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The B. anthracis Real-Time PCR Assay is an in vitro diagnostic test for the qualitative detection of plasmid and chromosomal DNA sequences from B. anthracis. The assay can be used to test human respiratory samples, whole blood, serum, plasma, swabs from lesions, cerebrospinal fluid, and bacterial culture isolates from individuals suspected of having anthrax.

Results generated from direct specimentesting are presumptive for the identification of B. anthracis. Results generated from culture isolate testing should be used in conventional methods for identification of Bacillus anthracis isolates as part of the LRN Bacillus anthracis Testing Algorithm. The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidences, in addition to the identification of B. anthracis from cultures or detection directly in clinical specimens.

Use is limited to Laboratory Response Network (LRN) designated laboratories.

The B. anthracis Real-time PCR Assay is also intended for environmental specimen testing for biothreat detection and response. FDA has not evaluated claims related to the use of this assay on environmental specimens.

The B. anthracis Real-time PCR Assay uses a fluorogenic probe, consisting of an oligonucleotide with a reporter dve (FAM) attached to the 5' end and a quencher dye (BHQ1) attached at or near the 3′ end. The probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5′ nuclease activity of Taq polymerase degrades the probe causing the reporter dye to separate from the quencher dye and a fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes and the fluorescence intensity is monitored during the PCR. The Tag polymerase used in this assay is inactive at room temperature. It must be activated by incubation at 95°C, which also minimizes the production of nonspecific amplification products.

Each extracted DNA sample is tested with three B. anthracis primer and probe sets run as individual reactions. The primer and probe sets target genes encoding virulence factors as well as conserved regions of DNA from the B. anthracis chromosome. All primer and probe sets must be positive for the overall result of the B. anthracis Real-time PCR Assay to be interpreted as positive. Any result that is positive for some, but not all target regions, is still considered equivocal and follow-up laboratory investigation should be performed per the LRN Bacillus anthracis testing algorithm.

The provided document is a 510(k) premarket notification for the B. anthracis Real-time PCR Assay. It indicates substantial equivalence to a predicate device and provides information on its intended use, device description, and a comparison with the predicate. However, it explicitly directs inquiries regarding performance characteristics (including analytical limit of detection, analytical sensitivity and specificity, and clinical performance) to the Centers for Disease Control and Prevention. Therefore, the acceptance criteria and the study that proves the device meets those criteria are not detailed within this document.

Here is a summary of the information that is available in the document, and what is explicitly stated as not available:

1. Table of Acceptance Criteria and Reported Device Performance

• Not available in the provided document. The document directs inquiries regarding performance characteristics to the Centers for Disease Control and Prevention.

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

• Not available in the provided document. The document states that inquiries regarding analytical LoD, analytical sensitivity and specificity, and clinical performance should be directed to the Centers for Disease Control and Prevention. This would include information about sample sizes and data provenance for validation studies.

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

• Not applicable / Not available in the provided document. For a PCR assay, the "ground truth" is typically established by definitive laboratory methods (e.g., bacterial culture, sequencing, or a reference method) rather than expert opinion on images or clinical assessments. The details of how ground truth was established for the analytical and clinical performance studies are not present.

4. Adjudication method for the test set

• Not applicable / Not available in the provided document. See point 3.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, and the effect size of how much human readers improve with AI vs without AI assistance

• Not applicable. This device is a diagnostic PCR assay, not an AI-assisted imaging or diagnostic tool that involves human readers in the traditional sense of an MRMC study.

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

• Yes, implicitly. The device is a Real-time PCR Assay, which is an in vitro diagnostic test. Its performance inherently refers to its standalone analytical and clinical performance in detecting target DNA sequences. While human operators are involved in running the assay and interpreting results, the "algorithm" (the PCR chemistry and detection) itself functions stand-alone. However, specific details of "standalone performance" metrics (like sensitivity, specificity, accuracy against a reference standard) are not provided and are referred to the CDC.

7. The type of ground truth used

• For a PCR assay detecting DNA sequences from B. anthracis, the ground truth for performance studies would typically be established by a "gold standard" laboratory method, such as:
  • Bacterial culture and identification: Confirmation of B. anthracis growth and subsequent identification using conventional microbiological methods (e.g., phenotypic differences, susceptibility testing).
  • Reference molecular methods: Other validated PCR assays or DNA sequencing for definitive identification.
• Specific details of how ground truth was established for the validation studies are not available in the provided document.

8. The sample size for the training set

• Not applicable. For a PCR assay, there isn't a "training set" in the machine learning sense. The assay is developed based on known genetic targets of B. anthracis. Development involves optimizing primer/probe design and assay conditions, but not "training" an algorithm on a dataset.

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

• Not applicable. See point 8.

