FILMARRAY RESPIRATORY PANEL (RP)
Device Facts
| Record ID | K103175 |
|---|---|
| Device Name | FILMARRAY RESPIRATORY PANEL (RP) |
| Applicant | Idaho Technology, Inc. |
| Product Code | OCC · Microbiology |
| Decision Date | Feb 17, 2011 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.3980 |
| Device Class | Class 2 |
Intended Use
The FilmArray Respiratory Panel (RP) is a multiplexed nucleic acid test intended for use with the FilmArray instrument for the simultaneous qualitative detection and identification of multiple respiratory viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections. The following virus types and subtypes are identified using the FilmArray RP: Adenovirus, Coronavirus HKU1, Coronavirus NL63, Human Metapneumovirus, Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza A subtype 2009 H1, Influenza B, Parainfluenza virus 3, Rhinovirus/Enterovirus, and Respiratory Syncytial Virus. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of a respiratory infection aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. Negative results do not preclude respiratory infection and should not be used as the sole basis for diagnosis, treatment or other management decisions. Positive results do not rule out bacterial infection or co-infection with other organisms. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) and clinical presentation must be taken into consideration in order to obtain the final diagnosis of respiratory infection. Due to seasonal prevalence, performance characteristics for Influenza A/H1, Influenza A/H3, Influenza A/2009 H1, and Influenza B were established primarily with retrospective clinical specimens. Due to the genetic similarity between human Rhinovirus and Enterovirus, the FilmArray RP cannot reliably differentiate them. A positive FilmArray RP Rhinovirus/Enterovirus result should be followed-up using an alternate method (e.g. cell culture or sequence analysis). The FilmArray RP detects Adenovirus species C serotype 6 with reduced sensitivity. It is recommended that specimens found to be negative for Adenovirus after examination using FilmArray RP be confirmed by an alternate method (e.g. FDA-cleared molecular test or cell culture). Performance characteristics for influenza A were established when influenza A/2009 H1N1, A/H1, and A/H3 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary. 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.
Device Story
FilmArray RP System is a multiplexed nucleic acid test for respiratory viral pathogens. Input: unprocessed nasopharyngeal swab (NPS) specimen mixed with buffer, loaded into a single-use pouch. Operation: pouch contains freeze-dried reagents; instrument uses inflatable bladders, seal points, and pneumatic pistons to move liquids and control reactions; two Peltier devices manage thermal cycling. Process: mechanical lysis and magnetic bead nucleic acid extraction; two-stage nested multiplex RT-PCR; high-resolution melting curve analysis. Output: automated identification of 12 viral targets (Influenza A/subtypes, B, Adenovirus, Coronaviruses, hMPV, PIV3, RSV, Rhinovirus/Enterovirus). Used in clinical settings; operated by laboratory personnel. Output provides rapid (under 1 hour) diagnostic aid to clinicians for patient management and infection control decisions.
Clinical Evidence
Prospective clinical study (853 subjects) and archived specimen study (349 confirmed specimens). Prospective study sensitivity ranged from 88.9% (Adenovirus, Flu A/2009 H1) to 100% (RSV); specificity ranged from 89.1% (RSV) to 100% (Flu A/H1, H3, B, CoV NL63). Archived study PPA ranged from 95.7% to 100%; NPA was 100% for all analytes. Bench testing included LoD, inclusivity, cross-reactivity, and interference studies.
Technological Characteristics
Multiplex nucleic acid test system. Components: FilmArray instrument, software, and single-use pouch. Pouch contains freeze-dried reagents for mechanical lysis, magnetic bead extraction, nested RT-PCR, and high-resolution melting analysis. Energy: electrical (Peltier heating/cooling, pneumatic actuators). Connectivity: preinstalled software on laptop. Sterilization: not specified.
Indications for Use
Indicated for individuals suspected of respiratory tract infections exhibiting signs and symptoms of respiratory infection. Used for qualitative detection and identification of respiratory viral nucleic acids in nasopharyngeal swabs (NPS).
Regulatory Classification
Identification
A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.
Special Controls
*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;” (2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and (3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.
Predicate Devices
- Luminex® xTAG™ Respiratory Viral Panel (RVP) (K063765, K081483, K091677)
Related Devices
- K110764 — FILMARRAY RP PANEL · Idaho Technology, Inc. · Apr 27, 2011
- K143080 — FilmArray Respiratory Panel (RP) for use with Multi-instrument FilmArray System · Biofire Diagnostics, LLC · Feb 17, 2015
- K152579 — FilmArray Respiratory Panel EZ (RP EZ) · Biofire Diagnostics, LLC · Oct 3, 2016
- K160068 — FilmArray Respiratory Panel (RP) for use with FilmArray Torch · Biofire Diagnostics, LLC · Feb 8, 2016
- K123620 — FILMARRAY RESPIRATORY PANEL (RP) · Biofire Diagnostics, Inc. · Feb 11, 2013
Submission Summary (Full Text)
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# 11 510(k) Summary
FFB 17 2011
#### 510(k) Summary Idaho Technology Inc. FilmArray RP Test System
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:
Idaho Technology Inc 390 Wakara Way Salt Lake City, UT 84108
Telephone: 801-736-6354 Facsimile: 801-588-0507
Contact: Beth Lingenfelter, ext. 407
Date Submitted: February 2, 2011
#### Device Name and Classification:
Trade Name: FilmArray RP System
Regulation Number: 21 CFR 866.3980
Classification Name: Respiratory Viral Panel Multiplex Nucleic Acid Assay
#### Predicate Device:
K063765, K081483, K091677 - Luminex® xTAG™ Respiratory Viral Panel (RVP).
#### Intended Use:
The FilmArray Respiratory Panel (RP) is a multiplexed nucleic acid test intended for use with the FilmArray instrument for the simultaneous qualitative detection and identification of multiple respiratory viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract infections. The following virus types and subtypes are identified using the FilmArray RP: Adenovirus, Coronavirus HKU1, Coronavirus NL63, Human Metapneumovirus, Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza A subtype 2009 H1, Influenza B, Parainfluenza virus 3, Rhinovirus/Enterovirus, and Respiratory Syncytial Virus. The detection and identification of specific viral nucleic acids from individuals exhibiting signs and
Idaho Technology Inc. 510(k) FilmArray Respiratory Panel System
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symptoms of a respiratory infection aids in the diagnosis of respiratory infection if used in conjunction with other clinical and epidemiological information. Negative results do not preclude respiratory infection and should not be used as the sole basis for diagnosis. treatment or other management decisions. Positive results do not rule out bacterial infection or co-infection with other organisms. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) and clinical presentation must be taken into considertation in order to obtain the final diagnosis of respiratory infection.
Due to seasonal prevalence, performance characteristics for Influenza A/H1, Influenza A/H3, Influenza A/2009 H1, and Influenza B were established primarily with retrospective clinical specimens.
Due to the genetic similarity between human Rhinovirus and Enterovirus, the FilmArray RP cannot reliably differentiate them. A positive FilmArray RP Rhinovirus/Enterovirus result should be followed-up using an alternate method (e.g. cell culture or sequence analysis).
The FilmArray RP detects Adenovirus species C serotype 6 with reduced sensitivity. It is recommended that specimens found to be negative for Adenovirus after examination using FilmArray RP be confirmed by an alternate method (e.g. FDA-cleared molecular test or cell culture).
