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The BIOFIRE FILMARRAY Pneumonia Panel (BIOFIRE Pneumonia Panel) is a multiplexed nucleic acid test intended for use with BIOFIRE FILMARRAY 2.0 (BIOFIRE 2.0) or BIOFIRE FILMARRAY TORCH (BIOFIRE TORCH) systems for the simultaneous detection of multiple respiratory viral and bacterial nucleic acids, as well as select antimicrobial resistance genes, in sputum-like speciorated sputum, or endotracheal aspirates) or bronchoalveolar lavage (BAL)-like specimens (BAL) obtained from individuals suspected of lower respiratory tract infection. The following bacteria are reported semi-quantitatively with bins representing approximately 10^4, 10^5, or ≥10°7 genomic copies of bacterial nucleic acid per milliliter (copies/mL) of specimen, to aid in estimating relative abundance of nucleic acid from these common bacteria within a specimen: Bacteria reported with bins of 10^4, 10^5, 10^6, or ≥10^7 copies/mL - · Acinetobacter calcoaceticus-baumannii complex - · Klebsiella oxytoca - · Serratia marcescens - · Enterobacter cloacae complex - Klebsiella pneumoniae group - · Staphylococcus aureus - · Escherichia coli - · Moraxella catarrhalis - · Streptococcus agalactiae - Haemophilus influenzae - · Proteus spp. - · Streptococcus pneumoniae - Klebsiella aerogenes - Pseudomonas aeruginosa - · Streptococcus pyogenes The following atypical bacteria, viruses, and antimicrobial resistance genes are reported qualitatively: #### Atypical Bacteria - Chlamydia pneumoniae - · Legionella pneumophila - Mycoplasma pneumoniae #### Viruses - · Adenovirus - Human rhinovirus/enterovirus - · Parainfluenza virus - · Coronavirus - · Influenza A virus - Respiratory syncytial virus • Human metapneumovirus - Influenza B virus Antimicrobial Resistance Genes - · CTX-M - IMP - КРС - NDM - OXA-48-like - VIM - · mecA/C and MREJ (MRSA) The detection and identification of specific viral and bacterial nucleic acids, as well as the estimation of relative abundance of nucleic acid from common bacterial analytes, within specimens collected from individuals exhibiting signs and/or symptoms of a respiratory infection, aids in the diagnosis of lower respiratory infection with other clinical and epidemiological information. The results of this test should not be used as 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, pathogens below the limit of detection, or in the case of bacterial analytes, present at levels below the lowest reported 10^4 copies/mL bin. Detection of analytes does not rule out co-infection with other organisms; the agent(s) detected by the BIOFIRE Pneumonia Panel 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 lower respiratory tract infection. Detection of bacterial nucleic acid may be indicative of colonizing or normal respiratory flora and may not indicate the causative agent of pneumonia. Semi-quantitative Bin (copies/mL) results generated by the BIOFIRE Pneumonia Panel are not equivalent to CFU/mL and do not consistently correlate with the quantity of bacterial analytes compared to CFU/mL. For specimens with multiple bacteria detected, the relative abundance of nucleic acids (copies/mL) may not correlate with the relative abundance of bacteria as determined by culture (CFU/mL). Clinical correlation is advised to determine significance of semi-quantitative Bin (copies/mL) for clinical management. The antimicrobial resistance gene detected may or may not be associated with the agent(s) responsible for disease. Negative results for these antimicrobial resistance gene assays do not indicate susceptibility to corresponding classes of antimicrobials, as multiple mechanisms of antimicrobial resistance exist. Antimicrobial resistance can occur via multiple mechanisms. A "Not Detected" result for a genetic marker of antimicrobial resistance does not indicate susceptibility to associated antimicrobial drugs or drug classes. A "Detected" result for a genetic marker of antimicrobial resistance cannot be definitively linked to the microorganism(s) detected. Culture is required to obtain isolates for antimicrobial susceptibility testing, and BIOFIRE Pneumonia Panel results should be used in conjunction with culture results for determination of bacterial susceptibility or resistance. Due to the genetic similarity between human rhinovirus and enterovirus, the test cannot reliably differentiate them. A positive Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required. Culture is required to identify pathogens not detected by the BIOFIRE Pneumonia Panel, to further speciate analytes in genus, complex, or group results if desired, to identify bacterial pathogens present below the 10°4 copies/mL bin if desired, and for antimicrobial susceptibility testing. BIOFIRE FILMARRAY Pneumonia Panel plus: The BIOFIRE FILMARRAY Pneumonia Panel plus (BIOFIRE Pneumonia Panel plus) is a multiplexed nucleic acid test intended for use with BIOFIRE FILMARRAY 2.0 (BIOFIRE 2.0) or BIOFIRE FILMARRAY TORCH (BIOFIRE TORCH) systems for the simultaneous detection and identification of nucleic acids from Middle East respiratory syndrome coronavirus (MERS-CoV) and multiple respiratory viral and bacterial nucleic acids, as well as select antimicrobial resistance genes, in sputum-like specimens (induced or expectorated sputum, or endotracheal aspirates) or bronchoalveolar lavage (BAL)-like specimens (BAL or mini-BAL) obtained from individuals meeting MERS-CoV clinical and/or epidemiological criteria. Testing with BIOFIRE Pneumonia Panel plus should not be performed unless the patient meets clinical and/or epidemiologic criteria for testing suspected MERS-CoV specimens. Thical signs and symptoms assocated with MERS-CoV infection, contact with a probable or confirmed MERS-CoV case, history of travel to geographic locations where MERS-CoV