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The TaqPath™ COVID-19 Diagnostic PCR Kit is a real-time reverse transcription polymerase chain reaction (RT-PCR) test intended for the qualitative detection of nucleic acid from SARS-CoV-2 in nasopharyngeal and anterior nasal specimens from individuals with signs and symptoms of respiratory tract infection.

The TaqPath™ COVID-19 Diagnostic PCR Kit is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical observations, epidemiological information and laboratory findings. The SARS-CoV-2 RNA is generally detectable in upper respiratory (anterior nasal and nasopharyngeal swabs) specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA. Positive results do not rule out bacterial infection or co-infection with other pathogens. The agent detected may not be the definite cause of disease.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient management decisions.

The TaqPath™ COVID-19 Diagnostic PCR Kit is intended for use by qualified clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.

The Applied BioSystems™ TaqPath™ COVID-19 Diagnostic PCR Kit (TaqPath™ COVID-19 Diagnostic PCR Kit) includes the assays and controls for a multiplex real-time RT-PCR test for the qualitative detection of RNA from SARS-CoV-2 in nasopharyngeal and anterior nasal specimens from individuals with signs and symptoms of respiratory tract infection.

Each kit includes the following components:

• Multiplexed assays that contain three primer/probe sets specific to different SARS-CoV-2 genomic regions and primers/probes for bacteriophage MS2
• MS2 Phage Control as an internal process control for nucleic acid extraction
• TaqPath™ COVID-19 Diagnostic PCR Control as a positive RNA control that contains targets specific to the SARS-CoV-2 genomic regions targeted by the assays.

The workflow begins with nucleic acid extraction from upper respiratory specimens (nasopharyngeal and anterior nasal swabs) that arrive in the testing site in transport media. Nucleic acids are isolated and purified from the specimens using the MagMAX™ Viral/Pathogen II Nucleic Acid Isolation Kit. Nucleic acid isolation is performed via an automated process using the KingFisher™ Flex Purification System. The nucleic acid is reverse transcribed into cDNA and amplified using the TaqPath™ COVID-19 Diagnostic PCR Kit and one of the following real-time PCR instruments:

• Applied Biosystems™ 7500 Fast Dx Real-Time PCR Instrument
• Applied Biosystems™ QuantStudio™ 5 Dx Real-Time PCR Instrument

In the process, the probes anneal to three (3) specific SARS-CoV-2 target sequences located between three (3) unique forward and reverse primers for the following genes:

• ORF1ab
• N gene
• S gene

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, generating a fluorescent signal. With each PCR cycle, additional reporter dye molecules are cleaved from their respective probes, increasing the fluorescent intensity, which is measured at each cycle by the real-time PCR instrument.

Following RT-PCR, the data from the instrument's data collection software are imported into COVID-19 Interpretive Software IVD Edition for analysis and interpretation. After data import, the software analyzes the run data, performs quality check (QC) analysis, and calculates the interpretive results for each sample and control. The imported data and interpretive results for each run are saved as a batch in the software. Results can be exported as CSV files and reports can be generated in PDF format.

Validation of the results is performed automatically by the COVID-19 Interpretive Software based on performance of the positive and negative controls. The following results are automatically generated using the calling rules, plate validity and the CT cutoff values for assay targets:

1. Repeat the test by re-extracting the
   original sample and repeating the
   RT-PCR.
   IMPORTANT! Samples with a
   result of SARS-CoV-2 Inconclusive
   shall be retested one time.
2. After retesting one time, report the
   results to the healthcare provider.
3. If the repeat result remains
   inconclusive, the healthcare
   provider should conduct additional
   confirmation testing with a new
   specimen, if clinically indicated. |
   | | Two or more SARS-CoV-2 targets
   = POS | | POS or
   NEG | VALID | Positive SARS-
   CoV-2 | REPORT
   Report the results to the healthcare
   provider. |

A minimum of one negative control and one positive control must be present for each run. Additional negative control wells shall be run for each extraction that is represented on a realtime RT-PCR plate. All control wells must pass for the real-time RT-PCR plate to be considered valid. Recommended actions of retest or report are also provided depending on the results generated.

The provided FDA 510(k) summary for the "Applied BioSystems™ TaqPath™ COVID-19 Diagnostic PCR Kit" details various studies to establish its performance. Here's a breakdown of the acceptance criteria and study proving device performance, as per your request:

Acceptance Criteria and Reported Device Performance

The acceptance criteria for each study are implicitly demonstrated by the reported performance meeting the standards required for the 510(k) clearance, primarily aiming for high Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to a composite comparator method, along with demonstrating robustness in analytical performance.

Here's a table summarizing key performance characteristics and the reported results:

Study Details to Prove Device Meets Acceptance Criteria

The studies primarily focus on analytical and clinical performance of the PCR kit itself, rather than an AI-driven device with human-in-the-loop interaction. Therefore, aspects related to MRMC studies or expert ground truth for image interpretation are not applicable here.

