Biofire RP2.1 Instructions for use & intended purpose & specs.pdf

423742

BioFire® Respiratory Panel 2.1 (RP2.1) BFR0000-8579-01 March 2021

BioFire® Respiratory Panel 2.1 (RP2.1)

Customer and Technical Support Information

*For more information on how to contact Customer and Technical Support, refer to Appendix B.

U.S. Customers Phone: 1-800-735-6544 (toll free) E-mail: support@BioFireDX.com Website: www.biofiredx.com

Outside of the U.S. Contact the local bioMérieux sales representative or an authorized distributor.

INTENDED PURPOSE

Intended Use The BioFire Respiratory Panel 2.1 (RP2.1) is a PCR-based multiplexed nucleic acid test intended for use with the BioFire® FilmArray® 2.0 or BioFire® FilmArray® Torch Systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections, including COVID-19.

The following organism types and subtypes are identified using the BioFire RP2.1:

Viruses Bacteria

Adenovirus Coronavirus 229E Coronavirus HKU1 Coronavirus NL63 Coronavirus OC43 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Human Metapneumovirus Human Rhinovirus/Enterovirus Influenza A, including subtypes H1, H3 and H1-2009 Influenza B Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Parainfluenza Virus 4 Respiratory Syncytial Virus

Bordetella parapertussis Bordetella pertussis Chlamydia pneumoniae Mycoplasma pneumoniae

Nucleic acids from the respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection is indicative of the presence of the identified

Rx Only Instructions for Use https://www.biofiredx.com/e-labeling/ITI0105

Quick Guide https://www.biofiredx.com/e-labeling/ITI0111

Safety Data Sheet (SDS) https://www.biofiredx.com/e-labeling/ITI0119

Pouch Module Software https://www.biofiredx.com/e-labeling/ITIFA20RP2110 mailto:support@BioFireDX.com http://www.biofiredx.com/ https://www.biofiredx.com/e-labeling/ITI0105 https://www.biofiredx.com/e-labeling/ITI0111 https://www.biofiredx.com/e-labeling/ITI0119 https://www.biofiredx.com/e-labeling/ITIFA20RP2110 microorganism and aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by an NPS specimen. Positive results do not rule out co-infection with other organisms. The agent(s) detected by the BioFire RP2.1 may not be the definite cause of disease.

Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.

Intended User and Use Environment The BioFire RP2.1 is intended for use by trained medical and laboratory professionals in a laboratory setting or under the supervision of a trained laboratory professional.

SUMMARY AND EXPLANATION OF THE TEST

Respiratory pathogens cause acute local and systemic disease, with the most severe cases occurring in children, the elderly, and immunocompromised individuals. Respiratory symptoms can include coughing, nasal discharge, congestion, fever, wheezing, shortness of breath, headache, and myalgia. Due to the similarity of diseases caused by many viruses and bacteria, diagnosis based on clinical symptoms alone is difficult. Identification of potential causative agents provides data to aid the physician in determining appropriate patient treatment and public health response for disease containment. The BioFire RP2.1 is a real-time, nested multiplexed polymerase chain reaction test designed to simultaneously identify nucleic acids from 22 different viruses and bacteria associated with respiratory tract infection from a single nasopharyngeal swab (NPS) specimen.

Summary of Detected Organisms Adenoviruses (AdV) are a diverse group of non-enveloped DNA viruses with seven species (A to G).1 Adenovirus species B, C, and E cause acute respiratory disease, but all types have been associated with human disease.2 Other Adenovirus species (A, D, F and G) can cause a variety of illnesses, including cystitis, gastroenteritis, and conjunctivitis 3, and may also be found in respiratory specimens. Outbreaks often occur in institutional settings such as military training, long-term care facilities, and pediatric tertiary-care hospitals, due to high rates of transmission in closed populations.4–6 Adenoviruses are shed for long periods of time and persist on surfaces in an infective state.6

Coronaviruses (CoV) - Human coronaviruses were established as respiratory pathogens in the 1960s and seven serological variants associated with human disease have been characterized to date: four types (coronaviruses 229E, OC43, HKU1, NL63) that regularly circulate in human populations, constituting about 15% of common colds7,8, and three strains (Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)) that have spread from animal to human populations since 20028 and represent the ongoing public health threat posed by emerging zoonotic pathogens.

