Aptima BV

Aptima ®
Aptima BV Assay ®
Instructions for Use

For in vitro diagnostic use
Rx only
General Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Summary and Explanation of the Test . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Principles of the Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Warnings and Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Reagent Storage and Handling Requirements . . . . . . . . . . . . . . . . . . . . 5
Specimen Collection and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Panther System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Reagents and Materials Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Materials Required and Available Separately . . . . . . . . . . . . . . . . . . . . . 9
Panther System Test Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Procedural Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Assay Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Negative and Positive Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Internal Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Test Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Panther System Expected Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Panther System Assay Performance . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Panther System Clinical Performance . . . . . . . . . . . . . . . . . . . . . . . . . 20
Performance Characteristics in Symptomatic Subjects . . . . . . . . . . . . 20
Positivity Rates in Asymptomatic Women . . . . . . . . . . . . . . . . . . . . . . 26
Invalid Rates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Panther System Analytical Performance . . . . . . . . . . . . . . . . . . . . . . . 27
Analytical Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Analytical Inclusivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Cross-Reactivity and Microbial Interference . . . . . . . . . . . . . . . . . . . . . 27
Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Within Laboratory Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Contact Information and Revision History . . . . . . . . . . . . . . . . . . . . . 34
Aptima BV Assay 1 AW-23712-001 Rev. 001

Aptima ® General Information
General Information
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.
Summary and Explanation of the Test

Vaginitis syndrome is characterized by a spectrum of conditions: vaginal and vulvar irritation,
odor, discharge, and pruritus (1). Causes of vaginitis include mechanical and chemical factors
(feminine hygiene products, contraceptive materials, etc.) as well as infectious agents (1).
Up to 90% of infectious vaginitis cases are caused by BV, vulvovaginal candidiasis (candida
vaginitis, CV) and trichomoniasis (trichomonas vaginalis vaginitis, TV) (2). BV has been
diagnosed in 22-50% of symptomatic patients, CV in 17-39%, and TV in 4-35% (1,2).
BV is responsible for the majority of infectious vaginitis cases. BV is characterized by a
change in the vaginal microbiota dominated by Lactobacillus species to a polymicrobial
anaerobe-dominated microbiota that includes Gardnerella vaginalis, Atopobium vaginae,
Prevotella, Bacteroides, Peptostreptococcus, Mobiluncus, Sneathia (Leptotrichia),
Mycoplasma, and BV associated bacteria (3). This change in vaginal microbiota is associated
with the onset of Amsel clinical signs, resulting from the biochemical and cytological changes
in the vaginal mileu that are pathognomonic for BV (11). BV has been associated with pelvic
inflammatory disease (4), cervicitis (5), elevated risk of acquisition of STIs, such as
chlamydia, gonorrhea, HSV, HIV (6,7,8), spontaneous abortion, and preterm birth (9,10).
Diagnosis of BV based on clinical criteria (vaginal pH, presence of clue cells, whiff test, and
discharge) has been proposed by Amsel (11). Nugent et al. proposed a classification for BV
based on microscopic description of observed types of bacteria via Gram stain in vaginal
swabs (12). Recent studies suggest that molecular diagnostic tools would be beneficial to
improve diagnosis of BV and that nucleic acid amplification, targeting several BV-associated
bacteria, could be utilized (13).
The Aptima BV assay is a real time TMA assay developed for use on the automated Panther
system that detects and discriminates RNA markers from the Lactobacillus species group
(L. gasseri, L. crispatus and L. jensenii), Gardnerella vaginalis, and Atopobium vaginae in
clinician-collected and patient-collected vaginal swab specimens from symptomatic females.
The Aptima BV assay uses an algorithm to report a qualitative result for BV based on
detection of target organisms. The Aptima BV assay includes an internal control (IC).

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General Information Aptima
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 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 to determine the
BV Positive or Negative status of each sample.
Warnings and Precautions

A. For in vitro diagnostic use.
B. For professional use.
C. To reduce the risk of invalid results, carefully read the entire package insert and the
Panther/Panther Fusion System Operator’s Manual prior to performing this assay.
D. Only personnel adequately trained in the use of the Aptima BV assay and in handling
potentially infectious materials should perform this procedure. If a spill occurs,
immediately disinfect following appropriate site procedures.
E. For additional specific warnings and precautions, refer to the Panther/Panther Fusion
System Operator’s Manual.
Laboratory Related
F. Use only supplied or specified disposable laboratory ware.
G. Use routine laboratory precautions. Do not pipette by mouth. Do not eat, drink or smoke
in designated work areas. Wear disposable, powderless gloves, protective eye wear, and

laboratory coats when handling specimens and kit reagents. Wash hands thoroughly after
handling specimens and kit reagents.
H. Work surfaces, pipettes, and other equipment must be regularly decontaminated with
2.5% to 3.5% (0.35 M to 0.5 M) sodium hypochlorite solution. Thoroughly clean and
disinfect all work surfaces.
I. Dispose of all materials that have come in contact with specimens and reagents in
accordance with applicable national, international, and regional regulations (14, 15, 16).
Thoroughly clean and disinfect all work surfaces.
Specimen Related
J. Expiration dates for the collection kits pertain to the collection of specimens and not to the
specimen testing. Samples collected any time prior to the expiration date of the collection
kit, and transported and stored in accordance with the package insert, are valid for testing
even if the expiration date on the collection tube has passed.
K. Specimens may be infectious. Use Universal Precautions when performing this assay
(14,15). Proper handling and disposal methods should be established according to local
regulations (16). Only personnel adequately trained in the use of the Aptima BV assay
and trained in handling infectious materials should perform this procedure.
L. Maintain proper storage conditions during specimen shipping to ensure the integrity of the
specimen. Specimen stability under shipping conditions other than those recommended
has not been evaluated.
M. Avoid cross-contamination during the specimen handling steps. Specimens can contain
extremely high levels of organisms. Ensure that specimen containers do not contact one
another, and discard used materials without passing over any open container. Change
gloves if they come in contact with a specimen.
N. Upon piercing, liquid can discharge from Aptima transfer tube caps under certain
conditions. Refer to the Panther System Test Procedure for more information.
O. If the lab receives an Aptima Multitest Swab Specimen Collection Kit transport tube with
no swab, two swabs, a cleaning swab, or a swab not supplied by Hologic, the specimen
must be rejected.
Assay Related
P. Do not interchange, mix, or combine assay reagents from kits with different master lot
numbers. Controls, the calibrator, and assay fluids may be interchanged.
Q. Cap and store reagents at the specified temperatures. The performance of the assay may
be affected by use of improperly stored reagents. See Reagent Storage and Handling
Requirements and Panther System Test Procedure for more information.
R. Do not combine any assay reagents or fluids without specific instruction. Do not top off
reagents or fluids. The Panther system verifies reagent levels.
S. Avoid microbial and nuclease contamination of reagents.

