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 GPP 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 noninfectious 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 test is indicated for use with the Luminex® 100/200™ and MAGPIX® instruments 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

The provided text describes the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) and various studies supporting its performance. I will extract the requested information based on the additional analytes (Adenovirus 40/41, Entamoeba histolytica, and Vibrio cholerae) while noting that much of the original device's performance data (from K121894) is still applicable.

Description of Acceptance Criteria and Studies Proving Device Performance

The Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid test designed for the qualitative detection and identification of multiple viral, bacterial, and parasitic nucleic acids in human stool specimens or human stool in Cary-Blair media. This submission (K140647) primarily focuses on the addition of Adenovirus 40/41, Entamoeba histolytica, and Vibrio cholerae to the panel, as well as the use of stool in Cary-Blair media.

The device performance was evaluated through analytical studies (Analytical Reactivity, Carry-over Contamination, Limit of Detection, Repeatability, Analytical Specificity/Interference, Fresh vs. Frozen Stool Evaluation, Reproducibility/Precision) and clinical studies (Detection in Asymptomatic Volunteers, Detection in Symptomatic Patients from prospective and retrospective cohorts, and simulated samples).

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for clinical performance are primarily inferred from the detailed reporting of Positive Percent Agreement (PPA) / Sensitivity and Negative Percent Agreement (NPA) / Specificity with their 95% Confidence Intervals (CI). Analytical performance criteria are noted qualitatively (e.g., "100% agreement," "no interference").

Clinical Performance Acceptance Criteria (Inferred from reporting, generally striving for high agreement):

Sensitivity/Positive Agreement: Typically >90% with lower bound 95% CI >80% (explicitly stated for Norovirus GI/GII and Clostridium difficile toxin A/B in Cary-Blair media, and implied for other analytes by robust performance).
Specificity/Negative Agreement: Typically >90% with lower bound 95% CI >90% (explicitly stated for all targets in Cary-Blair media, and implied for other analytes by robust performance).

Analyte (Additional)	Study Type	Acceptance Criteria (Inferred/Stated)	Reported Device Performance (Summary)
Analytical Studies
Adenovirus 40/41	Analytical Reactivity	Reactivity established at concentrations 2-3x LoD.	Reactivity demonstrated with various strains (Dugan, Tak) at 1.49E+07 and 1.43E+07 Copies/mL (CDC samples). 9 Adenovirus 40 and 28 Adenovirus 41 clinical samples positively detected and confirmed by sequencing.
Entamoeba histolytica	Analytical Reactivity	Reactivity established at concentrations 2-3x LoD.	Reactivity demonstrated with various ATCC strains ranging from 8.90E+01 to 2.88E+03 Copies/mL, generally corresponding to 0.4x to 6.7x LoD, with one strain (ATCC 50738) at 0.2x LoD.
Vibrio cholerae (ctx gene)	Analytical Reactivity	Reactivity established at concentrations 2-3x LoD.	Reactive with 12 out of 22 tested strains including O:1, O:139, and non-O:1 strains expressing ctx gene, at 7.02E+06 CFU/mL (3xLoD).
Adenovirus 40	Carry-over Contamination	No false positives from HP, no false negatives from HN.	100% of 144 high negative samples remained negative; 100% of 144 high positive samples remained positive. Lack of carryover contamination demonstrated.
Adenovirus 40/41, E. histolytica, V. chol	Limit of Detection (LoD)	LoD determined per analyte with CV%.	Adenovirus 40: 1.45x10^1 TCID50/mL (34.26% CV); Adenovirus 41: 7.69 TCID50/mL (20.27% CV); Entamoeba histolytica: 2.88x10^1 cells/mL (17.77% CV); Vibrio cholerae: 2.34x10^6 CFU/mL (23.94% CV).
Adenovirus 40/41, E. histolytica, V. chol	Repeatability	20/20 correct calls for Low and Moderate Positive.	Adenovirus 40/41: LP: 20/20 POS, MP: 20/20 POS; Entamoeba histolytica: LP: 20/20 POS, MP: 20/20 POS; Vibrio cholerae: LP: 20/20 POS, MP: 20/20 POS.
E. dispar (cross-reactivity)	Analytical Specificity	No cross-reactivity with commensal flora.	One strain (ATCC PRA-353) cross-reacted at 3.0E+05 cells/mL (>10^4 times LoD for E. histolytica), but not at 4-fold lower titer (7.50E+04 cells/mL). In silico analysis confirmed reverse primer mismatches for E. dispar reducing amplification efficiency, implying negligible risk of false positives at clinically relevant titers.
Adenovirus 40/41	Interference (non-panel)	No interference at LP with HP non-panel organisms.	No interference observed from Astrovirus (HP) on Adenovirus serotypes 40/41 (LP).
Adenovirus, E. histolytica, V. chol.	Interference (commensal)	No interference at LP with HP commensal organisms.	No interference found with xTAG GPP analytes Adenovirus, Entamoeba histolytica, and Vibrio cholerae when tested with 10 different common commensal bacteria, yeast, and parasites at high concentrations.
Adenovirus (competitive)	Interference (competitive)	No competitive interference with other panel pathogens.	No competitive interference observed between Adenovirus and Norovirus, Salmonella, or Campylobacter jejuni at LP/HP combinations.
E. histolytica (stability)	Fresh vs. Frozen Stool (1M)	Positive agreement ≥95% (lower bound 95% CI >85%).	Un-extracted: Not met. Pre-treated: Met. Extracted: Met. (Supplemental testing supported un-extracted stability for 1 month).
E. histolytica (stability)	Fresh vs. Frozen Stool (3M)	Positive agreement ≥95% (lower bound 95% CI >85%).	Un-extracted: Met. Pre-treated: Not reported. Extracted: Not met. (Overall data supports stability for 1 month).
Adenovirus 40/41, V. chol.	Fresh vs. Frozen Stool (1M)	Positive agreement ≥95% (lower bound 95% CI >85%).	Un-extracted: Met. Pre-treated: Met. Extracted: Met.
Adenovirus 40/41, V. chol.	Fresh vs. Frozen Stool (3M)	Positive agreement ≥95% (lower bound 95% CI >85%).	Un-extracted: Met. Pre-treated: Not reported. Extracted: Met.
Adenovirus 40/41, E. histolytica, V. chol	Reproducibility (Single)	LP: 82/90 to 90/90 correct calls; MP: 86/90 to 90/90 correct calls.	Adenovirus 40/41: LP: 89/90 (98.9%), MP: 89/90 (98.9%); Entamoeba histolytica: LP: 82/90 (91.1%), MP: 86/90 (95.6%); Vibrio cholerae: LP: 90/90 (100%), MP: 90/90 (100%). HN variability noted.
Adenovirus (dual analyte)	Reproducibility (Dual)	Positive call for HP (100%) and LP (high agreement).	HP: 100% positive calls. LP: 4/90 (C.difficile HP/Adenovirus LP) and 2/90 (Rotavirus HP/Adenovirus LP) generated negative calls for Adenovirus. Overall, adequate site-to-site reproducibility established.
C. difficile, Giardia, Norovirus GII	Stool in Cary-Blair LoD	Equivalent LoD between raw stool and Cary-Blair.	Equivalent LoD demonstrated for all three representative analytes (C. difficile Toxin A/B, Giardia lamblia, Norovirus GII) in raw stool vs. stool in Cary-Blair media.
Clinical Studies
Adenovirus 40/41 (Prospective Stool)	Diagnostic Accuracy	PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%).	PPA: 80% (4/5) with 95% CI: 37.5%-96.4%. NPA: 98.5% (1158/1175) with 95% CI: 97.7%-99.1%. (Note: Low positivity rates impacted CI width).
Entamoeba histolytica (Prospective Stool)	Diagnostic Accuracy	PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%).	PPA: N/A (0/0 positives by comparator). NPA: 98.3% (1154/1174) with 95% CI: 97.4%-98.9%. (Note: No positive by comparator).
Vibrio cholerae (Prospective Stool)	Diagnostic Accuracy	PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%).	PPA: N/A (0/0 positives by comparator). NPA: 99.7% (1171/1174) with 95% CI: 99.2%-99.9%. (Note: No positive by comparator).
Adenovirus 40/41 (Prospective Cary-Blair)	Diagnostic Accuracy	PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%).	PPA: 33.3% (2/6) with 95% CI: 9.7%-70.0%. NPA: 99.6% (1285/1290) with 95% CI: 99.1%-99.8%. (Note: PPA reduced due to low titer specimen and unavailable samples, supplemental data provided).
Entamoeba histolytica (Pre-selected)	Positive Agreement	PPA >90%.	100% (1/1) with 95% CI: 2.5%-100%. (Note: Very limited sample size).
Adenovirus 40/41 (Pre-selected)	Positive Agreement	PPA >90%.	100% (3/3) with 95% CI: 43.8%-100%. (Note: Limited sample size).
Adenovirus 40/41 (Botswana Pediatric)	Diagnostic Accuracy	PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%).	PPA: 67.3% (35/52) with 95% CI: 53.7%-78.5%. (Note: 17 false negatives were low viral titer by RT-PCR). NPA: 100% (255/255) with 95% CI: 98.5%-100%.
Entamoeba histolytica (Contrived Stool)	Positive/Negative Agreement	PPA >90% ; NPA >90%.	PPA: 100% (50/50) with 95% CI: 92.9%-100%. NPA: 100% (100/100) with 95% CI: 96.1%-100%.
Vibrio cholerae (Contrived Stool)	Positive/Negative Agreement	PPA >90% ; NPA >90%.	PPA: 98% (49/50) with 95% CI: 89.5%-99.7%. NPA: 100% (100/100) with 95% CI: 96.1%-100%.
Adenovirus 40/41 (Contrived Cary-Blair)	Positive Agreement	PPA >90%.	PPA: 100% (50/50 overall) with 95% CI: 92.9%-100%.
E. histolytica (Contrived Cary-Blair)	Positive Agreement	PPA >90%.	PPA: 96% (47/49 overall) with 95% CI: 86.3%-98.9%.
Vibrio cholerae (Contrived Cary-Blair)	Positive Agreement	PPA >90%.	PPA: 100% (50/50 overall) with 95% CI: 92.9%-100%.

