(225 days)
The xTAG® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid test intended for the simultaneous qualitative detection and identification of multiple viral, bacterial, and parasitic nucleic acids in human stool specimens or human stool in Cary-Blair media from individuals with signs and symptoms of infectious colitis or gastroenteritis. The following pathogen types, subtypes and toxin genes are identified using the xTAG GPP:
Viruses
. Adenovirus 40/41
Norovirus GI/GII ●
Rotavirus A
Bacteria
Campylobacter (C. jejuni, C. coli and C. lari only) ●
Clostridium difficile (C. difficile) toxin A/B
Escherichia coli (E. coli) 0157
Enterotoxigenic Escherichia coli (ETEC) LT/ST
. Salmonella
Shiga-like Toxin producing E. coli (STEC) stx 1/stx 2
Shigella (S. boydii, S. sonnei, S. flexneri and S. dysenteriae)
Vibrio cholerae (V. cholerae) cholera toxin gene (ctx) ●
Parasites
Cryptosporidium (C. parvum and C. hominis only) ●
Entamoeba histolytica (E. histolytica)
. Giardia (G. lamblia only, also known as G. intestinalis and G. duodenalis)
The detection and identification of specific gastrointestinal microbial nucleic acid from individuals exhibiting signs and symptoms of gastrointestinal infection aids in the diagnosis of gastrointestinal infection when used in conjunction with clinical evaluation, laboratory findings and epidemiological information. A gastrointestinal microorganism multiplex nucleic acid-based assay also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.
xTAG GPP positive results are presumptive and must be confirmed by FDA-cleared tests or other acceptable reference methods.
The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Confirmed positive results do not rule out co-infection with other organisms that are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative xTAG GPP results in the setting of clinical illness compatible with gastroenteritis may be due to infection by pathogens that are not detected by this test or noninfectious causes such as ulcerative colitis, irritable bowel syndrome, or Crohn's disease.
xTAG GPP is not intended to monitor or guide treatment for C. difficile infections.
The xTAG GPP test is indicated for use with the Luminex® 100/200™ and MAGPIX® instruments with xPONENT® software.
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).
The provided text describes the Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) and various studies supporting its performance. I will extract the requested information based on the additional analytes (Adenovirus 40/41, Entamoeba histolytica, and Vibrio cholerae) while noting that much of the original device's performance data (from K121894) is still applicable.
Description of Acceptance Criteria and Studies Proving Device Performance
The Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) is a multiplexed nucleic acid test designed for the qualitative detection and identification of multiple viral, bacterial, and parasitic nucleic acids in human stool specimens or human stool in Cary-Blair media. This submission (K140647) primarily focuses on the addition of Adenovirus 40/41, Entamoeba histolytica, and Vibrio cholerae to the panel, as well as the use of stool in Cary-Blair media.
The device performance was evaluated through analytical studies (Analytical Reactivity, Carry-over Contamination, Limit of Detection, Repeatability, Analytical Specificity/Interference, Fresh vs. Frozen Stool Evaluation, Reproducibility/Precision) and clinical studies (Detection in Asymptomatic Volunteers, Detection in Symptomatic Patients from prospective and retrospective cohorts, and simulated samples).
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria for clinical performance are primarily inferred from the detailed reporting of Positive Percent Agreement (PPA) / Sensitivity and Negative Percent Agreement (NPA) / Specificity with their 95% Confidence Intervals (CI). Analytical performance criteria are noted qualitatively (e.g., "100% agreement," "no interference").
Clinical Performance Acceptance Criteria (Inferred from reporting, generally striving for high agreement):
- Sensitivity/Positive Agreement: Typically >90% with lower bound 95% CI >80% (explicitly stated for Norovirus GI/GII and Clostridium difficile toxin A/B in Cary-Blair media, and implied for other analytes by robust performance).
- Specificity/Negative Agreement: Typically >90% with lower bound 95% CI >90% (explicitly stated for all targets in Cary-Blair media, and implied for other analytes by robust performance).