Performance characteristics for influenza A were established when influenza A/2009 H1N1, A/H1, and A/H3 were the predominant influenza A viruses in circulation. When other influenza A viruses are emerging, performance characteristics may vary. 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.
#### Device Description:
The FilmArray RP System is multiplex nucleic acid test system composed of the FilmArray instrument, the FilmArray software (preinstalled on a laptop computer) and the FilmArray RP pouch. The FilmArray RP pouch contains freeze-dried reagents to perform nucleic acid purification, reverse transcription, and nested, multiplex PCR with DNA melt analysis. The Respiratory Panel (RP) pouch identifies 12 common and emerging viral respiratory pathogens (see Table 1).
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Table 1. Viruses Detected by the FilmArray Respiratory Panel
| Viral Respiratory Pathogens |
|-----------------------------|
| Influenza A |
| H1 subtype |
| H3 subtype |
| 2009 H1 subtype |
| Influenza B |
| Adenovirus |
| Coronavirus HKU1 |
| Coronavirus NL63 |
| Human Metapneumovirus |
| Parainfluenza Virus 3 |
| Respiratory Syncytial Virus |
| Rhinovirus and Enterovirus |
A test is initiated by loading Hydration Solution and an unprocessed patient nasopharyngeal swab (NPS) specimen (i.e. specimen mixed with Sample Buffer) into the FilmArray RP pouch. 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 FilmArrav software guides the user though the steps of placing the pouch into the instrument, scanning the pouch barcode, entering the sample identification and initiating the run.
The FilmArray instrument contains a coordinated system of inflatable bladders and seal points, which act on the pouch to control the movement of liquid between the pouch blisters. When a bladder is inflated over a reagent blister, it forces liquid from the blister into connecting channels. Alternatively, when a seal is placed over a connecting channel it acts as a valve to open or close a channel. In addition, electronically controlled pneumatic pistons are positioned over multiple plungers in order to deliver the rehydrated reagents into the blisters at the appropriate times. Two Peltier devices control heating and cooling of the pouch to drive the reverse transcription reactions, the PCR reactions, and the melting curve analysis.
Nucleic acid extraction occurs within the FilmArray pouch using mechanical lysis and 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, Idaho Technology). This second master mix 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 second stage PCR, or nested PCR, is performed in singleplex fashion in each well of the array. At the conclusion of the 200 stage PCR, the array is interrogated by melting curve analysis for the detection of signature amplicons denoting the presence of specific viral or bacterial targets A digital camera placed in front of the second stage PCR captures fluorescent images of the PCR reactions in real time.
Idaho Technology Inc. 510(k) FilmArray Respiratory Panel System
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The FilmArray software automatically interprets the results of each DNA melting 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.
#### Substantial Equivalence:
The Luminex® xTAG™ RVP is a PCR-based system for detecting the presence of viral nucleic acid in nasopharyngeal swabs collected from individuals with signs and symptoms of respiratory illness. The similarities and differences between the xTAG RVP and the FilmArray RP are outlined below.
| Element | FilmArray Respiratory Panel Test<br>System | Luminex® xTAG™ RVP |
|-----------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------|
| Organisms<br>Detected | Influenza A, Influenza A subtype H1,<br>Influenza A subtype H3, Influenza B,<br>Respiratory Syncytial Virus, human<br>Metapneumovirus, Adenovirus,<br>Parainfluenza 3, and<br>Rhinovirus/Enterovirus | Same<br>See below for differences |
| Analyte | RNA/DNA | Same |
| Technological<br>Principles | multiplex nucleic acid | Same<br>See below for differences |
| Specimen<br>Types | Nasopharyngeal swabs | Same |
Table 2. Similarities between the xTAG RVP and the FilmArray RP
#### Table 3. Differences between the xTAG RVP and the FilmArray RP
| Element | FilmArray Respiratory Panel Test<br>System | Luminex® xTAG™ RVP |
|---------------------------------|---------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Organisms<br>Detected | Can distinguish Influenza A subtype 2009<br>H1 from Influenza A subtype H1.<br>Also detects Coronavirus NL63 and<br>Coronavirus HKU1. | Can distinguish Respiratory Syncytial Virus<br>Type A from Respiratory Syncytial Virus<br>Type B. Detects Parainfluenza virus 1 and<br>Parainfluenza virus 2. |
| Technological<br>Principles | Nested multiplex RT-PCR followed by high<br>resolution melting analysis to confirm<br>identity of amplified product. | Multiplex RT-PCR and multiplex TSPE<br>followed by Fluorescence-activated sorting<br>of labeled beads coupled to streptavidin-<br>conjugated biotinylated products |
| Instrumentation | FilmArray Instrument | PCR Thermocycler<br>Luminex® 100 IS or 200 system |
| Time to result | Less than 1 hour | Approximately 8 hours |
| Test<br>Interpretation | Automated test interpretation and report<br>generation. User cannot access raw data. | Semi-automated test interpretation. User<br>must review all "no call" results to<br>determine cause and retesting strategy. |
| Sample<br>Preparation<br>Method | Sample Processing is automated in the<br>FilmArray instrument. | Up front sample processing is required to<br>extract nucleic acid. |
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| Element | FilmArray Respiratory Panel Test<br>System | Luminex® xTAG™ RVP |
|--------------------|----------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------|
| Reagent<br>Storage | Reagents are stored at room temperature. | Reagents stored at 4°C and -20°C. |
| Controls | Two controls are included in each reagent<br>pouch to control for sample processing and<br>both stages of PCR and melt analysis. | Internal control added to each sample.<br>External control processed with each batch<br>of samples. |
| User<br>Complexity | Moderate/Low | High |
#### Summary of Performance Data
#### Clinical Performance
The clinical performance of the FilmArray RP system was established during a prospective study at 3 U.S. clinical sites over a 6 month time period (December 2009 through May 2010). Subjects with signs and/or symptoms of respiratory infection were invited to participate. Upon obtaining informed consent, NPS samples were collected for FilmArray and comparator testing; a second respiratory sample was collected from each subject for viral culture reference testing. A total of 857 subjects were initially enrolled in the study and four were withdrawn. Table 4, provides a summary of demographic information for the 853 subjects that participated in the prospective study.
| Sex | Number of Subjects |
|--------|--------------------|
| Male | 449 (53%) |
| Female | 404 (47%) |
| Age | |
| ≤5 | 484 (57%) |
| 6-21 | 95 (11%) |
| 22-49 | 190 (22%) |
| ≥50 | 84 (10%) |
#### Table 4. Demographic Summary for FilmArray RP Prospective Study
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Each NPS specimen was tested with the FilmArray RP. The performance of the FilmArray RP was evaluated by comparing the FilmArray RP test result for each member of the panel with the appropriate comparator/reference methods shown in Table 5.
| Organism/Virus | Reference/Comparator Method(s) |
|-----------------------------|--------------------------------------------------|
| Adenovirus | |
| Influenza A | Viral culture followed by DFA<br>identification1 |
| Influenza B | |
| Parainfluenza virus 3 | |
| Respiratory Syncytial Virus | |
| FluA/H1 subtyping | 1 PCR test of influenza A positive viral |
| FluA/H3 subtyping | culture with bi-directional sequence |
| FluA/2009 H1subtyping | confirmation2 |
| Human Rhinovirus | |
| Enterovirus | 2 PCR tests of patient specimen with bi- |
| Coronavirus NL63 | directional sequence confirmation3 |
| Coronavirus HKU1 | |
| Human Metapneumovirus | |
Table 5. Reference/Comparator Methods Used to Assess FilmArray RP Performance
1 Performance of the FilmArray RP system detecting Adenovirus, Flu A. Flu B. PIV3, or RSV. respectively, was compared to viral culture followed by fluorescent antibody identification. "True" Adenovirus, Influenza B, Parainfluenza virus 3, or RSV positively, were considered as any sample that tested positive for Adenovirus, Influenza A, Influenza B, Parainfluenza virus 3, or RSV, respectively, by viral culture followed by DFA testing. "True" Adenovirus, Influenza A. Influenza virus 3, or RSV negatives, respectively, were considered as any sample that tested negative for Adenovirus. Influenza B. Parainfluenza virus 3, or RSV. respectively, by viral culture followed by DFA testing.
