The FilmArray® Pneumonia Panel plus is a multiplexed nucleic acid test intended for use with FilmArray® 2.0, or FilmArray® Torch systems for the simultaneous detection 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) obtained from individuals meeting MERS-CoV clinical and/or epidemiological criteria.

Testing with FilmArray Pneumonia Panel plus should not be performed unless the patient meets clinical and/or epidemiologic criteria for testing suspecimens. This includes: clinical signs and symptoms associated 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, 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 influenza
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 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.

FilmArray 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.

FilmArray Pneumonia Panel plus MERS-CoV negative results, even in the context of a FilmArray 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 FilmArray 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 FilmArray 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 FilmArray 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 FilmArray Pneumonia Panel plus 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 plus results should be used in conjunction with culture results for deterial 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 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.

Device Description

The FilmArray Pneumonia Panel plus is designed to simultaneously identify Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and 26 other potential pathogens of lower respiratory tract infection (LRTI) and seven 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 meeting MERS-CoV clinical and/or epidemiological criteria in a time (~1 hour) that allows the test results to be used in determining appropriate patient treatment and management.

The FilmArray Pneumonia Panel plus is compatible with BioFire's PCR-based in vitro diagnostic FilmArray, 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.

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 plus 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 through the steps of placing the instrument, scanning the pouch barcode, entering the sample identification, and initiating the run.

The FilmArray instruments contain coordinated systems 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, electronicallycontrolled 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 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.

A new feature of the FilmArray Pneumonia Panel plus 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.

AI/ML Overview

The provided text describes the performance study for the FilmArray Pneumonia Panel plus, a multiplex nucleic acid test. Below is a structured summary addressing the requested points.

Acceptance Criteria and Device Performance Study

The acceptance criteria for the FilmArray Pneumonia Panel plus are indirectly demonstrated through the clinical and analytical performance studies, showing the device's ability to accurately detect a wide range of respiratory pathogens and antimicrobial resistance genes in specific clinical samples. The study proves the device meets these criteria through robust empirical testing and in silico analyses.

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" in a go/no-go format for each analyte, but rather presents the performance (Sensitivity/PPA and Specificity/NPA) from the clinical study as evidence of meeting regulatory requirements. The implicit acceptance criteria would likely be high agreement percentages and confidence intervals.

Table 1: Summary of Reported Device Performance (Clinical Study - BAL Specimens)

Analyte	Reference Method	Sensitivity/PPA (%)	95% CI (Sensitivity/PPA)	Specificity/NPA (%)	95% CI (Specificity/NPA)
MERS-CoV	PCR/Seq	-	-	100	99.5-100%
Acinetobacter calcoaceticus-baumannii complex	qRefCx	-	-	99.2	98.3-99.6%
Enterobacter cloacae complex	qRefCx	91.7	64.6-98.5%	98.6	97.5-99.2%
Escherichia coli	qRefCx	100	75.8-100%	99.0	98.1-99.5%
Haemophilus influenzae	qRefCx	100	72.2-100%	91.4	89.3-93.1%
Klebsiella aerogenes	qRefCx	85.7	48.7-97.4%	99.2	98.3-99.6%
Klebsiella oxytoca	qRefCx	100	34.2-100%	98.9	98.0-99.4%
Klebsiella pneumoniae group	qRefCx	100	79.6-100%	98.6	97.5-99.2%
Moraxella catarrhalis	qRefCx	-	-	96.6	95.1-97.6%
Proteus spp.	qRefCx	100	56.6-100%	99.5	98.8-99.8%
Pseudomonas aeruginosa	qRefCx	100	90.4-100%	95.3	93.6-96.6%
Serratia marcescens	qRefCx	100	61.0-100%	99.3	98.5-99.7%
Staphylococcus aureus	qRefCx	97.9	88.9-99.6%	91.2	89.1-93.0%
Streptococcus agalactiae	qRefCx	100	-	97.2	95.8-98.1%
Streptococcus pneumoniae	qRefCx	100	56.6-100%	97.1	95.8-98.1%
Streptococcus pyogenes	qRefCx	100	34.2-100%	99.3	98.5-99.7%
Chlamydia pneumoniae	PCR/Seq	-	-	99.9	99.3-100%
Legionella pneumophila	PCR/Seq	100	34.2-100%	100	99.5-100%
Mycoplasma pneumoniae	PCR/Seq	100	43.9-100%	99.9	99.3-100%
Adenovirus	PCR/Seq	100	67.6-100%	100	99.5-100%
Coronavirus	PCR/Seq	85.7	65.4-95.0%	98.4	97.3-99.1%
Human Metapneumovirus	PCR/Seq	100	67.6-100%	99.9	99.3-100%
Human Rhinovirus/Enterovirus	PCR/Seq	96.3	87.5-99.0%	98.6	97.5-99.2%
Influenza A	PCR/Seq	100	72.2-100%	99.6	98.9-99.9%
Influenza B	PCR/Seq	83.3	43.6-97.0%	99.9	99.3-100%
Parainfluenza Virus	PCR/Seq	88.9	67.2-96.9%	99.8	99.1-99.9%
Respiratory Syncytial Virus	PCR/Seq	100	43.9-100%	100	99.5-100%
CTX-M (AMR Gene)	qMol	85.7	48.7-97.4%	100	97.4-100%
IMP (AMR Gene)	qMol	-	-	100	97.5-100%
KPC (AMR Gene)	qMol	100	34.2-100%	99.3	96.3-99.9%
mecA/C and MREJ (AMR Gene)	qMol	88.9	76.5-95.2%	91.4	82.5-96.0%
NDM (AMR Gene)	qMol	0	-	99.3	96.3-99.9%
OXA-48-like (AMR Gene)	qMol	-	-	100	96.0-100%
VIM (AMR Gene)	qMol	-	-	100	97.5-100%

Note: Performance for Sputum specimens presented in Table 5.
Note: PPA = Positive Percent Agreement (Sensitivity), NPA = Negative Percent Agreement (Specificity).
Note: '-' indicates TP/(TP+FN) or TN/(TN+FP) was 0/0, thus percentage and CI could not be calculated.

2. Sample Sizes and Data Provenance

Test Set (Clinical Performance):
- Sample Size: 846 BAL specimens (including mini-BAL) and 836 sputum specimens (including ETA).
- Data Provenance: Multi-center study conducted at eight geographically distinct U.S. study sites. The study was prospective in nature, collecting specimens from October 2016 to July 2017. Additionally, archived retrospective specimens were used for low-prevalence analytes, including 8 BAL and 10 sputum specimens from a 2015 MERS-CoV outbreak in South Korea, and 171 archived specimens from external laboratories (139 BAL, 14 sputum, and negative controls). Contrived specimens were also used (1225 total, N=625 BAL, N=600 sputum for main contrived study; N=60 BAL, N=60 sputum for polymicrobial contrived study).

3. Number of Experts and Qualifications for Ground Truth

The document does not specify the exact number and qualifications of experts establishing the ground truth for the test set. Instead, it details the reference methods used for ground truth:

Bacterial Analytes: Quantitative reference culture (qRefCx) with a threshold of ≥ 3162 (10^3.5) CFU/mL. Discrepancies were further investigated using single PCR assays followed by quantitative molecular assay including sequencing (qMol), and additional molecular assays followed by sequence analysis where needed.
Atypical Bacteria and Viruses: Two conventional PCR assays followed by bidirectional sequencing. A specimen was considered positive if bidirectional sequencing data met predefined quality acceptance criteria and matched organism-specific sequences in the NCBI GenBank database.
AMR Genes: Single PCR assay followed by sequencing (qMol) from the specimen, combined with phenotypic AST (antimicrobial susceptibility testing) via qRefCx performed in conjunction with current CLSI guidelines for correlation.
MERS-CoV: For clinical specimens, it was assumed they were negative as the virus was not circulating in the US during enrollment. For archived specimens, previous laboratory results (from South Korea during the 2015 outbreak) served as ground truth.

4. Adjudication Method for the Test Set

The adjudication method for discrepant results between the FilmArray Pneumonia Panel plus and the primary comparator method varied:

For bacterial analytes, discrepancies were first examined to see if the qRefCx or FilmArray observed the analyte but below the detection threshold. If unresolved, qMol testing results were considered. If still unresolved, multiple additional molecular assays followed by sequence analysis were used.
For other analytes (atypical bacteria, viruses, AMR genes), molecular comparator results (two conventional PCR assays followed by bidirectional sequencing) were used. If the two assays agreed, that was the ground truth. If they disagreed, additional molecular assays followed by sequence analysis were performed. The results of "SOC testing" (Standard of Care) were also considered.

This suggests an algorithm-mediated adjudication with expert review for difficult cases (discrepancies), rather than a fixed "2+1" or "3+1" expert consensus model initially. The ground truth itself often involved molecular methods, which are considered highly accurate for nucleic acid detection.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

There is no MRMC comparative effectiveness study described in the provided text. The study focuses on comparing the device's performance against reference laboratory methods (molecular and culture-based) for direct detection, not on evaluating how human readers' performance might improve with or without AI assistance. The device is an in vitro diagnostic (IVD) test, not an AI-assisted diagnostic imaging tool.

6. Standalone (Algorithm Only) Performance

The entire performance study described is essentially a standalone (algorithm only without human-in-the-loop) performance evaluation for the FilmArray Pneumonia Panel plus. The results for sensitivity/PPA and specificity/NPA directly report the device's accuracy in detecting various pathogens and AMR genes compared to established reference methods.

7. Type of Ground Truth Used

The ground truth used varied by analyte:

Bacterial Analytes: Quantitative Reference Culture (qRefCx) for primary evaluation. Discrepancies were resolved using quantitative molecular assays (qMol) and subsequent sequencing/additional molecular assays.
Atypical Bacteria, Viruses (excluding MERS-CoV in US clinical samples): Two conventional PCR assays followed by bidirectional sequencing. Discrepancies resolved with additional molecular assays and sequencing.
MERS-CoV (archived specimens): Previous laboratory results (implicitly molecular/viral culture confirmation) from the outbreak.
Antimicrobial Resistance Genes: Single PCR assay followed by sequencing (qMol) directly from the specimen, and correlation with phenotypic AST from cultured isolates.

Therefore, the ground truth is a combination of expert-interpreted molecular data (PCR/sequencing), quantitative culture, and phenotypic AST, with further discrepancy resolution protocols.

8. Sample Size for the Training Set

The document focuses solely on the performance evaluation of the FilmArray Pneumonia Panel plus device. It states that the device is "designed to simultaneously identify Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and 26 other potential pathogens of lower respiratory tract infection (LRTI) and seven associated antimicrobial resistance (AMR) genes." It describes analytical and clinical validation studies but does not provide information on a "training set" sample size for an AI/ML model, as this is a molecular diagnostic device not explicitly using an AI/ML component in its core diagnostic algorithm. The phrase "FilmArray Software automatically interprets the results" implies a deterministic algorithm, not a trainable machine learning model.

9. How the Ground Truth for the Training Set was Established

Since no training set for an AI/ML model is mentioned, there is no information provided on how its ground truth was established. The document describes the validation/test set ground truth as outlined in point 7.

Summary

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November 15, 2018

BioFire Diagnostics, LLC Kristen Kanack Senior Vice President, Regulatory & Clinical Affairs 515 Colorow Drive Salt Lake City, Utah 84108

Re: K181324

Trade/Device Name: FilmArray Pneumonia Panel plus Regulation Number: 21 CFR 866.4001 Regulation Name: MERS-CoV and common respiratory pathogens multiplex nucleic acid detection system Regulatory Class: Class II Product Code: ODS Dated: May 17, 2018 Received: May 18, 2018

Dear Kristen Kanack:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food. Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part

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801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.html; good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4. Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Steven R. Gitterman -S for

Uwe Scherf, Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K181342

Device Name FilmArray Pneumonia Panel plus

Indications for Use (Describe)

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 influenza
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

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· 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

Viral culture should not be attempted on specimens with positive FilmArray 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 FilmArray 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.

The antimicrobial resistance gene detected may or may not be associated with the agent(s) responsible for disease.

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Negative results for these antimicrobial resistance gene assays do not indicate susceptibility to corresponding classes of antimicrobials, as multiple mechanisms of antimicrobial resistance exist.

Type of Use (Select one or both, as applicable)
☑ Prescription Use (Part 21 CFR 801 Subpart D)	☐ Over-The-Counter Use (21 CFR 801 Subpart C)

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510(k) Summary BioFire Diagnostics, LLC

FilmArray Pneumonia Panel plus

Introduction:

According to the requirements of 21 CFR 807.92, the following information provides sufficient detail to understand the basis for a determination of substantial equivalence.

Submitted by:

BioFire Diagnostics, LLC 515 Colorow Drive Salt Lake City, UT 84108

Telephone: 801-736-6354 Facsimile: 801-588-0507

Contact: Kristen J. Kanack, ext. 1330

Date Submitted: May 17, 2018

Device Name and Classification:

Trade Name: FilmArray Pneumonia Panel plus

Regulation Number: 21 CFR 866.4001

Classification Name: MERS-CoV and common respiratory pathogens multiplex nucleic acid detection system

Predicate Device:

DEN170017 - FilmArray Respiratory Panel 2 plus (RP2plus)

Intended Use:

The FilmArray® Pneumonia Panel plus is a multiplexed nucleic acid test intended for use with FilmArray®, FilmArray® 2.0, or FilmArray® 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 FilmArray Pneumonia Panel plus should not be performed unless the patient meets clinical and/or epidemiologic criteria for testing suspected MERS-CoV specimens. This includes: clinical signs and symptoms associated with MERS-CoV infection, contact with a probable or confirmed MERS-CoV

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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 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-baumanniicomplex	Klebsiella oxytoca	Serratia marcescens
Enterobacter cloacae complex	Klebsiella pneumoniaegroup	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
Middle East Respiratory Syndrome Coronavirus
Adenovirus	Human Rhinovirus/Enterovirus	Parainfluenza Virus
Coronavirus	Influenza A	Respiratory Syncytial Virus
Human Metapneumovirus	Influenza B
Antimicrobial Resistance Genes
CTX-M	NDM	mecA/C and MREJ
IMP	OXA-48-like
KPC	VIM

FilmArray Pneumonia Panel plus MERS-CoV negative results, even in the context of a FilmArray 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 Diagnostics 510(k) FilmArray Pneumonia Panel plus

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FilmArray 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 FilmArray 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 value. Detection of analytes does not rule out coinfection with other organisms; the agent(s) detected by the FilmArray 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 FilmArray 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 semiquantitative Bin (copies/mL) for clinical management.

Due to the genetic similarity between human rhinovirus and enterovirus, the test cannot reliably differentiate them. A positive Rhinovirus 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 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.

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Device Description:

MERS-CoV – Qualitative Results
Middle East Respiratory Syndrome Coronavirus
Other Common Lower Respiratory Pathogens
Bacteria -Results with Bin Values	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	Viruses
Proteus spp.	Adenovirus
Pseudomonas aeruginosa	Coronavirus
Serratia marcescens	Human Metapneumovirus
Streptococcus agalactiae	Human Rhinovirus/Enterovirus
Streptococcus pneumoniae	Influenza A
Streptococcus pyogenes	Influenza B
Bacteria (Atypical) - Qualitative Results	Parainfluenza Virus
Chlamydia pneumoniae	Respiratory Syncytial Virus
Legionella pneumophila

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Substantial Equivalence:

The FilmArray Pneumonia Panel plus is substantially equivalent to the FilmArray Respiratory Panel 2 plus (RP2plus) Application (DEN170017), which was cleared on November 24, 2017 and determined to be a Class II device under the classification product code QDS.

Table 1 compares the FilmArray Pneumonia Panel plus to the FilmArray Respiratory Panel 2 plus (RP2plus) and outlines the similarities and differences between the two systems.

Element	New Device:FilmArray Pneumonia Panel plus	Predicate:FilmArray Respiratory Panel 2 plus (RP2plus)(DEN170017)
Specimen Types	Sputum-like (induced or expectorated sputum,endotracheal aspirates) and BAL-like (BAL ormini-BAL) specimens	NPS in viral transport medium
Organisms Detected	Middle East Respiratory Syndrome Coronavirus(MERS-CoV)	Middle East Respiratory Syndrome Coronavirus(MERS-CoV)

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Element	New Device:FilmArray Pneumonia Panel plus	Predicate:FilmArray Respiratory Panel 2 plus (RP2plus)(DEN170017)
	Bacteria: Acinetobacter calcoaceticus-baumanniicomplex, Enterobacter cloacae complex,Escherichia coli , Haemophilus influenzae,Klebsiella oxytoca, Klebsiella aerogenes,Klebsiella pneumoniae group, Moraxellacatarrhalis, Proteus spp., Pseudomonasaeruginosa, Serratia marcescens, Staphylococcusaureus, Streptococcus agalactiae, Streptococcuspneumoniae, Streptococcus pyogenesAtypical Bacteria: Chlamydia pneumoniae,Legionella pneumophila, MycoplasmapneumoniaeAntimicrobial Resistance Genes: CTX-M, IMP,KPC, mecA/C + MREJ, NDM, Oxa48-like, VIMViruses: Adenovirus, Coronavirus, HumanMetapneumovirus, HumanRhinovirus/Enterovirus, Influenza A, Influenza B,Parainfluenza virus,Respiratory Syncytial virus	Bacteria: Bordetella parapertussis (IS 1001),Bordetella pertussis (ptxP), Chlamydiapneumoniae, Mycoplasma pneumoniaeViruses: Adenovirus, Coronavirus 229E,Coronavirus HKU1, Coronavirus NL63,Coronavirus OC43, Human Metapneumovirus,Human Rhinovirus/Enterovirus, Influenza A,including subtypes H1, H1-2009, and H3,Influenza B, Parainfluenza Virus 1, ParainfluenzaVirus 2, Parainfluenza Virus 3, ParainfluenzaVirus 4, Respiratory Syncytial Virus
Analyte	DNA/RNA	Same
TechnologicalPrinciples	Multiplex nucleic acid	Same
Result types	Detection with bin values (in 1-log roundedcopies/mL bins) for BacteriaQualitative for MERS-CoV, Atypical Bacteria,Antimicrobial Resistance Genes, and Viruses	Qualitative for all analytes
Instrumentation	FilmArray, FilmArray 2.0, or FilmArray Torch	FilmArray 2.0 or FilmArray Torch
Time to result	About 1 hour	~ 45 minutes
Reagent Storage	Room temperature	Same
Test Interpretation	Automated test interpretation and reportgeneration. User cannot access raw data.	Same
ControlsTwo controls are included in each reagent pouchto control for sample processing and both stagesof PCR and melt analysis.		Same
User Complexity	Moderate/Low	Same

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Summary of Performance Data

Clinical Performance

The clinical performance of the FilmArray Pneumonia Panel plus was established during a multi-center study conducted at eight geographically distinct U.S. study sites from October 2016 to July 2017. A total of 904 residual BAL (including mini-BAL) and 925 residual sputum (821 BAL and 83 mini-BAL) and 925 residual sputum (478 sputum and 447 ETA) specimens were acquired for the prospective clinical study. FilmArray Pneumonia Panel plus performance in BAL and mini-BAL was similar, as was performance in sputum and ETA; therefore these sample types are not stratified further in performance tables. A total of 58 BAL and 89 sputum specimens were excluded from the final data analysis. The most common reasons for specimen exclusion for both specimen types was reference culture unable to be performed, the specimen was found to not meet the inclusion criteria after the specimen had been enrolled, or the study site was unable to complete the Case Report Form (CRF). The final data set consisted of 846 BAL and 836 sputum specimens. Table 3 provides a summary of demographic information for the specimens included in the prospective study.

BAL
		Overall	Site 1ª	Site 2	Site 3	Site 4	Site 5	Site 6	Site 7	Site 8
Sex	Male	480 (57%)	80 (59%)	7 (54%)	138(55%)	21 (68%)	75 (61%)	82 (52%)	27 (61%)	50 (55%)
	Female	366 (43%)	55 (41%)	6 (46%)	113(45%)	10 (32%)	48 (39%)	76 (48%)	17 (39%)	41 (45%)
Age	≤ 5 years	23 (3%)	0 (0%)	5 (38%)	0 (0%)	15 (48%)	0 (0%)	3 (2%)	0 (0%)	0 (0%)
	6 - 17 years	27 (3%)	0 (0%)	8 (62%)	0 (0%)	13 (42%)	0 (0%)	4 (3%)	1 (2%)	1 (1%)
	18 - 34 years	70 (8%)	18 (13%)	0 (0%)	17 (7%)	3 (10%)	10 (8%)	10 (6%)	5 (11%)	7 (8%)
	35 - 65 years	470 (56%)	78 (58%)	0 (0%)	152(61%)	0 (0%)	70 (57%)	88 (56%)	27 (61%)	55 (60%)
	> 65 years	255 (30%)	38 (28%)	0 (0%)	82 (33%)	0 (0%)	43 (35%)	53 (34%)	11 (25%)	28 (31%)
Care Setting	Hospitalized	666 (79%)	116(86%)	12 (92%)	223(89%)	9 (29%)	82 (67%)	118(75%)	25 (57%)	81 (89%)
	Outpatient	159 (19%)	18 (13%)	0 (0%)	28 (11%)	22 (71%)	31 (25%)	39 (25%)	14 (32%)	7 (8%)
	Emergency	21 (2%)	1 (1%)	1 (8%)	0 (0%)	0 (0%)	10 (8%)	1 (1%)	5 (11%)	3 (3%)	Total	846	135	13	251	31	123	158
Emergency	21 (2%)	1 (1%)	1 (8%)	0 (0%)	0 (0%)	10 (8%)	1 (1%)	5 (11%)	3 (3%)
	Total	846	135	13	251	31	123	158	44	91

Table 2. Overall and Per Site Demographic Analysis for BAL Specimens

ª Subject age could not be determined for one specimen from Site 1

Table 3. Overall and Per Site Demographic Analysis for Sputum Specimens

	Sputum
	Overall	Site 1	Site 2	Site 3	Site 4	Site 5	Site 6	Site 7	Site 8
Sex	Male	481(58%)	66 (59%)	54 (54%)	136(56%)	97 (61%)	14 (82%)	31 (53%)	34 (74%)	49 (47%)
	Female	355(42%)	45 (41%)	46 (46%)	105(44%)	61 (39%)	3 (18%)	28 (47%)	12 (26%)	55 (53%)
Age	≤ 5 years	138(17%)	0 (0%)	49 (49%)	0 (0%)	80 (51%)	0 (0%)	0 (0%)	2 (4%)	7 (7%)
	6 - 17 years	107(13%)	0 (0%)	35 (35%)	0 (0%)	64 (41%)	0 (0%)	0 (0%)	2 (4%)	6 (6%)
	18 - 34 years	86 (10%)	15 (14%)	16 (16%)	20 (8%)	13 (8%)	1 (6%)	6 (10%)	5 (11%)	10 (10%)
	35 - 65 years	284(34%)	51 (46%)	0 (0%)	133(55%)	1 (1%)	6 (35%)	36 (61%)	20 (43%)	37 (36%)
	> 65 years	221(26%)	45 (41%)	0 (0%)	88 (37%)	0 (0%)	10 (59%)	17 (29%)	17 (37%)	44 (42%)
CareSettil	Hospitalized	682(82%)	106(95%)	64 (64%)	219(91%)	105(66%)	12 (71%)	52 (88%)	23 (50%)	101(97%)

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

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Sputum
	Overall	Site 1	Site 2	Site 3	Site 4	Site 5	Site 6	Site 7	Site 8
Outpatient	73 (9%)	2 (2%)	14 (14%)	18 (7%)	24 (15%)	2 (12%)	5 (8%)	7 (15%)	1 (1%)
Emergency	81 (10%)	3 (3%)	22 (22%)	4 (2%)	29 (18%)	3 (18%)	2 (3%)	16 (35%)	2 (2%)
Total	836	111	100	241	158	17	59	46	104

All specimens were evaluated with the FilmArray Pneumonia Panel plus at clinical study sites. Refrigerated specimen aliquots were sent to a central reference laboratory for quantitative reference culture (qRefCx) and frozen specimen aliquots were also sent to BioFire for evaluation by polymerase chain reaction (PCR)/sequencing-based comparator methods.

The reference methods used in this study were as follows:

Bacterial analytes were compared to qRefCx to evaluate sensitivity and specificity, and the method was considered positive for the presence of the organism of interest if it was recovered in culture and enumerated at a level of 3162 (10^3.5) CFU/mL or greater.

Bacterial analytes were also evaluated by comparison to a single PCR assay for the organism of interest followed by a quantitative molecular assay that included sequencing (qMol) to assess FilmArray bin reporting performance. Atypical bacteria and viruses were compared to two conventional PCR assays followed by bidirectional sequencing. For specimens with an applicable bacteria detected by FilmArray, AMR genes were compared to a single PCR assay (from the specimen) followed by sequencing. A specimen was considered to be positive for an analyte if bi-directional sequencing data meeting predefined quality acceptance criteria matched organism-specific sequences deposited in the NCBI GenBank database (www.ncbi.nlm.nih.gov) with acceptable E-values. When two PCR comparator assays were used, any specimen that tested negative by both of the comparator assays was considered Negative.

No reference testing was performed for MERS-CoV as this virus was not circulating in the United States at the time of enrollment; therefore all specimens were assumed to be negative.

Positive Percent Agreement (PPA) or Sensitivity for each analyte was calculated as 100% x (TP / (TP + FN)). True positive (TP) indicates that both the FilmArray Pneumonia Panel plus and the comparator method had a positive result for this specific analyte, and false negative (FN) indicates that the FilmArray Pneumonia Panel plus result was negative while the comparator result was positive. Negative Percent Agreement (NPA) or Specificity was calculated as 100% x (TN + FP)). True negative (TN) indicates that both the FilmArray Pneumonia Panel plus and the comparator method had negative results, and a false positive (FP) indicates that the FilmArray Pneumonia Panel plus result was positive but the comparator result was negative. The exact binomial two-sided 95% confidence interval was calculated. Samples for which false positive and/or false negative results (i.e., discrepant results) were obtained when comparing the FilmArray Pneumonia Panel plus results to the comparator method results were further investigated. The discrepancy investigations were primarily performed as follows: for discrepancies between the Pneumonia Panel plus and reference culture for bacterial analytes, discrepancies were first examined to see if qRefCx or FilmArray had observed the analyte but reported it as "negative" or "Not Detected" because it was below the detection threshold. If this did not resolve the discrepancy, the results of qMol testing were considered. And if these methods still did not resolve the discrepancy, it was then investigated in the same manner as other analytes that used molecular comparator (i.e. using multiple additional molecular assays followed by sequence analysis). The results of SOC testing were also

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considered. The prospective clinical study results are summarized in Table 5 for BAL and sputum specimens, respectively.

BAL
	Reference		Sensitivity/PPA		Specificity/NPA
Analyte	Method	TP/(TP +FN)	%	95%CI	TN/(TN +FP)	%	95%CI
Middle East Respiratory Syndrome Coronavirus(MERS-CoV)	PCR/Seq	0/0	-		846/846	100	99.5-100%
Bacteria
Acinetobacter calcoaceticus-baumannii complexb	qRefCx	0/0	-		839/846	99.2	98.3-99.6%
Enterobacter cloacae complexc	qRefCx	11/12	91.7	64.6-98.5%	822/834	98.6	97.5-99.2%
Escherichia colid	qRefCx	12/12	100	75.8-100%	826/834	99.0	98.1-99.5%
Haemophilus influenzaee	qRefCx	10/10	100	72.2-100%	764/836	91.4	89.3-93.1%
Klebsiella aerogenesf	qRefCx	6/7	85.7	48.7-97.4%	832/839	99.2	98.3-99.6%
Klebsiella oxytocag	qRefCx	2/2	100	34.2-100%	835/844	98.9	98.0-99.4%
Klebsiella pneumoniae grouph	qRefCx	15/15	100	79.6-100%	819/831	98.6	97.5-99.2%
Moraxella catarrhalisi	qRefCx	0/0	-		817/846	96.6	95.1-97.6%
Proteus spp.j	qRefCx	5/5	100	56.6-100%	837/841	99.5	98.8-99.8%
Pseudomonas aeruginosak	qRefCx	36/36	100	90.4-100%	772/810	95.3	93.6-96.6%
Serratia marcescensl	qRefCx	6/6	100	61.0-100%	834/840	99.3	98.5-99.7%
Staphylococcus aureusm	qRefCx	46/47	97.9	88.9-99.6%	729/799	91.2	89.1-93.0%
Streptococcus agalactiaen	qRefCx	1/1	100		821/845	97.2	95.8-98.1%
Streptococcus pneumoniaeo	qRefCx	5/5	100	56.6-100%	817/841	97.1	95.8-98.1%
Streptococcus pyogenesp	qRefCx	2/2	100	34.2-100%	838/844	99.3	98.5-99.7%
Atypical Bacteria
Chlamydia pneumoniaeq	PCR/Seq	0/0	-		844/845	99.9	99.3-100%
Legionella pneumophilar	PCR/Seq	2/2	100	34.2-100%	833/833	100	99.5-100%
Mycoplasma pneumoniaes	PCR/Seq	3/3	100	43.9-100%	841/842	99.9	99.3-100%
Viruses
Adenovirus	PCR/Seq	8/8	100	67.6-100%	837/837	100	99.5-100%
Coronavirust	PCR/Seq	18/21	85.7	65.4-95.0%	810/823	98.4	97.3-99.1%
Human Metapneumovirusu	PCR/Seq	8/8	100	67.6-100%	836/837	99.9	99.3-100%
Human Rhinovirus/Enterovirusv	PCR/Seq	52/54	96.3	87.5-99.0%	771/782	98.6	97.5-99.2%
Influenza Aw	PCR/Seq	10/10	100	72.2-100%	830/833	99.6	98.9-99.9%
Influenza Bx	PCR/Seq	5/6	83.3	43.6-97.0%	837/838	99.9	99.3-100%
Parainfluenza Virusy	PCR/Seq	16/18	88.9	67.2-96.9%	824/826	99.8	99.1-99.9%
Respiratory Syncytial Virus	PCR/Seq	3/3	100	43.9-100%	841/841	100	99.5-100%

Table 4. FilmArray Pneumonia Panel plus Clinical Performance Summary for BAL Specimens®

® The performance measures of sensitivity and specificity only refer to the bacterial analytes for which the gold-stand of qReferice method. Performance measures of PPA and NPA refer to all other analytes, for which PCR/sequencing assays were used as comparator methods.