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T. Do not use the reagent, control, or calibrator kits after the expiration date.
U. Some reagents used with the Aptima BV assay are labeled with risk and safety symbols.
Note: Hazard Communication information for labeling of globally marketed products reflects the US
and EU Safety Data Sheets (SDS) classifications. For hazard communication information specific to
your region, refer to the region specific SDS on the Safety Data Sheet Library at www.hologicsds.com.
For more information on the symbols, refer to the symbol legend on www.hologic.com/package-inserts.
EU Hazard Information
Promoter Reagent
MAGNESIUM CHLORIDE 35 - 40%
—
H412 - Harmful to aquatic life with long lasting effects.
P273 - Avoid release to the environment
P280 - Wear eye protection/ face protection.
Target Capture Reagent

HEPES 5 - 10%
EDTA 1 - 5%
— LITHIUM HYDROXIDE, MONOHYDRATE 1 - 5%
H412 - Harmful to aquatic life with long lasting effects

P273 - Avoid release to the environment
P280 - Wear eye protection/ face protection
Reagent Storage and Handling Requirements

A. The following table shows the storage conditions and stability for the reagents, the
calibrator, and the controls.
Open Kit (Reconstituted)
Reagent Unopened Storage Storage Stability

Amplification Reagent 2ºC to 8ºC
Amplification Reconstitution
15ºC to 30ºC 2ºC to 8ºC 30 days1
Solution
Enzyme Reagent 2ºC to 8ºC
Enzyme Reconstitution
Solution
Promoter Reagent 2ºC to 8ºC
Promoter Reconstitution
Solution
Target Capture Reagent 15ºC to 30ºC 15ºC to 30ºC2 30 days1
Positive Calibrator 2ºC to 8ºC Single use vial
Negative Control 2ºC to 8ºC Single use vial
Positive Control 2ºC to 8ºC Single use vial
Internal Control 2ºC to 8ºC Single use vial
1
When reagents are removed from the Panther system, they should be immediately returned to their appropriate storage
temperatures.

2
Storage condition for the working Target Capture Reagent (Target Capture Reagent with Internal Control added).
B. Discard any unused reconstituted reagents and working Target Capture Reagent (wTCR)
after 30 days or after the Master Lot expiration date, whichever comes first.
C. Reagents stored on the Panther system have 120 hours of onboard stability. Reagents
can be loaded onto the Panther system up to 8 times. The system logs each time the
reagents are loaded.
D. The Promoter Reagent and reconstituted Promoter Reagent are photosensitive. Protect
these reagents from light during storage and preparation for use.
E. Avoid cross-contamination during reagent handling and storage. Recap all reconstituted
reagents with new reagent caps prior to storage.
F. Do not freeze reagents.
Specimen Collection and Storage

Note: Handle all specimens as if they contain potentially infectious agents. Use Universal Precautions.
Note: Take care to avoid cross-contamination during sample handling steps. For example, discard
used material without passing over open tubes.
Vaginal swab specimens can be tested with the Aptima BV assay. Assay performance has
not been evaluated with specimens other than those collected with the following specimen
collection kit:
• Aptima Multitest Swab Specimen Collection Kit
A. Specimen collection
Refer to the appropriate specimen collection kit package insert for specific collection
instructions.
B. Specimen transport and storage before testing:

Only the following storage conditions should be used for specimens with the Aptima BV assay.
1. Swab specimens
a. After collection, swab specimens in transport tubes can be stored at 2ºC to 8ºC for
up to 30 days. If longer storage is needed, specimens may be stored at -20°C or
-70°C for an additional 60 days.
b. After collection, swab specimens in transport tubes can be stored at 15°C to 30°C for
up to 30 days.
C. Specimen storage after testing:

1. Specimens that have been tested must be stored upright in a rack.
2. The specimen transport tubes should be covered with a new, clean plastic film or foil
barrier.
3. If assayed samples need to be shipped, remove penetrable cap and place new non-
penetrable caps on the specimen transport tubes. If specimens need to be shipped for
testing at another facility, recommended temperatures should be maintained.

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4. Prior to uncapping, specimen transport tubes must be centrifuged for 5 minutes at

420 ± 100 relative centrifugal force (RCF) to bring all of the liquid down to the bottom of
the tube. Avoid splashing and cross-contamination.
Note: Specimens must be shipped in accordance with applicable national, international, and regional
transportation regulations.

Aptima ® Panther System
Panther System
Reagents for the Aptima BV assay are listed below for the Panther system. Reagent
identification symbols are also listed next to the reagent name.
Reagents and Materials Provided

Aptima BV Assay Kit
100 tests: 2 assay boxes, 1 calibrator kit, and 1 controls kit (Cat. No. PRD-05186)
Aptima BV Assay Refrigerated Box

(store at 2ºC to 8ºC upon receipt)
Symbol Component Quantity

A Amplification Reagent 1 vial
Non-infectious nucleic acids dried in buffered solution.
E Enzyme Reagent 1 vial
Reverse transcriptase and RNA polymerase dried in HEPES
buffered solution.
PRO Promoter Reagent 1 vial
Non-infectious nucleic acids dried in buffered solution.
IC Internal Control 1 x 0.3 mL
Non-infectious RNA nucleic acids in buffered solution.
Aptima BV Assay Room Temperature Box

AR Amplification Reconstitution Solution 1 x 7.2 mL

Aqueous solution containing glycerol and preservatives.
ER Enzyme Reconstitution Solution 1 x 5.8 mL
HEPES buffered solution containing a surfactant and glycerol.
PROR Promoter Reconstitution Solution 1 x 4.5 mL
Aqueous solution containing glycerol and preservatives.
TCR Target Capture Reagent 1 x 26.0 mL
Buffered salt solution containing non-infectious nucleic acids
and magnetic particles.
Reconstitution Collars 3
Master Lot Barcode Sheet 1 sheet

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Panther System Aptima
Aptima BV Assay Calibrator Kit (PRD-05188)


PCAL Positive Calibrator 5 x 2.8 mL
Non-infectious nucleic acids in buffered solution.
Calibrator Barcode Label 1 sheet
Aptima BV Assay Controls Kit (PRD-05187)


CONTROL- Negative Control 5 x 1.7 mL
Non-infectious L. crispatus cultured cells in buffered
solution.
CONTROL+ Positive Control 5 x 1.7 mL
Non-infectious G. vaginalis and A. vaginae cultured cells in
buffered solution.
Control Barcode Label 1 sheet
Materials Required and Available Separately

Note: Materials available from Hologic have catalog numbers listed, unless otherwise specified.
Material Cat. No.
Panther System 303095

Panther Run Kit for Real Time Assays (for real time assays only) PRD-03455 (5000 tests)
Aptima Assay Fluids Kit (also known as Universal Fluids Kit) 303014 (1000 tests)
Contains Aptima Wash Solution, Aptima Buffer for Deactivation Fluid, and
Aptima Oil Reagent
Multi-tube units (MTUs) 104772-02
Panther Waste Bag Kit 902731
Panther Waste Bin Cover 504405
Or, Panther System Run Kit 303096 (5000 tests)
When running non-real time-TMA assays in parallel with real time-TMA assays
Contains MTUs, waste bags, waste bin covers, auto detect, and assay fluids
Aptima Assay Fluids Kit 303014 (1000 tests)
Contains Aptima Wash Solution, Aptima Buffer for Deactivation Fluid, and Aptima
Oil Reagent
Multi-tube units (MTUs) 104772-02

