BioFire Defense RFIT-ASY-0094 FilmArray BioThreat Panel Instructions

BioFire Defense RFIT-ASY-0094 FilmArray BioThreat Panel

Product Information

Kit Part No: RFIT-ASY-0094
Product Name: BioThreat Panel
Manufacturer: BioFire Defense, LLC
Address: 79 West 4500 South, Suite 14 Salt Lake City, UT 84107 USA
Intended Use: For Environmental Surveillance and Research Use Only. Not for use in diagnostic procedures.

Product Usage Instructions

1. Refer to the Symbols Glossary on Page 23 of the user manual for explanations of the symbols used.
2. This product is intended for Environmental Surveillance and Research Use Only. It is not to be used for diagnostic procedures.
3. The FilmArray BioThreat Panel tests for sixteen (16) bacterial, viral, and toxin-encoding biothreats with a total of 26 assay targets. Refer to Table 1 for the list of targets detected.
4. The results for the FilmArray BioThreat Panel can be obtained in approximately one hour.
5. The entire run process takes about an hour. Additional details can be found in the BioFire FilmArray 2.0 Operator’s Manual.
6. During a run, the FilmArray system performs reverse transcription and a single, large-volume, highly-multiplexed reaction (PCR1), followed by multiple singleplex second-stage PCR reactions (PCR2) to amplify sequences within the PCR1 products.
7. Each FilmArray BioThreat Panel kit contains sufficient reagents to test 6 samples. Refer to the user manual for more information on the materials provided.
8. Additional documentation is available online at www.biofiredefense.com.
9. General precautions:
   • The FilmArray BioThreat Panel is for Environmental Surveillance and Research Use Only. It should not be used for diagnostic procedures.
   • This test is intended only for use in biological surveillance activities as described in the Intended Use section.
   • FilmArray BioThreat Panel pouches are only to be used with the BioFire FilmArray 2.0 System.
   • The FilmArray BioThreat Panel is a qualitative test and does not provide quantitative values for the targets in the sample.
   • Always check the expiration date on the pouch and do not use it after its expiration date.
   • BioFire FilmArray pouches are stored under a vacuum in individually wrapped canisters to preserve their integrity.

INTENDED USE

The FilmArray® BioThreat Panel is a qualitative, multiplexed, nucleic acid- based test intended for use with the BioFire® FilmArray® 2.0 instrument. The FilmArray BioThreat Panel detects bacterial and viral pathogens, and toxin- encoding genes, directly from environmental samples, including liquid, powder, and surface swabs. The pathogens and toxin-encoding genes identified by the FilmArray BioThreat Panel are listed in Table 1. Results from the FilmArray BioThreat Panel are not intended to be used for diagnosis, treatment, or other patient management decisions.
For Environmental Surveillance and Research Use Only. Not for use in diagnostic procedures.

SUMMARY AND EXPLANATION OF THE TEST

The FilmArray BioThreat Panel tests for sixteen (16) bacterial, viral, and toxin-encoding biothreats with a total of 26 assay targets (Table 1). Results for the FilmArray BioThreat Panel are available in about one hour.
Table 1. Targets Detected by the FilmArray BioThreat Panel

Bacteria

Bacillus spp.
Brucella spp.
Coxiella burnetii
Yersinia pestis
Rickettsia spp.
Viruses
Eastern equine encephalitis (EEE) virus
Orthopox viruses
Variola virus
V (VEE) virus
Western equine encephalitis (WEE) virus
Toxin-Encoding Genes 2
Botulinum toxin-encoding genes ( Clostridium botulinum )
Ricin toxin-encoding genes ( Ricinus communis )

1. The Burkholderia mallei/ psuedomallei assay may be cross-reactive with other Burkholderia species.
2. The FilmArray BioThreat Panel does not detect toxins directly. The panel only detects genes that encode these toxins.

PRINCIPLE OF THE PROCEDURE

The FilmArray BioThreat Panel 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 acids from multiple pathogens within a single sample. A variety of environmental samples can be tested by the FilmArray BioThreat Panel. After sample collection and preparation, the user injects FilmArray® Hydration Solution and sample combined with FilmArray® Sample Buffer into the pouch, places the pouch into a BioFire® FilmArray® 2.0 Instrument, and starts a run.
The entire run process takes about an hour. Additional details can be found in the BioFire FilmArray 2.0 Operator’s Manual.