2 Performance of the FilmArray RP system detecting Influenza A/H1, A/H3, or A/2009 H1, respectively, was compared to viral culture followed by one analytically validated PCR assay with bi-directional sequence confirmation. The comparator assays were designed to amplify a different sequence from that amplified by the FilmArray assay(s). None of the comparator PCR assays overlapped any FilmArray amplicon sequence even if the same gene was targeted. "True" Influenza A/H1, A/H3, or A/2009 H1 positively, were considered as any sample that tested positive for Influenza A by viral culture, and had bi-directional sequencing pre-defined quality acceptance criteria that matched Influenza A/H1, A/H3, or A/2009 H1 sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), respectively, with acceptable E-values. "True" Influenza A/H1, A/H3, or A/2009 H1 negatively, were considered as any sample that tested negative for Influenza A by viral culture, or any sample that tested positive for Influenza A virus by viral culture, but tested negative by the respective Influenza A subtype specific PCR assay.
3 Performance of the FilmArray RP system detecting Human Rhinovirus, Coronavirus NL63, Coronavirus HKU1. or Human Metapneumovirus, respectively, was compared to a predetermined algorithm that used composite comparator methods. The methods consist of two analytically validated PCR assays followed by bi-directional sequencing. The comparator assays were designed to amplify a different sequence from that amplified by the FilmArray assay(s). None of the comparator PCR assays overlapped any FilmArray amplicon sequence even if the same gene was targeted. "True" Human Rhinovirus, Enterovirus NL63, Coronavirus HKU1, or Human Metapneumovirus positives, respectively, were considered as any sample that had bi-directional sequencing data meeting pre-defined quality acceptance criteria that matched Human Rhinovirus, Coronavirus NL63, Coronavirus HKUI, or Human Metapneumovirus sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm,nih.gov), respectively, with acceptable E-values. "True" Human Rhinovirus, Enterovirus, Coronavirus NL63, Coronavirus HKU1, or Human Metapneumovirus negatives, respectively, were considered as any sample that tested negative by both of the comparator PCR assays.
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A total of 853 specimens were evaluated in this study. Clinical sensitivity or positive percent agreement (PPA) was calculated as 100% x (TP / TP + FN). True positive (TP) indicates that both the FilmArray RP and comparator method had a positive result for this specific analyte and false negative (FN) indicates that the FilmArray result was negative while the comparator result was positive. Specificity was calculated as 100% x (TN / TN + FP). True negative (TN) indicates that both the FilmArray RP and the comparator method had negative results and a false positive (FP) indicates that the FilmArray RP result was positive but the comparator results was negative. The exact binomial two-sided 95% confidence interval was calculated. The results are summarized in Table 6.
| | Sensitivity | 95% CI | Specificity | 95% CI | | |
|------------------------------|-------------|--------|--------------|----------|-------|--------------|
| Adenovirus | 24/27a | 88.9% | 70.8 - 97.7% | 812/826b | 98.3% | 97.2 - 99.1% |
| Influenza A | 9/10 | 90.0% | 55.5 - 99.8% | 841/843d | 99.8% | 99.2 - 100% |
| Influenza A/H1 | 0/0 | n/a | n/a | 853/853 | 100% | 99.6 - 100% |
| Influenza A/H3 | 0/0 | n/a | n/a | 853/853 | 100% | 99.6 - 100% |
| Influenza A/2009 H1 | 8/9 | 88.9% | 51.8 - 99.7% | 841/844d | 99.6% | 99.0 - 99.9% |
| Influenza B | 0/0 | n/a | n/a | 853/853 | 100% | 99.6 - 100% |
| Parainfluenza Virus 3 | 23/24e | 95.8% | 78.9 - 99.9% | 819/829f | 98.8% | 97.8 - 99.4% |
| Respiratory Syncytial Virus | 52/52 | 100% | 93.2 - 100% | 714/801g | 89.1% | 86.8 - 91.2% |
| | PPA | 95% CI | NPA | 95% CI | | |
| Coronavirus HKU1 | 23/24 | 95.8% | 78.9 - 99.9% | 827/829c | 99.8% | 99.1 - 100% |
| Coronavirus NL63 | 23/24 | 95.8% | 78.9 - 99.9% | 829/829 | 100% | 99.6 - 100% |
| Human Metapneumovirus | 88/93 | 94.6% | 87.9 - 98.2% | 754/760 | 99.2% | 98.3 - 99.7% |
| Human Rhinovirus/Enterovirus | 190/205 | 92.7% | 88.2 - 95.8% | 613/648 | 94.6% | 92.6 - 96.2% |
Table 6, Clinical Sensitivity and Specificity for the Film Array RP Prospective Clinical Study
4 The FilmArty RP system detected Adenovirus in 1/3 false negative specimens when retested. The Adenovirus in the retested specimen was identified as species C by bi-directional sequence analysis. The remaining two false negative specimens were identified as species C and species B.
8 Adenoviruses were identified in 13/14 false positive specimens using bi-directional sequence analysis. Ten were identified as species C, two as species B, and one as species E.
CoV-HKUI viruses were identified in 2/2 false positive specimens using bi-directional sequence analysis with an alternate assav.
4 Influenza A viruses (2009 H1 subtype) were identified in 3/3 false positive specimens (2/2 false positive compared to influenza A culture alone) using sequence analysis with an alternate assay.
& The FilmArray RP system detected PIV3 in the single false negative specimen when retested.
1 PIV3 viruses were identified in 10/10 false positive specimens using bi-directional sequence analysis.
8 RSV viruses were identified in 83/87 false positive specimens using bi-directional sequence analysis.
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The FilmArray RP system detected a total of 81 mixed infections in the prospective clinical evaluation. This represents 16.1% of the total positive specimens (81/502). Seventy-six (76/81; 93.8%) were double infections, and 5 (5/81; 6.2%) were triple infections. The total number of test results comprising these co-infections was 167. The single most common co-infection (21/81; 25.9%) was Human Rhinovirus/Enterovirus with Respiratory Syncytial Virus. These viruses were the most prevalent in the tested population. Out of the 81 co-infections, 47 contained one or more analytes that had not been detected with the reference/comparator methods, i.e. discrepant co-infection.