Material Cat. No.
Tips, 1000 μL filtered, conductive, liquid sensing, and disposable. 901121(10612513Tecan)

Not all products are available in all regions. Contact your 903031 (10612513 Tecan)
representative for region-specific information MME-04134 (30180117 Tecan)
MME-04128
Aptima Multitest Swab Specimen Collection Kit PRD-03546

Bleach, 5.0% to 8.25% (0.7 M to 1.16 M) sodium hypochlorite solution --
Disposable, powderless gloves --
Aptima penetrable caps 105668
Replacement non-penetrable caps 103036A
Reagent Replacement Caps
Amplification, Enzyme, and Promoter reagent reconstitution bottles CL0041 (100 caps)
TCR bottle 501604 (100 caps)
Plastic-backed laboratory bench covers --
Lint-free wipes --
Pipettor --
Tips --
Tube Rocker --
Panther System Test Procedure

Note: See the Panther/Panther Fusion System Operator’s Manual for additional Panther system
procedural information.
A. Work Area Preparation

1. Clean work surfaces where reagents will be prepared. Wipe down work surfaces with
2.5% to 3.5% (0.35 M to 0.5 M) sodium hypochlorite solution. Allow the sodium
hypochlorite solution to contact surfaces for at least 1 minute and then follow with a
deionized (DI) water rinse. Do not allow the sodium hypochlorite solution to dry.
2. Clean a separate work surface where samples will be prepared. Use the procedure
described above (Step A.1).
3. Clean any pipettors. Use the cleaning procedure described above (Step A.1).
B. Reagent Reconstitution/Preparation of a New Kit

Note: Reagent reconstitution should be performed prior to beginning any work on the
Panther system.
1. Prior to testing, Amplification, Enzyme, and Promoter Reagents must be reconstituted
by combining contents of the bottles of lyophilized reagent with the appropriate
reconstitution solution.
a. Allow the lyophilized reagents to reach room temperature (15°C to 30°C) before
use.
b. Pair each reconstitution solution with its lyophilized reagent. Before attaching the
reconstitution collar, ensure that the reconstitution solution and reagent have
matching label symbols.

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c. Check the lot numbers on the Master Lot Barcode Sheet to ensure that the
appropriate reagents are paired.
d. Open the lyophilized reagent vial and firmly insert the notched end of the
reconstitution collar into the vial opening (Figure 1, Step 1).
e. Open the matching reconstitution solution bottle, and set the cap on a clean,
covered work surface.
f. While holding the reconstitution solution bottle on the bench, firmly insert the other
end of the reconstitution collar into the bottle opening (Figure 1, Step 2).
g. Slowly invert the assembled bottles. Allow the solution to drain from the bottle into
the glass vial (Figure 1, Step 3).
h. Gently swirl the solution in the bottle to mix. Avoid creating foam while swirling the
bottle (Figure 1, Step 4).
i. Wait at least 15 minutes for the lyophilized reagent to go into solution, then invert the
assembled bottles again, tilting at a 45° angle to minimize foaming (Figure 1, Step
5). Allow all of the liquid to drain back into the plastic bottle.
j. Remove the reconstitution collar and glass vial (Figure 1, Step 6).
k. Recap the plastic bottle. Record operator initials and reconstitution date on the label
(Figure 1, Step 7).
l. Discard the reconstitution collar and glass vial (Figure 1, Step 8).
Option: Additional mixing of the Amplification, Enzyme, and Promoter Reagents
using a tube rocker is allowed. The reagents may be mixed by placing the
recapped plastic bottle on a tube rocker set to 20 RPM (or equivalent) for a
minimum of 5 minutes.
Warning: Avoid creating foam when reconstituting reagents. Foam compromises the
level-sensing in the Panther system.
Figure 1. Reagent Reconstitution Process
2. Prepare Working Target Capture Reagent (wTCR)

a. Pair the appropriate bottles of TCR and IC.
b. Check the reagent lot numbers on the Master Lot Barcode Sheet to make sure that
the appropriate reagents in the kit are paired.
c. Open the bottle of TCR, and set the cap on a clean, covered work surface.

d. Open the bottle of IC and pour the entire contents into the bottle of TCR. Expect a
small amount of liquid to remain in the IC bottle.
e. Cap the bottle and gently swirl the solution to mix the contents. Avoid creating foam
during this step.
f. Record operator initials and the current date on the label.
g. Discard the IC bottle and cap.
C. Reagent Preparation for Previously Prepared Reagents

1. Previously prepared Amplification, Enzyme, and Promoter reagents must reach room
temperature (15°C to 30°C) prior to the start of the assay.
Option: The reagents may be brought to room temperature by placing the
reconstituted Amplification, Enzyme, and Promoter Reagents on a tube rocker set to
20 RPM (or equivalent) for a minimum of 25 minutes.
2. If wTCR contains precipitate, warm wTCR at 42°C to 60°C for up to 90 minutes. Allow
the wTCR to equilibrate to room temperature prior to use. Do not use if precipitate
persists.
3. Verify that the reagents have not exceeded their storage stability times, including
onboard stability.
4. Thoroughly mix each reagent by gently inverting prior to loading on the system. Avoid
creating foam when inverting reagents.
5. Do not top off reagent bottles. The Panther system will recognize and reject bottles that
have been topped off.
D. Specimen Handling
1. Allow the specimens to reach room temperature prior to processing.
2. Do not vortex specimens.
3. Visually confirm that each specimen tube meets the following criteria:
a. The presence of a single pink Aptima collection swab in a swab specimen
transport tube.
4. Inspect specimen tubes before loading into rack:
a. If a specimen tube contains bubbles in the space between the liquid and the cap,
centrifuge the tube for 5 minutes at 420 RCF to eliminate the bubbles.
b. If a specimen tube has a lower volume than typically observed when collection
instructions have been followed, centrifuge the tube for 5 minutes at 420 RCF to
ensure that no liquid is in the cap.
Note: Failure to follow Steps 4a-4b may result in liquid discharge from the specimen tube cap.
Note: Up to 4 separate aliquots can be tested from each specimen tube. Attempts to pipette
more than 4 aliquots from the specimen tube can lead to processing errors.
E. System Preparation
1. Set up the system according to the instructions in the Panther/Panther Fusion System
Operator’s Manual and Procedural Notes. Make sure that the appropriately sized
reagent racks and TCR adapters are used.