Evidence of ACB complex was found in all seven FP specimens; one was enumerated below 10°3.5 CFUmL by qRefCx and six were detected by QMol.

& E. cloace complex was observed in the 10% bin by the FilmArray Preumonia Panel plus. Evidence of E. cloacae complex was found in all 12 FP specimens; six were enumerated below 10'3.5 CFU/mL by qRefCx, five were detected using an additional molecular method.

^ Evidence of E. coli was found in all eight FP speciment six were enumerated below 10°3.5 CFU/mL by qRefCx and two were detected by qMol.

Evidence of H. influenzae was found in all 72 FP specimented below 10°3.5 CFUmL by qRefCx, 56 were detected by aMol, eight were detected using an additional molecular method, and one was identified in SOC culture.

f K. aerogenes was identified in the single FN speciment in all seven FP specimens was found in all seven FP specimens; four were enumerated below 10^3.5 CFU/mL by qRefCx and three were detected by qMol.

9 Evidence of K. oxytoca was found in all nine FP speciments three were enumerated below 1043.5 CFU/mL by qRefCx, five were detected by qMol, and one was detected using an additional molecular method.

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" Evidence of K. pneumoniae group was found in all 12 FP speciment seven were enumerated below 10'3.5 CFU/mL by gRefCx, four were detected by qMol, and one was detected using an additional molecular method.

'Evidence of M. catarthalis was found in all 29 FP speciments two were enumerated below 10°3.5 CFUmL by qRefCx, 25 were detected by qMol, and two were detected using an additional molecular method.

'Evidence of Proteus spp. was found in all four FP speciments three were enumerated below 103.5 CFUmL by qRefCx and one was detected by QMol.

Evidence of P. aeruginosa was found in all 38 FP specimented below 10°3.5 CFUmL by qRefCx, 16 were detected by gMol, and three were detected using an additional molecular method

'Evidence of S. marcescens was found in all six FP specimens; four were enumerated below 10°3.5 CFUmL by qRefCx and two were detected by QMol.

™ S. aureus was detected in the single FN specifical molecular method. Evidence of S. aureus was found in 6970 FP specimens; 29 were enumerated below 10%.5 CFUmL by qRefCx, 30 were detected using an additional molecular method, and two were identified in SOC culture.

" Evidence of S. agalactiae was found in all 24 FP speciments seven were enumerated below 10°3.5 CFU/mL by qRefCv, 13 were detected by qMol, and four were detected using an additional molecular method.

° Evidence of S. pneumoniae was found in all 24 FP specimerated below 10°3.5 CFUmL by qReCx, 18 were detected by gMol, and one was detected using an additional molecular method.

P Evidence of S. pyogenes was found in all six FP speciments two were enumerated below 10°3.5 CFU/mL by qRefCx, three were was detected using an additional molecular method.

9 The single FP specimen was negative for C. pneumoniae when tested with additional methods during discrepancy investigation.

' The single FP specimen was negative for M. pneumoniae when tested with additional methods during discrepancy investigation.

$ CoV was detected in 2/3 FN and 8/13 FP specimens using an additional molecular method.

™ The single FP specimen was negative for hMPV when tested with additional methods during discrepancy investigation.

" HRV/EV was detected in both FN specimens using and HRV/EV was detected in 8/1 FP specimens during discrepancy investigation; seven were detected using an additional method and one was detected upon FilmArray Pneumonia Panel plus retest.

Y FluA was detected in 2/3 FP specimens using an additional molecular method.

" FluB was detected in the single FilmArray Pneumonia Panel plus retest. FluB was detected in the single FP specimen using an additional molecular method.

PIV was detected in both FN and both FP specimens using an additional molecular method.

Table 5. FilmArray Pneumonia Panel plus Clinical Performance Summary for Sputum Specimens®

Sputum
	Reference	Sensitivity/PPA			Specificity/NPA
Analyte	Method	TP/(TP +FN)	%	95%CI	TN/(TN +FP)	%	95%CI
Middle East Respiratory Syndrome Coronavirus(MERS-CoV)	PCR/Seq	0/0	-	-	836/836	100	99.5-100%
		Bacteria
Acinetobacter calcoaceticus-baumannii complexb	qRefCx	10/11	90.9	62.3-98.4%	807/825	97.8	96.6-98.6%
Enterobacter cloacae complexc	qRefCx	11/12	91.7	64.6-98.5%	803/824	97.5	96.1-98.3%
Escherichia colid	qRefCx	23/24	95.8	79.8-99.3%	787/812	96.9	95.5-97.9%
Haemophilus influenzaee	qRefCx	16/18	88.9	67.2-96.9%	727/818	88.9	86.5-90.9%
Klebsiella aerogenesf	qRefCx	3/4	75.0	30.1-95.4%	823/832	98.9	98.0-99.4%
Klebsiella oxytocag	qRefCx	9/9	100	70.1-100%	817/827	98.8	97.8-99.3%
Klebsiella pneumoniae grouph	qRefCx	21/23	91.3	73.2-97.6%	769/813	94.6	92.8-95.9%
Moraxella catarrhalisi	qRefCx	5/5	100	56.6-100%	761/831	91.6	89.5-93.3%
Proteus spp.j	qRefCx	15/15	100	79.6-100%	813/821	99.0	98.1-99.5%
Pseudomonas aeruginosak	qRefCx	103/106	97.2	92.0-99.0%	673/730	92.2	90.0-93.9%
Serratia marcescensl	qRefCx	26/27	96.3	81.7-99.3%	782/809	96.7	95.2-97.7%
Staphylococcus aureusm	qRefCx	111/112	99.1	95.1-99.8%	631/724	87.2	84.5-89.4%
Streptococcus agalactiaen	qRefCx	9/9	100	70.1-100%	793/827	95.9	94.3-97.0%
Streptococcus pneumoniaeo	qRefCx	16/16	100	80.6-100%	785/820	95.7	94.1-96.9%
Streptococcus pyogenesp	qRefCx	6/6	100	61.0-100%	825/830	99.4	98.6-99.7%
Atypical Bacteria
Chlamydia pneumoniae	PCR/Seq	0/0	-	-	835/835	100	99.5-100%
Legionella pneumophilaq	PCR/Seq	0/1	0	-	826/826	100	99.5-100%
Mycoplasma pneumoniaer	PCR/Seq	7/8	87.5	52.9-97.8%	827/827	100	99.5-100%
Viruses

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Sputum
Analyte	Reference Method	TP/(TP + FN)	%	95%CI	TN/(TN + FP)	%	95%CI
Adenoviruss	PCR/Seq	13/17	76.5	52.7-90.4%	815/817	99.8	99.1-99.9%
Coronavirust	PCR/Seq	28/32	87.5	71.9-95.0%	796/802	99.3	98.4-99.7%
Human Metapneumovirusu	PCR/Seq	20/21	95.2	77.3-99.2%	812/813	99.9	99.3-100%
Human Rhinovirus/Enterovirusv	PCR/Seq	96/96	100	96.2-100%	717/730	98.2	97.0-99.0%
Influenza Aw	PCR/Seq	13/13	100	77.2-100%	819/822	99.6	98.9-99.9%
Influenza Bx	PCR/Seq	12/12	100	75.8-100%	821/823	99.8	99.1-99.9%
Parainfluenza Virusy	PCR/Seq	28/29	96.6	82.8-99.4%	804/806	99.8	99.1-99.9%
Respiratory Syncytial Virusz	PCR/Seq	43/43	100	91.8-100%	787/791	99.5	98.7-99.8%

® The performance measures of sensitivity only refer to the bacterial analyes for which the gold-standard of qRefCx was used as the reference method. Performance measures of PPA and NPA refer to all other analytes, for which PCR/sequencing as comparator methods

· The isolate recovered from the single the molentified by oReCx; molecular testing of the isolate identified it as Pseudomonas fluorescens during discrepancy investigation. Evidence of ACB complex 15 were detected by gMol, two were detected using an additional molecular method, and one was identified in SOC culture.

· E. cloace complex was detected in the single FN specific an additional molecular method. Evidence of E. cloace complex was found in all 21 FP specimens; four were enumerated below 10°3.5 CFUmL by qReCx; 16 were detected using an additional molecular method.

d E. coli was observed in the single FN speciment of the FilmArray Pneumonia Panel plus. Evidence of E. coll was found in all 25 FP specimens; six were enumerated below 10°3.5 CFUlmL by qRefCx, 14 were detected using an additional molecular method

H. influenzae was delected in 1/2 FN speciment from the other FN specimen was misidentified by qReCx; molecular testing of the isolate identified it as Haemophilus haemolyticus during discription. Evidence of H. influenzae was found in all 91 F P specimens; four were enumerated below 10°3.5 CFUmL by qReCx, 78 were detected by gMol, seven were detected using an additional method, and two were identified in SOC culture.

TThe isolate recovered from the single intiled by qReCx; molecular testing of the isolate identified it as Hafria paralvei during discrepancy investigation. Evidence of K. aerogenes was found in all nine FP specimens; three were enumerated below 10°3.5 CFUmL by qRol, and one was detected using an additional molecular method.

® Evidence of K. oxytoca was found in all 10 FP speciments three were enumerated below 10°3.5 CFUmL by qRefCx, five were detected using an additional molecular method.

K. pneumoniae group was detected in 1/2 FN speciment to be a result of a specimen swap at the central reference laboratory. Evidence of K. pneumoriae group was found in 43/44 FP specimens; 15 were enumerated below 10°3.5 CFU/mL by qRefCx, 21 were detected by gMol, and seven were detected using an additional molecular method.

'Evidence of M. catarrhalis was found in all 70 FP speciments one was enumerated below 10°3.5 CFU/mL by gReCx, 63 were detected using an additional molecular method, and one was identified in SOC culture.

Evidence of Proteus sp. was found in all eight FP specimented below 10°3.5 CFUmL by gRefOx, four were delected by gMol, and wo were detected using an additional molecular method.

r P. aerginosa was observed in 1/3 FN speciment be 104 bin by the FilmAray Pneumonia Panel plus. The isolates recovered from the other two FN specimens were misidentified by qRefCx; nolecular testing one as Pseudomonas denitrificans and the other as Pseudomonas flurescens during discrepancy investigation. Evidence of P. aeruginosa was found in all 57 FP speciments; 21 were enumerated below 1093.5 CFUmL by gRefCx, 33 were detected by gMol. two were detected using an additional molecular method, and one was identified in SOC culture.

1 S. marcescens was observed in the single FN speciment of 104 bin by the Filmlerray Preumonia Panel plus. Evidence of S. marcessers was found in 2027 FP specimens; seven were enumerated below 10°3.5 CFU/mL by gReCx, 16 were detected using an additional molecular meltod.

™ S. aureus was observed in the single FN speciment the 10% bin by the FilmArray Pneumonia Panel plus. Evidence of S. aureus was found in all 93 FP specimens; 43 were enumerated below 10°3.5 CFUlmL by qReCx; 43 were detected using an additional molecular method, and four were identified in SOC culture.

" Evidence of S. agalactiae was found in all 34 FP speciments in below 10°3.5 CFUmL by qRefCx, 24 were detected by qMol, and five were detected using an additional molecular method

º Evidence of S. pneumoniae was found in all 35 FP speciments one was enumerated below 10°3.5 CFU/mL by qRefCx and 34 were detected by QMol.

P Evidence of S. pyogenes was found in all five FP speciments of the mas detected using an additional molecular method.

a L. pneumophila was detected in the single FN specimen using an additional molecular method.

' The single FN specimen was negative for M. pneumoniae when tested with an additional method during discrepancy investigation.

$ AdV was detected in all four FN and 1/2 FP specimens using an additional molecular method.

↑ CoV was detected in all four FN and 3/6 FP specimens using an additional molecular method.

" MPV was delected in the single FN specimen using an method. The single FP specimen was negative for MPV when tested with an additional molecular method during discrepancy investigation.

" HRV/EV was detected in 12/13 FP specimens during in were detected using an additional molecular method and one was delected upon FilmArray Pneumonia Panel plus retest.

FluA was detected in all three FP specimens using an additional molecular method.
Both FP specimens were negative for FluB when tested with additional methods during discrepancy investigation.

Y PIV was detected in the single FN and 1/2 FP specimens using an additional molecular method.

² RSV was detected in all four FP specimens using an additional molecular method.

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{16}------------------------------------------------

A total of 156 BAL specimens and 295 sputum specimens received a FilmArray Pneumonia Panel plus Detected result for at least one applicable gram-negative bacterium on the panel and reported results for CTX-M, IMP, KPC, NDM, OXA-48-like, and VIM; a total of 94 BAL specimens and 196 sputum specimens received a FilmArray Pneumonia Panel plus Detected result for at least one applicable gramnegative bacterium on the panel and reported results for OXA-48-like; and a total of 116 BAL specimens and 204 sputum specimens received a FilmArray Pneumonia Panel plus Staphylococcus aureus Detected result and reported results for mecA/C and MREJ. Performance of the Pneumonia Panel plus AMR gene assays was calculated by comparing results of qMol direct from these specimens and is shown in Table 6 (five BAL and four sputum specimens were excluded from qMol analysis due to invalid comparator results).

				BAL			Sputum
Analyte		PPA		NPA			PPA		NPA
	TP/(TP +FN)	%	95%CI	TN/(TN +FP)	%	95%CI	TP/(TP +FN)	%	95%CI	TN/(TN +FP)	%	95%CI
CTX-Mb	6/7	85.7	48.7-97.4%	144/144	100	97.4-100%	8/10	80.0	49.0-94.3%	280/281	99.6	98.0-99.9%
IMP	0/0	-	-	151/151	100	97.5-100%	0/0	-	-	291/291	100	98.7-100%
KPCc	2/2	100	34.2-100%	148/149	99.3	96.3-99.9%	7/7	100	64.6-100%	284/284	100	98.7-100%
mecA/Cand MREJd	40/45	88.9	76.5-95.2%	64/70	91.4	82.5-96.0%	94/98	95.9	90.0-98.4%	91/104	87.5	79.8-92.5%
NDMe	0/1	0	-	149/150	99.3	96.3-99.9%	0/0	-	-	291/291	100	98.7-100%
OXA-48-like	0/0	-	-	92/92	100	96.0-100%	0/0	-	-	195/195	100	98.1-100%
VIMf	0/0	-	-	151/151	100	97.5-100%	1/1	100	-	289/290	99.7	98.1-99.9%

Table 6. FilmArray Pneumonia Panel plus Clinical Performance Summary – AMR Genes (comparator method: qMol direct from specimen)3

a Performance in this summary table is calculated when any applicable organism is detected in the sample.

PCTX-M was detected in the single FN BAL and 1/2 FN sputum specimens . The single FP sputum specimen was negative for CTX-M when tested during discrepancy investigation. None of the applicable isolates identified by the FilmArray or gelection of ESBL activity or CTX-M presence.

· KPC was detected in the single FP BAL speciment method; the isolate recovered from this specimen (A. baumanii) exhibited carbapenem resistance but did not carry KPC.

ª Evidence of mecAC and/or SCCmecassette generits was found in all five FN sputum specimens by an additional molecular method; three of these also had a MRSA isolate recovered via qReck andor SCC nec cassette geneic elements was found in 56 FP BAL and all 13 FP sputum speciments in a ReCx or SOC culture, and nine additional specimens had evidence of meet C and/or SCCmec cassette genetic elements by an additional molecular method.

NDM was detected in the single FN BAL speciment molecular method; P. aeruginosa was recovered from the specimen and was resistant to carbapents but carried only KPC. The single FP BAL specimen was negative for NDM when tested with adscreancy investigation.

' The single FP sputum specimen was negative for VIM when tested with additional methods during discrepancy investigation.

qRefCx isolated one or more applicable gram-negative bacteria from 127 of the 156 BAL specimens and 230 of the 295 sputum specimens that received a FilmArray Pneumonia Panel plus Detected result for an applicable gram-negative bacterium for CTX-M, IMP, KPC, NDM, and VIM. For informational purposes, the method used to assess correlation of the CTX-M, IMP, KPC, NDM, and VIM results (Table 7 and Table 8) reported in the specimen by the FilmArray Pneumonia Panel plus to identification of the gene in the cultured isolates from that particular specimen was one conventional PCR assay followed by bidirectional sequencing, performed directly on the isolate.

{17}------------------------------------------------

						BAL
Applicable Bacteria Result(FilmArray)	N	CTX-M		IMP		KPC		NDM		VIM		Overall(any resistance gene)
		PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA
Overall(any applicable bacteriaDetected)a	127	4/4(100%)	121/123(98.4%)	0/0(-)	127/127(100%)	1/1(100%)	124/126(98.4%)	0/0(-)	127/127(100%)	0/0(-)	127/127(100%)	5/5(100%)[56.6-100%]	118/122(96.7%)[91.9-98.7%]
Acinetobacter calcoaceticus-baumannii complex	0	-	-	-	-	-	-	-	-	-	-	-	-
Enterobacter cloacae complex	9	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)
Escherichia coli	12	4/4(100%)	8/8(100%)	0/0(-)	12/12(100%)	0/0(-)	12/12(100%)	0/0(-)	12/12(100%)	0/0(-)	12/12(100%)	4/4(100%)	8/8(100%)
Klebsiella aerogenes	7	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)
Klebsiella oxytoca	2	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)
Klebsiella pneumoniae group	14	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)
Proteus spp.	6	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)
Pseudomonas aeruginosa	43	0/0(-)	42/43(97.7%)	0/0(-)	43/43(100%)	0/0(-)	43/43(100%)	0/0(-)	43/43(100%)	0/0(-)	43/43(100%)	0/0(-)	42/43(97.7%)
Serratia marcescens	6	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)
Polymicrobial specimens	28	0/0(-)	27/28b(96.4%)	0/0(-)	28/28(100%)	1/1c(100%)	25/27d(92.6%)	0/0(-)	28/28(100%)	0/0(-)	28/28(100%)	1/1(100%)	24/27(88.9%)

Table 7. CTX-M, IMP, KPC, NDM, and VIM Performance Table (PCR/seq on cultured isolate(s) from BAL specimens)

in additional nine speciment had no applicable belection is and and by QReC); no resimately and by QReC); no resistance markers were icentified in the colluced isstate(s) y from these specimens.

One specimen E. cloace complex and K. preumoniae group detected by qReC: CTX-M was not identified in this isolate by PCRseg)

^ E. cloacae complex and P. aeruginosa detect by FilmArray and isolated by qRefCx (KPC identified from the E. cloacae isolate by PCR/seq)

ී රාජ දෙදෙන්න A. adocedous tamilitary on presently Film ray (A. calcosetics familition production of the Cruce of Child on the seat of the sealary of the sealary of the sea specimen Proteus spp. and P. aeruginosa detected by FilmArray (P. aeruginosa isolated by qRefCx; KPC was not identified in this isolated by PCR(seq)

Table 8. CTX-M, IMP, KPC, NDM, and VIM Performance Table (PCR/seq on cultured isolate(s) from sputum specimens)
-----------------------------------------------------------------------------------------------------------------	--	--

Sputum
Applicable Bacteria Result	N	CTX-M		IMP		KPC		NDM		VIM		Overall(any resistance gene)
		PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA
Overall(any applicable bacteriaDetected)a	230	3/4(75.0%)	221/226(97.8%)	1/1(100%)	229/229(100%)	5/6(83.3%)	223/224(99.6%)	0/0(-)	230/230(100%)	1/1(100%)	229/229(100%)	9/11b(81.8%)[52.3-94.9%]	214/219(97.7%)[94.8-99.0%]
Acinetobacter calcoaceticus-baumannii complex	5c	0/0(-)	5/5(100%)	0/0(-)	5/5(100%)	0/0(-)	5/5(100%)	0/0(-)	5/5(100%)	0/0(-)	5/5(100%)	0/0(-)	5/5(100%)
Enterobacter cloacae complex	7d	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)
Escherichia coli	10	1/1(100%)	9/9(100%)	0/0(-)	10/10(100%)	0/0(-)	10/10(100%)	0/0(-)	10/10(100%)	0/0(-)	10/10(100%)	1/1(100%)	9/9(100%)
Klebsiella aerogenes	3	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{18}------------------------------------------------

Sputum
Applicable Bacteria Result	N	CTX-M		IMP		KPC		NDM		VIM		Overall(any resistance gene)
		PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA
Klebsiella oxytoca	4	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)
Klebsiella pneumoniae group	21	1/1(100%)	20/20(100%)	0/0(-)	21/21(100%)	1/1(100%)	20/20(100%)	0/0(-)	21/21(100%)	0/0(-)	21/21(100%)	2/2(100%)	19/19(100%)
Proteus spp.	6	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)
Pseudomonas aeruginosa	68	0/1(0%)	65/67(97.0%)	0/0(-)	68/68(100%)	0/1(0%)	67/67(100%)	0/0(-)	68/68(100%)	0/0(-)	68/68(100%)	0/2(0%)	64/66(97.0%)
Serratia marcescens	14e	0/0(-)	14/14(100%)	1/1(100%)	13/13(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	1/1(100%)	13/13(100%)
Polymicrobial specimens	92	1/1f(100%)	88/91g(96.7%)	0/0(-)	92/92(100%)	4/4h(100%)	87/88(98.9%)	0/0(-)	92/92(100%)	1/1j(100%)	91/91(100%)	5/5(100%)	84/87(96.6%)

i in additional 19 perimers had no applicated by Filmlery, but had one or more applicatle boteni solated by PCR son in resperiner (E. collisolated by PCR Source Specific (E. but no other resistance markers were identified in the cultured isolate(s) by PCR/seq from these specimens

b One specimen had presence of dual AMR genes (KPC and VIM)

^ An A. calcoaceticus-baumannii isolate was not recovered by qRefCx for one specimen

d An E. cloacae isolate was not recovered by qRefCx for one specimen

€ A S. marcescens isolate was not recovered by qRefCx for one specimen

1 E. coli and P. aeruginosa detected by FilmArray and isolated by qRefCx (CTX-M identified in the E. coli isolate by PCR/seq)

" One speimen A. calcaetius cannance group, Probas sp., and P. aerginosa deleted by FilmAray (K. peurnories group and P. aarginosa istatibled in either of these is and one speiment. Coll. K. preuninse group, and P. aevoinos isstead by Glorian in and in one of icentify in eller of these isolated by PCR socimen Proteus sp. and P. aeruginosa detected by Film kray ( P. aeruginosa isolated by qRefCx; CTX-M was not identified in this isolate of PCRsed

"One speimen E. coll and K. premoriae group of scialed by ReC. (KPC identified in the K. preuminer P. anginosa and S. nerososs and S. nerososs and S. nerososs and S. nerosos Fith kray and isstated by Reflection the S. narces in A. calceae in en A. calceerican propes, K. preunonise group, and P. annonomie group, and P. aarginosadeedd y Finh Hay (A. calcacetos-barnanii, K. preunosa issted by (ReC); (RC icentified in the K. premorise issult on esecines to accoacer compress (roup, Proteus sp., and P. aeruginosa detected by Filmly (K. aeuginosa isolated by qRefC: KPC identified in the K. pneumoniae isolate by PCRseq)

'One specimen K, pneumoniae group and P. aeruginosa isolated by qRefC; KPC was not identified in this isolate by PCRseg)

' One specinen A. adocastionalise you, and P. aeupinsa decised by Film Aray (A. calcacelias-taunanii) K. preuncirea and P. annunca isolad (y (ReC); VII identified in the P. aeruginosa isolate by PCR/seq)

{19}------------------------------------------------

qRefCx isolated one or more applicable gram-negative bacteria from 79 of the 94 BAL specimens that received a FilmArray Pneumonia Panel plus Detected result for an applicable gram-negative bacterium for OXA-48-like. For informational purposes, the method used to assess correlation of the OXA-48-like results (Table 9 and Table 10) reported in the FilmArray Pheumonia Panel plus to identification of the cultured isolates from that particular specimen was one conventional PCR assay followed by bidirectional sequencing, performed directly on the isolate.

BAL
Applicable Bacteria Result	Positive Percent Agreement			Negative Percent Agreement
	TP/(TP + FN)	%	95%CI	TN/(TN + FP)	%	95%CI
Overall(any applicable bacteria Detected)	0/0	-	-	79/79	100	95.4-100%
Enterobacter cloacae complex	0/0	-	-	10/10	100	72.2-100%
Escherichia coli	0/0	-	-	13/13	100	77.2-100%
Klebsiella aerogenes	0/0	-	-	7/7	100	64.6-100%
Klebsiella oxytoca	0/0	-	-	3/3	100	43.9-100%
Klebsiella pneumoniae group	0/0	-	-	15/15	100	79.6-100%
Proteus spp.	0/0	-	-	6/6	100	61.0-100%
Serratia marcescens	0/0	-	-	8/8	100	67.6-100%
Polymicrobial specimens	0/0	-	-	17/17	100	81.6-100%

		Table 9. OXA-48-like Performance Table (PCR/seq on cultured isolate(s) from BAL specimens)
--	--	--------------------------------------------------------------------------------------------

Sputum
Applicable Bacteria Result	Positive Percent Agreement			Negative Percent Agreement
	TP/(TP + FN)	%	95%CI	TN/(TN + FP)	%	95%CI
Overall(any applicable bacteria Detected)	0/0	-	-	131/131	100	97.2-100%
Enterobacter cloacae complex	0/0	-	-	9/9	100	70.1-100%
Escherichia coli	0/0	-	-	17/17	100	81.6-100%
Klebsiella aerogenes	0/0	-	-	4/4	100	51.0-100%
Klebsiella oxytoca	0/0	-	-	5/5	100	56.6-100%
Klebsiella pneumoniae group	0/0	-	-	25/25	100	86.7-100%
Proteus spp.	0/0	-	-	9/9	100	70.1-100%
Serratia marcescens	0/0	-	-	25/25	100	86.7-100%
Polymicrobial specimens	0/0	-	-	37/37	100	90.6-100%

Table 10. OXA-48-like Performance Table (PCR/seq on cultured isolate(s) from sputum specimens)

{20}------------------------------------------------

qRefCx isolated S. aureus from 75 of 116 BAL specimens that received a FilmArray Pneumonia Panel plas Staphylococus aureus Detected result. The method used to assess correlation of the mecA C and MREJ results (Table 12) reported in the specimen by the FilmArray Pneumonia Panel to identification of the gene in the cultured isolates from that particular specimen was one conventional PCR assay followed by bidirectional sequencing, performed directly on the isolate.

