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 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. The 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. The Aptima BV assay is provided as a 100-test kit containing 8 reagents, 1 calibrator, and 2 controls.

AI/ML Overview

Here's a summary of the acceptance criteria and study details for the Aptima BV Assay based on the provided FDA 510(k) summary:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are generally inferred from the "Brief Description of Analytical (Non-Clinical) Studies" and "Brief Description of Clinical Studies" sections, focusing on the demonstrated acceptable performance. Specific numerical acceptance criteria are not explicitly stated as pass/fail thresholds in percentage terms, but rather performance results are reported which are presumably acceptable for clearance.

Performance Metric	Inferred Acceptance Criteria (Type of Result)	Reported Device Performance
Analytical Studies:
Limit of Detection (LoD)	Ability to detect target organisms at low concentrations	- A. vaginae: 2901 CFU/mL (95% detection)
		- G. vaginalis: The content of the document in this context in not in English. It says This village has primary school, panchayat house, anganwadi, and milk dairy facilities. The main occupation of the people of this village is agriculture, farm labor CFU/mL (95% detection)
		- L. crispatus: The content of the document in this context in not in English. It says This village has primary school, panchayat house, anganwadi, and milk dairy facilities. The main occupation of the people of this village is agriculture, farm labor CFU/mL (95% detection)
		- L. gasseri: 2,207 CFU/mL (95% detection)
		- L. jensenii: 10 CFU/mL (95% detection)
Analytical Inclusivity	Detection of various strains of target organisms	- All 5 strains of G. vaginalis and A. vaginae detected at 3X C95.
		- All 5 strains of L. crispatus and L. gasseri detected at 3X LoD.
		- 3 of 5 strains of L. jensenii detected at 3X LoD, remaining 2 at 10X LoD.
Cross-Reactivity & Microbial Interference	No interference from common non-target organisms and substances	- No cross-reactivity or microbial interference observed for 62 organisms at specified concentrations (except Lactobacillus acidophilus at ≥1x10⁴ CFU/mL).
Interference	No interference from common endogenous and exogenous substances	- No interference observed from 20 tested substances at specified concentrations (except Mucus at ≥2% V/V, Tioconazole 6.5% Ointment at 5% W/V, Vaginal Moisturizing Gel at ≥0.5% W/V).
Within Laboratory Precision	Consistent performance across operators, instruments, days, lots, and runs	- BV percent positive results for panels ranged from 0% (negative) to 100% (positive) as expected.
		- Signal variability (Total CV) for Lactobacillus, G. vaginalis, and A. vaginae panel members ranged from 3.30% to 5.74%.
Clinical Studies (Symptomatic Subjects):
Sensitivity (Patient-collected)	High sensitivity for BV detection	97.3% (95% CI: 95.8-98.2)
Specificity (Patient-collected)	High specificity for BV detection	85.8% (95% CI: 83.1-88.2)
PPV (Patient-collected)	High PPV for BV detection	87.0% (95% CI: 84.8-88.9)
NPV (Patient-collected)	High NPV for BV detection	97.0% (95% CI: 95.5-98.1)
Sensitivity (Clinician-collected)	High sensitivity for BV detection	95.0% (95% CI: 93.1-96.4)
Specificity (Clinician-collected)	High specificity for BV detection	89.6% (95% CI: 87.1-91.6)
PPV (Clinician-collected)	High PPV for BV detection	89.8% (95% CI: 87.7-91.7)
NPV (Clinician-collected)	High NPV for BV detection	94.8% (95% CI: 93.1-96.3)
Reproducibility:
Agreement with Expected Results	100% agreement expected for controls and panels	100% (95% CI: 96.6-100) for all 7 panel members (True Neg, BV Neg, Gvag Low Pos, Avag Low Pos, BV Low Pos, Gvag Mod Pos, Avag MosPos).
Signal Variability	Low variability across sites, operators, etc.	- Total %CV for analyte-positive panels: 4.21% to 4.76%. Total SD values: ≤1.12.
		- Total %CV for controls and calibrators: 4.47% to 5.36%. Total SD values: ≤1.11.

