(79 days)
The AmpliVue® HSV 1+2 Assay is an in vitro diagnostic test for the direct, qualitative detection and differentiation of Herpes Simplex Virus 1 (HSV-1) and Herpes Simplex Virus 2 (HSV-2) DNA in cutaneous or mucocutaneous lesion specimens from symptomatic patients. The test is intended for use as an aid in diagnosis of HSV infection in symptomatic patients.
Warning: The AmpliVue HSV 1+2 Assay is not FDA cleared for use with cerebrospinal fluid (CSF). The assay is not intended for prenatal screening.
The AmpliVue HSV 1+2 Assay consists of three major steps: 1) specimen preparation, 2) isothermal Helicase-Dependent Amplification (HDA) of target amplicons specific to HSV-1 and HSV-2, and 3) detection of the amplified DNA by target-specific hybridization probes via a colorimetric reaction on a lateral-flow strip which is embedded in a self-contained disposable cassette to prevent amplicon contamination.
Specimen preparation involves one simple dilution step in which specimens in viral transport medium are diluted 80-fold in Dilution Tubes.
The diluted samples are transferred into a 0.2 mL Amplification Tube containing lyophilized HDA reagents. Incubation at 64°C for 45 minutes results in the release of the HSV DNA and subsequent isothermal amplification of the target sequence. The amplified DNA is detected by a set of specific detection probes included in the Amplification Tube: HSV-1 target hybridizes to two specific probes labeled with Biotin (BioTEG) and Digoxigenin (DIG) and HSV-2 target hybridizes to two specific probes labeled with Biotin (BioTEG) and Fluorescein isothiocyanate (FITC). A competitive internal control (IC) is included in the Amplification Tube to monitor inhibitory substances in clinical samples, reagent failure or device failure. The IC target is amplified by HSV-2 specific primers and hybridizes to the biotin-labeled HSV-2 probe and a 1C specific probe labeled with 2,4-dinitrophenyl (DNP-TEG).
Detection of the amplified DNA with specific probes is achieved by Type III BEStTM cassettes. The self-contained Type III BESTM cassettes carry lateral-flow DNA detection strips coated with anti-DNP antibodies (C line), anti-DIG antibodies (T1 line) and anti-FITC antibodies (T2 line). HSV-1 amplicon with BioTEG and DIG-labeled probes is captured by anti-DIG antibodies at the TI-Line and HSV-2 amplicon with BioTEG and FITC-labeled probes is captured by anti-FITC antibodies at the T2-Line, while the IC amplicon with BioTEG and DNP-labeled probes is captured by anti-DNP antibodies at the C-Line. The amplicon-probe complexes captures the streptavidin-conjugated color particles for visualization and the test result is shown as colored lines that are visually read.
A positive result for HSV-1 (detection of HSV-1 DNA) is reported when the T1 line is visible through the detection window of the cassette, while a positive result for HSV-2 (detection of HSV-2 DNA) is reported when the T2 line is visible through the detection window of the cassette. A positive result for both HSV-2 (detection of both HSV-1 and HSV-2 DNA) is reported when both the T1 line and the T2 line are visible through the detection window of the cassette. A negative result (no detection of HSV-2 DNA) is reported when only the C line is displayed. The assay result is regarded as invalid when the T1 line, T2 line and C line are not present and the assay should be repeated.
Here's an analysis of the acceptance criteria and study proving the device meets them, based on the provided text:
Device Name: AmpliVue® HSV 1+2 Assay
1. Table of Acceptance Criteria and Reported Device Performance
The provided document primarily focuses on demonstrating the substantial equivalence of the AmpliVue® HSV 1+2 Assay to a predicate device and presenting its analytical and clinical performance. While explicit "acceptance criteria" (numerical thresholds that must be met) are not clearly defined as a separate section with specific targets, the performance studies implicitly set the standard for acceptable performance compared to the gold standard.