| Organism Combinations | Number<br>of<br>Positive<br>Samples | Percentage<br>of Total<br>Samples<br>Tested | Organism | Number of<br>Mixed<br>Infections | Prevalence<br>in Mixed<br>Infections |
|-----------------------------------|-------------------------------------|---------------------------------------------|--------------|----------------------------------|--------------------------------------|
| HRV/Entero + RSV | 21 | 2.46% | Adenovirus | 16 | 19.75% |
| Adenovirus + HRV/Entero | 9 | 1.06% | CoVHKU1 | 10 | 12.35% |
| HRV/Entero + PIV3 | 8 | 0.94% | CoVNL63 | 13 | 16.05% |
| hMPV + HRV/Entero | 7 | 0.82% | hMPV | 22 | 27.16% |
| hMPV + RSV | 4 | 0.47% | HRV/Entero | 56 | 69.14% |
| CoVNL63 + HRV/entero | 4 | 0.47% | FluA/H1 | 0 | 0.00% |
| CoVHKU1 + hMPV | 3 | 0.35% | FluA/H1-2009 | 0 | 0.00% |
| CoVHKU1 + HRV/Entero | 3 | 0.35% | FluA/H3 | 0 | 0.00% |
| CoVHKU1 + RSV | 3 | 0.35% | FluB | 0 | 0.00% |
| CoVNL63 + hMPV | 3 | 0.35% | PIV3 | 14 | 17.28% |
| CoVNL63 + RSV | 3 | 0.35% | RSV | 36 | 44.44% |
| hMPV + PIV3 | 3 | 0.35% | | | |
| Adenovirus + HRV/Entero +<br>PIV3 | 2 | 0.23% | | | |
| Adenovirus + RSV | 2 | 0.23% | | | |
| Adenovirus + CoVNL63 | 1 | 0.12% | | | |
| Adenovirus + hMPV | 1 | 0.12% | | | |
| Adenovirus + PIV3 | 1 | 0.12% | | | |
| CoVNL63 + HRV/Entero + RSV | 1 | 0.12% | | | |
| CoVHKU1 + HRV/Entero +<br>RSV | 1 | 0.12% | | | |
| CoVNL63 + hMPV + RSV | 1 | 0.12% | | | |
| Total Mixed Infections | 81 | 9.50% | | | |
Table 7. Mixed Infections Detected by FilmArray RP and Prevalence of Individual Analytes in Mixed Infections
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Table 8 provides the prevalence and age distribution of FilmArray RP detected analytes in the clinical study. The most prevalent analytes were Human Rhinovirus/Enterovirus, Respiratory Syncytial Virus, and Human Metapneumovirus; these three analytes comprised 78% of all positive results. Positive results were obtained across all age groups tested; however, the majority was detected in children 5 years or younger.
| Analyte | Total<br>(Prevalence) | ≤ 5 years | 6-21<br>years | 22-49<br>years | ≥ 50<br>years |
|------------------------------|-----------------------|-----------|---------------|----------------|---------------|
| Adenovirus | 38 (4.5%) | 32 | 2 | 3 | 1 |
| Coronavirus HKU1 | 25 (2.9%) | 12 | 1 | 8 | 4 |
| Coronavirus NL63 | 23 (2.7%) | 17 | 2 | 2 | 2 |
| Human Metapneumovirus | 94 (11%) | 76 | 4 | 10 | 4 |
| Human Rhinovirus/Enterovirus | 225 (26.4%) | 161 | 24 | 29 | 11 |
| Influenza A (all subtypes) | 11 (1.3%) | 1 | 1 | 7 | 2 |
| Influenza A/H1 | 0 (0%) | 0 | 0 | 0 | 0 |
| Influenza A/2009 H1 | 11 (1.3%) | 1 | 1 | 7 | 2 |
| Influenza A/H3 | 0 (0%) | 0 | 0 | 0 | 0 |
| Influenza B | 0 (0%) | 0 | 0 | 0 | 0 |
| Parainfluenza Virus 3 | 33 (3.9%) | 31 | 1 | 0 | 1 |
| Respiratory Syncytial Virus | 139 (16.3%) | 127 | 3 | 4 | 5 |
Table 8. Prevalence and Age Distribution of Analytes in the Clinical Study
Several analytes, such as influenza viruses were either not encountered in the clinical study or had a low prevalence. To supplement the results of the prospective clinical study, an evaluation of preselected archived samples was performed.
#### Testing of Preselected Archived Specimens
In addition to the prospective clinical study, archived clinical NPS specimens were also tested using the FilmArray RP. The specimens were selected because they had previously tested positive for one of the following organisms: Adenovirus, Enterovirus, Influenzas A/H1, 2009 H1N1, and H3, Influenza B, and Parainfluenza Virus 3, or had been negative by previous testing methods. Prior to testing with the FilmArray RP, the presence or absence of the analyte of interest was confirmed in each specimen using analyte specific PCR and bi-directional sequencing. Of 400 specimens, 349 were confirmed to contain the analyte of interest (or lack thereof for negative specimens). The specimens were organized into "test panels" and randomized such that the users testing the samples with the FilmArray RP were blinded as to the expected test result. Each panel contained specimens known to be positive and negative for the specific analyte being evaluated allowing the calculation of a positive percent agreement (PPA) and a negative percent agreement (NPA). A summary of the available demographic information of the tested samples is provided in Table 9 and the results of the FilmArray testing are presented in Table 10.
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| Total Specimens | | 349 |
|-----------------|------------|------------|
| Sex | Female (%) | 82 (23.5%) |
| | Male (%) | 79 (22.6%) |
| | Unknown | 188(53.9%) |
| Age | Avg | 14.1 |
| | Median | 4.0 |
| | Min | 0.5 |
| | Max | 83.0 |
| Age Range | ≤5 | 89 (25.5%) |
| | 6-21 | 35 (10.0%) |
| | 22-49 | 23 (6.6%) |
| | ≥50 | 14 (4.0%) |
| | Unknowna | 188(53.9%) |
Table 9. Demographic Summary of FilmArray RP Archived Specimen Study
4 Demographic information was not provided for specimens from one source. Because the specimens were provided by a pediatric hospital, it is understood that the age range of specimens was from <1 yrs to 21 yrs.
| | | Table 10. FilmArray Archived Specimen Performance Data Summary | |
|--|--|----------------------------------------------------------------|--|
|--|--|----------------------------------------------------------------|--|
| | Positive Percent Agreement (PPA) | | | Negative Percent Agreement (NPA) | | |
|--------------|----------------------------------|---------|--------------|----------------------------------|---------|-------------|
| | TP/TP+FN | Percent | 95% CI | TN/TN+FP | Percent | 95% CI |
| Adenovirus | 27/27 | 100.0% | 87.2 - 100% | 28/28 | 100.0% | 87.7 - 100% |
| Enterovirus | 22/23 | 95.7% | 78.0 - 99.9% | 90/90 | 100.0% | 96.0 - 100% |
| FluA/H1 | 32/32 | 100.0% | 89.1 - 100% | 127/127 | 100.0% | 97.1 - 100% |
| FluA/H1-2009 | 34/34 | 100.0% | 89.7 - 100% | 125/125 | 100.0% | 97.1 - 100% |
| FluA/H3 | 54/54 | 100.0% | 93.4 - 100% | 105/105 | 100.0% | 96.5 - 100% |
| Influenza B | 30/30 | 100.0% | 88.4 - 100% | 129/129 | 100.0% | 97.2 - 100% |
| PIV3 | 36/36 | 100.0% | 90.3 - 100% | 93/93 | 100.0% | 96.1 - 100% |
{10}------------------------------------------------
#### Selected Analytic Studies
#### Limit of Detection
The analytical sensitivity or Limit of Detection (LoD) for each FilmArray RP analyte (except for Coronavirus HKU1) was determined by testing limiting dilutions of live, quantified viruses. The LoD for Coronavirus HKU1 was determined by testing limiting dilutions of a clinical specimen containing a high viral load of Coronavirus HKU1. LoD is defined as the lowest concentration at which the analyte is consistently detected (detection in ≥95% of samples tested). Simulated NPS sample matrix (cultured human cells in VTM) was spiked with one or more analytes and at least 20 replicates were tested at the LoD concentration. The LoD for each FilmArray RP analyte is listed in Table 11.
