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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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The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Anthrax Detection System is a real-time polymerase chain reaction (PCR) test system intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences on the pXO1 plasmid (Target 1) and the pXO2 plasmid (Target 2) from Bacillus anthracis. The system can be used to test the following:

• Human whole blood collected in sodium citrate from individuals suspected of having anthrax
• Positive blood cultures
• Cultured organisms grown on blood agar plates.
  The JBAIDS Anthrax Target 2 assay is used as a supplementary test only after a positive result with the Target 1 Assay.
  The JBAIDS Anthrax Target 1 and Target 2 Assays are run on the JBAIDS instrument using the Diagnostic Wizard. Results are for the presumptive identification of B. anthracis, in conjunction with culture and other laboratory tests. The following considerations also apply:
• The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence, in addition to the identification of pXO1 and pXO2 targets either from cultures or from direct blood specimens.
• The assays have not been evaluated with blood from individuals without clinical signs or symptoms who were presumed exposed and who subsequently developed anthrax (inhalation or other forms of the disease), or from individuals with any form of anthrax (inhalational, cutaneous, or gastrointestinal).
• The level of plasmid targets that would be present in blood from individuals with early systemic infection is unknown.
• The definitive identification of B. anthracis from colony growth, liquid blood culture growth, or from blood specimens requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required.
  The safety and effectiveness of other types of tests or sample types (not identified as "For in vitro diagnostic use") have not been established.

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Anthrax Detection System is a fully integrated IVD system composed of the portable JBAIDS instrument, laptop computer and software, the JBAIDS Anthrax Detection Kit with two different freeze-dried PCR assays for detection of pathogenic Bacillus anthracis DNA. The system has been validated using four different sample preparation kits for isolating DNA from whole blood (IT 1-2-3TM Platinum Path, QFLOWdma, FLOW Sample Purification Kits), positive blood cultures (IT I-2-3TM SWIPE Sample Purification Kit), and plate cultures (IT 1-2-3™ Platinum Path and SWIPE Sample Purification Kits). Use of the JBAIDS DNA Extraction Control Kit is also recommended.
Prior to testing, specimens are processed using BioFire Diagnostic's IT 1-2-3 Sample Purification Kits. The resulting purified sample is added to Target 1 Unknown and Target 1 Inhibition Control vials, along with reconstitution buffer. Target 1 Positive Control and Negative Control vials are prepared using reconstitution buffer and water. When B. anthracis DNA is present, a fragment of B. anthracis DNA is amplified. The amplicon is detected by fluorescence using a specific hydrolysis probe. Each probe is labeled on one end with a fluorescent reporter moiety (6-carboxyfluorescein (6-FAM)) and elsewhere with a quencher moiety (carboxy tetramethylrhodamine (TAMRA)). When the probe is intact, the quencher absorbs the light emitted by the reporter moiety. During PCR, the probe hybridizes to the target sequence before the exonuclease activity of Taq polymerase hydrolyzes the probe, separating the fluorophore from the quencher and permitting detection of the fluorescent signal generated by the reporter. The fluorescent signal increases as additional templates are amplified and more probes are hydrolyzed.
JBAIDS Software analyzes the fluorescence amplification curves and reports results as positive, negative, uncertain or inhibited. A failure of the Positive or Negative Control will result in the entire run being called invalid. Retesting is required to resolve uncertain, invalid or inhibited results. The Target 2 assay is used as a supplementary test only after a positive result is obtained with the Target 1 assay.

Here's a breakdown of the acceptance criteria and study information based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The submission primarily focuses on demonstrating equivalence of a new DNA extraction method (Platinum Path) to an existing one (QFLOWdna) for use with the JBAIDS Anthrax Detection Kit. Therefore, the "acceptance criteria" are implicitly defined by the performance of the predicate device with the QFLOWdna kit, and the "reported device performance" is how the new Platinum Path kit compares.

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

• Clinical Performance (Surrogate Whole Blood):

  • Sample Size: 100 surrogate whole blood specimens (50 spiked with B. anthracis, 50 not spiked). Each sample was processed by both new (Platinum Path) and old (QFLOWdna) extraction methods and then tested.
  • Data Provenance: Prospectively collected from febrile volunteers in the USA (November 2012 to April 2013). These were surrogate specimens spiked with inactivated B. anthracis, not true clinical anthrax samples.

