423745 (6 pack test)

423744 (30 pack test)

423748 (6 pack (+) control)

BioFire ®
COVID-19 Test
Instructions for Use

The Symbols Glossary is provided on Page 38 of this booklet.

For use under an Emergency Use Authorization (EUA) only
Please visit us at www.biofiredefense.com/covid-19test

Manufactured by
BioFire Defense, LLC
79 West 4500 South, Suite 14
Salt Lake City, UT 84107 USA
 TABLE OF CONTENTS
INTENDED USE ............................................................................................................. 1
SUMMARY AND EXPLANATION OF THE TEST ................................................................. 1
PRINCIPLE OF THE PROCEDURE ..................................................................................... 2
MATERIALS PROVIDED .................................................................................................. 3
MATERIALS REQUIRED BUT NOT PROVIDED .................................................................. 3
WARNINGS AND PRECAUTIONS .................................................................................... 4
General Precautions ........................................................................................................ 4
Safety Precautions........................................................................................................... 4
Laboratory Precautions ................................................................................................... 5
Precaution Related to Public Health in the United States ............................................... 6
REAGENT STORAGE, HANDLING, AND STABILITY ........................................................... 6
SAMPLE REQUIREMENTS............................................................................................... 7
BIOFIRE® COVID-19 TEST PROCEDURE............................................................................ 7
Preparing Specimens for Pooling .................................................................................... 7
Step 1: Prepare Pouch ..................................................................................................... 8
Step 2: Hydrate Pouch ..................................................................................................... 8
Step 3: Prepare Sample Mix ............................................................................................ 9
Step 4: Load Sample Mix ................................................................................................. 9
Step 5: Run Pouch ......................................................................................................... 11
FilmArray 2.0 .............................................................................................................. 11
FilmArray Torch .......................................................................................................... 12
QUALITY CONTROL ...................................................................................................... 13
Process Controls ............................................................................................................ 13
Monitoring Test System Performance .......................................................................... 13
BIOFIRE® COVID-19 TEST EXTERNAL CONTROLS ........................................................... 14
External Controls ........................................................................................................... 14
BioFire® COVID-19 Test External Control Procedure (+) ................................................ 15
BioFire® COVID-19 Test External Control Procedure (-) ................................................. 16
INTERPRETATION OF RESULTS ..................................................................................... 17
Assay Interpretation ...................................................................................................... 17
Organism Interpretation ............................................................................................... 18
SARS-CoV-2 ................................................................................................................. 18

BioFire Defense, LLC BioFire® COVID-19 Test - Instructions for Use i

 BioFire® COVID-19 Test Report ...................................................................................... 19
Run Summary ............................................................................................................. 20
Result Summary.......................................................................................................... 20
Run Details ................................................................................................................. 22
Change Summary ....................................................................................................... 22
Analysis of BioFire COVID-19 Test External Control (+) Assays ..................................... 22
LIMITATIONS............................................................................................................... 24
CONDITIONS OF AUTHORIZATION FOR THE LABORATORY........................................... 24
PERFORMANCE CHARACTERISTICS .............................................................................. 26
Clinical Summary ........................................................................................................... 26
Testing of Archived Clinical Specimens ......................................................................... 26
Testing of Contrived Clinical Specimens........................................................................ 26
Testing of Pooled Clinical Specimens ............................................................................ 27
Limit of Detection .......................................................................................................... 28
FDA SARS-CoV-2 Reference Panel Testing .................................................................... 28
Analytical Reactivity (in silico Inclusivity) ...................................................................... 29
Analytical Specificity (Exclusivity) .................................................................................. 30
Interference .................................................................................................................. 32
SPECIMEN POOLING .................................................................................................... 34
Pooling Implementation ................................................................................................ 34
Pooling Monitoring ....................................................................................................... 35
APPENDIX A ................................................................................................................ 37
Symbols Glossary........................................................................................................... 37
APPENDIX B ................................................................................................................ 38
Contact and Legal Information ...................................................................................... 38
Customer and Technical Support ................................................................................ 38
APPENDIX C ................................................................................................................ 39
References..................................................................................................................... 39

BioFire Defense, LLC BioFire® COVID-19 Test - Instructions for Use ii

INTENDED USE
The BioFire® COVID-19 Test is a nested multiplexed RT-PCR test intended for the qualitative detection of nucleic
acid from SARS-CoV-2 in nasopharyngeal swabs in transport media from individuals suspected of COVID-19 by
their healthcare provider. Testing of non-pooled specimens is limited to laboratories certified under the Clinical
Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet the requirements to perform
high or moderate complexity tests, and similarly qualified U.S. Department of Defense (DoD) and non-U.S.
laboratories.

The BioFire COVID-19 Test is also for the qualitative detection of nucleic acid from SARS-CoV-2 in pooled
samples containing up to eight nasopharyngeal swabs collected individually in transport media from individuals
suspected of COVID-19 by their healthcare provider. Testing of pooled specimens is limited to DoD laboratories
that meet the requirements to perform high complexity tests.

Specimens should only be pooled in areas with low SARS-CoV-2 prevalence, and when testing demand exceeds
laboratory capacity or reagent availability. For pooled specimen testing, authorized laboratories will adhere to a
protocol for ongoing monitoring of the pooling strategy or limit testing to individuals who are subjected to a
detailed infection prevention and control plan.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally detectable in
nasopharyngeal swabs in transport media during the acute phase of infection. Positive results are indicative of
the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other diagnostic information is
necessary to determine patient infection status. Positive results do not rule out bacterial infection or co-infection
with other viruses. Pooled samples with positive or equivocal results must be tested individually prior to reporting
results. The agent detected may not be the definite cause of disease. Laboratories within the United States and
its territories are required to report all results to the appropriate public health authorities.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for patient
management decisions. Negative results must be combined with clinical observations, patient history, and
epidemiological information. Negative results from pooled samples should be reported as presumptive.
Specimens with low viral genetic material may not be detected in pooled samples due to decreased sensitivity. If
clinical signs and symptoms are inconsistent with a negative result or results are necessary for patient
management, the patient should be considered for individual testing.

The BioFire COVID-19 Test is intended for use by laboratory personnel who have received specific training on
the use of the FilmArray® 2.0 and/or the FilmArray® Torch Instrument Systems. The BioFire COVID-19 Test is
only for use under the Food and Drug Administration’s Emergency Use Authorization.

For In Vitro Diagnostic Use.

SUMMARY AND EXPLANATION OF THE TEST

The BioFire® COVID-19 Test is a qualitative test on the FilmArray® 2.0 or FilmArray® Torch systems for the
detection of the 2019 coronavirus (SARS-CoV-2) RNA in nasopharyngeal swabs (NPS) in transport media.
Internal controls are used to monitor all stages of the test process. The BioFire® COVID-19 Test External
Control Kit (+) includes positive external control material and may be used for quality control and laboratory
verification.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 1

PRINCIPLE OF THE PROCEDURE
The BioFire COVID-19 Test is a closed system disposable that stores all the necessary reagents for sample
preparation, reverse transcription, polymerase chain reaction (PCR), and detection in order to isolate, amplify,
and detect nucleic acid from the SARS-CoV-2 virus within a single NPS specimen. After sample collection, the
user injects hydration solution, and sample combined with sample buffer into the pouch, places the pouch into
a FilmArray instrument, and starts a run. The entire run process takes about 50 minutes. Additional details can
be found in the appropriate FilmArray operator’s manual.

During a run, the FilmArray® system:

• Lyses the sample by agitation (bead beading).
• Extracts and purifies all nucleic acids from the sample using magnetic bead technology.
• Performs nested multiplex PCR by:
o First performing reverse transcription and a single, large volume, multiplexed reaction (PCR1).
o Then performing multiple singleplex second-stage PCR reactions (PCR2) to amplify sequences
within the PCR1 products.
• Uses endpoint melting curve data to detect and generate a result for each target assay on the BioFire
COVID-19 Test.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 2

MATERIALS PROVIDED
Each BioFire COVID-19 Test Kit contains sufficient reagents to test 6 samples (6-test kit; 423745) or
30 samples (30-test kit; 423744):
• Individually-packaged BioFire COVID-19 Test pouches
• Single-use (1.0 mL) Sample Buffer tubes
• Single-use pre-filled (1.5 mL) Hydration Injection Vials (blue)
• Single-use Sample Injection Vials (red)
• Individually-packaged Transfer Pipettes
• Instructions and Documents
o BioFire COVID-19 Test – Quick Guide
Each BioFire COVID-19 Test External Control Kit (+) contains sufficient reagents for six positive control runs
(6-control kit; 423748). Negative controls may be run using only the BioFire COVID-19 Test with no additional
materials as described in BioFire COVID-19 Test External Control Procedure.
• Individually-packaged BioFire COVID-19 Test External Control (+) Vials
• Instructions and Documents
o BioFire COVID-19 Test External Control Kit – Quick Guide
NOTE: Optional verification protocol (for laboratory verification which may not use External Control Kit) is
available online at www.biofiredefense.com/covid-19test

MATERIALS REQUIRED BUT NOT PROVIDED

• FilmArray system including:
o FilmArray® 2.0/Torch Instrument Systems and accompanying software
o FilmArray® Pouch Loading Station
• 10% bleach solution or a similar disinfectant
• Transport media or 0.9% saline solution (for External Control Testing)
• BioFire COVID-19 Additional Documentation
o BioFire COVID-19 Test – Patient Fact Sheet
o BioFire COVID-19 Test – Healthcare Provider Fact Sheet
NOTE: Additional labeling documents are available online at www.biofiredefense.com/covid-19test

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 3

WARNINGS AND PRECAUTIONS

General Precautions
1. For in vitro diagnostic (IVD) use under Emergency Use Authorization only.
2. Positive results are indicative of the presence of SARS-CoV-2 RNA.
3. Testing of pooled specimens should only be performed in high complexity laboratories.
4. Laboratories within the Unites States and its territories are required to report all results to the appropriate
public health authorities.
5. The BioFire COVID-19 Test has not been FDA cleared or approved but has been authorized by FDA
under an Emergency Use Authorization (EUA) for use by laboratories which are certified under the
Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, and meet the
requirements to perform moderate and high complexity tests.
6. The BioFire COVID-19 Test has been authorized only for the detection of nucleic acid from SARS-CoV-
2, not for any other viruses or pathogens.
7. The BioFire COVID-19 Test is only authorized for the duration of the declaration that circumstances exist
justifying the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of
COVID-19 under Section 564(b)(1) of the Federal Food, Drug and Cosmetic Act, 21 U.S.C. § 360bbb-
3(b)(1), unless the declaration is terminated or authorization is revoked sooner..
8. BioFire COVID-19 Test pouches are only for use with FilmArray 2.0 and FilmArray Torch systems.
9. BioFire COVID-19 External Control Kit (+) is only for use with FilmArray 2.0 and FilmArray Torch
systems.
10. Always check the expiration date on the pouch. Do not use a pouch after its expiration date.
11. FilmArray pouches are stored under vacuum in individually-wrapped canisters. To preserve the integrity
of the pouch vacuum for proper operation, be sure that a FilmArray instrument/module will be available
and operational before unwrapping any pouches for loading.
12. Bleach introduced in a sample may damage nucleic acids in the sample, which may lead to a false
negative result.
13. If infection with SARS-CoV-2 is suspected based on current clinical and epidemiological screening
criteria recommended by public health authorities, specimens should be collected with appropriate
infection control precautions.

