defined as follows:

High Positive (HP) viral cultures were prepared to a concentration of 10 PFU/mL (10 TCIDso/mL) or higher; High Positive (HP) bacterial cultures were prepared to a concentration of 10° CFU/mL or higher.

Each sample replicate underwent a single pre-treatment and extraction step. All samples were extracted using the NucliSENS easyMAG extraction method. Extracted material was kept frozen at -70 C until testing. A total of 90 replicates were tested for each single analyte and dual analyte sample (3 replicates per run x 5 runs per operators per site x 3 sites = 90 replicates). Reproducibility was assessed both in terms of calls and MFI values.

Single Analyte Results

For single analyte samples prepared at the MP level, depending on the microbial target, 89/90 (99%) to 90/90 (100%) replicates generated a positive result (after allowable re-runs). For LP dilutions, depending on the microbial target, the correct positive call was made in 85/90 (94%) to 90/90 (100%) replicates tested. For HN dilutions, depending on the target, the correct negative call was generated in as few as 67/90 (74%) replicates to as many as 90/90 (100%). Greater variability in the HN dilution, compared to the LP and MP dilution, was expected based on the fact that a target is present in these samples at levels sufficient to generate MFI values 20-30% below the cut-off MFI, and based on the stochastic nature of end-point PCR in the presence of low levels of targeted analytes. Accordingly, percent variability, measured as the coefficient of variation (CV) for MFI values were lowest at the MP dilution and highest at the HN dilution. Results for single analyte samples are presented in Table 19.

25

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Dual Analyte Results

For dual analyte samples tested for the additional targets (Table 20), all targets generated a positive call when present as a HP dilution. When present at the LP concentration, 3 of the 4 target combinations tested generated a positive call in 90/90 (100%) replicates tested. The 4 combinations were:

Rotavirus (HP) / Adenovirus (LP) Adenovirus (HP) / Rotavirus (LP)

C. difficile (HP) / Adenovirus (LP)

Adenovirus (HP)/ C. difficile (LP)

C. difficile has two probes resulting in a call for this target, (if either is positive, the target is positive). The following was observed for the remaining target present at LP concentration in the sample containing a second target at HP concentration:

. 1/90 replicates of the C. difficile (HP) /Adenovirus (LP) sample generated a negative call for Adenovirus
lt should be noted that although the C. difficile LP sample was 89/90 for probe 1, probe 2 made all the calls for the LP sample. Overall, adequate site-to-site reproducibility has been established for all targets that xTAG GPP has been designed to detect (also see results in K121454).

26

Luminex.

| | Panel Member ID | Adenovirus
40/41
Low Positive | Adenovirus
40/41
Medium
Positive | Adenovirus
40/41
High Negative | Entamoeba
histolytica
Low Positive | Entamoeba
histolytica
Medium
Positive | Entamoeba
histolytica
High Negative | Vibrio cholerae
Low Positive | Vibrio cholerae
Medium
Positive | Vibrio cholerae
High Negative | | | | | | | | | | | | | |
|--------------------------------|-----------------------------------------|-------------------------------------|-------------------------------------------|--------------------------------------|------------------------------------------|------------------------------------------------|-------------------------------------------|---------------------------------|---------------------------------------|----------------------------------|--------|-----------------------------------------|-----------------------------|-------------------------------------------------------------|------------------------------|-------------------------------------------------------------|-------------------------------------|--------------------------------------------------------------|----------------------|--------------------------------------|--------------------------------------------------------------|----------------------|---------|
| | Concentration | 1.45E+01
TCID50/mL | 5.8E+01
TCID50/mL | 1.81 TCID50/mL | 1.44E+01
Cells/mL | 5.76E+01
Cells/mL | 2.25E-01
Cells/mL | 9.37E+06
CFU/mL | 3.75E+07
CFU/mL | 5.86E+05
CFU/mL | | Panel Member ID | | Rotavirus A Low Positive/
Adenovirus 40/41 High Positive | | Rotavirus A High Positive/
Adenovirus 40/41 Low Positive | | Adenovirus 40/41 Low Positive/
C. difficile High Positive | | | Adenovirus 40/41 High Positive/
C. difficile Low Positive | | |
| | Agreement with Expected
Result | 30/30
100% | 30/30
100% | 22/30
73.3% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | | | Rotavirus A
Low Positive | Adenovirus
40/41 High
Positive | Rotavirus A
High Positive | Adenovirus
40/41
Low Positive | Adenovirus
40/41 Low
Positive | C. difficile
High Positive | | Adenovirus
40/41 High
Positive | C. difficile
Low Positive | | |
| | 25th Percentile MFI | 634.0 | 1333.3 | 110.5 | 573.0 | 1270.0 | 36.0 | 579.5 | 1206.0 | 50.0 | | | | | | | | | Probe 1 | Probe 2 | | Probe 1 | Probe 2 |
| Site 1 | Median MFI Value | 678.8 | 1392.0 | 133.0 | 614.8 | 1410.3 | 41.8 | 690.0 | 1269.0 | 57.3 | | Concentration | Indeterminate

```
     | $9.28E+02$
```

