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K Number
DEN130003

Validate with FDA (Live)

Device Name
XTAG GASTROINTESTINAL PATHOGEN PANEL (GPP) XTAG DATA ANALYSIS SOFTWARE FOR GPP (TDAS)
Manufacturer
LUMINEX MOLECULAR DIAGNOSTICS, INC.
Date Cleared
2013-01-14

(3 days)

Product Code
PCH,GAS,JJH,NSU
Regulation Number
866.3990
Type
Post-NSE
Panel
Microbiology
Reference & Predicate Devices
N/A
Predicate For
N/A
AI/MLSaMDIVD (In Vitro Diagnostic)TherapeuticDiagnosticis PCCP AuthorizedThirdpartyExpeditedreview
Intended Use

The xTAG® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple viral. parasitic, and bacterial nucleic acids in human stool specimens 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:

  • · 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)

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 coinfection with other organisms that are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative xTAGGastrointestinal Pathogen Panel results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

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

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

Device Description

The Luminex Molecular Diagnostics xTAG GPP consists of kit reagents and software. The reagents in conjunction with a thermal cycler are used to perform nucleic acid amplification (reverse transcription-polymerase chain reaction, or RT-PCR/PCR), and the protocol configuration file is used to generate results while the data analysis software (TDAS GPP (US)) is used to analyze the results from the Luminex Corporation Luminex 100/200 instrument system (which includes the xPONENT core software).

The components of the xTAG GPP kit are contained within 2 boxes (one that is frozen, and one that is refrigerated). The kit is shipped with the xTAG GPP CD which contains the xTAG GPP T-A (LX) protocol configuration file and the TDAS GPP (US) software. The instrument is shipped with the xPONENT software.

The xTAG Gastrointestinal Pathogen Panel (xTAG GPP) incorporates multiplex reverse transcription and polymerase chain reaction (RT-PCR / PCR) with Luminex's proprietary universal tag sorting system on the Luminex platform. The assay also detects an internal control (bacteriophage MS2) that is added to each sample prior to extraction. Each sample is pre-treated prior to extraction and is then put through extraction using the Biomerieux NucliSens EasyMag kit (product code JJH, class I, an IVD-labeled automated system for nucleic acid extraction).

Post-extraction, for each sample, 10 uL of extracted nucleic acid is amplified in a single multiplex RT-PCR/PCR reaction. Each target or internal control in the sample results in PCR amplicons ranging from 58 to 202 bp (not including the 24-mer tag). A five uL aliquot of the RT-PCR product is then added to a hybridization/detection containing bead populations coupled to sequences from the Universal Array ("antitags"), streptavidin, R-phycoerythrin conjugate. Each Luminex bead population detects a specific microbial target or control through a specific tag/anti-tag hybridization reaction. Following the incubation of the RT-PCR products with the xTAG GPP Bead Mix and xTAG Reporter Buffer, the Luminex instrument sorts and reads the hybridization/detection reactions.

A signal, or median fluorescence intensity (MFI), is generated for each bead population. These fluorescence values are analyzed to establish the presence of bacterial, viral or parasitic targets and/or controls in each sample. A single multiplex reaction identifies all targets.

The xTAG Data Analysis Software for the Gastrointestinal Pathogen Panel (TDAS GPP (US)) analyzes the data to provide a report summarizing which pathogens are present. 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). Consequently the remaining target results are masked and cannot be retrieved.

Target results above or equal to the cutoff are considered positive, while target results below the cutoff are considered negative. For each sample analyzed by TDAS GPP (US), there are individual results for each of the targets and the internal control (bacteriophage MS2).

AI/ML Overview

Acceptance Criteria & Study Results for xTAG® Gastrointestinal Pathogen Panel (GPP)

This document describes the acceptance criteria and study results for the xTAG® Gastrointestinal Pathogen Panel (GPP), a qualitative nucleic acid multiplex test for the simultaneous detection and identification of multiple viral, parasitic, and bacterial nucleic acids in human stool specimens.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for clinical performance are implicitly defined by the reported sensitivity and specificity (or Positive Percent Agreement and Negative Percent Agreement) for each analyte, with a 95% Confidence Interval (CI) lower bound typically being the minimum acceptable performance. The provided study data shows the performance relative to reference/comparator methods.

OrganismMetric (Clinical Study)Acceptance Criteria (Implicit from FDA Review)Reported Device Performance (Prospective Clinical Study, After Discrepant Investigation)
CampylobacterSensitivityLower bound of 95% CI to be acceptable100% (95% CI: 43.8% - 100%)
SpecificityLower bound of 95% CI to be acceptable98.2% (95% CI: 97.3% - 98.8%)
CryptosporidiumSensitivityLower bound of 95% CI to be acceptable92.3% (95% CI: 66.7% - 98.6%)
SpecificityLower bound of 95% CI to be acceptable95.5% (95% CI: 94.2% - 96.6%)
E. coli O157SensitivityLower bound of 95% CI to be acceptable100% (95% CI: 34.2% - 100%)
SpecificityLower bound of 95% CI to be acceptable99.2% (95% CI: 98.5% - 99.6%)
GiardiaSensitivityLower bound of 95% CI to be acceptable100% (95% CI: 51.0% - 100%)
SpecificityLower bound of 95% CI to be acceptable96.7% (95% CI: 95.5% - 97.6%)
SalmonellaSensitivityLower bound of 95% CI to be acceptable100% (95% CI: 72.2% - 100%)
SpecificityLower bound of 95% CI to be acceptable98.4% (95% CI: 97.6% - 99.0%)
STEC stx1/stx2SensitivityLower bound of 95% CI to be acceptable100% (95% CI: 20.7% - 100%)
SpecificityLower bound of 95% CI to be acceptable98.6% (95% CI: 97.8% - 99.2%)
ShigellaSensitivityLower bound of 95% CI to be acceptable100% (95% CI: 34.2% - 100%)
SpecificityLower bound of 95% CI to be acceptable98.5% (95% CI: 97.7% - 99.1%)
C. difficile Toxin A/BPositive Percent Agreement (PPA)Lower bound of 95% CI to be acceptable93.9% (95% CI: 87.9% - 97.0%)
Negative Percent Agreement (NPA)Lower bound of 95% CI to be acceptable89.8% (95% CI: 87.8% - 91.5%)
ETEC LT/STPositive Percent Agreement (PPA)Lower bound of 95% CI to be acceptable25.0% (95% CI: 7.1% - 59.1%)
Negative Percent Agreement (NPA)Lower bound of 95% CI to be acceptable99.7% (95% CI: 99.1% - 99.9%)
Norovirus GI/GIIPositive Percent Agreement (PPA)Lower bound of 95% CI to be acceptable94.9% (95% CI: 87.5% - 98.0%)
Negative Percent Agreement (NPA)Lower bound of 95% CI to be acceptable91.4% (95% CI: 89.6% - 92.9%)
Rotavirus APositive Percent Agreement (PPA)Lower bound of 95% CI to be acceptable100% (95% CI: 34.2% - 100%)
Negative Percent Agreement (NPA)Lower bound of 95% CI to be acceptable99.8% (95% CI: 99.4% - 100%)

Note on Acceptance Criteria: Explicit quantitative acceptance criteria (e.g., "Sensitivity must be >X%") are not explicitly stated in the provided text as an overarching target. However, the FDA's acceptance of the de novo classification implies that the presented clinical performance data, including these sensitivity/specificity/agreement ranges with their 95% CIs, were deemed sufficient for the intended use and risk-benefit profile. The FDA's review and ultimate acceptance of the de novo classification indicate that the device "meets the acceptance criteria" as evaluated against relevant standards and guidance documents. The FDA also notes concerns regarding "relatively low specificity of two of the analytes tested in the panel (C. difficile and Norovirus)," but considers these addressed by labeling requirements for confirmation.

2. Sample Sizes Used for the Test Set and Data Provenance

Prospective Clinical Study:

  • Sample Size: 1407 clinical stool specimens (from 1407 subjects) were included in the primary prospective study after exclusions. An additional 200 asymptomatic donor samples were tested for baseline levels.
  • Data Provenance: Prospective, collected from six clinical laboratories in North America (four sites in the U.S. and two sites in Canada) between June 2011 and February 2012.

Retrospective Clinical Study 1 (Pre-Selected Specimens):

  • Sample Size: 203 pre-selected positive clinical specimens and 277 "negative" clinical specimens (total 480) were tested.
  • Data Provenance: Retrospective, collected at multiple sites in North America and Europe.

Supplemental Clinical Study (Botswana Pediatric Stool Specimens):

  • Sample Size: 313 pediatric stool specimens.
  • Data Provenance: Prospective, collected between February 2011 and January 2012 from symptomatic pediatric patients admitted to two referral hospitals in Botswana, Africa.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document does not explicitly state the "number of experts" used to establish ground truth for the test sets in a single, overarching assessment. Instead, the ground truth was established through a combination of:

  • Routine diagnostic algorithms used at the clinical sites (e.g., bacterial culture, EIA/DFA, microscopic examination, real-time PCR, nucleic acid amplification tests followed by bi-directional sequencing). The qualifications of personnel performing these routine diagnostics are implied to be standard for clinical laboratory settings but not specified in detail (e.g., "trained clinical laboratory personnel").
  • Reference and comparator method testing conducted at central laboratories, independent of xTAG GPP testing sites. These methods included various FDA-cleared assays and analytically validated PCR/sequencing assays. The scientific rigor of these methods implies expert oversight in their performance and interpretation, but specific expert qualifications (e.g., "radiologist with 10 years of experience") are not provided for the individuals performing these reference tests or adjudicating results.

For the composite comparator methods (e.g., Norovirus, ETEC, Rotavirus), the interpretation algorithms and sequencing criteria suggest a structured, expert-defined process, but the number and specific qualifications of individuals involved in defining these are not enumerated.

4. Adjudication Method for the Test Set

The adjudication method employed was primarily discrepant analysis using analytically validated PCR/sequencing assays or FDA-cleared molecular assays.

  • In the Prospective Clinical Study, discrepant results between the xTAG GPP and the initial reference methods were evaluated using these advanced molecular methods. The final performance metrics (Sensitivity, Specificity, PPA, NPA) were calculated "After Discrepant Investigation," indicating that the results of the discrepant analysis were incorporated to refine the "true" status of the samples.
  • Similarly, in the Retrospective and Supplemental Studies, xTAG GPP positive results for analytes not initially targeted by the comparator were subjected to PCR/bi-directional sequencing for confirmation.

There is no mention of a specific "X+Y" type of adjudication involving multiple human readers beyond the standard clinical laboratory and molecular testing procedures.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. The study's focus was on the performance of the xTAG GPP device against established laboratory reference methods, not on comparing human reader performance with and without AI assistance. The xTAG GPP is an automated nucleic acid test, not an AI-powered diagnostic imaging tool that would typically involve human reader interpretation.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the xTAG GPP device's performance was evaluated in a standalone manner. The device performs nucleic acid amplification and detects specific markers, with its inherent software (TDAS GPP (US)) analyzing the data to provide qualitative results (positive/negative) for each pathogen. Human involvement is in sample preparation, loading, and interpreting the final report, but the "performance" data presented (sensitivity, specificity, agreement) directly reflects the algorithm's output compared to ground truth, without a human interpretation step that enhances or modifies the algorithm's initial call. The study assessed the device's ability to detect targets "as determined by the xTAG GPP" against comparator methods.

7. The Type of Ground Truth Used

The ground truth for the test sets was established through a combination of:

  • Expert Consensus (Implicit/Composite Comparator): For several analytes (e.g., Norovirus, Rotavirus, ETEC), a "composite comparator method" was used. This involved combining results from multiple individual reference assays (e.g., EIA + PCR/sequencing) and applying specific interpretive algorithms (as detailed for Norovirus) to determine the "true" infection status. This process implicitly relies on expert consensus in defining the gold standard for these complex cases.

  • Pathology/Laboratory Methods:

    • Bacterial Culture: For bacterial pathogens like Salmonella, Shigella, Campylobacter, and E. coli O157.
    • Microscopy: For parasites like Cryptosporidium and Giardia.
    • FDA-cleared Assays: For C. difficile (Bartels Cytotoxicity Assay) and some other analytes (e.g., ImmunoCard STAT EHEC for STEC, Premier Rotaclone EIA for Rotavirus).
    • Analytically Validated PCR/Sequencing: Used as a primary comparator for ETEC and for discrepant analysis across many pathogens.

  • Outcomes Data: Not explicitly mentioned as a primary method for establishing ground truth, though clinical signs and symptoms were collected and considered in patient selection for the prospective study. The ground truth was primarily based on laboratory detection of the pathogen.

8. The Sample Size for the Training Set

The document does not explicitly specify a "training set" for the xTAG GPP device in the context of machine learning. The device described appears to be a molecular diagnostic assay using pre-defined cut-offs rather than a machine learning algorithm that undergoes a training phase on a dataset.

However, the "Assay cut-off" section mentions that "Clinical specimens, cultured isolates spiked in a synthetic stool matrix sample and extraction controls...were used to establish cut-offs." This process, which involves empirically determining optimal thresholds for MFI values, serves a similar function to model calibration or "training" in traditional statistical modeling.

  • Samples used for cut-off establishment: Distinct sample sets comprising clinical specimens with known pathogen status (based on routine diagnostic algorithms), cultured isolates diluted into negative matrix, and extraction controls were used. The precise number of these samples is not explicitly aggregated and labeled as a "training set size."

9. How the Ground Truth for the Training Set Was Established

For the "training" data used to establish assay cut-offs (threshold-setting):

  • Known Sample Types/Results from Clinical Sites: Samples were assigned a "positive" or "negative" call based on the known sample types or results obtained at the clinical sites. These results were derived from routine diagnostic algorithms (e.g., bacterial culture, EIA/DFA, microscopic examination, real-time PCR, nucleic acid amplification tests followed by bi-directional sequencing).
  • Cultured Isolates: Serially diluted cultured isolates with confirmed viral, bacterial, or parasitic identity were used.
  • Extraction Controls: Negative matrix spiked with MS2 (internal control) were used and coded as negative for all targets.
  • Exclusion Criteria: If comparator results were not available for all 15 targets for a given sample, that target was excluded from the threshold-setting data sets for which its status was unknown.
  • Process: The process involved setting an initial cut-off range based on the 95th percentile of negative signals and 5th percentile of positive signals, then recommending optimized cut-offs via Receiver Operating Characteristic (ROC) analysis, and finally establishing the MFI cut-off through a Design Review Committee (DRC) assessment of ROC curves.

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EVALUATION OF AUTOMATIC CLASS III DESIGNATION (DE NOVO) FOR xTAG® GASTROINTESTINAL PATHOGEN PANEL (GPP) DECISION SUMMARY

  • K121454 A. 510(k) Number:
  • B. Purpose for Submission: Clearance of new assay C. Measurand: · 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 E. coli (ETEC) LT/ST • Giardia (G. lamblia only - also known as G. intestinalis and G. duodenalis) • Norovirus GI/GII • Rotavirus A · Salmonella (see Analytical Reactivity section for a list of serotypes detected)
    • · Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2
    • Shigella (S. boydii, S. sonnei, S. flexneri and S. dysenteriae) in human stool samples.
  • D. Type of Test: Qualitative nucleic acid multiplex test
  • E. Applicant: Luminex Molecular Diagnostics, Inc.
  • F. Proprietary and Established Names: xTAG® Gastrointestinal Pathogen Panel (GPP)
  • G. Regulatory Information:

FDA identifies this generic type of device as:

A gastrointestinal microorganism multiplex nucleic acid-based assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple gastrointestinal microbial nucleic acids extracted from human stool specimens. The device detects specific nucleic acid sequences for organism identification as well as for determining the presence of toxin genes. The detection and identification of a specific gastrointestinal microbial nucleic acid from individuals exhibiting signs and symptoms of gastrointestinal infection aids in the diagnosis of gastrointestinal infection when used in conjunction with clinical evaluation and other laboratory findings. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

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1. New Regulation Number:

21 CFR 866.3990 - Gastrointestinal microorganism multiplex nucleic acid-based assay

    1. Classification:
      Class II
    1. Product code:
      PCH, NSU, JJH
    1. Panel:
      Microbiology (83)

H. Intended Use:

    1. Intended use(s):
      The xTAG® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple viral. parasitic, and bacterial nucleic acids in human stool specimens 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:

  • · 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)

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-

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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 coinfection with other organisms that are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative xTAGGastrointestinal Pathogen Panel results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or non-infectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.

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

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

    1. Indication(s) for use:
      Same as intended use.
    1. Special conditions for use statement(s):
      For prescription use only. Manufacturer must provide device-specific user training to facilities prior to using the device.
    1. Special instrument requirements:
      Extraction: Biomerieux NucliSens® EasyMag® instrument

Analysis: Luminex® 100/200™ instruments with xPONENT® software

I. Device Description:

The Luminex Molecular Diagnostics xTAG GPP consists of kit reagents and software. The reagents in conjunction with a thermal cycler are used to perform nucleic acid amplification (reverse transcription-polymerase chain reaction, or RT-PCR/PCR), and the protocol configuration file is used to generate results while the data analysis software (TDAS GPP (US)) is used to analyze the results from the Luminex Corporation Luminex 100/200 instrument system (which includes the xPONENT core software).

The components of the xTAG GPP kit are contained within 2 boxes (one that is frozen, and one that is refrigerated). The kit is shipped with the xTAG GPP CD which contains the xTAG GPP T-A (LX) protocol configuration file and the TDAS GPP (US) software. The instrument is shipped with the xPONENT software.

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The xTAG Gastrointestinal Pathogen Panel (xTAG GPP) incorporates multiplex reverse transcription and polymerase chain reaction (RT-PCR / PCR) with Luminex's proprietary universal tag sorting system on the Luminex platform. The assay also detects an internal control (bacteriophage MS2) that is added to each sample prior to extraction. Each sample is pre-treated prior to extraction and is then put through extraction using the Biomerieux NucliSens EasyMag kit (product code JJH, class I, an IVD-labeled automated system for nucleic acid extraction).

Post-extraction, for each sample, 10 uL of extracted nucleic acid is amplified in a single multiplex RT-PCR/PCR reaction. Each target or internal control in the sample results in PCR amplicons ranging from 58 to 202 bp (not including the 24-mer tag). A five uL aliquot of the RT-PCR product is then added to a hybridization/detection containing bead populations coupled to sequences from the Universal Array ("antitags"), streptavidin, R-phycoerythrin conjugate. Each Luminex bead population detects a specific microbial target or control through a specific tag/anti-tag hybridization reaction. Following the incubation of the RT-PCR products with the xTAG GPP Bead Mix and xTAG Reporter Buffer, the Luminex instrument sorts and reads the hybridization/detection reactions.

A signal, or median fluorescence intensity (MFI), is generated for each bead population. These fluorescence values are analyzed to establish the presence of bacterial, viral or parasitic targets and/or controls in each sample. A single multiplex reaction identifies all targets.

The xTAG Data Analysis Software for the Gastrointestinal Pathogen Panel (TDAS GPP (US)) analyzes the data to provide a report summarizing which pathogens are present. 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). Consequently the remaining target results are masked and cannot be retrieved.

Target results above or equal to the cutoff are considered positive, while target results below the cutoff are considered negative. For each sample analyzed by TDAS GPP (US), there are individual results for each of the targets and the internal control (bacteriophage MS2).

J. Substantial Equivalence Information:

    1. Predicate device name(s): None
    1. Predicate 510(k) number(s): None
    1. Comparison with predicate: Not applicable

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K. Standard/Guidance Document Referenced (if applicable):

Standards Referenced

StandardsNo.RecognitionNumber(FDA)Standards TitleDate
1MM13-A7-191Collection, Transport, Preparation andStorage of Specimens03/18/2009
2EP15-A27-153User Verification of Performance forPrecision and Trueness (2nd edition)09/09/2008
3EP05-A27-110Evaluation of Precision Performance ofQuantitative measurement Methods (2nd ed.)10/31/2005
4EP07-A27-127Interference Testing in Clinical Chemistry(2nd edition)05/21/2007
5EP12-A27-152User Protocol for Evaluation f QualitativeTest Performance (2nd edition)09/09/2008
6EP17-A7-194Protocol for Determination of Limits ofDetection and Limits of Quantitation03/18/2009
7EP14-A27-128 and7-143Evaluation of Matrix Effects (2nd edition)06/01/2004
8MM03-A27-132Molecular Diagnostic Methods for InfectiousDiseases (2nd edition)09/09/2008
9CEN 136407-84Stability Testing of In Vitro DiagnosticReagents06/01/2004
10ISO 149715-40Application of Risk Management to MedicalDevices09/12/2007

Guidance Documents Referenced

TitleDate
1Establishing the Performance Characteristics of In Vitro Diagnostic11/29/10
Devices for the Detection of Clostridium difficile
2Class II Special Controls Guidance Document: Norovirus Serological03/09/12
Reagents
3Class II Special Controls Guidance Document: Instrumentation for03/10/05
Clinical Multiplex Test Systems - Guidance for Industry and FDA Staff
বGuidance for the Content of Premarket Submissions for Software5/11/05
Contained in Medical Devices
റGuidance document for Format for Traditional and Abbreviated 510(k)s08/12/05
6Guidance on the CDRH Premarket Notification Review Program,06/30/86
510(k) Memorandum #K86-3
7The New 510(k) Paradigm - Alternate Approaches to Demonstrating03/20/98
Substantial Equivalence in Premarket Notifications - Final Guidance
8The 510(k) Program: Evaluating Substantial Equivalence in Premarket12/27/11
Notifications [510(k)]
ರDraft Guidance for Industry and Food and Drug Administration Staff -10/17/12
eCopy Program for Medical Device Submissions
10Guidance for Industry and FDA Staff - Factors to Consider When03/28/12

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Making Benefit-Risk Determinations in Medical Device PremarketApproval and De Novo Classifications
11Draft Guidance for Industry and Food and Drug Administration Staff -De Novo Classification Process (Evaluation of Automatic Class IIIDesignation)10/03/11
12Guidance for Industry and Food and Drug Administration Staff - FDAand Industry Actions on Premarket Notification (510(k)) Submissions:Effect on FDA Review Clock and Goals10/15/12

L. Test Principle:

Human stool samples are pretreated and then subjected to nucleic acid extraction. For each sample, 10 uL of extracted nucleic acid is amplified in a single multiplex RT-PCR/PCR reaction. Each target or internal control in the sample results in PCR amplimers ranging from 58 to 202 bp (not including the 24-mer tag). A five uL aliquot of the RT-PCR product is then added to a hybridization/detection reaction containing bead populations coupled to sequences from the Universal Array ("antitags"), streptavidin, R-phycoerythrin conjugate. Each Luminex bead population detects a specific microbial target or control through a specific tag/anti-tag hybridization reaction. Following the incubation of the RT-PCR products with the xTAG GPP Bead Mix and xTAG Reporter Buffer, the Luminex instrument sorts and reads the hybridization/detection reactions. A signal or median fluorescence intensity (MFI) is generated for each bead population. These fluorescence values are analyzed to establish the presence or absence of bacterial, viral or parasitic targets and/or controls in each sample. A single multiplex reaction identifies all targets.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

Site-to-site reproducibility was assessed for each of the indicated microbial targets and for mixed analyte samples (representing co-infected samples). Replicates of simulated samples were tested across 3 sites by 2 operators at each site. 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

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    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° TCID30/mL) or higher; High Positive (HP) bacterial cultures were prepared to a concentration of 106 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 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.