cases were detected, or other epidemiological links for which MERS-CoV testing may be indicated. The following bacteria are reported semi-quantitatively with bins representing approximately 10^4, 10^5, or ≥10°7 genomic copies of bacterial nucleic acid per milliliter (copies/mL) of specimen, to aid in estimating relative abundance of nucleic acid from these common bacteria within a specimen: Bacteria reported with bins of 10^4, 10^5, 10^6, or ≥10^7 copies/mL - Acinetobacter calcoaceticus-baumannii complex - Enterobacter cloacae complex - Escherichia coli - Haemophilus influenzae - Klebsiella aerogenes - · Klebsiella oxytoca - · Klebsiella pneumoniae group - Moraxella catarrhalis - Proteus spp. - Pseudomonas aeruginosa - · Serratia marcescens - Staphylococcus aureus - Streptococcus agalactiae - · Streptococcus pneumoniae - · Streptococcus pyogenes The following atypical bacteria, viruses, and antimicrobial resistance genes are reported qualitatively: Atypical Bacteria - Chlamydia pneumoniae - · Legionella pneumophila - Mycoplasma pneumoniae Viruses - · Middle East respiratory syndrome coronavirus (MERS-CoV) - Adenovirus - Coronavirus - Human metapneumovirus - Human rhinovirus/enterovirus - · Influenza A virus - Influenza B virus - Parainfluenza virus - · Respiratory syncytial virus Antimicrobial Resistance Genes - CTX-M - IMP - · KPC - NDM - OXA-48-like - VIM - · mecA/C and MREJ (MRSA) The detection and identification of specific viral and bacterial nucleic acids from MERS-CoV and other respiratory pathogens, as well as the estimation of relative abundance of nucleic acid from common bacterial analytes, within specimens collected from individuals meeting MERS-CoV clinical and/or epidemiological criteria aids in the differential diagnosis of MERS-CoV infection, if used in conjunction with other clinical and epidemiological information in accordance with the guidelines provided by the appropriate public health authorities. BIOFIRE Pneumonia Panel plus MERS-CoV positive results are for the presumptive identification of MERS-CoV. The definitive identification of MERS-CoV requires additional testing and confirmation procedures in consultation with the appropriate public health authorities (e.g., local or state public health departments, etc.) for whom reporting is necessary. The diagnosis of MERS-CoV infection must be made based on history, signs, symptoms, exposure likelihood, and other laboratory evidence in addition to the identification of MERS-CoV. BIOFIRE Pneumonia Panel plus MERS-CoV negative results, even in the context of a BIOFIRE Pneumonia Panel plus positive result for one or more of the common respiratory pathogens, do not preclude MERS-CoV infection and should not be used as the sole basis for patient management decisions. The levels of MERS-CoV that would be present in sputum-like or BAL-like specimens from individuals with early infection and from asymptomatic MERS-CoV carriers are not well understood. A negative BIOFIRE Pneumonia Panel plus MERS-CoV result in an asymptomatic individual does not rule out the possibility of future illness and does not demonstrate that the individual is not infectious. Viral culture should not be attempted on specimens with positive BIOFIRE Pneumonia Panel plus results for MERS-CoV unless a BSL 3 facility is available to receive and culture specimens. Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, pathogens below the limit of detection, or in the case of bacterial analytes, present at levels below the lowest reported 10^4 copies/mL bin. Detection of analytes does not rule out co-infection with other organisms; the agent(s) detected by the BIOFIRE Pneumonia Panel plus 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 lower respiratory tract infection. Detection of bacterial nucleic acid may be indicative of colonizing or normal respiratory flora and may not indicate the causative agent of pneumonia. Semi-quantitative Bin (copies/mL) results generated by the BIOFIRE Pneumonia Panel plus are not equivalent to CFU/mL and do not consistently correlate with the quantity of bacterial analytes compared to CFU/mL. For specimens with multiple bacteria detected, the relative abundance of nucleic acids (copies/mL) may not correlate with the relative abundance of bacteria as determined by culture (CFU/mL). Clinical correlation is advised to determine significance of semi-quantitative Bin (copies/mL) for clinical management. The antimicrobial resistance gene detected may or may not be associated with the agent(s) responsible for disease. Negative results for these antimicrobial resistance gene assays do not indicate susceptibility to corresponding classes of antimicrobials, as multiple mechanisms of antimicrobial resistance exist. Antimicrobial resistance can occur via multiple mechanisms. A "Not Detected" result for a genetic marker of antimicrobial resistance does not indicate susceptibility to associated antimicrobial drugs or drug classes. A "Detected" result for a genetic marker of antimicrobial resistance cannot be definitively linked to the microorganism(s) detected. Culture is required to obtain isolates for antimicrobial susceptibility testing, and BIOFIRE Pneumonia Panel plus results should be used in conjunction with culture results for determination of bacterial susceptibility or resistance. Due to the genetic similarity between human rhinovirus and enterovirus, the test cannot reliably differentiate them. A positive Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required. Culture is required to identify pathogens not detected by the BIOFIRE Pneumonia Panel plus, to further speciate analytes in genus, complex, or group results if desired, to identify bacterial pathogens present below the 10°4 copies/mL bin if desired, and for antimicrobial susceptibility testing.

