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The DiaSorin Molecular Simplexa™ Bordetella Direct assay is an in vitro diagnostic test intended for use on the LIAISON® MDX instrument for the qualitative detection and differentiation of Bordetella pertussis and Bordetella parapertussis nucleic acids from nasopharyngeal swab (NPS) specimens from patients with signs and symptoms of Bordetella infection of the respiratory tract.

The Simplexa™ Bordetella Direct assay is performed on the LIAISON® MDX instrument and utilizes real-time PCR amplification to detect B. pertussis by targeting the IS481 insertional element of the B. pertussis genome and to detect B. parapertussis by targeting the IS1001 insertional element of the B. parapertussis genome. The IS481 insertional element can also be present in B. holmesii and B. bronchiseptica. Specimens collected from patients with respiratory infection caused by B. pertussis, B. holmesii or B. bronchiseptica may yield positive test results in IS481 assays. B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the Simplexa™ Bordetella Direct assay do not preclude Bordetella infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the Simplexa™ Bordetella Direct assay should be used with other clinical findings and epidemiological information as an aid in diagnosis of Bordetella infection. Test results should not be used as the sole basis for treatment or other patient management decisions.

Simplexa™ Bordetella Positive Control Pack

The Simplexa™ Bordetella Positive Control Pack is intended to be used as a control with the Simplexa™ Bordetella Direct kit.

This control is not intended for use with other assays or systems.

The Simplexa™ Bordetella Direct assay system is a real-time PCR assay that enables the direct amplification, detection and differentiation of Bordetella pertussis and Bordetella parapertussis DNA from unprocessed nasopharyngeal swabs (NPS) without nucleic acid extraction. The system consists of the Simplexa™ Bordetella Direct assay, the LIAISON® MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ Bordetella Direct assay, primers and fluorescent probes are used together to amplify and detect Bordetella pertussis, Bordetella parapertussis and internal control targets. Insertion sequences IS481 and IS1001 are targeted to identify Bordetella pertussis and Bordetella parapertussis DNA respectively in the specimen. An internal control is used to detect PCR failure and/or inhibition.

The provided text details the 510(k) summary for the Simplexa™ Bordetella Direct and Simplexa™ Bordetella Positive Control Pack, specifically focusing on the update to include fresh nasopharyngeal swab (NPS) samples. While the document mentions "acceptance criteria" and "design inputs" for the validation, it does not explicitly state the quantitative acceptance criteria for device performance (e.g., specific thresholds for PPA and NPA) in the provided sections. Instead, it presents the results of the clinical studies.

However, based on the provided data, we can infer that 100% PPA and a high NPA (above 95%) were considered acceptable, given the reported results.

Here's a breakdown of the available information:

1. Table of Acceptance Criteria and Reported Device Performance

As stated, the document does not explicitly list the acceptance criteria as specific numerical targets for PPA and NPA (e.g., "PPA must be ≥ 95%"). However, the reported performance is provided. We can infer that the observed performance was deemed acceptable by the FDA for the device's clearance.

Note: The confidence intervals are provided in the source document but are not included in this table for brevity.

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

Bordetella pertussis (Fresh Samples):

• Sample Size: 369 evaluable fresh samples.
• Data Provenance: Prospectively collected from five (5) geographically diverse sites in the US (inferred, as it's an FDA submission for a US company) from May 2018 to October 2018, from patients with signs and symptoms of Bordetella infection.

Bordetella parapertussis (Fresh Samples):

• Sample Size: 176 evaluable fresh samples (out of 178 collected).
• Data Provenance: Prospectively collected from six (6) geographically diverse sites in the US (inferred) between July 2017 and August 2017, from patients with signs and symptoms of Bordetella infections.

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

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

4. Adjudication Method for the Test Set

• For B. pertussis (Table 3): The reference method was an "FDA Cleared NAAT" (Nucleic Acid Amplification Test). The document does not describe the adjudication method if results between different reference tests varied or whether a composite ground truth derived from multiple tests was used, other than referencing a single FDA cleared NAAT.
• For B. parapertussis (Table 4): The reference method was a "composite reference method" consisting of "two well-characterized real-time PCR assays followed by confirmation of positive PCR amplification products with bidirectional sequencing, per target." Samples were characterized as positive if one or both composite reference methods were positive and confirmed by bi-directional sequencing. Samples were characterized as negative if both composite reference methods were negative. This describes an adjudication method that combines results from multiple tests.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done

No, a MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) test for qualitative detection of nucleic acids, not an imaging device requiring human reader interpretation. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable.

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

Yes, the performance presented is standalone algorithm performance. The Simplexa™ Bordetella Direct assay is an automated real-time PCR assay performed on the LIAISON® MDX instrument, which provides automated test interpretation and report generation. There is no human-in-the-loop performance described for the diagnostic decision-making process based on the assay's output.

7. The Type of Ground Truth Used

• For B. pertussis (Table 3): The ground truth was established by an "FDA Cleared reference method testing."
• For B. parapertussis (Table 4): The ground truth was established by a "composite reference method" combining two well-characterized real-time PCR assays and bidirectional sequencing confirmation. This is a form of expert-defined molecular diagnostic ground truth.

8. The Sample Size for the Training Set

The document does not provide information on a specific training set size. The studies described are for "Method Comparison" using prospectively collected samples, which typically serve as validation or test sets for device performance rather than training sets. For molecular diagnostic assays like this, the "training" usually involves optimizing primer/probe design and assay conditions, which isn't described in terms of a "training set" with ground truth in the same way an AI/ML model would be.

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

As no training set is described in the provided document, the method for establishing its ground truth is also not applicable/not provided.

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Simplexa™ Bordetella Direct MOL2750
The DiaSorin Molecular Simplexa™ Bordetella Direct MOL2750 assay is an in vitro diagnostic test intended for use on the LIAISON® MDX instrument for the qualitative detection and differentiation of Bordetella pertussis and Bordetella parapertussis nucleic acids from frozen nasopharyngeal (NPS) specimens from patients with signs and symptoms of Bordetella infection of the respiratory tract.
The Simplexa™ Bordetella Direct assay is performed on the LIAISON® MDX instrument and utilizes realtime PCR amplification to detect B. pertussis by targeting the IS481 insertional element of the B. pertussis genome and to detect B. parapertussis by targeting the IS1001 insertional element of the B. parapertussis genome. The IS481 insertional element can also be present in B. holmesii and B. bronchiseptica. Specimens collected from patients with respiratory infection caused by B. pertussis, B. holmesii or B. bronchiseptica may yield positive test results in IS481 assays. B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.
Negative results for the Simplexa™ Bordetella Direct assay do not preclude Bordetella infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the Simplexa™ Bordetella Direct assay should be used with other clinical findings and epidemiological information as an aid in diagnosis of Bordetella infection. Test results should not be used as the sole basis for treatment or other patient management decisions.
Simplexa™ Bordetella Positive Control Pack MOL 2760
The Simplexa™ Bordetella Positive Control Pack MOL2760 is intended to be used as a control with the Simplexa™ Bordetella Direct kit. This control is not intended for use with other assays or systems.