SARS-CoV was declared contained by the WHO in 2003, less than 12 months after its emergence, and no new cases have been reported since 2004. MERS-CoV, which was first described in 2012,9 continues to cause occasional outbreaks, which are characterized by animal to human transmissions followed by person-to-person transmission.10 SARS-CoV-2 is the novel coronavirus that causes COVID-19, an illness that reached a level of pandemic spread in the short time since its emergence in late 2019.11,12 Coronaviruses have been linked to croup and exacerbation of asthma.13,14 Infections with coronavirus 229E, OC43, HKU1, and NL63 occur more often in the winter, and there appears to be a periodicity of circulation.15 Illnesses caused by these coronaviruses are generally self-limiting.16 Though coronaviruses as a group are most commonly associated with upper respiratory tract infections; all human coronaviruses are also associated with lower respiratory tract infection and MERS-CoV, SARS-CoV, and SARS-CoV-2 can cause Acute Respiratory Distress Syndrome (ARDS), as well as significant rates of hospitalization, complications, and death, especially in patients with underlying health conditions.17

Note: MERS-CoV and SARS-CoV are not detected by the BioFire RP2.1.

Human Metapneumovirus (hMPV) is in the family Paramyxoviridae.18 HMPV was discovered in 2001 as a respiratory pathogen in children.19 Further studies confirmed hMPV infections in persons of all ages.20 The two genotypes, A and B, can circulate at the same time and do not appear to differ in the severity of illness.18 HMPV is the second leading cause of bronchiolitis in young children.18 Additionally, infection can result in a broad range of upper and lower respiratory symptoms:

cough, rhinorrhea, wheeze, dyspnea, and fever.21 HMPV is estimated to be responsible for 5-7% of respiratory tract infections in children and 3% among individuals of all ages.21 The seasonal peak of hMPV is winter and early spring and often co-occurs with the seasonal peak of Respiratory Syncytial Virus (RSV).22

Influenza A and B are RNA viruses in the Orthomyxoviridae family. During annual influenza epidemics, 5-20% of the population is affected with upper respiratory tract infections with rapid onset of fever.23 The dominant type of influenza virus varies often due to antigenic drift and shift.24 Influenza A can be subtyped by the hemagglutinin (H) and neuraminidase (N) genes; influenza A subtypes H1N1 and H3N2 are the strains that most commonly infect humans. More severe disease and increased mortality are associated with H3N2 subtype.24 During the 2009-10 Influenza season, influenza A (H1N1)pdm09

(H1- 2009,also known as “swine flu”) became the dominant circulating influenza virus, accounting for approximately 99% of reported influenza infections and has since replaced pre-2009 H1N1 strains (Table 1).25 Currently, at least four antiviral medications are available for influenza treatment – amantadine, rimantadine, zanamivir and oseltamivir – with type-specific efficacy and drug resistance arising with the spread of new strains of the virus.26 Complications with viral or bacterial pneumonia increase mortality from influenza infections.27

Table 1. Proportions of Influenza Subtype Infections in the United States (as reported by the US Centers for Disease Control)

Flu Season1 Influenza A % of Subtyped Influenza A Influenza B H1 H1-2009 H3 2020-20212,3 53.8% 0.0 47.1 52.9 46.2% 2019-2020 58.7% 0.0 92.8 7.2 41.3% 2018-2019 94.3% 0.0 55.5 44.5 5.7% 2017-2018 71.0% 0.0 15.5 84.5 29.0% 2016-20174 77.1% 0.0 3.1 96.9 22.9% 2015-20164 70.3% 0.0 78.4 21.6 29.7% 2014-2015 83.1% 0.0 0.5 99.5 16.9% 2013-2014 85.2% 0.0 87.3 12.7 14.8% 2012-2013 70.4% 0.0 5.2 94.8 29.6%

