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The EntericBio" Dx assay, performed on ABI 7500 Fast Dx real-time instrument, is an in vitro multiplexed nucleic acid test for the direct, simultaneous, qualitative detection and identification of multiple enteric pathogens in Cary-Blair preserved stool specimens from individuals with signs and symptoms of infectious colitis or gastroenteritis. The test is based on detection of nucleic acids from:

• . Salmonella enterica spp.
• Shigella spp./ Enteroinvasive E. coli (EIEC)
• Campylobacter spp. (jejuni, coli and lari)
• STEC (Shiga-like toxin-producing E. coli), stx1/stx2 genes
• Vibrio spp. (cholerae and parahaemolyticus)
• . Giardia lamblia (also known as G. intestinalis and G. duodenalis)
• Entamoeba histolytica

Testing is performed on Cary-Blair preserved diarrheal specimens from symptomatic patients with suspected acute gastroenteritis, enteritis or colitis of bacterial or parasitic origin. The test is performed directly on the specimen, utilizing real-time polymerase chain reaction (PCR) for the amplification of Salmonella-specific, Campylobacter-specific, Shigella/ EIEC-specific ipaH, stx1/stx2, Vibrio-specific, Entamoeba-specific and Giardia-specific gene sequences. The test utilizes fluorogenic sequence-specific hybridization probes for the detection of the amplified DNA.

This test is intended for use, in conjunction with clinical presentation, laboratory findings and epidemiological information, as an aid in the diagnosis of Salmonella, Shigella / EIEC, Shigalike toxin-producing E. coli, Campylobacter spp., Entamoeba histolytica and Giardia spp. infections in humans.

Results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. 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 EntericBio" Dx assay 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 EntericBio® Dx Assay provides PCR reagents to be used in conjunction with an automated pipetting system and the ABI 7500 Fast Dx instrument using standard filters. Results are interpreted using the EntericBio FastFinder plugin. The system provides automated, real-time amplification, detection and analysis and a user constructed template suitable for the EntericBio® Dx Assay.

The assay is composed of Stool Preparation Solution (SPS) tubes, PCR reagent strips containing lyophilized reagents, Resuspension Buffer (Negative Kit Control), Positive Kit Control containing DNA from all target analytes (with appropriate reconstitution buffer), and associated accessories,instruments and software for detection of bacterial and parasitic causes of gastroenteritis in humans.

The EntericBio" Dx assay detects target DNA from diarrheal Cary-Blair stool specimens from symptomatic individuals with suspected gastroenteritis or infectious colitis.

The assay works directly from a Cary-Blair preserved stool sample and does not require commercial nucleic acid extraction /purification. The PCR master mix with all the reagents required to perform each test is lyophilized into individual reaction wells on a strip. Each reaction well contains an Internal Amplification Control (IAC) to monitor for PCR inhibition.

Here's a breakdown of the acceptance criteria and the study proving the device's performance, based on the provided FDA 510(k) summary for the Serosep, Ltd. EntericBio® Dx Assay:

Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the performance targets outlined for the analytical and clinical studies. Due to the nature of this 510(k) summary, specific numerical acceptance thresholds for clinical performance (e.g., minimum sensitivity/specificity) are not explicitly stated, but rather the observed performance metrics are presented. The reproducibility study explicitly states acceptance criteria.

Table of Acceptance Criteria and Reported Device Performance

Study Details:

1. Sample Sizes Used for the Test Set and Data Provenance:

   • Reproducibility Study (Test Set): Contrived stool samples spiked with various combinations of Vibrio parahaemolyticus, Shigella sonnei, and Giardia lamblia. Each analyte evaluated at True Negative, Low Positive (1.5x LoD), and Moderate Positive (3x LoD).
     • Sample Size: For accuracy, 30 samples per concentration per site (90 total per concentration per analyte across 3 sites). For negative, 60 samples per site (180 total across 3 sites).
     • Data Provenance: In-house (simulating multi-site) from Serosep. Retrospective.
   • LoD Study (Test Set): Contrived samples with target analytes spiked into negative stool matrix.
     • Sample Size: 20 replicates for each sample type/concentration.
     • Data Provenance: In-house, retrospective.
   • Analytical Reactivity/Exclusivity/Interference/Competitive Inhibition/Carry-Over (Test Sets): Contrived samples spiked into negative stool matrix or genomic DNA/RNA.
     • Sample Size: Generally 3 replicates for analytical reactivity/exclusivity/interference/carry-over. Competitive inhibition tested in triplicate.
     • Data Provenance: In-house, retrospective.
   • Clinical Performance Study (Test Set):
     • Fresh Clinical Samples (Prospective): 1491 prospective, 32 select. Total 1523 enrolled, 1472 evaluable.
       • Data Provenance: Multi-site clinical study, including two (2) US clinical sites and one (1) non-US clinical site. Prospective collection from symptomatic patients.
     • Frozen Clinical Samples (Archived): 212 enrolled, 209 evaluable.
       • Data Provenance: Archived samples from unknown collection sites, selected based on previous positive testing. Retrospective.
     • Contrived Samples (Simulated): 310 evaluable samples.
       • Data Provenance: Prepared using residual fresh specimens that tested negative. Retrospective, in-house.

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

   • For the EntericBio® Dx Assay, the ground truth for the clinical study was established by comparator methods, not directly by human experts classifying images/data. The summary does not specify the number or qualifications of experts involved in running or interpreting the results of the FDA-cleared comparator assays, or for establishing the initial diagnosis for the archived samples. In diagnostics studies like this, the "ground truth" is typically another diagnostic method (often a laboratory reference method or a composite of multiple methods), which itself is run by trained laboratory personnel.

3. Adjudication Method for the Test Set:

   • For STEC and Campylobacter in fresh specimens, a composite comparator method was used: a specimen was positive if 2 out of 3 comparator assays were positive, and negative if 2 out of 3 were negative.
   • For all other target analytes and archived specimens, the comparator method consisted of one FDA-cleared assay.
   • This implies an adjudication method for the composite comparator (2/3 rule) but not for single comparator methods. There is no mention of human expert adjudication post-test to resolve 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:

   • No, an MRMC comparative effectiveness study was not done. This device is a molecular diagnostic assay (PCR-based), not an AI-assisted diagnostic imaging device that involves human "readers" interpreting images. Therefore, the concept of human readers improving with AI assistance is not applicable here.

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

   • The EntericBio® Dx Assay is a standalone diagnostic test in the sense that its performance is evaluated directly against comparator methods. It does not inherently involve a "human-in-the-loop" decision-making process in the same way an AI-CAD (Computer-Aided Detection) system for radiology would. The device provides a result (positive/negative) based on its PCR analysis, and clinical interpretation occurs downstream by lab personnel and clinicians. The clinical performance tables (e.g., Table 12a-g) represent this standalone performance of the assay.

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

   • The ground truth for the clinical performance study was established by comparator methods, specifically:
     • For STEC and Campylobacter (fresh specimens): A composite comparator method of three FDA-cleared assays (2/3 rule).
     • For other targets (fresh) and all archived specimens: One FDA-cleared assay.
     • For contrived samples: The known spike concentration/strain was the ground truth.

7. The Sample Size for the Training Set:

   • This document is a 510(k) summary for an in vitro diagnostic (IVD) assay based on PCR, not a machine learning/AI model. Therefore, the concept of a "training set" for an algorithm is not directly applicable in the same way it would be for AI/ML device development. The device's design and optimization (analogous to "training") would have occurred during its development phase, using proprietary methods, and is not explicitly detailed as a distinct 'training set' of clinical samples in this regulatory submission.

8. How the Ground Truth for the Training Set was Established:

   • As explained above, this product is not an AI/ML algorithm requiring a "training set" in the conventional sense. The "ground truth" for the analytical studies (e.g., LoD, exclusivity) was based on precisely prepared samples with known concentrations and identities of microorganisms. For the development and validation of the molecular targets (primers/probes), ground truth would have been established through extensive molecular biology techniques, including sequencing and controlled lab cultures. These methods are inherent to the development of robust PCR assays.

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The Great Basin Stool Bacterial Pathogens Panel is a multiplexed, qualitative test for the detection and identification of DNA targets of enteric bacterial pathogens. The Stool Bacterial Pathogens Panel detects nucleic acids from:

• · Campylobacter (C. coli/C. jejuni)
• · Salmonella
• · Shiga toxin 1 (stx1)
• Shiga toxin 2 (stx2)
• · Escherichia coli serotype 0157
• Shigella

Shiga toxin genes are found in Shiga toxin-producing strains of E. coli (STEC/EHEC/VTEC) and Shigella dysenteriae. The E. coli O157 test result is only reported if a Shiga toxin gene (stx1 and/or stx2) is also detected.

