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The Aptima Trichomonas vaginalis (TV) assay is an in vitro qualitative nucleic acid amplification test (NAAT) for the detection of ribosomal RNA (rRNA) from Trichomonas vaginalis to aid in the diagnosis of trichomoniasis using the Panther system.

The assay may be used to test the following specimens from symptomatic or asymptomatic individuals: clinician-collected endocervical swabs, clinician-collected and patient-collected vaginal swabs (in a clinical setting), female and male urine, and specimens collected in PreservCyt Solution.

The Aptima TV assay involves the technologies of target capture, transcription-mediated amplification (TMA), and hybridization protection assay (HPA). Specimens are collected and transferred into their respective specimen transport tubes. The transport solution in these tubes releases the rRNA target and protects it from degradation during storage. When the Aptima TV assay is performed in the laboratory, the target rRNA is isolated from the specimens using a specific capture oligomer and magnetic microparticles in a method called target capture. The capture oligomer contains a sequence complementary to a specific region of the target molecule as well as a string of deoxyadenosine residues. During the hybridization step, the sequence-specific region of the capture oligomer binds to a specific region of the target molecule. The capture oligomer:target complex is then captured out of solution by decreasing the temperature of the reaction to room temperature. This temperature reduction allows hybridization to occur between the deoxyadenosine region on the capture oligomer and the poly-deoxythymidine molecules that are covalently attached to the magnetic particles. The microparticles, including the captured target molecule bound to them, are pulled to the side of the reaction vessel using magnets and the supernatant is aspirated. The particles are washed to remove residual specimen matrix that may contain amplification inhibitors. After the target capture steps are completed, the specimens are ready for amplification.

The provided text describes the analytical and clinical studies performed for the Aptima Trichomonas vaginalis Assay. Here's a breakdown of the requested information:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria values for sensitivity, specificity, PPA, or NPA. Instead, it presents the achieved performance. However, implicit acceptance criteria for NAAT assays generally involve high sensitivity and specificity. The reproducibility study tables show numerical targets for agreement (e.g., >95% positivity for LoD, 90.7% to 100% agreement for reproducibility).

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

• Clinical Study 2 (Primary Test Set):
  • Total evaluable specimens: 5502 specimens from 3820 evaluable subjects.
  • Breakdown by specimen type:
    • 1785 patient-collected vaginal swab specimens
    • 1782 female urine specimens
    • 1935 male urine specimens
  • Data Provenance: Prospective, multicenter clinical study conducted at 11 geographically and ethnically diverse US clinical sites (obstetrics and gynecology, family planning, and STI clinics).
  • Retrospective/Prospective: Primarily prospective. Samples were collected from consenting subjects in a prospective 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 mention the use of human experts (e.g., radiologists, pathologists) to establish the ground truth. For this in vitro diagnostic (IVD) device, the ground truth was established by molecular testing.

4. Adjudication Method for the Test Set

The ground truth for the clinical test set was established using a "composite comparator method" or "patient infected status (PIS)" / "composite comparator algorithm (CCA)" based on results from up to three cleared NAATs.

• Method:
  • Specimens were categorized as infected (PIS) or positive (CCA) if a positive result occurred in at least two of the comparator NAATs.
  • Specimens were categorized as not infected or negative if at least 2 of the comparators results were negative.
  • A third (tie-breaker) comparator was only required if the first 2 comparator results were discordant.
  • Specimens that could not be categorized due to missing results from comparator assays were excluded.

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

No, an MRMC comparative effectiveness study was not applicable and therefore not performed. This is an in vitro diagnostic (IVD) device for the detection of ribosomal RNA from Trichomonas vaginalis. It is a lab-based assay, not an imaging device that requires human readers to interpret results, or AI assistance for human reader improvement.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was done

Yes, the performance reported (sensitivity, specificity, PPA, NPA) for the Aptima TV assay is its standalone performance. The assay itself is the "algorithm" in this context; it processes specimens and provides a qualitative result (positive/negative) without direct human interpretation of the assay's raw output for diagnosis. The study evaluates the device's ability to accurately detect the target in various specimens against the established ground truth.