Safety Precautions
1. Wear appropriate Personal Protective Equipment (PPE), including (but not limited to) disposable clean
powder-free gloves. Protect skin, eyes, and mucus membranes. Change gloves often when handling
reagents or samples.
2. Handle all samples and waste materials as if they were capable of transmitting infectious agents.
Observe safety guidelines such as those outlined in:
• CDC/NIH Biosafety in Microbiological and Biomedical Laboratories1
• CLSI Document M29 Protection of Laboratory Workers from Occupationally Acquired Infections.2
• Refer to Interim Laboratory Safety Guidelines for Handling and Processing Specimens Associated
with SARS-CoV-2 www.cdc.gov/coronavirus/2019-nCoV/lab-biosafety-guidelines.html.
3. Follow your institution's safety procedures for handling biological samples.
4. Dispose of materials used in this assay, including reagents, samples, and used buffer tubes, according
to federal, state, and local regulations.
5. Sample Buffer is assigned the following classifications:
• Acute toxicity (Category 4),

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 4

 • Serious eye damage (Category 1), and
• Skin irritation (Category 2).
Please refer to the BioFire COVID-19 Test Safety Data Sheet (SDS) for more information.
6. Sample Buffer will form hazardous compounds and fumes when mixed with bleach or other disinfectants.
WARNING: Never add bleach to Sample Buffer or sample waste.
7. Bleach, a recommended disinfectant, is corrosive and may cause severe irritation or damage to eyes
and skin. Vapor or mist may irritate the respiratory tract. Bleach is harmful if swallowed or inhaled.
• Eye contact: Hold eye open and rinse with water for 15-20 minutes. Remove contact lenses after the
first 5 minutes and continue rinsing eye. Seek medical attention.
• Skin contact: Immediately flush skin with plenty of water for at least 15 minutes. If irritation develops,
seek medical attention.
• Ingestion: Do not induce vomiting. Drink a glassful of water. If irritation develops, seek medical
attention.
• Please refer to the appropriate Safety Data Sheet (SDS) for more information.

Laboratory Precautions
1. Preventing Organism Contamination
Due to the sensitive nature of the BioFire COVID-19 Test, it is important to guard against contamination of
the sample and work area by carefully following the testing process outlined in this instruction document,
including these guidelines:
• Do not handle samples or pouches in a biosafety cabinet which is used for SARS-CoV-2 culture or
immunofluorescence testing.
• Prior to processing samples, thoroughly clean both the work area and the FilmArray® Pouch Loading
Station using a suitable cleaner such as freshly prepared 10% bleach or a similar disinfectant. To avoid
residue buildup and potential damage to the sample or interference from disinfectants, wipe disinfected
surfaces with water.
• Samples and pouches should be handled and/or tested one-at-a-time. Always change gloves and
clean the work area between each pouch and sample.
• Use clean gloves to remove materials from bulk packaging bags and reseal bulk-packaging bags when
not in use.

2. Preventing Amplicon Contamination

A common concern with PCR-based assays is false positive results caused by contamination of the work
area with PCR amplicon. Because the BioFire COVID-19 Test pouch is a closed system, the risk of
amplicon contamination is low if pouches remain intact after the test is completed. Adhere to the following
guidelines, in addition to those above, to prevent amplicon contamination:
• Discard used pouches in a biohazard container immediately after the run has completed.
• Avoid excessive handling of pouches after test runs.
• Change gloves after handling a used pouch.
• Avoid exposing pouches or sample injection vials to sharp edges or anything that might cause a
puncture.
• Change gloves after loading the External Control (+) material.
• Clean thoroughly after loading the External Control (+) material to avoid contamination with the
External Control (+).

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 5

 WARNING: If liquid is observed on the exterior of a pouch, the liquid and pouch should be
immediately contained and discarded in a biohazard container. The instrument and workspace must
be decontaminated as described below and in the appropriate FilmArray operator’s manual.
DO NOT PERFORM ADDITIONAL TESTING UNTIL THE AREA HAS BEEN DECONTAMINATED.

Precaution Related to Public Health in the United States

Local, state, and federal regulations for notification of reportable disease are continually updated and include a
number of organisms/viruses for surveillance and outbreak investigations.3,4 Laboratories are responsible for
following their state and/or local regulations and should consult their local and/or state public health
laboratories for isolate and/or clinical sample submission guidelines.

REAGENT STORAGE, HANDLING, AND STABILITY

1. Store the test and control kit, including reagent pouches and provided buffers, at room temperature (15-
30°C). DO NOT REFRIGERATE.
2. Avoid storage of any materials near heating or cooling vents, or in direct sunlight.
3. All kit components should be stored and used together. Do not use components from one kit with those of
another kit. Discard any extra components from the kit after all pouches have been consumed.
4. Do not remove pouches from their packaging until a sample is ready to be tested. Once the pouch
packaging has been opened, the pouch should be loaded as soon as possible (within approximately 30
minutes).
5. Once a pouch has been loaded, the test run should be started as soon as possible (within approximately 60
minutes). Do not expose a loaded pouch to temperatures above 40°C (104°F) prior to testing.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 6

SAMPLE REQUIREMENTS
See below for the recommended requirements for specimen collection, preparation, and handling that will help
ensure accurate test results.

Specimen Type Nasopharyngeal Swab (NPS) collected according to standard

technique and immediately placed in 1-3 mL of transport media.

Minimum Specimen Volume 0.3 mL (300 µL) per test

Specimens should be processed and tested with the BioFire

COVID-19 Test as soon as possible.
If storage is required, samples can be held:
Transport and Storage
• At room temperature for up to 4 hours (15-25ºC)
• Refrigerated for up to 3 days (2-8ºC)
• Frozen (≤-15ºC or ≤-70ºC) for up to 30 days

NOTE: NPS specimens should not be centrifuged before testing.

NOTE: Bleach can damage organisms/nucleic acids within the specimen, potentially causing false negative
results. Contact between bleach and specimens during collection, disinfection, and testing procedures should
be avoided.

BIOFIRE® COVID-19 TEST PROCEDURE

Use clean gloves and other Personal Protective Equipment (PPE) when handling pouches and samples. Only
prepare one BioFire COVID-19 Test pouch at a time and change gloves between samples and pouches. Once
sample is added to the pouch, promptly transfer to the instrument to start the run. After the run is complete,
discard the pouch in a biohazard container.

Refer to the BioFire COVID-19 Test Quick Guide or the appropriate FilmArray operator’s manual for more
details.

Preparing Specimens for Pooling

Prior to considering specimen pooling, laboratories should evaluate pooling strategies based on population
positivity rates (see section below on Specimen Pooling Implementation and Monitoring). Nasopharyngeal swabs
which have been collected individually in transport media may be pooled. Pools of up to 8 specimens may be
tested on the BioFire COVID-19 Test. Perform the following procedure when pooling specimens for testing.

1. Obtain an empty collection tube (collection tube is not provided).

2. Determine the appropriate volume of each specimen to add to the pool based on the number of specimens
that will be pooled. The final volume of the pooled sample should be at least 750µL (to allow for one re-test
as needed). Each specimen to be included in the pool should contribute an equal volume. For example, if
pooling three specimens, 250 µL of each specimen should be pooled.
3. Transfer the determined volume of each individual specimen to the collection tube.
4. Mix the prepared sample pool.
5. Test the prepared sample pool according to the BioFire COVID-19 Test Procedure. Sample IDs should
indicate that the sample was pooled.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 7

 Step 1: Prepare Pouch
1. Thoroughly clean the work area and the FilmArray Pouch Loading Station with freshly prepared 10%
bleach (or suitable disinfectant) followed by a water rinse.
2. Remove the pouch from its vacuum-sealed package by tearing or cutting the notched outer packaging
and opening the protective aluminum canister.
NOTE: The pouch may still be used even if the vacuum seal of the pouch is not intact. Attempt to
hydrate the pouch using the steps in the Hydrate Pouch section. If hydration is successful, continue with
the run. If hydration fails, discard the pouch and use a new pouch to test the sample.

3. Check the expiration date on the pouch. Do not use expired products.
4. Insert the pouch into the FilmArray Pouch Loading Station, aligning
the red and blue labels on the pouch with the red and blue arrows on
the FilmArray Pouch Loading Station.
5. Place a Sample Injection Vial (with red cover) into the red well of the
FilmArray Pouch Loading Station.
6. Place a Hydration Injection Vial (with blue cover) into the blue well of
the FilmArray Pouch Loading Station.

Step 2: Hydrate Pouch

Unscrew the Hydration Injection Vial from the blue cover.
Remove the Hydration Injection Vial, leaving the blue cover in the FilmArray
Pouch Loading Station.
Insert the Hydration Injection Vial cannula tip into the pouch hydration port
located directly below the blue arrow of the FilmArray Pouch Loading Station.
Forcefully push down in a firm and quick motion to puncture seal until a faint
“pop” is heard and there is an ease in resistance. Wait as the correct volume of Hydration Solution is
pulled into the pouch by vacuum.
If the hydration solution is not automatically drawn into the pouch, repeat Step 2 to verify that the seal
of the pouch hydration port was broken. If hydration solution is again not drawn into the pouch, discard
the current pouch, retrieve a new pouch, and repeat from Step 1: Prepare Pouch.
Verify that the pouch has been hydrated.
Flip the barcode label down and check to see that fluid has entered the reagent wells (located at the
base of the rigid plastic part of the pouch). Small air bubbles may be seen.
If the pouch fails to hydrate (dry reagents appear as white pellets), repeat Step 2 to verify that the seal
of the pouch hydration port was broken. If hydration solution is still not drawn into the pouch, discard
the current pouch, retrieve a new pouch, and repeat from Step 1: Prepare Pouch.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 8

 Step 3: Prepare Sample Mix
1. Thoroughly mix the NPS sample by vortex or inversion.
2. Use the Transfer Pipette provided in the test kit to draw the sample to the third line (approximately 0.3 mL)
of the Transfer Pipette.
3. Add the sample to the Sample Injection Vial.
4. Discard the Transfer Pipette in a biohazard waste container.