For single analyte samples prepared at the MP level, depending on the microbial target, 89/90 (99%) to 90/90 (100%) replicates generated a positive result. For LP dilutions, depending on the microbial target, the correct positive call was made in 80/90 (89%) to 90/90 (100%) replicates tested. The only exception in terms of LP detection was Cryptosporidium, due to the fact that the initial titer for this particular sample was below the targeted range of 1-5X the cut-off MFI. For HN dilutions, depending on the microbial target, the correct negative call was generated in as few as 54/90 (60%) 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 microbial 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.

For dual analyte samples, all microbial targets generated a positive call when present as a HP dilution. When present at the LP concentration, 3 of the 6 microbial target combinations tested generated a positive call in 90/90 (100%) replicates tested. The following was observed for the remaining 3 targets present at LP concentrations in samples containing a second microbial target at HP concentrations:

  • 2/90 replicates of the ETEC (HP) / Salmonella (LP) sample generated a negative . call for Salmonella
  • . 4/90 replicates of the Salmonella (HP) / Rotavirus (LP) sample generated a negative call for rotavirus
  • . 12/90 replicates of the Rotavirus (HP) / Norovirus GII (LP) sample generated a negative call for norovirus

{7}------------------------------------------------

{8}------------------------------------------------

Panel Member IDCampylob-actorLowPositiveCampylob-actorMediumPositiveCampylob-actorHighNegativeC. difficile Toxin A/BLow PositiveC. difficile Toxin A/BMedium PositiveC. difficile Toxin A/BHigh NegativeCryptospor-idium hominisLow PositiveCryptospor-idium hominisMedium PositiveCryptospor-idium hominisHigh NegativePanel Member IDE. coli O157Low PositiveE. coli O157Medium PositiveE. coli O157High NegativeETEC LT/STLow PositiveProbe 1ETEC LT/STLow PositiveProbe 2ETEC LT/STMedium PositiveProbe 1ETEC LT/STMedium PositiveProbe 2ETEC LT/STHigh NegativeProbe 1ETEC LT/STHigh NegativeProbe 2GiardiaLow PositiveGiardiaMedium PositiveGiardiaHigh NegativePanel Member IDNorovirus GI/GIILow PositiveNorovirus GI/GIIMedium PositiveNorovirus GI/GIIHigh NegativeRotavirus ALow PositiveRotavirus AMedium PositiveRotavirus AHigh NegativeSalmonella*Low PositiveSalmonella*Medium PositiveSalmonella*High Negative
Concentration$9.38x10^5$CFU/mL$3.75x10^6$CFU/mL$1.17x10^5$CFU/mL$3.75x10^6$CFU/mL$9.38x10^5$CFU/mL$1.50x10^7$CFU/mL$1.50x10^7$CFU/mL$2.34x10^5$CFU/mL$2.34x10^5$CFU/mL$6.21x10^3$Copies/mL$2.05x10^4$Copies/mL$6.37x10^2$Copies/mLConcentration$2.34x10^5$CFU/mL$3.75x10^6$CFU/mL$2.93x10^4$CFU/mL$2.93x10^4$CFU/mL$9.37x10^5$CFU/mL$3.75x10^6$CFU/mL$3.75x10^6$CFU/mL$7.32x10^3$CFU/mL$7.32x10^3$CFU/mL$8.79x10^2$Cells/mL$3.25x10^3$Cells/mL$2.74x10^1$Cells/mLProbe 1Probe 2Probe 1Probe 2Probe 1Probe 2Probe 1Probe 2Probe 1Probe 2Probe 1Probe 2
Agreement withExpected Result30/30100%30/30100%5/3016.7%30/30100%30/30100%30/30100%30/30100%30/30100%28/3093.3%17/3056.7%30/30100%30/30100%Agreement withExpected Result30/30100%30/30100%24/3080%30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%Concentration$1.2x10^3$Copies/mL$1.74x10^3$Copies/mL$4.64x10^3$Copies/mL$7.45x10^3$Copies/mL$5.84x10^1$Copies/mL$5.95x10^1$Copies/mL$2.24x10^4$Copies/mL$4.47x10^5$Copies/mL$1.29x10^3$Copies/mL$1.17x10^5$CFU/mL$1.17x10^5$CFU/mL$9.38x10^5$CFU/mL$9.38x10^5$CFU/mL$3.66x10^3$CFU/mL$3.66x10^3$CFU/mL
25th Percentile MFI1081.02022.0183.5259.5256.0918.01821.038.567.0165.0748.048.025th Percentile MFI546.02494.074.0294.01387.02203.02097.053.025.0541.01145.051.0Agreement withExpected Result30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%26/3086.7%30/30100%29/3096.7%30/30100%29/3096.7%30/30100%30/30100%24/3080%29/3096.7%
Site 1Median MFI Value1283.32269.0275.5363.0297.01113.01936.348.580.5265.0838.554.0Site 1Median MFI Value685.02585.0104.3344.01468.52287.82149.367.536.5657.81303.359.525th Percentile MFI477.0860.01405.51635.050.090.0212.5546.050.0831.5469.02602.02744.043.038.0
75th Percentile MFI1561.02520.5329.5413.5383.01317.02010.055.5105.0325.0895.566.575th Percentile MFI840.02673.0140.0408.51545.02356.02216.590.042.5742.01392.076.5Site 1Median MFI Value554.5941.81476.51797.564.3108.8315.8762.060.5938.5591.02681.32870.075.344.5
% CV23.4013.06N/A32.6631.1916.738.11N/AN/A35.3715.89N/A% CV27.644.82N/A18.497.756.276.32N/AN/A21.8713.80N/A75th Percentile MFI659.51177.01697.02059.078.0132.0480.01101.068.51189.0787.02843.03039.5146.078.0
Agreement withExpected Result27/3090%30/30100%19/3063.3%29/3096.7%30/30100%30/30100%30/30100%29/3096.7%17/3056.7%27/3090%29/3096.7%30/30100%Agreement withExpected Result30/30100%30/30100%23/3076.7%29/3096.7%30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%29/3096.7%30/30100%% CV26.1423.8712.9219.34N/AN/A59.7056.62N/A23.4837.046.016.07N/AN/A
25th Percentile MFI842.51924.599.0235.5315.0726.02529.029.085.0284.5827.049.0Site 225th Percentile MFI506.02618.069.0317.01617.02532.52443.051.536.5752.01506.041.0Agreement withExpected Result30/30100%30/30100%30/30100%30/30100%29/3096.7%30/30100%26/3086.7%30/30100%28/3093.3%30/30100%30/30100%30/30100%30/30100%23/3076.7%22/3073.3%
Site 2Median MFI Value1075.32086.0130.3362.8412.3988.52723.340.8141.0327.81059.056.5Median MFI Value707.83012.393.8419.31729.02641.02537.874.046.5901.51579.857.025th Percentile MFI492.0743.01337.01643.046.0104.0212.0888.054.0867.0565.53098.03289.042.030.0
75th Percentile MFI1520.02515.0200.0444.0509.01263.02864.045.0166.5535.51140.564.075th Percentile MFI947.03159.0120.0494.51867.02759.52619.0106.552.51053.01953.072.5Site 2Median MFI Value587.01004.01620.02012.558.8115.5399.51264.064.0992.0706.33272.83494.096.044.3
% CV51.6618.76N/A37.1736.6133.208.95N/AN/A34.4626.02N/A% CV42.4212.44N/A31.1818.926.267.77N/AN/A21.6325.88N/A75th Percentile MFI765.01288.51777.02201.077.0131.0675.01548.072.51143.5894.03393.03678.5191.0203.0
Agreement withExpected Result25/3083.3%30/30100%30/30100%21/3070%25/3083.3%29/3096.7%30/30100%30/30100%30/30100%2/306.7%30/30100%30/30100%Site 3Agreement withExpected Result27/3090%30/30100%30/30100%29/3096.7%30/30100%30/30100%30/30100%25/3083.3%30/30100%29/3096.7%30/30100%30/30100%% CV32.8627.6117.8215.20N/AN/A79.9635.73N/A28.8944.3515.2713.89N/AN/A
25th Percentile MFI160.0416.042.5145.0187.0620.01491.037.555.0122.0505.051.025th Percentile MFI229.01391.053.0425.51265.02058.51723.069.036.0278.5817.049.5Agreement withExpected Result30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%28/3093.3%30/30100%27/3090%30/30100%30/30100%30/30100%30/30100%30/30100%28/3093.3%
Site 3Median MFI Value258.5757.555.5195.3213.0790.01691.044.067.0160.8559.061.8Median MFI Value314.51595.367.5477.01384.52212.31903.395.043.0412.31065.058.525th Percentile MFI398.5614.01088.01231.049.592.0228.0628.058.0753.5641.02178.02383.046.036.5
75th Percentile MFI369.01086.565.0258.5250.0945.01851.056.076.0191.0652.075.075th Percentile MFI346.01773.578.0668.01473.02326.52028.0146.550.0574.51214.072.5Site 3Median MFI Value491.8676.51218.31353.062.0107.0332.5748.068.0936.0691.02323.52526.365.556.3
% CV48.9649.92N/A38.3528.3636.0723.66N/AN/A32.5219.57N/A% CV39.7934.37N/A37.5022.8029.9530.36N/AN/A35.2024.62N/A75th Percentile MFI603.5897.01324.01735.083.5136.0474.5978.588.01154.0925.02564.02773.599.091.5
Total Agreement withExpected Result82/9091.1%90/90100%54/9060%80/9088.9%85/9094.4%89/9098.9%90/90100%89/9098.9%75/9083.3%46/9051.1%89/9098.9%90/90100%Total Agreement withExpected Result87/9096.7%90/90100%77/9085.6%88/9097.8%90/90100%90/90100%90/90100%85/9094.4%90/90100%89/9098.9%89/9098.9%90/90100%% CV27.7922.9819.2121.50N/AN/A54.0933.58N/A26.0334.6220.4422.38N/AN/A
95% CI83.4%-95.4%95.9%-100.0%49.7%-69.5%80.7%-93.9%87.6%-97.6%94.0%-99.8%95.9%-100.0%94.0%-99.8%74.3%-89.6%41.0%-61.2%94.0%-99.8%95.9%-100.0%95% CI90.7%-98.9%95.9%-100.0%76.9%-91.4%92.3%-99.4%95.9%-100.0%95.9%-100.0%95.9%-100.0%87.6%-97.6%95.9%-100.0%94.0%-99.8%94.0%-99.8%95.9%-100.0%Total Agreement withExpected Result90/90100%90/90100%90/90100%90/90100%89/9098.9%90/90100%80/9088.9%90/90100%84/9093.3%90/90100%89/9098.9%90/90100%90/90100%77/9085.6%79/9087.8%
Overall 25th PercentileMFI298.01086.562.0218.0219.0781.01801.535.061.5161.0584.048.5Overall 25th PercentileMFI338.01773.564.0328.01387.02203.01978.055.533.0472.01089.045.095% CI95.9%-100.0%95.9%-100.0%95.9%-100.0%95.9%-100.0%94.0%-99.8%95.9%-100.0%80.4%-93.9%95.9%-100.0%86.2%-96.9%95.9%-100.0%94.0%-99.8%95.9%-100.0%95.9%-100.0%76.9%-91.4%79.4%-93.0%
Overall MedianMFI Value1003.01990.3121.5281.5283.0954.51966.343.579.3260.0801.358.0Overall Median MFIValue537.02548.382.8415.31499.52327.02170.876.341.3636.01310.059.0Overall 25th PercentileMFI440.0690.01233.01484.049.090.5221.0685.054.0807.0546.02401.52616.043.036.0
Overall 75th PercentileMFI1315.02326.0241.0382.5397.51193.52529.052.5121.0325.0944.070.0Overall 75th PercentileMFI764.52746.5116.0489.01650.52615.02456.5106.549.5827.51539.072.5Overall Median MFIValue548.3887.31439.01733.862.0112.8336.5861.863.0956.0690.02712.82986.572.848.0
Overall% CV66.0145.24N/A43.2144.3232.0125.52N/AN/A50.1330.95N/AOverall% CV51.2728.85N/A36.9021.6918.8420.99N/AN/A38.4329.58N/AOverall 75th PercentileMFI693.01093.01698.02059.080.0132.0510.51318.076.51154.0874.03131.03425.0139.5108.5
Overall% CV30.2928.2319.5821.71N/AN/A70.3046.14N/A26.0640.1520.0119.72N/AN/A

Reproducibility of Overall Total Raw Median MFI values for All Targets in xTAG GPP

{9}------------------------------------------------

{10}------------------------------------------------

*The Salmonella positive (Ne) calls presented in this able, represent when the signal from the individual Salmonella probe in question is either above or below the assy thres for a positive call, it does not represent a true as information from both probes is required to determine the call for this target.

{11}------------------------------------------------

Panel Member IDSTEC stx1/stx2 Low PositiveSTEC stx1/stx2 Medium PositiveSTEC stx1/stx2 High NegativeShigella Low PositiveShigella Medium PositiveShigella High Negative
Probe 1Probe 2Probe 1Probe 2Probe 1Probe 2
Concentration$9.38x10^5$ CFU/mL$2.34x10^5$ CFU/mL$3.75x10^6$ CFU/mL$3.75x10^6$ CFU/mL$2.93x10^4$ CFU/mL$2.93x10^4$ CFU/mL$7.32x10^3$ CFU/mL$2.93x10^4$ CFU/mL$2.29x10^2$ CFU/mL
Agreement with Expected Result30/30100%30/30100%30/30100%30/30100%30/30100%29/3096.7%29/3096.7%30/30100%27/3090%
25th Percentile MFI715.5296.51573.01756.042.058.5589.01102.043.0
Site 1Median MFI Value813.0414.81632.51830.357.085.3644.51171.051.0
75th Percentile MFI894.0509.51676.51881.076.0105.0730.01240.580.0
% CV18.7325.758.565.86N/AN/A25.2112.04N/A
Agreement with Expected Result30/30100%30/30100%30/30100%30/30100%28/3093.3%23/3076.7%30/30100%30/30100%25/3083.3%
25th Percentile MFI875.0455.51974.02086.045.064.0623.01255.042.0
Site 2Median MFI Value995.3542.02051.82286.358.0102.0707.31316.364.8
75th Percentile MFI1140.0607.02198.02387.0100.0148.0866.51375.0125.5
% CV23.3822.1511.048.77N/AN/A23.6810.25N/A
Agreement with Expected Result29/3096.7%30/30100%30/30100%30/30100%30/30100%23/3076.7%28/3093.3%30/30100%30/30100%
25th Percentile MFI412.0412.01027.51509.550.068.5307.5678.545.0
Site 3Median MFI Value494.5522.31136.51641.857.0103.3432.0826.356.0
75th Percentile MFI583.0597.01273.01726.071.0148.0495.0951.071.0
% CV29.2728.0729.2430.35N/AN/A41.8839.24N/A
Total Agreement with Expected Result89/9098.9%90/90100%90/90100%90/90100%88/9097.8%75/9083.3%87/9096.7%90/90100%82/9091.1%
95% CI94.0%-99.8%95.9%-100.0%95.9%-100.0%95.9%-100.0%92.3%-99.4%74.3%-89.6%90.7%-98.9%95.9%-100.0%83.4%-95.4%
Overall 25th Percentile MFI564.5386.51273.01663.545.065.0438.0901.043.0
Overall Median MFI Value769.0490.31622.31848.057.096.5623.81171.057.0
Overall 75th Percentile MFI958.0557.51984.52225.077.5134.0727.01313.096.5
Overall % CV34.8427.3928.4921.93N/AN/A35.1626.97N/A

{12}------------------------------------------------

Rotavirus A Low Positive/Norovirus GII High PositiveRotavirus A High Positive/Norovirus GII Low PositiveRotavirus A Low Positive/Salmonella* High PositiveRotavirus A High Positive/Salmonella* Low Positive
Panel Member IDRotavirus ALow PositiveNorovirus GIIHigh PositiveRotavirus AHigh PositiveNorovirus GIILow PositiveRotavirusA LowSalmonella*High PositiveRotavirusA HighSalmonella*Low Positive
Probe 2Probe 2PositiveProbe 1Probe 2PositiveProbe 1Probe 2
Concentration4.47x103Copies/mL2.94x104Copies/mL1.02x105 Copies/mL3.93x103 Copies/mL3.78x103Copies/mL3.75 x100CFU/mL3.75 x10°CFU/mL1.93x104Copies/mL1.17x103CFU/mL1.17x10CFU/mL
Agreement withExpected Result30/30100%30/30100%30/30100%30/30100%29/3096.7%30/30100%30/30100%30/30100%30/30100%30/30100%
25th Percentile MFI303.52181.01995.0707.0207.02958.03220.01583.01043.0586.0
Site 1Median MFI Value416.82306.02386.0905.0302.83062.53368.31944.31188.8664.0
75th Percentile MFI665.02548.02612.01053.0543.03200.03508.02344.01303.0835.0
% CV61.0513.2125.3924.8571.779.047.3731.0917.3732.68
Site 2Agreement withExpected Result30/30100%30/30100%30/30100%28/3093.3%29/3096.7%30/30100%30/30100%30/30100%30/30100%30/30100%
25th Percentile MFI460.01699.52424.0605.0311.03430.03549.02206.01235.5902.0
Median MFI Value625.02086.83164.5740.0432.03564.53770.82430.81363.01060.0
75th Percentile MFI1154.02382.03660.5877.0527.53633.53924.02708.51547.01398.0
% CVરતે તેવ19.5424.1731.4941.026.158.0720.7818.5137.00
Agreement withExpected Result30/30100%30/30100%30/30100%20/3066.7%28/3093.3%30/30100%30/30100%30/30100%30/30100%30/30100%
25th Percentile MFI402.51277.01290.5331.5257.02501.02759.01040.5782.0603.0
Site 3Median MFI Value625.51486.51843.0388.5357.82750.32985.51319.8918.5774.5
75th Percentile MFI796.02202.02538.0613.0601 -52934.53106.51765.01071.0899.0
% CV71.7930.6838.7839.0680.6615.4713.6050:3120.2033.99
Total Agreement withExpected Result90/90100%90/90100%90/90100%78/9086.7%86/9095.6%90/90100%90/90100%90/90100%90/90100%90/90100%
95% CI95.9%-100.0%95.9%-100.0%95.9%-100.0%78.1%-92.2%89.1%-98.3%95.9%-100.0%95.9%-100.0%95.9%-100.0%95.9%-100.0%95.9%-100.0%
Overall 25th PercentileMFI373.01675.01846.0432.0261.02782.03049.01439.0947.0608.0
Overall MedianMFI Value568.02181.02412.3700.0385.33079.83365.51964.51146.8791.3
Overall 75th PercentileMFI887.02386.03015.5890.5566.53462.03673.02455.01335.01065.5
Overall% CV67.1425.1434.5541.1967.2014.2513.4138.0124.6041.11

Reproducibility of Overall Total Raw Median MFI values for Mixed Analytes in xTAG GPP

*The Salmonella positive (Ne) calls presented in this table, represent when the signal from the individual Salmonella probe in question is ether above or below the assorthesh for a positive call, it does not represent a true as information from both probes is required to determine the call for this target.

{13}------------------------------------------------

Panel Member IDETEC Low PositiveSalmonella* High PositiveETEC High PositiveSalmonella* Low Positive
Probe 1Probe 2Probe 1Probe 2Probe 1Probe 2
Concentration$9.37x10^5$ CFU/mL$9.37x10^5$ CFU/mL$3.75 x10^6$ CFU/mL$7.50x10^6$ CFU/mL$7.50x10^6$ CFU/mL$1.17x10^5$ CFU/mL$1.17x10^5$ CFU/mL
Agreement withExpected Result30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%29/3096.7%
25th Percentile MFI1331.0516.03013.02477.02180.01111.5720.0
Site 1Median MFI Value1526.3588.03083.82559.02361.81269.0919.5
75th Percentile MFI1713.5654.03221.02723.52452.01501.01189.0
% CV13.7318.496.229.948.9122.2332.28
Agreement withExpected Result30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%
25th Percentile MFI2153.0883.03442.03175.02717.51315.0882.0
Site 2Median MFI Value2508.81104.83657.03399.02879.51378.01065.0
75th Percentile MFI2788.01378.03789.03519.03111.01641.01236.0
% CV18.3928.809.158.619.3124.4031.01
Agreement withExpected Result30/30100%30/30100%30/30100%30/30100%30/30100%30/30100%29/3096.7%
25th Percentile MFI1985.0660.52515.02530.01937.0816.0680.0
Site 3Median MFI Value2163.5741.52918.52736.52026.51072.5889.3
75th Percentile MFI2323.0943.53056.02930.52252.01221.01081.0
% CV14.7631.7612.7912.1815.9531.0836.57
Total Agreement withExpected Result90/90100%90/90100%90/90100%90/90100%90/90100%90/90100%88/9097.8%
95% CI95.9%-100.0%95.9%-100.0%95.9%-100.0%95.9%-100.0%95.9%-100.0%95.9%-100.0%92.3%-99.4%
Overall 25th PercentileMFI1578.0610.52927.52545.02162.01048.5764.0
Overall MedianMFI Value2039.8736.53083.82788.82390.51279.0961.0
Overall 75th PercentileMFI2398.0983.03442.03175.02717.51474.01216.0
Overall% CV24.9339.1313.8615.5118.5928.0233.74

*The Salmonella positive (Ne) alls presented in this table, represent when the signal from the individual Salmonella probe in question is either above or below the assystems for a positive call, it does not represent a true as information from both probes is required to determine the call for this target.

{14}------------------------------------------------

Overall, adequate site-to-site reproducibility has been established for the 11 viral, bacterial and parasitic targets that xTAG GPP has been designed to detect.

