Aptima CV/TV Assay
Device Facts
| Record ID | K190472 |
|---|---|
| Device Name | Aptima CV/TV Assay |
| Applicant | Hologic, Inc. |
| Product Code | PQA · Microbiology |
| Decision Date | May 16, 2019 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3975 |
| Device Class | Class 2 |
Indications for Use
The Aptima CV/TV assay is an in vitro nucleic acid amplification test for the detection of RNA from microorganisms associated with vulvovaginal candidiasis and trichomoniasis. The assay utilizes real time transcription-mediated amplification (TMA) to detect and qualitatively report results for the following organisms: Candida species group (C. albicans, C. tropicalis, C. parapsilosis, C. dubliniensis) Candida glabrata Trichomonas vaginalis The assay differentiates between Candida glabrata and the Candida species group (C spp) by targeting the RNA component of RNAse P ribonucleoprotein; the assay does not differentiate among C spp. For Trichomonas vaginalis, the assay targets ribosomal RNA (rRNA) and differentiates the result from results for Candida glabrata and C spp. The assay is intended to aid in the diagnosis of vulvovaginal candidiasis and trichomoniasis on the automated Panther system using clinician-collected and patient-collected vaginal swab specimens from females with a clinical presentation consistent with vaginitis or vulvovaginitis.
Device Story
Aptima CV/TV assay is an in vitro nucleic acid amplification test (NAAT) performed on the automated Panther system. Input: vaginal swab specimens (clinician or patient-collected) in specimen transport media. Process: target capture on magnetic microparticles; target amplification via transcription-mediated amplification (TMA) using MMLV reverse transcriptase and T7 RNA polymerase; real-time detection using fluorescent-labeled torches. Output: qualitative results for Candida species group, C. glabrata, and T. vaginalis. Used in clinical settings to aid diagnosis of vulvovaginal candidiasis and trichomoniasis. Healthcare providers use results to guide clinical management of symptomatic patients.
Clinical Evidence
Prospective multi-center clinical study of 1496 evaluable symptomatic subjects. Performance relative to composite reference (culture/sequencing for Candida, dual-assay molecular/culture for T. vaginalis). C spp sensitivity 91.7% (clinician) / 92.9% (patient), specificity 94.9% / 91.0%. C. glabrata sensitivity 84.7% / 86.2%, specificity 99.1% / 98.7%. T. vaginalis sensitivity 96.5% / 97.1%, specificity 95.1% / 98.9%.
Technological Characteristics
Real-time TMA assay; targets RNA (RNAse P for Candida, rRNA for T. vaginalis). Automated on Panther System. Uses magnetic microparticles for target capture and fluorescent torches for detection. Reagents include MMLV reverse transcriptase and T7 RNA polymerase. Qualitative reporting. Internal control included in every test.
Indications for Use
Indicated for females ≥14 years with clinical presentation consistent with vaginitis or vulvovaginitis. Used for detection of Candida species group, Candida glabrata, and Trichomonas vaginalis from clinician-collected or patient-collected vaginal swabs.
Regulatory Classification
Identification
A device that detects nucleic acid sequences from microorganisms associated with vaginitis and bacterial vaginosis is a qualitative in vitro diagnostic device intended for the detection of microbial nucleic acid sequences in vaginal specimens collected from patients with signs and symptoms of vaginitis or bacterial vaginosis. This device is intended to aid in the diagnosis of vaginitis or bacterial vaginosis when used in conjunction with clinical signs and symptoms and other laboratory findings.
Special Controls
*Classification.* Class II (special controls). The special controls for this device are:(1) Design verification and validation must include: (i) Documentation with a detailed device description of device components; ancillary reagents required but not provided; and explanation of the methodology including primer/probe sequence, design, and rationale for sequence selection. (ii) Documentation with information that demonstrates the performance characteristics of the device, including: (A) Limit of Detection; (B) Precision (reproductivity); (C) Analytical specificity; (D) Analytical reactivity (inclusivity); (E) Specimen stability; and (F) Effects of interfering substances. (iii) Detailed documentation from a prospective clinical study. As appropriate to the intended use, the prospective clinical study must be performed on an appropriate study population, including women of various ages and ethnicities. The prospective clinical study must compare the device performance to results obtained from well-accepted comparator methods. (iv) Detailed documentation for device software, including software applications and hardware-based devices that incorporate software. (2) The labeling required under § 809.10(b) of this chapter must include: (i) A detailed explanation of the interpretation of results and acceptance criteria; (ii) For devices with an intended use that includes detection of nucleic acid sequences from bacteria associated with bacterial vaginosis, clinical performance stratified by patient demographics such as race, ethnicity, age, and pregnancy status. (iii) For devices with an intended use that includes detection of nucleic acid sequences from bacteria associated with bacterial vaginosis, a summary of device results in an asymptomatic population with demographic characteristics appropriate to the intended use population. (iv) For devices with an intended use that includes detection of either Candida species or bacteria associated with bacterial vaginosis, a limitation that *Candida* species and bacterial compositions associated with bacterial vaginosis can be present as part of normal vaginal flora and results should be considered in conjunction with available clinical information.
Predicate Devices
- BD MAX Vaginal Panel (DEN160001)
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
A. 510(k) Number:
K190472
B. Purpose for Submission:
New device 510k clearance for the Aptima CV/TV assay performed on the Panther System
C. Measurand:
RNA component of RNAse P ribonucleoprotein of Candida glabrata and the Candida species group (C. albicans, C. tropicalis, C. parapsilosis, C. dubliniensis) (C spp); ribosomal RNA (rRNA) of Trichomonas vaginalis (TV)
D. Type of Test:
Qualitative Nucleic Acid Amplification - Real Time Transcription-Mediated Amplification (TMA)
E. Applicant:
Hologic, Inc.
F. Proprietary and Established Names:
Aptima CV/TV Assay
G. Regulatory Information:
1. Regulation section:
21 CFR 866.3975
Device that detects nucleic acid sequences from microorganisms associated with vaginitis and bacterial vaginosis
2. Classification:
Class II
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3. Product code:
PQA - Vaginitis and bacterial vaginosis nucleic acid detection system
NSU - Instrumentation for clinical multiplex test systems
4. Panel:
Microbiology (83)
### H. Intended Use:
1. Intended use(s):
The Aptima CV/TV assay is an in vitro nucleic acid amplification test for the detection of RNA from microorganisms associated with vulvovaginal candidiasis and trichomoniasis. The assay utilizes real time transcription-mediated amplification (TMA) to detect and qualitatively report results for the following organisms:
- Candida species group (C. albicans, C. tropicalis, C. parapsilosis, C. dubliniensis)
- Candida glabrata
- Trichomonas vaginalis
The assay differentiates between Candida glabrata and the Candida species group (C spp) by targeting the RNA component of RNAse P ribonucleoprotein; the assay does not differentiate among C spp. For Trichomonas vaginalis, the assay targets ribosomal RNA (rRNA) and differentiates the result from results for Candida glabrata and C spp. The assay is intended to aid in the diagnosis of vulvovaginal candidiasis and trichomoniasis on the automated Panther system using clinician-collected and patient-collected vaginal swab specimens from females with a clinical presentation consistent with vaginitis or vulvovaginitis.
2. Indication(s) for use:
Same as Intended Use(s)
3. Special conditions for use statement(s):
For prescription use only
4. Special instrument requirements:
Panther System
### I. Device Description:
The Aptima CV/TV assay is an in vitro nucleic acid amplification test for the detection and
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quantitation of RNA from microorganisms associated with vulvovaginal candidiasis and trichomoniasis, in women with a clinical presentation consistent with vaginitis and vulvovaginitis. The Aptima CV/TV assay utilizes the automated Panther system to provide qualitative results to aid in the diagnosis of vulvovaginal candidiasis and trichomoniasis.
## Reagents and Materials Provided
The Aptima CV/TV assay is provided as a 100-test kit. The Aptima CV/TV assay master kit contains eight reagents, one calibrator, and two controls required for sample processing. There are four boxes that make up the assay master kit. Boxes 1 and 2 contain the Aptima CV/TV assay reagents packaged according to storage conditions. Box 3 contains the calibrator, and Box 4 contains the controls when provided as part of the master kit. The Aptima CV/TV Calibrator and Controls kit may also be procured separately if customers need additional calibrators or controls. A listing of the components that are required to perform the Aptima CV/TV assay are detailed in Table 1 below.
Table 1: Reagents Required to Perform the Aptima CV/TV Assay (Test Kit)
| Box | Symbol | Component | Quantity |
| --- | --- | --- | --- |
| 1 | A | Amplification Reagent (Non-infectious nucleic acids dried in buffered solution.) | 1 vial |
| | E | Enzyme Reagent (Reverse transcriptase and RNA polymerase dried in HEPES buffered solution.) | 1 vial |
| | PRO | Promotor Reagent (Non-infectious nucleic acids dried in buffered solution.) | 1 vial |
| | IC | Internal Control (Non-infectious nucleic acids in buffered solution.) | 1 x 0.3 mL |
| 2 | AR | Amplification Reconstitution Solution (Aqueous solution containing glycerol and preservatives.) | 1 x 7.2 mL |
| | ER | Enzyme Reconstitution Solution (HEPES buffered solution containing a surfactant and glycerol.) | 1 x 5.8 mL |
| | PROR | Promotor Reconstitution Solution (Aqueous solution containing glycerol and preservatives.) | 1 x 4.5 mL |
| | TCR | Target Capture Reagent (Buffered salt solution containing non-infectious nucleic acids and magnetic particles.) | 1 x 26.0 mL |
| | | Reconstitution Collars | 3 |
| | | Master Lot Barcode Sheet | 1 sheet |
| 3 | PCAL | Positive Calibrator (Non-infectious nucleic acids in buffered solution.) | 5 x 2.8 mL |
| | | Calibrator Barcode Label | 1 sheet |
| 4 | CONTROL- | Negative Control (Buffered solution.) | 5 x 2.7 mL |
| | CONTROL+ | Positive Control (Non-infectious C. albicans, C. glabrata, and T. vaginalis cultured organisms in buffered solution.) | 5 x 1.7 mL |
| | | Control Barcode Label | 1 sheet |
## Instrumentation
The Aptima CV/TV 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 CV/TV 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.
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## Materials Required but Available Separately
A listing of the materials that are required to perform the Aptima CV/TV assay on the Panther system but available separately from the Aptima CV/TV assay test kit are detailed in Table 2 below.
