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The Alinity m HSV 1 & 2 / VZV assay is an in vitro real-time polymerase chain reaction (PCR) assay for the qualitative detection and differentiation of Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2) and Varicella Zoster Virus (VZV) DNA from clinician-collected cutaneous or mucocutaneous lesion swab specimens from symptomatic patients suspected of active herpes simplex virus 2 and/or varicella-zoster infection. The Alinity m HSV 1 & 2 / VZV assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster active cutaneous or mucocutaneous infections. Negative results do not preclude herpes virus type 1, herpes simplex virus type 2 or varicella-zoster virus infections and should not be used as the sole basis for diagnosis, treatment or other management decisions.

The Alinity m HSV 1 & 2 / VZV assay is not intended for use with cerebrospinal fluid (CSF) or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Alinity m HSV 1 & 2 / VZV assay is not intended for use in prenatal screening.

The Alinity m HSV 1 & 2 / VZV assay is an in vitro real-time polymerase chain reaction (PCR) assay for the qualitative detection and differentiation of Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2) and Varicella Zoster Virus (VZV) DNA from clinician--collected cutaneous or mucocutaneous lesion swab specimens from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster infection. This assay is intended for use with the automated Alinity m System.

The steps of the Alinity m HSV 1 & 2 / VZV assay consist of sample preparation, PCR assembly, amplification/detection, and result calculation and reporting. The steps involved in all stages of the Alinity m HSV 1 & 2 / VZV assay procedure are executed automatically by the Alinity m System. No intermediate processing or transfer steps are performed by the user. The Alinity m System is designed to be a random access analyzer that can perform the Alinity m HSV 1 & 2 / VZV assay in parallel with other Alinity assays on the same instrument.

The Alinity m HSV 1 & 2 / VZV assay requires two separate assay specific kits as follows:

1. The Alinity m HSV 1 & 2 / VZV AMP Kit (List No. 09N61-095) is comprised of 2 types of multi-well trays:

TRAY 1: Alinity m HSV 1 & 2 / VZV AMP TRAY 1 TRAY 2: Alinity m HSV 1 & 2 / VZV ACT TRAY 2.

TRAY 1 - Alinity m HSV 1 & 2 / VZV AMP is individually packed in a foil pouch and contains 48 unit-dose liquid amplification reagent wells and 48 unitdose liquid IC wells. One well of each is used per test.

· Amplification reagent wells consist of synthetic oligonucleotides, DNA Polymerase, dNTPs, and 0.15% ProClin® 950 in a buffered solution with a reference dye. IC wells consist of plasmid DNA with unrelated IC sequences and poly dA:dT in a TE buffer containing 0.15% ProClin 950 as a preservative.

TRAY 2 - Alinity m HSV 1 & 2 / VZV ACT is individually packed in a foil pouch and contains 48 unit-dose liquid activation reagent wells. One reagent well is used per test.

· Activation reagent wells consist of magnesium chloride, potassium chloride, and tetramethylammonium chloride. Preservative: 0.15% ProClin 950.

2. The Alinity m HSV 1 & 2 / VZV CTRL Kit (09N61-085) consists of negative controls and positive controls, each supplied as liquid in single-use tubes.

Alinity m HSV 1 & 2 / VZV Negative CTRL (List No. 9N61Z) consists of Negative Diluent / TE buffer (containing 0.085% Sodium Azide and 0.087% ProClin 950).

Alinity m HSV 1 & 2 / VZV Positive CTRL (List No. 9N61W) consists of linearized plasmid DNA containing HSV-1, HSV-2 and VZV DNA sequences in Negative Diluent / TE buffer (containing 0.085% Sodium Azide and 0.087% ProClin 950).

The Alinity m HSV 1 & 2 / VZV assay is to be run on the Alinity m System which is a fully integrated, sample to result automated system that performs real-time PCR test using the Alinity m HSV 1 & 2 / VZV AMP Kit along with the Alinity m HSV 1 & 2 / VZV CTRL Kit.

The provided text describes the performance of the "Alinity m HSV 1 & 2 / VZV" assay, which is an in vitro real-time PCR assay for detecting and differentiating Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), and Varicella Zoster Virus (VZV) DNA.

Here's an analysis of the acceptance criteria and study proving the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by the clinical agreement and analytical performance values demonstrated to establish substantial equivalence to a predicate device. For clinical agreement, the performance is measured by Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to a commercially available NAAT comparator method.

The acceptance criteria typically would be pre-defined statistical thresholds for PPA and NPA (e.g., PPA ≥ 95% and NPA ≥ 95%). Based on the reported results, the device successfully meets high performance metrics, suggesting that the implicit acceptance criteria were met for substantial equivalence.

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

• Prospective Study Test Set:
  • Sample Size: 1,258 results (1,257 for HSV-1, 1,257 for HSV-2, and 1,257 for VZV) from unique specimens.
  • Data Provenance: Multicenter, prospective clinical study conducted in the United States. Specimens were clinician-collected from symptomatic individuals.
• Retrospective Study Test Set:
  • Sample Size: 411 specimens (410 for HSV-1, 410 for HSV-2, and 411 for VZV).
  • Data Provenance: Retrospective study using archived, leftover lesion swab specimens from routine clinical testing. The location is not explicitly stated, but given it follows a prospective US study and is for US FDA clearance, it is highly likely to be from the US.

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

• The document does not specify the number or qualifications of experts used to establish the ground truth.
• The ground truth was established by comparison to a "commercially available NAAT comparator method (NAAT 1)". For discordant results, a "second commercially available RT-PCR comparator method (NAAT 2)" was used for informational purposes only (not for the primary analysis of device performance). This means the ground truth is essentially defined by the predicate/comparator device(s).

4. Adjudication Method for the Test Set

• The primary ground truth was established by a single comparator method (NAAT 1).
• For specimens with discordant results between the Alinity m assay and NAAT 1, a second NAAT method (NAAT 2) was used. However, the results of this second test were "not included in the analysis of device performance and are considered for information purposes only." This implies that the primary PPA/NPA calculations are based solely on the agreement with NAAT 1, and there was no formal 2+1 or 3+1 adjudication to establish a "resolved" ground truth for discordant cases that would then be used in the performance calculations.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, and what was the effect size of how much human readers improve with AI vs without AI assistance

• This is not applicable as the device is an in vitro diagnostic (IVD) assay detecting viral DNA, not an AI-assisted diagnostic imaging device requiring human reader interpretation. There are no "human readers" in the context of this device.

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

• Yes, this is a standalone device performance study. The Alinity m HSV 1 & 2 / VZV assay is an automated system described as "fully integrated, sample to result." The performance data presented (clinical agreement, analytical sensitivity, specificity, precision, reproducibility, carryover) represent the performance of the algorithm/assay system without direct human interpretation of raw data being a variable. The "results are calculated and reported" automatically by the Alinity m System.

7. The Type of Ground Truth Used

• The ground truth was established by comparison to a "commercially available NAAT comparator method (NAAT 1)." For discordant cases, a second NAAT (NAAT 2) was used, but its results were for informational purposes only. This is a form of clinical surrogate ground truth, relying on an established and legally marketed diagnostic method. It is not pathology, expert consensus (in the traditional sense of multiple human interpretations), or outcomes data.