The BIOFIRE® FILMARRAY® Pneumonia Panel and BIOFIRE® FILMARRAY® Pneumonia Panel plus use nested, multiplex reverse transcription polymerase chain reaction (PCR), followed by melting curve analysis for the detection of select organisms and antimicrobial resistance (AMR) genes in sputum-like (induced and expectorated sputum as well as endotracheal aspirate, ETA) and bronchoalveolar lavage (BAL)-like (BAL and mini-BAL) specimens. The panels allow for the identification of specific bacteria, atypical bacteria, viruses, and AMR genes as indicated in Table 1. The BIOFIRE Pneumonia Panel and BIOFIRE Pneumonia Panel plus pouches are identical, but the BIOFIRE Pneumonia Panel plus includes reporting of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which is not included in the BIOFIRE Pneumonia Panel. Reporting of MERS-CoV is controlled through software masking of the MERS-CoV result for the BIOFIRE Pneumonia Panel. The BIOFIRE Pneumonia Panels are compatible with bioMérieux's PCR-based in vitro diagnostic BIOFIRE® FILMARRAY® 2.0 and BIOFIRE® FILMARRAY® TORCH Systems for infectious disease testing. Specific software module (i.e. BIOFIRE Pneumonia Panel Pouch Module Software) are used to perform BIOFIRE Pneumonia Panels testing on these systems.

The FilmArray® Pneumonia Panel is a multiplexed nucleic acid test intended for use with FilmArray® 2.0 or FilmArray® Torch systems for the simultaneous detection of multiple respiratory viral and bacterial nucleic acids, as well as select antimicrobial resistance genes, in sputum-like specimens (induced or expectorated sputum, or endotracheal aspirates) or bronchoalveolar lavage (BAL)-like specimens (BAL or mini-BAL) obtained from individuals suspected of lower respiratory tract infection. The following bacteria are reported semi-quantitatively with bins representing approximately 10^4, 10^5 genomic copies of bacterial nucleic acid per milliliter (copies/mL) of specimen, to aid in estimating relative abundance of nucleic acid from these common bacteria within a specimen: Bacteria reported with bins of 10^4, 10^5, 10^6, or ≥10^7 copies/mL -Acinetobacter calcoaceticus-baumannii complex - -Enterobacter cloacae complex - -Escherichia coli - -Haemophilus influenzae - -Klebsiella aerogenes - -Klebsiella oxytoca - -Klebsiella pneumoniae group - -Moraxella catarrhalis - -Proteus spp. - -Pseudomonas aeruginosa - -Serratia marcescens - -Staphylococcus aureus - -Streptococcus agalactiae - -Streptococcus pneumoniae - -Streptococcus pyogenes The following atypical bacteria, viruses, and antimicrobial resistance genes are reported qualitatively: Atypical Bacteria -Chlamydia pneumoniae -Legionella pneumophila - -Mycoplasma pneumoniae Viruses -Adenovirus - -Coronavirus - -Human Metapneumovirus - -Human Rhinovirus/Enterovirus - -Influenza A - -Influenza B - -Parainfluenza Virus - -Respiratory Syncytial Virus Antimicrobial Resistance Genes -CTX-M -IMP -KPC -NDM -OXA-48-like -VIM -mecA/C and MREJ The detection and identification of specific viral and bacterial nucleic acids, as well as the estimation of relative abundance of nucleic acid from common bacterial analytes, within specimens collected from individuals exhibiting signs and/or symptoms of a respiratory infection, aids in the diagnosis of lower respiratory infection with other clinical and epidemiological information. The results of this test should not be used as 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, pathogens below the limit of detection, or in the case of bacterial analytes, present at levels below the lowest reported 10^4 copies/mL bin. Detection of analytes does not rule out co-infection with other organisms; the agent(s) detected by the FilmArray Pneumonia Panel 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 lower respiratory tract infection. Detection of bacterial nucleic acid may be indicative of colonizing or normal respiratory flora and may not indicate the causative agent of pneumonia. Semi-quantitative Bin (copies/mL) results generated by the FilmArray Pneumonia Panel are not equivalent to CFU/mL and do not consistently correlate with the quantity of bacterial analytes compared to CFUmL. For specimens with multiple bacteria detected, the relative abundance of nucleic acids (copies/mL) may not correlate with the relative abundance of bacteria as determined by culture (CFU/mL). Clinical correlation is advised to determine significance of semi-quantitative Bin (copies/mL) for clinical management. The antimicrobial resistance gene