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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 RNA (rRNA) from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC) to aid in the diagnosis of chlamydial and/or gonococcal disease using the Panther System as specified. On the Panther System, the assay may be used to test the following specimens from symptomatic and asymptomatic individuals: clinician-collected endocervical, PreservCyt® Solution liquid Pap specimens, vaginal, throat, rectal, and male urethral swab specimens; patient-collected vaginal swab specimens1, and female and male urine specimens.

The Aptima Combo 20 Assay is a target amplification nucleic acid probe test that utilizes target capture for the in vitro qualitative detection and differentiation of ribosomal RNA (rRNA) from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC) to aid in the diagnosis of chlamydial and/or gonococcal urogenital disease using the Tigris® DTS® Automated Analyzer or semi-automated instrumentation as specified. The assay may be used to test the following specimens from symptomatic individuals: clinician-collected endocervical, vaginal and male urethral swab specimens; and female and male urine specimens. The assay may be used to test the following specimens from asymptomatic individuals: clinician-collected endocervical, vaginal and male urethral swab specimens; patient-collected vaginal swab specimens'; and female and male urine specimens. The assay is also intended for use with the testing of gynecological specimens, from both symptomatic and asymptomatic patients, collected in the PreservCyt® Solution.

The Aptima Combo 2 Assay combines the technologies of target capture, TMA, and DKA. Specimens are collected and transferred into their respective specimen transport tubes. The transport solutions in these tubes release the rRNA targets and protect them from degradation during storage. When the Aptima Combo 2 Assay is performed in the laboratory, the target rRNA molecules are isolated from specimens by use of capture oligomers via target capture that utilizes magnetic microparticles. The capture oligomers contain sequences complementary to specific regions of the target molecules as well as a string of deoxyadenosine residues. A separate capture oligomer is used for each target. During the hybridization step, the sequence specific regions of the capture oligomers bind to specific regions of the target molecules. 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 molecules 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 reaction inhibitors. After the target capture steps are completed, the specimens are ready for amplification.

Target amplification assays are based on the ability of complementary oligonucleotide primers to specifically anneal and allow enzymatic amplification of the target nucleic acid strands. The Aptima Combo 2 Assay replicates a specific region of the 23S rRNA from CT and a specific region of the 16S rRNA from GC via DNA intermediates. A unique set of primers is used for each target molecule. Detection of the rRNA amplification product sequences (amplicon) is achieved using nucleic acid hybridization. Single-stranded nucleic acid chemiluminescent probes, which are complementary to a region of each target amplicon, are labeled with different acridinium ester molecules. The updated version of the Aptima Combo 2 assay incorporates a second CT probe, complementary to a unique region of the existing CT amplicon. This tandem probe provides detection coverage for the variant strains of C. trachomatis that emerged in 2019. The labeled probes combine with amplicon to form stable hybrids. The Selection Reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from unhybridized probe. During the detection step, light emitted from the labeled hybrids is measured as photon signals in a luminometer, and are reported as Relative Light Units (RLU). In DKA, differences in the kinetic profiles of the CT and GC labeled probes allow for the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time. The chemiluminescent detection for CT signal has very rapid kinetics and has the "flasher" kinetic type. The chemiluminescent detection reaction for GC signal is relatively slower and has the "glower" kinetic type. Assay results are determined by a cut-off based on the total RLU and the kinetic curve type.

The provided text describes a 510(k) premarket notification for a modified diagnostic device, the Aptima Combo 2 Assay, used for detecting Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). The modification involves a change in the Probe reagent to improve detection of emerging CT variants.

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

1. Table of Acceptance Criteria and Reported Device Performance

The FDA 510(k) submission process for in vitro diagnostic devices focuses on demonstrating substantial equivalence to a legally marketed predicate device. For modifications to an existing cleared device, the key acceptance criteria revolve around showing that the changes do not negatively impact the assay's performance, safety, and effectiveness, and specifically address the reason for the modification (improved CT variant detection).

