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The Applied Biosystems Bacillus anthracis 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 for Bacillus anthracis, or BA). The Applied Biosystems Bacillus anthracis Detection Kit is intended to test human whole blood (EDTA) specimens and blood culture specimens with growth detected by a continuous monitoring blood culture system. Blood culture specimens must be determined to contain gram-positive bacilli by Gram stain prior to testing of whole blood specimens must be performed concomitantly with standard of care blood culture.

The Applied Biosystems Bacillus anthracis Detection Kit is indicated for use in CLIA-certified high-complexity laboratories in response to a confirmed Bacillus anthracis event only in accordance with the guidelines provided by public health authorities prior to or during a public health emergency. Testing with the Applied Biosystems Bacillus anthracis Detection Kit must only be performed when public health authorities have determined the need for this test must only be used with specimens from individuals with clinical signs and symptoms of B. anthracis infection and who have either been exposed to B. anthracis or may have been exposed to B. anthracis.

The Applied Biosystems Bacillus anthracis Detection Kit is intended for use as an aid in the diagnosis of anthrax infection and results are for the presumptive identification of Bacillus anthracis. The diagnosis of B. anthracis infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence, in addition to the identification of B. anthracis from cultures or directly from clinical specimens. The definitive identification of B. anthracis requires additional testing and confirmation procedures in consultation with the appropriate public health authorities for whom reports may be required.

The Applied Biosystems Bacillus anthracis Detection Kit has not been clinically evaluated with specimens collected from individuals with B. anthracis infection or those presumed to B. anthracis. 'B. anthracis Not detected' results do not preclude infection with Bacillus anthracis and should not be used as for diagnosis, treatment, or other patient management decisions.

Laboratories implementing this test must have the appropriate biosafety equipment, personal protective equipment (PPE), containment facilities and personnel trained in the safe handling of diagnostic clinical specimens potentially containing B. anthracis. Anthrax is a nationally notifiable disease caused by a biothreat microbial agent and must be reported to public health authorities.

The distribution of in vitro diagnostic devices for Bacillus spp. detection is limited to laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.

The Applied Biosystems Bacillus anthracis Detection Kit is intended for use with the AB1 7500 Fast Dx Real-Time PCR Instrument with analysis using the Applied Biosystems Bacillus anthracis Interpretive Software (BaIS).

The Applied Biosystems™ Bacillus anthracis Detection Kit is a multiplexed real-time polymerase chain reaction (PCR) test kit intended for the qualitative in vitro diagnostic (IVD) detection of target DNA sequences for B. anthracis. Reagents are lyophilized in a 96-well plate format as a fully formulated Mastermix and are stable at room temperature for up to one year. The kit is specifically designed for performing real-time PCR using the Applied Biosystems (ABI) 7500 Fast Dx instrument and software, with nucleic acids extracted from clinical specimens using a Qiagen manual extraction method or Roche MagNA Pure automated extraction methods. An automated interpretative software component (BalS) is included in the kit but supplied separately and operates on a computer(s) that is separate from the ABI 7500 Fast Dx computer.

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

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly present a formal "Acceptance Criteria" table with pass/fail thresholds for all aspects. Instead, it describes performance studies and their outcomes. Based on the performance data provided, we can infer the de facto acceptance associated with demonstrating strong performance indicators.