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Procedural Notes
A. Calibrator and Controls
Allow the calibrator and controls to reach room temperature prior to processing.
1. The positive calibrator, positive control and negative control tubes can be loaded in
any rack position or in any Sample Bay Lane on the Panther system. Specimen
pipetting will begin when one of the following 2 conditions has been met:
a. The calibrator and controls are currently being processed by the system.
b. Valid results for the calibrator and controls are registered on the system.
2. Once the calibrator and control tubes have been pipetted and are processing for a
specific reagent kit, patient specimens can be tested with the associated kit up to 24
hours unless:
a. The calibrator result or control results are invalid.
b. The associated assay reagent kit is removed from the system.
c. The associated assay reagent kit has exceeded stability limits.
3. Each calibrator or each control tube can be used once. Attempts to use more than once
can lead to processing errors.
B. Temperature
Room temperature is defined as 15°C to 30°C.
C. Glove Powder
As in any reagent system, excess powder on some gloves may cause contamination of
opened tubes. Powderless gloves are recommended.

Aptima ® Quality Control
Quality Control
An operator may invalidate an individual specimen or an entire run if it was observed and
documented that a procedural, technical, or instrument-related error occurred while
performing the assay.
Assay Calibration
To generate valid results, an assay calibration must be completed. The calibrator is run in
triplicate each time a reagent kit is loaded on the Panther system. Once established, the
calibration is valid for up to 24 hours. Software on the Panther system alerts the operator
when a calibration is required. The operator scans the calibration coefficients found on the
Master Lot Barcode Sheet provided with each reagent kit.
During processing, criteria for acceptance of the calibrator is automatically verified by the
software on the Panther system. If less than two of the calibrator replicates are valid, the
software automatically invalidates the run. Samples in an invalidated run must be retested
using a freshly prepared calibrator and freshly prepared controls.
Negative and Positive Controls

To generate valid results, a set of assay controls must be tested. One replicate each of the
negative control and the positive control must be tested each time a reagent kit is loaded on
the Panther system. Once established, the controls are valid for up to 24 hours. Software on
the Panther system alerts the operator when controls are required.
During processing, criteria for acceptance of controls are automatically verified by software
on the Panther system. If any one of the controls has an invalid result, the software
automatically invalidates the run. Samples in an invalidated run must be retested using a
freshly prepared calibrator and freshly prepared controls.
Internal Control
An IC is added to each sample with the wTCR. During processing, IC acceptance criteria are
automatically verified by the Panther system software. If an IC result is invalid, the sample
result is invalidated. Every sample with an invalid IC result must be retested to obtain a valid
result.
The Panther system software is designed to accurately verify processes when procedures
are performed following the instructions provided in this package insert and the Panther/
Panther Fusion System Operator's Manual.
Test Interpretation
Test results are automatically determined by the assay software. The table below shows the
possible results reported in a valid run and result interpretations. Samples with invalid test
results should be retested.
Table 1: Result Interpretation
BV Result Interpretation
Positive Positive for BV

®
Limitations Aptima
Table 1: Result Interpretation
BV Result Interpretation
Negative Negative for BV
Invalid Invalid test
Limitations
A. Use of this assay is limited to personnel who have been trained in the procedure. Failure
to follow the instructions given in this package insert may result in erroneous results.
B. The effects of other potential variables such as vaginal discharge, use of tampons, and
specimen collection variables have not been determined.
C. Performance with specimen types other than vaginal swab specimens has not been
evaluated.
D. Reliable results are dependent on adequate specimen collection, transport, storage, and
processing. Failure to observe proper procedures in any one of these steps can lead to
incorrect results. Because the transport system used for this assay does not permit
microscopic assessment of specimen adequacy, training of clinicians in proper specimen
collection techniques is necessary. See Specimen Collection and Storage for instructions.
For detailed information, refer to the appropriate instructions for use.
E. Therapeutic failure or success cannot be determined with the Aptima BV assay since
nucleic acid may persist following appropriate antimicrobial therapy.
F. Bacterial species targeted by the Aptima BV assay may comprise part of the normal
microbiome for a significant number of women; a BV positive result should be interpreted
in conjunction with other clinical data available to the clinician.
G. A negative result does not preclude a possible infection. Test results may be affected by
improper specimen collection, technical error, or specimen mix-up.
H. The Aptima BV assay provides qualitative results. Therefore, a correlation cannot be

drawn between the magnitude of a positive assay signal and the number of organisms in
a specimen.
I. Performance of the assay has not been evaluated in individuals less than 14 years of
age.
J. Customers must independently validate an LIS transfer process.
K. The Aptima BV assay has not been evaluated for use with specimens collected by
patients at home.
L. Collection and testing of patient-collected vaginal swab specimens with the Aptima BV
assay is not intended to replace clinical examination.
M. Public health recommendations should be consulted regarding testing for additional

sexually transmitted infections (STI) for patients with a positive result with the Aptima BV
assay.

For Regulatory Submission Use Only
Aptima ®
N. Additional microorganisms not detected by the Aptima BV assay such as Prevotella

species and Mobiluncus species, Ureaplasma, Mycoplasma, and numerous fastidious or
uncultivated anaerobes have also been found in women with BV, but are less associated
with BV due to their relatively low prevalence, sensitivity, and/or specificity (17).
O. Interference with the Aptima BV assay was observed in the presence of the following
substances: Mucus (1.5% V/V), Vaginal Moisturizing Gel (0.5% W/V) and Tioconazole
(5% W/V).
P. Cross-reactivity was observed with the Aptima BV assay in the presence of Lactobacillus
acidophilus 1x104 CFU/mL).
Q. A positive test result does not necessarily indicate the presence of viable organisms. A
positive result is indicative of the presence of target RNA.

®
Panther System Expected Values Aptima
Panther System Expected Values

The prevalence of bacterial vaginosis in patient populations depends on age, race/ethnicity,
risk factors, the type of clinic, and the sensitivity of the test used to detect infections. A
summary of the BV positivity in symptomatic subjects, as determined by the Aptima BV assay
on the Panther system, is shown in Table 2 for the multi-center study, by clinical site and
overall.
Table 2: Positivity as Determined by the Aptima BV Assay in Symptomatic Women by

Specimen Type and Clinical Site
%Positivity (# positve/# tested with valid results
Site Clinician-collected Vaginal Swabs Patient-collected Vaginal Swabs
1 40.0 (6/15) 46.7 (7/15)
2 20.0 (1/5) 0.0 (0/5)
3 63.6 (14/22) 63.6 (14/22)
4 51.9 (108/208) 60.5 (124/205)
5 48.5 (64/132) 50.8 (66/130)
6 46.5 (33/71) 50.7 (36/71)
7 68.1 (130/191) 69.3 (131/189)
8 100.0 (1/1) 100.0 (1/1)
9 48.0 (49/102) 54.9 (56/102)
10 70.6 (12/17) 70.6 (12/17)
11 50.7 (34/67) 50.7 (34/67)
12 32.8 (41/125) 34.1 (42/123)
13 63.2 (43/68) 62.3 (43/69)
14 55.6 (5/9) 55.6 (5/9)
15 50.0 (2/4) 50.0 (2/4)
16 58.6 (17/29) 65.5 (19/29)
17 49.4 (39/79) 51.3 (41/80)
18 64.4 (56/87) 64.4 (56/87)
19 45.6 (31/68) 50.0 (34/68)
20 11.1 (4/36) 19.4 (7/36)
21 58.4 (45/77) 57.9 (44/76)
All 52.0 (735/1413) 55.1 (774/1405)

Aptima ® Panther System Assay Performance
Panther System Assay Performance
Reproducibility
Aptima BV assay reproducibility was evaluated on the Panther system at three US sites
using seven panel members. Two operators performed testing at each site. Each operator
performed one run per day over six days using one reagent lot over the course of testing.
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.
Signal variability of the Aptima BV assay was calculated for each target in analyte positive
panel members. Only samples with valid results were included in the analyses. Variability,
calculated between sites, between operators, between days, between runs, within run, and
overall, is shown in Table 3 to Table 5 for Lactobacillus, G. vaginalis, and A. vaginae positive
panel members, respectively.
Table 3: Signal Variability for Lactobacillus Positive Panel Members

Between Between Between Between Within
Total
Sites Operators Days Runs Run
Panel Mean
N SD CV (%) SD CV (%) SD CV (%) SD CV (%) SD CV (%) SD CV (%)
Description TTime1
L. crispatus
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
BV Negative2
L. jensenii
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
BV Low Positive2
CV = coefficient of variation, SD = standard deviation.

