(262 days)
The Aptima Herpes Simplex Viruses 1 & 2 assay (Aptima HSV 1 & 2 assay) is an in vitro diagnostic nucleic acid amplification test (NAAT), using real time transcription-mediated amplification (TMA), for the qualitative detection and differentiation of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) messenger RNA (mRNA) in clinician-collected swab specimens from anogenital skin lesions. The assay is intended for use with swab specimens placed in Aptima specimen transport medium (STM) or in viral transport media (VTM) that is immediately diluted into STM.
The Aptima HSV 1 & 2 assay is intended for use as an aid in the diagnosis of HSV-1 and/or HSV-2 infections in symptomatic male and female patients. The Aptima HSV 1 & 2 assay is indicated for use on the Panther® system.
The Aptima Herpes Simplex Virus 1 & 2 assay (Aptima HSV assay) is a nucleic acid amplification test (NAAT) developed for use on the fully automated Panther system that utilizes target capture, transcription mediated amplification (TMA), and real-time detection of HSV-1, HSV-2, and an internal control (IC). The Aptima HSV assay amplifies and detects mRNAs for HSV-1 and HSV-2. These RNAs are expressed from the viral genome during the infection cycle, and are packaged inside HSV-1 and HSV-2 viral particles prior to virus release from infected cells. The Aptima HSV assay therefore detects virus-infected cells and the mature virus particles themselves.
The Aptima HSV assay involves three main steps, which all take place in a single tube on the Panther® system: target capture, target amplification by TMA, and detection of the amplification products (amplicon) by the fluorescent labeled probes (torches). The assay incorporates an IC in every test to monitor targeted nucleic acid capture, amplification and detection.
When the Aptima HSV assay is performed, the targeted viral mRNA and IC are isolated using magnetic microparticles and target-specific capture oligomers, in a process called target capture. The capture oligomers contain sequences complementary to specific regions of the targeted RNA (HSV mRNA or IC) as well as a string of deoxyadenosine residues. During the hybridization step, the sequence-specific regions of the capture oligomers bind to specific regions of the RNA target molecules. The microparticles, including the captured RNA target molecules bound to them, are pulled to the side of the reaction tube using magnets and the supernatant is aspirated. The particles are washed to remove residual specimen matrix that may contain amplification inhibitors. After target capture steps are completed, the specimens are ready for amplification.
Target amplification occurs via TMA, which is a transcription-based nucleic acid amplification method that utilizes two enzymes, MMLV (Moloney murine leukemia virus) reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy (containing a promoter sequence for T7 RNA polymerase) of the target sequence. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template. Detection is achieved using single-stranded nucleic acid torches that are present during the amplification of the target and hybridize specifically to the amplicon in real time. Each torch has a fluorophore and a quencher. The quencher suppresses the fluorescence of the fluorophore as it is designed to be in close proximity when not hybridized to the amplicon. When the torch binds to the amplicon, the quencher is moved farther away from the fluorophore and it will emit a signal at a specific wavelength when excited by a light source. More torch hybridizes when more amplicon is present. The increase in fluorescent signal from progressive amplification is detected by fluorometers within the Panther system. The Panther system can detect and discriminate between the three fluorescent signals corresponding to HSV-1, HSV-2 and IC amplification products. The fluorescence (measured in relative fluorescence units [RFU]) is monitored over time to produce a real-time fluorescence emergence curve for each reporter dye. The Panther system software compares the fluorescence emergence curves to fixed cut off times to report results (TTime) for HSV-1, HSV-2 and IC.
Here's an analysis of the acceptance criteria and the study proving the device meets them, based on the provided text:
Acceptance Criteria and Device Performance for Aptima Herpes Simplex Viruses 1 & 2 Assay
1. Table of Acceptance Criteria and Reported Device Performance
The acceptance criteria are implied by the clinical performance targets presented in the study. While explicit pre-defined acceptance thresholds (e.g., "Sensitivity must be >90%") are not directly stated as pass/fail criteria, the reported performance metrics demonstrate the device's capability. For this analysis, I will use the clinical performance study results as the reported device performance against generally expected high standards for diagnostic accuracy.
Note: The document does not explicitly state the pre-defined "acceptance criteria" numerical targets. The reported performance below represents the observed results of the clinical study, which presumably met the internal performance requirements for the manufacturer and FDA review.
