Predicate Devices

Reference Devices

FilmArray Respiratory Panel; BioFire Diagnostics; K160068

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description focuses on the automated nucleic acid testing process using electrowetting and electrochemical detection, with no mention of AI or ML for data analysis or interpretation.

Therapeutic

No
Explanation: This device is an in vitro diagnostic test designed to detect and identify specific viral and bacterial nucleic acids to aid in the diagnosis of respiratory infections. It does not treat, mitigate, or prevent disease.

Diagnostic

Yes

The "Intended Use / Indications for Use" section explicitly states that the ePlex® Respiratory Pathogen (RP) Panel is an "in vitro diagnostic test" and that the detection and identification of nucleic acids "aids in the diagnosis of respiratory infection."

SaMD (Software as a Medical Device)

No

The device is an in vitro diagnostic test that requires a specific hardware instrument (ePlex Instrument) to perform nucleic acid extraction, amplification, and detection. It is not solely software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the ePlex® Respiratory Pathogen (RP) Panel is a "multiplexed nucleic acid in vitro diagnostic test".
Nature of the Test: The test is designed to detect and identify multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs. This is a laboratory test performed on a biological sample outside of the body, which is the definition of an in vitro diagnostic.
Purpose: The test is intended to aid in the diagnosis of respiratory infection when used in conjunction with other clinical and epidemiological information. This aligns with the purpose of IVD devices.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The ePlex® Respiratory Pathogen (RP) Panel is a multiplexed nucleic acid in vitro diagnostic test intended for use on the ePlex® Instrument for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals exhibiting signs and symptoms of respiratory tract infection.

The following virus types, subtypes, and bacteria are identified using the ePlex RP Panel: adenovirus, coronavirus, human metapneumovirus, human rhinovirus/enterovirus, influenza A, influenza A H1, influenza A H1-2009, influenza A H3, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus (RSV) A, respiratory syncytial virus (RSV) B, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory tract infection aids in the diagnosis of respiratory infection when used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex RP Panel may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.

Due to the genetic similarity between human rhinovirus and enterovirus, the ePlex RP Panel cannot reliably differentiate them. If differentiation is required, an ePlex RP Panel positive human rhinovirus/enterovirus result should be followed-up using an alternative method (e.g., cell culture or sequence analysis).

Performance characteristics for influenza A were established when influenza A H1-2009 and A H3 were the predominant influenza A viruses in circulation. Performance of detecting influenza A may vary if other influenza A strains are circulating or a novel influenza A virus emerges. 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.

Product codes

OCC, OEM, OEP, OOU, OTG, OZE, OZY, OZX, OQW, NSU

Device Description

The ePlex RP Panel is an automated qualitative nucleic acid multiplex in vitro diagnostic test for simultaneous detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS). The test is able to detect 15 respiratory viral targets and 2 bacterial targets as summarized in the table below. This test is performed on the ePlex Instrument.

The ePlex Instrument automates all aspects of nucleic acid testing including extraction, amplification, and detection, combining electrowetting and GenMark's eSensor® technology in a single-use cartridge. eSensor technology is based on the principles of competitive DNA hybridization and electrochemical detection. which is highly specific and is not based on fluorescent or optical detection.

Electrowetting, or digital microfluidics, uses electrical fields to directly manipulate discrete droplets on the surface of a hydrophobically coated printed circuit board (PCB). Sample and reagents are moved in a programmable fashion in the ePlex cartridge to complete all portions of the sample processing from nucleic acid extraction to detection.

A sample is loaded onto the ePlex cartridge and nucleic acids are extracted and purified from the specimen via magnetic solid phase extraction. For RNA targets, a reverse transcription step is performed to generate complementary DNA from the RNA. followed by PCR to amplify the targets. Exonuclease digestion creates single-stranded DNA in preparation for eSensor detection.

