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The Verigene Enteric Pathogens Nucleic Acid Test (EP) is a multiplexed, qualitative test for simultaneous detection and identification of common pathogenic enteric bacteria, viruses and genetic virulence markers from liquid or soft stool preserved in Cary-Blair medium, collected from individuals with signs and symptoms of gastrointestinal infection. The test is performed on the automated Nanosphere Verigene System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific gastrointestinal microbial nucleic acid gene sequences associated with the following pathogenic bacteria and viruses:

• · Campylobacter Group (composed of C. coli, C. jejuni, and C. lari)
• · Salmonella species
• · Shigella species (including S. dysenteriae, S. boydii, S. sonnei, and S. flexneri)
• · Vibrio Group (composed of V. cholerae and V. parahaemolyticus)
• · Yersinia enterocolitica
• Norovirus GI/GII
• Rotavirus A

In addition, EP detects the Shiga toxin 1 gene and Shiga toxin 2 gene virulence markers. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins 1 and 2.

EP is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness, in conjunction with other clinical, laboratory, and epidemiological information; however, is not to be used to monitor these infections. EP also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

Due to the limited number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Yersinia enterocolitica, Vibrio Group and Shigella species were primarily established with contrived specimens.

Concomitant culture is necessary for organism recovery and further typing of bacterial agents.

EP results should not be used as the sole basis for diagnosis, treatment 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 EP 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.

The Verigene Enteric Pathogens Nucleic Acid Test (EP) is a molecular assay that relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial or viral nucleic acid sequences detected by EP, unique Capture and Mediator oligonucleotides are used, with gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides are covalently bound to the microarray substrate and hybridize to a specific portion of the nucleic acid targets. The Mediator oligonucleotides have a region that binds to a different portion of the same nucleic acid targets and also have a sequence that allows binding of a gold nanoparticle probe. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency and provide accurate detection of target capture.

The EP test is performed on the Verigene System, a "sample-to-result," fully automated, benchtop molecular diagnostics workstation. The System enables automated nucleic acid extraction from unformed stool specimens (liquid or soft) preserved in Cary-Blair media and detection of analyte-specific target nucleic acids. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP.

The Reader is the Verigene System's user interface and serves as the central control unit for all aspects of test processing, automated imaging, and result generation using a touch-screen control panel and a barcode scanner. The Verigene Processor SP executes the test procedure, automating the steps of (1) Sample Preparation and Target Amplification - cell lysis and magnetic bead-based bacterial and viral nucleic acid isolation and amplification, and (2) Hybridization- detection and identification of analyte-specific nucleic acid in a microarray format by using gold nanoparticle probe-based technology. Once the specimen is loaded by the operator, all other fluid transfer steps are performed by an automated pipette that transfers reagents between wells of the trays and finally loads the specimen into the Test Cartridge for hybridization. Single-use disposable test consumables and a self-contained Verigene Test Cartridge are used for each sample tested with the EP assay.

To obtain the test results after test processing is complete, the user removes the Test Cartridge from the Processor SP, and inserts the substrate holder into the Reader for analysis. Light scatter from the capture spots is imaged by the Reader and intensities from the microarray spots are used to make a determination regarding the presence (Detected) or absence (Not Detected) of a targeted nucleic acid sequence/analyte. This determination is made by means of software-based decision algorithm resident in the Reader.

Here's an analysis of the provided text regarding the acceptance criteria and study for the Verigene® Enteric Pathogens Nucleic Acid Test (EP):

1. Table of Acceptance Criteria and Reported Device Performance

The document primarily focuses on overall agreement rates rather than explicitly stated acceptance criteria with specific thresholds for positive and negative agreement. However, we can infer performance targets from the reported results and the nature of medical device studies aiming for high accuracy.

Notes on Acceptance Criteria: Based on typical FDA clearance for diagnostic devices, the acceptance criteria would generally be a pre-defined lower bound for the confidence interval of the positive and negative agreement (e.g., >90% or >95% for the lower bound of the 95% CI). While not explicitly stated here, the reported percentages (and confidence intervals where provided) demonstrate that the device aimed for and largely achieved very high agreement rates. The low numbers of positive clinical specimens for some targets (Rotavirus A, Shigella spp., Vibrio spp., Y. enterocolitica) led to the use of contrived specimens to establish performance, which is a common practice when clinical prevalence is low.

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

• Total Valid Specimens: 1940

  • 1294 prospectively-collected fresh specimens
  • 34 prospectively-collected frozen specimens
  • 203 selected samples
  • 409 simulated specimens

• Data Provenance:

  • Country of Origin: United States (multi-site prospective investigation study at eight (8) U.S. institutions).
  • Retrospective/Prospective: Primarily prospective (1294 fresh, 34 frozen specimens). There were also "selected samples" (203) and "simulated specimens" (409), which would not be considered purely prospective clinical data. The phrase "selected samples" often implies retrospectively identified samples with known disease status.

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

The document does not specify the number of experts used or their qualifications for establishing ground truth.