NOTE: DO NOT use the Transfer Pipette to mix the sample once it is loaded into the
Sample Injection Vial.

5. Add Sample Buffer to the Sample Injection Vial.

• Hold the Sample Buffer Tube with the tip facing up.
NOTE: Avoid touching the tube tip during handling, as this may introduce contamination.
• Firmly pinch at textured plastic tab on the side of the tube until the seal snaps.
• Invert the tube over the red-capped Sample Injection Vial and dispense Sample
Buffer using a slow, forceful squeeze followed by a second squeeze.
NOTE: Avoid squeezing the tube additional times. This will generate foam, which
should be avoided.
WARNING: The Sample Buffer is harmful if swallowed and can
cause serious eye damage and skin irritation.

6. Tightly close the lid of the Sample Injection Vial.

7. Remove the Sample Injection Vial from the FilmArray Pouch Loading Station
and invert the vial at least 3 times to mix.
8. Return the Sample Injection Vial to the red well of the FilmArray Pouch
Loading Station.

Step 4: Load Sample Mix

1. Slowly twist to unscrew the Sample Injection Vial from the red cover and wait for 5 seconds with the vial
resting in the cover.

NOTE: Waiting 5 seconds decreases the risk of dripping and contamination from
the sample.

2. Lift the Sample Injection Vial, leaving the red cover in the well of the FilmArray
Pouch Loading Station, and insert the Sample Injection Vial cannula tip into the
pouch sample port located directly below the red arrow of the FilmArray Pouch
Loading Station.
3. Forcefully push down in a firm and quick motion to puncture seal (a faint “pop” is heard) and sample is
pulled into the pouch by vacuum.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 9

 4. Verify that the sample has been loaded.
• Flip the barcode label down and check to see that fluid has entered the reagent well next to the sample
loading port.
• If the pouch fails to pull sample from the Sample Injection Vial, the pouch should be discarded.
Retrieve a new pouch and repeat from Step 1: Prepare Pouch.
5. Discard the Sample Injection Vial and the Hydration Injection Vial in a biohazard sharps container.
6. Record the Sample ID in the provided area on the pouch label (or affix a barcoded Sample ID) and remove
the pouch from the FilmArray Pouch Loading Station.
NOTE: Optional added operator protection: Before removal from biosafety cabinet, run a bleach wipe, a
paper towel with 10% bleach (one part bleach to nine parts water), across the top of the pouch from the
hydration port to the sample port, and follow with a water wipe. This reduces the potential for contact with
small amounts of sample mixed with sample buffer that may be retained at the sample injection port.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 10

 Step 5: Run Pouch
The FilmArray Software includes step-by-step on-screen instructions that guide the operator through performing
a run. Brief instructions for FilmArray 2.0 and FilmArray Torch systems are given below. Refer to the appropriate
FilmArray operator’s manual for more detailed instructions.

FilmArray 2.0
1. Ensure that the FilmArray 2.0 system (instrument and computer) is powered on and the software is launched.
2. Follow on-screen instructions and procedures described in the FilmArray 2.0 operator’s manual to place
the pouch in an instrument, enter pouch, sample, and operator information.
3. Pouch identification (Lot Number and Serial Number), Pouch Type and Protocol, will be automatically
entered when the barcode is scanned. If it is not possible to scan the barcode, the pouch Lot Number,
Serial Number, and Pouch Type can be manually entered from the information provided on the pouch label
into the appropriate fields. To reduce data entry errors, it is strongly recommended that the pouch
information be entered by scanning the barcode.

NOTE: When selecting a Pouch Type manually, ensure that the Pouch Type matches the label on the
BioFire COVID-19 Test pouch.

4. Enter the Sample ID. The Sample ID can be entered manually or scanned in by using the barcode scanner
when a barcoded Sample ID is used.
5. Select and/or confirm the appropriate protocol for your sample type from the Protocol drop down list. The
BioFire COVID-19 Test has a single NPS2 protocol available from the drop down list.
6. Enter a user name and password in the Name and Password fields.

NOTE: The font color of the user name is red until the user name is recognized by the software.

7. Review the entered run information on the screen. If correct, select Start Run.

Once the run has started, the screen displays a list of the steps being performed by the instrument and the
number of minutes remaining in the run.

NOTE: The bead-beater apparatus can be heard as a high-pitched noise during the first minute of
operation.

8. When the run is finished, follow the on-screen instructions to remove the pouch, then immediately discard it
in a biohazard waste container.
9. The run file is automatically saved in the FilmArray database, and the test report can be printed, viewed,
and/or saved as a PDF file.
10. To view run data, double click on a run file, select the interpretation tab and click on a specific assay result.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 11

 FilmArray Torch
1. Ensure that the FilmArray Torch system is powered on.
2. Select an available Module (instrument) on the touch screen or scan the barcode on the FilmArray pouch
using the barcode scanner.
3. Pouch identification (Lot Number and Serial Number), Pouch Type, and Protocol information will be
automatically entered when the barcode is scanned. If it is not possible to scan the barcode, the pouch Lot
Number, Serial Number, Pouch Type, and Protocol can be manually entered from the information provided
on the pouch label into the appropriate fields. To reduce data entry errors, it is strongly recommended that
the pouch information be entered by scanning the barcode.
NOTE: When selecting a Pouch Type manually, ensure that the Pouch Type matches the label on the
BioFire COVID-19 Test pouch.
4. Enter the Sample ID. The Sample ID can be entered manually or scanned in by using the barcode scanner
when a barcoded Sample ID is used.
5. Insert the pouch into the available Module (instrument).
Ensure that the pouch fitment label is lying flat on top of pouch and not folded over. As the pouch is
inserted, the Module (instrument) will grab onto the pouch and pull it into the chamber.
6. If necessary, select and/or confirm the appropriate protocol for your sample type from the Protocol drop
down list. The BioFire COVID-19 Test has a single NPS2 protocol available from the drop down list.
7. Enter operator user name and password, then select Next.

NOTE: The font color of the user name is red until the user name is recognized by the software.

8. Review the entered run information on the screen. If correct, select Start Run.

Once the run has started, the screen displays a list of the steps being performed by the Module
(instrument) and the number of minutes remaining in the run.

NOTE: The bead-beater apparatus can be heard as a high-pitched noise during the first minute of
operation.

9. At the end of the run, remove the partially ejected pouch, then immediately discard it in a biohazard waste
container.
10. The run file is automatically saved in the FilmArray database, and the test report can be viewed, printed,
and/or saved as a PDF file.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 12

QUALITY CONTROL

Process Controls
Two process controls are included in each pouch:

1. RNA Process Control

The RNA Process Control assay targets an RNA transcript from the yeast Schizosaccharomyces pombe.
The yeast is present in the pouch in a freeze-dried form and becomes rehydrated when sample is loaded.
The control material is carried through all stages of the test process, including lysis, nucleic acid
purification, reverse transcription, PCR1, dilution, PCR2, and DNA melting. A positive control result
indicates that all steps carried out in the BioFire COVID-19 Test were successful.

2. PCR2 Control
The PCR2 Control assay detects a DNA target that is dried into wells of the array along with the
corresponding primers. A positive result indicates that PCR2 was successful.

Both control assays must be positive for the test run to pass. If controls fail, the sample should be retested
using a new pouch.

Monitoring Test System Performance

The FilmArray software will automatically fail the run if the melting temperature (Tm) for either the RNA
Process Control or the PCR2 Control is outside of an acceptable range (80.3-84.4°C for the RNA Process
Control and 73.8-78.2°C for the PCR2 Control). If required by local, state, or accrediting organization quality
control requirements, users can monitor the system by trending Tm values for the control assays and
maintaining records according to standard laboratory quality control practices.5, 6 Refer to the appropriate
FilmArray operator’s manual for instructions on obtaining control assay Tm values. The PCR2 Control is used
in several FilmArray pouch types (e.g., RP2, BCID, GI, ME) and can therefore be used to monitor the system
when multiple pouch types are used on the same FilmArray system or instrument.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 13

BIOFIRE® COVID-19 TEST EXTERNAL CONTROLS

External Controls
For quality control and laboratory test verification, BioFire Defense provides an optional external positive assayed
control kit to monitor the performance of in vitro laboratory nucleic acid testing procedures for the qualitative
detection of the BioFire COVID-19 Test performed on FilmArray 2.0 and FilmArray Torch systems. Offered
separately, the BioFire COVID-19 Test External Control (+) kit is a surrogate control material comprised of dried
synthetic RNA in buffer and stabilizer, supplied in an External Control Vial that is used directly with the BioFire
COVID-19 Test. The BioFire COVID-19 Test External Control (+) Kit contains six (6) BioFire COVID-19 Test
External Control (+) Vials. The RNA in the external control includes RNA segments to monitor whether the PCR
primers for each SARS-CoV-2 assay are present for both stages of the nested PCR.

The BioFire COVID-19 Test External Control (+) Kit contains no biological hazards and is 100% non-infectious.
This control is stored at 15-30°C. To run a positive external control, reference BioFire COVID-19 Test External
Control Procedure (+) below or BioFire COVID-19 External Control Kit (+) Quick Guide. To run a negative
external control, use the BioFire COVID-19 Test and reference BioFire COVID-19 External Control Procedure (-)
below.

An optional verification protocol may be obtained from the BioFire Defense Product Support webpage
www.biofiredefense.com/covid-19test. Customers may also use these BioFire COVID-19 Test External Control
Kit (+) or an alternate Quality Control material for verification testing.

Good laboratory practice recommends running positive and negative external controls regularly. Evaluation of
external controls is recommended prior to using a new shipment or new lot of BioFire COVID-19 Test Kits.
Evaluation of external controls is also recommended when there is a new operator, and following
replacement/repair of a FilmArray® 2.0 or FilmArray® Torch system.

External controls may also be used in initial laboratory validations of the FilmArray 2.0 or FilmArray Torch system
used with the BioFire COVID-19 Test in accordance with appropriate federal, state, and local guidelines or
accreditation requirements, as applicable.