The target DNA is mixed with ferrocene-labeled signal probes that are complementary to the specific targets on the panel. Target DNA hybridizes to its complementary signal probe and capture probes, which are bound to gold-plated electrodes. The presence of each target is determined by voltammetry which generates specific electrical signals from the ferrocene-labeled signal probe.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Nasopharyngeal swabs (NPS)

Indicated Patient Age Range

All ages

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Clinical Performance - Prospective Clinical Samples:

Sample Size: 2462 evaluable samples out of 2469 eligible prospectively-collected samples (2732 total samples collected). 511 samples tested fresh and 1951 tested after being previously frozen.
Data Source: Clinical nasopharyngeal swab samples in VTM prospectively-collected at 8 clinical sites. Phase 1: March 2013-August 2014 (2218 samples collected and frozen). Phase 2: September 2016-October 2016 (514 samples collected and tested fresh).
Annotation Protocol: Comparator methods included an FDA-cleared multiplexed molecular respiratory pathogen panel and analytically validated PCR tests with bi-directional sequencing for confirmation of RSV subtypes. TP matched ePlex RP Panel and comparator, TN matched negative ePlex RP Panel and negative comparator.

Clinical Performance - Retrospective Clinical Samples:

Sample Size: 446 evaluable samples out of 535 retrospectively collected.
Data Source: Retrospectively collected nasopharyngeal swab in VTM samples from 6 sites that had previously tested positive for one or more target organisms during standard-of-care (SOC) testing.
Annotation Protocol: Composition and integrity of retrospective samples confirmed with the same comparator method used in the prospective clinical study (FDA-cleared multiplexed respiratory pathogen panel). Excluded samples where original SOC positive results for intended organisms were not confirmed by comparator method or for other protocol non-compliance.

Contrived Sample Performance:

Sample Size: 326 evaluable samples out of 327 contrived samples.
Data Source: 104 contrived samples contained one or more low prevalence organisms, and 223 were negative for the contrived organisms.
Annotation Protocol: Not explicitly stated, but implies evaluation against known composition of contrived samples.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Clinical Performance:

Study Type: Prospective, multicenter clinical study and retrospective clinical study.
Sample Size:
- Prospective: 2462 evaluable samples
- Retrospective: 446 evaluable samples
- Contrived: 326 evaluable samples
Key Results (Prospective - Fresh Samples, N=511):
- Adenovirus: PPA 75.0 (40.9-92.9), NPA 99.2 (98.0-99.7)
- Coronavirus: PPA 100 (64.6-100), NPA 99.8 (98.9-100)
- Human Metapneumovirus: PPA ---, NPA 100 (99.3-100)
- Human Rhinovirus/Enterovirus: PPA 96.2 (92.3-98.1), NPA 96.3 (93.7-97.9)
- Influenza A: PPA ---, NPA 100 (99.3-100)
- Influenza A H1: PPA ---, NPA 100 (99.3-100)
- Influenza A H1-2009: PPA ---, NPA 100 (99.3-100)
- Influenza A H3: PPA ---, NPA 100 (99.3-100)
- Influenza B: PPA 100 (20.7-100), NPA 99.8 (98.9-100)
- Parainfluenza Virus 1: PPA 100 (20.7-100), NPA 100 (99.3-100)
- Parainfluenza Virus 2: PPA 92.3 (66.7-98.6), NPA 99.8 (98.9-100)
- Parainfluenza Virus 3: PPA 100 (56.6-100), NPA 100 (99.2-100)
- Parainfluenza Virus 4: PPA 100 (43.9-100), NPA 99.0 (97.7-99.6)
- RSV A: PPA 88.9 (56.5-98.0), NPA 100 (99.2-100)
- RSV B: PPA 90.0 (59.6-98.2), NPA 100 (99.2-100)
- Chlamydia pneumoniae: PPA ---, NPA 100 (99.3-100)
- Mycoplasma pneumoniae: PPA 100 (43.9-100), NPA 99.8 (98.9-100)
Key Results (Prospective - Previously Frozen Samples, N=1951):
- Adenovirus: PPA 90.6 (79.7-95.9), NPA 98.7 (98.1-99.1)
- Coronavirus: PPA 80.9 (72.6-87.2), NPA 99.3 (98.8-99.6)
- Human Metapneumovirus: PPA 94.7 (88.9-97.5), NPA 99.7 (99.3-99.9)
- Human Rhinovirus/Enterovirus: PPA 94.3 (91.3-96.4), NPA 95.6 (94.5-96.5)
- Influenza A: PPA 95.5 (89.9-98.1), NPA 99.8 (99.4-99.9)
- Influenza A H1: PPA ---, NPA 100 (99.8-100)
- Influenza A H1-2009: PPA 98.6 (92.4-99.8), NPA 99.7 (99.3-99.9)
- Influenza A H3: PPA 91.9 (78.7-97.2), NPA 100 (99.8-100)
- Influenza B: PPA 89.2 (79.4-94.7), NPA 99.8 (99.5-99.9)
- Parainfluenza Virus 1: PPA 95.8 (79.8-99.3), NPA 99.9 (99.7-100)
- Parainfluenza Virus 2: PPA 100 (70.1-100), NPA 99.9 (99.7-100)
- Parainfluenza Virus 3: PPA 90.4 (83.2-94.7), NPA 99.7 (99.4-99.9)
- Parainfluenza Virus 4: PPA 100 (56.6-100), NPA 99.9 (99.6-100)
- RSV A: PPA 87.1 (71.1-94.9), NPA 99.9 (99.7-100)
- RSV B: PPA 94.2 (87.1-97.5), NPA 99.9 (99.6-100)
- Chlamydia pneumoniae: PPA 40.0 (11.8-76.9), NPA 99.9 (99.7-100)
- Mycoplasma pneumoniae: PPA 80.0 (37.6-96.4), NPA 99.9 (99.7-100)
Key Results (Retrospective Samples, N=446):
- Adenovirus: PPA 98.2 (90.6-99.7), NPA 99.0 (97.4-99.6)
- Coronavirus: PPA 87.7 (81.2-92.2), NPA 100 (98.8-100)
- Human Metapneumovirus: PPA 71.4 (35.9-91.8), NPA 100 (99.1-100)
- Human Rhinovirus/Enterovirus: PPA 90.2 (77.5-96.1), NPA 95.5 (93.0-97.1)
- Influenza A: PPA 91.5 (83.4-95.8), NPA 100 (99.0-100)
- Influenza A H1: PPA ---, NPA 100 (99.1-100)
- Influenza A H1-2009: PPA 87.1 (71.1-94.9), NPA 100 (99.1-100)
- Influenza A H3: PPA 88.2 (76.6-94.5), NPA 100 (99.0-100)
- Influenza B: PPA 100 (20.7-100), NPA 100 (99.1-100)
- Parainfluenza Virus 1: PPA 89.6 (77.8-95.5), NPA 99.7 (98.6-100)
- Parainfluenza Virus 2: PPA 90.2 (79.0-95.7), NPA 100 (99.0-100)
- Parainfluenza Virus 3: PPA 100 (34.2-100), NPA 100 (99.1-100)
- Parainfluenza Virus 4: PPA 90.0 (69.9-97.2), NPA 100 (99.1-100)
- RSV A: PPA 92.6 (76.6-97.9), NPA 100 (99.1-100)
- RSV B: PPA 95.5 (78.2-99.2), NPA 100 (99.1-100)
- Chlamydia pneumoniae: PPA 100 (20.7-100), NPA 100 (99.1-100)
- Mycoplasma pneumoniae: PPA 100 (64.6-100), NPA 100 (99.1-100)
Key Results (Contrived Samples):
- Chlamydia pneumoniae: PPA 100 (93.1-100), NPA 100 (98.6-100)
- Influenza A H1: PPA 100 (93.0-100), NPA 100 (98.6-100)
Clinical Study ePlex Instrument Performance:
- 95.3% (95% CI: 94.5%-96.0%) generated valid results on the first attempt.
- Final validity rate after re-test: 99.8% (95% CI: 99.5%-99.9%).