| Analyte (Additional) | Study Type | Acceptance Criteria (Inferred/Stated) | Reported Device Performance (Summary) |
|---|---|---|---|
| Analytical Studies | |||
| Adenovirus 40/41 | Analytical Reactivity | Reactivity established at concentrations 2-3x LoD. | Reactivity demonstrated with various strains (Dugan, Tak) at 1.49E+07 and 1.43E+07 Copies/mL (CDC samples). 9 Adenovirus 40 and 28 Adenovirus 41 clinical samples positively detected and confirmed by sequencing. |
| Entamoeba histolytica | Analytical Reactivity | Reactivity established at concentrations 2-3x LoD. | Reactivity demonstrated with various ATCC strains ranging from 8.90E+01 to 2.88E+03 Copies/mL, generally corresponding to 0.4x to 6.7x LoD, with one strain (ATCC 50738) at 0.2x LoD. |
| Vibrio cholerae (ctx gene) | Analytical Reactivity | Reactivity established at concentrations 2-3x LoD. | Reactive with 12 out of 22 tested strains including O:1, O:139, and non-O:1 strains expressing ctx gene, at 7.02E+06 CFU/mL (3xLoD). |
| Adenovirus 40 | Carry-over Contamination | No false positives from HP, no false negatives from HN. | 100% of 144 high negative samples remained negative; 100% of 144 high positive samples remained positive. Lack of carryover contamination demonstrated. |
| Adenovirus 40/41, E. histolytica, V. chol | Limit of Detection (LoD) | LoD determined per analyte with CV%. | Adenovirus 40: 1.45x10^1 TCID50/mL (34.26% CV); Adenovirus 41: 7.69 TCID50/mL (20.27% CV); Entamoeba histolytica: 2.88x10^1 cells/mL (17.77% CV); Vibrio cholerae: 2.34x10^6 CFU/mL (23.94% CV). |
| Adenovirus 40/41, E. histolytica, V. chol | Repeatability | 20/20 correct calls for Low and Moderate Positive. | Adenovirus 40/41: LP: 20/20 POS, MP: 20/20 POS; Entamoeba histolytica: LP: 20/20 POS, MP: 20/20 POS; Vibrio cholerae: LP: 20/20 POS, MP: 20/20 POS. |
| E. dispar (cross-reactivity) | Analytical Specificity | No cross-reactivity with commensal flora. | One strain (ATCC PRA-353) cross-reacted at 3.0E+05 cells/mL (>10^4 times LoD for E. histolytica), but not at 4-fold lower titer (7.50E+04 cells/mL). In silico analysis confirmed reverse primer mismatches for E. dispar reducing amplification efficiency, implying negligible risk of false positives at clinically relevant titers. |
| Adenovirus 40/41 | Interference (non-panel) | No interference at LP with HP non-panel organisms. | No interference observed from Astrovirus (HP) on Adenovirus serotypes 40/41 (LP). |
| Adenovirus, E. histolytica, V. chol. | Interference (commensal) | No interference at LP with HP commensal organisms. | No interference found with xTAG GPP analytes Adenovirus, Entamoeba histolytica, and Vibrio cholerae when tested with 10 different common commensal bacteria, yeast, and parasites at high concentrations. |
| Adenovirus (competitive) | Interference (competitive) | No competitive interference with other panel pathogens. | No competitive interference observed between Adenovirus and Norovirus, Salmonella, or Campylobacter jejuni at LP/HP combinations. |
| E. histolytica (stability) | Fresh vs. Frozen Stool (1M) | Positive agreement ≥95% (lower bound 95% CI >85%). | Un-extracted: Not met. Pre-treated: Met. Extracted: Met. (Supplemental testing supported un-extracted stability for 1 month). |
| E. histolytica (stability) | Fresh vs. Frozen Stool (3M) | Positive agreement ≥95% (lower bound 95% CI >85%). | Un-extracted: Met. Pre-treated: Not reported. Extracted: Not met. (Overall data supports stability for 1 month). |
| Adenovirus 40/41, V. chol. | Fresh vs. Frozen Stool (1M) | Positive agreement ≥95% (lower bound 95% CI >85%). | Un-extracted: Met. Pre-treated: Met. Extracted: Met. |
| Adenovirus 40/41, V. chol. | Fresh vs. Frozen Stool (3M) | Positive agreement ≥95% (lower bound 95% CI >85%). | Un-extracted: Met. Pre-treated: Not reported. Extracted: Met. |
| Adenovirus 40/41, E. histolytica, V. chol | Reproducibility (Single) | LP: 82/90 to 90/90 correct calls; MP: 86/90 to 90/90 correct calls. | Adenovirus 40/41: LP: 89/90 (98.9%), MP: 89/90 (98.9%); Entamoeba histolytica: LP: 82/90 (91.1%), MP: 86/90 (95.6%); Vibrio cholerae: LP: 90/90 (100%), MP: 90/90 (100%). HN variability noted. |