1 CDC FluView data accessed on December 16, 2020.

2 Cumulative results through December 5, 2020.

3 Season during which BioFire RP2.1 prospective clinical data described in this submission were accumulated.

4 Season during which BioFire RP2 prospective clinical data described in this submission were accumulated.

Parainfluenza Viruses (PIVs) are RNA viruses in the Paramyxoviridae family. In the 1950s, parainfluenza viruses were determined to be respiratory pathogens different from influenza viruses.28 Parainfluenza viruses are divided into four types (parainfluenza viruses 1, 2, 3, and 4). Parainfluenza virus 1 causes biennial epidemics in the fall, with 50% of croup cases attributed to this virus.28 Parainfluenza virus 2 causes of epidemics every one to two years, which may alternate with parainfluenza virus 1 circulation.28 Children less than six months old are particularly susceptible to parainfluenza virus 3 infection, with outbreaks occurring in neonatal intensive care units. PIV3 is associated with the highest mortality and morbidity of all strains29 and epidemics are most common in the spring and summer.28 Parainfluenza virus 4 infection affects all age groups but because of infrequent detection periodicity of infection has not been established.30,31

Respiratory Syncytial Virus (RSV) is a member of the RNA viruses in the Paramyxoviridae family, related to human metapneumoviruses and parainfluenza viruses.32 RSV has two major subtypes (A and B), which vary annually in their prevalence.33 RSV is the most common cause of severe respiratory disease in infants, with acute bronchiolitis as the major cause of hospitalization.32 RSV is now also recognized as an important pathogen in adults, although adult infections are in general less severe and limited to the upper respiratory tract.34 Peak activity of RSV is typically in January and February.35

Rhinoviruses and Enteroviruses are related RNA viruses in the Picornaviridae family.36 There are more than 100 serotypes of human rhinovirus based on the serology of the capsid protein.36 Rhinovirus is noted as causing the “common cold”, but may also be involved in precipitating asthma attacks and severe complications.36 Enteroviruses are divided into four species that include a total of at least 89 distinct types. Individual types can be associated with different clinical manifestations, including nonspecific respiratory illnesses in infants or adults.37 Both rhinoviruses and enterovirus are prevalent year round.38,39

Bordetella pertussis, a gram-negative bacterium, is the predominant causative agent of whooping cough or pertussis, a vaccine-preventable, highly infectious disease that is reportable to public health organizations.40–42 Pertussis occurs most commonly in children but also occurs in adolescents and adults and outbreaks have been documented in fully vaccinated populations due to waning immunity (immunity has been shown to decrease 5-10 years after vaccination).42,43 Early (catarrhal) pertussis disease is non-specific, and classic signs of pertussis (paroxysmal coughing, inspiratory ‘whoop’, post-tussive emesis, as well as apnea or cyanosis in infants) do not arise until approximately two weeks after the initial onset of symptoms. Bordetella parapertussis is known to cause a milder pertussis-like disease.42 No peak season has been defined for Bordetella infections.

Chlamydia pneumoniae (previously known as Chlamydophila pneumoniae) is an obligate intracellular bacterium that causes acute respiratory infections and is a common cause of community-acquired atypical (walking) pneumonia and bronchitis.44–46 C. pneumoniae has an incubation period of approximately three weeks and can be transmitted from asymptomatic carriers.46 Outbreaks occur in schools, military barracks, and nursing homes.47 No peak season has been identified for C. pneumoniae infections.

Mycoplasma pneumoniae is another bacterial agent of community-acquired atypical pneumonia, occurring frequently in outbreak situations.48,49 Incubation time for M. pneumoniae infection is approximately 1 to 4 weeks.50 M. pneumoniae respiratory disease does not have a defined season of highest incidence but epidemics have a periodicity of 3-7 years.49

PRINCIPLE OF THE PROCEDURE

The BioFire RP2.1 pouch 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 multiple respiratory pathogens within a single NPS specimen. After sample collection, the user injects hydration solution and sample combined with BioFire® FilmArray® Sample Buffer into the pouch, places the pouch into a BioFire® FilmArray® System instrument module, and starts a run. The entire run process takes about 45 minutes. Additional detail can be found in the appropriate BioFire System Operator’s Manual.

During a run, the BioFire 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:

o First performing reverse transcription, followed by a multiplexed first stage PCR reaction (PCR1).

o Then performing multiple simultaneous second-stage PCR reactions (PCR2) in the array to amplify sequences within the PCR1 products.

• Uses endpoint melting curve data to detect target-specific amplicons and analyses the data to generate a result for each analyte.