Selected Analytical Studies:

Limit of Detection (LoD): Identified and verified for each viral and bacterial target using quantified reference strains/isolates, tested at least 20 replicates per target. LoD defined as lowest concentration detected at least 95% of the time. (Specific LoD concentrations are provided in Table 14).
Analytical Reactivity (Inclusivity): 101 strains/isolates representing genetic, temporal, and geographic diversity evaluated. All 101 strains/isolates detected. (Results are shown in Tables 15-25).
Analytical Specificity (Cross-Reactivity and Exclusivity):
- Cross-Reactivity with ePlex RP Panel Target Organisms: Evaluated at high concentrations (1 x 10^5 TCID50/mL or >1 x 10^5 EID50/mL for viruses, 1 x 10^6 CFU/mL or CCU/mL for bacterial isolates, or 1 x 10^6 copies/mL for in vitro transcripts). No cross-reactivity observed between on-panel viruses or bacteria. (Results in Table 26).
- Exclusivity with Organisms Not Detected by the ePlex RP Panel: Evaluated at high concentrations (1 x 10^5 TCID50/mL or copies/mL for viruses, 1 x 10^6 CFU/mL for bacterial and yeast isolates, or 1 x 10^6 copies/mL for plasmid DNA or genomic RNA). No cross-reactivity observed between off-panel viruses, bacteria, or fungi with the ePlex RP Panel targets. (Results in Table 27).
Reproducibility: Multisite study (3 sites, 2 external, 1 internal) with 3 or 4 towers per instrument, performed by 2 operators on 6 days using 3 unique lots. Reproducibility panel with 6 organisms (7 targets) at 3 concentrations (moderate positive, low positive, negative) tested in triplicate.
- Percent agreement with expected results was 100% for all 7 targets for moderate positive and negative panels.
- For low positive panel: 100% agreement for Coronavirus, Human Metapneumovirus, Influenza A, Influenza A H3, Parainfluenza 1, and RSV A. 91.6% agreement for Adenovirus. (Detailed results in Tables 28-34, and Table 35 for targets not included in the reproducibility panel).
Samples with Co-Detected Organisms: Evaluated detection of two RP Panel organisms, one at low concentration (1-3x LoD) and the second at high concentration (1 x 10^5 TCID/mL). Demonstrated ability to detect both organisms. (Results in Table 36).
Sample Matrix Equivalency: Demonstrated equivalency between natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples) and viral transport media for targets spiked at approximately 10x LoD. No difference observed.
Interfering Substances: Evaluated common substances found in respiratory samples or medications. Quantified representative viral and bacterial strains diluted to 1x LoD and tested in triplicate. All substances and organisms tested were compatible. No interfering substances found to inhibit the ePlex RP Panel at concentrations tested. (Substances listed in Table 37).
Carryover and Cross-contamination: Tested in a checkerboard approach over 5 separate runs on 5 days using high positive (Parainfluenza virus 3 at 20,000x LoD) and negative samples. 100% of PIV 3 positive samples detected and 100% of PIV 3 negative samples were "No Target Detected". No carryover or cross-contamination observed.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Sensitivity: Reflected by Positive Percent Agreement (PPA).
Specificity: Reflected by Negative Percent Agreement (NPA).
Positive Percent Agreement (PPA): Calculated by dividing True Positive (TP) results by (TP + False Negative (FN)) results.
Negative Percent Agreement (NPA): Calculated by dividing True Negative (TN) results by (TN + False Positive (FP)) results.
Prevalence: Provided for each organism in tables 7 and 8.
Confidence Intervals (CI): Two-sided 95% confidence intervals are provided for PPA and NPA.

