Aptima BV Assay

K190452 · Hologic, Inc. · PQA · May 23, 2019 · Microbiology

Device Facts

• Record ID: K190452
• Device Name: Aptima BV Assay
• Applicant: Hologic, Inc.
• Product Code: PQA · Microbiology
• Decision Date: May 23, 2019
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3975
• Device Class: Class 2

Intended Use

The Aptima BV assay is an in vitro nucleic acid amplification test that utilizes real time transcription-mediated amplification (TMA) for detection and quantitation of ribosomal RNA from bacteria associated with bacterial vaginosis (BV), including Lactobacillus (L. gasseri, L. crispatus, and L. jensenii), Gardnerella vaginalis, and Atopobium vaginae. The assay reports a qualitative result for BV and does not report results for individual organisms. The assay is intended to aid in the diagnosis of BV on the automated Panther system using clinician-collected and patient-collected vaginal swab specimens from females with a clinical presentation consistent with vaginitis and/or vaginosis.

Device Story

Aptima BV assay is an in vitro nucleic acid amplification test (NAAT) performed on the automated Panther system; detects and quantitates rRNA from bacteria associated with bacterial vaginosis (Lactobacillus spp., G. vaginalis, A. vaginae). Input: vaginal swab specimens in transport media. Process: target capture on magnetic microparticles; transcription-mediated amplification (TMA) using MMLV reverse transcriptase and T7 RNA polymerase; real-time detection via fluorescent-labeled torches. Output: qualitative BV positive/negative result. Used in clinical settings; operated by laboratory personnel. Software automates sample processing, amplification, detection, and data reduction. Benefits: provides objective, automated diagnosis of BV from symptomatic patients, aiding clinical decision-making.

Clinical Evidence

Prospective clinical study of 1,519 symptomatic and 174 asymptomatic women. Performance evaluated against Nugent score (with Amsel for intermediates). In symptomatic subjects (n=1417 evaluable), patient-collected swabs showed 97.3% sensitivity and 85.8% specificity; clinician-collected swabs showed 95.0% sensitivity and 89.6% specificity. Study included demographic and clinical condition sub-analyses (e.g., pregnancy, menses, post-menopausal).

Technological Characteristics

Nucleic acid amplification test (NAAT) using real-time Transcription-Mediated Amplification (TMA). Analyte: ribosomal RNA. Instrumentation: Panther System (automated). Detection: fluorescent-labeled torches. Software: assay-specific parameters for incubation/processing. Sterilization: N/A (reagents). Connectivity: standalone instrument, no wireless transmission.

Indications for Use

Indicated for females with clinical presentation consistent with vaginitis/vaginosis. Used for detection and quantitation of rRNA from bacteria associated with BV (Lactobacillus spp., G. vaginalis, A. vaginae) using clinician- 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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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

May 23, 2019

Hologic, Inc. Jeffrey Hergesheimer Regulatory Affairs Manager 10210 Genetic Center Drive San Diego, California 92121

## Re: K190452

Trade/Device Name: Aptima BV Assay, Aptima BV Controls Kit, Aptima BV Calibrator Kit Regulation Number: 21 CFR 866.3975 Regulation Name: Device that detects nucleic acid sequences from microorganisms associated with vaginitis and bacterial vaginosis. Regulatory Class: Class II Product Code: PQA, NSU, PMN Dated: February 22, 2019 Received: February 25, 2019

Dear Jeffrey Hergesheimer:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part

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801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.htm); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

for

Uwe Scherf, Ph.D. Director Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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# 510(k) SUMMARY Aptima BV Assay

# I. SUBMITTER

Hologic, Inc. 10210 Genetic Center Drive San Diego, CA 92121

| Contact Person: | Jeffrey Hergesheimer, MS, RAC<br>Regulatory Affairs Manager<br>[e-mail]<br>Phone: [phone]<br>Fax: [phone] |
|-----------------|-------------------------------------------------------------------------------------------------------------------------------------------------|
|-----------------|-------------------------------------------------------------------------------------------------------------------------------------------------|

Date Prepared: February 19, 2019

# II. DEVICE

| Proprietary Name of Device: | Aptima BV Assay |
|-----------------------------|------------------------------------------------------------------------------------------------------------------|
| Classification Name: | Device that detects nucleic acid sequences from microorganisms associated with vaginitis and bacterial vaginosis |
| Regulation Number: | 21 CFR 866.3975 |
| Regulatory Class: | Class II |
| Product Code: | PQA, NSU, PMN |

# III. PREDICATE DEVICE

The predicate device is the BD MAX Vaginal Panel (DEN160001; approved 10/28/2016, BD Diagnostics).

# IV. DEVICE DESCRIPTION

The Aptima BV assay is an in vitro nucleic acid amplification test for the detection and quantitation of rRNA from bacteria associated with bacterial vaginosis in women with a clinical presentation consistent with vaginitis/vaginosis. The Aptima BV assay utilizes the automated Panther system to provide qualitative results to aid in the diagnosis of bacterial vaginosis.

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#### Principles of the Procedure

The Aptima BV 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 cells, releases the RNA, and protects it from degradation during storage. When the Aptima BV 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 Lactobacillus group, Atopobium vaginae, Gardnerella vaginalis and IC amplification products. The Panther system software compares signal emergence times for each target organism to calibration information in order to determine the BV Positive or Negative status of each sample.

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#### Assay Components

The Aptima BV assay is provided as a 100-test kit. The Aptima BV assay master kit contains 8 reagents, 1 calibrator, and 2 controls required for sample processing. There are 4 boxes that make up the assay master kit. Boxes 1 and 2 contain the Aptima BV 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 BV 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 BV assay are detailed in Table 1. In addition, there is one ancillary kit required to run the assay, and one collection kit utilized for collection of specimens (Table 2).

| Box | Components Description |
|-----|---------------------------------------|
| 1 | Amplification Reagent |
| | Enzyme Reagent |
| | Promoter Reagent |
| | Internal Control |
| 2 | Amplification Reconstitution Solution |
| | Enzyme Reconstitution Solution |
| | Promoter Reconstitution Solution |
| | Target Capture Reagent |
| 3 | Positive Calibrator |
| 4 | Negative Control |
| | Positive Control |

Table 1: Reagents Required to Perform the Aptima BV Assay

| Table 2: Ancillary and Collection Kits Required to Perform the Aptima BV Assay |
|--------------------------------------------------------------------------------|
| Aptima Assay Fluids Kit |
| Aptima Multitest Swab Specimen Collection Kit |

Instrumentation

The Aptima BV 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 BV 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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## V. INDICATIONS FOR USE

The Aptima BV assay is an in vitro nucleic acid amplification test that utilizes real time transcription-mediated amplification (TMA) for detection and quantitation of ribosomal RNA from bacteria associated with bacterial vaginosis (BV), including Lactobacillus (L. gasseri, L. crispatus, and L. jensenii), Gardnerella vaginalis, and Atopobium vaginae. The assay reports a qualitative result for BV and does not report results for individual organisms. The assay is intended to aid in the diagnosis of BV on the automated Panther system using clinician-collected and patient-collected vaginal swab specimens from females with a clinical presentation consistent with vaginitis and/or vaginosis.