During a run, the FilmArray system:

• Lyses the sample by agitation (bead beating) in addition to chemical lysis mediated by the Sample Buffer.
• Extracts and purifies all nucleic acids from the sample using magnetic bead technology.
• Performs nested multiplex PCR by:
  • First performing reverse transcription and a single, large volume, highly-multiplexed reaction (PCR1).
  • Then 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 on the FilmArray BioThreat Panel array.

MATERIALS PROVIDED

Each FilmArray BioThreat Panel kit contains sufficient reagents to test 6 samples (Part No. RFIT-ASY-0094). Materials include:

• Individually packaged FilmArray BioThreat Panel pouches
• Single-use (1.0 mL) FilmArray® Sample Buffer tubes
• Single-use pre-filled (1.5 mL) FilmArray® Hydration Injection Vials (blue)
• Individually packaged FilmArray® Sample Injection Vials (red)
• Individually packaged Transfer Pipettes
• Instructions available online at https://www.biofiredefense.com/product-support/filmarray-support/
  • FilmArray BioThreat Panel – Instructions for Use
  • FilmArray BioThreat Panel – Quick Guide
    NOTE : Additional documentation is available online at www.biofiredefense.com

MATERIALS REQUIRED BUT NOT PROVIDED

• BioFire® FilmArray® 2.0 System including:
  • BioFire® FilmArray® 2.0 instrument and computer with accompanying software
  • BioFire® FilmArray® Pouch Loading Station
  • FilmArray® BioThreat Panel Pouch Module Software is required to run the FilmArray BioThreat Panel and is available by request at www.biofiredefense.com if not already installed on the instrument system
• 10% bleach solution or a similar disinfectant

OPTIONAL MATERIALS

• For dry air filter samples:

  • 50 mL conical tube
  • PBS with 0.1% Triton X-100

• For powder or surface swab samples:
  Option 1

  • Sterile, single-use synthetic sample swab (dacron, rayon, macrofoam, polyester, etc)

  • Sterile scissors, to cut handles of swabs without molded breakpoint
    Option 2 (Non-sterile)

  • BioFire® FilmArray® Powder or Surface Swab Accessory Kit (Part No. NGDS-ASY-0012)

WARNINGS AND PRECAUTIONS

General Precautions

1. The FilmArray BioThreat Panel is for Environmental Surveillance and Research Use Only. This product is not intended for use in diagnostic procedures.
2. This test is intended only for use in biological surveillance activities as described in the Intended Use.
3. FilmArray BioThreat Panel pouches are only for use with BioFire FilmArray 2.0 System.
4. The FilmArray BioThreat Panel is a qualitative test and does not provide quantitative values for the targets in the sample.
5. Always check the expiration date on the pouch. Do not use a pouch after its expiration date.
6. BioFire 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 BioFire FilmArray 2.0 instrument will be available and operational before unwrapping any pouches for loading.
7. Components from this kit should not be stored or used with any other kit. Discard any extra components from the kit after all pouches have been used.
8. Bleach introduced in a sample may damage nucleic acids in the sample, which may inhibit detection of the targets in the sample.

Safety Precautions

1. Wear appropriate Personal Protective Equipment (PPE), including (but not limited to) disposable clean powder-free gloves and lab coats. 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 Infections2

3. Follow your organization’s safety procedures for handling biological samples.

4. Dispose of materials used in this test (including reagents, samples, and used buffer tubes) according to federal, state, and local regulations.

5. Sample Buffer is assigned the following classifications:
   The following statements apply:

   • Acute toxicity (Category 4),
   • Serious eye damage (Category 1), and
   • Skin irritation (Category 2).
     Please refer to the appropriate Safety Data Sheet (SDS) for more information: https://www.biofiredefense.com/product-support/safety-data-sheets/

6. Sample Buffer will form hazardous compounds and fumes when mixed with bleach or other disinfectants.
   WARNING : To avoid generating chlorine gas, 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 Sample Contamination
   Due to the sensitive nature of the FilmArray BioThreat Panel, 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:

   • To avoid potential contamination, samples should be processed in a biosafety cabinet. If a biosafety cabinet is not available, a dead air box or protective shield should be used when preparing sample for testing.
   • Do not handle samples or pouches in a biosafety cabinet that is used for manipulating pathogen culture.
   • Prior to processing samples, thoroughly clean both the work area and the BioFire 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 contamination of the work area with PCR amplicon. Because the FilmArray BioThreat Panel pouch is a closed system, the risk of amplicon contamination is low, provided that the recommended procedures are followed, and 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 been completed.
   • Avoid excessive handling of pouches after test runs.
   • Change gloves after handling a used pouch.
   • Avoid exposing pouches to sharp edges or anything that might cause a puncture.
     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 BioFire FilmArray Operator’s Manual.
     DO NOT PERFORM ADDITIONAL TESTING UNTIL THE AREA HAS BEEN DECONTAMINATED.