MATERIALS PROVIDED

Each kit contains sufficient reagents to test 30 samples (30-test kit – REF# 423742):

• Individually packaged BioFire RP2.1 pouches

• Single-use (1.0 mL) Sample Buffer ampoules

• Single-use pre-filled (1.5 mL) BioFire® FilmArray® Hydration Injection Vials (blue)

• Single-use BioFire® FilmArray® Sample Injection Vials (red)

• Individually packaged Transfer Pipettes

• BioFire RP2.1 Pouch Module Software This software is required to run the BioFire RP2.1 and can be downloaded at https://www.biofiredx.com/e-labeling/ITIFA20RP2110 if not already installed on the BioFire 2.0 or BioFire Torch Systems.

MATERIALS REQUIRED BUT NOT PROVIDED

• BioFire System including:

o BioFire2.0 or BioFire Torch Systems including accompanying system-specific core software o BioFire® FilmArray® Pouch Loading Station

• 10% bleach solution or a similar disinfectant

WARNINGS AND PRECAUTIONS

General Precautions

1. A trained healthcare professional should carefully interpret the results from the BioFire RP2.1 in conjunction with a patient’s signs and symptoms, results from other diagnostic tests, and relevant epidemiological information.

2. BioFire RP2.1 pouches are only for use with BioFire 2.0 and BioFire Torch Systems.

3. Always check the expiration date on the pouch. Do not use a pouch after its expiration date.

4. BioFire RP2.1 pouches are stored under vacuum in individually wrapped canisters. To preserve the integrity of the pouch vacuum for proper operation, be sure that an instrument/module will be available and operational before unwrapping any pouches for loading.

Safety Precautions 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.

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 Laboratories52

• CLSI Document M29 Protection of Laboratory Workers from Occupationally Acquired Infections53

• 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 or more current guidelines specific for SARS-CoV-2.

Follow your institution's safety procedures for handling biological samples.

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. Viral culture should not be attempted in cases of positive results for SARS-CoV-2 and/or any similar microbial agents unless a facility with an appropriate level of laboratory biosafety (e.g., BSL 3 and BSL 3+, etc.) is available to receive and culture specimens.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to a state or local health department for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

Dispose of materials used in this assay, including reagents, samples, and used buffer vials, according to federal, state, and local regulations.

Sample Buffer contains Guanidinium chloride and Triton X100. The following statements apply:

The following statements apply.

• Health Hazards o Acute Toxicity, oral (Category 4)

H302 – Harmful if swallowed.

o Skin corrosion/irritation (Category 2)

H315 - Causes skin irritation.

o Serious eye damage/eye irritation (Category 1)

H318 - Causes serious eye damage.

• Environment Hazards o Hazardous to the aquatic environment, acute aquatic hazard (Category 1)

H400 - Very toxic to aquatic life.

o Hazardous to the aquatic environment, long-term aquatic hazard (Category 1)

H410 - Very toxic to aquatic life with long lasting effects.

• Precautionary Statements o Prevention

P273 – Avoid release to the environment.

P280 – Wear protective gloves/protective clothing/eye protections/face protection.

o Response

P391 - Collect spillage.

P332 + P313 - If skin irritation occurs: Get medical advice/attention.

P305 + P351 + P338 - IF IN EYES: Rinse cautiously with water for several minutes.

Remove contact lenses, if present and easy to do. Continue rinsing.

P301 + P312 - IF SWALLOWED: Call a POISON CENTRE/doctor if you feel unwell.

P337 + P313 - If eye irritation persists: Get medical advice/attention.

http://www.cdc.gov/coronavirus/2019-nCoV/lab-biosafety-guidelines.html

Please refer to the BioFire RP2.1 Safety Data Sheet (SDS) for more information: https://www.biofiredx.com/e-labeling/ITI0119.

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.

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 RP2.1, 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:

• Laboratory personnel may carry or shed common respiratory pathogens asymptomatically and can inadvertently contaminate the specimen while it is being processed. Careful adherence to the sample processing steps described in this document is recommended to avoid possible contamination. Samples should be processed in a clean biosafety cabinet if available, or according to local laboratory guidelines. If a biosafety cabinet is not used, a dead air box (e.g., AirClean PCR workstation), a splash shield (e.g., Bel-Art Scienceware Splash Shields), or a face shield can be used when preparing samples instead.