Table 2: Materials Required to Perform the Aptima CV/TV Assay but Available Separately
| Maretial |
| --- |
| Aptima Multitest Swab Specimen Collection Kit |
| Panther Run Kit for Real Time Assays (for real time assays only) - Aptima Assay Fluids Kit (also known as Universal Fluids Kit). Contains Aptima Wash Solution, Aptima Buffer for Deactivation Fluid, and Aptima Oil Reagent - Multi-tube units (MTUs) - Panther Waste Bag Kit - Panther Waste Bin Cover |
| Or, Panther System Run Kit, when running non-real time-TMA assays in parallel with real time-TMA assays Contains MTUs, waste bags, waste bin covers, auto detect, and assay fluids |
| Aptima Assay Fluids Kit |
| Contains Aptima Wash Solution, Aptima Buffer for Deactivation Fluid, and Aptima Oil Reagent |
| Multi-tube units (MTUs) |
| Tips, 1000 μL conductive, liquid sensing |
| Aptima penetrable caps |
| Replacement non-penetrable caps |
| Reagent Replacement Caps (for Amplification, Enzyme, and Promoter reagent reconstitution bottles, and TCR bottle) |
| Tube Rocker |
| Plastic-backed laboratory bench covers |
| Lint-free wipes |
| Pipettor and tips |
| Bleach, 5.0% to 7.0% (0.7 M to 1.0 M) sodium hypochlorite solution |
| Disposable, powderless gloves |
## Quality Control
### Assay Calibration
To generate valid results, an assay calibration must be completed. The calibrator is run in triplicate each time a reagent kit is loaded on the Panther system. Once established, the calibration is valid for up to 24 hours. Software on the Panther system alerts the operator when a calibration is required. The operator scans the calibration coefficients found on the Master Lot Barcode Sheet provided with each reagent kit.
During processing, criteria for acceptance of the calibrator is automatically verified by the software on the Panther system. If less than two of the calibrator replicates are valid, the software automatically invalidates the run. Samples in an invalidated run must be retested using a freshly prepared calibrator and freshly prepared controls.
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### *Negative and Positive Controls*
To generate valid results, a set of assay controls must be tested. One replicate each of the negative control and positive control must be tested each time a reagent kit is loaded on the Panther system. Once established, the controls are valid for up to 24 hours. Software on the Panther system alerts the operator when controls are required.
During processing, criteria for acceptance of controls are automatically verified by software on the Panther system. If any one of the controls has an invalid result, the software automatically invalidates the run. Samples in an invalidated run must be retested using a freshly prepared calibrator and freshly prepared controls.
### *Internal Control (IC)*
Each sample contains an IC. During processing, IC acceptance criteria are automatically verified by the Panther system software. If an IC result is invalid, the sample result is invalidated. Every sample with an invalid IC result must be retested to obtain a valid result. The Panther system software is designed to accurately verify processes when procedures are performed following the instructions provided in this package insert and the Panther System Operator's Manual.
### *Sample and Assay Run Validity*
The Panther software assesses the validity of all samples, calibrators and controls. Only the results of specimens that are valid will be reported to the user.
In the Aptima CV/TV assay, only the C spp channel is subjected to in-run calibration. If the reagents have valid calibration and controls, the Panther system calculates the specimen results automatically for the user.
Sample Validity is determined from several factors:
- Samples are invalidated if the estimated background relative fluorescence units (RFU) signal exceeds a predefined background threshold.
- For samples determined to be positive for one or more analytes (C spp, *C. glabrata* or *T. vaginalis*), the IC signal is not utilized in determining sample validity.
- For samples determined to be negative in all three analyte channels, the sample IC must be valid for the negative result to be valid. To be valid, the IC amplification curve time of emergence (TTime), emergence rate, and relative fluorescence units (RFU) must be within specified ranges. If the IC is not within these ranges, the sample is invalidated by the Panther software.
Run Validity is determined by the calibrator and control performance:
- Calibration is evaluated on the FAM (C spp) channel only. Replicates of the calibrator must meet RFU range, TTime, and emergence rate criteria to be valid. Outlier analysis from the three replicates of calibrator may be triggered by predefined
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%CV criteria. Calibrator replicates remaining after outlier removal are used along with the calibration coefficient found on the Master Lot Barcode Sheet to generate a calibration curve. Each calibrator replicate is quantitated and must fall within an acceptable deviation for the calibration curve to be valid. A failing replicate may be removed and the curve regenerated as long as two valid replicates remain. If there are less than two valid calibrator replicates, the calibration fails and the run is invalid.
- The CV/TV negative control must meet amplification criteria (RFU range, TTime, and emergence rate) for the Cy5.5 (IC) channel, and no amplification on the FAM (C spp), HEX (C.glabrata), or ROX (T. vaginalis) channels.
- The CV/TV positive control must report POS for C spp, C.glabrata, and T. vaginalis analytes, i.e., it must meet amplification criteria (RFU range, TTime, and emergence rate) for the FAM, the HEX, and the ROX channels. In addition, the calculated log copy value for C spp must fall within a specified range.
An operator may also manually invalidate an individual specimen or an entire run if it was observed and documented that a procedural, technical, or instrument-related error occurred while performing the assay.
## Results Generation
Assay results are automatically generated by the Panther system Aptima CV/TV assay software.
A qualitative result is separately reported for C spp (positive or negative). Calibration of the C spp channel is used to determine if the C spp concentration is above or below the C spp positivity assay cutoff.
Qualitative results are also separately reported for C. glabrata and T. vaginalis. Calibration is not utilized for these analytes. To determine positivity, minimum specifications for amplification based on TTime, emergence rate, and RFU range must be met.
When a sample is co-infected with C spp and a high concentration of T. vaginalis (as determined by T. vaginalis TTime), C spp positivity is determined by the detection of C spp, regardless of C spp concentration as determined by calibration. In this scenario, C spp positivity must meet minimum specifications for amplification based on TTime, emergence rate, and RFU range.
Test results may also be masked by the user (No Test); the user may pre-select to not report (mask) one or more analyte results.
## Results Interpretation
The user interprets the results generated by the Panther system based on Table 3 below.
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Table 3: Aptima CV/TV Assay Result Interpretation
| C spp Result | C. glabrata Result | T. vaginalis Result | Results Valid or Invalid | Interpretation |
| --- | --- | --- | --- | --- |
| Positive | Negative | Negative | Valid | Candida species group RNA detected; Candida glabrata RNA and Trichomanas vaginalis RNA not detected. |
| Positive | Positive | Negative | Valid | Candida species group RNA and Candida glabrata RNA detected; Trichomanas vaginalis RNA not detected. |
| Positive | Negative | Positive | Valid | Candida species group RNA and Trichomanas vaginalis RNA detected; Candida glabrata RNA not detected. |
| Positive | Positive | Positive | Valid | Candida species group RNA, Candida glabrata RNA, and Trichomanas vaginalis RNA detected. |
| Negative | Positive | Negative | Valid | Candida glabrata RNA detected; Candida species group RNA and Trichomanas vaginalis RNA not detected. |
| Negative | Negative | Positive | Valid | Trichomanas vaginalis RNA detected; Candida species group RNA and Candida glabrata RNA not detected. |
| Negative | Positive | Positive | Valid | Candida glabrata RNA and Trichomanas vaginalis RNA detected; Candida species group RNA not detected. |
| Negative | Negative | Negative | Valid | Candida species group RNA, Candida glabrata RNA, and Trichomonas vaginalis RNA not detected. |
| Invalid | Invalid | Invalid | Invalid | Invalid: there was an error in the generation of the result. Specimen should be retested. |
Note: Candida species group = C. albicans, C. parapsilosis, C. dubliniensis, and/or C. tropicalis
### J. Substantial Equivalence Information:
1. Predicate device name(s):
BD MAX Vaginal Panel
2. Predicate 510(k) number(s):
DEN160001
3. Comparison with predicate:
| Similarities and Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | Aptima CV/TV Assay (on the Pather System) K190472 | BD MAX Vaginal Panel (on the BD MAX System) DEN160001 |
| Intended Use | The Aptima CV/TV assay is an in vitro nucleic acid amplification test for the detection of RNA from microorganisms associated with vulvovaginal candidiasis and trichomoniasis. The assay utilizes real time transcription-mediated amplification (TMA) to detect and qualitatively report results for the following organisms:• Candida species group (C. albicans, C. tropicalis, C. parapsilosis, C. dubliniensis) | The BD MAX Vaginal Panel performed on the BD MAX System is an automated qualitative in vitro diagnostic test for the direct detection of DNA targets from bacteria associated with bacterial vaginosis (qualitative results reported based on detection and quantitation of targeted organism markers), Candida species associated with vulvovaginal candidiasis, and Trichomonas vaginalis from vaginal swabs in patients who are symptomatic for vaginitis/vaginosis. The test utilizes real-time polymerase chain reaction (PCR) for the amplification of specific DNA |
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| Similarities and Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | **Aptima CV/TV Assay (on the Pather System) K190472** | **BD MAX Vaginal Panel (on the BD MAX System) DEN160001** |
| | - *Candida glabrata* - *Trichomonas vaginalis* The assay differentiates between *Candida glabrata* and the *Candida* species group (C spp) by targeting the RNA component of RNAse P ribonucleoprotein; the assay does not differentiate among C spp. For *Trichomonas vaginalis*, the assay targets ribosomal RNA (rRNA) and differentiates the result from results for *Candida glabrata* and C spp. The assay is intended to aid in the diagnosis of vulvovaginal candidiasis and trichomoniasis on the automated Panther system using clinician-collected and patient-collected vaginal swab specimens from females with a clinical presentation consistent with vaginitis or vulvovaginitis. | targets and utilizes fluorogenic target-specific hybridization probes to detect and differentiate DNA from - Bacterial vaginosis markers (Individual markers not reported) - *Lactobacillus* spp. (*L. crispatus* and *L. jensenii*) - *Gardnerella vaginalis* - *Atopobium vaginae* - Bacterial Vaginosis Associated Bacteria-2 (BVAB-2) - *Megasphaera-1* - *Candida* spp. (*C. albicans*, *C. tropicalis*, *C. parapsilosis*, *C. dubliniensis*) - *Candida glabrata* - *Candida krusei* - *Trichomonas vaginalis* The BD MAX Vaginal Panel is intended to aid in the diagnosis of vaginal infections in women with a clinical presentation consistent with bacterial vaginosis, vulvovaginal candidiasis and trichomoniasis. |
| **Sample Type** | Vaginal swabs in female patients who are symptomatic for vaginitis or vulvovaginitis | Same |
| **Patient Population** | Women with a clinical presentation consistent with vaginitis or vulvovaginitis | Same |
| **Organisms Detected** | *Candida* species (*C. albicans*, *C. tropicalis*, *C. parapsilosis*, *C. dubliniensis*); *Candida glabrata*; *Trichomonas vaginalis* | *Lactobacillus* (*L. crispatus*, and *L. jensenii*), *Gardnerella vaginalis*, *Atopobium vaginae*, Bacterial Vaginosis Associated Bacteria-2 (BVAB-2), *Megasphaera-1*, *Candida* (*C. albicans*, *C. tropicalis*, *C. parapsilosis*, *C. dubliniensis*), *Candida glabrata*, *Candida krusei*, *Trichomonas vaginalis* |
| **Platform/Technology Principle of Operation** | Panther System/Real-time Transcription Mediated Amplification (TMA) | BD MAX System/Real-time polymerase chain reaction (PCR) |
| **Analyte** | RNA | DNA |
| **Assay Controls** | Incorporates an Internal Control in every test. Uses external positive and negative controls. | Same |
| **Assay Calibrators** | Positive Calibrator for the FAM (C spp) channel | None |
### K. Standard/Guidance Document Referenced (if applicable):
None
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# L. Test Principle:
The Aptima CV/TV assay involves three main steps, all of which take place in a single tube on the Panther system: target capture, target amplification by TMA, and detection of the amplification products (amplicon) by fluorescent labeled probes (torches). The assay incorporates an internal control (IC) in every test to monitor nucleic acid capture, amplification, and detection.