• Limit of Detection:

  • Sample Size: 20 independent whole blood specimens, each spiked with B. anthracis at the LoD level.
  • Data Provenance: Not explicitly stated, but implied to be laboratory-prepared samples for analytical validation.

• Reproducibility:

  • Sample Size: A panel of 12 blood samples (4 medium positive, 4 low positive, 4 negative). Each sample was tested twice a day for four days at each of three testing sites. Total individual tests for each target would be 12 samples * 2 tests/day * 4 days * 3 sites = 288 for each target (Target 1 and Target 2).
  • Data Provenance: Multicenter study, locations are "Site 1, Site 2, Site 3." Country of origin not explicitly stated, but typically assumed to be domestic for FDA submissions unless otherwise specified.

• Detection of Direct Culture Samples:

  • Sample Size: 10 B. anthracis colonies and 10 non-B. anthracis colonies.
  • Data Provenance: Not explicitly stated, but implied to be laboratory-prepared cultures.

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

The ground truth for the surrogate clinical specimens and analytical studies was established by:

• Spiking Status: Knowing which samples were spiked with inactivated B. anthracis and at what concentration, and which were not. This is a controlled laboratory process, not dependent on human expert interpretation for definitive classification.
• Predicate Device Performance: For the surrogate clinical specimens, the "correct result" was defined by the JBAIDS result when processed with the QFLOWdna kit, which is the predicate/established method. This avoids the need for external expert consensus on the B. anthracis status of a sample that has already been spiked.

Therefore, no external clinical experts were used to establish the ground truth in the traditional sense for these studies. The ground truth was based on the controlled spiking of samples and comparison to an established method.

4. Adjudication Method for the Test Set

Not applicable in the traditional sense. The "ground truth" for the surrogate clinical specimens was determined by the results from the predicate extraction method (QFLOWdna), and for the analytical studies, it was determined by the known spiking status of the samples. There was no need for expert adjudication of conflicting results as the comparison was against a predetermined "truth."

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 a molecular diagnostic test for B. anthracis DNA, not an imaging device that would typically involve human readers interpreting images. The study focuses on the analytical and clinical (surrogate) performance of the extraction method and PCR kit.

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

Yes, the studies presented are standalone (algorithm only). The JBAIDS system, with its software, analyzes the fluorescence amplification curves and reports results (positive, negative, uncertain, inhibited). The operators were blinded to the analyte content, meaning their human "interpretation" was limited to running the instrument and following its automated output. The performance metrics (PPA, NPA, LoD, reproducibility) are based directly on the device's output.

7. The Type of Ground Truth Used

• Clinical Performance (Surrogate Whole Blood): Known spiking status of the samples from a laboratory perspective, and the performance of the predicate DNA extraction method (QFLOWdna) as the "correct result" for comparison.
• Limit of Detection: Known concentration of spiked B. anthracis in laboratory-prepared whole blood specimens.
• Reproducibility: Known concentration of spiked B. anthracis (medium, low, or unspiked).
• Detection of Direct Culture Samples: Known identity of the bacterial colonies (either B. anthracis or non-B. anthracis) through laboratory culture and identification.

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" for the device itself. This submission is for modifications to an existing device (the JBAIDS Anthrax Detection Kit) by adding compatibility with a new sample purification kit (IT 1-2-3 Platinum Path). The JBAIDS system and its algorithm were presumably developed and validated previously. The studies described here are a validation of the new sample preparation method, not the training of a new algorithm.

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

Since there is no explicit mention of a "training set" for a new algorithm within this submission, this question is not directly applicable. The JBAIDS Anthrax Detection System already existed and was previously cleared (K051713 and K071188). The ground truth for its initial development and validation would have followed similar principles of known positive and negative samples, likely through spiking and confirmed cultures, as is typical for molecular diagnostic assays.