While explicit enumerated "acceptance criteria" with numerical thresholds are not presented in a table format as might be seen for a new device, the document implies the following performance criteria were met:

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

• Limit of Detection (LoD) Study:

  • Sample Size: 30 replicates of each dilution were tested for each specimen type (urine, ThinPrep, simulated swab matrix). This was done with 3 reagent lots on both Panther and Tigris systems.
  • Total replicates: 30 (replicates/dilution) * 3 (specimen types) * 3 (lots) * 2 (systems) = 540 replicates per target organism (CT/GC).
  • Data Provenance: In vitro transcripts diluted in negative urine specimens, negative ThinPrep specimens, and simulated swab matrix specimens. This is laboratory-derived analytical data, not directly stated to be from a particular country. It is essentially prospective in nature for validating the new formulation.

• Clinical Comparability Study:

  • Sample Size: Not explicitly stated as a total count, but implied by the comparison tables below Table 4 and Table 5.
    • CT: 49 CT positive, 273 CT negative, 3 discordant (updated positive, current negative). Total: 325 remnant samples.
    • GC: 47 GC positive, 275 GC negative, 1 discordant (updated positive, current negative). Total: 323 remnant samples.
  • Data Provenance: Remnant swab specimens collected from patients undergoing CT and/or GC screening. The origin country is not specified, but it's retrospective use of existing clinical samples.

• CT/GC Clinical Panel Agreement Study:

  • Sample Size: 20 prepared CT/GC clinical panels. Each panel was tested in triplicate, in two runs per day, on three Panther systems, by two operators, using three lots of reagents over seven days.
  • Total tests: 20 (panels) * 3 (replicates) * 2 (runs/day) * 3 (systems) * 2 (operators) * 3 (lots) * 7 (days) = This calculation seems off and yields a very large number. More simply, if each panel was tested in triplicate across the stated conditions, the total number of data points specifically for these panels would be substantial. The document states "Each of the 20 panels were tested in triplicate...over seven days", implying multiple runs under varying conditions, leading to hundreds of data points (e.g., 20 panels * 3 replicates * 2 runs * 3 systems * 3 lots * 2 operators * 7 days if all combinations were run, which is impractical; more likely "across" these variables). Let's interpret "in triplicate...over seven days" as at least 3 runs per panel across variable conditions for a total of 60 tests (20 panels * 3 replicates) per condition (e.g., per system/lot/operator combination). The key is "The results show 100%...total CT and GC agreement to the expected panel result for the updated AC2 assay."
  • Data Provenance: Prepared clinical panels containing known concentrations of wild type CT, FI-nvCT, and GC in urine specimens. This is an expertly constructed analytical study, effectively prospective in its execution for validation.

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

The document does not mention the use of human experts (e.g., radiologists) for establishing ground truth for a test set. This is a molecular diagnostic assay, not an imaging device. Ground truth for these studies is typically established by:

• Analytical studies: Known concentrations of purified nucleic acids (e.g., in vitro transcripts) or spiked microorganisms.
• Clinical comparability: Agreement with a legally marketed predicate device (the "current" AC2 assay) on remnant clinical samples. The predicate device itself was cleared based on its own performance studies.
• Clinical panels: Known concentrations of target organisms in simulated or real matrices.

Therefore, the concept of "experts" in the context of ground truth establishment for this specific device would relate to the scientific team involved in designing the analytical panels and interpreting the molecular results, rather than clinical experts adjudicating cases.

4. Adjudication Method for the Test Set

Not applicable in the typical sense for this type of device. There's no "adjudication" of images or clinical cases by multiple readers. The output of the device is a qualitative "positive" or "negative" result based on Relative Light Units (RLU) and kinetic curve type. Equivalence is determined by statistical agreement (percent agreement) between the test device and the predicate or expected panel results.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No. An MRMC study is relevant for imaging devices where human readers interpret medical images, often with and without AI assistance, to assess diagnostic performance. This document concerns a molecular diagnostic assay where a laboratory instrument provides a qualitative result. There is no human "reading" of the assay outcome in the same way an image is read.

6. If a Standalone (Algorithm Only Without Human-in-the-Loop Performance) Was Done

Yes, implicitly. The performance data presented (LoD, clinical comparability, clinical panel agreement, cross-reactivity) are all "standalone" in the sense that they demonstrate the analytical and clinical performance of the assay system itself (reagent + instrument) without any direct human interpretation of raw data (beyond standard laboratory procedures for operating the instrument and interpreting its final qualitative output, which is not "in-the-loop" AI assistance). The device is intended to provide a diagnostic result directly.