# VI. COMPARISON OF TECHNOLOGICAL CHARACTERISTICS WITH THE PREDICATE DEVICE

A comparison of the Aptima BV assay to the predicate device, BD MAX Vaginal Panel, is summarized in Table 4 (similarities) and Table 5 (differences).

| Item | BD MAX Vaginal Panel<br>(DEN160001) | Aptima BV Assay<br>(Subject Device) |
|--------------------|-----------------------------------------------------------------------------------------------------|-------------------------------------|
| Patient Population | Women with a clinical presentation<br>of vaginitis/vaginosis | Same |
| Specimen Types | Vaginal swabs in patients who are<br>symptomatic for vaginitis/vaginosis | Same |
| Assay Controls | Incorporates an Internal Control in<br>every test. Uses external positive<br>and negative controls. | Same |

Table 4: Similarities Between Predicate Device and Subject Device

# Table 5: Differences Between Predicate Device and Subject Device

| Item | BD MAX Vaginal Panel<br>(DEN160001) | Aptima BV Assay<br>(Subject Device) |
|--------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Organisms Detected | Lactobacillus (L. crispatus, and L.<br>jensenii), Lactobacillus (L. gasseri,<br>L. crispatus, and L. jensenii),<br>Atopobium vaginae, Bacterial<br>Vaginosis Associated Bacteria-2<br>(BVAB-2), Megasphaera-1,<br>Candida (C. albicans, C.<br>tropicalis, C. parapsilosis, C.) | Lactobacillus (L. gasseri, L.<br>crispatus, and L. jensenii),<br>Gardnerella vaginalis, and<br>Atopobium vaginae |
| Item | BD MAX Vaginal Panel<br>(DEN160001) | Aptima BV Assay<br>(Subject Device) |
| | dubliniensis), Candida glabrata,<br>Candida krusei, Trichomonas<br>vaginalis | |
| Platform/Technology<br>Principle of<br>Operation | BD MAX System/<br>Real-time polymerase chain<br>reaction (PCR) | Panther System/<br>Real-time Transcription Mediated<br>Amplification (TMA) |
| Analyte | DNA | ribosomal RNA |
| Assay Calibrators | None | Positive Calibrator |
| Intended Use | The BD MAX Vaginal Panel<br>performed on the BD MAX System<br>is an automated qualitative <i>in vitro</i><br>diagnostic test for the direct<br>detection of DNA targets from<br>bacteria associated with bacterial<br>vaginosis (qualitative results<br>reported based on detection and<br>quantitation of targeted organism<br>markers), <i>Candida</i> species<br>associated with vulvovaginal<br>candidiasis, and <i>Trichomonas<br/>vaginalis</i> from vaginal swabs in<br>patients who are symptomatic for<br>vaginitis/vaginosis. The test<br>utilizes real-time polymerase chain<br>reaction (PCR) for the<br>amplification of specific DNA<br>targets and utilizes fluorogenic<br>target-specific hybridization probes<br>to detect and<br>differentiate DNA from:<br>- Bacterial vaginosis markers<br>(Individual markers not<br>reported)<br><i>Lactobacillus</i> spp. ( <i>L. crispatus</i><br>and <i>L. jensenii</i> ) <i>Gardnerella<br/>vaginalis</i><br><i>Atopobium vaginae</i><br>Bacterial Vaginosis Associated<br>Bacteria-2 (BVAB-2)<br><i>Megasphaera</i> -1<br><i>Candida</i> spp. ( <i>C. albicans, C.</i> | The Aptima BV assay is an <i>in vitro</i><br>nucleic acid amplification test that<br>utilizes real time transcription-<br>mediated amplification (TMA) for<br>detection and quantitation of<br>ribosomal RNA from bacteria<br>associated with bacterial vaginosis<br>(BV), including <i>Lactobacillus</i> ( <i>L.<br/>gasseri, L. crispatus, and L.<br/>jensenii</i> ), <i>Gardnerella vaginalis</i> ,<br>and <i>Atopobium vaginae</i> . The assay<br>reports a qualitative result for BV<br>and does not report results for<br>individual organisms. The assay is<br>intended to aid in the diagnosis of<br>BV on the automated Panther<br>system using clinician-collected<br>and patient-collected vaginal swab<br>specimens from females with a<br>clinical presentation consistent<br>with vaginitis and/or vaginosis. |
| Item | BD MAX Vaginal Panel<br>(DEN160001) | Aptima BV Assay<br>(Subject Device) |
| | tropicalis, C. parapsilosis, C.<br>dubliniensis)<br>• Candida glabrata<br>• Candida krusei<br>• Trichomonas vaginalis<br>The BD MAX Vaginal Panel is<br>intended to aid in the diagnosis of<br>vaginal infections in women with a<br>clinical presentation consistent<br>with bacterial vaginosis,<br>vulvovaginal candidiasis and<br>trichomoniasis. | |

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# VII. PERFORMANCE DATA

The following performance data were provided in support of the substantial equivalence determination.

# Brief Description of Analytical (Non-Clinical) Studies

The following analytical (non-clinical) studies were conducted to support the clearance of the Aptima BV assay on the Panther system.

# Analytical Sensitivity

The analytical sensitivity (Limit of Detection or LoD) and BV positivity limits of the Aptima BV assay were determined by testing a series of panels consisting of L. crispatus, L. gasseri, L. jensenii, G. vaginalis, or A. vaginae cell lysates diluted into simulated vaginal swab matrix (SVSM). A minimum of 20 replicates of each panel member were tested with each of two reagent lots for a minimum of 40 replicates per panel member. The predicted detection limits for each organism calculated using Probit analysis are shown in Table 6.

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| Organism | Predicted Detection<br>Limit | CFU/mL |
|--------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--------|
| A. vaginae | 95% | 2901 |
| G. vaginalis | તે તે જેની જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી ત | રતા |
| L. crispatus | તે તે જેની જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં પ્રાથમિક શાળા, પંચાયતઘર, આંગણવાડી તેમ જ દૂધની ડેરી જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી | 143 |
| L. gasseri | 95% | 2,207 |
| L. jensenii | 95% | 10 |

Table 6: Limit of Detection of the Aptima BV Assay

1 Predicted BV Positivity Limits (C95) for A. vaginae and G. vaginalis in the Aptima BV assay are approximately 5.10 log CFU/mL and 4.86 log CFU/mL, respectively.