The Simplexa™ Bordetella Direct assay system is a real-time PCR assay that enables the direct amplification, detection and differentiation of Bordetella pertussis and Bordetella parapertussis DNA from unprocessed nasopharyngeal swabs (NPS) without nucleic acid extraction. The system consists of the Simplexa™ Bordetella Direct assay, the LIAISON® MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc and associated accessories.
In the Simplexa™ Bordetella Direct assay, primers and fluorescent probes are used together to amplify and detect Bordetella pertussis, Bordetella parapertussis and internal control targets. Insertion sequences IS481 and IS1001 are targeted to identify Bordetella pertussis and Bordetella parapertussis DNA respectively in the specimen. An internal control is used to detect PCR failure and/or inhibition.
The DiaSorin Molecular Simplexa™ Bordetella Direct kit contains sufficient reagents for 24 reactions. Upon receipt, store at -10 to -30ºC (do not use a frost-free freezer). Each vial contains sufficient material for a single reaction. Use within 30 minutes of thawing.

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

Acceptance Criteria and Reported Device Performance

Study Details from the Provided Text:

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

• Test Set Sample Size (Clinical Performance/Method Comparison):
  • 1113 evaluable prospectively collected frozen nasopharyngeal swab (NPS) samples.
  • An additional 112 samples: 56 contrived Bordetella parapertussis samples (at 2-50 X LoD) and 56 negative samples, randomized together.
• Data Provenance:
  • Country of Origin: Not explicitly stated, but samples were "prospectively collected and frozen from five (5) geographically diverse sites" (Page 7). This often implies within the USA for FDA submissions, but not definitively confirmed.
  • Retrospective or Prospective: Primarily prospective.
    • "One thousand one hundred and forty-two (1142) samples were prospectively collected and frozen..." (Page 7).
    • "The Bordetella pertussis prospectively banked frozen sample results are shown in Table 1." (Page 7).
    • "The Bordetella parapertussis prospectively banked frozen sample results are shown in Table 2 and the Bordetella parapertussis contrived sample results are shown in Table 3." (Page 7).

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

• The text does not specify the number or qualifications of experts directly establishing ground truth. Instead, it describes a "composite reference method."

4. Adjudication method for the test set:

• The ground truth was established using a "composite reference method [which] consisted of two well-characterized real-time PCR assays followed by confirmation of positive PCR amplification products with bi-directional sequencing, per target."
• "Samples were characterized as positive if one or both composite reference methods were positive and confirmed by bi-directional sequencing. Samples were characterized as negative if both composite reference methods were negative." (Page 7).
• This suggests a form of consensus/confirmation, but not a 2+1 or 3+1 expert adjudication in the typical human-in-the-loop sense for imaging. It's an algorithmic/laboratory "adjudication" against established molecular methods.

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:

• No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) PCR assay, not an AI-assisted imaging device. Its performance is evaluated directly against a reference method, not in comparison to human readers.

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

• Yes, the primary clinical performance ("Method Comparison") and all analytical studies (Reproducibility, LoD, Reactivity, Specificity, Interference, etc.) represent the standalone performance of the Simplexa™ Bordetella Direct assay. It's an automated molecular diagnostic assay, designed to operate without human interpretation of the primary signal for diagnosis.

7. The type of ground truth used:

• The ground truth was established using a composite reference method consisting of:
  • Two "well-characterized real-time PCR assays."
  • "Confirmation of positive PCR amplification products with bi-directional sequencing, per target." (Page 7).

8. The sample size for the training set:

• The document describes performance studies for a finalized device. It does not provide information about a separate "training set" in the context of machine learning, because this is a PCR assay with defined primers and probes, not a machine learning model that requires training data in that sense. The analytical and clinical studies serve to validate the assay's performance.

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

• Not applicable, as this is not a machine learning device with a distinct "training set" ground truth. The development of the assay (designing primers, probes, optimizing conditions) is an engineering process, not a data-driven model training process.

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The ARIES® Bordetella Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and identification of Bordetella pertussis (B. pertussis) and Bordetella parapertussis (B. parapertussis) nucleic acid in nasopharyngeal swab (NPS) specimens obtained from individuals suspected of having a respiratory tract infection attributable to B. pertussis or B. parapertussis.

The ARIES® Bordetella Assay targets the B. pertussis toxin promoter and the B. parapertussis IS1001 insertion element in the genomes. When clinical factors suggest that B. pertussis or B. parapertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the ARIES® Bordetella Assay do not preclude B. pertussis infection and positive results do not rule out co-infections with other respiratory pathogens. The direction and identification of B. pertussis and B. parapertussis nucleic acids from symptomatic patients aids in the diagnosis of B. perfussis and B. parapertussis respiratory infection with other clinical findings and epidemiological information.

The ARIES® Bordetella Assay is indicated for use with the ARIES® Systems.

The ARIES® Bordetella Assay is a polymerase chain reaction (PCR)-based qualitative in vitro diagnostic test system that consists of the ARIES® System or the ARIES® M1 System with their included ARIES® Software, an assay-specific cassette, and an assay-specific protocol file. The ARIES® Bordetella Assay cassette is a disposable, single-use cassette containing nucleic acid purification reagents, internal sample process control (SPC), and an assay-specific master mix capable of performing the designated assay on one sample. The ARIES® Bordetella Assay cassette directly detects and identifies B. pertussis and B. parapertussis DNA from nasopharyngeal swab (NPS) specimens collected from the human nasopharynx region.

Nasopharyngeal swab specimens are collected from patients using a commercially available E-Swab™ (Nylon® Flocked Swab along with modified Liquid Amies) or a commercially available nasopharyngeal swab (i.e. rayon, flocked, nylon, plastic shaft, etc.) placed into an approved transport media (i.e UTM, M5, M6, or equivalent). The specimen is then transported to the laboratory for testing. The specimen is lysed and nucleic acid is extracted using an ARIES® System. An extractable sample processing control (SPC) target is present in the ARIES® Bordetella Assay cassette and is processed with the specimen. The SPC controls for specimen lysis, for recovery of extracted nucleic acid, for inhibitory substances and for PCR reagent and instrument integrity. The Ct value of the SPC is designed to verify proper specimen lysis and nucleic acid extraction, to identify PCR inhibition, if any, and verify proper function of the extraction system and real-time instrument. The Tm value of the SPC is used as a reference for determining the target Tm.