BAL
S. aureusmecA/C and MREJ		qRefCx: S. aureusPCR/seq: mecA/C
		Org+ / Res+	Org+ / Res-	Org -	Total
FilmArrayResult	Org+ / Res+	19	2	25	46
	Org+ / Res-	1	24	45	70
	Org -	0	1	729	730
		Total	20	27	799	846
Performance		Agreement		%	95%CI
		Org+ / Res+	19/20	95.0%	76.4-99.1%
		Org+ / Res-	24/27	88.9%	71.9-96.1%
		Org -	729/799	91.2%	89.1-93.0%
Interpretation				PPA	NPA	Prevalence
		MRSA		19/20(95.0%)	799/826(96.7%)	46/846(5.4%)
		MSSA		24/27(88.9%)	773/819(94.4%)	70/846(8.3%)
		S. aureus		46/47(97.9%)	729/799(91.2%)	116/846(13.7%)

Table 11. mecA/C and MREJ 3x3 Performance Table (qRefCx & PCR/seq on cultured isolate(s) from BAL specimens)

Table 12. mecA/C and MREJ 3x3 Performance Table (qRefCx & PCR/seq on cultured isolate(s)
from sputum specimens)

from sputum specimens)
Sputum
	S. aureusmecA/C and MREJ	qRefCx: S. aureusPCR/seq: mecA/C
		Org+ / Res+	Org+ / Res-	Org -	Total
FilmArrayResult	Org+ / Res+	58	4	45	107
	Org+ / Res-	0	49	48	97
	Org -	0	1	631	632
	Total	58	54	724	836
Performance		Agreement	%	95%CI
Org+ / Res+		58/58	100%	93.8-100%
Org+ / Res-		49/54	90.7%	80.1-96.0%
Org -		631/724	87.2%	84.5-89.4%
Interpretation		PPA	NPA	Prevalence
MRSA		58/58(100%)	729/778(93.7%)	107/836(12.8%)
MSSA		49/54(90.7%)	734/782(93.9%)	97/836(11.6%)
S. aureus		111/112(99.1%)	631/724(87.2%)	204/836(24.4%)

{21}------------------------------------------------

FilmArray Pneumonia Panel plus CTX-M reporting was also compared to the standard phenotypic extended spectrum (8-lactamase (ESBL) activity testing methods performed in conjunction with aReCx. Standard phenotypic ESBL activity was only reported for E. coli and Klebsiella spp. by the central reference laboratory.

Of the 156 BAL specimens that received a FilmArray Pneumonia Panel plus Detected result for at least one applicable gram-negative bacterium on the panel, 53 specimens received a Detected result for E. coli, K. preumoniae; qRefCx isolated E. coli, K. orxtoca, and or K. pneumoniae from 43 of these specimens. Of the 295 sputum specimens that received a FilmArray Pheumonia Panel for at least one applicable gram-negative bacel. 114 specimens received a Detected result for E. coli. K. oxytoca, and or K. pneumoniae; aRefCx isolated E. coli, K. oxytoca, and/or K. pneumoniae from 71 of these specimens. The correlation between FilmArray Pneumonia Panel plus reporting of CTX-M in a particular specimen as compared to the phenotypic AST results of ison the same specimen are stratified by each applicable associated organism in Table 13 and Table 14.

BAL
	Positive Percent Agreement			Negative Percent Agreement
Applicable Bacteria Result	TP/(TP + FN)	%	95%CI	TN/(TN + FP)	%	95%CI
Overall(any applicable bacteria Detected)	4/5	80.0	37.6-96.4%	38/38	100	90.8-100%
Escherichia coli	4/4	100	51.0-100%	11/11	100	74.1-100%
Klebsiella oxytoca	0/0	-	-	5/5	100	56.6-100%
Klebsiella pneumoniae group	0/1	0	-	17/17	100	81.6-100%
Polymicrobial specimens	0/0	-	-	5/5	100	56.6-100%

Table 13. CTX-M Performance Table (comparison to phenotypic AST methods for BAL specimens)

Table 14. CTX-M Performance Table (comparison to phenotypic AST methods for sputum specimens)

Sputum
Applicable Bacteria Result	TP/(TP + FN)	%	95%CI	TN/(TN + FP)	%	95%CI
Overall(any applicable bacteria Detected)	4/7	57.1	25.1-84.2%	63/64	98.4	91.7-99.7%
Escherichia coli	2/3	66.7	20.8-93.9%	17/17	100	81.6-100%
Klebsiella oxytoca	0/0	-	-	9/9	100	70.1-100%
Klebsiella pneumoniae group	1/2	50.0	-	26/27	96.3	81.7-99.3%
Polymicrobial specimens	1/2a	50.0	-	11/11	100	74.1-100%

ª One specimen E. coll and K. oxytoca detected by qReCx (ESBL activity identified in the E. coli isolate by qRefCx AST); one speciment E. coli and K. pneumoniae group detected by FilmArray (E. coli isolated by gRefCx and ESBL activity identified by qRefCx AST).

{22}------------------------------------------------

The FilmArray Pneumonia Panel plus carbance gene reporting was also compared to the standard phenotypic carbapenem susceptbility testing methods performed in conjunction with current CLSI guidelines, standard phenotype ertapenen susceptibility is not reported for A. calcoaceticus-baunannii complex, therefore carbapenem susceptibility is only based on meropenem susceptibility for this organism. Resistance to either ertapenem or meropenem or meropenem constituted carbapenem resistance for this analysis. The correlation between FilmArray Pheumonia Panel plus reporting of the carbance genes in a particular specimen as compared to the phenotypic AST results of isolates recovered from the same specimen are stratified by each applicable associated organism in Table 15 and Table 16.

BAL
Applicable Bacteria Result		IMP		KPC		NDM		VIM		Overall(any carbapenemresistance gene)
	N	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA
Overall(any applicable bacteria Detected)	126a	0/17(0%)	109/109(100%)	3/17(17.6%)	109/109(100%)	0/17(0%)	109/109(100%)	0/17(0%)	109/109(100%)	3/17(17.6%)[6.2-41.0%]	109/109(100%)[96.6-100%]
Acinetobacter calcoaceticus-baumannii complex	0	-	-	-	-	-	-	-	-	-	-
Enterobacter cloacae complex	9	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)	0/0(-)	9/9(100%)
Escherichia coli	12	0/0(-)	12/12(100%)	0/0(-)	12/12(100%)	0/0(-)	12/12(100%)	0/0(-)	12/12(100%)	0/0(-)	12/12(100%)
Klebsiella aerogenes	7	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)	0/0(-)	7/7(100%)
Klebsiella oxytoca	2	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)	0/0(-)	2/2(100%)
Klebsiella pneumoniae group	14	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)	0/0(-)	14/14(100%)
Proteus spp.	6	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)
Pseudomonas aeruginosa	42	0/12(0%)	30/30(100%)	0/12(0%)	30/30(100%)	0/12(0%)	30/30(100%)	0/12(0%)	30/30(100%)	0/12(0%)	30/30(100%)
Serratia marcescens	6	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)
Polymicrobial specimens	28	0/5(0%)	23/23(100%)	3/5(60.0%)	23/23(100%)	0/5(0%)	23/23(100%)	0/5(0%)	23/23(100%)	3/5b(60.0%)	23/23(100%)

Table 15. IMP, KPC, NDM, and VIM Performance Table (comparison to phenotypic AST methods for BAL specimens)

a The isolate recovered from one specimen (P. aeruginosa) did not yield valid AST results on the VITEK instrument

* Of the tree specifies that wee concorner in comperand K. preumoniae grup detected by Finhray (A. calosesticus-raumanii issted by (Red), carageren essistance identified by che is and RC not identifican in expeniner. Cocase complex and P. aerginces detected by FilmAray and issance in essiance identified in the E. cloace is and AC identified in the E. claace issues on e caeugines detected by Fitner (P. eneuginos delected by Fimeray (P. eneuginosa solael by RedC), caracener resistance in the Croit in the isstate of the issues by PCRee). Of the wo specifies in especifies in especifies in experiment complex and P aevginosa dected by Filmlery (P. aevginosa issistanti identified by (ReCx AST, and KPC not isottified in the isolae by PCRSeq); one specified in the issease opnex and K. aerocenes detected by FilmArray (E. cloace isolated by gRefCx AST and KPC nd KPC nd KPC not identified in the isolate by PCR(seg).

{23}------------------------------------------------

Sputum
Applicable Bacteria Result	N	IMP		KPC		NDM		VIM		Overall(any carbapenemresistance gene)
		PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA	PPA	NPA
Overall(any applicable bacteria Detected)	229a	0/35(0%)	194/194(100%)	6/35(17.1%)	193/194(99.5%)	0/35(0%)	194/194(100%)	1/35(2.9%)	194/194(100%)	6/35b(17.1%)[8.1-32.7%]	193/194(99.5%)[97.1-99.9%]
Acinetobacter calcoaceticus-baumannii complex	5c	0/2(0%)	3/3(100%)	0/2(0%)	3/3(100%)	0/2(0%)	3/3(100%)	0/2(0%)	3/3(100%)	0/2(0%)	3/3(100%)
Enterobacter cloacae complex	7d	0/1(0%)	6/6(100%)	0/1(0%)	6/6(100%)	0/1(0%)	6/6(100%)	0/1(0%)	6/6(100%)	0/1(0%)	6/6(100%)
Escherichia coli	10	0/0(-)	10/10(100%)	0/0(-)	10/10(100%)	0/0(-)	10/10(100%)	0/0(-)	10/10(100%)	0/0(-)	10/10(100%)
Klebsiella aerogenes	3	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)	0/0(-)	3/3(100%)
Klebsiella oxytoca	4	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)	0/0(-)	4/4(100%)
Klebsiella pneumoniae group	21	0/2(0%)	19/19(100%)	1/2(50.0%)	19/19(100%)	0/2(0%)	19/19(100%)	0/2(0%)	19/19(100%)	1/2(50.0%)	19/19(100%)
Proteus spp.	6	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)	0/0(-)	6/6(100%)
Pseudomonas aeruginosa	67	0/16(0%)	51/51(100%)	0/16(0%)	51/51(100%)	0/16(0%)	51/51(100%)	0/16(0%)	51/51(100%)	0/16(0%)	51/51(100%)
Serratia marcescens	14e	0/1(0%)	13/13(100%)	1/1(100%)	13/13(100%)	0/1(0%)	13/13(100%)	0/1(0%)	13/13(100%)	1/1(100%)	13/13(100%)
Polymicrobial specimens	92	0/13(0%)	79/79(100%)	4/13(30.8%)	78/79(98.7%)	0/13(0%)	79/79(100%)	1/13(7.7%)	79/79(100%)	4/13f(30.8%)	78/79(98.7%)

Table 16. IMP. KPC. NDM. and VIM Performance Table (comparison to phenotypic AST methods for sputum specimens)

ª The isolate recovered from one specimen (P. aeruginosa) did AST results on the VITEK instrument

b One specimen had presence of dual AMR genes (KPC and VIM)

· An A. calcoaceticus-baumannii isolate was not recovered by qRefCx for one specimen

d An E. cloacae isolate was not recovered by qRefCx for one specimen

e A S. marcescens isolate was not recovered by qRefCx for one specimen

Cothe four speciment one spearnen A catcacetics - armanii concele, K. aeropere, K. preumoniae group, and P. aerupinsa delected by Film-leroy / L. aacacelius couranii, K. preu and P. aeugince is and by the Career resident in all the issues by ReC. AST. KPC internation in the K. oneumone issuate by P. Story and VM identified in the P. aspurisos is d PCR Searcher A calcaced courranni complex, K. preunosa detected by Film ray (K. preumoniae and P. aeuginosa islated by ReC., cades en resistane icentied in the K. peamoniae istatibed in the K. preumoniae istate on PCRSed; one speamen E. coll and K. pneumoniae group deeded by ReCC (carbaceren resistance in the K. preumaria in the isstem in the isstem in the isstem in P. aequinces and S. marcessers detect by Filmal rand solated by Filmal rand solated by (carbaceren) resistance in the S. narces en stilled in the issue of PC icentified in the solerings that wee not concribed in essessions to anominationel specifical program an ගා ගැන අගමය sp. deated by Film nay and islaad by ඇිඳිට ( patcapean resistance in the A. cabsactional indel by ReC. AST and KPC not teather in either in either in either in one speciment E. cloace complex and P. aewinosa istated by of eC. cadaperen resistance itentified by (ReC). AST, and KP ont itentified in the issuale of the issuale one of P speciment of Carrylines detected by Film kray and isstance in electified in the P. apryinces isstance in the P. as running in effect AST and KPC not teatlied in ether includ speciner K. preumane group and P. aergines desday phellion (catapenern resisted in the K. presmoner is startiled in the K. presidentified in of KC not i dentified in other is PCR Son); one speciment Protes spp. and P. aerginosa and states in catagement resistance identifies in the P. aerginess inder by ReCC AST and KPC not icentified in eller isol by P P P Reserime P. aequinosa and S. marces end sistem on issiated by (ReC.) (carbagerer resistention in the S. marcessers is date by ReC.X AST and Kerine in eliber isdate by P.R.Septime P. aeuginosa and S. marces est by Film-ing JP . aeuginosa islance by ReCC. AST, and KPC not icentified in to issuale by PCRson), one specifies is a ranni complex, E. coli, K. aeuginosa dected by Filmlery (E. col and P. aengines istated by G.eC., cabaperen resistante in the P. aeruginosa isolae by Rella in ether issable by PCRSeg), one specimen A. calcoeetics-barnanni complex, Protes spp., P. aerujnces, and S. marcesons deteals by Finh vay (Proteus spo. and P. aeruginosa isolated by oRef. catalied in the P. aerupinosa isolate by gReCx AST and KPC not identified in either isolate by PCRseg).

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{24}------------------------------------------------

BAL
	Positive Percent Agreement			Negative Percent Agreement
Applicable Bacteria Result	TP/(TP + FN)	%	95%CI	TN/(TN + FP)	%	95%CI
Overall(any applicable bacteria Detected)	0/2	0	-	77/77	100	95.2-100%
Enterobacter cloacae complex	0/1	0	-	9/9	100	70.1-100%
Escherichia coli	0/0	-	-	13/13	100	77.2-100%
Klebsiella aerogenes	0/0	-	-	7/7	100	64.6-100%
Klebsiella oxytoca	0/0	-	-	3/3	100	43.9-100%
Klebsiella pneumoniae group	0/0	-	-	15/15	100	79.6-100%
Proteus spp.	0/0	-	-	6/6	100	61.0-100%
Serratia marcescens	0/0	-	-	8/8	100	67.6-100%
Polymicrobial specimens	0/1a	0	-	16/16	100	80.6-100%

Table 17. OXA-48-like Performance Table (comparison to phenotypic AST methods for BAL specimens)

ª E. cloacee complex and K. aerogenes detected by FilmArray (E. oloace complex isolated by qRefCx and carbapenem resistance identified by qRefCx AST)

Table 18. OXA-48-like Performance Table (comparison to phenotypic AST methods for sputum specimens)

Sputum
Applicable Bacteria Result	Positive Percent Agreement			Negative Percent Agreement
	TP/(TP + FN)	%	95%CI	TN/(TN + FP)	%	95%CI
Overall(any applicable bacteria Detected)	0/10	0	-	121/121	100	96.9-100%
Enterobacter cloacae complex	0/1	0	-	8/8	100	67.6-100%
Escherichia coli	0/0	-	-	17/17	100	81.6-100%
Klebsiella aerogenes	0/0	-	-	4/4	100	51.0-100%
Klebsiella oxytoca	0/0	-	-	5/5	100	56.6-100%
Klebsiella pneumoniae group	0/3	0	-	22/22	100	85.1-100%
Proteus spp.	0/0	-	-	9/9	100	70.1-100%
Serratia marcescens	0/3	0	-	22/22	100	85.1-100%
Polymicrobial specimens	0/3a	0	-	34/34	100	89.8-100%

^ One speciment. onlind K. preumonial on the Cr. (carapenen resistance identified in the K. preumoniae group isolate by ReC. AST); one sperimen K. aergenes and K. preumoniae group delected by Film is and carbapenen resistance identified by gRelCx AST); one specifier. N preumoniae group and Proteus spp. detected by FilmArray (K. pneumoniae group isolated by qRefCx and carbapenen resistance identified by qRefCx AST)

{25}------------------------------------------------

FilmArray Pneumonia Panel plus mece/C and MREJ reporting was also compared to the standard phenotypic cefoxitin susceptibility testing methods performed in conjunction with qRefCx. The correlation between FilmArray Pheumonia Panel plus reporting of mecA/C and MREJ in a particular specimen as compared to the phenotypic AST results of isolates recovered from the same specimen is shown in Table 19 and Table 20.

BAL
S. aureusmecA/C and MREJ		qRefCx: S. aureusqRefCx Phenotypic AST: cefoxitin susceptibility
		Org+ / Res+	Org+ / Res-	Org -	Total
	Org+ / Res+	18	3	25	46
FilmArray	Org+ / Res-	1	24	45	70
Result	Org -	0	1	729	730
	Total	19	28	799	846
Performance			Agreement	%	95%CI
Org+ / Res+			18/19	94.7%	75.4-99.1%
Org+ / Res-			24/28	85.7%	68.5-94.3%
Org -			729/799	91.2%	89.1-93.0%
Interpretation			PPA	NPA	Prevalence
			18/19	799/827	46/846
		MRSA	(94.7%)	(96.6%)	(5.4%)
			24/28	772/818	70/846
		MSSA	(85.7%)	(94.4%)	(8.3%)
			46/47	729/799	116/846
		S. aureus	(97.9%)	(91.2%)	(13.7%)

Table 19. mecA/C and MREJ 3x3 Performance Table (qRefCx & phenotypic AST on cultured isolate(s) from BAL specimens)

Table 20. mecA/C and MREJ 3x3 Performance Table (qRefCx & phenotypic AST on cultured isolate(s) from sputum specimens)

Sputum
S. aureusmecA/C and MREJ	qRefCx: S. aureusqRefCx Phenotypic AST: cefoxitin susceptibility
	Org+ / Res+	Org+ / Res-	Org -	Total
Org+ / Res+	59	3	45	107
Org+ / Res-	1	48	48	97
Org -	0	1	631	632
Total	60	52	724	836
Performance
		Agreement	%	95%CI
Org+ / Res+		59/60	98.3%	91.1-99.7%
Org+ / Res-		48/52	92.3%	81.8-97.0%
Org -		631/724	87.2%	84.5-89.4%
Interpretation
		PPA	NPA	Prevalence
MRSA		59/60(98.3%)	728/776(93.8%)	107/836(12.8%)
MSSA		48/52(92.3%)	735/784(93.8%)	97/836(11.6%)
S. aureus		111/112(99.1%)	631/724(87.2%)	204/836(24.4%)

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The FilmArray Pneumonia Panel plus bin performance compared to the quantitative molecular assay (qMol) comparator is shown for BAL (Table 21) and sputum (Table 22). The qMol values are broken into one-log ranges correlating to the reported semi-quantitative FilmArray Pneumonia Panel plus bins. The relationship between qMol quantitative bins in copies/mL and traditional culture quantification in CFU/mL is unknown.

	BAL
qMol Binned Valuesa(copies/mL)		ND to<10^3.5	10^4.0	10^5.0	10^6.0	≥10^7.0	Total
	ND	12025	35	5	0	2	12067
FilmArray Bin(copies/mL)	10^4	47	48	21	0	1	117
	10^5	5	23	57	22	2	109
	10^6	3	3	29	40	13	88
	≥10^7	2	0	4	41	112	159
	% concordant	12025/12082(99.5%)	48/109(44.0%)	57/116(49.1%)	40/103(38.8%)	112/130(86.2%)	12540

Table 21. FilmArray Pneumonia Panel plus Overall Bin Performance Summary for BAL Specimens (qMol)

a Shaded cells indicate results concordant between the FilmArray Pneumonia Panel plus and qMol.

Table 22. FilmArray Pneumonia Panel plus Overall bin performance for Sputum Specimens (qMol)
----------------------------------------------------------------------------------------------	--	--

Sputum
qMol Range of Values a(copies/mL)		ND to<103.5	<104.0	<105.0	<106.0	≥107.0	Total
FilmArray Bin(copies/mL)	ND	11392	85	17	2	2	11498
	104	79	87	41	7	0	214
	105	12	33	104	43	5	197
	106	2	4	39	88	41	174
	≥107	4	0	1	44	288	337
% concordant		11392/11489(99.2%)	87/209(41.6%)	104/202(51.5%)	88/184(47.8%)	288/336(67.9%)	12420
567/931 (60.9%)

a Shaded cells indicate results concordant between the FilmArray Pneumonia Panel and qMol.

The FilmArray Pneumonia Panel bin performance compared to qRefCx quantification and semiquantification values provided by the source lab is shown for BAL and sputum in Table 23 - Table 34. Data is shown for overall performance, as well as for some individual organisms. In these tables, the values reported by culture are broken into ranges. A FilmArray Pneumonia Panel bin plus result is considered concordant if the culture value is within 0.5 log of the bin boundary. For example, the 10^5 FilmArray Pneumonia Panel plus bin (10^4.5-10^5.5) is concordant with the culture range of 10^4-10^6 CFU/mL.

Table 23. FilmArray Pneumonia Panel plus Overall bin performance for BAL Specimens (qRefCx)

BAL
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to$<10^3.5$(ND)	$10^3.5$ to$<10^4.0$( $10^4$ )	$10^4.0$ to$<10^5.0$( $10^4$ or $10^5$ )	$10^5.0$ to$<10^6.0$( $10^5$ or $10^6$ )	$10^6.0$ to$<10^7.0$( $10^6$ or $10^7$ )	≥ $10^7.0$(≥ $10^7$ )
FilmArray Bin	ND	12202	1	2	0	0	0
	$10^4$	116	1	3	0	0	0

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{27}------------------------------------------------

		90	10	11	0	1	0
	10^6	61	10	17	2	1	0
	≥10^7	61	10	36	32	11	12
% concordant		12202/12530(97.4%)	1/32(3.1%)	14/69(20.3%)	2/34(5.9%)	12/13(92.3%)	12/12(100%)
					41/160 (25.6%)

Table 24. FilmArray Pneumonia Panel plus Overall bin performance for Sputum Specimens (qRefCx)

Sputum
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
FilmArray Bin(copies/mL)	ND	11596	4	6	2	0	1
	10^4	193	14	9	0	0	0
	10^5	139	19	28	14	1	0
	10^6	94	19	36	21	4	1
	≥10^7	121	8	88	53	49	20
% concordant		11596/12143(95.5%)	14/64(21.9%)	37/167(22.2%)	35/90(38.9%)	53/54(98.1%)	20/22(90.9%)
						159/397 (40.1%)

Table 25. FilmArray Pneumonia Panel plus H. influenzae bin performance for BAL Specimens (qRefCx)

BAL
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
FilmArray Bin(copies/mL)	ND	764	0	0	0	0	0
	10^4	17	0	0	0	0	0
	10^5	12	0	0	0	0	0
	10^6	13	2	0	0	0	0
	≥10^7	30	0	2	5	0	1
% concordant		764/836(91.4%)	0/2(0%)	0/2(0%)	0/5(0%)	0/0(-)	1/1(100%)
					1/10 (10.0%)

Table 26. FilmArray Pneumonia Panel plus H. influenzae bin performance for Sputum Specimens (qRefCx)

Sputum
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
FilmArray Bin(copies/mL)	ND	727	0	1	1	0	0
	10^4	21	0	0	0	0	0
	10^5	19	0	0	1	0	0
	10^6	13	0	1	0	0	0
	≥10^7	38	0	10	2	2	0
% concordant		727/818(88.9%)	0/0(-)	0/12(0%)	1/4(25.0%)	2/2(100%)	0/0(-)
3/18 (16.7%)

{28}------------------------------------------------

Table 27. FilmArray Pneumonia Panel plus P. aeruginosa bin performance for BAL Specimens (qRefCx)

BAL
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
FilmArray Bin(copies/mL)	ND	772	0	0	0	0	0
	10^4	12	0	1	0	0	0
	10^5	14	2	1	0	0	0
	10^6	5	1	2	1	1	0
	≥10^7	7	1	11	12	1	2
% concordant		772/810(95.3%)	0/4(0.0%)	2/15(13.3%)	1/13(7.7%)	2/2(100%)	2/2(100%)
					7/36 (19.4%)

Table 28. FilmArray Pneumonia Panel plus P. aeruginosa bin performance for Sputum Specimens (qRefCx)

Sputum
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
FilmArray Bin(copies/mL)	ND	673	2	1	0	0	0
	10^4	16	3	1	0	0	0
	10^5	17	3	6	2	0	0
	10^6	12	4	8	3	1	0
	≥10^7	12	3	26	19	18	6
% concordant		673/730(92.2%)	3/15(20.0%)	7/42(16.7%)	5/24(20.8%)	19/19(100%)	6/6(100%)
						40/106 (37.7%)

Table 29.FilmArray Pneumonia Panel plus S. aureus bin performance for BAL Specimens (qRefCx)

	BAL
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
FilmArray Bin(copies/mL)	ND	729	1	0	0	0	0
	10^4	33	0	0	0	0	0
	10^5	23	3	0	0	0	0
	10^6	13	2	7	1	0	0
	≥10^7	1	5	12	8	5	3
% concordant		729/799(91.2%)	0/11(0.0%)	0/19(0.0%)	1/9(11.1%)	5/5(100%)	3/3(100%)
9/47 (19.1%)

Table 30. FilmArray Pneumonia Panel plus S. aureus bin performance for Sputum Specimens (qRefCx)

Sputum
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)	ND to$<10^3.5$(ND)	$10^3.5$ to$<10^4.0$( $10^4$ )	$10^4.0$ to$<10^5.0$( $10^4$ or $10^5$ )	$10^5.0$ to$<10^6.0$( $10^5$ or $10^6$ )	$10^6.0$ to$<10^7.0$( $10^6$ or $10^7$ )	≥ $10^7.0$(≥ $10^7$ )
FilmArray Bin(copies/mL)	ND	631	1	0	0	0	0
	$10^4$	39	1	3	0	0	0
	$10^5$	33	7	8	4	1	0
	$10^6$	12	7	13	9	1	1

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{29}------------------------------------------------

Sputum
	qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)	ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
	≥10^7	9	2	21	15	11	7
% concordant		631/724(87.2%)	1/18(5.6%)	11/45(24.4%)	13/28(46.4%)	12/13(92.3%)	7/8(87.5%)
44/112 (39.3%)

Table 31. FilmArray Pneumonia Panel plus S. pneumoniae bin performance for BAL Specimens (qRefCx)

BAL
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
FilmArray Bin(copies/mL)	ND	817	0	0	0	0	0
	10^4	8	1	0	0	0	0
	10^5	7	0	1	0	0	0
	10^6	5	0	1	0	0	0
	≥10^7	4	1	0	1	0	0
% concordant		817/841(97.1%)	1/2(50.0%)	1/2(50.0%)	0/1(0.0%)	0/0(-%)	0/0(-%)
						2/5 (40%)

Table 32. FilmArray Pneumonia Panel plus S. pneumoniae bin performance for Sputum Specimens (qRefCx)

Sputum
qRefCx Range of Values[CFU/mL](Predicted FilmArray Bin)		ND to<10^3.5(ND)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)
ND		785	0	0	0	0	0
FilmArray Bin(copies/mL)	10^4	10	2	0	0	0	0
	10^5	8	0	2	0	0	0
	10^6	9	1	0	0	0	0
	≥10^7	8	0	4	2	4	1
	% concordant		785/820(95.7%)	2/3(66.7%)	2/6(33.3%)	0/2(0.0%)	4/4(100%)
9/16 (56.3%)

Table 33. FilmArray Pneumonia Panel plus Bin Performance Summary for BAL Specimens comparted to qRefCx

BAL
qRefCx Range of Values(Concordant FilmArray Bin)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)	Overall
Acinetobacter calcoaceticus-baumannii complex	0/0(-)	0/0(-)	0/0(-)	0/0(-)	0/0(-)	0/0(-)
Enterobacter cloacae complex	0/1(0%)	1/7(14.3%)	0/3(0%)	0/0(-)	1/1(100%)	2/12(16.7%)
Escherichia coli	0/4(0%)	1/7(14.3%)	0/0(-)	0/0(-)	1/1(100%)	2/12(16.7%)
Haemophilus influenzae	0/2(0%)	0/2(0%)	0/5(0%)	0/0(-)	1/1(100%)	1/10(10.0%)
Klebsiella aerogenes	0/1(0%)	1/2(50.0%)	0/1(0%)	2/3(66.7%)	0/0(-)	3/7(42.9%)
Klebsiella oxytoca	0/0(-)	1/2(50.0%)	0/0(-)	0/0(-)	0/0(-)	1/2(50.0%)

{30}------------------------------------------------

BAL
qRefCx Range of Values(Concordant FilmArray Bin)	$10^3.5$ to$<10^4.0$( $10^4$ )	$10^4.0$ to$<10^5.0$( $10^4$ or $10^5$ )	$10^5.0$ to$<10^6.0$( $10^5$ or $10^6$ )	$10^6.0$ to$<10^7.0$( $10^6$ or $10^7$ )	$\ge 10^7.0$( $\ge 10^7$ )	Overall
Klebsiella pneumoniae group	0/5(0%)	3/5(60.0%)	0/0(-)	2/2(100%)	3/3(100%)	8/15(53.3%)
Moraxella catarrhalis	0/0(-)	0/0(-)	0/0(-)	0/0(-)	0/0(-)	0/0(-)
Proteus spp.	0/1(0%)	1/1(100%)	0/1(0%)	1/1(100%)	1/1(100%)	3/5(60.0%)
Pseudomonas aeruginosa	0/4(0%)	2/15(13.3%)	1/13(7.7%)	2/2(100%)	2/2(100%)	7/36(19.4%)
Serratia marcescens	0/1(0%)	1/4(25.0%)	0/1(0%)	0/0(-)	0/0(-)	1/6(16.7%)
Staphylococcus aureus	0/11(0.0%)	0/19(0%)	1/9(11.1%)	5/5(100%)	3/3(100%)	9/47(19.1%)
Streptococcus agalactiae	0/0(-)	0/1(0%)	0/0(-)	0/0(-)	0/0(-)	0/1(0%)
Streptococcus pneumoniae	1/2(50.0%)	1/2(50.0%)	0/1(0%)	0/0(-)	0/0(-)	2/5(40.0%)
Streptococcus pyogenes	0/0(-)	2/2(100%)	0/0(-)	0/0(-)	0/0(-)	2/2(100%)