detected may or may not be associated with the agent(s) responsible for disease. Negative results for these antimicrobial resistance gene assays do not indicate susceptibility to corresponding classes of antimicrobials, as multiple mechanisms of antimicrobial resistance exist. Antimicrobial resistance can occur via multiple mechanisms. A "Not Detected" result for a genetic marker of antimicrobial resistance does not indicate susceptibility to associated antimicrobial drugs or drug classes. A "Detected" result for a genetic marker of antimicrobial resistance cannot be definitively linked to the microorganism(s) detected. Culture is required to obtain isolates for antimicrobial susceptibility testing, and FilmArray Pneumonia Panel results should be used in conjunction with culture results for determination of bacterial susceptibility or resistance. Due to the genetic similarity between human rhinovirus and enterovirus, the test cannot reliably differentiate them. A positive Rhinovirus Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required. Culture is required to identify pathogens not detected by the FilmArray Pneumonia Panel, to further speciate analytes in genus, complex, or group results if desired, to identify bacterial pathogens present below the 10^4 copies/mL bin if desired, and for antimicrobial susceptibility testing.

The FilmArray Pneumonia (PN) Panel is designed to simultaneously identify 26 potential pathogens of lower respiratory tract infection (LRTI) and associated antimicrobial resistance (AMR) genes from a sputum-like (induced and expectorated sputum as well as endotracheal aspirate, ETA) or bronchoalveolar lavage (BAL)-like (BAL and mini-BAL) specimens obtained from individuals with signs and/or symptoms of lower respiratory tract infection in a time (~1 hour) that allows the test results to be used in determining appropriate patient treatment and management. FilmArray PN Panel is compatible with BioFire Diagnostics' (BioFire) PCR-based in vitro diagnostic FilmArray 2.0 (K143178) and FilmArray Torch (K160068) systems for infectious disease testing. A specific software module (i.e. FilmArray PN Panel pouch module) is used to perform FilmArray PN Panel testing on these systems. Bacteria - Quantitative Results: Acinetobacter calcoaceticus-baumannii complex, Enterobacter cloacae complex, Escherichia coli, Haemophilus influenzae, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group, Moraxella catarrhalis, Proteus spp., Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes Bacteria (Atypical) - Qualitative Results: Chlamydia pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae Antimicrobial Resistance Genes: blaCTX-M (Extended spectrum beta-lactamase (ESBL)), blaIMP (Carbapenem resistance), blaKPC (Carbapenem resistance), mecA/mecC and MREJ (Methicillin resistance), blaNDM (Carbapenem resistance), blaOXA48-like (Carbapenem resistance), blaVIM (Carbapenem resistance) Viruses: Adenovirus, Coronavirus, Human Metapneumovirus, Human Rhinovirus/Enterovirus, Influenza A, Influenza B, Parainfluenza Virus, Respiratory Syncytial Virus A test is initiated by loading Hydration Solution into one port of the FilmArray pouch and a sputum-like or BAL-like sample mixed with the provided Sample Buffer into the other port of the FilmArray PN Panel pouch and placing it in a FilmArray instrument. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the FilmArray Software guides the user though the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run. The FilmArray instrument contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis. Nucleic acid extraction occurs within the FilmArray pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the FilmArray performs a nested multiplex PCR that is executed in two stages. During the first stage, the FilmArray performs a single, large volume, highly multiplexed reverse transcription PCR (rt-PCR) reaction. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Diagnostics). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, is performed in single plex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data. The FilmArray Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel. A feature of the FilmArray PN Panel is the reporting of organism abundance for common bacteria in discrete bins representing 10^4, 10^5, 10^6, and >10^7 genomic copies/mL. The panel accomplishes this by comparing the amplification of the bacterial assays with that of a Quantified Standard Material (QSM) present in the pouch.

