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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 specimens , and female and male urine specimens.
¹Patient-collected vaginal swab specimens are an option for screening women when a pelvic exam is not otherwise indicated. The Aptima Multitest Swab Specimen Collection Kit has not been evaluated for home use.

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 Tigris® DTS® Automated Analyzer. 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.
1 Patient-collected vaginal swab specimens are an option for screening women when a pelvic exam is not otherwise indicated. The Aptima Multitest Swab Specimen Collection Kit is not for home use.

The Aptima Trichomonas vaginalis 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 women: clinician-collected endocervical swabs, clinician-collected vaginal swabs, and specimens collected in PreservCyt Solution.

The Aptima Trichomonas vaginalis 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 Tigris® DTS® System. The assay may be used to test the following specimens from symptomatic or asymptomatic women: clinician-collected endocervical swabs, clinician-collected vaginal swabs, female urine specimens, and specimens collected in PreservCyt Solution.

The Aptima Combo 2 Assay (AC2) 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 polydeoxythymidine 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 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 Trichomonas vaginalis Assay (ATV) 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 Trichomonas vaginalis Assay is performed in the laboratory, the target rRNA is isolated from the specimens by the use of 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.
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 Hologic TMA reaction amplifies a specific region of the small ribosomal subunit from T. vaginalis via DNA and RNA intermediates and generates RNA amplicon molecules. Detection of the rRNA amplification product sequences is achieved using nucleic acid hybridization (HPA). A single stranded chemiluminescent DNA probe, which is complementary to a region of the target amplicon, is labeled with an acridinium ester molecule. The labeled DNA probe combines 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).

Acceptance Criteria and Device Performance for Aptima Combo 2 Assay and Aptima Trichomonas Vaginalis Assay (RMR Probe Reagent Update)

This document describes the acceptance criteria and the studies that demonstrate the device meets those criteria, specifically concerning the manufacturing change to the Ready-Made Reagents (RMR) Probe Reagent for the Aptima Combo 2 Assay (AC2) and Aptima Trichomonas Vaginalis Assay (ATV). The core of this submission is to prove that the removal of the lyophilization step for the RMR Probe Reagent does not negatively impact assay performance compared to the previously cleared predicate devices.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implicitly defined by demonstrating comparability to the predicate devices. The key performance metrics evaluated are Intended Use (overall agreement with expected positivity) and Limit of Detection (LoD). For clinical performance, the acceptance criteria are 100% agreement between the predicate and the modified RMR assay. For LoD, the acceptance criterion is that the RMR assay's LoD is within ½ log of the predicate assay's LoD.

2. Sample Size for the Test Set and Data Provenance

Intended Use Study:

• Aptima Combo 2 Assay (Panther): Negative and positive panels (exact number of panels or individual samples not specified, but stated to include CT positive, FI-nvCT positive, and CT/GC dual positive panels).
• Aptima Combo 2 Assay (Tigris): Negative and positive panels (exact number of panels or individual samples not specified, but stated to include CT positive, FI-nvCT positive, and CT/GC dual positive panels).
• Aptima Trichomonas Vaginalis Assay (Panther): Negative, TV positive, and TV low positive panels (exact number of panels or individual samples not specified).
• Aptima Trichomonas Vaginalis Assay (Tigris): Negative, TV positive, and TV low positive panels (exact number of panels or individual samples not specified).
• Data Provenance: Not explicitly stated, but these appear to be contrived panels (controlled positive and negative samples) rather than directly clinical specimens for this specific study.

Limit of Detection (LoD) Study:

• Aptima Combo 2 Assay (Panther & Tigris): Stocks of CT and GC organisms, and FI-nvCT in vitro transcript. These were run in negative clinical liquid pap specimens collected in PreservCyt solution (ThinPrep).
• Aptima Trichomonas Vaginalis Assay (Panther & Tigris): Stocks of TV organisms in negative clinical liquid pap specimens collected in PreservCyt solution (ThinPrep).
• Data Provenance: The base matrix used for spiking was "negative clinical liquid pap specimens," suggesting these are retrospective clinical samples from an unspecified origin, used in a prospective manner for LoD determination.

Clinical Performance Study:

• Aptima Combo 2 Assay (Panther): 219 remnant clinical swab specimens.
• Aptima Combo 2 Assay (Tigris): 200 remnant clinical swab specimens.
• Data Provenance: "remnant clinical swab specimens". This indicates these are retrospective samples collected from prior clinical testing, with the country of origin not specified.

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

The ground truth for these studies is established by the performance of the predicate AC2 assay or ATV assay, which was previously cleared by the FDA. The document does not describe the involvement of additional human experts for the ground truth of these specific comparability studies, as the goal is to show the new RMR Probe Reagent performs equivalently to the already established predicate. The reliability of the predicate assays themselves would have been established through prior studies reviewed by the FDA, presumably involving expert consensus or validated methods.

4. Adjudication Method for the Test Set

Adjudication methods were not applicable in these studies. The assessment compares the performance of the modified AC2/ATV RMR assays directly against the predicate AC2/ATV assays. The predicate assay's result is used as the reference/ground truth for comparison. There is no mention of a separate expert adjudication process for discordant results between the predicate and the modified device in these comparability studies. In the LoD studies, the ground truth for spiked samples is defined by the known concentration of the spiked organisms/transcripts.

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

No MRMC comparative effectiveness study was done. This study concerns an in vitro diagnostic (IVD) device (nucleic acid amplification test) for direct detection of pathogens, not an AI-assisted diagnostic tool for human readers. Therefore, there is no human-in-the-loop performance to evaluate, and thus no effect size for human reader improvement with AI assistance.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance)

The studies presented are effectively standalone performance evaluations of the modified IVD assays. The assays are fully automated on the Panther and Tigris systems, and the results are interpreted based on predefined cut-offs (Relative Light Units and kinetic curve type). There is no human intervention in the result determination process once the assay is run. The comparison is between two versions of an automated assay.

7. Type of Ground Truth Used

• Intended Use Study: The "expected positivity results" indicate that calibrated positive and negative control panels (contrived ground truth) were used. These panels simulate clinical conditions but are created in a controlled laboratory setting.
• Limit of Detection (LoD) Study: The ground truth for LoD was spiked negative clinical specimens with known concentrations of target organisms/transcripts. This is a form of contrived ground truth based on quantitative standards.
• Clinical Performance Study: The ground truth for the clinical comparability study was the result of the predicate AC2 assay. This means the predicate assay's output was considered the reference standard, rather than an independent expert consensus or pathology review of the remnant specimens themselves for this specific comparability study. The performance of the predicate itself would have been validated against clinical outcomes or a gold standard during its initial clearance.

8. Sample Size for the Training Set

No training set is explicitly mentioned or relevant for this submission. This submission describes a manufacturing change to a reagent component of already cleared IVD assays. The assays are based on established molecular biology principles and do not involve machine learning algorithms that require a training set to "learn" patterns or make predictions. The "development activities" mentioned relate to design control and verification testing, not algorithmic training.

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

As there is no training set for an algorithm, this question is not applicable.

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