#### Analytical Inclusivity

Five strains of each target organism were tested using lysate targeting 3X C95 for G. vaginalis and A. vaginae, and 3X LoD for Lactobacillus species (L. crispatus, L. gasseri, and L. jensenii) in SVSM. The Aptima BV Assay was BV positive for all five strains of G. vaginalis and A. vaginae at 3X C95. All five strains of L. crispatus and L. gasseri were detected at 3X LoD. Three of the five strains of L. jensenii were detected at 3X LoD, and the remaining two strains at 10X LoD .

#### Cross-Reactivity and Microbial Interference

Cross-reactivity and microbial interference with the Aptima BV assay was evaluated in the presence of non-targeted organisms. A panel consisting of 62 organisms (Table 7) was tested in SVSM in the absence or in the presence of L. crispatus at 3X LoD, G. vaginalis at 3X C95, or A. vaginae at 3X C95. No cross-reactivity or microbial interference was observed for any of the 62 organisms tested in the Aptima BV assay at the following concentrations.

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| Microorganism | Concentration | Microorganism | Concentration |
|------------------------------|-----------------|----------------------------|-----------------|
| Acinetobacter lwoffii | 1x106 CFU/mL | Herpes simplex virus I | 1x104 TCID50/mL |
| Actinomyces israelii | 1x106 CFU/mL | Herpes simplex virus II | 1x104 TCID50/mL |
| Alcaligenes faecalis | 1x106 CFU/mL | HIV | 1x105 copies/mL |
| Atopobium minutum | 1x106 CFU/mL | Klebsiella pneumoniae | 1x106 CFU/mL |
| Atopobium parvulum | 1x106 CFU/mL | Lactobacillus acidophilus | 1x103 CFU/mL2 |
| Atopobium rimae | 1x106 CFU/mL | Lactobacillus iners | 1x106 CFU/mL |
| Bacteroides fragilis | 1x106 CFU/mL | Lactobacillus mucosae | 1x106 CFU/mL |
| Bifidobacterium adolescentis | 1x106 CFU/mL | Leptotrichia buccalis | 1x106 CFU/mL |
| Bifidobacterium breve | 1x106 CFU/mL | Listeria monocytogenes | 1x106 CFU/mL |
| BVAB-11 | 1x106 copies/mL | Megasphaera Type 11 | 1x106 copies/mL |
| BVAB-21 | 1x106 copies/mL | Mobiluncus curtisii | 1x106 CFU/mL |
| Campylobacter jejuni | 1x106 CFU/mL | Mycoplasma genitalium | 1x106 CFU/mL |
| Candida albicans | 1x106 CFU/mL | Mycoplasma hominis | 1x106 CFU/mL |
| Candida dubliniensis | 1x106 CFU/mL | Neisseria gonorrhoeae | 1x106 CFU/mL |
| Candida glabrata | 1x106 CFU/mL | Pentatrichomonas hominis | 1x105 cells/mL |
| Candida krusei | 1x106 CFU/mL | Peptostreptococcus magnus | 1x106 CFU/mL |
| Candida lusitaniae | 1x106 CFU/mL | Pichia fermentans | 1x106 CFU/mL |
| Candida orthopsilosis | 1x106 CFU/mL | Prevotella bivia | 1x106 CFU/mL |
| Candida parapsilosis | 1x106 CFU/mL | Propionibacterium acnes | 1x106 CFU/mL |
| Candida tropicalis | 1x106 CFU/mL | Proteus vulgaris | 1x106 CFU/mL |
| Chlamydia trachomatis | 1x106 IFU/mL | SiHa cells | 1x104 cells/mL |
| Clostridium difficile | 1x106 CFU/mL | Sneathia amnii | 1x106 CFU/mL |
| Corynebacterium genitalium | 1x106 CFU/mL | Staphylococcus aureus | 1x106 CFU/mL |
| Cryptococcus neoformans | 1x106 CFU/mL | Staphylococcus epidermidis | 1x106 CFU/mL |
| Eggerthella lenta | 1x106 CFU/mL | Streptococcus agalactiae | 1x106 CFU/mL |
| Enterobacter cloacae | 1x106 CFU/mL | Streptococcus pyogenes | 1x106 CFU/mL |
| Enterococcus faecalis | 1x106 CFU/mL | Treponema pallidum1 | 1x106 copies/mL |
| Escherichia coli | 1x106 CFU/mL | Trichomonas tenax | 1x105 cells/mL |
| Fusobacterium nucleatum | 1x106 CFU/mL | Trichomonas vaginalis | 1x105 cells/mL |
| Haemophilus ducreyi | 1x106 CFU/mL | Ureaplasma parvum | 1x106 CFU/mL |
| HeLa cells | 1x104 cells/mL | Ureaplasma urealyticum | 1x106 CFU/mL |

# Table 7: Cross-Reactivity and Microbial Interference Panel

CFU = Colony Forming Units; IFU = Inclusion Forming Units; TCID50 = Median Tissue Culture Infectious Dose 1 In Vitro Transcript tested.

2 Lactobacillus acidophilus affects BV positivity at 1x104 CFU/mL or higher.

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## Interference

Potentially interfering substances were tested in the Aptima BV assay. Panels were built in SVSM and evaluated for potential effects on assay sensitivity and specificity. Sensitivity performance was evaluated separately for L. crispatus by spiking lysate at 3X LoD, and for G. vaginalis and A. vaginae by spiking lysate at 3X C95. 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 8.

| Substance | Final Concentration1 |
|---------------------------|----------------------|
| Whole Blood | 5% V/V |
| Leukocytes | 1x106 cells/mL |
| Mucus2 | 1.5% V/V |
| Seminal Fluid | 5% V/V |
| Contraceptive Foam | 5% W/V |
| Contraceptive Film | 5% W/V |
| Tioconazole3 | 1% 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 Gel4 | 0.4% W/V |
| Lubricant | 5% V/V |
| Spermicide | 5% W/V |
| Anti-fungal | 5% W/V |
| Deodorant/Spray | 5% W/V |
| Glacial Acetic Acid | 5% V/V |
| Vagisil Cream | 5% W/V |

# Table 8: Interfering Substances Panel

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 Interference was observed with Mucus at ≥2% V/V and not observed at 1.5% V/V.