8. The Sample Size for the Training Set

• The document does not provide information regarding a specific training set size or how the device's algorithms (if any, beyond PCR signal processing) were trained. Regulatory submissions for IVD assays primarily focus on analytical and clinical validation of the final, locked-down device performance, rather than algorithm development specifics. The device seems to be a PCR assay, where the "training" would be more akin to assay design and optimization, not machine learning model training on large datasets in the way an AI imaging model is.

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

• As the document does not mention a training set in the context of machine learning, there is no information on how a ground truth for such a set would have been established. For a PCR assay, the development process involves extensive analytical characterization, optimization, and verification using characterized samples (e.g., known positive/negative samples, spiked samples, reference strains) to establish assay parameters and thresholds. This is distinct from establishing ground truth for a machine learning training dataset.

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The Savanna HSV 1+2/VZV Assay is an automated, rapid multianalyte real-time PCR test for the simultaneous qualitative detection and differentiation of herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus DNA isolated from human cutaneous or mucocutaneous lesion samples obtained from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster infection. This in vitro diagnostic test is intended to aid in the diagnosis of patients with signs or symptoms of herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus infection.

The Savanna HSV 1+2/VZV Assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and varicella-zoster virus active infections. The results of this test should not be used as the sole basis for diagnosis, treatment or other management decisions and must be combined with clinical observations, patient history and/or epidemiological information. Negative results do not preclude herpes simplex virus type 1, herpes simplex virus type 2, or varicella-zoster virus infection that is not detected by a cutaneous or mucocutaneous lesion swab specimen. Positive results do not rule out co-infection with other organisms. Additional laboratory testing (e.g., viral culture, immunoassay, serology) may be necessary for patient evaluation. Savanna HSV 1+2/VZV Assay is for professional use. The Savanna HSV 1+2/VZV Assay is intended for use only with the Savanna instrument.

Warning: The Savanna HSV 1+2/VZV Assay is not intended for use with the cerebrospinal fluid (CSF) or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Savanna HSV 1+2/VZV Assay is not intended for use in prenatal screening.

The Savanna HSV 1+2/VZV Assay consists of a single, self-contained assay cartridge employing real-time PCR technology for use with the Savanna instrument to detect and differentiate DNA from herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus. In approximately 24 minutes, this platform extracts, amplifies and detects DNA present in cutaneous or mucocutaneous lesion swab specimens obtained from symptomatic patients and placed in transport media.

To initiate the assay, a patient cutaneous or mucocutaneous lesion swab specimen in transport medium is transferred via the supplied transfer pipette to the Liguid Sample Port of the Test Cartridge. The user closes the Sample Port and inserts the Test Cartridge into the Savanna instrument, initiating sample processing. The sample containing human DNA is pushed out of the Sample Port by lysis buffer, which then rehydrates the Process Internal Control (IC), and together with the paramagnetic nucleic acid binding particles, are pumped into the extraction chamber. The solution is mixed, and virus and/ or bacteria are further lysed by sonication within the extraction chamber. Specimen and IC DNA are bound to, washed and then eluted off the paramagnetic particles. The purified specimen DNA and IC solution is used to rehydrate four individual lyophilized master mixes. Each master mix is pumped into a PCR chamber and Taq-man® multiplex real-time PCR reactions are carried out under optimized conditions, generating amplicons for the targeted pathogen (if present) and the Process Internal Control (IC).

Each master mix contains primers and dual-labeled probes unique for the pathogen targets and the IC. The probes are labeled with a fluorophore on one end and a quencher on the other end. Target DNA sequences are amplified by pathogen-specific primers and detected by correspondingly specific fluorescence probes. The IC targets are also amplified by specific primers and detected by an IC-specific fluorescence probe. A polymerase included in the master mix cleaves the probes bound to complementary DNA sequences, separating the fluorophore from the quencher. This step generates a signal and if it surpasses multiple defined thresholds, the sample is reported as positive for the detected target sequence.

The Savanna instrument will display the test results (Positive or Invalid) on the main bay screen.

The provided text describes the performance characteristics and clinical studies of the Savanna HSV 1+2/VZV Assay, which is an in vitro diagnostic (IVD) device for the detection and differentiation of herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus DNA. It is not an AI/ML device in the context of typical FDA AI/ML medical device submissions, as it is a molecular diagnostic test. Therefore, many of the requested points regarding AI/ML device evaluation (like human-in-the-loop, expert consensus for ground truth, MRMC studies, number of experts for ground truth, adjudication methods, and sample size for training set) are not applicable to this type of device.

However, I can extract the acceptance criteria and performance data for this diagnostic assay as presented. The study aims to demonstrate substantial equivalence to a predicate device, the Solana HSV 1+2/VZV Assay (K162451), not to prove performance of an AI model.

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state "acceptance criteria" in a tabulated format for the clinical performance. Instead, it presents the results of the clinical studies with Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) values with their confidence intervals, comparing the Savanna Assay to a commercially available RT-PCR comparator method. For laboratory studies, the acceptance criteria are implied by the results (e.g., >95% positivity for LoD, 100% detection for inclusivity, no interference/cross-reactivity).

I will present the clinical performance results as the "reported device performance" against an implied high standard of agreement with the comparator method.

Table: Reported Device Performance (Clinical Study 1 & 2)

2. Sample size used for the test set and the data provenance

• Clinical Study 1 (Fresh Samples):

  • Sample Size: 590 residual specimens (207 cutaneous, 383 mucocutaneous).
  • Data Provenance: United States, from symptomatic patients suspected of HSV-1, HSV-2, or VZV infection. Retrospective (residual specimens).

• Clinical Study 2 (Frozen Residual Samples):

  • Sample Size: 154 evaluable samples.
  • Data Provenance: Not explicitly stated but implies U.S. as it's a supplement to Clinical Study #1. Retrospective (residual frozen samples).

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

This is not applicable as this is an IVD device, not an AI/ML device relying on human expert labels for ground truth. The ground truth is established by a comparator FDA-cleared nucleic acid amplification test.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

Not applicable. The ground truth is determined by the results of an FDA-cleared comparator nucleic acid amplification test, not by human adjudication of images.

5. 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

Not applicable. This is an IVD device, not an AI/ML device intended to assist human readers.

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

The device is a standalone diagnostic assay (real-time PCR test on the Savanna instrument). Its performance is evaluated independently against a comparator method. The results presented (PPA, NPA) represent this standalone performance.

7. The type of ground truth used

The ground truth for the clinical studies was established by comparison to FDA-cleared nucleic acid amplification tests (comparator method).

8. The sample size for the training set

Not applicable. This is not an AI/ML device that requires a "training set" in the context of machine learning model development. The robust performance of an IVD often relies on extensive analytical studies (LoD, inclusivity, cross-reactivity, precision, etc.) and clinical validation.

9. How the ground truth for the training set was established

Not applicable, as there is no "training set" in the AI/ML sense. The analytical performance was established through various laboratory studies (e.g., Limit of Detection, Inclusivity, Cross-Reactivity, Precision) using well-characterized viral strains and matrices.