| Organism | Strain Identification | Limit of Detection |
|-----------------------------|------------------------------|-------------------------|
| Adenovirus | Serotype 1 (Species C) | 300 TCID50/mL |
| Coronavirus HKU1a | Clinical Specimen (Type B ) | 1.9 x 106 RNA copies/mL |
| Coronavirus NL63 | NR-470 | 5 TCID50/mL |
| Human Metapneumovirus | Type A1 (hMPV-16, IA10-2003) | 2 TCID50/mL |
| Enterovirus | Echovirus 6 | 30,000 TCID50/mL |
| Human Rhinovirus | A1 | 1 TCID50/mL |
| Influenza A/H1 | A/Brisbane/59/07 | 200 TCID50/mL |
| Influenza A/H1 | A/New Caledonia/20/99 | 2,000 TCID50/mL |
| Influenza A/2009 H1 | A/SwineNY/03/2009 | 100 TCID50/mL |
| Influenza A/H3 | A/Wisconsin/67/2005 | 5 TCID50/mL |
| Influenza A/H3 | A/Port Chalmers/1/73 | 50 TCID50/mL |
| Influenza B | B/FL/04/06 | 60 TCID50/mL |
| Influenza B | B/Taiwan/2/62 | 60 TCID50/mL |
| Parainfluenza Virus 3 | Type 3 | 10 TCID50/mL |
| Respiratory Syncytial Virus | Type A | 2 TCID50/mL |
#### Table 11. LoD for Analytes Detected by FilmArray RP
" Coronavirus HKU1 was quantified by a non-FilmArray real-time PCR assay against a standard curve of synthetic Coronavirus HKUI RNA transcript to obtain quantification of the viral nucleic acid in the clinical specimen (RNA copies/mL).
NOTE: Most analytes were re-grown and quantified in TCID-s(50% Tissue Culture Infectious Dose). The unit TCID-so is a measure of infectivity or cytotoxicity rather than number of organisms or copies of nucleic acid. Variability in TCIDs/mL may not accurately reflect differences in the relative of detection between different organisms or different strains of the same organism.
{11}------------------------------------------------
#### Analytical Reactivity (Inclusivity)
The analytical reactivity of the FilmArray RP system assays was evaluated with an inclusivity panel consisting of 99 strains or isolates that represent the genetic, temporal. and geographic diversity of the FilmArray RP analytes. The tested organisms include: 17 Adenovirus, 4 Coronavirus (3 HKU1 and 1 NL63), 10 human Metapneumovirus, 12 Enterovirus, 14 Rhinovirus, 22 Influenza A (including 10 Influenza A/H1, 3 Influenza A/2009 H1 and 9 Influenza A/H3), 11 Influenza B, 3 Parainfluenza virus 3 and 6 Respiratory Syncytial Virus. Each organism was initially tested in a simulated NPS sample matrix at or near the system LoD. Higher concentrations were tested if the analyte was not detected at LoD.
Each of the 99 strains tested in this study were reactive with the FilmArray RP system. Reactivity of the FilmArray RP system with Adenovirus C serotype 6 was 10.000-fold less than the system LoD due to sequence variation in the region targeted by the FilmArray RP Adenovirus assay.
Additional clinical data analyses, and in silico analyses were also carried out to supplement the testing of the inclusivity panel.
Results from inclusivity testing are presented below. The concentration and multiple of LoD at which each strain was detected by the FilmArray RP system is indicated.
| Species | Serotype (Isolate) | Concentration | Multiple of LoD |
|---------|-----------------------|---------------------|-----------------|
| A | 31 | 300 TCID50/mL | 1x |
| | 3 | 300 TCID50/mL | 1x |
| | 7a | 300 TCID50/mL | 1x |
| | 7d2 (Iowa/2001) | 300 TCID50/mL | 1x |
| B | 7h (Iowa/1999) | 300 TCID50/mL | 1x |
| | 11 (Wisconsin/2005) | 3,000 TCID50/mL | 10x |
| | 14 (Missouri/2005) | 300 TCID50/mL | 1x |
| | 21 (Missouri/2005) | 300 TCID50/mL | 1x |
| | 34 (Texas/2005) | 300 TCID50/mL | 1x |
| C | 1 | 300 TCID50/mL | 1x |
| | 2 (New York/2004) | 30,000 TCID50/mL | 100x |
| | 5 | 3,000 TCID50/mL | 10x |
| | 6 (Colorado/2005)* | 3,000,000 TCID50/mL | 10,000x |
| D | 8 | 3,000 TCID50/mL | 10x |
| E | 4a (S Carolina/2004) | 300 TCID50/mL | 1x |
| | 4p3 (New Jersey/2005) | 300 TCID50/mL | 1x |
Table 12. Results of Inclusivity Testing for Adenovirus
{12}------------------------------------------------
| Species | Serotype (Isolate) | Concentration | Multiple of LoD |
|---------|--------------------|---------------|-----------------|
| F | 41 (Indiana/2004) | 300 TCID50/mL | 1x |
*Due to sequence variation, the FilmArray RP Adenovirus assay reacts with adenovirus serotype 6 (species C) less efficiently than with other adenovirus serotypes.
#### Supplemental Adenovirus Reactivity Information (Clinical Data and in silico analyses):
A subset of prospective and retrospective archived clinical specimens that were FilmArray positive for Adenovirus was subjected to PCR and bi-directional sequence analysis. BLAST analysis of the sequence data obtained for a region of the Adenovirus polymerase gene identified adenovirus species B (serotypes 3, 3+11p, 7, 16 and 21), species C (serotypes 1, 2, and 5), and species E (serotype 4) in these clinical specimens. Species B, C and E are the most common Adenovirus species associated with respiratory illness. Species A, D, and F (often associated with conjunctivitis and gastroenteritis) were not identified in clinical specimens.
In addition to laboratory testing, bioinformatics resources were also used to predict reactivity of additional Adenovirus species and serotypes with the FilmArray RP System. Simulated reactivity was based on the number and location of mismatches between the target sequence and the assay primer(s). Table 13 lists the adenovirus types that were not tested by the FilmArray system either in analytical testing. Based on the bioinformatics analysis, the FilmArray RP system is predicted to react with all of the indicated Adenovirus species and serotypes.