3 Interference was observed with Tioconazole 6.5% Ointment at 5% W/V and not observed at 1% W/V.

4 Interference was observed with Vaginal Moisturizing Gel at ≥0.5% W/V and not observed at 0.4% W/V.

#### Within Laboratory Precision

Within Lab Precision was evaluated on three Panther systems at one site. Three operators performed testing across 21 days and three reagent lots. Each operator performed two runs

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per day using an 11 member panel. Each run consisted of three replicates of each panel member. The panel members were made using SVSM negative for Lactobacillus species. G. vaginalis, and A. vaginae. Ten panel members contained cell lysates of at least 1 of the following organisms: L. crispatus, L. jensenii, G. vaginalis, or A. vaginae; different bacterial combinations were prepared to represent the variety of targeted BV organism combinations present in vaginal specimens. Ten panel members targeted BV Negative (<5% BV Positive), BV High Negative (20-80% BV positive), BV Low Positive ( 95% BV positive) and BV Moderate Positive (100% BV positive) results. One negative panel member contained matrix with no added target analytes.

BV percent positive results for each panel are presented in Table 9. Signal variability (TTime) of the Aptima BV assay was calculated for each target in analyte positive panel members. Variability calculated between operators, between instruments, between days, between lots, between runs, within run, and overall, is shown in Tables 10 through 12.

| Panel Description | BV Positive/<br>Total n | Expected BV<br>Positivity | BV Positivity<br>(95% CI) |
|------------------------------------------------------------|-------------------------|---------------------------|---------------------------|
| SVSM | 0/168 | 0% | 0<br>(0.0-1.6) |
| L. crispatus, A. vaginae<br>BV Negative | 0 /168 | <5% | 0<br>(0.0-1.6) |
| L. crispatus, G. vaginalis<br>BV High Negative | 76 /168 | 20-80% | 45.2<br>(37.9-52.8) |
| L. crispatus, G. vaginalis, A. vaginae<br>BV High Negative | 131/165 | 20-80% | 79.4<br>(72.6-84.9) |
| G. vaginalis<br>BV Low Positive | 168/168 | ≥95% | 100<br>(98.4-100.0) |
| A. vaginae<br>BV Low Positive | 168/168 | ≥95% | 100<br>(98.4-100.0) |
| L. jensenii, A. vaginae<br>BV Low Positive | 168/168 | ≥95% | 100<br>(98.4-100.0) |
| G. vaginalis, A. vaginae<br>BV Low Positive | 168/168 | ≥95% | 100<br>(98.4-100.0) |
| L. crispatus, G. vaginalis, A. vaginae<br>BV Low Positive | 168/168 | ≥95% | 100<br>(98.4-100.0) |
| G. vaginalis<br>BV Mod Positive | 168/168 | 100% | 100<br>(98.4-100.0) |
| A. vaginae<br>BV Mod Positive | 168/168 | 100% | 100<br>(98.4-100.0) |

Table 9: BV Positivity of Precision Panels

1 Three invalid results were excluded from the analysis.

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| | | Between<br>Operators | | Between<br>Instruments | | Between<br>Days | | Between<br>Lots | | Between<br>Runs | | Within<br>Run | | Total | | | |
|--------------------------------------------------------------------------------------------------|------|----------------------|------|------------------------|------|-----------------|------|-----------------|------|-----------------|------|---------------|------|--------|------|--------|--|
| Panel<br>Description | N | Mean<br>TTime1 | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | |
| L. crispatus<br>BV Negative2 | 168 | 19.87 | 0.10 | 0.49 | 0.16 | 0.80 | 0.14 | 0.71 | 1.03 | 5.18 | 0.17 | 0.09 | 0.18 | 0.93 | 1.08 | 5.46 | |
| L. crispatus<br>BV High Negative2 | 168 | 23.95 | 0.11 | 0.47 | 0.12 | 0.52 | 0.19 | 0.79 | 1.22 | 5.11 | 0.18 | 0.77 | 0.28 | 1.15 | 1.29 | 5.40 | |
| L. crispatus<br>BV High Negative3 | 1654 | 22.40 | 0.09 | 0.40 | 0.17 | 0.74 | 0.20 | 0.87 | 1.22 | 5.47 | 0.09 | 0.39 | 0.27 | 1.21 | 1.29 | 5.74 | |
| L. jensenii<br>BV Low Positive2 | 168 | 24.80 | 0.10 | 0.38 | 0.14 | 0.57 | 0.14 | 0.57 | 1.33 | 5.35 | 0.17 | 0.69 | 0.25 | 1.01 | 1.38 | 5.56 | |
| L. crispatus<br>BV Low Positive316823.510.150.630.090.400.170.731.365.770.100.440.311.311.426.02 | | | | | | | | | | | | | | | | | |

Table 10: Signal Variability of Lactobacillus Panel Members

CV = Coefficient of variation

1 TTime is shown for Lactobacillus only .

2 Panel member contains 2 different organisms: results are shown for only the Lactobacillus component.

3 Panel member contains 3 different organisms: results are shown for only the Lactobacillus component.

4 Three invalid results were excluded from the analysis.

Note: Variability from some factors may be numerically negative. This can occur if the variability due to those factors is very small. In these cases, SD and CV are shown as 0.00.

| | | | Between<br>Operators | | Between<br>Instruments | | Between<br>Days | | Between<br>Lots | | Between<br>Runs | | Within<br>Run | | Total | |
|-----------------------------------|------|----------------|----------------------|--------|------------------------|--------|-----------------|--------|-----------------|--------|-----------------|--------|---------------|--------|-------|--------|
| Panel<br>Description | N | Mean<br>TTime1 | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| G. vaginalis<br>BV High Negative2 | 168 | 17.11 | 0.00 | 0.00 | 0.18 | 1.08 | 0.17 | 0.99 | 0.47 | 2.75 | 0.17 | 0.96 | 0.16 | 0.94 | 0.58 | 3.39 |
| G. vaginalis<br>BV High Negative3 | 1654 | 15.71 | 0.00 | 0.00 | 0.19 | 1.19 | 0.18 | 1.12 | 0.48 | 3.05 | 0.11 | 0.72 | 0.12 | 0.79 | 0.57 | 3.62 |
| G. vaginalis<br>BV Low Positive | 168 | 15.80 | 0.00 | 0.00 | 0.16 | 1.00 | 0.14 | 0.89 | 0.43 | 2.70 | 0.15 | 0.97 | 0.15 | 0.92 | 0.52 | 3.30 |
| G. vaginalis<br>BV Mod Positive | 168 | 14.46 | 0.00 | 0.00 | 0.17 | 1.18 | 0.05 | 0.35 | 0.38 | 2.63 | 0.16 | 1.09 | 0.18 | 1.25 | 0.48 | 3.35 |
| G. vaginalis<br>BV Low Positive2 | 168 | 15.01 | 0.00 | 0.00 | 0.14 | 0.93 | 0.14 | 0.91 | 0.40 | 2.67 | 0.16 | 1.08 | 0.13 | 0.86 | 0.49 | 3.28 |
| G. vaginalis<br>BV Low Positive3 | 168 | 14.06 | 0.00 | 0.00 | 0.16 | 1.11 | 0.15 | 1.09 | 0.39 | 2.75 | 0.14 | 0.99 | 0.16 | 1.16 | 0.49 | 3.51 |