7. The Type of Ground Truth Used

• Analytical Studies (LoD, Cross-Reactivity, Microbial Interference): "Spiked" or "known concentration" ground truth. For example, FI-nvCT in vitro transcripts at varying known concentrations, or panels of known microorganisms.
• Clinical Comparability: The results from the predicate device (current AC2 assay) on remnant clinical samples served as the de-facto ground truth for evaluating equivalence. While not "absolute" ground truth (like pathology), it's the standard for substantial equivalence in device modifications.
• Clinical Panel Agreement: Expected panel result based on known concentrations of spiked organisms. This is a form of engineered, highly controlled ground truth.

8. The Sample Size for the Training Set

The document does not directly disclose the sample size for the training set. This is a common characteristic of medical device submissions, as the focus is on validation (test set performance) rather than the proprietary details of model training (if applicable to this type of assay, though molecular assays often rely on established biochemical principles rather than "training" in the machine learning sense). The development of the reformulated probe reagent would have involved laboratory optimization and characterization experiments, which are analogous to a "training" phase.

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

Since this is a molecular diagnostic assay and not an AI/ML product requiring extensive "training" data, the concept of "ground truth for the training set" isn't directly applicable in the same way. The development process for the reformulated probe would have involved:

• Understanding the genetic variants: Identifying the specific mutations in the C. trachomatis variants (e.g., FI-nvCT) that caused detection issues with the previous probe.
• Rational design: Designing new probe sequences that would bind effectively to both the original target and the new variants.
• Iterative testing and optimization: Extensive laboratory testing of various probe formulations using cultured organisms, synthetic nucleic acids, and potentially some clinical samples to ensure desired performance (sensitivity, specificity, variant detection) during the development phase. This iterative process, guided by known target sequences and laboratory results, serves as the "ground truth" for optimizing the assay components.

In summary, the submission demonstrates that the modified Aptima Combo 2 Assay maintains or improves its performance compared to the predicate device, particularly in its ability to detect emerging CT variants, without compromising its overall accuracy or introducing new risks. The studies are analytical and comparative, appropriate for a molecular diagnostic device modification rather than an imaging AI solution.

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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 RNA (rRNA) from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC) to aid in the diagnosis of chlamydial and/or gonococcal disease using the Panther® System as specified.

On the Panther System, the assay may be used to test the following specimens from symptomatic and asymptomatic individuals: clinician-collected endocervical, vaginal, throat, rectal and male urethral swab specimens, clinician-collected gynecological specimens collected in the PreservCyt® Solution, patient-collected vaginal swab specimens, and female and male urine specimens.

Clearance of this pre-market application will add extra-genital (throat and rectal) swab specimens as acceptable specimen types using the Aptima Combo 2 assay on the Panther system.

The Aptima Combo 2 Assay combines the technologies of target capture. Transcription-Mediated Amplification (TMA), and Dual Kinetic Assay (DKA). Specimens are collected and transferred into their respective specimen transport tubes. The transport solutions in these tubes release the rRNA targets and protect them from degradation during storage. When the Aptima Combo 2 Assay is performed in the laboratory, the target rRNA molecules are isolated from specimens by use of capture oligomers via target capture that utilizes magnetic microparticles. The capture oligomers contain sequences complementary to specific regions of the target molecules as well as a string of deoxyadenosine residues. A separate capture oligomer is used for each target. During the hybridization step, the sequence specific regions of the capture oligomers bind to specific regions of the target molecules. 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 molecules 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 reaction inhibitors. After the target capture steps are completed, the specimens are ready for amplification.