(Specific values are listed in the 'Summary of Performance Studies' section, taken from Tables 7, 8, 10, and 11).

Predicate Device(s)

FilmArray Respiratory Panel; BioFire Diagnostics; K160068

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 866.3980 Respiratory viral panel multiplex nucleic acid assay.

(a)
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.
(b)
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.

Summary

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo is circular and contains the words "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" around the perimeter. Inside the circle is an abstract symbol that resembles a stylized caduceus or a series of human profiles.

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

June 9, 2017

GenMark Diagnostics, Incorporated Alan Maderazo, Ph.D., RAC Vice President, Quality, Regulatory and Clinical Affairs 5964 La Place Court Carlsbad, CA 92008

Re: K163636

Trade/Device Name: ePlex® Respiratory Pathogen (RP) Panel Regulation Number: 21 CFR 866.3980 Regulation Name: Respiratory Viral Panel Multiplex Nucleic Acid Assay Regulatory Class: II Product Code: OCC, OEM, OEP, OOU, OTG, OZE, OZX, OZY, OQW, NSU Dated: May 3, 2017 Received: May 5, 2017

Dear Dr. Maderazo:

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. 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 Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. 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 the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours.

Tamara V. Feldblyum -S for

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

Enclosure

2

Indications for Use

510(k) Number (if known) K163636

Device Name

ePlex® Respiratory Pathogen (RP) Panel

Indications for Use (Describe)

The ePlex® Respiratory Pathogen (RP) Panel is a multiplexed nucleic acid in vitro diagnostic test intended for use on the ePlex® Instrument for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals exhibiting signs and symptoms of respiratory tract infection.

The following virus types, subtypes, and bacteria are identified using the ePlex RP Panel: adenovirus, coronavirus, human metapneumovirus, human rhinovirus/enterovirus, influenza A, influenza A H1, influenza A H1-2009, influenza A H3, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus (RSV) A, respiratory syncytial virus (RSV) B, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory tract infection aids in the diagnosis of respiratory infection when used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms; the organism(s) detected by the ePlex RP Panel may not be the definite cause of disease. Additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.

Due to the genetic similarity between human rhinovirus and enterovirus, the ePlex RP Panel cannot reliably differentiate them. If differentiation is required, an ePlex RP Panel positive human rhinovirus/enterovirus result should be followed-up using an alternative method (e.g., cell culture or sequence analysis).

Performance characteristics for influenza A were established when influenza A H1-2009 and A H3 were the predominant influenza A viruses in circulation. Performance of detecting influenza A may vary if other influenza A strains are circulating or a novel influenza A virus emerges. 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.

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Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)

| | Over-The-Counter Use (21 CFR 801 Subpart C)

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510(k) Summary

Summary of Safety and Effectiveness

Submitter Information
Submitter:	GenMark Diagnostics, Incorporated
	5964 La Place Court
	Carlsbad, CA 92008
Manufacturer:	GenMark Diagnostics, Incorporated
	5964 La Place Court
	Carlsbad, CA 92008
Establishment Registration Number:	3008632402
Contact:	Alan Maderazo, Ph.D., RAC
	Vice President, Quality, Regulatory and Clinical Affairs
Phone:	760-448-4308
Fax:	760-683-6961
E-mail:	Al.Maderazo@genmarkdx.com
Alternate Contact:	Joseph McMullen
	Consultant, Regulatory Affairs
Phone:	760-410-5052
Fax:	760-683-6961
E-mail:	Joseph.McMullen@genmarkdx.com
Date Prepared:	December 21, 2016

Name of Device and Classification

Product Name:	ePlex® Respiratory Pathogen (RP) Panel
Device Classification:	866.3980, Respiratory viral panel multiplex nucleic acid assay, Class I
Product Code(s):	OCC, OEM, OOU, OEP, OTG, OQW, OZE, OZY, OZX, NSU

Predicate Device

Predicate: FilmArray Respiratory Panel; BioFire Diagnostics; K160068

5

Device Description

The ePlex RP Panel is an automated qualitative nucleic acid multiplex in vitro diagnostic test for simultaneous detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS). The test is able to detect 15 respiratory viral targets and 2 bacterial targets as summarized in the table below. This test is performed on the ePlex Instrument.