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The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Plague Detection Kit is a real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences of Yersinia pestis. The kit can be used to test human whole blood collected in sodium citrate or sputum collected aseptically from individuals greater than 18 years of age suspected of having septic or pneumonic plague. In addition, positive blood cultures and colonies may be tested. The JBAIDS Plague Target 2 assay is used as a supplementary test only after a positive result with the Target 1 Assay.
The JBAIDS Plague Target 1 and Target 2 assays are run on the JBAIDS instrument using the Diagnostic Wizard. Results are for the presumptive identification of Y. pestis in conjunction with culture and other laboratory tests. The definitive identification of Y. pestis from colony growth, liquid blood culture growth, or from blood or sputum specimens requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required.

The diagnosis of plague must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence in addition to the identification of Y. pestis from cultures or directly from whole blood or sputum specimens.

The JBAIDS Plague Detection Kit is intended for use by trained clinical laboratory personnel who have received specific training on the use of the JBAIDS Plague Detection Kit. The level of Y. pestis that would be present in blood or sputum from individuals with early systemic infection is unknown. Due to the difficulty in obtaining clinical specimens, these assays were not evaluated with blood or sputum from individuals with septic or pneumonic plague.

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Plague Detection Kit is a real-time polymerase chain reaction (PCR) reagent kit, which, when used with the JBAIDS instrument and software, allows the qualitative in vitro diagnostic (IVD) detection of target DNA sequences within the pathogenic bacterium, Yersinia pestis, the causative agent of plague. The kit contains two assays, Plague Target 1 and Target 2, each of which consists of oligonucleotide primers and a fluorescent-labeled target assay probe that specifically detect Y. pestis DNA. The Target 2 assay is reserved for samples that test positive with the Target 1 assay. The kit is designed for use with the JBAIDS instrument, a portable thermocycler and real-time fluorimeter that performs PCR in glass capillaries.

Before testing, samples are purified using Idaho Technology's 1-2-3TM Sample Purification Kits (or validated equivalent). The resulting purified sample is added to an Unknown reagent vial and an Inhibition Control reagent vial, along with reconstitution buffer. When the organism is present, a fragment of Y. pestis DNA is amplified. The amplicon is detected by fluorescence using a specific hydrolysis probe. The hydrolysis probe contains a short oligonucleotide that hybridizes to an internal sequence of the amplified fragment during the annealing phase of the PCR cycle. This probe has the 5' and 3' ends labeled with a reporter dye and a quenching dye, respectively. When the probe hybridizes to the specific DNA target, the Taq polymerase enzyme replicating the target-specific DNA hydrolyzes the probe, separating the two fluorophores and allowing the reporter dye to fluoresce.

The JBAIDS instrument measures the level of fluorescence from each unknown sample and control. JBAIDS Software analyzes the fluorescence amplification curves and reports results as positive, negative, inhibited, or uncertain. A failure of the Positive or Negative Control will result in the entire run being called invalid. Failure of the Inhibition Control yields an inhibited result when the associated sample has a negative result for the target assay and requires retesting of that sample.

Due to the nature of the provided document (a 510(k) summary for a diagnostic test kit), the study performed is a diagnostic accuracy study rather than a study evaluating the performance of an AI/ML device. Therefore, many of the requested fields (MRMC study, effect size of human readers improving with AI, number of experts for ground truth establishment, etc.) are not applicable and will be marked as "N/A".

Here's an analysis of the provided text based on your request:

JBAIDS Plague Detection Kit Performance Study Analysis

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" for the clinical trial in terms of specific performance metrics that needed to be met for FDA clearance. However, it details the design of the clinical specificity study and the resulting performance. For analytical studies, it provides "LOD" (Limit of Detection) and inclusivity/exclusivity data which serve as performance measures.

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

• Analytical Test Set:
  • Inclusivity: 18 isolates of virulent Y. pestis (subtypes and strains)
  • Exclusivity: A "panel" of 24 isolates (phylogenetically related and unrelated organisms)
  • Data Provenance: Not explicitly stated, but typically research laboratory settings for analytical studies. Retrospective in nature.
• Clinical Test Set:
  • Whole Blood: 132 samples
  • Sputum: 36 samples
  • Data Provenance: The study was a "multisite clinical trial". The samples were obtained from "subjects with clinical signs and symptoms consistent with systemic plague and for whom a blood and/or sputum culture had been ordered." Since clinical plague is rare, and the document explicitly states "Due to the difficulty in obtaining clinical specimens, these assays were not evaluated with blood or sputum from individuals with septic or pneumonic plague," these samples were likely from individuals suspected of plague but ultimately found to be negative. Thus, these are clinical samples, but representative of a "negative" population in the context of plague in humans. Retrospective in nature.