Specimens are collected in a tube containing specimen transport media (STM) that lyses the organisms, releases the RNA, and protects it from degradation during storage. When the assay is performed, capture oligonucleotides hybridize to highly conserved regions of the target RNA, if present, in the test specimen. The hybridized target is then captured onto magnetic microparticles that are separated from the specimen in a magnetic field. Wash steps remove extraneous components from the reaction tube. Target amplification occurs via TMA, a transcription-based nucleic acid amplification method that utilizes two enzymes, Moloney murine leukemia virus (MMLV) reverse transcriptase and T7 RNA polymerase. The reverse transcriptase is used to generate a DNA copy of the target RNA sequence, adding a promoter sequence for T7 RNA polymerase. T7 RNA polymerase produces multiple copies of RNA amplicon from the DNA copy template.
Detection is achieved using single-stranded nucleic acid torches that are present during the amplification of the target and hybridize specifically to the amplicon in real time. Each torch has a fluorophore and a quencher. The quencher suppresses the fluorescence of the fluorophore when the torch is not hybridized to the amplicon. When the torch binds to the amplicon, the fluorophore is separated from the quencher and emits a signal at a specific wavelength when excited by a light source. The Panther system detects and discriminates between four fluorescent signals corresponding to C spp, C. glabrata, TV, and IC amplification products. The Panther system software uses an Aptima CV/TV assay-specific algorithm to generate a Positive or Negative status for each target organism in the sample.
# M. Performance Characteristics (if/when applicable):
# 1. Analytical performance:
a. Precision/Reproducibility:
Within Laboratory Precision Study
Within Laboratory Precision was evaluated on three Panther systems at one testing site. Three operators performed testing across 22 days and three reagent lots. Each operator performed two runs per day using a seven-member sample panel. Each run consisted of three replicates of each panel member. The panel members were prepared with C. albicans, C. glabrata or T. vaginalis in simulated vaginal swab matrix (SVSM). The six positive panel members targeted C. albicans at Low (approximately 2x C95) and Moderate (3x C95) Positive levels, C. glabrata at Low (approximately 2x
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LoD) and Moderate (3x LoD) Positive levels, and T. vaginalis at Low (approximately 2x LoD) and Moderate (3x LoD) Positive levels. One Negative panel member contained SVSM with no added target analytes.
The study results are presented in Table 4 below. Signal variability of the Aptima CV/TV assay in terms of amplification curve time of emergence (TTime) was also calculated for analyte positive panel members. Variability calculated between instruments, between operators, between lots, between days, between runs, within runs, and overall, is shown in Table 5 below.
Table 4: Within Laboratory Precision - Agreement of Aptima CV/TV Assay with Expected Results
| Panel Member (Analyte Composition) | Positive/Total N | Expected Positivity | Percent Positivity (95% CI) |
| --- | --- | --- | --- |
| Negative (SVSM) | 0/162 | 0% | 0 (0.0-2.3) |
| Low Positive (C. albicans) | 162/162 | ≥ 95% | 100 (97.7-100.0) |
| Low Positive (C. glabrata) | 162/162 | ≥ 95% | 100 (97.7-100.0) |
| Low Positive (T. vaginalis) | 162/162 | ≥ 95% | 100 (97.7-100.0) |
| Moderate Positive (C. albicans) | 162/162 | 100% | 100 (97.7-100.0) |
| Moderate Positive (C. glabrata) | 162/162 | 100% | 100 (97.7-100.0) |
| Moderate Positive (T. vaginalis) | 162/162 | 100% | 100 (97.7-100.0) |
Table 5: Within Laboratory Precision - Signal Varability of the Aptima CV/TV Assay by Analyte Positive Panel Member
| | | | Between Days | | Between Instruments | | Between Operators | | Between Lots | | Between Runs | | With Runs | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member (Analyte Composition) | N | Mean TTime\( ^{1} \) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Low Positive (C. albicans) | 162 | 14.96 | 0.12 | 0.82 | 0.00 | 0.00 | 0.24 | 1.59 | 0.54 | 3.58 | 0.23 | 1.52 | 0.28 | 1.84 | 0.70 | 4.66 |
| Low Positive (C. glabrata) | 162 | 21.07 | 0.00 | 0.00 | 0.15 | 0.69 | 0.25 | 1.18 | 0.14 | 0.65 | 0.19 | 0.89 | 0.40 | 1.91 | 0.55 | 2.59 |
| Low Positive (T. vaginalis) | 162 | 24.09 | 0.00 | 0.00 | 0.33 | 1.38 | 0.22 | 0.93 | 0.01 | 0.05 | 0.21 | 0.87 | 0.59 | 2.46 | 0.75 | 3.09 |
| Moderate Positive (C. albicans) | 162 | 14.62 | 0.11 | 0.72 | 0.00 | 0.00 | 0.22 | 1.47 | 0.43 | 2.95 | 0.26 | 1.77 | 0.24 | 1.62 | 0.60 | 4.14 |
| Moderate Positive (C. glabrata) | 162 | 20.63 | 0.00 | 0.00 | 0.00 | 0.00 | 0.26 | 1.27 | 0.31 | 1.50 | 0.26 | 1.25 | 0.52 | 2.51 | 0.71 | 3.42 |
| Moderate Positive (T. vaginalis) | 162 | 22.73 | 0.00 | 0.00 | 0.12 | 0.54 | 0.24 | 1.08 | 0.18 | 0.80 | 0.28 | 1.23 | 0.41 | 1.79 | 0.59 | 2.61 |
CV = coefficient of variation, SD = standard deviation, TTime = amplification curve time of emergence (above a specific threshold) Note: If variability from a factor was numerically negative, SD and CV are shown as 0.00.
\( ^{1} \) The assay reports TTime for each assay analyte separately; the mean and signal variability reported are for the TTime corresponding to the analyte present in each panel member.
### Multi-Site Reproducibility
Aptima CV/TV assay reproducibility was evaluated at three US sites testing a seven-member sample panel. Testing was performed using one lot of assay reagents and six operators (two at each site). At each site, testing was performed for at least six days. Each operator performed one run per day, and each run had three replicates of each panel member. This study design allowed the generation of 108 data points for each panel member (i.e., 3 replicates/run/operator x 1 run/day x 2 operators/site x 6 days x 3
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sites = 108).
The panel members were prepared using a simulated vaginal swab matrix (SVSM) negative for Candida species (including C. glabrata) and T. vaginalis. Six panel members contained cell lysates of one of the following organisms: C. albicans (used as a representative of the Candida species group), C. glabrata, or T. vaginalis. Low Positive and Moderate Positive concentrations of each analyte were tested. One negative panel member contained only the SVSM with no added target analytes. The six positive panel members targeted C. albicans at Low (approximately 2x C95) and Moderate (3x C95) Positive, C. glabrata at Low (approximately 2x LoD) and Moderate (3x LoD) Positive, and T. vaginalis at Low (approximately 2x LoD) and Moderate (3x LoD) Positive.
While the study design allowed repeat testing of invalid runs in accordance with the Aptima CV/TV Assay Instructions for Use using a new set of assay calibrator, controls, and samples, tested samples with invalid results and samples with “error” results in a valid run were not retested in this study. Samples with invalid and “error” results were excluded from the performance analyses.
A total of 36 runs were performed during this study. Of the 36 runs, all 36 (100%, 95% CI: 90.4% - 100%) were valid. Of the 756 samples tested in valid runs, 751 samples (99.3%, 95% CI: 98.5% - 99.7%) had valid Aptima CV/TV assay results, and 5 samples (0.7%, 95% CI: 0.3% - 1.5%) had invalid results due to hardware error and were excluded from the performance analyses.
The reproducibility study results are presented in Table 6 and Table 7 below. Signal variability of the Aptima CV/TV assay in terms of amplification curve time of emergence (TTime) was also calculated for analyte positive panel members. Variability calculated between sites, between operators, between days, between runs, within runs, and overall, is shown in Table 8 below.
