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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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The DiaSorin Molecular Simplexa™ COVID-19 Direct is real-time RT-PCR assay intended for use on the LIAISON® MDX instrument for the in vitro qualitative detection of nucleic acid from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs (NPS) and nasal swabs (NS) from symptomatic individuals suspected of COVID 19 by their healthcare provider. The Simplexa™ COVID-19 Direct assay is an aid in the diagnosis of SARS-CoV-2 infection.

Positive results are indicative of the presence of SARS-CoV-2 RNA. Clinical correlation with patient history and other diagnostic information is necessary to determine patient infection status. Positive results do not rule out co-infection with other pathogens. Negative results do not preclude SARS-CoV-2 infection and should not be used as tor patient management decisions.

Results are meant to be used in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the relevant public health authorities.

The Simplexa COVID-19 Direct is a real-time RT-PCR (rRT-PCR) system that enables the direct amplification and detection of SARS-CoV-2 (COVID-19) RNA from nasopharyngeal swab or nasal swab that has not undergone nucleic acid extraction. The system consists of the Simplexa COVID-19 Direct reaction mix, the LIAISON MDX (with LIAISON MDX Studio Software), the Direct Amplification Disc and associated accessories. The assay uses forward and reverse primers and associated fluorescent probe(s) included in the reaction mix to amplify SARS-CoV-2 cDNA reverse transcribed from RNA. The primers and probe sets are designed to detect SARS-CoV-2 ORF 1ab and S gene from the viral RNA in nasopharyngeal swab or nasal swab. An RNA internal control, with associated primers and a fluorescent probe, is included in the reaction mix to detect RT-PCR failure and/or inhibition.

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

Device: Simplexa™ COVID-19 Direct

1. Table of Acceptance Criteria and Reported Device Performance

For the Simplexa™ COVID-19 Direct assay, the primary acceptance criteria revolve around its accuracy in detecting SARS-CoV-2 (COVID-19) RNA in patient samples, as well as its reproducibility, analytical sensitivity (Limit of Detection), analytical reactivity (ability to detect various strains), and specificity (cross-reactivity and interference).

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

• Clinical Agreement Test Set:

  • Total Samples: 1,150 prospective (fresh and/or frozen) samples collected.
  • Samples analyzed: 1,011 samples (114 excluded due to insufficient evidence for media types, 24 invalid results, 1 indeterminate CRM result).
  • Breakdown: 299 Nasopharyngeal Swabs (NPS) and 712 Nasal Swabs (NS).
  • Provenance: Collected from four (4) geographically diverse collection sites, one of which was outside the United States (OUS). Samples were prospective (fresh and/or frozen).
  • Timeframe: October 2020 to April 2021.

• Reproducibility Test Set:

  • Total replicates: 90 replicates per panel member (4 panel members), totaling 360 individual tests.
  • Panel members: 2 contrived low positive (LP), 2 contrived moderate positive (MP), 1 positive control, 1 negative (UTM).
  • Provenance: Tested at two (2) external clinical sites and one (1) internal site.
  • Study Design: Each panel member tested in triplicate per run, for 2 runs per day, for 5 non-consecutive testing days. Each site had two operators.

• Analytical Sensitivity (LoD) Test Set:

  • NPS: 40 replicates for confirmation.
  • NS: 20 replicates for confirmation.
  • WHO International Standard: 40 replicates for confirmation.

• Analytical Reactivity (Wet testing) Test Set:

  • 3 replicates per strain for 5 SARS-CoV-2 strains.

• Cross-Reactivity Test Set:

  • 3 replicates per organism for 47 different viruses, bacteria, and fungi (some 6 replicates for Leptospira interrogans).

• Potential Interfering Substances Test Set:

  • 3 replicates per substance (some 6 replicates for saliva and Zanamivir).

• Interference by Other Microorganisms Test Set:

  • 3 replicates per organism (some 6 replicates for Lactobacillus plantarum 17-5).