4. Adjudication Method for the Test Set

The document does not specify an adjudication method. It states that "The viral comparator methods were a composite of a real-time RT-PCR assay and conventional PCR assays with confirmatory bi-directional sequencing." This implies a reference standard rather than an adjudication process between multiple readers.

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. This study is for a diagnostic test (Verigene EP) and compares its performance against reference laboratory methods, not human readers.

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

Yes, the study described is a standalone performance study. The Verigene EP test is an automated system that produces results directly from the specimen ("sample-to-result") without human interpretation of images or complex data that would necessitate a "human-in-the-loop" assessment. The performance is assessed by comparing the device's output to a reference method.

7. The Type of Ground Truth Used

The ground truth for the clinical study was established using PCR-based viral reference methods, specifically:

• A composite of a real-time RT-PCR assay.
• Conventional PCR assays with confirmatory bi-directional sequencing.

For bacterial targets and Shiga toxin genes, while not explicitly detailed in the clinical study section, it can be inferred that similar robust molecular or culture-based methods served as the reference standard.

8. The Sample Size for the Training Set

The document does not explicitly state the sample size for a training set. The clinical study described is primarily for performance evaluation (test set). For a molecular diagnostic device like this, the development and training of the underlying detection algorithm would typically occur during the assay development phase, using internal validation samples, and may not have a "training set" in the same way an AI/ML image analysis algorithm would. Analytical performance studies (LoD, inclusivity, cross-reactivity) are part of establishing the robustness of the assay's detection mechanism.

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

As no specific training set is outlined in the context of this 510(k) summary, the method for establishing its ground truth is also not described. For the analytical studies (e.g., LoD, inclusivity, cross-reactivity), the "ground truth" is inherently defined by using characterized microbial strains, known concentrations, and well-established molecular and microbiological techniques to prepare the samples.

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The Verigene® Enteric Pathogens Nucleic Acid Test (EP) is a multiplexed. qualitative test for simultaneous detection and identification of common pathogenic enteric bacteria and genetic virulence markers from liquid or soft stool preserved in Cary-Blair media, collected from individuals with signs and symptoms of gastrointestinal infection. The test is performed on the automated Nanosphere Verigene System utilizing reverse transcription (RT), polymerase chain reaction (PCR), and array hybridization to detect specific gastrointestinal microbial nucleic acid gene sequences associated with the following pathogenic bacteria:

• Campylobacter Group (comprised of C. coli. C. jejuni. and C. lari) .
• . Salmonella species
• Shigella species (including S. dysenteriae, S. boydii, S. sonnei, and S. flexneri) .
• Vibrio Group (comprised of V. cholerae and V. parahaemolyticus) .
• . Yersinia enterocolitica

In addition, EP detects the Shiga toxin 1 gene and Shiga toxin 2 gene virulence markers. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins l and 2.

EP is indicated as an aid in the diagnosis of specific agents of gastrointestinal illness, in conjunction with other clinical, laboratory, and epidemiological information; however, is not to be used to monitor these infections. EP also aids in the detection and identification of acute gastroenteritis in the context of outbreaks.

Due to the limited number of positive specimens collected for certain organisms during the prospective clinical study, performance characteristics for Yersinia enterocolitica, Vibrio Group and Shigella species were primarily established with contrived specimens.

Concomitant culture is necessary for organism recovery and further typing of bacterial agents.

EP results 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 EP 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.

The Verigene Enteric Pathogens Nucleic Acid Test (EP) is a molecular assay which relies on detection of specific nucleic acid targets in a microarray format. For each of the bacterial nucleic acid sequences detected by EP, unique Capture and Mediator oligonucleotides are utilized, with gold nanoparticle probe-based endpoint detection. The Capture oligonucleotides are covalently bound to the microarray substrate and hybridize to a specific portion of the nucleic acid targets. The Mediator oligonucleotides have a region which bind to a different portion of the same nucleic acid targets and also have a sequence which allows binding of a gold nanoparticle probe. Specific silver enhancement of the bound gold nanoparticle probes at the capture sites results in gold-silver aggregates that scatter light with high efficiency and provide accurate detection of target capture.

The EP test is performed on the Verigene System, a "sample-to-result", fully automated, bench-top molecular diagnostics workstation. The System enables automated nucleic acid extraction from unformed stool specimens (liquid or soft) preserved in Cary-Blair media and detection of bacterial-specific target DNA. The Verigene System consists of two components: the Verigene Reader and the Verigene Processor SP.

The Reader is the Verigene System's user interface, which serves as the central control unit for all aspects of test processing, automated imaging, and result generation using a touchscreen control panel and a barcode scanner. The Verigene Processor SP executes the test procedure, automating the steps of (1) Sample Preparation and Target Amplification – cell lysis and magnetic bead-based bacterial DNA isolation and amplification, and (2) Hybridizationdetection and identification of bacterial-specific DNA in a microarray format by using gold nanoparticle probe-based technology. Once the specimen is loaded by the operator, all other fluid transfer steps are performed by an automated pipette that transfers reagents between wells of the trays and finally loads the specimen into the Test Cartridge for hybridization. Single-use disposable test consumables and a self-contained Verigene Test Cartridge are utilized for each sample tested with the EP assay.