1 TTime is shown for Lactobacillus only.
2
Panel member contains 2 different organisms; results are shown for only the Lactobacillus component.
Note: In the event that variability from some factors is numerically negative, SD and CV are shown as 0.00.
Table 4: Signal Variability for G. vaginalis Positive Panel Members

Total
Panel Mean
Description TTime1
G. vaginalis
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
Low Positive
G. vaginalis
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
Moderate Positive

1
TTime is shown for G. vaginalis only.

®
Panther System Assay Performance Aptima
Table 5: Signal Variability for A. vaginae Positive Panel Members

Total
Panel Mean
Description TTime1
A. vaginae
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
BV Negative2
A. vaginae
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
Low Positive
A. vaginae
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
BV Low Positive2
A. vaginae
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
Moderate Positive

1
TTime is shown for A. vaginae only.
2 Panel member contains 2 different organisms; results are shown for only the A. vaginae component.

Aptima ® Panther System Clinical Performance
Panther System Clinical Performance
Performance Characteristics in Symptomatic Subjects

A prospective, multi-center clinical study was conducted to establish the clinical performance
characteristics of the Aptima BV assay on the Panther system. Female subjects presenting
with symptoms of vaginitis were enrolled at 21 geographically and ethnically diverse US
clinical sites, including private and academic family practice, obstetric-gynecologic, family
planning, public health, STI, medical group clinics, and clinical research centers.
Three (3) vaginal swab samples were collected from each subject: one clinician-collected
swab sample and one patient-collected swab sample were collected using the Aptima
Multitest Swab Specimen Collection Kit for Aptima BV assay testing, and one clinician-
collected swab sample was collected for reference method testing. Aptima samples were
tested with the Aptima BV assay on the Panther system at three sites. BV infection status
was determined using a combination of Nugent interpretations and Amsel criteria from the
final vaginal swab sample.
• Samples with normal flora as per the Nugent interpretation were considered negative;
samples positive for BV flora were considered positive.
• Samples with intermediate Nugent interpretations were classified as positive or
negative for BV using modified Amsel criteria. Samples positive for ≥20% clue cells
and at least 1 of the 2 following criteria were considered Amsel positive: vaginal
pH >4.5 and positive whiff test.
• Samples that were unable to be assessed for the Nugent criteria, and samples with
indeterminate Nugent interpretation for which a modified Amsel result was not
available, were considered to have unknown BV infection status.
Performance characteristics for each sample, with corresponding 2-sided 95% Score
confidence intervals (CIs), were estimated relative to the BV infection status.
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 subjects were
evaluable for at least one of the sample types. Table 6 shows the demographics of evaluable
subjects.

®
Panther System Clinical Performance Aptima
Table 6: Demograhics of Evaluable Subjects
Characteristics Total
Total, N N 1417
Age (years) Mean ± SD 34.7 ± 11.11
Median 33.0
Range 14-75
Age category (years), n (%) 14-17 4 (0.3)
18-29 537 (37.9)
30-39 469 (33.1)
40-49 235 (16.6)
>50 172 (12.1)
Race/Ethnicity, n (%) Asian 67 (4.7)
Black or African American 731 (51.6)
White (Hispanic or Latino) 248 (17.5)
White (Not Hispanic or Latino) 307 (21.7)
Other1 64 (4.5)
1 Includes patient-reported other, mixed, and unknown races.
For the 1417 evaluable subjects, 1413 clinician-collected vaginal swab samples and 1405
patient-collected vaginal swab samples were included in the analyses. The sensitivity and
specificity of the Aptima BV assay for the detection of BV are shown for both sample types
overall and by site in Table 7. Assay performance is shown stratified by race/ethnicity in
Table 8, and by clinical condition in Table 9.

Table 7: Performance Characteristics by Collection Site in Symptomatic Women
Clinician-collected Vaginal Swabs Patient-collected Vaginal Swabs
Prev (%) Sensitivity % Specificity % Sensitivity % Specificity %

Site N (95% CI)1 (95% CI)1 N Prev (%) (95% CI)1 (95% CI)1
95.0 89.6 97.3 85.8
(93.1-96.4) (87.1-91.6) (95.8-98.2) (83.1-88.2)
All 1413 49.2 660/6952 643/7183 1405 49.3 673/6924 612/7135
100 100 100 88.9
1 15 40.0 (61.0-100) (70.1-100) 15 40.0 (61.0-100) (56.5-98.0)
6/6 9/9 6/6 8/9
100 100 0.0 100
2 5 20.0 (20.7-100) (51.0-100) 5 20.0 (0.0-79.3) (51.0-100)
1/1 4/4 0/1 4/4
100 88.9 100 88.9
3 22 59.1 (77.2-100) (56.5-98.0) 22 59.1 (77.2-100) (56.5-98.0)
13/13 8/9 13/13 8/9
89.2 90.7 96.4 81.1
4 208 53.4 (82.0-93.7) (83.3-95.0) 205 53.7 (91.0-98.6) (72.0-87.7)
99/111 88/97 106/110 77/95
96.2 82.5 98.1 80.8
5 132 39.4 (87.0-98.9) (72.7-89.3) 130 40.0 (89.9-99.7) (70.7-88.0)
50/52 66/80 51/52 63/78
90.6 89.7 100 89.7
6 71 45.1 (75.8-96.8) (76.4-95.9) 71 45.1 (89.3-100) (76.4-95.9)
29/32 35/39 32/32 35/39
97.6 89.2 98.4 86.2
7 191 66.0 (93.2-99.2) (79.4-94.7) 189 65.6 (94.3-99.6) (75.7-92.5)
123/126 58/65 122/124 56/65
100 100
8 1 100.0 (20.7-100) NC 1 100.0 (20.7-100) NC
1/1 1/1
87.8 88.7 95.9 83.0
9 102 48.0 (75.8-94.3) (77.4-94.7) 102 48.0 (86.3-98.9) (70.8-90.8)
43/49 47/53 47/49 44/53
92.3 100 92.3 100
10 17 76.5 (66.7-98.6) (51.0-100) 17 76.5 (66.7-98.6) (51.0-100)
12/13 4/4 12/13 4/4
96.8 88.9 96.8 88.9
11 67 46.3 (83.8-99.4) (74.7-95.6) 67 46.3 (83.8-99.4) (74.7-95.6)
30/31 32/36 30/31 32/36
94.3 91.1 91.7 89.7
12 125 28.0 (81.4-98.4) (83.4-95.4) 123 29.3 (78.2-97.1) (81.5-94.5)
33/35 82/90 33/36 78/87
100 83.3 97.4 80.6
13 68 55.9 (90.8-100) (66.4-92.7) 69 55.1 (86.5-99.5) (63.7-90.8)
38/38 25/30 37/38 25/31
100 80.0 100 80.0
14 9 44.4 (51.0-100) (37.6-96.4) 9 44.4 (51.0-100) (37.6-96.4)
4/4 4/5 4/4 4/5
100 66.7 100 66.7
15 4 25.0 (20.7-100) (20.8-93.9) 4 25.0 (20.7-100) (20.8-93.9)
1/1 2/3 1/1 2/3
93.8 84.6 100 76.9
16 29 55.2 (71.7-98.9) (57.8-95.7) 29 55.2 (80.6-100) (49.7-91.8)
15/16 11/13 16/16 10/13