| Virus | Species | Serotype | GenBank ID | Simulated FilmArray<br>Adenovirus Result |
|---------------------|---------|----------|------------|------------------------------------------|
| Human<br>Adenovirus | A | 12 | AB330093 | Positive |
| | A | 18 | DQ149610 | Positive |
| | | 16 | AB330097 | Positive |
| | B | 35 | AB052912 | Positive |
| | B | 50 | DQ149643 | Positive |
| | D | 9 | AB330090 | Positive |
| | D | 10 | DQ149615 | Positive |
| | D | 13 | DQ149616 | Positive |
| | D | 15 | DQ149617 | Positive |
| | D | 17 | AB330098 | Positive |
| | D | 19 | DQ149618 | Positive |
| | D | 20 | DQ149619 | Positive |
| | D | 22 | DQ149620 | Positive |
| | D | 23 | DQ149621 | Positive |
| | D | 24 | DQ149622 | Positive |
Table 13. Simulated FilmArray RP Reactivity with Untested Adenovirus Serotypes
{13}------------------------------------------------
| Virus | Species | Serotype | GenBank ID | Simulated FilmArray<br>Adenovirus Result |
|-------|---------|----------|------------|------------------------------------------|
| | | 25 | DQ149623 | Positive |
| | | 26 | DQ149624 | Positive |
| | | 27 | DQ149625 | Positive |
| | | 28 | DQ149626 | Positive |
| | | 29 | DQ149627 | Positive |
| | | 30 | DQ149628 | Positive |
| | | 32 | DQ149629 | Positive |
| | | 33 | DQ149630 | Positive |
| | | 36 | DQ149631 | Positive |
| | | 37 | DQ149632 | Positive |
| | | 38 | DQ149633 | Positive |
| | | 39 | DQ149634 | Positive |
| | | 42 | DQ149635 | Positive |
| | | 43 | DQ149636 | Positive |
| | | 44 | DQ149637 | Positive |
| | | 45 | DQ149638 | Positive |
| | | 46 | DQ149639 | Positive |
| | | 47 | DQ149640 | Positive |
| | | 48 | AB330129 | Positive |
| | | 49 | DQ149641 | Positive |
| | | 51 | DQ149642 | Positive |
| | | 53 | FJ169625 | Positive |
| | F | 40 | AB330121 | Positive |
| | G | 52 | DQ923122 | Positive |
Table 13. Simulated FilmArray RP Reactivity with Untested Adenovirus Serotypes
Table 14. Results of Inclusivity Testing for Coronaviruses
| Type | Strain / Isolate | Concentration ab | Multiple of LoD |
|------|---------------------------------|------------------------------------------------------------|-----------------|
| | Clinical Sample #1120 | $2.08 x 10^6$<br>RNA copies/mL | 1.1x |
| HKU1 | Clinical Sample # 6123 | $1.41 x 10^4$ TCID50/mL<br>~ $1.9 x 10^6$<br>RNA copies/mL | 1x |
| | Clinical Sample #6213 (Type B)c | $1.9 x 10^6$<br>RNA copies/mL | 1x |
| NL63 | BEI Resources NR-470 | 50 TCID50/mL | 1x |
4 Virus contained in Clinical Sample #6123 was grown in culture and quantified (TCIDs) by infectivity assay. b Quantification of the viral RNA contained in clinical specimens containing Coronavirus HKU1 was performed using real-time RT-PCR against a standard curve generated from a synthetic RNA template.
6 Phylogenetic information for Coronavirus HKU1 Clinical Sample #6213 was obtained from bi-directional sequence analysis of the nucleocapsid (N) gene.
{14}------------------------------------------------
| Subtype | | Strain ID | Concentration | Multiple of LoD |
|---------|----|------------|---------------|-----------------|
| A1 | 9 | IA3-2002 | 2 TCID50/mL | 1x |
| | 16 | IA10-2003 | 2 TCID50/mL | 1x |
| A2 | 20 | IA14-2003 | 2 TCID50/mL | 1x |
| | 27 | IA27-2004 | 2 TCID50/mL | 1x |
| B1 | 3 | Peru2-2002 | 2 TCID50/mL | 1x |
| | 5 | Peru3-2003 | 2 TCID50/mL | 1x |
| | 13 | IA7-2003 | 2 TCID50/mL | 1x |
| | 18 | IA18-2003 | 2 TCID50/mL | 1x |
| B2 | 8 | Peru6-2003 | 2 TCID50/mL | 1x |
| | 22 | IA16-2003 | 2 TCID50/mL | 1x |
Table 15. Results of Inclusivity Testing for Human Metapneumovirus
Table 16. Results of Inclusivity Testing for Enterovirus and Rhinovirus
| Species | Strain | Concentrationa | Multiple of LoD |
|---------------|------------------------------------------------|-------------------------------|-----------------|
| Enterovirus A | Coxsackievirus A10 ATCC VR-168 | 30,000 TCID50/mL | 1x |
| | Enterovirus 71 ATCC VR-1432 | 1:30,000<br>dilution of stock | n/a |
| | Enterovirus 71 | 9400 TCID50/mLb | <1x |
| Enterovirus B | Coxsackievirus A9 | 9400 TCID50/mLb | <1x |
| | Coxsackievirus B3 | 30,000 TCID50/mL | 1x |
| | Coxsackievirus B4 | 30,000 TCID50/mL | 1x |
| | Echovirus 6 | 30,000 TCID50/mL | 1x |
| | Echovirus 9 | 9400 TCID50/mLb | <1x |
| | Echovirus 11 | 300,000 TCID50/mL | 10x |
| Enterovirus C | Coxsackievirus A21 /Kuykendall<br>ATCC VR-850 | 30,000 TCID50/mL | 1x |
| | Coxsackievirus A24 DN-19<br>ATCC VR-583 | 30,000 TCID50/mL | 1x |
| Enterovirus D | Enterovirus 68 (F02-3607 corn)<br>ATCC VR-1197 | 30,000 TCID50/mL | 1x |
| Rhinovirus A | A1 | 1 TCID50/mL | 1x |
| | A2 (HGP) ATCC VR-482 | 10 TCID50/mL | 10x |
| | A7 (68-CV11) ATCC VR-1601 | 1 TCID50/mL | 1x |
| | A16 (11757) ATCC VR-283 | 10 TCID50/mL | 10x |
| | A34 (137-3) ATCC VR-507 | 1 TCID50/mL | 1x |
| | A57 (Ch47) ATCC VR-1600 | 100 TCID50/mL | 100x |
{15}------------------------------------------------
| Species | Strain | Concentrationa | Multiple of LoD |
|--------------|--------------------------------|----------------|-----------------|
| | A77 (130-63) ATCC VR-1187 | 1 TCID50/mL | 1x |
| | A85 (50-525-CV54) ATCC VR-1195 | 10 TCID50/mL | 10x |
| | B3 (FEB) ATCC VR-483 | 1 TCID50/mL | 1x |
| | B14 (1059) ATCC VR-284 | 1 TCID50/mL | 1x |
| Rhinovirus B | B17 (33342) ATCC-282 | 100 TCID50/mL | 100x |
| | B27 (5870) ATCC VR-1137 | 1 TCID50/mL | 1x |
| | B42 (56822) ATCC VR-338 | 10 TCID50/mL | 10x |
| | B83 (Baylor 7) ATCC VR-1193 | 1 TCID50/mL | 1x |
ª The LoD for Enterovirus is 30,000 TCID50mL. The LoD for Rhinovirus is 1 TCID50mL.
b Strains were tested below the LoD concentration due to a lesser concentration of virus in the culture fluid.
