(265 days)
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
| Category | Metric/Target | Acceptance Criteria (If explicitly stated) | Reported Device Performance (EntericBio® Dx Assay) |
|---|---|---|---|
| Analytical Performance | |||
| Reproducibility (Accuracy) | Agreement with expected results for target analytes (Moderate Positive) | 100% agreement | Shigella sonnei: 100% (90/90) |
| Vibrio parahaemolyticus: 100% (90/90) | |||
| Giardia lamblia: 100% (90/90) | |||
| Reproducibility (Accuracy) | Agreement with expected results for target analytes (Low Positive) | ≥95% agreement | Shigella sonnei: 100% (90/90) |
| Vibrio parahaemolyticus: 100% (90/90) | |||
| Giardia lamblia: 98% (89/90) | |||
| Reproducibility (Precision) | Cq Value Coefficient of Variation (%CV) | Not explicitly stated as an acceptance criterion for individual Cq values, but presented as a measure of precision. | Shigella sonnei: Moderate Positive (2.44% CV), Low Positive (1.97% CV) |
| Vibrio parahaemolyticus: Moderate Positive (1.21% CV), Low Positive (1.82% CV) | |||
| Giardia lamblia: Moderate Positive (3.53% CV), Low Positive (4.05% CV) | |||
| Limit of Detection (LoD) | Lowest concentration of analyte consistently detected | ≥95% detection rate | Salmonella spp.: 8 x 10^4 CFU/mL |
| Shigella spp./EIEC: 1.25 x 10^4 - 1 x 10^4 CFU/mL | |||
| Campylobacter spp.: 1 x 10^4 - 4 x 10^4 CFU/mL | |||
| E. coli (STEC): 5 x 10^5 - 1 x 10^6 CFU/mL | |||
| Vibrio spp.: 1 x 10^4 CFU/mL | |||
| Giardia lamblia: 25-100 cells/mL | |||
| Entamoeba histolytica: 25-100 cells/mL | |||
| Analytical Reactivity | Detect all tested strains within target diversity | All organisms tested detectable over three replicates. | All 101 target organisms were detectable. |
| Analytical Exclusivity | No cross-reactivity with non-target organisms (or limited to closely related organisms with shared sequence similarity) | Not explicitly stated, but implies minimal/no false positives from unrelated organisms. | Cross-reactivity observed with Vibrio campbellii, V. fluvialis, V. furnissii, V. mimicus, V. fischeri, V. natriegens (all cross-reacted with Vibrio target). Other noted "cross-reactivity" issues were attributed to potential sample contamination or expected in-silico findings for some highly related organisms (e.g., Cryptosporidium viatorum/Shigella, Cryptosporidium cuniculus/Giardia). |
| Microbial Interference | No interference from high concentrations of common microorganisms. | No interference in presence of 3X LoD of each target analyte. | No interference observed with 11 tested microorganisms. |
| Interfering Substances | No interference from common exogenous/endogenous substances. | Detection of targets/IAC in presence of substances. | Most substances (21/23) showed no interference. False negative observed for V. parahaemolyticus with hemorrhoidal cream (1/3 replicates) and C. jejuni with tetracycline (2/3 replicates). IAC failures with >1% Benzalkonium chloride and >1% hemorrhoidal cream. |
| Competitive Inhibition | No competitive inhibition when multiple target analytes are present. | No competitive inhibition. | Competitive inhibition observed for Giardia lamblia in presence of Entamoeba histolytica. (Note: Not evaluated for Well B analytes: Vibrio and STEC). |
| Carry-over/Cross-Contamination | No carry-over/cross-contamination. | No carryover or cross-contamination. | None observed. |
| Specimen Stability | Determine recommended storage conditions. | Established storage conditions. | 5 days at 2-8°C for Cary-Blair preserved stool. |
| Real Time Stability | Determine shelf life. | Established shelf life. | 6 months at 2-8°C. |
| Clinical Performance | |||
| Overall Agreement (Positive) | Agreement with comparator method for target detection for Fresh, Archived, and Contrived samples. (Individual values depend on analyte) | Not explicitly stated, but implied to be high enough for substantial equivalence. | Salmonella: Fresh (92.3%), Select (90.0%), Frozen (85.7%). Campylobacter: Fresh (91.1%), Select (90.0%), Frozen (93.8%). Shigella/EIEC: Fresh (100%), Select (100%), Frozen (90.9%). STEC: Fresh (100%), Select (100%), Frozen (94.6%). Vibrio: Fresh (0.0% - due to low prevalence), Simulated (100%). Giardia: Fresh (85.7%), Select (100%), Frozen (100%). Entamoeba: Fresh (NA - no positives), Frozen (0.0% - no positives), Simulated (98.6%). |
| Overall Agreement (Negative) | Agreement with comparator method for non-detection for Fresh, Archived, and Contrived samples. (Individual values depend on analyte) | Not explicitly stated, but implied to be high enough for substantial equivalence. | Salmonella: Fresh (100%), Select (100%), Frozen (100%). Campylobacter: Fresh (99.7%), Select (100%), Frozen (100%). Shigella/EIEC: Fresh (100%), Select (100%), Frozen (100%). STEC: Fresh (99.9%), Select (100%), Frozen (100%). Vibrio: Fresh (100%), Simulated (100%). Giardia: Fresh (99.9%), Select (100%), Frozen (100%). Entamoeba: Fresh (100%), Frozen (100%), Simulated (100%). |
| Invalid Rate | Rate of invalid results. | Not explicitly stated, but low rate desirable. | Fresh: Initial 1.9% (resolved to 0.6%). Frozen: Initial 0.9% (resolved to 0.5%). Total: Initial 1.7% (resolved to 0.6%). |
| Indeterminate Rate | Rate of indeterminate results. | Not explicitly stated, but low rate desirable. | Fresh: Initial 0.1% (remained 0.1%). Frozen: Initial 0.9% (remained 0.9%). Total: Initial 0.2% (remained 0.2%). |
Study Details:
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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.
- Fresh Clinical Samples (Prospective): 1491 prospective, 32 select. Total 1523 enrolled, 1472 evaluable.
- 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).
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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.
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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.
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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.
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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.
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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.
- The ground truth for the clinical performance study was established by comparator methods, specifically:
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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.
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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.
§ 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).