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The DiaSorin Molecular Simplexa™ VZV Swab Direct assay is intended for use on the LIAJSON® MDX instrument for the qualitative detection of varicella-zoster virus (VZV) DNA present in cutaneous and mucocutaneous lesion swabs from patients with signs and symptoms of VZV infection. This test is intended as an aid in the diagnosis of VZV infection. Negative results do not preclude VZV infection and should not be used as the sole basis for treatment or other patient management decisions.

The Simplexa™ VZV Positive Control Pack is intended to be used as a control with the Simplexa™ VZV Direct kit and the Simplexa™ VZV Swab Direct kit on the LIAISON® MDX Instrument. It is not intended for use with other assays or systems.

The Simplexa™ VZV Swab Direct assay is a real-time PCR system that enables the direct amplification and detection of VZV DNA from unprocessed cutaneous and mucocutaneous lesion swab specimens without nucleic acid extraction. The system consists of the Simplexa™ VZV Swab Direct assay, the LIAISON® MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc (DAD) and associated accessories.

In the Simplexa™ VZV Swab Direct assay, fluorescent probes are used together with corresponding forward and reverse primers to amplify VZV and internal control targets. A well-conserved region of the VZV DNA polymerase gene is targeted to identify VZV DNA in the specimen. An internal control is used to detect PCR failure and/or inhibition.

This document describes the validation of the DiaSorin Molecular Simplexa™ VZV Swab Direct assay. The assay is intended for the qualitative detection of varicella-zoster virus (VZV) DNA directly from cutaneous and mucocutaneous lesion swabs.

1. Table of Acceptance Criteria and Reported Device Performance:

The document implicitly defines acceptance criteria through the results of the clinical and analytical studies, which consistently demonstrate high agreement and sensitivity/specificity.

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The HSV 1&2 ELITe MGB® Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of Herpes Simplex Virus 1 and 2 (HSV-2) DNA in cutaneous or mucocutaneous lesion swab specimens from patients with signs and symptoms of HSV-2 infection. This test is an aid in the differential diagnosis of HSV-1 and HSV-2 infections.

The HSV 1&2 ELITe MGB Assay is not FDA cleared for use with cerebrospinal fluid (CSF) specimens. The assay is not intended to be used for prenatal screening or for screening blood or blood products.

The HSV 1&2 ELITe MGB Assay is a qualitative in vitro diagnostic Real-Time PCR Assay for the direct detection of Herpes Simplex Virus (HSV) DNA (glycoprotein D gene for HSV-1 and glycoprotein G gene for HSV-2) in symptomatic male and female patients using DNA purified from swab specimens collected from individuals with cutaneous or mucocutaneous herpetic lesions.

The HSV 1&2 ELITe MGB Assay system is comprised of three major processes: (1) automated preparation of unprocessed sample to extract nucleic acids from primary swab specimens using the ELITe InGenius SP 200 Extraction Cartridge, (2) PCR amplification of target DNA sequences using HSV-1 and HSV-2 specific primers, and (3) real-time detection of fluorescent-labeled HSV-1 and HSV-2 specific oligonucleotide detection probes.

An Internal Control (IC), containing unrelated randomized DNA sequence, is added to all samples prior to extraction and monitors the integrity of the reagents, equipment function, and the presence of inhibitors in the samples. A positive signal in the Internal Control channel in the absence of HSV DNA indicates that the PCR has not been inhibited.

The amplification reagents, Positive Control and Internal Control are packaged as part of the HSV 1&2 ELITe MGB Assay.

Here's a breakdown of the acceptance criteria and study information for the HSV 1&2 ELITe MGB Assay, based on the provided text:

Acceptance Criteria and Reported Device Performance

The acceptance criteria for the HSV 1&2 ELITe MGB Assay are presented in terms of Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) against a composite reference method.

Study Information

1. Sample Size Used for the Test Set and Data Provenance:

   • Test Set Sample Size: 1,174 prospectively collected archived swab samples were initially collected. After exclusions:
     • 1,171 samples were analyzed for HSV-1.
     • 1,170 samples were analyzed for HSV-2.
     • A separate contrived oral panel study involved 75 individual negative cheek swab samples spiked with HSV-2, plus 10 HSV-1 positive samples and 15 HSV-1/HSV-2 negative oral samples.
   • Data Provenance: The samples were "left-over prospectively collected archived swab samples" from symptomatic patients. The country of origin is not explicitly stated, but the context of an FDA submission generally implies US-based data.

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

   • The document does not explicitly state the number of experts or their qualifications for establishing the ground truth. The ground truth was established using a composite reference method.

3. Adjudication Method for the Test Set:

   • The adjudication method for the test set was a "composite reference method" defined as: "an FDA cleared assay and a validated HSV 1&2 PCR followed by bi-directional sequencing of gel electrophoresis-positive samples."
   • "A positive result by the composite reference method is defined as a positive by the FDA cleared PCR or the validated sequencing. Two negative results are needed to confirm a negative." This implies a type of adjudicted consensus, where the "truth" is determined by agreement or hierarchical resolution between different methods, rather than human experts per se.

4. If a Multi Reader Multi Case (MRMC) Comparative Effectiveness Study Was Done:

   • No. This document describes the analytical and clinical performance of an in vitro diagnostic (IVD) assay, which is an algorithm-only (standalone) test. It does not involve human readers in the primary diagnostic decision-making process that would warrant an MRMC study comparing human performance with and without AI assistance.

5. If a Standalone Performance Study Was Done:

   • Yes. The entire clinical performance section (Section 11.b) is a standalone performance study where the HSV 1&2 ELITe MGB Assay's performance is compared directly against a composite reference method. The assay operates without human intervention in the interpretation of results.

6. The Type of Ground Truth Used:

   • Composite Reference Method: This ground truth combined an FDA-cleared PCR assay and a validated HSV 1&2 PCR followed by bi-directional sequencing. This type of ground truth integrates highly sensitive and specific molecular methods, reflecting a robust laboratory-based gold standard.

7. The Sample Size for the Training Set:

   • The document does not explicitly mention a "training set" in the context of an AI/ML algorithm. This device is a real-time PCR assay, which is a chemical and enzymatic reaction rather than a machine learning model that would typically require a separate training set. The various analytical performance studies (Limit of Detection, Analytical Reactivity, Specificity, etc.) serve to characterize the assay's performance across different conditions, which is analogous to how a system is "trained" or refined during development, but not in the sense of a distinct machine learning training set. The "assay cut-off" was established using a separate set of 141 clinical samples, which could be considered a form of calibration/training for the threshold of positivity.

8. How the Ground Truth for the Training Set Was Established:

   • As noted above, a traditional "training set" for an AI model is not applicable here. For the establishment of the assay cut-off (using 141 clinical samples), the ground truth was established using the same "composite reference method" (FDA-cleared real-time PCR assay combined with PCR amplification and bidirectional sequencing). For other analytical studies (like LoD, inclusivity, cross-reactivity), quantitated viral strains or characterized organisms were used as the reference.

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The DiaSorin Molecular Simplexa™ HSV 1 & 2 Direct assay is intended for use on the LIAISON® MDX instrument for the qualitative detection and differentiation of herpes simplex virus (HSV-2) DNA present in mucocutaneous and cutaneous lesion swabs from patients with signs and symptoms of HSV-1 or HSV-2 infection. This test is an aid in the differential diagnosis of HSV-1 and HSV-2 infections.