• Whole Blood: 150 CFU/mL
• Blood Culture, Aerobic: 10,000 CFU/mL
• Blood Culture, Anaerobic: 10,000 CFU/mL
  Roche MagNA Pure:
• Whole Blood: 50 CFU/mL
• Blood Culture, Aerobic: 2270 CFU/mL
• Blood Culture, Anaerobic: 3040 CFU/mL
  (All values are presumptively "met" the >95% detection criteria given they are reported as LODs). |
  | Analytical Inclusivity| 100% detection rate for B. anthracis strains carrying both plasmid targets. Correctly identify 'Bacillus anthracis suspected' for strains with only one plasmid target. | 100% detection rate for B. anthracis strains that carry both plasmid targets. For three strains known to carry only one plasmid target, the assay generated 'Bacillus anthracis suspected' results as expected. |
  | Analytical Exclusivity| High no detection rate (close to 100%) for non-target organisms. No false positives. | Initial no detection rate for exclusivity testing was 95.68%. Repeat testing was conducted, resulting in a final no detection rate of 100%. Some known Bacillus cereus strains (BAG1X1-1, 03BB102, and G-9241) that contain a pXO1-like plasmid were correctly identified as 'Bacillus anthracis suspected'. |
  | Interfering Substances| No significant interference from common substances unless noted with appropriate labeling limitations. | Identified specific technique-specific substances that interfere (e.g., MagNA Pure Wash Buffer, 10% Bleach, Ethanol >5%). Appropriate limitations were added to product labeling. |
  | Microbial Interference| 100% detection rate of B. anthracis in the presence of other clinically-relevant organisms. | 100% detection rate of B. anthracis in the presence of potentially interfering organisms in whole blood and blood culture. |
  | Reproducibility | Consistent results across operators, instruments, and reagent lots. Limited false negatives/positives in control samples. Positive percent agreement metrics indicate reproducibility. | One low positive sample returned negative. One high positive sample returned 'Bacillus anthracis suspected' due to pXO1 assay failure, but returned 'Bacillus anthracis detected' on repeat. No false positive events out of 270 negative PCR tests. 24 sample replicates (across all three lots) returned initial indeterminate results, all determined negative by supervisor review. No invalid results. |
  | Carry-Over/Cross-contamination| Minimal to no false positive events due to carry-over/cross-contamination. | Automated Method (MagNA Pure): 2 false positive events out of 135 negative samples (Specificity: 98.5%).
  Manual Method (Qiagen DSP): No false positive events out of 139 negative samples (Specificity: 100%).
  Contamination events identified on surfaces during testing, highlighting areas of risk. |
  | Clinical Specificity | 100% Negative Percent Agreement (NPA) for B. anthracis negative samples. | For 401 blood culture specimens and 439 whole blood specimens: 100% Negative Percent Agreement (NPA) when compared to the expected negative result. |
  | Clinical Sensitivity | High Positive Percent Agreement (PPA) for B. anthracis positive samples. | 96% Positive Percent Agreement (PPA) (CI: 90.4-96.5%) for 87 low-positive whole blood specimens. Three specimens initially generated 'Bacillus anthracis suspected' results. |

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

• Limit of Detection (LOD): 1612 technical replicates were tested. Data provenance is not specified by country, but it's analytical data likely generated in-house at MRIGlobal (developer). Prospective (controlled spike-in experiments).
• Analytical Inclusivity: 24 different Bacillus anthracis strains tested in triplicate (24 strains * 2 extraction methods * 3 replicates = 144 technical replicates)
• Analytical Exclusivity: 154 non-target organisms tested in wet lab.
• Interfering Substances: 50 potentially interfering substances. Each substance tested in triplicate-paired samples (with and without B. anthracis). 50 * 2 * 3 = 300 samples (assuming paired means 3 concentrations per, and triplicate-paired means 3 replicates per substance per condition).
• Reproducibility: 7 panel members tested twice a day by three teams on five non-consecutive days. Details not fully specified for total sample count, but likely included multiple replicates for each panel member/condition. (e.g., 7 panel members * 2 times/day * 3 teams * 5 days = 210 runs, each run likely involving multiple replicates). "No false positive events occurred out of the 270 PCR tests of negative samples."
• Carry-Over/Cross-contamination:
  • Automated method (MagNA Pure): 9 runs, each with 31 samples, for a total of 279 samples. 135 negative samples.
  • Manual method (Qiagen DSP): 12 batches, total of 279 samples. 139 negative samples.
• Clinical Specificity: 401 blood culture specimens and 439 whole blood specimens. Samples were "left-over fresh and frozen blood culture samples," "randomly accessed, residual blood," and "febrile whole blood samples." Collected prospectively and serially from three point-of-care collection sites within the US.
• Clinical Sensitivity: 87 low-positive whole blood specimens (simulated/contrived). Each aliquot was spiked with 1 of 18 Bacillus anthracis strains. "Febrile whole blood specimens ... determined to be negative for Bacillus anthracis" were used as the matrix for spiking. Samples were contrived (prospective experimental design using collected negative clinical matrix). Clinical performance testing was conducted at three laboratories within the US.