REAGENT STORAGE, HANDLING, AND STABILITY

1. Store the test kit, including reagent pouches and buffers, at room temperature (18-25°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). A pouch may be run when the vacuum seal is broken if hydration is successful. If hydration is not successful, the pouch must be discarded.
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.
6. Always check the kit expiration date and do not use reagents beyond the expiration date printed on the pouch or kit.

SAMPLE AND SAMPLE COLLECTION

The following table describes recommendations for sample collection, preparation, and handling that will help ensure accurate test results. Samples should be tested with the FilmArray BioThreat Panel as soon as possible. Users collecting suspicious powder and swab samples for on-site assessment should follow industry best practices in accordance with ASTM E2458-17 and their organization’s procedures.
Table 2. Recommendations for Sample Collection.

| Potential Sample Types
---|---
Liquid Samples (including Air-into- PBS / Dry Filter / Liquid)| ****

Powder Samples

| ****

Surface Swab Samples

| ****

ASTM E2458-17

Method B Swab

Recommended Sample Collection

| ****

Collect ~300 µL of liquid sample.

| Wet a sample swab with sample buffer. Touch wetted swab to a powder pile or source, then add to Sample Injection Vial.| Drag a dry sample swab across the surface suspected of contamination, then add to the Sample Injection Vial.| Add the collected swab to the Sample Injection Vial, cutting off the tip so it remains in the vial.

NOTE : Bleach can damage organisms/nucleic acids within the sample, potentially causing interference or failure to detect the intended targets. Contact between bleach and samples during collection, disinfection, and testing procedures should be avoided.

The sample collecting instructions below are recommendations. Follow your organization’s guidelines for sample collection as requirements may vary. For questions about compatibility of sample collection methods contact BioFire Defense Technical Support.

Dry Air Filter

1. Place the dry air filter into a 50 mL conical tube with the collection side facing away from the side of the tube.
2. Add 10 mL PBS with 0.1% Triton X-100 to the conical tube.
3. Cap the tube and shake by hand for 2 minutes.

For Powder Samples:
Wet the tip of the swab. Touch the tip of the sample swab to the powder sample.

For Surface Swabs Samples:
Wet the tip of the swab. Wipe the swab over the sampling area using horizontal S-strokes or Z-strokes over the surface. The recommended maximum surface area should be 20 by 20 cm (8 by 8 in.).

PROCEDURE

• Samples should be collected prior to opening a pouch. Use clean gloves and other Personal Protective Equipment (PPE) when handling pouches and samples. Only prepare one FilmArray BioThreat pouch at a time and change gloves between samples and pouches. Particular attention should be given to the laboratory precautions noted under the Warnings and Precautions section to avoid potential contamination of the sample or testing area.
• Once a sample is added to the pouch, promptly transfer it to the instrument to start the run. After the run is complete, discard the pouch in a biohazard container.
• Refer to the appropriate BioFire FilmArray 2.0 Operator’s Manual for more details.

Step 1: Prepare Pouch

1. Thoroughly clean the work area and the BioFire 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 Pouch Loading Station, aligning the red and blue labels on the pouch with the red and blue arrows on the BioFire FilmArray Pouch Loading Station.

5. Remove the Sample Injection Vial from its package by tearing or cutting the notched outer packaging. Remove the clear cap from the end of the Sample Injection Vial (red cover). Place a Sample Injection Vial (with red cover) into the red well of the Pouch Loading Station.

6. Place a Hydration Injection Vial (with a blue cover) into the blue well of the Pouch Loading Station.

Step 2: Hydrate Pouch

1. Unscrew the Hydration Injection Vial from the blue cover.
2. Remove the Hydration Injection Vial, leaving the blue cover in the Pouch Loading Station.
3. Insert the Hydration Injection Vial cannula into the pouch hydration port located directly below the blue arrow of the Pouch Loading Station.
4. Forcefully push down in a firm and quick motion to puncture the 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, re-insert the Hydration Injection Vial to ensure that the seal of the pouch hydration port was broken. If the Hydration Solution is again not drawn into the pouch, discard the current pouch, retrieve a new pouch, and repeat from Step 1: Prepare Pouch.
5. 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), re-insert the Hydration Injection Vial to ensure that the deal of the pouch hydration port was broken. If the Hydration Solution is still not drawn into the pouch, discard the current pouch, retrieve a new pouch, and repeat from Step 1: Prepare the Pouch.