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

Clinical Performance Study (Symptomatic Subjects):
- Evaluable Subjects: 1417 symptomatic women.
- Patient-collected Aptima vaginal swab samples: 1405
- Clinician-collected Aptima vaginal swab samples: 1413
- Data Provenance: Prospectively-collected patient- and clinician-collected vaginal swab samples from women ≥14 years in 21 geographically diverse US private and academic family practice, obstetric-gynecologic, family planning, public health, STI, medical group clinics, and clinical research centers.
Clinical Performance Study (Asymptomatic Subjects):
- Evaluable Subjects: 172 asymptomatic women.
- Clinician-collected Aptima vaginal swab samples: 172
- Data Provenance: Prospectively-collected clinician-collected vaginal swab samples from asymptomatic women in 21 geographically diverse US sites (same as symptomatic subjects).
Within Laboratory Precision:
- Replicates: A minimum of 20 replicates per panel member for LoD. For precision, each operator performed 2 runs per day, with 3 replicates of each of the 11 panel members per run, across 21 days. (Total N = 168 or 165 for some panels due to exclusions).
- Data Provenance: Laboratory study using synthetic panels (SVSM).
Reproducibility:
- Replicates: 3 replicates of each of the 7 panel members per run. Testing performed for at least six days at each of three sites. (Total N = 108 for each panel member).
- Data Provenance: Multi-site laboratory study using synthetic panels (SVSM).

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

The document does not explicitly state the number or specific qualifications of experts (e.g., "radiologist with 10 years of experience"). However, it mentions that for the clinical performance study, the ground truth for BV infection status was established using Nugent score evaluation, and modified Amsel criteria if necessary. This implies trained laboratory personnel and clinicians performing these established diagnostic methods.

4. Adjudication Method for the Test Set

Clinical Performance Study:
- "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."
- This indicates a hierarchical adjudication or ground truth establishment method where Nugent scoring was primary, and Amsel criteria served as a secondary method for intermediate Nugent results. The document does not describe a multi-reader adjudication process in the sense of multiple experts independently reviewing and then reaching a consensus for the ground truth.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

No, the document does not describe a multi-reader multi-case (MRMC) comparative effectiveness study comparing human readers with AI assistance versus without AI assistance. The study evaluates the standalone performance of the Aptima BV Assay against established clinical criteria (Nugent score/Amsel criteria).

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the studies presented are standalone performance evaluations of the Aptima BV Assay (an in vitro nucleic acid amplification test performed on the automated Panther system). The results (BV positive or negative status) are generated by the system's software based on signal emergence times and calibration information, without human interpretation of the assay's output for diagnostic purposes in the performance studies. Its performance is compared to a ground truth established by clinical methods.

7. The Type of Ground Truth Used

Clinical Performance Study:
- The primary ground truth for Bacterial Vaginosis (BV) infection status was established using Nugent score evaluation.
- For cases with intermediate Nugent interpretations, modified Amsel criteria were used to establish the BV reference status.
Analytical and Reproducibility Studies:
- Ground truth was based on the known composition of the synthetic panels (Simulated Vaginal Swab Matrix - SVSM) spiked with specific concentrations of target organism lysates.

8. The Sample Size for the Training Set

The document does not provide information on the sample size used for the training set of the Aptima BV Assay. This is because the Aptima BV Assay is a nucleic acid amplification test, a type of IVD, that relies on biochemical reactions and calibrated thresholds rather than machine learning algorithms that typically require explicit training data sets for model development. The "calibration information" mentioned in the description of the Panther system suggests a pre-defined set of parameters, likely established through analytical studies.

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

As noted above, for this type of in-vitro diagnostic device, there isn't typically a "training set" in the machine learning sense with an associated ground truth established by experts.

Instead, the assay's operational parameters (e.g., thresholds for positivity based on signal emergence times) would be established through a rigorous process of analytical validation (like the LoD and precision studies described) using samples with known concentrations of targets. This ensures the assay's performance characteristics (sensitivity, specificity) meet predefined criteria. The "calibration information" is central to how the device makes its determination.

Summary

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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 (DICE@fda.hhs.gov) or phone (1-800-638-2041 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, RACRegulatory Affairs ManagerJeffrey.Hergesheimer@Hologic.comPhone: (858) 410-8536Fax: (858) 410-5557
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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

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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