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The B. anthracis Real-Time PCR Assay is an in vitro diagnostic test for the qualitative detection of plasmid and chromosomal DNA sequences from B. anthracis. The assay can be used to test human respiratory samples, whole blood, serum, plasma, swabs from lesions, CSF, pleural fluid, and bacterial culture isolates from individuals suspected of having anthrax.

Results generated from direct specimen testing are presumptive for the identification of B. anthracis. Results generated from culture isolate testing should be used in conjunction with other conventional methods for identification of Bacillus anthracis isolates as part of the LRN Bacillus anthracis Testing Algorithm. The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidences in addition to the identification of B. anthracis from cultures or detection directly in clinical specimens.

Use is limited to Laboratory Response Network (LRN) designated laboratories.

The B. anthracis Real-time PCR Assay is also intended for environmental specimen testing for biothreat detection and response. FDA has not evaluated claims related to the use of this assay on environmental specimens.

The B. anthracis Real-time PCR Assay uses a fluorogenic probe, consisting of an oligonucleotide with a reporter dye (FAM) attached to the 5' end and a quencher dye (BHQ1) attached at or near the 3' end. The probe anneals to a specific target sequence located between the forward and reverse primers. During the extension phase of the PCR cycle, the 5′ nuclease activity of Tag polymerase degrades the probe causing the reporter dye to separate from the quencher dye and a fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes and the fluorescence intensity is monitored during the PCR. The Tag polymerase used in this assay is inactive at room temperature. It must be activated by incubation at 95°C, which also minimizes the production of nonspecific amplification products.

Each extracted DNA sample is tested with three B. anthracis primer and probe sets run as individual reactions. The primer and probe sets target genes encoding virulence factors as well as conserved regions of DNA from the B. anthracis chromosome. All three primer and probe sets must be positive for the overall result of the B. anthracis Real-time PCR Assay to be interpreted as positive. Any result that is positive for some, but not all three target regions, is still considered equivocal and follow-up laboratory investigation should be performed per the LRN Bacillus anthracis Testing Algorithm.

Here's an analysis of the acceptance criteria and study information for the B. anthracis Real-time PCR Assay, based on the provided 510(k) summary:

1. Acceptance Criteria and Reported Device Performance

The provided document does not explicitly state quantitative acceptance criteria (e.g., sensitivity, specificity thresholds with confidence intervals) for the B. anthracis Real-time PCR Assay. However, the performance characteristics were established by demonstrating its ability to differentiate B. anthracis from other Bacillus species. The document details the types of studies conducted to establish performance but does not provide specific numerical outcomes.

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

The document does not explicitly state the specific sample sizes used for the test sets in each of the clinical performance evaluations (historical collection of Bacillus sp. isolates, known B. cereus isolates, known bacterial isolates).

• Data Provenance: The studies utilized "a historical collection of Bacillus sp. isolates," "known B. cereus isolates," and "known bacterial isolates." The direct country of origin for these specific collections is not mentioned, but the submitting organization is the Centers for Disease Control and Prevention (CDC) in the USA, suggesting the data is likely from the USA or samples curated by the CDC. The studies appear to be retrospective as they involve testing historical and known isolate collections.

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" involved in establishing the ground truth for the test set.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method (such as 2+1 or 3+1) for the test set.

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

A Multi-Reader Multi-Case (MRMC) comparative effectiveness study comparing human readers with and without AI assistance was not done. This device is an in vitro diagnostic test (a PCR assay), not an AI-assisted diagnostic tool that would typically involve human readers interpreting results.

6. Standalone Performance Study (Algorithm Only Without Human-in-the-Loop Performance)

Yes, a standalone performance study was done. The B. anthracis Real-time PCR Assay is an automated PCR-based diagnostic test. Its performance characteristics (Limit of Detection, Analytical Sensitivity, Analytical Specificity, and Clinical Performance) were established by evaluating the assay's ability to detect B. anthracis DNA in various samples. This represents the intrinsic performance of the diagnostic algorithm/assay itself, without direct human interpretation of raw data beyond reading the assay's output.

7. Type of Ground Truth Used

The ground truth for the clinical performance evaluations was established using a "battery of tests performed by the CDC, including the gamma phage lysis, conventional culture, microbiological, and biochemical testing." This indicates the use of expert consensus based on established microbiological and biochemical methods as the reference standard.

8. Sample Size for the Training Set

The document does not specify a "training set" or its sample size. As a PCR assay, its development typically involves primer and probe design and optimization, which isn't usually framed in terms of a "training set" in the same way machine learning models are. The performance characteristics were established using "in-house and multicenter sensitivity studies" and evaluations on historical and known bacterial isolate collections.

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

Since the concept of a "training set" in the context of machine learning isn't directly applicable here, the method of establishing ground truth for development/optimization wasn't explicitly stated. However, similar to the test set, it would logically involve established microbiological and molecular techniques to confirm the identity of B. anthracis and other Bacillus species used during the assay's development and validation.

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