Step 3: Prepare Sample Mix
Add Sample Buffer:

1. Hold the Sample Buffer Tube with the tip facing up.
   NOTE : Avoid touching the tube tip during handling, as this may introduce contamination.

2. Firmly pinch at the textured plastic tab on the side of the tube until the seal snaps.

3. Invert the tube over the Sample Injection Vial and dispense the Sample Buffer using a slow, forceful squeeze followed by a second squeeze. NOTE: Avoid squeezing the tube additional times. This will generate foaming, which should be avoided.
   WARNING : The Sample Buffer is harmful if swallowed and can cause serious eye damage and skin irritation.

For Liquid Samples (including Air-into-PBS / Dry Filter / Liquid):
NOTE : Gently invert the sample container until thoroughly mixed

1. After collecting the liquid sample, use the Transfer Pipette to draw filter wash or liquid to the 3rd line (approx. 300 uL) and add to the Sample Injection Vial.
2. Tightly close the lid of the Sample Injection Vial and mix sample by gently inverting 3 times.
3. Return the Sample Injection Vial to the red-well of the Pouch Loading Station.

For Swab Samples (including Powder / Surface):

1. After collecting the sample, place the swab into the Sample Injection Vial and swirl for 5 seconds.

2. Break the swab handle at the molded break point, leaving the tip of the swab in the Sample Injection Vial.
   NOTE : If the swab handle does not have a molded break point, cut the tip of the swab with clean scissors, leaving the tip of the swab in the Sample Injection Vial.

3. Discard the handle in an appropriate waste container.

4. Tightly close lid of the Sample Injection Vial and mix sample by gently inverting 3 times.

5. Return the Sample Injection Vial to the red-well of the 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 Pouch Loading Station, and insert the Sample Injection Vial into the pouch sample port located directly below the red arrow of the Pouch Loading Station.

3. Forcefully push down in a firm and quick motion to puncture the seal (a faint “pop” is heard) and the sample is pulled into the pouch by vacuum.

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 the sample from the Sample Injection Vial, the pouch should be discarded. Discard the Sample Injection Vial and the Hydration Injection Vial in a biohazard container. Retrieve a new pouch and repeat from Step 1: Prepare Pouch.

5. Screw the injection vials back into their plastic covers in the Pouch Loading Station before disposing of them in a biohazard 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 Pouch Loading Station.
   NOTE : Optional added operator protection: Before removal from the biosafety cabinet, run a bleach wipe, and a paper towel with 10% bleach (one part bleach to nine parts water), across the top of the pouch from the pouch hydration port to the pouch 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 pouch sample port.

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

1. Ensure that the BioFire 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 appropriate BioFire FilmArray 2.0 operator’s manual to place the pouch in an instrument and enter pouch, sample, and operator information.

3. Pouch identification (Lot Number and Serial Number) and Pouch Type 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 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 FilmArray BioThreat Panel 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 the BT PBS v3.0 protocol.

6. Enter a username and password in the Name and Password fields.
   NOTE : The font color of the username is red until the username 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 makes an audible, 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 BioFire FilmArray Software database, and the test report can be 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 an analyte for a specific assay.

QUALITY CONTROL

Process Controls
Three internal process controls are included in each pouch:

1. RNA and DNA Process Controls
   The RNA and DNA Process Control assays target an RNA transcript and genomic region, respectively, 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. Positive control results indicate that all steps carried out in the FilmArray BioThreat Panel pouch were successful.

2. PCR2 Control
   The PCR2 Control assaCy 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.

The RNA Process control assay must be positive for the FilmArray run to pass. If the RNA Process control fails, all results will be listed as ‘Invalid’ and the sample should be retested with a new pouch. DNA process control and PCR2 controls are included for troubleshooting purposes.

INTERPRETATION OF RESULTS
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 the FilmArray 2.0 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 falls inside the assay- specific Tm range, the melt curve is called positive. If the software determines that the melt 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 replicates for each assay to determine the assay result. For an assay to be called positive, at least two associated melt curves must be called positive, and both Tm values must be similar. Assays that do not meet these criteria are called negative.
• The FilmArray BioThreat Panel automatically interprets and returns results (Detected or Not Detected) for each pathogen. The interpretation is based on the results of one or more assays for each pathogen as shown in Table 3. In cases where the RNA Process control assay has failed all analyte results are reported as Invalid (Figure 3).