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Table 6: Multi-Site Reproducibility Study - Agreement of Aptima CV/TV Assay Results With Expected Results on the Panther System by Site
| | | Site 1 | | Site 2 | | Site 3 | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Target | Concentration | Agreed N/Total N | Agreement(%) (95% CI) | Agreed N/Total N | Agreement(%) (95% CI) | Agreed N/Total N | Agreement(%) (95% CI) |
| C. albicans | Negative | 180/180 | 100 (97.9-100) | 180/180 | 100 (97.9-100) | 176/176 | 100 (97.9-100) |
| | Low Positive (approximately 2x \( C_{95} \)) | 36/36 | 100 (90.4-100) | 36/36 | 100 (90.4-100) | 36/36 | 100 (90.4-100) |
| | Moderate Positive (3x \( C_{95} \)) | 36/36 | 100 (90.4-100) | 36/36 | 100 (90.4-100) | 35/35 | 100 (90.1-100) |
| C. glabrata | Negative | 180/180 | 100 (97.9-100) | 180/180 | 100 (97.9-100) | 178/178 | 100 (97.9-100) |
| | Low Positive (approximately 2x LoD) | 36/36 | 100 (90.4-100) | 36/36 | 100 (90.4-100) | 34/34 | 100 (89.8-100) |
| | Moderate Positive (3x LoD) | 36/36 | 100 (90.4-100) | 36/36 | 100 (90.4-100) | 35/35 | 100 (90.1-100) |
| T. vaginalis | Negative | 180/180 | 100 (97.9-100) | 180/180 | 100 (97.9-100) | 176/176 | 100 (97.9-100) |
| | Low Positive (approximately 2x LoD) | 36/36 | 100 (90.4-100) | 36/36 | 100 (90.4-100) | 36/36 | 100 (90.4-100) |
| | Moderate Positive (3x LoD) | 36/36 | 100 (90.4-100) | 36/36 | 100 (90.4-100) | 35/35 | 100 (90.1-100) |
Table 7: Multi-Site Reproducibility Study - Agreement of Aptima CV/TV Assay Results With Expected Results on the Panther System by Panel Member
| Target | Concentration | Agreed N/Total N | Agreement (%) (95% CI) |
| --- | --- | --- | --- |
| C. albicans | Negative\( ^{1} \) | 536/536 | 100 (99.3-100) |
| | Low Positive (approximately 2x \( C_{95} \)) | 108/108 | 100 (96.6-100) |
| | Moderate Positive (3x \( C_{95} \)) | 107/107 | 100 (96.5-100) |
| C. glabrata | Negative\( ^{1} \) | 538/538 | 100 (99.3-100) |
| | Low Positive (approximately 2x LoD) | 106/106 | 100 (96.5-100) |
| | Moderate Positive (3x LoD) | 107/107 | 100 (96.5-100) |
| T. vaginalis | Negative\( ^{1} \) | 536/536 | 100 (99.3-100) |
| | Low Positive (approximately 2x LoD) | 108/108 | 100 (96.6-100) |
| | Moderate Positive (3x LoD) | 107/107 | 100 (96.5-100) |
\( ^{1} \) Includes all panel members without the target present.
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Table 8: Multi-Site Reproducibility - Signal Variability of the Aptima CV/TV Assay by Analyte Positive Panel Member
| | | | Between Sites | | Between Operators | | Between Days | | Between Runs | | With Runs | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Panel Member (Analyte Composition) | N | Mean TTime^{1} | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Low Positive (*C. albicans*) | 108 | 14.68 | 0.66 | 4.47 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.30 | 2.02 | 0.83 | 5.64 |
| Moderate Positive (*C. albicans*) | 107 | 14.37 | 0.66 | 4.58 | 0.14 | 0.99 | 0.00 | 0.00 | 0.00 | 0.00 | 0.28 | 1.98 | 0.81 | 5.64 |
| Low Positive (*C. glabrata*) | 106 | 21.36 | 0.84 | 3.94 | 0.18 | 0.84 | 0.00 | 0.00 | 0.00 | 0.00 | 0.62 | 2.89 | 1.26 | 5.88 |
| Moderate Positive (*C. glabrata*) | 107 | 20.54 | 0.99 | 4.83 | 0.30 | 1.46 | 0.00 | 0.00 | 0.00 | 0.00 | 0.48 | 2.34 | 1.37 | 6.68 |
| Low Positive (*T. vaginalis*) | 108 | 24.32 | 1.16 | 4.77 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.60 | 2.48 | 1.59 | 6.54 |
| Moderate Positive (*T. vaginalis*) | 107 | 23.09 | 1.18 | 5.13 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | 0.56 | 2.41 | 1.56 | 6.77 |
CV = coefficient of variation, SD = standard deviation, TTime = amplification curve time of emergence (above a specific threshold)
Note: If variability from a factor was numerically negative, SD and CV are shown as 0.00.
$^{1}$ The assay reports TTime for each assay analyte separately; the mean and signal variability reported are for the TTime corresponding to the analyte presents in each panel member.
### b. Linearity/assay reportable range:
Although a final qualitative result is reported for C spp (positive or negative), calibration of the C spp (FAM) channel is initially used to generate a quantitative concentration value in log copies/mL, and then a determination is made whether the C spp concentration in log copies/mL is above or below the C spp positivity assay cutoff.
The linearity of the four *Candida* species detected in the C spp (FAM) channel, *C. albicans*, *C. parapsilosis*, *C. dubliniensis*, and *C. tropicalis*, was assessed by testing serial dilutions of lysate in SVSM for each of the four *Candida* species, and performing a polynomial regression analysis to determine an acceptable degree of non-linearity for each *Candida* species detected in the C spp (FAM) channel. The serial dilutions spanned the clinically relevant range of the assay. Testing was performed on one Panther system using one reagent lot.
For each of the four *Candida* species detected in the C spp (FAM) channel, six serial dilutions in total were prepared to show a larger dynamic range of linearity for each organism, and concentrations were adjusted to maintain 100% positivity for all dilutions. Cell lysate stocks with a confirmed CFU/mL concentration for each organism were used to build the serial dilutions at the concentrations listed in Table 9 below:
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Table 9: Linearity Study Dilutions per Candida Species
| Dilution Concentrations (CFU/mL) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Dilution # | Candida albicans | Dilution # | Candida parapsilosis | Dilution # | Candida dubliniensis | Dilution # | Candida tropicalis |
| 1 | \( 3.0 \times 10^{5} \) | 1 | \( 3.0 \times 10^{5} \) | 1 | \( 1.0 \times 10^{5} \) | 1 | \( 1.0 \times 10^{5} \) |
| 2 | \( 1.0 \times 10^{5} \) | 2 | \( 1.0 \times 10^{5} \) | 2 | \( 3.0 \times 10^{4} \) | 2 | \( 3.0 \times 10^{4} \) |
| 3 | \( 3.0 \times 10^{4} \) | 3 | \( 3.0 \times 10^{4} \) | 3 | \( 1.0 \times 10^{4} \) | 3 | \( 1.0 \times 10^{4} \) |
| 4 | \( 1.0 \times 10^{4} \) | 4 | \( 1.0 \times 10^{4} \) | 4 | \( 3.0 \times 10^{3} \) | 4 | \( 3.0 \times 10^{3} \) |
| 5 | \( 3.0 \times 10^{3} \) | 5 | \( 3.0 \times 10^{3} \) | 5 | \( 1.0 \times 10^{3} \) | 5 | \( 1.0 \times 10^{3} \) |
| 6 | \( 2.0 \times 10^{3} \) | 6 | \( 1.0 \times 10^{3} \) | 6 | \( 3.0 \times 10^{2} \) | 6 | \( 7.5 \times 10^{2} \) |
Five replicates per dilution were tested. Dilutions were randomized within run. Only valid data was used in data analysis.
Determination of linearity was performed by polynomial regression analysis per CLSI Guideline EP6-A2. The acceptance criteria for the linearity study were 1) degree of non-linearity should not exceed the \(\pm 0.50\) log copies/mL range at all concentrations tested; and 2) standard deviation of the observed log copies/mL value should be \(\leq 0.25\) at all concentrations tested to meet the repeatability requirements. The linearity study results for C. albicans are presented in Table 10 to Table 12 below.
Table 10: Degree of Non-Linearity for C. albicans
| Best Fit Polynomial | Dilution # | N | Linear Fit Calculated Mean (Log copies/mL) | Best Fit Polynomial Calculated Mean (Log copies/mL) | Non-Linearity Best Fit Polynomial Calculated Mean – Linear Fit Calculated Mean (Log copies/mL) | Degree of Non-Linearity is within ± 0.5 Log copies/mL (Yes or No) |
| --- | --- | --- | --- | --- | --- | --- |
| \( {3}^{\text{rd }} \) Degree Polynomial | 1 | 5 | 4.819 | 4.811 | -0.008 | Yes |
| | 2 | 5 | 4.297 | 4.225 | -0.072 | Yes |
| | 3 | 5 | 3.774 | 3.810 | 0.036 | Yes |
| | 4 | 5 | 3.252 | 3.383 | 0.130 | Yes |
| | 5 | 5 | 2.730 | 2.756 | 0.026 | Yes |
| | 6 | 5 | 2.521 | 2.409 | -0.113 | Yes |
Table 11: Repeatability for C. albicans
| Candida Species | Dilution # | N | Observed Mean (Log copies/mL) | Observed Standatd Deviation (Log copies/mL) | Standard Deviation ≤ 0.25 Log copies/mL (Yes or No) |
| --- | --- | --- | --- | --- | --- |
| C. albicans | 1 | 5 | 4.799 | 0.081 | Yes |
| | 2 | 5 | 4.267 | 0.103 | Yes |
| | 3 | 5 | 3.757 | 0.090 | Yes |
| | 4 | 5 | 3.401 | 0.138 | Yes |
| | 5 | 5 | 2.777 | 0.191 | Yes |
| | 6 | 5 | 2.391 | 0.176 | Yes |
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Table 12: C. albicans – Deviations between the Observed Mean and the Linear Fit Calculated Mean
| Candida Species | Dilution # | N | Observed Mean (Log copies/mL) | Observed Standatd Deviation (Log copies/mL) | Linear Fit Calculated Mean (Log copies/mL) | Deviation Observed Mean - Best Fit Polynomial Calculated Mean (Log copies/mL) |
| --- | --- | --- | --- | --- | --- | --- |
| C. albicans | 1 | 5 | 4.799 | 0.081 | 4.819 | -0.020 |
| | 2 | 5 | 4.267 | 0.103 | 4.297 | -0.030 |
| | 3 | 5 | 3.757 | 0.090 | 3.774 | -0.017 |
| | 4 | 5 | 3.401 | 0.138 | 3.252 | 0.149 |
| | 5 | 5 | 2.777 | 0.191 | 2.730 | 0.047 |
| | 6 | 5 | 2.391 | 0.176 | 2.521 | -0.130 |
Note: The bolded value indicates the maximum deviation between the Observed Mean in log copies/mL and the Linear Fit Calculated Mean in log copies/mL.
C. parapsilosis did not have a statistically significant \( 2^{nd} \) or \( 3^{rd} \) degree polynomial model fit, therefore, the linear fit was determined to be the best fit for this species. The linearity study results for C. parapsilosis are presented in Table 13 and Table 14 below.