Repeatability

Repeatability was assessed for each microbial 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. Due to a limitation in the sample volume available for the Cryptosporidium analyte, and for the Rotavirus analyte, the Moderate Positive dilution level was not assessed for these targets. Results of testing were as follows:

AnalyteDilution LevelConcentrationxTAG GPP CallsMean MFI Value% CV
CampylobacterModerate2.34x105 CFU/mL20 of 2089612.91%
CampylobacterLow5.86x104 CFU/mL20 of 2038325.85%
C. difficile Toxin A/BModerate1.50x107 CFU/mL20 of 20122425.56%
Probe 1 Low3.75x106 CFU/mL20 of 2045019.68%
C. difficile Toxin A/BProbe 2 Moderate3.75x106 CFU/mL20 of 20112611.10%
Probe 2 Low9.38x105 CFU/mL20 of 2036224.83%
Cryptosporidium hominisModerate PositiveNot Assessed^Not AssessedNot AssessedNot Assessed
Cryptosporidium hominisLow Positive/LoD3.51x104 Copies/mL (extracted DNA)19 of 20 POS81024.17%
E. coli O157Moderate9.38x105 CFU/mL20 of 20167413.82%
E. coli O157Low2.34x105 CFU/mL20 of 2058528.45%
ETEC LT/STProbe 1 Moderate9.38x105 CFU/mL20 of 2093019.89%
Probe 1 Low2.34x105 CFU/mL20 of 2032127.28%
ETEC LT/STProbe 2 Moderate7.50x106 CFU/mL20 of 2017417.55%
Probe 2 Low3.75x106 CFU/mL20 of 2051520.89%
GiardiaModerate8.81x102 cells/mL20 of 20191320.97%
GiardiaLow2.20x102 cells/mL20 of 20124318.97%
Norovirus GI/GIIProbe 1 Moderate Positive1.95x106 Copies/mL (extracted RNA)20 of 20 POS175618.37%
Probe 1 Low Positive/LoD6.56x105 Copies/mL (extracted RNA)20 of 20 POS99121.53%
Norovirus GI/GIIProbe 2 Moderate Positive2.44x106 Copies/mL (extracted RNA)20 of 20 POS102532.96%
Probe 2 Low Positive/LoD1.15x106 Copies/mL (extracted RNA)20 of 20 POS80828.87%

Assay Repeatability

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Rotavirus AModeratePositiveNot available§20 of 20POS98016.37%
Low Positive/LoD6.84x104 Copies/mL(extracted RNA)19 of 20POS48620.70%
SalmonellaProbe 1Moderate9.38x105 CFU/mL20 of 2021006.42%
Low2.34x105 CFU/mL20 of 20137717.87%
Probe 2Moderate9.38x105 CFU/mL20 of 20191611.20%
Low2.34x105 CFU/mL20 of 20100525.29%
ShigellaModerate2.93x104 CFU/mL20 of 2017156.26%
Low3.67x103 CFU/mL20 of 2079528.19%
STEC stx1/stx2Probe 1Moderate3.75x106 CFU/mL20 of 20127110.29%
Low9.38x105 CFU/mL20 of 2050317.11%
Probe 2Moderate9.38x105 CFU/mL20 of 20100211.65%
Low2.34x105 CFU/mL20 of 2033431.91%

^Due to limited sample volume (pooled positive clinical material was used) &A clinical specimen was used for which the concentration was not available

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

  • b. Linearity/assay reportable range:
    Not applicable, qualitative assay.

  • c. Traceability, Stability, Expected values (controls, calibrators, or methods):
    Before using the Luminex system to read samples prepared by the xTAG assay, prepare and calibrate the Luminex instrument system following the procedures in the appropriate system user manual.

Negative Controls - Negative controls are defined as either RNase-free water added to the RT-PCR/PCR step (amplification/detection negative control) or lysis buffer that has undergone the entire assay procedure (pretreatment/extraction/amplification/detection negative control). At least one negative control that underwent extraction process must be included in each batch of specimens run on xTAG GPP. The recommended number of negative controls to be included in a batch is dependent on batch size. For batches of 1-30 samples, one negative control must be included. For batches of 31-61 samples, two negative controls are recommended. For batches of 62-92 samples, three negative controls are recommended. When running multiple negative controls disperse the controls throughout the batch.

NOTE: Users will need to identify all the negative controls (including extraction controls) from the TDAS software before the test data is analyzed. If a negative control has a significant signal detected for an analyte, the TDAS software will generate a 'no

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call' for the samples that were positive for the specific analyte and they will need to be retested.

External Positive Controls - Known strains or positive clinical samples with known results for the targeted viruses, bacteria or parasites should be included in routine quality control procedures ("external controls") as positive controls for the assay. At least one of these external controls are analyte positive controls and should be included with each batch of patient specimens and controls positive for different targets should be rotated from batch to batch. External controls should be prepared, extracted and tested in the same manner as patient samples. Results from external controls should be examined before the results from the patient samples. The interpretation of the correct positive control results is performed by the user and not the data analysis software (TDAS). If a given analyte control does not perform as expected, all results for that analyte in the batch of samples should be examined to determine if a re-run is required. If any unexpected calls occur where one or more analytes with signal exceeding the thresholds are detected in any of the positive controls (i.e. non-specific positive signals) for a given run then samples that were positive for the specific analyte(s) that triggered a control failure will need to be re-run. At least one positive control per PCR run must pass, i.e. all expected calls made in order to report any results from the plate.

Internal Control - Bacteriophage MS2 is the internal control for the assay. This internal positive control is added to each patient specimen prior to extraction. This internal control allows the user to ascertain whether the assay is functioning properly. Failure to generate a PRES (present) call for the MS2 control indicates a failure at either the extraction step, and/or the reverse-transcription step, and/or the PCR step, and may be indicative of the presence of amplification inhibitors, which can lead to false negative results.

d. Detection limit:

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 were as follows:

AnalyteStrain IDTitre(corresponding tothe estimated LoD)Average MFIValue%CV
CampylobacterCampylobacter jejuni, 49943(Strain LRA 094.06.89)$5.86x10^4$ CFU/mL38325.85%
C. difficile Toxin A/BClostridium difficile, BAA-1805 (toxinotype III A+B+)$9.38x10^5$ CFU/mL36224.83%
Clostridium difficile, 43255(toxinotype 0 A+B+)$3.75x10^6$ CFU/mL52719.63%
CryptosporidiumhominisClinical sample$3.51x10^4$ Copies/mL81024.17%

Summary Limit of Detection (LoD) for GPP Analytes

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E. coli O157E. coli O157, 0801622(EDL933; O157:H7; STECToxin I+II+)$2.34x10^5$ CFU/mL58528.45%
ETEC LT/STE. coli, 35401 (O78:H11;ST+LT+)$2.34x10^5$ CFU/mL32127.28%
GiardiaGiardia lamblia, PRA-243$2.20x10^2$ cells/mL124318.97%
Norovirus GI/GIIClinical sample – GI$6.56x10^5$ Copies/mL99121.53%
Norovirus GI/GIIClinical sample – GII$1.15x10^6$ Copies/mL80828.87%
Rotavirus AClinical sample$6.84x10^4$ Copies/mL48620.70%
SalmonellaSalmonella enterica, 13311(Serotype Typhimurium)$2.34x10^5$ CFU/mLProbe 1=1377Probe 2=1005Probe 1=17.87%Probe 2=25.29%
STEC stx1/stx2E. coli O157, 0801622(EDL933; O157:H7; STECToxin I+II+)$2.34x10^5$ CFU/mL33431.91%
ShigellaShigella sonnei, 25931(Subgroup D)$3.67x10^3$ CFU/mL79528.19%

The data summarized above establish a limit of detection for each indicated analyte.

e. Analytical specificity:

Analytical Reactivity

Analytical reactivity was assessed through empirical testing of a wide range of clinically relevant GI pathogen strains, genotypes, serotypes and isolates representing temporal and geographical diversity for each analyte. (Note: Some differences in sensitivity may be expected as a result of sequence diversity within the gene targeted by the GPP assay primers.) Pathogens were diluted two to three times (2x-3x) the claimed Limit of Detection (LoD) in a negative clinical matrix (stool) prior to pre-treatment and extraction. An internal control (MS2) was added to each diluted pathogen sample prior to pretreatment. Extraction was performed with the Biomerieux Nuclisens EasyMag extraction method. Through testing of 265 unique samples covering all intended use pathogens, reactivity was established at concentrations 2 to 3 times the limit of detection. The following table lists the samples tested:

Reactivity
PathogenATCC/OtherReferencePathogenATCC/OtherReference
Campylobacter jejuniATCC 29428Salmonella entericasubsp. enterica, SerovarTyphimuriumATCC 14028
Campylobacter jejunisubsp. JejuniATCC 33291Salmonella entericasubsp. enterica, SerotypeDublinATCC 15480
Campylobacter coliATCC 33559Salmonella entericasubsp. Enterica, serotypeTyphiATCC 19430
Campylobacter jejunisubsp. jejuniATCC 33560Salmonella bongori typestrainATCC 43975 / NCTC12419
Campylobacter lariATCC 35221Salmonella entericasubsp. enterica, SerotypeVirchowATCC 51955
Campylobacter larisubsp. lariATCC 35223Salmonella entericasubsp. enterica, SerotypeHadarATCC 51956
Campylobacter jejunisubsp. jejuniATCC 35920Salmonella entericasubsp. enterica, SerotypeAgonaATCC 51957
Campylobacter coliATCC 43473Salmonella entericasubsp. Enterica,Serotype Paratyphi Bvariant JavaATCC 51962
Campylobacter coliATCC 43474Salmonella entericasubsp. Enterica,Serotype DerbyATCC 6960
Campylobacter coliATCC 43482Salmonella entericasubsp. Enterica,Serotype NewportATCC 6962
Campylobacter coliATCC 43485Salmonella entericasubsp. enterica, SerotypeBraenderupATCC 700136
Campylobacter lariATCC 43675Salmonella entericasubsp. enterica, SerotypeCholeraesuisATCC 7001
Campylobacter lariATCC BAA-1060Salmonella entericasubsp. Enterica,Serotype StanleyATCC 7308
Campylobacter coliATCC BAA-372Salmonella entericasubsp. enterica, SerotypePanamaATCC 7378
CampylobacterZeptometrix 0801650Salmonella entericasubsp. Enterica, SerotypeHeidelbergATCC 8326
Clostridium difficiletoxin A/BATCC 9689Salmonella entericasubsp. enterica, SerotypeMontevideoATCC 8387
Clostridium difficiletoxin A/BATCC 17857 (870)Salmonella entericasubsp. enterica, SerotypeMuenchenATCC 8388
Clostridium difficiletoxin A/BATCC 17858 (1253)Salmonella entericasubsp. enterica, SerotypeThompsonATCC 8391
Clostridium difficiletoxin A/BATCC 43594Salmonella entericasubsp. Enterica,Serotype Paratyphi BATCC 8759
Clostridium difficiletoxin A/BATCC 43596Salmonella entericasubsp. Enterica,Serotype BareillyATCC 9115
Clostridium difficileATCC 43598Salmonella entericaATCC 9239
Clostridium difficiletoxin A/BATCC 43599Salmonella entericasubsp. Enterica,Serotype OranienburgATCC 9263
Clostridium difficiletoxin A/BATCC 43600Salmonella entericasubsp. enterica, SerotypeKentuckyATCC 9270
Clostridium difficiletoxin A/BATCC 51695Salmonella entericasubsp. Enterica,Serotype AnatumATCC 9712
Clostridium difficiletoxin A/BATCC 700792Salmonella entericasubsp. Enterica,Serotype SaintpaulATCC BAA-1675
Clostridium difficiletoxin A/BATCC BAA-1382Salmonella entericasubsp. enterica, SerotypeInfantisCDC_Salmonella A
Clostridium difficiletoxin A/BATCC BAA-1803Salmonella entericasubsp. entericaCDC_Salmonella B
Clostridium difficiletoxin A/BATCC BAA-1814Salmonella entericasubsp. Enterica,Serotype Paratyphi Bvar. L(+) tartrate+07-7741, CNR#
Clostridium difficiletoxin A/BATCC BAA-1870Salmonella entericasubsp. enterica, 4:i:-07-2537, CNR
Clostridium difficiletoxin A/BATCC BAA-1871Salmonella entericasubsp. enterica, 4:i:-05-960, CNR
Clostridium difficiletoxin A/BATCC BAA-1872Salmonella entericasubsp. enterica, Agona1137/72, CNR
Clostridium difficiletoxin A/BATCC BAA-1875Salmonella entericasubsp. enterica, Agona84K, CNR
Clostridium difficiletoxin A/BATCC BAA-2155Salmonella entericasubsp. Enterica, Anatum08-2926, CNR
Clostridium difficiletoxin A/BATCC BAA-2156Salmonella entericasubsp. Enterica, Anatum49K, CNR
Clostridium difficiletoxin A/BZeptometrix 0801620Salmonella entericasubsp. enterica,Braenderup24K, CNR
Cryptosporidium parvumWaterborne Inc.Salmonella entericasubsp. enterica,Brandenburg2/84, CNR
Cryptosporidium hominisWaterborne Inc.Salmonella entericasubsp. enterica,Choleraesuis var Decatur36K, CNR
Cryptosporidium parvumZeptometrix 0801700Salmonella entericasubsp. Enterica,Choleraesuis varKunzendorf34K, CNR
Cryptosporidium parvumATCC PRA-67Dsubsp. enterica,Choleraesuis var sensustricto263K, CNR
Cryptosporidium parvumATCC 87668Salmonella entericasubsp. Enterica,Corvallis20K, CNR
Cryptosporidium parvumATCC 87712Salmonella entericasubsp. enterica, Derby354/67, CNR
Cryptosporidium parvumATCC 87763Salmonella entericasubsp. enterica, Dublin05-1078, CNR
Cryptosporidium parvumATCC 87765Salmonella entericasubsp. enterica, Dublin65K, CNR
Escherichia coli O157ATCC 43888Salmonella entericasubsp. enterica,Enteritidis89-323, CNR
Escherichia coli O157ATCC 43890Salmonella entericasubsp. enterica,Enteritidis02-131, CNR
Escherichia coli O157ATCC 43894Salmonella entericasubsp. enterica,Enteritidis02-9053, CNR
Escherichia coli O157ATCC 43895Salmonella entericasubsp. enterica,Enteritidis89-329, CNR
Escherichia coli 091(Produces shiga-liketoxin II)ATCC 51435Salmonella entericasubsp. enterica,Enteritidis5-56, CNR
Escherichia coli O157ATCC 700376Salmonella entericasubsp. enterica,Enteritidis03-3527, CNR
Escherichia coli O157ATCC 700377Salmonella entericasubsp. enterica,Enteritidis02-4884, CNR
Escherichia coli O157ATCC 700378Salmonella entericasubsp. enterica, Hadar02-2760, CNR
Escherichia coli 0113(Produces shiga toxin 2)ATCC BAA-176Salmonella entericasubsp. enterica, Hadar2-74, CNR
Escherichia coli 0113(Produces shiga toxin 1and 2)ATCC BAA-177Salmonella entericasubsp. enterica,Heidelberg16K, CNR
Escherichia coli 0111ATCC BAA-181Salmonella enterica08-2380, CNR
(Produces shiga toxin 1and 2)subsp. enterica,Heidelberg
Escherichia coli O104(produces shiga toxin 2)ATCC BAA-182Salmonella entericasubsp. enterica, Infantis158K, CNR
Escherichia coli O26ATCC BAA-1653Salmonella entericasubsp. enterica, Infantis05-6334, CNR
Escherichia coli O104(produces shiga toxin 2)ATCC BAA-2326Salmonella entericasubsp. enterica, Javiana4-57, CNR
Escherichia coli078:H11 (produces LTand ST)ATCC 35401Salmonella entericasubsp. enterica, Javiana214K, CNR
Escherichia coliO25:K98:NM (producesLT)ATCC 43886Salmonella entericasubsp. enterica,Kentucky98K, CNR
Escherichia coliZeptometrix 0801624Salmonella entericasubsp. enterica,Kentucky07-6574, CNR
Escherichia coli078::K80H12 (producesST)ATCC 43896Salmonella entericasubsp. enterica,Kentucky06-5737, CNR
Giardia intestinalisATCC 30888Salmonella entericasubsp. Enterica,Mississippi1933/77, CNR
Giardia intestinalisATCC 30957Salmonella entericasubsp. enterica,Montevideo126K, CNR
Giardia intestinalisATCC 50114Salmonella entericasubsp. enterica,Montevideo06-7410, CNR
Giardia intestinalisATCC 50137Salmonella entericasubsp. enterica,Montevideo46K, CNR
Giardia intestinalisATCC 50581Salmonella entericasubsp. enterica,Montevideo06-8080, CNR
Giardia intestinalisATCC 50584 (JH)Salmonella entericasubsp. enterica,Montevideo06-8107, CNR
Giardia intestinalisATCC 50585Salmonella entericasubsp. enterica,Montevideo05-8072, CNR
Giardia lambliaATCC PRA-242Salmonella entericasubsp. enterica54K, CNR
Muenchen
Giardia lambliaATCC PRA-244Salmonella entericasubsp. enterica, Newport05-815, CNR
Giardia lambliaATCC PRA-247Salmonella entericasubsp. enterica, Newport50K, CNR
Giardia lambliaATCC PRA-249Salmonella entericasubsp. enterica, Newport04-2487, CNR
Giardia lambliaWaterborne Inc.Salmonella entericasubsp. enterica, Newport01-2174, CNR
Norovirus GICDC – GP-001Salmonella entericasubsp. enterica, Newport02-7891, CNR
Norovirus GICDC – GP-003Salmonella entericasubsp. Enterica,Oranienburg42K, CNR
Norovirus GICDC – GP-005Salmonella entericasubsp. enterica, Panama73K, CNR
Norovirus GICDC – GP-007Salmonella entericasubsp. enterica,Paratyphi A1K, CNR
Norovirus GICDC – GP-008Salmonella entericasubsp. enterica,Paratyphi A06-2065, CNR
Norovirus GICDC – GP-009Salmonella entericasubsp. enterica,Paratyphi BCIPA214, CNR
Norovirus GICDC – GP-010Salmonella entericasubsp. enterica,Paratyphi B05-4862, CNR
Norovirus GICDC – GP-012Salmonella entericasubsp. enterica,Paratyphi B02-9348, CNR
Norovirus GICDC – GP-013Salmonella entericasubsp. enterica,Paratyphi B5K, CNR
Norovirus GICDC – GP-015Salmonella entericasubsp. enterica,Paratyphi B02-2529, CNR
Norovirus GICDC – GP-016Salmonella entericasubsp. enterica,Paratyphi B6332/88-1, CNR
Norovirus GICDC – GP-018Salmonella entericasubsp. enterica,Paratyphi C32K, CNR
Norovirus GICDC – GP-020Salmonella enterica subsp. enterica, Saintpaul108K, CNR
Norovirus GIICDC – GP-023Salmonella enterica subsp. enterica, Saintpaul05-5166, CNR
Norovirus GIICDC – GP-024Salmonella enterica subsp. Enterica, Stanley15K, CNR
Norovirus GIICDC – GP-025Salmonella enterica subsp. Enterica, Stanley397K, CNR
Norovirus GIICDC – GP-027Salmonella enterica subsp. Enterica, Tennessee142K, CNR
Norovirus GIICDC – GP-030Salmonella enterica subsp. enterica, Thompson40K, CNR
Norovirus GIICDC – GP-033Salmonella enterica subsp. enterica, Typhimurium38 (98) MN, CNR
Norovirus GIICDC – GP-034Salmonella enterica subsp. enterica, Typhimurium49 (98) MN, CNR
Norovirus GIICDC – GP-035Salmonella enterica subsp. enterica, Typhimurium150 (98) MN, CNR
Norovirus GIICDC – GP-036Salmonella enterica subsp. enterica, Typhimurium226 (97) MN, CNR
Norovirus GIICDC – GP-038Salmonella enterica subsp. enterica, Typhimurium31 (98) MN, CNR
Norovirus GIICDC – GP-039Salmonella enterica subsp. enterica, Typhimurium02-1180, CNR
Norovirus GIICDC – GP-041Salmonella enterica subsp. enterica, Typhimurium14-58, CNR
Norovirus GIICDC – GP-042Salmonella enterica subsp. enterica, Typhimurium00-7866, CNR
Norovirus GIICDC – GP-045Salmonella enterica subsp. enterica, Typhimurium75-2099, CNR
Norovirus GIICDC - GP-047Salmonella entericasubsp. enterica,Typhimurium75/67, CNR
Norovirus GIICDC - GP-048Salmonella entericasubsp. enterica,TyphimuriumSonLa1/Hoang63,CNR
Norovirus GIICDC - GP-049Salmonella entericasubsp. enterica,Typhimurium02-3215, CNR
Norovirus GIICDC - GP-050Salmonella entericasubsp. enterica,Typhimurium02-4577, CNR
Norovirus GIICDC - GP-053Salmonella entericasubsp. enterica,TyphimuriumDK4, CNR
Norovirus GIICDC - GP-054Salmonella entericasubsp. enterica,TyphimuriumLT2, CNR
Norovirus GIICDC - GP-056Salmonella entericasubsp. enterica,Typhimurium01-1639, CNR
Norovirus GIICDC - GP-057Salmonella entericasubsp. enterica,Typhimurium02-4496, CNR
Norovirus GIICDC - GP-058Salmonella entericasubsp. enterica, Virchow41K, CNR
Norovirus GIICDC - GP-059Salmonella entericasubsp. enterica, Virchow03-5167, CNR
Norovirus GIICDC - GP-060Salmonella entericasubsp. arizonae,53:g,z51:-SO 8/9, CNR
Norovirus GIICDC - GP-063Salmonella entericasubsp. Diarizonae,17:z10:e,n,z151458/74, CNR
Norovirus GIICDC - GP-064Salmonella entericasubsp. salamae,11:1,z28:enx1368K, CNR
Norovirus GIICDC - GP-067Salmonella entericasubsp. houtenae,6,7:z4,z24:-575K, CNR
Rotavirus Group AATCC VR-1546Salmonella entericasubsp. indica, 11🅱️1,7437/68, CNR
Rotavirus Group AATCC VR-2018Salmonella bongori1900/76, CNR
66:z35:-
Rotavirus Group AATCC VR-2272Shigella dysenteriae,(Subgroup A)ATCC 11835
Rotavirus Group AATCC VR-2273Shigella flexneri(Subgroup B, serotype 3)ATCC 11836
Rotavirus Group AATCC VR-2274Shigella dysenteriae(Subgroup A, serotype 8)ATCC 12021
Rotavirus Group AATCC VR-2275Shigella flexneri(Subgroup B, serotype 4a)ATCC 12023
Rotavirus Group AATCC VR-2417Shigella flexneri(Subgroup B, serotype 6)ATCC 12025
Rotavirus Group AATCC VR-2550Shigella boydii(Subgroup C, serotype 8)ATCC 12028
Rotavirus Group AATCC VR-2551Shigella boydii(Subgroup C, serotype 10)ATCC 12030
Rotavirus Group ACDC – GP079Shigella boydii(Subgroup C, serotype 11)ATCC 12031
Rotavirus Group ACDC – GP080Shigella dysenteriae(Subgroup A, serotype 9)ATCC 12037
Salmonella entericasubsp. enterica, SerotypeCholeraesuisATCC 10708Shigella dysenteriae(Type strain, SubgroupA, serotype 1)ATCC 13313
Salmonella entericasubsp. Enterica, SerotypeJavianaATCC 10721Shigella sonnei ,Subgroup DATCC 29029
Salmonella entericasubsp. Enterica, SerotypeTennesseeATCC 10722Shigella sonnei,Subgroup DATCC 29030
Salmonella entericasubsp. Enterica, SerotypeParatyphi AATCC 11511Shigella dysenteriae(Subgroup A, serotype 11)ATCC 49547
Salmonella entericasubsp. enterica, SerotypeEnteritidisATCC 13076Shigella dysenteriae,(Subgroup A, serotype 1)ATCC 9361
Salmonella entericasubsp. enterica, SerotypeTyphimuriumATCC 13311Shigella sonnei0801627
Salmonella entericasubsp. ArizonaeATCC 13314 / NCTC8297Shigella flexneri0801757
Salmonella entericasubsp. Enterica, SerotypeParatyphi CATCC 13428

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#CNR – The French National Reference Center

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Analytical Specificity and Potential Interfering Agents

Analytical specificity was assessed with respect to the following parameters:

    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.
    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.
    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.