• Laboratory personnel with active respiratory symptoms (runny nose, cough) should wear a standard surgical mask (or equivalent) and should avoid touching the mask while handling specimens.

• It is recommended to avoid handling specimens or pouches in an area used to routinely process respiratory pathogen culture, and/or immunofluorescence testing, unless the area is thoroughly cleaned first.

• Prior to processing specimens, thoroughly clean both the work area and the BioFire® Pouch Loading Station using a suitable cleaner such as freshly prepared 10% bleach or a similar disinfectant. To avoid residue build-up and potential damage to the specimen or interference from disinfectants, wipe disinfected surfaces with water.

• Specimens and pouches should be handled and/or tested one-at-a-time. Always change gloves and clean the work area between each pouch and specimen.

• Use clean gloves when removing Sample Buffer ampoules and Sample/Hydration Injection Vials from bulk packaging bags and reseal bulk packaging bags when not in use.

• Avoid collecting or handling specimens in areas that are exposed to vaccine material for pathogens detected by the BioFire RP2.1 (e.g. influenza, poliovirus, SARS-CoV-2, and Bordetella pertussis), if possible. Particular care should be taken during these processes to avoid contamination. Some B. pertussis acellular vaccines (i.e.

Pentacel®, Daptacel®, and Adacel®) contain PCR-detectable DNA. Contamination of specimens or testing materials with vaccine can cause false-positive B. pertussis results (http://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-pcr-bestpractices.html).

https://www.biofiredx.com/e-labeling/ITI0119 https://www.biofiredx.com/e-labeling/ITI0119 http://www.cdc.gov/pertussis/clinical/diagnostic-testing/diagnosis-pcr-bestpractices.html

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 RP2.1 pouch is a closed system, the risk of amplicon contamination is low provided that 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 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 in the appropriate BioFire System Operator’s Manual.

DO NOT PERFORM ADDITIONAL TESTING UNTIL THE AREA HAS BEEN DECONTAMINATED.

3. Transport media or saline may contain non-viable organisms and/or nucleic acids at levels that can be detected by the BioFire RP2.1.

The presence of non-viable organisms and/or nucleic acids in transport media or saline may lead to false positive test results.

Precautions Related to Public Health Reporting Local, state, and federal regulations for notification of reportable disease are continually updated and include a number of organisms for surveillance and outbreak investigations. 51,52 Additionally, the Centers for Disease Control and Prevention (CDC) recommends that when pathogens from reportable diseases are detected by a culture independent diagnostic test (CIDT), the laboratory should facilitate obtaining the isolate or clinical materials for submission to the appropriate public health laboratory to aid in outbreak detection and epidemiological investigations. 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.

Pertussis is a nationally notifiable infectious condition in the U.S. If Bordetella pertussis is detected, notify the state and/or local health departments.

Laboratories in the U.S. are required to report all positive SARS-CoV-2 results to the appropriate public health authorities.

REAGENT STORAGE, HANDLING, AND STABILITY

Store the test kit, including reagent pouches and buffers, at room temperature (15–25 ºC). DO NOT

REFRIGERATE.

Avoid storage of any materials near heating or cooling vents or in direct sunlight.

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.

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).

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.

SAMPLE REQUIREMENTS

The following table describes the 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 up to 3 mL of transport media or saline.

Minimum Sample Volume 0.3 mL (300 µL)

Transport and Storage

Specimens should be tested with the BioFire RP2.1 as soon as possible.

If storage is required, specimens can be held:

• At room temperature for up to 4 hours (15-25 °C)

• Refrigerated for up to 3 days (2-8 °C)

• Frozen (≤-15 °C or ≤-70°C) (for up to 30 days)a a Frozen storage for up to 30 days was evaluated for this sample type. However, longer frozen storage at -70°C or lower may be acceptable. Please follow your institution’s rules and protocols regarding sample storage validation.

NOTE: 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.

PROCEDURE

Use clean gloves and other Personal Protective Equipment (PPE) when handling pouches and samples. Only prepare one BioFire RP2.1 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.

Step 1: Prepare Pouch Thoroughly clean the work area and the BioFire Pouch Loading Station with freshly prepared 10% bleach (or suitable disinfectant) followed by a water rinse.