Target amplification assays are based on the ability of complementary oligonucleotide primers to specifically anneal and allow enzymatic amplification of the target nucleic acid strands. The Aptima Combo 2 Assay replicates a specific region of the 23S rRNA from CT and a specific region of the 16S rRNA from GC via DNA intermediates. A unique set of primers is used for each target molecule. Detection of the rRNA amplification product sequences (amplicon) is achieved using nucleic acid hybridization. Single-stranded chemiluminescent DNA probes, which are complementary to a region of each target amplicon, are labeled with different acridinium ester molecules. The labeled DNA probes combine with amplicon to form stable RNA:DNA hybrids. The Selection Reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from unhybridized probe. During the detection step, light emitted from the labeled RNA:DNA hybrids is measured as photon signals in a luminometer, and are reported as Relative Light Units (RLU). In DKA, differences in the kinetic profiles of the CT and GC labeled probes allow for the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time. The chemiluminescent detection for CT signal has very rapid kinetics and has the "flasher" kinetic type. The chemiluminescent detection reaction for GC signal is relatively slower and has the "glower" kinetic type. Assay results are determined by a cut-off based on the total RLU and the kinetic curve type.

The Aptima Combo 2 assay has been designed for and validated on the Panther system. The Panther system is an integrated hardware and software system that together with the Aptima Combo 2 assay fully automates all the steps necessary to perform the assay from sample preparation through amplification of nucleic acid, detection, data reduction and amplicon inactivation.

Here's a summary of the acceptance criteria and study details for the Aptima Combo 2 Assay:

Aptima Combo 2 Assay (Panther System) - Acceptance Criteria and Study Details

The Aptima Combo 2 Assay is a target amplification nucleic acid probe test for the in vitro qualitative detection and differentiation of ribosomal RNA (rRNA) from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC) using the Panther System. This submission specifically addresses the addition of extra-genital (throat and rectal) swab specimens as acceptable specimen types.

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as numerical thresholds in the provided text. However, the study aims to demonstrate "comparable performance to the predicate device" and support a "substantial equivalence decision." The clinical study evaluates the device's sensitivity and specificity against an "anatomic site infected status (ASIS)" established by reference NAATs.

Based on the provided tables (Table 10 and Table 11), the reported device performance for sensitivity and specificity (with 95% Confidence Intervals) are as follows:

Note: The superscripts in the table refer to footnotes in the original document regarding equivocal results and their impact on recalculating sensitivity and specificity.

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

• Sample Size (Evaluable Subjects): 2591 subjects had at least one sample type tested.
  • Throat Samples: 2585 for CT performance, 2579 for GC performance (after exclusions).
  • Rectal Samples: 2562 for CT performance, 2569 for GC performance (after exclusions).
• Data Provenance: Prospectively-collected samples from adult (≥18 years) participants seeking STI testing, with or without symptoms, attending nine (9) participating US medical facilities.

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

The ground truth (anatomic site infected status - ASIS) was established using reference NAATs. The document does not specify the number or qualifications of experts involved in performing or interpreting these reference NAATs. It suggests that the ASIS was determined algorithmically based on the results of the reference NAATs rather than through human expert consensus, stating: "Subjects were categorized as infected if a positive result occurred in at least two reference NAATs, and as not infected if at least 2 of the reference results were negative."

4. Adjudication Method for the Test Set

The adjudication method for establishing the ASIS (ground truth) was a 2+1 rule based on reference NAATs.

• Infected: Positive result in at least two reference NAATs.
• Not Infected: Negative result in at least two reference NAATs.
• Tie-breaker: A third reference NAAT was used if the first two reference results were discordant.

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

No MRMC comparative effectiveness study was mentioned. The study evaluates the standalone performance of the Aptima Combo 2 Assay against a reference standard. It does not compare human readers with AI assistance versus without AI assistance.

6. Standalone Performance Study

Yes, a standalone performance study was conducted. The clinical performance of the Aptima Combo 2 Assay (algorithm only) was evaluated against the Anatomic Site Infected Status (ASIS) for each specimen type-organism combination. The results for sensitivity and specificity presented in Tables 10 and 11 represent the standalone performance of the device.

7. Type of Ground Truth Used

The ground truth used was "anatomic site infected status (ASIS)" established by multiple reference Nucleic Acid Amplification Tests (NAATs). These reference NAATs were "cleared for the detection of urogenital CT/GC infection and validated for use in rectal swab and throat swab specimens." This is a form of consensus-based ground truth derived from other laboratory tests, not pathology or outcomes data.

8. Sample Size for the Training Set

The document does not provide information on the sample size used for the training set. The provided performance data (sensitivity, specificity) relates to the clinical validation study using prospectively collected samples, which serves as the test set for device clearance. As a diagnostic assay rather than an AI/ML device, a distinct 'training set' in the context of machine learning model development is not typically applicable in the same way. The analytical studies (LoD, cross-reactivity, interfering substances) would inform the assay's development and optimization, but specific training set sizes for algorithmic learning are not detailed.