The assay is not intended for use as a screening test for the presence of HSV-2 in blood or blood products. The assay is for professional use only.

Simplexa™ HSV 1 & 2 Positive Control Pack MOL2160

The Simplexa™ HSV 1 & 2 Positive Control Pack is intended to be used as a control with the Simplexa™HSV 1 & 2 Direct kit.

This control is not intended for use with other assays or systems.

The Simplexa™ HSV 1 & 2 Direct assay system is a real-time PCR that enables the direct amplification, detection and differentiation of HSV-1 and/or HSV-2 DNA from unprocessed cutaneous and mucocutaneous lesion swab specimens without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct assay, the LIAISON® MDX (with LIAISON® MDX Studio Software), the Direct Amplification Disc and associated accessories.

In the Simplexa™ HSV 1 & 2 Direct assay, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1, HSV-2 and internal control targets. Well conserved regions of the HSV-1 and HSV-2 DNA polymerase genes are targeted to identify HSV-1 and HSV-2 DNA respectively in the specimen. An internal control is used to detect PCR failure and/or inhibition.

Here's a breakdown of the acceptance criteria and study information for the Simplexa™ HSV 1 & 2 Direct device, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria (e.g., "PPA must be ≥ 95%"). Instead, it presents the "Clinical Agreement" results from validation studies and implies that these figures demonstrate acceptable performance for clearance. Therefore, the "acceptance criteria" listed below are inferred from the reported performance, representing the observed outcomes deemed sufficient for the device to be considered substantially equivalent.

Note on "Acceptance Criteria": The document provides the study results directly. For diagnostics, these agreement percentages are typically compared against pre-specified acceptance criteria. The absence of explicit acceptance criteria in the provided text implies that the reported performance was considered adequate for FDA clearance.

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

• Prospective Study 1 (K150962 data, re-used):
  • Original Collected: 718 samples
  • Evaluated: 696 samples (after removals for various reasons like not tested, invalid results, internal control failure, wrong sample type, insufficient volume, control issues, non-enrollable)
  • Data Provenance: From 6 geographically diverse locations, prospectively collected from May 28, 2014, through December 4, 2014.
• Prospective Study 2 (K173798 data):
  • Original Collected: 514 samples
  • Evaluated:
    • 511 samples for Simplexa™ HSV 1 & 2 Direct
    • 512 samples for culture method
    • 510 samples for bi-directional sequencing method (after removals for EC505 codes, insufficient volume, daily control issues, non-enrollable)
  • Data Provenance: From 4 geographically diverse sites, prospectively collected from July 24, 2017, through October 11, 2017.
• Retrospective Study:
  • Evaluated: 174 Cutaneous HSV-1 swabs, 174 Mucocutaneous HSV-2 swabs, and 17 samples from unknown locations.
  • Data Provenance: Retrospectively collected from June 6, 2011, to May 17, 2014, and February 21, 2017, through July 17, 2017.

All clinical samples were cutaneous and mucocutaneous lesion swabs from patients with signs and symptoms of HSV-1 or HSV-2 infection.

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

The document does not specify the number or qualifications of experts for establishing ground truth. The ground truth was established using a "composite comparator algorithm" rather than expert opinion alone.

4. Adjudication Method for the Test Set

The ground truth was established using a composite comparator algorithm based on a "2 out of 3 rule":

• Components: Culture, bi-directional sequencing, and an FDA-cleared NAAT (Nucleic Acid Amplification Test).
• Method: Any sample yielding a positive result by either sequencing or culture was then tested on an FDA-cleared NAAT. A "2 out of 3 rule" (implying agreement between at least two of the three methods) was used to determine the final composite results.
• Note: For culture, HSV-2 was tested first. If positive for HSV-2, no further culture testing was done (meaning dual positives could not be identified by culture alone).

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

No, an MRMC comparative effectiveness study was not explicitly done. The study focuses on evaluating the device's performance against a composite reference method, not on human reader performance with or without AI assistance. The device is a molecular diagnostic assay, not an AI-assisted diagnostic tool for human readers.

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

Yes, the studies presented are for the standalone performance of the Simplexa™ HSV 1 & 2 Direct assay, which is a real-time PCR system. This system performs direct amplification, detection, and differentiation of HSV-1 and HSV-2 DNA from unprocessed swab specimens, without requiring human interpretation of results beyond reading the instrument's output. The "algorithm" here refers to the PCR assay's mechanics and the LIAISON® MDX instrument's software for detection.

7. The Type of Ground Truth Used

The ground truth for the clinical studies was a composite comparator algorithm consisting of:

• Cell culture
• Bi-directional sequencing
• An FDA-cleared NAAT

This is considered a robust method for establishing ground truth in molecular diagnostics, combining phenotypic (culture) and genotypic (sequencing, NAAT) evidence.

8. The Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of machine learning, as this is a molecular diagnostic assay cleared through substantial equivalence, not an AI/ML-based device requiring separate training and test sets in that sense. The analytical studies (reproducibility, LoD, cross-reactivity, interference) use contrived samples and defined panels. The "clinical agreement" uses the prospective and retrospective patient samples as the validation/test sets to demonstrate performance against the composite reference standard.

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

As stated above, no "training set" in the AI/ML context is described. For the analytical studies, the "ground truth" (e.g., presence/absence and concentration of a virus, presence of cross-reactants/interferents) was established by using:

• Quantified stocks of HSV-1 and HSV-2 (for Limit of Detection, Analytical Reactivity).
• Known concentrations of various microorganisms and substances (for Cross-Reactivity and Interference studies).
• Contrived sample pools with known viral loads (for Reproducibility and Competitive Interference).

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The Solana® HSV 1+2/VZV Assay is an in vitro diagnostic test, using isothermal amplification technology (helicase-dependent amplification, HDA), for the qualitative detection and differentiation of herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus DNA isolated and purified from cutaneous or mucocutaneous lesion samples obtained from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster infection. The Solana® HSV 1+2/VZV Assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and varicella-zoster virus active cutaneous or mucocutaneous infections. Negative results do not preclude herpes simplex virus 1, herpes simplex virus 2 and varicella-zoster virus infections and should not be used as the sole basis for diagnosis, treatment or other management decisions. The Solana® HSV 1+2/VZV Assay is intended for use only with the Solana® instrument.

Warning: The Solana® HSV 1 + 2/VZV Assay is not intended for use with cerebrospinal fluid or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Solana® HSV 1 + 2/VZV Assay is not intended for use in prenatal screening.

The Solana® HSV 1+2/VZV Assay amplifies and detects viral DNA isolated from cutaneous or mucocutaneous lesion samples obtained from symptomatic patients suspected of active herpes simplex virus 1, herpes simplex virus 2 and/or varicella-zoster infection.

The assay consists of two (2) major steps: 1) specimen preparation, and 2) amplification and detection of target sequence specific to HSV-1, HSV-2 and/or VZV using isothermal Helicase-Dependent Amplification (HDA) in the presence of target-specific fluorescence probe.