This study was mainly conducted at LMD (Toronto) with some runs performed at (1) the National Calicivirus Laboratory, Center for Disease Control (CDC) in Atlanta, (2) Scott & White Hospital, Temple, Texas, and (3) Luminex Headquarters, Austin, Texas, Viral cultures were prepared by growing the virus in the appropriate cell host, to a titer of 10 pfu/mL (10 TCIDs/mL) or higher, if available (high positive (HP) sample).

Bacterial cultures were prepared at concentrations of 10 cfu/mL or higher (high positive (HP) sample). Parasites were tested at a clinically relevant level as supported by literature or clinical trial data (e.g. a high titer clinical sample). Low positive samples (LP) were prepared at a concentration that gave MFI values approximately 1-5 times the assay cutoff (depending on the target). Non-microbial agents were prepared at the concentration noted in the table. Microbial and non-microbial agents were prepared in negative clinical matrix.

Results for the 3 categories of testing outlined above were as follows:

  1. There was no cross-reactivity observed in the majority (84) of the 86 relevant pathogen strains, genotypes, serotypes and isolates tested. Note that 9 of the 84 samples that did not cross-react did generate a positive call as they include analytes that are detected by the assay (i.e. they were included to show non-cross reactivity with another analyte). The remaining 2 cross-reacting species are described below and will be addressed in product labeling:

a) Campylobacter fetus subsp. fetus (NCTC 10842, type strain [ATCC 27374]) at a concentration of 6.00E+08 cfu/mL resulted in a positive call for Campylobacter and b) Escherichia coli (Migula) Castellani and Chalmers strain CDC EDL 1284 [929-78] (serotype O124:NM [ATCC 43893]) (enteroinvasive) resulted in a positive call for Shigella.

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Pathogenic FloraATCC/OtherReferenceTiter TestedCross-ReactiveYes (Y) / No (N)
Acinetobacter baumanniiATCC196062.4 x 10^9 cfu/mLN
Adenovirus serotype 1^ATCC VR-11.58 x 10^7TCID50/mLN
Adenovirus serotype 3Zeptometrix 0810062CF5.89 x 10^7TCID50/mLN
Adenovirus serotype 4Zeptometrix 0810070CF7.24 x 10^5TCID50/mLN
Adenovirus serotype 5Zeptometrix 0810020CF1.02 x 10^8TCID50/mLN
Adenovirus serotype 8Zeptometrix 0810069CF3.16 x 10^5TCID50/mLN
Adenovirus serotype 14ATCC VR-151.58 x 10^9TCID50/mLN
Adenovirus serotype 18ATCC VR-10953.16 x 10^6TCID50/mLN
Adenovirus serotype 31GP-092 (CDC)Not knownN
Aeromonas hydrophilaATCC 356546 x 10^8 cfu/mLN
Aichi virusSO603Dijon (CDC)1.00 x 10^8copies/uLN
Arcobacter butzleriATCC 496166 x 10^8 cfu/mLN
Arcobacter cryaerophilusATCC 43158>10^6 cfu/vialN
Astrovirus Type 1GP-086 (CDC)6.00 x 10^7copies/uLN
Astrovirus Type 2GP-087 (CDC)6.00 x 10^7copies/uLN
Bacillus cereusATCC 145796 x 10^8 cfu/mLN
Bacillus cereusATCC 64646 x 10^8 cfu/mLN
Campylobacter fetus subsp. fetus(NCTC 10842, type strain)ATCC 273746 x 10^8 cfu/mLYwith Campylobacter(C. jejuni, C. coli, andC. lari only)
Campylobacter fetus subsp. fetusATCC 332464.43 x 10^5copies/mLN
Campylobacter fetus subsp. fetusATCC 332474.25 x 10^4copies/mLN
Campylobacter fetus subsp. venerealisATCC 194384.11 x 10^4copies/mLN
Campylobacter fetus subsp. venerealisATCC 335614.10 x 10^4copies/mLN
Campylobacter hyointestinalisATCC 352176 x 10^8 cfu/mLN
Pathogenic flora evaluated for potential cross reactivitv
Y (expected)
Campylobacter jejuni subsp. jejuni*ATCC 332916 x 10^8 cfu/mLwith Campylobacter(C. jejuni, C. coli, andC. lari only)
Campylobacter upsaliensisATCC 439542.57 x 10^9copies/mLN
Chlamydia trachomatisATCC VR-3462.81 x 10^6TCID50/mLN
Clostridium perfringensATCC 131246 x 10^8 cfu/mLN
Clostridium septicumATCC 124646 x 10^8 cfu/mLN
Clostridium sordelliiATCC 97146 x 10^8 cfu/mLN
Clostridium tertiumATCC 145736 x 10^8 cfu/mLN
Clostridium tetaniATCC 194066 x 10^8 cfu/mLN
Coxsackie virusATCC VR-288.89 x 10^7TCID50/mLN
Cronobacter sakazakiiZeptometrix 08015332.83 x 10^9 cfu/mLN
Cryptosporidium meleagridisWaterborne, Cat #SPECIAL 18672.50 x 10^5oocysts/mLN
Cryptosporidium murisWaterborne P-104-1X10-6-L2.50 x 10^5 cells/mLN
CytomegalovirusATCC VR-1590Not knownN
CytomegalovirusZeptometrix 0810003CF9.55 x 10^6TCID50/mLN
EchovirusATCC VR-418.89 x 10^6TCID50/mLN
Edwardsiella tardaATCC 159476 x 10^8 cfu/mLN**
Enterovirus (Human enterovirus D(Enterovirus Type 70)), strain J670/71ATCC VR-8368.89 x 10^6TCID50/mLN
Enterovirus (Sabin 3)GP-090 (CDC), cellcultureNot knownN
Escherichia blattaeATCC 299076 x 10^8 cfu/mLN
Escherichia coli (Migula) Castellaniand Chalmers strain CDC EDL 1284[929-78] (serotype O124:NM)(enteroinvasive)ATCC 438936 x 10^8 cfu/mLYwith Shigella
Escherichia coli (Migula) Castellaniand Chalmers strain CFT073(uropathogenic strain)ATCC 7009286 x 10^8 cfu/mLN
Escherichia coli (Migula) Castellaniand Chalmers (serotypeO16:K1(L):NM)ATCC 235116 x 10^8 cfu/mLN
Escherichia coli (Migula) Castellani andChalmers serotype 0111:H8 strainCDC 1999-3249) (Produces Shigatoxin 1 and 2)ATCC BAA-1811 x 10^7 cfu/mLY (expected)with STEC stx1 /stx2
Escherichia fergusoniiATCC 354696 x 10^8 cfu/mLN
Escherichia hermaniiATCC 336506 x 10^8 cfu/mLN
Escherichia vulnerisATCC 338216 x 10^8 cfu/mLN
Gardnerella vaginalisATCC 140196 x 10^8 cfu/mLN
Helicobacter felisATCC 491796 x 10^8 cfu/mLN
Helicobacter pyloriATCC 43504No titer availableN
Helicobacter pyloriZeptometrix 08014863.57 x 10^6 cfu/mLN
Hepatitis A virusGP-088, strain HM175(CDC)2.00 x 10^6 pfu/mLN
Klebsiella oxytocaATCC 131826 x 10^8 cfu/mLN
Klebsiella ozaenae (K. pneumoniasubsp. ozaenae)ATCC 112966 x 10^8 cfu/mLN
Listeria grayiATCC 191206 x 10^8 cfu/mLN
Listeria monocytogenesATCC BAA-8396 x 10^8 cfu/mLN
Norovirus GIVGP-068, Clinical stoolsample collected during anoutbreak (CDC)Not knownN
Plesiomonas shigelloidesATCC 140296 x 10^8 cfu/mLN
Porphyromonas asaccharolyticaATCC 252606 x 10^8 cfu/mLN
Providencia alcalifaciensATCC 98866 x 10^8 cfu/mLN
Providencia rettgeriATCC 92506 x 10^8 cfu/mLN
Providencia stuartiiATCC 336726 x 10^8 cfu/mLN
Rotavirus A (strain WA)*ATCC VR-20181.58 x 10^8 TCID50/mLY (expected)with Rotavirus A
Rotavirus Group BCDC, clinical stoolsample collected during anoutbreakNot knownN
Rotavirus Group CCDC, cell cultureNot knownN
Salmonella enterica subsp. entericaserovar Choleraesuis*ATCC 70016 x 10^8 cfu/mLY (expected)with Salmonella
Salmonella enterica subsp. entericaserovar Typhimurium (formerlySalmonella choleraesuis subsp.Choleraesuis serotype Typhimurium)*ATCC 518126 x 10^8 cfu/mLY (expected)with Salmonella
Salmonella enterica subsp. entericaserovar Typhimurium (formerly Salmonellacholeraesuis subsp.Choleraesuis serotype Typhimurium)*ATCC 195856 x 10^8 cfu/mLY (expected)with Salmonella
Sapovirus GIGP-082, clinical isolate(CDC)1.00 x 10^5copies/uLN
Sapovirus GIIGP-083, clinical isolate(CDC)1.00 x 10^3copies/uLN
Sapovirus GIVGP-085, clinical stoolsample (CDC)1.00 x 10^6copies/uLN
Serratia liquefaciensATCC 355516 x 10^8 cfu/mLN
Serratia marcescens subsp.marcescensATCC 138803.8 x 10^9bacteria/mLN
Shigella boydii*ATCC 120286 x 10^8 cfu/mLY (expected)with Shigella
Shigella dysenteriae serotype 1 ***strain AMC 43-A-14ATCC 93611.00 x 10^7 cfu/mLY (expected)with Shigella andSTEC stx1 / stx2
Shigella sonnei*ATCC 259316 x 10^8 cfu/mLY (expected)with Shigella
Stenotrophomonas maltophiliaATCC 136376 x 10^8 cfu/mLN
Streptococcus dysgalactiae subsp.dysgalactiaeATCC 430786 x 10^8 cfu/mLN
Streptococcus pyogenesATCC 515005.85 x 10^7cells/mLN
Vibrio parahaemoliticusATCC 178026 x 10^8 cfu/mLN
Yersinia bercovieriATCC 439706 x 10^8 cfu/mLN
Yersinia pseudotuberculosisATCC 298336 x 10^8 cfu/mLN
Yersinia rohdeiATCC 433806 x 10^8 cfu/mLN
Commensal FloraATCC/OtherReferenceTiter TestedCross-ReactiveYes (Y) / No (N)
Abiotrophia defectiva†ATCC 491766 x 10^8 cfu/mLN
Acinetobacter haemolyticusATCC 179061.64 x 10^7 cells/mLN
Acinetobacter lwoffiiATCC 153096 x 10^8 cfu/mLN
Actinomyces naeslundiiATCC 121046 x 10^8 cfu/mLN
Akkermansia muciniphilaATCC BAA-8356 x 10^8 cfu/mLN
Alcaligenes faecalis subsp. FaecalisATCC 155546 x 10^8 cfu/mLN
Anaerococcus tetradiusATCC 350986 x 10^8 cfu/mLN
Atopobium vaginaeATCC BAA-556 x 10^8 cfu/mLN
Bacillus subtilis subsp. SpizizeniiATCC 66331.9 x 10^7 cfu/mLN
Bacillus subtilis subsp. SubtilisATCC 60516 x 10^8 cfu/mLN
Bacteroides caccaeATCC 431856 x 10^8 cfu/mLN
Bacteroides fragilisATCC 252856 x 10^8 cfu/mLN
Bacteroides stercorisATCC 431836 x 10^8 cfu/mLN
Bacteroides thetaiotaomicronATCC 291486 x 10^8 cfu/mLN
Bacteroides vulgatusATCC 84826 x 10^8 cfu/mLN
Bifidobacterium adolescentisATCC 157036 x 10^8 cfu/mLN
Bifidobacterium bifidumATCC 295216 x 10^8 cfu/mLN
Bifidobacterium longum subsp.LongumATCC 157076 x 10^8 cfu/mLN
Blastocystis hominisATCC 50587≥ 10^6 cells/mLN
Blastocystis hominisATCC 506082 x 10^7 cells/mLN
Campylobacter concisusATCC 332373.11 x 10^5 copies/mLN
Campylobacter curvusATCC 352241.71 x 10^5 copies/mLN
Campylobacter gracilisATCC 332361.41 x 10^5 copies/mLN
Campylobacter helveticusATCC 512094.64 x 10^7 copies/mLN
Campylobacter hominisATCC BAA-3816.61 x 10^3 copies/mLN
Campylobacter rectusATCC 332381.18 x 10^5 copies/mLN
Campylobacter showaeATCC 511462.49 x 10^3 copies/mLN
Campylobacter sputorum biovarSputorumATCC 359801.56 x 10^6 copies/mLN
Candida albicansATCC 102316 x 10^8 cfu/mLN
Candida catenulateATCC 105656 x 10^8 cfu/mLN
Capnocytophaga gingivalisATCC 336246 x 10^8 cfu/mLN
Cedecea davisaeATCC 334316 x 10^8 cfu/mLN
Chryseobacterium gleumATCC 359106 x 10^8 cfu/mLN
Citrobacter amalonaticusZeptometrix 08017181.35 x 10^10 cfu/mLN
Citrobacter freundiiATCC 80901.3 x 10^8 bacteria/mLN
Citrobacter koseriATCC 270286 x 10^8 cfu/mLN
Citrobacter sedlakiiATCC 511156 x 10^8 cfu/mLN
Clostridium beijerinckiiATCC 82606 x 10^8 cfu/mLN
Clostridium bifermentansATCC 6286 x 10^8 cfu/mLN
Clostridium bolteaeATCC BAA-6136 x 10^8 cfu/mLN
Clostridium butyricumATCC 193986 x 10^8 cfu/mLN
Clostridium chauvoeiATCC 119576 x 10^8 cfu/mLN
Clostridium difficile (non-toxigenic)ATCC 435936 x 10^8 cfu/mLN
Clostridium difficile (non-toxigenic)ATCC 436016 x 10^8 cfu/mLN
Clostridium difficile (non-toxigenic)ATCC 7000576 x 10^8 cfu/mLN
Clostridium fallaxATCC 194006 x 10^8 cfu/mLN
Clostridium haemolyticumATCC 96506 x 10^8 cfu/mLN
Clostridium histolyticumATCC 194016 x 10^8 cfu/mLN
Clostridium innocuumATCC 145016 x 10^8 cfu/mLN
Clostridium methylpentosumATCC 438296 x 10^8 cfu/mLN
Clostridium nexileATCC 277576 x 10^8 cfu/mLN
Clostridium novyiATCC 35406 x 10^8 cfu/mLN
Clostridium paraputrificumATCC 257806 x 10^8 cfu/mLN
Clostridium ramosumATCC 255826 x 10^8 cfu/mLN
Clostridium scindensATCC 357046 x 10^8 cfu/mLN
Clostridium sphenoidesATCC 194036 x 10^8 cfu/mLN
Clostridium sporogenesATCC 35846 x 10^8 cfu/mLN
Clostridium symbiosumATCC 149406 x 10^8 cfu/mLN
Corynebacterium genitaliumATCC 330303.53 x 10^7 cells/mLN
Corynebacterium glutamicumATCC 130326 x 10^8 cfu/mLN
Desulfovibrio pigerATCC 290986 x 10^8 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) strain CrooksATCC 87396 x 10^8 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) serotype O26:K60(B6)ATCC 127956 x 10^8 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) O Group 26ATCC 118406 x 10^8 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) serotype 0103:K:H8ATCC 239826 x 10^8 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) serotype 0111:NMZeptometrix 08017471.05 x 10^10 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) – feces, human (feces from ahealthy human), strain HGH21ATCC BAA-976 x 10^8 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) - adult, human New York,strain ECOR2ATCC 353216 x 10^8 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) - adult, human Sweden, ECOR9 (reference strain)ATCC 353286 x 10^8 cfu/mLN
E. coli (strain: (Migula) Castellani andChalmers) – adult, human Tonga, ECOR41 (reference strain)ATCC 353606 x 10^8 cfu/mLN
Eggerthella lentaATCC 255596 x 10^8 cfu/mLN
Entamoeba disparATCC PRA-2606.80 x 10^6 copies/mLN
Entamoeba moshkovskiiATCC 50004Not knownN
Enterobacter aerogenesATCC 350286 x 10^8 cfu/mLN
Enterobacter cloacae subsp. cloacaeATCC 130476 x 10^8 cfu/mLN
Enterococcus casseliflavusATCC 257886 x 10^8 cfu/mLN
Enterococcus cecorumATCC 431986 x 10^8 cfu/mLN
Enterococcus disparATCC 512666 x 10^8 cfu/mLN
Enterococcus faecalisATCC 194336 x 10^8 cfu/mLN
Enterococcus faecalis vanBATCC 512991.1 x 10^9 bacteria/mLN
Enterococcus faeciumATCC 194346 x 10^8 cfu/mLN
Enterococcus faecium vanAATCC 7002216 x 10^8 cfu/mLN
Enterococcus gallinarumATCC 495736 x 10^8 cfu/mLN
Enterococcus hiraeATCC 80435.8 x 10^9 bacteria/mLN
Enterococcus raffinosusATCC 494276 x 10^8 cfu/mLN
Eubacterium rectaleATCC 336566 x 10^8 cfu/mLN
Faecalibacterium prausnitzii (formerlyFusobacterium prausnitzii)ATCC 277666 x 10^8 cfu/mLN
Fusobacterium variumATCC 85016 x 10^8 cfu/mLN
Gemella morbillorumATCC 278246 x 10^8 cfu/mLN
Hafnia alveiATCC 133376 x 10^8 cfu/mLN
Helicobacter fennelliaeATCC 356836 x 10^8 cfu/mLN
Homo sapiensATCC MGC-15492Titer not available;used from stockN
Klebsiella pneumoniae subsp.pneumoniaeATCC 138836 x 10^8 cfu/mLN
Lactobacillus acidophilusATCC 43566 x 10^8 cfu/mLN
Lactobacillus caseiATCC 3936 x 10^8 cfu/mLN
Lactobacillus reuteriATCC 232726 x 10^8 cfu/mLN
Lactococcus lactis subsp. lactisATCC 114549 x 10^8 cfu/mLN
Leminorella grimontiiATCC 339996 x 10^8 cfu/mLN
Listeria innocuaATCC 330906 x 10^8 cfu/mLN
Mycoplasma fermentansATCC 19989Titer not available;used from stockN
Peptoniphilus asaccharolyticusATCC 149636 x 10^8 cfu/mLN
Peptostreptococcus anaerobiusATCC 273376 x 10^8 cfu/mLN
Porphyromonas leviiATCC 291476 x 10^8 cfu/mLN
Prevotella melaninogenicaATCC 258453.2 x 10^7 bacteria/mLN
Proteus mirabilisATCC 46306 x 10^8 cfu/mLN
Proteus penneriATCC 351986 x 10^8 cfu/mLN
Proteus vulgarisATCC 63806 x 10^8 cfu/mLN
Pseudomonas aeruginosaATCC 278536 x 10^8 cfu/mLN
Pseudomonas putidaATCC 470546 x 10^8 cfu/mLN
Ruminococcus bromiiATCC 27255Not knownN
Salmonella subterranea**ATCC BAA-8366 x 10^8 cfu/mLYwith Shigella
Staphylococcus aureus subsp. Aureusstrain FDA 209ATCC 65386 x 10^8 cfu/mLN
Staphylococcus aureus subsp. aureus,Cowan's serotype 1 (contains aprotein A)ATCC 125986 x 10^8 cfu/mLN
Staphylococcus epidermidisATCC 122286 x 10^8 cfu/mLN
Streptococcus intermediusATCC 273356 x 10^8 cfu/mLN
Streptococcus salivariusATCC 70736 x 10^8 cfu/mLN
Streptococcus sp.ATCC 129736 x 10^8 cfu/mLN
Streptococcus uberisATCC 194366 x 10^8 cfu/mLN
Trabulsiella guamensisATCC 494901.84 x 10^8 cfu/mLN
Veillonella atypicaATCC 126416 x 10^8 cfu/mLN

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*Although these analytes are probed by xTAG GPP, they have been included in this study as it has been recommended in the FDA Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection of Clostridium difficile guidance document to test for cross-reactivity

** Both replicates showed high MFI for Salmonella probe 2 (1895, 1779.5). However, this sample is called NEG because in order to make the Salmonella call either probe 1 is ≥1400 or both probe 1 and probe 2 must be ≥200.

*** Although this analyte is probed by xTAG GPP, it has been included in this study to evaluate potential cross-reactivity of this organism with the STEC stx 1 toxin gene.

There was no cross-reactivity observed with 120 of the 121 commensal flora tested. A false positive call for Shigella was obtained when Salmonella subterranea was tested. Salmonella subterranea (ATCC BAA-836), a facultatively anaerobic, acid-resistant bacterium, was originally isolated from a low-pH, nitrate- and U(VI)-contaminated subsurface sediment (Shelobolina et al. 2004). However, according to the latest White-Kauffman-Le Minor Scheme maintained by Institut Pasteur, the species called Salmonella subterranea (Appl. Environ. Microbiol., 2004, 70, 2959-2965) does not belong in the genus Salmonella (Grimont. A.D., Weill. F-X. 2007. Antigenic Formulae of the Salmonella Serovars, 9th edition, Pasteur Institute, Paris France, available at http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s-000036-089). Unfortunately, the only sequence available in GenBank at this time is a partial 16S sequence (AY373829.2) making it difficult to determine the basis of the cross-reactivity with Shigella. A dilution study was performed to see at what concentration the cross-reactivity occurred. Salmonella subterranea (ATCC BAA-836) cross-reactivity

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with Shigella was detected at a concentration of 6.0 x 10° cfu/mL, but was no longer observed at a concentration of 1.5 x 10° cfu/mL or lower. This information will be included in product labeling.

Commensal flora evaluated for potential cross-reactivity

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{34}------------------------------------------------

Note: Streptococcus faccalis is another name for Enterococcus faecalis. Therefore, only one of the two (Enterococcus faecalis) were tested.

† - Added following release of the C. difficile FDA guidance

document Nov. 29, 2010.

** Salmonella subterranea is closely related to Escherichia hermanii and does not belong to the genus Salmonella.

^ One of eight replicates cross-reacted with Shigella.

An additional 20 pathogens were not attainable but were evaluated 'in silico' to assess the potential for cross-reactivity that could lead to false positive results. While 2 of these 20 could potentially cross-react based on BLAST analysis (Entamoeba coli and Taenia saginata), positive detection of these pathogens by xTAG GPP is highly unlikely based

{35}------------------------------------------------

on either thermodynamic (Tm) analysis of the pathogen sequence with the kit primers or lack of incorporation of biotin required to produce a signal.

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

In silico evaluation of pathogens for potential cross-reactivity

Interference

There was no interference observed for analytes probed by the assay when low positive concentrations of these analytes (Norovirus GI/GII, Rotavirus A and C. difficile) were assayed in the presence of high concentrations of the 4 non-panel gastrointestinal pathogens listed below.