Table 3. Assay Number and Interpretation Rules for the FilmArray BioThreat Panel

Organism

| ****

No. of Assays

| ****

Assay Interpretation Rules

---|---|---
BACTERIA
Bacillus anthracis / Bacillus spp.| 3| See Table 4
Brucella melitensis / Brucella spp.| 2| See Table 5
Burkholderia mallei / pseudomallei 1| 2| Any Positive = Detected
Coxiella burnetiid| 2| Any Positive = Detected
Francisella tularensis| 2| Any Positive = Detected
Rickettsia prowazekii / Rickettsia spp.| 2| See Table 6
Yersinia pestis| 2| Any Positive = Detected
VIRUSES
Ebola Zaire| 1| Positive = Detected
Marburg virus| 2| Any Positive = Detected
Orthopox genus virus| 2| Any Positive = Detected

Variola virus

| ****

2

| Both Assays Positive AND Both Orthopox Genus Virus Assays Positive = Detected
EEE Virus| 1| Positive = Detected
VEE Virus| 2| Any Positive = Detected
WEE Virus| 1| Positive = Detected
TOXIN-ENCODING GENES
Clostridium botulinum| 1| Positive = Detected
Ricinus communis| 1| Positive = Detected

1 Cross-reactivity with other Burkholderia spp. may be observed.
Table 4.  Bacillus species Calling Scheme

pXO1

| ****

pXO2

| Chromosome Element| ****

Interpretation

| ****

Call

Pos| Pos| Pos| Bacillus anthracis| Detected
Neg| Neg| Neg| Bacillus anthracis| Not Detected
Neg| Neg| Pos| Bacillus species| Detected
Neg| Pos| Neg| Bacillus species| Detected
Neg| Pos| Pos| Bacillus species| Detected
Pos| Neg| Neg| Bacillus species| Detected
Pos| Neg| Pos| Bacillus species| Detected
Pos| Pos| Neg| Bacillus species| Detected

Table 5. Brucella species Calling Scheme

Genus

| ****

Species-Specific

| ****

Interpretation

| ****

Call

Pos| Neg| Brucella species| Detected
Neg| Pos| Brucella| Detected
Pos| Pos| Brucella| Detected
Neg| Neg| Brucella| Not Detected

Table 6. Rickettsia species Calling Scheme

Genus

| ****

Species-Specific

| ****

Interpretation

| ****

Call

Pos| Neg| Rickettsia species| Detected
Neg| Pos| Rickettsia prowazekii| Detected
Pos| Pos| Rickettsia prowazekii| Detected
Neg| Neg| Rickettsia prowazekii| Not Detected

Bacteria Reporting

• Bacillus anthracis / Bacillus species
  The FilmArray BioThreat Panel contains three (3) assays for the detection of Bacillus spp. Two (2) assays target regions on the pXO1 and pXO2 virulence plasmids, respectively, and the third targets a chromosomal region of the Bacillus genus. If at least one of the assays is positive, but not all three, the result is reported as Bacillus species Detected. If all three (3) assays are positive the result is reported as Bacillus anthracis Detected. See Table

• Brucella melitensis / Brucella species
  The FilmArray BioThreat Panel contains two (2) assays for the detection of Brucella meliensis/Brucella spp. One Brucella genus level assay and one assay specific to it. If the B. assay is positive, the result is reported as Brucella Detected. If only the genus level assay is positive, the result is reported as Brucella species Detected. See Table 5.

• Burkholderia mallei / pseudomallei
  The FilmArray BioThreat Panel contains two (2) assays for the detection of Burkholderia mallei. If either assay is positive the result is reported as Burkholderia mallei/Detected.
  NOTE : This assay may be cross-reactive with other species in the Burkholderia genus.

• Coxiella burnetii
  The FilmArray BioThreat Panel contains two (2) assays for the detection of. If either assay is positive the result is reported as Coxiella Detected.

• Francisella tularensis
  The FilmArray BioThreat Panel contains two (2) assays for the detection of Francisella tularensis. If either assay is positive the result is reported as Francisella tularensis Detected.

• Rickettsia species
  The FilmArray BioThreat Panel contains two (2) assays for the detection of. One Rickettsia genus level assay and one assay specific to . If the R. assay is positive, the result is reported as Rickettsia Detected. If only the genus level assay is positive, the result is reported as Rickettsia species Detected. See Table 6.