Patient specimen is transferred to a Process Tube, subjected to heat treatment at 95°C for 5 minutes and mixed and vortexed. The processed sample is transferred to a Reaction Tube and mixed. The Reaction Tube contains lyophilized HDA reagents, dNTPs, primers and probes. Once rehydrated with the diluted sample, the Reaction Tube is placed in Solana for amplification and detection of specific target sequence. In Solana, the target sequences are amplified by HSV-1, HSV-2 and/or VZV specific primers and detected by HSV-1, HSV-2 and/or VZV specific fluorescence probes included in the Reaction Tube. A competitive process control (PRC) is included in the Process Tube to monitor sample processing, inhibitory substances in clinical samples, reagent failure or device failure. The PRC target is amplified by specific primers and detected by a PRC specific fluorescence probe.

The target and PRC probes are labeled with a quencher on one end and a fluorophore on the other end. In addition, the target and PRC probes carry a ribonucleic acid. Upon annealing to HSV-1, HSV-2, VZV or PRC amplicons, the fluorescence probes are cleaved by RNaseH2 and the fluorescence signal increases due to physical separation of fluorophore from quencher. Solana measures and interprets the fluorescent signal, using on-board method-specific algorithms. Solana will then report the test results to the user on its display screen, and the results can be printed via an attached printer.

{
  "1. A table of acceptance criteria and the reported device performance": {
    "HSV-1 Cutaneous Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "100% (95% CI: 85.1% to 100%)",
        "Specificity": "97.8% (95% CI: 95.0% to 99.1%)"
      }
    },
    "HSV-2 Cutaneous Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "92.3% (95% CI: 75.9% to 97.9%)",
        "Specificity": "94.4% (95% CI: 90.8% to 96.6%)"
      }
    },
    "VZV Cutaneous Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "100% (95% CI: 85.1% to 100%)",
        "Specificity": "96.5% (95% CI: 93.0% to 98.3%)"
      }
    },
    "HSV-1 Mucocutaneous Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "100% (95% CI: 96.7% to 100%)",
        "Specificity": "96.4% (95% CI: 94.0% to 97.8%)"
      }
    },
    "HSV-2 Mucocutaneous Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "99.1% (95% CI: 95.0% to 99.8%)",
        "Specificity": "97.2% (95% CI: 95.4% to 98.3%)"
      }
    },
    "VZV Mucocutaneous Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "100% (95% CI: 51.0% to 100%)",
        "Specificity": "98.6% (95% CI: 96.9% to 99.4%)"
      }
    },
    "HSV-1 Uncategorized Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "100% (95% CI: 86.7% to 100%)",
        "Specificity": "97.6% (95% CI: 93.1% to 99.2%)"
      }
    },
    "HSV-2 Uncategorized Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "100% (95% CI: 82.4% to 100%)",
        "Specificity": "96.6% (95% CI: 92.3% to 98.6%)"
      }
    },
    "VZV Uncategorized Lesions": {
      "Acceptance Criteria": {
        "Sensitivity": "≥ 85%",
        "Specificity": "≥ 95%"
      },
      "Reported Device Performance": {
        "Sensitivity": "100% (95% CI: 81.6% to 100%)",
        "Specificity": "94.1% (95% CI: 87.8% to 97.3%)"
      }
    }
  },
  "2. Sample sized used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)": {
    "Total Sample Size": "1062 fresh lesion specimens (combined Cutaneous, Mucocutaneous, and Uncategorized)",
    "Data Type": "Prospective study",
    "Country of Origin": "United States (three sites across the United States)",
    "Specific Sample Sizes by Lesion Type for Clinical Performance": {
      "Cutaneous Lesions (HSV-1)": "249 specimens",
      "Cutaneous Lesions (HSV-2)": "275 specimens",
      "Cutaneous Lesions (VZV)": "222 specimens",
      "Mucocutaneous Lesions (HSV-1)": "501 specimens",
      "Mucocutaneous Lesions (HSV-2)": "610 specimens",
      "Mucocutaneous Lesions (VZV)": "372 specimens",
      "Uncategorized Lesions (HSV-1)": "148 specimens",
      "Uncategorized Lesions (HSV-2)": "166 specimens",
      "Uncategorized Lesions (VZV)": "119 specimens"
    }
  },
  "3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)": "Not specified. The ground truth was established by comparator methods (ELVIS cell culture system for HSV-1/2 and H&V mixed cells with DFA for VZV), which are FDA-cleared laboratory methods, rather than expert consensus on images or other forms of data typically requiring expert review.",
  "4. Adjudication method (e.g. 2+1, 3+1, none)  for the test set": "For discrepant results between the Solana assay and the primary comparator method, an additional RT-PCR assay was used for secondary adjudication. The document notes how many of the discrepant cases were resolved by this additional RT-PCR assay (e.g., 'Three (3) of the five (5) positives was positive by an additional RT-PCR assay' for HSV-1 cutaneous lesions). This indicates a form of adjudication involving a third, independent test.",
  "5. 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": "Not applicable. This is a molecular diagnostic assay, not an imaging device that uses AI for interpretation by human readers. The clinical study compares the device's performance against established laboratory methods.",
  "6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done": "Yes, a standalone study was conducted. The Solana® HSV 1+2/VZV Assay is an in vitro diagnostic test that processes specimens and provides results using on-board method-specific algorithms on the Solana® instrument. The reported performance characteristics (clinical sensitivity and specificity) are for the device operating as a standalone system against the comparator methods.",
  "7. The type of ground truth used (expert concensus, pathology, outcomes data, etc)": "Laboratory comparator methods: ELVIS cell culture system for HSV-1 and HSV-2, and H&V mixed cells with DFA cell culture systems for VZV. Discrepancies were resolved using an additional RT-PCR assay.",
  "8. The sample size for the training set": "Not explicitly stated for a distinct training set. The descriptions focus on the performance evaluation of the final device. For analytical performance (e.g., LOD, cross-reactivity, interference), contrived samples and specific strains were used for testing, but these are not referred to as a 'training set' in the context of machine learning. Clinical studies used fresh lesion specimens as described in section 2.",
  "9. How the ground truth for the training set was established": "Not applicable, as a distinct training set (in the machine learning sense) with established ground truth is not described for this diagnostic device. Analytical sensitivities (LOD) were determined using quantified viral cultures (TCID50/mL), which would serve as a 'truth' for analytical performance testing."
}

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The ARIES® HSV 1&2 Assay is a real-time polymerase chain reaction (PCR) based qualitative in vitro diagnostic test for the direct detection and differentiation of Herpes Simplex Virus 1 and HSV 2) DNA in cutaneous or mucocutaneous lesion specimens from symptomatic patients. The test is indicated for use as an aid in diagnosis of HSV infection in symptomatic patients. The ARIES® HSV 1&2 Assay is indicated for use on the ARIES® System.

WARNING: The ARIES® HSV 1&2 Assay is not FDA cleared for use with cerebrospinal fluid (CSF). The assay is not intended to be used for prenatal screening.