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xTAG GPP Analyte(concentration)SourcePotentiallyInterferingOrganism(concentration)SourceInterferenceYes (Y) /No (N)
Norovirus GI (LP)(6.56 x 10^5 copies/mL)CDCNoneN
Aichi virus (HP)(1.00 x 10^8 pfu/mL)CDCN
Astrovirus (HP)(6.00 x 10^10 copies/mL)CDCN
Sapovirus (HP)(5.00E+08 copies/mL)CDCN
Norovirus GII (LP)(1.01 x 10^8 copies/mL)CDCNoneN
Aichi virus (HP)(1.00 x 10^8 pfu/mL)CDCN
Astrovirus (HP)(6.00 x 10^10 copies/mL)CDCN
Sapovirus (HP)(5.00 x 10^8 copies/mL)CDCN
Rotavirus (LP)(4.85 x 10^9 copies/mL)CDCNoneN
Aichi virus (HP)(1.00 x 10^8 pfu/mL)CDCN
Astrovirus (HP)(6.00 x 10^10 copies/mL)CDCN
Sapovirus (HP)(5.00 x 10^8 copies/mL)CDCN
Clostridium difficile toxin A/B (LP)(3.75 x 10^6 cfu/mL)ATCCNoneN
Enterococcus faecium,vancomycin resistant (HP)(6.00 x 10^8 cfu/mL)ATCC 700221N

Non-panel GI pathogens tested for potential interference

None of the ten common non-panel commensal bacteria, yeast and parasites listed below interfered with the detection of the panel analytes (Campylobacter, C. difficile, Cryptosporidium, E. coli 0157, ETEC LT/ST, Giardia, Norovirus GI/GII, Rotavirus A, Salmonella, STEC stx1/stx2, and Shigella).

Common commensal bacteria, yeast and parasites tested for interference

Bacteroides thetaiotaomicron (ATCC 29148)
Citrobacter koseri (ATCC 27028)
Clostridium sporogenes (ATCC 3584)
E. coli strain ECOR2 (ATCC 35321)

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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)

There was no interference observed with the 18 non-microbial agents tested. In addition, none of the non-microbial agents tested in the presence of C. difficile inhibited the detection of the C. difficile Toxin A and B analytes.

Non-microbial agentsBrandLot NumberCross-ReactiveYes (Y) / No (N)
Whole blood (40% v/v)BioreclamationBRH288023N
Mucin (3.5% w/v)Sigma-Aldrich039k7003vN
Fecal fat - triglcerides (4.8% w/v)SuplecoLB81189N
Fecal fat - cholesterol (4.8% w/v)Sigma-Aldrich061m53001vN
Hemoglobin (tarry stool) (12.5% w/v)Sigma-Aldrich051m7004vN
Pepto-Bismol (5% w/v) (Bismuthsubsalicylate)Pepto-Bismol1151171951N
Kaopectate (5 mg/mL) (Attapulgite)KaopectateL0705N
Imodium (5% w/v) (Loperamidehydrochloride)ImodiumCNERN
Nystatin† (50% w/w) (antifungal)Ratio-nystatin655900N
Hydrocortisone† (50% w/v)Rexall Hydrocortisonecream USPF1022N
Calcium Carbonate† (5% w/v)(antacids)Tums1C21N
Magnesium Hydroxide, AluminumHydroxide† (5% v/v) (antacids)Maalox10114204N
Mineral Oil† (50% v/v)RexallMineralOilheavy ISP150-1N
Sennosides† (5% w/v) (laxative)SennokotF328N
Naproxen Sodium† (2170 umol/L)(non-steroid anti-inflammatory)Rexall Naproxenp6172N

Non-microbial agents evaluated for interference

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Benzalkonium Chloride, Ethanol† (50%v/v) (moist towellets)Sigma-Aldrich,Commercial alcoholsszba3280, 9163N
Ampicillin sodium salt† (152 $µ$ mol/L)(antibiotic)Sigma-Aldrichbcbf5293vN
Polymyxin B sulfate, bacitracin zinc†,(50% w/v) (antibiotic, topical)Polysporin1410N

† - Added following release of the C. difficile FDA guidance document

Nov. 29, 2010

Competitive Interference

There was no competitive interference observed between pathogens probed by xTAG GPP when testing was carried out with the mixed analyte samples described below.

Mixed analyte samples tested for competitive interference

xTAG GPP Analyte #1xTAG GPP Analyte #2
Campylobacter jejuni (HP)(6.00E+08 cfu/mL)No Analyte #2
Shigella sonnei (LP)(1.01E+04 cfu/mL)
Campylobacter jejuni (LP)(2.93E+05 cfu/mL)No Analyte #2
Shigella sonnei (HP)(6.00E+08 cfu/mL)
Cryptosporidium parvum (HP)(2.50E+05 oocysts/mL)No Analyte #2
Giardia lamblia (LP)(1.10E+03 cells/mL)
Cryptosporidium parvum (LP)(6.25E+04 oocysts/mL)No Analyte #2
Giardia lamblia (HP)(9.00E+06 cells/mL)
E. coli (enterotoxic) (HP)(6.00E+08 cfu/mL)No Analyte #2
Shigella sonnei (LP)*(1.01E+04 cfu/mL)
E. coli (enterotoxic) (LP)(3.51E+05 cfu/mL)Campylobacter jejuni (LP)(2.93E+05 cfu/mL)
No Analyte #2
Shigella sonnei (HP)(6.00E+08 cfu/mL)
Norovirus (HP)(stock)Campylobacter jejuni (HP)(6.00E+08 cfu/mL)
No Analyte #2
Clostridium difficile (LP) (3.75E+06 cfu/mL)**
Norovirus (LP)(dil 3 = 160x dilution of stockconcentration)No Analyte #2
Clostridium difficile (HP) (6.00E+08 cfu/mL)
Rotavirus (HP)No Analyte #2

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xTAG GPP Analyte #1xTAG GPP Analyte #2
(1.58E+06 TCID50/mL)Shigella sonnei (LP)
(1.01E+04 cfu/mL)
Campylobacter jejuni (LP)
(2.93E+05 cfu/mL)
Giardia lamblia (HP)
(1.10E+03 cells/mL)
Cryptosporidium hominis (LP)
(2.15E+04 copies/mL)
E. coli (enterotoxic) (LP)
(3.51E+05 cfu/mL)
Clostridium difficile (LP)
(3.75E+06 cells/mL)
No Analyte #2
Shigella sonnei (HP)
(6.00E+08 cfu/mL)
Campylobacter jejuni (HP)
(6.00E+08 cfu/mL)
Rotavirus (LP)Giardia lamblia (HP)
(5.27E+05 TCID50/mL)(9.00E+06 cells/mL)
Cryptosporidium parvum (HP)
(2.50E+05 oocysts/mL)
E. coli (enterotoxic) (HP)
(6.00E+08 cfu/mL)
Clostridium difficile (HP)
(6.00E+08 cells/mL)

*In addition, no interference with Shigella at a concentration of 5.04 x 10^3 cfu/mL

**Clostridium difficile was also tested at 8.33 x 10^6 cfu/mL

Carry-over Contamination

The likelihood of carry-over contamination events was assessed by testing 2 representative pathogens: bacterial (C. difficile), and parasitic (Giardia). These analytes were 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). Each target was examined in a set of 2 identical runs (including the pre-treatment and extraction steps) arranged in a checkerboard manner on a 96-well plate.

Results showed that all 96 high negative samples remained negative when run on the Luminex 100/200 instrument for both targets (100% HN). In addition, results show that all 96 high positive samples remained positive when run on the Luminex 100/200 instrument for both targets (100% HP). Therefore a lack of carryover contamination has been demonstrated.

f. Assay cut-off:

Clinical specimens, cultured isolates spiked in a synthetic stool matrix sample and extraction controls (negative matrix spiked with MS2) were used to establish cut-offs.

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These cutoff values are hard-coded into the TDAS software (US IVD) and can not be modified.

Assay cut-off determination (threshold-setting) consists of three steps for each analyte:

    1. Setting an initial cut-off range based on the 95th percentile of signals for the NEG samples and 5th percentile of signals for the POS samples.
    1. Recommending optimized cut-offs within this range based on Receiver Operating Characteristic (ROC) analysis of empirical data, and
    1. Establishing an MFI cut-off value through a Design Review Committee (DRC) assessment of ROC curves.

Distinct sample sets were used for setting initial cut-offs (step 1 above) and for finding the optimized cut-offs (step 2 above) for GPP. Samples were assigned a "positive" or "negative" call for the analyte in question based on the known sample types or results obtained at the clinical sites. These results were based on the routine diagnostic algorithm at the collection sites (e.g. bacterial culture, EIA/DFA, microscopic examination, real-time PCR, nucleic acid amplification tests followed by bi-directional sequencing). For some samples, comparator results were not available for all 15 targets in the x T A G GPP assay, but rather than drop or lose these samples, these data points were highlighted. When the comparator result was not available for a particular target, the target for that sample was excluded from the threshold-setting data sets.

The sample set used in these two cut-off determination steps also included cultured isolates with confirmed viral, bacterial or parasitic identity which were serially diluted into negative matrix. Finally, the sample set was supplemented with extraction controls (negative matrix spiked with MS2) that were coded as negative for all targets. All samples were extracted using the Biomerieux EasyMag® method prior to being tested with xTAG GPP.

The table below details the final cutoff values selected for each of the targets probed by the xTAG GPP assay. For most targets that have a single probe, sample results above or equal to the cutoff value are considered positive, while sample results below the cutoff value are considered negative. Please note that for multi-probe targets, like C. Difficile, Norovirus, Enterotoxigenic E. coli (ETEC) LT/ST and Shiga-like toxin producing E. Coli (STEC), a single qualitative POS (positive) call is made if either one of their probes is above or equal to the cutoff value, otherwise a single qualitative NEG (negative) call is made. For Salmonella, a single qualitative POS (positive) call is made when Probe-1 signal is above or equal to 1400, and a single qualitative NEG (negative) call is made when its signal is less than 200. Probe-2 signal will only be used to determine the final call when the Probe-1 signal falls within the equivocal zone, i.e. signals greater than or equal to 200 but less than 1400.

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AnalyteFinal Cut-off (MFI) for LX 100/200
Campylobacter≥ 150 (POS)
C. difficile Probe-1≥ 150 (POS)
C. difficile Probe-2≥ 150 (POS)
Cryptosporidium≥ 250 (POS)
E. coli O157≥ 150 (POS)
ETEC Probe-1≥ 200 (POS)
ETEC Probe-2≥ 200 (POS)
Giardia≥ 250 (POS)
Norovirus Probe-1≥ 200 (POS)
Norovirus Probe-2≥ 350 (POS)
Rotavirus A≥ 150 (POS)
Salmonella Probe-1≥200 (NEG), ≥1400 (POS)
Salmonella Probe-2≥ 200 (POS)
STEC Probe-1≥ 150 (POS)
STEC Probe-2≥ 150 (POS)
Shigella≥ 150 (POS)

xTAG GPP Analyte Cutoff Values for all targets probed by the assay

Fresh vs. Frozen

The purpose of this evaluation was to generate data to support the hypothesis that no significant difference in the performance of xTAG GPP would be observed 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 analyte target probed by the assay was 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.

The results of this study will be used to support (or reject) the inclusion of frozen clinical specimens in the multi-site method comparison clinical evaluation of xTAG GPP and will support sample storage claims in the instructions for use.

Following the selection of the appropriate dilution to represent the three different levels (Low Positive, Moderate Positive and High Positive), identical sets of the simulated specimens were prepared for each analyte target so they could be examined at the following intervals: baseline (fresh), 1 months (un-extracted specimen and

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nucleic acid extracts only) and 6 months after freezing at -70°C to -80°C.

This study examined the stability of un-extracted specimens as well as pre-treated material and nucleic acid extracts (see three horizontal red block arrows to the right side of the Figure) after being stored frozen at -70°C to -80°C for up to 3 months. The first block arrow shows the 'un-extracted stool' material. The second block arrow shows the 'pre-treated' material (prior to extraction). The third block arrow (after nucleic acid extraction) shows the 'extracted' nucleic acid material. Un-extracted, pre-treated and extracted specimen stability will also be examined after storage at -70°C to -80°C for 6 months.

Image /page/42/Figure/2 description: The image shows a diagram of a pretreatment process for nucleic acid extraction. The process begins with 100-150 mg/100 uL stool, 1 mL easyMAG Lysis Buffer, and 10 uL xTAG MS2 being added to a SK38 Bead Tube. The mixture is then vortexed for 5 minutes, incubated for 10-15 minutes at room temperature, and centrifuged for 2 minutes at 1400 rpm. Finally, 200 uL of supernatant is extracted for nucleic acid extraction, with the entire pretreatment process taking 30-45 minutes for 24 samples.

Instructions for Use and Samples Tested (Red Block Arrows) in this Study

For each analyte, HP, MP and LP un-extracted stocks were prepared in negative stool matrix and split into 5 aliquots. Two (2) aliquots, sufficient volume for 36 pre-treatments and extractions of HP, 44 of MP, and 40 of LP, were immediately extracted (no freezethaw). When pooled in pairs, the volume for each of these aliquots was enough for 18 pre-treatments and extractions of HP, 22 of MP and 20 of LP. The remaining 3 aliquots were stored at -70 to -80°C for later stability testing (see Figure below).

For each dilution, two aliquots of extracted nucleic acid were pooled and pooled material was split into four aliquots. One aliquot was immediately tested by xTAG GPP (no freeze-thaw) to generate "Baseline" values for all sample types (i.e. un-extracted, pretreated and extracted stool). The remaining three aliquots ("Nucleic Acids") were stored at -80°C for later stability testing at 1-month and 6-month stability time points.

In the same manner, two Pre-treated samples ("Pre-treated Stool") were also pooled,

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split into aliquots and stored -70 to -80°C for stability testing at 1-month and 6-month stability time points.

Frozen un-treated specimens were pre-treated, extracted and tested by xTAG GPP at each designated stability time point (see Figure below).

Study workflow of the stability of fresh ("Baseline") and frozen ("un-extracted Stool") specimens.

Image /page/43/Figure/3 description: The image shows a flowchart describing the process of splitting a simulated specimen into aliquots for different treatments and storage conditions. Initially, the specimen is divided into two paths: one for fresh aliquots and another for frozen aliquots. The fresh aliquots undergo immediate pre-treatment and extraction, followed by pooling and splitting into at least 4 aliquots for testing, while the frozen aliquots are stored at -70°C to -80°C and thawed at different time intervals (1, 3, and 6 months) for pre-treatment, extraction, and testing.

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In order to assess pre-treated sample and extracted nucleic acid stability for each analyte target, the remaining three aliquots of pre-treated material and extracted nucleic acid from the "Fresh" arm in the Figure above were tested by xTAG GPP at the following time points post freezing: 1 month (both) and 3 months (extracted nucleic acid only). Pretreated and extracted sample stability will also be examined after storage at -70°C to -80°C for 6 months.

Data generated at each time point (1 month and 3 months) on frozen un-extracted specimens, nucleic acid extracts and pre-treated material were compared to the data generated at baseline (time 0 or Fresh). The 6- month time-point is not yet available. It is not expected that clinical specimens will be stored for longer than 30 days (1 month) in clinical practice.

Image /page/44/Figure/2 description: This image shows a flowchart of the remaining pre-treated material from a fresh sample in Figure 2. At time 1 month, the material is thawed, extracted, and test aliquot #1 is taken. At time 6 months, the material is thawed, extracted, and test aliquot #3 is taken.

Study workflow of stability of the pre-treated material ("Pre-treated Stool").

Acceptance Criteria

In order to demonstrate no significant difference in assay performance between fresh and frozen un-extracted specimens and the stability of frozen pre-treated material and nucleic acid at each time point, the positive agreement (i.e. the agreement between positive results generated in fresh aliquots compared to positive results generated in frozen aliquots) should be ≥ 95% with a lower bound of the 95% (two- sided) confidence interval exceeding 85% for each claimed analyte.

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1-Month Stability Results

1-month stability acceptance criteria were met for all of the targets except the following:

  • Campylobacter (un-extracted and extracted specimens only) .
  • Giardia (un-extracted and pre-treated specimens only) .
  • Norovirus GII (un-extracted specimen only) .

For those targets that met the 1-month stability acceptance criteria, MFIs generated on HP, MP and LP replicates of frozen un-extracted, extracted and extracted specimens were generally close to those generated at baseline.

For Campylobacter, MFIs generated on HP, MP and LP replicates of frozen unextracted and extracted specimens were well below those generated at baseline. The same observation was made for HP, MP and LP replicates of Giardia (un-extracted and pre-treated specimens only). In addition, internal control (MS2) values generated on frozen replicates of un-extracted and pre-treated samples were generally lower for these 3 analytes compared to baseline MS2 values suggesting that sub-optimal extraction may be the cause for these results.

For Norovirus GII, although the 1-month stability acceptance criteria was not met for unextracted specimens, MFI generated from HP and MP replicates of both fresh and frozen specimens were similar. The mean MFI value generated on frozen LP replicates was 360 (1x assay cut-off) compared to 868 for fresh specimens (2.5x assay cut-off).

Luminex was unable to source suitable stock material of cryptosporidium to generate enough replicates of HP. MP and LP concentrations for the study. Therefore, only LP dilutions were generated for this target. xT A G GPP only generated 40/60 positive results at baseline and MFIs ranged from 0.7x to 2.6x the assay cut-off. Although 1month stability criteria were not met for this target, MFIs generated on frozen unextracted, pre-treated and extracted specimens ranged from 0.35x - 1.66x, 0.33x - 0.36x and 0.6x - 1.74x the assay cut-off respectively suggesting that these results are most likely due to low starting titer rather than specimen stability.

3-Month Stability Results

To date, 3-month stability results for un-extracted and extracted specimens are available for Campylobacter, C. difficile, E. coli O157, ETEC, Giardia, Norovirus GII, Rotavirus A, Salmonella, STEC and Shigella.

3-month stability acceptance criteria for frozen un-extracted specimens were met for all targets tested to date with the exception of Norovirus GII. For this target, MFI values generated for LP replicates bracket the assay cut-off.

3-month stability acceptance criteria for frozen extracted specimens were met for all targets tested to date with the exception of Norovirus GII and Giardia.

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3-month stability results of Campylobacter, Norovirus GII and Giardia are of particular interest as they do not reflect the 1-month stability results. That is, study acceptance criteria were met for Campylobacter un-extracted and extracted specimens at the 3month stability time point but not at the 1-month time point. Similarly, study acceptance criteria were met for Giardia and Norovirus GII un-extracted specimens at the 3-month stability time point but not at the 1-month time point. One possible explanation for the discrepant results generated on frozen replicates of un-extracted samples at 1-month and 3-month time points is sub-optimal extraction. This hypothesis is supported by the fact that the internal control (MS2) values generated on frozen replicates of unextracted samples, in particular Giardia, were generally lower at Month 1 compared to baseline and Month 3. LMD is unable to explain the discrepant results observed at Month 1 and Month 3 for Campylobacter nucleic acid extracts.

Supplemental Stability Results - Cryptosporidium (pre-treated and extracted)

In order to verify that the results obtained at the Month-1 time point for Cryptosporidium un-extracted, pre-treated and extracted specimens were attributed to sample titer rather than to stability, LP and MP results generated as part of the multi-site reproducibility study at site 1 (Luminex) were re-analyzed. All LP and MP un-extracted, pre-treated and extracted specimen remnants were also re-tested by xTAG GPP at a later date.

Results generated on LP specimens in terms of calls and MFIs are consistent with those generated as part of the Fresh vs Frozen study. However, mean MFI values generated on MP dilutions of un-extracted, pre- treated and extracted left-over specimens at different time points were similar and ranged from 2.2x to 4.4x the assay cut-off MFI. These results suggest that un-extracted, pre-treated and extracted Cryptosporidium specimens prepared at a concentration 1-5x the assay cut-off MFI are stable for at least 1-month when stored frozen at -70°C to -80°C.

Conclusion for Fresh vs. Frozen Study

Stability results generated to date support the inclusion of frozen clinical specimens positive for all targets in the multi-site clinical evaluation of the xTAG GPP. Results generated to date also indicate that pre-treated material and nucleic acid extracts of all targets evaluated to date are stable for at least 1 month post freezing (with the exception of Giardia pre-treated material).

Analyte TargetUn-extracted1 monthUn-extracted3 monthsPre-Treated1 monthExtracted1 monthExtracted3 months
CampylobacterX√√X√
C. difficile Toxin A/B√√√√√
Cryptosporidium√^Pending√^√^Pending
E. coli O157√√√√√

Summary of Stability Results

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ETEC (LT/ST)ﮧN
GiardiaXNVA1Pending
Norovirus GIVPending1/1Pending
Norovirus GIIX11ﮨﮯX
Rotavirus AﮧV
Salmonella1/
STEC (stx1/stx2)11
Shigellaﻢく

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

Comparator Assays Analytical Validation Studies

PCR followed by bi-directional sequencing assays (PCR/sequencing) are used as a comparator method and to resolve discordant results to establish analyte identity during the clinical evaluation of xTAG assays. They are validated to evaluate certain performance characteristics including analytical sensitivity (limit of detection), analytical reactivity and specificity (cross-reactivity).

The primers were chosen to perform sequencing as a comparator method for Campylobacter, Enterotoxigenic Escherichia Coli (ETEC) LT and ST, and Rotavirus A targets of the xTAG Gastrointestinal Pathogen Panel (xTAG GPP). Two different primer sets were designed and validated for ETEC LT and one primer set was designed and validated for Campylobacter, and Rotavirus A.

To the extent possible, the sequencing primers were designed to amplify regions of the genomic sequence that are not covered by the xTAG GPP kit primers. The second set of sequencing primers designed for ETEC LT and ETEC ST targets were designed to flank the GPP kit amplicon. Bi-directional (both forward and reverse sequences of the produced amplicon) Sanger dideoxy - sequencing method and BLAST analysis were used to confirm sequence identity.

Sequencing primers were validated using samples from the following sources:

  • Representative Clinical Sample: Wherever possible, known positive 1. clinical samples were tested with the sequencing primers to evaluate detection from an extracted clinical stool sample.
  • Limit of Detection (LoD): Serial dilutions of the target analytes were tested 2. to establish the lower limit of primer sensitivity. Samples tested for "Evaluation of the Limit of Detection and Repeatability of xTAG Gastrointestinal Pathogen Panel (FDA)," study were used here.
    1. Cross-reactivity: For the xTAG GPP panel targets, samples representing all the targtes in the xTAG GPP panel, were tested at the highest available titres. For the xTAG GPP non-panel cross-reactivity targets, BLAST analysis was preformed with each sequencing primer. If both the forward and reverse sets contained an 11 base pair match up to the 3' end (Kwok S, 1994) of the primer with any of the non-panel cross-reactivity species, then a

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representative sample for that strain was tested to evaluate cross-reactivity.

    1. Reactivity: Various strains for each target were analyzed to evaluate the strain coverage of the sequencing primers. Samples tested for "Evaluation of Analytical Reactivity of the xTAG Gastrointestinal Pathogen Panel (FDA)" study were used here.
      Detailed descriptions of the types of samples tested are listed below:

  • · Clinical Sample: Pre-characterized target-specific clinical samples for Rotavirus and Campylobacter were tested with the sequencing primers. For ETEC, since there is no validated comparator other than the sequencing method, clinical samples positive for either ETEC LT or ST by the xTAG GPP assay were used.