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(DEN160001)	Aptima BV Assay(Subject Device)
Patient Population	Women with a clinical presentationof vaginitis/vaginosis	Same
Specimen Types	Vaginal swabs in patients who aresymptomatic for vaginitis/vaginosis	Same
Assay Controls	Incorporates an Internal Control inevery test. Uses external positiveand 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(DEN160001)	Aptima BV Assay(Subject Device)
Organisms Detected	Lactobacillus (L. crispatus, and L.jensenii), Lactobacillus (L. gasseri,L. crispatus, and L. jensenii),Atopobium vaginae, BacterialVaginosis Associated Bacteria-2(BVAB-2), Megasphaera-1,Candida (C. albicans, C.tropicalis, C. parapsilosis, C.)	Lactobacillus (L. gasseri, L.crispatus, and L. jensenii),Gardnerella vaginalis, andAtopobium vaginae
Item	BD MAX Vaginal Panel(DEN160001)	Aptima BV Assay(Subject Device)
	dubliniensis), Candida glabrata,Candida krusei, Trichomonasvaginalis
Platform/TechnologyPrinciple ofOperation	BD MAX System/Real-time polymerase chainreaction (PCR)	Panther System/Real-time Transcription MediatedAmplification (TMA)
Analyte	DNA	ribosomal RNA
Assay Calibrators	None	Positive Calibrator
Intended Use	The BD MAX Vaginal Panelperformed on the BD MAX Systemis an automated qualitative in vitrodiagnostic test for the directdetection of DNA targets frombacteria associated with bacterialvaginosis (qualitative resultsreported based on detection andquantitation of targeted organismmarkers), Candida speciesassociated with vulvovaginalcandidiasis, and Trichomonasvaginalis from vaginal swabs inpatients who are symptomatic forvaginitis/vaginosis. The testutilizes real-time polymerase chainreaction (PCR) for theamplification of specific DNAtargets and utilizes fluorogenictarget-specific hybridization probesto detect anddifferentiate DNA from:- Bacterial vaginosis markers(Individual markers notreported)Lactobacillus spp. ( L. crispatusand L. jensenii ) GardnerellavaginalisAtopobium vaginaeBacterial Vaginosis AssociatedBacteria-2 (BVAB-2)Megasphaera -1Candida spp. ( C. albicans, C.	The Aptima BV assay is an in vitronucleic acid amplification test thatutilizes real time transcription-mediated amplification (TMA) fordetection and quantitation ofribosomal RNA from bacteriaassociated with bacterial vaginosis(BV), including Lactobacillus ( L.gasseri, L. crispatus, and L.jensenii ), Gardnerella vaginalis ,and Atopobium vaginae . The assayreports a qualitative result for BVand does not report results forindividual organisms. The assay isintended to aid in the diagnosis ofBV on the automated Panthersystem using clinician-collectedand patient-collected vaginal swabspecimens from females with aclinical presentation consistentwith vaginitis and/or vaginosis.
Item	BD MAX Vaginal Panel(DEN160001)	Aptima BV Assay(Subject Device)
	tropicalis, C. parapsilosis, C.dubliniensis)• Candida glabrata• Candida krusei• Trichomonas vaginalisThe BD MAX Vaginal Panel isintended to aid in the diagnosis ofvaginal infections in women with aclinical presentation consistentwith bacterial vaginosis,vulvovaginal candidiasis andtrichomoniasis.

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

Table 9: BV Positivity of Precision Panels

1 Three invalid results were excluded from the analysis.

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		BetweenOperators		BetweenInstruments		BetweenDays		BetweenLots		BetweenRuns		WithinRun		Total
PanelDescription	N	MeanTTime1	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)
L. crispatusBV 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. crispatusBV 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. crispatusBV 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. jenseniiBV 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. crispatusBV 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.

			BetweenOperators		BetweenInstruments		BetweenDays		BetweenLots		BetweenRuns		WithinRun		Total
PanelDescription	N	MeanTTime1	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)
G. vaginalisBV 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. vaginalisBV 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. vaginalisBV 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. vaginalisBV 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. vaginalisBV 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. vaginalisBV 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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			BetweenOperators		BetweenInstruments		BetweenDays		BetweenLots		BetweenRuns		WithinRun		Total
PanelDescription	N	MeanTTime1	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)	SD	CV (%)
A. vaginaeBV 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. vaginaeBV 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. vaginaeBV 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. vaginaeBV 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. vaginaeBV 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. vaginaeBV 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. vaginaeBV 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

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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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SpecimenType	N	TP	FP	TN	FN	Prevalence1(95% CI)2	Sensitivity(95% CI)2	Specificity(95% CI)2	PPV(95% CI)3	NPV(95% CI)3
Patient-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-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(95% CI)2	Specificity(95% CI)2	PPV(95% CI)3	NPV(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.

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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.

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

Subjects	% Positivity(# 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.

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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.

		BetweenSites		BetweenOperators		BetweenDays		BetweenRuns		WithinRuns		Total
Panel Description	N	MeanTTime1	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).

			BetweenSites		BetweenOperators		BetweenDays		WithinRuns		Total
Control	N	MeanCt	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

	BetweenSites		BetweenOperators		BetweenDays		BetweenRuns		WithinRuns		Total
Analyte	N	MeanTTime	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 deviation, TTime = energence time of a signal (above a specific threshold)

VIII. CONCLUSIONS

The analytical and clinical study results demonstrate that the Aptima BV assay on the Panther system performs comparably to the predicate device that is currently marketed for the same intended use. Hardware and software verification and validation demonstrate that the Aptima BV assay on the Panther system will perform as intended.

Regulation Number and Section

§ 866.3975 Device that detects nucleic acid sequences from microorganisms associated with vaginitis and bacterial vaginosis.

(a)
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.(b)
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.