To obtain the test results after test processing is complete, the user removes the Test Cartridge from the Processor SP, and inserts the substrate holder into the Reader for analysis. Light scatter from the capture spots is imaged by the Reader and intensities from the microarray spots are used to make a determination regarding the presence (Detected) or absence (Not Detected) of a bacterial nucleic acid sequence/analyte. This determination is made by means of software-based decision algorithm resident in the Reader.

Here's a breakdown of the acceptance criteria and the study proving the device meets them, based on the provided text:

Device: Verigene® Enteric Pathogens Nucleic Acid Test (EP)
Purpose: Multiplexed, qualitative test for simultaneous detection and identification of common pathogenic enteric bacteria and genetic virulence markers from liquid or soft stool preserved in Cary-Blair media.

1. Acceptance Criteria and Reported Device Performance

The document describes analytical and clinical performance studies, which serve to establish the device's meeting of performance criteria. The tables provided in the original document directly illustrate the reported performance against implied acceptance metrics (e.g., target agreement percentages).

Table of Acceptance Criteria and Reported Device Performance:

Since specific acceptance criteria values (e.g., "must achieve X% sensitivity") are not explicitly stated as distinct criteria, I will list the performance metrics presented in the document as reported performance, implying they met the internal acceptance thresholds for regulatory submission. The precision and reproducibility results are strong indicators of meeting defined criteria for consistency.

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

• Clinical Study (Method Comparison):

  • Total Specimens Tested with EP Test: 1975
  • Valid/Evaluable Specimens: 1852 (after excluding 98 specimens and 25 indeterminate "No Call" specimens).
  • Data Provenance:
    • Country of Origin: United States (7 U.S. institutions involved in prospective investigation study).
    • Retrospective/Prospective: Primarily prospective collection of fresh and frozen Cary-Blair specimens. Additionally, simulated frozen seeded Cary-Blair specimens were used (408 specimens, prepared from deidentified prospectively-collected glycerol stocks from 12 clinical specimen acquisition sites).

• Precision Study (Internal): 14-member simulated sample panel tested daily in duplicate by 2 operators for 4 non-consecutive days, yielding 224 total results.

• Reproducibility Study (External): 14 unique samples tested daily in triplicate by 2 operators for 5 non-consecutive days at 3 external sites, yielding 1260 total results.

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

The document does not specify the number or qualifications of experts used to establish the ground truth. It states that the EP test results were compared to "reference methods, including bacterial culture and automated phenotype identification for the bacterial targets and broth enrichment followed by EIA and PCR amplification/BDS for Stx1/Stx2 typing." This implies laboratory professionals following established protocols, but no specifics on "experts" or their qualifications for ground truth establishment beyond standard lab procedures.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for discrepancies in the clinical test set results (e.g., by multiple experts). It simply states the comparison to reference methods. For the analytical studies (LoD, reactivity), "expected results" were confirmed by replicate testing and quantitative definitions.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

There is no indication of a multi-reader multi-case (MRMC) comparative effectiveness study being done, nor any mention of human readers assisting or being assisted by the AI/device. This device is a diagnostic test intended to be performed by laboratory personnel, not an AI for image interpretation or clinical decision support with physician assistance. Therefore, an effect size of human readers improving with AI vs. without AI assistance is not applicable to this type of device.

6. Standalone Performance

The entire clinical performance section (pages 10-11) describes the standalone performance of the Verigene EP test against reference methods. This means the algorithm/device's performance (results from the Verigene System) without human intervention in result interpretation beyond what is normally done in a lab setting (e.g., verifying automated calls as per standard operating procedures).

7. Type of Ground Truth Used

The ground truth for the clinical study was established using reference laboratory methods, specifically:

• Bacterial targets: Bacterial culture and automated phenotype identification.
• Stx1/Stx2 typing: Broth enrichment followed by EIA and PCR amplification/BDS.

For artificial/simulated specimens, the ground truth was the known composition of the seeded samples.

8. Sample Size for the Training Set

The document describes the analytical and clinical validation of the device. There is no information provided regarding a "training set" or the process of machine learning model development. This is a molecular diagnostic test, likely based on established probes and detection algorithms, not a deep learning AI model that requires a distinct training phase. The "Cutoff Verification" section mentions assessing 3800 data points (1120 expected positive) from LoD testing to verify the assay cutoff, which is more akin to internal algorithm parameter tuning rather than machine learning training.

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

As noted above, there is no explicit mention of a training set in the context of machine learning model development. If "training" refers to internal development and optimization of the assay's detection parameters and algorithms, the ground truth would have been established through controlled laboratory experiments (e.g., precisely known concentrations of target organisms, pure cultures, etc.) similar to the analytical studies described.

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