# Supplemental Human Rhinovirus/Enterovirus Reactivity Information
## (clinical data and in silico analyses):
In addition to the analytical inclusivity testing, BLAST analysis was performed on sequence data (5' UTR) obtained from prospective and retrospective archived clinical specimens that were FilmArray positive for Human Rhinovirus/Enterovirus. The following species and subtypes were identified in clinical specimens:
| Enterovirus Species A: | Enterovirus serotype 71<br>Coxsackievirus A2 and A6 |
|------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Enterovirus Species B: | Echovirus serotypes 3, 6, 7, 11, 15, 21 and 30<br>Coxsackievirus B1, B3 and A9<br>Enterovirus serotypes 81 and 88 |
| Rhinovirus Species A: | Human Rhinovirus serotypes 1B, 8, 9,10, 13, 19, 21, 22, 23,<br>28, 30, 32, 34, 38, 39, 40, 46, 47, 49, 51, 54, 56, 58, 59, 61, 62,<br>66, 68, 75, 77, 78, 80, 82, 98 and 100 |
| Rhinovirus Species B: | Human Rhinovirus serotypes 27, 69, 83 and 91 |
| Rhinovirus Species C: | at least 3 individual strains and 12 distinct isolates |
ªHuman Rhinovirus species C (also known as Enterovirus species D) has not been classified into serotypes.
{16}------------------------------------------------
Enterovirus species C includes Poliovirus types 1-3 (PV1, PV2, and PV3). Simulated reactivity of the FilmArray RP Human Rhinovirus/Enterovirus assays with Enterovirus Species C Poliovirus sequences was generated using a bioinformatics approach. Alignment of assay primer sequences with the GenBank sequences indicates the FilmArray RP assay will react with Poliovirus types 1, 2 and 3, giving a Human Rhinovirus/Enterovirus result.
| Strain | GenBank ID | Simulated FilmArray RP Result |
|--------------------------------------------------------|------------|-------------------------------|
| Human poliovirus 1 strain<br>CHN8264c/GZ/CHN/2004 | FJ769385 | Human Rhinovirus/Enterovirus |
| Human poliovirus 2, complete genome | AY177685 | Human Rhinovirus/Enterovirus |
| Human poliovirus 3 strain IRA10853, complete<br>genome | EU684056 | Human Rhinovirus/Enterovirus |
Table 17. Simulated Reactivity of the FilmArray RP HRV and Entero Assays with Poliovirus Sequences
| Type | Strain | Concentration | Multiple of LoD |
|----------------------------|-------------------------------------|----------------|-----------------|
| Influenza A<br>(H1N1) | A/Brisbane/59/07 | 200 TCID50/mL | 1x |
| | A/Solomon Islands/3/2006 | 200 TCID50/mL | 1x |
| | A/Hawaii/15/01<br>CDC#2001701117 | 1:300 ª | n/a |
| | A/New Caledonia/20/99 | 200 TCID50/mL | 1x |
| | A1/Denver/1/57 | 200 TCID50/mL | 1x |
| | A/Mal/302/54 | 200 TCID50/mL | 1x |
| | A1/FM/1/47 | 200 TCID50/mL | 1x |
| | A/Weiss/43 | 200 TCID50/mL | 1x |
| | A/PR/8/34 | 2000 TCID50/mL | 10x |
| | A/NWS/33 | 200 TCID50/mL | 1x |
| | Swine NY/01/2009 | 100 TCID50/mL | 1x |
| Influenza A<br>(H1N1-2009) | Swine NY/02/2009 | 100 TCID50/mL | 1x |
| | Swine NY/03/2009 | 100 TCID50/mL | 1x |
| | Swine NY/03/2009 | 100 TCID50/mL | 1x |
| Influenza A<br>(H3N2) | A/Brisbane/10/07 | 5 TCID50/mL | 1x |
| | A/Wisconsin/67/2005 | 5 TCID50/mL | 1x |
| | A/NewYork/55/2005<br>CDC#2005705561 | 1:300,000 ª | n/a |
| | A/Victoria/3/75 | 5 TCID50/mL | 1x |
Table 18. Results of Inclusivity Testing for Influenza A
{17}------------------------------------------------
| Type | Strain | Concentration | Multiple of LoD |
|------|------------------------------------------------|---------------|-----------------|
| | A/Port Chalmers/1/73 | 5 TCID50/mL | 1x |
| | A/Aichi/2/68 | 50 TCID50/mL | 10x |
| | A/Hong Kong/8/68 | 5 TCID50/mL | 1x |
| | Alice (vaccine) A/England/42/72<br>ATCC VR-776 | 5 TCID50/mL | 1x |
| | MRC-2 Recombinant strain<br>ATCC VR-777 | 5 TCID50/mL | 1x |
4 Strain was not re-cultured and titered. Dilutions of the original culture/titer from the CDC were used. The HA titer for A/Hawaii/15/01 is unknown and the HA titer for A/NewYork/55/2005 was 256.
#### Supplemental Reactivity Information for Influenza Strains of Human, Swine and Avian Origin (analytical testing and in silico analyses):
Results of testing viral isolates or nucleic acid from viral culture indicate that the FilmArray RP pan-influenza and subtyping assays react with virus of swine and avian origin as expected (Table 19).
| Host | Subtype | Isolate / Strain | Test<br>Concentration | FilmArray<br>Result |
|-------|---------|-----------------------------------------------------------------------|-----------------------|---------------------|
| Swine | H1N1 | Influenza A/Swine/1976/31 | ~1 x 106 EID50/mL | Influenza A H1 a |
| | H1N1 | Influenza A/Swine/Iowa/15/30 | ~1 x 107 EID50/mL | |
| Avian | H1N2 | Kilbourne F63<br>A/NWS/34 (HA) x A/Rockefeller<br>Institute/5/57 (NA) | 14.8 ng RNA b | Influenza A H1 |
Table 19. Results of Inclusivity Testing with Swine and Avian Isolates of Influenza A
ª No reactivity was observed with the 2009 H1 subtyping assay or other FilmArray RP assays.
b Purified and quantified RNA from avian influenza culture was obtained from BEI Resources.
Laboratory testing of influenza A strains was supplemented with in silico predictions of reactivity using bioinformatics and sequence alignments between FilmArray RP assay primers and sequences for influenza A strains of human, swine, and avian origin. For each strain. multiple (3) GenBank IDs were evaluated, corresponding to the gene segments targeted by the FilmArray RP assays (matrix (MA), non-structural (NS) and hemagglutinin(HA)). Simulated reactivity was determined based on the number and location of mismatches in the targeted region. The strains listed in Table 20 are predicted to react with the FilmArray RP pan-influenza A and H1, H1-2009 or H3 subtyping assays as indicated.
