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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

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.

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The APTIMA Combo 2 Assay is a target amplification nucleic acid probe test that utilizes target capture for the in vitro qualitative detection and differentiation of ribosomal ribonucleic acid (rRNA) from Chlamydia trachomatis and/or Neisseria gonorrhoeae in clinician-collected endocervical, vaginal, and male urethral swab specimens, patient-collected vaginal swab specimens* and male and female urine specimens. The assay may be used to test specimens from symptomatic and asymptomatic individuals to aid in the diagnosis of gonococcal and/or chlamydial urogenital disease.

*Patient-collected vaginal swab specimens are an option for screening women when a pelvic exam is not otherwise indicated. The vaginal swab specimen collection kit is not for home use.

The APTIMA Vaginal Swab Specimen Collection Kit is for use with the APTIMA Assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The APTIMA Vaginal Swab Specimen Collection Kit is intended to be used for clinician and patient collection of vaginal swab specimens according to the instructions provided. Patient-collected vaginal swab specimens are an option for screening women when a pelvic exam is not otherwise indicated. The APTIMA Vaginal Swab Specimen Collection Kit is not for home use.

Clearance of this premarket notification extends the clinical performance claims of the commercially available GEN-PROBE APTIMA Combo 2 Assay to include clinician-collected and patient-collected vaginal swabs (in a medical setting) as acceptable testing specimens. The ancillary kit formulated for this specific application is the GEN-PROBE APTIMA Vaginal Swab Specimen Collection Kit. The components of the APTIMA Vaginal Swab Specimen Collection Kit include: (1) a sterile swab for the collection of vaginal specimens and (2) a transport tube containing transport media with a penetrable cap.

The APTIMA Vaginal Swab Specimen Collection Kit is for use with the APTIMA Assays for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The APTIMA Vaginal Swab Specimen Collection Kit is intended to be used for clinician and patient collection of vaginal swab specimens according to the instructions provided. The APTIMA Vaginal Swab Specimen Collection Kit not for home use.

The APTIMA Combo 2 Assay incorporates the technologies of target capture, in vitro nucleic acid amplification, and hybridization of target amplicons with acridinium ester-labeled DNA probes to specifically detect and differentiate both C. trachomatis and N. gonorrhoeae nucleic acids in clinical specimens. GEN-PROBE's proprietary technologies are combined in this product to allow qualitative detection of C. trachomatis rRNA and N. gonorrhoeae rRNA.

Here's a breakdown of the acceptance criteria and study details for the GEN-PROBE® APTIMA® Combo 2 Assay, based on the provided text:

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied through the sensitivity and specificity values reported, which are compared against a "patient infected status algorithm" as the ground truth. While explicit numerical acceptance thresholds are not stated in the provided text (common in older 510(k) summaries which often relied on substantial equivalence to predicate devices), the study aims to demonstrate high performance.

Chlamydia trachomatis (CT)

Neisseria gonorrhoeae (NG)

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

The study was a multi-center clinical study involving 1,464 symptomatic and asymptomatic female subjects.

• Sample Size:
  • For CT analysis: 2,868 vaginal swab test results.
  • For NG analysis: 2,867 vaginal swab test results.
  • These numbers represent the combined results from patient-collected and clinician-collected vaginal swabs.
• Data Provenance: The data was collected from subjects attending STD, OB/GYN, teen, and family planning clinics. The text does not explicitly state the country of origin, but given it's a US FDA submission, it's highly likely to be a prospective multi-center clinical study conducted in the United States.

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

The ground truth was established using an algorithm, not directly by human experts interpreting images or test results. Therefore, the concept of "number of experts" and "qualifications of those experts" as typically applied to expert review in imaging studies, does not directly apply here. The accuracy of the reference standard relies on the performance of the commercially available NAATs used.

4. Adjudication Method for the Test Set

The adjudication method for establishing the patient infected status was algorithmic (rule-based):

• Subjects were considered infected with C. trachomatis or N. gonorrhoeae if two of the four reference NAAT results were positive (one specimen testing positive in each NAAT: APTIMA Combo 2 Assay and another commercially available NAAT, using endocervical swab and urine specimens).
• Subjects were considered non-infected if less than two reference NAAT results were positive.

This method uses concordant results from multiple tests/specimens to define the "true" infection status.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not performed. This study is for an in vitro diagnostic (IVD) assay detecting biomarkers, not an AI-assisted diagnostic imaging device that involves human reader interpretation. Therefore, the concept of human readers improving with AI vs. without AI assistance does not apply.

6. Standalone Performance Study

Yes, a standalone performance study was done for the algorithm (the APTIMA Combo 2 Assay). The entire clinical study described evaluates the performance of the device on its own, comparing its results from vaginal swab specimens to the defined patient infected status algorithm. The reported sensitivity and specificity values are for the device operating as a standalone diagnostic tool for vaginal swab specimens.

7. Type of Ground Truth Used

The ground truth used was an algorithmic consensus based on multiple reference nucleic acid amplification tests (NAATs). Specifically:

• Endocervical swab and urine specimen results from the commercially-available APTIMA Combo 2 Assay and another commercially-available NAAT were used.
• Infection status was determined by requiring a positive result from both reference NAATs.
• Culture was not used as a reference test for this specific study, as the APTIMA Combo 2 Assay had previously been evaluated against culture for other specimen types (as per K003395).

8. Sample Size for the Training Set

The document does not provide information regarding a distinct "training set" sample size. For an IVD assay like this, development typically involves analytical validation (sensitivity, specificity, interference etc.) and then clinical validation with a distinct set of clinical samples. The analytical studies (like analytical sensitivity, specificity, recovery, interference) might be considered analogous to early-stage development/testing, but a formal "training set" in the machine learning sense is not explicitly mentioned or typically applicable for this type of assay in this context. The clinical study described served as the primary performance evaluation.

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

As noted above, the concept of a separate "training set" with established ground truth in the machine learning context is not detailed. The primary clinical study used the algorithmic consensus described in point 4 as its ground truth for evaluating the device's performance.

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