TCID50/mL | Indeterminate

```
      | $2.17E+01$
```

TCID50/mL | $2.17E+01$
TCID50/mL | $6.00E+07$
CFU/mL | $6.00E+07$
CFU/mL | $9.28E+02$
TCID50/mL | $7.50E+06$
CFU/mL | $7.50E+06$
CFU/mL | |
| | 75th Percentile MFI | 727.5 | 1428.0 | 151.5 | 775.0 | 1516.0 | 50.5 | 763.0 | 1364.0 | 74.0 | Site 1 | Agreement with
Expected Result | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | |
| | % CV | 12.26 | 6.34 | N/A | 23.47 | 14.51 | N/A | 20.16 | 10.06 | N/A | | 25th Percentile MFI | 409.0 | 1883.0 | 1269.0 | 466.0 | 485.0 | 1818.0 | 2596.5 | 1932.0 | 426.5 | 1193.5 | |
| | Agreement with Expected
Result | 30/30
100% | 30/30
100% | 15/30
50% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 21/30
70% | | Median MFI Value | 899.0 | 1955.5 | 1557.0 | 512.3 | 552.0 | 2196.8 | 2702.0 | 2005.5 | 551.5 | 1380.5 | |
| | 25th Percentile MFI | 770.0 | 1623.0 | 124.0 | 319.0 | 1111.0 | 33.0 | 860.5 | 1612.5 | 62.0 | | 75th Percentile MFI | 1410.0 | 2051.0 | 1833.5 | 564.0 | 639.0 | 2409.5 | 2766.0 | 2213.0 | 766.0 | 1604.0 | |
| Site 2 | Median MFI Value | 929.0 | 1706.3 | 151.0 | 422.5 | 1370.5 | 42.0 | 1327.3 | 1897.8 | 111.5 | | % CV | 73.05 | 5.86 | 29.42 | 17.82 | 21.03 | 17.79 | 6.39 | 9.21 | 46.02 | 25.12 | |
| | 75th Percentile MFI | 1178.5 | 1903.0 | 255.5 | 659.0 | 1661.0 | 48.0 | 1574.0 | 2294.0 | 162.0 | | Agreement with
Expected Result | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | |
| | % CV | 28.63 | 11.45 | N/A | 41.15 | 25.39 | N/A | 45.08 | 30.80 | N/A | | 25th Percentile MFI | 478.5 | 2064.5 | 1607.5 | 413.0 | 441.0 | 2233.0 | 3001.0 | 2369.0 | 587.0 | 1764.5 | |
| | Agreement with Expected
Result | 30/30
100% | 30/30
100% | 30/30
100% | 25/30
83.3% | 29/30
96.7% | 30/30
100% | 30/30
100% | 30/30
100% | 29/30
96.7% | Site 2 | Median MFI Value | 828.0 | 2438.5 | 1854.8 | 510.8 | 558.0 | 2514.5 | 3185.8 | 2521.8 | 687.5 | 1992.5 | |
| | 25th Percentile MFI | 245.0 | 618.5 | 54.0 | 277.0 | 749.0 | 40.0 | 270.0 | 757.0 | 37.0 | | 75th Percentile MFI | 1670.0 | 2616.0 | 2160.5 | 606.0 | 720.0 | 2705.0 | 3394.0 | 2709.0 | 957.0 | 2276.5 | |
| Site 3 | Median MFI Value | 275.3 | 715.3 | 64.0 | 364.3 | 986.3 | 47.0 | 326.3 | 924.0 | 47.5 | | % CV | 80.25 | 11.69 | 26.10 | 26.82 | 34.32 | 19.83 | 11.54 | 9.97 | 45.91 | 18.23 | |
| | 75th Percentile MFI | 340.0 | 860.0 | 77.0 | 446.5 | 1135.0 | 55.0 | 522.5 | 1031.0 | 56.0 | | Agreement with
Expected Result | 30/30
100% | 30/30
100% | 30/30
100% | 30/30
100% | 29/30
96.7% | 30/30
100% | 30/30
100% | 30/30
100% | 29/30
96.7% | 30/30
100% | |
| | % CV | 24.57* | 41.66* | N/A | 34.84 | 37.32 | N/A | 48.47 | 24.88 | N/A | | 25th Percentile MFI | 430.5 | 1452.0 | 1093.0 | 206.0 | 222.0 | 1088.0 | 2118.0 | 1431.0 | 274.5 | 1088.0 | |
| | Total Agreement with
Expected Result | 90/90
100% | 90/90
100% | 67/90
74.4% | 85/90
94.4% | 89/90
98.9% | 90/90
100% | 90/90
100% | 90/90
100% | 80/90
88.9% | Site 3 | Median MFI Value | 689.5 | 1527.5 | 1538.0 | 264.5 | 235.5 | 1510.5 | 2404.5 | 1550.0 | 414.3 | 1247.5 | |
| | 95% CI | 95.9%-100.0% | 95.9%-100.0% | 64.6%- 82.3% | 87.6%- 97.6% | 94.0%- 99.8% | 95.9%-100.0% | 95.9%-100.0% | 95.9%-100.0% | 80.7%- 93.9% | | 75th Percentile MFI | 1067.0 | 1645.0 | 1726.0 | 292.0 | 269.5 | 1837.0 | 2613.5 | 1630.0 | 523.0 | 1425.0 | |
| | Overall 25th Percentile MFI | 340.0 | 860.0 | 72.5 | 330.5 | 1007.0 | 36.0 | 420.5 | 1006.0 | 47.0 | | % CV | 64.25 | 11.30 | 35.50 | 23.76 | 22.74 | 32.71 | 15.68 | 13.12 | 48.98 | 24.47 | |
| | Overall Median MFI Value | 672.5 | 1394.0 | 112.5 | 489.3 | 1240.5 | 43.5 | 672.8 | 1258.0 | 58.5 | | Total Agreement with
Expected Result | 90/90
100% | 90/90
100% | 90/90
100% | 90/90
100% | 89/90
98.9% | 90/90
100% | 90/90
100% | 90/90
100% | 89/90
98.9% | 90/90
100% | |
| | Overall 75th Percentile MFI | 824.0 | 1659.5 | 151.5 | 631.0 | 1473.5 | 51.0 | 975.5 | 1612.5 | 90 | | 95% CI | 95.9%- 100.0% | 95.9%- 100.0% | 95.9%- 100.0% | 95.9%- 100.0% | 94.0%- 99.8% | 95.9%-
100.0% | 95.9%-
100.0% | 95.9%- 100.0% | 94.0%-
99.8% | 95.9%-
100.0% | |
| | Overall % CV | 51.30* | 35.00* | N/A | 40.94 | 31.45 | N/A | 66.46 | 42.16 | N/A | | Overall 25th Percentile
MFI | 430.5 | 1645.0 | 1361.0 | 292.0 | 269.5 | 1648.0 | 2483.0 | 1630.0 | 406.0 | 1219.0 | |
| Overall Median
MFI Value | 741.8 | 1948.8 | 1614.8 | 444.3 | 447.0 | 2122.3 | 2706.3 | 2004.8 | 575.8 | 1452.8 | | | | | | | | | | | | | |
| Overall 75th Percentile
MFI | 1251.5 | 2128.5 | 1884.5 | 543.0 | 603.0 | 2422.0 | 3023.0 | 2377.5 | 766.0 | 1849.0 | | | | | | | | | | | | | |
| Overall
% CV | 75.04 | 20.54 | 32.39 | 37.71 | 44.38 | 30.51 | 16.62 | 23.01 | 53.59 | 30.19 | | | | | | | | | | | | | |

Table 19: Summary of Overall Total Raw Median MFI values for the Three Targets in xTAG GPP after Reruns

This %CV value includes the re-run

27

Luminex

Table 20: Summary of Overall Total Raw Median MFI values for Mixed Analytes in xTAG GPP after Reruns

*Real-time PCR failed to return a meaningful result. The sample is the same as the amount used in equivalent Rotavirus dilutions used in the Repeatability study.

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Stool in Cary-Blair Media Limit of Detection Study Results

The purpose of this analytical study was to evaluate the equivalency in the limit of detection (LD) between the two sample types: raw stool (sample type from K121454) and stool in Cary-Blair transport medium (additional sample type commonly collected) in a representative sub-set of the xTAG GPP targets. One analyte from each of three pathogen classes (bacterial, parasitic, and viral) was examined in the form of simulated stool samples in Cary-Blair media. The simulated samples were prepared as a dilution series using high titre stocks. The three representative analytes tested in this study were: Clostridium difficile, Giardia lamblia and Norovirus Gll. Results of testing presented in Table 21 demonstrate that raw stool samples in Cary-Blair media have equivalent limits of detection.

| | | Raw Stool | | Stool in Cary-Blair | | LoD Difference
between Stool
and Stool in Cary-Blair |
|---------------------------|-------------------------------------------------------------------|-----------------------------------------|--------------------------------|-----------------------------------------|---------------------------------|------------------------------------------------------------|
| Analyte | Strain ID | Titre at
limit of
detection | Average MFI
Value (n=20) | Titre at
limit of
detection | Average MFI
Value (n=20) | |
| C. difficile
Toxin A/B | Clostridium
difficile,
BAA-1805
(toxinotype
III A+B+) | $4.69E+05$
CFU/mL | Probe 1 = 383
Probe 2 = 862 | $4.69E+05$
CFU/mL | Probe 1 = 460
Probe 2 = 1128 | None |
| Giardia | Giardia
lamblia,
PRA-243 | $2.20E+02$
cells/mL | 1069 | $2.20E+02$
cells/mL | 1008 | None |
| Norovirus
GI/GII | Norovirus
GII, Clinical
sample,
source
Toronto | $4.75E+02$
copies/mL
(Ct = 32.23) | 1466 | $4.75E+02$
copies/mL
(Ct = 32.23) | 2299 | None |