Table 13: Repeatability for C. parapsilosis
| Candida Species | Dilution # | N | Observed Mean (Log copies/mL) | Observed Standatd Deviation (Log copies/mL) | Standard Deviation ≤ 0.25 Log copies/mL (Yes or No) |
| --- | --- | --- | --- | --- | --- |
| C. parapsilosis | 1 | 5 | 4.214 | 0.208 | Yes |
| | 2 | 5 | 3.834 | 0.037 | Yes |
| | 3 | 5 | 3.280 | 0.154 | Yes |
| | 4 | 5 | 2.806 | 0.186 | Yes |
| | 5 | 5 | 2.301 | 0.117 | Yes |
| | 6 | 5 | 1.819 | 0.108 | Yes |
Table 15: C. parapsilosis – Deviations between the Observed Mean and the Linear Fit Calculated Mean
| Candida Species | Dilution # | N | Observed Mean (Log copies/mL) | Observed Standatd Deviation (Log copies/mL) | Linear Fit Calculated Mean (Log copies/mL) | Deviation (Observed Mean - Linear Fit Calculated Mean) (Log copies/mL) |
| --- | --- | --- | --- | --- | --- | --- |
| C. parapsilosis | 1 | 5 | 4.214 | 0.208 | 4.260 | -0.046 |
| | 2 | 5 | 3.834 | 0.037 | 3.773 | 0.061 |
| | 3 | 5 | 3.280 | 0.154 | 3.286 | -0.006 |
| | 4 | 5 | 2.806 | 0.186 | 2.799 | 0.007 |
| | 5 | 5 | 2.301 | 0.117 | 2.312 | -0.011 |
| | 6 | 5 | 1.819 | 0.108 | 1.824 | -0.005 |
Note: The bolded value indicates the maximum deviation between the Observed Mean in log copies/mL and the Linear Fit Calculated Mean in log copies/mL.
The linearity study results for C. dubliniensis are presented in Table 16 to Table 18 below.
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Table 16: Degree of Non-Linearity for C. dubliniensis
| Best Fit Polynomial | Dilution # | N | Linear Fit Calculated Mean (Log copies/mL) | Best Fit Polynomial Calculated Mean (Log copies/mL) | Non-Linearity Best Fit Calculated Mean - Linear Fit Calculated Mean (Log copies/mL) | Degree of Non-Linearity is within ± 0.5 Log copies/mL (Yes or No) |
| --- | --- | --- | --- | --- | --- | --- |
| \( {2}^{\text{nd }} \) Degree Polynomial | 1 | 5 | 5.040 | 4.886 | -0.154 | Yes |
| | 2 | 5 | 4.470 | 4.501 | 0.031 | Yes |
| | 3 | 5 | 3.900 | 4.023 | 0.123 | Yes |
| | 4 | 5 | 3.330 | 3.453 | 0.123 | Yes |
| | 5 | 5 | 2.760 | 2.791 | 0.031 | Yes |
| | 6 | 5 | 2.190 | 2.035 | -0.154 | Yes |
Table 17: Repeatability for C. dubliniensis
| Candida Species | Dilution # | N | Observed Mean (Log copies/mL) | Observed Standatd Deviation (Log copies/mL) | Standard Deviation ≤ 0.25 Log copies/mL (Yes or No) |
| --- | --- | --- | --- | --- | --- |
| C. dubliniensis | 1 | 5 | 4.937 | 0.018 | Yes |
| | 2 | 5 | 4.380 | 0.141 | Yes |
| | 3 | 5 | 4.117 | 0.088 | Yes |
| | 4 | 5 | 3.389 | 0.056 | Yes |
| | 5 | 5 | 2.868 | 0.138 | Yes |
| | 6 | 5 | 1.999 | 0.185 | Yes |
Table 18: C. dubliniensis –Deviations between the Observed Mean and the Linear Fit Polynomial Calculated Mean
| Candida Species | Dilution # | N | Observed Mean (Log copies/mL) | Observed Standatd Deviation (Log copies/mL) | Linear Fit Calculated Mean (Log copies/mL) | Deviation (Observed Mean - Best Fit Polynomial Calculated Mean) (Log copies/mL) |
| --- | --- | --- | --- | --- | --- | --- |
| C. dubliniensis | 1 | 5 | 4.937 | 0.018 | 5.040 | -0.103 |
| | 2 | 5 | 4.380 | 0.141 | 4.470 | -0.090 |
| | 3 | 5 | 4.117 | 0.088 | 3.900 | 0.217 |
| | 4 | 5 | 3.389 | 0.056 | 3.330 | 0.059 |
| | 5 | 5 | 2.868 | 0.138 | 2.760 | 0.108 |
| | 6 | 5 | 1.999 | 0.185 | 2.190 | -0.191 |
Note: The bolded value indicates the maximum deviation between the Observed Mean in log copies/mL and the Linear Fit Calculated Mean in log copies/mL.
C. tropicalis did not have a statistically significant \( 2^{nd} \) or \( 3^{rd} \) degree polynomial model fit, therefore, the linear fit was determined to be the best fit for this species. The linearity study results for C. tropicalis are presented in Table 19 and Table 20 below.
Table 19: Repeatability for C. tropicalis
| Candida Species | Dilution # | N | Observed Mean (Log copies/mL) | Observed Standatd Deviation (Log copies/mL) | Standard Deviation ≤ 0.25 Log copies/mL (Yes or No) |
| --- | --- | --- | --- | --- | --- |
| C. tropicalis | 1 | 5 | 4.839 | 0.066 | Yes |
| | 2 | 5 | 4.452 | 0.071 | Yes |
| | 3 | 5 | 3.801 | 0.167 | Yes |
| | 4 | 5 | 3.605 | 0.124 | Yes |
| | 5 | 5 | 2.987 | 0.036 | Yes |
| | 6 | 5 | 2.859 | 0.122 | Yes |
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Table 20: C. tropicalis – Deviations between the Observed Mean and the Linear Fit Calculated Mean
| Candida Species | Dilution # | N | Observed Mean (Log copies/mL) | Observed Standard Deviation (Log copies/mL) | Linear Fit Calculated Mean (Log copies/mL) | Deviation Observed Mean - Best Fit Polynomial Calculated Mean (Log copies/mL) |
| --- | --- | --- | --- | --- | --- | --- |
| C. tropicalis | 1 | 5 | 4.839 | 0.066 | 4.862 | -0.023 |
| | 2 | 5 | 4.452 | 0.071 | 4.381 | 0.071 |
| | 3 | 5 | 3.801 | 0.167 | 3.936 | -0.135 |
| | 4 | 5 | 3.605 | 0.124 | 3.455 | 0.150 |
| | 5 | 5 | 2.987 | 0.036 | 3.010 | -0.023 |
| | 6 | 5 | 2.859 | 0.122 | 2.899 | -0.040 |
Note: The bolded value indicates the maximum deviation between the Observed Mean in log copies/mL and the Linear Fit Calculated Mean in log copies/mL.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
# External Quality Controls
To generate valid results, a set of assay controls must be tested. One replicate each of negative control and positive control must be tested each time a reagent kit is loaded on the Panther system. Once established, the controls are valid for up to 24 hours. Software on the Panther system alerts the operator when controls are required. During processing, criteria for acceptance of controls are automatically verified by software on the Panther system. If any one of the controls has an invalid result, the software automatically invalidates the run. Samples in an invalidated run must be retested using a freshly prepared calibrator and freshly prepared controls.
During the prospective clinical study conducted, a total of 55 positive controls and 55 negative controls were tested in valid runs on the Panther System. For the positive controls, a median TTime of 13.6 minutes with a range of 12.7 minutes to 16.4 minutes and a 4.85% CV were observed for the C spp (FAM) channel; a median TTime of 19.2 minutes with a range of 18.0 minutes to 28.6 minutes and a 9.84% CV were observed for the C. glabrata (HEX) channel; a median TTime of 17.3 minutes with a range of 16.3 minutes to 24.0 minutes and a 8.26% CV were observed for the T. vaginalis (ROX) channel; and a median TTime of 17.9 minutes with a range of 16.8 minutes to 26.6 minutes and a 10.31% CV were observed for the IC (Cy5.5) channel. For the negative controls, a median TTime of 17.0 minutes with a range of 16.0 minutes to 23.9 minutes and a 7.78% CV were observed for the IC (Cy5.5) channel.
# Effectiveness of External Quality Control (Run Controls), Kit Calibrator and Internal Control
The Panther software enforces kit calibrator and run control addition after reagent kit loading and prior to processing samples. The assay also features an internal control. The kit calibrator, run controls, and the internal control are used to determine whether a run and/or a specimen result is valid. An analytical study was performed to assess whether the Aptima CV/TV assay run controls and/or calibration are properly
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invalidated under operator-induced fault conditions that are not detected by instrument process controls.
The study results are presented in Table 21 below.
Table 21: List of Falt Conditions Tested, Run Validity Expectation, and Run Validity Result
| Condition | Run Set Up | Run Validity Expection | Run Validity Result | Total Test^{1} | Invalid^{1} Tests | Run Error Codes |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | Normal Condition (Control) | Invalid | Invalid | 80 | 80 | NA |
| 2 | IC not added to TCR | Invalid | Invalid | 80 | 80 | p, x |
| 3 | Amplification reconstitution buffer used directly (no lyophilized reagent added) | Invalid | Invalid | 80 | 80 | f, p, x |
| 4 | Promotor reconstitution buffer used directly (no lyophilized reagent added) | Invalid | Invalid | 80 | 80 | f, p, x |
| 5 | Enzyme reconstitution buffer used directly (no lyophilized reagent added) | Invalid | Invalid | 80 | 80 | f, p, x, ebl |
| 6 | Amplification lyophilized reagent reconstituted with enzyme buffer; Enzyme lyophilized reagent reconstituted with Amplification buffer; Promoter reagent normal | Invalid | Invalid | 80 | 80 | f, p, x |
| 7 | Enzyme lyophilized reagent reconstituted with promoter buffer; Promoter lyophilized reagent reconstituted with enzyme buffer; Amplification reagent normal | Invalid | Invalid | 80 | 80 | f, p, x, FLSOR |
f = calibration failed; p = assay processing error; x = control invalidated; ebl = the calculated estimated baseline is below the threshold value; FLSOR = a fluorometer has an off-target error
$^{1}$ Excluding kit calibrator and controls replicates.
The results of this study demonstrated that the Aptima CV/TV assay kit calibrators and controls are effective at properly invalidating operator induced fault conditions.
### Vaginal Swab Specimen Stability
An analytical study was performed to establish the stability of clinical vaginal swab specimens for Aptima CV/TV assay testing. Clinical vaginal swab specimen stability was demonstrated by spiking whole organism lysate into individual naturally occurring negative vaginal swab specimens collected using the Aptima Multitest Swab Specimen Collection Kit, targeting 3x C$_{95}$ or 3x LoD for each analyte. These specimens were tested with a minimum of 20 replicates under the following storage conditions:
- Storage at 2-8°C for a minimum of 30 days.