The Unyvero LRT BAL Application is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of nucleic acid sequences from the following microorganisms (N = 20) and antibiotic resistance markers (N = 10) in bronchoalveolar lavage (BAL)-like specimens (BAL or mini-BAL) from adult hospitalized patients with suspected lower respiratory tract infections. The Unyvero LRT BAL Application performed on the Unyvero System is indicated as an aid in the diagnosis of lower respiratory tract infection in adult hospitalized patients with signs and symptoms of lower respiratory infection; results should be used in conjunction with other clinical and laboratory findings. As BAL specimens may contain colonizing microorganisms, detection of Unyvero LRT BAL microbial targets does not indicate that the microorganism is the disease. Unyvero positive results do not rule out co-infection with other microorganisms. Negative results do not preclude lower respiratory infection, as the causative agent may be a microorganism not detected by this test. A negative result for any antibiotic resistance marker does not indicate that detected microorganisms are susceptible to applicable antimicrobial agents. Detected antibiotic resistance markers cannot be definitively linked to specific microorganisms, and may be present in organisms that are not detected by the Unyvero LRT BAL Application. Microbiology cultures of BALs should be performed to obtain isolates for species identification and antimicrobial susceptibility testing and to identify potential microorganisms not targeted by the Unyvero LRT BAL Application.

The Unyvero LRT BAL Application automates and integrates DNA purification and eight parallel multiplex endpoint PCR reactions. It provides qualitative detection of nucleic acids from multiple lower respiratory pathogens using hybridization on PCR chamber arrays in a single use cartridge from a single bronchoalveolar lavage (BAL)-like specimen (BAL or mini-BAL). The Unyvero LRT BAL Application identifies 20 microorganisms and 10 antibiotic resistance markers as listed in the Intended Use Statement, below. The Unyvero LRT BAL Application uses a multiplex PCR approach following array hybridization which targets 30 individual analytes (microorganisms (N = 20) and antibiotic resistance markers (N = 10) divided into eight separate PCR reactions that are performed in individual reaction chambers simultaneously on a Unyvero LRT BAL Application cartridge. Multiplex compositions are designed to avoid any expected common occurrence of certain analytes within the same multiplex to largely reduce competitive PCR inhibition. Individual analyte assays of the Unyvero LRT BAL panel are designed to exhibit low or absent cross-reactivity with the relevant bronchoalveolar lavage (BAL)like specimens (BAL or mini-BAL) sample matrix or 'close neighbor' strains. Array oligonucleotides are designed for similar hybridization and melting temperatures (approx. 65 - 80 ℃, varying by amplicon). Hybridization and melting temperatures are used to exclude non-specific hybridization signals for improved signal specificity. The instrumentation consists of one (or more) Unyvero L4 Lysator, one (or more) Unyvero A50 Analyzer, a Unyvero C8 Cockpit, and four single-use consumables: the Unyvero LRT BAL Cartridge, the Unyvero Sample Tube, Sample Tube Cap and the Unyvero Master Mix. A Unyvero Sample Tube Holder is supplied as accessory to simplify the sample filling step. - Unyvero LRT BAL Cartridge contains DNA isolation and purification reagents, . primers, hybridization and wash buffers, and oligonucleotides for detection. - Unyvero T1 Sample Tube and Transport Cap contains glass beads and buffers to lyse ● microorganisms and liquefy the sample. - . Unyvero T1 Sample Tube Cap seals the Unyvero Sample Tube and contains Proteinase K and a synthetic control gene for process monitoring. - Unyvero M1 Master Mix Tube contains reagents for DNA amplification. ● An internal control (a synthetic gene without any homology to known sequences) is processed in every chamber in order to verify the DNA purification, array hybridization, and detection. Other than the built-in controls, no external materials are supplied with Unyvero LRT BAL Application devices and consumables. Good laboratory practice recommends running external positive and negative controls using samples cultured in the laboratory. No additional reagents are required to perform the Unyvero LRT BAL Application; all reagents are supplied within the cartridge or within the other consumables with the exception of the polymerase Master Mix, which is provided separately (frozen). How to perform a Unyvero LRT BAL test: - 1. Remove the Unyvero Sample Tube from its packaging and slide it in the Unyvero Sample Tube Holder in the upright position with the barcoded end at the bottom. - 2. Remove the Transport Cap from the Sample Tube by pulling it upward. - 3. Pipette 180 uL of vortexed patient specimen into the Sample Tube and close it using the Unyvero Cap provided in the LRT BAL kit: align the small nodules on the neck of the Sample Tube with the Tube with the openings on the Cap and press down to lock in place. - 4. Scan the Sample Tube and place it into the Lysator. Close the Lysator lid to start the Lysator. - 5. Remove Master Mix from freezer and thaw at room temperature (15 ℃ 25 ℃) for approximately 30 minutes. - 6. When lysis is complete, remove the Sample Tube from the Lysator and place it into the labeled position on the left-hand side of the Unyvero LRT BAL Cartridge. - 7. Place the thawed Master Mix into the labeled position on the right-hand side of the Cartridge. - 8. Scan the Cartridge on the Cockpit and insert it into the position indicated on the Analyzer. The software provides on-screen instructions to start the test. - 9. View results after completion of the run. During the automated analysis (Step 8), which is entirely controlled by the A50 Analyzer, the sample is mixed with ethanol and then transferred onto the DNA purification