The extracted nucleic acid and SPC are transferred via magnetic beads through the cassette to the ARIES® Bordetella Assay lyophilized PCR reagents in the PCR tube that contains primer pairs specific to the B. pertussis toxin promoter (ptxA-pr), the B. parapertussis IS1001 insertion element, and the SPC sequence. Each of the primer pairs are labeled with a distinct fluorophore and detected in distinct channels of the ARIES® Systems. PCR amplification is performed and assay fluorescence is monitored. Incorporation of a quencher-labeled nucleotide results in a decrease in fluorescence for the associated primer pair. Following amplification, the reaction is slowly heated to separate the fluorescent-labeled strand from the quencher-labeled strand, a process that results in an increase in the fluorescence signal. The reaction fluorescence is measured during this process and the temperature at which the change in fluorescence is the maximum Tm of the amplicon. The strands of the amplicons will separate at a specific melting temperature (Tm) and an increase in fluorescence is observed. The instrument fluorescence output is analyzed and test results are determined using the ARIES® System software and the ARIES® Bordetella Assay protocol and run files. ARIES® Bordetella Assay results may be reported from the ARIES® Software or from the optional SYNCT® Software.

Here's an analysis of the acceptance criteria and study detailed in the provided document, structured as requested:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria provided are predominantly for analytical performance (reproducibility) and general performance goals for clinical studies (e.g., 95% confidence intervals). Specific quantitative acceptance criteria for clinical performance (PPA, NPA) are not explicitly stated as distinct numerical targets in the text, but rather implied by the successful outcomes of the studies.

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

• Test Set (Clinical Performance Study):

  • Prospective Cohort: 1052 unique nasopharyngeal swab (NPS) specimens.
    • Positive B. pertussis: 32 specimens
    • Positive B. parapertussis: 2 specimens
    • Negative for both: 1018 specimens (calculated from B. pertussis NPA of 1009/1020 and B. parapertussis NPA of 1048/1050, roughly consistent with the total size)
  • Supplemental Cohort (Banked/Pre-selected):
    • 37 B. pertussis positive specimens
    • 20 B. parapertussis positive specimens
    • Paired with an equal number of unique negative clinical specimens (not explicitly stated for each organism, but implied for blinded testing).
  • Supplemental Cohort (Contrived):
    • 50 B. parapertussis contrived specimens
  • Data Provenance:
    • Country of Origin: United States. Specimens were collected from five (5) geographically distinct clinical sites within the United States.
    • Retrospective/Prospective: The study combines both prospective and pre-selected (banked) specimens.
      • "Leftover de-identified nasopharyngeal swab (NPS) specimens prospectively collected from pediatric and adult patients suspected of having respiratory tract infection attributable to B. pertussis or B. parapertussis."
      • "Due to the low prevalence B. pertussis and B. parapertussis observed in the prospective study, the clinical sample set was supplemented with banked (pre-selected) B. pertussis (N=37) and B. parapertussis (N=20) positive specimens as well as contrived B. parapertussis specimens (N=50)."

• Test Set (Analytical Studies):

  • Reproducibility (Site): 30 replicates per condition (low positive, moderate positive, negative) per site (3 sites), across 5 days (e.g., 30/30 positive reported for each condition per site, meaning 30 tests performed per condition). Total = 90 tests per condition for overall result.
  • Reproducibility (Lot-to-Lot): 15 replicates per condition (low positive, moderate positive, negative) per lot (3 lots). Total = 45 tests per condition for overall result.
  • Within-Laboratory Precision: 30 replicates per condition (low positive, moderate positive, negative) (30/30 positive/negative reported).
  • Limit of Detection: 20 replicates per strain/concentration per organism type (e.g., 19/20 or 20/20 positive reported).
  • Inclusivity: 3 replicates per strain per organism (3/3 positive/negative reported).
  • Interfering Substances: 3 replicates per Bordetella type (with interferant) and 3 replicates of negative matrix (with interferant) for each of 19 substances.
  • Cross-Reactivity (Exclusivity): 3 replicates for each of 71 unique microorganisms.
  • Microbial Interference/Co-Infection: 3 replicates per target strain + CRO combination, plus specific re-testing for certain CROs.

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

The ground truth for the clinical test set was established using a "composite comparator assay consisting of two well-characterized real-time PCR assays (for each bacterial pathogen) followed by confirmation of positive PCR amplification product with bi-directional sequencing."

The document does not explicitly state the "number of experts" or their "qualifications" involved in establishing this ground truth. It refers to "well-characterized real-time PCR assays" and "bi-directional sequencing" performed at a "centralized testing facility," implying laboratory professionals skilled in molecular diagnostics. It does not mention clinical experts (e.g., radiologists, pathologists) directly establishing the ground truth for this in vitro diagnostic device.

4. Adjudication Method for the Test Set

The adjudication method for determining the composite comparator ground truth was:

• Positive: If one out of two comparator PCR assays was positive (Ct values ≤40) and confirmed by bi-directional sequencing, OR if both comparator PCR assays were positive.
• Negative: If one out of two comparator PCR assays was negative (Ct values >40) and confirmed by bi-directional sequencing, OR if both comparator PCR assays were negative.

This is a form of multimethod consensus rather than human expert adjudication.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) test, specifically a real-time PCR assay. It does not involve human "readers" interpreting images or clinical data in the way AI in diagnostics typically does. Therefore, the concept of human readers improving with or without AI assistance is not applicable to this type of device.

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

Yes, the studies presented here are primarily standalone (algorithm only) performance studies. The ARIES® Bordetella Assay is an automated PCR-based system. Its "performance" refers to its analytical and clinical accuracy in detecting specific nucleic acid sequences directly, interpreted by the ARIES® System software. There is no human-in-the-loop performance component in the sense of a diagnostic interpretation loop that the device assists. Operators load samples, but the detection and interpretation are automated by the instrument and its software, making it a standalone device performance evaluation.

7. The Type of Ground Truth Used

The ground truth used for the clinical performance study was a composite comparator method. This method consisted of:

• Two independent, well-characterized real-time PCR assays (for each bacterial pathogen).
• Followed by confirmation of positive PCR amplification product with bi-directional sequencing.