The Luminex ARIES® HSV 1 & 2 Kit is a polymerase chain reaction (PCR)-based qualitative in vitro diagnostic test for the direct detection and differentiation of herpes simplex virus (HSV) DNA using cutaneous and mucocutaneous lesion swab specimens. Patient lesion swab specimens are collected in Copan Universal Transport Medium, or identical Copan manufactured media formulations (Becton Dickinson Universal Viral Transport Media, Copan branded Universal Transport Medium for LabCorp, and the Quest Viral Culture Media) and transported to the laboratory. The specimen is pipetted into a cassette specific to the ARIES® HSV 1&2 Assay. In the cassette the specimen is lysed and nucleic acid is extracted using the ARIES® System and an extraction/ PCR cassette specific to the ARIES® HSV 1&2 Kit. An extractable sample processing control (SPC) target is present in the ARIES® HSV 1&2 assay cassette and is processed with the specimen. The Ct value of the SPC is designed to verify proper specimen lysis and nucleic acid extraction, to identify PCR inhibition, if any, and verify proper function of the extraction system and real-time instrument. The Tm value of the SPC is used as a reference for determining the target Tm.

The extracted nucleic acid is transferred via magnetic beads to the ARIES® HSV 1 & 2 Kit lyophilized PCR reagents in the cassette that contain a primer pair specific to HSV 1 and a primer pair specific to the SPC sequence. The specific primer pairs are labeled with distinct fluorophore labels. PCR amplification is performed and assay fluorescence is monitored on the Luminex ARIES® System. Incorporation of the quencher-labeled nucleotide causes a decrease in assay fluorescence. Following amplification, the reaction is slowly heated and fluorescence is monitored. The strands of the amplification products will separate at a specific melting temperature (Tm) that is determined by an increase in fluorescence as the strands are separated. The sequences between the PCR primer binding sites of the HSV 1 and HSV 2 amplicons have different base compositions that are distinguished by their different Tm. The instrument fluorescence output is analyzed and test results are determined using the ARIES® HSV 1 & 2 Kit assay protocol file. A printed results report is generated.

The Luminex ARIES® HSV 1&2 Assay chemistry is based on an expanded genetic alphabet technology, consisting of synthetic DNA base pair 2'-deoxy-5-methyl-isocytidine (iC): 2'deoxyisoguanosine (iG). The isobases ( iC and iG) pair specifically with each other and not with natural nucleotides. In addition isobases are efficiently incorporated during PCR. During PCR amplification, a quencher-modified iGTP is incorporated by the polymerase opposite an iC and a fluorophore reporter attached to a PCR primer. If the target is present and is amplified, assay fluorescence decreases with every cycle as amplification product accumulates. The decrease in assay fluorescence is monitored in real time using the Luminex ARIES® Instrument. Following PCR, the amplification products are thermally denatured and assay fluorescence is monitored. The strands of the amplification products are separated and assay fluorescence increases, thus enabling determination of the melting temperature (Tm) of the amplicon.

Here's an analysis of the acceptance criteria and study details for the ARIES® HSV 1&2 Assay, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" in a single, defined table. However, analytical and clinical performance metrics are provided and can be inferred as the criteria against which the device's performance was judged.

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

• Clinical Test Set: A total of 1963 left-over clinical specimens were initially included.
  • 1500 specimens were prospectively collected ("all comers").
  • 463 specimens were pre-selected (also prospectively collected) to ensure representation of under-represented lesion types.
  • 62 specimens were excluded from accuracy determinations (55 undeclared anatomical sites, 4 invalid upon re-testing, 3 unavailable for re-testing).
  • Actual N values for clinical performance tables vary (e.g., N=347 for HSV-1 Cutaneous, N=1190 for HSV-1 Mucocutaneous, N=448 for HSV-2 Cutaneous, N=1453 for HSV-2 Mucocutaneous), reflecting subsets analyzed.
• Data Provenance: The clinical study was conducted at three (3) geographically diverse clinical sites in the United States. The data were collected prospectively.

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

The document does not specify the number or qualifications of experts used to establish the ground truth. The "reference/comparative method" used was the ELVIS HSV ID and D3 Typing Test System (a viral culture-based method). For discrepant results, bi-directional sequencing analysis using analytically validated primers was used as a supplemental reference. This implies that the ground truth was established by laboratory testing methods rather than by expert clinical consensus from a panel.

4. Adjudication Method for the Test Set

The primary reference method was the ELVIS viral culture. When the ARIES® HSV 1&2 Assay results differed from the ELVIS method, bi-directional sequencing analysis was performed as a confirmatory method for discrepancy resolution. This acts as a form of "adjudication" where a third, presumably more definitive, method is used to resolve conflicts between the index test and the primary reference.

5. 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 MRMC study was described. This device is a fully automated in vitro diagnostic test for direct detection of viral DNA, not an imaging or diagnostic aid that requires human interpretation. Therefore, the concept of "human readers improving with AI" is not applicable here.

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

Yes, the clinical performance study describes the standalone performance of the ARIES® HSV 1&2 Assay, comparing its results directly against the reference method (ELVIS viral culture, with sequencing for discrepancy resolution). The device itself is an automated system (ARIES® System) that performs nucleic acid extraction, amplification, and detection without human intervention in the result generation process.

7. The Type of Ground Truth Used

The ground truth for the clinical study was established using a reference laboratory method:

• Primary Reference: ELVIS HSV ID and D3 Typing Test System (viral culture).
• Discrepancy Resolution/Confirmatory: Bi-directional sequencing analysis using analytically validated primers that targeted genomic regions distinct from the ARIES® HSV 1&2 Assay. This can be considered a highly accurate laboratory-based ground truth.

8. The Sample Size for the Training Set

The document provided is a 510(k) summary for a diagnostic assay, which typically doesn't involve "training sets" in the same way machine learning algorithms do. The "assay" defines the biochemical and molecular components (primers, probes, reagents, protocol file) and the "system" (ARIES® System) is the instrument. These are developed and validated through analytical studies (LoD, specificity, reproducibility, etc.) and then clinically evaluated.

Therefore, there is no explicit "training set" sample size mentioned for a machine learning model, as this is a traditional in vitro diagnostic device, not an AI/ML-based one. The development of such assays involves extensive analytical testing of reagents and instrument parameters, which could be seen as an iterative "training" process for the assay design, but this is not reported in terms of "training set" sample sizes in the context of typical AI device submissions.

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

Since there is no "training set" in the AI/ML sense, this question is not directly applicable. The assay's performance characteristics (e.g., optimal primer design, reaction conditions, melting temperature thresholds) would have been established during the development and optimization phases before the formal validation studies, using internal R&D samples and controls. The specifics of how these internal "ground truths" were established for assay development are not detailed in this regulatory submission.

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The illumigene HSV 1&2 DNA amplification assay, performed on the illumipro-10™, is a qualitative in vitro diagnostic test for the direct detection and differentiation of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) DNA in cutaneous and mucocutaneous lesion specimens from male and female patients suspected of Herpetic infections.

illumigene HSV 1&2 utilizes loop-mediated isothermal DNA amplification (LAMP) technology to detect HSV-1 and HSV-2 by targeting segments of the herpes simplex virus 1 and herpes simplex virus 2 genomes. Results from illumigene HSV 1&2 are used as an aid in the diagnosis of HSV infection in symptomatic patients.

The assay is intended for use in hospital, reference or state laboratory settings. This device is not intended for nonlaboratory point-of-care use.

WARNING: illumigene HSV 1&2 is not FDA cleared for use with cerebrospinal fluid (CSF) or to aid in the diagnosis of HSV infections of the central nervous system (CNS). The device is not intended for prenatal screening.