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

As mentioned above, specific information regarding a 'training set' and its ground truth establishment in the context of machine learning is not provided for this in vitro diagnostic device. The assay development would have involved internal validation and optimization, but the document focuses on the clinical validation (test set) for regulatory submission.

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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 RNA (rRNA) from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC) to aid in the diagnosis of chlamydial and/or gonococcal urogenital disease using the Panther® System as specified.

On the Panther System, the assay may be used to test the following specimens from symptomatic and asymptomatic individuals: clinician-collected endocervical, vaginal and male urethral swab specimens, clinician-collected gynecological specimens collected in the PreservCyt® Solution, patient-collected vaginal swab specimens, 1 and female and male urine specimens.

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

Clearance of this pre-market application will add female urine as an acceptable specimen type using the Aptima Combo 2 assay on the Panther system.

The Aptima Combo 2 Assay combines the technologies of target capture, Transcription-Mediated Amplification (TMA), and Dual Kinetic Assay (DKA). Specimens are collected and transferred into their respective specimen transport tubes. The transport solutions in these tubes release the rRNA targets and protect them from degradation during storage. When the Aptima Combo 2 Assay is performed in the laboratory, the target rRNA molecules are isolated from specimens by use of capture oligomers via target capture that utilizes magnetic microparticles. The capture oligomers contain sequences complementary to specific regions of the target molecules as well as a string of deoxyadenosine residues. A separate capture oligomer is used for each target. During the hybridization step, the sequence specific regions of the capture oligomers bind to specific regions of the target molecules. 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 molecules 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 reaction inhibitors. After the target capture steps are completed, the specimens are ready for amplification.

Target amplification assays are based on the ability of complementary oligonucleotide primers to specifically anneal and allow enzymatic amplification of the target nucleic acid strands. The Aptima Combo 2 Assay replicates a specific region of the 23S rRNA from CT and a specific region of the 16S rRNA from GC via DNA intermediates. A unique set of primers is used for each target molecule. Detection of the rRNA amplification product sequences (amplicon) is achieved using nucleic acid hybridization. Single-stranded chemiluminescent DNA probes, which are complementary to a region of each target amplicon, are labeled with different acridinium ester molecules. The labeled DNA probes combine with amplicon to form stable RNA:DNA hybrids. The Selection Reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from unhybridized probe. During the detection step, light emitted from the labeled RNA:DNA hybrids is measured as photon signals in a luminometer, and are reported as Relative Light Units (RLU). In DKA, differences in the kinetic profiles of the CT and GC labeled probes allow for the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time. The chemiluminescent detection for CT signal has very rapid kinetics and has the "flasher" kinetic type. The chemiluminescent detection reaction for GC signal is relatively slower and has the "glower" kinetic type. Assay results are determined by a cut-off based on the total RLU and the kinetic curve type.

The Aptima Combo 2 assay has been designed for and validated on the Panther system. The Panther system is an integrated hardware and software system that together with the Aptima Combo 2 assay fully automates all the steps necessary to perform the assay from sample preparation through amplification of nucleic acid, detection, data reduction and amplicon inactivation.

Here's an analysis of the provided text regarding the Aptima Combo 2 Assay (Panther System), focusing on acceptance criteria and the supporting study:

The provided document describes a 510(k) premarket notification for the Aptima Combo 2 Assay (Panther System) to add female urine as an acceptable specimen type. The study aimed to demonstrate substantial equivalence to the predicate device, which already included other specimen types.

1. Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a table format with numerical targets. Instead, it presents the Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of the device compared to a Composite Comparator Algorithm (CCA) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC) in female urine samples. For regulatory clearance, these performance metrics are implicitly the acceptance criteria; the observed performance must be deemed sufficient for the intended use and comparable to similar marketed devices.

Based on the performance tables provided, here's a summary of the reported device performance, which likely served as the basis for acceptance:

Reported Performance of Aptima Combo 2 Assay (Panther System) for Female Urine

Note: The confidence intervals provide the range within which the true PPA/NPA is likely to fall. For asymptomatic GC, the PPA has a wider confidence interval due to a smaller number of positive cases.