®
Table 7: Performance Characteristics by Collection Site in Symptomatic Women (continued)
Clinician-collected Vaginal Swabs Patient-collected Vaginal Swabs
Prev (%) Sensitivity % Specificity % Sensitivity % Specificity %

Site N (95% CI)1 (95% CI)1 N Prev (%) (95% CI)1 (95% CI)1
97.2 90.7 100 88.6
17 79 45.6 (85.8-99.5) (78.4-96.3) 80 45.0 (90.4-100) (76.0-95.0)
35/36 39/43 36/36 39/44
98.1 88.2 100 91.2
18 87 60.9 (90.1-99.7) (73.4-95.3) 87 60.9 (93.2-100) (77.0-97.0)
52/53 30/34 53/53 31/34
100 94.9 100 87.2
19 68 42.6 (88.3-100) (83.1-98.6) 68 42.6 (88.3-100) (73.3-94.4)
29/29 37/39 29/29 34/39
66.7 100 66.7 90.0
20 36 16.7 (30.0-90.3) (88.6-100) 36 16.7 (30.0-90.3) (74.4-96.5)
4/6 30/30 4/6 27/30
100 91.4 97.6 88.6
21 77 54.5 (91.6-100) (77.6-97.0) 76 53.9 (87.4-99.6) (74.0-95.5)
42/42 32/35 40/41 31/35
CI = confidence interval, NC = not calculable, Prev = prevalence.

1 Score CI.
2 Of the 35 false negatives results, 10 subjects were Nugent intermediates and had BV infection status determined by Amsel
criteria, and 15 were negative by Amsel.
3 Of the 75 false positive results, 46 subjects were Nugent intermediates and had BV infection status determined by Amsel
criteria, and 6 were positive by Amsel.
4 Of the 19 false negative results, 6 subjects were Nugent intermediates and had BV infection status determined by Amsel
criteria, and 7 were negative by Amsel.
5 Of the 101 false positive results, 55 subjects were Nugent intermediates and had BV infection status determined by Amsel
criteria, and 9 were positive by Amsel.

Table 8: Performance Characteristics by Race/Ethnicity in Symptomatic Women

Sensitivity % Specificity %
Specimen Type Race/Ethnicity N Prev (%) (95% CI)1 (95% CI)1
95.0 (93.1-96.4) 89.6 (87.1-91.6)

All 1413 49.2
660/695 643/718
95.2 (77.3-99.2) 91.3 (79.7-96.6)

Asian 67 31.3
20/21 42/46
95.5 (93.2-97.1) 89.1 (84.9-92.2)

Black/African-American 729 61.0
425/445 253/284
Clinician-collected 96.5 (91.3-98.6) 86.5 (79.6-91.3)

Vaginal Swabs White (Hispanic/Latino) 247 46.2
110/114 115/133
White 88.6 (80.3-93.7) 91.7 (87.3-94.7)

306 28.8
(Not Hispanic/Latino) 78/88 200/218
100 (87.5-100) 89.2 (75.3-95.7)

Other2 64 42.2
27/27 33/37
97.3 (95.8-98.2) 85.8 (83.1-88.2)

All 1405 49.3
673/692 612/713
95.0 (76.4-99.1) 86.7 (73.8-93.7)

Asian 65 30.8
19/20 39/45
97.5 (95.6-98.6) 84.8 (80.1-88.5)

Black/African-American 727 61.2
Patient-collected 434/445 239/282
Vaginal Swabs
99.1 (95.2-99.8) 83.5 (76.2-88.8)
White (Hispanic/Latino) 246 45.9
112/113 111/133
White 93.1 (85.8-96.8) 87.5 (82.4-91.3)

303 28.7
(Not Hispanic/Latino) 81/87 189/216
100 (87.5-100) 91.9 (78.7-97.2)

Other2 64 42.2
27/27 34/37
CI = confidence interval, Prev = prevalence

1 Score CI.

®
Table 9: Performance Characteristics by Clinical Condition in Symptomatic Women

Sensitivity % Specificity %
Collection Type Clinical Condition N1 Prev (%) (95% CI)2 (95% CI)2
95.0 (93.1-96.4) 89.6 (87.1-91.6)
All 1413 49.2
660/695 643/718
100 (20.7-100) 100 (34.2-100)
Use of antibiotics 3 33.3
1/1 2/2
100 (34.2-100) 100 (61.0-100)
Use of antifungals 8 25.0
2/2 6/6
100 (34.2-100)
Use of estrogen therapy 2 0.0 NC
2/2
Recurrent symptoms of vaginitis 95.2 (92.7-96.9) 88.8 (85.4-91.4)
832 49.8
Clinician-collected in the last 12 months 394/414 371/418
Vaginal Swabs Unprotected intercourse in the last 92.6 (82.4-97.1) 85.0 (70.9-92.9)
94 57.4
24 hours 50/54 34/40
100 (70.1-100) 100 (74.1-100)
Pregnant 20 45.0
9/9 11/11
96.2 (87.0-98.9) 86.4 (75.5-93.0)
With Menses 111 46.8
50/52 51/59
95.6 (93.7-97.0) 89.3 (86.6-91.6)
Without Menses 1177 50.6
569/595 520/586
85.4 (72.8-92.8) 93.5 (85.7-97.2)
Post-menopausal 125 38.4
41/48 72/77
97.3 (95.8-98.2) 85.8 (83.1-88.2)
All 1405 49.3
673/692 612/713
100 (20.7-100) 100 (34.2-100)
Use of antibiotics 3 33.3
1/1 2/2
100 (34.2-100) 100 (61.0-100)
Use of antifungals 8 25.0
2/2 6/6
100 (34.2-100)
Use of estrogen therapy 2 0.0 NC
2/2
Recurrent symptoms of vaginitis 98.1 (96.2-99.0) 85.1 (81.3-88.2)
828 49.9
Patient-collected in the last 12 months 405/413 353/415
Vaginal Swabs Unprotected intercourse in the last 98.1 (90.2-99.7) 75.0 (59.8-85.8)
94 57.4
24 hours 53/54 30/40
100 (70.1-100) 90.9 (62.3-98.4)
Pregnant 20 45.0
9/9 10/11
100 (93.1-100) 84.2 (72.6-91.5)
With Menses 109 47.7
52/52 48/57
97.5 (95.9-98.5) 85.4 (82.3-88.0)
Without Menses 1175 50.6
579/594 496/581
91.3 (79.7-96.6) 90.7 (82.0-95.4)
Post-menopausal 121 38.0
41/46 68/75
CI = confidence interval, NC = not calculable, Prev = prevalence

1 Subjects may report multiple clinical conditions; sum of subject numbers in all subgroups does not equal the total number of
subjects.
2 Score CI.