It is ultimately the responsibility of each laboratory to determine the frequency and type of material used for
external control testing as part of the laboratory’s Quality Control program.

Information on how to obtain optional external control material is posted on the BioFire Defense webpage.
BioFire COVID-19 Test External Control Kit (+)
Part Number: 423748

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 14

 BioFire® COVID-19 Test External Control Procedure (+)
1. Follow Step 1 and Step 2 from the BioFire COVID-19 Test Procedure to prepare and hydrate the
pouch.
2. Use the Transfer Pipette provided in the test kit to draw the transport media or saline to the third
line (approximately 0.3mL) of the Transfer Pipette. Add to the Sample Injection Vial.
3. Using clean gloves, obtain a Sample Buffer Tube from the BioFire COVID-19 Test Kit.
4. Uncap the External Control Vial (+) and place the cap on a clean surface (a paper towel
may be used).
5. Add Sample Buffer to the External Control Vial (+).
• Hold the Sample Buffer Tube tip facing up and firmly pinch at textured plastic tab
on the side of the tube until the seal snaps.
• Invert the Sample Buffer tube over the uncapped External Control Vial (+)
and dispense Sample Buffer using a slow, forceful squeeze followed by a
second squeeze.

NOTE: Avoid generating excessive foam.

6. Recap the External Control Vial (+) and mix by gently inverting three (3) times.
7. Pour the rehydrated External Control (+) into the Sample Injection Vial and
immediately dispose of the External Control Vial (+).
• Change gloves.
8. Tightly close lid of Sample Injection Vial and mix by gently inverting at least three (3)
times.
9. Return Sample Injection Vial to red well of Pouch Loading Station.
10. Continue at Step 4 of the BioFire COVID-19 Test Panel Procedure to load the
pouch and run it on the FilmArray.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 15

 BioFire® COVID-19 Test External Control Procedure (-)
1. Follow Step 1 and Step 2 from the BioFire COVID-19 Test Procedure to prepare and hydrate the
pouch.
2. Use the Transfer Pipette provided in the test kit to draw the transport media or saline to
the third line (approximately 0.3mL) of the Transfer Pipette. Add to the Sample Injection
Vial.
3. Using clean gloves, obtain a Sample Buffer Tube from the BioFire COVID-19 Test Kit.
4. Add Sample Buffer to the Sample Injection Vial.
• Hold the Sample Buffer Tube tip facing up and firmly pinch at textured plastic tab on the
side of the tube until the seal snaps.
• Invert the Sample Buffer Tube over the Sample Injection Vial and dispense Sample Buffer
using a slow, forceful squeeze followed by a second squeeze.

NOTE: Avoid generating excessive foam.

5. Tightly close the lid of the Sample Injection Vial.

6. Remove the Sample Injection Vial from the FilmArray Pouch Loading Station
and invert the vial at least three (3) times to mix.

7. Return the Sample Injection Vial to the red well of the FilmArray Pouch
Loading Station.
8. Continue at Step 4 of the BioFire COVID-19 Test Procedure to load the
pouch and run the FilmArray.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 16

INTERPRETATION OF RESULTS
The BioFire COVID-19 Test consists of three independent and non-overlapping assays targeting two SARS-CoV-
2 open reading frame sequences: ORF1ab and ORF8. The target of each assay is shown in Table 1 below. The
assays are designed to detect SARS-CoV-2 specifically. Detection of SARS-CoV-2 is based on the combined
results of the three assays as described below.

Table 1. Gene targets for assays on the BioFire COVID-19 Test.

Assay Name SARS-COV-2 Genomic Region

SARS-CoV-2a ORF1ab
SARS-CoV-2d ORF1ab
SARS-CoV-2e ORF8

Assay Interpretation
When PCR2 is complete, the FilmArray instrument performs a DNA melting analysis on the PCR products and
measures the fluorescence signal generated in each well (for more information see appropriate FilmArray
operator’s manual). The FilmArray Software then performs several analyses and assigns a final assay result
for every well. The steps in the analyses are described below.
Analysis of Melt Curves. The FilmArray Software evaluates the DNA melt curve for each well of the PCR2 array
to determine if a PCR product was present in that well. If the melt profile indicates the presence of a PCR
product, then the analysis software calculates the melting temperature (Tm) of the curve and compares it against
the expected Tm range for the assay. If the software determines that the Tm of the curve is within the assay
specification Tm range, the melt curve is called positive. If the software determines that the Tm of the curve is not
in the appropriate Tm range, the melt curve is called negative.
Analysis of Replicates. Once positive melt curves have been identified, the software evaluates the three
replicates for each assay to determine the assay result. For an assay to be called positive, at least two of the
three associated melt curves must be called positive, and the Tm for at least two of the three positive melt curves
must be similar (within 1.0°C). Assays that do not meet these criteria are called negative.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 17

 Organism Interpretation
SARS-CoV-2
The BioFire COVID-19 Test contains three different assays (SARS-CoV-2a, SARS-CoV-2d, SARS-CoV-2e) for
the detection of SARS-CoV-2. The FilmArray Software interprets each of these assays independently (as
described above) and the results are combined as a final test result for the virus. For interpretation of the
results, refer to Table 2 if testing individual specimens, or Table 3 if testing pooled specimens.

Interpretation When Testing Individual Specimens

If two or more assays are ‘Detected’, the result on the test report will be SARS-CoV-2 ‘Detected’. If all assays
are ‘Not Detected’, the result on the test report will be SARS-CoV-2 ‘Not Detected’. If only one of the assays is
‘Detected’, the test report result will be SARS-CoV-2 ‘Equivocal’. If an ‘Equivocal’ result is obtained, retest the
original sample using a new pouch. If the result of the retest is ‘Equivocal’ or ‘Detected’, the overall
interpretation will be ‘Detected’. If the retest is ‘Not Detected’, seek confirmatory testing. In cases where either
or both the control assays have failed, all results are reported as ‘Invalid’ and retesting is required.

Table 2. Interpretation Rules for Individual Specimens

SARS-CoV-2 Interpretation Assay Results Action

3/3 Assays ‘Detected’

Detected Report the Results
2/3 Assays ‘Detected’

Retest the original sample and report the

results of the retest. If the result of the retest is
Equivocal 1/3 Assays ‘Detected’ ‘Equivocal’ or ‘Detected’, the overall
interpretation will be ‘Detected’. If the retest is
‘Not Detected’, seek confirmatory testing.

Not Detected 0/3 Assays ‘Not Detected’ Report the Results

Retest the original sample. If repeated errors

Invalid Invalid occur, contact the BioFire Defense Customer
Support Team.

Interpretation When Testing Pooled Specimens

If two or more assays are ‘Detected’, the test report result will be SARS-CoV-2 ‘Detected’. If only one of the
assays is ‘Detected’, the test report result will be SARS-CoV-2 ‘Equivocal’. If either a ‘Detected’ or an
‘Equivocal’ result is obtained, individual reflex testing must be performed (each specimen included in the
pooled sample must be tested following individual testing procedure). If all assays are ‘Not Detected’, the result
on the test report will be SARS-CoV-2 ‘Not Detected’. A ‘Not Detected’ result should be considered
presumptive. Specimens with low viral loads may not be detected when pooling samples due to decreased
sensitivity. If clinical signs and symptoms are inconsistent with a negative result, the patient should be
considered for individual testing. In cases where either or both control assays have failed, all results are
reported as ‘Invalid’ and the pooled sample should be retested. If the result of the retest is ‘Invalid’, each
specimen included in the pooled sample should be retested individually.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 18

 Table 3. Interpretation Rules for Pooled Samples

SARS-CoV-2 Interpretation Assay Results Action

Perform individual specimen reflex testing.

3/3 Assays ‘Detected’
Detected Retest all specimens included in the sample
2/3 Assays ‘Detected’
pool individually.

Perform individual specimen reflex testing.

Equivocal 1/3 Assays ‘Detected’ Retest all specimens included in the sample
pool individually.

Not Detected 0/3 Assays ‘Not Detected’ Report the Results

Retest sample pool. If sample pool fails a

second time, retest individual specimens. See
Table 4, Interpretation of Internal Controls
Invalid Invalid
Field on the BioFire Test Report for
instruction. If repeated errors occur, contact
the BioFire Defense Customer Support Team.

BioFire® COVID-19 Test Report

The BioFire COVID-19 test report is automatically displayed upon completion of a run and can be printed or
saved as a PDF file. Each report contains a Run Summary, a Result Summary, and a Run Details section.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 19

 Run Summary
The Run Summary section of the test report provides the Sample ID, time and date of the run, internal control
results, and an overall summary of the test results. If the SARS-CoV-2 interpretation is ‘Detected’, it will be
listed in the ‘Detected’ field. If all of the assays are ‘Not Detected’ then ‘None’ will be displayed in the Detected
field. Internal controls are listed as ‘Passed’, ‘Failed’, or ‘Invalid’. Table 4 provides additional information for
each of the possible internal control field results.

Table 4. Interpretation of Internal Controls Field on the BioFire COVID-19 Test Report
Internal
Controls Explanation Action
Result
The run was successfully
completed
None
Passed AND Report the results provided on the test
report.
Both pouch controls were
successful.
The run was successfully
completed

BUT Repeat the test using a new pouch.

Failed If the error persists, contact BioFire Defense Customer
At least one of the pouch Support for further instruction.
controls (RNA Process
Control and/or PCR2
Control) failed.
Note any error codes displayed during the run and the Run
The controls are invalid
Status field in the Run Details section of the report. Refer to
because the run did not
the appropriate FilmArray operator’s manual or contact BioFire
Invalid complete.
Defense Customer Support for further instruction.
(Typically, this indicates a
Once the error is resolved, repeat the test or repeat the test
software or hardware error.)
using another instrument.

Result Summary
The Result Summary section of the test report lists the result for the overall target and each individualized
assay result. Possible results for each assay are ‘Detected’, ‘Equivocal’, ‘Not Detected’, or ‘Invalid’. Table 5
provides an explanation for each interpretation and any follow-up necessary to obtain a final result. The SARS-
CoV-2 target and the three associated assays are listed in the Results Summary section. According to the
result for the target, ‘Detected’, ‘Not Detected’, ‘Equivocal’, or ‘Invalid’ will be indicated to the left of the target
name. According to the result for each associated assay, ‘Detected’, ‘Not Detected’, or ‘Invalid’ will be
indicated to the left of each assay name.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 20

 Table 5. Reporting of Results and Required Actions

SARS-CoV-2
Explanation Action
Results
The run was successfully completed

AND Individual Specimen: Report results.