2. Sample Size and Data Provenance

• Sample Size for Test Set:
  • Total subjects initially enrolled: 2640
  • Subjects with valid Aptima Combo 2 Assay results on Panther System: 2581
  • Evaluable subjects for performance (conclusive CCA status): 2580
  • Final Sample Size for CT performance: 2572 (1379 symptomatic, 1193 asymptomatic) after accounting for equivocal results and non-evaluable subjects.
  • Final Sample Size for GC performance: 2579 (1383 symptomatic, 1196 asymptomatic) after accounting for equivocal results and non-evaluable subjects.
• Data Provenance: Retrospective study.
  • Specimens originated from women enrolled in a previously completed prospective study.
  • The study participants (women) were enrolled from 17 geographically and ethnically diverse US clinical sites, including family planning, academic centers, and public health clinics.

3. Number of Experts and Qualifications for Ground Truth

The document does not specify the number of experts used to establish the ground truth or their qualifications. The ground truth (Composite Comparator Algorithm, CCA) was established using multiple FDA-cleared NAATs, not directly by human experts adjudicating individual cases based on clinical information or pathology.

4. Adjudication Method for the Test Set

The adjudication method used to establish the Composite Comparator Algorithm (CCA) for the ground truth was:

• 2 out of 3 rule: When 2 out of 3 FDA-cleared CT/GC NAATs were positive, the CCA was considered positive. When 2 out of 3 NAATs were negative, the CCA was considered negative.
• Tie-breaker: If the results from the initial two comparator NAATs did not determine the CCA, a third FDA-cleared CT/GC NAAT was performed using remnant urine samples to determine the CCA.

This is a form of consensus-based ground truth, but using other diagnostic tests rather than direct clinical expert consensus.

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

No MRMC comparative effectiveness study was done. This study evaluated the standalone performance of a diagnostic assay (Aptima Combo 2 Assay) against a reference standard (CCA), not the effect of AI assistance on human readers.

6. Standalone Performance

Yes, a standalone performance study was done. The entire clinical study described evaluates the performance of the Aptima Combo 2 assay on the Panther System (the algorithm/device itself) directly against the Composite Comparator Algorithm (CCA) without human interpretation as part of the primary outcome measure. The PPA and NPA values reported are the standalone performance metrics.

7. Type of Ground Truth Used

The ground truth used was a Composite Comparator Algorithm (CCA), which was derived from the results of multiple (up to 3) FDA-cleared nucleic acid amplification tests (NAATs). While this is a common method for establishing a "gold standard" in diagnostic test evaluations, it is not direct pathology, clinical outcomes data, or expert consensus in the traditional sense of clinicians reviewing patient records or images. It establishes the "truth" based on a highly sensitive and specific panel of existing diagnostic tools.

8. Sample Size for the Training Set

The document does not provide information on the sample size for a training set. This is a diagnostic assay (a lab test), not an AI/machine learning model in the typical sense that would require a separate, explicit "training set" for model parameters. The "development" or "training" of such an assay involves reagent formulation, assay protocol optimization, and establishing cut-offs, typically done using characterized samples, but not usually reported with a distinct "training set" size in the same manner as an AI algorithm. The study described is a clinical validation or "test set" evaluation.

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

As mentioned above, there is no explicit "training set" described in the context of this 510(k) submission. The performance study evaluated the device against a CCA, as detailed in point 4 and 7.

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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 RNA (rRNA) from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC) to aid in the diagnosis of chlamydial and/or gonococcal urogenital disease using the PANTHER System as specified.

On the PANTHER System, the assay may be used to test the following specimens from symptomatic and asymptomatic individuals: clinician-collected endocervical, vaginal and male urethral swab specimens, clinician-collected gynecological specimens collected in the PreservCyt Solution, patient-collected vaginal swab specimens, and male urine specimens.

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 Combo 2 Assay combines the technologies of target capture, transcriptionmediated amplification (TMA), and dual kinetic assay (DKA).