Table 34. FilmArray Pneumonia Panel plus Bin Performance Summary for Sputum Specimens comparted to qRefCx

qRefCx Range of Values(Concordant FilmArray Bin)	10^3.5 to<10^4.0(10^4)	10^4.0 to<10^5.0(10^4 or 10^5)	10^5.0 to<10^6.0(10^5 or 10^6)	10^6.0 to<10^7.0(10^6 or 10^7)	≥10^7.0(≥10^7)	Overall
Acinetobacter calcoaceticus-baumannii complex	1/5(20.0%)	1/1(100%)	0/2(0%)	3/3(100%)	0/0(-)	5/11(45.5%)
Enterobacter cloacae complex	0/1(0%)	3/6(50.0%)	1/3(33.3%)	1/1(100%)	1/1(100%)	6/12(50.0%)
Escherichia coli	2/3(66.7%)	1/13(7.7%)	2/5(40.0%)	1/1(100%)	2/2(100%)	8/24(33.3%)
Haemophilus influenzae	0/0(-)	0/12(0%)	1/4(25.0%)	2/2(100%)	0/0(-)	3/18(16.7%)
Klebsiella aerogenes	1/1(100%)	0/1(0%)	0/1(0%)	1/1(100%)	0/0(-)	2/4(50.0%)
Klebsiella oxytoca	1/3(33.3%)	0/3(0%)	2/2(100%)	1/1(100%)	0/0(-)	4/9(44.4%)
Klebsiella pneumoniae group	3/6(50.0%)	2/7(28.6%)	5/7(71.4%)	2/2(100%)	0/1(0%)	12/23(52.2%)
Moraxella catarrhalis	0/1(0%)	0/1(0%)	0/1(0%)	1/1(100%)	1/1(100%)	2/5(40.0%)
Proteus spp.	0/1(0%)	5/10(50.0%)	2/3(66.7%)	1/1(100%)	0/0(-)	8/15(53.3%)
Pseudomonas aeruginosa	3/15(20.0%)	7/42(16.7%)	5/24(20.8%)	19/19(100%)	6/6(100%)	40/106(37.7%)
Serratia marcescens	0/4(0%)	4/12(33.3%)	3/6(50.0%)	4/4(100%)	1/1(100%)	12/27(44.4%)
Staphylococcus aureus	1/18(5.6%)	11/45(24.4%)	13/28(46.4%)	12/13(92.3%)	7/8(87.5%)	44/112(39.3%)
Streptococcus agalactiae	0/3(0%)	1/5(20.0%)	0/1(0%)	0/0(-)	0/0(-)	1/9(11.1%)
Streptococcus pneumoniae	2/3(66.7%)	2/6(33.3%)	0/2(0%)	4/4(100%)	1/1(100%)	9/16(56.3%)
Streptococcus pyogenes	0/0(-)	0/3(0%)	1/1(100%)	1/1(100%)	1/1(100%)	3/6(50.0%)

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A 'rank order' analysis was performed on polymicrobial specimens to compare the relative abundance of each analyte within a specimen as reported by qRefCx to the order reported by the FilmArray Pneumonia Panel plus. In this analysis, there were 20 specimens with two or more organisms reported by qRefCx for BAL and 84 for sputum. In these specimens, the FilmArray Pneumonia Panel plus was in agreement with SOC for the most abundant organism 78.8% of the time (41/52) for BAL and 85.9% of the time (85/99) for sputum. For the second most abundant organism, the FilmArray Pneumonia Panel plus was in agreement qRefCx for the most abundant organism 45.0% of the time (9/20) for BAL and 41.7% of the time (35/84) for sputum. For the second most abundant organism, the FilmArray Pneumonia Panel was in agreement with qRefCx 30.0% of the time (6/20) for BAL and 26.2% of the time (22/84) for sputum, and was in agreement for the third most abundant organism 7.7% of the time (1/13) for BAL and 3.8% of the time (2/52) for sputum.

Ranking Performance		BAL		Sputum
	Correct	Total	%	Correct	Total	0/0
Most Abundant	g	20	45.0%	રૂડે	84	41.7%
Second Most Abundant	6	20	30.0%	22	84	26.2%
Third Most Abundant		13	7.7%	2	52	3.8%

Table 35. Concordance of Abundance in Polymicrobial Specimens (as compared to gRefCx)

'Rank concordance' analysis was performed on polymicrobial specimens to determine the ability of the FilmArray Pneumonia Panel to measure relative abundance of the nucleic acid for an organism with respect to other organisms in the specimen, as compared to qRefCx. In this analysis, the detected organisms from individual polymicrobial specimens (110 BAL and 246 sputum) were ranked in descending order based on their quantification values from qRefCx. The rank determined by the FilmArray Pneumonia Panel bin result was compared to the qRefCx ranking. False positives results were always considered discordant (i.e. only exact rank matches are considered concordant).

Table 36. Concordance of Organism Ranking in Polymicrobials Specimens (as compared to qRefCx); false positive results considered discordant

Ranking Performance	BAL			Sputum
	Concordant	Total	%	Concordant	Total	%
Acinetobacter calcoaceticus-baumannii complex	1	6	16.7%	7	25	28.0%
Enterobacter cloacae complex	9	14	64.3%	9	25	36.0%
Escherichia coli	6	13	46.2%	14	39	35.9%
Haemophilus influenzae	9	47	19.1%	10	62	16.1%
Klebsiella aerogenes	4	9	44.4%	4	9	44.4%
Klebsiella oxytoca	3	10	30.0%	5	17	29.4%
Klebsiella pneumoniae group	8	16	50.0%	15	43	34.9%
Moraxella catarrhalis	0	15	0.0%	3	59	5.1%
Proteus spp.	2	6	33.3%	5	20	25.0%
Pseudomonas aeruginosa	17	25	68.0%	59	107	55.1%
Serratia marcescens	5	10	50.0%	17	43	39.5%
Staphylococcus aureus	30	55	54.5%	59	141	41.8%
Streptococcus agalactiae	6	19	31.6%	10	35	28.6%
Streptococcus pneumoniae	4	23	17.4%	7	37	18.9%
Streptococcus pyogenes	1	5	20.0%	2	5	40.0%

{32}------------------------------------------------

The overall success rate for initial specimen tests in the prospective study was 98.1% (1764/1798); 34 tests were unsuccessful (two due to an incomplete test and 32 due to control failures). Two tests (2/1798; 0.1%) did not complete on the initial run, resulting in an instrument success rate of 99.9% (1796/1798) for initial specimen tests. Both specimens were able to be retested and valid results were produced after a single retest.

Thirty-two (32) tests (32/1764; 1.8%) did not produce valid pouch controls, resulting in a pouch control success rate of 98.2% (1764/1796) for completed runs in the initial specimen tests. Twenty-eight (28) of the 32 invalid specimens were able to be retested; 25 produced valid control results after a single retest, while the remaining three did not produce valid control results after retesting and were not able to be retested further due to insufficient specimen volume; four were not able to be retested at all due to insufficient specimen volume. Three additional studies were also conducted to demonstrate all aspects of clinical performance (see Testing of Preselected Archived Specimens - MERS-CoV, Testing of Preselected Archived Specimens - Common Lower Respiratory Specimens, and Testing of Contrived Specimens below).

Testing of Preselected Archived Specimens – MERS-CoV

Some of the analytes on the FilmArray Pneumonia Panel plus, including MERS-CoV, were of low prevalence and were not encountered in large enough numbers during the prospective study to adequately demonstrate system performance. To supplement the results of the prospective clinical study, an evaluation of preselected archived retrospective specimens was performed. Due to the BSL3 requirements for handling specimens that are positive for MERS-CoV, testing of archived specimens containing this analyte was performed as a separate study. A summary of testing of for the archived specimens can be found in the Testing of Preselected Archived Specimens - Common Lower Respiratory Specimens section below.

In this study, eight bronchoalveolar lavage (BAL) and ten sputum specimens that tested positive for MERS-CoV during a 2015 outbreak in South Korea were evaluated using the FilmArray Pneumonia Panel plus. Testing was performed in the BSL3 laboratory at Seoul National University Hospital (Seoul, South Korea). At the completion of testing, one BAL specimen was excluded due to a MERS-CoV Equivocal result that could not be retested due to insufficient volume.

The FilmArray Pneumonia Panel plus demonstrated 100% positive percent agreement (PPA) with previous laboratory results for MERS-CoV for both BAL and sputum. NPA was not evaluated in this study. The performance for MERS-CoV is shown in Table 37.

Analyte	PPA
	TP/(TP + FN)	%	95% CI
BAL
MERS-CoV	7/7	100	64.6-100
Sputum
MERS-CoV	10/10	100	72.3-100

			Table 37. FilmArray Pneumonia Panel plus MERS-CoV Archived Specimen Performance Data Summary

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Testing of Preselected Archived Specimens – Common Lower Respiratory Specimens

Some of the analytes on the FilmArray Pneumonia Panel plus were of low prevalence and were not encountered in large enough numbers during the prospective study to adequately demonstrate system performance. To supplement the results of the prospective clinical study, an evaluation of preselected archived retrospective specimens was performed at BioFire.

A total of 171 frozen archived specimens were received for testing from external laboratories. Eighteen (18) specimens were negatives (13 BAL and 5 sputum) and 153 specimens (139 BAL and 14 sputum) contained at least one analyte of interest. Twenty-two (22) specimens contained two or more analytes of interest. The set included specimens known to be positive for: Acinetobacter calcoaceticus-baumannii complex, Klebsiella aerogenes, Klebsiella oxytoca, Proteus spp., Serratia marcescens, Streptococus pyogenes, Chlamydia pneumoniae, Legionella pneumophila, adenovirus, human metapneumovirus, influenza A, influenza B, parainfluenza virus (PIV), respiratory syncytial virus, various gram-negative bacteria with extended-spectrum ß-lactamase (ESBL) phenotype, and various gram-negative bacteria with a carbapenem resistant phenotype.

Prior to testing with the FilmArray Pneumonia Panel plus, the composition/integrity of the specimens was first confirmed with confirmatory molecular methods. At the completion of testing, four specimens were excluded due to invalid confirmation tests; these specimens had insufficient volume for retesting. Results from the remaining 18 negative and 149 positive specimens (containing 173 analytes) are presented here.

The reported analyte (as determined by the source laboratory) was confirmed for 117 analytes (107 in BAL and 10 in sputum) of the expected positive results (117/173; 67.6 %). More than three quarters of the unconfirmed analytes were specimens previously identified as positive for gram-negative bacteria exhibiting phenotypic ESBL or carbapenamase activity (44/57; 77.2%). This is expected since this phenotypic activity can be conferred by alternative mechanisms beyond the antibiotic resistance genes found on the FilmArray Pneumonia Panel plus. Specimens with unconfirmed (or unexpected) analytes were excluded from performance calculations for that particular analyte and FilmArray test results are reported separately.

The FilmArray Pneumonia Panel plus demonstrated positive percent agreement (PPA) of 100% with previous laboratory results for 11 of 14 analytes tested in BAL specimens (Table 38) and all of the analytes tested in sputum specimens (Table 39). The exceptions were Klebsiella aerogenes, Proteus spp. and RSV with a PPA of 50.0%, 80.0%, and 93.8% respectively, due to one false negative (FN) for each analyte. While PPA for most analytes was 100%, an insufficient number of specimens were tested for all but influenza A, parainfluenza virus, and respiratory syncytial virus in BAL. Contrived specimen testing was used to demonstrate performance for these analytes in an additional contrived study (see Testing of Contrived Specimens). Negative percent agreement (NPA) was 100% for all analytes except parainfluenza virus in BAL and influenza A in sputum; however, NPA was more thoroughly evaluated in the prospective clinical study.

Table 38. FilmArray Pneumonia Panel plus Archived BAL Specimen Performance Data Summary

Analyte	PPA			NPA
	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Quantitative Bacteria
Acinetobacter calcoaceticus-baumanniicomplex	4/4	100	51.0-100%	53/53	100	93.2-100%
Klebsiella aerogenes	1/2	50.0	-	53/53	100	93.2-100%
Proteus spp.	4/5	80.0	37.6-96.4%	48/48	100	92.6-100%

BioFire Diagnostics 510(k)

FilmArray Pneumonia Panel plus

{34}------------------------------------------------

	PPA			NPA
Analyte	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Serratia marcescens	10/10	100	72.2–100%	46/46	100	92.3–100%
Streptococcus pyogenes	1/1	100	-	57/57	100	93.7–100%
Antimicrobial Resistance Genes
CTX-M	7/7	100	64.6–100%	29/29	100	88.3–100%
Atypical Bacteria
Chlamydia pneumoniae	1/1	100	-	90/90	100	95.9–100%
Legionella pneumophila	1/1	100	-	57/57	100	93.7–100%
Viruses
Adenovirus	8/8	100	67.6–100%	81/81	100	95.5–100%
Human metapneumovirus	11/11	100	74.1–100%	77/77	100	95.2–100%
Influenza A	21/21	100	84.5–100%	69/69	100	94.7–100%
Influenza B	3/3	100	43.9–100%	86/86	100	95.7–100%
Parainfluenza virus	17/17	100	81.6–100%	68/69	99.0	92.2–99.7%
Respiratory syncytial virus	15/16	93.8	71.7–98.9%	74/74	100	95.1–100%

Table 39. FilmArray Pneumonia Panel plus Archived Sputum Specimen Performance Data Summary

	PPA			NPA
Analyte	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Quantitative Bacteria
Streptococcus pyogenes	7/7	100	64.6-100%	8/8	100	67.6-100%
Antimicrobial Resistance Genes
CTX-M	1/1	100	-	12/12	100	75.8-100%
Viruses
Influenza A	2/2	100	34.2-100%	0/1	0	-

Testing of Contrived Specimens

A prospective clinical evaluation of the FilmArray Pneumonia Panel plus was performed during the 2016-2017 respiratory infection season at several geographically diverse clinical laboratories. Over 1600 specimens were analyzed from subjects whose specimens were submitted for microbial evaluation of lower respiratory tract pathogens. In the prospective study, some analytes were of insufficient prevalence to adequately demonstrate system performance and additional archived, preselected positive specimens containing rare analytes were also tested. Several analytes were so rare that both prospective and archived testing efforts were insufficient to demonstrate system performance. In this study, contrived clinical specimens were created to evaluate the sensitivity and specificity of the FilmArray Pneumonia Panel plus assays for these rare analytes (Table 40).

Table 40. Contrived Clinical Specimen Analytes

Analyte	Matrix
	BAL	Sputum
Middle East Respiratory Syndrome Coronavirus	X	X
Bacteria
Acinetobacter calcoaceticus-baumannii complex	X

{35}------------------------------------------------

Klebsiella oxytoca	X
Proteus spp.	X
Serratia marcescens	X
Streptococcus pyogenes	X	X
Atypical Bacteria
Chlamydia pneumoniae	X	X
Legionella pneumophila	X	X
Mycoplasma pneumoniae	X	X
Viruses
Adenovirus	X	X
Human metapneumovirus	X	X
Influenza A	X	X
Influenza B	X	X
Antibiotic Resistance Markers
CTX-M	X	X
IMP	X	X
KPC	X	X
NDM	X	X
OXA-48 like	X	X
VIM	X	X

Contrived specimens (N=1225) were spiked using residual clinical samples that were pre-screened with the FilmArray Pneumonia Panel plus and found to be negative for the analytes of interest. Specimens were spiked with a variety of different isolates/strains for each organism at concentrations spanning observed ranges in clinical specimens. Different isolates of organisms were used from those used in analytical testing when possible. Samples positive for one analyte served as negatives for other analytes.

For the majority of analytes reported qualitatively, at least 25 of the contrived positive specimens had analyte concentrations at 2 x the limit of detection (LoD), while the remaining specimens were tested at additional concentrations that spanned clinically observed ranges. The "clinically observed range" was based on data from previous FilmArray Pneumonia Panel plus positive test results (e.g. observations from the prospective or archived studies). If a clinically observed range could not be determined for a particular analyte, specimens were spiked at various factors of LoD. If the stock concentration of organism did not allow for spiking at the highest level, the highest achievable level was used. For bacteria reported with binned values, specimens were spiked at various concentrations starting just below and then spanning the reported levels (i.e. 10^3 to ≥10^7 copies per milliliter (mL)).

Specimens were prepared and randomized at BioFire such that the analyte status of each contrived specimen was unknown to the users performing the testing. BAL and sputum specimens were analyzed separately; however, the preparation and testing for both matrices were identical. Contrived specimens were frozen, then distributed to prospective study sites and tested according to the prospective clinical study protocol alongside clinical (non-contrived) specimens.

The positive percent agreement (PPA) and negative percent (NPA) for the FilmArray Pneumonia Panel plus assays were determined using standard binomial sampling statistics. In this study, a success was defined as agreement between the known composition of the contrived specimen and the BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{36}------------------------------------------------

FilmArray Pneumonia Panel plus result; i.e., a positive FilmArray Pneumonia Panel plus result for spiked samples (True Positive, TP) and a negative FilmArray Pneumonia Panel plus result for un-spiked samples (True Negative, TN).

The results of the 1225 specimens tested in this study are summarized in Table 41 for BAL and Table 42 for sputum below.

The majority of analytes in both specimen types met the performance goals of 90% PPA with an 80% lower bound of the 95% Cl and 98% NPA with a 95% lower bound of the 95% CI. The exceptions being influenza A spiked into BAL and Klebsiella aerogenes spiked into BAL and sputum. Influenza A spiked in BAL demonstrated 86% PPA in part due to two missed detections at 0.2 × LoD and two additional missed detections at 2 × LoD from a strain that may have been under-quantified. However, FilmArray performance goals for influenza A in BAL were achived study. Klebsiella aerogenes spiked in both sample types demonstrated 85.5% PPA in part due to five missed detections in BAL and four missed detections in sputum at a range of concentrations from an Klebsiella aerogenes strain that demonstrated poor reactivity with the FilmArray Pneumonia Panel plus.

Samples spiked with bacterial analytes just below the 10°4 copies/mL binned value (i.e., near 1.00E+03), and samples spiked with other analytes below their LoD (i.e., near 0.2 × LoD), produced the expected unreliable detection.

	Sensitivity/PPA			Specificity/NPA
Analyte	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Middle East Respiratory Syndrome Coronavirus	50/50	100	92.9-100%	604/604	100	99.4-100%
Bacteria
Acinetobacter calcoaceticus-baumannii complex	47/50	94.0	83.8-97.9%	598/598	100	99.4-100%
Klebsiella aerogenesa	47/55	85.5	73.8-92.4%	592/594	99.7	98.8-99.9%
Klebsiella oxytoca	46/50	92.0	81.2-96.8%	604/604	100	99.4-100%
Proteus spp.	48/50	96.0	86.5-98.9%	603/603	100	99.4-100%
Serratia marcescens	49/50	98.0	89.5-99.6%	604/604	100	99.4-100%
Streptococcus pyogenes	49/50	98.0	89.5-99.6%	597/597	100	99.4-100%
Atypical Bacteria
Chlamydia pneumoniae	47/50	94.0	83.8-97.9%	604/604	100	99.4-100%
Legionella pneumophila	50/50	100	92.9-100%	599/599	100	99.4-100%
Mycoplasma pneumoniae	48/50	96.0	86.5-98.9%	603/604	99.8	99.1-100%
Viruses
Adenovirus	50/53	94.3	84.6-98.1%	568/569	99.8	99.0-100%
Human metapneumovirus	50/50	100	92.9-100%	597/598	99.8	99.1-100%
Influenza Ab	43/50	86.0	73.8-93.0%	585/585	100	99.3-100%
Influenza Bc	47/50	94.0	83.8-97.9%	588/589	99.8	99.0-100%
Antibiotic Resistance Markers
CTX-M	130/130	100	97.1-100%	323/324	99.7	98.3-100%
IMP	45/45	100	92.1-100%	412/412	100	99.1-100%
KPC	53/53	100	93.2-100%	400/400	100	99.1-100%
NDM	53/53	100	93.2-100%	404/404	100	99.1-100%

Table 41. FilmArray Pneumonia Panel plus Performance of Contrived BAL Specimens

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{37}------------------------------------------------

	Sensitivity/PPA			Specificity/NPA
Analyte	TP/(TP + FN)	%	95% Cl	TN/(TN + FP)	%	95% Cl
OXA-48 like	53/53	100	93.2-100%	307/307	100	98.8-100%
VIM	58/58	100	93.8-100%	399/399	100	99.0-100%

f Five FV specimens were spiked with a K. aerogenes) strain (ATCC, 29751) that demonstrated poor readinity with the FilmAray Pneumonia Panel plus (see Table 47).

b Two FN specimens were spiked with an influenza A strain that may have been under-quantified

· Three FN specimens were spiked with an influenza B strain that may have been under-quantified

Table 42. FilmArray Pneumonia Panel plus Performance of Contrived Sputum Specimens

	Sensitivity/PPA			Specificity/NPA
Analyte	TP/(TP + FN)	%	95% CI	TN/(TN + FP)	%	95% CI
Middle East Respiratory Syndrome Coronavirus	49/49	100	92.7-100%	521/521	100	99.3-100%
	Bacteria
Klebsiella aerogenesa	47/55	85.5	73.8-92.4%	513/513	100	99.3-100%
Streptococcus pyogenes	48/50	96.0	86.5-98.9%	516/516	100	99.3-100%
	Atypical Bacteria
Chlamydia pneumoniae	49/50	98.0	89.5-99.6%	521/521	100	99.3-100%
Legionella pneumophila	50/50	100	92.9-100%	521/521	100	99.3-100%
Mycoplasma pneumoniae	48/50	96.0	86.5-98.9%	521/521	100	99.3-100%
	Viruses
Adenovirus	50/52	96.2	87.0-98.9%	494/494	100	99.2-100%
Human metapneumovirus	51/51	100	93.0-100%	520/520	100	99.3-100%
Influenza Ab	47/50	94.0	83.8-97.9%	517/521	99.2	98.0-99.7%
Influenza Bº	48/51	94.1	84.1-98%	516/517	99.8	98.9-100%
Antibiotic Resistance Markers
CTX-M	121/122	99.2	95.6-99.9%	289/290	99.7	98.1-99.9%
IMP	43/44	97.7	88.2-99.6%	381/381	100	99.0-100%
KPC	54/54	100	93.4-100%	360/361	99.7	98.4-100%
NDM	53/53	100	93.2-100%	372/372	100	99.0-100%
OXA-48 like	51/51	100	93.0-100%	232/232	100	99.0-100%
VIM	56/56	100	93.6-100%	369/369	100	99.0-100%

^ Four FN specimens were spiked with a K. aerogenes) strain (ATCC, 29751) that demonstrated poor reactivity with the Film-ray Pneumonia Panel plus (see Table 47).

b Two FN specimens were spiked with an influenza A strain that may have been under-quantified

· Two FN specimens were spiked with an influenza B strain that may have been under-quantified

Testing of Polymicrobial Contrived Specimens

Additionally, two sets of individual BAL (N=60) and sputum (N=60) specimens were multi-spiked with randomized low, medium and high relative concentrations of either A. baumannii, E. cloacae, and E. coli or K. oxytoca, P. mirabilis, and S. marcescens. As shown in Table 44, the majority of the spiked organisms were reported at the expected relative low, medium, or high bin level by the FilmArray Pneumonia Panel plus. In four BAL specimens, E. cloacae was intended to be spiked at a medium level but was reported in a in a high (≥10°7) bin. Also, one specimen spiked with a high level of P. mirabilis was not detected (i.e. a false negative result for P. mirabilis).

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Organism Spiked Into Sputum
Spike Level		FilmArray Result		Total
		Low	Medium	High
	Low(104 copies/mL)	60	0	0	60
	Medium(105.5 copies/mL)	0	60	0	60
	High(107 copies/mL)	0	0	60	60

Table 43. Polymicrobial Sputum Specimen Results

Table 44. Polymicrobial BAL Specimen Results

Organism Spiked Into BAL
	FilmArray Result
Spike Level	Low	Medium	High	Total
Low(104 copies/mL)	60	0	0	60
Medium(105.5 copies/mL)	0	56	4a	60
High(107 copies/mL)	0	0	59	59b

ªQuantified at the bin boundary and reported as>=10^7 bOne false negative result

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Selected Analytical Studies

Limit of Detection

A limit of detection (LoD) was established for atypical bacteria and viruses detected by the FilmArray Pneumonia Panel plus. LoD was estimated by testing dilutions of contrived BAL or sputum samples containing known concentrations of organisms. Confirmation of LoD was achieved by testing at least 20 replicates per samples type on FilmArray 2.0 and FilmArray Torch systems (60 replicates total per sample type). LoD concentration was confirmed when the analyte was detected in at least 95% of the replicates tested.

The confirmed LoD for each atypical bacterium or virus (including a LoD for more than one isolate of the more genetically diverse viruses) is listed in Table 45. LoD concentration is based on quantification of each culture in viable units (TCIDso/mL or CFU/mL) and a corresponding molecular LoD concentration (DNA or RNA copies/mL) is provided based on quantitative real-time or digital PCR.

Analyte	IsolateStrain/Serotype/Source ID	LoD Concentrationa
		Viable Units	Molecular (DNA or RNA)
Atypical Bacteria
Chlamydia pneumoniae	TW183ATCC VR-2282	5.0E-01 TCID50/mLb	3.3E+02 copies/mLb
Legionella pneumophila	Philadelphia-1ATCC 33152	5.0E+02 CFU/mL	1.6E+03 copies/mL
Mycoplasma pneumoniae	M129Zeptometrix 0801579	7.5E+01 TCID50/mLb	3.5E+03 copies/mLb
Viruses
Middle East RespiratoryCoronavirus	EMC/2012BEI NR-50171(heat inactivated)	5.0E+01 TCID50/mLb	3.2E+03 copies/mLb
Adenovirus	Species A (A18)ATCC VR-19	5.0E+01 TCID50/mL	9.2E+03 copies/mL
	Species B (B3)Zeptometrix 0810062CF	1.0E+00 TCID50/mL	1.8E+03 copies/mL
	Species C (C2)ATCC VR-846	5.0E+00 TCID50/mL	7.5E+03 copies/mL
	Species D (D37)Zeptometrix 0810119CF	2.5E-01 TCID50/mLb	2.9E+03 copies/mLb
	Species E (E4)Zeptometrix 0810070CF	1.0E-01 TCID50/mLb	3.5E+04 copies/mLb
	Species F (F41)ATCC VR-930	5.0E+00 TCID50/mL	5.5E+03 copies/mL
	229EATCC VR-740	5.0E-01 TCID50/mL	8.1E+01 copies/mL
Coronavirus	HKU1Clinical Specimend	-	1.0E+04 copies/mL
	NL63BEI NR-470	2.5E+00 TCID50/mLc	5.4E+02 copies/mLc
	OC43ATCC VR-759	5.0E+02 TCID50/mLc	9.3E+03 copies/mLc
Human Metapneumovirus	16 Type A1Zeptometrix 0810161CF	5.0E+01 TCID50/mL	5.9E+03 copies/mL
HumanRhinovirus/Enterovirus	RhinovirusType 1AZeptometrix 810012CFN	1.5E+01 TCID50/mLb	6.6E+03 copies/mLb
	Echovirus 6Zeptometrix 0810076CF	1.0E+02 TCID50/mL	5.7E+02 copies/mL

Table 45. Summary of Limit of Detection (LoD) for FilmArray Pneumonia plus Atypical Bacteria and Viruses

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Analyte	IsolateStrain/Serotype/Source ID	LoD Concentrationa
		Viable Units	Molecular (DNA or RNA)
Influenza A	H1N1pdm09A/SwineNY/03/09Zeptometrix 0810249CF	2.5E+00 TCID50/mLc	1.7E+03 copies/mLc
Influenza A	H3N2A/Port Chalmers/1/73ATCC VR-810	1.0E+00 TCID50/mLb	2.1E+02 copies/mLb
Influenza B	B/FL/04/06Zeptometrix 0810255CF	5.0E+00 TCID50/mLc	4.2E+02 copies/mLc
Parainfluenza Virus	Type 1Zeptometrix 0810014CF	2.5E+01 TCID50/mL	5.2E+03 copies/mL
	Type 2Zeptometrix 0810015CF	2.5E+01 TCID50/mLc	1.5E+03 copies/mLc
	Type 3Zeptometrix 0810016CF	2.5E+01 TCID50/mLc	3.8E+02 copies/mLc
	Type 4AZeptometrix 0810060CF	2.5E+02 TCID50/mL	8.1E+03 copies/mL
Respiratory SyncytialVirus	Type AZeptometrix 0810040ACF	1.0E+00 TCID50/mL	4.3E+02 copies/mL

4 The listed concentration was confirmed with ≥95% detection on each FilmArray system in artificial BAL (aBAL) and/or sputum.

b LoD confirmation (≥95% detection) was achieved at a 2 to 5-fold lower concentration in aBAL.