The Stool Bacterial Pathogens Panel is performed directly from Cary Blair or C&S Medium preserved stool specimens from symptomatic patients with suspected acute gastroenteritis, or colitis and is performed on the Portrait™ Analyzer.

The test is intended for use as an aid in the diagnosis of gastrointestinal illness in conjunction with clinical and epidemiological information; however, it is not to be used to monitor these infection . Positive results do not rule out co-infection with other organisms and may not be the definitive cause of patient illness. Negative test 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. Concomitant culture is necessary if organism recovery or further typing of bacterial agents is desired.

The Great Basin Stool Bacterial Pathogens Panel on the PA500 Portrait™ System utilizes automated, hot-start PCR amplification technology to amplify specific nucleic acid sequences that are then detected using hybridization probes immobilized on a modified silicon chip surface, in a single-use, self-contained test cartridge.

An aliquot of the specimen (stool preserved in stool transport media) is first processed using the Sample Preparation Device (SPD). An aliquot of the eluate obtained from the SPD is loaded into the sample port of the SBPP Test Cartridge.

Genomic DNA is extracted from microbial cells and diluted to reduce potential inhibitors of the PCR. During the PCR process, biotin-labeled primers direct the amplification of specific nucleic acid sequences within a conserved region for identification of: a bacterial sample processing control (SPC), Campylobacter coli/Campylobacter jejuni, Salmonella spp., Shiga toxin 1, Shiqa toxin 2, and E. coli serotype 0157.

Following PCR, biotin-labeled, amplified target DNA sequences are hybridized to sequence specific probes immobilized on the silicon chip surface, and incubated with antibody conjugated to the horseradish peroxidase enzyme (HRP). The unbound conjugate is washed away, and tetramethylbenzidine (TMB) is added to produce a colored precipitate at the location of the probe/target sequence complex. The resulting signal is detected by the automated Portrait™ Optical Reader within the PA500 Portrait™ Analyzer System. The SPC undergoes the same extraction, amplification, and detection steps as the sample in order to inhibitory substances, as well as process inefficiency due to instrument or reagent failure. No operator intervention is required once the sample is loaded into the sample port, and the Stool Bacterial Pathogens Panel cartridge is loaded into the Portrait™ Analyzer.

The PA500 Portrait™ Analyzer System is a fully automated system that includes: the Portrait™ Analyzer, single-use Stool Bacterial Pathogen Panel Cartridges, and the Portrait™ Data Analysis Software Program. The Portrait™ System is designed to perform automated sample preparation, PCR, and optical chip-based detection with integrated data analysis in less than two hours.

This detailed document outlines the performance characteristics of the Great Basin Stool Bacterial Pathogens Panel (SBPP). While it does not include AI-specific performance criteria, it provides a comprehensive overview of the device's analytical and clinical validation, which are analogous to acceptance criteria and study data for traditional medical devices. I will extract the relevant information and present it in the requested format, interpreting "acceptance criteria" as the performance benchmarks demonstrated in the studies and "device performance" as the results achieved.

For AI-specific questions (Adjudication method, MRMC, Standalone performance, Training set details), the document does not contain this information as it pertains to a nucleic acid-based assay and not an AI/ML-driven device. I will explicitly state "Not applicable" for these points.

Acceptance Criteria and Device Performance for Great Basin Stool Bacterial Pathogens Panel

1. Table of Acceptance Criteria and the Reported Device Performance

Since this is a diagnostic assay and not an AI-driven image analysis tool, the acceptance criteria are based on analytical and clinical performance metrics. The document describes several studies (Analytical Sensitivity, Analytical Reactivity, Analytical Specificity, Competitive Inhibition, Interfering Substances, Microbial Interference, Carry-over/Cross Contamination, Reproducibility, Specimen Stability, and Clinical Studies).

For simplicity and relevance to a typical "acceptance criteria" table for a diagnostic device, I will focus on the key performance indicators from the reproducibility and clinical studies. The implicit "acceptance criteria" are the demonstrated performance percentages, often with 95% confidence intervals where available.