Specimens are collected and transferred into their respective specimen transport tubes. The transport solutions in these tubes release the rRNA targets and protect them from degradation during storage. When the APTIMA Combo 2 Assay is performed in the laboratory, the target rRNA molecules are isolated from specimens by use of capture oligomers via target capture that utilizes magnetic microparticles. The capture oligomers contain sequences complementary to specific regions of the target molecules as well as a string of deox yadenosine residues. A separate capture oligomer is used for each target. During the hybridization step, the sequence specific regions of the capture oligomers bind to specific regions of the target molecules. 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 molecules 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 reaction inhibitors. After the target capture steps are completed, the specimens are ready for amplification.

Target amplification assays are based on the ability of complementary oligonucleotide primers to specifically anneal and allow enzymatic amplification of the target nucleic acid strands. The APTIMA Combo 2 Assay replicates a specific region of the 23S rRNA from CT and a specific region of the 16S rRNA from GC via DNA intermediates. A unique set of primers is used for each target molecule. Detection of the rRNA amplification product sequences (amplicon) is achieved using nucleic acid hybridization. Single-stranded chemiluminescent DNA probes, which are complementary to a region of each target amplicon, are labeled with different acridinium ester molecules. The labeled DNA probes combine with amplicon to form stable RNA:DNA hybrids. The Selection Reagent differentiates hybridized from unhybridized probe, eliminating the generation of signal from unhybridized probe. During the detection step, light emitted from the labeled RNA:DNA hybrids is measured as photon signals in a luminometer, and are reported as Relative Light Units (RLU). In DKA, differences in the kinetic profiles of the CT and GC labeled probes allow for the differentiation of signal; kinetic profiles are derived from measurements of photon output during the detection read time. The chemiluminescent detection for CT signal has very rapid kinetics and has the "flasher" kinetic type. The chemiluminescent detection reaction for GC signal is relatively slower and has the "glower" kinetic type. Assay results are determined by a cut-off based on the total RLU and the kinetic curve type.

APTIMA Combo 2® Assay (on PANTHER® System) - Acceptance Criteria and Study Details

The APTIMA Combo 2® Assay is a nucleic acid amplification test for the qualitative detection and differentiation of ribosomal RNA from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC) to aid in the diagnosis of chlamydial and/or gonococcal urogenital disease. This 510(k) submission (K132251) specifically focuses on clearing the assay for use with male urine specimens on the PANTHER System.

1. Acceptance Criteria and Reported Device Performance

The provided document details the performance characteristics for male urine, alongside other specimen types, in terms of sensitivity and specificity based on two clinical studies. While explicit "acceptance criteria" in a numeric format (e.g., minimum sensitivity of X%) are not directly stated, the reported performance metrics demonstrate the device's efficacy across various specimen types and symptom statuses. The regulatory approval implies these performance levels met the FDA's requirements for substantial equivalence.

Reported Device Performance for Male Urine (from Table 4 for CT and Table 7 for GC):

Additional detailed performance by symptom status is provided in Table 5 (CT) and Table 8 (GC) and by individual study in Table 6 (CT) and Table 9 (GC).

2. Sample Sizes and Data Provenance for Test Set

The clinical performance data was derived from two multi-center clinical studies conducted in the United States. The data is prospective, as specimens were collected from enrolled symptomatic and asymptomatic individuals for the purpose of the study.

Test Set Sample Sizes:

• Clinical Study 1: Included male urethral swab, vaginal swab, PreservCyt Solution liquid Pap, female endocervical swab, and male urine samples.
  • Male subjects (urine): 580 enrolled. 580 male urine samples were tested.
  • For CT performance analysis, 1799 male urine samples were included (data from Clinical Study 1 and 2 combined, see Table 4).
  • For GC performance analysis, 1797 male urine samples were included (data from Clinical Study 1 and 2 combined, see Table 7).
• Clinical Study 2: Primarily focused on male urine specimens.
  • Male subjects: 1492 enrolled, 1478 male urine samples from non-withdrawn subjects were tested.
  • For CT performance analysis, 1799 male urine samples were included (data from Clinical Study 1 and 2 combined, see Table 4).
  • For GC performance analysis, 1797 male urine samples were included (data from Clinical Study 1 and 2 combined, see Table 7).