Target	Classification (Genome Type)
Adenovirus (A-F)	Adenovirus (DNA)
Coronavirus	Coronavirus (RNA)
(229E, HKU1, NL63, OC43)
Human Metapneumovirus	Paramyxovirus (RNA)
Human Rhinovirus/
Enterovirus	Picornavirus (RNA)
Influenza A
Influenza A H1
Influenza A H1-2009	Orthomyxovirus (RNA)
Influenza A H3
Influenza B
Parainfluenza Virus 1
Parainfluenza Virus 2	Paramyxovirus (RNA)
Parainfluenza Virus 3
Parainfluenza Virus 4
Respiratory Syncytial Virus A	Paramyxovirus (RNA)
Respiratory Syncytial Virus B
Chlamydia pneumoniae	Bacterium (DNA)
Mycoplasma pneumoniae	Bacterium (DNA)

Targets Detected by the ePlex RP Panel

The ePlex Instrument automates all aspects of nucleic acid testing including extraction, amplification, and detection, combining electrowetting and GenMark's eSensor® technology in a single-use cartridge. eSensor technology is based on the principles of competitive DNA hybridization and electrochemical detection. which is highly specific and is not based on fluorescent or optical detection.

Electrowetting, or digital microfluidics, uses electrical fields to directly manipulate discrete droplets on the surface of a hydrophobically coated printed circuit board (PCB). Sample and

6

reagents are moved in a programmable fashion in the ePlex cartridge to complete all portions of the sample processing from nucleic acid extraction to detection.

A sample is loaded onto the ePlex cartridge and nucleic acids are extracted and purified from the specimen via magnetic solid phase extraction. For RNA targets, a reverse transcription step is performed to generate complementary DNA from the RNA. followed by PCR to amplify the targets. Exonuclease digestion creates single-stranded DNA in preparation for eSensor detection.

The target DNA is mixed with ferrocene-labeled signal probes that are complementary to the specific targets on the panel. Target DNA hybridizes to its complementary signal probe and capture probes, which are bound to gold-plated electrodes. The presence of each target is determined by voltammetry which generates specific electrical signals from the ferrocenelabeled signal probe.

Intended Use/Indications for Use

The ePlex® Respiratory Pathogen (RP) Panel is a multiplexed nucleic acid in vitro diagnostic test intended for use on the ePlex Instrument for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals exhibiting signs and symptoms of respiratory tract infection.

The following virus types, subtypes, and bacteria are identified using the ePlex RP Panel: adenovirus, coronavirus, human metapneumovirus, human rhinovirus/enterovirus, influenza A, influenza A H1, influenza A H1-2009, influenza A H3, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, respiratory syncytial virus (RSV) A, respiratory syncytial virus (RSV) B, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory tract infection aids in the diagnosis of respiratory infection when used in conjunction with other clinical and epidemiological

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information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions.

Negative results in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Positive results do not rule out co-infection with other organisms: the organism(s) detected by the ePlex RP Panel may not be the definite cause of disease. Additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) may be necessary when evaluating a patient with possible respiratory tract infection.

Due to the genetic similarity between human rhinovirus and enterovirus, the ePlex RP Panel cannot reliably differentiate them. If differentiation is required, an ePlex RP Panel positive human rhinovirus/enterovirus result should be followed-up using an alternative method (e.g., cell culture or sequence analysis).

Performance characteristics for influenza A were established when influenza A H1-2009 and A H3 were the predominant influenza A viruses in circulation. Performance of detecting influenza A may vary if other influenza A strains are circulating or a novel influenza A virus emerges. 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.