Here's a summary of the key performance metrics reported in the document:
| Performance Metric Category | Specific Metric | Acceptance Criteria (Implied) | Reported Device Performance |
|---|---|---|---|
| Reproducibility (Overall) | HSV-1 Low Positive Rate of Detection / Agreement | High (e.g., nearing 100%) | 89/90 (99%) Detection, 94-100% CI |
| HSV-1 Moderate Positive Rate of Detection / Agreement | High (e.g., nearing 100%) | 90/90 (100%) Detection, 96-100% CI | |
| HSV-2 Low Positive Rate of Detection / Agreement | High (e.g., nearing 100%) | 90/90 (100%) Detection, 96-100% CI | |
| HSV-2 Moderate Positive Rate of Detection / Agreement | High (e.g., nearing 100%) | 90/90 (100%) Detection, 96-100% CI | |
| Positive Control Agreement | 100% | 100% (both HSV-1 and HSV-2 sections) | |
| Negative Control Agreement | 100% | 100% (both HSV-1 and HSV-2 sections) | |
| Reproducibility (High Negative) | HSV-1+2 High Negative Rate of Detection / Agreement (HSV-1 calculation) | Undefined (expected low detection, high agreement for negativity) | 45/90 (50%) Detection, 50% Agreement, 40-60% CI |
| HSV-1+2 High Negative Rate of Detection / Agreement (HSV-2 calculation) | Undefined (expected low detection, high agreement for negativity) | 50/90 (44%) Detection, 44% Agreement, 35-55% CI | |
| Repeatability (Within Lab) | HSV-1 Low Positive Agreement | High (e.g., nearing 100%) | 100% (95-100% CI) |
| HSV-1 Moderate Positive Agreement | High (e.g., nearing 100%) | 100% (95-100% CI) | |
| HSV-2 Low Positive Agreement | High (e.g., nearing 100%) | 100% (95-100% CI) | |
| HSV-2 Moderate Positive Agreement | High (e.g., nearing 100%) | 100% (95-100% CI) | |
| Positive Control Agreement | 100% | 100% | |
| Negative Control Agreement | 100% | 100% | |
| Level of Detection (LoD) | Positivity Rate at LoD | ≥95% (as per study definition) | HSV-1: 1.1 x 10^5 TCID50/mL (100% at this level) |
| HSV-2: 1.1 x 10^4 TCID50/mL (100% at this level for MS, 97% for G) | |||
| Analytical Specificity/Cross-Reactivity | No interference/cross-reactivity with specified microorganisms | 100% no interference | None of 89 microorganisms interfered or cross-reacted. |
| Interfering Substances | No interference with specified substances at 3xLoD | 100% no interference | No interference observed with 33 substances and 5 viral transport media. |
| Competitive Inhibition | No inhibition with coinfection at certain concentrations | Undefined, but expected for clinical utility | Not observed with one target at 3xLoD and other up to 300xLoD. Observed at 3000xLoD. Not observed with equal concentrations (3xLoD-3000xLoD). |
| Carry-Over and Cross Contamination | No carry-over in alternating high pos/neg samples | 100% absence | Confirmed no carry-over or cross-contamination. |
| Clinical Performance (Sensitivity) | HSV-1 Cutaneous Lesions | High (Implied by equivalence to gold standard) | 100% (95% CI: 88.6%-100%) |
| HSV-1 Mucocutaneous Lesions | High (Implied by equivalence to gold standard) | 94.9% (95% CI: 90.3%-97.4%) | |
| HSV-2 Cutaneous Lesions | High (Implied by equivalence to gold standard) | 98.3% (95% CI: 91.0%-99.7%) | |
| HSV-2 Mucocutaneous Lesions | High (Implied by equivalence to gold standard) | 97.4% (95% CI: 93.4%-99.0%) | |
| Clinical Performance (Specificity) | HSV-1 Cutaneous Lesions | High (Implied by equivalence to gold standard) | 97.1% (95% CI: 94.6%-98.5%) |
| HSV-1 Mucocutaneous Lesions | High (Implied by equivalence to gold standard) | 96.5% (95% CI: 94.8%-97.7%) | |
| HSV-2 Cutaneous Lesions | High (Implied by equivalence to gold standard) | 95.6% (95% CI: 92.8%-97.3%) | |
| HSV-2 Mucocutaneous Lesions | High (Implied by equivalence to gold standard) | 95.8% (95% CI: 94.2%-97.0%) |
Note on Acceptance Criteria: The document describes the results of the studies and states that they "demonstrate that the AmpliVue® HSV 1+2 Assay is substantially equivalent to the predicate device." This implies that the performance shown met the internal criteria for substantial equivalence, which often means demonstrating non-inferiority or comparable performance to a legally marketed predicate. Specific numerical thresholds for "acceptance" are not explicitly listed in an "Acceptance Criteria" section but are inherent in the successful outcomes of these analytical and clinical studies. For example, for LoD, a positivity rate of ≥95% was defined within the study itself as the criterion for determining the LoD.
2. Sample Size Used for the Test Set and Data Provenance
The primary clinical performance study (test set) involved:
- Sample Size: A total of 1355 specimens from symptomatic male and female patients were tested. 19 tests were considered invalid and removed, resulting in 1336 specimens for the initial analysis. For HSV-1 performance calculation, 211 HSV-2 positive specimens were removed, leaving 1125 specimens.