In essence, it's a molecular gold standard combining highly sensitive and specific laboratory techniques.

8. The Sample Size for the Training Set

The document does not explicitly state a separate "training set" sample size for the ARIES® Bordetella Assay. For IVD submissions like this, the "training" aspect often refers to internal optimization and verification studies that determine assay parameters (like Ct cut-offs) before formal performance evaluation. The text mentions:

• "Initial Assay Protocol File parameters were set during internal optimization studies"
• "The final Assay Protocol File parameters were then established during internal verification studies"

These internal studies likely used various samples for development and refinement, but a distinct, quantified "training set" in the context of machine learning model development is not detailed or specified. The clinical trial data (prospective, pre-selected, contrived) is presented primarily as a test set for validation.

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

Given that a specific, quantified "training set" is not detailed in the document in the context of a machine learning paradigm, the method for establishing its ground truth is also not explicitly described. However, if "training set" conceptually refers to the samples used during "internal optimization studies" and "internal verification studies" mentioned in section M.1.g, it can be inferred that these would have used:

• Well-characterized bacterial strains: Diluted into appropriate matrices (like native nasopharyngeal matrix) at known concentrations.
• Known negative samples: Such as culture media or confirmed pathogen-free clinical matrices.
• The ground truth for these samples would be based on known organism identity and concentration (e.g., quantified using standard culture techniques like CFU/mL, verified by plating and colony counting). This is how analytical studies (like LoD, inclusivity) established their ground truth.
• For any clinical samples used in early internal verification, the ground truth would likely have been established using similar reference methods (e.g., culture, reference PCR, sequencing) as those later used for the clinical test set.

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The Great Basin Bordetella Direct Test is a qualitative in vitro diagnostic test for the detection of Bordetella pertussis DNA from nasopharyngeal swab specimens obtained from patients suspected of having a respiratory tract infection attributable to B. pertussis.

The Bordetella Direct Test is performed on the PA500 Portrait Analyzer and utilizes PCR amplification of the insertion sequence IS481. The IS481 sequence is also found in other organisms including Bordetella holmesii or Bordetella bronchiseptica. Respiratory infection with B. pertussis, B. holmesii ot B. bronchiseptica may yield positive test results with IS481 assays. B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the Great Basin Bordetella Direct Test do not preclude B. pertussis infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the Great Basin Bordetella Direct Test should be used in conjunction with information obtained during the patient's clinical evaluation as an aid in diagnosis of Bordetella pertussis infection and should not be used as the sole basis for treatment or other patient management decisions.

The Great Basin Bordetella Direct Test on the PA500 Portrait™ Analyzer System utilizes automated hot-start PCR technology to target and amplify the IS481 insertion sequence of B. pertussis. Genomic DNA is extracted from microbial cells and diluted to reduce potential inhibitors of PCR. During PCR, double-stranded DNA is separated and the target nucleic acid sequence is amplified by thermal cycling using biotin-labeled primers that target the IS481 sequence for identification of B. pertussis. Following PCR, biotin-labeled amplicon is hybridized to sequence-specific capture probes immobilized on the silicon chip surface, then incubated with anti-biotin antibody conjugated to the horseradish peroxidase enzyme (HRP). The unbound conjugate is washed away and tetramethylbenzidine (TMB) is added to produce a visible 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 Specimen Processing Control (SPC), undergoes the extraction, and detection steps to monitor for inhibitory substances as well as process inefficiency due to instrument or reagent failure. No operator intervention is necessary once the clinical sample is loaded into the sample port and the Bordetella Direct Test cartridge is loaded into the Portrait Analyzer.

The PA500 Portrait™ Analyzer System is a fully automated system that includes: The Portrait™ Analyzer, single-use Bordetella Direct Test 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 documentation describes acceptance criteria and the study that proves the Great Basin Bordetella Direct Test meets these criteria. The device is a qualitative in vitro diagnostic test for the detection of Bordetella pertussis DNA from nasopharyngeal swab specimens.

Here's the detailed breakdown of the acceptance criteria and study information:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the Great Basin Bordetella Direct Test are implicitly defined by the performance observed in the analytical and clinical studies, which are designed to demonstrate the device's acceptable functioning for its intended use. While explicit numerical acceptance criteria (e.g., "PPA must be >X%") are not directly stated as pass/fail thresholds in the provided text, the reported performance metrics are presented as the results that demonstrate substantial equivalence.

• ATCC 8467: 3.3 x 10^3 CFU/mL (20/20 detected)
• ATCC 9797: 1.6 x 10^3 CFU/mL (20/20 detected)
• ATCC BAA-589: 2.3 x 10^3 CFU/mL (19/20 detected) |
  | Analytical Reactivity (Inclusivity) | Correct detection of additional B. pertussis strains. | All 8 additional B. pertussis strains tested were correctly detected (3/3 positive for each strain). |
  | Analytical Specificity (Exclusivity) | No cross-reactivity with non-target organisms commonly found in the human respiratory system, except for known IS481-positive Bordetella species, which should be noted as limitations. | Most non-target organisms (48 bacteria, 20 viruses, 2 yeast, 1 human genomic DNA) showed no cross-reactivity (100% expected negative results for all tested).
  Observed cross-reactivity:
• Bordetella bronchiseptica: 1 of 3 strains showed cross-reactivity (ATCC 4617).
• Bordetella holmesii: All 3 strains showed cross-reactivity.
• Bordetella hinzii: 1 of 2 strains showed cross-reactivity (ATCC 51784).
  These noted cross-reactivities for Bordetella species are consistent with the presence of the IS481 insertion sequence also detected by the test. |
  | Microbial Interference | No interference (i.e., correct detection of B. pertussis) when B. pertussis is present with high concentrations of other organisms. | 83 of 84 tested organisms (48 bacteria, 19 viruses, 2 yeast, 14 Bordetella non-B. pertussis strains, 1 human genomic DNA) showed no interference (100% expected positive results). One exception: 1 replicate for M. tuberculosis initially reported 'NOT DETECTED', but subsequent retesting confirmed no interference. |
  | Interfering Substances | No interference with common chemical substances found in patients with upper respiratory conditions. | All 19 tested chemical substances showed no interference (100% expected positive and negative results). |
  | Carry-over/Cross-Contamination | No false positives in negative samples following high positive samples. | No carry-over or cross-contamination observed (100% correct results for alternating high positive and negative samples). |
  | Swab, Transport Media, Elution Buffer Equivalency | Device compatibility with various swab types, transport media, and elution buffers. | All tested swab types (Flocked Nylon, Polyester, Rayon), transport media (Remel M4/M4RT/M5/M6 VTM, BD Universal VTM, ESwab), and elution buffers (Molecular grade water, PBS, Saline, TE Buffer) showed no interference (3/3 expected results for each).
  Equivalence study confirmed 100% agreement when using different combinations of swab types and transport/elution media. |
  | Sample Stability and Storage | Specimen stability for specified storage conditions (room temperature and refrigerated). | 100% agreement with expected results for samples stored up to 72 hours at room temperature and up to 168 hours (7 days) at 2-8°C, supporting specimen storage claims of 48 hours at room temperature or ≤120 hours at 2-8°C. |
  | Reproducibility | Consistent results across different sites, operators, and reagent lots. | 100% agreement observed across replicates, runs, operators (6), and sites (3 clinical sites) for low positive, moderate positive, and negative samples. |
  | Clinical Studies | | |
  | Positive Percent Agreement (PPA) | High agreement with the Reference NAAT for positive samples, demonstrating clinical sensitivity. (Implicitly, the confidence interval should be acceptable, likely meeting a lower bound, although not explicitly stated). | Prospective Study (Fresh): 85.7% (95% CI: 65.4% - 95.0%) (18/21 true positives)
  Frozen Retrospective Study: 94.6% (95% CI: 86.1% - 98.3%) (56/59 true positives) |
  | Negative Percent Agreement (NPA) | High agreement with the Reference NAAT for negative samples, demonstrating clinical specificity. (Implicitly, the confidence interval should be acceptable, likely meeting a lower bound, although not explicitly stated). | Prospective Study (Fresh): 99.6% (95% CI: 98.9% - 99.8%) (890/894 true negatives)
  Frozen Retrospective Study: 100.0% (95% CI: 94.3% - 100.0%) (63/63 true negatives) |

