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The Focus Diagnostics Simplexa™ HSV 1 & 2 Direct is intended for use on the 3M Integrated Cycler instrument for the qualitative detection and differentiation of HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF) samples from patients suspected of herpes simplex virus (HSV) infections of the central nervous system (CNS). This test is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS.

Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.

The assay is not intended for use as a donor screening test. The assay is for professional use only.

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 CSF specimens without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct assay, the 3M Integrated Cycler (with 3M Integrated Cycler 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.

The provided text describes a 510(k) summary for the Simplexa™ HSV 1 & 2 Direct and Simplexa™ HSV 1 & 2 Positive Control Pack, which is a modification to a previously cleared device (K133621). The changes are specifically related to the Integrated Cycler Studio Software version 6.0.

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

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

The document mentions that "Verification activities for the Integrated Cycler Studio software version 6.0 included development of verification test plans with defined acceptance criteria (design inputs), conducting and documenting verification testing and review of the verification results as they compared to the verification test plans predetermined acceptance criteria (design outputs)." It also states that "Integrated Cycler Studio software version 6.0 was validated in a similar fashion with the development of validation test plans with defined acceptance criteria (design inputs), conducting validation testing and review of the validation results as they compared to the validation test plans predetermined acceptance criteria (design outputs)."

However, the specific acceptance criteria (e.g., percentage agreement, sensitivity, specificity, or specific values for these metrics) for the software's performance with the assay, and the reported device performance against these criteria, are not explicitly detailed in the provided text. The text only states that "The results of verification and validation of Integrated Cycler Studio software version 6.0 show the results met the predetermined acceptance criteria." and "The results of assays ran with the Integrated Cycler Studio software version 6.0 demonstrate that the results obtained with the previously released versions of Integrated Cycler Studio software were equivalent to the results obtained using Integrated Cycler Studio software version 6.0."

Without the actual defined acceptance criteria and the quantitative or qualitative results against them, a detailed table cannot be created from this document.

2. Sample size used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

The document focuses on software changes and comparison to a predicate device. It indicates that "The results of assays ran with the Integrated Cycler Studio software version 6.0 demonstrate that the results obtained with the previously released versions of Integrated Cycler Studio software were equivalent to the results obtained using Integrated Cycler Studio software version 6.0." This suggests that a comparison study was performed, likely using existing assay data or running new assays with the modified software.

However, the sample size for any test set, the specific data provenance (country of origin), and whether the data was retrospective or prospective are not mentioned in the provided text.

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)

This information is not provided in the text. The device is an in vitro diagnostic for detecting HSV-1 and HSV-2 DNA. The "ground truth" for such devices is typically established through molecular reference methods or clinical diagnosis, not usually expert consensus in the same way as imaging analysis.

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

This information is not applicable or provided in the text. Adjudication methods are typically used in studies involving human interpretation (e.g., radiologists reviewing images), where disagreement among experts needs resolution. For a PCR-based diagnostic device, the assessment of results against a ground truth would follow established laboratory protocols for molecular diagnostics, not human adjudication.

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

An MRMC study is not applicable here as the device is a direct molecular diagnostic test, not an AI-assisted human reader interpretation system. The device directly detects viral DNA.

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

The device is inherently a "standalone" algorithm (PCR assay interpreted by software) in the sense that it performs the detection and differentiation of HSV DNA without a human interpreting the primary assay signal directly for diagnosis. The software (Integrated Cycler Studio) processes the raw data from the PCR reaction to deliver a qualitative result (presence/absence of HSV-1/HSV-2 DNA). The statement that "The results of assays ran with the Integrated Cycler Studio software version 6.0 demonstrate that the results obtained with the previously released versions of Integrated Cycler Studio software were equivalent to the results obtained using Integrated Cycler Studio software version 6.0" implies an assessment of the software's performance on its own.

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

Given that this is a molecular diagnostic for HSV DNA, the ground truth would typically be established by a highly sensitive and specific reference molecular method (e.g., an FDA-cleared or a well-validated in-house laboratory developed test with stringent analytical performance characteristics, or potentially by clinical diagnosis combined with other laboratory findings). The specific method for establishing ground truth is not explicitly stated in the provided text.

8. The sample size for the training set

The document describes software version changes and verification/validation activities. There is no mention of a "training set" in the context of machine learning or AI. The software is designed to interpret PCR assay results.

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

As there is no mention of a "training set" or AI/machine learning development, this question is not applicable based on the provided text.

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The Focus Diagnostics Simplexa™ HSV 1 & 2 Direct is intended for use on the 3M Integrated Cycler instrument for the qualitative detection and differentiation of HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF) samples from patients suspected of Herpes Simplex Virus (HSV) infections of the central nervous system (CNS). This test is intended as an aid in the diagnosis of HSV-1 and HSV-2 infections of the CNS.

Negative results do not preclude HSV-1 or HSV-2 infection and should not be used as the sole basis for treatment or other patient management decisions.