2. Sample size used for the test set and the data provenance

Asymptomatic Volunteers (Test Set): 200 clinical stool samples.
- Data Provenance: Retrospective, collected from healthy, asymptomatic donors. Country of origin not specified, but collected for this study.
Symptomatic Patients (Prospective Clinical Study in Stool Specimens) (Test Set): 1407 clinical stool specimens.
- Data Provenance: Prospective, collected between June 2011 and February 2012. Origin: Four laboratories in the United States (Arizona, Missouri, Tennessee, Texas) and two in Southern Ontario (Canada).
Symptomatic Patients (Prospective Clinical Study in Stool in Cary-Blair Media) (Test Set): A subset of the 1407 prospective samples for which adequate sample was available. The exact number varied per analyte for each comparison (e.g., Adenovirus 40/41 had N=6 positives in Cary-Blair for PPA calculation).
- Data Provenance: Prospective, from the same cohort described above.
Pre-selected stool specimens Retrospective Study (Test Set): 207 archived stool specimens positive for various pathogens (low prevalence in prospective study), plus negative clinical specimens.
- Data Provenance: Retrospective, collected at multiple sites in North America, Africa, and Europe.
Pre-selected Stool in Cary-Blair Specimens Retrospective Study (Test Set): Remnants of available pre-selected frozen stool specimens mixed proportionally with Cary-Blair medium.
- Data Provenance: Retrospective, from the same pre-selected cohort.
Botswana Pediatric Stool Specimens (Test Set): 313 pediatric stool specimens.
- Data Provenance: Prospective, collected between February 2011 and January 2012 from symptomatic pediatric patients in Botswana, Africa.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

The document does not explicitly state the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience") for establishing ground truth. Instead, it refers to "reference/comparator methods" or "comparator methods" performed at "central laboratories independent of xTAG GPP testing sites." For Entamoeba histolytica, microscopy was part of the comparator method, implying trained microscopists. For other analytes, molecular methods (EIA, amplification + sequencing, bacterial culture) were used, suggesting laboratory professionals proficient in these techniques.

4. Adjudication method for the test set

The document describes the establishment of ground truth for clinical studies using "Comparator methods."

For Adenovirus 40/41: Composite comparator consisting of Premier Adenoclone Type 40/41 EIA and Amplification + sequencing.
For Entamoeba histolytica: Microscopy followed by amplification + sequencing (for positive samples by microscopy only).
For Vibrio cholerae: Bacterial culture.
For other analytes (from K121894), similar molecular and culture-based comparator methods were used.

A "true positive" or "true negative" result for xTAG GPP was assigned if it agreed with the comparator method. In cases of discrepancies (e.g., xTAG GPP positive, comparator negative), confirmatory testing (e.g., bi-directional sequencing) was performed to determine if the xTAG GPP result was a true positive or false positive. For example, in the pre-selected study, confirmatory testing by NAAT and bi-directional sequencing was done on "additional positive calls" (xTAG GPP positive but not pre-selected).

This represents a form of discrepancy resolution, where a higher-tier reference method (like sequencing) is used to adjudicate disagreements. It is not an "expert consensus" adjudication in the sense of multiple independent experts reviewing and voting, but rather a technical adjudication against a more definitive laboratory standard.

5. 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 document describes a diagnostic device (multiplex nucleic acid test) for pathogen detection, not a system involving human readers interpreting images or data with and without AI assistance. Therefore, an MRMC comparative effectiveness study regarding human reader improvement with AI assistance is not applicable to this device and was not conducted. The "readers" here are automated instruments and software, and the "human-in-the-loop" refers to laboratory technicians performing the assay and interpreting the automated output based on predefined algorithms and cut-offs.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, the analytical and clinical performance studies described represent a standalone (algorithm only) performance evaluation of the xTAG GPP device. The system automates nucleic acid extraction, amplification, detection, and analysis (TDAS GPP software applies algorithms to MFI values to generate qualitative results, which are then compiled into a report). The studies assess the accuracy of these automated results against comparator methods. While human operators perform sample preparation and run the instruments, the critical detection and identification steps, as well as the interpretation of raw data into qualitative positive/negative calls, are performed by the device's integrated system and software.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The ground truth used was a combination of:

Reference Laboratory Methods / Comparator Methods: These included FDA-cleared EIAs, bacterial cultures, microscopy, and Nucleic Acid Amplification Tests (NAATs) followed by bi-directional sequencing for specific gene targets. These are often considered the gold standard or best available methods in microbiology diagnostics.
Discrepancy Resolution using Sequencing: For discrepant results (e.g., xTAG GPP positive, comparator negative), bi-directional sequencing, which is a highly specific molecular method, was used as a definitive arbiter to confirm the presence or absence of the target nucleic acid.
Clinical Outcomes Data: This was not explicitly stated as a direct method for establishing ground truth for individual pathogens within the scope discussed. The focus was on direct pathogen detection.

8. The sample size for the training set

The document does not explicitly delineate a "training set" in the context of machine learning. The studies described are for analytical validation and clinical performance evaluation of the completed device.

However, the "design history file" and "in silico analysis" mentioned (e.g., for primer cross-reactivity) imply that analytical methods were developed and refined based on bioinformatics and laboratory testing during the device's development phase. The analytical reactivity studies, for instance, involve empirical testing of a wide range of strains to establish reactivity, which could be seen as part of the developmental data used to "train" or optimize the assay's ability to detect targets. The document focuses on the validation data for the regulatory submission.

9. How the ground truth for the training set was established

As noted above, a formal "training set" with established ground truth, as understood in machine learning, is not described. The analytical studies (e.g., Limit of Detection, Analytical Reactivity) used well-characterized strains, genotypes, isolates, or clinical specimens with known positivity/negativity established by commercial sources (e.g., ATCC, Zeptometrix) or validated laboratory methods (e.g., quantitative PCR, cell culture infectious dose determination). This type of well-characterized material would be analogous to ground truth for developing and optimizing the assay's performance characteristics.

Summary

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

October 24, 2014

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

Re: K140647 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: September 15, 2014 Received: September 16, 2014

Dear Ms. Ip:

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

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

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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,

Stephen J. Lovell -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

510(k) Number (if known) K140647

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 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. 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 dethe sole or definitive cause of patient illness. Negative xTAG GPP results in the setting of clinical illness compatible with gastroently 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 test is indicated for use with the Lumines® 100/200™ and MAGPIX® instruments 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 Summary of 510(k) information is being submitted in accordance with the requirements of 21 CFR 807.92.