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

• Prospective Study (Fresh Samples):
  • Sample Size: 915 samples (936 collected, 21 excluded due to improper storage or failed QC).
  • Data Provenance: U.S. clinical study sites (four external, geographically-diverse sites: Northwest, Southwest, Midwest, West). Samples collected prospectively from July 2016 to January 2017. These were excess, de-identified nasopharyngeal (NP) swab specimens submitted for standard of care B. pertussis testing.
• Frozen Retrospective Study:
  • Sample Size: 122 samples (124 initially, 2 excluded due to failed QC).
  • Data Provenance: Archived frozen NP swab specimens from institutions across the U.S. (implied from the context of clinical studies and specimen collection methods), retrospective in nature. These were de-identified specimens previously characterized by a nucleic acid amplification test at their originating institution.

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

This document describes a diagnostic test for Bordetella pertussis using molecular methods (NAAT). The "ground truth" for the clinical test sets (both prospective and retrospective) was established by comparison to an FDA-cleared Nucleic Acid Amplification Test (Reference NAAT).

• No human experts (e.g., radiologists) were used to establish the ground truth for classification of positive/negative B. pertussis status in the clinical studies.
• The ground truth relies on the performance of the established Reference NAAT.

4. Adjudication Method for the Test Set

• For the clinical performance studies, discrepant results between the Great Basin Bordetella Direct Test and the initial Reference NAAT were investigated by testing in a second FDA-cleared NAAT (NAAT 2) which also detects Bordetella pertussis. This acts as an adjudication step.
• The document states: "In total, there were six (6) false negative and four (4) false positive results. Two (2) of the six (6) false negatives were also negative by a second FDA cleared NAAT and two (2) of the four (4) false positives were also positive by the second FDA cleared NAAT." This indicates the NAAT 2 was used to help understand the nature of the discrepancies, but it does not specify if the NAAT 2 result was definitively taken as the final "truth" for the agreement calculations in all cases. Typically, in diagnostic studies, a third, independent method or a consensus of multiple reference methods is used for resolving discrepancies, but the exact arbitration rule (e.g., 2-out-of-3, or specific tie-breaking rules) is not detailed for the final truth determination for PPA/NPA beyond the investigation by NAAT 2.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done

• No, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) test, specifically a nucleic acid amplification test (NAAT). The performance is measured by its ability to detect specific DNA directly from a patient sample, not by a human interpreting images or data to make a diagnosis. Therefore, comparative effectiveness with human readers (e.g., radiologists, pathologists) is not relevant for this type of device. The studies focused on its analytical performance and its agreement with a reference molecular diagnostic test.

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

• Yes, the primary performance evaluation of the Great Basin Bordetella Direct Test is effectively "standalone" performance. This means the device itself, the automated PA500 Portrait Analyzer and its associated assay, is evaluated for its ability to produce positive or negative results. There is no human interpretation or intervention in the diagnostic output generation described beyond loading the sample into the automated system. The results are generated by the instrument's automated software.

7. The Type of Ground Truth Used

• For the clinical studies, the ground truth for classifying samples as positive or negative for B. pertussis was established by a Reference FDA-cleared Nucleic Acid Amplification Test (NAAT). Discrepancies were investigated by a second FDA-cleared NAAT.
• For the analytical studies (LoD, inclusivity, exclusivity, interference), the ground truth was established by known concentrations of cultured bacterial strains (CFU/mL) or genomic copies/TCID50 for viruses and other microbes, which were spiked into negative clinical matrix.

8. The Sample Size for the Training Set

• The provided document is a 510(k) premarket notification summary for a test that is likely based on traditional molecular biology techniques (PCR) rather than a machine learning/AI algorithm that requires a separate training set.
• Therefore, the concept of a "training set" in the context of machine learning, where an algorithm learns patterns from data, does not directly apply to the development and validation of this specific in vitro diagnostic device. Its "training" is more akin to traditional assay development and optimization to ensure primers, probes, and reaction conditions are specific and sensitive.
• The document does not mention a machine learning component or a distinct "training set" data size.

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

• As explained in point 8, the concept of a "training set" for a machine learning algorithm is not applicable here. The device's underlying technology is PCR amplification, which is based on established biological and chemical principles rather than machine learning from a specific training dataset. Development and optimization of such assays involve
  • Designing and testing primers/probes: Based on known genetic sequences of B. pertussis.
  • Optimizing reaction conditions: To achieve desired sensitivity and specificity using known positive and negative controls/samples.
  • Analytical validation: As described in the document (LoD, inclusivity, exclusivity) using characterized bacterial strains and other microbes.
• The "ground truth" during the development and optimization phases would have been established by precisely characterized biological materials, such as pure cultures of B. pertussis at known concentrations, or panels of well-characterized positive and negative patient samples.