Table 1: Acceptance Criteria (Implied) and Reported Device Performance
| Metric | Target (Implied Acceptance) | Reported Device Performance (Combined, VTM) | Reported Device Performance (Combined, STM) |
|---|---|---|---|
| HSV-1 Sensitivity | High sensitivity, typically >90% for diagnostic assays. | 93.4% (95% CI: 85.5-97.2) | 94.7% (95% CI: 87.1-97.9) |
| HSV-1 Specificity | High specificity, typically >95-98% for diagnostic assays. | 99.8% (95% CI: 98.8 - >99.9) | 99.6% (95% CI: 98.4-99.9) |
| HSV-2 Sensitivity | High sensitivity, typically >90% for diagnostic assays. | 96.9% (95% CI: 94.0-98.4) | 98.4% (95% CI: 96.1-99.4) |
| HSV-2 Specificity | High specificity, typically >95-98% for diagnostic assays. | 97.5% (95% CI: 94.9-98.8) | 92.8% (95% CI: 89.1-95.3) |
| Reproducibility | Consistent results across sites, operators, and reagent lots, especially at low concentrations. | Agreement with expected results generally high, with some variability at concentrations near or below LoD (e.g., 46.3% - 100%). | Agreement with expected results generally high, with some variability at concentrations near or below LoD (e.g., 46.3% - 100%). |
| Limit of Detection (LoD) | Low detection limit to ensure detection of low viral loads. | HSV-1: 60.6-186.9 TCID50/mL (depending on strain/media) | HSV-2: 18.2-128.8 TCID50/mL (depending on strain/media) |
| Interfering Substances | No significant impact on assay sensitivity or specificity. | No effect observed for tested substances at specified concentrations. | No effect observed for tested substances at specified concentrations. |
| Cross-Reactivity | No cross-reactivity with non-target microorganisms. | No evidence of cross-reactivity or microbial interference (except for Streptococcus pneumoniae at 1x10^6 CFU/mL where cross-reactivity was observed). | No evidence of cross-reactivity or microbial interference (except for Streptococcus pneumoniae at 1x10^6 CFU/mL where cross-reactivity was observed). |
| Co-Infection Detection | Ability to detect both HSV-1 and HSV-2 when present. | 100% detection for both HSV-1 and HSV-2 in co-infected panels. | 100% detection for both HSV-1 and HSV-2 in co-infected panels. |
2. Sample size used for the test set and the data provenance
- Clinical Test Set Sample Size:
- Total Subjects: 544 evaluable subjects (195 males and 349 females).
- Evaluated for HSV-1: 528 VTM specimens and 531 STM specimens.
- Evaluated for HSV-2: 533 VTM specimens and 535 STM specimens.
- Data Provenance:
- Country of Origin: United States. The study was conducted at 17 US clinical sites.
- Retrospective or Prospective: Prospective. The study is described as a "prospective, multicenter clinical study."
3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts
The document does not specify the number of experts used to establish the ground truth or their specific qualifications (e.g., "radiologist with 10 years of experience").
However, it describes the methods used for the composite reference method:
- ELVIS HSV ID and D3 Typing Test system viral culture
- A validated bidirectional PCR/sequencing procedure
- A third FDA-cleared assay for HSV-1 and HSV-2 was used for final composite reference interpretation when the initial methods disagreed or when PCR/sequencing detected both types.
This suggests that the ground truth was established through a combination of highly reliable laboratory tests, implying a rigorous approach to defining true positive/negative cases, rather than relying solely on individual expert interpretation without further clarification.
4. Adjudication method (e.g., 2+1, 3+1, none) for the test set
The document describes an adjudication method for the ground truth:
- "A third FDA-cleared assay for HSV-1 and HSV-2, was used to determine the final composite reference interpretation when the ELVIS D3 culture and PCR/sequencing results did not agree on the type of HSV detected or when PCR/sequencing detected both HSV-1 and HSV-2."
This indicates a hierarchical or tie-breaking system rather than a simple 2+1 or 3+1 consensus among human readers. It relies on a "composite reference method" combining results from multiple validated laboratory tests.
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
This is a diagnostic assay for detecting viral RNA, not an imaging device that involves human readers interpreting images with or without AI assistance. Therefore, no MRMC comparative effectiveness study involving human readers with AI assistance was performed or reported in this submission. The device (Aptima HSV 1 & 2 Assay) is designed to provide a direct qualitative result (positive/negative for HSV-1 and/or HSV-2) from a processed specimen.
6. If a standalone (i.e. algorithm only without human-in-the loop performance) was done
Yes, a standalone performance study was done in the sense that the Aptima HSV 1 & 2 Assay (the "algorithm" in this context) directly processes specimens and generates results without requiring human interpretation for its output. The clinical performance study directly evaluated the accuracy of the assay's results against a composite reference standard. The "human-in-the-loop" would be the clinician collecting the sample and laboratory technicians running the test and reporting the results, but the analytical output itself is determined by the assay system.
7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)
The ground truth used was a composite reference method combining:
- ELVIS HSV ID and D3 Typing Test system viral culture
- A validated bidirectional PCR/sequencing procedure
- A third FDA-cleared assay for HSV-1 and HSV-2 (used for tie-breaking/discrepancy resolution)
This is a robust form of ground truth based on multiple established laboratory diagnostic methods.
8. The sample size for the training set
The document does not report a sample size for a training set. This is expected for a diagnostic assay of this type, as it's not a machine learning or AI algorithm that requires a separate "training set" in the conventional sense. The assay's design and optimization (e.g., probe sequences, amplification conditions) would have been developed iteratively, but a distinct "training set" for performance evaluation is not applicable here. The analytical studies (LoD, cross-reactivity, etc.) and the clinical performance study represent the validation of the finalized assay.
9. How the ground truth for the training set was established
As there is no explicit "training set" in the context of machine learning, this question is not directly applicable. The assay's components and parameters would have been optimized using internal development processes and validated through analytical studies. For these analytical studies (e.g., LoD, cross-reactivity), the "ground truth" (i.e., known-positive or known-negative samples, specific viral strains/concentrations) would have been established through well-characterized laboratory standards, spiked samples, and reference materials.
§ 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).