column, where buffers purify and elute the DNA. Eluted DNA is transferred to a chamber, where mixing with the Master Mix takes place. This mixture is distributed into eight separate PCR reaction chambers each containing multiple primer pairs, consisting of one labeled and one non-labeled primer for the respective multiplex endpoint PCR. After specific amplification, PCR products are hybridized to the corresponding array probes. Each array has been manufactured with probes corresponding to the amplicons for the targeted microorganism sequences described above. A total of up to 49 spots per array allows for redundant detection with at least four spots per analyte, as well as spots for intensity calibration, and orientation markers for the image processing software. Binding of amplicons to specific probes is detected by analyzing fluorescence images of the arrays. Result data are displayed on the C8 Cockpit for visualization, printout, temporary storage and electronic data export. A test run is completed after 4 to 5 hrs, and results for panel microorganisms and corresponding antibiotic resistance markers are displayed on the Cockpit screen. Four screens are provided: - . The Result Summary screen provides a quick overview of all detected LRT BAL panel microorganisms, together with all detected corresponding antibiotic resistance markers. - . The Microorganisms screen provides a list of all panel microorganisms grouped in Grampositive bacteria, non-fermenting bacteria, Enterobacteriaceae and other microorganisms together with the analysis result (reported as detected, not detected or invalid). - The Result Details screen provides a list of all analyzed microorganisms and the ● corresponding antibiotic resistance markers together with the analysis result (reported as detected, not detected, invalid or NA). - Test Details screen showing user name, lot numbers of used consumables, expiration dates of ● the consumables and start and stop times and dates of the test. Results can be reviewed on the cockpit or, optionally, be printed out. All results are saved in a database on the Unyvero Cockpit for later review and printing. The Unyvero software is designed to: - Manage analysis workflow (Cockpit) ● - Carry out sample lysis (Lysator) ● - Execute the analysis and generate the analytical result (Analyzer) . - Manage communication among units (Cockpit, Lysator, Analyzer) ● - Monitor internal mechanical / electrical actuators (Lysator, Analyzer) ● - Present analysis results (Cockpit) . - Store analysis results (Cockpit) ● Each device (Cockpit, Lysator. Analyzer) is a subsystem within the overall system, and each consists of hardware and software components. The different devices are interconnected by an Ethernet based communication interface, and system functionality is provided by the interaction of all three device types. Only the Cockpit presents a rich user interface and allows interaction with the operator. The Lysator and Analyzer units include a simple display for showing device status. Optional HIS/LIS connectivity allows transferring results to a hospital or laboratory information system.

The FilmArray® Pneumonia Panel is a multiplexed nucleic acid test intended for use with FilmArray® 2.0, or FilmArray® Torch systems for the simultaneous detection of multiple respiratory viral and bacterial nucleic acids, as well as select antimicrobial resistance genes, in sputum-like specimens (induced or expectorated sputum, or endotracheal aspirates) or bronchoalveolar lavage (BAL)-like specimens (BAL or mini-BAL) obtained from individuals suspected of lower respiratory tract infection. The following bacteria are reported semi-quantitatively with bins representing approximately 10°4 10°5, 10°6, or ≥10°7 genomic copies of bacterial nucleic acid per milliliter (copies/mL) of specimen, to aid in estimating relative abundance of nucleic acid from these common bacteria within a specimen: Bacteria reported with bins of 10^4 10^5, 10^6, or ≥10^7 copies/mL - Acinetobacter calcoaceticus-baumannii complex - Enterobacter cloacae complex - · Escherichia coli - Haemophilus influenzae - Klebsiella aerogenes - Klebsiella oxytoca - Klebsiella pneumoniae group - Moraxella catarrhalis - · Proteus spp. - Pseudomonas aeruginosa - Serratia marcescens - Staphylococcus aureus - Streptococcus agalactiae - Streptococcus pneumoniae - Streptococcus pyogenes The following atypical bacteria, viruses, and antimicrobial resistance genes are reported qualitatively: Atypical Bacteria - Chlamydia pneumoniae - Legionella pneumophila - Mycoplasma pneumoniae Viruses - Adenovirus - Coronavirus - Human Metapneumovirus - · Human Rhinovirus/Enterovirus - · Influenza A - · Influenza B - Parainfluenza Virus - Respiratory Syncytial Virus Antimicrobial Resistance Genes - CTX-M - IMP - КРС - NDM - · OXA-48-like - VIM - · mecA/C and MREJ The detection and identification of specific viral and bacterial nucleic acids, as well as the estimation of relative abundance of nucleic acid from common bacterial analytes, within specimens collected from individuals exhibiting signs and/or symptoms of a respiratory infection, aids in the diagnosis of lower respiratory infection if used in conjunction with other clinical and epidemiological information. The results of this test should not be used as 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, pathogens below the limit of detection, or in the case of bacterial analytes, present at levels below the lowest reported 10°4 copies/mL bin. Detection of analytes does not rule out co-infection with other organisms: the agent(s) detected by the Film Array Pneumonia Panel 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 lower respiratory tract infection. Detection of bacterial nucleic acid may be indicative of colonizing or normal respiratory flora and may not indicate the causative agent of pneumonia. Semi-quantitative bin (copies/mL) results generated by the FilmArray Pneumonia Panel are not equivalent to CFU/mL and do not consistently