| Adenovirus (dual analyte) | Reproducibility (Dual) | Positive call for HP (100%) and LP (high agreement). | HP: 100% positive calls. LP: 4/90 (C.difficile HP/Adenovirus LP) and 2/90 (Rotavirus HP/Adenovirus LP) generated negative calls for Adenovirus. Overall, adequate site-to-site reproducibility established. |
| C. difficile, Giardia, Norovirus GII | Stool in Cary-Blair LoD | Equivalent LoD between raw stool and Cary-Blair. | Equivalent LoD demonstrated for all three representative analytes (C. difficile Toxin A/B, Giardia lamblia, Norovirus GII) in raw stool vs. stool in Cary-Blair media. |
| Clinical Studies | |||
| Adenovirus 40/41 (Prospective Stool) | Diagnostic Accuracy | PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%). | PPA: 80% (4/5) with 95% CI: 37.5%-96.4%. NPA: 98.5% (1158/1175) with 95% CI: 97.7%-99.1%. (Note: Low positivity rates impacted CI width). |
| Entamoeba histolytica (Prospective Stool) | Diagnostic Accuracy | PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%). | PPA: N/A (0/0 positives by comparator). NPA: 98.3% (1154/1174) with 95% CI: 97.4%-98.9%. (Note: No positive by comparator). |
| Vibrio cholerae (Prospective Stool) | Diagnostic Accuracy | PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%). | PPA: N/A (0/0 positives by comparator). NPA: 99.7% (1171/1174) with 95% CI: 99.2%-99.9%. (Note: No positive by comparator). |
| Adenovirus 40/41 (Prospective Cary-Blair) | Diagnostic Accuracy | PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%). | PPA: 33.3% (2/6) with 95% CI: 9.7%-70.0%. NPA: 99.6% (1285/1290) with 95% CI: 99.1%-99.8%. (Note: PPA reduced due to low titer specimen and unavailable samples, supplemental data provided). |
| Entamoeba histolytica (Pre-selected) | Positive Agreement | PPA >90%. | 100% (1/1) with 95% CI: 2.5%-100%. (Note: Very limited sample size). |
| Adenovirus 40/41 (Pre-selected) | Positive Agreement | PPA >90%. | 100% (3/3) with 95% CI: 43.8%-100%. (Note: Limited sample size). |
| Adenovirus 40/41 (Botswana Pediatric) | Diagnostic Accuracy | PPA >90% (lower bound 95% CI >80%); NPA >90% (lower bound 95% CI >90%). | PPA: 67.3% (35/52) with 95% CI: 53.7%-78.5%. (Note: 17 false negatives were low viral titer by RT-PCR). NPA: 100% (255/255) with 95% CI: 98.5%-100%. |
| Entamoeba histolytica (Contrived Stool) | Positive/Negative Agreement | PPA >90% ; NPA >90%. | PPA: 100% (50/50) with 95% CI: 92.9%-100%. NPA: 100% (100/100) with 95% CI: 96.1%-100%. |
| Vibrio cholerae (Contrived Stool) | Positive/Negative Agreement | PPA >90% ; NPA >90%. | PPA: 98% (49/50) with 95% CI: 89.5%-99.7%. NPA: 100% (100/100) with 95% CI: 96.1%-100%. |
| Adenovirus 40/41 (Contrived Cary-Blair) | Positive Agreement | PPA >90%. | PPA: 100% (50/50 overall) with 95% CI: 92.9%-100%. |
| E. histolytica (Contrived Cary-Blair) | Positive Agreement | PPA >90%. | PPA: 96% (47/49 overall) with 95% CI: 86.3%-98.9%. |
| Vibrio cholerae (Contrived Cary-Blair) | Positive Agreement | PPA >90%. | PPA: 100% (50/50 overall) with 95% CI: 92.9%-100%. |
2. Sample size used for the test set and the data provenance
- Asymptomatic Volunteers (Test Set): 200 clinical stool samples.
- Data Provenance: Retrospective, collected from healthy, asymptomatic donors. Country of origin not specified, but collected for this study.
- Symptomatic Patients (Prospective Clinical Study in Stool Specimens) (Test Set): 1407 clinical stool specimens.
- Data Provenance: Prospective, collected between June 2011 and February 2012. Origin: Four laboratories in the United States (Arizona, Missouri, Tennessee, Texas) and two in Southern Ontario (Canada).
- Symptomatic Patients (Prospective Clinical Study in Stool in Cary-Blair Media) (Test Set): A subset of the 1407 prospective samples for which adequate sample was available. The exact number varied per analyte for each comparison (e.g., Adenovirus 40/41 had N=6 positives in Cary-Blair for PPA calculation).
- Data Provenance: Prospective, from the same cohort described above.
- Pre-selected stool specimens Retrospective Study (Test Set): 207 archived stool specimens positive for various pathogens (low prevalence in prospective study), plus negative clinical specimens.
- Data Provenance: Retrospective, collected at multiple sites in North America, Africa, and Europe.
- Pre-selected Stool in Cary-Blair Specimens Retrospective Study (Test Set): Remnants of available pre-selected frozen stool specimens mixed proportionally with Cary-Blair medium.