7. The Type of Ground Truth Used

The ground truth used was established using a composite comparator method (sometimes referred to as a "gold standard" or "reference standard" in IVD studies), which relied on the results of multiple (up to three) cleared Nucleic Acid Amplification Tests (NAATs). This is a form of expert consensus among molecular assays.

8. The Sample Size for the Training Set

The document does not explicitly state the sample size for a separate "training set." For IVD assays, particularly those based on well-established molecular biology principles (like NAATs), development and optimization (analogous to "training") often use specific analytical panels or earlier development runs rather than a distinct, large "training set" of clinical samples as seen in machine learning/AI models. The studies described are primarily for validation.

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

Since a distinct "training set" with established ground truth is not explicitly mentioned as a separate phase of the pivotal study, the establishment of ground truth for any developmental or optimization work would likely follow similar principles as the validation ground truth: using reference materials, spiked samples, or well-characterized clinical samples confirmed by established laboratory methods or multiple comparator assays. The document focuses on the validation and reproducibility of the assay.

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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 AmpliVue® Trichomonas Assay is an in vitro diagnostic test, uses isothermal amplification technology (helicase-dependent amplification, HDA) for the qualitative detection of Trichomonas vaginalis nucleic acids isolated from clinician-collected vaginal swab specimens obtained from symptomatic or asymptomatic females to aid in the diagnosis of trichomoniasis.

The AmpliVue® Trichomonas Assay combines simple processing, an isothermal amplification technology named Helicase-Dependent Amplification (HDA), and a selfcontained disposable amplicon detection device for the detection of T. vaginalis in clinician-collected vaginal swabs from symptomatic and asymptomatic women. The assay targets a conserved multi-copy sequence of the T. vaginalis DNA. The vaginal swab is eluted in a lysis tube, and the cells are lysed by simple heat treatment. After heat treatment, an aliquot of the lysed specimen is transferred into a dilution tube. An aliquot of the diluted sample is added to a reaction tube containing a lyophilized mix of HDA reagents including primers specific for the amplification of a conserved DNA sequence only found in T. vaginalis. The assay also includes an internal control to confirm the integrity of the assay reagents and cassette detection as well as to control for (or determine whether) HDA-inhibitors that may be present within the clinical specimens. The HDA reaction is asymmetric so that an excess of single stranded DNA (amplicon) is formed. The sequence specific capture probes as well as a biotinylated detection probe shared by both target and internal control bind to the corresponding single-stranded amplicons, forming dual labeled probe-amplicon hybrid. After completion of the HDA reaction, the reaction tube is transferred to a cassette for rapid detection with the test result displayed as test and/or control lines in the window of the cassette. The dual-labeled probe-amplicon hybrid is then detected by the lateral flow strip within the cassette. The bottom line captures the test amplicon and the top line captures the control amplicon. The biotin label binds the streptavidin-conjugated color particles for visualization and the test result is shown as colored lines visible to the naked eye. The cassette is comprised of two individual components: an amplicon cartridge that holds the running buffer and a single 0.2 mL thin wall reaction tube containing the amplified product; and the detection chamber which houses the amplicon cartridge and a verticalflow DNA detection strip embedded into the cassette. The DNA detection strip is coated with different anti-hapten antibodies that serve as the T. vaginalis test (T) line and the control (C) line in the assay. A razor blade and a plastic pin located at the bottom of the detection chamber opens the HDA reaction tube and the running buffer bulb when the handle of the cassette is closed. The mixture flows through a fiberglass paper connected to the DNA detection strip that contains a fiberglass pad pre-loaded with streptavidinconjugated color particles for color visualization. Detection of T. vaginalis DNA is reported whenever the T2 (Test line 2) is visible through the detection window of the cassette. The presence of the C line is not required for positive results. No detection of T. vaginalis DNA is reported when only the C line is displayed. The assay is regarded as invalid when neither line is displayed.