Table 21: Summary of the Limit of Detection (LoD) for GPP Analytes in Stool and Stool in Cary-Blair Media

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Summary of negative control failures and sample re-run rates for analytical performance studies

Including all analytes in the xTAG GPP test intended use, there were a total of 278 xTAG GPP runs performed over the course of analytical performance studies. Each xTAG run has at least one no template negative on batch size. Of the 278 runs, 12 (4.32%) had one or more negative control (NC) failures. These are summarized in the table below.

| Study | Total # of runs
(including
allowable re-runs) | Total # of runs
with at least one
NC failure | % total
runs with
at least
one NC
failure | Total No. of
NCs included
in runs and
allowable re-runs | Total No.
of NC
failures | % total NC s included
which failed in xTAG runs
/ allowable re-runs |
|--------------------------------------------|-----------------------------------------------------|----------------------------------------------------|-------------------------------------------------------|------------------------------------------------------------------|--------------------------------|---------------------------------------------------------------------------|
| Multi-site reproducibility | 95 | 6 | 6.32% | 248 | 7 | 2.82% |
| Matrix equivalence | 3 | 0 | 0 | 9 | 0 | 0 |
| Limit of detection | 38 | 0 | 0 | 136 | 0 | 0 |
| Carry-over contamination | 9 | 0 | 0 | 0 | 0 | 0 |
| Analytical specificity and
interference | 25 | 1 | 4.00% | 101 | 1 | 0.99% |
| Analytical reactivity | 36 | 2 | 5.56% | 212 | 3 | 1.42% |
| Evaluation of fresh vs.
frozen stool | 72 | 3 | 4.17% | 216 | 3 | 1.39% |
| Overall | 278 | 12 | 4.32% | 922 | 14 | 1.52% |

Table 22: Summary of Negative Control Failures for Analytical Performance Studies

Included in the 278 xTAG runs summarized above were 14960 specimens. Of these, 99.81% (14931/14960) vielded valid results on the first attempt. The remaining 29 specimens generated valid results following allowable re-runs. Sample re-run ratized in the table below.

30

Luminex.

| Studies | Total # of
specimens
tested | Total # of
invalid results
prior to re-run | % invalid
results prior
to re-run | Invalid results
after re-run | % invalid results
after re-run |
|--------------------------------------------|-----------------------------------|--------------------------------------------------|-----------------------------------------|---------------------------------|-----------------------------------|
| Multi-site reproducibility | 5065 | 25 | 0.49% | 0 | 0.00% |
| Matrix equivalence | 180 | 0 | 0.00% | 0 | 0.00% |
| Limit of detection | 992 | 1 | 0.10% | 0 | 0.00% |
| Carry-over contamination | 864 | 0 | 0.00% | 0 | 0.00% |
| Analytical specificity and
interference | 1472 | 0 | 0.00% | 0 | 0.00% |
| Analytical reactivity | 2225 | 1 | 0.04% | 0 | 0.00% |
| Evaluation of fresh vs.
frozen stool | 4162 | 2 | 0.05% | 0 | 0.00% |
| Overall | 14960 | 29 | 0.19% | 0 | 0.00% |

Table 23: Summary of Sample Re-Run Rates for Analytical Performance Studies

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Clinical Performance:

Matrix comparison study

Unchanged, reference results in K121454

Detection in asymptomatic volunteers

In order to determine baseline levels for each analyte included in xTAG GPP for individuals who are not exhibiting signs and symptoms of infectious gastroenteritis, 200 clinical stool samples were collected from healthy, asymptomatic donors. Asymptomatic donors from various age groups were included in this study. Results presented below include the additional analytes in the xTAG GPP test. PCR inhibition, as determined by results for the internal control used with xTAG GPP (bacteriophage MS2), was observed in 23 of the 200 samples tested (11.5%). After rerunning these specimens in accordance with the instructions for use, PCR inhibition was still observed in 8 samples (4%). The absence of a detectable internal control signal in these samples meant that negative results for the indicated microbial targets could not be reported. Therefore, the final data analysis was conducted on 192 of the 200 samples collected for this study.

| Target | Percent Negative Results by
xTAG GPP for all samples | Percent Negative Results by xTAG GPP
for samples negative by sequencing |
|-------------------------------|---------------------------------------------------------|----------------------------------------------------------------------------|
| Adenovirus 40/41 | 100.0% (192/192) | 100.0% (192/192) |
| Campylobacter | 100.0% (192/192) | 100.0% (192/192) |
| C. difficile toxin A/B | 98.4% (189/192)1 | 99.5% (189/190) |
| Cryptosporidium | 100.0% (192/192) | 100.0% (192/192) |
| E. histolytica | 99.5% (191/192)2 | 99.5% (191/192) |
| E. coli O157 | 100.0% (192/192) | 100.0% (192/192) |
| ETEC LT/ST | 100.0% (192/192) | 100.0% (192/192) |
| Giardia | 99.0% (190/192)3 | 99.0% (190/192) |
| Norovirus GI/GII | 98.4% (189/192)*4 | 98.4% (189/192) |
| Rotavirus A | 100.0% (192/192) | 100.0% (192/192) |
| Salmonella | 97.4% (187/192)5 | 97.4% (187/192) |
| STEC stx1/stx2 | 100.0% (192/192) | 100.0% (192/192) |
| Shigella | 100.0% (192/192) | 100.0% (192/192) |
| V. cholerae | 100.0% (192/192) | 100.0% (192/192) |

Table 24: Percent Negative Results (including Adenovirus 40/41, E. histolytica and V. cholerae) and the Analytes Previously Presented in the Decision Summary for K121454

*NOTE: Sample 216 was positive by xTAG GPP for both Norovirus GII and C. Difficile

1 Two (2) out of 3 xTAG GPP C. Difficile positive samples were confirmed as positive by sequencing analysis.

2 The (1) xTAG GPP E. histolytica positive sample was not confirmed as positive by sequencing analysis.

3 None of the 2 xTAG GPP Giardia positive samples was confirmed as positive by sequencing analysis.

4 None of the 3 xTAG GPP Noroviris GI/GII positive samples was confirmed as positive by sequencing analysis.

5 None of the 5 xTAG GPP Salmonella positive samples was confirmed as positive by sequencing analysis.

As described in submission K121454, results of the study demonstrated ≥97% negative percent agreement across all analytes in the 192 samples (at the specimen level) that

32

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were positive by xTAG GPP but negative by sequencing were considered false positives (12/192). These samples had MFI values that were relatively close to the cut-offs. 2 samples at the specimen level that were called positive by xTAG GPP were also positive by sequencing for C. difficile. These two samples positive for C. difficile by both xTAG GPP and sequencing probably represent asymptomatic infections.

Clinical Cutoff

Not applicable

Detection in symptomatic patients (prospective clinical study in stool specimens)

The clinical performance of xTAG GPP for each analyte probed by the assay was evaluated in clinical specimens (stools) prospectively collected between June 2011 and February 2012. A total of 1407 clinical specimens were collected from pediatric and adult patients and submitted for testing at six (6) independent laboratories. Four (4) of the laboratories were located in the United States (Arizona, Missouri, Tennessee and Texas) and two (2) were in Southern Ontario (Canada). Demographic details for this prospective data set were summarized in the original submission K121454. In this submission, results for the additional analytes (Adenovirus 40/41, Entamoeba histolytica and Vibrio cholerae) are provided for the prospective clinical study for samples collected in stool. Additionally, these same samples were also stored in Cary-Blair media, and results of testing these are also provided.

