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.

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).

AI/ML Overview

Here's a summary of the acceptance criteria and study information for the xTAG Gastrointestinal Pathogen Panel (GPP) device, based on the provided text:

Acceptance Criteria and Device Performance

The acceptance criteria are generally established as ranges or thresholds for performance metrics. For specific analytes, detailed ranges for Mean MFI and %CV are provided as device performance, particularly in the reproducibility studies for Low Positive (LP) and Moderate Positive (MP) samples. Overall, the qualitative agreement with expected results and confidence intervals (95% CI) are the primary indicators of acceptance for both analytical and clinical performance.

Note on Table Content: Due to the extensive nature of the data, a comprehensive single table with all acceptance criteria and reported performance for every analyte across all studies would be extremely large. The table below focuses on general acceptance criteria mentioned for clinical performance and provides examples of reported performance for the additional analytes emphasized in this submission (Adenovirus 40/41, Entamoeba histolytica, Vibrio cholerae). For full details on all analytes from the original submission (K121454), one would need to consult that document.

Metric / Acceptance Criteria	Reported Device Performance (Examples for New Analytes)
Analytical Performance
Analytical Reactivity: Reactivity established at concentrations 2 to 3 times the limit of detection for a wide range of clinically relevant strains.	Adenovirus 40: Reactivity at 1.49E+07 Copies/mL (POS). Adenovirus 41: Reactivity at 1.43E+07 Copies/mL (POS). (Lowest reactivity titers found were 3x and 1x LoD level, respectively). Numerous clinical specimens also positive by sequencing. *E. histolytica: Reactivity titers mostly 0.4x to 6.7x LoD; one strain (ATCC 50738) at 0.2x LoD (all POS). V. cholerae*: Reactivity at 7.02E+06 CFU/mL for toxinogenic and certain non-O1 strains (all POS).
Carry-over Contamination: High Negative (HN) samples remain negative, High Positive (HP) samples remain positive (100% agreement).	Adenovirus 40: 100% of 144 HN samples remained negative, 100% of 144 HP samples remained positive, demonstrating lack of carryover contamination. (Previously demonstrated for C. difficile and Giardia).
Limit of Detection (LoD): Defined by specific titers for each analyte.	Adenovirus 40: 1.45E+01 TCID50/mL. Adenovirus 41: 7.69 TCID50/mL. E. histolytica: 2.88E+01 cells/mL. V. cholerae: 2.34E+06 CFU/mL.
Repeatability: Correct qualitative result obtained for ≥ 19 of 20 replicates at low positive level and 20 of 20 replicates at moderate positive level. Acceptable %CV for MFI values.	Adenovirus: 20/20 POS at moderate, 20/20 POS at low positive. E. histolytica: 20/20 POS at moderate, 20/20 POS at low positive. V. cholerae: 20/20 POS at moderate, 19/20 POS at low positive. (%CVs reported range from 7.83% to 34.09% for positive samples).
Analytical Specificity (Cross-reactivity/Interference): No false positives with non-probed pathogens/commensal flora; no false negatives with panel analytes in presence of high concentrations of interfering organisms or competitive interference.	*E. dispar* (Commensal flora): One strain (ATCC PRA-353) showed cross-reactivity at 3.0E+05 cells/mL, but not at 7.50E+04 cells/mL. In silico analysis explained the unlikely clinical relevance due to target mismatches. Astrovirus (Interfering pathogen): No interference with Adenovirus 40/41. Common Commensal bacteria/yeast/parasites: No interference found. Competitive Interference: No competitive interference observed between tested panel pathogens (e.g., Rotavirus/Adenovirus, C. difficile/Adenovirus). In silico analysis for other pathogens: Most showed insufficient homology. E. coli was deemed unlikely to cross-react due to Tm analysis of primer binding.
Fresh vs. Frozen Stability: Positive agreement between fresh and frozen un-extracted specimens ≥ 95% with a lower bound of 95% (two-sided) confidence interval exceeding 85%. Similar criteria for pre-treated and extracted specimens.	1-Month Stability: Adenovirus 40/41 and V. cholerae met criteria for all types. E. histolytica failed initial un-extracted, but met for pre-treated and extracted. 3-Month Stability: Adenovirus 40/41 and V. cholerae met for un-extracted and extracted. E. histolytica met for un-extracted but failed for extracted nucleic acid stability at 3 months. Overall data supports 1-month stability for un-extracted and extracted E. histolytica at -70°C to -80°C.