The pouch controls were successful (Passed)

Detected
AND
Sample Pool: Perform individual
Two or three assays for the virus were ‘Detected’ specimen reflex testing. Retest all
(i.e., met the requirements for a positive result specimens included in the sample pool
described in the Assay Interpretation section individually.
above)
The run was successfully completed

AND Individual Specimen: Report results.

The pouch controls were successful (Passed)

Not Detected AND

The three assays for the virus were ‘Not

Detected’ Sample Pool: Report results.
(i.e., did not meet the requirements for a positive
result described in the Assay Interpretation
section above)

The run was successfully completed Individual Specimen: Retest the

original specimen using a new pouch
AND and report the results of the retest. If
the retest is ‘Equivocal’ or ‘Detected’,
report the results as ‘Detected’. If the
The pouch controls were successful (Passed)
result is ‘Not Detected’ seek
Equivocal confirmatory testing.
AND
Sample Pool: Perform individual
Only one of three assays was ‘Detected’ for the specimen reflex testing. Retest all
virus. The combination of ‘Detected’ and ‘Not specimens included in the sample pool
Detected’ assay results were inconclusive individually.

Individual Specimen: See Table 4,

The pouch controls were not successful (Failed) Interpretation of Internal Controls Field
on the BioFire Test Report for
OR instruction.
Invalid Sample Pool: Retest sample pool. If
The run was not successful sample pool fails a second time, retest
(Run Status displayed as: Aborted, Incomplete, individual specimens. If repeated errors
Instrument Error or Software Error) occur, contact the BioFire Defense
Customer Support Team.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 21

 Run Details
The Run Details section provide additional information about the run including: pouch information (type, lot
number, and serial number), run status (Completed, Incomplete, Aborted, Instrument Error, Instrument
Communication Error, or Software Error), the protocol that was used to perform the test, the identity of the
operator that performed the test, and the instrument used to perform the test.

Change Summary
It is possible to edit the Sample ID once a run has completed. If this information has been changed, an
additional section called Change Summary will be added to the test report. This Change Summary section
lists the field that was changed, the original entry, the revised entry, the operator that made the change, and
the date that the change was made. Sample ID is the only field of the report that can be changed.

Analysis of BioFire COVID-19 Test External Control (+) Assays

The BioFire COVID-19 External Control (+) passes when all three SARS-CoV-2 assays are ‘Detected’.
Positivity is evaluated by opening the report and confirming that ‘Detected’ is indicated to the left of each of the
three assay names listed in the Result Summary.
If any of the three SARS-CoV-2 assays have a ‘Not Detected’ result, the External Control (+) fails and should
be repeated. If the failure persists, contact BioFire Defense Customer Support for further instruction. Refer to
Table 6 for interpreting the report Result Summary.
Laboratories may decide to perform negative control testing on the system. In this case, after testing negative
material (e.g., transport media or saline), the user should open the report and confirm that ‘Not Detected’ is
indicated to the left of all three assay names listed in the Result Summary. If any of the three SARS-CoV-2
assays have a ‘Detected’ result, the External Control (-) fails and should be repeated after a thorough cleaning
of the area. If the error persists, contact BioFire Defense Customer Support for further instruction.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 22

 Table 6. BioFire COVID-19 Test External Control (+) Interpretation of Results
Result Interpretation Result Analysis Action

External Control (+)

Report the results.
Passes

Repeat External Control (+)

Testing.
BioFire COVID-19 External
Test Control (+) Failed Result If the error persists, contact
BioFire Defense Customer
Support for further instruction.

Repeat External Control (+)

Testing.
BioFire COVID-19 External
Control (+) Invalid Result If the error persists, contact
BioFire Defense Customer
Support for further instruction.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 23

LIMITATIONS
1. For In Vitro Diagnostic (IVD) Use under Emergency Use Authorization (EUA).
2. BioFire COVID-19 Test performance has only been established on the FilmArray 2.0 and FilmArray Torch
systems.
3. The BioFire COVID-19 Test is a qualitative test and does not provide a quantitative value for the virus in
the sample.
4. The BioFire COVID-19 Test has not been validated for testing of samples other than nasopharyngeal swab
(NPS) specimens in transport media.
5. A false negative BioFire COVID-19 Test result may occur when the concentration of virus in the sample is
below the device limit of detection.
6. The detection of viral nucleic acid is dependent upon proper sample collection, handling, transportation,
storage and preparation. Failure to observe proper procedures in any one of these steps can lead to
incorrect results.
7. There is a risk of false positive and false negative results caused by improperly collected, transported, or
handled samples. The RNA process control and the PCR2 control will not indicate whether or not nucleic
acid has been lost due to inadequate collection, transport, or storage of samples.
8. As with any molecular test, mutations within the target regions of SARS-CoV-2 could affect primer binding,
resulting in failure to detect the presence of virus.
9. All three assays show 80% or greater homology to Bat coronavirus RaTG13 (accession: MN996532). In
addition, the SARS-CoV-2e assay shows greater than 80% homology to Pangolin coronavirus isolate
MP789 (accession: MT084071). It is unlikely that these isolates would be found in our sample matrix of
nasopharyngeal swabs; however, little is known about their potential to infect a human host, or their
evolutionary relationship to SARS-CoV-2.

CONDITIONS OF AUTHORIZATION FOR THE LABORATORY

The BioFire COVID-19 Test Letter of Authorization, along with the authorized Fact Sheet for Healthcare Providers,
the authorized Fact Sheet for Patients and authorized labeling are available on the FDA website:
https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergency-use-
authorizations#covid19ivd.

To assist clinical laboratories running the BioFire COVID-19 Test, the relevant Conditions of Authorization are
listed below, and are required to be met by laboratories performing the EUA test.

A. Authorized laboratories1 using the BioFire COVID-19 Test will include with test result reports, all
authorized Fact Sheets. Under exigent circumstances, other appropriate methods for disseminating these
Fact Sheets may be used, which may include mass media.

B. Authorized laboratories using the BioFire COVID-19 Test will use the BioFire COVID-19 Test as outlined
in the Instructions for Use. Deviations from the authorized procedures, including the authorized
instruments, authorized extraction methods, authorized clinical specimen types, authorized control
materials, authorized other ancillary reagents and authorized materials required to use the BioFire
COVID-19 Test are not permitted.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 24

 C. Authorized laboratories that receive the BioFire COVID-19 Test will notify the relevant public health
authorities of their intent to run your product prior to initiating testing.

D. Authorized laboratories using the BioFire COVID-19 Test will have a process in place for reporting test
results to healthcare providers and relevant public health authorities, as appropriate.

E. Authorized laboratories will collect information on the performance of the BioFire COVID-19 Test and
report to DMD/OHT7-OIR/OPEQ/CDRH (via email: [email protected]) and BioFire
Defense Product Support website https://www.biofiredefense.com/product-support/filmarray-
support/adverse-reporting-biofire-covid19-test/ any suspected occurrence of false positive or false
negative results and significant deviations from the established performance characteristics of the BioFire
COVID-19 Test of which they become aware.

F. All laboratory personnel using the BioFire COVID-19 Test must be appropriately trained in RT-PCR
techniques and use appropriate personal protective equipment when handling this kit, and use the BioFire
COVID-19 Test in accordance with the authorized labeling.

G. For pooled specimen testing, authorized laboratories will adhere to a protocol for ongoing monitoring of
the pooling strategy or limit testing to individuals who are subjected to a detailed infection prevention and
control plan.

H. Authorized laboratories using specimen pooling strategies when testing patient specimens with the
BioFire COVID-19 Test will include with test result reports for specific patients whose specimen(s) were
the subject of pooling, a notice that pooling was used during testing and that “Patient specimens with low
viral loads may not be detected in sample pools due to the decreased sensitivity of pooled testing.”

I. Authorized laboratories implementing pooling strategies for testing patient specimens must use the
“Specimen Pooling Implementation and Monitoring” recommendations available in the authorized labeling
to evaluate the appropriateness of continuing to use such strategies based on the recommendations in the
protocol.

J. Authorized laboratories will keep records of specimen pooling strategies implemented including type of
strategy, date implemented, and quantities tested, and test result data generated as part of the “Specimen
Pooling Implementation and Monitoring”. For the first 12 months from the date of their creation, such
records will be made available to FDA within 48 business hours for inspection upon request, and will be
made available within a reasonable time after 12 months from the date of their creation.

K. BioFire Defense, LLC, authorized distributors, and authorized laboratories using the BioFire COVID-19
Test will ensure that any records associated with this EUA are maintained until otherwise notified by FDA.
Such records will be made available to FDA for inspection upon request.
1
For ease of reference, the letter of authorization refers to “authorized laboratories” as follows: Testing of non-pooled specimens is limited to
laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet the requirements to
perform high or moderate complexity tests, and similarly qualified U.S. Department of Defense (DoD) and non-U.S. laboratories. Testing of
pooled specimens is limited to DoD laboratories that meet the requirements to perform high complexity tests.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 25

PERFORMANCE CHARACTERISTICS

Clinical Summary
The clinical performance was evaluated by testing individual archived nasopharyngeal swab specimens collected
in transport media. The overall clinical performance including only individual clinical specimens is summarized in
Table 7. The studies contributing to this summary are detailed below.

Table 7. Summary of Individual Clinical Specimens Evaluated with the BioFire COVID-19 Test
PPA % NPA %
Agreement 29/30 96.7% 71/71 100%
95% CI [83.3-99.4%] [94.9-100%]

Testing of Archived Clinical Specimens

Clinical testing was performed using ten positive and five negative NPS specimens stored in transport media. The
positive samples were collected from patients presenting with signs or symptoms of COVID-19, and previously
identified as positive for SARS-CoV-2 by another test (nine specimens were determined positive by a validated
laboratory developed test (NECOV19) and one was determined positive by the Roche cobas SARS-CoV-2 EUA
Test). The negative samples were collected in 2018, and therefore were presumed negative for SARS-CoV-2. All
samples were de-identified before testing on the BioFire COVID-19 Test.

Positive Percent Agreement (PPA) was calculated as 100% x (TP / (TP +FN)). Negative Percent Agreement
(NPA) was calculated as 100% x (TN / (TN+FP)). Nine of out ten positive samples were Detected by the BioFire
COVID-19 Test and five out of five negative samples were Not Detected, resulting in 90% PPA and 100% NPA
(Table 8).