All prospective clinical specimens were analyzed by reference/comparator at central laboratories independent of xTAG GPP testing sites. Comparator methods were described in the original submission K121454 apart from the 3 listed below. For the additional analytes, the comparator methods are described in Table 25.

xTAG GPP analytes	Comparator Method
Adenovirus 40/41	Composite comparator consisting of Premier Adenoclone Type 40/41 EIA (Meridian
Bioscience, K881894)^ directly on the stool specimen and Amplification + sequencing directly
from clinical specimen using one NAAT+
Entamoeba
histolytica	Microscopy followed by amplification + sequencing directly from clinical specimens using one
NAAT+ (positive specimens by microscopy only)
Vibrio cholerae	Bacterial culture

Table 25: Comparator Methods

^ Meridian Bioscience acquired Cambridge Bioscience Corp. products

NAAT, nucleic acid amplification test - see detailed description below

Clinical runs and re-runs using xTAG GPP were carried out on clinical specimens that had been extracted from the fresh or frozen state using the NucliSENS easyMAG method (bioMérieux, Inc., Durham, NC) according to the manufacturer's instructions. Total extracted nucleic acid material was stored at -70℃ prior to testing with xTAG GPP at each of the clinical sites. xTAG GPP positive

33

Lumi

results (expected values) for each individual target were summarized per age group in submission K121454, and are now summarized for the additional analytes in Table 26.

| Target
(Analyte) | Overall
(n=1407) | | 0-1 year (n=6) | | >1-5 years (n=20) | | >5-21 years (n=76) | | >21-65 years
(n=879) | | >65 years
(n=426) | |
|--------------------------|---------------------|-------------------|----------------|-------------------|-------------------|-------------------|--------------------|-------------------|-------------------------|-------------------|----------------------|-------------------|
| | No. | Expected
Value | No. | Expected
Value | No. | Expected
Value | No. | Expected
Value | No. | Expected
Value | No. | Expected
Value |
| Adenovirus
40/41 | 17 | 1.2% | 0 | 0.0% | 2 | 10.0% | 0 | 0.0% | 10 | 1.1% | 5 | 1.2% |
| Entamoeba
histolytica | 19 | 1.4% | 0 | 0.0% | 0 | 0.0% | 1 | 1.3% | 14 | 1.6% | 4 | 0.9% |
| Vibrio
cholerae | 1 | 0.1% | 0 | 0.0% | 0 | 0.0% | 0 | 0.0% | 1 | 0.1% | 0 | 0.0% |

Table 26: Expected Values in Stool Specimens (As determined by xTAG GPP) - Summary by Age Groups for the xTAG GPP Prospective Clinical Evaluation (June 2011 – February 2012)

Accuracy determinations (diagnostic sensitivity and specificity, positive and negative agreement) were based on the fraction of comparator positive (or negative) results which were also positive (or negative) by xTAG GPP. Sensitivity (or positive agreement) was calculated by dividing the total number of "true positive" xTAG GPP results (TP) by the sum of the TP and "false negative" (FN) xTAG GPP results. Specificity (or negative agreement) was calculated by dividing the total number of "true negative" xTAG GPP results (TN) by the sum of the TN and "false positive" (FP) xTAG GPP results. An xTAG GPP result was considered to be a TP or TN result only in the event that it agreed with the comparator method result for the analyte in question. 95% confidence intervals were calculated using the Wilson score method.

Since the reagents in the xTAG Kit remain the same, data from the original clinical study (K121454) are still applicable. Tables 27-29 present the stool results for each of the additional analyte targets added to the intended use of xTAG GPP for the clinical prospective sample set (N=1407).

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xTAG GPP	Primary Comparator
	Positive	Negative	Invalid	TOTAL
Positive	4	13	0	17
Negative	11	1154	0	1155
Invalid	2	233	0	235
TOTAL	7	1400	0	1407
		95% CI
Sensitivity	80%	37.5% - 96.4%
Specificity	98.9%	98.1% - 99.3%
Invalid Rate	16.7%

Table 27: 3X3 for Adenovirus 40/41 (stool)

1 The one specimen that was positive for Adenovirus 40/41 by comparator but negative by xTAG GPP was positive by bi-directional sequencing only (i.e. FDA-cleared EIA negative).

	Table 28: 3X3 for Entamoeba histolytica (stool)

xTAG GPP	Primary Comparator
	Positive	Negative	Invalid	TOTAL
Positive	0	19	0	19
Negative	0	1149	0	1149
Invalid	0	239	0	239
TOTAL	0	1407	0	1407
		95% CI
Sensitivity	N/A	N/A
Specificity	98.4%	97.5% – 99%
Invalid Rate	17.0%

Table 29: 3X3 for Vibrio cholerae (stool)

xTAG GPP	Primary Comparator
	Positive	Negative	Invalid	TOTAL
Positive	0	1	0	1
Negative	0	1166	0	1166
Invalid	0	240	0	240
TOTAL	0	1407	0	1407
		95% CI
Sensitivity	N/A	N/A
Specificity	99.9%	99.5% – 100%
Invalid Rate	17.0%

A summary of the prospective clinical performance data in human stool specimens (from K121454 and this submission) is presented for each of the analytes in Table 30 below.

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Table 30: Summary of Prospective Performance Data (N=1407) Testing Human Stool Specimens Including Adenovirus 40/41, E. histolytica and V. cholerae, with the Results from K121454

| Sensitivity | | | | Specificity | | | | Number Invalid
xTAG GPP
Results due to
PCR Inhibition |
|----------------------------|-----------------|---------|---------------|--------------------|---------|---------------|-----|----------------------------------------------------------------|
| Analyte | TP /
(TP+FN) | percent | 95%CI | TN /
(TN+FP) | percent | 95%CI | | |
| Campylobacter | 3/3 | 100% | 43.9% - 100% | 1155/1176 | 98.2% | 97.3% - 98.8% | 228 | |
| Cryptosporidium | 12/13 | 92.3% | 66.7% - 98.6% | 1131/1184 | 95.5% | 94.2% - 96.6% | 210 | |
| E. coli 0157 | 2/2 | 100% | 34.2% - 100% | 1158/1167 | 99.2% | 98.5% - 99.6% | 238 | |
| Entamoeba
histolytica | 0/0 | N/A | N/A | 1149/1168 | 98.4% | 97.5% - 99% | 239 | |
| Giardia | 4/4 | 100% | 51.0% - 100% | 1132/1171 | 96.7% | 95.5% - 97.6% | 232 | |
| Salmonella | 10/10 | 100% | 72.2% - 100% | 1143/1161 | 98.4% | 97.6% - 99.0% | 236 | |
| Shigella | 2/2 | 100% | 34.2% - 100% | 1154/1171 | 98.5% | 97.7% - 99.1% | 234 | |
| Vibrio cholerae | 0/0 | N/A | N/A | 1166/1167 | 99.9% | 99.5% - 100% | 240 | |
| Positive Agreement | | | | Negative Agreement | | | | Number Invalid
xTAG GPP
Results due to
PCR Inhibition |
| Analyte | TP /
(TP+FN) | percent | 95%CI | TN /
(TN+FP) | percent | 95%CI | | |
| Adenovirus
40/41 | 4/5 | 80% | 37.5% - 96.4% | 1154/1167 | 98.9% | 98.1% - 99.3% | 235 | |
| C. difficile Toxin
A/B1 | 107/114 | 93.9% | 87.9% - 97.0% | 922/1027 | 89.8% | 87.8% - 91.5% | 171 | |
| ETEC | 2/8 | 25.0% | 7.1% - 59.1% | 1156/1160 | 99.7% | 99.1% - 99.9% | 239 | |
| Norovirus Gi/Gll | 74/78 | 94.9% | 87.5% - 98.0% | 1023/1119 | 91.4% | 89.6% - 92.9% | 210 | |
| Rotavirus A | 2/2 | 100% | 34.2% - 100% | 1162/1164 | 99.8% | 99.4% - 100% | 241 | |
| STEC | 1/1 | 100% | 20.7% - 100% | 1153/1169 | 98.6% | 97.8% - 99.2% | 237 | |

1 total of 95 specimens generated a "Nonspecific reaction, not characteristic of Clostridium difficile toxin. A titration test was performed on all 95 specimens and it was determined that in each case, the cytotoxicity reaction was not typical of C. difficile toxin.