Precision / Reproducibility (Single Analyte): For MP, 89/90 (99%) to 90/90 (100%) positive calls. For LP, 85/90 (94%) to 90/90 (100%) positive calls. For HN, correct negative calls. Acceptable %CV for MFI values.	Overall Agreement with Expected: Adenovirus 40/41: 100% (MP, LP), 74.4% (HN). E. histolytica: 98.9% (MP), 94.4% (LP), 100% (HN). V. cholerae: 100% (MP, LP), 88.9% (HN). Lower %CV for MP, increasing for LP and HN as expected.
Precision / Reproducibility (Dual Analyte): All HP targets generate positive calls. LP targets in dual analyte tests achieve high positive call rates.	All HP targets generated positive calls (100%). For LP in dual analyte: Rotavirus (HP)/Adenovirus (LP) - 100%; Adenovirus (HP)/Rotavirus (LP) - 100%; C. difficile (HP)/Adenovirus (LP) - 98.9%; Adenovirus (HP)/C. difficile (LP) - 98.9%.
Stool in Cary-Blair LoD Equivalency: Demonstrate equivalent LoD between raw stool and stool in Cary-Blair for representative analytes.	Clostridium difficile, Giardia lamblia, and Norovirus GII confirmed equivalent LoD and similar average MFI values between raw stool and stool in Cary-Blair.
Clinical Performance
Asymptomatic Volunteers (Negative Percent Agreement): ≥97% negative percent agreement across all analytes at the specimen level.	For additional analytes, all were 100% negative (192/192) by xTAG GPP (Adenovirus 40/41, V. cholerae). E. histolytica was 99.5% (191/192). One E. histolytica positive by xTAG GPP was not confirmed by sequencing. The study confirmed ≥97% negative percent agreement overall.
Prospective Clinical Study (Stool): Sensitivity (Positive Agreement) and Specificity (Negative Agreement) with 95% CI. Quantitative cutoffs for sensitivity and specificity were generally desired to be high, though with caveats for low prevalence analytes.	Adenovirus 40/41: Sensitivity 80% (37.5%-96.4%), Specificity 98.9% (98.1%-99.3%). E. histolytica: Sensitivity N/A (0/0 positives), Specificity 98.4% (97.5%-99%). V. cholerae: Sensitivity N/A (0/0 positives), Specificity 99.9% (99.5%-100%). For low prevalence analytes, 90% positive agreement with a lower bound 95% CI was targeted in subsequent Cary-Blair studies for Norovirus and C. difficile.
Prospective Clinical Study (Stool in Cary-Blair): Clinical sensitivity (positive percentage agreement) and specificity (negative percentage agreement) acceptance criterion of 90% with a lower bound 95% confidence interval for Norovirus GI/GII and C. difficile toxin A/B. Acceptance criteria for other targets would be similar to unpreserved stool performance.	Adenovirus 40/41: Positive Agreement 40% (11.8%-76.9%), Negative Agreement 99.8% (99.3%-99.9%). E. histolytica: Positive Agreement N/A, Negative Agreement 98.4% (97.6%-99.0%). V. cholerae: Positive Agreement N/A, Negative Agreement 99.9% (99.6%-100%). Explicit 90% sensitivity/specificity with 95% CI lower bound for Norovirus and C. difficile were met as equivalent to unpreserved stool.
Retrospective Study (Pre-selected Stool): High positive agreement with reference/comparator methods.	Adenovirus 40/41: Positive agreement 100% (3/3). E. histolytica: Positive agreement 100% (1/1). V. cholerae: No positive specimens available.
Supplemental Clinical Data (Simulated Stool/Cary-Blair): High concordance with expected positive results (e.g., 100% or close to it) and expected negative results, with acceptable 95% CI.	Simulated Stool: E. histolytica 100% overall positive agreement (92.9%-100% CI), 100% negative agreement (96.1%-100% CI). V. cholerae 98% overall positive agreement (89.5%-99.7% CI), 100% negative agreement (96.1%-100% CI). Simulated Cary-Blair (Adenovirus 40/41): 100% overall positive agreement (86.7%-100% CI for each type, and 92.9%-100% overall). *Simulated Cary-Blair (E. histolytica/V. cholerae):* E. histolytica 96% overall positive agreement (86.3%-98.9% CI). V. cholerae 100% overall positive agreement (92.9%-100% CI).
Botswana Pediatric Stool Specimens: High positive and negative agreement with comparator methods.	Adenovirus 40/41: Positive Agreement 65.4% (51.8%-76.8%), Negative Agreement 100% (98.5%-100%). Lower positive agreement attributed to low viral titer in discrepant samples. No positive E. histolytica or V. cholerae specimens were detected in this cohort by xTAG GPP.