• Yersinia pestis
  The FilmArray BioThreat Panel contains two (2) assays for the detection of Yersinia pestis. If either assay is positive the result is reported as Yersinia pestis Detected.

Virus Reporting

• Ebola Zaire
  The FilmArray BioThreat Panel contains one (1) assay for the detection of Ebola Zaire. If the assay is positive the result is reported as Ebola Zaire Detected.

• Marburg virus
  The FilmArray BioThreat Panel contains two (2) assays for the detection of Marburg viruses. If either assay is positive the result is reported as Marburg virus Detected.

• Orthopoxvirus genus
  The FilmArray BioThreat Panel contains two (2) assays for the detection of Orthopox genus viruses. If either assay is positive the result is reported as Orthopox genus Virus Detected.

• Variola virus
  The FilmArray BioThreat Panel contains two (2) assays for the detection of variola virus. Both variola virus assays AND both Orthopox virus genus assays must be positive for the result to be reported as Variola virus Detected.

• EEE Virus
  The FilmArray BioThreat Panel contains one (1) assay for the detection of EEE Virus. If the assay is positive the result is reported as EEE Virus Detected.

• VEE Virus
  The FilmArray BioThreat Panel contains two (2) assays for the detection of VEE Virus. If either assay is positive the result is reported as VEE virus Detected.

• WEE Virus
  The FilmArray BioThreat Panel contains one (1) assay for the detection of WEE Virus. If the assay is positive the result is reported as WEE virus Detected.

Toxin-Encoding Genes
Clostridium botulinum
The FilmArray BioThreat Panel contains one (1) assay for the detection of botulinum toxin-encoding genes. If the assay is positive the result is reported as Clostridium botulinum Detected.
NOTE : The FilmArray BioThreat Panel does not detect botulinum toxins directly.

Ricinus communis
The FilmArray BioThreat Panel contains one (1) assay for the detection of ricin toxin-encoding genes. If the assay is positive the result is reported as Ricinus communis Detected.
NOTE : The FilmArray BioThreat Panel does not detect ricin toxins directly.

FilmArray BioThreat Panel Test Report
The FilmArray BioThreat Panel test report is automatically displayed upon completion of a run and contains three sections: the Run Summary, the Result Summary, and the Run Details. The test report can be saved as a file or printed (see Figure 1)

The Run Summary section of the test report (Figure 1) is displayed at the top of the page. It provides information about the run including the Sample ID, date and time of the run, the target(s) detected, and the control results. Control results are listed as Passed, Failed, or Invalid. The Control field will display Passed only if the run was completed successfully (no instrument or software errors) and the RNA Process Control assay was successful. The Control field will display Failed if the run was completed successfully (no instrument or software errors) but the RNA Process Control assay failed. If the internal control result fails, then the result for all of the targets on the panel is displayed as Invalid and the sample will need to be retested with a new pouch. If repeated internal control failures are observed, consider any potential inhibitory effects the sample input may have or contact BioFire Defense Technical Support for assistance. Table 7 provides additional information for each of the possible control field results. See Table 8 for complete result interpretation and required actions.

Table 7. Interpretation of Internal Control Field on the BioThreat Panel Test Report.

RNA Process Control Result| ****

Explanation

---|---

Pass

| The run was successfully completed AND

RNA Process control was successful.

Fail

| The run was successfully completed BUT

RNA Process Control failed.

Invalid

| ****

The RNA Process control is invalid because the run failed (typically, a software or hardware error).

• The Results Summary section of the test report lists each target tested as either Detected, Not Detected, or Invalid. See the Results Explanation section below for detailed information about the interpretation of test results and appropriate follow-up for Invalid results.
• The Run Details section provides additional information about the pouch including the pouch type, serial number, and lot number.
• Once a run has been completed, it is possible to edit the Sample ID. 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 (Figure 2). Sample ID is the only field of the report that can be changed.

Results Explanation and Required Actions
The Results Summary section provides a complete list of all test results. Possible results include Detected, Not Detected, and Invalid. Table 8 provides an explanation for each interpretation and any follow-up necessary to obtain a final result.
Table 8. Interpretation of Results on the FilmArray BioThreat Panel Test Report

Not Detected

| The run was successfully completed AND

RNA Process control was successful (Pass) AND

The assay(s) for the organism were NEGATIVE

| ****

The results are valid.

Detected

| The run was successfully completed AND

RNA Process control was successful (Pass) AND

The assay(s) for the organism were POSITIVE

| ****

Results are valid.

Fail

| ****

Run completed and control failed OR

Run did not complete

| All results are invalid because the run failed.

