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The Prodesse® ProGastro SSCS Assav is a multiplex real time PCR in vitro diagnostic test for the qualitative detection and differentiation of Salmonella, Shigella, and Campylobacter (C. jejimi and C. coli only, undifferentiated) nucleic acids and Shiga Toxin 1 (stxl) and Shiga Toxin 2 (stx2) genes. Shiga toxin producing E. coli (STEC) typically harbor one or both genes that encode for Shiga Toxins I and 2. Nucleic acids are isolated and purified from preserved stool specimens obtained from symptomatic patients exhibiting signs and symptoms of gastroenteritis. This test is intended for use, in conjunction with clinical presentation and epidemiological risk factors, as an aid in the differential diagnosis of Salmonella, Shigella, Campylobacter jejuni/Campylobacter coli, and STEC infections in humans.

The 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 are not detected by this test, and may not be the sole or definitive cause of patient illness. Negative ProGastro SSCS 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 ProGastro SSCS Assay enables detection and differentiation of Salmonella, Shigella, Campylobacter (C. jejuni and C. coli only, undifferentiated) and an Internal Control in the SSC Mix and Shiga Toxin Producing E. coli (STEC, stx1 and stx2 differentiated) and an Internal Control in the STEC Mix.

An overview of the procedure is as follows:

• 1. Collect raw stool specimens from symptomatic patients and place into Cary Blair Transport Medium or ParaPak C&S (C&S) Transport Medium .
• 2. Add the Gastro RNA/DNA Internal Control (GIC) to every sample to monitor for inhibitors present in the specimens.
• 3. Perform isolation and purification of nucleic acids using a NucliSENS easyMAG System and the Automated Magnetic Extraction Reagents (bioMérieux).
• 4. Add purified nucleic acids to the SSC Mix included in the ProGastro SSCS Assay Kit. The SSC Mix contains target-specific oligonucleotide primers and probes for detection of Salmonella, Shigella, and Campylobacter (C. jejuni and C. coli only). The primers and probes are complementary to highly conserved regions of genetic sequences for these organisms. The probes are dual-labeled with a reporter dye and a quencher (see table below).
• 5. Add purified nucleic acids to the STEC Mix included in the ProGastro SSCS Assay Kit. The STEC Mix contains target-specific oligonucleotide primers and probes for detection of Shiga Toxin 1 and 2 genes (stxl and stx2). The primers and probes are complementary to highly conserved regions of these genes. The probes are dual-labeled with a reporter dye and a quencher (see table below).
• 6. Perform amplification of DNA in a Cepheid SmartCycler II instrument. In this process, the probe anneals specifically to the template followed by primer extension and amplification. The ProGastro SSCS Assay is based on Tagman reagent chemistry, which utilizes the 5' - 3' exonuclease activity of Taq polymerase to cleave the probe thus separating the reporter dye from the quencher. This generates an increase in fluorescent signal upon excitation from a light source. With each cycle, additional reporter dye molecules are cleaved from their respective probes, further increasing fluorescent signal. The amount of fluorescence at any given cycle is dependent on the amount of amplification products present at that time. Fluorescent intensity is monitored during each PCR cycle by the real-time instrument.

Here's a breakdown of the acceptance criteria and study information for the ProGastro SSCS Assay:

The document provided details the clinical performance and analytical performance (reproducibility and precision) of the ProGastro SSCS Assay. The primary acceptance criteria for clinical performance are presented as Sensitivity, Specificity, Positive Percent Agreement (PPA), and Negative Percent Agreement (NPA). For analytical performance, it focuses on the agreement with expected results and the Coefficient of Variation (CV) for Ct values.

1. Table of Acceptance Criteria and Reported Device Performance

Clinical Performance (Prospective Study):

Clinical Performance (Retrospective Study):

Reproducibility (Across 3 Sites, 2 Operators/Site, 5 Days - Total 90 runs per condition):

Precision (Internal, 2 Operators, 12 Days - Total 72 runs per condition):

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

• Prospective Clinical Study:

  • Initial samples: 1214 patients.
  • Eligible samples for analysis: 1139 (after excluding 61 for protocol deviations and 14 for the SSC Mix and 14 for the STEC Mix due to "Unresolved" results and no successful retest).
  • Data Provenance: Prospective, collected from July 2011 - November 2011 and May 2012 - July 2012, and tested November 2011 - August 2012. Conducted at four U.S. clinical laboratories. Samples were "excess remnants of stool specimens that were prospectively collected from symptomatic individuals suspected of gastrointestinal infection, and were submitted for routine care or analysis by each site, and that otherwise would have been discarded."

