(3 days)
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.
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).
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.
| Organism | Metric (Clinical Study) | Acceptance Criteria (Implicit from FDA Review) | Reported Device Performance (Prospective Clinical Study, After Discrepant Investigation) |
|---|---|---|---|
| Campylobacter | Sensitivity | Lower bound of 95% CI to be acceptable | 100% (95% CI: 43.8% - 100%) |
| Specificity | Lower bound of 95% CI to be acceptable | 98.2% (95% CI: 97.3% - 98.8%) | |
| Cryptosporidium | Sensitivity | Lower bound of 95% CI to be acceptable | 92.3% (95% CI: 66.7% - 98.6%) |
| Specificity | Lower bound of 95% CI to be acceptable | 95.5% (95% CI: 94.2% - 96.6%) | |
| E. coli O157 | Sensitivity | Lower bound of 95% CI to be acceptable | 100% (95% CI: 34.2% - 100%) |
| Specificity | Lower bound of 95% CI to be acceptable | 99.2% (95% CI: 98.5% - 99.6%) | |
| Giardia | Sensitivity | Lower bound of 95% CI to be acceptable | 100% (95% CI: 51.0% - 100%) |
| Specificity | Lower bound of 95% CI to be acceptable | 96.7% (95% CI: 95.5% - 97.6%) | |
| Salmonella | Sensitivity | Lower bound of 95% CI to be acceptable | 100% (95% CI: 72.2% - 100%) |
| Specificity | Lower bound of 95% CI to be acceptable | 98.4% (95% CI: 97.6% - 99.0%) | |
| STEC stx1/stx2 | Sensitivity | Lower bound of 95% CI to be acceptable | 100% (95% CI: 20.7% - 100%) |
| Specificity | Lower bound of 95% CI to be acceptable | 98.6% (95% CI: 97.8% - 99.2%) | |
| Shigella | Sensitivity | Lower bound of 95% CI to be acceptable | 100% (95% CI: 34.2% - 100%) |
| Specificity | Lower bound of 95% CI to be acceptable | 98.5% (95% CI: 97.7% - 99.1%) | |
| C. difficile Toxin A/B | Positive Percent Agreement (PPA) | Lower bound of 95% CI to be acceptable | 93.9% (95% CI: 87.9% - 97.0%) |
| Negative Percent Agreement (NPA) | Lower bound of 95% CI to be acceptable | 89.8% (95% CI: 87.8% - 91.5%) | |
| ETEC LT/ST | Positive Percent Agreement (PPA) | Lower bound of 95% CI to be acceptable | 25.0% (95% CI: 7.1% - 59.1%) |
| Negative Percent Agreement (NPA) | Lower bound of 95% CI to be acceptable | 99.7% (95% CI: 99.1% - 99.9%) | |
| Norovirus GI/GII | Positive Percent Agreement (PPA) | Lower bound of 95% CI to be acceptable | 94.9% (95% CI: 87.5% - 98.0%) |
| Negative Percent Agreement (NPA) | Lower bound of 95% CI to be acceptable | 91.4% (95% CI: 89.6% - 92.9%) | |
| Rotavirus A | Positive Percent Agreement (PPA) | Lower bound of 95% CI to be acceptable | 100% (95% CI: 34.2% - 100%) |
| Negative Percent Agreement (NPA) | Lower bound of 95% CI to be acceptable | 99.8% (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.
§ 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).