- Data Provenance:
- Country of Origin: United States.
- Retrospective or Prospective: Not explicitly stated as retrospective or prospective, but the description "evaluated at five geographically diverse locations within the United States" and involving "symptomatic male and female patients" suggests a prospective collection and testing of clinical samples.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
- Number of Experts: Not applicable in the context of this diagnostic assay.
- Qualifications of Experts: The ground truth was established by a "gold standard/reference method" which is the ELVIS® HSV ID and D³ Typing Test System (cell culture using an enzyme linked virus inducible system). This is a laboratory-based reference method, not an expert panel.
4. Adjudication Method for the Test Set
- Adjudication Method: Not applicable. The ground truth was determined by a single reference method (ELVIS® viral culture), not by a panel of human adjudicators. Discrepancies would likely be investigated by re-testing or deeper analysis against the reference method. The document notes that the ELVIS culture inability to detect co-infected specimens led to the removal of HSV-2 positive samples when calculating HSV-1 performance, which served as a form of handling limitations of the ground truth.
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
- There was no MRMC comparative effectiveness study done.
- This device is an in vitro diagnostic (IVD) assay for the direct detection of viral DNA, not an AI-assisted diagnostic tool that involves human readers interpreting images or data. Therefore, the concept of human readers improving with AI assistance is not relevant to this device.
6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done
- This question is partially applicable. The AmpliVue® HSV 1+2 Assay is an "algorithm only" in the sense that it is a chemical and molecular diagnostic test with a qualitative visual readout (colored lines). There is no "human-in-the-loop" interpretation beyond reading the positive/negative lines based on established rules.
- The entire clinical performance study (sensitivity and specificity tables) represents the standalone performance of the assay compared to the reference method.
7. The Type of Ground Truth Used
- Ground Truth Type: Expert reference method / Gold standard.
- Specifically, the ground truth was established using the ELVIS® HSV ID and D³ Typing Test System, which is described as the "gold standard/reference method i.e., cell culture using an enzyme linked virus inducible system."
8. The Sample Size for the Training Set
- Sample Size for Training Set: The document does not explicitly mention a separate "training set" as commonly understood in machine learning contexts. This is an IVD assay, and the development process would involve analytical studies (LoD, cross-reactivity, interference, etc.) and then clinical validation. The samples used for initial assay development and optimization (analogous to a training set) are not detailed in terms of specific numbers.
- However, the analytical performance section describes studies that would have informed the assay's design and operational characteristics, such as:
- Level of Detection (LoD): Used serially diluted viral stocks (30 replicates across 3 lots for initial LoD determination, then 20 replicates for confirmation). This involves a number of samples to establish analytical sensitivity.
- Analytical Specificity/Cross-Reactivity: Tested 89 microorganisms (each in triplicate).
- Interfering Substances: Tested 33 substances and 5 viral transport media (each in triplicate).
9. How the Ground Truth for the Training Set was Established
- As noted above, a distinct "training set" with its own ground truth establishment method isn't explicitly described. However, for the analytical studies that would inform assay development (like LoD, specificity, and interference), the ground truth was established by:
- Quantified viral stocks: For LoD studies, Herpes Simplex Virus 1 (HSV-1) and Herpes Simplex Virus 2 (HSV-2) viral stocks were quantified (TCID50/mL). These quantified stocks served as the known "true" concentrations for determining the assay's detection limit.
- Known microorganisms and substances: For analytical specificity and interfering substances studies, known microorganisms (purified genomic DNA, quantified cultures) and specific chemical/biological substances were introduced at known concentrations. The "ground truth" here is the precise identity and concentration of the added components, and the expectation is zero interference or cross-reactivity.
§ 866.3305 Herpes simplex virus serological assays.
(a)
Identification. Herpes simplex virus serological assays are devices that consist of antigens and antisera used in various serological tests to identify antibodies to herpes simplex virus in serum. Additionally, some of the assays consist of herpes simplex virus antisera conjugated with a fluorescent dye (immunofluorescent assays) used to identify herpes simplex virus directly from clinical specimens or tissue culture isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by herpes simplex viruses and provides epidemiological information on these diseases. Herpes simplex viral infections range from common and mild lesions of the skin and mucous membranes to a severe form of encephalitis (inflammation of the brain). Neonatal herpes virus infections range from a mild infection to a severe generalized disease with a fatal outcome.(b)
Classification. Class II (special controls). The device is classified as class II (special controls). The special control for the device is FDA's revised guidance document entitled “Class II Special Controls Guidance Document: Herpes Simplex Virus Types 1 and 2 Serological Assays.” For availability of the guidance revised document, see § 866.1(e).