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

• N/A. For this in vitro diagnostic (IVD) device, the ground truth for analytical studies is established by known bacterial stocks and for clinical studies, by established laboratory culture methods.
• The ground truth for the clinical study was "laboratory culture results" which were considered the "gold standard." These methods are typically performed by trained microbiologists or clinical laboratory scientists, not "experts" in the sense of physicians adjudicating medical imaging or complex interpretations.

4. Adjudication Method for the Test Set

• N/A. For analytical studies, the ground truth (presence/absence of specific bacteria, concentration) is determined by established microbiological methods and known samples, without adjudication.
• For the clinical specificity study, the ground truth was "laboratory culture results." These are typically definitive for bacterial presence/absence. No adjudication among experts is mentioned or typically needed for this type of ground truth.

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

• No. This is an in vitro diagnostic (IVD) device, not an AI/ML device requiring human-in-the-loop performance evaluation. The device provides an automated "positive, negative, inhibited, or uncertain" result.
• Effect size of how much human readers improve with AI vs. without AI assistance: N/A.

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

• Yes, in essence. The JBAIDS system operates as a standalone diagnostic test. The "JBAIDS Software analyzes the fluorescence amplification curves and reports results as positive, negative, inhibited, or uncertain." This is the standalone performance of the analytical system.
• While the kit is "intended for use by trained clinical laboratory personnel," their role is to operate the instrument and interpret the automated results within a clinical context, not to perform a subjective interpretation of a raw signal that the algorithm then enhances.

7. The Type of Ground Truth Used

• Analytical Studies: Known strains/isolates of Y. pestis and other bacteria, with confirmed identification and concentrations (e.g., CFU/mL).
• Clinical Studies: "Laboratory culture results," which are considered the "gold standard" for bacterial identification in clinical samples.

8. The Sample Size for the Training Set

• Not explicitly stated in terms of a "training set." For IVDs, assay development and optimization (which can be considered analogous to training) involve numerous preliminary experiments with various dilutions, strains, and conditions, but these aren't typically documented as a formal "training set" with a defined size in the same way an AI/ML model would. The document describes the assay's design and then its validation/testing.

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

• N/A (as no formal "training set" is described). However, for the development of PCR assays (like this kit), the ground truth for optimizing primers and probes would be established by:
  • Using purified genomic DNA of Yersinia pestis and other closely related and common organisms.
  • Sequencing to confirm target regions.
  • Utilizing known bacterial cultures and dilutions with confirmed concentrations.
  • These foundational truths are based on established molecular biology and microbiology techniques.

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The JBAIDS Anthrax Detection System is a real-time polymerase chain reaction (PCR) test system intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences on the pXO1 plasmid (Target 1) and the pXO2 plasmid (Target 2) from Bacillus anthracis. The system can be used to test human whole blood collected in sodium citrate from individuals suspected of having anthrax, positive blood cultures, and cultured organisms grown on blood agar plates. The JBAIDS Anthrax Target 2 assay is used as a supplementary test only after a positive result with the Target 1 Assay.

The JBAIDS Anthrax Target 1 and Target 2 Assays are run on the JBAIDS instrument using the Diagnostic Wizard.

Results are for the presumptive identification of B. anthracis, in conjunction with culture and other laboratory tests. The following considerations also apply:

• The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence, in addition to the identification of pXO1 and pXO2 targets either from cultures or from direct blood specimens.
• The assays have not been evaluated with blood from individuals without clinical signs or symptoms who were presumed exposed and who subsequently developed anthrax (inhalation or other forms of the disease), or from individuals with any form of anthrax (inhalational, cutaneous, or gastrointestinal).
• The level of plasmid targets that would be present in blood from individuals with early systemic infection is unknown.
• The definitive identification of B. anthracis from colony growth, liquid blood culture growth, or from blood specimens requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required.