2. Sample sizes used for the test set and the data provenance:

• Clinical Test Sets:

  • Prospective Study (Fresh Samples): 1479 samples included in the analysis (1506 collected, 24 excluded). Collected from four external, geographically-diverse U.S. clinical study sites (Midwest, Northeast, Southwest and West) from July 2016 to November 2016. These were excess remnants of preserved stool samples. Prospective data.
  • Frozen Retrospective Sample Study: 150 frozen archived de-identified specimens initially (for the general panel), with specific numbers for each analyte:
    • Salmonella: 206 samples
    • Shiga Toxin 1: 206 samples
    • Shiga Toxin 2: 206 samples
    • E. coli O157: 48 samples
    • Shigella: 206 samples
      These were de-identified specimens previously characterized (historical result). Retrospective data.
  • Selected Fresh Positive Salmonella Samples Study: 28 additional fresh samples. Collected from Intermountain Healthcare (IMC) in Salt Lake City, UT. Prospective data.

• Analytical Test Sets:

  • Analytical Sensitivity (LoD): 10 bacterial strains, serially diluted.
  • Analytical Reactivity (Inclusivity): 91 well-characterized bacterial strains, multiple replicates per strain (at least 3).
  • Analytical Specificity (Exclusivity): 100 non-target organisms (84 bacterial, 3 yeast, 3 parasites, 9 viruses) and human genomic DNA, multiple replicates per organism (minimum of 3).
  • Competitive Inhibition: 48 unique combinations of pathogens, each tested in triplicate.
  • Interfering Substances: 19 different substances, tested with 8 target organisms, minimum of 3 replicates per substance/organism combination.
  • Microbial Interference: 29 non-target organisms tested in presence of 8 target analytes, minimum of 3 replicates.
  • Carry-over/Cross Contamination: 40 high positive samples and 40 negative samples (total 80 tests).
  • Reproducibility: 7 different samples tested in triplicate over 5 non-consecutive days by 6 operators, across 3 sites (90-92 replicates per positive analyte, 450 replicates for negative).

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

The document describes the ground truth for clinical samples as "standard stool culture-based methods" and "standard of care method used at the institution (historical result)," further confirmed by "FDA cleared Nucleic Acid Amplification Test (NAAT)" for retrospective samples. The experts are implicitly the microbiologists and clinical laboratory personnel at the clinical sites performing these standard methods. No specific number or explicit qualifications (e.g., years of experience) for these "experts" are provided in the document, which is typical for diagnostic assay submissions relying on established clinical laboratory practices.

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

None described. The ground truth for the clinical studies relied on standard microbiological culture methods and FDA-cleared NAATs as reference methods. Discrepant results were investigated by further testing with other FDA-cleared NAATs (BioFire Film Array GI Panel or Nanosphere Verigene® EP test). This is a discrepant analysis approach, not an adjudication process by human experts re-interpreting initial data.

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:

Not applicable. This device is a molecular diagnostic assay, not an AI-driven imaging or diagnostic tool intended for human-in-the-loop assistance.

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

Yes, the device operates as a standalone automated system. The entire analytical and clinical performance evaluation described in the document represents the standalone performance of the SBPP, as it integrates sample preparation, PCR, and detection with automated software interpreting results. It is an "algorithm only" in the sense of a laboratory assay's automated result interpretation, although not a machine learning algorithm.

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

The ground truth for the clinical studies was established primarily by:

• Standard stool culture-based methods (for prospective samples).
• Historical results from clinical sites using their standard of care methods, then confirmed by FDA cleared Nucleic Acid Amplification Tests (NAATs) (for frozen retrospective samples).
• Discrepant analysis was performed using other FDA-cleared NAATs (BioFire Film Array GI Panel or Nanosphere Verigene® EP test) for unconcordant results.

Thus, the ground truth is a combination of established microbiological standards and validated molecular diagnostic tests.

8. The sample size for the training set:

Not applicable. This is a nucleic acid-based diagnostic device, not an AI/ML device that requires a "training set" in the conventional sense. The development of the assay (primer/probe design, assay conditions) would be based on scientific knowledge and wet-lab experiments, not a machine learning training data set.

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

Not applicable. See point 8. The "ground truth" for developing the assay would be pure cultures of target and non-target organisms, along with their known biochemical and genetic characteristics, established through standard microbiology laboratory practices.

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