NOTE: If any error codes are displayed, refer to the BioFire FilmArray Operator’s Manual for more information. If the error persists, contact Technical Support for further instructions.

Retest the sample.

WARNING : Detection of three or more pathogens may indicate a possible contamination event. Therefore, it is recommended to retest a sample that has three or more pathogen detected to confirm the result. If the three or more positive results are not duplicated, contact BioFire Technical Support and discontinue testing until the test area has been decontaminated.

CLEANING MATERIALS

This list provides items that are necessary in a laboratory to keep contamination to a minimum.

1.  10% bleach solution in a squeeze or spray bottle (1 part bleach to 9 parts water)
2. Distilled, de-ionized, sterile, or molecular-grade water in a squeeze or spray bottle
3. DNAZap™ or equivalent DNA degrading system
4. Paper towels
5. Bleach wipes

DECONTAMINATION PROCEDURES

• The decontamination and cleaning procedures listed are intended to limit spread of contaminants as a result of a BioThreat Panel Detected pathogen, a suspected positive pouch, or a broken or leaked pouch. A suspected positive sample is one that the user strongly suspects may be positive for an analyte on the FilmArray BioThreat Panel.
• If a pouch leak or breakage occurs, change gloves and other potentially contaminated personal protective equipment (PPE). Change gloves often during the decontamination process, especially during the first steps of decontamination and before touching any clean surface. All PPE should be disposed of after decontamination.

CAUTION : It is important that contamination from leaking and/or punctured pouches be contained and cleaned immediately. Pouches that break after PCR contain amplified nucleic acid material that can contaminate future pouch runs. This material, although noninfectious, is easily spread if precautions are not taken. Very small (molecular) quantities can be amplified by PCR in future runs, which can result in false positives. Treat all broken pouches as capable of contaminating the work area.

BIOLOGICAL RISKS: If the pouch contains potentially infectious material, the risk of biohazard contamination exists in addition to sample contamination.
NOTE : Use of distilled water is recommended for cleaning.

BioFire FilmArray Pouch Loading Station Decontamination
Routine cleaning of the BioFire FilmArray Pouch Loading Station includes a 10% bleach wipe followed by two water wipes before each new pouch is loaded.
In the event of work with a BioThreat Panel Detected pathogen, a suspected positive sample, or contamination from a sample spill, or a pouch leak, perform the following decontamination procedures:

1. Put on clean PPE, such as lab coat and gloves.
2. Fill a sink or bin with water and add bleach to create a 10% bleach solution.
3. Submerge the Pouch Loading Station until completely covered with bleach solution. Soak for 15 minutes.
4. Remove the Pouch Loading Station from the sink or bin. Replace the bleach solution with distilled water.
5. Rinse the Pouch Loading Station by completely submerging in water two additional times.

Contact BioFire Defense Technical Support to obtain a replacement Pouch Loading Station, if necessary.

Decontamination Related to Detected Pathogen or Toxin-Encoding Gene, or Pouch Leakage
If a pouch was loaded with a FilmArray BioThreat Panel detected pathogen, a suspected positive sample, or if the pouch leaks, take the following precautions to avoid contamination:

1. Put on clean PPE, such as a lab coat, gloves, and eye protection.
2. Ensure no one uses the instrument or potentially contaminated areas until the decontamination is complete.
3. Decontaminate the instrument and work area and dispose of the pouch using the following steps:
   • a. Dispose of potentially contaminated gloves and put on clean gloves.
   • b. Dispose of the potentially contaminated lab coat and put on a clean lab coat.
   • c. Discard the leaking pouch in a biohazard container.
   • d. Change gloves.
   • e. Clean the instrument and affected work areas per the guidelines below.
     CAUTION : Use only 10% bleach solution, water, and/or DNAZap to decontaminate the instrument and Pouch Loading Station.

BioFire FilmArray 2.0 Instrument Decontamination
Pouch Loading Chamber Decontamination 

1. Put on clean PPE, such as a lab coat and gloves.
2. Remove the pouch from the instrument and discard in a biohazard waste container. Change gloves and any contaminated PPE.
3. Wet a paper towel with 10% bleach (one part bleach to nine parts water) and wipe the inner chamber and under the lid. Let it stand for at least 3 minutes to allow the bleach solution to react with any contaminants. Discard paper towel in biohazard waste. Change gloves.
4. Repeat Step 3 twice with fresh paper towels for a total of three bleach wipes.
5. Wet a paper towel with water and wipe the inner chamber.
6. Repeat Step 5 with fresh gloves and a paper towel.