# Table 11: Signal Variability of G. vaginalis Panel Members

CV = Coefficient of variation, Mod = moderate

1 TTime is shown for G. vaginalis only.

2 Panel member contains 2 different organisms: results are shown for only the G. vaginalis component.

3 Panel member contains 3 different organisms: results are shown for only the G. vaginalis component.

4 Three invalid results were excluded from the analysis.

Note: Variability from some factors may be numerically negative. This can occur if the variability due to those factors is very small. In these cases, SD and CV are shown as 0.00.

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| | | | Between<br>Operators | | Between<br>Instruments | | Between<br>Days | | Between<br>Lots | | Between<br>Runs | | Within<br>Run | | Total | |
|---------------------------------|------|----------------|----------------------|--------|------------------------|--------|-----------------|--------|-----------------|--------|-----------------|--------|---------------|--------|-------|--------|
| Panel<br>Description | N | Mean<br>TTime1 | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| A. vaginae<br>BV Negative2 | 168 | 18.20 | 0.02 | 0.11 | 0.25 | 1.36 | 0.15 | 0.84 | 0.58 | 3.17 | 0.19 | 1.02 | 0.19 | 1.05 | 0.70 | 3.84 |
| A. vaginae<br>BV High Negative3 | 1654 | 16.56 | 0.00 | 0.00 | 0.25 | 1.53 | 0.18 | 1.11 | 0.56 | 3.38 | 0.13 | 0.79 | 0.12 | 0.70 | 0.67 | 4.02 |
| A. vaginae<br>BV Low Positive | 168 | 15.11 | 0.00 | 0.00 | 0.19 | 1.25 | 0.15 | 0.97 | 0.51 | 3.40 | 0.12 | 0.82 | 0.12 | 0.78 | 0.59 | 3.92 |
| A. vaginae<br>BV Low Positive2 | 168 | 15.13 | 0.00 | 0.00 | 0.20 | 1.30 | 0.12 | 0.80 | 0.51 | 3.34 | 0.14 | 0.89 | 0.16 | 1.07 | 0.59 | 3.92 |
| A. vaginae<br>BV Mod Positive | 168 | 14.13 | 0.08 | 0.54 | 0.21 | 1.50 | 0.17 | 1.21 | 0.51 | 3.63 | 0.08 | 0.57 | 0.20 | 1.40 | 0.62 | 4.41 |
| A. vaginae<br>BV Low Positive2 | 168 | 15.78 | 0.03 | 0.16 | 0.17 | 1.09 | 0.10 | 0.65 | 0.50 | 3.17 | 0.16 | 1.00 | 0.12 | 0.75 | 0.57 | 3.64 |
| A. vaginae<br>BV Low Positive3 | 168 | 15.61 | 0.00 | 0.00 | 0.23 | 1.47 | 0.15 | 0.94 | 0.51 | 3.29 | 0.10 | 0.66 | 0.18 | 1.15 | 0.62 | 3.95 |

Table 12: Signal Variability of A. vaginae Panel Members

CV = Coefficient of variation, Mod = moderate

1 TTime is shown for A. vaginae o n 1 y .

2 Panel member contains 2 different organisms: results are shown for only the A. vaginae component.

3 Panel member contains 3 different organisms: results are shown for only the A. vaginae component.

4 Three invalid results were excluded from the analysis.

Note: Variability from some factors may be numerically negative. This can occur if the variability due to those factors is very small. In these cases, SD and CV are shown as 0.00.

#### Brief Description of Clinical Studies

Clinical testing of the Aptima BV assay on the Panther system included performance and reproducibility testing. Substantial equivalence is based in part on the performance study.

#### Clinical Performance Study

This study was performed to demonstrate clinical performance characteristics for the Aptima BV assay. A multicenter study was conducted using prospectively-collected patient- and cliniciancollected vaginal swab samples from women ≥14 years who were asymptomatic for or who exhibited signs and/or symptoms of vaginitis (ie, symptomatic). Twenty-one participating geographically diverse US private and academic family practice, obstetric-gynecologic, family planning, public health, STI, medical group clinics, and clinical research centers obtained vaginal

{14}------------------------------------------------

swab samples from 1519 symptomatic women, 174 asymptomatic women, and 4 women with unknown symptom status.

For each symptomatic subject, two vaginal swab samples (one patient-collected, one cliniciancollected) were tested with the investigational Aptima BV assay. One clinician-collected vaginal swab sample was used for Nugent score evaluation, and modified Amsel criteria if necessary, to determine BV infection status. A Nugent interpretation established positive and negative BV reference status, except in cases of intermediate determinations. For intermediate Nugent interpretations, BV reference status was established using modified Amsel criteria. For each asymptomatic subject, one (1) clinician-collected vaginal swab sample was collected and tested with the investigational Aptima BV assay.

The clinical performance of the Aptima BV assay in symptomatic subjects (ie, the intended use population) was estimated relative to the BV infection status; sensitivity, specificity, PPV, and NPV were calculated for each Aptima sample type, along with corresponding 2-sided 95% Cls. Positivity rates were calculated for asymptomatic subjects.

Of the 1519 symptomatic subjects enrolled, 102 were not evaluable due to withdrawal (n = 17) or unknown BV infection status (n = 85). The remaining 1417 symptomatic subjects were evaluable for at least one of the sample types. Of the 174 asymptomatic subjects, 2 were not evaluable due to withdrawal; the remaining 172 asymptomatic subjects were evaluable. Table 13 shows the demographic and baseline clinical characteristics of evaluable subjects.

Of the 1502 non-withdrawn symptomatic subjects, 1417 subjects were evaluable for the performance analyses for BV detection; results from 1405 patient-collected Aptima vaginal swab samples (99.2%, 1405/1417) and 1413 clinician-collected Aptima vaginal swab samples (99.7%, 1413/1417) were included in the performance analyses. All 172 non-withdrawn asymptomatic subjects were evaluable for the performance analyses for BV detection; results from the 172 clinician-collected Aptima vaginal swab samples were included in the positivity analysis.