  • · Limit of Detection Study: The same sample sets prepared for the Evaluation of the Limit of Detection and Repeatability of xTAG GPP study, were used for this Sequencing Primer Validation study. Briefly, stock solutions were diluted to a starting concentration and dilution series were prepared by making sequential 4fold dilutions to about 10 dilution levels. Sample dilutions were prepared and tested in triplicates.

  • · Cross-reactivity: To test for cross-reactivity of the sequencing primers the following studies were conducted.

    • For the xTAG GPP panel targets, samples representing all the targets in o the xTAG GPP panel, were tested at the highest available titers.
    • For the xTAG GPP non-panel cross-reactivity targets, BLAST analysis O was preformed with each sequencing primer. If both the forward and reverse sets contained an 11 base pair match up to the 3' end (Kwok S, 1994) of the primer with any of the non-panel cross-reactivity species, then a representative sample for that strain was tested to evaluate crossreactivity.

  • · Reactivity: A variety of strains, genotypes and serotypes for ETEC, Rotavirus, and Campylobacter used in the Analytical Reactivity study were tested with each sequencing primer set.

Categorizing Sequencing Results

Positive - Samples were considered positive by sequencing if the following criteria were met:

  • The generated sequences, from bidirectional sequencing, should be at least o 200 bases of an acceptable quality, defined as a minimum of 90% of the total bases (20 bases per 200bp read) with PHRED quality score of 20 or higher (accuracy of base call is ≥ 99%)
  • For sequences containing ambiguous base calls such as "N"s, the total O number of ambiguous bases in the acceptable quality sequences generated using bidirectional sequencing should not exceed 5% of total bases (or 10

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bases per 200 bp read).

  • Blast analysis of the acceptable quality sequences generated by bidirectional o sequencing should have at least 95% query coverage compared to reference and at least 95% identity to reference.
  • Sequence matches the reference or sequence generates an Expected Value O (E-Value) < 10-30 for the specific target following a BLAST search in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/).

Negative - Samples were considered negative by sequencing if any one of the above criteria were not met.

Acceptance Criteria

  • Clinical Sample: The clinical sample of known identity, if available, must be . positive by sequencing for the expected target.
  • Limit of Detection Study: At least, 2 of the 3 extraction replicates must be . positive by sequencing at the equivalent or lower titer than the established limit of detection recorded for the xTAG GPP analyte.
  • . Cross-reactivity Study: All samples tested should generate no sequencing reactions of acceptable quality.
  • Reactivity: Strains, genotypes and serotypes should generate positives results . with their respective sequencing primers.

Conclusion

All sequencing primers met the acceptance criteria for all studies.

RotavirusETEC LTETEC STCampylobacter
PartiallyFlanking2F/3ROutside101Flanking2F/2BRPartiallyFlanking102F/1BRFlanking7AF/12.1ROutside 101
Limit ofDetectionEqual tokitMoreSensitivethan kitEqual tokitMoreSensitivethan kitMoreSensitivethan kitMore Sensitivethan kit
Cross-ReactivityNoneNoneNoneNoneNoneNone
Reactivity80f9strainsreacted '6of7 LTpositivestrainsreacted-6of7 LTpositivestrainsreacted25of6 STpositivestrainsreacted25of6 STpositivestrainsreacted214of14 strainsreacted

Summary of Sequencing Primers Validation Studies

  • The 1 strain of Rotavirus that did not react was ATCC 2275

2 The 1 strain that did not react (ATCC 43896) with the ETEC primers also did not react with the ETEC LT and ST targets of the GPP kit

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

There were a total of 217 xTAG GPP runs performed over the course of analytical performance studies. Each xTAG run has at least one no template negative control depending on batch size. Of the 217 runs, 11 (5.07%) had one or more negative control (NC) failures. These are summarized in the table below.

StudyTotal # ofruns(includingallowablere-runs)Total # ofruns withat leastone NCfailure% totalruns withat leastone NCfailureTotal No. ofNCs includedin runs andallowable re-runsTotalNo. ofNCfailures% total NC sincluded whichfailed in xTAG runs/ allowable re-runs
Multi-site reproducibility6469.38%18873.72%
Matrix equivalence300900
Limit of detection290010800
Carry-over contamination600000
Analytical specificity andinterference2314.34%9111.10%
Analytical reactivity3126.45%20431.47%
Evaluation of fresh vs.frozen stool6123.28%18821.06%
Overall217115.07%788131.65%
Summary of Negative Control Failures for Analytical Performance Studies

Included in the 217 xTAG runs summarized above were 12473 specimens. Of these, 99.79% (12447/12473) yielded valid results on the first attempt. The remaining 26 specimens generated valid results following allowable re-runs. Sample re-run rates are summarized in the table below.

Summary of Sample Re-Run Rates for Analytical Performance Studies

StudiesTotal # ofspecimenstestedTotal # ofinvalidresults priorto re-run% invalidresults priorto re-runInvalidresultsafter re-run% invalidresults afterre-run
Multi-site reproducibility4230220.52%00.00%
Matrix equivalence18000.00%00.00%
Limit of detection74010.14%00.00%
Carry-over contamination57600.00%00.00%
Analytical specificity andinterference131900.00%00.00%
Analytical reactivity186610.05%00.00%
Evaluation of fresh vs.frozen stool356220.06%00.00%
Overall12473260.21%00.00%

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2. Comparison studies:

  • a. Method comparison with predicate device:
    Not applicable. Refer to the Clinical Studies section of this document.

  • b. Matrix comparison:

Matrix Equivalency

The purpose of this study was to determine if the performance of the xTAG Gastrointestinal Panel (xTAG GPP) in stool re-suspended in pre-treatment buffer (designated as PT buffer) prior to spiking known concentrations of analytes is equivalent to that of native (raw and untreated) stool (designated as NS) spiked with known concentrations of analytes before the pre-treatment step. The performance of xTAG GPP in these two matrices (raw untreated stool re-suspended in pre- treatment buffer) was assessed by comparing serial dilution curves of analyte targets generated using a single lot of xTAG GPP.

Based on comparative analysis of dilution curves, this study suggests that xTAG GPP performance is equivalent between samples prepared in native stool and stool resuspended in pre-treatment buffer when extracted with the Biomerieux NucliSens® EasyMag® system. Thus, negative stool re-suspended in pre-treatment buffer as a base matrix can be used for all analytical studies of xTAG GPP.

    1. Clinical studies:
    • a. Clinical Sensitivity:

Microbial 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.

Demographic information for the asymptomatic donors is shown in the table below. Demographic Information for Asymptomatic Donors

GenderNumber of Subjects
Male92 (46%)
Female108 (54%)
Total200
Age
0 - 15 (2.5%)

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2 - 57 (3.5%)
6 - 2143 (21.5%)
22 - 60111 (55.5%)
≥6134 (17.0%)

PCR inhibition, as determined by results for the internal control used with xTAG GPP (bacteriophage MS2), was observed in 23 of the 200 samples tested (11.5%). After re-running these specimens in accordance with the instructions for use, PCR inhibition was still observed in eight samples (4%). The absence of a detectable internal control signal in these samples meant that negative results for the indicated microbial targets could not be reported. Therefore, the final data analysis was conducted on 192 of the 200 samples collected for this study.

A total of 13 samples that were positive by xTAG GPP were sequenced. Two (2) out of 13 samples were positive by sequencing (C. Difficile Toxin A/B), while 11 of 13 samples were not positive by sequencing.

Asymptomatic Donor Results for xTAG GPP
TargetPercent Negative Results by xTAGGPP for all samples
Campylobacter100.0% (192/192)
C. difficile toxin A/B98.4% (189/192) 1
Cryptosporidium100.0% (192/192)
E. coli 0157100.0% (192/192)
ETEC LT/ST100.0% (192/192)
Giardia99.0% (190/192) 2
Norovirus GI/GII98.4% (189/192) 3
Rotavirus A100.0% (192/192)
Salmonella97.4% (187/192) 4
STEC stx1/stx2100.0% (192/192)
Shigella100.0% (192/192)

These results are summarized in the table below:

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

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

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

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

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

Samples (at the specimen level) that were positive by xTAG GPP but negative by sequencing were considered false positives (11/192, 5.3%). These samples had MFI values that were relatively close to the cut-offs. Two samples at the specimen level that were called positive by xTAG GPP were also positive by sequencing analysis for C. difficile. These two samples positive for C. difficile by both xTAG GPP and sequencing may represent asymptomatic infections.

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Prospective Clinical Study

The clinical performance of the xTAG GPP was evaluated during prospective studies at six clinical laboratories in North America (four sites in the U.S. and two sites in Canada). Stool specimens were collected and tested at the six clinical laboratories (Sites 1, 2, 3, 4, 5, and 6) during June 2011 thru February 2012. Clinical study sites were selected based on the types of patients usually referred (e.g. pediatrics, adults), conditions often treated (e.g. C. difficile colitis), as well as the geographical prevalence of particular targeted pathogens.

Six geographically separated clinical study sites participated in the clinical evaluation of the xTAG GPP. The study sites were located in East-Central Canada (Toronto, Ontario and Hamilton, Ontario), and Southeast (Nashville, TN), Southwest (Temple, TX and Tucson, AZ), and Midwest (St Louis, MN) of the U.S. Each study location was representative of the intended use setting (clinical laboratories) and testing was performed by trained clinical laboratory personnel.

The table below summarized the total number of patients recruited at each site:

Site ## PatientsRecruited
l461
2449
3188
4295
റ97
б44
1534

Number of Patients Per Site

Patient specimens (one specimen from each of the recruited patients) that met all of the following characteristics were eligible for the study.

    1. An exemption from the requirement for Informed Consent had been granted by the site IRB to include the left-over stool specimen in the study.
    1. The specimen was from a pediatric or adult, male or female subject who was either hospitalized, admitted to a hospital emergency department, visiting an outpatient clinic or resident of a long-term care facility.
    1. The specimen was from a patient for whom a requisition had been made for testing of microbial pathogens suspected of gastrointestinal tract infections.
  • The specimen was from a patient exhibiting clinical signs and symptoms of 4. infectious colitis (including C. difficile colitis) or gastroenteritis (including traveler's diarrhea), such as diarrhea, nausea and vomiting, loss of appetite, fever, abdominal pain and tenderness, cramping, bloating, flatulence, bloody stools, fainting and weakness.
    1. The volume of the specimen was ≥ 8.5 ML or ≥ 6 g.

Patient specimens with any one of the following characteristics was not eligible for study entry:

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    1. The specimen was collected at a site which was not covered under the study IRB.
    1. The specimen was a preserved stool, stool in Cary-Blair media or rectal swab.
    1. The specimen was from an individual who did not exhibit clinical signs and symptoms of infectious colitis or gastroenteritis.
    1. Based on available clinical information, the specimen was from an individual with known and documented non-infectious conditions such as ulcerative colitis, irritable bowel syndrome and/or Crohn's disease.
    1. The specimen was not properly collected, transported, processed or stored according to the instructions provided by the sponsor.
    1. The specimen could not be tested by the relevant comparator assays within 72 hours of collection.

Of the 1534 stool specimens, 127 were excluded from the study. The reasons for exclusion are summarized in the table below.

Summary of Excluded Specimens (N=127)
Reason for Specimen ExclusionExclusion Criteria# Excluded Specimens
The specimen was collected from a site not covered under the study IRB15 (0.3%)
The specimen was from an individual with known and documented non-infectious conditions such as ulcerative colitis, irritable bowel syndrome and/or Crohn's disease467 (4.3%)
The specimen was not properly collected, transported, processed or stored according to the instructions provided by the sponsor550 (3.2%)
The specimen could not be tested by the relevant comparator assays within 72 hours of collection64 (0.2%)
Other: multiple extraction failuresN/A1 (0.05%)
Total127

Summary of Excluded Specimens (N=127)

The following table provides a summary of demographic information for the 1407 subjects whose stool specimens were included in the prospective study.

SexNumber of Subjects
Male632 (44.9%)
Female775 (55.1%)
Total1407
Age (yrs)
0 - 16 (0.4%)
>1 - 520 (1.4%)
>5 - 1225 (1.8%)
>12 - 2151 (3.6%)
>21 - 65879 (62.5%)
>65426 (30.3%)
Total1407
Subject Status

General Demographic Details for the Prospective Data Set (N=1407)

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Outpatients421 (29.9%)
Hospitalized804 (57.1%)
Emergency Department118 (8.4%)
Long Term Care Facility18 (1.3%)
Not Determined46 (3.3%)
Total1407
Immune Status
Immuno-compromised493 (35.0%)
Immuno-competent758 (53.9%)
Not Determined156 (11.1%)
Total1407

In addition to patients' demographic details, every effort was made to ensure that information on clinical signs and symptoms of infectious colitis or gastroenteritis was available on all subjects enrolled in the prospective study. This information was collected by way of chart reviews. Chart reviews were conducted by an individual at the sites who was not directly involved in the study (e.g. research nurse) so that information was collected in a manner that did not make the specimen source identifiable to the investigator or any other individual involved in the investigation including the Sponsor. Local IRB approval for the study was obtained prior to study start. If available, the following information was also collected:

  • · Stool consistency (based on Bristol Stool Scale)
  • · Clinical signs and symptoms of infectious colitis or gastroenteritis such as diarrhea, nausea and vomiting, loss of appetite, fever, abdominal pain and tenderness, cramping, bloating, flatulence, bloody stools, fainting and weakness
  • · Duration and severity of symptoms prior to enrolment
  • · Method of transmission (e.g. food-borne outbreak or close contact method)
  • · Prior and concomitant medications including dose, type, frequency and duration.
  • · Other orally ingested substances (e.g. fiber, stool bulking agents), including dose, type, frequency and duration
  • · Other laboratory results (e.g. viral/bacterial culture, gram positive/negative infection, hematology and serum chemistry etc.)

Wherever available in the medical charts, the duration and severity of each specific sign or symptom was also recorded.

Stool consistency (based on the Bristol Stool Form Scale) was recorded for each clinical specimen included in the prospective clinical study. A summary of this information is provided in the table below.

Stool consistency (N=1107)
Stool Consistency# Specimens (%)
Type 1 Separate hard lumps8 (0.5%)
Type 2 Sausage-shaped but lumpy24 (1.7%)
Type 3 Like a sausage but with cracks26 (1.8%)
Type 4 Like sausage/snake, smooth, soft77 (5.5%)

Stool consistency (N=1407)

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Type 5 Soft blobs with clear-cut edges160 (11.4%)
Type 6 Fluffy pieces with ragged edges354 (25.2%)
Type 7 Watery, no solid pieces758 (53.9%)

Information on clinical signs and symptoms of infectious colitis or gastroenteritis were available on 918 patients (65.2%). A summary of the findings from the patient medical charts is provided in the table below.

Clinical Signs and Symptoms# Events Reported (%)Duration Reported
Diarrhea807 (87.9%)1 day to 6 months
Nausea327 (35.6%)1 day to 6 months
Vomiting228 (24.8%)1 to 30 days
Loss of appetite179 (19.4%)1 day to 2 months
Fever170 (18.5%)1 day to 2 weeks
Abdominal pain284 (30.9%)1 day to 6 months
Tenderness118 (12.8%)1 day to 4 months
Cramping101 (11.0%)1 day to 4 months
Bloating62 (6.7%)1 day to 6 months
Flatulence50 (5.4%)1 day to 3 months
Bloody stool89 (9.7%)1 day to 4 months
Weakness159 (17.3%)1 day to 4 months
Other (e.g. Constipation)87 (9.5%)1 to 25 days

Summary of Clinical Signs and Symptoms (N=918)

All prospective clinical specimens were submitted fresh to the sites and were processed according to their routine algorithm and as ordered by the referring physician. Upon receipt at the laboratory, any left-over stool specimen that met the study inclusion / exclusion criteria was placed into the following six containers.

    1. Meridian sterile, leak-proof, wide-mouthed empty container (unpreserved stools)
  • Meridian container containing Cary-Blair holding medium (Para-Pak® C&S) 2.
  • Meridian container containing PVA fixative (Para-Pak® LV-PVA Fixative) 3.
    1. Meridian container containing formalin (Para-Pak® 10% Buffered Neutral Formalin)
  • న. Container containing ACTD medium (swab)
  • Sterile container for xTAG GPP testing (unpreserved stools) 6.

The time from collection to processing into the appropriate containers was kept to a minimum (<24 hours). Prior to study initiation, processing instructions as well as shipping details were provided to each clinical site by the central laboratories carrying out reference and comparator method testing. Specimens were shipped to the central laboratories within 24 hours of processing. Prospective clinical specimens were then processed for both comparator testing and xTAG GPP testing as described below.

For all prospective specimens, reference and comparator method testing was performed at central laboratories independent of xTAG GPP testing sites. Reference/comparator testing was performed for all analytes on all prospectively collected specimens. In the event that comparator results were not available for all targets on a given specimen, then

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the specimen in question was excluded from performance calculations of xTAG GPP.

Reference and comparator methods for each analyte target are listed in the table below.

xTAG GPPAnalytesReference/Comparator MethodShippingRequirements
Rotavirus AComposite comparator consistingof PremierRotaclone EIA (MeridianBioScience K852969) directly onthe stool specimen and one PCR/sequencing assay directly fromclinical specimen1Unpreserved stool in sterile tubes
NorovirusComposite comparator consistingof CDC real-time PCR andconventional PCR followed by bi-directional sequencing assaysdirectly from clinical specimen1Unpreserved stool in sterile tubes
Clostridium difficile Toxin A/BBartels CytotoxicityAssay for Clostridium difficileToxin (Bartels K833447) usingdiluted stool filtrate processeddirectly from clinical specimenUnpreserved stool in sterile tubes
SalmonellaBacterial cultureStool in Cary-Blair holding medium
ShigellaBacterial cultureStool in Cary-Blair holding medium
CampylobacterBacterial culture(A PCR/Sequencing assay wasalso performed directly on clinicalspecimens that were testedpositive by culture for speciesidentification only)Stool in Cary- Blair holdingmedium
E. coli O157Bacterial cultureStool in Cary-Blair holding medium
Shiga-Like Toxin Producing E.coli (STEC)Broth enrichment followed byImmunoCard STAT EHEC(Meridian BioScience, K062546)Unpreserved stool in sterile tube
Enterotoxigenic E. coli (ETEC)LT/STComposite comparator consistingof PCR/sequencing directly fromclinical specimen using fourPCR/sequencing assays, twoassays each for the LT and the STgene1Unpreserved stool in sterile tube
CryptosporidiumMicroscopyPreserved stool in 10% Formalin
GiardiaMicroscopyPreserved stool in PVA fixative

Reference/Comparator Methods and Shipping Requirements

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1 Refer to more detailed descriptions below.

Performance of the xTAG GPP detecting ETEC-LT and ETEC-ST was compared to a composite comparator method consisted of four separate analytically validated PCR followed by bi-directional sequencing assays (two for ETEC-LT and two for ETEC-ST). "True" ETEC positives were considered as any sample that was tested positive for LT or ST by any of the four PCR/sequencing assays. "True" ETEC negatives were considered as any sample that was tested negative for LT and ST by all four PCR/sequencing assays. PCR/sequencing assays were performed on nucleic acid extracted directly from clinical specimens using primers that targeted different genomic regions from the ones probed by xTAG GPP. Generated sequence results were analyzed as follows:

  • · For a given base from the consensus sequence generated from bi-directional sequencing, the PHRED score was calculated by averaging the PHRED quality score from the forward and reverse sequencing.
  • The generated sequence should be at least 200 bases of an acceptable quality, defined as a minimum of 90% of the total bases with PHRED quality score of 20 or higher.
  • · Blast analysis of the consensus sequence generated by bi-directional sequencing should have at least 95% query coverage compare to reference, at least 95% identity to reference and an Expected Value (E-Value) of at least 10-30
  • · For sequences containing 'N's, the consensus generated using bi-directional sequencing should correspond to the strand including the high quality base instead of the strand including the "N" called base. In addition, the total number of N's should not exceed 5% of total bases (or 10 bases per 200 bp read).

Performance of the xTAG GPP detecting rotavirus was compared to a composite comparator method consisted of an FDA cleared EIA test and one analytically validated PCR followed by bi-directional sequencing assay. "True" rotavirus positives were considered as any sample that was tested positive for rotavirus by the EIA and/or the PCR/sequencing assay. "True" rotavirus negatives were considered as any sample that was tested negative for rotavirus by both the EIA and the PCR/sequencing assay. PCR/sequencing was performed on nucleic acid extracted directly from clinical specimens using primers that targeted different genomic regions from the ones probed by xTAG GPP. Generated sequence results were analyzed described above.

Performance of the xTAG GPP for norovirus was assessed by comparing test results to the "patient norovirus infected status" of each specimen. The "patient norovirus infected

1 The E-Value from NCBI BLAST Alignment indicates the statistical significance of a given pair-wise alignment and reflects the size of the database and the scoring system used. The lower the E-Value, the more significant the hit. A sequence alignment that has an E-Value of 1e-3 means that this similarity has a 1 in 1000 chance of occurring by chance alone. (http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=handbook.section.614).

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status" was determined using a composite comparator method consisting of the CDC norovirus real-time Taqman RT-PCR assay and the CDC Conventional RT-PCR (Region-C and D primers) followed by bi-directional sequencing assays. The following interpretation algorithm was used to determine the "patient norovirus infected status":

Composite Comparator Algorithm for Norovirus

CDC Norovirus Real- TimeTaqman RT-PCR ResultCDC ConventionalRT-PCR Result (Region C)Followed by Bi-DirectionalSequencingCDC ConventionalRT-PCR Result (Region D)Followed by Bi-DirectionalSequencingFinal CompositeComparator Result
PositivePositiveN/APositive
NegativePositiveN/APositive
PositiveNegativePositivePositive
PositiveNegativeNegativeNegative
NegativeNegativeN/ANegative

Clinical runs and re-runs (per the instructions provided in the product package insert)

using xTAG GPP were carried out on left-over clinical specimens that had been extracted from the fresh or frozen state using the NucliSENS EasyMAG method (BioMérieux, Inc.. Durham, NC) according to the manufacturer's instructions. Total extracted nucleic acid material was stored at -70℃ prior to testing with xTAG GPP.

PCR negative (water blanks, NTC) control and external rotating positive controls (RC) representing analytes probed by the assay were also included with each xTAG GPP run. The external positive controls used in the study are listed in the table below and, for the most part (except for Cryptosporidium), consisted of chemically-inactivated bacteria, viruses and parasites from ZeptoMetrix. These controls were used to control the entire assay process including nucleic acid extraction, amplification, and detection. The external positive controls contained low organism copy numbers and were designed to mimic patient specimens. These were run as separate samples, concurrently with patient specimens. External positive controls were included in each assay plate in a rotating manner.

External Positive ControlSourceDilution Factor
CampylobacterNatrol (ZeptoMetrix)Stock*
C difficile Toxin A/BNatrol (ZeptoMetrix)1/100
CryptosporidiumPooled clinical specimensStock**
E. coli 0157 / STECNatrol (ZeptoMetrix)1/100
ETECNatrol (ZeptoMetrix)1/10
GiardiaPRA-243 (ATCC)Stock
Norovirus GINatrol (ZeptoMetrix)1/100
Norovirus GIINatrol (ZeptoMetrix)1/1000
RotavirusNatrol (ZeptoMetrix)1/10
SalmonellaNatrol (ZeptoMetrix)1/10
ShigellaNatrol (ZeptoMetrix)1/1000

External Positive Controls

  • Stock material was used as MFI signals generated for campylobacter in the initial clinical runs using1/10

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dilution of the stock were too close to the assay cut-off.