correlate with the quantity of bacterial analytes compared to CFU/mL. For specimens with multiple bacteria detected, the relative abundance of nucleic acids (copies/mL) may not correlate with the relative abundance of bacteria as determined by culture (CFU/mL). Clinical correlation is advised to determine significance of semi-quantitative bin (copies/mL) for clinical management. The antimicrobial resistance gene detected may or may not be associated with the agent(s) responsible for disease. Negative results for these antimicrobial resistance gene assays do not indicate susceptibility to corresponding classes of antimicrobials, as multiple mechanisms of antimicrobial resistance exist. Antimicrobial resistance can occur via multiple mechanisms. A "Not Detected" result for a genetic marker of antimicrobial resistance does not indicate susceptibility to associated antimicrobial drugs or drug classes. A "Detected" result for a genetic marker of antimicrobial resistance cannot be definitively linked to the microorganism(s) detected. Culture is required to obtain isolates for antimicrobial susceptibility testing, and FilmArray Pneumonia Panel results should be used in conjunction with culture results for determination of bacterial susceptibility or resistance. Due to the genetic similarity between human rhinovirus and enterovirus, the test cannot reliably differentiate them. A positive Rhinovirus/Enterovirus result should be followed up using an alternate method (e.g., cell culture or sequence analysis) if differentiation is required. Culture is required to identify pathogens not detected by the FilmArray Pneumonia Panel, to further speciate analytes in genus, complex, or group results if desired, to identify bacterial pathogens present below the 10°4 copies/mL bin if desired, and for antimicrobial susceptibility testing.

The FilmArray Pneumonia Panel is designed to simultaneously identify 26 potential pathogens of lower respiratory tract infection (LRTI) and associated antimicrobial resistance (AMR) genes from a sputumlike (induced and expectorated sputum as well as endotracheal aspirate, ETA) or bronchoalveolar lavage (BAL)-like (BAL and mini-BAL) specimens obtained from individuals with signs and/or symptoms of lower respiratory tract infection in a time (~1 hour) that allows the test results to be used in determining appropriate patient treatment and management. FilmArray Pneumonia Panel is compatible with BioFire Diagnostics' (BioFire) PCR-based in vitro diagnostic FilmArray 2.0, and FilmArray Torch systems for infectious disease testing. A specific software module (i.e. FilmArray Pneumonia Panel pouch module) is used to perform FilmArray Pneumonia Panel testing on these systems. Bacteria - Quantitative Results | Antimicrobial Resistance Genes -----------------------------------------------|---------------------------------------------------- Acinetobacter calcoaceticus-baumannii complex | blaCTX-M (Extended spectrum beta-lactamase (ESBL)) Enterobacter cloacae complex | blaIMP (Carbapenem resistance) Escherichia coli | blaKPC (Carbapenem resistance) Haemophilus influenzae | mecA/mecC and MREJ (Methicillin resistance) Klebsiella aerogenes | blaNDM (Carbapenem resistance) Klebsiella oxytoca | blaOXA48-like (Carbapenem resistance) Klebsiella pneumoniae group | blaVIM (Carbapenem resistance) Moraxella catarrhalis | Proteus spp. | Adenovirus Pseudomonas aeruginosa | Coronavirus Serratia marcescens | Human Metapneumovirus Staphylococcus aureus | Human Rhinovirus/Enterovirus Streptococcus agalactiae | Influenza A Streptococcus pneumoniae | Influenza B Streptococcus pyogenes | Bacteria (Atypical) - Qualitative Results | Viruses Chlamydia pneumoniae | Parainfluenza Virus Legionella pneumophila | Respiratory Syncytial Virus Mycoplasma pneumoniae | A test is initiated by loading Hydration Solution into one port of the FilmArray pouch and a sputum-like or BAL-like sample mixed with the provided Sample Buffer into the other port of the FilmArray Pneumonia Panel pouch and placing it in a FilmArray instrument. The pouch contains all of the reagents required for specimen testing and analysis in a freeze-dried format; the addition of Hydration Solution and Sample/Buffer Mix rehydrates the reagents. After the pouch is prepared, the FilmArray Software guides the user though the steps of placing the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run. The FilmArray instrument contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically-controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the PCR reactions and the melt curve analysis. Nucleic acid extraction occurs within the FilmArray pouch using mechanical and chemical lysis followed by purification using standard magnetic bead technology. After extracting and purifying nucleic acids from the unprocessed sample, the FilmArray performs a nested multiplex PCR that is executed in two stages. During the filmArray performs a single, large volume, highly multiplexed reverse transcription PCR (rt-PCR) reaction. The products from first stage PCR are then diluted and combined with a fresh, primer-free master mix and a fluorescent double stranded DNA binding dye (LC Green® Plus, BioFire Diagnostics). The solution is then distributed to each well of the array. Array wells contain sets of primers designed specifically to amplify sequences internal to the PCR products generated during the first stage PCR reaction. The 2nd stage PCR, or nested PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 2nd stage PCR, the array is interrogated by melt curve analysis for the detection of signature amplicons denoting the presence of specific targets. A digital camera placed in front of the 2nd stage PCR captures fluorescent images of the PCR reactions and software interprets the data. The FilmArray Software automatically interprets the results of each DNA melt curve analysis and combines the data with the results of the internal pouch controls to provide a test result for each organism on the panel.