510(k) Number: K140647

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)

Regulatory Information:

Product Code	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)

Device Components

Product	Description
xTAG® GPP Kit	Unchanged from K121894
xTAG® GPP TDAS (Software CD)	Revised CD, containing data acquisition protocol and data analysissoftware (updated to include Adenovirus 40/41, V. cholerae and E.histolytica)
Luminex® MAGPIX® instrument	Unchanged from K121894
xPONENT® Software	xPONENT® Software unchanged from K121894

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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.

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

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The xTAG GPP test is indicated for use with the Luminex® 100/200™ and MAGPIX® instruments with xPONENT® software.

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

Special instrument requirements: Luminex MAGPIX instrument with xPONENT software.

Device Description:

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Substantial Equivalence Information:

Item	New Device (K140647) xTAG GPP	Predicate (K121894) 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 OneStep	xTAG GPP Primer Mix, xTAG OneStep
	Enzyme Mix, xTAG® OneStep Buffer,	Enzyme Mix, xTAG OneStep Buffer,
	xTAG RNase-Free Water, xTAG BSA,	xTAG RNase-Free Water, xTAG BSA,
	xTAG MS2, xTAG® GPP Bead Mix, xTAG	xTAG MS2, xTAG GPP Bead Mix, xTAG
	Reporter Buffer, xTAG 0.22 SAPE	Reporter Buffer, xTAG 0.22 SAPE
Test Format (Same)	Multiplex MAGPLEX bead-based	Multiplex MAGPLEX bead-based
	universal array	universal array
Detection Method (Same)	Fluorescence based	Fluorescence based
Quality Control (Same)	Internal Control (MS2), rotating analyte	Internal Control (MS2), rotating
	controls and negative control (RNAse-	analyte controls and negative control
	free water)	(RNAse-free water)
Results (Same)	Qualitative	Qualitative
Instrument SoftwareSystem	Luminex MAGPIX with xPONENTSoftware	Luminex MAGPIX with xPONENTSoftware

Table 1: Similarities between New Device and Predicate

Table 2: Differences between New Device and Predicate

Item	New Device (K140647)xTAG GPP	Predicate (K121894)xTAG GPP
SpecimenTypes	Human stool specimens and human stool inCary-Blair media	Human stool specimens
Software	Updated assay protocol to acquire and showdata for additional 3 analytes: Adenovirus40/41, Entamoeba histolytica (E. histolytica),and Vibrio cholerae (V. cholerae).	Assay protocol file excludes analytesAdenovirus 40/41, Entamoeba histolytica (E.histolytica), and Vibrio cholerae (V. cholerae)
IntendedUse	See above. Addition of sample type humanstool in Cary-Blair media and addition ofanalytes Adenovirus 40/41, Entamoebahistolytica (E. histolytica), and Vibrio cholerae(V. cholerae) cholera toxin gene (ctx).Specified software used with Luminex 100/200(K140377) and MAGPIX instruments.Organized analytes listed under sub-heading ofviruses, bacteria and parasites.	The xTAG Gastrointestinal Pathogen Panel(GPP) is a multiplexed nucleic acid test intendedfor the simultaneous qualitative detection andidentification of multiple viral, parasitic, andbacterial nucleic acids in human stoolspecimens from individuals with signs andsymptoms of infectious colitis or gastroenteritis.The following pathogen types, subtypes andtoxin genes are identified using the xTAG® GPP:• Campylobacter (C. jejuni, C. coli and C. larionly)• Clostridium difficile (C. difficile) toxin A/B• Cryptosporidium (C. parvum and C. hominisonly)• Escherichia coli (E. coli) O157• Enterotoxigenic Escherichia coli (ETEC) LT/ST• Giardia (G. lamblia only - also known as G.lamblia and G. duodenalis)
Item	New Device (K140647)	Predicate (K121894)
	xTAG GPP	xTAG GPP
		• Norovirus GI/GII
		• Rotavirus A
		• Salmonella
		• Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2
		• Shigella (S. boydii, S. sonnei, S. flexneri and S. dysenteriae)
		The detection and identification of specific gastrointestinal microbial nucleic acid from individuals exhibiting signs and symptoms of gastrointestinal infection aids in the diagnosis of gastrointestinal infection when used in conjunction with clinical evaluation, laboratory findings and epidemiological information. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.
		xTAG GPP positive results are presumptive and must be confirmed by FDA-cleared tests or other acceptable reference methods.
		The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Confirmed positive results do not rule out co-infection with other organisms that are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative xTAG Gastrointestinal Pathogen Panel results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease. xTAG GPP is not intended to monitor or guide treatment for C. difficile infections.
		The xTAG GPP is indicated for use with the Luminex MAGPIX instrument.
TargetsReported	Adenovirus 40/41, Campylobacter (C. jejuni, C. coli and C. lari only), Clostridium difficile (C. difficile) toxin A/B, Cryptosporidium (C. parvum and C. hominis only), Escherichia coli (E. coli) 0157, Enterotoxigenic Escherichia coli (ETEC) LT/ST, Entamoeba histolytica (E. histolytica), Giardia (G. lamblia only - also known as G. intestinalis and G. duodenalis), Norovirus GI/GII, Rotavirus A, 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)	Campylobacter (C. jejuni, C. coli and C. lari only), Clostridium difficile (C. difficile) toxin A/B, Cryptosporidium (C. parvum and C. hominis only), Escherichia coli (E. coli) O157, Enterotoxigenic Escherichia coli (ETEC) LT/ST, Giardia (G. lamblia only - also known as G. intestinalis and G. duodenalis), Norovirus GI/GII, Rotavirus A, Salmonella, Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2, Shigella (S. boydii, S. sonnei, S. flexneri and S. dysenteriae)

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xTAG® GPP with Luminex® MAGPIX® Traditional 510(k) Submission

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Standards/Guidance Documents referenced (if applicable):

Table 3: Guidance Documents

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

Table 4: Standards

	StandardNo.	RecognitionNumber(FDA)	Standards Title	Date
1	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
3	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
5	EP15-A2	7-153	User Verification of Performance for Precision andTrueness (2nd edition)	09/09/2008
6	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
9	MM03-A2	7-132	Molecular Diagnostic Methods for InfectiousDiseases (2nd edition)	09/09/2008

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xTAG® GPP with Luminex® MAGPIX® 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 K121894 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 K121894 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 K121894 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 TCID5g/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	ReactivityTiter(Copies/mL)	Results Summary
Analytical reactivity_II	Adenovirus40	CDC – GP-093	DuganpCMK₂Gr₁₀, 9/23/91	1.49E+07	POS
Analytical reactivity_II	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 strains were in the range of 0.4x to 6.7x multiple of LoD level for E.histolytica. The reactivity titer