{18}------------------------------------------------
| Host | Subtype | Strain | GenBankID | Simulated<br>FilmArray RP<br>Reactivity |
|-------|-----------|----------------------------------------------------------------------|----------------------------------------------------------------------|-----------------------------------------------------------|
| Human | H1N1 | A/California/UR06-0393/2007(H1N1) | CY026540<br>CY026543<br>CY026539 | Influenza A H1 |
| | H1N1-2009 | A/Aalborg/INS133/2009(H1N1) | CY063606<br>CY063610<br>CY063607 | Influenza A H1-2009 |
| | H1N2 | A/New York/297/2003(H1N2) | CY002668<br>CY002665<br>CY002664 | Influenza A H1 |
| | H2N2 | A/Albany/20/1957(H2N2) | CY022013<br>CY022014<br>CY022017 | Influenza A<br>(no subtype<br>detected) |
| | H3N2 | A/Boston/38/2008(H3N2) | CY044581<br>CY044584<br>CY044580 | Influenza A H3 |
| | H5N1 | A/Cambodia/R0405050/2007(H5N1) | HQ200572<br>HQ200573<br>FJ225472 | Influenza A<br>(no subtype<br>detected) |
| | H5N1 | A/Hong Kong/486/97(H5N1) | AF084281<br>AF255368<br>AF115289 | Influenza A<br>(no subtype<br>detected) |
| | H7N2 | A/New York/107/2003(H7N2) | EU587368<br>EU587373<br>EU587374 | Influenza A<br>(no subtype<br>detected) |
| | H7N3 | A/Canada/rv504/2004(H7N3) | CY015006<br>CY015007<br>CY015010 | Influenza A<br>(no subtype<br>detected) |
| Swine | H7N7 | A/Netherlands/219/03(H7N7) | AY340089<br>AY342422<br>AY338459 | Influenza A<br>(no subtype<br>detected) |
| | H9N2 | A/Hong Kong/1073/99(H9N2) | AJ404626<br>AJ278647<br>AJ278649 | Influenza A<br>(no subtype<br>detected) |
| | H1N1 | A/swine/Wisconsin/1/1971(H1N1) | CY022414<br>CY022417<br>CY022413 | Influenza A H1 |
| | H1N2 | A/swine/Hong Kong/NS857/2001(H1N2)<br>A/swine/Sweden/1021/2009(H1N2) | GQ229348<br>GQ229350<br>GQ229347<br>GQ495135<br>GQ495136<br>GQ495132 | Influenza A H1<br>Influenza A<br>(no subtype<br>detected) |
| Avian | H5N1 | A/swine/East Java/UT6010/2007(H5N1) | HM440124<br>HM440111<br>HM440123 | Influenza A<br>(no subtype<br>detected) |
| | H2N2 | A/chicken/New York/13828-3/1995(H2N2)<br>A/Japan/305/1957(H2N2) | CY014822<br>CY014825<br>CY014821<br>CY045804<br>CY014977<br>CY014980 | Influenza A<br>(no subtype<br>detected) |
| Host | Subtype | Strain | GenBankID | Simulated<br>FilmArray RP<br>Reactivity |
| | | A/Korea/426/1968(H2N2) | CY031595 | Influenza A |
| | | | CY031596 | (no subtype<br>detected) |
| | | | CY031599 | |
| | H3N1 | A/blue-winged teal/ALB/452/1983(H3N1) | CY004635 | |
| | | | CY004638 | Influenza A H3 |
| | | | CY005940 | |
| | H3N2 | A/American black duck/North Carolina/675-075/2004(H3N2) | GU051135 | Influenza A |
| | | | GU051136 | (no subtype<br>detected) |
| | | | GU051137 | |
| | H3N5 | A/mallard/Netherlands/2/1999(H3N5) | CY060261 | |
| | | | CY060264 | Influenza A H3 |
| | | | CY060265 | |
| | H3N6 | A/American black duck/New<br>Brunswick/25182/2007(H3N6) | CY047696 | Influenza A |
| | | | CY047697 | (no subtype<br>detected) |
| | | | CY047700 | |
| | H3N7 | A/northern<br>shoveler/California/HKWF1367/2007(H3N7) | CY033372 | |
| | | | CY033375 | Influenza A H3 |
| | | | CY033376 | |
| | H3N8 | A/American black<br>duck/Washington/699/1978(H3N8) | GU052299 | |
| | | | GU052302 | Influenza A H3 |
| | | | GU052300 | |
| | H4N6 | A/blue-winged teal/Minnesota/Sg-<br>00043/2007(H4N6) | CY063977 | Influenza A |
| | | | CY063978 | (no subtype<br>detected) |
| | | | CY063981 | |
| | | A/rook/Rostov-on-Don/26/2007(H5N1) | EU814503 | Influenza A<br>(no subtype<br>detected) |
| | | | EU814504 | |
| | | | EU814507 | |
| | H5N1 | A/turkey/VA/505477-18/2007(H5N1) | GU186509 | Influenza A |
| | | | GU186510 | (no subtype<br>detected) |
| | | | GU186513 | |
| | | A/chicken/Bangladesh/1151-10/2010(H5N1) | HQ156765 | Influenza A |
| | | | HQ156766 | (no subtype<br>detected) |
| | | | HQ156764 | |
| | H5N2 | A/duck/Pennsylvania/10218/1984(H5N2) | AB295603 | Influenza A |
| | | | AB286120 | (no subtype<br>detected) |
| | | | AB286652 | |
| | H5N3 | A/duck/Singapore/F119/3/1997(H5N3) | GU052802 | Influenza A |
| | | | GU052803 | (no subtype<br>detected) |
| | | | GU052805 | |
| | H6N1 | A/duck/PA/486/1969(H6N1) | EU743286 | Influenza A |
| | | | EU743287 | (no subtype<br>detected) |
| | | | EU743289 | |
| | H6N2 | A/mallard/Czech Republic/15902-<br>17K/2009(H6N2) | HQ244430 | Influenza A |
| | | | HQ244433 | (no subtype<br>detected) |
| | | | HQ244434 | |
| | H7N7 | A/mallard/Korea/GH171/2007(H7N7) | FJ750872 | Influenza A<br>(no subtype<br>detected) |
| | | | FJ959087 | |
| | | | FJ959090 | |
| Host | Subtype | Strain | GenBankID | Simulated<br>FilmArray RP<br>Reactivity |
| | | | CY014664 | (no subtype<br>detected) |
| | | | CY014667 | |
| | H10N7 | A/chicken/Germany/N/1949(H10N7) | GQ176136 | Influenza A |
| | | | GQ176135 | (no subtype<br>detected) |
| | | | GQ176132 | |
| | H11N9 | A/duck/Memphis/546/1974(H11N9) | CY014691 | Influenza A |
| | | | GQ257441 | (no subtype<br>detected) |
| | | | CY014687 | |
Table 20. Simulated Reactivity of FilmArray Influenza A Assays with Human, Swine, and Avian Influenza Strains ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ -
{19}------------------------------------------------
Table 20. Simulated Reactivity of FilmArray Influenza A Assays with Human, Swine, and Avian Influenza Strains
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Table 20. Simulated Reactivity of FilmArray Influenza A Assays with Human, Swine, and Avian Influenza Strains
#### Table 21. Results of Inclusivity Testing for Influenza B
| Strain | Concentration | Multiple of LoD |
|-------------------------------------|----------------|-----------------|
| B/FL/04/06 | 60 TCID50/mL | 1x |
| B/Ohio/01/2005<br>CDC#2005743348 | 1:3,000,000 a | n/a |
| B/Florida/07/04 | 60 TCID50/mL | 1x |
| B/Malaysia/2506/04 | 600 TCID50/mL | 10x |
| B/Hong Kong/5/72 ATCC VR-823 | 60 TCID50/mL | 1x |
| B/Taiwan/2/62 ATCC VR-295 | 60 TCID50/mL | 1x |
| B/Maryland/1/59 ATCC VR-296 | 600 TCID50/mL | 10x |
| B/GL/1739/54 ATCC VR-103 | 60 TCID50/mL | 1x |
| B/Allen/45 ATCC VR-102 | 6,000 EID50/mL | n/a |
| B/Lee/40ATCC VR-101 | 60 TCID50/mL | 1x |
| B/Brigit Recombinant<br>ATCC VR-786 | 60 TCID50/mL | 1x |
a Strain was not re-cultured and titered. Dilutions of the original culture/titer from the CDC were used. The HA titer for B/Ohio/01/2005 was 128.
| | | | | Table 22. Results of Inclusivity Testing for Parainfluenza Virus 3 | |
|--|--|--|--|--------------------------------------------------------------------|--|
|--|--|--|--|--------------------------------------------------------------------|--|
| Type | Strain…