· LoD confirmation (≥95% detection) was achieved at a 2 to 5-fold lower concentration in sputum.

d No cultured isolates of Coronavirus HKU1 were available for testing.

Note: LoD concentrations of the cultured viruses and the obligate intracellular atypical bacteria (C. pneumoniae and M. pneumoniae) are provided in units of TCID50 (50% Tissue Culture Infectious Dose). TCIDso is an indirect measure of viral or bacterial concentration based on infectivity and cytotoxicity and will therefore vary considerably depending on technique and methodology (including cell type, culture media and conditions, cytotoxicity of the virus, etc.). It is not appropriate to make determinations on relative sensitivity of different molecular assays for detection of viruses and bacteria based on LoD values measured in TCIDso/mL. Concentrations are also presented in copies/mL based upon independent quantitative PCR assays (qPCR) or digital PCR. Note that the accuracy of qPCR assays may also be affected by assay conditions, the standard reference material, and sequence variance between strains.

No assay-specific LoD concentrations were determined for the bacterial analytes. For bacteria, the FilmArray Pneumonia Panel plus reports a Detected result when the estimated bacterial nucleic acid abundance is >10^3.5 copies/mL, and the panel reports a Not Detected result if there is no amplification or the estimated bacterial nucleic acid abundance is <10^3.5 copies/mL. Each assay was determined to be linear in relation to input concentration (slope ~ 1.0 and coefficient of determination (Adj R2) >0.95) and estimates of nucleic acid abundance and corresponding bin results were determined to be accurate within 0.5-logio copies/mL when compared to a copies/mL input concentration determined by digital PCR.

Antimicrobial resistance (AMR) genes are reported as Detected when an applicable bacterium is detected and the assay for the AMR gene is positive. No AMR gene assay-specific LoD concentrations were determined for the AMR gene, but positive AMR gene assay results were recorded in ≥95% of 90 replicates of applicable bacteria tested at concentrations of 1.0E+04 copies/mL or less in the precision evaluation (see Precision below).

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Analytical Reactivity (Inclusivity) for MERS-CoV

Due to limited availability of well-characterized MERS-CoV strains, empirical testing of MERS-CoV strains for assessment of analytical reactivity was also limited (one cultured MERS-CoV strain tested in the LoD study, an external quality assessment (EQA) panel from Quality Control Molecular Diagnostics (QCMD), and seventeen MERS-CoV positive clinical specimens collected and archived from the 2015 outbreak in South Korea).

Organism	Strain/Location/Year	Source	Test Concentration		Result
			(copies/mL)	xLoD
Middle EastRespiratorySyndromeCoronavirus	EMC/2012	BEI NR-50171a	3.2E+03	1x	Middle EastRespiratorySyndromeCoronavirus(MERS-CoV)Detected
	unknown	Quality Control Molecular Diagnostics (QCMD)2017 MERS-CoV External Quality Assessment (EQA)	variousb
	South Korea 2015	7 clinical BAL specimens10 clinical sputum specimens	unknown

Table 46. Middle East Respiratory Syndrome Coronavirus Isolates Tested and Detected

ª Organism obtained through BEI Resources, NIAD, NH: Middle East Respiratory (MERS-CoV), EMC/2012, Heat-Inactivated, NR-50171. · FilmArray Pneumonia Panel plus results for MERS-CoV and other coronaviruses was concordant with the indicated.

Analytical reactivity of the FilmArray Pneumonia Panel plus MERS-CoV assays was further assessed by conducting in silico analyses of all publicly available virus sequences (as of March 2018) of human and camel host origin.

Based on an in silico analysis of 230 publicly available MERS-CoV sequences of human host that align with the MERS1 (M gene) assay primers and 221 publicly available MERS-CoV sequences of human host that align with the MERS2 (E gene) assay primers, there is no evidence of sequence variants that would contribute to altered or impaired reactivity with the MERS1 assay, and only two sequences with a >400 bp deletion between ORF5 and the E protein that could prevent reactivity with the MERS2 assay. The two variant sequences were obtained from a specimen taken from a single patient, and wild-type virus was also identified in this patient1.

FilmArray Pneumonia Panel plus MERS1 and MERS2 assay primer sequences were also aligned with 240 and 241 (respectively) publicly available MERS-CoV sequences from camels (the suspected animal host reservoir for the virus). This analysis revealed only five sequences under the MERS1 assay primers (5/240) and one sequence under the MERS2 assay primers (1/241) with a single base mismatch near the 3' end of a primer that could moderately impair reactivity and detection at low viral concentrations.

Analytical Reactivity (Inclusivity) for Other Analytes

Analytical reactivity of FilmArray Pneumonia Panel plus assays was evaluated via a combination of empirical (wet) testing and in silico analysis of sequences available in public databases. Testing was performed on a collection of more than 350 genetically diverse viruses, bacteria, and antimicrobial resistance genes. The tested isolates representing relevant species, strains, serotypes, or genotypes as well as temporal and geographic diversity for each of the panel analytes. Each isolate was tested in triplicate at concentrations near LoD or the lowest reportable level for the analyte. In silico analyses of sequence data from was also used to make predictions of assay reactivity for less common strains or serotypes and AMR gene types that were not tested but that may be detected by the FilmArray Pneumonia Panel plus assays.

Atypical bacteria and viruses were tested and detected at concentrations within 3× LoD (Table 48 -Table 58). Bacteria were tested at a concentration of 1.0E+04 copies/mL (based on digital PCR of a single-copy BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{42}------------------------------------------------

gene in the bacterial chromosome) and the majority of isolates (94.4%) were detected with the expected bin result (Table 59 - Table 73) and when the bacterium was detected, the appropriate associated AMR gene(s) were also detected (Table 74 - Table 81).

Limitations on assay reactivity (based on wet testing observations) with specific viral and bacterial isolates or sequences and AMR gene types or sequences are noted in Table 47. Most limitations are associated with single-base sequence variants under one or more assay primers. Additional predicted limitations on reactivity based on sequence analysis are provided in the analyte-specific tables below.

Note: FilmArray Pneumonia Panel plus Influenza A and Influenza B assays are predicted to react with attenuated viruses used in vaccines.

Table 47. Limitations on Analytical Reactivity of FilmArray Pneumonia Panel plus Assays
Limitation	Observed/PredictedResulta	Analyte	Strain/Isolate/Variant
Minor	Detectedmay be under-reported byone bin (≤10-fold)	Enterobacter cloacae complex	Enterobacter hormaechei(ATCC 49162)a
	Klebsiella pneumoniae group	Klebsiella quasipneumoniae subsp.quasipneumoniae(DSM 28211)b
	Moraxella catarrhalis	Moraxella catarrhalis(ATCC 23246)c
	Streptococcus pyogenes	Streptococcus pyogenes(ATCC 19615)
Major	Detectedmay be under-reported bytwo or more bins (>10-fold)	Enterobacter cloacae complex	Enterobacter asburiae(ATCC 35953, 35954, 35955, and 35957)e
	Klebsiella aerogenes	Klebsiella (Enterobacter) aerogenes(ATCC 29751)d
	mecA/C and MREJ	MREJ type xv
	Acinetobacter calcoaceticus-baumannii complex	Acinetobacter nosocomialis(ATCC 700472)f
	Not Detected	Pseudomonas aeruginosa	Pseudomonas aeruginosa(ATCC 25619)g
	mecA/C and MREJ	MREJ type xviiiMREJ type xix

Table 47. Limitations on Analytical Reactivity of FilmArray Pneumonia Panel plus Assays

1 Minor limitation observed for this isotate variant under a primer. Similar limitation predicted for two E. hornaecher sequences (3.0%) from public databases

Minor limitation observed for this isolae due to sequence variant unter a primer. Smilar limitation predicted for one K. quasipeumoriae sequence (20.0%) from public databases. Additional minor or major limitation predicted for four K. pneumoniae sequences (0.3%) from public databases.

^ Minor limitation observed for this isolate. Additional mitation predicted for two M. catarrhalis sequences (3.3%) from public databases.

1 Major limitation observed or predicted for these is a requence variance under primer. Similar limitation predicted for fine E. asburiae sequences (10.9%), eight E. cloacae sequences (2.2%), and two E. ludwigii sequences (16.7%) from public databases.

® Major limitation observed for this isolate variance under primers. Similar limitation predicted for six K. aerogenes sequences (4.3%) from public databases

f ATCC 700472 could not be confirmed as A. nosoconialis by sequence and may be a non-Acinetobacter calcaes that has been mis-identified.

9 Major limitation observed for this isolate variant under a primer. Similar limitation predicted for one P. aeruginosa sequence (0.7%) from public databases.

Table 48. Adenovirus Isolates Tested and Detected

Organism	Species	Serotypea	Source [Strain/Location/Year]	Test Concentration		Result
Adenovirus	A	18	ATCC VR-19 [Washington D.C./1954]	9.2E+03	1x	AdenovirusDetected
		12	ATCC VR-863 [Huie/Massachusetts]	2.7E+04	3x
		31	Zeptometrix 0810073CF	2.7E+04	3x
	B	3	Zeptometrix 0810062CF	1.8E+03	1x
		7	ATCC VR-7 [Gomen/California/1954]	5.3E+03	3x
		7A	Zeptometrix 0810021CF	5.3E+03	3x
		7d/d2	Univ of Iowa Research Foundation [Iowa/2001]	5.3E+03	3x
		7h	Univ of Iowa Research Foundation [Iowa/1999]	5.3E+03	3x
		11	ATCC VR-12 [Slobitski/Massachusetts]	5.3E+03	3x
		14	ATCC VR-15 [De Wit/Netherlands/1955]	5.3E+03	3x

{43}------------------------------------------------

Organismm	Species	Serotypea	Source [Strain/Location/Year]	Test Concentration
		16	ATCC VR-17 [CH.79/Saudi Arabia/1955]	5.3E+03	3x
		21	ATCC VR-1833 [128/Saudi Arabia/1956]	5.3E+03	3x
		34	ATCC VR-716 [Compton/1972]	5.3E+03	3x
		35	ATCC VR-718 [Holden]	5.3E+03	3x
		50	ATCC VR-1602 [Wan/Amsterdam/1988]	5.3E+03	3x
		2	ATCC VR-846 [Adenoid 6]	7.5E+03	1x
C		1	Zeptometrix 0810050CF	2.3E+04	3x
		5	Zeptometrix 0810020CF	2.3E+04	3x
		6	ATCC VR-6 [Tonsil 99]	2.3E+04	3x
	D	37	Zeptometrix 08100119CF	5.8E+02	1x
	D	8	Zeptometrix 0810069CF	1.7E+03	3x
		20	Zeptometrix 0810115CF	1.7E+03	3x
	E	4	Zeptometrix 0810070CF	1.7E+04	1x
E		4	ATCC VR-1572 [RI-67/Missouri/1952-1953]	1.0E+04	0.6x
		4a	Univ of Iowa Research Foundation [S Carolina/2004]	1.0E+04	0.6x
		41	ATCC VR-930 [Tak 73-3544/ Netherlands/1973]	5.5E+03	1x
	F	40	NCPV 0101141v	1.6E+04	3x
		40	Zeptometrix 0810084CF	1.6E+04	3x
		41	Zeptometrix 0810085CF	1.6E+04	3x

4 In silico analysis of available sequences preumonia Panel plus will also react with Adenovirus B55, C57, all species D serotypes, and G52.

		Table 49. Coronavirus Isolates Tested and Detected
--	--	----------------------------------------------------	--	--	--	--

Organism	Type	Source [Location/Year]	Test Concentration		Result
			(copies/mL)	xLoD
Coronavirus	229E	ATCC VR-740	8.1E+01	1x	CoronavirusDetected
	229E	Zeptometrix 0810229CF	2.4E+02	3x
	HKU1a	Clinical Specimen [Utah/2015]	1.0E+04	1x
	HKU1a	Clinical Specimen [Detroit/2010]	3.0E+04	3x
	HKU1a	Clinical Specimen [Utah/2015]	3.0E+04	3x
	HKU1a	Clinical Specimen [Utah/2015]	3.0E+04	3x
	HKU1a	Clinical Specimen [S Carolina/2010]	3.0E+04	3x
	NL63	BEI NR-470b [Amsterdam/2003]	2.7E+02	1x
	NL63	Zeptometrix 0810228CF	8.0E+02	3x
	OC43	ATCC VR-759c	4.6E+03	1x
OC43	Zeptometrix 0810024CF	1.4E+04	3x

ª No cultured isolates of Coronavirus HKU1 were available Specimens containing Coronavirus HKU1 were collected from different regions of the US in 2010 and 2015, quantified molecularly, and tested.

Organism obtained through the NIH Biodefense and Emerging Infections Resources Repository, NIAD, NH: Human Coronavirus NL63, NR-470. © Discontinued part #. See ATCC VR-1558.

Table 50. Human Metapneumovirus Isolates Tested and Detected

Organism	Genotype	Serotype	Source [Location/Year]	Test Concentration		Result
				(TCID50/mL)	xLoD
HumanMetapneumovirus	A1	16	Zeptometrix 0810161CF [Iowa10/2003]	5.0E+01	1x	HumanMetapneumovirusDetected
		9	Zeptometrix 0810160CF [Iowa3/2002]	1.5E+02	3x
	A2	20	Zeptometrix 0810163CF [Iowa14/2003]	1.5E+02	3x
		27	Zeptometrix 0810164CF [Iowa27/2004]	1.5E+02	3x
	B1	3	Zeptometrix0810156CF [Peru2/2002]	1.5E+02	3x
		5	Zeptometrix 0810158CF [Peru3/2003]	1.5E+02	3x
	B2	8	Zeptometrix 0810159CF [Peru6/2003]	1.5E+02	3x
		4	Zeptometrix 0810157CF [Peru1/2002]	1.5E+02	3x
		18	Zeptometrix 0810162CF [Iowa18/2003]	1.0E+02	3x

Table 51. Human Rhinovirus and Enterovirus Isolates Tested and Detected

Species	Serotype	Source [Strain/Location/Year]	Test Concentration		Result
Human Rhinovirusa
A	1	Zeptometrix 0810012CFN [1A]	2.2E+03	1x	HumanRhinovirus/Enterovirus
	2	ATCC VR-482 [HGP]	1.7E+03	3x
	7	ATCC VR-1601 [68-CV11]	1.7E+03	3x
	16	ATCC VR-283 [11757/Washington DC/1960]	1.7E+03	3x
	34	ATCC VR-507b [137-3]	1.7E+03	3x	Detected
	57	ATCC VR-1600 [Ch47]	1.7E+03	3x
	77	ATCC VR-1187 [130-63]	1.7E+03	3x

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{44}------------------------------------------------

Species	Serotype	Source [Strain/Location/Year]	Test Concentration(copies/mL)	xLoD	Result
B	85	ATCC VR-1195 [50-525-CV54]	1.7E+03	3x
B	3	ATCC VR-483 [FEB]	1.7E+03	3x
B	14	ATCC VR-284 [1059/S Carolina/1959]	1.7E+03	3x
B	17	ATCC VR-1663 [33342/N Carolina/1959]	1.7E+03	3x
B	27	ATCC VR-1137 [5870]	1.7E+03	3x
B	42	ATCC VR-338 [56822]	1.7E+03	3x
B	83	ATCC VR-1193 [Baylor 7]	1.7E+03	3x
Enterovirus
A	Coxsackievirus 10	ATCC VR-168 [NY/1950]	1.7E+03	3x	HumanRhinovirus/EnterovirusDetected
A	Enterovirus 71	ATCC VR-1432 [H]	1.7E+03	3x
B	Coxsackievirus A9	Zeptometrix 0810017CF	1.7E+03	3x
B	Coxsackievirus B3	Zeptometrix 0810074CF	1.7E+03	3x
B	Coxsackievirus B4	Zeptometrix 0810075CF	1.7E+03	3x
B	Echovirus 6	Zeptometrix 0810076CF	5.7E+02	1x
B	Echovirus 9	Zeptometrix 0810077CF	1.7E+03	3x
B	Echovirus 11	Zeptometrix 0810023CF	1.7E+03	3x
C	Coxsackievirus A21	ATCC VR-850 [Kuykendall/California/1952]	1.7E+03	3x
C	Coxsackievirus A24	ATCC VR-583 [DN-19/Texas/1963]	1.7E+03	3x
D	Enterovirus 68	ATCC VR-1823 [US/MO/2014-18947]	1.7E+03	3x

ª The concentration used for Human Rhinovirus isolate testing was based on 3x the Enterovirus LoD concentration (5.7E+02 copies/mL).

b Discontinued part #; see ATCC VR-1365.

Table 52. Influenza A Isolates Tested and Detected

Organism	Subtype	Source [Strain/Location/Year]	Test Concentration (copies/mL)	xLoD	Result
Influenza A	Human
	H1N1	ATCC VR-219 [NWS/1933]	3.1E+02	3x
		ATCC VR-95 [PR/8/1934a]	1.0E+03	1.5xa
		ATCC VR-96 [Wiess/1943]	3.1E+02	3x
		ATCC VR-97 [FM/1/1947]	3.1E+02	3x
		ATCC VR-98 [Mal/302/1954]	3.1E+02	3x
		ATCC VR-546 [Denver/1/1957]	3.1E+02	3x
		Zeptometrix 0810036CF [New Caledonia/20/1999]	3.1E+02	3x
		Zeptometrix 0810036CFN [Solomon Islands/3/2006]	3.1E+02	3x
		Zeptometrix 0810244CF [Brisbane/59/2007]	3.1E+02	3x	Influenza A Detected
	H1N2	BEI NR-3478b [Kilbourne F63 A/NWS/1934 (HA) xA/Rockefeller Institute/5/1957 (NA)]	3.1E+02	3x
		Zeptometrix 0810249CF [SwineNY/03/2009]	6.6E+02	1x
		Zeptometrix 0810109CFJ [Canada/6294/2009]	3.1E+02	3x
H1N1pdm09	Zeptometrix 0810165CF [California/07/2009]	3.1E+02	3x
		Zeptometrix 0810166CF [Mexico/4108/2009]	3.1E+02	3x
		BEI NR-19823c [Netherlands/2629/2009]	3.1E+02	3x
		BEI NR-42938d [Georgia/F32551/2012]	3.1E+02	3x
		BEI NR-44345e [Hong Kong/H090-761-V1(0)/2009]	1.0E+03	1.5xa
H3N2	ATCC VR-810 [Port Chalmers/1/1973]	1.0E+02	1x
		ATCC VR-776 [Alice (live attenuated vaccine)]	3.1E+02	3x
		Zeptometrix 0810238CF [Texas /50/2012]	3.1E+02	3x
		ATCC VR-547 [Aichi/2/1968]	3.1E+02	3x
		ATCC VR-544 [Hong Kong/8/1968]	3.1E+02	3x
		ATCC VR-822 [Victoria/3/1975]	3.1E+02	3x
		Zeptometrix 0810252CF [Wisconsin/67/2005]	3.1E+02	3x
		Zeptometrix 0810138CF [Brisbane/10/2007]	3.1E+02	3x
H3N2v	ODHL1 [Ohio/2012]	3.1E+02	3x
Avian
H2N2	BEI NR-2775f [Japan/305/1957]	3.1E+02	3x
H2N3	MRIGlobalg [Mallard/Alberta/79/2003]	3.1E+02	3x
H5N1	MRIGlobalg [Chicken/Yunnan/1251/2003]	3.1E+02	3x
H5N2	MRIGlobalg [Northern pintail/Washinton/40964/2014]	3.1E+02	3x
H5N3	BEI NR-9682h [Duck/Singapore/645/97]	3.1E+02	3x
H5N8	MRIGlobalg [Gyrfalcon/Washing-ton/41088-6/2014]	3.1E+02	3x
H7N7	MRIGlobalg [Netherlands/219/2003]	3.1E+02	3x
H7N9	MRIGlobalg [Anhui/01/2013]	3.1E+02	3x
H10N7	BEI NR-2765i [Chicken/Germany/N/49]	3.1E+02	3x

{45}------------------------------------------------

Organism	Subtype	Source [Strain/Location/Year]	Test Concentration(copies/mL)	xLoD	Result
	H1N1	ATCC VR-333 [Swine/Iowa/15/1930]	3.1E+02	3x
	Swine	ATCC VR-99 [Swine/1976/1931]	3.1E+02	3x

ª 1.5x the LoD for Influenza A H1N1pdm09 Zeptometrix 0810109CFN [SwineNY/03/2009] (6.6E+02 copies/mL).

Genomic RNA obtained through BEI Resources NAID, NH: Kilbourne F63: ANWS/1934 (HA) x ARockefeller Institute/5/1957 (NA) (H1N2), Reasortant NIVS-F, NR-9677.

് Virus obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/Netherlands/2629/2009 (H1N1)pdm09, NR-19823

d Virus obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/Georgia/F32551/2012 (H1N1)pdm09, NR-42938.

® Virus obtained through BEI Resources, NIAID, NIH: Influenza A Virus, A/Hong Kong/H090-761-V1(0)/2009 (H1N1)pdm09, NR-44345.

f Genomic RNA obtained through BEI Resources, NIAD, NH: Genomic RNA from Influenza A Virus, AJapan/305/1957 (H2N2), NR-2775.

9 Isolate provided and tested by MRI Global, Kansas City, MO.

Genomic RNA obtained through BEI Resources, NIAD, NH: Genomic RNA from Influenza A Virus, Alduck/Singapore/645/1997 (H5N3), Wild Type, NR-9682. | Genomic RNA obtained through BEI Resources, NIAD, NH: Genomic RNA from Influenza A Virus, A/chicker/Germany/N/1949 (H10N7), NR-2765.

Table 53. Influenza B Isolates Tested and Detected

Organism	Lineage	[Strain/Location/Year], Source	Test Concentration(copies/mL)	xLoD	ReportedResult
Influenza B	N/A	ATCC VR-101 [Lee/1940]	6.3E+02	3x	Influenza BDetected
	N/A	ATCC VR-102 [Allen/1945]	6.3E+02	3x
	N/A	ATCC VR-103 [GL/1739/1954]	6.3E+02	3x
	N/A	ATCC VR-296 [1/Maryland/1959]	6.3E+02	3x
	N/A	ATCC VR-295 [2/Taiwan/1962]	6.3E+02	3x
	N/A	ATCC VR-786 [Brigit/Russia/1969]	6.3E+02	3x
	Victoria	ATCC VR-823 [5/Hong Kong/1972]	6.3E+02	3x
	Victoria	Zeptometrix 0810258CF [2506/Malaysia/2004]	6.3E+02	3x
	Victoria	CDC 2005743348 [1/Ohio/2005]	6.3E+02	3x
	Yamagata	Zeptometrix 0810256CF [07/Florida/2004]	6.3E+02	3x
		Zeptometrix 0810255CF [04/Florida/2006]	2.1E+02	1x
		Zeptometrix 0810241CF [1/Wisconsin/2010]	6.3E+02	3x
		Zeptometrix 0810239CF [2/Massachusetts/2012]	6.3E+02	3x

Table 54. Parainfluenza Virus Isolates Tested and Detected

Organism	Type	Source [Strain/Location/Year]	Test Concentration(copies/mL)		Result
ParainfluenzaVirus	1	Zeptometrix 0810014CF	5.2E+03	1x	ParainfluenzaVirusDetected
	1	BEI NR-48680a [FRA/29221106/2009]	1.6E+04	3x
	1	ATCC VR-94 [C-35/Washington DC/1957]	1.6E+04	3x
	2	Zeptometrix 0810015CF	1.5E+03	1x
	2	ATCC VR-92 [Greer/Ohio/1955]	8.9E+02	0.6x
	3	Zeptometrix 0810016CF	1.9E+02	1x
	3	BEI NR-3233b [NIH 47885, Wash/47885/57]	5.7E+02	3x
	3	ATCC VR-93 [C-243/Washington DC/1957]	5.7E+02	3x
	4	Zeptometrix 0810060CF	8.1E+03	1x
	4	ATCC VR-1378 [M-25/1958]	2.4E+04	3x
	4	Zeptometrix 0810060BCFATCC VR-1377 [CH-19503/Washington DC/1962]	2.4E+04	3x

ª Virus obtained through BEI Resources, NIAID, NH: Human Parainfluenza Virus 1, HPV1/FRA/29221106/2009, NR-48660.

b Virus obtained through BEI Resources, NIAID, NIH: Human Parainfluenza Virus 3, NIH 47885, NR-3233.

Table 55. Respiratory Syncytial Virus Isolates Tested and Detected

Organism	Type	Source [Strain/Location/Year]	Test Concentration		Result
			(copies/mL)	xLoD
RespiratorySyncytial Virus	A	Zeptometrix 0810040ACF [2006]	4.3E+02	1x	RespiratorySyncytial VirusDetected
	A	ATCC VR-26 [Long/Maryland/1956]	1.3E+03	3x
	A	ATCC VR-1540 [A2/Melbourne/1961]	1.3E+03	3x
	B	Zeptometrix 0810040CF [Ch-93 (18)-18]	1.3E+03	3x
	B	ATCC VR-1400 [WV/14617/1985]	1.3E+03	3x
	B	ATCC VR-955 [9320/Massachusetts/1977]	1.3E+03	3x
	B	ATCC VR-1580 [18537/Washington DC/1962]	1.3E+03	3x

Table 56. Chlamydia pneumoniae Isolates Tested and Detected

Organism	Source [Strain]	Test Concentration		Result
		(copies/mL)	xLoD
	ATCC VR-2282 [TW-183/Taiwan/1965]	6.7E+01	1x

{46}------------------------------------------------

Organism	Source [Strain]	Test Concentration		Result
		(copies/mL)	xLoD
Chlamydiapneumoniae	ATCC VR-1310 [CWL-029]	2.0E+02	3x	Chlamydiapneumoniae
	ATCC VR-1360 [CM-1/Georgia]	2.0E+02	3x	Chlamydiapneumoniae
	ATCC 53592 [AR-39/Seattle/1983]	2.0E+02	3x	Detected

Table 57. Legionella pneumophila Isolates Tested and Detected

Species/Subspecies	Serogroup	Source [Strain]	Test Concentration		Result
			(CFU/mL)	xLoD
L. pneumophila	1	ATCC 33152 [Philadelphia-1]	5.0E+02	1x	LegionellapneumophilaDetected
L. pneumophila	3	ATCC 33155 [Bloomington-2]	1.5E+03	3x
L. pneumophila subsp. fraseri	4	ATCC 33156 [Los Angeles-1]	1.5E+03	3x
L. pneumophila subsp. pascullei	5	ATCC 33216 [Dallas 1E]	1.5E+03	3x
L. pneumophila subsp. pascullei	5	ATCC 33737 [U8W]	1.5E+03	3x
L. pneumophila subsp. pneumophila	10	ATCC 43283 [Leiden 1]	1.5E+03	3x
L. pneumophila subsp. pneumophila	14	ATCC 43703 [1169-MN-H]	1.5E+03	3x

Table 58. Mycoplasma pneumoniae Isolates Tested and Detected

Organism	Type	Source [Strain]	Test Concentration		Result
			(copies/mL)	xLoD
Mycoplasmaapneumoniae		Zeptometrix 0801579 [M129]	1.2E+03	1x	Mycoplasmapneumoniae
	1	ATCC 29342 [M129-B7]	3.5E+03	3x
		ATCC 29085 [PI 1428]	3.5E+03	3x
	2	ATCC 15531-TTR [FH strain of Eaton Agent [NCTC 10119]]	3.5E+03	3x
		ATCC 15492 [Mac]	3.5E+03	3x
		ATCC 15293 [M52]	3.5E+03	3x	Detected
	unknown	ATCC 15377 [Bru]	3.5E+03	3x
		ATCC 39505 [Mutant 22]	3.5E+03	3x
		ATCC 49894 [UTMB-10P]	3.5E+03	3x

Table 59. Acinetobacter calcoaceticus-baumannii complex Isolates Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
A. baumannii	ATCC 9955 [6-561]	1.0E+04
A. baumannii	ATCC 19606 [2208 Type strain]	1.0E+04
A. baumannii	ATCC 17961 [CDC 7788]	1.0E+04
A. baumannii	AR-Bank #0033	1.0E+04
A. baumannii	GRE 1153064	1.0E+04	Acinetobacter calcoaceticus-baumannii complex Detected
A. baumannii	GRE 1062081	1.0E+04
A. baumannii	ATCC 51432	1.0E+04
A. calcoaceticus	ATCC 23055 [46]	1.0E+04
A. calcoaceticus	ATCC 14987 [HO-1]	1.0E+04
A. calcoaceticus subsp. anitratus	ATCC 15308 [NCTC 7844]	1.0E+04
A. pittii	ATCC 19004 [57.071.228]	1.0E+04
A. nosocomialisa	ATCC 17903 [NCTC 8102]	1.0E+04
A.seifertii	CCUG 34785	1.0E+04

a A. nosocomialis ATCC 700472 was not detected at any concentration. Sequencing suggests the isolate may be mis-identified.