The assay is not intended for use as a donor screening test. The assay is for professional use only.

The Positive Control is intended to be used as a control with the Simplexa™ HSV 1 & 2 Direct. This control is not intended for use with other assays or systems.

The Simplexa™ HSV 1 & 2 Direct system is a real-time PCR that enables the direct amplification, detection and differentiation of HSV-2 DNA from unprocessed CSF samples without nucleic acid extraction. The system consists of the Simplexa™ HSV 1 & 2 Direct, the 3M Integrated Cycler (with 3M Integrated Cycler Studio Software), the Direct Amplification Disc and associated accessories.

In the SimplexaTM HSV 1 & 2 Direct, bi-functional fluorescent probe-primers are used together with corresponding reverse primers to amplify HSV-1, HSV-2 and DNA internal control (IC) 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. The IC is used to detect PCR failure and/or inhibition.

The 3M Integrated Cycler is a real-time PCR thermocycler which uses real-time fluorometric detection to identify targets within the sample wells. The instrument is controlled by an external computer running the Integrated Cycler Studio Software.

The Simplexa™ HSV 1 & 2 Direct assay reaction takes place in the DAD consumable. The DAD consumable is compartmentalized into 8 separate wedges and up to 8 separate samples or controls may be processed on each disc. Each wedge can be used only once, however, the disc may be reused until all wedges have been utilized. Each wedge contains sample and reagent input wells, microfluidic channels and laser activated valves to control the fluid flow. and a reaction chamber. The disc is specifically designed to meter the amount of reagent (Reaction Mix) and sample that are placed into specific wells in the disc. To start processing a patient sample, a foil seal is lifted and the user adds 50 µL of Reaction Mix to the reagent input well using a fixed volume pipette. Next, the user adds 50 uL of unextracted specimen to the sample input well.

The SimplexaTM HSV 1 & 2 Direct kit contains reagents for 24 reactions. Each vial contains sufficient material for a single reaction. The kit contains the Reaction Mix, the Simplexa™ HSV 1 & 2 Direct Barcode Card with assay specific information, and the Package Insert.

This document describes the evaluation of the Simplexa™ HSV 1 & 2 Direct device, a real-time PCR test for detecting and differentiating HSV-1 and HSV-2 DNA in cerebrospinal fluid (CSF). The evaluation supports its classification as a Class II device with special controls.

Here's an analysis of the acceptance criteria and the studies that prove the device meets them:

1. Table of Acceptance Criteria and Reported Device Performance:

The document does not explicitly state "acceptance criteria" in a singular table for all performance measures. Instead, performance is demonstrated through various analytical and clinical studies, with implicit acceptance criteria being met if the results are favorable (e.g., high agreement rates, low variability, no cross-reactivity). For the clinical study, the performance is measured by Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) compared to a comparator method.

Let's summarize the key performance aspects that serve as de facto acceptance criteria based on the presented data:

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

• Reproducibility Test Set:

  • 6 samples (high negative, low positive, moderately positive for HSV-1 and HSV-2, and a positive control)
  • Total of 90 replicates per sample panel member (3 sites x 5 days x 2 runs x 3 replicates) = 540 replicates per analyte group (HSV-1, HSV-2, IC).
  • Data Provenance: Not explicitly stated, but implies multi-site, laboratory-based testing for analytical performance validation.

• Detection Limit (LoD) Test Set:

  • Two primary strains (HSV-1 McIntyre, HSV-2 G): Each dilution assayed in 32 replicates across 32 DAD runs.
  • Two additional strains (HSV-1 HF, HSV-2 MS): 3-32 replicates per dilution/concentration for screening and confirmation.
  • Data Provenance: Laboratory-generated samples (viral stock spiked into negative human CSF matrix).

• Analytical Reactivity Test Set:

  • 2 additional HSV-1 strains. Each spiked into negative CSF, assayed in triplicate. (3 replicates per strain, with 2 repeated; total 12 technical replicates for HSV-1 strains).
  • Data Provenance: Laboratory-generated samples.

• Analytical Specificity (Cross-Reactivity) Test Set:

  • 51 potential cross-reactants.
  • Each assayed in triplicate. (3 replicates per cross-reactant).
  • Data Provenance: Laboratory-generated samples (organisms spiked into negative CSF).

• Interference Test Set:

  • Interfering Substances: 7 substances tested. Each in low positive HSV-1 and HSV-2 sample, assayed in triplicate (3 replicates per interferent).
  • Competitive Interference: Samples contrived with low and high concentrations of HSV-1 and HSV-2. Replicates ranged from 3 to 8 depending on the condition.
  • Inhibition by Other Microorganisms: 51 microorganisms. Initially tested in triplicate, then 5 additional replicates if any "Not Detected" (total 8 replicates if retested).
  • Data Provenance: Laboratory-generated samples (substances/organisms spiked into CSF).