A breakdown of male urine samples by study and symptom status for performance evaluation:

• CT (Table 6):
  • Study 1 Asymptomatic: 323 samples
  • Study 2 Asymptomatic: 979 samples
  • Study 2 Symptomatic: 497 samples
  • Total (Study 1 + Study 2) for male urine (Table 4): 1799 samples
• GC (Table 9):
  • Study 1 Asymptomatic: 320 samples
  • Study 2 Asymptomatic: 980 samples
  • Study 2 Symptomatic: 497 samples
  • Total (Study 1 + Study 2) for male urine (Table 7): 1797 samples

3. Number of Experts and Qualifications for Ground Truth

The document does not explicitly state the number of "experts" used to establish the ground truth or their specific qualifications (e.g., radiologist with X years of experience), as this is a diagnostic assay for infectious disease rather than image-based diagnosis.

Instead, the ground truth ("infected status") was established using cleared nucleic acid amplification tests (NAATs). The reference methods are described as:

• "cleared nucleic acid amplification tests (NAATs)" for Clinical Study 1 (male urethral swab, male and female urine, and PreservCyt Solution liquid Pap samples).
• "cleared NAATs" for Clinical Study 2 (male urethral swab and urine samples). Specifically, the infected status algorithm used "urethral swab and urine sample results from one reference CT and GC NAAT and urine sample results from two additional reference CT and GC NAATs to generate four reference results for each analyte."

The qualifications of the individuals performing these reference NAATs are not specified but would presumably be laboratory professionals trained in molecular diagnostics.

4. Adjudication Method for the Test Set

The adjudication method for establishing the "infected status" (ground truth) for the clinical test set was based on an algorithm using multiple reference NAATs.

• Clinical Study 1: "Subjects were categorized as infected if a positive result occurred in each of the two reference NAATs." (See Tables 10, 11, 13, and 14 for specific algorithms for different specimen types and analytes). For female subjects, if positive NAAT results occurred only in urine, they were considered infected for urine evaluation but non-infected for other non-urine specimens.
• Clinical Study 2 (Male Urine): "Subjects were categorized as infected if a positive result occurred in at least two of the reference NAATs." (See Tables 13 and 15).

This method is a form of "consensus" or "composite comparator" ground truth, where multiple established diagnostic tests are used to determine the true infection status in the absence of a single universally accepted gold standard.

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

No Multi-Reader Multi-Case (MRMC) comparative effectiveness study was mentioned or performed. This device is an in vitro diagnostic assay, not an imaging device requiring human reader interpretation or AI assistance for human readers. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable here. The assay provides a qualitative result directly.

6. Standalone (Algorithm Only) Performance

Yes, a standalone performance study was conducted. The performance data presented in the tables (Tables 4, 5, 6, 7, 8, 9) for sensitivity, specificity, PPV, and NPV represent the standalone performance of the APTIMA Combo 2 Assay on the PANTHER System, without human-in-the-loop interpretation being part of the result generation. The device itself performs the detection and differentiation of rRNA and provides a qualitative result.

7. Type of Ground Truth Used

The type of ground truth used was "composite comparator" or "reference NAAT consensus". For both clinical studies, the "infected status" was established by comparing results from two or more FDA-cleared nucleic acid amplification tests (NAATs) performed on the same or corresponding samples, rather than pathology (histology), clinical outcomes data, or a single expert's opinion.

8. Sample Size for the Training Set

The document does not explicitly state a separate "training set" sample size in the context of device development. For in vitro diagnostic devices, "training" often refers to internal analytical studies and optimization during development, rather than a distinct clinical "training set" in the way it's used for AI or machine learning models. The provided clinical studies (Clinical Study 1 and Clinical Study 2) represent the validation or test sets used to establish clinical performance.

Analytical sensitivity (Limit of Detection) studies were conducted using dilutions of CT organisms and GC organisms (7). These analytical studies involve controlled samples to determine the detection limits and would be part of the internal development and analytical verification processes, but are not typically referred to as a "training set" in this context.

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

As noted above, a distinct "training set" with established ground truth in the clinical context is not explicitly described. For the analytical sensitivity studies, the "ground truth" (i.e., known concentration of organisms) was established by spiking known concentrations of CT and GC organisms into various matrices (e.g., Specimen Transport Medium, urine) and testing dilutions. This allows for the determination of the limit of detection (LOD) for the assay.

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