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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 Titer(Cells orCopies/mL)	ResultsSummary
20120216_JF_GPP_Reactivity_MP	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_MP	Entamoebahistolytica	ATCC 30190	(HB-301:NIH, fecesfrom adult humanmale with amebicdysentery, Burma,1960); test tube	1.07E+03Copies/mL	POS
20120216_JF_GPP_Reactivity_MP	Entamoebahistolytica	ATCC 30457	(HU-21:AMC, colonicbiopsy from malechild with amebicdysentery, LittleRock, AR, 1970); testtube	1.68E+03Copies/mL	POS
20120216_JF_GPP_Reactivity_MP	Entamoebahistolytica	ATCC 30458	(200:NIH); frozen	1.83E+02 Cells/mL,or 2.42E+03Copies/mL	POS
20120216_JF_GPP_Reactivity_MP	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_MP	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_MP	Entamoebahistolytica	ATCC 30923	(HU-2:MUSC)	1.61E+03Copies/mL	POS
20120207_JF_GPP_Reactivity_MP	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_MP	Entamoebahistolytica	ATCC 50007	DKB	2.88E+03Copies/mL	POS
20120411 JF GPP React MP	Entamoebahistolytica	ATCC 50481	SD157	1.36E+03Copies/mL	POS
20120411 JF GPP React MP	Entamoebahistolytica	ATCC 50738	Rahman	8.90E+01Copies/mL	POS
20120411 JF GPP React MP	Entamoebahistolytica	ATCC 50454	HB-301:NIH	1.08E+03Copies/mL	POS
Run Batch ID	Target	Source	Strain or Serotype	Reactivity Titer(CFU/mL)	ResultsSummary
20120827-JX-V cholera-AR-MP	Vibrio choleraePacini	NCTC 30	Non-O:1, ATCC4735;MARTIN 1	6.00E+08	NEG
20120827-JX-V cholera-AR-MP	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-MP	Vibrio cholerae	NCTC 7260	O:1, EGYPT 117	7.02E+06	POS
20120827-JX-V cholera-AR-MP	Vibrio cholerae	NCTC 11500	Non-O:1, VL 7050	6.00E+08	NEG
20120827-JX-V cholera-AR-MP	Vibrio cholerae	NCTC 11507	Non-O:1, VL 1941	6.00E+08	NEG
20120827-JX-V cholera-AR-MP	Vibrio cholerae	NCTC 11510	O:1, VL 01211	7.02E+06	POS
20120827-JX-V cholera-AR-MP	Vibrio cholerae	NCTC 12945	0:139 (Non-O:1(NAG) — referencestrain for 0:139serovar	7.02E+06	POS
20120827-JX-V cholera-AR-MP	Vibrio cholerae	NCTC 12946	0:139 (Non-O:1(NAG))	7.02E+06	POS
20120406-JX-AnaReact-Vibrio2-MP	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-MP	Vibrio choleraeasiaticae (Trevisan)Pfeiffer	ATCC 14035	O:1, Serotype Ogawa[7787]	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-MP	Vibrio choleraePacini	ATCC 14101	O:1, SerotypeOgawa, clinicalspecimen – human([185754] choleraepidemic circa 1960,Calcutta) CalcuttaIndia	7.02E+06	POS
20120406-JX-AnaReact-Vibrio2-MP	Vibrio choleraePacini	ATCC 14374	Non-O:1 (NAG),5035; R. Hugh 1513	1.50E+08	NEG
20120921-MB-VibrioAnalytical-MP	Vibrio choleraePacini	ATCC 14730	Non-O:1 (SerovarO:2), biovar El Tor,Subgroup III ofGardner andVenkatraman, NCTC4711, NANKING	6.00E+08	NEG
Run Batch ID	Target	Source	Strain or Serotype	Reactivity Titer(CFU/mL)	ResultsSummary
			32/123
20120921-MB-VibrioAnalytical-MP	Vibrio choleraePacini	ATCC 14731	Non-O:1, (SerovarO:3), biovar El Tor,Subgroup V ofGardner andVenkatraman, NCTC4715, El Tor 34-D23;CN 3426	7.02E+06	NEG
20120921-MB-VibrioAnalytical-MP	Vibrio choleraePacini	ATCC 14732	Non-O:1 (SerovarO:4), biovar El Tor,Subgroup VI ofGardner andVenkatraman, NCTC4716, KASAULI 73	6.00E+08	NEG
20120921-MB-VibrioAnalytical-MP	Vibrio choleraePacini	ATCC 14733	Non-O:1 (SerovarO:7), biovar El Tor,Subgroup II ofGardner andVenkatraman, NCTC8042, NANKING32/124	6.00E+08	NEG
20120404-JX-AnaReact-Vibrio-MP	Vibrio choleraePacini	ATCC 25870	O:1, Serotype Inaba	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-MP	Vibrio choleraePacini	ATCC 25872	Non-O:1 (NAG),Isolated from apatient with clinicalcholera	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-MP	Vibrio choleraePacini	ATCC 25873	Non-O:1 (NAG),Isolated from apatient with clinicalcholera	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-MP	Vibrio choleraePacini	ATCC 51394	0:139 (Non-O:1[NAG]), Cholerapatient, Madras,India	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-MP	Vibrio choleraePacini	ATCC 51395	0:139 (non 0:1[NAG]), clinicalspecimen - human(cholera patient,Madras, India)	7.02E+06	POS
20120404-JX-AnaReact-Vibrio-MP	Vibrio cholerae	ATCC BAA-2163	O:1, Isolated from apatient in ArtiboniteDepartment, Haiti,October 2010,Serotype Ogawa,Biogroup El Torcholera toxin positiveCDC Isolate 2010 EL-1786	7.02E+06	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 cholerae strains that do express cholera toxin gene, ctx (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 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-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.

Table 8: Vibrio cholerae Reactivity List

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xTAG® GPP with Luminex® MAGPIX® Traditional 510(k) Submission

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

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Luminex.

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

Pathogen	ATCC / 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-0:1 [NAG])	ATCC 51394
Adenovirus 41	GPP03-048B	Vibrio cholerae Pacini, serovar O:139 (Non-0:1 [NAG])	ATCC 51395
Adenovirus 41	GPP03-055B	Vibrio cholera, serovar 0:1, serotype Ogawa,biovar El Tor, cholera toxin positive	ATCC BAA-2163
Adenovirus 41	GPP03-060B

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

Table 10: Vibrio cholerae Strains that did not React with xTAG GPP

Pathogen	ATCC / Other	Pathogen	ATCC / Other
----------	--------------	----------	--------------

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xTAG® GPP with Luminex® MAGPIX® Traditional 510(k) Submission

	Reference		Reference
Vibrio cholerae Pacini, SerovarNon-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 ElTor, Subgroup III of Gardner andVenkatraman	ATCC 14730
Vibrio cholerae, Serovar Non-O:1	NCTC 11500	Vibrio cholerae Pacini, Serovar O:3, biovarElTor, Subgroup V of Gardner andVenkatraman	ATCC 14731
Vibrio cholerae, Serovar Non-O:1	NCTC 11507	Vibrio cholerae Pacini, Serovar O:4, biovar ElTor; Subgroup VI of Gardner andVenkatraman	ATCC 14732
Vibrio cholerae Pacini, Serovar 01,biotype El Tor, serotype Inaba,non-toxinogenic	ATCC 14033	Vibrio cholerae Pacini, Serovar O:7, biovar ElTor; Subgroup II of Gardner and Venkatraman	ATCC 14733

Carry-over Contamination

The likelihood of carry-over contamination events was initially assessed and presented in K121894 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 K121894 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 K121894, the LoD was assessed by analyzing serial dilutions of simulated samples made from high-titer stocks of commercial strains or high-titer 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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		Titer (correspondingto the estimated	AverageMFI
Analyte	Strain ID	LoD)	Value	%CV
Adenovirus40/41	Adenovirus 40, 0810084CF (Dugan)	$1.45x10^1$ TCID50/mL	686	34.26%
40/41	Adenovirus 41, 0810085CF (Tak)	7.69 TCID50/mL	389	20.27%
Entamoebahistolytica	Entamoeba histolytica, 30890	$2.88x10^1$ cells/mL	1103	17.77%
Vibriocholerae	Vibrio cholerae, 14101 (SerovarO:1)	$2.34x10^6$ CFU/mL	309	23.94%

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

Repeatability

As in the original study results presented for K121894, 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
Adenovirus40/41	Moderate Positive	$5.80x10^1$ TCID50/mL	20 of 20 POS	1562	8.60%
Adenovirus40/41	Low Positive/LoD	$1.45x10^1$ TCID50/mL	20 of 20 POS	686	33.39%
Entamoebahistolytica	Moderate Positive	$5.76x10^1$ cells/mL	20 of 20 POS	886	8.73%
Entamoebahistolytica	Low Positive/LoD	$2.88x10^1$ cells/mL	20 of 20 POS	1103	17.32%
Vibriocholerae	Moderate Positive	$4.68x10^6$ CFU/mL	20 of 20 POS	504	15.48%
Vibriocholerae	Low Positive/LoD	$2.34x10^6$ CFU/mL	20 of 20 POS	309	23.33%

Table 12: Assay Repeatability Assessed by Confirmation of Calls

The correct qualitative result was obtained for 20 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 titers.
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 K121894.