- Storage at 30°C for a minimum of 30 days.
- Storage at -20°C for a minimum of 60 days.
- Storage at 2-8°C for a minimum of 30 days followed by storage at -20°C for 60 days.
- Storage at 30°C for a minimum of 30 days followed by storage at -20°C for 60 days.
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- Storage at -20°C through at least 3 freeze/thaw (F/T) cycles.
The vaginal swab specimen stability study results are presented in Table 22 below.
Table 22: Clinical Vaginal Swab Specimen Stability Results
| Specimen | Testing Time Point | Storage Conditions | Samples Tested (N) | % Aptima CV/TV Assay Positive |
| --- | --- | --- | --- | --- |
| C. albicans (3x C_{95}) | T=0 (Baseline) | NA | 24 | 100% |
| | T=30 Days | 2-8°C | 23 | 100% |
| | | 30°C | 23 | 95.7%^{1} |
| | T=60 Days | -20°C | 23 | 100% |
| | T=90 Days | 2-8°C for 30 days followed by -20°C for 60 days | 23 | 100% |
| | | 30°C for 30 days followed by -20°C for 60 days | 23 | 100% |
| | After three rounds of freeze/thaw | -20°C freeze/thaw | 23 | 100% |
| C. glabrata (3x LoD) | T=0 (Baseline) | NA | 24 | 100% |
| | T=30 Days | 2-8°C | 23 | 100% |
| | | 30°C | 23 | 100% |
| | T=60 Days | -20°C | 23 | 100% |
| | T=90 Days | 2-8°C for 30 days followed by -20°C for 60 days | 23 | 100% |
| | | 30°C for 30 days followed by -20°C for 60 days | 23 | 100% |
| | After three rounds of freeze/thaw | -20°C freeze/thaw | 23 | 100% |
| T. vaginalis (3x LoD) | T=0 (Baseline) | NA | 24 | 100% |
| | T=30 Days | 2-8°C | 23 | 100% |
| | | 30°C | 23 | 100% |
| | T=60 Days | -20°C | 23 | 100% |
| | T=90 Days | 2-8°C for 30 days followed by -20°C for 60 days | 23 | 100% |
| | | 30°C for 30 days followed by -20°C for 60 days | 23 | 100% |
| | After three rounds of freeze/thaw | -20°C freeze/thaw | 23 | 100% |
Note: At baseline, 24 samples were tested for each analyte. One T. vaginalis positive sample demonstrated background amplification for C. albicans in the FAM channel. All samples prepared using this natural negative donor were eliminated from further testing in this study, resulting in 23 samples being tested per subsequent time point per analyte.
$^{1}$ One sample was negative due to the log copy/mL value being below the C spp assay cutoff.
The clinical vaginal swab specimen stability study results met the acceptance criteria (i.e., ≥ 95% for all analytes at each condition evaluated) for this study for all conditions evaluated.
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An additional analytical study was also performed to establish the stability of clinical vaginal swab specimens at 2-8°C for a minimum of 60 days for Aptima CV/TV assay testing. Clinical vaginal swab specimen stability at 2-8°C for a minimum of 60 days was demonstrated by spiking whole organism lysate into individual naturally occurring negative vaginal swab specimens collected using the Aptima Multitest Swab Specimen Collection Kit, targeting 3x C95 or 3x LoD for each analyte. These specimens were tested with 24 replicates.
Results of this additional vaginal swab specimen stability study also met the acceptance criteria; all replicates (100%) were detected for all analytes at the condition evaluated in this study.
## Matrix Equivalency Study
A simulated vaginal swab matrix (SVSM) was developed for use in the precision and reproducibility studies and several other analytical studies in order to mitigate the effect of Aptima CV/TV assay targeted analytes introduced by use of natural vaginal swab matrix (NVSM).
The SVSM consisted of 2.9 mL Aptima specimen transport medium (STM) combined with 150 uL of simulated vaginal fluid (SVF). The composition of the SVF is provided in Table 23 below.
Table 23: Simulated Vaginal Fluid (SVF) Composition
| Composition (in water) | Concentration |
| --- | --- |
| Sodium chloride | 3.51 g/L |
| Potassium hydroxide | 1.40 g/L |
| Calcium hydroxide | 0.222 g/L |
| Bovine serum albumin | 0.018 g/L |
| Lactic acid | 2.00 g/L |
| Acetic acid | 1.00 g/L |
| Glycerol | 0.16 g/L |
| Urea | 0.40 g/L |
| Glucose | 5.00 g/L |
| C33A cells | 33,000 cells/mL |
| pH 4.2 | pH 4.2 |
| Mucin | 1.5% (w/v) |
In order to utilize the SVSM as a simulated matrix in preparing contrived samples to be tested in precision/reproducibility studies and other analytical studies, an analytical study was performed to evaluate the performance using SVSM against a true negative NVSM with the Aptima CV/TV assay. Specimen Panels spiked with C. albicans, C. glabrata, or T. vaginalis lysate were prepared in both SVSM and NVSM at <1x, 1-2x, and 5x the LoD (for C. glabrata and T. vaginalis), or at <1x, 1-2x, and 5x the C95 (for C. albicans) concentrations. A minimum of 10 samples per spiking concentration per analyte for both SVSM and NVSM were tested with the Aptima CV/TV assay in this study.
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Results of this study are presented in Table 24 below.
Table 24: Matrix Equivalency Study Results – SVSM vs. NVSM
| Analyte | Concentration (multiples of LoD or C95) | Expected % Positive | N | Positive NVSM (N) | Positive SVSM (N) | % Positive NVSM | % Positive SVSM | Average TTime NVSM | Average TTime SVSM | Relevant Channel |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| C. albicans | Negative | 0 | 10 | 0 | 0 | 0 | 0 | NA | NA | FAM |
| | < 1x C95 | 10-90 | 20 | 12 | 14 | 60% | 70% | 15.91 | 16.06 | |
| | 1-2x C95 | ≥ 95 | 30 | 30 | 30 | 100% | 100% | 15.22 | 15.30 | |
| | 5x C95 | 100 | 10 | 10 | 10 | 100% | 100% | 14.15 | 14.21 | |
| C. glabrata | Negative | 0 | 10 | 0 | 0 | 0 | 0 | NA | NA | HEX |
| | < 1x C95 | 10-90 | 10 | 3 | 4 | 30% | 40% | 22.87 | 22.78 | |
| | 1-2x C95 | ≥ 95 | 30 | 30 | 30 | 100% | 100% | 22.08 | 21.65 | |
| | 5x C95 | 100 | 10 | 10 | 10 | 100% | 100% | 19.95 | 19.80 | |
| T. vaginalis | Negative | 0 | 10 | 0 | 0 | 0 | 0 | NA | NA | ROX |
| | < 1x C95 | 10-90 | 10 | 1 | 6 | 10% | 60% | 27.70 | 27.68 | |
| | 1-2x C95 | ≥ 95 | 30 | 30 | 30 | 100% | 100% | 25.00 | 23.79 | |
| | 5x C95 | 100 | 10 | 10 | 10 | 100% | 100% | 22.60 | 22.06 | |
The data support equivalency between NVSM and SVSM for preparation of samples for the Aptima CV/TV assay analytical study and precision/reproducibility study.
## Kit Calibration Interval
An analytical study was conducted to establish allowable kit calibration interval for the Aptima CV/TV assay on the Panther System.
The assay reagents were stored on-board the Panther instrument and tested at 0 (baseline), 24, and 30 hours using positive and negative samples. Results at 24 and 30 hours were analyzed using calibration established at the baseline.
The study results are presented in Table 25 below.
Table 25: Kit Calibration Study Results
| Condition | Sample | Tested N | Reported Positive N | Reported Negative N | % Positive | Reported Valid N | Reported Invalid N | % Valid |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 0 Hour | Kit Calibrator | 3 | 3 | 0 | 100% | 3 | 0 | 100% |
| | Positive Control | 1 | 1 | 0 | 100% | 1 | 0 | 100% |
| | Negative Control | 1 | 0 | 1 | 0% | 1 | 0 | 100% |
| | Positive Control as Sample | 5 | 5 | 0 | 100% | 5 | 0 | 100% |
| | Negative Control as Sample | 5 | 0 | 5 | 0% | 5 | 0 | 100% |
| 24 Hours | Positive Control as Sample | 5 | 5 | 0 | 100% | 5 | 0 | 100% |
| | Negative Control as Sample | 5 | 0 | 5 | 0% | 5 | 0 | 100% |
| 30 Hours | Positive Control as Sample | 5 | 5 | 0 | 100% | 5 | 0 | 100% |
| | Negative Control as Sample | 5 | 0 | 5 | 0% | 5 | 0 | 100% |
The study results showed no difference between testing at baseline (0 hour), 24 hours, and 30 hours after calibration, supporting the claim that Aptima CV/TV assay reagent kit calibration is valid for at least 24 hours when reagents are stored on-board the Panther System.
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#### d. Detection limit:
The limit of detection (LoD) for each of the Aptima CV/TV assay analytes was determined by testing serial dilutions consisting of target organisms diluted in pooled negative natural vaginal swab matrix (NVSM) or simulated vaginal swab matrix (SVSM). A minimum of 20 replicates of each dilution were tested with each of the two reagent lots, for a minimum of 40 replicates per dilution. Probit analysis was performed to generate the 95% predicted detection limit for each organism. The predicted detection limits are shown in Table 26 below.
Table 26: Predicated Limit of Detection (LoD) of the Aptima CV/TV Assay by Probit Analysis
| Organism | Predicted Detection Limit | Concentration | Units |
| --- | --- | --- | --- |
| Candida albicans1 | 95% | 4439 | CFU/mL |
| Candida glabrata1 | 95% | 41 | CFU/mL |
| Candida parapsilosis2 | 95% | 9416 | CFU/mL |
| Candida tropicalis2 | 95% | 811 | CFU/mL |
| Candida dubliniensis2 | 95% | 1176 | CFU/mL |
| Trichomonas vaginalis1 | 95% | 0.0024 | Cells/mL |
\( ^{1} \) Tested in natural vaginal swab matrix (NVSM).
\( ^{2} \) Tested in simulated vaginal swab matrix (SVSM).