Table 60. Isolates Enterobacter cloacae Complex Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
E. cloacae	ATCC 49141 [AmMs 204]	1.0E+04	Enterobacter cloacaecomplex Detected
E. cloacae	ATCC BAA-1143 [Entb 55M]	1.0E+04
E. cloacae	ATCC BAA-2341 [1101152]	1.0E+04
E. cloacae	AR-Bank #0154	1.0E+04
E. cloacae	NCTC 13464	1.0E+04
E. cloacae subsp. cloacae	ATCC 13047 [Type Strain]	1.0E+04
E. cloacae subsp. dissolvens	ATCC 23373Da [ICPB ED105]	1.0E+04
E. asburiae	ATCC 35953b [CDC 1497-78 Type Strain ]	1.0E+06b
E. asburiae	ATCC 35957b [CDC 570-83 ]	1.0E+06b
E. hormaechei	ATCC 49162b [CDC 992-77]	1.0E+05b
E. hormaechei	ATCC BAA-2082	1.0E+04
E. kobei	CCUG 59410	1.0E+04
E. ludwigii	CCUG 23050	1.0E+04

ª Genomic DNA from E. cloacae subsp. dissolvens.

b See Table 47 for limitation.

{47}------------------------------------------------

Table 61. Escherichia coli Isolates and Cross-Reactive Species Tested and Detected
------------------------------------------------------------------------------------	--	--

Organism	Source [Strain]	Test concentration(copies/mL)	Reported Result
E. coli	ATCC 25922 [FDA strain Seattle 1946]	1.0E+04
	ATCC 43888 [CDC B568-73]	1.0E+04
	AR-Bank #0061	1.0E+04
	AR-Bank #0086	1.0E+04
	AR-Bank #0137	1.0E+04
	AR-Bank #0150	1.0E+04
	AR-Bank #0162	1.0E+04	Escherichia coliDetected
	GRE 1062016	1.0E+04
	GRE 1252008	1.0E+04
	GRE 1252009	1.0E+04
	GRE 1256018	1.0E+04
	Zeptometrix 0801905 [Z136]	1.0E+04
	ATCC 29930 [WRAIR I virulent]	1.0E+04

Table 62. Haemophilus influenzae Isolates Tested and Detected

Organism	Serotype	Source [Strain/Location/Year]	Test concentration(copies/mL)	Result
H. influenzaea	Type a	ATCC 9006 [AMC 36-A-3 [610, PCM 2436]]	1.0E+04	HaemophilusinfluenzaeDetected
	Type b	ATCC 10211 [AMC 36-A-1 [572]], Biotype 1	1.0E+04
	Type c	ATCC 49699 [C 9007]	1.0E+04
	Type d	ATCC 9008 [AMC 36-A-6 [611]]	1.0E+04
	Type e	ATCC 8142 [AMC 36-A-7 [595, NCTC 8472]]	1.0E+04
	Type f	ATCC 700223 [GA1264]	1.0E+04
	Biogroup aegyptius	ATCC 11116 [180-a [NCTC 8502]]	1.0E+04
	Non-typeable	ATCC 51907 [Rd [KW20]]	1.0E+04

NOTE: The Hinfluenzae assay will not react with strains that do not carry the hpd gene².

Table 63. Klebsiella (Enterobacter) aerogenes Isolates Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
K. aerogenes a	ATCC 13048 [NCTC 10006]	1.0E+04	Klebsiella aerogenesDetected
	AR-Bank #0062	1.0E+04
	AR-Bank #0074	1.0E+04
	AR-Bank #0161	1.0E+04
	GRE 1254066	1.0E+04
	ATCC 29751a [MULB-250]	1.0E+07b

ª Previously known as Enterobacter aerogenes

b See Table 47 for limitation.

Table 64. Klebsiella oxytoca Isolates Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
K. oxytoca	ATCC 13182 [479-2 Type strain]	1.0E+04	Klebsiella oxytocaDetected
	ATCC 43086 [Pasco 201]	1.0E+04
	ATCC 49131 [AmMS 101]	1.0E+04
	ATCC 700324 [LBM 90.11.033]	1.0E+04
	ATCC 8724 [NRRL B-199]	1.0E+04
	AR-Bank #0147	1.0E+04
	JMI 2523	1.0E+04
	JMI 2661	1.0E+04
	JMI 7818	1.0E+04
	JMI 10678	1.0E+04
	JMI 14611	1.0E+04
	GRE 1254054	1.0E+04

Table 65. Klebsiella pneumoniae Isolates Tested and Detected

Organism	Source [Strain]	Test concentration (copies/mL)	Result
K. pneumoniae	ATCC BAA-1705 [ART 2008133]	1.0E+04	KlebsiellapneumoniaeDetected
	AR-Bank #0068	1.0E+04
	AR-Bank #0075	1.0E+04
	AR-Bank #0076	1.0E+04
	AR-Bank #0079	1.0E+04
	AR-Bank #0080	1.0E+04
	AR-Bank # 0097	1.0E+04

BioFire Diagnostics 510(k) FilmArray Pneumonia Panel plus

{48}------------------------------------------------

Organism	Source [Strain]	Test concentration(copies/mL)
	AR-Bank #0107	1.0E+04
	AR-Bank #0153	1.0E+04
	GRE 1062084	1.0E+04
	GRE 1355030	1.0E+04
	JMI 328	1.0E+04
	JMI 766	1.0E+04
	NCTC 13465	1.0E+04
	Zeptometrix 0801886	1.0E+04
K. pneumoniae subsp. ozaenae	ATCC 11296 [AMC 35-E-5]	1.0E+04
K. pneumoniae subsp. pneumoniae	ATCC 13883 [NCTC 9633]	1.0E+04
K. pneumoniae subsp. rhinoscleromatis	ATCC 13884 [NCTC 5046]	1.0E+04
K. quasipneumoniae subsp. quasipneumoniae	DSM 28211a [01A030, SB11]	1.0E+05a
K. quasipneumoniae subsp. simipneumoniae	DSM 28212 [07A044, SB30]	1.0E+04
K. variicola	ATCC BAA-830 [F2R9]	1.0E+04

ª See Table 47 for limitation.

Table 66. Moraxella catarrhalis Isolates Tested and Detected

Organism	Source [Strain]	Test concentration (copies/mL)	Result
M. catarrhalis	ATCC 25238 [Ne 11]	1.0E+04	Moraxella catarrhalisDetected
	ATCC 25240 [N9]	1.0E+04
	ATCC 8176 [20]	1.0E+04
	ATCC 23246a [NCTC 4103]	1.0E+05a
	ATCC 49143 [Am MS 116]	1.0E+04

a See Table 47 for limitation.

Table 67. Proteus spp. Isolates Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
P. mirabilis	ATCC 29906 [1003]	1.0E+04
	ATCC 33583 [571101]	1.0E+04
	ATCC 35659 [LRA 08 01 73]	1.0E+04
	AR-Bank #0156	1.0E+04
	AR-Bank #0159	1.0E+04
	GRE 1254053	1.0E+04
P. hauseri	ATCC 13315 [NCTC 4175 Strain Lehmann]	1.0E+04	Proteus spp.Detected
P. hauseri	ATCC 700826 [CDC 1732-80]	1.0E+04
P. penneri	ATCC 33519 [Type Strain CDC 1808-73]	1.0E+04
P. penneri	ATCC 35197 [CDC 1655-67]	1.0E+04
P. vulgaris	ATCC 29905	1.0E+04
	ATCC 33420	1.0E+04
	ATCC 27973 [CDC 1787-64-SC1]	1.0E+04

Table 68. Pseudomonas aeruginosa Isolates Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
P. aeruginosa a	ATCC 10145 [ MDB strain BU 277 type strain]	1.0E+04	Pseudomonasaeruginosa Detected
	ATCC BAA-1744 [109246]	1.0E+04
	ATCC 19429 [NCTC 6750]	1.0E+04
	ATCC 27853 [Boston 41501]	1.0E+04
	AR-Bank #0054	1.0E+04
	AR-Bank #0092	1.0E+04
	AR-Bank #0100	1.0E+04
	AR-Bank #0103	1.0E+04
	AR-Bank #0111	1.0E+04
	Creighton University PS28	1.0E+04
	NCTC 13437	1.0E+04

ª P. aeruginosa ATCC 25619 was not detected at any concentration tested. See Table 47 for limitation.

Table 69. Serratia marcescens Isolates Tested and Detected
------------------------------------------------------------

Organism	Source [Strain]	Test concentration(copies/mL)	Result
S. marcescens	ATCC 13880 [Type strain]	1.0E+04	Serratia marcescens
S. marcescens	ATCC 27137 [CDC 3100-71]	1.0E+04	Detected

{49}------------------------------------------------

Organism	Source [Strain]	Test concentration(copies/mL)	Result
	ATCC 43297 [3G]	1.0E+04
	ATCC BAA-885 [Type strain KRED]	1.0E+04
	GRE 1659005	1.0E+04
	GRE 1659004	1.0E+04
	JMI 697	1.0E+04

Table 70. Staphylococcus aureus Isolates Tested and Detected
--------------------------------------------------------------

Organism	Source [Strain] (PFGE Type if applicable)	Test concentration(copies/mL)	Result
Staphylococcus aureus representing PFGE Types USA100-USA1200
S. aureus	NARSA NRS705 [PFGE USA100]	1.0E+04
	NARSA NRS701 [PFGE USA200]	1.0E+04
	ATCC BAA-1717 [PFGE USA300]	1.0E+05ª
	NARSA NRS683 [PFGE USA300]	1.0E+04
	NARSA NRS662 [PFGE USA300]	1.0E+04
	NARSA NRS707 [PFGE USA300]	1.0E+04
	ATCC BAA-1707 [PFGE USA400]	1.0E+04
	NARSA NRS691 [PFGE USA500]	1.0E+04
	NARSA NRS648 [PFGE USA600]	1.0E+04
	NARSA NRS689 [PFGE USA700]	1.0E+04
	NARSA NRS668 [PFGE USA800]	1.0E+04
	ATCC BAA-1749 [PFGE USA900 96:308]	1.0E+04
	ATCC BAA-1759 [PFGE USA900 N7129]	1.0E+04
	ATCC BAA-1700 [PFGE USA1000]	1.0E+04
	BEI NR-46081 [PFGE USA1100 HIP12899]	1.0E+04
	ATCC BAA-1765 [PFGE USA1200 102-04]	1.0E+04
	ATCC BAA-1691 [Not USA100-1100]	1.0E+04
Methicillin Sensitive Staphylococcus aureus (MSSA)
	ATCC 10832 [Wood 46]	1.0E+04
	ATCC 14154 [Rose]	1.0E+04
	ATCC 12600 [NCTC Type strain]	1.0E+04
	ATCC 25923 [Seattle/1945]	1.0E+04
	ATCC 29213 [Wichita]	1.0E+04
	ATCC BAA-2421 [Mass/2010]	1.0E+04
	Rennes 1060728	1.0E+04
	GRE 1062519 [SCCmec Type: III / MREJ xix]b	1.0E+04
Borderline Resistant Staphylococcus aureus (BORSA)			StaphylococcusaureusDetected
	SUN1 [Sunnybrook]	1.0E+04
	Methicillin Resistant Staphylococcus aureus (MRSA)
	ATCC 43300 [F182 Kansas / SCCmec Type: II]	1.0E+04
	ATCC BAA-2422 [Worcester MA/2010 / SCCmec Type: II]	1.0E+04
	ATCC BAA-1720 [MRSA252 / SCCmec Type: II / PFGE USA200]	1.0E+04
	NARSA NRS745 [CA-629 / SCCmec Type: V]	1.0E+04
	ATCC BAA-38 [E2125 / SCCmec Type: I]	1.0E+04
	NARSA NRS686 [MREJ type i]	1.0E+04
	ATCC BAA-44 [HPV107 / SCCmec Type: I / PFGE: Iberian]	1.0E+04
	ATCC BAA-41 [NYBK2464 / SCCmec Type: II / PFGE 100]	1.0E+04
	NARSA NRS385 [MREJ type ii]	1.0E+04
	ATCC BAA-42 [HDE288 / SCCmec: Type VI / PFGE 800]	1.0E+04
	ATCC BAA-39 [HUSA304 / SCCmec Type: III]	1.0E+04
	ATCC BAA-40 [CPS22 / SCCmec Type: III]	1.0E+04
	GRE 1062264 [SCCmec Type: IV / MREJ type iv]	1.0E+04
	GRE 1055015 [SCCmec Type: IVa / MREJ type vi]	1.0E+04
	GRE 0759084 [SCCmec Type: IV / MREJ type v]	1.0E+04
	GRE 0860042 [SCCmec Type: III / MREJ type vii]	1.0E+04
	GRE 1052034 [MREJ ix]	1.0E+04
	GRE 1151100 [SCCmec Type: IV / MREJ type xi]	1.0E+04
	GRE 0960006 [MREJ type xii]	1.0E+04
	GRE 1055017 [SCCmec Type: IVa / MREJ type xiii]	1.0E+04
	GRE 0759163 [MREJ type xiv]	1.0E+04
	GRE 1062373 [MREJ type xv]	1.0E+04
	GRE 1057114 [MREJ type xvii]	1.0E+04
	GRE 1062292 [MREJ type xviii]	1.0E+04
	Methicillin Resistant Staphylococcus aureus (MRSA) - mecC+
	ATCC BAA-2313 [M10/0148 / SCCmec Type: XI / mecC ]	ATCC BAA-2312 [M10/0061 / SCCmec Type: XI / mecC]	1.0E+04	1.0E+04

{50}------------------------------------------------

³ Staphylococus aureus ATCC BAA-1717 was not detected at 1.0E+05 copies/mL with acurate bin results. No limitation on reactivity could be identified based on the isolate sequence.

b MREJ type xix characterized as MSSA.3

Table 71. Streptococcus agalactiae Isolates Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
S. agalactiae	NCTC 8017 [MK 104 P]	1.0E+04	Streptococcus agalactiae Detected
	ATCC 13813 [la/c Type Strain]	1.0E+04
	ATCC 12403 [III Typing Strain D136C]	1.0E+04
	ATCC 12386 [Grouping strain O90R]	1.0E+04
	ATCC BAA-611 [V 2603 V/R]	1.0E+04
	ATCC BAA-2669 [VIII 5030-08]	1.0E+04
	Clinical Isolate [Utah/2010/Cl03]	1.0E+04

Table 72. Streptococcus pneumoniae Isolates Tested and Detected

Organism	Serotype	Source [Strain]	Test concentration(copies/mL)	Result
S. pneumoniae	3	ATCC 6303	1.0E+04	Streptococcus pneumoniaeDetected
	5	ATCC BAA-341 [SPN1439-106]	1.0E+04
	11A	NCTC 11900 [Gorman]	1.0E+04
	14	ATCC 700672 [VH14]	1.0E+04
	19A	ATCC 700673 [Hungary 19A-6]	1.0E+04
	Non-capsulated	ATCC BAA-255 [R6]	1.0E+04
	unknown	ATCC BAA-1409 [62076]	1.0E+04

Table 73. Streptococcus pyogenes Isolates Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
S. pyogenes	ATCC 12344 [Typing strain T1, NCIB 11841, SF 130]	1.0E+04	StreptococcuspyogenesDetected
	ATCC 12348 [Typing strain S43 Type 6]	1.0E+04
	ATCC 12384 [Typing strain C203 Type 3]	1.0E+04
	ATCC 19615a [Bruno]	1.0E+06a
	ATCC 700294 [SF370; M1 GAS [M-type 1 T-type 1]]	1.0E+05b
	ATCC 49399 [QC A62]	1.0E+04
	ATCC BAA-595 [MGAS 315, serotype M3]	1.0E+04
	ATCC BAA-947 [MGAS 5005, serotype M1]	1.0E+04

ª See Table 47 for limitation.

· Streptococus pyggenes ATCC 700294 was detected in 3/5 replicates at 1.0E+04 copies/mL bin results and 3/3 replicates at 1.0E+05 copies/mL with 10°5 copies/mL bin results. No limitation on reactivity could be identified based on isolate sequence.

The following tables (Table 74 - Table 81) describe the reactivity of the AMR genes assays with different AMR gene types in various host bacteria. Results are shown for the isolates tested as well as predictions of reactivity with untested AMR gene types based on in silico analysis of sequences retrieved from public databases from June 2016 to Sept 2016.

Table 74. Isolates Containing mecA/C and MREJ Tested and Detected

Organism	Source [Strain]	Test concentration(copies/mL)	Result
S. aureus	Methicillin Sensitive Staphylococcus aureus (MSSA)containing SCCmec cassette (non-functional mecA variant)
	ATCC BAA-2421 [Mass/2010]	1.0E+04
	Methicillin Resistant Staphylococcus aureus (MRSA)(Characterized SCCmec Types)
	NARSA NRS705 [NY-12 / SCCmec Type: II]	1.0E+04	mecA/C and MREJDetected
S. aureus	NARSA NRS701 [MN-082 / SCCmec Type: II]	1.0E+04
	ATCC BAA-1717 [TCH1516 / SCCmec Type: IVa]	1.0E+05a
	NARSA NRS683 [GA-298 / SCCmec Type: IV]	1.0E+04
	NARSA NRS662 [CO-34 / SCCmec Type: IV]	1.0E+04
	NARSA NRS707 [NY-155 / SCCmec Type: IV]	1.0E+04
	ATCC BAA-1707 [MW2 / SCCmec Type: IV]	1.0E+04
	NARSA NRS691 [GA-62 / SCCmec Type: IV]	1.0E+04
	NARSA NRS648 [CA-347 / SCCmec Type:II or IV]	1.0E+05a
	NARSA NRS689 [GA-442 / SCCmec Type: IV]	1.0E+04
	NARSA NRS668 [CO-72 / SCCmec Type: IV]	1.0E+04
	ATCC BAA-1700 [HEH-33798 / SCCmec Type: IVb]	1.0E+04

{51}------------------------------------------------

Organism	Source [Strain]	Test concentration(copies/mL)	Result
	BEI NR-46081a (NRSA NRS484) [HIP12899 / SCCmec Type: IV]	1.0E+05a
	ATCC BAA-1691 [HFH-30137 / SCCmec Type: IV]	1.0E+04
	ATCC 43300 [F182 Kansas / SCCmec Type: II ]	1.0E+04
	ATCC BAA-2422 [Worcester MA/2010 / SCCmec Type: II]	1.0E+04
	ATCC BAA-1720 [MRSA252 / SCCmec Type: II]	1.0E+04
	NARSA NRS745 [CA-629 / SCCmec Type: IV or V]	1.0E+04
	Methicillin Resistant Staphylococcus aureus (MRSA)(Characterized MREJ Types)
	ATCC BAA-38 [MREJ type i]	1.0E+04
	NARSA NRS686 [MREJ type i]	1.0E+04
	ATCC BAA-44 [MREJ type ii]	1.0E+04
	ATCC BAA-41 [MREJ type ii]	1.0E+04
	NARSA NRS385 [MREJ type ii]	1.0E+04
	ATCC BAA-42 [MREJ type ii]	1.0E+04
	ATCC BAA-39 [MREJ type iii]	1.0E+04
	ATCC BAA-40 [MREJ type iv]	1.0E+04
	GRE 1062264 [MREJ type iv]	1.0E+04
	GRE 1055015 [MREJ type vi]	1.0E+04
	GRE 0860042 [MREJ type vii]	1.0E+04
	GRE 1052034 [MREJ type ix]	1.0E+04
	GRE 1151100 [MREJ type xi]	1.0E+04
	GRE 0960006 [MREJ type xii]	1.0E+04
	GRE 1055017 [MREJ type xiii]	1.0E+04
	GRE 0759163 [MREJ type xiv]	1.0E+04
	GRE 1062373 [MREJ type xv]b	1.0E+06b
	GRE 1057114 [MREJ type xvii]	1.0E+04
	GRE 1062292 [MREJ type xviii]c	3.3E+08	mecA/C and MREJ
	GRE 1062519 [MREJ type xix]cd	1.0E+07	Not Detected
	Methicillin Resistant Staphylococcus aureus (MRSA)(SCCmec Type: XI / mecC / mecALGA251 variants)
	ATCC BAA-2312 [M10/0061 / SCCmec Type: XI / mecC]	1.0E+04	mecA/C and MREJ
	ATCC BAA-2313 [M10/0148 / SCCmec:Type XI / mecC ]	1.0E+04	Detected
S. argenteus	Methicillin Resistant Staphylococcus argenteusDSM 28299 [MSHB-1132]	1.0E+05

ª mec4/C and MREJ assays positive in less than three replicates at 1.0E+04 copies/mL, no sequence based limitation on reactivity identified.

b Bacteria obtained through NARSA for distribution by BEI Resources, NIAID, NIH: Staphylococcus aureus, Strain HIP12899, NR-46081

6 See Table 47 for limitation.

d MREJ type xix characterized MSSA.3

Table 75. In Silico Reactivity Predictions for mecA/C and MREJ

mecA/Ca,b		MREJd
Detected	Reduced Reactivityor Not Detected	Detected	Reduced Reactivityor Not Detected	UnknownReactivity(no sequences)
mecA in S. aureusc	mecA in someisolates of S. capitis,S. kloosii and S.vitulinus	MREJ i, ia – viie	MREJ ixf	MREJ viii
mecC in S. aureus		MREJ xi-xiv	MREJ xvg	MREJ x
		MREJ xvi – xvii	MREJ xviii
mecA and mecC innon-aureus staphylococci(including S. argenteus)	mecC in S. sciuri	MREJ inS. argenteus	MREJ xix, xxhMREJ in non-aureusstaphylococci andother speciesd

ª July 2016; analysis of 1,257 database mecA sequences from S. aureus, as well as mecA and mecC sequences from nonaureus staphylococci.

b mecC is also referenced as SCCmec XI and mecALGA251.

° Limited or reduced reactivity predicted for 2/1,257 mecA sequences from S. aureus (0.2%).

4 June 2016; analysis of approximately 1,450 typed MREJ database sequences from S. aureus non-aureus staphylococi and non-staphylococus species (Bacillus thuringiensis, Marrococus caseolyticus, Clostidium acidurici, and Rummelibacillus stabekisi).

® Limited or reduced reactivity predicted for 1/141 MREJ iii sequences (0.7%); normal reactivity observed for the inited in the Pay (see Table 74).

^ Limited or reduced reactivity prediced for 2/8 MREJ ix sequences (25.0%); normal readivity observed for the isolated (see Table 74).

9 Reduced reactivity predicted by in silico analysis and observed with the isolate of MREJ xv tested (see Table 74).

h MREJ xix and xx were not included in the assay design because they were identified from methicillin-sensitive S. aureus3

{52}------------------------------------------------

CTX-M Type	Organism	Source	Test concentration(copies/mL)	Result
CTX-M	E. coli	AR-Bank #0137a	1.0E+04	CTX-M Detected
	K. oxytoca	GRE 1254054	1.0E+04
	K. pneumoniae	AR-Bank #0068 a	1.0E+04
	K. pneumoniae	AR-Bank #0153 a	1.0E+04
		GRE 1355030	1.0E+04
CTX-M-1	E. coli	AR-Bank #0162	1.0E+04
CTX-M-2	K. pneumoniae	AR-Bank #0107	1.0E+04
CTX-M-8	K. aerogenes	GRE 1254066	1.0E+04
CTX-M-9	E.coli	AR-Bank #0086	1.0E+04
	E.cloacae	NCTC 13464	1.0E+04
CTX-M-14	K. pneumoniae	AR-Bank #0079	1.0E+04
CTX-M-15	E.coli	Zeptometrix 0801905	1.0E+04
CTX-M-22	P. mirabilis	GRE 1254053	1.0E+04
CTX-M-25	K. pneumoniae	NCTC 13465	1.0E+04
In silico Reactivity Predictionsa
Detected		Not Detected	Unknown Reactivity(no sequences)
CTX-M-1 – CTX-M-117	CTX-M-121 – CTX-M-126	CTX-M-151	CTX-M-118	CTX-M-143
CTX-M-129 – CTX-M-132	CTX-M-150		CTX-M-119	CTX-M-145
CTX-M-134	CTX-M-152		CTX-M-120	CTX-M-146
CTX-M-136 – CTX-M-139	CTX-M-155 – CTX-M-177		CTX-M-127	CTX-M-149
CTX-M-141 – CTX-M-142	CTX-M-179 – CTX-M-185		CTX-M-128	CTX-M-153
CTX-M-144			CTX-M-133	CTX-M-154
CTX-M-147 – CTX-M-148			CTX-M-135	CTX-M-178
			CTX-M-140

Table 76. Isolates Containing the blacky gene Tested and In Silico Reactivity Predictions

a July 2016; analysis of over 1,200 database CTX-M sequences (typed and untyped).

Table 77. Isolates Containing the blame gene Tested and In Silico Reactivity Predictions

IMP Type	Organism	Source	Test concentration(copies/mL)	Result
IMP	K. aerogenes	AR-Bank #0161	1.0E+04
IMP	E. coli	GRE 1062016	1.0E+04
IMP	K. pneumoniae	AR-Bank #0080	1.0E+04
IMP-1a	P. aeruginosa	AR-Bank #0103	1.0E+04
IMP-3a	E. coli	GRE 1252008	1.0E+04	IMP Detected
IMP-4	A. baumannii	GRE 1062081	1.0E+04
IMP-8	K. pneumoniae	GRE 1062084	1.0E+04
IMP-9	E. coli	GRE 1252009	1.0E+04
IMP-14	P. aeruginosa	AR-Bank #0092	1.0E+04
In silico Reactivity Predictionsb
Detected			Reduced Reactivityor Not Detected	Unknown Reactivity(no sequences)
IMP-1 – IMP-30a			IMP-31	IMP-36
IMP-40 – IMP-45			IMP-35	IMP-39
IMP-58 – IMP-60				IMP-46
IMP-32 – IMP-34				IMP-47
IMP-48 – IMP-49				IMP-50
IMP 37 – IMP-38				IMP-57
IMP-51 – IMP-56

ª Limited or reduced reactivity predicted for 1/36 (2.8%) of IMP-1 and 1/3 (33.3%) of IMP-3 sequences.

ఠ June 2016; analysis of over 220 database IMP sequences (typed and untyped).

Table 78. Isolates Containing the blakes gene Tested and In Silico Reactivity Predictions

KPC Type	Organism	Source	Test concentration(copies/mL)	Result
KPC	E. cloacae	ATCC BAA-2341	1.0E+04	KPC Detected
	E. hormaechi	ATCC BAA-2082	1.0E+04
	P. mirabilis	AR-Bank #0156	1.0E+04
	K. oxytoca	AR-Bank #0147	1.0E+04
	K. pneumoniae	AR-Bank #0097	1.0E+04
	K. oxytoca	JMI 2523	1.0E+04
KPC-2	K. oxytoca	JMI 7818	1.0E+04
	K. pneumoniae	Zeptometrix 0801886	1.0E+04
	K. pneumoniae	JMI 328	1.0E+04
	K. pneumoniae	ATCC BAA-1705	1.0E+04

{53}------------------------------------------------

	S. marcescens	JMI 697	$1.0E+04$
KPC-3	E. coli	AR-Bank #0061	$1.0E+04$
	K. oxytoca	JMI 2661	$1.0E+04$
KPC-4	K. pneumoniae	JMI 766	$1.0E+04$
KPC-5	P. aeruginosa	Creighton University PS28	$1.0E+04$
In silico Reactivity Predictionsa
Detected		Not Detected	Unknown Reactivity(no sequences)
KPC-1-19	KPC-21-22	KPC-24-26	None	KPC-20

a August 2016; analysis of approximately 1,125 database KPC sequences (typed and untyped).