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

The ground truth for the clinical agreement test set was established using a "Composite Reference Method (CRM)" based on three (3) COVID-19 EUA approved NAAT assays. The rule for CRM agreement was: "Two out of three positive results determined 'Detected' CRM and two out of three negative results determined 'Not Detected' CRM."

The document does not specify the number or qualifications of experts (e.g., medical technologists, clinical lab scientists, or physicians) who performed these NAAT assays or interpreted their results for the CRM. It's implied that these were standard laboratory personnel qualified to run EUA-approved molecular diagnostic tests.

For analytical studies (LoD, reproducibility, reactivity, cross-reactivity, interference), the ground truth was based on the known composition and concentration of the samples (e.g., spiked RNA, cultured organisms, negative matrix). No external experts beyond the study design team would have been needed for this type of ground truth establishment.

4. Adjudication Method for the Test Set

For the clinical agreement test set, the adjudication method for the ground truth (CRM) was clearly defined: "Two out of three positive results determined 'Detected' CRM and two out of three negative results determined 'Not Detected' CRM." This is a form of consensus-based adjudication, specifically a majority rule.

For other analytical studies, adjudication was not described as it involved pre-defined positive/negative samples rather than interpretive human judgment.

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 in vitro diagnostic performance of a molecular assay (RT-PCR) in a laboratory setting, not on the interpretative performance of human readers (e.g., radiologists) with or without AI assistance. Therefore, there is no discussion of human readers or an effect size of AI assistance.

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

Yes, the primary clinical performance and analytical studies are standalone (algorithm only) performance. The Simplexa™ COVID-19 Direct is an RT-PCR assay. Its "performance" refers to its ability to detect SARS-CoV-2 RNA based on its set algorithms for signal detection (Ct values for S gene and ORF1ab targets) and interpretation. The results (detected/not detected) are determined directly by the instrument and its software, not by a human interpreting images or complex patterns. The human involvement is in sample preparation and loading, and reviewing the qualitative output from the instrument.

7. The Type of Ground Truth Used

• Clinical Agreement Test Set: Composite Reference Method (CRM) using results from three (3) COVID-19 EUA approved NAAT assays, with a "two out of three" majority rule for determining "Detected" or "Not Detected." This is a form of expert consensus based on other validated diagnostic tests.
• Analytical Studies (Reproducibility, LoD, Reactivity, Cross-Reactivity, Interference): Known analytical truth established by spiking known concentrations of inactivated viral particles or other organisms into negative matrices. This is a laboratory-controlled ground truth.

8. The Sample Size for the Training Set

The provided document describes a premarket notification (510(k)) for an in vitro diagnostic device. For such devices, particularly RT-PCR assays, the "training set" typically refers to internal development and optimization data, rather than a distinct, formally defined "training set" for machine learning algorithms that would be tested on a separate "test set."

The document does not specify a numerical sample size for a training set. The assay's design (primers, probes, conditions) would have been developed and optimized internally by DiaSorin Molecular using various samples and experiments, but these are not enumerated as a specific "training set" in this regulatory submission. The "in silico inclusivity analysis" section points to the use of GISAID databases (millions of sequences) which could be considered a form of "training data" for validating the generalizability of the primer/probe design, but not as a conventional, labeled "training set" in a machine learning context.

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

Since a formal "training set" with established ground truth is not explicitly detailed in the way a machine learning model's training data would be, we can infer the following:

• Assay Development & Optimization: The ground truth for the development phase would have been based on known positive and negative samples, viral loads, and various SARS-CoV-2 strains or synthetic genetic material. This involves standard molecular biology techniques where the presence or absence of the target nucleic acid, and its concentration, are experimentally determined and controlled.
• In Silico Inclusivity: For the evaluation of primer/probe design against genetic variants, the "ground truth" is the published, annotated SARS-CoV-2 genome sequences available in the GISAID database. This involves bioinformatic analysis to determine sequence homology and potential binding efficacy.

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