Study Details

Sample Size used for the test set and the data provenance:
- Analytical Reactivity: Empirical testing of a wide range of clinically relevant GI pathogen strains, genotypes, and isolates. For new analytes, specific strains (e.g., Adenovirus 40/41 from Zeptometrix, CDC; E. histolytica from ATCC; V. cholerae from NCTC, ATCC) were used. Additionally, sequencing analysis was performed on 9 Adenovirus 40 and 28 Adenovirus 41 clinical samples.
- Carry-over Contamination: 144 High Negative samples and 144 High Positive samples for Adenovirus 40, run in a checkerboard manner in duplicate after 6 independent extractions.
- Limit of Detection (LoD): Serial dilutions of simulated samples in negative clinical stool matrix, confirmed with at least 20 replicates of the selected dilution for each analyte.
- Repeatability: 20 replicates for each of two different analyte concentrations (LoD and 5x-10x above cut-off MFI).
- Analytical Specificity: Organisms (cross-reactivity, interference) tested at high positive titers, panel analytes at low positive concentrations. Specific numbers of organisms are not consistently provided for all tests but mentioned for E. dispar strains (ATCC PRA-353, PRA-368).
- Fresh vs. Frozen Stability: Sets of simulated specimens prepared at Low Positive, Moderate Positive, and High Positive concentrations. Specific numbers are not explicitly stated for each level but indicate replicates were run.
- Precision/Reproducibility: 90 replicates for each single analyte and dual analyte sample (3 replicates per run x 5 runs per operators per site x 3 sites).
- Stool in Cary-Blair Media LoD Study: Serial dilutions for Clostridium difficile, Giardia lamblia, Norovirus GII (representative analytes). 20 replicates for each LoD titer.
- Asymptomatic Volunteers (Clinical Study): 200 clinical stool samples. Data was analyzed for 192 samples due to PCR inhibition.
- Prospective Clinical Study (Stool): 1407 clinical specimens. Collected prospectively between June 2011 and February 2012 from pediatric and adult patients. Data provenance: 4 laboratories in the United States (Arizona, Missouri, Tennessee, Texas) and 2 in Southern Ontario (Canada).
- Prospective Clinical Study (Stool in Cary-Blair): A subset of samples from the 1407 prospective study for which adequate sample was available.
- Pre-selected Stool Specimens (Retrospective Study): 207 archived stool specimens. Collected retrospectively from multiple sites in North America, Africa, and Europe.
- Pre-selected Stool in Cary-Blair Specimens (Retrospective Study): Remnants of available pre-selected frozen stool specimens mixed proportionally with Cary-Blair.
- Supplemental Clinical Data (Simulated Stool/Cary-Blair): 50 negative, 50 positive E. histolytica, 50 positive V. cholerae simulated stool specimens. 50 negative, 50 positive Adenovirus 40/41, 50 positive E. histolytica, 50 positive V. cholerae simulated stool in Cary-Blair specimens.
- Botswana Pediatric Stool Specimens (Retrospective Study): 313 pediatric stool specimens. Collected prospectively between February 2011 and January 2012 from symptomatic pediatric patients in Botswana, Africa.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience):

The document does not explicitly state the number or specific qualifications (e.g., years of experience) of experts involved in establishing ground truth. However, it indicates:
- Reference/Comparator methods: For the clinical studies, reference/comparator methods were used at central laboratories independent of xTAG GPP testing sites. These methods included Premier Adenoclone Type 40/41 EIA, Amplification + sequencing (one NAAT+), Microscopy, and Bacterial culture.
- Sequencing analysis: Bi-directional sequencing with analytically validated primers was performed. This implies expertise in molecular biology and sequence analysis.
- Clinical evaluation: The study was conducted at 6 independent laboratories (4 in US, 2 in Canada) for the prospective study, and 4 sites for the retrospective study (3 external to Luminex Molecular Diagnostics - LMD). This suggests that the ground truth assessment, particularly for clinical diagnosis and interpretation of comparator methods, involved multiple trained laboratory personnel and clinicians.
Adjudication method (e.g. 2+1, 3+1, none) for the test set:

The document describes confirmatory testing for discrepant results, particularly in the retrospective and Botswana studies. This often implies an adjudication process:
- For positive xTAG GPP results not pre-selected at the banking site, confirmatory testing by nucleic acid amplification followed by bi-directional sequencing was performed to determine if these were True Positive or False Positive.
- For certain analytes in the Botswana study, a random subset of xTAG GPP negative samples was also assessed by nucleic acid amplification and bi-directional sequencing.
- 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).
- These actions suggest a form of discrepant analysis, where a more definitive "gold standard" (like sequencing) is used to adjudicate differences. However, a formal "m+n" type adjudication (e.g., 2+1, 3+1) involving a panel of experts for every case is not explicitly stated. The comparator methods themselves serve as the primary reference standard, with sequencing as a confirmatory tool for discrepancies.
If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