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 K121894. 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 titer (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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Table 14: Interference with Non-Panel Gastrointestinal Pathogens
------------------------------------------------------------------	--

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)(1.43 E+07 copies/mL)	CDC	None		N
		Astrovirus (HP)(6.00 E+10 copies/mL)	CDC	N

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

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

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

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

Table 16: Competitive Interference with Panel Pathogens

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The pathogens listed in Table 17 were not attainable. However, an in silico analysis was performed to assess the potential for non-specific cross-reactivity of these microbial pathogens with the primers used in xTAG GPP (BLAST results located in the design history file). 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

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stable interaction with the xTAG primers which could possibly result in cross reactivity with the xTAG GPP kit. Mismatches would negatively impact the Tm of the primers and Entamoebo 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 K121894, 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.

Three Month Stability Results

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

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

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

Supplemental Stability Results - Entamoeba histolytica (un-extracted)

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

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

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

	Table 18: Summary of Stability Results Additional Analytes xTAG GPP (also see K121894)
--	----------------------------------------------------------------------------------------	--

^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.

Precision / Reproducibility

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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 K121894. 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 operator x 2 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, 86/90 (95.6%) 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 82/90 (91.1%) to 90/90 (100%) replicates tested. For HN dilutions, depending on the target, the correct negative call was generated in as few as 62/90 (68.9%) 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.

Dual Analyte Results

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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, 2 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 single 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:

. 4/90 replicates of the C. difficile (HP) /Adenovirus (LP) sample generated a negative call for Adenovirus
. 2/90 replicates of the Rotavirus (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 K121894).

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	Panel Member ID	Adenovirus40/41Low Positive	Adenovirus40/41MediumPositive	Adenovirus40/41HighNegative	EntamoebahistolyticaLow Positive	EntamoebahistolyticaMediumPositive	EntamoebahistolyticaHighNegative	VibriocholeraeLow Positive	VibriocholeraeMediumPositive	VibriocholeraeHighNegative
	Concentration	1.45x101TCID50/mL	5.8x101TCID50/mL	1.81 TCID50/mL	1.44x101Cells/mL	5.76x101Cells/mL	2.25x10-1Cells/mL	9.37x106CFU/mL	3.75x107CFU/mL	5.86x105CFU/mL
	Agreement withExpected Result	30/30100%	30/30100%	20/3066.7%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%
	25th PercentileMFI	732.5	1594.0	118.0	596.0	1366.0	43.0	616.5	1219.5	52.0
Site 1	Median MFIValue	797.0	1642.0	130.5	677.3	1475.5	45.5	691.3	1277.3	57.0
	75th PercentileMFI	880.0	1692.0	160.0	783.5	1621.0	53.0	737.5	1364.0	69.0
	% CV	12.08	5.34	N/A	23.66	14.90	N/A	17.81	8.43	N/A
	Agreement withExpected Result	30/30100%	30/30100%	13/3043.3%	30/30100%	30/30100%	30/30100%	30/30100%	30/30100%	20/3066.7%
	25th PercentileMFI	740.0	1602.0	131.0	291.0	988.0	41.0	958.0	1579.0	66.0
Site 2	Median MFIValue	872.3	1748.8	170.3	423.3	1253.3	46.0	1256.5	1765.3	117.0
	75th PercentileMFI	1046.0	1806.5	272.0	600.0	1573.5	58.0	1490.0	2001.5	172.0
	% CV	27.53	11.01	N/A	40.13	25.18	N/A	34.56	22.27	N/A
	Agreement withExpected Result	29/3096.7%	29/3096.7%	29/3096.7%	22/3073.3%	26/3086.7%	30/30100%	30/30100%	30/30100%	30/30100%
Site 3	25th PercentileMFI	227.0	481.0	60.0	249.0	603.0	42.0	303.5	843.0	43.0
	Median MFIValue	287.0	648.5	69.0	352.8	778.5	43.5	373.5	1110.8	47.0
	75th PercentileMFI	338.0	770.0	85.0	446.0	979.0	52.0	559.0	1210.0	58.0
	% CV	24.72	36.80	N/A	42.16	41.48	N/A	48.95	24.82	N/A
	Total Agreementwith ExpectedResult	89/9098.9%	89/9098.9%	62/9068.9%	82/9091.1%	86/9095.6%	90/90100%	90/90100%	90/90100%	80/9088.9%

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

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xTAG® GPP with Luminex® MAGPIX® Traditional 510(k) Submission

Panel Member IDConcentration	Adenovirus40/41Low Positive$1.45x10^1$TCID50/mL	Adenovirus40/41MediumPositive$5.8x10^1$TCID50/mL	Adenovirus40/41HighNegative$1.81$ TCID50/mL	EntamoebahistolyticaLow Positive$1.44x10^1$Cells/mL	EntamoebahistolyticaMediumPositive$5.76x10^1$Cells/mL	EntamoebahistolyticaHighNegative$2.25x10^{-1}$Cells/mL	VibriocholeraeLow Positive$9.37x10^6$CFU/mL	VibriocholeraeMediumPositive$3.75x10^7$CFU/mL	VibriocholeraeHighNegative$5.86x10^5$CFU/mL
95% CI	94.0%-99.8%	94.0%-99.8%	58.7%-77.5%	83.4%-95.4%	89.1%-98.3%	95.9%-100.0%	95.9%-100.0%	95.9%-100.0%	80.7%- 93.9%
Overall 25thPercentile MFI	338.0	770.0	80.5	319.0	937.0	42.0	435.0	1148.0	49.0
Overall MedianMFI Value	732.8	1596.0	127.8	459.5	1185.5	44.5	684.3	1297.8	58.5
Overall 75thPercentile MFI	874.0	1722.0	167.5	667.0	1487.5	55.0	993.0	1610.5	89.0
Overall % CV	48.08	38.52	N/A	44.47	35.79	N/A	52.32	30.36	N/A

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	Rotavirus A Low Positive/		Rotavirus A High Positive/		Adenovirus 40/41 Low Positive/			Adenovirus 40/41 High Positive/
		Adenovirus 40/41 High Positive			Adenovirus 40/41 Low Positive	C. difficile High Positive			C. difficile Low Positive
	Panel Member ID		Adenovirus40/41 High	Rotavirus A	Adenovirus	Adenovirus	C. difficile		Adenovirus	C. difficile
		Rotavirus A			40/41	40/41 Low		High Positive	40/41 High		Low Positive
		Low Positive	Positive	High Positive	Low Positive	Positive	Probe 1	Probe 2	Positive	Probe 1	Probe 2
		Indeterminate	9.28x10-	Indeterminate	2.17×10	2.17x10-	6.00x10	6.00x10'	9.28x10-	7.50x10°	7.50x10°
	Concentration	*	TCID50/mL	*	TCID50/mL	TCID50/mL	CFU/mL	CFU/mL	TCID50/mL	CFU/mL	CFU/mL
	Agreement with	30/30	30/30	30/30	30/30	30/30	30/30	30/30	30/30	30/30	30/30
	Expected Result	100%	100%	100%	100%	100%	100%	100%	100%	100%	100%
Site	25" Percentile MFI	480.0	2198.0	1467.0	557.5	654.0	2216.5	3042.0	2529.0	577.0	1415.0
	Median MFI Value	1050.8	2313.0	1777.3	612.3	698.0	2540.0	3255.8	2618.0	730.0	1765.5
	75" Percentile MFI	1544.0	2407.5	1973.0	659.0	842.0	2756.5	3383.5	2785.0	875.0	1945.0
	% CV	71.36	5.85	28.29	15.37	21.80	17.84	7.66	6.38	40.08	23.43
	Agreement with	30/30	30/30	30/30	30/30	30/30	30/30	30/30	30/30	30/30	30/30
	Expected Result	100%	100%	100%	100%	100%	100%	100%	100%	100%	100%
Site :	25" Percentile MFI	443.5	2262.0	1626.0	413.5	400.0	2207.0	3043.0	2428.0	525.0	1636.0
	Median MFI Value	779.3	2501.0	1820.0	530.0	582.8	2547.5	3225.0	2540.3	599.0	1784.8
	75" Percentile MFI	1614.5	2709.0	2056.0	663.5	738.0	3029.0	3375.5	2827.0	1014.0	2012.5
	% CV	87.56	12.46	25.33	28.58	35.82	24.55	12.70	9.62	49.15	17.54
	Agreement with	30/30	30/30	30/30	28/30	26/30	30/30	30/30	30/30	29/30	30/30
	Expected Result	100%	100%	100%	93.3%	86.7%	100%	100%	100%	96.7%	100%
Site 3	25" Percentile MFI	440.0	1517.5	1130.0	198.0	218.0	972.0	2347.0	1570.5	281.5	1143.0
	Median MFI Value	719.5	1626.5	1299.5	239.8	259.5	1548.5	2516.0	1724.0	449.3	1278.5
	75" Percentile MFI	1153.0	1770.0	1577.5	280.0	280.0	1744.0	2632.0	1862.5	563.0	1427.0
	% CV	56.30	13.34	36.58	27.00	28.44	36.44	17.21	15.06	54.03	23.07
	Total Agreement with	90/90	90/90	90/90	88/90	86/90	90/90	90/90	90/90	89/90	90/90
	Expected Result	100%	100%	100%	97.8%	95.6%	100%	100%	100%	98.9%	100%
	95% Cl	95.9%- 100.0%	95.9%- 100.0%	95.9%- 100.0%	92.3%-	89.1%- 98.3%	95.9%-	95.9%-	95.9%- 100.0%	94.0%-	95.9%-
					99.4%		100.0%	100.0%		99.8%	100.0%
	Overall 25th PercentileMFI	443.5	1770.0	1291.0	280.0	280.0	1599.0	2580.0	1862.5	454.0	1311.0
	Overall MedianMFI Value	762.5	2239.5	1662.5	470.5	512.0	2216.8	3042.5	2485.3	588.5	1639.3
	Overall 75" PercentileMFI	1207.0	2412.5	1943.5	624.0	710.5	2686.0	3305.0	2715.0	859.5	1890.0
	Overall% CV	76.97	20.60	31.77	41.43	49.01	34.27	18.18	21.27	50.99	25.77