Positivity Rates in Asymptomatic Women

The detection of an imbalance in the vaginal microbiome is relevant for treatment decisions.
Although the Aptima BV assay is not intended for use in testing samples from asymptomatic
women, organisms associated with BV infection and detected by the Aptima BV assay also
may be present in asymptomatic women. Presence of the Aptima BV assay bacterial targets
was assessed in clinician-collected vaginal swab samples from 172 asymptomatic women. A
summary of the BV detection rates, as determined by the Aptima BV assay, is shown in
Table 10 for the multi-center study overall and by race/ethnicity.
Table 10: Positivity as Determined by the Aptima BV Assay in Asymptomatic Women
Race/Ethnicity % Positivity (# positive/# tested with valid results)
All 40.7% (70/172)
Asian 40.0% (2/5)
Black/African American 52.0% (39/75)
White
43.9% (18/41)
(Hispanic/Latino)
White
15.9% (7/44)
(Not Hispanic/Latino)
Other1 57.1% (4/7)
Invalid Rates
A total of 3175 clinician- and patient-collected samples from symptomatic and asymptomatic
subjects were processed in valid Aptima BV runs to establish clinical performance. Of these,
0.7% had initial invalid results. Upon retest, 0.1% remained invalid and were excluded from
all analyses.

®
Panther System Analytical Performance Aptima
Panther System Analytical Performance
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 11.
Table 11: Limit of Detection of the Aptima BV Assay
Organism Predicted Detection Limit CFU/mL
A. vaginae 95% 2901

G. vaginalis 95% 551
L. crispatus 95% 143
L. gasseri 95% 2,207
L. jensenii 95% 10
1 Predicted BV Positivity Limits (C ) for A. vaginae and G. vaginalis in the Aptima BV assay are
95
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 were evaluated in the
presence of non-targeted organisms. A panel consisting of 62 organisms (Table 12) 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 concentrations listed in
Table 12.

Aptima ® Panther System Analytical Performance
Table 12: Cross-Reactivity and Microbial Interference Panel

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
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.

®
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 13.
Table 13: Interfering Substances Panel

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
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
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 14. 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 Table 15 through Table 17.
Table 14: BV Positivity of Precision Panels
Panel BV Positive/ Expected BV BV Positivity

Description Total n Positivity (95% CI)
0
SVSM 0/168 0%
(0.0-1.6)
L. crispatus, A. vaginae 0
0 /168 <5%
BV Negative (0.0-1.6)
L. crispatus, G. vaginalis 45.2
76 /168 20-80%
BV High Negative (37.9-52.8)
L. crispatus, G. vaginalis, A. vaginae 79.4
131/1651 20-80%
BV High Negative (72.6-84.9)
G. vaginalis 100
168/168 ≥95%
BV Low Positive (98.4-100.0)
A. vaginae 100
168/168 ≥95%
L. jensenii, A. vaginae 100
168/168 ≥95%
G. vaginalis, A. vaginae 100
168/168 ≥95%
L. crispatus, G. vaginalis, A. vaginae 100
168/168 ≥95%
G. vaginalis 100
168/168 100%
BV Mod Positive (98.4-100.0)
A. vaginae 100
168/168 100%
BV Mod Positive (98.4-100.0)
1
Three invalid results were excluded from the analysis.

®
Table 15: Signal Variability of Lactobacillus Panel Members
Between Between Between Between Between Within

Total
Operators Instruments Days Lots Runs Run
Panel Mean
N SD CV (%) SD CV (%) SD CV (%) SD CV (%) SD CV (%) SD CV (%) SD CV (%)
Description TTime1
L. crispatus
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
BV Negative2
L. crispatus
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
BV High Negative2
L. crispatus
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
BV High Negative3
L. jensenii
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
BV Low Positive2
L. crispatus
168 23.51 0.15 0.63 0.09 0.40 0.17 0.73 1.36 5.77 0.10 0.44 0.31 1.31 1.42 6.02
BV Low Positive3
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.
Table 16: Signal Variability of G. vaginalis Panel Members

Total
Panel Mean
Description TTime1
G. vaginalis
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
BV High Negative2
G. vaginalis
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
BV High Negative3
G. vaginalis
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
BV Low Positive
G. vaginalis
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
BV Mod Positive
G. vaginalis
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
BV Low Positive2
G. vaginalis
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
BV Low Positive3
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.

Table 17: Signal Variability of A. vaginae Panel Members

Total
Panel Mean
Description TTime1
A. vaginae
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
BV Negative2
A. vaginae
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
BV High Negative3
A. vaginae
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
BV Low Positive
A. vaginae
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
BV Low Positive2
A. vaginae
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
BV Mod Positive
A. vaginae
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
BV Low Positive2
A. vaginae
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
BV Low Positive3
CV = Coefficient of variation, Mod = moderate