The Unyvero LRT Application is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of nucleic acid sequences from the following microorganisms and antibiotic resistance markers in endotracheal aspirates from adult hospitalized patients with suspected lower respiratory tract infections. The Unyvero LRT Application performed on the Unyvero System is indicated as an aid in the diagnosis of lower respiratory tract infection in adult hospitalized patients with signs and symptoms of lower respiratory infection: results should be used in conjunction with other clinical and laboratory findings. As tracheal aspirates commonly contain colonizing microorganisms, detection of Unyvero LRT microbial targets does not indicate that the microorganism is the cause of the disease. Unyvero positive results do not rule out co-infection with microorganisms not detected by the Unyvero LRT Application. Negative results do not preclude lower respiratory infection, as the causative agent may be a microorganism not detected by this test. A negative result for any antibiotic resistance marker does not indicate that detected microorganisms are susceptible to applicable antimicrobial agents. Detected resistance markers cannot be definitively linked to specific microorganisms, and may be present in organisms that are not detected by the Unyvero LRT Application such as organisms present as colonizing or normal flora. Microbiology cultures of aspirates should be performed to obtain isolates for species identification and antimicrobial susceptibility testing, to differentiate quantities of identified microorganisms as well as normal flora present in the specimen and to identify potential microorganisms not targeted by the Unyvero LRT Application.

The Unyvero LRT Application is a qualitative test that includes specimen processing, genomic bacterial DNA isolation and purification, multiplex PCR and array hybridization and detection. The Unyvero LRT Application performed using the Unyvero System detects specific nucleic acid sequences from microorganisms and resistance markers in tracheal aspirates collected from patients with signs and symptoms of lower respiratory infection. The Unyvero LRT Application consists of the following components: - Unyvero LRT Cartridge: Contains DNA isolation and purification reagents, a DNA isolation column, eight separate PCR chambers with eight corresponding detection arrays. The Cartridge also contains fluorescently-labeled primers, hybridization and wash buffers and oligonucleotide probes for detection of targeted PCR products using array hybridization technology. - Unyvero T1 Sample Tube: Contains glass beads and buffers to lyse bacteria and liquefy the sample. - Unyvero T1 Sample Tube Cap (with Internal Control): Contains proteinase K and a synthetic internal control gene for process monitoring. The T1 Sample Tube Cap seals the Unyvero Sample Tube after which the internal control is combined with each patient specimen. The internal control DNA sequence does not have significant homology to targeted sequences and is amplified independently in each of the eight PCR chambers and the amplified internal control product is hybridized on each array. - Unyvero M1 Master Mix: Contains reagents for DNA amplification. - Unyvero T1 Transfer Tool: The Transfer tool can be used to transfer viscous specimens from the primary sample container to the Unyvero Sample Tube. The Unyvero System consists of the following components: - Unyvero Lysator: The Lysator lyses the specimen and can process up to four specimens simultaneously in four separate slots. - Unyvero Analyzer: The Analyzer automates DNA purification, amplification and detection. Each Analyzer can simultaneously process up to two Unyvero Cartridges with each slot available using random access. - Unyvero Cockpit: The Cockpit provides the main user interface for the Unyvero System, guides the user through the steps to run the Unvvero LRT Application and automatically generates and displays test results. The Cockpit is equipped with a high-resolution touch screen and a barcode reader. - Unyvero Sample Tube Holder: The Sample Tube holder holds the Sample Tube securely while the specimen is transferred into the Sample Tube.