Remove the pouch from its vacuum-sealed package by tearing or cutting the notched outer packaging and opening the protective 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.

Check the expiration date on the pouch. Do not use expired pouches.

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 Pouch Loading Station.

Place a red-capped Sample Injection Vial into the red well of the Pouch Loading Station.

Place a blue-capped Hydration Injection Vial into the blue well of the Pouch Loading Station.

Step 2: Hydrate Pouch Unscrew the Hydration Injection Vial from the blue cap.

Remove the Hydration Injection Vial, leaving the blue cap in the BioFire Pouch Loading Station.

Insert the Hydration Injection Vial’s cannula tip into the pouch hydration port located directly below the blue arrow of the 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.

Step 3: Prepare Sample Mix Add Sample Buffer to the Sample Injection Vial.

• Hold the Sample Buffer ampoule with the tip facing up.

NOTE: Avoid touching the ampoule tip during handling, as this may introduce contamination.

• Firmly pinch at textured plastic tab on the side of the ampoule until the seal snaps.

• Invert the ampoule 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 ampoule 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.

Thoroughly mix the NPS specimen by vortex or inversion.

Use the transfer pipette provided in the test kit to draw specimen to the third line (approximately 0.3 mL) of the transfer pipette.

Add the specimen to the Sample Buffer in the Sample Injection Vial.

Tightly close the lid of the Sample Injection Vial and 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.

Remove the Sample Injection Vial from the Pouch Loading Station and invert the vial at least 3 times to mix.

Return the Sample Injection Vial to the red well of the Pouch Loading Station.

Step 4: Load Sample Mix Slowly twist to unscrew the Sample Injection Vial from the red cap and wait for 5 seconds with the vial resting in the cap.

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

Lift the Sample Injection Vial, leaving red cap in the well of the Pouch Loading Station, and insert the Sample Injection Vial cannula tip into the pouch sample port located directly below the red arrow of the Pouch Loading Station.

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.

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.

Discard the Sample Injection Vial and the Hydration Injection Vial in appropriate biohazard sharps container.

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.

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 2.0 and BioFire Torch Systems are given below. Refer to the appropriate BioFire System Operator’s Manual for more detailed instructions.

BioFire 2.0 Ensure that the system (instrument and computer) is powered on and the software is launched.

Follow on-screen instructions and procedures described in the Operator’s Manual to place the pouch in a module, enter pouch, sample, and operator information.

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 RP2.1 pouch.

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.

If necessary, select and/or confirm the appropriate protocol for your sample type from the Protocol drop down list.

The BioFire RP2.1 has a single protocol available in the drop down list.

Enter a user name and password in the Name and Password fields.

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

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.

When the run is finished, follow the on-screen instructions to remove the pouch, then immediately discard it in a biohazard waste container.

The run file is automatically saved in the BioFire Software database, and the test report can be viewed, printed, and/or saved as a PDF file.

BioFire Torch Ensure that the system is powered on.

Select an available module on the touch screen or scan the barcode on the pouch using the barcode scanner.

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 RP2.1 pouch.

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.

Insert the pouch into the available module.

• Ensure that the pouch fitment label is lying flat on top of pouch and not folded over. As the pouch is inserted, the module will grab onto the pouch and pull it into the chamber.

If necessary, select and/or confirm the appropriate protocol for your sample type from the Protocol drop down list.

The BioFire RP2.1 has a single protocol available in the drop down list.

Enter operator user name and password, then select Next.

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

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 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.

At the end of the run, remove the partially ejected pouch, then immediately discard it in a biohazard waste container.

The run file is automatically saved in the BioFire Software database, and the test report can be viewed, printed, and/or saved as a PDF file.

QUALITY CONTROL

Process Controls Two process controls are included in each pouch:

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 RP2.1 Panel pouch were successful.

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 the controls fail, the sample should be retested using a new pouch.

Monitoring Test System Performance The 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 (80.3-84.3°C for the RNA Process Control and 73.8-77.8°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.56,57

Refer to the appropriate BioFire System Operator’s Manual for instructions on obtaining control assay Tm values. The PCR2 Control is used in several BioFire pouch types and can, therefore, be used to monitor the system when multiple pouch types are used on the same BioFire System.