Summary of Technological Characteristics of the Device Compared to the Predicate Device

The GenMark ePlex Respiratory Pathogen (RP) Panel ("Proposed Device") and the legally marketed device, FilmArray Respiratory Panel ("Predicate Device") are described below:

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Characteristic	Proposed Device	Predicate Device
Product Name	ePlex Respiratory Pathogen (RP) Panel	FilmArray Respiratory Panel
Manufacturer	GenMark Diagnostics	BioFire Diagnostics
Regulation	866.3980
Respiratory viral panel multiplex
nucleic acid assay	866.3980
Respiratory viral panel multiplex
nucleic acid assay
Product Code(s)	OCC, OEM, OOU, OEP, OTG, OZE,
OQW, OZY, OZX, NSU	OCC, OEM, OOU, OEP, OTG,
OQW, OOI, OZZ, OZY, OZX
Device Class	Class II	Class II
Organisms
Detected	Influenza A
Influenza A subtype H1
Influenza A subtype H3
Influenza A subtype H1-2009
Influenza B
Respiratory Syncytial Virus A and B
Parainfluenza Virus 1, 2, 3, 4
Human Metapneumovirus
Adenovirus
Human Rhinovirus/Enterovirus
Coronavirus
Chlamydia pneumoniae
Mycoplasma pneumoniae	Influenza A
Influenza A subtype H1
Influenza A subtype H3
Influenza A subtype H1-2009
Influenza B
Respiratory Syncytial Virus
Parainfluenza Virus 1, 2, 3, 4
Human Metapneumovirus
Adenovirus
Human Rhinovirus/Enterovirus
Coronavirus HKU1, NL63, 229E,
OC43
Bordetella pertussis
Chlamydia pneumoniae
Mycoplasma pneumoniae
Analyte	RNA/DNA	RNA/DNA
Technological
Principles	Multiplex nucleic acid amplification test
(NAAT)	Multiplex nucleic acid amplification
test (NAAT)
Specimen Type	Nasopharyngeal swabs (NPS) in VTM	Nasopharyngeal swabs (NPS) in
VTM
Chemistry	Reagents contained within cartridge to
allow: sample lysis and nucleic acid
extraction, RT-PCR amplification, and
hybridization-based electrochemical
detection reagents.	Nested multiplex RT-PCR followed
by high resolution melting analysis to
confirm identity of amplified
product.
Instrumentation	ePlex Instrument	FilmArray Instrument
Test
Interpretation	Automated test interpretation and report
generation. User cannot access raw data.	Automated test interpretation and
report generation. User cannot
access raw data.
Sample
Preparation
Method	Sample processing is automated in the
ePlex RP cartridge.	Sample processing is automated in
the FilmArray RP pouch.
Controls	Eight internal controls are used to
demonstrate that sample extraction,
amplification and detection processes
functioned as intended.	Two controls are included in each
reagent pouch to control for sample
processing and both stages of PCR
and melt analysis.
Characteristic	Proposed Device	Predicate Device
User Complexity	Moderate	Moderate
Reagent Storage	Cartridge (which contains reagents) is
stored at refrigerated (2-8°C) temperature.	Reagents are stored at room
temperature.

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Analysis of the similarities and differences indicate that the devices are substantially equivalent in their intended uses/indications for use, and are generally the same regarding user process, ease of use and general operator protocol. Comparison of technological similarities and differences between the proposed device and the predicate do not raise new or different questions of safety and effectiveness, and therefore render the proposed device as substantially equivalent to the predicate device.

Expected Values

A prospective, multicenter clinical study was conducted to evaluate the clinical performance of the ePlex RP Panel in nasopharyngeal swab samples. 2462 nasopharyngeal swab samples were prospectively-collected at 8 collection sites in 2 phases from patients of all ages and genders presenting with signs and/or symptoms of respiratory infection. In the first phase from March 2013 through August 2014, 1951 samples were prospectively-collected and frozen; from September 2016 through October 2016, 511 samples were prospectively-collected and tested fresh (never frozen). The expected values of individual analytes based on ePlex RP Panel results in prospective samples for each phase are summarized in Tables 1-4.