This device is a diagnostic assay (in vitro diagnostic device), not an AI-assisted imaging or diagnostic tool that involves human readers interpreting results with or without AI assistance. Therefore, a multi-reader multi-case (MRMC) comparative effectiveness study focusing on human reader improvement with AI vs. without AI assistance was not performed. The evaluation is focused on the standalone performance of the assay itself compared to existing reference methods.
If a standalone (i.e. algorithm only without human-in-the loop performance) was done:

Yes, a standalone performance evaluation was done. The entire analytical and clinical performance studies described (analytical reactivity, LoD, repeatability, specificity, stability, reproducibility, clinical prospective, retrospective, and supplemental studies) are all assessments of the algorithm-only performance of the xTAG GPP device. The device automates the detection and identification of nucleic acids from pathogens, and the results are qualitatively interpreted by the xTAG Data Analysis Software (TDAS GPP (US)) which applies algorithms to MFI values to generate a "positive" or "negative" result. Human involvement is in sample preparation, running the instrument, and reviewing the generated report, but the "performance" discussed is that of the assay and its associated software for detection, not human interpretation aided by AI.
The type of ground truth used (expert consensus, pathology, outcomes data, etc):

The ground truth was primarily established through reference/comparator methods, which varied by pathogen:
- Composite Comparator: For Adenovirus 40/41 (Premier Adenoclone Type 40/41 EIA + Amplification + sequencing).
- Bacterial Culture: For Vibrio cholerae.
- Microscopy followed by Amplification + Sequencing: For Entamoeba histolytica (positive specimens by microscopy only).
- Nucleic Acid Amplification Tests (NAATs) followed by bi-directional sequencing: Used for confirmatory testing of discrepant results across various analytes.
- In-house real-time PCR: Used for some confirmations (e.g., Giardia, Adenovirus).
  These methods represent established diagnostic assays and molecular techniques, often considered "gold standards" or highly accurate reference methods in clinical microbiology.
The sample size for the training set:

The document describes various analytical and clinical studies, but it does not explicitly mention a "training set" in the context of machine learning model development. This is typical for traditional in vitro diagnostic devices. The performance evaluation focuses on validation, not iterative training and testing of an AI model.

However, if we broadly interpret "training set" as data used during the development and initial optimization before formal validation:
- The document states that the reagents were unchanged from a previous submission (K121454). This implies development and some level of internal testing and optimization (akin to a training phase) would have occurred prior to that submission using various pathogen strains and clinical samples to establish the assay's parameters.
- The "Analytical Reactivity" studies involve testing a wide range of strains to establish reactivity, which could be considered part of an iterative development process that feeds into refinement.
Without specific details on Luminex's internal development process, a distinct "training set" with a defined sample size for an AI algorithm is not identified in this regulatory submission.
How the ground truth for the training set was established:

Since a discrete "training set" for an AI algorithm is not explicitly identified, the method for establishing ground truth for such a set is also not detailed.
However, for the general development and optimization of the assay:
- Known strains and characterized clinical specimens: Analytical studies like analytical reactivity and LoD determination use well-characterized, often commercially available, strains (e.g., ATCC, Zeptometrix, NCTC) and high-titer clinical specimens where the presence and concentration of the pathogen are already known through established microbiological and molecular techniques. These serve as the "ground truth" for calibrating and optimizing assay parameters (like MFI cut-offs) during development.
- Reference methods: Any prior development would have relied on the same types of reference methods (culture, EIA, PCR/sequencing) that were used to establish ground truth in the formal validation studies.

Summary

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

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

September 16, 2014

Re: K140377

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

Dear Ms. Ip:

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

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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

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Sincerely yours,

Uwe Scherf -S for

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

Enclosure

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Indications for Use

Form Approved: OMB No. 0910-0120

510(k) Number (if known)

Device Name

xTAG® Gastrointestinal Pathogen Panel (GPP)

Indications for Use (Describe)

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

Viruses

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

Bacteria

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

Parasites

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

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

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

The results of this test should not be used as for diagnosis, treatment, or other patient management decisions. Confirmed positive results do not rule out co-infection with other organisms that are not detected by this test, and may not be the sole of patient illness. Negative xTAG Gastronitestinal Pathogen Panel results in the setting of cliness compatible with gastroenteriis may be due to infection by pathogens that are notinfectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