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

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

• For analytical studies (LOD, Inclusivity, Exclusivity, Interference, Reproducibility, Carry-Over), ground truth is established by the known concentration/presence of cultured organisms or purified DNA, or by the known absence of the target.
• For clinical specificity, samples were "assumed to be negative for Bacillus anthracis" based on their collection context (e.g., routine CBC, fever of unknown origin that would not indicate B. anthracis exposure, or confirmed to be positive for other bacterial species but not B. anthracis). No independent expert assessment or gold standard positive confirmation was used for these negative samples.
• For clinical sensitivity, the ground truth was established by artificial spiking of known concentrations of Bacillus anthracis into negative clinical samples.

4. Adjudication Method for the Test Set

The document mentions adjudication in two instances:

• Reproducibility: "24 sample replicates (across all three reagent lots) that returned initial indeterminate results. All were determined to be negative by supervisor review." This suggests a form of expert review for indeterminate results.
• Analytical Exclusivity: "Some known Bacillus cereus strains... Detection of these strains was indicated by a test result of ‘Bacillus anthracis suspected’ with amplification of the plasmid manually confirmed by the supervisor." This also indicates supervisor review for specific expected "suspected" results.

No broad 2+1 or 3+1 adjudication method for clinical test sets is described. For the clinical performance, the results are directly compared to the assumed/contrived ground truth.

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

No MRMC comparative effectiveness study was done. This device is an in vitro diagnostic (IVD) PCR test kit, not an imaging AI designed to assist human readers. Its performance is assessed as a standalone diagnostic tool.

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

Yes, the studies presented (Analytical Sensitivity, Inclusivity, Exclusivity, Interference, Microbial Interference, Reproducibility, Carry-Over/Cross-contamination, Clinical Specificity, Clinical Sensitivity) represent the standalone performance of the "Applied Biosystems™ Bacillus anthracis Detection Kit" which includes the AB1 7500 Fast Dx Real-Time PCR Instrument and the Applied Biosystems Bacillus anthracis Interpretive Software (BaIS). It is an automated test with automated interpretation and report generation, so its performance is inherently a "standalone" or "algorithm only" type of assessment.

7. The Type of Ground Truth Used

The ground truth varied by study type:

• Analytical Studies (LOD, Inclusivity, Exclusivity, Interference, Microbial Interference, Reproducibility, Carry-Over): Controlled laboratory settings with known concentrations of purified DNA or cultured organisms. For negative controls, known absence of target.
• Clinical Specificity: "Assumed to be negative for Bacillus anthracis" for clinical samples collected from individuals not suspected of anthrax, or confirmed to have other bacteria. This is a clinical "expected negative" based on the patient population and standard clinical practice, rather than an independent gold standard for B. anthracis negativity.
• Clinical Sensitivity: Contrived samples where known concentrations of Bacillus anthracis were spiked into clinical samples previously determined to be negative for B. anthracis. This is a simulated positive ground truth.

8. The Sample Size for the Training Set

The document describes premarket validation studies for a diagnostic kit. It does not provide information about a "training set" size for model development, as this device is a PCR assay with interpretive software, not a machine learning model that undergoes a separate training phase with a distinct dataset of this nature. The "training" in this context refers to the development and optimization process, which is not typically quantified in terms of a "training dataset size" in the same way as for AI/ML algorithms.

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

As above, the concept of a "training set" and its ground truth establishment, as it applies to AI/ML, is not directly applicable to this PCR diagnostic kit. The "ground truth" during the development phase would have involved known positive and negative controls, spiked samples, and characterized bacterial strains, similar to the analytical studies performed for validation.

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