Table 79. Isolates Containing the blawn gene Tested and In Silico Reactivity Predictions

(copies/mL)	xLoD	(copies/mL)	xLoD	(copies/mL)
NDM	E. coli	AR-Bank #0162	$1.0E+04$
NDM	K. pneumoniae	AR-Bank #0153	$1.0E+04$
NDM	K. pneumoniae	AR-Bank #0068	$1.0E+04$
NDM	P. mirabilis	AR-Bank #0159	$1.0E+04$
NDM-1	A. baumannii	AR-Bank #0033	$1.0E+04$	NDM Detected
NDM-2	A. baumannii	GRE 1153064	$1.0E+04$
NDM-5	E. coli	AR-Bank #0150	$1.0E+04$
NDM-6	E. coli	AR-Bank #0137	$1.0E+04$
In silico Reactivity Predictionsb
Detected			Not Detected
NDM-1a	NDM-7	NDM-13	None
NDM-2	NDM-8	NDM-14
NDM-3	NDM-9	NDM-15
NDM-4	NDM-10	NDM-16
NDM-5	NDM-11
NDM-6	NDM-12

a Limited or reduced reactivity is predicted for 3/430 NDM-1 sequences (0.7%).

b June 2016; analysis of 900 database NDM sequences (typed and untyped).

Table 80. Isolates Containing the black and like genes Tested and In Silico Reactivity Predictions

OXA Typea	Organism	Source	Test concentration(copies/mL)	Result
OXA-48	K. aerogenes	AR-Bank #0074	1.0E+04
	S. marcescens	GRE 1411136	1.0E+04
OXA-48-like	S. marcescens	GRE 1411137	1.0E+04	Gram negative &
OXA-162	K. pneumoniae	GRE 1355030	1.0E+04	OXA-48-Like Detected
OXA-181	K. pneumoniae	AR-Bank #0068	1.0E+04
OXA-232	K. pneumoniae	AR-Bank #0075	1.0E+04
In silico Reactivity Predictionsa
Detected		Not Detectedb
OXA-48	OXA-204	OXA-370	OXA-163c	OXA-438c
OXA-48-like	OXA-232	OXA-484	OXA-247c	OXA-439c
OXA-162	OXA-244	OXA-505	OXA-405c
OXA-181	OXA-245		OXA-416
OXA-199	OXA-252		OXA-436c

a June 2016; analysis of 165 database OXA-48-like sequences (typed and untyped).

Sequence analysis predicts that the listed OXA-48-like types (e.g. OXA-23-like, OXA-4024-like, OXA-4024-like, OXA-51-ike, and OXA-58-like, OXA-143a-like and OXA-143-like) will also not be detected.

^ Deletion variants with altered carbapenem hydrolysis activity, as described for OXA-1631.

Table 81. Isolates Containing the blam gene Tested and Detected, and In Silico Reactivity Predictions

VIM Type	Organism	Source	Test concentration(copies/mL)	Result
VIM	E. cloacae	AR-Bank #0154	1.0E+04
VIM	P. aeruginosa	AR-Bank #0111	1.0E+04
VIM	K. pneumoniae	AR-Bank 0076	1.0E+04
VIM-2	P. aeruginosa	AR-Bank #0100	1.0E+04	VIM Detected
VIM-4	P. aeruginosa	AR-Bank #0054	1.0E+04
VIM-7	E. coli	GRE 1256018	1.0E+04
VIM-10	P. aeruginosa	NCTC 13437	1.0E+04
In silico Reactivity Predictionsb
	Detected		Reduced Reactivityor Not Detected	Unknown Reactivity(no sequences)

{54}------------------------------------------------

VIM-1 – VIM-20ª	VIM-23 – VIM-47	VIM-49 – VIM-51	VIM-39	VIM-21
			VIM-45	VIM-22
			VIM-46	VIM-48

a Limited or reduced reactivity is predicted for 2/177 VIM-2 sequences (1.1%).

b September 2016; analysis of over 600 database VIM sequences (typed and untyped).

Analytical Specificity (Cross-Reactivity and Exclusivity)

There is a risk of false positive results due to non-specific amplification and/or cross-reactivity with organisms found in the respiratory tract. The potential for non-specific amplification and detection by the FilmArray Pneumonia Panel plus assays was evaluated by in silico analyses of available sequences and by empirical (wet) testing of high concentrations of organisms in contrived samples and the observed and predicted cross-reactivities for organisms closely related to those detected by the panel and unrelated organisms that may present in lower respiratory specimens summarized in Table 82. Erroneous results due to cross-reactivity with organisms that were not evaluated or new variant sequences that emerge is also possible.

On-panel organisms were tested to assess the potential for intra-panel cross-reactivity (Table 83). Offpanel organisms included species of the same genus or otherwise genetically related to organisms detected by the panel, as well as normal flora and pathogens that may be present in sputum-like and BAL-like specimens (Table 84). Antimicrobial resistance genes were also evaluated in conjunction with on and off panel host organisms.

The final concentration of analyte in the sample (typically ≥1.0E+07 CFU/mL for bacteria and fungi and >1.0E+05 TCID30/mL for viruses) represented levels ~100 - 100,000 fold higher than the LoD or lowest reportable level of the FilmArray Pneumonia Panel plus assays.

FilmArray Pneumonia Panel plus Result	Cross-Reactive Organism
Closely-Related Species
Escherichia coli	Escherichia fergusoniia
	Shigella species (S. boydii, S. dysenteriae, S. flexneri, S. sonnei)a
Klebsiella oxytoca	Klebsiella michiganensisa
Staphylococcus aureus	Staphylococcus argenteusb
	Staphylococcus schweitzeric
Pseudomonas aeruginosa	Pseudomonas putidad
Unrelated Species
Human Rhinovirus/Enterovirus	Bordetella speciese
	Aspergillus niger
Parainfluenza Virusf	Cryptococcus laurentii
	Cryptococcus uniguttulatus
Adenovirus	Stenotrophomonas acidaminiphilag
CTX-Mh	Acinetobacter schindleri
	Burkholderia vietnamiensisi
	Lelliottia amnigena (Enterobacter amnigenus)
	Enterobacter kobei
Escherichia colij,k	Enterobacter ludwigii
	Enterobacter cloacae

Table 82. Observed and Predicted Cross-Reactivity of FilmArray Pneumonia Panel plus Assays

Genetically or phenotypically indistinguished by standard laboratory techniques. Detected at a concentrations ≥1.0E+04 copies/mL

^ Genetically or phenotypically indisting is and often misidentified by standard laboratory techniques. Detected a a concentrations ≥1.0E+06 copies/mL.

d Cross-reactivity possible at concentrations >1.0E+07 copies/mL.

· Cross-reactivity with B. perussis confirmed at ≥1.0E+06 CFUmL. Cross-reactivity with B. bronchiseptica was not observed at 1.0E+06 CFU/mL, but possible based on sequence analysis.

I Cross-reactivity was observed with A. niger, C. laurentrations >1.0E+06 copies/mL. Cross-reactivity with other Cryptococus species may be possible based on sequence analysis.

9 S. acidaminiphila has not been isolated from human clinical specimens, no cross-reactivity observed with other Steners species

™ Cross-reactive product observed only at concentrations >4.5E+07 CFUmL and only reported if an applicable gram-negative bacterium is also detected

Genetically or phenotypically indistinguised by standard laboratory techniques. Detected at a concentrations ≥1.0E+05 copies/mL

{55}------------------------------------------------

¹ Not tested. Predicted by in silico analysis.

i If observed, results will be reported as Escherichia coli 10^4 copies/mL.

" Based on in silico analysis, cross-reactivity is also possible at high concentrations (>1.0E+07 copies) (E. hornaeche, K. aerogenes, E. lingolyticus), Citrobacter koseri, Escherichia vulneris, and Leclercia adecaboxylata.

Table 83. On Panel Organisms Tested for Evaluation of FilmArray Pneumonia Panel plus Analytical Specificity

False positive results were observed when testing the species shown in bold.

ON-PANEL
Bacteria
Acinetobacter baumannii	Enterobacter kobeia	Klebsiella quasipneumoniae	Pseudomonas aeruginosa
Acinetobacter calcoaceticus	Enterobacter ludwigiia	Klebsiella variicola	Serratia marcescens
Acinetobacter nosocomialis	Escherichia coli	Moraxella catarrhalis	Staphylococcus aureus
Acinetobacter pittii	Haemophilus influenzae	Proteus hauseri	Streptococcus agalactiae
Enterobacter asburiae	Klebsiella aerogenes	Proteus mirabilis	Streptococcus pneumoniae
Enterobacter cloacae	Klebsiella oxytoca	Proteus penneri	Streptococcus pyogenes
Enterobacter hormaechei	Klebsiella pneumoniae	Proteus vulgaris
Atypical Bacteria
Chlamydia pneumoniae	Legionella pneumophila	Mycoplasma pneumoniae
Viruses
Middle East RespiratorySyndrome Coronavirus (MERS-CoV)	Coronavirus HKU1	Human Metapneumovirus	Parainfluenza Virus 2
Adenovirus B	Coronavirus NL63	Influenza A	Parainfluenza Virus 3
Adenovirus C	Coronavirus OC43	Influenza B	Parainfluenza Virus 4
Adenovirus E	Enterovirus	Parainfluenza Virus 1	Respiratory Syncytial Virus
Coronavirus 229E	Human Rhinovirus
Antimicrobial Resistance Genes
CTX-M(Klebsiella oxytoca)	KPC(Klebsiella pneumoniae)	OXA-48-like(Serratia marcescens)	mecA and MREJ(Staphylococcus aureus)
IMP(Pseudomonas aeruginosa)	NDM(Acinetobacter baumannii)	VIM(Enterobacter cloacae)

ª See Table 82 for cross-reactivity information.

Table 84. Off-Panel Bacteria Tested or Evaluated by In Silico Analysis for FilmArray Pneumonia Panel plus Analytical Specificity False positive results were observed when testing the species shown in bold.

OFF-PANEL
Bacteria
Abiotrophia defectiva	Escherichia fergusoniiª	Mycobacterium tuberculosis	Shigella boydiiª
Achromobacterxylosoxidans	Escherichia hermanii	Mycoplasma bovis	Shigella dysenteriae ª
Acinetobacter haemolyticus	Escherichia vulneris	Mycoplasma genitalium	Shigella flexneriª
Acinetobacter johnsonii	Fluoribacter dumoffei	Mycoplasma hominis	Shigella sonnei ª
Acinetobacter junii	Fusobacterium varium	Mycoplasma orale	Staphylococcus argenteus ª
Acinetobacter Iwolfii	Gemella morbillorum	Neisseria gonorrhoeae	Staphylococcus capitis
Acinetobacter radioresistens	Granulicatella adiacens	Neisseria lactamica	Staphylococcus caprae
Acinetobacter schindleriª	Haemophilus ducreyi	Neisseria meningitidis	Staphylococcus cohnii
Acinetobacter ursingii	Haemophilus haemolyticus	Neisseria mucosa	Staphylococcus haemolyticus
Actinobacillusactinomycetemcomitans	Haemophilusparahaemolyticus	Neisseria sicca	Staphylococcusepidermidis(mecA)
Actinobacillus hominis	Haemophilus parainfluenzae	Nocardia asteroides	Staphylococcus hominis
Actinobacillus ureae	Haemophilus parasuis	Nocardia brasilensis	Staphylococcus intermedius
Actinomyces isrealii	Haemophilus sputorum	Pantoea agglomerans	Staphylococcus lugdunensis
Actinomyces naeslundii	Hafnia alvei	Pasteurella multocida	Staphylococcus lutrae
Bacillus cereus	Hafnia paralvei	Pediococcus acidilactici	Staphylococcus pasteuri
Bacteriodes fragilis	Helicobacter pylori	Peptostreptococcus anaerobius	Staphylococcuspseudointermedius
Bordatella bronchiseptica	Kingella kingae	Pluralibacter gergoviae	Staphylococcus saprophyticus
Bordatella parapertussis	Klebsiella michiganensis ª	Porphyromonas gingivalis	Staphylococcus schleiferi
Bordatella pertussis ª	Kluyvera intermedia	Prevotella intermedia	Staphylococcusschweitzeri a
Burkholderia cepacia	Kluyvera ascorbata	Prevotella melaninogenica	Staphylococcus sciuri
Burkholderia mallei	Lactobacillus acidophilus	Prevotella oralis	Staphylococcus warneri
Burkholderia multivorans	Leclercia adecarboxylata	Propionibacterium acnes	Staphylococcus xylosus
Burkholderia pseudomallei	Legionellabozemanii	Providencia rettgeri(OXA-48-like)	Stenotrophomonasacidaminiphila ª
Cardiobacterium hominis	Legionella cincinnatiensis	Providencia stuartii	Stenotrophomonas maltophilia
Cedecea davisae	Legionella feeleii	Pseudomonas fluorescens	Stenotrophomonasnitritireducens
Chlamydia trachomatis	Legionella lansingensis	Pseudomonas luteola	Stenotrophomonas rhizophila

{56}------------------------------------------------

OFF-PANEL
Chlamydophila psittaci	Legionella longbeachae	Pseudomonas nitroreducens	Streptococcus equi subsp. Zooepidemicus
Citrobacter freundii (KPC)	Legionella micdadei	Pseudomonas oryzihabitans	Streptococcus mitis
Citrobacter koseri(OXA-48-like)	Legionella wadsworthii	Pseudomonas pertucinogena	Streptococcus mutans
Citrobacter sedlakii	Lelliottia nimipressuralis	Pseudomonas putidaa (IMP)	Streptococcus oralis
Citrobacter werkmanii (VIM)	Lelliottia amnigena a(Enterobacter amnigenus)	Pseudomonas stutzeri	Streptococcus parasanguinis
Clostridium difficile	Leuconostoc lactis	Ralstonia pickettii	Streptococcuspseudopneumoniae
Clostridium perfringens	Listeria monocytogenes	Raoultella ornithinolytica	Streptococcus salivarius
Corynebacterium diptheriae	Macrococcus caseolyticus	Raoultella planticola	Streptococcus sanguinis
Corynebacterium genitalium	Micrococcus luteus	Raoultella terrigena	Streptococcus tigurinus
Corynebacteriumpseudodiptherticum	Moraxella equi	Rhodococcus equi	Streptomyces anulatus
Corynebacterium urealyticum	Moraxella lacunata	Rothia mucilaginosa	Treponema denticola
Cronobacter sakazakii	Moraxella lincolnii	Salmonella enterica (CTX-M)	Ureaplasma parvum
Eikenella corrodens	Moraxella nonliquiefaciens	Serratia fonticola	Ureaplasma urealyticum
Enterobacter cancerogenus	Morganella morganii (NDM)	Serratia liquefaciens	Vagococcus fluvialis
Enterobacter massiliensis	Mycobacterium africanumb	Serratia odorifera	Veillonella parvula
Enterobacter soli	Mycobacterium bovis	Serratia plymuthica	Yersinia enterocolitica
Enterococcus faecium	Mycobacterium caprae	Serratia rubidaea	Yersinia pseudotuberculosis
Enterococcus faecalis	Mycobacterium microtib
		Viruses
Bocavirus	Hantavirusb	Human Papillomavirus (HPV)	Varicella zoster virus
Cytomegalovirus	Herpes simplex virus 1	Influenza Cb	Severe Acute RespiratorySyndrome Coronavirus(SARS-CoV)
Epstein Barr virus	Human ImmunodeficiencyVirus (HIV)	Mumps virus
German Measles Virus (Rubella)	Measles Virus (Rubeola)
		Fungi/Yeast
Aspergillus flavus	Coccidioides posadasii	Fusarium kyushense	Pneumocystis carinii
Aspergillus fumigatus	Cryptococcus albidus	Histoplasma capsulatumc	Pneumocystis jirovecii
Aspergillus nigera	Cryptococcus gattii	Paecilomyces variottii	Pneumocystis murina
Aspergillus terreus	Cryptococcus laurentiia	Paracoccidodes brasiliensisb	Rhizopus microsporus
Blastomyces dermatitidis	Cryptococcus neoformans	Penicillium chrysogenumc	Scedosporium apiospermum
Candida albicans	Cryptococcus uniguttalatusa	Penicillium marneffei	Scedosporium prolificans
Candida glabrata	Filobasidium capsuligenum
Antimicrobial Resistance Genes
AmpC(Klebsiella (Enterobacter)aerogenes)	OXA-24/40 (non-48-like)(Acinetobacter baumannii)	SME (Serratia marcescens)	TEM (Escherichia coli)
CMY (II) (Escherichia coli)	SHV (Klebsiella pneumoniae)	SPM(Pseudomonas aeruginosa)	VAN(Staphylococcus aureus)
ompK36 [SHV-12, OMPC]a(Klebsiella pneumoniae)	SCCmec variant lacking mecA or mecC (Staphylococcus aureus)d

a See Table 82 for cross-reactivity information.

Analytical specificity was evaluated only by in silico analysis of whole genome sequences in public databases. No cross-reactivity is predicted based on the sequences analyzed.

® Tested at a concentration less than 1.0E+07 CFUmL analysis. Cross-eactivity was not observed in testing nor prediced based on the sequences analyzed.

^ Methicillin-sensitive isolate of S. aureus (Rennes MREJ sequence but no mecA or mecC gene (empty cassette). Staphylooocus aureus was reported as Detected and the mecA/C and MREJ result was Not Detected.

Precision (Reproducibility)

Precision (Reproducibility) testing was performed with contrived BAL samples over multiple days at three laboratory locations (sites) on a combination of FilmArray, FilmArray 2.0 and FilmArray Torch systems. The testing incorporated a range of potential variation introduced by operator, system, instrument or Torch module, concentration and reagent lot, for a total of 30 tests per system and 90 total replicates per sample/concentration.

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Evaluation of the reproducibility of Detected results for atypical bacteria and viruses included samples containing combinations of five different analytes, at Negative, Low Positive (1×LoD), and Moderate Positive (3×LoD) concentrations. Negative results were obtained from samples that were not spiked with the analyte (see evaluation of precision for bacterial analytes below).

A summary of results (percent (%) agreement with the expected or Not Detected result) for atypical bacteria and viruses (by site and system) is provided in Table 85.

Analyte	Concentration Tested	ExpectedResult	Agreement with Expected Result			AllSites/Systems[95% CI]
			FilmArraySite A	FilmArray 2.0Site B	FilmArray TorchSite C
Atypical Bacteria
Chlamydia pneumoniae	None(No Analyte)	Not Detected	780/780100%	780/780100%	780/780100%	2,340/2,340100%[99.8%-100%]
Legionella pneumophilaPhiladelphia-1ATCC 33152	Moderate Positive3x LoD$1.5E+03$ CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%[96.0%-100%]
	Low Positive1x LoD$5.0E+02$ CFU/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%[96.0%-100%]
	None(No Analyte)	Not Detected	720/720100%	720/720100%	720/720100%	2,160/2,160100%[99.8%-100%]
Mycoplasma pneumoniae	None(No Analyte)	Not Detected	780/780100%	780/780100%	780/780100%	2,340/2,340100%[99.8%-100%]
	Viruses
Middle East RespiratorySyndrome Coronavirus	None(No Analyte)	Not Detected	780/780100%	780/780100%	780/780100%	2,340/2,340100%[99.8%-100%]
	AdenovirusSpecies B Serotype 3ZeptoMetrix 0810062CF	Moderate Positive3x LoD$3.0E+00$ TCID50/mL	Detected	30/30100%	30/30100%	30/30100%
Low Positive1x LoD$1.0E+00$ TCID50/mL		Detected	30/30100%	30/30100%	30/30100%	90/90100%[96.0%-100%]
None(No Analyte)		Not Detected	720/720100%	720/720100%	720/720100%	2,160/2,160100%[99.8%-100%]
Coronavirus	None(No Analyte)	Not Detected	780/780100%	776/78099.5%	780/780100%	2,336/2,34099.8%[99.6%-100%]
	Human Metapneumovirus16 Type A1ZeptoMetrix 0810161CF	Moderate Positive3x LoD$1.5E+02$ TCID50/mL	Detected	30/30100%	30/30100%	30/30100%
Low Positive1x LoD$5.0E+01$ TCID50/mL		Detected	30/30100%	29/3096.7%	30/30100%	89/9098.9%[94.0%-100%]
None(No Analyte)		Not Detected	720/720100%	720/720100%	720/720100%	2,160/2,160100%[99.8%-100%]
HumanRhinovirus/Enterovirus	None(No Analyte)	Not Detected	779/78099.9%	780/780100%	779/78099.9%	2,338/2,34099.9%[99.7%-100%]
	Influenza AH3N2A/Port Chalmers/1/73ATCC VR-810	Moderate Positive3x LoD$1.5E+00$ TCID50/mL	Detected	30/30100%	30/30100%	30/30100%
Low Positive1x LoD$0.5E-01$ TCID50/mL		Detected	30/30100%	29/3096.7%	30/30100%	89/9098.9%[94.0%-100%]
None(No Analyte)		Not Detected	720/720100%	720/720100%	720/720100%	2,160/2,160100%[99.8%-100%]

		Table 85. Reproducibility of FilmArray Pneumonia Panel plus Atypical Bacteria and Virus Results

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	Concentration Tested	ExpectedResult	Agreement with Expected Result
Analyte			FilmArray	FilmArray 2.0	FilmArray Torch	All
			Site A	Site B	Site C	Sites/Systems[95% [CI]]
Influenza B	None(No Analyte)	Not Detected	780/780100%	780/780100%	780/780100%	2,340/2,340100%[99.8%-100%]
Parainfluenza VirusType 2ZeptoMetrix 0810015CF	Moderate Positive3 $\times$ LoD7.5E+01 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%[96.0%-100%]
	Low Positive1 $\times$ LoD2.5E+01 TCID50/mL	Detected	30/30100%	30/30100%	30/30100%	90/90100%[96.0%-100%]
	None(No Analyte)	Not Detected	720/720100%	720/720100%	720/720100%	2,160/2,160100%[99.8%-100%]
Respiratory SyncytialVirus	None(No Analyte)	Not Detected	780/780100%	780/780100%	780/780100%	2,340/2,340100%[99.8%-100%]

Precision for bacterial analytes was measured at each concentration as 1) precision of bin results and 2) reproducibility of analyte detection. When a sample containing one or more bacteria is tested repeatedly. the precision of the bin results (probability that each replicate will result) will vary based on the concentration of nucleic acid measured and the relation of that concentration to the limits of each bin. Bin precision may be as low as 50% for values at a bin limit and precision will increase (up to 90% or higher) as the distance of the measured value from a bin limit increases. The precision of FilmArray Pneumonia Panel plus bin results will follow the model illustrated in Figure 1:

90% at a bin center (Scenario 1) ●
~60 90% between a bin limit and bin center (Scenario 2) ●
~50% at bin limits (Scenario 3) ●

Image /page/58/Figure/5 description: The image shows a graph of the probability of a reported bin result versus the measured value. The x-axis is the measured value in log10 copies/mL, and the y-axis is the probability of the reported bin result. There are vertical lines at 4.0, 5.0, 6.0, and 7.0, which correspond to the boundaries between the bins. Below the graph are three scenarios showing the mean measured value and the probability of the reported bin result.

Figure 1. Model for Precision of FilmArray Pneumonia Panel plus Bin Results

Top: The probability of the same bin results for each replicate tested on proximity of the measured value to a bin limit. Bottom: Expected distribution of bin results at different mean measured values.

Samples containing bacteria and corresponding antimicrobial (AMR) genes were tested at six different concentrations over the reportable range and below. A summary of the bin precision (percent (%) of

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replicates reported in each bin) and the reproducibility of detection is shown at each concentration tested in Table 86.

Table 86. Reproducibility of FilmArray Pneumonia Panel plus Bacterial Bin Results on FilmArray Torch

Grey shading indicates the expected bin results based on the analyte concentration and bold font indicates the bin with the greatest percentage of results at each concentration.

Analyte	Concentration(log₁₀ copies/mL)	% Replicates Reported in Each Bin Result					TotalDetected
		≥10^7	10^6	10^5	10^4	ND
Acinetobacterbaumannii(NDM-1)AR-BANK#0033	7.5	90/90(100%)	-	-	-	-	90/90100%
	6.5	87/90(96.7%)	3/90(3.3%)	-	-	-	90/90100%
	5.5	-	82/90(91.1%)	8/90(8.9%)	-	-	90/90100%
	4.5	-	1/90(1.1%)	80/90(88.9%)	9/90(10.0%)	-	90/90100%
	3.5	-	-	1/90(1.1%)	74/90(82.2%)	15/90(16.7%)	75/9083.3%
	2.5	-	-	-	1/90(1.1%)	89/90(98.9%)	1/901.1%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
Enterobacter cloacae(VIM)AR-BANK#0154	7.0	90/90(100%)	-	-	-	-	90/90100%
	6.0	4/90(4.4%)	86/90(95.6%)	-	-	-	90/90100%
	5.0	-	6/90(6.7%)	80/90(88.9%)	-	4/90(4.4%)	86/9095.6%
	4.0	-	-	6/90(6.7%)	83/90(92.2%)	1/90(1.1%)	89/9098.9%
	3.0	-	-	1/90(1.1%)	4/90(4.4%)	85/90(94.4%)	5/905.6%
	2.0	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
Escherichia coli(IMP)GRE 1062016	7.0	90/90(100%)	-	-	-	-	90/90100%
	6.0	7/90(7.8%)	82/90(91.1%)	-	-	1/90(1.1%)	89/9098.9%
	5.0	-	10/90(11.1%)	80/90(88.9%)	-	-	90/90100%
	4.0	-	-	12/90(13.3%)	77/90(86.7%)	1/90(1.1%)	89/9098.9%
	3.0	-	-	1/90(1.1%)	15/90(16.7%)	74/90(82.2%)	16/9017.8%
	2.0	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
Haemophilus influenzaeATCC 10211	7.0	89/90(98.9%)	1/90(1.1%)	-	-	-	90/90100%
	6.0	35/90(48.9%)	55/90(61.1%)	-	-	-	90/90100%
	5.0	-	49/90(54.4%)	40/90(44.4%)	1/90(1.1%)	-	90/90100%
	4.0	-	-	41/90(45.6%)	49/90(54.4%)	-	90/90100%
	3.0	-	-	-	42/90(46.7%)	48/90(53.3%)	42/9046.7%
	2.0	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
Klebsiella aerogenes(Enterobacter	7.5	90/90(100%)	-	-	-	-	90/90100%
Analyte	Concentration(log₁₀ copies/mL)	% Replicates Reported in Each Bin Result					TotalDetected
		≥10^7	10^6	10^5	10^4	ND
aerogenes)ATCC 13048	6.5	65/90(72.2%)	25/90(27.8%)	-	-	-	90/90100%
	5.5	-	52/90(57.8%)	38/90(42.2%)	-	-	90/90100%
	4.5	-	-	38/90(42.2%)	51/90(56.7%)	1/90(1.1%)	89/9098.9%
	3.5	-	-	-	33/90(36.7%)	57/90(63.3%)	33/9036.7%
	2.5	-	-	-	1/90(1.1%)	89/90(98.9%)	1/901.1%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
	7.5	90/90(100%)	-	-	-	-	90/90100%
	6.5	90/90(100%)	-	-	-	-	90/90100%
Klebsiella oxytoca(CTX-M)GRE 1254054	5.5	1/90(1.1%)	84/90(93.3%)	3/90(3.3%)	-	2/90(2.2%)	88/9097.8%
	4.5	-	-	89/90(98.9%)	-	1/90(1.1%)	89/9098.9%
	3.5	-	-	-	90/90(100%)	-	90/90100%
	2.5	-	-	1/90(1.1%)	1/90(1.1%)	88/90(97.8%)	2/902.2%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
	7.00	90/90(100%)	-	-	-	-	90/90100%
	6.00	12/90(13.3%)	78/90(86.7%)	-	-	-	90/90100%
	5.00	-	15/90(16.7%)	75/90(83.3%)	-	-	90/90100%
Klebsiella pneumoniae(KPC)AR-BANK#0097	4.00	-	-	23/90(25.6%)	66/90(73.3%)	1/90(1.1%)	89/9098.9%
	3.00	-	-	-	15/90(16.7%)	75/90(83.3%)	15/9016.7%
	2.00	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
	7.0	90/90(100%)	-	-	-	-	90/90100%
	6.0	26/90(28.9%)	64/90(71.1%)	-	-	-	90/90100%
Moraxella catarrhalisATCC 8176	5.0	-	6/90(6.7%)	83/90(92.2%)	1/90(1.1%)	-	90/90100%
	4.0	-	-	4/90(4.4%)	86/90(95.6%)	-	90/90100%
	3.0	-	-	-	4/90(4.4%)	86/90(95.6%)	4/904.4%
	2.0	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
	7.0	88/90(97.8%)	-	-	-	2/90(2.2%)	88/9097.8%
	6.0	27/90(30.0%)	63/90(70.0%)	-	-	-	90/90100%
Proteus mirabilisATCC 35659	5.0	-	26/90(28.9%)	64/90(71.1%)	-	-	90/90100%
	4.0	-	-	14/90(15.6%)	75/90(83.3%)	1/90(1.1%)	89/9098.9%
	3.0	-	-	-	28/90(31.1%)	62/90(68.9%)	28/9031.1%
	2.0	-	-	-	-	90/90(100%)	0/900.0%
	Concentration	% Replicates Reported in Each Bin Result					Total
Analyte	(log10 copies/mL)	≥10^7	10^6	10^5	10^4	ND	Detected
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
	7.0	90/90(100%)	-	-	-	-	90/90100%
	6.0	20/90(22.2%)	70/90(77.8%)	-	-	-	90/90100%
	5.0	-	24/90(26.7%)	66/90(73.3%)	-	-	90/90100%
Pseudomonasaeruginosa	4.0	-	-	16/90(17.8%)	74/90(82.2%)	-	90/90100%
ATCC 10145	3.0	-	-	-	14/90(15.6%)	76/90(84.4%)	14/9015.6%
	2.0	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
	7.0	90/90(100%)	-	-	-	-	90/90100%
	6.0	2/90(2.2%)	88/90(97.8%)	-	-	-	90/90100%
	5.0	-	7/90(7.8%)	83/90(92.2%)	-	-	90/90100%
Serratia marcescens(OXA-48-like)	4.0	-	-	6/90(6.7%)	83/90(92.2%)	1/90(1.1%)	89/9098.9%
GRE 1659005	3.0	-	-	-	6/90(6.7%)	84/90(93.3%)	6/906.7%
	2.0	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
	7.0	90/90(100%)	-	-	-	-	90/90100%
	6.0	-	90/90(100%)	-	-	-	90/90100%
	5.0	-	-	90/90(100%)	-	-	90/90100%
Staphylococcus aureussubsp. aureus	4.0	-	-	-	89/90(98.9%)	1/90(1.1%)	89/9098.9%
(mecA/C and MREJ)ATCC 43300	3.0	-	-	-	-	90/90(100%)	0/900.0%
	2.0	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	2/1260(0.2%)	1258/1260(99.8%)	2/12600.2%
	7.8	89/90(98.9%)	1/90(1.1%)	-	-	-	90/90100%
	6.8	89/90(98.9%)	-	-	-	1/90(1.1%)	89/9098.9%
	5.8	-	88/90(97.8%)	1/90(1.1%)	1/90(1.1%)	-	90/90100%
Streptococcusagalactiae	4.8	-	-	89/90(98.9%)	1/90(1.1%)	-	90/90100%
ATCC 13813	3.8	-	-	-	86/90(95.6%)	4/90(4.4%)	86/9095.6%
	2.8	-	-	-	3/90(3.3%)	87/90(96.7%)	3/903.3%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
	6.5	90/90(100%)	-	-	-	-	90/90100%
Streptococcus	5.5	-	90/90(100%)	-	-	-	90/90100%
pneumoniaeATCC 6303	4.5	-	-	89/90(98.9%)	1/90(1.1%)	-	90/90100%
	3.5	-	-	-	89/90(98.9%)	1/90(1.1%)	89/9098.9%
Analyte	Concentration(log10 copies/mL)	% Replicates Reported in Each Bin Result	TotalDetected
		≥10^7	10^6	10^5	10^4	ND
	2.5	-	-	-	-	90/90(100%)	0/900.0%
	1.5	-	-	-	-	90/90(100%)	0/900.0%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%
StreptococcuspyogenesATCC 49399	7.8	90/90(100%)	-	-	-	-	90/90100%
	6.8	90/90(100%)	-	-	-	-	90/90100%
	5.8	5/90(5.6%)	84/90(93.3%)	1/90(1.1%)	-	-	90/90100%
	4.8	-	4/90(4.4%)	86/90(95.6%)	-	-	90/90100%
	3.8	-	-	3/90(3.3%)	87/90(96.7%)	-	90/90100%
	2.8	-	-	-	16/90(18.9%)	74/90(81.1%)	16/9017.8%
	None(No Analyte)	-	-	-	-	1800/1800(100%)	0/18000.0%

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The precision of the antimicrobial resistance (AMR) genes was measured as the reproducibility of analyte detection on each system and overall, presented in Table 87 as the percent of replicates that are detected at concentrations of the associated bacterium that are within the reportable range, or below the reportable range, as well as the percent agreement with the expected result in unspiked samples.