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

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

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

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

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

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

FOR FDA USE ONLY

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

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510(k) Summary

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

510(k) Number: K140377

Submission Type: Traditional 510(k), New Device

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

Type of Test: Qualitative nucleic acid multiplex test

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

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

ProductCode	Classification	Regulation Section	Review Panel
PCH	II	21CFR866.3990 Gastrointestinal Pathogen PanelMultiplex 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 K121454xPONENT 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:

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)

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

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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:

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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 andAmplification Method(Same)	Multiplex end point RT-PCR	Multiplex end point RT-PCR
Kit Reagents (Same)	xTAG GPP Primer Mix, xTAG OneStepEnzyme Mix, xTAG OneStep Buffer,xTAG RNase-Free Water, xTAG BSA,xTAG MS2, xTAG GPP Bead Mix, xTAGReporter Buffer, xTAG 0.22 SAPE	xTAG GPP Primer Mix, xTAG OneStepEnzyme Mix, xTAG OneStep Buffer,xTAG RNase-Free Water, xTAG BSA,xTAG MS2, xTAG GPP Bead Mix, xTAGReporter Buffer, xTAG 0.22 SAPE
Test Format (Same)	Multiplex MAGPLEX bead-baseduniversal array	Multiplex MAGPLEX bead-baseduniversal array
Detection Method (Same)	Fluorescence based	Fluorescence based
Quality Control (Same)	Internal Control (MS2), rotating analytecontrols and negative control (RNAse-free water)	Internal Control (MS2), rotatinganalyte controls and negative control(RNAse-free water)
Results (Same)	Qualitative	Qualitative
Instrument SoftwareSystem	Luminex 100/200 with xPONENTSoftware	Luminex 100/200 with xPONENTSoftware

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. Iarionly)
		• 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 soleor 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 DiagnosticDevices for the Detection of Clostridium difficile	Nov. 29, 2010
2	Class II Special Controls Guidance Document: Norovirus SerologicalReagents	Mar. 9, 2012
3	Class II Special Controls Guidance Document: Instrumentation forClinical Multiplex Test Systems - Guidance for Industry and FDA Staff	Mar. 10, 2005
4	Guidance for the Content of Premarket Submissions for SoftwareContained 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 DemonstratingSubstantial Equivalence in Premarket Notifications - Final Guidance	Mar. 20, 1998
8	The 510(k) Program: Evaluating Substantial Equivalence in PremarketNotifications [510(k)]	Dec. 27, 2011
9	Guidance for Industry and Food and Drug Administration Staff - eCopyProgram for Medical Device Submissions	Oct. 10, 2013
10	Guidance for Industry and Food and Drug Administration Staff - FDA andIndustry Actions on Premarket Notification (510(k)) Submissions: Effecton FDA Review Clock and Goals	Oct. 15, 2012

Table 4: Standards

	StandardNo.	RecognitionNumber(FDA)	Standards Title	Date
।	EP05-A2	7-110	Evaluation of Precision Performance of Quantitativemeasurement Methods (2nd ed.)	10/31/2005
2	EP07-A2	7-127	Interference Testing in Clinical Chemistry (2ndedition)	05/21/2007
ന	EP12-A2	7-152	User Protocol for Evaluation f Qualitative TestPerformance (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 andTrueness (2nd edition)	09/09/2008
ട	EP17-A	7-194	Protocol for Determination of Limits of Detectionand Limits of Quantitation(NOTE: Original studies included this standard)	03/28/2009
7	EP17-A2	7-233	Evaluation of Detection Capability for ClinicalLaboratory 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 InfectiousDiseases (2nd edition)	09/09/2008