1 TTime is shown for A. vaginae only.
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.

®
Bibliography Aptima
Bibliography
1. Hainer BL, Gibson MV. Vaginitis: Diagnosis and Treatment. Am Fam Physician. 2011 Apr 1;83(7):807-815.
2. Granato PA. Vaginitis: Clinical and Laboratory Aspects for Diagnosis. Clin Microbiol Newsletter. 2010 Aug 1,(15): 111-116.
3. Onderdonk AB, Delaney ML, Fichorova RN. The Human Microbiome during Bacterial Vaginosis. Clin Microbiol Rev. 2016
Apr;29(2):223-38. doi: 10.1128/CMR.00075-15.
4. Haggerty CL, Hillier SL, Bass DC, Ness RB; PID Evaluation and Clinical Health study investigators. Bacterial vaginosis and
anaerobic bacteria are associated with endometritis. Clin Infect Dis. 2004 Oct 1;39(7):990-5. Epub 2004 Sep 2.
5. Marrazzo JM, Wiesenfeld HC, Murray PJ, Busse B, Meyn L, Krohn M, Hillier SL. Risk factors for cervicitis among women with
bacterial vaginosis. J Infect Dis. 2006 Mar 1;193(5):617-624. Epub 2006 Feb 2.
6. Bautista CT, Wurapa EK, Sateren WB, Morris SM, Hollingsworth BP, Sanchez JL. Association of Bacterial Vaginosis with
Chlamydia and Gonorrhea Among Women in the U.S. Army. Am J Prev Med. 2017;52(5):632-639. doi: 10.1016/
j.amepre.2016.09.016.
7. Cherpes TL, Meyn LA, Krohn MA, Lurie JG, Hillier SL. Association between acquisition of herpes simplex virus type 2 in
women and bacterial vaginosis. Clin Infect Dis. 2003 Aug 1;37(3):319-325.
8. Cohen CR, Lingappa JR, Baeten JM, et al. Bacterial vaginosis associated with increased risk of female-to-male HIV-1
transmission: a prospective cohort analysis among African couples. PLoS Med. 2012;9(6):e1001251. doi: 10.1371/
journal.pmed.1001251.
9. Işik G, Demirezen, Dönmez HG, Beksaç MS. Bacterial vaginosis in association with spontaneous abortion and recurrent
pregnancy losses. J Cytol. 2016 Jul-Sep;33(3):135-140.
10. Donders GG, Van Calsteren K, Bellen G, et al. Predictive value for preterm birth of abnormal vaginal flora, bacterial vaginosis
and aerobic vaginitis during the first trimester of pregnancy. BJOG. 2009 Sep;116(10):1315-24.
11. Amsel R, Totten PA, Spiegel CA, Chen KCS, Eschenbach DA, Holmes KK. Nonspecific vaginitis: diagnostic criteria and
epidemiologic associations. Am J Med. 1983 74:14–22.
12. Nugent RP, Krohn MA, Hillier SL. Reliability of Diagnosing Bacterial Vaginosis Is Improved by a Standardized Method of Gram
Stain Interpretation. J Clin Microbiol. Feb 1991, 29(2): 297-301.
13. Plummer EL, Garland SM, Bradshaw CS, et al. Molecular diagnosis of bacterial vaginosis: Does adjustment for total bacterial
load or human cellular content improve diagnostic performance? J Microbiol Methods. 2017 Feb;133:66-68. doi: 10.1016/
j.mimet.2016.12.024. Epub 2016 Dec 29.
14. Clinical and Laboratory Standards Institute. 2006. Collection, Transport, Preparation, and Storage of Specimens for Molecular
Methods; Approved Guideline. CLSI Document MM13. Wayne, PA.
15. Centers for Disease Control and Prevention/National Institutes of Health. 2009. Biosafety in Microbiological and Biomedical
Laboratories (BMBL).
16. Clinical and Laboratory Standards Institute. 2011. Clinical Laboratory Waste Management. CLSI Document GP5. Villanova,
PA.
17. Centers for Disease Control and Prevention. 2015. United States Morbidity and Mortality Weekly Report. Recommendations
and Reports, Vol. 64, No. 3.

Aptima ® Contact Information and Revision History
Contact Information and Revision History
Australian Sponsor Address:

Hologic (Australia & New Zealand) Pty Ltd
Hologic, Inc.
10210 Genetic Center Drive Macquarie Park NSW 2113
San Diego, CA 92121 USA
For country-specific Technical Support and Customer Service email address and telephone number, visit www.hologic.com/
support.
Serious incidents occurring in relation to the device in the European Union should be reported to the manufacturer and the
competent authority of the Member State in which the user and/or the patient is established.
Hologic, Aptima, TMA, Panther, and associated logos are trademarks and/or registered trademarks of Hologic, Inc. and/or its
subsidiaries in the United States and/or other countries.
All other trademarks, registered trademarks, and product names that may appear in this package insert are the property of their
respective owners.
This product may be covered by one or more U.S. patents identified at www.hologic.com/patents.
©2022 Hologic, Inc. All rights reserved.
AW-23712-001 Rev. 001
2022-08
Revision History Date Description

• Created Aptima Bacterial Vaginosis (BV)
assay IFU AW-23712 Rev. 001 based on
AW-18811 Rev. 003 for regulatory
compliance with IVDR.
AW-23712 Rev. 001 August 2022 • Updated Hazard information.
• Updated contact information including: EC
Rep, CE Mark, Australian Rep information,
and technical support.
• Miscellaneous style and formatting updates.

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Instructions for Use

Aptima BV Assay 1 AW-23712-001 Rev. 001

Summary and Explanation of the Test

Aptima BV Assay 2 AW-23712-001 Rev. 001

Principles of the Procedure

Warnings and Precautions

B. For professional use.

F. Use only supplied or specified disposable laboratory ware.

Aptima BV Assay 3 AW-23712-001 Rev. 001

S. Avoid microbial and nuclease contamination of reagents.

Aptima BV Assay 4 AW-23712-001 Rev. 001

Target Capture Reagent

H412 - Harmful to aquatic life with long lasting effects

Reagent Storage and Handling Requirements

Open Kit (Reconstituted)

Reagent Unopened Storage Storage Stability

Aptima BV Assay 5 AW-23712-001 Rev. 001

F. Do not freeze reagents.

Specimen Collection and Storage

B. Specimen transport and storage before testing:

C. Specimen storage after testing:

Aptima BV Assay 6 AW-23712-001 Rev. 001

4. Prior to uncapping, specimen transport tubes must be centrifuged for 5 minutes at

Aptima BV Assay 7 AW-23712-001 Rev. 001

Reagents and Materials Provided

Aptima BV Assay Refrigerated Box

Symbol Component Quantity

Aptima BV Assay Room Temperature Box

Symbol Component Quantity

AR Amplification Reconstitution Solution 1 x 7.2 mL

Aptima BV Assay 8 AW-23712-001 Rev. 001

Aptima BV Assay Calibrator Kit (PRD-05188)

Symbol Component Quantity

Aptima BV Assay Controls Kit (PRD-05187)

Symbol Component Quantity

Materials Required and Available Separately

Material Cat. No.

Panther System 303095

Aptima BV Assay 9 AW-23712-001 Rev. 001

Material Cat. No.

Tips, 1000 μL filtered, conductive, liquid sensing, and disposable. 901121(10612513Tecan)

Aptima Multitest Swab Specimen Collection Kit PRD-03546

Panther System Test Procedure

A. Work Area Preparation

B. Reagent Reconstitution/Preparation of a New Kit

Aptima BV Assay 10 AW-23712-001 Rev. 001

Figure 1. Reagent Reconstitution Process

2. Prepare Working Target Capture Reagent (wTCR)

Aptima BV Assay 11 AW-23712-001 Rev. 001

C. Reagent Preparation for Previously Prepared Reagents

Aptima BV Assay 12 AW-23712-001 Rev. 001

Aptima BV Assay 13 AW-23712-001 Rev. 001

Negative and Positive Controls

Aptima BV Assay 14 AW-23712-001 Rev. 001

Table 1: Result Interpretation

H. The Aptima BV assay provides qualitative results. Therefore, a correlation cannot be

J. Customers must independently validate an LIS transfer process.

M. Public health recommendations should be consulted regarding testing for additional

Aptima BV Assay 15 AW-23712-001 Rev. 001