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The illumigene Pertussis DNA Amplification Assay, performed on the illumipro-10™, is a qualitative in vitro diagnostic test for the direct detection of Bordetella pertussis in human nasopharyngeal swab samples taken from patients suspected of having respiratory tract infection attributable to Bordetella pertussis.

The illumigene Pertussis DNA Amplification Assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to pertussis by targeting the IS481 insertional element of the B. pertussis genome. The 18481 insertional element can also be found in B. holmesii and some B. bronchiseptica strains. Respiratory infections with B. pertussis, B. holmesii or B. bronchiseptica may yield positive test results infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the illumigene Pertussis DNA Amplification Assay do not precude Bordetella pertussis infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the illumigene Pertussis assay should be used in conjunction with information obtained during the patient's clinical evaluation as an aid in diagnosis of B. pertussis infection and should not be used as the sole basis for treatment or other patient management decisions.

illumigene Pertussis DNA Amplification Assay is intended for use in hospital, reference or state laboratory settings. The device is point-of-care use.

The illumigene Pertussis DNA Amplification Assay, performed on the illumipro-10™, is a qualitative in vitro diagnostic test for the direct detection of Bordetella pertussis in human nasopharyngeal swab samples. The assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to target the IS481 insertional element of the B. pertussis genome.

Here's an analysis of the provided text regarding the illumigene Pertussis DNA Amplification Assay, structured to answer your questions about acceptance criteria and the supporting study:

The provided document is an FDA 510(k) clearance letter for the illumigene Pertussis DNA Amplification Assay. It primarily outlines the device's intended use and FDA's determination of substantial equivalence. Crucially, this document does NOT contain detailed information about the acceptance criteria or the specific study results proving the device meets those criteria.

Generally, for an in vitro diagnostic (IVD) like this, acceptance criteria would involve performance metrics such as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) compared to a reference standard. The study data would then quantify these metrics.

Since the provided text does not contain this information, I will indicate where the information is not provided by the document.

Acceptance Criteria and Study Details for illumigene Pertussis DNA Amplification Assay

1. Table of Acceptance Criteria and Reported Device Performance

2. Sample size used for the test set and the data provenance

• Sample Size for Test Set: Not provided in this document.
• Data Provenance (e.g., country of origin of the data, retrospective or prospective): Not provided in this document. Typically, clinical studies for IVDs involve prospective collection of samples from diverse geographic locations to ensure generalizability.

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

This information is typically relevant for image-based diagnostic devices or those requiring expert interpretation. For a DNA amplification assay, the "ground truth" for the test set is established by a reference method (e.g., culture, another highly sensitive PCR method with sequencing confirmation). Therefore, the concept of "experts establishing ground truth" in the same way it would apply to imaging interpretation is not directly applicable here.

• Number of experts: Not applicable in the context of this type of diagnostic test's ground truth.
• Qualifications of experts: Not applicable.

4. Adjudication method for the test set

As the ground truth for a DNA amplification assay is typically established through laboratory reference methods (e.g., bacterial culture, validated PCR with sequencing), an "adjudication method" involving human readers (like 2+1, 3+1 for imaging) is not applicable. Discrepancies would be resolved by retesting or using a higher-level confirmatory test.

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

• MRMC Study: No, an MRMC study is not applicable for a standalone in vitro diagnostic (IVD) test like a DNA amplification assay. This type of study is designed for diagnostic tools that involve human interpretation, such as medical imaging analysis, often comparing AI-assisted performance against unassisted human performance. The illumigene Pertussis DNA Amplification Assay is a laboratory test that provides a quantitative or qualitative result directly, without requiring human "reading" in the same diagnostic sense.

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

Yes, for an IVD like the illumigene Pertussis DNA Amplification Assay, the performance data presented to the FDA always represents the standalone performance of the device (i.e., "algorithm only" in the context of an automated assay). The device generates a result which is then interpreted by a healthcare professional in conjunction with clinical information, but the core performance characteristics (sensitivity, specificity) are intrinsic to the assay itself.

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

For molecular diagnostic assays like the illumigene Pertussis DNA Amplification Assay, the ground truth is typically established using:

• Reference Culture: For bacterial infections, culture is often considered the gold standard, although its sensitivity for Bordetella pertussis can be low, especially in later stages of infection.
• Another highly sensitive and specific molecular method (e.g., a validated laboratory-developed PCR test or a previously cleared FDA PCR test), often with Sanger sequencing confirmation: This is frequently used as a composite reference standard when culture sensitivity is a concern.

The specific ground truth method used for this device is not provided in this document.

8. The sample size for the training set

• Training Set Sample Size: Not provided in this document. For molecular diagnostic assays, there isn't a "training set" in the machine learning sense. Instead, there are often development and validation phases where different sets of samples (sometimes called analytical and clinical validation sets) are used to establish performance. The term "training set" is more applicable to AI/ML devices.

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

As mentioned in point 8, the concept of a "training set" with ground truth in the AI/ML context doesn't directly apply here. For the various validation studies (analytical and clinical), ground truth would be established by the reference methods described in point 7. The specific methodology for establishing ground truth for any development or validation samples is not provided in this document.

In summary: The provided FDA 510(k) clearance document confirms the device's legal market entry based on substantial equivalence for its stated Indications for Use. However, it does not detail the specific performance acceptance criteria or the comprehensive study design and results (e.g., sample sizes, ground truth methodology, actual sensitivity/specificity values) that were submitted to justify that equivalence. Such detailed information would typically be found in the full 510(k) summary or the device's instructions for use.

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The AmpliVue® Bordetella Assay is an in vitro diagnostic test for the qualitative detection of Bordetella pertussis nucleic acids isolated from nasopharyngeal swab specimens suspected of having respiratory tract infection attributable to Bordetella pertussis.

The AmpliVue® Bordetella Assay utilizes helicase-dependent amplification (HDA) of the insertion sequence IS481 and a selfcontained disposable amplification device that allows for manual evaluation of assay results. The IS48 sequence can also be found in strains of other organisms (i.e., B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the AmpliVue® Bordetella Assay do not prection and positive results do not rule out coinfection with other respiratory pathogens. Results from the AmpliVue® Bordetella Assay should be used in conjunction with information obtained during the patient's clinical evaluation as an aid in diagnosis infection and should not be used as the sole basis for treatment or other patient management decisions.