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

StandardNo.	RecognitionNumber(FDA)	Standards Title	Date
10	MM13-A	7-191	Collection, Transport, Preparation and Storage ofSpecimens	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	ReactivityTitre(Copies/mL)	Results Summary
Analytical reactivity_II_LX200	Adenovirus40	CDC – GP-093	DuganpCMK2Gr10, 9/23/91	1.49E+07	POS
Analytical reactivity_II_LX200	Adenovirus41	CDC – GP-094	TakHeLa2Gr10, 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 orCopies/mL)	ResultsSummary
20120216_JF_GPP_Reactivity_LX	Entamoebahistolytica	ATCC 30015	(HK-9, colonic biopsyfrom adult humanmale with amebicdysentery, Korea);frozen	2.86E+00 Cells/mL,or 1.82E+02Copies/mL	POS
20120216_JF_GPP_Reactivity_LX	Entamoebahistolytica	ATCC 30190	(HB-301:NIH, fecesfrom adult humanmale with amebicdysentery, Burma,1960); test tube	1.07E+03Copies/mL	POS
20120216_JF_GPP_Reactivity_LX	Entamoebahistolytica	ATCC 30457	(HU-21:AMC, colonicbiopsy from malechild with amebicdysentery, LittleRock, AR, 1970); testtube	1.68E+03Copies/mL	POS
20120216_JF_GPP_Reactivity_LX	Entamoebahistolytica	ATCC 30458	(200:NIH); frozen	1.83E+02 Cells/mL,or 2.42E+03Copies/mL	POS
20120216_JF_GPP_Reactivity_LX	Entamoebahistolytica	ATCC 30459	(HM-1:IMSS [ABRM];feces from adulthuman male,asymptomatic cystpasser, England,1972); test tube	1.83E+02 Cells/mL,or 1.10E+03Copies/mL	POS
20120314_JF_GPP_React_LX	Entamoebahistolytica	ATCC 30889	(H-458:CDC[ATCC30217], fecesfrom human adultfemale with amebicdysentery, Asia (?),(patient in U.S. fortreatment), 1971);test tube	8.78E+02Copies/mL	POS
20120411_JF_GPP_React_LX	Entamoebahistolytica	ATCC 30923	(HU-2:MUSC)	4.98E+02Copies/mL	POS
20120207_JF_GPP_Reactivity	Entamoebahistolytica	ATCC 30925	(HU-1:CDC, feces offemale child,asymptomatic, sero-negative cyst passer,Cherokee, NC, 1978)	1.89E+02Copies/mL	POS
20120411_JF_GPP_React_LX	Entamoebahistolytica	ATCC 50007	DKB	2.88E+03Copies/mL	POS
20120411_JF_GPP_React_LX	Entamoebahistolytica	ATCC 50481	SD157	1.36E+03Copies/mL	POS
20120411_JF_GPP_React_LX	Entamoebahistolytica	ATCC 50738	Rahman	8.90E+01Copies/mL	POS
20120411_JF_GPP_React_LX	Entamoebahistolytica	ATCC 50454	HB-301:NIH	1.08E+03Copies/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)	ResultsSummary
20120827-JX-V cholera-AR-LX	Vibrio choleraePacini	NCTC 30	Non-O:1, ATCC4735;MARTIN 1	6.00E+08	NEG
20120827-JX-V cholera-AR-LX	Vibrio cholerae	NCTC 4714	Non-O:1, Isolatedfrom pilgrims in ElTor 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) — referencestrain for 0:139serovar	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 choleraePacini	ATCC 14033	O:1, El Tor DO1930;CN 5774;R.Hugh 1092, SerotypeInaba, Non-toxinogenic	1.50E+08	NEG
20120404-JX-AnaReact-Vibrio-LX	Vibrio choleraeasiaticae (Trevisan)Pfeiffer	ATCC 14035	O:1, Serotype Ogawa[7787]	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-LX	Vibrio choleraePacini	ATCC 14101	O:1, SerotypeOgawa, clinicalspecimen – human([185754] choleraepidemic circa 1960,Calcutta) CalcuttaIndia	7.02E+06	POS
20120406-JX-AnaReact-Vibrio2-LX	Vibrio choleraePacini	ATCC 14374	Non-O:1 (NAG),5035; R. Hugh 1513	1.50E+08	NEG
20120921-MB-VibrioAnalytical-LX	Vibrio choleraePacini	ATCC 14730	Non-O:1 (SerovarO:2), biovar El Tor,Subgroup III ofGardner andVenkatraman, NCTC4711, 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)	ResultsSummary
20120921-MB-VibrioAnalytical-LX	Vibrio choleraePacini	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 choleraePacini	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 choleraePacini	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 choleraePacini	ATCC 25870	O:1, Serotype Inaba	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-LX	Vibrio choleraePacini	ATCC 25872	Non-O:1 (NAG), Isolated from a patient with clinical cholera	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-LX	Vibrio choleraePacini	ATCC 25873	Non-O:1 (NAG), Isolated from a patient with clinical cholera	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-LX	Vibrio choleraePacini	ATCC 51394	O:139 (Non-O:1 [NAG]), Cholera patient, Madras, India	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-LX	Vibrio choleraePacini	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

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

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

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

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

Carry-over Contamination

The likelihood of carry-over contamination events was initially assessed and presented in K121454 by testing 2 representative pathogens (a bacteria and a parasite): C. difficile, and Giardia respectively. In this study, a representative virus (Adenovirus 40) was tested. This analyte was examined in the form of simulated samples prepared at concentrations just below the assay cut-off (High Negative, HN) and well above the assay cut-off (High Positive, HP). The target was examined in a set of 6 independent extractions. Each extraction was assayed in duplicate arranged in a checkerboard manner on a 96-well plate using xTAG GPP. As with the results in K121454 for the representative bacteria (C. difficile) and parasite (Giardia), results with the virus (Adenovirus 40) showed that all 144 high negative samples remained negative when run on the Luminex 100/200 instrument for all three targets (100% HN). In addition, results for Adenovirus 40 showed that all 144 high positive samples remained positive when run on the Luminex 100/200 instrument (100% HP), as with the targets previously tested. Therefore a lack of carryover contamination has been demonstrated.