When all of the analyte data is combined, xTAG GPP detected a total of 98 mixed infections in the prospective clinical evaluation. This represents 20.2% of the total number of xTAG GPP positive specimens (98/486). 62 (62/98; 63.3%) were double infections, 23 (23/98; 23.5%) were triple infections, 8 (8/98; 8.2%) were quadruple infections, 2 (2/98; 2.0%) were quintuple infections, 1 (1/98; 1.0%) was sextuple infection, 1 was septuple infection (1/98; 1.0%) and 1 was octuple infection (1/98; 1.0%). The single most common co-infection (20/98; 20.4%%) was Norovirus GI/GII with C. difficile Toxin A/B. Out of the 98 co-infections, 93 contained one or more analytes that had not been detected with the reference/comparator methods, i.e. discrepant co-infections.

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Detection in symptomatic patients (prospective clinical study in Stool in Cary-Blair Media)

Original comparator method test results for all samples in the prospective study (see K121454) were utilized for comparison to stool samples in Cary-Blair media for which adequate sample was available. The purpose of the study was to establish diagnostic accuracy of xTAG GPP in stool specimens in Cary-Blair medium. Clinical performance (sensitivity/positive percentage agreement and specificity/negative percentage agreement) of xTAG GPP on stool in Cary-Blair medium is summarized for each individual target in Table 31 below. For comparison purposes, clinical performance results generated from the unpreserved stool as part of the original clinical study (see report TDR-736-189-001) are also presented.

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Table 31: Summary of xTAG GPP Clinical Performance

Sensitivity					Specificity
Target	TP/(TP+FN)	%	95% CI	TP/(TP+FN)	%	95% CI	TN/(TN+FP)	%	95% CI	TN/(TN+FP)	%	95% CI
Campylobacter	3/3	100.0%	43.9% - 100%	3/3	100.0%	43.9% - 100%	1155/1176	98.2%	97.3% - 98.8%	1268/1277	99.3%	98.7% - 99.6%
Cryptosporidium	12/13	92.3%	66.7% - 98.6%	12/13	92.3%	66.7% - 98.6%	1131/1184	95.5%	94.2% - 96.6%	1253/1274	98.4%	97.5% - 98.9%
Entamoeba
histolytica	n/a			n/a			1149/1168	98.4%	97.5% - 99.0%	1264/1284	98.4%	97.6% - 99.0%
E. coli O157	2/2	100.0%	34.2% - 100%	2/2	100.0%	34.2% - 100%	1158/1167	99.2%	98.5% - 99.6%	1269/1278	99.3%	98.7% - 99.6%
Giardia	4/4	100.0%	51.0% - 100%	4/4	100.0%	51.0% - 100%	1132/1171	96.7%	95.5% - 97.6%	1259/1281	98.3%	97.4% - 98.9%
Norovirus GI/GII	74/78	94.9%	87.5% - 98.0%	70/73	95.9%	88.6% - 98.6%	1023/1119	91.4%	89.6% - 92.9%	1144/1210	94.5%	93.1% - 95.7%
Salmonella	10/10	100.0%	72.2% - 100%	10/10	100.0%	72.2% - 100%	1143/1161	98.4%	97.6% - 99.0%	1237/1273	97.2%	96.1% - 98.0%
Shigella	2/2	100.0%	34.2% - 100%	2/2	100.0%	34.2% - 100%	1154/1171	98.5%	97.7% - 99.1%	1275/1279	99.7%	99.2% - 99.9%
Vibrio cholera	n/a			n/a			1166/1167	99.9%	99.5% - 100%	1279/1280	99.9%	99.6% - 100%
				Positive Agreement					Negative Agreement
Target	TP/(TP+FN)	%	95% CI	TP/(TP+FN)	%	95% CI	TN/(TN+FP)	%	95% CI	TN/(TN+FP)	%	95% CI
Adenovirus 40/411	4/5	80.0%	37.5% - 96.4%	2/5	40.0%	11.8% - 76.9%	1154/1167	98.9%	98.1% - 99.3%	1272/1275	99.8%	99.3% - 99.9%
Clostridium difficile
toxin A/B	107/114	93.9%	87.9% - 97.0%	98/107	91.6%	84.8% - 95.5%	922/1027	89.8%	87.8% - 91.5%	1020/1109	92.0%	90.2% - 93.4%
ETEC LT/ST2	2/8	25.0%	7.1% - 59.1%	2/9	22.2%	6.3% - 54.7%	1156/1160	99.7%	99.1% - 99.9%	1266/1271	99.6%	99.1% - 99.8%
Rotavirus A	2/2	100.0%	34.2% - 100%	2/2	100.0%	34.2% - 100%	1162/1164	99.8%	99.4% - 100%	1277/1278	99.9%	99.6% - 100%
STEC	1/1	100.0%	20.7% - 100%	1/1	100.0%	20.7% - 100%	1153/1169	98.6%	97.8% - 99.2%	1273/1280	99.5%	98.9% - 99.7%

'n the case of Adenovins 40/41, one of the clinical positive in the original GPP runs performed on raw stool yielded a negative result when tested in Cary-Blair. MF generated in the original stool run were closed of (195) suggesting a low ther specimen that was inlibited in the original on raw stool

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

vielded a negative result in the Car-Bair runs positive for Adenovirus 0/41 by composite comparator was unavarable for re-testing in the Car-Blair study. For these reasons, positive ageement of x AG GPP for 80% (4/5) in the raw stody to 40% (2/5 in the Cary Blair evaluation. Refer to the results of urther evaluation testing contrived Cary-Blair samples close to the limit of detection (LoD) for Adenovirus 40/41.

ÉTEC comparator results were calculated and our well characterized nucleic acid ampification tects (NATS) followed by bi-directional sequencing, Al socimens hat were false hegative by xTAG GPP for ETEC were postion NATS. Repeat sequencing of these specified by all four MAT, except or one sample which was positive by one NAAT.

Clinical sensitivity or positive agreement acceptance criterion of 90% with a lower bound 95% confidence interval for Norovirus G/Gll and Clotridium difficile toxin A/B on stool in Cary-Blair media. The results were equivalent to those obtained for unpreserved stool specimens. Similar to the unpreserved stool, the lower bound 95% confidence interval for sensibility was not met for all other targets probed by xTAG GPP on stool in Cary-Blair media. This can be explained by the low positivity rate in the prospective sample set.

Although a smaller sample set was used for the study, positive agreement between comparator and xTAG GPP results was 100% for all pre-selected targets tested. Clinical specificity or negative percentage arreement acceptance criterion of 90% with a lower bound 95% confidence interval of at least 90% were achieved for all targets probed by xTAG GPP.