The safety and effectiveness of other types of tests or sample types (not identified as "For in vitro diagnostic use") have not been established.

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Anthrax Detection System is a fully integrated in vitro diagnostic (IVD) system composed of the following:

• JBAIDS instrument with laptop computer
• Software
• Two different freeze-dried reagent assays (in one kit) for the qualitative detection of pathogenic Bacillus anthracis
• Four different sample preparation protocols, two for isolating target DNA from whole blood, one for processing blood culture, and another for processing colonies.

The JBAIDS instrument, using Polymerase Chain Reaction (PCR) technology, is a portable thermocycler and real-time fluorimeter. The JBAIDS Anthrax Detection Kit is specially designed for PCR in glass capillaries using the JBAIDS instrument and hydrolysis probes for sequence-specific detection of B. anthracis DNA found on the pX01 plasmid (Target 1) and the pX02 plasmid (Target 2).

The reagent kit contains four different types of freeze-dried reagent vials: Positive Controls, Negative Controls, Inhibition Controls, and Unknowns (used for testing the patient sample). Each JBAIDS assay requires a Positive and Negative Control, and each sample is tested using both an Inhibition Control vial and an Unknown reagent vial.

Before testing, whole-blood samples are purified using the Idaho Technology IT 1-2-3 FLOW or QFLOWdir Sample Purification Kit (or validated equivalent), while blood culture and direct culture specimens are prepared using the IT 1-2-3 SWIPE Sample Purification Kit (or validated equivalent). The resulting purified sample is added to an Unknown reagent vial and an Inhibition Control reagent vial, along with reconstitution buffer. A Positive Control and a Negative Control vial are prepared using reconstitution buffer and reagent grade water. Aliquots from each reagent vial are transferred to two reaction capillaries that are tested together in the JBAIDS instrument. The instrument is programmed to perform heating and cooling cycles that drive the PCR process. The heating and cooling cycles are generated using a heating coil and varying fan speeds. Fluorescence emission is monitored over one of three wavelengths, and the instrument software interprets the change in fluorescence to determine whether the target DNA is present.

When the organism is present, a fragment of B. anthracis DNA is amplified using specific primers. The amplicon is detected by fluorescence using a specific hydrolysis probe. The hydrolysis probe contains a short oligonucleotide that hybridizes to an internal sequence of the amplified fragment during the annealing phase of the PCR cycle. This probe has the 5' and 3' ends labeled with a reporter dye and a quenching dye, respectively. When the probe hybridizes to the specific DNA target, the Taq polymerase enzyme, replicating the target-specific DNA, hydrolyzes the probe, which separates the two fluorophores, thus allowing the reporter dye to fluoresce.

The level of fluorescence from each unknown sample and control is measured by the JBAIDS instrument. JBAIDS Software analyzes fluorescence amplification curves and reports results as "Positive," "Negative," "Inhibited," or "Uncertain." A failure of the Positive or Negative Control will result in the entire run being called "Invalid." Failure of the Inhibition Control yields an Inhibited result for the associated sample and requires retesting of that sample.

The JBAIDS Anthrax Detection System is a real-time PCR test system intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences on the pXO1 plasmid (Target 1) and the pXO2 plasmid (Target 2) from Bacillus anthracis.

1. Acceptance Criteria and Reported Device Performance:

The provided document describes a method comparison and carry-over study rather than specific acceptance criteria thresholds with a pass/fail outcome. The studies aim to demonstrate equivalence between a new sample purification kit (IT 1-2-3 QFLOWdna) and an existing one (IT 1-2-3 FLOW).

2. Sample Sizes and Data Provenance:

• Method Comparison - Spiked Samples: 6 citrated whole blood samples spiked with B. anthracis at the Limit of Detection (LOD).
• Method Comparison - Negative Samples: 16 normal healthy donors (citrated whole blood samples).
• Carry-over Study:
  • Strongly positive samples (spiked at 5 x 10" CFU/mL) processed next to negative (unspiked) samples.
  • QFLOW™ protocol: 42 negative capillaries were assessed for carry-over.
  • IT 1-2-3 FLOW protocol: 12 negative capillaries were assessed for carry-over.
• Data Provenance: Not explicitly stated (e.g., country of origin). The studies appear to be prospective as they were specifically performed to evaluate the new sample purification kit.