Limit of Detection

As in the original study results presented for K121454, the LoD was assessed by analyzing serial dilutions of simulated samples made from high-titre stocks of commercial strains or high-titre clinical specimens (when commercial strains were not available). All simulated specimens were prepared in negative clinical matrix (stool). The data from serial dilutions were confirmed in at least 20 replicates of the selected dilution for each analyte target. Results of testing for the three additional analytes were as follows:

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		Titre (correspondingto the estimated	AverageMFI
Analyte	Strain ID	LoD)	Value	%CV
Adenovirus40/41	Adenovirus 40, 0810084CF (Dugan)	$1.45E+01$ TCID50/mL	548	34.09%
Adenovirus40/41	Adenovirus 41, 0810085CF (Tak)	$7.69$ TCID50/mL	360	22.04%
Entamoebahistolytica	Entamoeba histolytica, 30890	$2.88E+01$ cells/mL	883	16.96%
Vibriocholerae	Vibrio cholerae, 14101 (SerovarO: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 GPPCalls	Mean MFIValue	%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 InterferingOrganism (concentration)	Source	InterferenceYes (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 #2Norovirus (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 #2Norovirus (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

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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-extracted1 month	Un-extracted3 months	Pre-Treated1 month	Extracted1 month	Extracted3 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.

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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.

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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).

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Luminex.

	Panel Member ID	Adenovirus40/41Low Positive	Adenovirus40/41MediumPositive	Adenovirus40/41High Negative	EntamoebahistolyticaLow Positive	EntamoebahistolyticaMediumPositive	EntamoebahistolyticaHigh Negative	Vibrio choleraeLow Positive	Vibrio choleraeMediumPositive	Vibrio choleraeHigh Negative
	Concentration	1.45E+01TCID50/mL	5.8E+01TCID50/mL	1.81 TCID50/mL	1.44E+01Cells/mL	5.76E+01Cells/mL	2.25E-01Cells/mL	9.37E+06CFU/mL	3.75E+07CFU/mL	5.86E+05CFU/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 ExpectedResult	30/30100%	30/30100%	22/3073.3%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%			Rotavirus ALow Positive	Adenovirus40/41 HighPositive	Rotavirus AHigh Positive	Adenovirus40/41Low Positive	Adenovirus40/41 LowPositive	C. difficileHigh Positive		Adenovirus40/41 HighPositive	C. difficileLow 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 withExpected Result	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%
	% 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 ExpectedResult	30/30100%	30/30100%	15/3050%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	21/3070%		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 withExpected Result	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%
	% 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 ExpectedResult	30/30100%	30/30100%	30/30100%	25/3083.3%	29/3096.7%	30/30100%	30/30100%	30/30100%	29/3096.7%	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 withExpected Result	30/30100%	30/30100%	30/30100%	30/30100%	29/3096.7%	30/30100%	30/30100%	30/30100%	29/3096.7%	30/30100%
	% 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 withExpected Result	90/90100%	90/90100%	67/9074.4%	85/9094.4%	89/9098.9%	90/90100%	90/90100%	90/90100%	80/9088.9%	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 withExpected Result	90/90100%	90/90100%	90/90100%	90/90100%	89/9098.9%	90/90100%	90/90100%	90/90100%	89/9098.9%	90/90100%
	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 PercentileMFI	430.5	1645.0	1361.0	292.0	269.5	1648.0	2483.0	1630.0	406.0	1219.0
Overall MedianMFI Value	741.8	1948.8	1614.8	444.3	447.0	2122.3	2706.3	2004.8	575.8	1452.8
Overall 75th PercentileMFI	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

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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 Differencebetween Stooland Stool in Cary-Blair
Analyte	Strain ID	Titre atlimit ofdetection	Average MFIValue (n=20)	Titre atlimit ofdetection	Average MFIValue (n=20)
C. difficileToxin A/B	Clostridiumdifficile,BAA-1805(toxinotypeIII A+B+)	$4.69E+05$CFU/mL	Probe 1 = 383Probe 2 = 862	$4.69E+05$CFU/mL	Probe 1 = 460Probe 2 = 1128	None
Giardia	Giardialamblia,PRA-243	$2.20E+02$cells/mL	1069	$2.20E+02$cells/mL	1008	None
NorovirusGI/GII	NorovirusGII, Clinicalsample,sourceToronto	$4.75E+02$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(includingallowable re-runs)	Total # of runswith at least oneNC failure	% totalruns withat leastone NCfailure	Total No. ofNCs includedin runs andallowable re-runs	Total No.of NCfailures	% total NC s includedwhich 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 andinterference	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.