Table 8. BioFire COVID-19 Test Performance Summary

PPA % NPA %
Agreement 9/10 a 90.0% 5/5 100%
95% CI [59.6-98.2%] [56.6-100%]
a
FN specimen had a late Ct value when originally evaluated on the NECOV19 test. When the FN sample was
retested on the NECOV19 test, the result was negative. These results indicate a near-LoD level of SARS-CoV-2
virus and/or sample degradation after the first NECOV19 test and prior to the BioFire COVID-19 Test.

Testing of Contrived Clinical Specimens

Contrived testing was performed using 4 unspiked negative clinical specimens and 30 negative contrived clinical
specimens spiked with live SARS-CoV-2 virus (cultured from the USA_WA1/2020 strain obtained from World
Reference Center for Emerging Viruses and Arboviruses (WRCEVA)). The thirty (30) individual unique clinical
samples were contrived at 1× LoD (N=20), 10× LoD (N=5), and at 100× LoD (N=5), and tested with the four (4)
negative (unspiked) specimens. These 34 samples were tested randomized and in a blinded fashion. In addition,
sixty-two (62) additional negative individual unique clinical specimens were also evaluated. All test results were
as expected. The Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) was determined by
comparing the observed test results to the expected result. PPA and NPA are shown in Table 9.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 26

 Table 9. Clinical Contrived and Negative Testing with the BioFire COVID-19 Test
Agreement with Known Analyte
Composition
PPA % NPA %
Agreement 30/30 100% 66/66a 100%

95% CI [88.6 – 100] [94.5-100]

a
For comparison, 52 of the 66 negative samples were evaluated on the FilmArray 2.0 and FilmArray Torch systems. No
unexpected results were observed on either system. The NPA was 100% for the negative samples on both systems.

Testing of Pooled Clinical Specimens

Archived specimens previously characterized as part of standard of care were used in testing. Twenty (20)
specimens that returned ‘SARS-CoV-2 Detected’ results when tested on the CDC 2019-nCoV test were selected
to represent a range of clinically relevant concentrations based on Ct values. An additional 160 specimens that
returned ‘SARS-CoV-2 Not Detected’ results when tested on the CDC 2019-nCoV test were also selected.

Positive specimens were re-tested individually on the BioFire COVID-19 Test. Single individual positive
specimens were combined with the negative specimens in pools of 5 and 8 specimens. Twenty pools of each
size were tested. Pooled test results were compared to individual test results to evaluate the effect of pooling on
SARS-CoV-2 detection. Results are shown in Table 10.

Table 10. Detection of SARS-CoV-2 in Pools of 5 or 8 Specimens (Binned by Ct Value)

Ct Value a Individual Known
Pools of 5 Specimens Pools of 8 Specimens
Bins for Positive Samples
Positive Detection Rate Detection Rate Detection Rate
Samples 95% CI 95% CI 95% CI
(PPA) (PPA) (PPA)
5/5 5/5 5/5
Ct ≥ 35 56.6-100% 56.6-100% 56.6-100%
(100%) (100%) (100%)
5/5 5/5 4/5
30 ≤ Ct < 35 56.6-100% 56.6-100% 37.6-96.4%
(100%) (100%) (80%)
5/5 5/5 5/5
25 ≤ Ct < 30 56.6-100% 56.6-100% 56.6-100%
(100%) (100%) (100%)
5/5 5/5 5/5
Ct < 25 56.6-100% 56.6-100% 56.6-100%
(100%) (100%) (100%)
20/20 20/20 19/20
Overall 83.9-100% 83.9-100% 76.4-99.1%
(100%) (100%) (95%)
a
Ct values are from reconfirmation testing with the CDC 2019-nCoV test.

SARS-CoV-2 was detected by the BioFire COVID-19 Test in 20/20 (100% PPA) of the pools of 5 specimens and
in 19/20 (95% PPA) of the pools of 8 specimens. For the single 8-pooled sample run in which SARS-CoV-2 was
not detected, the positive specimen included in this pool had late amplification when tested individually and when
included in a pool of 5 specimens, indicating analyte levels near the Limit of Detection (LoD).

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 27

 Limit of Detection
Tentative Limit of Detection (LoD) for the BioFire COVID-19 Test was determined by testing three-fold dilutions
of quantified live SARS-CoV-2 virus (cultured from the USA_WA1/2020 strain obtained from World Reference
Center for Emerging Viruses and Arboviruses (WRCEVA)). Viral genomic copies per mL (GC/mL) for SARS-
CoV-2 virus stock was determined by quantitative RT-PCR using the WHO protocol
(https://www.who.int/docs/default-source/coronaviruse/protocol-v2-1.pdf). Subsequent testing at the tentative
LoD was conducted to confirm reliable detection (≥95%) at LoD, and loss of detection (<95%) when tested 10-
fold below LoD (0.1×LoD). LoD testing was performed using the FilmArray 2.0 system. Results for LoD testing
are shown in Table 11. The LoD was determined to be 3.3E+02 GC/mL (2.2E-02 TCID50/mL), with a detection
rate of 20/20 at 1×LoD, and 14/20 at 0.1×LoD (3.3E+01 GC/mL; 2.2E-03 TCID50/mL).

Table 11. SARS-CoV-2 Virus Test Results at 1× and 0.1× LoD for the BioFire COVID-19 Test
Concentration Tested Test Result (% Detection)
×LoD
Genomic Copies/mL TCID50/mL Detected Equivocal Not Detected
1× 3.3E+02 2.2E-02 20/20 (100%) 0/20 (0%) 0/20 (0%)

0.1× 3.3E+01 2.2E-03 14/20 (70%) 5/20 (25%) 1/20 (5%)

FDA SARS-CoV-2 Reference Panel Testing

SARS-CoV-2 sensitivity and MERS-CoV cross-reactivity were evaluated using the FDA SARS-CoV-2
Reference Panel according to the standard protocol provided by the US FDA. The evaluation was performed
using reference material (T1) and blinded samples. The study included a range finding study and a
confirmatory study for LoD. Blinded sample testing was used to establish specificity and to confirm the LoD.
The product LoD when using the FDA Reference Panel is presented in Table 12. No cross-reactivity with
MERS-CoV was reported.

Table 12: Summary of LoD Confirmation Result using the FDA SARS-CoV-2 Reference Panel
Reference Materials Cross-
Specimen Type Product LoD
Provided by FDA Reactivity
SARS-CoV-2 5.4E+03 NDU/mL1 N/A2
NPS in transport medium
MERS-CoV N/A2 ND3
1
NDU: Nucleic acid amplification test (NAAT) Detectable Units
2
N/A: Not applicable
3
ND: Not detected

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 28

 Analytical Reactivity (in silico Inclusivity)
Inclusivity of the BioFire COVID-19 Test was analyzed in silico using bioinformatics to align all unique genomes
from both NCBI and GISAID EpiCoV databases.

As of March 16, 2020, the majority of assay primers showed 100% homology to all available SARS-CoV-2
genome sequences. Four primers, across 2 of the 3 BioFire COVID-19 Test assays, showed single mismatches
to sequences of SARS-CoV-2. In the majority of these cases these were single isolate sequences. In both
assays the mismatches were single base pair differences. Such mismatches are well tolerated on the BioFire
platform and the assays are predicted to be reactive in the BioFire COVID-19 test despite such mismatches.

Analytical Specificity (in silico Exclusivity)

An in silico analysis was performed on the organisms listed in Table 13.

Table 13. Organisms Tested for Evaluation of BioFire COVID-19 Test in silico Cross-Reactivity
Recommended Organisms Additional Organisms
Human coronavirus 229E Parechovirus
Human coronavirus OC43 Corynebacterium diphtheria
Human coronavirus HKU1 Bacillus anthracis
Human coronavirus NL63 Moraxella catarrhalis
SARS-coronavirus Neisseria elongata
MERS-coronavirus Neisseria meningitidis
Adenovirus Pseudomonas aeruginosa
Human Metapneumonovirus (hMPV) Leptospira
Parainfluenza virus 1-4 Chlamydia psittaci
Influenza A & B Coxiella burnetii
Enterovirus Staphylococcus aureus
Respiratory syncytial virus Homo sapiens
Rhinovirus SARS-coronavirus
Chlamydia pneumoniae Coronavirus
Haemophilus influenza Recombinant SARSr-CoV
Legionella pneumophila SARS2
Mycobacterium tuberculosis SARS coronavirus ExoN1
Streptococcus pneumonia SARS coronavirus wtic-MB
Streptococcus pyogenes SARS coronavirus MA15
Bordetella pertussis SARS coronavirus MA15 ExoN1
Mycoplasma pneumoniae Bat Betacoronavirus SARS related virus
Pneumocystis jirovecii Coronaviridae
Candia albicans Coronavirinae
Pseudomonas aeruginosa
Staphylococcus epidermidis
Staphylococcus salivarius

All assays show 80% or greater homology to Bat coronavirus RaTG13 (accession: MN996532). In addition, the
SARS-CoV-2e assay shows greater than 80% homology to Pangolin coronavirus isolate MP789 (accession:
MT084071). It is unlikely that these isolates would be found in our sample matrix of nasopharyngeal swabs;
however, little is known about their potential to infect a human host, or their evolutionary relationship to SARS-
CoV-2. No other significant amplification of non-target sequences is predicted.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 29

 Analytical Specificity (Exclusivity)
Six viruses that are closely related to SARS-CoV-2 were tested, and none of the BioFire COVID-19 Test
assays were cross-reactive to any of these viruses. More than 30 additional organisms were also tested, and
none of the assays cross-reacted with any of these organisms. Results are shown in Table 14 below.