#### e. Analytical Reactivity
Five strains of each Candida target organism were tested using lysate targeting 3x LoD or C95 for C. albicans, C. parapsilosis, C. tropicalis, C. dubliniensis, and C. glabrata in SVSM. Nine strains of T. vaginalis including a metronidazole resistant strain were tested with cell lysate targeting 3x LoD in SVSM. The Aptima CV/TV assay was positive for all Candida strains tested at 3x LoD or C95. Eight of the nine T. vaginalis strains, including the metronidazole resistant strain, were detected at 3x LoD. One strain of T. vaginalis was detected at 4x LoD.
Results of the analytical inclusivity study are presented in Table 27 below.
Table 27: Results of the Analytical Inclusivity Study
| Species | ATCC (Or Other Strain ID) | Replicates N | % Positive C spp (FAM) | % Positive C. glabrata (HEX) | % Positive T. vaginalis (ROX) | Test Concentration |
| --- | --- | --- | --- | --- | --- | --- |
| C. albicans | 18804 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| | 14053 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| | 11006 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| | 24433 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| | 36232 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| C. parapsilosis | 22019 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| | 58895 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| | 7330 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| | MYA-4646 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
| | 96137 | 20 | 100% | 0% | 0% | 3x \( C_{95} \) |
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| C. tropicalis | 750 | 20 | 100% | 0% | 0% | 3x C95 |
| --- | --- | --- | --- | --- | --- | --- |
| | 42678 | 20 | 100% | 0% | 0% | 3x C95 |
| | 13803 | 20 | 100% | 0% | 0% | 3x C95 |
| | 34139 | 20 | 100% | 0% | 0% | 3x C95 |
| | CDC: MAS92-384 | 20 | 100% | 0% | 0% | 3x C95 |
| C. dubliniensis | MYA-580 | 20 | 100% | 0% | 0% | 3x C95 |
| | MYA-582 | 20 | 100% | 0% | 0% | 3x C95 |
| | MYA-581 | 20 | 100% | 0% | 0% | 3x C95 |
| | MYA-583 | 20 | 100% | 0% | 0% | 3x C95 |
| | MYA-180 | 20 | 100% | 0% | 0% | 3x C95 |
| C. glabrata | 2001 | 20 | 0% | 100% | 0% | 3x LoD |
| | 48435 | 20 | 0% | 100% | 0% | 3x LoD |
| | CDC: MAS92-2 | 20 | 0% | 100% | 0% | 3x LoD |
| | CDC: MAS92-115 | 20 | 0% | 100% | 0% | 3x LoD |
| | CDC: MAS92-262 | 20 | 0% | 100% | 0% | 3x LoD |
| T. vaginalis | 30184 | 20 | 0% | 0% | 100% | 3x LoD |
| | 30185^{1} | 20 | 0% | 0% | 100% | 4x LoD |
| | 30187 | 20 | 0% | 0% | 100% | 3x LoD |
| | 30188 | 20 | 0% | 0% | 100% | 3x LoD |
| | 30236 | 20 | 0% | 0% | 100% | 3x LoD |
| | 50143^{2} | 20 | 0% | 0% | 100% | 3x LoD |
| | 30092 | 20 | 0% | 0% | 100% | 3x LoD |
| | 50144 | 20 | 0% | 0% | 100% | 3x LoD |
| | 50146 | 20 | 0% | 0% | 100% | 3x LoD |
$^{1}$ T. vaginalis ATCC 30185 passed the analytical reactivity criteria at 4x LoD.
$^{2}$ T. vaginalis strain ATCC 51043 is a metronidazole-resistant strain.
### f. Analytical specificity
#### Cross-Reactivity Study
This study was performed to evaluate the potential of the Acucy Influenza A&B Test to cross-react with closely related and non-targeted microbial organisms, resulting in a false positive result. A total of 64 organisms and human cell lines prepared in SVSM were tested in triplicate with the Aptima CV/TV assay.
Results of the cross-reactivity study are summarized in Table 28 below.
Table 28: Summary Results of the Cross-Reactivity Study
| Organism | Final Concentration Tested | C spp Positive Replicates | C. glabrata Positive Replicates | T. vaginalis Positive Replicates |
| --- | --- | --- | --- | --- |
| Acinetobacter lwoffii | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Actinomyces israelii | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Alcaligenes faecalis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Atopobium vaginae | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Bacteroides fragilis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Bifidobacterium adolescentis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| BVAB-1^{1} | 1x10^{6} copies/mL | 0/3 | 0/3 | 0/3 |
| BVAB-2^{1} | 1x10^{6} copies/mL | 0/3 | 0/3 | 0/3 |
| Campylobacter jejuni | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Candida catenulata | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Candida famata^{2} | 5x10^{5} CFU/mL | 0/3 | 0/3 | 0/3 |
| Candida guilliermondii | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Candida haemulonii | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
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| Candida inconspicua | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| --- | --- | --- | --- | --- |
| Candida kefyr | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Candida krusei | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Candida lusitaniae | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Candida norvegica | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Candida orthopsilosis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Chlamydia trachomatis | 1x10^{6} IFU/mL | 0/3 | 0/3 | 0/3 |
| Clostridium difficile | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Corynebacterium genitalium | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Cryptococcus neoformans | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Eggerthella lenta | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Enterobacter cloacae | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Enterococcus faecalis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Escherichia coli | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Fusobacterium nucleatum | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Gardnerella vaginalis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Haemophilus ducreyi | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| HeLa cells | 1x10^{4} Cells/mL | 0/3 | 0/3 | 0/3 |
| HIV | 1x10^{5} copies/mL | 0/3 | 0/3 | 0/3 |
| Herpes simplex virus 1 | 1x10^{4} TCID_{50}/mL | 0/3 | 0/3 | 0/3 |
| Herpes simplex virus 2 | 1x10^{4} TCID_{50}/mL | 0/3 | 0/3 | 0/3 |
| Klebsiella pneumoniae | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Lactobacillus acidophilus | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Lactobacillus crispatus | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Lactobacillus gasseri | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Lactobacillus iners | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Lactobacillus jensenii | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Lactobacillus mucosae | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Leptotrichia buccalis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Listeria monocytogenes | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Megasphaera Type 1^{1} | 1x10^{6} copies/mL | 0/3 | 0/3 | 0/3 |
| Mobiluncus curtisii | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Mycoplasma genitalium | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Mycoplasma hominis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Neisseria gonorrhoeae | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Peptostreptococcus magnus | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Pentatrichomonas hominis | 1x10^{5} cells/mL | 0/3 | 0/3 | 0/3 |
| Pichia fermentans | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Prevotella bivia | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Propionibacterium acnes | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Proteus vulgaris | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| SiHa cells | 1x10^{4} cells/mL | 0/3 | 0/3 | 0/3 |
| Sneathia amnii | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Staphylococcus aureus | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Staphylococcus epidermidis | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Streptococcus agalactiae | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Streptococcus pyogenes | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Treponema pallidum^{1} | 1x10^{6} copies/mL | 0/3 | 0/3 | 0/3 |
| Trichomonas tenax | 1x10^{5} cells/mL | 0/3 | 0/3 | 0/3 |
| Ureaplasma parvum | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
| Ureaplasma urealyticum | 1x10^{6} CFU/mL | 0/3 | 0/3 | 0/3 |
CFU = Colony Forming Units; IFU = Inclusion Forming Units; TCID$_{50}$ = Median Tissue Culture Infectious Dose
$^{1}$ In Vitro Transcript tested.
2 Cross-reactivity with Candida famata was seen at concentrations higher than \(5\times 10^{5}\) CFU/mL.
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No cross-reactivity was observed for any of the 64 organisms and human cell lines tested in the Aptima CV/TV assay at the concentrations listed in Table 28 above. Cross-reactivity with Candida famata was observed at concentrations higher than \( 5 \times 10^{5} \) CFU/mL, and is noted in the Limitations section of the package insert.
### Microbial Interference Study
Microbial interference was assessed for the Aptima CV/TV assay using the same 64-member test panel as the one used in the Cross-Reactivity Study above. A total of 64 organisms and human cell lines prepared in SVSM were tested in the presence of 3x LoD or C95 C. albicans, C. glabrata or T. vaginalis in triplicate with the Aptima CV/TV assay.
Results of the Microbial Interference Study are summarized in Table 29 below.
Table 29: Summary Results of the Microbial Interference Study
| Organism | Final Concentration Tested | C. albicans (3xC95) | C. glabrata (3xLoD) | T. vaginalis (3xLoD) |
| --- | --- | --- | --- | --- |
| | | C spp Positive Replicates | C. glabrata Positive Replicates | T. vaginalis Positive Replicates |
| Acinetobacter lwoffii | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Actinomyces israelii | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Alcaligenes faecalis | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Atopobium vaginae | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Bacteroides fragilis | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Bifidobacterium adolescentis | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| BVAB-1\( ^{1} \) | \( 1 \times 10^{6} \) copies/mL | 3/3 | 3/3 | 3/3 |
| BVAB-2\( ^{1} \) | \( 1 \times 10^{6} \) copies/mL | 3/3 | 3/3 | 3/3 |
| Campylobacter jejuni | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida catenulata | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida famata | \( 5 \times 10^{5} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida guilliermondii | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida haemulonii | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida inconspicua | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida kefyr | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida krusei | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida lusitaniae | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida norvegica | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Candida orthopsilosis | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Chlamydia trachomatis | \( 1 \times 10^{6} \) IFU/mL | 3/3 | 3/3 | 3/3 |
| Clostridium difficile | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Corynebacterium genitalium | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Cryptococcus neoformans | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Eggerthella lenta | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Enterobacter cloacae | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Enterococcus faecalis | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Escherichia coli | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Fusobacterium nucleatum | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Gardnerella vaginalis | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| Haemophilus ducreyi | \( 1 \times 10^{6} \) CFU/mL | 3/3 | 3/3 | 3/3 |
| HeLa cells | \( 1 \times 10^{4} \) Cells/mL | 3/3 | 3/3 | 3/3 |
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| HIV | \( 1 \times {10}^{5} \) copies/mL | 3/3 | 3/3 | 3/3 |
| --- | --- | --- | --- | --- |
| Herpes simplex virus 1 | \( 1 \times {10}^{4}{\mathrm{{TCID}}}_{50}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Herpes simplex virus 2 | \( 1 \times {10}^{4}{\mathrm{{TCID}}}_{50}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Klebsiella pneumoniae | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Lactobacillus acidophilus | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Lactobacillus crispatus | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Lactobacillus gasseri | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Lactobacillus iners | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Lactobacillus jensenii | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Lactobacillus mucosae | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Leptotrichia buccalis | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Listeria monocytogenes | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Megasphaera Type 1\( ^{1} \) | \( 1 \times {10}^{6} \) copies/mL | 3/3 | 3/3 | 3/3 |
| Mobiluncus curtisii | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Mycoplasma genitalium | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Mycoplasma hominis | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Neisseria gonorrhoeae | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Peptostreptococcus magnus | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Pentatrichomonas hominis | \( 1 \times {10}^{5}\mathrm{{cells}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Pichia fermentans | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Prevotella bivia | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Propionibacterium acnes | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Proteus vulgaris | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| SiHa cells | \( 1 \times {10}^{4}\mathrm{{cells}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Sneathia amnii | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Staphylococcus aureus | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Staphylococcus epidermidis | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Streptococcus agalactiae | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Streptococcus pyogenes | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Treponema pallidum\( ^{1} \) | \( 1 \times {10}^{6}\mathrm{{copies}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Trichomonas tenax | \( 1 \times {10}^{5}\mathrm{{cells}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Ureaplasma parvum | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
| Ureaplasma urealyticum | \( 1 \times {10}^{6}\mathrm{{CFU}}/\mathrm{{mL}} \) | 3/3 | 3/3 | 3/3 |
CFU = Colony Forming Units; IFU = Inclusion Forming Units; TCID \( _{50} \) = Median Tissue Culture Infectious Dose
\( ^{1} \) In Vitro Transcript tested.