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Studies	Total # ofspecimenstested	Total # ofinvalid resultsprior to re-run	% invalidresults priorto re-run	% invalid resultsafter re-run
Multi-site reproducibility	5065	25	0.49%	0.00%
Matrix equivalence	180	0	0.00%	0.00%
Limit of detection	992	1	0.10%	0.00%
Carry-over contamination	864	0	0.00%	0.00%
Analytical specificity andinterference	1472	0	0.00%	0.00%
Analytical reactivity	2225	1	0.04%	0.00%
Evaluation of fresh vs.frozen stool	4162	2	0.05%	0.00%
Overall	14960	29	0.19%	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 byxTAG GPP for all samples	Percent Negative Results by xTAG GPPfor 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

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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 (MeridianBioscience, K881894)^ directly on the stool specimen and Amplification + sequencing directlyfrom clinical specimen using one NAAT+
Entamoebahistolytica	Microscopy followed by amplification + sequencing directly from clinical specimens using oneNAAT+ (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

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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.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue
Adenovirus40/41	17	1.2%	0	0.0%	2	10.0%	0	0.0%	10	1.1%	5	1.2%
Entamoebahistolytica	19	1.4%	0	0.0%	0	0.0%	1	1.3%	14	1.6%	4	0.9%
Vibriocholerae	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 InvalidxTAG GPPResults due toPCR 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
Entamoebahistolytica	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 InvalidxTAG GPPResults due toPCR Inhibition
Analyte	TP /(TP+FN)	percent	95%CI	TN /(TN+FP)	percent	95%CI
Adenovirus40/41	4/5	80%	37.5% - 96.4%	1154/1167	98.9%	98.1% - 99.3%	235
C. difficile ToxinA/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%
Entamoebahistolytica	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 difficiletoxin 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 AgreementTP / (TP+FN)	Positive AgreementPercent	95%CI for PositiveAgreement	Number Invalid xTAG GPFResults
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
Entamoebahistolytica	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%ConfidenceInterval (CI)	Number ofInvalidResults
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.

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Target	Concentration	Agreement withExpected Result	MeanMFIValue	% CV	95% CI*
Entamoeba histolytica	5.76E+01cells/mL	25/25 (100%)	1926	59.6%
	1.23E+02cells/mL	1/1 (100%)	2206	N/A
	3.96E+02cells/mL	1/1 (100%)	2811	N/A
	1.23E+03cells/mL	2/2 (100%)	2647	N/A
	1.23E+04cells/mL	2/2 (100%)	2681	N/A
	1.65E+04cells/mL	5/5 (100%)	2891	9.5%
	4.00E+04cells/mL	4/4 (100%)	2287	27.1%
	1.20E+05cells/mL	4/4 (100%)	2322	26.2%
	4.00E+05cells/mL	3/3 (100%)	2977	10.2%
	4.00E+06cells/mL	3/3 (100%)	2601	4.7%
Entamoeba histolytica OverallPositive Percent Agreement					50/50 (100%)	92.9%-100%
Negative Percent Agreement						100/100 (100%)	96.1%-100%
Vibrio cholerae	4.86E+06CFU/mL	25/25 (100%)	1607	16.7%
	1.00E+07CFU/mL	4/5 (80%)	1352	55.0%
	3.00E+07CFU/mL	5/5 (100%)	1679	9.4%
	1.00E+08CFU/mL	5/5 (100%)	1419	24.5%
	3.00E+08CFU/mL	5/5 (100%)	1509	11.3%
	6.00E+08CFU/mL	5/5 (100%)	1549	7.9%
Vibrio cholerae OverallPositive 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(approximatedbased on real-time PCR assay)	Number ofContrivedSamples	Agreement withExpectedPositive Results	95% ConfidenceInterval (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 withExpected Result	Mean MFIValue	% CV	95% CI*
Entamoebahistolytica	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.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue
Adenovirus 40/41	17	1.2%	8	1.8%	7	1.6%	0	0.0%	2	0.8%	0	0.0%	0	0.0%
Entamoebahistolytica	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.

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).