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Other supportive clinical data

Pre-selected stool specimens Retrospective Study

A total of 207 archived stool specimens that were positive by reference/comparator for pathogens that were of low prevalence in the prospective clinical study were collected at multiple sites in North America, Africa and Europe. Luminex was unable to source any stool specimens that tested positive for Vibrio cholerae by reference method for the pre-selected arm of the study. As previously noted, the range of analyte concentrations in these pre-selected specimens represented the clinically relevant range of concentrations observed in patients with gastrointestinal infection. All pre-selected positive specimens were tested with xTAG GPP at 4 sites (3 of which were external to LMD), along with negative clinical specimens in a randomized, blinded fashion. The "negative" designation for these specimens was based on the routine algorithms used at the banking site (e.g. bacterial culture, EIA, microscopy, in-house real time PCR). These algorithms did not test for all pathogen targets probed by xTAG GPP. Table 32 summarizes the positive agreement between reference/comparator and xTAG GPP for all preselected targets evaluated.

| Target | Positive Agreement
TP / (TP+FN) | Positive Agreement
Percent | 95%CI for Positive
Agreement | Number Invalid xTAG GPF
Results |
|--------------------------|------------------------------------|-------------------------------|---------------------------------|------------------------------------|
| Adenovirus 40/41 | 3/3 | 100% | 43.8% - 100% | 0 |
| Campylobacter | 40/41 | 97.6% | 87.4% - 99.6% | 0 |
| Cryptosporidium | 12/12 | 100% | 75.7% - 100% | 1 |
| Entamoeba
histolytica | 1/1 | 100% | 2.5% - 100% | 0 |
| E. coli O1571 | 14/14 | 100% | 78.5% - 100% | 0 |
| ETEC | 38/39 | 97.4% | 86.8% - 99.5% | 0 |
| Giardia2 | 15/16 | 93.7% | 71.7% - 98.9% | 1 |
| Rotavirus A | 28/28 | 100% | 87.9% - 100% | 0 |
| Salmonella | 24/27 | 88.89% | 71.9% - 96.1% | 0 |
| STEC3 | 18/18 | 100% | 82.4% - 100% | 0 |
| Shigella | 20/20 | 100% | 83.9% - 100% | 0 |

Table 32: Positive Percent Agreement of xTAG GPP in the Pre-selected Stool Data Set

1- Eight (8)/8 E. coli 0157 were also positive for STEC by xTAG GPP. Sample remnants of all 8 E. coli 0157 specimens were tested for the presence of stx1 and stx 2 genes by bi-directional sequencing and the results added to those obtained for STEC.

2- One (1) false negative Giardia specimen was reported. This specimen was also negative for Giardia by in-house real-time PCR performed at the site.

3- Six (6)/10 STEC were also positive for E. coli 0157 by xTAG GPP. Sample remnants of all 10 STEC specimens were assessed by bi-directional sequencing for E. coli 0157 and the results added to those obtained for E. coli 0157.

Confirmatory testing by nucleic acid amplification followed by bi-directional sequencing using analytically validated primers was also conducted on all available specimens tested in the pre-

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selected arm of the clinical study. More specifically, confirmatory testing was performed for those analytes that were positive by xTAG GPP but not pre-selected at the banking site in order to determine whether these additional positive calls represented True Positive (TP) or False Positive (FP) clinical results. To the extent possible, sequencing primers targeted genomic regions distinct from those of the kit primers. xTAG GPP generated 114 additional positive calls (after allowable re-runs) for analytes that were not pre-selected at the banking site. Results of confirmatory testing from the preselected study were presented in the submission summary K121454, and the additional analyte results only are presented here. Sequencing primer validation studies were also presented in the submission summary K121454 and are not repeated here.

	Table 33: 3X3 Table for Additional Adenovirus 40/41 Confirmatory Testing Results – Pre-selected
Stool Sample Set

xTAG GPP	PCR/Bi-directional Sequencing
	Positive	Negative	Not Done	TOTAL
Additional Positive	5	3	0	8
Negative	N/A	N/A	402	402
Invalid	N/A	N/A	67	67
TOTAL	5	3	469	477

Confirmed Positive	62.5%
Invalid Rate (N=480)	13.9%

3 specimens were pre-selected for Adenovirus 40/41.

Table 34: 3X3 Table for Additional Entamoeba histolytica Confirmatory Testing Results – Preselected Stool Sample Set

xTAG GPP	PCR/Bi-directional Sequencing
	Positive	Negative	Not Done	TOTAL
Additional Positive	1	7	0	8
Negative	N/A	N/A	403	403
Invalid	N/A	N/A	68	68
TOTAL	1	7	471	479*

Confirmed Positive	12.5%
Invalid Rate (N=480)	14.2%

1 specimen was pre-selected for Entamoeba histolytica.

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Table 35: 3X3 Table for Additional Vibrio Cholerge Confirmatory Testing Results – Pre-selected Stool Sample Set

xTAG GPP	PCR/Bi-directional Sequencing
	Positive	Negative	Not Done	TOTAL
Additional Positive	0	0	0	0
Negative	N/A	N/A	412	412
Invalid	N/A	N/A	68	68
TOTAL	0	0	480	480

Confirmed Positive	N/A
Invalid Rate (N=480)	14.2%

xTAG GPP detected a total of 71 mixed infections in the pre-selected arm of the clinical study. This represents 29.8% of the total number of xTAG GPP positive specimens (71/238). 56 (56/71: 78.9%) were double infections, 14 (14/71: 19.7%) were triple infections and 1 was quadruple infection (1/71; 1.4%). The single most common co-infections (excluding E. coli 0157 with STEC; N=12) was ETEC with Shigella (6/71; 8.4%). Out of the 71 co-infections, 25 contained one or more analytes that was not confirmed by bi-directional sequencing, i.e. discrepant co-infections. All mixed infection combinations detected by the reference/comparator methods were detected by xTAG GPP.

Pre-selected stool in Cary-Blair specimens Retrospective Study

Remnants of available pre-selected frozen stool specimens tested as part of the original clinical study were mixed proportionally with Cary-Blair medium and tested in a randomized, blinded fashion. Results are presented in the table below.

| | | Positive Agreement | | 95%
Confidence
Interval (CI) | Number of
Invalid
Results |
|--------------|------------|--------------------|--------------|------------------------------------|---------------------------------|
| Target | TP/(TP+FN) | Percentage | | | |
| | | Campylobacter | 40/40 | 100.0% | 91.3% - 100% |
| E. coli O157 | 2/2 | 100.0% | 34.2% - 100% | 0 | |
| Salmonella | 26/26 | 100.0% | 87.1% - 100% | 0 | |
| Shigella | 13/13 | 100.0% | 77.2% - 100% | 0 | |

Table 36: Positive percent agreement of xTAG GPP in the pre-selected Cary-Blair

Supplemental Clinical Data (Simulated Stool Specimen Results)

Due to difficulties in sourcing a sufficient number of retrospective stool specimens positive by reference method for Entamoeba histolytica and Vibrio cholerae, the performance of the xTAG GPP assay for these targets was further evaluated on contrived samples made using individual stool matrix spiked with varying levels of pathogen representing both the clinically relevant concentrations and concentrations that challenge the Limit of Detection (LoD) of the xTAG GPP assay. The results of testing are provided below (Table 37) and met study acceptance criteria.