External Controls External controls should be used in accordance with laboratory protocols and the appropriate accrediting organization requirements, as applicable. Transport media or saline can be used as an external negative control. Previously characterized positive samples or negative samples spiked with well-characterized organisms can be used as external positive controls. Commercial external control materials may be available from other manufacturers; these should be used in accordance with the manufacturers’ instructions and appropriate accrediting organization requirements, as applicable.

NOTE: Contamination can cause unexpected positive results in negative or positive external controls. If unexpected positive results are observed, thoroughly clean and decontaminate the work space and contact customer support if the unexpected results persist.

INTERPRETATION OF RESULTS

Assay Interpretation When PCR2 is complete, the instrument performs a high-resolution DNA melting analysis on the PCR products and measures the fluorescence signal generated in each well (for more information see appropriate BioFire System Operator’s Manual). The BioFire Software then performs several analyses and assigns a final assay result. The steps in the analyses are described below.

Analysis of melt curves. The BioFire 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 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°C). Assays that do not meet these criteria are called negative.

Organism Interpretation For most organisms detected by the BioFire RP2.1, the organism is reported as Detected if a single corresponding assay is positive. For example, Human Metapneumovirus will have a test report result of Human Metapneumovirus Detected if the hMPV assay is positive (at least two of the three hMPV assay wells on the array have similar positive melt peaks with Tm values that are within the assay-specific Tm range). The test results for Adenovirus, and Influenza A, and SARS-CoV-2 depend on the interpretation of results from more than one assay. Interpretation and actions for the multi-assay results are provided below.

Adenovirus The BioFire RP2.1 pouch contains five assays (Adeno2, Adeno3, Adeno6, Adeno7.1, and Adeno8) for the detection of Adenovirus. The BioFire Software interprets each of these assays independently (as described above) and the results are combined as a final test result for the virus. If one assay or any combination of assays is positive, the test report result will be Adenovirus Detected. If all assays are negative, the test report result will be Adenovirus Not Detected.

Influenza A The assays in the BioFire RP2.1 are designed to both detect Influenza A and to differentiate the common hemagglutinin subtypes. To accomplish this, the BioFire RP2.1 uses two Influenza A assays, (FluA-pan-1 and FluA-pan-2) and three subtyping assays directed at the hemagglutinin gene (FluA-H1-2, FluA-H1-2009, and FluA-H3). Each of the individual assays is interpreted independently (as described above) and the test result reported for Influenza A is based on the combined results of the five assays as outlined in Table 2. An Influenza A (or subtyping) Equivocal result could occur when the titer of the virus in the specimen is low and not detected by one or more required assays (Table 2). An Influenza A Equivocal result could also indicate the presence of an atypical Influenza A subtype (e.g. avian H7N9 or H5N1 types), or a novel Influenza A strain. Specimens with an Equivocal result or multiple Influenza A subtypes detected should be retested once.

Table 2. Possible Assay Results for Influenza A and the Corresponding Interpretation

Assay Result

FluA-pan Assays (n=2) FluA-H1-2 FluA-H1-2009 FluA-H3 Action

Influenza A Not Detected Negative Negative Negative Negative

None Influenza A H1 ≥1 positive Positive Negative Negative

Influenza A H3 ≥1 positive Negative Negative Positive

Influenza A H1-2009 ≥1 positive Any result Positive Negative

Influenza A H1 Influenza A H3 ≥1 positive Positive Negative Positive Multiple infections are possible but rare a, retest ONCE to confirm result b Influenza A H1-2009

Influenza A H3 ≥1 positive Any result Positive Positive

Influenza A (no subtype detected) 2 positive Negative Negative Negative Retest (see below)

Influenza A Equivocal 1 positive Negative Negative Negative

Retest once (see Result Summary section below for further instruction).

Influenza A H1 Equivocal Negative Positive Negative Negative

Influenza A H3 Equivocal Negative Negative Negative Positive

Influenza A H1-2009 Equivocal Negative Any result Positive Negative a The BioFire RP2.1 can simultaneously detect multiple influenza viruses contained in multivalent vaccines (see Limitations).

b Repeated multiple subtype positives should be further confirmed by other FDA cleared Influenza subtyping tests.