*Real-time PCR failed to return a meaningful result. The amount of Rotavirus added to this 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 K121894) 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 titer 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.

Analyte	Strain ID	Titer atlimit ofdetection	Raw StoolAverage MFI Value (n=20)	Stool in Cary-BlairTiter atlimit ofdetection	Stool in Cary-BlairAverage MFI Value (n=20)	LoD Differencebetween Stooland Stool in Cary-Blair
C. difficileToxin A/B	Clostridiumdifficile,BAA-1805(toxinotypeIII A+B+)	$4.69x10^5$CFU/mL	Probe 1 = 363Probe 2 = 784	$4.69x10^5$CFU/mL	Probe 1 = 454Probe 2 = 1097	None
Giardia	Giardialamblia,PRA-243	$2.20x10^2$cells/mL	658	$2.20x10^2$cells/mL	633	None
NorovirusGI/GII	NorovirusGII, Clinicalsample,sourceToronto	$4.75x10^2$copies/mL(Ct = 32.23)	1586	$4.75x10^2$copies/mL(Ct = 32.23)	2781	None

Table 21: Summary of the Limit of Detection (LoD) for GPP analytes in 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 284 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 284 runs, 15 (5.28%) 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	96	8	8.33%	249	10	4.02%
Matrix equivalence	3	0	0	9	0	0
Limit of detection	36	2	5.56%	119	2	1.68%
Carry-over contamination	9	0	0	0	0	0
Analytical specificity andinterference	25	1	4.00%	101	1	0.99%
Analytical reactivity	34	1	2.94%	191	3	1.57%
Evaluation of fresh vs.frozen stool	81	3	3.70%	249	3	1.20%
Overall	284	15	5.28%	918	19	2.07%

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

Included in the 284 xTAG runs summarized above were 15455 specimens. Of these, 99.62% (15396/15455) yielded valid results on the first attempt. The remaining 59 specimens generated valid results following allowable re-runs. Sample re-run ratized in the table below.

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Studies	Total # of specimens tested	Total # of invalid results prior to re-run	% invalid results prior to re-run	% invalid results after re-run
Multi-site reproducibility	5065	25	0.49%	0.00%
Matrix equivalence	180	0	0.00%	0.00%
Limit of detection	972	2	0.21%	0.00%
Carry-over contamination	864	0	0.00%	0.00%
Analytical specificity and interference	1472	0	0.00%	0.00%
Analytical reactivity	2156	3	0.14%	0.00%
Evaluation of fresh vs. frozen stool	4746	29	0.61%	0.00%
Overall	15455	59	0.38%	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 K121894

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 30 of the 200 samples tested (15.0%). After rerunning these specimens in accordance with the instructions for use, PCR inhibition was still observed in 7 samples (3.5%). 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 193 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% (193/193)	100.0% (193/193)
Campylobacter	100.0% (193/193)	100.0% (193/193)
C. difficile toxin A/B	97.9% (189/193)1	99.0% (189/191)
Cryptosporidium	100.0% (193/193)	100.0% (193/193)
E. histolytica	99.5% (192/193)2	99.5% (192/193)
E. coli 0157	100.0% (193/193)	100.0% (193/193)
ETEC LT/ST	100.0% (193/193)	100.0% (193/193)
Giardia	99.5% (192/193)3	99.5% (192/193)
Norovirus GI/GII	97.9% (189/193)*4	97.9% (189/193)
Rotavirus A	100.0% (193/193)	100.0% (193/193)
Salmonella	97.4% (188/193)5	97.4% (188/193)
STEC stx1/stx2	100.0% (193/193)	100.0% (193/193)
Shigella	99.5% (192/193)	99.5% (192/193)
V. cholerae	100.0% (193/193)	100.0% (193/193)

Table 24: Percent negative results (including Adenovirus 40/41, E. histolytica and V. cholerae) and the analytes previously presented in the decision summary for K121894

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

1 Two (2) out of 4 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 Norovirus Gl/Gll 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.

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As described in submission K121894, results of the study demonstrated ≥97% negative percent agreement across all analytes in the 193 samples (at the specimen level) that were positive by xTAG GPP but negative by sequencing were considered false positives (12/193). 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 K121894. 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 K121894 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.,

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Lumine

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 results (expected values) for each individual target were summarized per age group in submission K121894, and are now summarized for the additional analytes in Table 26.

	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)
Target(Analyte)	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue	No.	ExpectedValue
Adenovirus40/41	21	1.5%	0	0.0%	2	10.0%	0	0.0%	12	1.4%	7	1.6%
Entamoebahistolytica	20	1.4%	0	0.0%	0	0.0%	1	1.3%	14	1.6%	5	1.2%
Vibriocholerae	3	0.2%	0	0.0%	0	0.0%	0	0.0%	2	0.2%	1	0.2%

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 (K121894) 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).

xTAG GPP
	Positive	Primary ComparatorNegative	Invalid	TOTAL
Positive	4	17	0	21
Negative	11	1158	0	1159
Invalid	2	225	0	227
TOTAL	7	1400	0	1407
		95% CI
PositiveAgreement	80%	37.5% - 96.4%
NegativeAgreement	98.5%	97.7% - 99.1%
Invalid Rate	16.1%

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).

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xTAG GPP	Primary Comparator			TOTAL
	Positive	Negative	Invalid
Positive	0	20	0	20
Negative	0	1154	0	1154
Invalid	0	233	0	233
TOTAL	0	1407	0	1407
		95% CI
Sensitivity	N/A	N/A
Specificity	98.3%	97.4% – 98.9%
Invalid Rate	16.6%

Table 28: 3X3 for Entamoeba histolytica (stool)

Table 29: 3X3 for Vibrio cholerae (stool)

xTAG GPP	Primary Comparator
	Positive	Negative	Invalid	TOTAL
Positive	0	3	0	3
Negative	0	1171	0	1171
Invalid	0	233	0	233
TOTAL	0	1407	0	1407
		95% CI
Sensitivity	N/A	N/A
Specificity	99.7%	99.2% – 99.9%
Invalid Rate	16.6%

A summary of the prospective clinical performance data in human stool specimens (from K121894 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 K121894