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| | Symptomatic | Asymptomatic |
|------------------------------------------------------------|--------------|--------------|
| Total, N | 1417 | 172 |
| Age, years | | |
| Mean ± SD | 34.7 ± 11.11 | 41.1 ± 13.22 |
| Median | 33.0 | 40.0 |
| Range | 14-75 | 18-73 |
| Age category (years), n (%) | | |
| 14-17 | 4 (0.3) | 0 (0.0) |
| 18-29 | 537 (37.9) | 42 (24.4) |
| 30-39 | 469 (33.1) | 42 (24.4) |
| 40-49 | 235 (16.6) | 36 (20.9) |
| >50 | 172 (12.1) | 52 (30.2) |
| Race/Ethnicity, n (%) | | |
| Asian | 67 (4.7) | 5 (2.9) |
| Black or African American | 731 (51.6) | 75 (43.6) |
| White (Hispanic or Latino) | 248 (17.5) | 41 (23.8) |
| White (Not Hispanic or Latino) | 307 (21.7) | 44 (25.6) |
| Other¹ | 64 (4.5) | 7 (4.1) |
| ¹Includes patient-reported other, mixed, and unknown races | | |

Table 13: Summary of Demographics of Evaluable Subjects in the Aptima BV Assay Evaluation

Performance characteristics for detection of BV infection for patient-collected and cliniciancollected vaginal swab samples from symptomatic subjects were calculated overall (see Table 14), by race/ethnicity (see Table 15), and by clinical condition (see Table 16).

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| Specimen<br>Type | N | TP | FP | TN | FN | Prevalence1<br>(95% CI)2 | Sensitivity<br>(95% CI)2 | Specificity<br>(95% CI)2 | PPV<br>(95% CI)3 | NPV<br>(95% CI)3 |
|-------------------------|------|-----|------|-----|-----|--------------------------|--------------------------|--------------------------|------------------|------------------|
| Patient-<br>collected | 1405 | 673 | 1014 | 612 | 195 | 49.3 | 97.3 (95.8-98.2) | 85.8 (83.1-88.2) | 87.0 (84.8-88.9) | 97.0 (95.5-98.1) |
| Clinician-<br>collected | 1413 | 660 | 756 | 643 | 357 | 49.2 | 95.0 (93.1-96.4) | 89.6 (87.1-91.6) | 89.8 (87.7-91.7) | 94.8 (93.1-96.3) |

Table 14: Aptima BV Assay Performance Relative to BV Infection Status in Symptomatic Subjects

FN = false negative. FP = false positive value. PPV = positive predictive value. TP = true positive. TN = true negative.

'Study prevalence reported. 'Score C. "PPV 95% CI for positive likelihood rato; NPV 95% Cl computed from the 95% for the negative likelihood ratio

4 Of the 101 false positive results, 55 subjects were Nugent interion status determined by Amsel criteria, and 9 were positive by Amsel.

5 Of the 19 false negative results, 6 subjects were Nigent internediates and had BV infection status determined by Amsel.

6 Of the 75 false positive results, 46 subjects were Nued BV infection status determined by Amsel criteria, and 6 were positive by Amsel. 7 Of the 35 false negative results, 10 subjects were Nugent internetiates and had BV infection status determined by Amsel criteria, and 15 were negative by Amsel.

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| Race/Ethnicity | N | TP | FP | TN | FN | Prev1 (%) | Sensitivity<br>(95% CI)2 | Specificity<br>(95% CI)2 | PPV<br>(95% CI)3 | NPV<br>(95% CI)3 |
|-----------------------------|-----|-----|----|-----|----|-----------|--------------------------|--------------------------|------------------|------------------|
| Patient-collected | | | | | | | | | | |
| Asian | 65 | 19 | 6 | 39 | 1 | 30.8 | 95.0 (76.4-99.1) | 86.7 (73.8-93.7) | 76.0 (61.6-88.7) | 97.5 (89.3-99.9) |
| Black / African American | 727 | 434 | 43 | 239 | 11 | 61.2 | 97.5 (95.6-98.6) | 84.8 (80.1-88.5) | 91.0 (88.6-93.1) | 95.6 (92.6-97.7) |
| White (Hispanic/Latino) | 246 | 112 | 22 | 111 | 1 | 45.9 | 99.1 (95.2-99.8) | 83.5 (76.2-88.8) | 83.6 (78.0-88.6) | 99.1 (95.6- 100) |
| White (Not Hispanic/Latino) | 303 | 81 | 27 | 189 | 6 | 28.7 | 93.1 (85.8-96.8) | 87.5 (82.4-91.3) | 75.0 (68.1-81.5) | 96.9 (94.0-98.8) |
| Other4 | 64 | 27 | 3 | 34 | 0 | 42.2 | 100 (87.5-100) | 91.9 (78.7-97.2) | 90.0 (76.9-97.7) | 100 (91.3- 100) |
| Clinician-collected | | | | | | | | | | |
| Asian | 67 | 20 | 4 | 42 | 1 | 31.3 | 95.2 (77.3-99.2) | 91.3 (79.7-96.6) | 83.3 (68.2-94.3) | 97.7 (89.8-99.9) |
| Black / African American | 729 | 425 | 31 | 253 | 20 | 61.0 | 95.5 (93.2-97.1) | 89.1 (84.9-92.2) | 93.2 (90.9-95.2) | 92.7 (89.4-95.2) |
| White (Hispanic/Latino) | 247 | 110 | 18 | 115 | 4 | 46.2 | 96.5 (91.3-98.6) | 86.5 (79.6-91.3) | 85.9 (80.3-90.8) | 96.6 (92.3-99.0) |
| White (Not Hispanic/Latino) | 306 | 78 | 18 | 200 | 10 | 28.8 | 88.6 (80.3-93.7) | 91.7 (87.3-94.7) | 81.3 (73.9-87.5) | 95.2 (92.1-97.5) |
| Other4 | 64 | 27 | 4 | 33 | 0 | 42.2 | 100 (87.5-100) | 89.2 (75.3-95.7) | 87.1 (74.2-96.0) | 100 (91.1- 100) |

Table 15: Aptima BV Assay Performance Relative to BV Infection Status in Symptomatic Subjects, by Race/Ethnicity

FN = false negative, FP = false positive, NPV = nesitive value, PV = positive predictive value, Prev = prevalence TP = true negative

- Study prevalence reported. 35core CI. PPV 95% CI for positive likelihood ratio; NPV 95% CI computed from the 95% for the negative likelihood ratio. 4Includes patient-reported other, mixed, and unknown races.