** Pooled clinical specimens positive for Crytopsoridium hominis were used as positive control for this target. MFI values generated were however close to the assay cut-off and, in a number of clinical runs were below the threshold for a positive call.

Clinical specimens were tested in accordance with the package insert for xTAG GPP assay and were tested by a single operator at each of the clinical sites.

The xTAG GPP assay includes an internal control (MS2 bacteriophage) that is added to each sample prior to extraction. In the event that none of the pathogen targets probed by xTAG GPP were detected in a clinical specimen and the MS2 call in that specimen was "Absent", a 1/10 dilution of the nucleic acid remnant (from the original extraction) was prepared and tested by xTAG GPP. Two outcomes of running a 1/10 dilution were addressed in the following manner:

  • . If the MS2 call was "Present" following a 1/10 dilution of the original extract, it is likely that the original result was due to PCR inhibition. All additional positive results generated in this scenario were reported as "Positive" in the calculation of sensitivity and specificity (or positive and negative agreement). Negative results generated in this scenario were reported as "inhibited" and excluded from the calculation of sensitivity and specificity (or positive and negative agreement) for the targets in question. However, inhibited results are presented in the performance tables as "invalid" for each microbial target.
  • If the MS2 signal was "Absent" following a 1/10 dilution of the original extract and ● none of the pathogen targets were detected, then the sample was re-tested with xTAG GPP, starting from the extraction step. If MS2 signal was "Present" after retesting from the extraction step, it is likely that the original result was due to suboptimal extraction. Negative and positive results generated in this allowable re-run were included in the calculation of sensitivity and specificity (or positive / negative agreement) for each individual target. If MS2 signal was still "Absent" after retesting from the extraction step and none of the pathogen targets were detected, then the sample was coded as "inhibited" and was excluded from the calculation of sensitivity and specificity (or positive and negative agreement) for the targets in question. However, inhibited results are presented in the performance tables as "invalid" for each microbial target.

In the event that an unexpected positive call was made in any of the assay controls included in the xTAG GPP run (negative or external positive control), then all clinical specimens that tested positive for the analyte(s) in question were re-tested by xTAG GPP. Negative and positive results generated in this allowable re-run were included in the calculation of sensitivity and specificity (or positive and negative percent agreements) for each individual target.

Discrepant results between the xTAG GPP and the reference methods were also evaluated using analytically validated PCR/sequencing assays or FDA cleared molecular assays (i.e., for C. difficile Toxin), and results are footnoted in the performance tables below.

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The prospective performance data (all sites combined) are presented in the following tables by analyte:

xTAG GPPReference
PositiveNegativeInvalidTOTAL
Positive3212024
Negative0115501155
Invalid02280228
TOTAL31140401407
95% CI
Sensitivity100%43.8% - 100%
Specificity98.2%97.3% - 98.8%
Invalid Rate due to PCRInhibition16.2%

Campylobacter

Sequencing results from these specimens revealed that all three were campylobacter jejuni.

2 total of six Campylobacter xTAG GPP positive specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

Clostridium difficile Toxin A/B

xTAG GPPComparator
PositiveNegativeInvalidTOTAL
Positive107105182203
Negative792262991
Invalid117025196
TOTAL11511979521407
95% CI
Positive Percent Agreement93.9%87.9% - 97.0%
Negative Percent Agreement89.8%87.8% - 91.5%
Invalid Rate due to PCRInhibition13.9%

A total of 48 C. difficile Toxin A/B xTAG GPP positive specimens that were negative by the comparator method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP, or FDA cleared C. difficile Toxin molecular assays.

2 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. This finding is consistent with the expected values for invalid results noted in the Bartels Cytotoxicity Assay for Clostridium difficile Toxin.

A total of 151 (151/220, 68.7%) C. difficile Toxin A/B xTAG GPP positive specimens were positive for both the Toxin A and B gene targets by the xTAG GPP Test. A total of 57 (57/220, 25.9%) C. difficile Toxin A/B xTAG GPP positive specimens were positive for the Toxin B target and 12 (12/220, 5.4%) were positive for the Toxin A target.

Cryptosporidium
xTAG GPPReference
PositiveNegativeInvalidTOTAL
Positive12532065
Negative1113101132
Invalid02100210
TOTAL131139401407
95% CI

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Sensitivity92.3%66.7% - 98.6%
Specificity95.5%94.2% - 96.6%
Invalid Rate due to PCRInhibition14.9%

1 All 13 Cryptosporidium reference positive specimens were collected during a single outbreak which occurred at Site 2 and were typed as Cryptosporidium hominis.

2A total of eight Crytosporidium xTAG GPP positive specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

E. coli 0157

xTAG GPPReference
PositiveNegativeInvalidTOTAL
Positive291011
Negative0115801158
Invalid02380238
TOTAL22140501407
95% CI
Sensitivity100%34.2% - 100%
Specificity99.2%98.5% - 99.6%
Invalid Rate due to PCRInhibition16.9%

' A total of four E. coli O157 xTAG GPP positive specimens that were negative by the comparator method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

2 Both reference positive E. coli 0157 specimens were also positive for STEC by xTAG GPP. Only one was positive for STEC by the reference culture and EIA.

ETEC

xTAG GPPComparator
PositiveNegativeInvalidTOTAL
Positive21406
Negative62115601162
Invalid12380239
TOTAL9139801407
95% CI
Positive Percent Agreement25.0%7.1% - 59.1%
Negative Percent Agreement99.7%99.1% - 99.9%
Invalid Rate due to PCRInhibition17.0%

1 One sample was positive for LT by both ETEC-LT PCR/sequencing assays. The other sample was positive for ST by both ETEC-ST PCR/sequencing assays.

2 ETEC performance were calculated against a comparator consisting of four well-characterized PCR/bidirectional sequencing assays, two ETEC-LT PCR/sequencing assays and two ETEC-ST PCR/sequencing assays. All six specimens were positive by only one of the four PCR/sequencing assays.

Giardia

xTAG GPPReference
PositiveNegativeInvalidTOTAL
Positive439043
Negative0113201132
Invalid02320232

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TOTAL4140301407
95% CI
Sensitivity100%51.0% - 100%
Specificity96.7%95.5% - 97.6%
Invalid Rate due to PCR Inhibition16.5%

Norovirus GI/GII

xTAG GPPComparator
PositiveNegativeInvalidTOTAL
Positive74960170
Negative4¹102301027
Invalid02100210
TOTAL78²132901407
95% CI
Positive Percent Agreement94.9%87.5% - 98.0%
Negative Percent Agreement91.4%89.6% - 92.9%
Invalid Rate due to PCRInhibition14.9%

1 All four xTAG GPP false negative Norovirus specimens were Norovirus GII.

2 Five of the 78 Norovirus comparator positive specimens were typed as GI at the CDC by sequencing, and 73 of the 78 Norovirus comparator positive specimens were typed as GII at the CDC by sequencing.

Rotavirus A

Rotavirus A
xTAG GPPComparator
PositiveNegativeInvalidTOTAL
Positive2204
Negative0116201162
Invalid02410241
TOTAL2140501407
95% CI
Positive Percent Agreement100%34.2% - 100%
Negative Percent Agreement99.8%99.4% - 100%
Invalid Rate due to PCRInhibition17.1%

Salmonella

xTAG GPPReference
PositiveNegativeInvalidTOTAL
Positive10182028
Negative0114301143
Invalid02360236
TOTAL101139701407
95% CI
Sensitivity100%72.2% - 100%
Specificity98.4%97.6% - 99.0%
Invalid Rate due to PCRInhibition16.8%

1 Cultured isolates from all 10 salmonella reference positive clinical specimens were typed at the Ontario Public Health Laboratory in Toronto. Three specimens were typed as Salmonella enterica, subsp. enterica,

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Typhimurium; one specimen as Salmonella enterica, Typhi; one specimen as Salmonella enterica subsp. enterica, Salamae; one specimen as Salmonella enterica, Javiana; one specimen as Salmonella enterica, Bredeney; one specimen as Salmonella enterica subsp. enterica, Mississippi; one specimen as Salmonella enterica, Heidelberg; one specimen as Salmonella enterica subsp. enterica, Muenchen.

2A total of two salmonella xTAG GPP positive specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

xTAG GPPReference
PositiveNegativeInvalidTOTAL
Positive1162017
Negative0115301153
Invalid02370237
TOTAL11140601407
95% CI
Sensitivity100%20.7% - 100%
Specificity98.6%97.8% - 99.2%
Invalid Rate due to PCRInhibition16.9%

Shiga-Like Toxin Producing E. coli (STEC) stx1/stx2

1 This STEC reference positive specimen was typed a Shiga-like toxin 2 using the ImmunoCard STAT EHEC.

2 A total of one STEC xTAG GPP positive specimen that was negative by the reference method was confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

Shigella

xTAG GPPReference
PositiveNegativeInvalidTOTAL
Positive2172019
Negative0115401154
Invalid02340234
TOTAL21140501407
95% CI
Sensitivity100%34.2% - 100%
Specificity98.5%97.7% - 99.1%
Invalid Rate due to PCRInhibition16.6%

l Two clinical specimens tested positive for shigella by bacterial culture; one was reported as Shigella flexneri while the other one was reported as Shigella sonnei.

2A total of two shigella xTAG GPP positive specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

The prospective performance data (all sites combined) are presented in the following table by organism:

OrganismSensitivity95% CISpecificity95% CI
Campylobacter3/343.8% - 100%1155/11761 98.2%97.3% - 98.8%
Cryptosporidium12/1366.7% - 98.6%1131/11842 95.5%94.2% - 96.6%
E. coli O1572/234.2% - 100%1158/11673 99.2%98.5% - 99.6%
Giardia4/451.0% - 100%1132/1171 96.7%95.5% - 97.6%
Salmonella10/1072.2% - 100%1143/11614 98.4%97.6% - 99.0%

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STEC1/1100%20.7% - 100%1153/116998.6%97.8% - 99.2%
Shigella2/2100%34.2% - 100%1154/1171698.5%97.7% - 99.1%
OrganismPositive PercentAgreement95% CINegative PercentAgreement95% CI
C. difficile Toxin A/B107/11493.9%87.9% - 97.0%922/1027789.8%87.8% - 91.5%
ETEC2/825.0%7.1% - 59.1%1156/116099.7%99.1% - 99.9%
Norovirus GI/GII74/7894.9%87.5% - 98.0%1023/111991.4%89.6% - 92.9%
Rotavirus A2/2100%34.2% - 100%1162/116499.8%99.4% - 100%

1 A total of six Campylobacter xTAG GPP positive specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

2 A total of eight Crytosporidium xTAG GPP positive specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analysis using analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

3 A total of four E. coli O157 xTAG GPP positive specimens that were negative by the comparator method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

4 A total of two Salmonella xTAG GPP positive specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

3 A total of one STEC xTAG GPP positive speciment that was negative by the reference method was confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

6 A total of two Shigella xTAG GPP positive specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analytically validated primers that targeted genomic regions distinct from the xTAG GPP.

7 A total of 48 C. difficile Toxin A/B xTAG GPP positive specimens that were negative by the comparator method were confirmed as positive by bi-directional sequencing analysis using analytically validated primers that targeted genomic regions distinct from the xTAG GPP, or FDA cleared C. difficile Toxin molecular assay.

Prospective Clinical Study Mixed Infection Analysis

xTAG GPP detected a total of 91 mixed infections in the prospective clinical evaluation. This represents 18.7% of the total number of xTAG GPP positive specimens (91/486). (62/91; 68.1%) were double infections, 21 (21/91; 23.1%) were triple infections, four (4/91; 4.4%) were quadruple infections, two (2/91; 2.2%) were quintuple infections, one (1/91; 1.1%) was sextuple infection and one was septuple infection (1/91; 1.1%). The single most common co-infections (24/91; 26.4%) was Norovirus GI/GII with C. difficile Toxin A/B. Out of the 91 co-infections, 86 contained one or more analytes that had not been detected with the reference/comparator methods, i.e. discrepant co-infections. Distinct co-infection combinations detected by xTAG GPP in the prospective clinical study are summarized in the table below.

Distinct Co-infection Combinations Detected by the xTAG GPP in the Prospective Clinical Trial

Distinct Co-infection CombinationsDetected by xTAG GPP
Analyte 1Analyte 2Analyte 3Analyte 4Analyte 5Analyte 6Analyte 7TotalCo-infectionsNumber ofDiscrepantCo-infections aDiscrepant Analyte(s) a
Campyl.Crypto.11All
Campyl.Giardia22Campyl. (x2); Giardia (x1)
C. diff.Crypto.33All
C. diff.E. coli O157STEC11All
C. diff.Giardia22All
C. diff.STECl1STEC (x1);
C. diff.STECCrypto.22All
C. diff.ETEC11C. diff. (x1);
E. coli 0157STEC21E coli O157 (x1); STEC (x1);
GiardiaCrypto.l1All
NorovirusCampyl.C. diff.l1Campyl. (x1); C diff. (x1);
NorovirusCampyl.C. diff.Crypto.22All
NorovirusCampyl.C. diff.Crypto.STEC22All
NorovirusCampyl.Crypto.44All
NorovirusCampyl.Giardia1lNorovirus (x1); Giardia (x1);
NorovirusC. diff.2420Norovirus (x15); C diff. (x12);
NorovirusC. diff.E. coli 0157Giardia11All
NorovirusCrypto.99Norovirus (x9); Crypt. (x6);
NorovirusGiardiaETECl1ETEC (x1); Giardia (x1);
NorovirusGiardiaббNorovirus (x3); Giardia (x6);
NorovirusE. coli 0157STECl1STEC (x1);
NorovirusGiardiaCrypto.22All
NorovirusCrypto.STEC11All
NorovirusGiardiaSTEC11STEC (x1); Giardia (x1);
NorovirusSalmonellaShigella11Norovirus (x1); Shigella (x1);
NorovirusShigellaC. diff.l1Norovirus (x1); Shigella (x1);
NorovirusShigellaC. diff.Campyl.STECCrypto.ETEC11Norovirus (x1); Shigella (x1);Campyl. (x1); ETEC (x1);STEC (x1); Crypto. (x1)
NorovirusShigellaCampyl.E. coli 0157Crypto.ETECl1Shigella (x1); Campyl. (x1);ETEC (x1); E coli O157 (x1);Crypto. (x1);
RotavirusC. diff.1All
RotavirusNorovirusGiardia11All
STECCrypto.11All
SalmonellaC. diff.22Salmonella (x2); C diff. (x1);
SalmonellaC. diff.E. coli 015711C diff. (x1); E coli O157 (x1);
SalmonellaC. diff.STECCrypto.11All
SalmonellaCrypto.22All
SalmonellaC. diff.Crypto.l1All
SalmonellaShigella11All
SalmonellaGiardial1All
SalmonellaSTEC11STEC (x1);
SalmonellaShigellaGiardia11All
ShigellaGiardia11All

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Total Co-infections9186
Total Double Infections6257
Total Triple Infections2121
Total Quadruple infections44
Total Quintuple infections22
Total Number of sextuple infections11
Total Number of septuplet infections11

4 A discrepant co-infection or discrepant analyte was defined as one that was detected by the xTAG GPP but not detected by the reference/comparator methods.

b One Norovirus /C. difficile Tox A/B. one Norovirus /E. coli 0157/STEC and one Salmonella/C.difficile Tox A/B xTAG GPP reported co-infected specimens that were negative by the reference method were confirmed as positive by bi-directional sequencing analysis using analytically validated primers that targeted genomic regions distinct from the xTAG GPP

Additional Distinct Co-infection Combinations Detected by the Reference/Comparator Methods, But Not Detected by the xTAG GPP in the Prospective Clinical Trial

Distinct Co-infection Combinations aTotalCo-infectionsNumber ofDiscrepantCo-infectionsDiscrepant Analyte(s) b
Analyte 1Analyte 2
NorovirusC. diff.11C. diff.
NorovirusETEC22ETEC (x2)

a This table includes only distinct co-infections that were detected by the reference/comparator method but not by the xTAG GPP; the remaining co-infections detected by the reference methods are already represented in the table above.

b Discrepant analyte is defined as one that is detected by the reference/comparator but not detected by the xTAG GPP.

Of the 1407 clinical specimens included in the data analysis, 91 (6.5%) were identified as positive for more than one target by xTAG GPP. In most cases, bacteria presented with viruses (N=29, 31.9%), followed by bacteria + parasites (N=18, 19.8%), viruses + parasites (N=18, 19.8%), bacteria + viruses + parasites (N=15, 16.5%), bacteria + bacteria (N=10, 11.0%), and parasite + parasite (N=1, 1.1%). All enteric pathogens probed by xTAG GPP were implicated in co-infections. Results for co- infections are summarized in the table below.

Prevalence of Individual Analytes in Mixed Infections Detected by the xTAG GPP during the Prospective Clinical Study

TargetNumber Implicatedin Co-InfectionsPercent of Total Co-InfectedSpecimens (N=91)
Campylobacter1516.5%
C. difficile4852.7%
Cryptosporidium3437.4%
E. coli 015777.7%
ETEC44.4%
Giardia2123.1%
Norovirus GI/GII6167.0%

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Rotavirus22.2%
Salmonella1213.2%
Shigella77.7%
STEC1516.3%

Prospective Clinical Study Per Specimen/Patient Summary Results

Prospective study results were also analyzed on a per sample/patient basis. Results of this analysis are summarized in the table below both without taking into consideration the discrepant analysis by PCR/bi-directional sequencing or FDA cleared molecular assays (Primary Reference/Comparator) and taking into consideration this discrepant analysis (After Discrepant Investigation).

Per Sample/Patient Summary Results - Prospective Sample Set (N=1407)

AnalysesPrimaryReference/ComparatorAfter Discrepant Investigation
# Specimens with at least one pathogenpositive by xTAG GPP486486
# Specimens with at least one pathogenpositive by xTAG GPP and confirmed byreference/comparator217286
# Specimens with at least one pathogenpositive by xTAG GPP but none confirmedby reference/comparator269200
# Specimens with at least one pathogenpositive by reference/comparator but nonewas positive by xTAG GPP1717

Prospective Clinical Study Contaminated Runs

Unexpected positive call(s) in negative (NTC) or external rotating positive control(s) (RC) were reported in 10 out of 49 xTAG GPP runs (10/49, 20.4%) during the prospective clinical study. A total of 49 clinical specimens included in these contaminated runs tested positive for analytes that were unexpectedly present in assay controls (49/1407; 3.8%).

Retrospective Clinical Study 1 - Pre-Selected Clinical Specimens

Due to low prevalence observed for most of the xTAG GPP analytes in the prospective clinical study (see above), xTAG GPP performance detecting the following microbial targets was further evaluated in a retrospective clinical study testing pre-selected clinical specimens.

Campylobacter (C. jejuni, C. coli and C. lari only)
Cryptosporidium (C. parvum and C. hominis only)
E. coli 0157
Enterotoxigenic E. coli (ETEC) LT/ST
Giardia
Rotavirus A

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Salmonella
Shiga-like toxin producing E. coli (STEC) stx1/stx2
Shigella

Pre-selected stool specimens were collected at multiple sites in North America and Europe. Demographic information (age and gender) was collected on all pre-selected specimens for which these data were available and is summarized in the table below.

SexNumber of Subjects
Male106 (52.2%)
Female83 (40.9%)
Not known14 (6.9%)
Total203
Age (yrs)
0 - 136 (17.7%)
>1 - 525 (12.3%)
>5 - 1213 (6.4%)
>12 - 2111 (5.4%)
>21 - 6590 (44.3%)
>6514 (6.0%)
Not known14 (6.9%)
Total203

General Demographic Details for the Pre-Selected Data Set (N=203)

The table below outlines the number of pre-selected positive specimens included in the retrospective clinical study for each analyte target as well as the characterization method used.

Pre-selected Target# Specimens IncludedCharacterization Method (Comparator)
Campylobacter41Bacterial culture
Cryptosporidium13 (9 Cryptosporidium parvumand 4 Cryptosporidiumhominis)FDA cleared DFA or microscopy
E. coli O15781Bacterial culture
ETEC39PCR/sequencing directly from clinical specimen usingfour PCR/sequencing assays (two for LT and two for ST)
Giardia17FDA cleared DFA or microscopy
Rotavirus A28FDA cleared EIA or PCR followed bybi-directional sequencing using the same analyticallyvalidated primers as those used in the ProspectiveClinical Study
Salmonella27Bacterial culture
STEC102FDA cleared EIA

Pre-selected Specimen Information (N=203)

{70}------------------------------------------------

ShigellaBacterial culture
All and E. and 0157 alingal googleed by DCD followed by bi directional gamongang for STDC

All eight E. colt 0157 clinical specimens were also assessed by PCR tollowed by bi-directional sequencing for S 2 All 10 STEC clinical specimens were also assessed by PCR followed by bi-directional sequencing for E. coli 0157.

These pre-selected positive specimens were tested with xTAG GPP at three clinical sites along with 277 "negative" clinical specimens in a randomized, blinded fashion. The "negative" designation for these 277 specimens was based on the routine algorithms used at the clinical 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.

The table below summarizes the positive percent agreement between comparator and xTAG GPP for all pre-selected targets evaluated.

AnalytePositive PercentAgreement95%CI for PositivePercentAgreementNumber of “Invalid”xTAG GPP Results
TP /(TP+FN)percent
Campylobacter40/4197.6%87.4% - 99.6%0
Cryptosporidium12/12100%75.7% - 100%1
E. coli O157 114/14100%78.5% - 100%0
ETEC38/3997.4%86.8% - 99.5%0
Giardia15/1693.7%71.7% - 98.9%1
Rotavirus A28/28100%87.9% - 100%0
Salmonella24/2788.9%71.9% - 96.1%0
STEC 218/18100%82.4% - 100%0
Shigella20/20100%83.9% - 100%0

Positive Percent Agreement of xTAG GPP in the Pre-Selected Data Set

4 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 stxl and stx 2 genes by bi-directional sequencing and the results added to those obtained for STEC

2 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.

Nucleic acid amplification followed by bi-directional sequencing using analytically validated primers was also performed on all available pre-selected clinical specimens that were positive by xTAG GPP for other analytes. 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 98 additional positive calls (after allowable re-runs) for analytes that were not pre-selected at the banking site. A summary of these additional calls and confirmatory testing results are provided in the tables below.

{71}------------------------------------------------

Campylobacter

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive3104
NegativeNANA369369
InvalidNANA6666
TOTAL31435439*
Confirmed xTAG GPPPositives/All xTAG GPPPositives75.0%
Invalid Rate due to PCRInhibition (N=480)13.7%

*41 specimens were pre-selected for Campylobacter. Results are presented in the "Positive Percent Agreement of xTAG GPP in the Pre-Selected Data Set" table.