• Sample Stability Test Set:

  • A panel of 120 pairs of contrived samples (60 for HSV-1, 60 for HSV-2). Each pair consisted of a fresh and a frozen aliquot.
  • Data Provenance: Laboratory-contrived samples (spiked in negative human CSF matrix).

• Carry-over Contamination Test Set (from K120413 study):

  • 60 negative samples and 60 high positive samples tested across 17 runs.
  • Data Provenance: Not specified, but likely laboratory-contrived positive and negative samples for the previous device clearance.

• Clinical Studies Test Set:

  • Prospective Samples: 164 CSF samples.
  • Retrospective/Preselected Positive Samples: 55 CSF samples.
  • Total Clinical Samples: 219 CSF samples.
  • Data Provenance: Collected from eight external sites. Patients with signs and symptoms of Herpes Simplex Virus (HSV) central nervous system (CNS) infection. Submitted to Focus Diagnostics (frozen). Tested at five external sites (Simplexa™) and Focus Diagnostics (comparator). Retrospective samples were collected between 2004 and 2013, indicating retrospective data collection for these specific samples.

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

• Analytical Studies (Reproducibility, LoD, Reactivity, Specificity, Interference, Stability, Carry-over): The ground truth for these studies was established by the precise formulation and characterization of the contrived samples (e.g., known viral strains at specific concentrations, known presence/absence of interferents). No "experts" in the clinical sense were used to establish ground truth for these analytical tests.

• Clinical Studies:

  • The ground truth for the clinical samples was established using a "comparator" method: two PCR/bi-directional sequencing assays targeting two distinct regions of the HSV genome.
  • The interpretation of clinical diagnosis (e.g., "Final Diagnosis positive for HSV infection") used "the patient's attending physician and other clinical information such as chemistries, bacterial culture, MRI/CT scans and in-house PCR results." This implies expertise by the treating physicians, but the ground truth for comparison with the device was the defined PCR/sequencing method.
  • Number of experts and specific qualifications (e.g., radiologist with 10 years of experience) for establishing the comparator ground truth are not specified. It's implicitly handled by the laboratory performing the two PCR/bi-directional sequencing assays following established protocols.

4. Adjudication Method for the Test Set:

• Analytical Studies: Adjudication method is not applicable as the ground truth is pre-defined by the experimental setup. Results are evaluated based on agreement with expected outcomes.

• Clinical Studies:

  • The "comparator" method itself involved two PCR/bi-directional sequencing assays. A positive result was defined as positive by at least one of these two assays. A negative result required both assays to be negative.
  • This is a form of "rule-based adjudication" to derive a single ground truth from a multi-component comparator. It's not a consensus of human readers, but rather a defined algorithm for combining results from two independent laboratory assays.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance:

• No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done.
• This device is an in vitro diagnostic (IVD) assay (a laboratory test) for direct detection of viral DNA, not an artificial intelligence (AI) solution intended to assist human readers in interpreting images or other data. Therefore, the concept of human readers improving with AI assistance is not applicable to this submission.

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

• Yes, the primary performance evaluation of the Simplexa™ HSV 1 & 2 Direct assay is a standalone, algorithm-only performance.
• The device performs the amplification, detection, and differentiation of HSV-1 and HSV-2 DNA automatically based on the real-time PCR method. The results are calculated and displayed by the Integrated Cycler Studio Software. The performance characteristics (e.g., sensitivity, specificity, reproducibility, LoD) are determined for the device operating independently without subjective human interpretation of the primary signal generation. Human involvement is limited to sample preparation, loading, and interpreting the final "Detected," "Not Detected," or "Invalid" report from the software.

7. The Type of Ground Truth Used:

• Analytical Studies: The ground truth used for analytical studies (LoD, reactivity, specificity, interference) was laboratory-contrived samples with known concentrations of viral targets or known presence/absence of interferents/cross-reactants. For reproducibility, the expected result for each sample was known.
• Clinical Studies: The ground truth for clinical studies was established by a comparator method consisting of two PCR/bi-directional sequencing assays. This is a molecular/laboratory-based ground truth, not pathology or direct outcomes data, although clinical information was collected to characterize the patient population.

8. The Sample Size for the Training Set:

• This submission describes the evaluation and approval of an in vitro diagnostic (IVD) assay, not a machine learning or AI algorithm. Therefore, there is no explicit "training set" in the context of machine learning.
• The development of the assay (e.g., primer design, probe selection, cut-off determination) would have involved internal optimization and verification studies, but these are not typically referred to as a "training set" in the same way as for AI. The document mentions "feasibility and verification studies" for determining the assay cut-off.

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

• As noted above, there is no explicit "training set" for a machine learning algorithm.
• For the development and optimization of the PCR assay itself, the "ground truth" (e.g., optimal primer/probe sequences, reaction conditions, Ct cut-offs) would have been established through iterative laboratory experimentation, validation against characterized viral stocks, and empirical performance testing to achieve desired sensitivity and specificity. The document mentions that the cut-off was determined during "feasibility and verification studies" using LoD verification study replicates.

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