Instrument Exterior Decontamination

1. Put on clean PPE, such as a lab coat and gloves.
2. Wet a paper towel with the 10% bleach solution and wipe all exterior surfaces of the instrument, including the bottom and the bench top where the instrument had contact. Let it stand for at least 3 minutes to allow the bleach solution to react with any contaminants. Discard paper towels in biohazard waste. Change gloves.
3. Repeat Step 2 twice with fresh paper towels and clean gloves, for a total of three bleach wipes.
4. Change gloves, then wet a new paper towel with water and wipe all surfaces of the exterior of the instrument, including the bottom and the bench top where the instrument had contact.
5. Repeat Step 4 with fresh gloves.

Decontamination of Bench Tops and Other Areas

1. Put on clean PPE, such as a lab coat and gloves.
2. Spray the 10% bleach solution on the area that may have been contaminated. Let it stand for at least three minutes to allow the bleach solution to react with any contaminants on the surface.
3. Wipe the area with a clean paper towel. Change gloves.
4. Repeat Steps 2 and 3 twice, for a total of three wipes.
5. Change gloves. Spray the area with distilled water.
6. Wipe the area dry with a new paper towel. Change gloves.
7. Spray the area with DNAZapTM or an equivalent product. Follow the product’s instructions for correct use. Change gloves.
8. Rinse the area by spraying it with distilled water and wiping it dry.

Check the Function of Decontaminated Instrument

1. Test a negative sample by preparing a pouch, using water as the sample. Use distilled, sterile, or molecular-grade water for the test.
2. If run is successful and all results are negative, continue using the instrument as normal.
3. If unexpected positive results are obtained or the run fails, please contact BioFire Defense Technical Support for further instructions.

Check for Environmental Contamination
After decontaminating the instrument as described above, use environmental swabs to check for contamination by following the protocol below:

1. Prepare four aliquots of 0.2 mL of molecular-grade water.
2. Place one environmental swab in each aliquot and let soak it for five minutes.
3. Thoroughly swab the exterior of instruments and accessories, including laptops, especially areas of operator contact.
4. Return each swab to its original aliquot and mix the sample well.
5. Dispose of the swabs and combine the four aliquots into one.
6. Load pouch as described in the Procedure section of this document.
   • a. Load 0.2 mL of combined swabbing aliquot as the sample using Transfer Pipette, by drawing liquid up to the 2nd line.
   • b. Add sample to Sample Injection Vial.
   • c. Proceed with the normal pouch loading procedure.
7. Run the pouch using the BT PBS v3.0 protocol.
8. If a positive result is found, repeat the decontamination step, and contamination testing until no contamination is detected.
9. If problems persist, contact BioFire Defense Technical Support for further instructions.

APPENDIX A
Symbols Glossary
The following symbols can be found on labeling for the BioFire FilmArray 2.0, FilmArray BioThreat Panel kit, kit components, and accompanying packaging.

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

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

| Contact Us by Phone

1-801-262-3592 – US and Canada 1-801-262-3592 – International

 | Contact Us by Fax
 | 1-801-447-6907

BioFire Defense, LLC
79 West 4500 South, Suite 14 Salt Lake City, UT 84107 USA
© Copyright 2023, BioFire Defense, LLC. All rights reserved.
DFA2-PRT-0220-01, August 2023

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.
BioFire FilmArray Software, Detector, and Metacall software modules © 2002–2023 BioFire Diagnostics, LLC and/or BioFire Defense, LLC.
BioFire Defense, BioFire®, the BioFire logo, FilmArray®, and BIOFIRE® SHIELD™ 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.

APPENDIX C
References

1. Biosafety in Microbiological and Biomedical Laboratories. June 2020. https://www.cdc.gov/labs/pdf/SF__19_308133-A_BMBL6_00-BOOK-WEB-final-3.pdf. Accessed July 27, 2023.
2. CLSI. Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline-Fourth Edition. 2014. CLSI document M29-A4. Wayne, PA: Clinical and Laboratory Standards Institute;
3.  Standard Practices for Bulk Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being Biological Agents and Toxins from Nonporous Surfaces. April 2019. ASTM Int’l,

Revision History

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

References

• Safeguarding Humanity - BioFire Defense
• Legal Notices - BioFire Defense
• FilmArray® Global Fever Panel RUO v1.1 Pouch Module - Download Form - BioFire Defense
• Safety Data Sheets - BioFire Defense

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