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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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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, and patient-collected vaginal swab specimens.

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 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.

This FDA 510(k) summary describes the APTIMA Combo 2® Assay on the PANTHER System, a device for in vitro qualitative detection and differentiation of ribosomal RNA from Chlamydia trachomatis (CT) and/or Neisseria gonorrhoeae (GC). The submission is for clearing the assay for use on the PANTHER System, as it was previously cleared on the TIGRIS System.

Here's an analysis of the provided information:

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria for this device are implicitly tied to the performance characteristics, specifically sensitivity and specificity, as demonstrated in the clinical study. While explicit "acceptance criteria" values for sensitivity and specificity are not directly stated in the summary, the reported performance characteristics are presented with 95% confidence intervals, suggesting that the observed values met predefined thresholds for regulatory approval. The fact that the device received 510(k) clearance further indicates that its performance was deemed acceptable by the FDA.

Table of Performance for APTIMA Combo 2® Assay on the PANTHER System (Clinical Study Results)

Analytical Sensitivity (Limits of Detection):

• CT: Claimed 1 IFU/assay (7.25 IFU/swab, 9.75 IFU/mL, PreservCyt Solution liquid Pap). 100% positivity was observed in samples containing CT concentrations of 0.03 IFU/mL.
• GC: Claimed 50 cells/assay (362 cells/swab, 488 cells/mL PreservCyt Solution liquid Pap). 100% positivity was observed in samples containing GC concentrations of 0.04 CFU/mL.

Within Laboratory Precision (STM matrix, selected rows as an example):

Carryover Study: Overall carryover rate was 0% with a 95% confidence interval of 0-0.1%. This meets an implicit acceptance criterion of negligible or acceptable carryover.

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

The clinical study was a prospective, multicenter clinical study conducted across 7 geographically and ethnically diverse US clinical sites.

Test Set Sample Sizes:

• Male subjects: 580 enrolled, 567 evaluable male urethral swab samples.
  • 18 male urethral swab specimens had final invalid results and were excluded.
• Female subjects: 1332 enrolled.
  • 1319 evaluable vaginal swab samples (clinician-collected/patient-collected combined).
  • 1330 evaluable PreservCyt Solution liquid Pap samples.
  • 1310 evaluable endocervical swab samples.
  • 1 vaginal swab and 1 endocervical swab had final CT equivocal results and were excluded.
  • 1 PreservCyt and 1 endocervical swab had final GC equivocal results and were excluded.
  • Female urine specimens were collected but used as part of the "infected status algorithm" (ground truth definition) rather than directly tested as a primary specimen type for performance against the APTIMA Combo 2 Assay on PANTHER for this 510(k).

Data Provenance: United States (7 US clinical sites). The study was prospective.

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

The ground truth was established using an "infected status algorithm" based on results from two specimen types and two reference NAATs (Nucleic Acid Amplification Tests). The specific number and qualifications of experts directly involved in adjudicating the "infected status" or interpreting the reference NAATs are not explicitly stated in the provided text.

However, for a device like this, standard practice for establishing ground truth for NAATs in clinical trials typically involves:

• Use of one or more FDA-cleared and/or highly sensitive and specific reference NAATs.
• Concordance of positive results across multiple tests/specimens to define an "infected status."
• Discrepancies often resolved by a third, highly sensitive method or expert clinical review, though this detail is not provided.

The text states: "Subjects were categorized as infected if a positive result occurred in each of the 2 reference NAATs." This implies a rule-based algorithm for ground truth rather than individual expert adjudication for each case.

4. Adjudication Method for the Test Set

The adjudication method for determining the "infected status" (ground truth) was an algorithm-based approach:

• For male subjects and female subjects, if the positive NAAT results occurred only in the urine specimens and not in the PreservCyt specimens, the subject was categorized as infected; however, for the evaluation of the non-urine specimen types, the specimens were considered non-infected.
• "Subjects were categorized as infected if a positive result occurred in each of the 2 reference NAATs."

This indicates a 2-out-of-2 (or 2+0) concordance rule for positivity to establish infection status. The text doesn't mention a tie-breaking or expert review process if results were discordant between the two reference NAATs, but specimens with "invalid, equivocal, or error results" were retested, and those with final invalid/equivocal results were excluded from analysis.

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

No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done.

This device is an in vitro diagnostic test, specifically a nucleic acid amplification test (NAAT). These types of tests are designed to provide a qualitative result (positive/negative) based on an analytical assay run on an automated system, not to be interpreted by human readers in the same way an imaging device or pathology slide might be. Therefore, the concept of "human readers improve with AI vs. without AI assistance" does not apply to this type of device. The "AI" (algorithm) here is the test.

6. Standalone (Algorithm Only) Performance

Yes, standalone performance was done.

The entire clinical study described, including the sensitivity and specificity values provided in Tables 6, 7, 8, 9, 10, and 11, represents the standalone performance of the APTIMA Combo 2 Assay on the PANTHER System. The results are compared against an "infected status algorithm" which serves as the ground truth, not against human interpretation of raw assay data. The device's output is the final diagnostic result.

7. Type of Ground Truth Used

The ground truth used was an algorithm-based "infected status" derived from the results of two reference nucleic acid amplification tests (NAATs) on two different specimen types (e.g., male urethral swab and male urine for males, and PreservCyt and urine for females).

Specifically:

• "Male urethral swab, male and female urine, and PreservCyt samples were tested with cleared nucleic acid amplification tests (NAATs) to establish the infected status."
• "The infected status algorithm used results from two specimen types and two reference NAATs. Subjects were categorized as infected if a positive result occurred in each of the 2 reference NAATs."
• "For female subjects, if the positive NAAT results occurred only in the urine specimens and not in the PreservCyt specimens, the subject was categorized as infected; however, for the evaluation of the non-urine specimen types, the specimens were considered non-infected."

8. Sample Size for the Training Set

The provided text does not specify a sample size for a training set. This document is a 510(k) summary for a diagnostic test, not a submission for a de novo machine learning algorithm that typically requires a distinct training and test set with explicit disclosure of training data. The "device" in this context is the analytical assay and automated system. While the assay itself (APTIMA Combo 2) was developed and validated, the data tables presented relate to the performance evaluation (test set) for the new platform (PANTHER System).

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

As no explicit training set is detailed for the purpose of a machine learning algorithm in this 510(k) summary, the process for establishing ground truth for a training set is not applicable here. The provided data focuses on the validation of the device on a new platform (PANTHER System) for regulatory clearance, where the clinical study (test set) is the primary evidence for performance.

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