C. Difficile Toxin A/B

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive1690251
NegativeNANA394394
InvalidNANA6161
TOTAL169455480
Confirmed xTAG GPPPositives/All xTAG GPPPositives64.0%
Invalid Rate due to PCRInhibition (N=480)12.7%

1 A total of 17 (17/25, 68.0%) C. difficile Toxin A/B xTAG GPP positive specimens were positive for both the Toxin A and B gene targets by the xTAG GPP Test. A total of 7 (7/25, 28.0%) C. difficile Toxin A/B xTAG GPP positive specimens were positive for the Toxin B target and 1 (1/25, 4.0%) were positive for the Toxin A target.

Cryptosporidium

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive1001
NegativeNANA401401
InvalidNANA6565
TOTAL10466467*
Confirmed xTAG GPPPositives/All xTAG GPPPositives100%
Invalid Rate due to PCRInhibition (N=480)13.5%

*13 specimens were pre-selected for Cryptosporidium. Results are presented in the "Positive Percent of xTAG GPP in the Pre-Selected Data Set" table.

E. coli o157

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL

{72}------------------------------------------------

Positive1012
NegativeNANA397397
InvalidNANA6767
TOTAL10465466*
Confirmed xTAG GPPPositives/All xTAG GPPPositives50%
Invalid Rate due to PCRInhibition (N=480)13.9%

*14 specimens were pre-selected for E. coli O157. Results are presented in the "Positive Percent Agreement of xTAG GPP in the Pre-Selected Data Set" table.

ETEC

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive4408
NegativeNANA369369
InvalidNANA6464
TOTAL44433441*
Confirmed xTAG GPPPositives/All xTAG GPPPositives50%
Invalid Rate due to PCRInhibition (N=480)13.3%

*39 specimens were pre-selected for ETEC. Results are presented in the "Positive Percent Agreement of xTAG GPP in the Pre-Selected Data Set" table.

Giardia

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive0505
NegativeNANA395395
InvalidNANA6363
TOTAL05458463*
Confirmed xTAG GPPPositives/All xTAG GPPPositives0%
Invalid Rate due to PCRInhibition (N=480)13.1%

*17 specimens were pre-selected for Giardia. Results are presented in the "Positive Percent of xTAG GPP in the Pre-Selected Data Set" table.

Norovirus

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive27817
NegativeNANA396396
InvalidNANA6767

{73}------------------------------------------------

TOTAL27471480
Confirmed xTAG GPPPositives/All xTAG GPPPositives11.8%
Invalid Rate due to PCRInhibition (N=480)13.9%

Rotavirus

xTAG GPPPCR/Bi-directional SequencingTOTAL
PositiveNegativeNot Done
Positive6006
NegativeNANA379379
InvalidNANA6767
TOTAL60446452*
Confirmed xTAG GPPPositives/All xTAG GPPPositives100%
Invalid Rate due to PCRInhibition (N=480)13.9%

*28 specimens were pre-selected for Rotavirus. Results are presented in the "Positive Percent of xTAG GPP in the Pre-Selected Data Set" table.

Salmonella

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive46010
NegativeNANA382382
InvalidNANA6161
TOTAL46443453*
Confirmed xTAG GPPPositives/All xTAG GPPPositives40.0%
Invalid Rate due to PCRInhibition (N=480)12.7%

*27 specimens were pre-selected for Salmonella. Results are presented in the "Positive Percent Agreement of xTAG GPP in the Pre-Selected Data Set" table.

STEC
xTAG GPPPositiveNegativeNot DoneTOTAL
Positive3306
NegativeNANA390390
InvalidNANA6666
TOTAL33456462*
Confirmed xTAG GPPPositives/All xTAG GPPPositives50.0%NANANA

{74}------------------------------------------------

Invalid Rate due to PCRInhibition (N=480)13.7%
------------------------------------------------------------

*18 specimens were pre-selected for STEC. Results are presented in the "Positive Percent Agreement of xTAG GPP in the Pre-Selected Data Set" table.

Shigella

xTAG GPPPCR/Bi-directional SequencingTOTAL
PositiveNegativeNot Done
Positive112114
NegativeNANA379379
InvalidNANA6767
TOTAL112447460*
Confirmed xTAG GPPPositives/All xTAG GPPPositives78.6%
Invalid Rate due to PCRInhibition (N=480)13.9%

*20 specimens were pre-selected for Shigella. Results are presented in the "Positive Percent of xTAG GPP in the Pre-Selected Data Set" table.

Retrospective Clinical Study 1 (Pre-Selected Clinical Specimens) Contaminated Runs

Unexpected positive call(s) in negative (NTC) or external rotating positive control(s) (RC) were reported in three out of 15 pre-selected xTAG GPP runs (3/15, 20.0%). A total of 21 clinical specimens included in these runs tested positive by xTAG GPP for analytes that were unexpectedly present in assay controls (21/480; 4.4%).

Supplemental Clinical Study - Botswana Pediatric Stool Specimens

The clinical performance of xTAG GPP for Rotavirus, ETEC, Cryptosporidium and Gardia 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. General demographic details for these patients are summarized in the table below.

SexNumber of Subjects
Male186 (59.4%)
Female127(40.6%)
Total313
Age (yrs)
< 1231 (73.8%)
162 (19.8%)
211 (3.5%)
33 (0.9%)
43 (0.9%)

General demographic details of Botswana Sample Set (N=313)

{75}------------------------------------------------

3 (0.9%)
TotalALAڼۍ ﻟﻪ ﻟﯥ ﻟﻪ ﺩﯤ ﺩﻱ ﭼﯥ ﺩ ﺍﻭ ﺩ ﺍﻭ ﺩ ﺍﻭ ﺩ ﺍﻭ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ ﺩ ﺍﻓﻐﺎﻧﺴﺘﺎﻥ

All specimens were shipped frozen to one of the study sites in Ontario, Canada for xTAG GPP testing. Stools were extracted by the Biomerieux NucliSENS EasyMag and tested using the xTAG GPP per the instructions provided in the product package insert.

Comparator testing by nucleic acid amplification followed by bi-directional sequencing using analytically validated primers was performed on samples positive for Rotavirus, ETEC, Cryptosporidium and Giardia by xTAG GPP. In order to minimize bias, a random subset of the 313 Botswana specimens that tested negative by xTAG GPP was also assessed by the same nucleic acid amplification followed by bi-directional sequencing method for Rotavirus, ETEC, Cryptosporidium and Giardia. In the case of Cryptosporidium and Giardia, the number of xTAG GPP negative specimens assessed was equal to or greater than the number of specimens identified as positive by xTAG GPP. In the case of ETEC, the number of xTAG GPP negative specimens assessed was slightly less than the number of specimens identified as positive by xTAG GPP. Since 178 of 313 specimens tested positive by xTAG GPP for Rotavirus, the number of negative Rotavirus specimens tested by nucleic acid amplification followed by sequencing was less than the number of positive Rotavirus specimens tested by this comparator method. Comparator testing by nucleic acid amplification followed by bidirectional sequencing using analytically validated primers was performed on a total of 308, 56, 24, and 20 specimens for Rotavirus, ETEC, Cryptosporidium, and Giardia, respectively.

The Botswana Study performance data are presented in the following tables by analyte: Rotavirus A

xTAG GPPComparator
PositiveNegativeInvalidTOTAL
Positive17530178
Negative181080126
Invalid0404
TOTAL1931150308
95% CI
Positive Percent Agreement90.7%85.7% - 94.0%
Negative Percent Agreement97.3%92.4% - 99.1%
Invalid Rate due to PCRInhibition1.3%

1 Four out of a total of 313 samples tested by the xTAG GPP generated an "invalid" result for Rotavirus A.

ETEC

xTAG GPPComparator
PositiveNegativeInvalidTOTAL
Positive263029
Negative26027
Invalid0000
TOTAL27290ર્ રહ
95% CI
Positive Percent Agreement96.3%81.7% – 99.3%

{76}------------------------------------------------

Negative Percent Agreement89.7%73.6% - 96.4%
Invalid Rate due to PCRInhibition11.6%

1 Five out of a total of 313 samples tested by the xTAG GPP generated an "invalid" result for ETEC

Cryptosporidium

xTAG GPPComparator
PositiveNegativeInvalidTOTAL
Positive110011
Negative112013
Invalid0000
TOTAL1212024
95% CI
Positive Percent Agreement91.7%64.6% - 98.5%
Negative Percent Agreement100%75.7% - 100%
Invalid Rate due to PCRInhibition11.6%

4 Five out of a total of 313 samples tested by the xTAG GPP generated an "invalid" result for Cryptosporidium.

Giardia

xTAG GPPComparator
PositiveNegativeInvalidTOTAL
Positive91010
Negative010010
Invalid0000
TOTAL911020
95% CI
Positive Percent Agreement100%70.1% - 100%
Negative Percent Agreement90.9%62.3% - 98.4%
Invalid Rate due to PCRInhibition 11.6%

Five out of a total of 313 samples tested by the xTAG GPP generated an "invalid" result for Giardia.

The table below summarizes the positive and negative agreement (PPA and NPA) between PCR/bi-directional sequencing results and xTAG GPP for Rotavirus, Cryptosporidium and Giardia.

OrganismPPA95% CINPA95% CI
Rotavirus A175/19390.7%85.7% - 94.0%108/11197.3%92.4% - 99.1%
ETEC26/2796.3%81.7% - 99.3%26/2989.7%73.6% - 96.4%
Cryptosporidium11/1291.7%64.6% – 98.5%12/12100%75.7% - 100%
Giardia9/9100%70.1% - 100%10/1190.9%62.3% - 98.4%

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 (i.e., Campylobacter, C. difficile Toxin A/B, E. coli O157, Norovirus, Salmonella, Shigella, and STEC) in order to determine whether these additional positive calls represented True Positive (TP) or False Positive (FP) clinical results. The tables below summarize the confirmed xTAG GPP positive rate (i.e., confirmed xTAG GPP positives/all xTAG GPP positives) by PCR/bi-directional

{77}------------------------------------------------

sequencing for Campylobacter, C. difficile Toxin A/B, E. coli O157, Norovirus, Salmonella, Shigella, and STEC.

Campylobacter

xTAG GPPPCR/Bi-directional SequencingTOTAL
PositiveNegativeNot Done
Positive471149
NegativeNANA258258
InvalidNANA66
TOTAL471265313
Confirmed xTAG GPPPositives/All xTAG GPPPositives95.9%
Invalid Rate due to PCRInhibition1.9%

C. Difficile Toxin A/B

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive93315
NegativeNANA292292
InvalidNANA66
TOTAL93301313
Confirmed xTAG GPPPositives/All xTAG GPPPositives60.0%
Invalid Rate due to PCRInhibition1.9%

1 total of 9 (9/15, 60.0%) C. difficile Toxin A/B xTAG GPP positive specimens were possitive for both the Toxin A and B gene targets by the xTAG GPP Test. A total of 3 (3/15, 20.0%) C. difficile Toxin A/B xTAG GPP positive specimens were positive for the Toxin B target and 3 (3/15, 20.0%) were positive for the Toxin A target.

E. coli 0157

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive4015
NegativeNANA303303
InvalidNANA55
TOTAL40309313
Confirmed xTAG GPPPositives/All xTAG GPPPositives80.0%
Invalid Rate due to PCRInhibition1.6%

{78}------------------------------------------------

Norovirus

xTAG GPPPCR/Bi-directional SequencingTOTAL
PositiveNegativeNot DoneTOTAL
Positive299644
NegativeNANA263263
InvalidNANA66
TOTAL299275313
Confirmed xTAG GPPPositives/All xTAG GPPPositives65.9%
Invalid Rate due to PCRInhibition1.9%

Salmonella

xTAG GPPPCR/Bi-directional SequencingTOTAL
PositiveNegativeNot DoneTOTAL
Positive67417
NegativeNANA290290
InvalidNANA66
TOTAL67300313
Confirmed xTAG GPPPositives/All xTAG GPPPositives35.3%
Invalid Rate due to PCRInhibition1.9%

Shigella

xTAG GPPPCR/Bi-directional SequencingTOTAL
PositiveNegativeNot DoneTOTAL
Positive322236
NegativeNANA271271
InvalidNANA66
TOTAL322279313
Confirmed xTAG GPPPositives/All xTAG GPPPositives88.9%
Invalid Rate due to PCRInhibition1.9%

STEC

xTAG GPPPCR/Bi-directional Sequencing
PositiveNegativeNot DoneTOTAL
Positive3115
NegativeNANA302302
InvalidNANA66
TOTAL31309313

{79}------------------------------------------------

Confirmed xTAG GPPPositives/All xTAG GPPPositives60.0%
Invalid Rate due to PCRInhibition1.9%

Supplemental Clinical Study (Botswana Pediatric Stool Specimens) Contaminated Runs

Unexpected positive call(s) in negative (NTC) or external rotating positive control(s) (RC) were reported in 2 out of 5 Botswana xTAG GPP runs (40%). A total of 5 clinical specimens included in these runs tested positive by xTAG GPP for analytes that were unexpectedly present in assay controls (5/313; 1.6%).

4. Clinical cut-off:

Not applicable

5. Expected values/Reference range:

Expected Value (As Determined by the xTAG GPP) Summary by Site for the xTAG GPP Prospective Clinical Evaluation (June 2011 - February 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)
No.ExpectedValueNo.ExpectedValueNo.ExpectedValueNo.ExpectedValueNo.ExpectedValueNo.ExpectedValueNo.ExpectedValue
Campylobacter241.7%51.2%153.5%21.3%20.8%00.0%00.0%
Cryptosporidium654.6%112.5%4811.2%00.0%62.3%00.0%00.0%
E. coli 0157110.8%20.5%20.5%31.9%20.8%22.3%00.0%
ETEC LT/ST60.4%20.5%30.7%00.0%00.0%11.1%00.0%
Giardia lamblia433.1%133.0%174.0%31.9%83.1%22.3%00.0%
Salmonella282.0%112.5%112.6%21.3%41.5%00.0%00.0%
STEC (stx1/stx 2)171.2%92.1%51.2%10.6%10.4%11.1%00.0%
Shigella191.4%30.7%122.8%42.6%00.0%00.0%00.0%
C. difficile Toxin A/B22015.6%5713.1%6314.7%2818.1%4216.2%2123.9%921.4%
Norovirus GI/GII17012.1%245.5%7617.8%127.7%4115.8%1415.9%37.1%
Rotavirus A40.3%20.5%10.2%10.6%00.0%00.0%00.0%

Expected Value (As Determined by the xTAG GPP) Summary by Age Group for the xTAG GPP Prospective Clinical Evaluation (June 2011 - February 2012)

Overall (n=1407)0-1 year (n=6)>1-5 years (n=20)>5-21 years (n=76)>21-65 years (n=879)>65 years (n=426)
No.ExpectedValueNo.ExpectedValueNo.ExpectedValueNo.ExpectedValueNo.ExpectedValueNo.ExpectedValue
Campylobacter241.7%00.0%210.0%00.0%151.7%71.6%
Cryptosporidium654.6%00.0%420.0%22.6%465.2%133.1%
E. coli 0157110.8%116.7%00.0%22.6%60.7%20.5%
ETEC LT/ST60.4%00.0%00.0%11.3%30.3%20.5%
Giardia lamblia433.1%00.0%00.0%22.6%263.0%153.5%
Salmonella282.0%00.0%15.0%33.9%182.0%61.4%
STEC (stx1/stx 2)171.2%00.0%00.0%33.9%80.9%61.4%
Shigella191.4%00.0%00.0%00.0%121.4%71.6%
C. difficile Toxin A/B22015.6%233.3%210.0%1317.1%12013.7%8319.5%
Norovirus GI/GII17012.1%116.7%630.0%1114.5%10111.5%5112.0%
Rotavirus A40.3%00.0%00.0%22.6%10.1%10.2%

{80}------------------------------------------------

N. Instrument Name:

Luminex 100/200

O. System Descriptions:

    1. Modes of Operation:
      Batch
    1. Software:
      FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product types:

Yes _____________ or No ______________________________________________________________________________________________________________________________________________________

    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.

4. Specimen Sampling and Handling:

DNA is extracted using the Biomerieux NucliSens EasyMag system. Samples are manually prepared for amplification according to assay package insert and, once amplified, are transferred to a 96-well microtiter plate for analysis on the Luminex system.

5. Calibration:

xMAP Calibrator Microspheres, Classification (CAL1) and Reporter (CAL2) serve as system calibrators for Luminex xMAP technology based detectors and are intended to normalize the settings for both the classification channel (CL1, CL2), the doublet discriminator channel (DD), and the reporter channel (RP1). They are not intended to be used as calibrators for a given assay.

    1. Quality Control:
      xMAP Control Microspheres, Classification (CON1) and Reporter (CON2) are intended to verify the calibration and optical integrity for the Luminex 100/200 System. Classification Control Microspheres verify both classification channels and the doublet discriminator channel (DD). Reporter Control Microspheres verify the reporter channel. They are not intended to be used as controls for a given assay which are described in the specific assay package insert.

{81}------------------------------------------------

P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:

Instrument Performance Assurance:

Due to the open system design of the platform there is a potential for contamination, the Intended Use of this device states that all positive results are presumptive and need to be confirmed by another FDA-cleared or approved assay or acceptable reference method. The benefit of this test lies in its ability to rule out infection of a patient with the 11 pathogens on the panel. The following mitigations were instituted for the xTAG GPP:

    1. Proficiency Panel and Training-A formal training and certification program will be provided by Luminex with mandatory proficiency testing for end users that they would need to complete before running the xTAG GPP.
    1. Trending and Reporting Positivity Rates-As part of the formal training program, Luminex will include training that specifically focuses on maintaining and monitoring data related to positivity rates for the xTAG GPP. Labs running the xTAG GPP would establish a procedure to monitor unusual spikes in positivity rates and would use this procedure in determining how to report these spikes to Luminex through their existing complaint handling system.
    1. Environmental Monitoring and Cleaning Process-As part of the formal training progam, Luminex will instruct laboratories to create a procedure that specifically describes an xTAG GPP environmental monitoring program. This procedure would instruct the user to include the appropriate controls on the plate, to swab surfaces in the processing areas and run them with the xTAG GPP at least once per month, to monitor results of this swabbing, and initiate a cleaning protocol in the event of a positive finding. Increased frequency of swabbing would be recommended until the contamination has been adequately addressed.

Q. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

R. Risks to Health:

FDA has identified the risks that require special controls to be the following: failure of the device to detect and identify a targeted organism when such organism is present in the specimen (i.e., false negative test result for presence of organism) and detection of the targeted microorganism when such organism is not present in the specimen (i.e., false positive test result for presence of organism), both of which can lead to individual and/or public health consequences, and failure to correctly interpret test results

Failure of the device to detect and identify a targeted organism when such organism is present in the specimen (false negative result) may lead to a delay in finding the true cause of the gastrointestinal infection, additional diagnostic tests, and unnecessary treatment or to

{82}------------------------------------------------

inappropriate antibiotic use. For certain microorganisms detected by the device, failure of detection may contribute to incorrect patient management to prevent transmission of infection, or delay recognition of an outbreak. An incorrect positive test result (false positive result) also may lead to unnecessary or ineffective antibiotic therapy and delay in determining the true cause of the patient's illness. which for some microorganisms may lead to a more serious infection. Additionally, in the context of public health, a false positive teste result may lead to misallocation of resources used for disease surveillance and prevention.

Failure to correctly interpret test results in the context of other clinical and laboratory findings may lead to inappropriate or delayed treatment. For example, a microorganism present as a colonizer may be correctly detected, but not be the true cause of illness. Although this identical risk would be present from use of any microbiological assay in this setting, simultaneous testing of multiple analytes in a multiplex assay may be more likely to detect an unanticipated colonizer that might not be tested for individually.

The special controls necessary to address the risks posed by this device are identified in the special controls guideline 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," which includes mitigation measures relating to device characteristics, device specific performance characteristics, and device specific labeling.

S. Benefit/Risks Analysis

We considered the following factors in our analysis of benefit: the ability to more rapidly rule out potentially significant gastrointestinal pathogens in the setting of acute gastroenteritis, the ability of laboratorians to alter workflow, e.g., obviating the need for setting up multiple assays to detect the pathogens included in this panel, more sensitive detection of certain pathogens relative to existing FDA cleared assays (e.g., Norovirus), and potential improved tracking or sentinel detection of acute gastroenteritis outbreaks.

As noted earlier, the risks from this device include failure of the device to detect and identify a targeted organism when such organism is present in the specimen (i.e., false negative test result for presence of organism) and detection of the targeted microorganism when such organism is not present in the specimen (i.e., false positive test result for presence of organism), both of which can lead to individual and/or public health consequences, and failure to correctly interpret test results.

There is a concern with relatively low specificity of two of the analytes tested in the panel (C. difficile and Norovirus), but this is addressed by the labelling requirement that all positive test results tests be confirmed by other cleared or reference assays and by consideration of the risks from inaccurate results for each pathogen. The low incidence of many of the pathogens in the panel, despite high specificity, yields a low positive value in most clinical settings; however, this is also addressed by the need for confirmatory testing, clinician evaluation, and the results additional diagnostic testing. The sponsor has also mandated operator training prior to device use to mitigate risks of device contamination and

{83}------------------------------------------------

false positive results, a risk present in open nucleic acid amplification platforms.

There is the additional impact of false positive and/or false negative results as regards infection control within an institution or unrecognized spread of disease, or (in the extreme case where confirmation is not performed), false outbreak identification (i.e., a pseudooutbreak).

It should be recognized that the device is intended for use as an 'aid in the diagnosis' of gastroenteritis in conjunction with clinical presentation and the results of other laboratory tests. Both clinical presentation and other results would likely substantially mitigate concerns with both false positive and false negative test results; for example, a significantly ill patient with frank dysentery is unlikely to have Norovirus infection, or if so, there is likely to be a co-pathogen or a second concomitant illness. Similarly, an ill patient with a negative GPP result is likely to undergo additional conventional testing since all potential GI pathogens are not tested by this panel, and for sufficiently ill patients, empiric antibiotic use is likely.

It is also important to recognize the potential value of Norovirus testing in this panel; this panel is the first FDA-cleared device for nucleic acid-based testing of Norovirus. Norovirus is a major cause of outbreaks of gastrointestinal disease in closed populations such as nursing homes or cruise ships, and this panel may serve an important role in increasing the confirmed diagnosis of the entity, allowing more rapid intervention.

The benefits of the GPP assay outweigh the risks. The ability to more rapidly rule out potentially significant gastrointestinal pathogens in the setting of acute gastroenteritis is particularly beneficial. The identified risks posed by the device are adequately mitigated.

T. Conclusion:

The petition for Evaluation of Automatic Class III Designation for this device is accepted. The device is classified as Class II under regulation 21 CFR 866.3990 with special controls. The special control guidance document "Class II Special Controls Guidance Document: Gastrointestinal Microorganism Multiplex Nucleic Acid-Based Assays for Detection and Identification of Microorganisms and Toxin Genes from Human Stool Specimens." will be available shortly. The device is classified under the following:

Product Code: PCH, Gastrointestinal microorganism multiplex nucleic acid-based assay Device Type: Gastrointestinal microorganism multiplex nucleic acid-based assay Class: II Regulation: 21 CFR 866.3990

§ 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).