The illumigene Molecular Diagnostic Test System is comprised of the illumigene® HSV 1&2 DNA Amplification Assay Test Kit, the illumigene HSV 1&2 External Controls Kit, and the illumipro-10™ Automated Isothermal Amplification and Detection System.

The illumigene HSV 1&2 molecular assay utilizes loop-mediated amplification (LAMP) technology to detect herpes simplex virus in cutaneous and mucocutaneous lesion swab specimens. Each illumigene HSV 1&2 assay is completed using illumigene Sample Preparation Apparatus III (SMP PREP III), illumigene HSV 1 Test Devices, illumigene HSV 2 Test Devices, Mineral Oil, illumigene Heat Treatment Tubes, and transfer pipettes. Using a transfer pipette, specimens are added to SMP PREP III and dispensed into Heat Treatment Tubes. Samples are heat-treated to make target and control DNA available for amplification. Each heat-treated sample is added to one illumigene HSV 1 and one illumigene HSV 2 Test Device. Mineral oil is added to each illumigene Test Device to prevent evaporation.

The illumipro-10™ heats each illumigene HSV 1 and HSV 2 Test Device containing prepared sample and control material, facilitating amplification of target DNA. When HSV-1 or HSV-2 is present in the specimen, a 208 base pair (bp) sequence of the HSV-1 glycoprotein G (US4) gene or a 189 bp sequence of the HSV-2 glycoprotein G (US4) gene is amplified and magnesium pyrophosphate is generated. Magnesium pyrophosphate forms a precipitate in the reaction mixture.

The illumipro-10™ monitors the absorbance characteristics of the reaction solutions at the assay Run Start (Signal initial, S initial) and at the assay Run End (Signal final, S final). The illumipro-10™ calculates the change in light transmission between Run End and Run Start (S final; S initial) and compares the ratio to an established cut-off value. The illumipro-10™ performs this ratio calculation to both the TEST chamber and the CONTROL chamber.

Fixed cut-off values for the TEST chamber are used to report sample results. TEST chamber S final:S initial ratios less than 82% are reported as 'POSITIVE'; TEST chamber S final:S initial ratios greater than or equal to 82% are reported as 'NEGATIVE'. Numerical values are not reported.

Fixed cut-off values for the CONTROL chamber are used to determine validity. CONTROL chamber S final:S initial ratios less than 90% are considered valid and allow for reporting of TEST chamber results (POSITIVE). CONTROL chamber S final:S initial ratios greater than or equal to 90% are considered invalid and prevent reporting of TEST chamber results. Invalid CONTROL chamber reactions are reported as "INVALID'. More stringent cut-off criteria are applied to the CONTROL chamber reaction is not inhibited, reagents are performing as intended and that sample processing was performed appropriately.

The illumigene HSV 1&2 External Controls Kit contains a combined HSV-1 and HSV-2 Positive Control and a Negative Control (Negative Control IV) for use in routine Quality Control testing. External Control reagents are provided to aid the user in detection of reagent deterioration, adverse environmental or test conditions, or variance in operator performance that may lead to test errors.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as distinct pass/fail thresholds in percentage terms for sensitivity and specificity. However, the clinical study results represent the device's performance against a reference method (ELVIS® HSV ID and D3 Typing Test System) and can be considered the demonstrated performance for substantial equivalence. The document describes combined site performance for both cutaneous and mucocutaneous lesions for HSV-1 and HSV-2. Because the data is presented separately for HSV-1 cutaneous, HSV-1 mucocutaneous, HSV-2 cutaneous, and HSV-2 mucocutaneous, I will aggregate the positive and negative counts to provide an overall performance.

Overall Aggregated illumigene® HSV 1&2 DNA Amplification Assay Performance vs. ELVIS® HSV ID and D3 Typing Test System (Clinical Study)

Breakdown by Lesion Type (as provided in the document)

2. Sample size used for the test set and the data provenance

• Sample Size (Test Set):
  • Clinical Study: A total of 1158 leftover, de-identified lesion swab specimens were initially evaluated.
  • For HSV-1 performance analysis, 974 specimens were included (after exclusions).
  • For HSV-2 performance analysis, 1155 specimens were included (after exclusions).
• Data Provenance: The document does not explicitly state the country of origin. The study was conducted between October 2014 and March 2015. The specimens were "leftover, de-identified lesion swab specimens," indicating a retrospective study using previously collected samples.

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

The ground truth for the clinical performance data was established by comparison to the ELVIS® HSV ID and D3 Typing Test System. This is a laboratory-based diagnostic system, not a visual assessment by individual human experts. Therefore, the concept of "number of experts" and "qualifications of those experts" for establishing ground truth in the traditional sense (e.g., radiologists) does not apply here. The "expert" in this context is the validated reference diagnostic system itself.

4. Adjudication method for the test set

Not applicable. The ground truth was established by comparing the device's results to a predicate diagnostic system (ELVIS® HSV ID and D3 Typing Test System). There was no adjudication process involving human experts resolving discrepancies for the ground truth of the clinical samples. For discrepancies between the illumigene assay and the ELVIS system, some samples were re-tested with an "alternative, FDA-cleared molecular assay," but this is a reconciliation method, not an adjudication of primary ground truth experts.

5. 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

Not applicable. This device is an in vitro diagnostic (IVD) assay for molecular detection of HSV DNA, not an AI-powered image analysis tool or system designed to assist human readers. Therefore, an MRMC study and evaluation of human reader improvement with/without AI assistance are not relevant to this submission.

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

Yes, the clinical evaluation of the illumigene® HSV 1&2 DNA Amplification Assay against the ELVIS® HSV ID and D3 Typing Test System represents a standalone performance study of the algorithm (the assay itself) as an in vitro diagnostic device without human-in-the-loop intervention for result interpretation; the illumipro-10™ system automatically interprets signals based on fixed cut-off values.

7. The type of ground truth used

The ground truth used for the clinical performance study was the ELVIS® HSV ID and D3 Typing Test System, which is a legally marketed predicate diagnostic device for HSV detection and typing. This is a form of reference standard comparison. Additionally, for discordant results, an "alternative, FDA-cleared molecular assay" was used for confirmation, further supporting the reference standard approach.

8. The sample size for the training set

The document does not explicitly state a separate "training set" sample size for the clinical study directly tied to an external ground truth comparison. However, the "Assay cut-off" section mentions that "Development optimization includes evaluation of characterized positive and negative clinical specimens. Amplification reagent concentrations are adjusted during design as needed to ensure illumigene results are aligned with clinical specimen reported results." This implies internal development and optimization based on clinical specimens, but a specific "training set" as commonly defined in machine learning or AI context, with a stated size, is not provided.

9. How the ground truth for the training set was established

As inferred above, the ground truth for any internal optimization/development (which might be considered analogous to a training set) was established through "characterized positive and negative clinical specimens." The characterization method for these specimens is not detailed beyond "reported results," but it would logically rely on established diagnostic methods.