3. Number of Experts and Qualifications for Ground Truth: No information provided. This is a molecular diagnostic test, and ground truth would typically be established based on the known spiked concentration of B. anthracis or confirmed negative status of healthy donor samples, rather than expert interpretation of images or clinical data.

4. Adjudication Method: Not applicable. For this type of molecular test, objective results (positive/negative/inhibited) are generated by the instrument based on fluorescence curves and software analysis, not human interpretation requiring adjudication.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study: No. This is not an imaging device or one that relies on human interpretation for diagnosis; therefore, an MRMC study comparing human readers with and without AI assistance is not applicable.

6. Standalone Performance: Yes, the device's performance results (detection of B. anthracis DNA, specificity in negative samples, carry-over rate) are reported for the algorithm (JBAIDS Anthrax Detection System) based on its analysis of samples. The system reports "Positive," "Negative," "Inhibited," or "Uncertain" results without human intervention in the interpretation of the raw data.

7. Type of Ground Truth Used:

• For the method comparison using spiked samples: The ground truth was based on the known presence of B. anthracis DNA at a defined concentration (LOD).
• For the method comparison using healthy donor samples: The ground truth was the known absence of B. anthracis DNA in these healthy individuals.
• For the carry-over study: The ground truth for positive samples was their known high concentration of B. anthracis, and for negative samples, it was the known absence of B. anthracis.

8. Sample Size for the Training Set: Not specified. This document describes performance validation for a specific modification (new sample purification kit) rather than the initial development and training of the PCR assay's core algorithms. PCR assays are typically developed based on laboratory experiments to define primer/probe specificity and amplification efficiency, rather than machine learning on a "training set" in the traditional sense.

9. How the Ground Truth for the Training Set Was Established: Not specified. As mentioned above, for a PCR assay, "training" involves optimizing molecular biology parameters, and ground truth is based on known biological characteristics of the target organism and non-target organisms.

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The JBAIDS Anthrax Detection System is a real-time polymerase chain reaction (PCR) test system intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences on the pXO1 plasmid (Target 1) and the pXO2 plasmid (Target 2) from Bacillus anthracis. The system can be used to test human whole blood collected in sodium citrate from individuals suspected of having anthrax, positive blood cultures, and cultured organisms grown on blood agar plates. The JBAIDS Anthrax Target 2 assay is used as a supplementary test only after a positive result with the Target 1 Assay.
The JBAIDS Anthrax Target 1 and Target 2 Assays are run on the JBAIDS instrument using the Diagnostic Wizard.
Results are for the presumptive identification of B. anthracis, in conjunction with culture and other laboratory tests. The following considerations also apply:

• The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, other laboratory evidence, in addition to the identification of pXO1 and pXO2 targets either from cultures or from direct blood specimens.
• The assays have not been evaluated with blood from individuals without clinical signs or symptoms who were presumed exposed and who subsequently developed anthrax (inhalation or other forms of the disease), or from individuals with any form of anthrax (inhalational, cutaneous, or gastrointestinal).
• The level of plasmid targets that would be present in blood from individuals with early systemic infection is unknown.
• The definitive identification of B. anthracis from colony growth, liquid blood culture growth, or from blood specimens requires additional testing and confirmation procedures in consultation with public health or other authorities for whom reports are required.
  The safety and effectiveness of other types of tests or sample types (not identified as "For in vitro diagnostic use") have not been established.

