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

Viruses

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

Bacteria

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

Parasites

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

The detection and identification of specific gastrointestinal microbial nucleic acid from individuals exhibiting signs and symptoms of gastrointestinal infection aids in the diagnosis of gastrointestinal infection when used in conjunction with clinical evaluation, laboratory findings and epidemiological information. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

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

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

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

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

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

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

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

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

Acceptance Criteria and Device Performance

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

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

Study Details

1. Sample Size used for the test set and the data provenance:

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

2. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience):

   The document does not explicitly state the number or specific qualifications (e.g., years of experience) of experts involved in establishing ground truth. However, it indicates:

   • Reference/Comparator methods: For the clinical studies, reference/comparator methods were used at central laboratories independent of xTAG GPP testing sites. These methods included Premier Adenoclone Type 40/41 EIA, Amplification + sequencing (one NAAT+), Microscopy, and Bacterial culture.
   • Sequencing analysis: Bi-directional sequencing with analytically validated primers was performed. This implies expertise in molecular biology and sequence analysis.
   • Clinical evaluation: The study was conducted at 6 independent laboratories (4 in US, 2 in Canada) for the prospective study, and 4 sites for the retrospective study (3 external to Luminex Molecular Diagnostics - LMD). This suggests that the ground truth assessment, particularly for clinical diagnosis and interpretation of comparator methods, involved multiple trained laboratory personnel and clinicians.

3. Adjudication method (e.g. 2+1, 3+1, none) for the test set:

   The document describes confirmatory testing for discrepant results, particularly in the retrospective and Botswana studies. This often implies an adjudication process:

   • For positive xTAG GPP results not pre-selected at the banking site, confirmatory testing by nucleic acid amplification followed by bi-directional sequencing was performed to determine if these were True Positive or False Positive.
   • For certain analytes in the Botswana study, a random subset of xTAG GPP negative samples was also assessed by nucleic acid amplification and bi-directional sequencing.
   • One specimen that was positive for Adenovirus 40/41 by comparator but negative by xTAG GPP was positive by bi-directional sequencing only (i.e. FDA-cleared EIA negative).
   • These actions suggest a form of discrepant analysis, where a more definitive "gold standard" (like sequencing) is used to adjudicate differences. However, a formal "m+n" type adjudication (e.g., 2+1, 3+1) involving a panel of experts for every case is not explicitly stated. The comparator methods themselves serve as the primary reference standard, with sequencing as a confirmatory tool for discrepancies.

4. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:

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

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

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

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

   The ground truth was primarily established through reference/comparator methods, which varied by pathogen:

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

7. The sample size for the training set:

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

   However, if we broadly interpret "training set" as data used during the development and initial optimization before formal validation:

   • The document states that the reagents were unchanged from a previous submission (K121454). This implies development and some level of internal testing and optimization (akin to a training phase) would have occurred prior to that submission using various pathogen strains and clinical samples to establish the assay's parameters.
   • The "Analytical Reactivity" studies involve testing a wide range of strains to establish reactivity, which could be considered part of an iterative development process that feeds into refinement.

   Without specific details on Luminex's internal development process, a distinct "training set" with a defined sample size for an AI algorithm is not identified in this regulatory submission.

8. How the ground truth for the training set was established:

   Since a discrete "training set" for an AI algorithm is not explicitly identified, the method for establishing ground truth for such a set is also not detailed.
   However, for the general development and optimization of the assay:

   • Known strains and characterized clinical specimens: Analytical studies like analytical reactivity and LoD determination use well-characterized, often commercially available, strains (e.g., ATCC, Zeptometrix, NCTC) and high-titer clinical specimens where the presence and concentration of the pathogen are already known through established microbiological and molecular techniques. These serve as the "ground truth" for calibrating and optimizing assay parameters (like MFI cut-offs) during development.
   • Reference methods: Any prior development would have relied on the same types of reference methods (culture, EIA, PCR/sequencing) that were used to establish ground truth in the formal validation studies.

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