The results showed that the tested organisms and human cell lines at the concentrations tested in this study did not interfere with the detection of C. albicans, C. glabrata, or T. vaginalis samples at close to the LoD concentrations.
### g. Competitive Interference Study
Since the Aptima CV/TV assay detects three analytes (C spp, C. glabrata, and T. vaginalis), a competitive interference study was conducted to determine whether target analytes at a high concentration would interfere with the detection of a second target analyte at near LoD concentrations in co-infected samples. Low concentration of one target lysate and high concentration of another target lysate in SVSM were tested in combination in 10 replicates per combination with the Aptima CV/TV assay.
Panel composition and concentrations are listed in Table 30 below.
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Table 30: Co-Infection Test Panel
| Panel Member | C. albicans Concentration | C. glabrata Concentration | T. vaginalis Concentration |
| --- | --- | --- | --- |
| C. albicans Low; C. glabrata High | 3x \( C_{95} \) | \( 1 \times 10^{6} \) CFU/mL | N/A |
| C. albicans Low; T. vaginalis High | 3x \( C_{95} \) | N/A | \( 1 \times 10^{5} \) cells/mL |
| C. glabrata Low; T. vaginalis High\( ^{1} \) | N/A | 3x LoD | \( 1 \times 10^{3} \) cells/mL |
| C. albicans High; C. glabrata Low | \( 1 \times 10^{6} \) CFU/mL | 3x LoD | N/A |
| C. albicans High; T. vaginalis Low | \( 1 \times 10^{6} \) CFU/mL | N/A | 3x LoD |
| C. glabrata High; T. vaginalis Low | N/A | \( 1 \times 10^{6} \) CFU/mL | 3x LoD |
CFU = Colony Forming Units
\( ^{1} \) Competitive interference was observed for the combinations of low C. glabrata (3x LoD) and high T. vaginalis (1x10 \( ^{5} \) cells/mL), and low C. glabrata (3x LoD) and high T. vaginalis (1x10 \( ^{4} \) cells/mL). Further testing was conducted and resulted in 100% detection for the combination of low C. glabrata (3x LoD) and high T. vaginalis (1x10 \( ^{3} \) cells/mL).
All testing resulted in 100% detection for both target analytes present except for the combination of low C. glabrata (3x LoD) and high T. vaginalis (1x10 \( ^{5} \) or 1x10 \( ^{4} \) cells/mL). Further testing was conducted and resulted in 100% detection for the combination of low C. glabrata (3x LoD) and high T. vaginalis (1x10 \( ^{3} \) cells/mL). The observed competitive interference is noted in the Limitations section of the package insert.
### h. Potentially Interfering Substances Study
Potentially interfering substances were tested in the Aptima CV/TV assay. Panels were built in SVSM and evaluated in triplicate for potential effects on assay sensitivity and specificity. Sensitivity performance was evaluated separately for C. albicans, C. glabrata, and T. vaginalis by spiking lysate at 3x LoD (3x C \( _{95} \) for C spp). Negative panels containing each substance were also evaluated for specificity.
No interference was observed in the presence of the following exogenous and endogenous substances tested at the concentrations listed in Table 31 below. Interference was observed in the presence of Tioconazole 6.5% Ointment, Vaginal Moisturizing Gel, and Glacial Acetic Acid, at higher concentrations (see Table 31 footnotes), and is noted in the Limitations section of the package insert.
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Table 31: Interfering Substances Pabel
| Substance | Final Concentration1 |
| --- | --- |
| Whole Blood | 5% V/V |
| Leukocytes | \( 1 \times 10^{6} \) cells/mL |
| Mucus | 5% V/V |
| Seminal Fluid | 5% V/V |
| Contraceptive Foam | 5% W/V |
| Contraceptive Film | 5% W/V |
| Tioconazole 6.5%2 | 2% W/V |
| Douche | 5% W/V |
| Progesterone | 5% W/V |
| Estradiol | 5% W/V |
| Acyclovir | 5% W/V |
| Metronidazole | 5% W/V |
| Hemorrhoidal Cream | 5% W/V |
| Vaginal Moisturizing Gel3 | 0.5% W/V |
| Lubricant | 5% V/V |
| Spermicide | 5% W/V |
| Anti-fungal | 5% W/V |
| Deodorant/Spray | 5% W/V |
| Glacial Acetic Acid4 | 4% V/V |
| Vagisil Cream | 5% W/V |
W/V = weight by volume; V/V = volume by volume
\( ^{1} \) Final Concentration represents final concentration in the sample when tested on the Panther instrument.
\( ^{2} \) Tioconazole 6.5% Ointment: Interference was observed at \( \geq \) 3% W/V for all analytes. No interference was observed at 2% W/V for all analytes.
\( ^{3} \) Vaginal Moisturizing Gel: Interference was observed at 5% W/V for C. glabrata, \( \geq \) 3% W/V for T. vaginalis, and \( \geq \) 1% W/V for C. albicans. No interference was observed at 0.5% W/V for C. albicans, 4% W/V for C. glabrata, and 2% W/V for T. vaginalis.
\( ^{4} \) Glacial Acetic Acid: Interference was observed at 5% V/V for C. albicans. No interference was observed at 4% V/V for C. albicans, 5% V/V for C. glabrata, and 5% V/V for T. vaginalis.
#### i. Carry-Over Study
Since carry-over rates on the Panther System had already been established for several other FDA cleared assays, such as the Aptima Trichomonas vaginalis Assay (K122062) and the Aptima Combo 2 Assay (K132251 and K111409), no additional testing on instrument carryover/cross-contamination for the Aptima CV/TV assay on the fully automated Panther System was performed.
#### j. Assay cut-off
#### Minimum Specifications for an Amplified Reaction
For Aptima CV/TV assay amplification, the C. glabrata (HEX) and T. vaginalis (ROX) minimum specifications for an amplified reaction also serve as the specifications for a positive reaction. For C spp (FAM), these channel minimum specifications for an amplified reaction serve as the specifications for a positive reaction only when the sample is co-infected with a high concentration of T. vaginalis exceeding a predetermined threshold (as determined by TTime in the ROX channel).
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The parameters that determine a legitimate amplified reaction for each channel are as follows:
- RFU Range – Relative Fluorescent Unit (RFU) is a measure of the intensity of the fluorescent signal, specific for each channel. The RFU Range is the difference between maximum and minimum fluorescent signal seen in a sample during amplification. The RFU range specification for each channel represents the minimum fluorescent threshold for an amplification curve to generate a TTime.
- TTime Max – The TTime parameter is a measure of the time (in minutes) for the real-time amplified fluorescent curve to emerge from the background signal. TTime values are generated based on the normalized curve from the Panther System TTime algorithm. The TTime max value is the maximum amount of time allowed for an amplification curve to generate a TTime. If the TTime exceeds this threshold, amplification has not occurred, and the specimen is negative.
- Emergence Rate - Emergence Rate is the background subtracted slope of the emerging fluorescent curve, calculated by linear regression of N data points before and after the established TTime.
The minimum specifications for an amplified reaction for each of the four channels are presented in Table 32 below.
Table 32: Specifications for an Amplified Reaction
| Analyte | Channel | RFU Range | TTime Max | Emergence Rate |
| --- | --- | --- | --- | --- |
| C spp | FAM | 1000 | 45 | 40 |
| C. glabrata | HEX | 1000 | 45 | 50 |
| T. vaginalis | ROX | 1000 | 50 | 80 |
| Internal Control | Cy5.5 | 1000 | 50 | 30 |
To determine the appropriate minimum specifications for an amplified reaction, logistic regression and receiver operating characteristic (ROC) analyses were initially performed with data generated from a minimum of 635 positive and a minimum of 594 negative samples for each channel during assay development. All parameter values were subsequently validated during assay development by running an independent set of 617 clinical samples with the proposed specifications and analyzed relative to composite culture and bi-directional sequencing comparator or NAAT assay comparator results to ensure the selected values maximized assay sensitivity and specificity. The selected specifications were further validated during the prospective clinical study and the contrived clinical specimens testing.
### Assay Cut-off for the C spp (FAM) Channel
During Aptima CV/TV assay development, preliminary analysis of the C spp assay results compared to Candida culture showed that the Limit of Detection (LoD) for the
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C spp assay (FAM) was sensitive enough to detect low level Candida infections; i.e. negative by culture (culture status below +1). The reference used for Candida species group positivity is Candida culture with Sabouraud Dextrose Agar and CHROMagar with +1 culture or above (on a scale of +1 to +4). An assay cutoff for the C spp assay (FAM) was therefore developed to better align the Aptima CV/TV assay detection of C spp with the comparator method of culture in order to achieve the desired specificity.
A calibration system was developed for the C spp assay (FAM channel) in order to optimize the C spp assay cutoff and adjust for run-to-run differences. Prior to establishing the C spp assay cutoff during early assay development, the specificity of the C spp assay against Candida culture was less than 90% in 430 clinical samples. The C spp assay cutoff was developed by running clinical sample collections with known culture status along with standards of known concentration (in vitro transcript calibrators). Linear regression was performed with the calibration standards and the TTimes of each characterized sample converted to a relative unit of l…
