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The IsoAmp® HSV Assay is an in vitro diagnostic test for the direct, qualitative detection of herpes simplex virus (HSV-1 & HSV-2) DNA in male and female genital and oral lesions. The test is intended for use as an aid in diagnosis of HSV infection in symptomatic patients.

Warning: The IsoAmp® HSV Assay is not FDA cleared for use with cerebrospinal fluid (CSF). The assay does not provide specific typing information to differentiate HSV-1 and HSV-2. The assay is not intended to be used for prenatal screening.

The IsoAmp® HSV Assay consists of three major steps: 1) specimen preparation: 2) isothermal Helicase-Dependent Amplification (HDA) of the HSV glycoprotein B (gB) gene using biotinylated primers; and 3) detection of the amplified DNA by a target-specific hybridization probe via a colorimetric reaction on a lateral-flow strip which is embedded in a self-contained disposable cassette to prevent amplicon contamination.

Specimen preparation includes a simple dilution step in which specimens in viral transport medium are diluted 40-fold in dilution buffer. The diluted samples are mixed with HDA reagents. Incubation at 64°C results in the release of the HSV DNA and subsequent isothermal amplification of the target sequence. A competitive internal control (IC) is included in the Amplification Reagents to monitor inhibitory substances in negative samples, reagent failure or device failure.

After incubation for one hour, the amplified DNA is detected by two detection probes, one labeled with fluorescein isothiocyanate (FITC) for hybridizing to the HSV target and the other labeled with digoxigenin (DIG) for binding to the IC target. The hybrid of FITC-labeled probe and HSV amplicon is captured at the Test Line (T-Line) on the lateral-flow strip by anti-FITC antibodies, while the DIG-labeled IC amplicon is captured at the Control Line (C-Line) on the strip by anti-DIG antibodies. The biotin label in each amplicon captures the streptavidinconjugated color particles for visualization and the test result is shown as colored lines that are visually read.

The self-contained Type II BESt™ cassettes contain lateral-flow DNA detection strips coated with anti-FITC antibodies and anti-DIG antibodies that serve as T line and C line respectively in the assay. A positive result (detection of HSV DNA) is reported when the T line is visible through the detection window of the cassette. A negative result (no detection of HSV DNA) is reported when only the C line is displayed. The assay result is regarded as invalid when both the T line and C line are not present and the assay should be repeated.

Here's a breakdown of the acceptance criteria and the study details for the IsoAmp® HSV Assay, based on the provided document:

1. Table of Acceptance Criteria and Reported Device Performance

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

• Clinical Performance Test Set (Prospective and Retrospective combined): 994 swab samples.
  • Prospective Samples: 962 samples (803 genital, 159 oral).
  • Retrospective Samples: 32 samples (15 genital, 17 oral) tested at a single study site.
• Data Provenance:
  • Country of Origin: United States.
  • Retrospective or Prospective: A mix of both. The majority (962) were prospectively collected, and a smaller number (32) were retrospectively collected.

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

• The document does not specify the number of experts or their qualifications for establishing the ground truth using the reference method (Cell Culture based ELVIS® HSV ID/Typing Test System). The ELVIS system is a commercial diagnostic kit, and its results would be interpreted according to its own instructions, potentially by trained laboratory personnel rather than clinical "experts" in the sense of physicians or radiologists.
• For the discordant samples, "bidirectional sequence analysis" was used to resolve discrepancies, but the personnel performing and interpreting this analysis are not described.

4. Adjudication Method for the Test Set

• The primary method for establishing ground truth was comparison to a "gold standard/reference method," the Cell Culture based ELVIS® HSV ID/Typing Test System.
• For discordant results between the IsoAmp® HSV Assay and the ELVIS system, bidirectional sequence analysis was used as an adjudication method.
  • For genital samples: 35 samples discordant by the IsoAmp assay were tested by sequencing.
  • For oral samples: 14 samples discordant by the IsoAmp assay were tested by sequencing.
  • This suggests a process where sequencing served as a tie-breaker or a higher-fidelity reference for ambiguous cases.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

• No, an MRMC comparative effectiveness study was not done.
• This study evaluated a diagnostic assay (a lab test), not an AI algorithm assisting human readers in interpreting images or other complex data. Therefore, the concept of "how much human readers improve with AI vs. without AI assistance" does not apply here.

6. Standalone Performance (Algorithm Only Without Human-in-the-Loop Performance)

• Yes, a standalone performance was done. The entire study tests the performance of the IsoAmp® HSV Assay as a complete system, which operates independently to produce a result, with visual reading of the lateral flow strip. There isn't a human interpreting images or data that the algorithm generates and then making a judgment. The visual reading of the T-line and C-line on the lateral flow strip could be considered the "human-in-the-loop" component for result interpretation, but the core performance data (sensitivity, specificity, LoD, etc.) reflects the assay's output as an isolated system. The results presented are for the device's output itself.

7. Type of Ground Truth Used

• Reference Method: Cell Culture based ELVIS® HSV ID/Typing Test System. This is a recognized laboratory "gold standard" for HSV detection.
• Adjudication for Discordant Results: Bidirectional sequence analysis. This is a highly accurate molecular method used to confirm the presence and type of viral DNA.

8. Sample Size for the Training Set

• The document does not explicitly mention a distinct "training set" for the IsoAmp® HSV Assay. This is common for molecular diagnostic assays like this, which are developed through iterative R&D processes (optimizing reagents, protocols, etc.) rather than supervised machine learning where a specific "training set" is used to teach an algorithm.
• The analytical performance studies (LoD, cross-reactivity, interference) use spiked samples and cultured organisms, which contribute to the development and validation of the assay but are not typically referred to as a "training set" in the AI/ML sense.

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

• As no explicit "training set" is described for an AI/ML context, this question is not directly applicable.
• For the analytical studies (e.g., LoD, cross-reactivity), the "ground truth" for the samples used was established by:
  • Quantified cultures: For LoD studies, HSV strains were precisely quantified (TCID₅₀/mL).
  • Known organisms/DNA: For cross-reactivity, purified DNA and cultured organisms of known identity and concentration were used.
  • HSV-negative matrix pools: Used as negative controls and for diluting samples.
  • These are standard methods in in vitro diagnostic assay development to create samples with a known status.

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