Table 14. Organisms Tested for Evaluation of BioFire COVID-19 Test Analytical Specificity

Assay Detections

SARS-CoV-2d
SARS-CoV-2a

SARS-CoV-2e
SARS-CoV-2
Organism ID Test Concentration
Test Result

Human coronavirus Zeptometrix

1.26E+06 TCID50/mL 0/3 0/3 0/3 Not Detected
229E 0810229CF
Human coronavirus Zeptometrix
9.55E+06 TCID50/mL 0/3 0/3 0/3 Not Detected
OC43 0810024CF
Human coronavirus
Clinical Isolate
HKU1 ~1.0E+08 copies/mLa 0/3 0/3 0/3 Not Detected
(NPS)
(clinical specimen)
Human coronavirus Zeptometrix
2.51E+05 TCID50/mL 0/3 0/3 0/3 Not Detected
NL63 0810228CF
SARS-coronavirus Culture (MRI
5.3E+08 GE/mL 0/3 0/3 0/3 Not Detected
(BSL3) Global)
MERS-coronavirus Culture (MRI
2.7E+08 GC/mL 0/3 0/3 0/3 Not Detected
(BSL3) Global)
Chlamydia pneumoniae ATCC 53592 2.90E+07 IFU/mL 0/3 0/3 0/3 Not Detected
4.20E+08 CFU/mL
Haemophilus influenzae ATCC 700223 0/3 0/3 0/3 Not Detected
(7.42E+08 copies/mL)
Zeptometrix
Legionella pneumophila 2.63E+09 CFU/mL 0/3 0/3 0/3 Not Detected
0801530
Mycobacterium
Zeptometrix
tuberculosis (attenuated 3.04E+07 CFU/mL 0/3 0/3 0/3 Not Detected
0801660
strain)
Streptococcus
ATCC 6303 8.90E+07 CFU/mL 0/3 0/3 0/3 Not Detected
pneumoniae
4.65E+08 CFU/mL
Streptococcus pyogenes ATCC 49399 0/3 0/3 0/3 Not Detected
(8.25E+08 copies/mL)
Zeptometrix
Bordetella pertussis 6.70E+09 CFU/mL 0/3 0/3 0/3 Not Detected
0801459
Mycoplasma Zeptometrix
3.98E+07 CCU/mL 0/3 0/3 0/3 Not Detected
pneumoniae 0801579
Pseudomonas
ATCC 10145 5.68E+08 CFU/mL 0/3 0/3 0/3 Not Detected
aeruginosa
Staphylococcus
ATCC 29887 7.43E+09 CFU/mL 0/3 0/3 0/3 Not Detected
epidermidis
Streptococcus salivarius ATCC 13419 7.38E+09 CFU/mL 0/3 0/3 0/3 Not Detected
Adenovirus 1 Zeptometrix
3.39E+07 TCID50/mL 0/3 0/3 0/3 Not Detected
(species C) 0810050CF
Zeptometrix 7.05E+04
Adenovirus 4 (species E) 0/3 0/3 0/3 Not Detected
0810070CF TCID50/mL

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 30

 Assay Detections

SARS-CoV-2d
SARS-CoV-2a

SARS-CoV-2e
SARS-CoV-2
Organism ID Test Concentration
Test Result

Zeptometrix 5.10E+07
Adenovirus 7 (species B) 0/3 0/3 0/3 Not Detected
0810021CF TCID50/mL
Human
Zeptometrix
Metapneumovirus 1.78E+05 TCID50/mL 0/3 0/3 0/3 Not Detected
0810161CF
(hMPV)
Parainfluenza virus 1 BEI NR-48681 8.0E+05 TCID50/mL 0/3 0/3 0/3 Not Detected
Zeptometrix
Parainfluenza virus 2 1.10E+06 TCID50/mL 0/3 0/3 0/3 Not Detected
0810504CF
5.10E+07 TCID50/mL
Parainfluenza virus 3 BEI NR-3233 0/3 0/3 0/3 Not Detected
(7.0E+08 copies/mL)
Zeptometrix
Parainfluenza virus 4 1.70E+07 TCID50/mL 0/3 0/3 0/3 Not Detected
08010060BCF
Zeptometrix
Influenza A subtype H1 7.05E+04 TCID50/mL 0/3 0/3 0/3 Not Detected
0810036CFN
Zeptometrix 7.05E+04 TCID50/mL
Influenza A subtype H3 0/3 0/3 0/3 Not Detected
0810252CF (1.92E+08 copies/mL)
Zeptometrix
Influenza B 4.78E+06 TCID50/mL 0/3 0/3 0/3 Not Detected
0810239CF
Enterovirus species A NCPV 5.0E+08 TCID50/mL
0/3 0/3 0/3 Not Detected
(EV71) 0812215v (3.8E+08 copies/mL)
Enterovirus species B Zeptometrix 5.10E+07 TCID50/mL
0/3 0/3 0/3 Not Detected
(Echovirus 6) 0810076CF (1.10E+08 copies/mL)
Enterovirus species C 7.90E+05 TCID50/mL
ATCC VR-1023 0/3 0/3 0/3 Not Detected
(Coxsackievirus A17) (3.17E+06 copies/mL)
Enterovirus species D Zeptometrix
1.58E+06 TCID50/mL 0/3 0/3 0/3 Not Detected
(68) 0810237CF
Respiratory syncytial Zeptometrix
1.05E+06 TCID50/mL 0/3 0/3 0/3 Not Detected
virus 0810040ACF
Zeptometrix
Rhinovirus 1.26E+06 TCID50/mL 0/3 0/3 0/3 Not Detected
0810012CFN
Pneumocystis jirovecii
ATCC PRA-159 1E+07 CFU/mL 0/3 0/3 0/3 Not Detected
(PJP)
ATCC MYA-
Candida albicans 7.88E+08 CFU/mL 0/3 0/3 0/3 Not Detected
2876
Pooled human nasal
- - - - - -
wash b
a
The human coronavirus HKU1 used in this study was a previously collected clinical specimen. The concentration of virus in the sample was
estimated based on the results of a previously performed real-time PCR test.
b
Pooled nasal wash was not evaluated in this study; however, approximately 50 negative residual NPS samples were evaluated during the
clinical evaluation of the test, and no cross-reactivity of test assays to flora present in NPS samples was observed.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 31

 Interference
Potentially interfering substances that could be present in NPS specimens or introduced during specimen
collection and testing were evaluated previously on the FilmArray® Respiratory 2 (RP2) Panel for their effect on
pouch performance. The RP2 Panel and the BioFire COVID-19 Test use the same sample type; no
interference testing has been performed for the BioFire COVID-19 Test. The data from the RP2 Panel
interfering substances evaluation are summarized in Table 15a.

Substances listed below include endogenous substances that may be found in specimens at normal or
elevated levels (e.g., blood, mucus/mucin, human genomic DNA), medications, washes or topical applications
for the nasal passage, various swabs and transport media for specimen collection, and substances used to
clean, decontaminate, or disinfect work areas. The concentration of substance added to the samples was
equal to or greater than the highest level expected to be in NPS specimens.

None of the substances were shown to interfere with the RP2 Panel function and are not expected to interfere
with the BioFire COVID-19 Test. However, it was observed that exposure of samples to bleach prior to testing
could damage the organisms/nucleic acids in the sample, leading to inaccurate test results (lack of analyte
detection). The effect of bleach was dependent on the concentration and/or length of time the bleach was
allowed to interact with the sample.

Various commensal or infectious microorganisms typically found in NPS specimens were tested and did not
interfere with the performance of the RP2 Panel Internal Controls. These organisms have not been tested on
the BioFire COVID-19 Test but due to similarities in the internal control, they are not expected to interfere with
the BioFire COVID-19 Test Internal controls. See Table 15b for a list of competitive organisms tested.

Table 15a. Substances Tested Demonstrating No Panel Interference in

FilmArray® RP2 Panel ab
Substance Tested
Endogenous Substances
Human Whole Blood
Human Mucin (Sputum)
Human Genomic DNA
Exogenous Substances
Tobramycin (systemic antibiotic)
Mupirocin
(active ingredient in anti-bacterial ointment)
Saline Nasal Spray with Preservatives
(0.65% NaCl, Phenylcarbinol, Benzalkonium chloride)
Nasal Decongestant Spray
(Oxymetazoline HCl 0.05%, Benzalkonium chloride, phosphate)
Analgesic ointment (Vicks®VapoRub®)
Petroleum Jelly (Vaseline®)
Snuff (Tobacco)
Disinfecting/Cleaning Substances
Bleachb
Disinfecting wipes (ammonium chloride)
Ethanol
DNAZap (Ambion™ AM9891G & AM9892G)
RNaseZap (Ambion™ AM9782)

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 32

 Substance Tested
Specimen Collection Materials
Rayon Swabs (Copan 168C)
Nylon Flocked Swabs (Copan 553C)
Polyester Swabs (Copan 175KS01)
Calcium Alginate Swabs (Puritan 25-801 A 50)
M4® Transport Medium
(Remel R12500, 3mL/tube)
M4-RT® Transport Medium
(Remel R12506, 3 mL/tube)
M5® Transport Medium
(Remel R12516, 3 mL/tube)
M6™ Transport Medium
(Remel R12535, 1.5 mL/tube)
Universal Viral Transport vial
(BD 220220, 3 mL/tube)
Sigma-Virocult™ Viral Collection and Transport System – Swabs and
Transport Medium (Medical Wire MW951SENT)
ESwab™ Sample Collection and Delivery System – Swabs and Liquid
Amies Medium (Copan 482C)
a
Interfering substances were tested on the FilmArray RP2 Panel and have not been evaluated
with the BioFire COVID-19 Test
b
‘Not Detected’ results were reported for several FilmArray RP2 Panel analytes after incubation
of the sample with 2% bleach for 10 minutes or overnight. It was concluded that interference
resulted primarily from damage to the organism/nucleic acids in the sample, rather than
inhibition or interference with pouch functions.

Table 15b. Competitive Microorganisms Tested on FilmArray® RP2 Panel

Substance Tested Concentration Tested

Competitive Microorganisms typically found in NPS

Coronavirus 229E 1.7 x 104 TCID50/mL
Adenovirus A12 8.9 x 105 TCID50/mL
Parainfluenza Virus 3 6.6 x 105 TCID50/mL
Bordetella pertussis 5.8 x 108 CFU/mL
Enterovirus D68 1.6 x 107 TCID50/mL
Echovirus 6 1.0 x 107 TCID50/mL
Respiratory Syncytial Virus 4.2 x 104 TCID50/mL
Staphylococcus aureus 2.5 x 107 CFU/mL
Streptococcus pneumoniae 1.7 x 107 CFU/mL
Haemophilus influenzae 6.2 x 107 CFU/mL
Candida albicans 1.0 x 106 CFU/mL
Herpes Simplex Virus 1 1.6 x 106 TCID50/mL
Cytomegalovirus (CMV) 1.2 x 106 TCID50/mL

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 33

SPECIMEN POOLING

Pooling Implementation
Pooling must only be performed by U.S. Department of Defense on individuals who are subjected to a detailed
infection prevention and control plan prior to and during operations, or by laboratories that can adhere to a full
protocol for ongoing monitoring of the pooling strategy per these Instructions for Use. Pooling of specimens
allows for testing of more individuals with fewer reagents. When resource availability is sufficient to meet testing
demand, laboratories should reconsider whether the risks of reduced test sensitivity with pooling continue to
outweigh the benefits of resource conservation. Pooling of specimens should also be considered in context of the
SARS-CoV-2 positivity rate within the test population. Higher positivity rates generally decrease the efficiency of
pooling samples because specimens in positive pools must be retested individually. The BioFire COVID-19 Test
has been validated for pooling up to eight samples.