		Sensitivity				Specificity
Analyte	TP /(TP+FN)	percent	95%CI	TN /(TN+FP)	percent	95%CI	Number InvalidxTAG GPPResults due toPCR Inhibition
Campylobacter	3/3	100%	43.9% - 100%	1161/1183	98.1%	97.2% - 98.8%	221
Cryptosporidium	12/13	92.3%	66.7% - 98.6%	1132/1189	95.2%	93.8% - 96.3%	205
E. coli 0157	2/2	100%	34.2% - 100%	1163/1174	99.1%	98.3% - 99.5%	231
Entamoebahistolytica	0/0	N/A	N/A	1154/1174	98.3%	97.4% - 98.9%	233
Giardia	4/4	100%	51.0% - 100%	1138/1175	96.9%	95.7% - 97.7%	228
Salmonella	10/10	100%	72.2% - 100%	1145/1164	98.4%	97.5% - 99.0%	233
Shigella	2/2	100%	34.2% - 100%	1160/1178	98.5%	97.6% - 99.0%	227
Vibrio cholerae	0/0	N/A	N/A	1171/1174	99.7%	99.2% - 99.9%	233
		Positive Agreement				Negative Agreement
Analyte	TP /(TP+FN)	percent	95%CI	TN /(TN+FP)	percent	95%CI	Number InvalidxTAG GPPResults due toPCR Inhibition
Adenovirus40/41	4/5	80%	37.5% - 96.4%	1158/1175	98.5%	97.7% - 99.1%	227
C. difficile ToxinA/B1	107/114	93.9%	87.9% - 97.0%	921/1035	89.0%	86.9% - 90.8%	163
ETEC	2/8	25.0%	7.1% - 59.1%	1161/1166	99.6%	99.0% - 99.8%	233
Norovirus GI/GII	74/78	94.9%	87.5% - 98.0%	1022/1121	91.2%	89.4% - 92.7%	208
Rotavirus A	2/2	100%	34.2% - 100%	1167/1170	99.7%	99.2% - 99.9%	235
STEC	1/1	100%	20.7% - 100%	1160/1175	98.7%	97.9% - 99.2%	231

A 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 97 mixed infections in the prospective clinical evaluation. This represents 19.4% of the total number of xTAG GPP positive specimens (97/501). 58 (58/97; 59.8%) were double infections, 26 (26/97; 26.8%) were triple infections, 7 (7/97; 7.2%) were quadruple infections, 2 (2/97; 2.1%) was sextuple infection and 4 were infected by 7 or more pathogens (4/97; 4.1%). The single most common co-infections (23/97; 23.7%) was Norovirus GI/Gll with C. difficile Toxin A/B. Out of the 97 co-infections, 92 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 K121894) 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 are also presented.

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Table 31: Summary of xTAG GPP Clinical Performance

Table S1: Summary of XTAG GPP Clinical Performance
Target	Sensitivity						Specificity
	Unpreserved Stool			Stool in Cary-Blair Media			Unpreserved Stool			Stool in Cary-Blair Media
	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%	1161/1183	98.1%	97.2% - 98.8%	1284/1293	99.3%	98.7% - 99.6%
Cryptosporidium	12/13	92.3%	66.7% - 98.6%	12/13	92.3%	66.7% - 98.6%	1132/1189	95.2%	93.8% - 96.3%	1259/1288	97.7%	96.8% - 98.4%
Entamoebahistolytica		n/a			n/a		1154/1174	98.3%	97.4% - 98.9%	1276/1298	98.3%	97.5% - 98.9%
E. coli O157	2/2	100.0%	34.2% - 100%	2/2	100.0%	34.2% - 100%	1163/1174	99.1%	98.3% - 99.5%	1287/1294	99.5%	98.9% - 99.7%
Giardia	4/4	100.0%	51.0% - 100%	4/4	100.0%	51.0% - 100%	1138/1175	96.9%	95.7% - 97.7%	1275/1298	98.2%	97.4% - 98.8%
Salmonella	10/10	100.0%	72.2% - 100%	10/10	100.0%	72.2% - 100%	1145/1164	98.4%	97.5% - 99.0%	1255/1289	97.4%	96.3% - 98.1%
Shigella	2/2	100.0%	34.2% - 100%	2/2	100.0%	34.2% - 100%	1160/1178	98.5%	97.6% - 99.0%	1291/1296	99.6%	99.1% - 99.8%
Vibrio cholerae		n/a			n/a		1160/1178	98.5%	97.6% - 99.0%	1296/1296	100%	99.7% -100%
Target	Positive Agreement						Negative Agreement
	Unpreserved Stool			Stool in Cary-Blair Media			Unpreserved Stool			Stool in Cary-Blair Media
	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%	2/6	33.3%	9.7% - 70.0%	1158/1175	98.5%	97.7% - 98.1%	1285/1290	99.6%	99.1% - 99.8%
Clostridium difficiletoxin A/B	107/114	93.9%	87.9% - 97.0%	98/108	90.7%	83.8% - 98.9%	921/1035	89.0%	86.9% - 90.8%	1027/1118	91.9%	90.1% - 93.3%
ETEC LT/ST²	2/8	25.0%	7.1% - 59.1%	2/9	22.2%	6.3% - 54.7%	1161/1166	99.6%	99.0% - 99.8%	1283/1287	99.7%	99.2% - 99.9%
Norovirus GI/GII	74/78	94.9%	87.5% - 98.0%	70/73	95.9%	88.6% - 98.6%	1022/1121	91.2%	89.4% - 92.7%	1153/1224	94.2%	92.8% - 95.4%
Rotavirus A	2/2	100.0%	34.2% - 100%	2/2	100.0%	34.2% - 100%	1167/1170	99.7%	99.2% - 99.9%	1294/1294	100%	99.7% - 100%
STEC	1/1	100.0%	20.7% - 100%	1/1	100.0%	20.7% - 100%	1160/1175	98.7%	97.9% - 99.2%	1290/1296	99.5%	99.0% - 99.8%

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xTAG® GPP with Luminex® MAGPIX® Traditional 510(k) Submission

In the case of Adenovirus 4041, one of the clinical cositive in the original GPP runs performed on raw stool yielded a negative result when tested in Cary Blair. MFI generated in the assay cut off (176) suggesting a low titer specimen. Two other specimens that were inhibited in the original stool runs performed on raw stock in the Cary-Blair uns. Lastly, one specimen that was positive for Adenovius 4041 by composite comparator was unarailable for re-testing in these reasons, positive agreement of xTAG GPP for Adenovirus 4041 droped from 80% (4/5) in the raw stool study to 33.3% (2/6) in the Cary-Blair evaluation.

"ETEC comparator results were calculated againsting of four well characterized nucleic acid amplification tests (NATS) followed by bi-diectional sequencing. All specimens that were false by xTAG GPP for ETEC were positive by only one out of four comparator NAATs. Repeat sequencing of these specimens were negative by all four NAAT, except for one sample which was positive by one NAAT.

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Clinical sensitivity or positive agreement acceptance criterion of 90% with a lower bound 95% confidence interval of at least 80% was achieved for Norovirus GI/GII and Clostridium 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 sensitivity 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 pre-selected arm of the study, positive agreement between comparator and xTAG GPP results was 100% for all pre-selected targets tested. Clinical specificity or negative percentage agreement acceptance criterion of 90% with a lower bound 95% confidence interval of at least 90% were achieved for all targets probed by xTAG GPP.

Other Supportive Clinical Data

Pre-selected stool specimens Retrospective Study

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

Target	Positive Agreement		95%CI for PositiveAgreement	Number Invalid xTAG GPResults
	TP / (TP+FN)	Percent
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

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

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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

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 stx2 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 preselected 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 122 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 K121894, and the additional analyte results only are presented here. Sequencing primer validation studies were also presented in the submission summary K121894 and are not repeated here.

xTAG GPP	PCR/Bi-directional Sequencing
	Positive	Negative	Not Done	TOTAL
Additional Positive	5	2	0	7
Negative	N/A	N/A	403	403
Invalid	N/A	N/A	67	67
TOTAL	5	2	470	477
Confirmed Positive	71.4%
Invalid Rate (N=480)	13.9%

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

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Lumin

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	6	1	8
Negative	N/A	N/A	404	404
Invalid	N/A	N/A	67	67
TOTAL	1	6	472	479*

Confirmed Positive	12.5%
Invalid Rate (N=480)	13.9%

1 specimen was pre-selected for Entamoeba histolytica.