42

| Target | Concentration | Agreement with
Expected Result | Mean
MFI
Value | % CV | 95% CI* | | |
|--------------------------------------------------------------------|----------------------|-----------------------------------|----------------------|-------|--------------|----------------|------------|
| Entamoeba histolytica | 5.76E+01
cells/mL | 25/25 (100%) | 1926 | 59.6% | | | |
| | 1.23E+02
cells/mL | 1/1 (100%) | 2206 | N/A | | | |
| | 3.96E+02
cells/mL | 1/1 (100%) | 2811 | N/A | | | |
| | 1.23E+03
cells/mL | 2/2 (100%) | 2647 | N/A | | | |
| | 1.23E+04
cells/mL | 2/2 (100%) | 2681 | N/A | | | |
| | 1.65E+04
cells/mL | 5/5 (100%) | 2891 | 9.5% | | | |
| | 4.00E+04
cells/mL | 4/4 (100%) | 2287 | 27.1% | | | |
| | 1.20E+05
cells/mL | 4/4 (100%) | 2322 | 26.2% | | | |
| | 4.00E+05
cells/mL | 3/3 (100%) | 2977 | 10.2% | | | |
| | 4.00E+06
cells/mL | 3/3 (100%) | 2601 | 4.7% | | | |
| Entamoeba histolytica Overall
Positive Percent Agreement | | | | | 50/50 (100%) | 92.9%-100% | |
| Negative Percent Agreement | | | | | | 100/100 (100%) | 96.1%-100% |
| Vibrio cholerae | 4.86E+06
CFU/mL | 25/25 (100%) | 1607 | 16.7% | | | |
| | 1.00E+07
CFU/mL | 4/5 (80%) | 1352 | 55.0% | | | |
| | 3.00E+07
CFU/mL | 5/5 (100%) | 1679 | 9.4% | | | |
| | 1.00E+08
CFU/mL | 5/5 (100%) | 1419 | 24.5% | | | |
| | 3.00E+08
CFU/mL | 5/5 (100%) | 1509 | 11.3% | | | |
| | 6.00E+08
CFU/mL | 5/5 (100%) | 1549 | 7.9% | | | |
| Vibrio cholerae Overall
Positive Percent Agreement | | | | | 49/50 (98%) | 89.5%-99.7% | |
| Negative Percent Agreement | | | | | | 100/100 (100%) | 96.1%-100% |

Table 37: Summary of the Results Obtained for the Analyte Positive Contrived Specimens

*Confidence intervals (CI) calculated using Cl calculator available online at http://www.vassarstats.net/prop1.html

The 50 Entamoeba histolytica contrived stool specimens had 100% (50/50) concordance with the expected positive result. The signals for the Entamoeba histolytica positive calls ranged from 225 MFI – 3752 MFI and the internal control signal (MS2) was present for all specimens. The 50

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Vibrio cholerae contrived stool specimens had 98% (49/50) concordance with the expected positive result. The one sample that did not call positive hand a signal of 91 MFI, which is near the positive call threshold of 150 MFI. The signal range for all Vibrio cholerge contrived stool specimens was 91 MFI - 2038 MFI; the signal range for the specimens which called positive for Vibrio cholerae was 834 MFI – 2038 MFI. All contrived negative stool specimens (N=50) produced the expected negative result for all analytes. The internal control was present in all the negative contrived stool specimens and produced a signal range of 180 MFI – 1266 MFI.

Supplemental Clinical Data (Simulated Stool in Cary-Blair Specimen Results)

In order to assess whether Cary-Blair results generated for Adenovirus 40/41 in the prospective study were an accurate representation of the performance of xTAG GPP for this target, contrived specimens made from individual negative stool specimens in Cary-Bair were prepared at concentration spanning the analytical detection range of the assay and tested in a randomized fashion with negative specimens. Both Adenovirus 40 and 41 cultured isolates were tested and 50% of the samples were prepared at a concentration of 2XLoD. Results of this evaluation are presented in the table below.

| Target | Source | Strain | Titer
(TCID50/mL) | Multiples of LoD
(approximated
based on real-
time PCR assay) | Number of
Contrived
Samples | Agreement with
Expected
Positive Results | 95% Confidence
Interval (CI) |
|--------------------------|-------------|--------------------|----------------------|------------------------------------------------------------------------|-----------------------------------|------------------------------------------------|---------------------------------|
| Adenovirus 40 | ATCC | Type 40
(Dugan) | 2.90 x 10+ | 2X | 13 | 100% (13/13) | |
| | | | 2.32 x 10 | 16x | 6 | 100% (6/6) | |
| | | | 9.28 x 10- | 64X | 6 | 100% (6/6) | |
| Adenovirus 40 Overall | | | | | 25 | 100% (25/25) | 86.7% -100% |
| Adenovirus 41 | Zeptometrix | Type 41
(Tak) | 1.54 x 10+ | 2X | 12 | 100% (12/12) | |
| | | | 1.23 x 10- | 16x | 7 | 100% (7/7) | |
| | | | 4.92 x 10€ | 64X | 6 | 100% (6/6) | |
| Adenovirus 41 Overall | | | | 25 | 100% (25/25) | 86.7% -100% | |
| Adenovirus 40/41 Overall | | | | 50 | 100% (50/50) | 92.9% - 100% | |

		Table 38. Summary of the results for Adenovirus 40/41 stool in Cary-Blair contrived samples
--	--	---------------------------------------------------------------------------------------------	--	--

In addition, due to the limited number of Entamoeba histolytica and Vibrio cholerae clinical samples available for testing during the clinical study, an additional study of contrived specimens in Cary-Blair was performed. A total of 150 stool in Cary-Blair contrived specimens consisting of 50 negative specimens, 50 specimens positive for Entamoeba histolytica and 50 specimens positive for Vibrio cholerae were analyzed with the xTAG GPP assay. Contrived specimens in Cary-Blair were prepared in the same manner as contrived stool specimens (see above). Results of this evaluation are summarized in the table below.

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Table 39. Summary of the results obtained for the analyte positive stool in Cary-Blair contrived specimens

| Target | Concentration | Agreement with
Expected Result | Mean MFI
Value | % CV | 95% CI* |
|-------------------------------|-------------------|-----------------------------------|-------------------|------|---------------|
| Entamoeba
histolytica | 5.76x101 Cells/mL | 22/24 (92%) | 1558 | 58% | |
| | 4.61x102 Cells/mL | 5/5 (100%) | 2249 | 29% | |
| | 9.22x102 Cells/mL | 5/5 (100%) | 2939 | 2% | |
| | 1.84x103 Cells/mL | 5/5 (100%) | 2900 | 7% | |
| | 1.00x104 Cells/mL | 5/5 (100%) | 2621 | 22% | |
| | 3.00x104 Cells/mL | 5/5 (100%) | 2660 | 34% | |
| Entamoeba histolytica Overall | | 47/49 (96%) | | | 86.3% - 98.9% |
| Vibrio cholerae | 4.68x106 CFU/mL | 25/25 (100%) | 1464 | 21% | |
| | 1.00x107 CFU/mL | 5/5 (100%) | 1818 | 3% | |
| | 3.00x107 CFU/mL | 5/5 (100%) | 1826 | 7% | |
| | 1.00x108 CFU/mL | 9/9 (100%) | 1945 | 12% | |
| | 3.00x108 CFU/mL | 6/6 (100%) | 1759 | 27% | |
| Vibrio cholerae Overall | | 50/50 (100%) | | | 92.9% - 100% |

Supplemental clinical data (Botswana Pediatric Stool Specimens)