1. Sample sizes used for the test set and the data provenance:

   • Limit of Detection (LoD):
     • Preliminary LoD: Serial dilutions (number of replicates not specified beyond "three (3) replicates per concentration level" in the WHO Standard test).
     • Confirmatory LoD: 20 replicates at three concentration levels (3x, 1x, 0.33x LoD) for both SARS-CoV-2 USA-WA1/2020 and WHO Standard.
     • Provenance: Contrived samples using inactivated SARS-CoV-2 virus spiked into pooled negative NP specimen matrix. No geographical provenance for these samples is specified, but likely from US. Retrospective in nature as samples are manipulated.
   • Reproducibility and Within-Laboratory Precision: 270 replicates per sample at each test level (0x LoD, 1.5x LoD, 5x LoD) on each PCR instrument model.
     • Provenance: Contrived samples (inactivated SARS-CoV-2 virus in pooled negative NP specimen matrix). Testing performed at three sites (two external, one internal), indicating multi-center analytical study. No geographical provenance for specimens specified. Retrospective.
   • Interfering Substances: 3 replicates per substance level (positive and negative). Some substances tested with 6 replicates at certain concentrations (e.g., Oxymetazoline, Triamcinolone).
     • Provenance: Contrived samples (inactivated SARS-CoV-2 virus at 3x LoD or negative matrix) spiked with interferents. Negative pooled NP samples were used as matrix.
   • Cross-Reactivity and Microbial Interference: 3 replicates of each of 46 microorganisms.
     • Provenance: Microorganisms spiked into pooled negative NP specimen matrix (for cross-reactivity) or contrived positive NP specimen matrix (for microbial interference).
   • Analytical Reactivity (Inclusivity): 3 replicates for each of 15 SARS-CoV-2 variants.
     • Provenance: Contrived samples (inactivated SARS-CoV-2 virus spiked into negative pooled NP specimen).
   • Carry-Over Cross-Contamination: 470 negative samples tested (47 replicates across 10 extraction runs x 2 instruments).
     • Provenance: Contrived samples with high viral titers and negative NP samples.
   • Specimen Storage Stability: Not explicitly stated, but implies multiple replicates at various time points for both positive and negative contrived samples.
     • Provenance: Contrived positive and negative NP samples in VTM.
   • Fresh vs Frozen Equivalency Study: 10 replicates for 0x and 5x LoD; 40 replicates for 1.5x LoD.
     • Provenance: Contrived NP samples in VTM.
   • Clinical Validation (Primary Test Set):
     • Total Subjects: 1076 subjects enrolled across 11 geographically diverse sites in the U.S. (April 2023 - August 2023).
     • Evaluable Samples: 1055 nasopharyngeal swabs (NP) and 1052 anterior nasal swabs (AN). Exclusions resulted in 1053 evaluable NP specimens and 1049 (7500 Fast Dx) / 1052 (QS5 Dx) evaluable AN specimens.
     • Provenance: Prospectively collected from individuals with signs and symptoms of respiratory tract infection in the U.S. "All-comers fashion". Both fresh (tested within 72 hours) and frozen (stored ≤-70°C, tested within 30 days) Category I and Category II specimens. This is prospective data from the US.

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

   • For this PCR diagnostic kit, the "ground truth" for clinical validation is established by a composite comparator method using multiple highly sensitive SARS-CoV-2 molecular assays (other FDA-cleared or authorized PCR tests). Qualified clinical laboratory personnel presumably performed these comparator tests. There is no mention of human expert consensus/adjudication typically seen in AI imaging studies.

3. Adjudication method for the test set:

   • Clinical Validation: A composite comparator approach was used. Samples were tested by two comparator assays (Test A and Test B). If there was discordance between the first two assays, a third comparator assay (Test C) was used to resolve the discrepancy. The "two-out-of-three" rule determined the composite comparator result, which served as the ground truth.

4. 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. This is a diagnostic PCR kit, not an AI-based imaging or interpretive device that requires human readers. Therefore, an MRMC study is not applicable. The device provides a direct biological result (presence/absence of viral RNA).

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

   • Yes, in essence. The analytical performance studies (LoD, reproducibility, cross-reactivity, inclusivity, etc.) evaluate the kit's performance independently of human interpretation nuances beyond standard laboratory procedures and instrument operation. The clinical validation also assesses its performance against a composite ground truth, effectively as a "standalone" diagnostic test. The "COVID-19 Interpretive Software IVD Edition" analyzes data and generates results automatically, which is an algorithmic standalone component.

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

   • A composite comparator method using other molecular diagnostic (RT-PCR) assays. This is considered the clinical reference standard for nucleic acid detection of SARS-CoV-2.

7. The sample size for the training set:

   • For a molecular diagnostic kit like this, a traditional "training set" in the machine learning sense isn't explicitly defined as it would be for an AI algorithm. The development and optimization of the primers, probes, and reaction conditions (which is analogous to "training" in developing molecular assays) are based on extensive genomic data of SARS-CoV-2 and related viruses. The document describes validation studies, not a separate training phase. The "control" elements (positive/negative controls, MS2 Phage internal control) are part of the kit's design to ensure proper function and validate each run.

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

   • Not applicable as there is no explicitly defined "training set" in the context of an AI algorithm or a separately identified ground truth for such a set. The "training" for such a device is likely the iterative design and optimization of the molecular components to achieve specificity and sensitivity based on known viral sequences and biological principles.

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