The AmpliVue® Bordetella Assay is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

The AmpliVue® Bordetella Assay combines simple processing, an isothermal amplification technology named helicase-dependent amplification (HDA), and a selfcontained disposable amplicon detection device, for the detection of Bordetella pertussis from nasopharyngeal swabs.

Patient samples are collected using a nasopharyngeal swab and placed into a liguid medium. Fifty microliters (50 µL) of the sample are then transferred to a process buffer that is provided with the kit and mixed. The Process Buffer tubes are heated at 95 °C for 10 minutes. Fifty microliters (50 µL) of the Process Buffer containing sample is added to a reaction tube containing lyophilized mix of HDA reagents. Included in the reaction mix are the isothermal polymerase, helicase and single stranded binding protein. After completion of the HDA reaction the reaction tube is transferred to the amplicon cartridge containing the running buffer. The amplicon cartridge is closed and inserted into the detection chamber. The detection chamber is activated by depressing the detection chamber handle. Upon activation, the reservoir containing the running buffer and the 0.2 mL tube containing the amplicon is punctured and the solutions are wicked to the lateral flow strip.

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Device Name: AmpliVue® Bordetella Assay

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the reported performance metrics of the clinical study and the analytical performance studies. The clinical study's primary metrics are Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to a Composite Reference Method. The analytical studies establish limits of detection, inclusivity, and specificity.

2. Sample size used for the test set and the data provenance

• Sample Size (Clinical): 842 fresh nasopharyngeal swab specimens were initially collected. After removing 6 invalid specimens, 836 specimens were included in the final analysis.
• Data Provenance: The specimens were collected from patients in the United States (at four locations). The study was prospective, collecting samples in Spring to Summer 2014 (April to August 2014) from patients suspected of having respiratory tract infection attributable to Bordetella pertussis.

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

The ground truth for the test set was established using a Composite Reference Method (CRM) involving molecular assays rather than human experts reviewing images or clinical data in an adjudicated manner.

• The CRM included two manufacturer-validated, IS481-targeted real-time PCR assays (PCR1 and PCR2).
• This was followed by bi-directional sequencing from PCR-positive specimens.

Therefore, the concept of "number of experts" and "qualifications of those experts" for establishing ground truth, as typically understood in fields like radiology or pathology, does not directly apply here. The ground truth was based on a laboratory-based molecular diagnostic gold standard.

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

The adjudication method was based on a Composite Reference Method (CRM):

• Specimens were considered positive when bi-directional sequence sequencing results from either comparator PCR assay confirmed the presence of Bordetella pertussis amplicon.
• Specimens were considered negative when neither comparator PCR assay produced Bordetella pertussis amplicon at the end of the 37 cycles.

This is a defined algorithmic (molecular) adjudication process rather than human expert consensus.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is an in vitro diagnostic assay for the qualitative detection of nucleic acids, not an imaging device or AI-assisted diagnostic tool that would involve human readers interpreting results.

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

The clinical study evaluated the standalone performance of the AmpliVue Bordetella Assay. The device itself performs the detection and provides a result (visible lines in the cassette window indicating detection or no detection). While human observation is required to read the lines, the assay itself is an "algorithm only" in the sense that its output is directly analogous to a positive/negative result from a fully automated system. The performance metrics (PPA, NPA) reflect this standalone performance against the Composite Reference Method.

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

The ground truth used was a Composite Reference Method (CRM) consisting of:

• Two manufacturer-validated, IS481-targeted real-time PCR assays.
• Bi-directional sequencing for confirmation of PCR-positive specimens.

This is a highly sensitive and specific molecular-based ground truth, often considered a "gold standard" for nucleic acid detection.

8. The sample size for the training set

The document does not report a sample size for a training set. This device uses Helicase-Dependent Amplification (HDA) with a lateral flow strip for detection. It is a molecular diagnostic assay that does not typically involve machine learning with distinct training and test sets in the same way an AI/CADe device would. The development of the assay (primers, probes, reaction conditions) would involve optimization, but this is not typically referred to as a "training set" in this context.

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

As there is no explicit training set discussed in the context of machine learning, the question of how its ground truth was established is not applicable. The underlying components of the assay (primers, probes) are designed based on known biological sequences and Bordetella pertussis characteristics.

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The illumigene® Pertussis DNA Amplification Assay, performed on the illumipro-10™, is a qualitative in vitro diagnostic test for the direct detection of Bordetella pertussis in human nasopharyngeal swab samples taken from patients suspected of having respiratory tract infection attributable to Bordetella pertussis.

The illumigene Pertussis assay utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect Bordetella pertussis by targeting the IS481 insertional element of the Bordetella pertussis genome. The IS481 insertional element can also be found in Bordetella holmesii and Bordetella bronchiseptica strains. Respiratory infection with Bordetella pertussis, Bordetella bronchiseptica may yield positive test results in IS481 assays. B. holmesii infection may cause clinical illness similar to B. pertussis, and mixed outbreaks involving both B. pertussis and B. holmesii infection have been reported. Additional testing should be performed if necessary to differentiate B. holmesii and B. pertussis. B. bronchiseptica is a rare cause of infection in humans. When clinical factors suggest that B. pertussis may not be the cause of respiratory infection, other clinically appropriate investigation(s) should be carried out in accordance with published guidelines.

Negative results for the illumigene Pertussis DNA Amplification Assay do not preclude Bordetella pertussis infection and positive results do not rule out co-infection with other respiratory pathogens. Results from the illumigene Pertussis assay should be used in conjunction obtained during the patient's clinical evaluation as an aid in diagnosis of Bordetella pertussis infection and should not be used as for treatment or other patient management decisions.

illumigene Pertussis is intended for use in hospital, reference or state laboratory settings. The device is not intended for point-of-care use.

The illumigene Molecular Diagnostic Test System is comprised of the illumigene® Pertussis DNA Amplification Assay Test Kit, the illumigene® Pertussis External Control Kit and the illumipro-10™ Automated Isothermal Amplification and Detection System.

The illumigene Pertussis assay utilizes loop-mediated isothermal amplification (LAMP) technology to detect the presence of Bordetella pertussis in human nasopharyngeal swab specimens. Each illumigene Pertussis assay is completed using an illumigene Assay Control Reagent containing Control material, an illumigene Sample Buffer, an illumigene Pertussis Test Device and Mineral Oil. Nasopharyngeal swab specimens are eluted with illumigene Sample Buffer. The illumigene Assay Control Reagent is added to the eluted sample and heat-treated. Target and Control DNA are made available for isothermal amplification via heat-treatment. The heat-treated Specimen/Control sample is added to the illumigene Test Device. Mineral oil is added to the illumigene Test Device to prevent evaporation. DNA amplification occurs in the illumigene Test Device.