The clinical performance of xTAG GPP for Adenovirus 40/41, Rotavirus, ETEC, Cryptosporidium and Giardia was also evaluated in a set of pediatric stool specimens (N=313) prospectively collected between February 2011 and January 2012 from symptomatic pediatric patients admitted to two referral hospitals in Botswana, Africa. All pediatric patients included in this evaluation presented with diarrhea and/or vomiting. All specimens were shipped frozen to a testing site located in Southern Ontario (Canada). As described and presented in K121454, comparator testing by nucleic acid amplification followed by bi-directional sequencing using analytically validated primers was performed on samples positive for Adenovirus 40/41, Rotavirus, ETEC, Cryptosporidium and Giardia by xTAG GPP. In order to minimize bias, a random subset of the Botswana cohort that tested negative by xTAG GPP was assessed by the same nucleic acid amplification followed by bi-directional sequencing method for Rotavirus, ETEC, Cryptosporidium and Giardia. In addition, all available clinical specimens (N=311) were assessed for Adenovirus 40/41 using the same FDA-cleared EIA as that used in the prospective study (Premier Adenoclone Type 40/41 EIA, Meridian Bioscience, K881894). Results for Adenovirus are presented below. Results for other analytes were previously presented in the submission summary for K121454.

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xTAG GPP	Adenovirus Type 40/41 EIA
	Positive	Negative	Invalid	TOTAL
Positive	342	0	0	34
Negative	181	255	0	273
Invalid	1	5	0	6
TOTAL	53	2603	0	313
	95% CI
Positive Agreement	65.4%	51.8% - 76.8%
Negative Agreement	100%	98.5% - 100%
Invalid Rate4	1.9%

² All 18 specimens that were positive for Adenovirus 40/41 by comparator but negative by xTAG GPP were positive by bidirectional sequencing only (i.e. FDA-cleared EIA negative). All these 18 specimens were assessed by real-time PCR for Adenovirus (all sub-types) at the laboratory testing site. The mean Ct value for these 18 specimens was 32.9; indicating low viral titer in these specimens, which is less clinically relevant.

² All these 34 specimens were also assessed by real-time PCR for Adenovirus (all sub-types) at the laboratory testing site. In contrast to the 18 specimens in footnote 1 above, the mean Ct value for the 34 adenovirus samples positive by the PCR/Bidirectional sequencing assay and detected by xTAG GPP in this cohort was 22.81; indicating higher viral titer in these specimens, which is more clinically relevant.

3 222 of the comparator negative Adenovirus 40/41 specimens were assessed by FDA-cleared EIA only.

4 Six out of a total of 313 samples tested by the xTAG GPP generated an "invalid" result for Adenovirus 40/41.

Nucleic acid amplification followed by bi-directional sequencing using analytically validated primers was also performed on all available clinical specimens that were positive by xTAG GPP for other analytes. The tables below summarize the confirmed xTAG GPP positive rate (i.e., confirmed xTAG GPP positives/all xTAG GPP positives) by PCR/bi-directional sequencing for Entamoeba Histolytica and Vibrio Cholerae. Results for Campylobacter, C. difficile Toxin A/B, E. coli 0157, Norovirus, Salmonella, Shigella, and STEC were previously presented in the submission summary for K121454.

xTAG GPP	PCR/Bi-directional Sequencing			TOTAL
	Positive	Negative	Not Done
Positive	0	0	0	0
Negative	NA	NA	307	307
Invalid	NA	NA	6	6
TOTAL	0	0	313	313
Confirmed Positive Rate	N/A
Invalid Rate	1.9%

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xTAG GPP	PCR/Bi-directional Sequencing
	Positive	Negative	Not Done	TOTAL
Positive	0	0	1	1
Negative	NA	NA	306	306
Invalid	NA	NA	6	6
TOTAL	0	0	313	313
Confirmed Positive Rate	N/A
Invalid Rate	1.9%

Table 42: 3X3 Table for Vibrio cholerae - Botswana Stool Sample Set

xTAG GPP detected a total of 110 mixed infections in the Botswana study. This represents 38.9% of the total number of xTAG GPP positive specimens (110/283). 8 1 (81/110; 73.6%) were double infections, 21 (21/110; 19.1%) were triple infections, 5 (5/110; 4.5%) were quadruple infections and 3 (3/110; 2.7%) were quintuple infections. The single most common co-infection was Rotavirus with Campylobacter (18/110; 16.4%). Out of the 110 co-infections, 21 contained one or more analytes that was not confirmed by bi-directional sequencing, i.e. discrepant co-infections.

A summary of the specimen failure rates for each clinical study are presented in Table 43 below.

Clinical Studies	Total # of specimens tested	Sample Failure due to PCR Inhibition		Sample Failure due to PCR Contamination
		# Re-runs	% Re-runs	# Re-runs	% Re-runs
Prospective Study	1407	241	17.1%	49	3.5%
Pre-selected Study	480	68	14.2%	21	4.4%
Botswana Study	313	6	1.9%	5	1.6%

Table 43: Summary of Sample Failure Rates in Clinical Performance Studies

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Expected Values / Reference Range

In addition to the Expected Values information for additional analytes presented in Table 26 above (summary by age groups), Table 44 details the expected values by site. Expected values for other analytes were presented in the decision summary for K121454.

Table 44: Expected Values (As determined by xTAG GPP) – Summary by Site for the xTAG GPP Prospective Clinical Evaluation (Jun 2011 to Feb. 2012)

	Overall (n=1407)		Site 1 (n=434)		Site 2 (n=428)		Site 3 (n=155)		Site 4 (n=260)		Site 5 (n=88)		Site 6 (n=42)
Target (Analyte)	No.	Expected
Value	No.	Expected
Value	No.	Expected
Value	No.	Expected
Value	No.	Expected
Value	No.	Expected
Value	No.	Expected
Value
Adenovirus 40/41	17	1.2%	8	1.8%	7	1.6%	0	0.0%	2	0.8%	0	0.0%	0	0.0%
Entamoeba
histolytica	19	1.4%	5	1.2%	7	1.6%	3	1.9%	3	1.2%	1	1.1%	0	0.0%
Vibrio cholerae	1	0.1%	0	0.0%	1	0.2%	0	0.0%	0	0.0%	0	0.0%	0	0.0%

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Instrument and System Information

Luminex 100/200 with xPONENT Software

1. Modes of Operation: Batch

Software: Hazard Analysis included in submission documentation
Specimen Identification: Users must fill in Batch Information by providing a unique batch Name, Description and Creator. Users have to enter appropriate patient information, i.e. number of samples, and sample IDs.
Specimen Sampling and Handling: DNA is extracted using the Biomérieux NucliSENS easyMAG system. Samples are manually prepared for amplification according to assay package insert and, once amplified, are transferred to a 96-well microtiter plate for analysis on the Luminex system. 5. Calibration: xMAP Calibrator Microspheres, Classification (CAL1) and Reporter (CAL2) serve as system calibrators for Luminex xMAP technology based detectors and are intended to normalize the settings for both the classification channel (CL1, CL2), the doublet discriminator channel (DD), and the reporter channel (RP1). They are not intended to be used as calibrators for a given assay.
Quality Control: xMAP Control Microspheres, Classification (CON1) and Reporter (CON2) are intended to verify the calibration and optical integrity for the Luminex 100/200 System. Classification Control Microspheres verify both classification channels and the doublet discriminator channel (DD). Reporter Control Microspheres verify the reporter channel. They are not intended to be used as controls for a given assay which are described in the specific assay package insert.