Influenza A (no subtype detected) If both FluA-pan assays are positive, but none of the hemagglutinin subtyping assays are positive, then the interpretation is Influenza A (no subtype detected). This result could occur when the titer of the virus in the specimen is low and not detected by the subtyping assays. This result could also indicate the presence of a novel Influenza A strain. In both cases, the sample in question should be retested. If the retest provides a different result, test the sample a third time to ensure the accuracy of the result. If the retest provides the same result, then the function of the BioFire RP2.1 pouches should be verified by testing with appropriate external control materials (known positive samples for Influenza A H1, Influenza A H3 and Influenza A H1-2009), and a negative control should also be run to test for PCR-product contamination. If the BioFire RP2.1 accurately identifies the external and negative controls, contact the appropriate public health authorities for confirmatory testing.

SARS-CoV-2 The BioFire RP2.1 pouch contains two different assays for the detection of the SARS-CoV-2. The target of each assay is shown in Table 3 below. The BioFire Software interprets each assay independently and if either one or both of the assays is positive, the test report will show Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as Detected. If both assays are negative, the test report result will be Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Not Detected.

Table 3. Gene Targets for SARS-CoV-2 Assays on the BioFire RP2.1 Assay Name Gene Target

SARSCoV2-1 Spike protein (S) gene

SARSCoV2-2 Membrane protein (M) gene

BioFire RP2.1 Test Report The BioFire RP2.1 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.

Run Summary The Run Summary section of the test report provides the Sample ID, time and date of the run, control results and an overall summary of the test results. Any organism with a Detected result will be listed in the corresponding field of the summary. If all of the organism assays were negative then ‘None’ will be displayed in the Detected field. Controls are listed as Passed, Failed, or Invalid. Table 4 provides additional information for each of the possible control field results.

Table 4. Interpretation of Controls Field on the BioFire RP2.1 Test Report

Control Result Explanation Action

Passed The run was successfully completed

AND

Both pouch controls were successful.

None Report the results provided on the test report

Failed

The run was successfully completed

BUT

At least one of the pouch controls (RNA Process Control and/or PCR2 Control) failed.

Repeat the test using a new pouch.

If the error persists, contact Technical Support for further instruction.

Invalid

The controls are invalid because the run did not complete.

(Typically this indicates a software or hardware error).

Note any error codes displayed during the run and the Run Status field in the Run Details section of the report.

Refer to the appropriate FilmArray operator’s manual or contact Technical Support for further instruction.

Once the error is resolved, repeat the test or repeat the test using another instrument.

Result Summary The Result Summary section of the test report lists the result for each target tested by the panel. Possible results for each organism are Detected, Not Detected, or Invalid (Equivocal is also a possible result for Influenza A and its subtypes). Table 5 provides an explanation for each interpretation and any follow-up necessary to obtain a final result.

Table 5. Reporting of Results and Required Actions

Result Explanation Action

Detecteda

The run was successfully completed

AND

The pouch controls were successful (Passed)

AND

The assay(s) for the organism were POSITIVE (i.e., met the requirements for a positive result described in the Assay Interpretation section above)

Report results.

Not Detected

The run was successfully completed

AND

The pouch controls were successful (Passed)

AND

The assay(s) for the organism were NEGATIVE (i.e., did not meet the requirements for a positive result described in the Assay Interpretation section above)

Report results.

Equivocal

The run was successfully completed

AND

The pouch controls were successful (Passed)

AND

The combination of positive and negative assay results for Influenza A were inconclusive (see Table 2)

Retest the original sample ONCE and report the result of the retestb.

Invalid

The pouch controls were not successful (Failed)

OR

The run was not successful (Run Status displayed as: Aborted, Incomplete, Instrument Error or Software Error)

See Table 4 , Interpretation of Control Field on the FilmArray

Test Report for instruction.

a If four or more organisms are detected in a specimen, retesting is recommended to confirm the polymicrobial result.

b Equivocal results can occur when the titer of the virus in the specimen is low (below LoD). Equivocal results could also indicate the presence of a novel Influenza A strain or reactivity with non-human influenza A viruses or rare human influenza A viruses that are not H1, H1-2009 or H3. Such strains generally produce Influenza A Equivocal or Influenza A (no subtype detected) results.

Run Details The Run Details section provides additional information about the run including: pouch information…