Table 35: 3X3 Table for Additional Vibrio Cholerae 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	413	413
Invalid	N/A	N/A	67	67
TOTAL	0	0	480	480

Confirmed Positive	N/A
Invalid Rate (N=480)	13.9%

xTAG GPP detected a total of 73 mixed infections in the pre-selected arm of the clinical study. This represents 29.9% of the total number of xTAG GPP positive specimens (73/244). 59 (59/73: 80.8%) were double infections, 13 (13/73: 17.8%) were triple infections and 1 was quadruple infection (1/73; 1.4%). The single most common co-infections (excluding E. coli 0157 with STEC; N=12) was ETEC with Shigella (6/73; 8.2%). Out of the 73 co-infections, 26 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.

Target	Positive Agreement		95%ConfidenceInterval (CI)	Number ofInvalidResults
	TP/(TP+FN)	Percentage
Campylobacter	40/40	100.0%	91.3% - 100%	0
E. coli O157	2/2	100.0%	34.2% - 100%	0

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

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Salmonella	26/26	100.0%	87.1% - 100%	0
Shigella	13/13	100.0%	77.2% - 100%	0

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.

Target	Concentration	Agreement withExpected Result	MeanMFIValue	% CV	95% CI*
Entamoebahistolytica	5.76x101 cells/mL	25/25 (100%)	2419	76.7%
	1.23x102 cells/mL	1/1 (100%)	2330	N/A
	3.96x102 cells/mL	1/1 (100%)	2973	N/A
	1.23x103 cells/mL	2/2 (100%)	2444	N/A
	1.23x104 cells/mL	2/2 (100%)	2383	N/A
	1.65x104 cells/mL	5/5 (100%)	3086	18.1%
	4.00x104 cells/mL	4/4 (100%)	2367	24.5%
	1.20x105 cells/mL	4/4 (100%)	2290	30.1%
	4.00x105 cells/mL	3/3 (100%)	2813	27.2%
	4.00x106 cells/mL	3/3 (100%)	2673	21.8%
	Entamoeba histolytica OverallPositive Percent Agreement		50/50 (100%)		92.9%-100%
	Negative Percent Agreement		100/100 (100%)		96.1%-100%
Vibriocholerae	4.86x106 CFU/mL	25/25 (100%)	1619	26.4%
	1.00x107 CFU/mL	4/5 (80%)	1448	55.9%
	3.00x107 CFU/mL	5/5 (100%)	1821	19.6%
	1.00x108 CFU/mL	5/5 (100%)	1405	27.2%
	3.00x108 CFU/mL	5/5 (100%)	1563	27.7%
	6.00x108 CFU/mL	5/5 (100%)	1429	25.8%
	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

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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 299 MFI – 5327 MFI and the internal control signal (MS2) was present for all specimens. The 50 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 73 MFI, which is near the positive call threshold of 150 MFI. The signal range for all Vibrio cholerae contrived stool specimens was 73 MFI – 2328 MFI; the signal range for the specimens which called positive for Vibrio cholerae was 899MFI – 2328MFI. All contrived negative stool specimens (N=50) produced the expected negative result for all analytes. The internal control (MS2) was present in all the negative contrived stool specimens and produced a signal range of 210 MFI – 1463 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 2x LoD. Results of this evaluation are presented in the table below (Table 38).

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)	$2.90 x 10^1$	2X	13	100% (13/13)
			$2.32 x 10^2$	16X	6	100% (6/6)
			$9.28 x 10^2$	64X	6	100% (6/6)
Adenovirus 40 Overall					25	100% (25/25)	86.7% -100%
Adenovirus 41	Zeptometrix	Type 41(Tak)	$1.54 x 10^1$	2X	12	100% (12/12)
			$1.23 x 10^2$	16X	7	100% (7/7)
			$4.92 x 10^2$	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% Cl*
	5.76x10- Cells/mL	22/24 (92%)	1581	58%
	4.61x10 - Cells/mL	5/5 (100%)	2652	28%
Entamoeba	9.22x102 Cells/mL	5/5 (100%)	3386	5%
histolytica	1.84x103 Cells/mL	5/5 (100%)	3257	17%
	1.00x104 Cells/mL	5/5 (100%)	2958	31%
	3.00x104 Cells/mL	5/5 (100%)	3063	38%
Entamoeba histolytica Overall		47/49 (96%)			86.3% - 98.9%
	4.68x106 CFU/mL	25/25 (100%)	1360	30%
Vibrio cholerae	1.00x107 CFU/mL	5/5 (100%)	2245	2%
	3.00x107 CFU/mL	5/5 (100%)	2107	23%
	1.00x10°CFU/mL	9/9 (100%)	2238	22%
	3.00x10°CFU/mL	6/6 (100%)	2079	26%
Vibrio cholerae Overall		50/50 (100%)			92.9% - 100%

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

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 K121894, 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 40/41 are presented below. Results for other analytes were previously presented in the submission summary for K121894.

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xTAG GPP	Adenovirus Type 40/41 EIA
	Positive	Negative	Invalid	TOTAL
Positive	35	0	0	35
Negative	17	255	0	272
Invalid	1	5	0	6
TOTAL	53	260	0	313
	95% CI
Positive Agreement	67.3%	53.7% - 78.5%
Negative Agreement	100%	98.5% - 100%
Invalid Rate 2	1.9%

Table 40: 3X3 Table for Adenovirus 40/41 - Botswana Stool Sample Set

1 All 17 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 17 specimens were assessed by real-time PCR for Adenovirus (all sub-types) at the laboratory testing site. The mean Ct value for these 17 specimens was 33.1; indicating low viral titer in these specimens, which is less clinically relevant.

All these 35 specimens were also assessed by real-time PCR for Adenovirus (all sub-types) at the laboratory testing site. In contrast to the 17 specimens in footnote 1 above, the mean Ct value for the 35 adenovirus samples positive by the PCR/Bidirectional sequencing assay and detected by xTAG GPP in this cohort was 21.38; 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 K121894.

xTAG GPP	PCR/Bi-directional Sequencing
	Positive	Negative	Not Done	TOTAL
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%

Table 41: 3X3 Table for Entamoeba Histolytica - Botswana Stool Sample Set

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

xTAG GPP	PCR/Bi-directional Sequencing
	Positive	Negative	Not Done	TOTAL
Positive	0	0	0	0
Negative	NA	NA	307	307
Invalid	NA	NA	6	6
TOTAL	0	0	313	313

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Confirmed Positive Rate	N/A
Invalid Rate	1.9%

xTAG GPP detected a total of 115 mixed infections in the Botswana study. This represents 40.5% of the total number of xTAG GPP positive specimens (115/284). 8 8 (88/115; 76.5%) were double infections, 20 (20/115; 117.4%) were triple infections, 5 (5/115; 4.3%) were quadruple infections and 2 (2/115; 1.7%) were quintuple infections. The single most common co-infection was Rotavirus with Campylobacter (18/115; 15.6%). Out of the 115 co-infections, 22 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 are summarized for each clinical study in Table 43 below.

Clinical Studies	Total # ofspecimenstested	Sample Failure due toPCR Inhibition		Sample Failure due to PCRContamination
		# Re-runs	% Re-runs	# Re-runs	% Re-runs
Prospective Study	1407	236	16.8%	56	4.0%
Pre-selected Study	480	67	14.0%	30	6.2%
Botswana Study	313	5	1.6%	80	25.6%

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 K121894.

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	21	1.5%	9	2.1%	9	2.1%	0	0.0%	3	1.2%	0	0.0%	0	0.0%
Entamoebahistolytica	20	1.4%	6	1.4%	8	1.9%	2	1.3%	2	0.8%	2	2.3%	0	0.0%
Vibrio cholerae	3	0.2%	0	0.0%	3	0.7%	0	0.0%	0	0.0%	0	0.0%	0	0.0%

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Instrument and System Information

Luminex MAGPIX with xPONENT Software

Modes of Operation: See Device Description above.
Software: Hazard Analysis included in original K121894 submission documentation

1. 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:

The Luminex MAGPIX Calibration Kit is intended to calibrate the optics of the MAGPIX instrument. During calibration, the system adjusts LED current and calibration factors for CL1, CL2, and RP1 until those values match the imported target values, thus calibrating the classification map. This product is not intended to be used in place of the assay calibrators or assay controls that are required to verify the proper function of a given assay.

Quality Control:

The Luminex MAGPIX Performance Verification Kit is intended to verify the optical calibration of the MAGPIX instrument. This product is not intended to be used in place of the assay calibrators or assay controls that are required to verify the proper function of a given assay.

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).