{18}------------------------------------------------

| Clinical Condition | N | TP | FP | TN | FN | Prev1 (%) | Sensitivity (95% CI)2 | Specificity (95% CI)2 | PPV (95% CI)3 | NPV (95% CI)3 |
|-------------------------------------------------------|------|-----|----|-----|----|-----------|-----------------------|-----------------------|------------------|------------------|
| Patient-collected | | | | | | | | | | |
| Use of antibiotics | 3 | 1 | 0 | 2 | 0 | 33.3 | 100 (20.7-100) | 100 (34.2-100) | 100 ( 7.8- 100) | 100 (45.1- 100) |
| Use of antifungals | 8 | 2 | 0 | 6 | 0 | 25.0 | 100 (34.2-100) | 100 (61.0-100) | 100 (33.3- 100) | 100 (77.4- 100) |
| Use of estrogen therapy | 2 | 0 | 0 | 2 | 0 | 0.0 | NC | 100 (34.2-100) | NC | 100 ( NC ) |
| Recurrent symptoms of vaginitis in the last 12 months | 828 | 405 | 62 | 353 | 8 | 49.9 | 98.1 (96.2-99.0) | 85.1 (81.3-88.2) | 86.7 (83.9-89.3) | 97.8 (95.9-99.0) |
| Unprotected intercourse in the last 24 hours | 94 | 53 | 10 | 30 | 1 | 57.4 | 98.1 (90.2-99.7) | 75.0 (59.8-85.8) | 84.1 (76.5-91.0) | 96.8 (85.6-99.9) |
| Pregnant | 20 | 9 | 1 | 10 | 0 | 45.0 | 100 (70.1-100) | 90.9 (62.3-98.4) | 90.0 (66.5-99.7) | 100 (77.9- 100) |
| With Menses | 109 | 52 | 9 | 48 | 0 | 47.7 | 100 (93.1-100) | 84.2 (72.6-91.5) | 85.2 (76.6-92.4) | 100 (93.6- 100) |
| Without Menses | 1175 | 579 | 85 | 496 | 15 | 50.6 | 97.5 (95.9-98.5) | 85.4 (82.3-88.0) | 87.2 (84.9-89.3) | 97.1 (95.4-98.3) |
| Post-menopausal | 121 | 42 | 7 | 68 | 4 | 38.0 | 91.3 (79.7-96.6) | 90.7 (82.0-95.4) | 85.7 (75.5-93.2) | 94.4 (88.1-98.3) |
| Clinician-collected | | | | | | | | | | |
| Use of antibiotics | 3 | 1 | 0 | 2 | 0 | 33.3 | 100 (20.7-100) | 100 (34.2-100) | 100 ( 7.8- 100) | 100 (45.1- 100) |
| Use of antifungals | 8 | 2 | 0 | 6 | 0 | 25.0 | 100 (34.2-100) | 100 (61.0-100) | 100 (33.3- 100) | 100 (77.4- 100) |
| Use of estrogen therapy | 2 | 0 | 0 | 2 | 0 | 0.0 | NC | 100 (34.2-100) | NC | 100 ( NC ) |
| Recurrent symptoms of vaginitis in the last 12 months | 832 | 394 | 47 | 371 | 20 | 49.8 | 95.2 (92.7-96.9) | 88.8 (85.4-91.4) | 89.3 (86.6-91.8) | 94.9 (92.5-96.7) |
| Unprotected intercourse in the last 24 hours | 94 | 50 | 6 | 34 | 4 | 57.4 | 92.6 (82.4-97.1) | 85.0 (70.9-92.9) | 89.3 (81.2-95.4) | 89.5 (78.4-96.6) |
| Pregnant | 20 | 9 | 0 | 11 | 0 | 45.0 | 100 (70.1-100) | 100 (74.1-100) | 100 (74.2- 100) | 100 (78.4- 100) |
| With Menses | 111 | 50 | 8 | 51 | 2 | 46.8 | 96.2 (87.0-98.9) | 86.4 (75.5-93.0) | 86.2 (77.6-93.1) | 96.2 (88.7-99.5) |
| Without Menses | 1177 | 569 | 62 | 520 | 26 | 50.6 | 95.6 (93.7-97.0) | 89.3 (86.6-91.6) | 90.2 (88.0-92.2) | 95.2 (93.3-96.8) |
| Post-menopausal | 125 | 41 | 5 | 72 | 7 | 38.4 | 85.4 (72.8-92.8) | 93.5 (85.7-97.2) | 89.1 (79.1-95.8) | 91.1 (84.7-95.9) |

#### Table 16: Aptima BV Assay Performance Relative to BV Infection Status in Symptomatic Subjects, by Clinical Condition

FN = false negative, FP = false positive, NC = not calculable, NPV = positive predictive value, PPV = positive predictive value, Prev = prevalence TP = true positive, TN = true negative

- Study prevalence reported. 35core Cl - PPV 95% Cl for positive likelihod ratio; NPV 95% Cl computed from the 95% for the negative likelihood ratio.

{19}------------------------------------------------

Positivity rates were estimated for clinician-collected vaginal swab samples from asymptomatic subjects overall and by race/ethnicity (see Table 17).

| Subjects | % Positivity<br>(# positive/# tested with valid results) |
|-----------------------------|----------------------------------------------------------|
| All | 40.7% (70/172) |
| Asian | 40.0% (2/5) |
| Black/African American | 52.0% (39/75) |
| White (Hispanic/Latino) | 43.9% (18/41) |
| White (Not Hispanic/Latino) | 15.9% (7/44) |
| Other | 57.1% (4/7) |

Table 17: Positivity of BV Infection as Determined by the Aptima BV Assay in Asymptomatic Subjects

#### Reproducibility

Aptima BV assay reproducibility was evaluated at three US sites using seven panel members. 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 run had three replicates of each panel member.

The panel members were made using a simulated vaginal swab matrix ('SVSM', which contains specimen transport media (STM) spiked with simulated vaginal fluid) negative for Lactobacillus species, G. vaginalis, and A. vaginae. Six panel members contained cell lysates of at least 1 of the following organisms: L. crispatus, L. jensenii, G. vaginalis, or A. vaginae; different bacterial combinations were prepared to represent the variety of targeted BV organism combinations present in vaginal specimens. One negative panel member contained only the matrix with no added target analytes.

The agreement with expected results was 100% for all panel members containing L. crispatus, L. jensenii, G. vaginalis, or A. vaginae, as shown in Table 18.