Before implementing a pooling strategy, laboratories should determine the percent positivity rate of the testing
population and choose an appropriate pooling sample size that is within the maximum validated pool size of eight
samples.

Using historical data for individual specimens from the previous 7-10 days, the percent positivity rate (Pindividual)
can be determined by dividing the number of positive specimens by the total number of specimens tested during
that date range.

(Pindividual) = (Number of positive specimens / Number of specimens tested) * 100

Refer to Table 16 to identify which pooling sample size provides the greatest testing efficiency for the determined
Pindividual within the validated pool sizes for the assay. If Pindividual is 2% or less, then the largest validated pool size
(n=8) should be used to maximize efficiency. If the Pindividual is greater than 25%, then pooling is not efficient and
should not be implemented. An example of the efficiency calculation for 5-sample pooling uses the formula F=1/
(1+ 1/5-(1-P)5), when P individual is 1%, the efficiency F is 3.46 for n=5. It means that 1,000 tests can cover
testing of 3,460 patients on average.

Table 16. Testing Efficiency of Pooling

Efficiency of n-Sample Pooling
n Corresponding to the
Pindividual (maximum increase in number of
Maximal Efficiency
tested patients)
1%–2% 8 5.11–3.65
3%–4% 6 3.00–2.60
5%–6% 5 2.35–2.15
7%–12% 4 1.99–1.54
13%–25% 3 1.48–1.10

If historical data for individual specimens from the previous 7-10 days are not available for a laboratory as
described above, pooling may be implemented with the maximum pool size of (n=8). However, efficiency may not
be maximized if Pindividual has not been determined.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 34

 Pooling Monitoring
Following the implementation of a pooling strategy, laboratories should evaluate performance of the strategy
regularly to determine if the desired testing efficiency is still being achieved. Determination of the percent
positivity rate in pools (Ppools) is required.

(Ppools) = (Number of positive specimens in pools / Total number of specimens tested in pools) * 100

For DoD Laboratories that Can Adhere to a Full Protocol for Ongoing Monitoring of the
Pooling Strategy
Continue to monitor n-sample pooling strategy by calculating the positivity rate among patient samples during n-
sample pooling (Ppools-x) for subsequent 7-10 day period based on n-sample pool testing. (Ppools-x) should be
updated daily using a moving average.

Compare Ppools-initial to Ppools-x. If Ppools-x is less than 90% of Ppools-initial. (Ppools-x < 0.90 × Ppools-initial), it is
recommended that:

• The n-samples pooling should be re-assessed periodically by conducting a re-assessment study

(described below).
• If Ppools is greater than 25%, pooling of patient samples is not efficient and should be discontinued until the
percent positivity rate drops below.

Pooling Re-Assessment Study

Note: Individual testing as part of either re-assessment study option may be performed using a
different and higher throughput EUA COVID-19 test.

Option 1 Stop n-sample pooling and return to individual testing. Patient samples should be prospectively
individually tested until 10 consecutive positive samples have been collected. These individually tested
samples should then be re-tested in a pool with one positive and n-1 negative samples.
Option 2 Continue n-sample pooling. Individual testing should be performed in parallel to the pooled testing
until 10 consecutive positive samples are obtained. These positive samples should include both positive
individual results generated from individual testing of samples from the non-negative sample pools following
the n-sample pooling and deconvoluting workflow, and positive individual results obtained from individual
testing of samples from the negative sample pools for the time period. Because non-negative pools require
individual testing of samples included in the pool (samples in the positive pools will be tested as a part of
normal n-sample pooling workflow), the study essentially consists of additionally testing individual samples
from the pools with negative results.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 35

 For both options the following should be applied:
If the PPA between pooled-testing results and individual-testing results is ≥ 90% (9 or 10 out of 10), then
implementation of testing using n-sample pooling is acceptable.

If the PPA between pooled-testing results and individual-testing results is less than 90% then:

• If PPA ≤70% (7 out of 10), reduce the pool size (consider a new n as n-1)

• If PPA is 80% (8 out of 10), to compensate for lost sensitivity, reduce the pool size (consider a new n
as n-1) and continue with the reassessment testing until PPA of pooled compared to individual testing
is not less than 90%. OR collect an additional 10 consecutive individually positive samples. Then,
calculate the PPA from the combined data of 20 samples, between pooled-testing results and
individual-testing results. If the PPA is ≥ 85%, then implementation of testing using n-sample pooling is
acceptable.
• If PPA of at least 85% cannot be reached, cease pooling patient specimens.

If n-sample pooling is acceptable based on re-assessment, re-establish Pindividual in your laboratory by

estimating the positivity rate from individual testing in the population from which the 10 (or 20) consecutive
individual positive samples were collected. If the total number of samples (N*) that needed to be tested to
obtain the 10 (or 20) consecutive positive samples is stopped at the 10th (or 20th) positive sample, then
the positivity rate of 10/N* (or 20/N*) is overestimated. The positivity rate should be corrected by the
following corresponding multiplier:

• Positivity rate for 10 samples is (10/N*) × (10/11)

• Positivity rate for 20 samples is (20/N*) × (20/21).

This updated new positivity rate should be used as Pindividual in the future laboratory monitoring.

For DoD Operations Unable to Adhere to a Full Protocol for Ongoing Monitoring of the
Pooling Strategy
Individuals should be subjected to a detailed infection prevention and control plan prior to and during operations.
This may include for example: restriction of movement, quarantine, isolation, continuous health monitoring
programs and regular molecular SARS-CoV-2 surveillance testing by pooled or individual sample testing with the
BioFire COVID-19 or other authorized molecular SARS-CoV-2 testing.

Continue to monitor n-sample pooling strategy by calculating the positivity rate among patient samples during n-
sample pooling (Ppools-x) for subsequent 7-10 day period based on n-sample pool testing. (Ppools-x) should be
updated daily using a moving average.

Compare Ppools-initial to Ppools-x. If Ppools-x is less than 90% of Ppools-initial. (Ppools-x < 0.90 × Ppools-initial), pooling may
continue, but a new n-sample pooling size may need to be considered. If Ppools-x is greater than 25%, pooling of
patient samples is not efficient and should be discontinued until the percent positivity rate drops below.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 36

APPENDIX A

Symbols Glossary
The following symbols can be found on labeling for the FilmArray 2.0, FilmArray Torch, and BioFire COVID-19
Test Kits, kit components, BioFire COVID-19 Test External Control Kits (+), and throughout accompanying
packaging.

ISO 15223-1
Graphical symbols for use on equipment – Registered Symbols
5.1.1 5.1.4 5.1.5
Use-By date Batch Code
Manufacturer (YYYY-MM-DD) (Lot Number)

5.1.6 5.1.7 5.2.8 Do Not Use if

Catalog Number Serial Number Package Is
Damaged

5.3.2 5.3.7 5.4.2

Keep Away from
Temperature Limit Do not re-use
Sunlight

5.4.3 5.5.1 5.5.5

Consult Instructions for In vitro Diagnostic Contains sufficient for
Use Medical Device <n> tests
n

United Nations Globally Harmonized System of Classification and Labeling of chemicals (GHS) (ST/SG/AC.10/30)

Exclamation Mark
Corrosive (Skin Hazardous to the
(Irritant, Acute Toxicity,
Corrosion/Burns, Eye aquatic environment,
Narcotic Effects,
Damage, Corrosive to long-term hazard
Metals) Respiratory Tract
Irritant)

81 FR 38911

Caution: Federal law restricts this device to sale by or on the order of a licensed healthcare
practitioner.

Manufacturer Symbols (BioFire Defense, LLC)

Positive Control COVID-19 symbol

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 37

APPENDIX B

Contact and Legal Information

Customer and Technical Support

Contact Us on the Web Contact Us by E-mail

http://www.BioFireDefense.com [email protected]

Contact Us by Mail Contact Us by Phone

79 West 4500 South, Suite 14 1-801-262-3592 – US and Canada
Salt Lake City, Utah USA
1-801-262-3592 – International
84107

Contact Us by Fax
1-801-447-6907

BioFire Defense, LLC

79 West 4500 South, Suite 14
Salt Lake City, UT 84107 USA

© Copyright 2020, BioFire Defense, LLC All rights reserved. DFA2-PRT-0080-04, October 2020

The information contained in this document is subject to change without notice. No part of this document may be
reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the express
written permission of BioFire Defense, LLC.

FilmArray Software, Detector, and Metacall software modules © 2002–2020 BioFire Defense, LLC.

BioFire Defense, BioFire®, the BioFire logo, FilmArray® and LCGreen® are trademarks of BioFire Diagnostics, LLC
and/or BioFire Defense, LLC and are registered trademarks in the United States. All other names of products and
brands appearing in this manual are trademarks or registered trademarks of their respective owners.

The purchase of this product includes a limited, non-transferable license under specific claims of one or more U.S.
patents as listed on BioFire Defense’s website (http://www.biofiredefense.com/LegalNotices/) and owned by the
University of Utah Research Foundation and/or BioFire.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 38

APPENDIX C

References

1. Biosafety in Microbiological and Biomedical Laboratories. December 2009.

http://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf. Accessed May 24, 2018.
2. CLSI. Protection of Laboratory Workers From Occupationally Acquired Infections; Approved
Guideline-Fourth Edition. CLSI document M29-A4. Wayne, PA: Clinical and Laboratory
Standards Institute; 2014.
3. Summary of Notifiable Diseases. MMWR available at https://www.cdc.gov/.
4. CIFOR Analysis of State Legal Authorities available at http://www.cifor.us/.
5. CLSI. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline-Second
Edition. CLSI document EP12-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
6. CLSI. Statistical Quality Control for Quantitative Measurement Procedures: Principles and
Definitions; Approved Guideline-Fourth Edition. CLSI document C24-Ed4. Wayne, PA: Clinical
and Laboratory Standards Institute; 2016.

BioFire Defense, LLC BioFire® COVID-19 Test – Instructions for Use 39

For additional information regarding our products and applications,
contact BioFire Defense Customer Support.