Here's an analysis of the acceptance criteria and the study that proves the device meets them, based on the provided text:

Description of Acceptance Criteria and Device Performance Study for AmpliVue® Trichomonas Assay

1. Table of Acceptance Criteria and Reported Device Performance:

The document does not explicitly state formal "acceptance criteria" in terms of specific performance targets set prior to the study. However, the study aims to demonstrate substantial equivalence to a predicate device, and the performance characteristics reported serve as the de facto demonstration of meeting the required clinical performance for this type of in vitro diagnostic device. The performance characteristics of the predicate device (APTIMA Trichomonas vaginalis Assay) are also provided for comparison.

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

• Sample Size for Test Set: 992 clinician-collected vaginal swab specimens.
• Data Provenance: The study was a multi-center study performed at four locations in the United States and one location in Canada. It was a prospective study, with specimens obtained from subjects after informed consent between April and November 2014.

3. Number of Experts and Qualifications for Ground Truth in the Test Set:

• The document states that the reference method for establishing ground truth was a composite of Wet Mount and InPouch TV culture. It does not specify the number or qualifications of experts involved in interpreting these reference methods. However, these methods are standard clinical laboratory procedures, implying trained laboratory personnel (e.g., medical technologists, clinical microbiologists) would have performed and interpreted them.

4. Adjudication Method for the Test Set:

• The ground truth was established using a composite reference method: Wet Mount and InPouch TV culture. A specimen was considered positive if either test was positive. This acts as a form of "OR" adjudication or a composite gold standard, rather than a direct agreement between multiple independent experts on the same test.
• For discordant results where the composite reference method was negative but the AmpliVue assay was positive, the FDA-cleared molecular device collection swab was used for discordant testing. Specifically, "Eight (8) of sixteen (16) Composite negative/AmpliVue positive specimens were positive by a FDA-cleared Trichomonas vaginalis molecular device." This implies a form of tie-breaking or re-evaluation using a third, highly sensitive method a posteriori.

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

• No, an MRMC comparative effectiveness study was not done. This study focuses on the standalone performance of the AmpliVue® Trichomonas Assay compared to a composite reference method, and its substantial equivalence to a predicate device. It does not evaluate human reader performance with or without AI assistance.

6. Standalone Performance Study:

• Yes, a standalone performance study was done. The entire clinical study described evaluates the performance of the AmpliVue® Trichomonas Assay (algorithm only, as it's an in vitro diagnostic test) in detecting Trichomonas vaginalis nucleic acids from vaginal swab specimens. The reported sensitivity, specificity, PPV, and NPV are all measures of this standalone performance.

7. Type of Ground Truth Used:

• The primary ground truth used was a composite reference method consisting of:
  • Wet Mount
  • InPouch TV Culture
• For discordant cases (AmpliVue positive, composite negative), an FDA-cleared Trichomonas vaginalis molecular device was used for further investigation, suggesting a hierarchical or confirmatory approach for specific discrepancies.

8. Sample Size for the Training Set:

• The document does not specify a sample size for a training set. The AmpliVue® Trichomonas Assay is an in vitro diagnostic test based on Helicase-Dependent Amplification (HDA) technology. This type of assay does not typically involve traditional "training sets" in the same way machine learning algorithms do. Its development involves analytical validation (LoD, cross-reactivity, interference, inclusivity) and then clinical validation with a distinct set of patient samples, rather than a machine learning training/test split.

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

• As the device is an in-vitro diagnostic assay rather than an AI/ML algorithm requiring a training set, the concept of "ground truth for the training set" does not directly apply here. The assay's design and analytical parameters (e.g., specific primers, probes, Lysis Buffer, Dilution Buffer, Reaction Tubes quantities) were established through laboratory development and analytical studies (LoD, cross-reactivity, inclusivity, interference) which confirm the assay's ability to detect the target DNA sequence under various conditions.

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