• Retrospective Study:

  • Sample size: 105 stool samples.
  • Data Provenance: Retrospective, collected from 2007 - 2011. Conducted at two clinical sites. These samples were previously determined positive or negative by culture and/or Broth Enrichment/EIA.

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

The document does not explicitly state the number or qualifications of individual "experts" used to establish ground truth for the test set.

Instead, the ground truth was established by:

• Reference Methods:
  • Culture: For Campylobacter, Salmonella, and Shigella. This typically involves trained microbiologists following established laboratory protocols, but specific expert qualifications are not detailed.
  • Broth enrichment followed by FDA cleared EIA test: For Shiga Toxin producing E. coli (STEC). Similar to culture, this relies on standard laboratory procedures and trained personnel.
• Confirmatory Methods for Discrepancies:
  • PCR followed by bi-directional sequencing: Used to confirm the presence of stx1 and/or stx2 genes in samples positive for STEC by broth/EIA and/or the ProGastro SSCS Assay.
  • Analytically validated PCR/sequencing assays: Used to evaluate discrepant results between the ProGastro SSCS Assay and the initial reference methods. This suggests a rigorous molecular biology approach.

4. Adjudication Method for the Test Set

The adjudication method involved confirmatory testing for discrepant results:

• For STEC: Samples positive by either the broth/EIA method or the ProGastro SSCS Assay underwent PCR followed by bi-directional sequencing to confirm stx1 and/or stx2 genes. "True" STEC positives were considered any sample testing positive by broth/EIA, while "True" stx2 positives were defined as positive by broth/EIA and PCR/sequencing. This suggests a hierarchical adjudication focused on confirming the genes.
• For all targets: Discrepant results between the ProGastro SSCS Assay and the initial reference methods were evaluated using analytically validated PCR/sequencing assays. The results of these confirmatory assays are footnoted in the performance tables, indicating they informed the final determination of "true" status for discrepant samples.

There is no mention of a traditional expert panel consensus (e.g., 2+1, 3+1) for initial ground truth establishment; rather, it relies on established laboratory methodologies and subsequent molecular confirmation for discrepancies.

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 evaluates the performance of an in vitro diagnostic test (a PCR assay), not a device that is read or interpreted by human readers in a diagnostic imaging or similar context. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" is not applicable here. The assay provides a qualitative result (positive/negative) for specific nucleic acids.

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

Yes, the study primarily represents standalone performance.

The ProGastro SSCS Assay is an in vitro diagnostic test. Its performance (detection of nucleic acids) is assessed as an algorithm only, without direct human cognitive input for interpretation of the primary result (e.g., "reading" a PCR curve to decide positive/negative beyond a set threshold). The technical procedure is performed by trained laboratory personnel, but the "performance" data (Sensitivity, Specificity, PPA, NPA) presented are solely based on the assay's output compared to the ground truth, reflecting the diagnostic capability of the test itself. The intended use states it is "an aid in the differential diagnosis" and "should not be used as the sole basis for diagnosis," implying it contributes to a physician's overall assessment, but its technical performance is standalone.

7. The Type of Ground Truth Used

The ground truth for the clinical studies was established using a combination of:

• Culture: For bacterial pathogens (Campylobacter, Salmonella, Shigella). This is a widely accepted gold standard for bacterial identification.
• FDA Cleared EIA Test (with broth enrichment): For Shiga Toxin producing E. coli (STEC). This is a recognized method for STEC detection.
• PCR followed by bi-directional sequencing: This molecular method served as a confirmatory gold standard for discrepant results and for the specific identification of stx1 and stx2 genes. This represents a highly specific and sensitive molecular confirmation.

So, the ground truth is a hybrid of established microbiological culture/EIA methods and molecular sequencing confirmation.

8. The Sample Size for the Training Set

The document does not report a separate training set or details on how the training of any underlying algorithm (if present, which is unlikely for a PCR assay) was performed.

For in vitro diagnostic assays like PCR tests, the "training" equivalent typically involves extensive analytical validation (e.g., limit of detection, inclusivity, exclusivity, interference studies) to establish the assay's performance characteristics, rather than a machine learning training set as understood for AI algorithms. The clinical studies (prospective and retrospective) are primarily for validation of performance.

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

As noted above, a distinct "training set" is not mentioned in the context of this PCR assay. The establishment of ground truth for the validation/test sets is detailed in point 7 (Culture, FDA cleared EIA, and PCR/sequencing).

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