- Data Provenance: Retrospective, from the same pre-selected cohort.
- Botswana Pediatric Stool Specimens (Test Set): 313 pediatric stool specimens.
- Data Provenance: Prospective, collected between February 2011 and January 2012 from symptomatic pediatric patients in Botswana, Africa.
3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts
The document does not explicitly state the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience") for establishing ground truth. Instead, it refers to "reference/comparator methods" or "comparator methods" performed at "central laboratories independent of xTAG GPP testing sites." For Entamoeba histolytica, microscopy was part of the comparator method, implying trained microscopists. For other analytes, molecular methods (EIA, amplification + sequencing, bacterial culture) were used, suggesting laboratory professionals proficient in these techniques.
4. Adjudication method for the test set
The document describes the establishment of ground truth for clinical studies using "Comparator methods."
- For Adenovirus 40/41: Composite comparator consisting of Premier Adenoclone Type 40/41 EIA and Amplification + sequencing.
- For Entamoeba histolytica: Microscopy followed by amplification + sequencing (for positive samples by microscopy only).
- For Vibrio cholerae: Bacterial culture.
- For other analytes (from K121894), similar molecular and culture-based comparator methods were used.
A "true positive" or "true negative" result for xTAG GPP was assigned if it agreed with the comparator method. In cases of discrepancies (e.g., xTAG GPP positive, comparator negative), confirmatory testing (e.g., bi-directional sequencing) was performed to determine if the xTAG GPP result was a true positive or false positive. For example, in the pre-selected study, confirmatory testing by NAAT and bi-directional sequencing was done on "additional positive calls" (xTAG GPP positive but not pre-selected).
This represents a form of discrepancy resolution, where a higher-tier reference method (like sequencing) is used to adjudicate disagreements. It is not an "expert consensus" adjudication in the sense of multiple independent experts reviewing and voting, but rather a technical adjudication against a more definitive laboratory standard.
5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance
This document describes a diagnostic device (multiplex nucleic acid test) for pathogen detection, not a system involving human readers interpreting images or data with and without AI assistance. Therefore, an MRMC comparative effectiveness study regarding human reader improvement with AI assistance is not applicable to this device and was not conducted. The "readers" here are automated instruments and software, and the "human-in-the-loop" refers to laboratory technicians performing the assay and interpreting the automated output based on predefined algorithms and cut-offs.
6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done
Yes, the analytical and clinical performance studies described represent a standalone (algorithm only) performance evaluation of the xTAG GPP device. The system automates nucleic acid extraction, amplification, detection, and analysis (TDAS GPP software applies algorithms to MFI values to generate qualitative results, which are then compiled into a report). The studies assess the accuracy of these automated results against comparator methods. While human operators perform sample preparation and run the instruments, the critical detection and identification steps, as well as the interpretation of raw data into qualitative positive/negative calls, are performed by the device's integrated system and software.
7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)
The ground truth used was a combination of:
- Reference Laboratory Methods / Comparator Methods: These included FDA-cleared EIAs, bacterial cultures, microscopy, and Nucleic Acid Amplification Tests (NAATs) followed by bi-directional sequencing for specific gene targets. These are often considered the gold standard or best available methods in microbiology diagnostics.
- Discrepancy Resolution using Sequencing: For discrepant results (e.g., xTAG GPP positive, comparator negative), bi-directional sequencing, which is a highly specific molecular method, was used as a definitive arbiter to confirm the presence or absence of the target nucleic acid.
- Clinical Outcomes Data: This was not explicitly stated as a direct method for establishing ground truth for individual pathogens within the scope discussed. The focus was on direct pathogen detection.
8. The sample size for the training set
The document does not explicitly delineate a "training set" in the context of machine learning. The studies described are for analytical validation and clinical performance evaluation of the completed device.
However, the "design history file" and "in silico analysis" mentioned (e.g., for primer cross-reactivity) imply that analytical methods were developed and refined based on bioinformatics and laboratory testing during the device's development phase. The analytical reactivity studies, for instance, involve empirical testing of a wide range of strains to establish reactivity, which could be seen as part of the developmental data used to "train" or optimize the assay's ability to detect targets. The document focuses on the validation data for the regulatory submission.
9. How the ground truth for the training set was established
As noted above, a formal "training set" with established ground truth, as understood in machine learning, is not described. The analytical studies (e.g., Limit of Detection, Analytical Reactivity) used well-characterized strains, genotypes, isolates, or clinical specimens with known positivity/negativity established by commercial sources (e.g., ATCC, Zeptometrix) or validated laboratory methods (e.g., quantitative PCR, cell culture infectious dose determination). This type of well-characterized material would be analogous to ground truth for developing and optimizing the assay's performance characteristics.
§ 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).