Table 87. Reproducibility of FilmArray Pneumonia Panel plus Antimicrobial Resistance Gene Results on FilmArray 2.0 and FilmArray Torch

AMR GeneOrganism	Concentration ofOrganismlog10	ExpectedResult	Agreement with the Expected Result			All Systems/Sites[95% CI]
			FilmArraySite A	FilmArray2.0Site B	FilmArrayTorchSite C
CTX-MKlebsiella oxytocaGRE 1254054	Reportable Range(3.5 - 7.5)	Detected	150/150100%	149/150a99.3%	150/150100%	449/450a99.8%[98.8%-99.9%]
CTX-MKlebsiella oxytocaGRE 1254054	Below ReportableRange(2.5)	Detected(Variable)	0/300.0%	1/303.3%	0/300.0%	1/901.1%[0.03%-6.0%]
CTX-MKlebsiella oxytocaGRE 1254054	None(No Analyte)	N/A orNot Detected	600/600100%	599/60099.8%	600/600100%	1799/180099.9%[99.7%-100%]
IMPEscherichia coliGRE 1062016	Reportable Range(4.0 - 7.0)	Detected	120/120100%	120/120100%	120/120100%	360/360100%[99.0%-100%]
IMPEscherichia coliGRE 1062016	Below ReportableRange(2.0-3.0)	Detected(Variable)	10/6016.7%	9/6015.0%	3/605.0%	22/18012.2%[7.8%-17.9%]
IMPEscherichia coliGRE 1062016	None(No Analyte)	N/A orNot Detected	600/600100%	600/600100%	600/600100%	1800/1800100%[99.8%-100%]
KPCKlebsiellapneumoniaeAR-Bank#0097	Reportable Range(4.0 - 7.0)	Detected	120/120100%	119/120b99.2%	120/120100%	359/360b99.7%[98.5%-100%]
KPCKlebsiellapneumoniaeAR-Bank#0097	Below ReportableRange(2.0 - 3.0)	Detected(Variable)	14/6023.3%	12/6020.0%	9/6015.0%	35/18019.4%[13.9%-25.0%]
KPCKlebsiellapneumoniaeAR-Bank#0097	None(No Analyte)	N/A orNot Detected	600/600100%	600/600100%	600/600100%	1800/1800100%[99.8%-100%]
mecA/C and MREJStaphylococcusaureusATCC 43300	Reportable Range(4.0 - 7.0)	Detected	119/120b99.2%	118/120b98.3%	120/120100%	357/360b99.2%[97.6%-99.8%]
mecA/C and MREJStaphylococcusaureusATCC 43300	Below ReportableRange	Detected(Variable)	0/600.0%	0/600.0%	0/600.0%	0/1800%

{63}------------------------------------------------

AMR GeneOrganism	Concentration ofOrganismlog10	ExpectedResult	Agreement with the Expected Result			All Systems/Sites[95% CI]
	(2.0 - 3.0)		FilmArraySite A	FilmArray2.0Site B	FilmArrayTorchSite C	[0.0%-2.0%]
	None(No Analyte)	N/A orNot Detected	420/420100%	420/420100%	420/420100%	1260/1260100%[99.7%-100%]
NDMAcinetobacterbaumanniiAR-Bank#0033	Reportable Range(3.5 - 7.5)	Detected	150/150100%	149/150a99.3%	150/150100%	449/450a99.8%[98.8%-100%]
	Below ReportableRange(2.5)	Detected(Variable)	1/303.3%	1/303.3%	0/300.0%	2/902.2%[0.3%-7.8%]
	None(No Analyte)	N/A orNot Detected	599/60099.8%	600/600100%	600/600100%	1799/180099.9%[99.7%-100%]
OXA-48-likeSerratia marcescensGRE 1659005	Reportable Range(4.0 - 7.0)	Detected	120/120100%	119/120b99.2%	120/120100%	359/360b99.70%[98.5%-100%]
	Below ReportableRange(2.0 - 3.0)	Detected(Variable)	14/6023.3%	12/6020.0%	9/6015.0%	35/18019.4%[13.9%-26.0%]
	None(No Analyte)	N/A orNot Detected	598/60099.7%	600/600100%	600/600100%	1798/180099.9%[99.6%-100%]
VIMEnterobactercloacaeAR-BANK#0154	Reportable Range(4.0 - 7.0)	Detected	120/120100%	120/120100%	120/120100%	360/360100%[99.0%-100%]
	Below ReportableRange(2.0 - 3.0)	Detected(Variable)	10/6016.7%	9/6015.0%	3/605.0%	22/18012.2%[7.8%-17.9%]
	None(No Analyte)	N/A orNot Detected	599/60099.8%	600/600100%	600/600100%	1799/180099.9%[99.7%-100%]

a CTX-M and NDM Not Detected results observed at the corresponding bacterial concentration of 4.5 logo copies/mL.

· KPC, mec4/C and MREJ, and OXA-48-like Not Detected results observed at the corresponding bacterial concentration of 4.0 logo copies/mL

Interference

Potentially interfering substances that could be present in BAL-like or sputum-like specimens or that may be introduced during specimen collection and testing were evaluated for their effect on FilmArray Pneumonia Panel plus performance. Substances included endogenous substances that may be found in specimens at normal or elevated levels (e.g. blood, mucus/mucin, human genomic DNA), various commensal or infectious microorganisms, medications, a variety of sample processing substances and substances used to clean, decontaminate, or disinfect work areas. The performance of the FilmArray Pneumonia Panel plus has not been established with all potentially interfering medications for the treatment of lower respiratory tract infections. The effect of interfering substances has only been evaluated for those listed in Table 88. Interference from substances that were not evaluated could lead to erroneous results.

Each substance was added to contrived samples containing representative qualitatively reported organisms and representative organisms with bin reporting. Qualitatively reported organisms were at concentrations near (2-3×) LoD and those with bin reporting were present at 4.0 log10 (copies/mL) (e.g. in the lowest reported bin). The concentration of substance added to the samples (Table 88) was equal to or greater than the highest level expected to be in BAL-like or Sputum-like specimens.

Four of the evaluated substances were found to interfere with the ability of the FilmArray Pneumonia Panel plus to report accurate analyte results; Bleach, MycoPrep, 2% NaOH, and 5% Oxalic acid. Each of these substances contain chemicals known to react with nucleic acids, altering their chemical structure. BioFire Diagnostics 510(k)

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The interference observed was related to the inability to detect the chemically modified nucleic acids. Treatment of specimens with these substances prior to FilmArray Pneumonia Panel plus testing may result in loss of analyte detection, therefore samples that have been in contact with these substances should not be tested using the FilmArray Pneumonia Panel plus. None of the other substances were shown to interfere with the FilmArray Pneumonia Panel plus results, however, testing of specimens that have been centrifuged or pre-treated by addition of enzyme, media, mucolytic agent, or decontaminating substances is not recommended..

Substance	Concentration Tested	Testing Outcome
Endogenous Substances
Blood	10% v/v	No Interference
Albumin	60 mg/mL	No Interference
HCI (gastric acid)	5 mmol/L	No Interference
Hemoglobin	2 mg/mL	No Interference
Human Cells (K-562 cell line)	3.8E+06 cells/mL	No Interference
Immunoglobulins (IgG)	60 mg/mL	No Interference
Mucin	16 mg/mL	No Interference
Exogenous Substances
Albuterol (bronchodilator)	1.7 µmol/L	No Interference
Benzocaine (Orajel)	1.0 % w/v	No Interference
Epinephrine (hormone, bronchodilator)	8.3 µg/mL	No Interference
Galphimia glauca (Homeopathic remedy)	1.0 % w/v	No Interference
Guaifenesin (expectorant)	15.2 mmol/L	No Interference
Lidocaine	5.1 mmol/L	No Interference
Menthol and cetylpyridinium chloride(Cepacol Mouthwash)	1.0% v/v	No Interference
Mupirocin (antibiotic)	6.0 ng/mL	No Interference
Nicotine	6.2 µmol/L	No Interference
Pentamidine (antimicrobial)	1.5 mg/mL	No Interference
Phenylephrine hydrochloride (decongestant)	0.3 mg/mL	No Interference
Tobramycin sulfate (antibiotic)	30 mg/mL	No Interference
Zanamivir (influenza antiviral)	426 ng/mL	No Interference
Competitive Microorganisms
Actinobacillus actinomycetecomitans	3.8E+07 CFU/mL	No Interference
Aspergillus fumigatus	5.5E+07 CFU/mL	No Interference
Burkholderia cepacia	1.7E+07 CFU/mL	No Interference
Cryptococcus neoformans	2.5E+05 CFU/mL	No Interference
Enterovirus D68	1.4E+06 copies/mL	No Interference
Haemophilus influenzae	1.8E+07 CFU/mL	No Interference
Legionella pneumophila	8.1E+06 CFU/mL	No Interference
Respiratory Syncytial Virus	3.5E+04 copies/mL	No Interference
Staphylococcus epidermidis	1.9E+07 CFU/mL	No Interference
Streptococcus mutans	5.9E+06 CFU/mL	No Interference
Streptococcus pyogenes	5.5E+06 CFU/mL	No Interference
Varicella Zoster Virus	8.7E+07copies/mL	No Interference
Disinfection/Cleaning Substances
Reagent Alcohol	7.0%	No Interference
Bleach	1.0% v/v(600 ppm chlorine)	Interference Observed a
Sample Processing Materials a
Copan Snotbuster (active ingredient DTT)	50.0% v/v	No Interference
Sputolysin (active ingredient DTT)	50.0% v/v	No Interference
SPUTASOL (active ingredient DTT + salts)	50.0% v/v	No Interference
MycoPrep (active ingredient NaOH + NALC)	50.0% v/v	Interference Observed b
NaOH (decontaminant)	1.0%	Interference Observed b
Oxalic Acid (decontaminant)	2.5%	Interference Observed b

Table 88. Evaluation of Potentially Interfering Substances on the FilmArray Pneumonia
---------------------------------------------------------------------------------------	--	--

® FilmArray Pneumonia Panel plus testing of lower respiratory specimens that have been processed or treated with these or other substances (e.g. trypsin) has not been validated and is not recommended.

BioFire Diagnostics 510(k)

FilmArray Pneumonia Panel plus

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· Pouch controls passed but Not Detected results were reported for incubation of the sample with substance. Substance(s) are known to chemically interact with and damage nucleic acids (DNA and/or RNA) to prevent amplification.

External Control Material

External Controls should be used in accordance with laboratory protocols and the appropriate accrediting organization requirements, as applicable. Molecular grade water or saline can be used as an external negative control. Previously characterized positive samples spiked with well characterized organisms can be used as external positive controls.

Alternatively, Maine Molecular Quality Controls, Inc. provides an external positive and negative assayed quality control panel designed to monitor the performance of in vitro laboratory nucleic acid testing procedures for the detection of FilmArray Pneumonia Panel plus assays on the FilmArray® 2.0 or the FilmArray® Torch Systems. The FilmArray Pneumonia Panel plus Control is composed of synthetic nucleic acid specifically designed for and intended to be used solely with the FilmArray Pneumonia Panel and FilmArray Pneumonia Panel plus. This material is composed of synthetic nucleic acid specific for all analytes targeted by the FilmArray Pneumonia Panel and FilmArray Pneumonia Panel plus assays, including a MERS-CoV synthetic nucleic acid of less than 500 bases. The material is provided as a liquid in a stabilizing matrix. To use the product, the operator opens the tube and uses the Sample Swab to deliver the same volume of material as in the actual test, and otherwise runs the test according to protocol. This control is shipped and stored at -20°C. This product is not intended to replace manufacturer internal controls provided with the test system.

The MMQCI external control material is available for purchase directly from:

Maine Molecular Quality Controls, Inc. 23 Mills Brook Road Saco, Maine 04072 Phone: (207) 885-1072 http://www.mmqci.com

FilmArray® Pneumonia/Pneumonia plus Control M340

It is ultimately the responsibility of each laboratory to determine the frequency of external control testing with the FilmArray Pneumonia Panel plus as part of the laboratory's Quality Control program.

References

1. Xie, Q. et al. Two deletion variants of Middle East respiratory syndrome coronavirus found in a patient with characteristic symptoms. Arch. Virol. 162, 2445-2449 (2017).
1. Smith-Vaughan, H. C. et al. Absence of an Important Vaccine and Diagnostic Target in Carriage- and Disease-Related Nontypeable Haemophilus influenzae. Clin. Vaccine Immunol. 21, 250-252 (2014).
1. Huletsky, A. & Giroux, R. Sequences for detection and identification of methicillin-resistant staphylococcus aureus (MRSA) of MREJ type XX.
1. Poirel, L. et al. OXA-163, an OXA-48-Related Class D B-Lactamase with Extended Activity Toward Expanded-Spectrum Cephalosporins. Antimicrob. Agents Chemother. 55, 2546-2551 (2011).

Regulation Number and Section

§ 866.4001 A multiplex respiratory panel to detect and identify emerging respiratory pathogen(s) and common respiratory pathogens in human clinical specimens.

(a)
Identification. A multiplex respiratory panel to detect and identify emerging respiratory pathogen(s) and common respiratory pathogens in human clinical specimens is identified as an in vitro diagnostic device intended for the qualitative detection and identification of both emerging and common respiratory pathogens from individuals meeting specific emerging respiratory pathogen clinical and/or epidemiological criteria. For example, clinical signs and symptoms associated with infection of the emerging respiratory pathogen, contact with a probable or confirmed emerging respiratory pathogen case, history of travel to geographic locations where cases of the emerging respiratory pathogen were detected, or other epidemiological links for which testing of the emerging respiratory pathogen may be indicated. A device to detect and identify emerging respiratory pathogen(s) and common respiratory pathogens in human clinical specimens, and in turn to distinguish emerging respiratory pathogen(s) from common respiratory pathogens, is intended to aid in the differential diagnosis of the emerging respiratory pathogen infection, in conjunction with other clinical, epidemiologic, and laboratory data, in accordance with the guidelines provided by the appropriate public health authorities.(b)
Classification. Class II (special controls). The special controls for this device are:(1) The intended use for the labeling required under § 809.10 of this chapter must include a description of what the device detects and measures, the specimen types, the results provided to the user, the clinical indications for which the test is to be used, the specific intended population(s), the testing location(s) where the device is to be used (if applicable), and other conditions of use as appropriate.
(2) The labeling required under § 809.10 of this chapter must include:
(i) A device description, including the parts that make up the device, ancillary reagents required but not provided, and an explanation of the methodology.
(ii) Performance characteristics from analytical studies, including cut-off (if applicable), analytical sensitivity (
i.e., limit of detection), inclusivity, reproducibility, interference, cross-reactivity, instrument carryover/cross-contamination (if applicable), and specimen stability.(iii) Detailed instructions for minimizing the risk of potential users' exposure to the emerging respiratory pathogen(s) that may be present in test specimens and those used as control materials.
(iv) Detailed instructions for minimizing the risk of generating false positive test results due to carry-over contamination from positive test specimens and/or positive control materials.
(v) A warning statement that the interpretation of test results requires experienced healthcare professionals who have training in principles and use of infectious disease diagnostics and reporting of results, in conjunction with the patient's medical history, clinical signs and symptoms, and the results of other diagnostic tests.
(vi) A warning statement that culture should not be attempted in cases of positive results for an emerging respiratory pathogen unless a facility with an appropriate level of laboratory biosafety (
e.g., BSL 3 and BSL 3+) is available to receive and culture specimens.(vii) A warning statement that device positive results for one or more common respiratory pathogens do not rule out bacterial infection, or co-infection with other common respiratory pathogens.
(viii) A warning statement that respiratory pathogen(s) detected may not be the definite cause of disease.
(ix) A warning statement that the use of additional laboratory testing (
e.g. bacterial culture, immunofluorescence, x-ray findings) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of a respiratory infection.(x) A limiting statement that device negative results for the common respiratory pathogens do not preclude infection of a respiratory pathogen and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.
(xi) A limiting statement that analyte targets (
e.g., pathogen nucleic acid sequences or other molecular signatures) may persist in vivo, independent of organism viability. Detection of analyte target(s) does not imply that the corresponding pathogen(s) is infectious, nor is the causative agent(s) for clinical symptoms.(xii) A limiting statement that detection of pathogen nucleic acid sequences or other molecular signatures is dependent upon proper specimen collection, handling, transportation, storage and preparation. Failure to observe proper procedures in any one of these steps can lead to incorrect results. There is a risk of false negative values resulting from improperly collected, transported, or handled specimens.
(xiii) A limiting statement that there is a risk of false positive values resulting from cross-contamination by target organisms, their nucleic acids or amplified product, or from non-specific signals in the assay.
(xiv) A limiting statement that there is a risk of false negative results due to the presence of nucleic acid sequence variants in the pathogen targets of the device.
(xv) A limiting statement that device performance was not established in immunocompromised patients.
(xvi) A limiting statement that positive and negative predictive values are highly dependent on prevalence. The device performance was established during one or more specific respiratory seasons. The performance for some respiratory pathogens may vary depending on the prevalence and patient population tested. False positive test results are likely when prevalence of disease due to a particular respiratory pathogen is low or non-existent in a community.
(xvii) In situations where the performance of the device was estimated based largely on testing pre-selected banked retrospective clinical specimens and/or contrived clinical specimen, a limiting statement that the estimated device performance of that specific pathogen or pathogen subtype may not reflect the performance or prevalence in the intended use population.
(xviii) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that testing with the device should not be performed unless the patient meets clinical and/or epidemiologic criteria for testing suspected specimens of the emerging respiratory pathogen.
(xix) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that positive results obtained with the device for the emerging respiratory pathogen are for the presumptive identification of that pathogen and that the definitive identification of the emerging respiratory pathogen requires additional testing and confirmation procedures in consultation with the appropriate public health authorities (
e.g., local or state public health departments) for whom reporting is necessary.(xx) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that negative results for the emerging respiratory pathogen, even in the context of device positive results for one or more of the common respiratory pathogens, do not preclude infection with the emerging respiratory pathogen and should not be used as the sole basis for patient management decisions.
(xxi) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that negative results for the emerging respiratory pathogen may be due to infection of the emerging respiratory pathogen at a specific respiratory tract location that may not be detected by a particular clinical specimen type. A negative result for the emerging respiratory pathogen in an asymptomatic individual does not rule out the possibility of future illness and does not demonstrate that the individual is not infectious.
(xxii) For devices with an intended use that includes detection of emerging respiratory pathogen(s), a limiting statement that a nationally notifiable Rare Disease of Public Health Significance caused by an emerging respiratory pathogen must be reported, as appropriate, to public health authorities in accordance with local, state, and federal law.
(3) Design verification and validation must include:
(i) Performance results of an appropriate clinical study (
e.g., a prospective clinical study) for each specimen type, and, if appropriate, results from additional characterized samples. The clinical study must be performed on a study population consistent with the intended use population and must compare the device performance to results obtained using FDA-accepted comparator methods or to expected negative results if the infection is not generally expected in the intended use population. Clinical specimens evaluated in the study must contain relevant organism concentrations applicable to the specimen type(s) and the targeted analyte(s). Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.(ii) For devices with an intended use that includes detection of emerging respiratory pathogen(s) for which an FDA recommended panel is available, design verification and validation must include the performance results of an analytical study testing an FDA recommended reference panel of characterized samples that contain the emerging respiratory pathogen. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(iii) An appropriate risk mitigation strategy, including a detailed description of all procedures and methods, for the post-market identification of genetic mutations and/or novel respiratory pathogen isolates or strains (
e.g., regular review of published literature and annual in silico analysis of target sequences to detect possible mismatches. The required documentation for this device must also include all of the results, including any findings, from the application of this post-market mitigation strategy.(iv) For devices with an intended use that includes detection of multiple common respiratory pathogens, in addition to detecting emerging respiratory pathogen(s) in human clinical specimens, a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the common respiratory pathogens that the device is designed to detect is addressed. Also, address in detail how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory diagnosis of respiratory tract infection. Perform an evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(v) A detailed device description, including the parts that make up the device, ancillary reagents required but not provided, and a detailed explanation of the methodology, including molecular target(s) for each analyte, design of target detection reagents, rationale for target selection, limiting factors of the device (
e.g., saturation level of hybridization and maximum amplification and detection cycle number), internal and external controls, and computational path from collected raw data to reported result (e.g., how collected raw signals are converted into a reported signal and result), as applicable and appropriate.(vi) A detailed description of the device software, including software applications and hardware-based devices that incorporate software.
(vii) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiate between the Influenza A virus subtypes in human clinical specimens, in addition to detecting emerging respiratory pathogen(s), a detailed description of the identity, phylogenetic relationship, or other recognized characterization of the Influenza A and B viruses that the device is designed to detect, a description of how the device results might be used in a diagnostic algorithm and other measures that might be needed for a laboratory identification of Influenza A or B virus and of specific Influenza A virus subtypes, and a description of the clinical and epidemiological parameters that are relevant to a patient case diagnosis of Influenza A or B and of specific Influenza A virus subtypes. Perform an evaluation of the device compared to a currently appropriate and FDA accepted comparator method. Detailed documentation must be kept of that study and its results, including the study protocol, study report for the proposed intended use, testing results, and results of all statistical analyses.
(4) For devices with an intended use that includes detection of Influenza A and Influenza B viruses and/or detection and differentiate between the Influenza A virus subtypes in human clinical specimens, in addition to detecting emerging respiratory pathogen(s), the labeling required under § 809.10 of this chapter must include the following:
(i) Where applicable, a limiting statement that performance characteristics for Influenza A were established when Influenza A/H3 and A/H1-2009 (or other pertinent Influenza A subtypes) were the predominant Influenza A viruses in circulation. When other Influenza A viruses are emerging, performance characteristics may vary.
(ii) Where applicable, a warning statement that reads 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 state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.
(iii) Where the device results interpretation involves combining the outputs of several targets to get the final results, such as a device that both detects Influenza A and differentiates all known Influenza A subtypes that are currently circulating, the device's labeling required under § 809.10(b)(9) of this chapter must include a clear interpretation instruction for all valid and invalid output combinations, and recommendations for any required follow up actions or retesting in the case of an unusual or unexpected device result.
(iv) A limiting statement that if a specimen yields a positive result for Influenza A, but produces negative test results for all specific influenza A subtypes intended to be differentiated (
e.g., H1-2009 and H3), this result requires notification of appropriate local, state, or federal public health authorities to determine necessary measures for verification and to further determine whether the specimen represents a novel strain of Influenza A.(5) The manufacturer must perform annual analytical reactivity testing of the device with contemporary influenza strains. This annual analytical reactivity testing must meet the following criteria:
(i) The appropriate strains to be tested will be identified by FDA in consultation with the Centers for Disease Control and Prevention (CDC) and sourced from CDC or an FDA designated source. If the annual strains are not available from CDC, FDA will identify an alternative source for obtaining the requisite strains.
(ii) The testing must be conducted according to a standardized protocol considered and determined by FDA to be acceptable and appropriate.
(iii) By July 31 of each calendar year, the results of the last 3 years of annual analytical reactivity testing must be included as part of the device's labeling. If a device has not been on the market long enough for 3 years of annual analytical reactivity testing to have been conducted since the device received marketing authorization from FDA, then the results of every annual analytical reactivity testing since the device received marketing authorization from FDA must be included. The results must be presented as part of the device's labeling in a tabular format, which includes the detailed information for each virus tested as described in the certificate of authentication, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that physically accompanies the device in a separate section of the labeling where the analytical reactivity testing data can be found; or
(B) In the device's label or in other labeling that physically accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's home page, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the web page containing this information and must allow unrestricted viewing access.
(6) If one of the actions listed at section 564(b)(1)(A)-(D) of the FD&C Act occurs with respect to an influenza viral strain, or if the Secretary of Health and Human Services (HHS) determines, under section 319(a) of the Public Health Service Act, that a disease or disorder presents a public health emergency, or that a public health emergency otherwise exists, with respect to an influenza viral strain:
(i) Within 30 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation, the manufacturer must have testing performed on the device with those viral samples in accordance with a standardized protocol considered and determined by FDA to be acceptable and appropriate. The procedure and location of testing may depend on the nature of the emerging virus.
(ii) Within 60 days from the date that FDA notifies manufacturers that characterized viral samples are available for test evaluation and continuing until 3 years from that date, the results of the influenza emergency analytical reactivity testing, including the detailed information for the virus tested as described in the certificate of authentication, must be included as part of the device's labeling in a tabular format, either by:
(A) Placing the results directly in the device's labeling required under § 809.10(b) of this chapter that physically accompanies the device in a separate section of the labeling where analytical reactivity testing data can be found, but separate from the annual analytical reactivity testing results; or
(B) In a section of the device's label or in other labeling that physically accompanies the device, prominently providing a hyperlink to the manufacturer's public website where the analytical reactivity testing data can be found. The manufacturer's home page, as well as the primary part of the manufacturer's website that discusses the device, must provide a prominently placed hyperlink to the web page containing this information and must allow unrestricted viewing access.