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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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