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The DiaSorin Molecular Simplexa™ COVID-19 & Flu A/B Direct is a real-time RT-PCR assay intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection and differentiation of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza B virus in nasopharyngeal swabs (NPS) from individuals with signs and symptoms of respiratory tract infection.

The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use as an aid in the differential diagnosis of SARS-CoV-2, influenza A and influenza B infection.

Negative results do not preclude SARS-CoV-2, influenza B infection and should not be used as the sole basis for patient management decisions. Positive results do not rule out coinfection with other organisms. Results should be combined with clinical observations, patient history, and epidemiological information.

The Simplexa™ COVID-19 & Flu A/B Direct assay is intended for use by qualified and trained clinical laboratory personnel specifically instructed and trained in the techniques of real-time PCR and in vitro diagnostic procedures.

The Simplexa™ COVID-19 & Flu A/B Direct assay system is a real-time RT-PCR system that enables the direct amplification, detection and differentiation of SARS-CoV-2 RNA, human influenza A (Flu A) virus RNA and human influenza B (Flu B) virus RNA from unprocessed nasopharyngeal swabs (NPS) that have not undergone nucleic acid extraction. The system consists of the Simplexa™ COVID-19 & Flu A/B Direct assay, the LIAISON® MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ COVID-19 & Flu A/B Direct assay, fluorescent probes are used together with corresponding forward and reverse primers to amplify SARS-CoV-2, Flu A, Flu B and internal control RNA targets. For COVID-19 detection, the assay targets two different regions specific to the SARS-CoV-2 genome; the S gene which encodes the spike glycoprotein and the ORF1ab region which encodes wellconserved non-structural proteins and therefore is less susceptible to recombination. For Flu detection the assay targets conserved regions of influenza A viruses (matrix gene) and influenza B viruses (matrix gene). The assay provides three results; COVID-19 (ORF1ab and/or S gene detection), influenza A viruses (matrix gene detection) and influenza B viruses (matrix gene detection). An RNA internal control is used to detect RT-PCR failure and/or inhibition.

This document describes the analytical and clinical performance studies for the DiaSorin Molecular Simplexa™ COVID-19 & Flu A/B Direct assay.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the clinical performance are generally indicated by the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) with their corresponding 95% Confidence Intervals (CI).

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

• Prospective Samples: Over 1400 total specimens (nasopharyngeal swabs (NPS)) were collected between August 2021 and March 2022 from six geographically diverse clinical sites within the United States. The exact number of prospective samples used for each target in the agreement analysis can be inferred from the TP/(TP+FN) and TN/(TN+FP) values in Table 2:
  • Influenza A: 1168 (62 positive, 1106 negative, plus 5 and 2 discrepant)
  • Influenza B: 1165 (all negative)
  • SARS-CoV-2: 496 (68 positive, 428 negative, plus 1 and 11 discrepant)
• Retrospective Samples: 82 positive influenza B specimens and 62 negative specimens were used. These were blinded and randomized for the study. The exact number of retrospective samples used for each target in the agreement analysis can be inferred from the TP/(TP+FN) and TN/(TN+FP) values in Table 3:
  • Influenza A: 258 (82 positive, 176 negative)
  • Influenza B: 258 (114 positive, 144 negative)
  • SARS-CoV-2: 252 (all negative)

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 of experts or their specific qualifications (e.g., radiologist with 10 years of experience) used to establish the ground truth for the test set.

4. Adjudication Method for the Test Set

• SARS-CoV-2: For SARS-CoV-2, a composite reference method (CRM) was used. This involved three COVID-19 Emergency Use Authorized (EUA) NAAT assays. The adjudication method was:
  • "Detected" CRM if two out of three EUA assays were positive.
  • "Not Detected" CRM if two out of three EUA assays were negative.
• Influenza A and B: For influenza A and B, the comparator was an FDA-cleared NAAT. There is no mention of a multi-assay composite reference method, suggesting a single FDA-cleared NAAT was used as the ground truth. Discrepancy analysis involved additional FDA cleared NAATs and PCR followed by BDS.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. This study focuses on the standalone performance of the diagnostic assay rather than human reader performance with or without AI assistance.

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

Yes, a standalone performance study was done. The entire clinical performance section evaluates the Simplexa™ COVID-19 & Flu A/B Direct assay's ability to detect and differentiate nucleic acids directly, without human interpretation in the results reporting, other than laboratory personnel operating the instrument. The results are presented as the assay's agreement with reference methods.

7. The Type of Ground Truth Used

• SARS-CoV-2: Composite reference method (CRM) based on the consensus of three COVID-19 Emergency Use Authorized (EUA) NAAT assays.
• Influenza A and B: An FDA-cleared NAAT was used as the primary comparator. In cases of discrepancy, additional FDA-cleared NAATs and PCR followed by Bidirectional Sequencing (BDS) were used for confirmation.

8. The Sample Size for the Training Set

The document describes the clinical performance (test set) and analytical studies. It does not explicitly mention a "training set" in the context of machine learning. The assay is a real-time RT-PCR assay, which typically relies on pre-defined primer and probe sequences rather than a machine learning model that requires a distinct training set in the conventional sense. The development and optimization of the primer/probe sets (e.g., analytical reactivity, inclusivity) can be considered analogous to a "training" or development phase, but no specific dataset labeled as such is provided.

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

As noted in point 8, a traditional "training set" with ground truth establishment in the context of machine learning is not applicable to this RT-PCR assay. The analytical studies (Limit of Detection, Analytical Reactivity/Inclusivity, Cross-Reactivity, Interfering Substances, Competitive Interference, Microbial Interference) use quantitated viral stocks, cultured isolates, purified nucleic acids, or in silico analysis against public strain databases (e.g., GISAID) to demonstrate the assay's analytical performance across a wide range of relevant targets and conditions. This ensures the assay's biochemical design (primers, probes) is sound and effective.

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