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Device Anthrax Detection System is a fully integrated in-vitro diagnostic (IVD) system composed of the JBAIDS instrument with laptop computer, software, 2 different freeze-dried reagent assays (in one kit) for the qualitative detection of pathogenic Bacillus anthracis, and 3 different sample preparation protocols for isolating target DNA from whole blood, blood culture, or direct culture.
The JBAIDS instrument, using Polymerase Chain Reaction (PCR) technology, is a portable thermocycler and real-time fluorimeter. The JBAIDS Anthrax Detection Kit is specially designed for PCR in glass capillaries using the JBAIDS instrument and hydrolysis probes for detection of the pXO1 plasmid (Target 1) and the pXO2 plasmid (Target 2) DNA sequences. A fragment of plasmid DNA is amplified using specific primers, creating amplicon. The amplicon is detected using a specific hydrolysis probe, which is a short oligonucleotide that hybridizes to an internal sequence of the amplified fragment during the annealing phase of the PCR cycle. This probe has the 5' and 3' ends labeled with a reporter dye and a quenching dye, respectively. When the probe hybridizes to the specific DNA target, the Taq polymerase enzyme replicating the target-specific DNA hydrolyzes the probe, separating the two fluorophores, thus allowing the reporter dye to fluoresce.
The reagent kit contains 4 different types of freeze-dried reagent vials: Positive Controls, Negative Controls, Inhibition Controls, and Unknowns (used for testing the patient sample). Each JBAIDS run requires a Positive and Negative Control, and each sample is tested using both an Inhibition Control vial and an Unknown reagent vial. The characteristics of the amplification curves from the positive control (PC), negative control (NC), inhibition controls (IC) and from each unknown sample are analyzed by the JBAIDS Software, and results are reported as Positive. Negative, Inhibited or Uncertain. When PCs or NCs are unacceptable, the test results for all samples in the JBAIDS run are considered invalid and must be repeated.
Prior to testing, whole-blood samples are purified using the Idaho Technology IT 1-2-37M FLOW Sample Purification Kit (or validated equivalent), while blood culture and direct culture specimens are prepared using the IT 1-2-31M SWIPE Sample Purification Kit (or validated equivalent). The resulting purified sample is added to an Unknown reagent vial and an Inhibition Control reagent vial, along with reconstitution buffer.

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

JBAIDS Anthrax Detection System Acceptance Criteria and Study Details

1. Table of Acceptance Criteria and Reported Device Performance

The provided document does not explicitly state formal "acceptance criteria" with numerical thresholds for sensitivity and specificity in a table format for the JBAIDS Anthrax Detection System. Instead, it describes performance characteristics that demonstrate substantial equivalence to a predicate device and good analytical and clinical performance. The closest interpretation of acceptance criteria would be that the device needs to achieve high sensitivity and specificity in detecting B. anthracis targets, similar to or exceeding the predicate method.

Here's a summary of the stated performance:

• Direct Culture Panel: 23/23 (100%) detected
• Blood Culture Panel: 11/11 (100%) detected
• Whole Blood Spiked Samples: 67/68 (98.5%) detected (at limit of detection levels) |
  | Analytical Specificity: | No cross-reactivity with non-B. anthracis organisms |
• Direct Culture Panel: 34/37 (91.9%) negative for non-B. anthracis strains
• Blood Culture Panel: 12/12 (100%) negative for non-B. anthracis strains
• Note: Cross-reacted with 3 virulent B. cereus strains known to cause anthrax-like illness. |
  | Clinical Specificity: | No false positives in samples from individuals suspected of anthrax (but without confirmed anthrax) | 150/150 (100%) negative in blood samples from hospitalized subjects (95% CI, 98%-100%) |
  | Equivalence to Predicate (CDC Gamma Phage Lysis Assay): | As effective as predicate, with additional benefits | "As effective as the predicate assay," "easier to use," "highly sensitive and specific." |

2. Sample Sizes and Data Provenance

The provided document indicates the following sample sizes and data provenance:

• Test Set (Analytical Studies):

  • Direct Culture Panel: 23 virulent B. anthracis strains (positive detection) + 37 non-B. anthracis strains (negative detection) = 60 samples.
  • Blood Culture Panel: 11 virulent B. anthracis strains (positive detection) + 12 non-B. anthracis strains (negative detection) = 23 samples.
  • Whole Blood Spiked Samples: 68 samples spiked with limit of detection levels of live B. anthracis.
  • Data Provenance: The document does not explicitly state the country of origin. It refers to "virulent strains of B. anthracis tested" and "non-B. anthracis strains." Given the context of CDC involvement and FDA submission, it's highly likely these were laboratory-controlled samples and reference strains, possibly from a national or international repository, and not necessarily patient data from a specific country. This appears to be retrospective testing of characterized samples.