The illumipro-10 heats each illumigene Pertussis Test Device containing prepared Sample and Control material, facilitating amplification of target DNA. When B. pertussis is present in the specimen, a 198 base pair sequence located within the IS481 insertional element of the B. pertussis genome is amplified and magnesium pyrophosphate is generated. Magnesium pyrophosphate forms a precipitate in the reaction mixture.

The illumipro-10 monitors the absorbance characteristics of the reaction solutions at the assay Run Start (Signal Initial or Si) and at the assay Run End (Signal final or Sf). The illumipro-10 calculates the ratio of the Run End (Signal final or Sf) reads with the Run Start (Signal Initial or Si) reads and compares the ratio to an established cut-off value. The illumipro-10 performs this ratio calculation to both the TEST chamber and the CONTROL chamber.

Fixed cut-off values for the CONTROL chamber are used to determine validity. CONTROL chamber Sf:Si ratios less than 90% are considered valid and allow for reporting of TEST chamber results (POSITIVE, NEGATIVE). CONTROL chamber Sf:Si ratios greater than or equal to 90% are considered invalid. Results are reported as 'INVALID'; Test chamber results are not reported. More stringent cut-off criteria are applied to the Control chamber reaction to ensure amplification is not inhibited, reagents are performing as intended and that sample processing was performed appropriately.

Fixed cut-off values for the TEST chamber are used to report sample results. TEST chamber Sf:Si ratios less than 82% are reported as 'POSITIVE'; TEST chamber Sf:Si ratios greater than or equal to 82% are reported as 'NEGATIVE'. Numerical values are not reported.

The illumigene Pertussis External Control Kit contains a Positive Control Reagent. The illumigene Assay Control/Negative Control Reagent provided in the illumigene Pertussis kit serves as the External Negative Control Reagent. External Control reagents are provided to aid the user in detection of reagent deterioration, adverse environmental or test conditions, or variance in operator performance that may lead to test errors. External Control reagents are provided for use in routine Quality Control testing.

illumigene® Pertussis DNA Amplification Assay - Acceptance Criteria and Study Summary

Here's an analysis of the illumigene® Pertussis DNA Amplification Assay's acceptance criteria and the study data presented:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state formal "acceptance criteria" with numerical thresholds for Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA). However, the device's performance is compared against a Composite Comparator Reference Method. Based on the clinical study results, the reported performance is:

Note: The document presents separate performance for prospective, retrospective (all comers), and retrospective (selected) samples. The "Overall" values for PPA and NPA (based on "Composite Method Comparator, All Comers" Total) are used here as the primary performance metrics from the clinical study.

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

The test set refers to the clinical study samples used for performance evaluation.

• Sample Size: A total of 729 qualified nasopharyngeal (NP) swab specimens.

  • Prospective: 508 specimens (69.7%)
  • Retrospective: 221 specimens (30.3%) (includes "all comers" and "selected" retrospective samples)

• Data Provenance:

  • Country of Origin: The samples were collected from four independent clinical test sites located in the Midwestern, Northern, and Eastern regions of the United States.
  • Retrospective or Prospective: Both retrospective and prospective specimens were included in the clinical study.

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

The document does not specify a number of "experts" in the traditional sense (e.g., radiologists) for establishing ground truth. Instead, the ground truth was established using a Composite Comparator Reference Method:

• This method involved two manufacturer-validated, real-time PCR Assays (PCR1 and PCR2).
• These were followed by bi-directional sequencing.
• Qualifications (Implied): The PCR assays were "manufacturer validated," implying they were developed and verified by experts in molecular diagnostics. Bi-directional sequencing is a standard molecular biology technique, performed and interpreted by trained personnel.

4. Adjudication Method for the Test Set

The adjudication method for the ground truth was as follows:

• Sequential Adjudication (Implicit):
  • Initial screening by two manufacturer-validated, real-time PCR Assays (PCR1 and PCR2), both targeting unique sequences within the IS481 insertional element.
  • Specimens producing positive Bordetella pertussis results from either comparator PCR assay were then sent for bi-directional sequencing.
  • Final Ground Truth Definition: Only samples that matched sequences within the Bordetella pertussis genome with pre-defined quality scores (PHRED20 and E-values) were considered true positives. Samples were considered negative when neither comparator PCR assay returned positive Bordetella pertussis results.

This can be seen as a form of "consensus with confirmation" or a multi-step adjudication process.

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

No. A Multi-Reader Multi-Case (MRMC) comparative effectiveness study, as typically understood for evaluating human reader performance with and without AI assistance, was not performed. This study evaluated the automated device's performance against a reference method, not against human readers.

6. Standalone Performance

Yes. The study primarily evaluated the standalone performance of the illumigene® Pertussis DNA Amplification Assay (algorithm only, with instrumentation for detection) without human-in-the-loop adjustments, beyond the initial sample preparation and loading. The results reported (PPA, NPA) directly reflect the device's accuracy in identifying Bordetella pertussis from nasopharyngeal swab samples.

7. Type of Ground Truth Used

The type of ground truth used was expert consensus / reference method based on molecular testing. Specifically:

• A Composite Comparator Reference Method was used.
• This involved two manufacturer-validated, real-time PCR Assays (PCR1 and PCR2).
• Confirmation of positive results was done via bi-directional sequencing with pre-defined quality scores.

This is a robust method leveraging highly sensitive and specific molecular techniques, which is considered a strong proxy for definitive diagnosis in this context.

8. Sample Size for the Training Set

The document does not specify a sample size for a "training set." This type of diagnostic assay, particularly for molecular tests, often relies on wet-lab optimization and development using characterized samples, rather than a distinct "training set" in the machine learning sense. The "Development optimization" section mentions "evaluation of characterized positive and negative clinical specimens" but without quantifying these for a training set.

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

As no specific "training set" is described, the method for establishing its ground truth is also not explicitly stated. However, the "Assay cut-off" section mentions:

• "Development optimization includes evaluation of characterized positive and negative clinical specimens."
• "Amplification reagent concentrations are adjusted during design as needed to ensure illumigene results are aligned with clinical specimen reported results."

This implies that the assay's parameters (like cut-off values) were refined using characterized clinical specimens, presumably with their Bordetella pertussis status determined by established laboratory methods, potentially similar to or derivative of the Composite Comparator Reference Method used for the main clinical study, or other well-accepted diagnostic techniques.

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