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The AnyplexTM II HSV-1/2 Assay is a real-time polymerase chain reaction (PCR)-based in vitro diagnostic test intended for the qualitative detection and differentiation of Herpes Simplex Virus Type-1 (HSV-1) and Herpes Simplex Virus Type-2 (HSV-2) DNA from female skin lesions from anogenital sites. The test is intended for use as and in the diagnosis of anogenital HSV infection in symptomatic patients.

WARNING: The AnyplexTM II HSV-1/2 Assay is not indicated for use with cerebrospinal fluid (CSF). The assay is not intended to be used for prenatal screening.

The Anyplex™ II HSV-1/2 Assay uses PCR to generate amplified product from HSV-1 and HSV-2 present in clinical specimens. The presence of HSV-1 and/or HSV-2 target DNA is indicated by the fluorescent signal generated through the use of fluorescently-labeled oligonucleotide probes (duplex Catcher) on the Cepheid SmartCycler® II Dx instrument. The probes do not generate a signal unless they are specifically bound to the amplified product. A preparation of HSV-1 and HSV-2 plasmids is included as the positive control in the Anyplex™ II HSV-1/2 Assay. Run as a separate control, the positive control serves to demonstrate that the HSV-1/2 PCR reagents are functional, and discriminate the validity of the run. In addition, the positive control functions as a process control, demonstrating that sample preparation has proceeded correctly during the run. An internal control (IC) is also included in the assay kit. The IC is added to each sample specimen during sample preparation, and is also used to create a Blank Negative Control by adding a set amount to viral transport media to serve as an extraction control. In addition, the RNase-free water is used to create the Negative Control by adding a set volume to the prepared master mix. Users are instructed to include all three controls, Positive, Negative, and Blank Negative Control with each test run.

The document describes the performance of the Anyplex™ II HSV-1/2 Assay, a real-time PCR-based in vitro diagnostic test for the qualitative detection and differentiation of Herpes Simplex Virus Type-1 (HSV-1) and Herpes Simplex Virus Type-2 (HSV-2) DNA from female skin lesions from anogenital sites.

Here's an analysis of the acceptance criteria and study details 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., "The device must achieve sensitivity of X% and specificity of Y%"). Instead, it presents the results of its performance studies, which are implicitly understood to demonstrate adequate performance for regulatory approval based on comparison to a predicate device. The clinical performance metrics are the most relevant in this context.

Implicit Acceptance Criteria (based on reported clinical performance): The device is expected to demonstrate high sensitivity and specificity for both HSV-1 and HSV-2 detection when compared to a reference method in symptomatic patients with anogenital lesions.

Analytical Performance (examples from the document):

• Precision/Repeatability: Achieved 100% agreement with expected results for HSV-1 Low Positive, HSV-1 High Positive, HSV-2 Low Positive, HSV-2 High Positive, and Negative samples in an in-house study. Ct %CV values were low (e.g., 1.34% - 2.56%).
• Reproducibility: Across three sites, overall agreement for positive controls (3X LoD and 1X LoD) was high (e.g., 100% for 3X LoD for both HSV-1 and HSV-2; 96.67% for HSV-1 1X LoD; 97.8% for HSV-2 1X LoD). Negative controls showed 99.4% agreement.
• Limit of Detection (LoD): Varied by strain (e.g., HSV-1 MacIntyre: 3.75x10^2 TCID50/mL; HSV-2 MS: 3.75x10^1 TCID50/mL).
• Cross-Reactivity/Microbial Interference: No cross-reactivity or interference observed with a panel of 50 organisms.
• Interfering Substances: None of 22 tested substances showed detectable interference.

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

• Sample Size: A total of 656 valid prospective specimens were included in the final data set for clinical performance analysis.
  • For HSV-1 performance calculation, 550 specimens were used (specimens positive for HSV-2 by the reference method were removed due to the reference method's inability to detect co-infections).
  • For HSV-2 performance calculation, all 656 specimens were used.
• Data Provenance: The study was conducted at three geographically diverse locations within the United States from 2013-2014. The data is prospective, as indicated by "656 prospective specimens included in the study."

3. Number of Experts and their Qualifications for Ground Truth

The document does not explicitly state the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience") used to establish the ground truth.

4. Adjudication Method for the Test Set

The primary adjudication method for discordant results in the clinical performance study was bidirectional sequencing.

• For HSV-1:
  • 29 samples positive by Anyplex but negative by reference method: HSV-1 was detected in 18 cases by bidirectional sequencing. The remaining 11 remained discordant.
  • 1 sample negative by Anyplex but positive by reference method: HSV-1 was detected in this sample by bidirectional sequencing.
• For HSV-2:
  • 35 samples positive by Anyplex but negative by reference method: HSV-2 was detected in 21 cases by bidirectional sequencing. The remaining 14 remained discordant.
  • 3 samples negative by Anyplex but positive by reference method: HSV-2 was not detected in any of these by bidirectional sequencing (meaning the reference method's positive call was likely a false positive, and Anyplex was correct).

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. This device is an in-vitro diagnostic assay (laboratory test), not an imaging or interpretation device that typically involves human readers. The clinical performance study compares the device's results against a laboratory-based reference method, not against human interpretation with or without AI assistance.

6. Standalone Performance

Yes, a standalone performance study was done. The entire clinical performance section (Tables 6 and 7) and the analytical performance sections (precision, reproducibility, LoD, cross-reactivity, interfering substances) describe the performance of the Anyplex™ II HSV-1/2 Assay algorithm/device only, without human intervention in the interpretation of the output. The assay provides a qualitative "POS" or "NEG" result for HSV-1 and HSV-2.

7. Type of Ground Truth Used

The ground truth for the clinical performance study was established using the ELVIS® (Enzyme Linked Virus Inducible System) HSV ID and D Typing Test System (Diagnostic Hybrids, Athens, OH), which is described as the "reference ELVIS viral culture method." For discordant results, bidirectional sequencing was used as a confirmatory method to resolve discrepancies.

8. Sample Size for the Training Set

The document does not specify the sample size used for a training set. This is typical for a traditional IVD device using PCR technology, where "training" in the machine learning sense isn't applicable. The development of such assays involves designing primers and probes, and then validating their performance across various analytical and clinical studies.

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

As noted above, the concept of a "training set" with ground truth in the AI/machine learning sense is not applicable to this type of device. The assay design (e.g., selection of primers and probes) is based on known genetic sequences of HSV-1 and HSV-2. The extensive analytical performance studies (precision, LoD, cross-reactivity) ensure the assay's fundamental ability to detect targets accurately, and the clinical performance study validates this in human samples against a recognized reference method.

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