• Test Set (Clinical Specificity Study):

  • Sample Size: 150 blood samples.
  • Data Provenance: "Blood samples from hospitalized subjects with clinical signs and symptoms consistent with inhalation or systemic anthrax and for whom a blood culture had been ordered." This indicates a prospective data collection from a clinical setting, presumably within the US. The document does not specify the exact location(s).

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number of experts used or their specific qualifications (e.g., "radiologist with 10 years of experience") for establishing ground truth in a systematic way for the test set.

However, for the predicate device and confirmation methods:

• Predicate Method: The predicate for the JBAIDS Anthrax Detection Kit is "traditional microbiological identification of the organism (preamendment methods) with confirmation by the Centers for Disease Control Laboratory Response Network Gamma Phage Lysis assay." This implies that the ground truth for B. anthracis identification was established by microbiologists and experts within the CDC's Laboratory Response Network, following standard microbiological identification criteria and the gamma phage lysis assay procedure.
• Confirmation for Analytical Studies: For the direct culture and blood culture panels, "all of the samples were confirmed as B. anthracis using standard biochemical identification and tested positive using the CDC Reference Laboratory Procedure for Identification of B. anthracis Using Lysis by Gamma Phage." This again points to verification by microbiology experts using established CDC protocols.

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method (e.g., 2+1, 3+1). The ground truth for the analytical studies was established by standard biochemical identification and the CDC Reference Laboratory Procedure for Identification of B. anthracis Using Lysis by Gamma Phage, which are definitive laboratory methods rather than subjective expert consensus requiring adjudication.

For the clinical specificity study, the ground truth for ruling out anthrax was "not identified in any of the blood cultures." This implies a definitive laboratory result rather than a consensus decision requiring adjudication.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. The JBAIDS Anthrax Detection System is an in-vitro diagnostic (IVD) PCR system that provides an automated result (Positive, Negative, Inhibited, Uncertain). It's an algorithm-only device without a human-in-the-loop component in its primary function, thus improvements in human reader performance with or without AI assistance are not applicable.

6. Standalone (Algorithm Only) Performance

Yes, a standalone (algorithm only) performance study was done for the JBAIDS Anthrax Detection System. The device itself is an automated system that uses PCR technology to detect specific DNA sequences and its software then analyzes the amplification curves and reports results as Positive, Negative, Inhibited, or Uncertain (as described in the "Description" section).

The performance metrics reported (sensitivity, specificity, detection rates for various samples) are directly from the JBAIDS system's automated output.

7. Type of Ground Truth Used

The ground truth used was:

• Microbiological Identification/Pathology: For the analytical studies, ground truth for B. anthracis presence was established by "standard biochemical identification and ... the CDC Reference Laboratory Procedure for Identification of B. anthracis Using Lysis by Gamma Phage." For non-B. anthracis strains, their identification as non-anthrax was also based on standard microbiological characterization.
• Outcomes Data (Indirect/Surrogate): For the clinical specificity study, the ground truth for the absence of anthrax was based on "blood cultures" being negative for B. anthracis. This serves as a clinical outcome surrogate to confirm the absence of the infection in symptomatic patients.

8. Sample Size for the Training Set

The document does not specify the sample size for a training set. This is common for PCR-based IVD systems, where the "training" (development and optimization) often relies on well-characterized laboratory strains and known genetic sequences rather than a distinct "training set" in the machine learning sense. The device is designed to detect known specific DNA sequences rather than learn patterns from a broad dataset.

9. How Ground Truth for the Training Set Was Established

As no specific "training set" is mentioned, the method for establishing ground truth for training data is not provided. However, the development of the assays (e.g., designing primers and probes for pXO1 and pXO2) would rely on the established genomic sequences of B. anthracis, confirmed by techniques such as genomic sequencing, traditional microbiological identification, and confirmed virulence studies from expert laboratories like the CDC. The document highlights that the targets (pXO1 and pXO2 plasmids) are "essential for the organism's pathogenicity," implicitly referencing well-established biological facts about B. anthracis.

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