{20}------------------------------------------------

| Panel Description | Expected Results | N | Agreement, % (95% CI) |
|-------------------|------------------|-------|-----------------------|
| True Neg | Negative | 36/36 | 100 (96.6-100) |
| BV Neg1 | Negative | 36/36 | 100 (96.6-100) |
| Gvag Low Pos | Positive | 36/36 | 100 (96.6-100) |
| Avag Low Pos | Positive | 36/36 | 100 (96.6-100) |
| BV Low Pos1 | Positive | 36/36 | 100 (96.6-100) |
| Gvag Mod Pos | Positive | 36/36 | 100 (96.6-100) |
| Avag MosPos | Positive | 36/36 | 100 (96.6-100) |

Table 18: Agreement of Aptima BV Assay Results With Expected Results

Avag = A. vaginae, CI = Score confidence interval, Gvag = G. vaginalis, Mod=moderate, Neg = negative, Pos = positive 1 Panel member contains 2 different organisms.

Across organisms/panel members, the total %CV values ranged from 4.21% to 4.76%; total SD values were ≤1.12. For most panel members, the "between sites," "between operators," and "between runs" factors were the largest contributors to total variability; for all other sources of variation, SD values were ≤0.18 (%CV values were ≤1.13%), as shown in Table 19.

| | | Between<br>Sites | | Between<br>Operators | | Between<br>Days | | Between<br>Runs | | Within<br>Runs | | Total | | |
|-------------------|-----|------------------|------|----------------------|------|-----------------|------|-----------------|------|----------------|------|--------|------|--------|
| Panel Description | N | Mean<br>TTime1 | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Lcrisp BV Neg2 | 108 | 19.73 | 0.30 | 1.53 | 0.61 | 3.07 | 0.13 | 0.64 | 0.63 | 3.17 | 0.12 | 0.62 | 0.94 | 4.76 |
| Ljen Low Pos2 | 108 | 24.31 | 0.00 | 0.00 | 0.77 | 3.16 | 0.00 | 0.00 | 0.80 | 3.28 | 0.15 | 0.62 | 1.12 | 4.60 |
| Gvag Low Pos | 108 | 15.69 | 0.35 | 2.26 | 0.40 | 2.52 | 0.00 | 0.00 | 0.38 | 2.43 | 0.15 | 0.96 | 0.67 | 4.28 |
| Gvag Mod Pos | 108 | 14.33 | 0.30 | 2.07 | 0.37 | 2.58 | 0.00 | 0.00 | 0.35 | 2.41 | 0.14 | 0.98 | 0.60 | 4.21 |
| Avag BV Neg2 | 108 | 18.01 | 0.39 | 2.15 | 0.44 | 2.46 | 0.08 | 0.45 | 0.47 | 2.59 | 0.18 | 0.97 | 0.78 | 4.30 |
| Avag Low Pos | 108 | 14.95 | 0.38 | 2.52 | 0.41 | 2.75 | 0.00 | 0.00 | 0.39 | 2.61 | 0.14 | 0.93 | 0.69 | 4.64 |
| Avag Low Pos2 | 108 | 14.94 | 0.41 | 2.76 | 0.37 | 2.51 | 0.00 | 0.00 | 0.37 | 2.45 | 0.17 | 1.13 | 0.69 | 4.60 |
| Avag Mod Pos | 108 | 13.99 | 0.29 | 2.08 | 0.36 | 2.60 | 0.03 | 0.18 | 0.39 | 2.82 | 0.14 | 1.00 | 0.63 | 4.48 |

Table 19: Signal Variability of the Aptima BV Assay by Analyte-positive Panel Member

Avag = A. vaginae, CV = coefficient of vag = G. vaginalis, Lcrisp = L. crispatus, Ljen = L. jensenii, Mod = moderate, Neg = negative, Pos = positive, SD = standard deviation, TTime = emergence time of a signal (above a specific threshold)

Note: If variability from a factor was numerically negative, SD and CV are shown as 0.0.

2 Panel member contains 2 different organisms; results are shown for only the component shown.

¹ The assay reports TTime for each assay analyte separately; the mean and signal variability reported are for the analyte(s) present in each panel member.

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For Aptima BV assay controls and positive calibrators, the total %CV values ranged from 4.47% to 5.36%; total SD values were ≤1.11 (see Tables 20 and 21, respectively).

| | | | Between<br>Sites | | Between<br>Operators | | Between<br>Days | | Within<br>Runs | | Total | |
|--------------|----|------------|------------------|--------|----------------------|--------|-----------------|--------|----------------|--------|-------|--------|
| Control | N | Mean<br>Ct | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Neg - Lcrisp | 36 | 20.34 | 0.24 | 1.16 | 0.74 | 3.63 | 0.00 | 0.00 | 0.71 | 3.48 | 1.05 | 5.16 |
| Pos - Gvag | 36 | 17.52 | 0.05 | 0.28 | 0.60 | 3.41 | 0.00 | 0.00 | 0.52 | 2.96 | 0.79 | 4.52 |
| Pos - Avag | 36 | 13.79 | 0.27 | 1.97 | 0.38 | 2.77 | 0.00 | 0.00 | 0.41 | 2.97 | 0.62 | 4.51 |

Table 20: Signal Variability of the Aptima BV Assav Positive Controls

Avag = A. vaginae, CV = coefficient of vag = G. vaginalis, Lcrisp = L. crispatus, Ljen = L. jensenii, Mod = moderate, Neg = negative, Pos = positive, SD = standard deviation, TTime = emergence time of a signal (above a specific threshold)

| | Between<br>Sites | | Between<br>Operators | | Between<br>Days | | Between<br>Runs | | Within<br>Runs | | Total | | | |
|---------|------------------|---------------|----------------------|--------|-----------------|--------|-----------------|--------|----------------|--------|-------|--------|------|--------|
| Analyte | N | Mean<br>TTime | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Lcrisp | 108 | 20.75 | 0.00 | 0.00 | 0.78 | 3.77 | 0.00 | 0.00 | 0.78 | 3.75 | 0.13 | 0.64 | 1.11 | 5.36 |
| Gvag | 108 | 19.03 | 0.34 | 1.78 | 0.61 | 3.19 | 0.00 | 0.00 | 0.56 | 2.96 | 0.15 | 0.79 | 0.91 | 4.76 |
| Avag | 108 | 18.46 | 0.41 | 2.23 | 0.48 | 2.62 | 0.08 | 0.42 | 0.51 | 2.76 | 0.11 | 0.58 | 0.83 | 4.47 |

Table 21. Signal Variability of the Antima RV Assay Positive Calibrators

Avag = A. vaginae, CV = coefficient of variation, Gvag = G. vaginalis, Lcrispatus, SD = standard d…