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The Lyra™ Direct HSV 1 + 2/VZV Assay is an in vitro multiplex Real-Time PCR test for qualitative detection and differentiation of herpes simplex virus type 1, herpes simplex virus type 2, and varicella-zoster virus DNA isolated and purified from cutaneous or mucocutaneous lesion samples obtained from symptomatic patients suspected of active herpes simplex virus 1. herpes simplex virus 2 and/or varicella-zoster infection. The Lyra™ Direct HSV 1 + 2/VZV Assay is intended to aid in the diagnosis of herpes simplex virus 1, herpes simplex virus 2 and varicella-zoster virus active cutaneous or mucocutaneous infections. Negative results do not preclude herpes simplex virus 1, herpes simplex virus 2 and varicella-zoster virus infections and should not be used as the sole basis for diagnosis, treatment or other management decisions. The Lyra™ Direct HSV 1 + 2/VZV Assay is not intended for use with cerebrospinal fluid or to aid in the diagnosis of HSV or VZV infections of the central nervous system (CNS). The Lyra™ Direct HSV 1 + 2/VZV Assay is not intended for use in prenatal screening. The device is not intended for point-of-care use.

The Lyra™ Direct HSV 1 + 2/VZV Assay detects viral nucleic acids from a patient sample. A multiplex Real-Time PCR reaction is carried out under optimized conditions in a single tube or well generating amplicons for HSV-1, HSV-2, VZV, and the Process Control (PRC). Identification of amplicons for HSV-1, HSV-2, VZV, and the PRC occurs by the use of target-specific primers and fluorescent-labeled probes that hybridize to conserved regions in the genomes of HSV-1, HSV-2, and VZV and to the PRC, respectively.

Here's an analysis of the acceptance criteria and study details for the Lyra™ Direct HSV 1 + 2/VZV Assay, formatted as requested:

Acceptance Criteria and Device Performance for Lyra™ Direct HSV 1 + 2/VZV Assay

The Lyra™ Direct HSV 1 + 2/VZV Assay is an in vitro multiplex Real-Time PCR test for qualitative detection and differentiation of HSV-1, HSV-2, and VZV DNA from cutaneous or mucocutaneous lesion samples. The regulatory classification is Class II with special controls (21 CFR 866.3309).

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria with numerical thresholds in a single table. Instead, performance is presented through various analytical and clinical studies. We will infer the de facto acceptance criteria based on the demonstrated performance that led to the device's classification. For clinical performance, the comparable effectiveness is evaluated against established methods (ELVIS® HSV ID and D3 Typing Test for HSV and DSFA and Culture with DFA for VZV). Successful performance in these comparisons implies the acceptance criteria were met.

Inferred Acceptance Criteria Table and Reported Device Performance:

Study Proving Acceptance Criteria Met:

The device's performance characteristics, including both analytical and clinical studies, were conducted to demonstrate it meets the requirements for a Class II designation with special controls.

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

Test Set Sample Sizes (Clinical Study):

The clinical study was a multi-center study conducted between April 2013 and October 2013.

• Life Technologies QuantStudio™ Dx:
  • Cutaneous Lesions: 279 specimens initially collected (278 for HSV-1/HSV-2, 222 for VZV after exclusions).
  • Mucocutaneous Lesions: 650 specimens initially collected (646 for HSV-1/HSV-2, 432 for VZV after exclusions).
• Applied Biosystems® 7500 Fast Dx:
  • Cutaneous Lesions: 279 specimens (279 for HSV-1/HSV-2, 223 for VZV after exclusions).
  • Mucocutaneous Lesions: 650 specimens initially collected (644 for HSV-1/HSV-2, 430 for VZV after exclusions).
• Cepheid SmartCycler® II:
  • Cutaneous Lesions: 279 specimens initially collected (278 for HSV-1/HSV-2, 222 for VZV after exclusions).
  • Mucocutaneous Lesions: 650 specimens initially collected (643 for HSV-1/HSV-2, 429 for VZV after exclusions).

Data Provenance:

• Country of Origin: Not explicitly stated, but the submission is to the FDA, suggesting United States given the typical regulatory context for De Novo submissions. The multi-center nature implies data was collected from different clinical sites within the same regulatory jurisdiction.
• Retrospective or Prospective: The clinical study description states: "A multi-center study was performed between April, 2013 and October, 2013 to evaluate the Lyra™ Direct HSV 1 + 2/VZV Assay using lesion swab specimens obtained from cutaneous or mucocutaneous lesions and submitted for HSV and/or VZV culture." This clearly indicates a prospective collection of specimens for the purpose of the study.

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

The ground truth for the clinical test set was established using FDA-cleared comparator methods, not directly by human experts in the sense of independent adjudication.

• For HSV-1 and HSV-2, the comparator method was the ELVIS® HSV ID and D3 Typing Test, an FDA-cleared cell culture system.
• For VZV, the comparator method involved staining cells present in the samples with an FDA-cleared VZV detection reagent (DSFA) and culturing the specimen using a mixed cell culture (H&V mixed cells) followed by staining with the same FDA-cleared reagent used for DSFA.

Therefore, there were no direct human experts establishing ground truth in the typical sense of radiologists or pathologists. The ground truth was based on the results of established, FDA-cleared laboratory methods, operated by trained laboratory personnel.

4. Adjudication Method for the Test Set

The primary adjudication method for discrepancies between the Lyra™ Direct Assay and the comparator methods was the use of an additional RT-PCR assay.

• For discrepancies in HSV-1 (e.g., Lyra™ positive, comparator negative), these cases were "positive by an additional RT-PCR assay."
• Similarly for HSV-2 and VZV discrepancies, "positives were positive by an additional RT-PCR assay."
• In some cases where Lyra™ was negative but the comparator was positive, these were also re-evaluated by an additional RT-PCR assay, with the statement "negatives were positive by an additional RT-PCR assay."

This acts as a tie-breaker or confirmatory test for discordant results, enhancing the robustness of the ground truth derived from the comparator methods.

5. 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, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic (IVD) PCR assay, not an AI/CADe medical imaging device that assists human readers. Its performance is evaluated compared to established laboratory methods, not human interpretation of images. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable here.

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

Yes, the primary clinical study represents standalone performance. The Lyra™ Direct HSV 1 + 2/VZV Assay is an automated real-time PCR test. Its results (positive/negative for each virus) are generated directly by the instrument and its software, without human interpretation of raw signals to determine the final viral status. The clinical sensitivity and specificity reported are the performance of the algorithm/device alone compared to the ground truth.

7. The Type of Ground Truth Used (Expert Consensus, Pathology, Outcomes Data, etc.)

The ground truth used for the clinical study was based on FDA-cleared conventional laboratory methods:

• Cell Culture with Immunofluorescence Staining (or similar detection):
  • HSV-1 and HSV-2: ELVIS® HSV ID and D3 Typing Test.
  • VZV: Direct Smear Fluorescent Antibody (DSFA) and mixed cell culture with DFA staining.
• Confirmatory RT-PCR/molecular assay: Used as an adjudication method for discordant results between the Lyra™ Direct Assay and the primary comparator methods.

8. The Sample Size for the Training Set

The document does not explicitly describe a separate "training set" for the clinical evaluation in the way machine learning algorithms typically use them. For IVD devices like this, the "development" or "training" process involves optimizing the assay components (primers, probes, reaction conditions) and setting analytical cut-offs based on analytical studies (e.g., Limit of Detection, reactivity, specificity studies) using characterized samples.

The reproducibility and precision studies used simulated samples (medium positive, low positive, high negative, negative, 5x LoD, 2x LoD,

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