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By Age Group in the Prospective Clinical Evaluation (Phase 1: March 2013 – August 2014)
	All Ages
(N=1951)	Age 0-1
(N=315)	Age >1-5
(N=250)	Age >5-21
(N=246)	Age >21-65
(N=745)	Age >65
(N=395)
Organism	n (%)	n (%)	n (%)	n (%)	n (%)	n (%)
Adenovirus	72 (3.7)	31 (9.8)	24 (9.6)	7 (2.8)	7 (0.9)	3 (0.8)
Coronavirus	102 (5.2)	19 (6.0)	18 (7.2)	16 (6.5)	32 (4.3)	17 (4.3)
Human Metapneumovirus	113 (5.8)	22 (7.0)	28 (11.2)	6 (2.4)	31 (4.2)	26 (6.6)
Human
Rhinovirus/Enterovirus	388 (19.9)	113 (35.9)	94 (37.6)	58 (23.6)	87 (11.7)	36 (9.1)
Influenza A	110 (5.6)	6 (1.9)	18 (7.2)	20 (8.1)	49 (6.6)	17 (4.3)
Influenza A H1	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza A H1-2009	76 (3.9)	4 (1.3)	13 (5.2)	14 (5.7)	37 (5.0)	8 (2.0)
Influenza A H3	34 (1.7)	1 (0.3)	5 (2.0)	6 (2.4)	12 (1.6)	10 (2.5)
Influenza B	62 (3.2)	4 (1.3)	9 (3.6)	10 (4.1)	24 (3.2)	15 (3.8)
Parainfluenza Virus 1	24 (1.2)	4 (1.3)	12 (4.8)	4 (1.6)	3 (0.4)	1 (0.3)
Parainfluenza Virus 2	10 (0.5)	4 (1.3)	4 (1.6)	0 (0.0)	2 (0.3)	0 (0.0)
Parainfluenza Virus 3	99 (5.1)	31 (9.8)	20 (8.0)	3 (1.2)	27 (3.6)	18 (4.6)
Parainfluenza Virus 4	7 (0.4)	3 (1.0)	2 (0.8)	1 (0.4)	1 (0.1)	0 (0.0)
RSV A	28 (1.4)	13 (4.1)	6 (2.4)	3 (1.2)	2 (0.3)	4 (1.0)
RSV B	83 (4.3)	33 (10.5)	19 (7.6)	6 (2.4)	15 (2.0)	10 (2.5)
Chlamydia pneumoniae	3 (0.2)	0 (0.0)	0 (0.0)	1 (0.4)	1 (0.1)	1 (0.3)
Mycoplasma pneumoniae	5 (0.3)	1 (0.3)	1 (0.4)	2 (0.8)	1 (0.1)	0 (0.0)

Table 1: Expected Value (As Determined by ePlex RP Panel) Summary
Eroup in the Prospective Clinical Fraluation (Phase 1: March 2013 – Aug Ry Age August 2014)

Table 2: Expected Value (As Determined by ePlex RP Panel) Summary By Age Group in the Prospective Clinical Evaluation (Phase 2: September 2016 – October 2016)

By Age Group in the Prospective Clinical Evaluation (Phase 2: September 2016 – October 2016)
Organism	All Ages
(N=511)
n (%)	Age 0-1
(N=73)
n (%)	Age >1-5
(N=75)
n (%)	Age >5-21
(N=75)
n (%)	Age >21-65
(N=181)
n (%)	Age >65
(N=107)
n (%)
Adenovirus	10 (2.0)	3 (4.1)	4 (5.3)	1 (1.3)	1 (0.6)	1 (0.9)
Coronavirus	8 (1.6)	2 (2.7)	0 (0.0)	1 (1.3)	4 (2.2)	1 (0.9)
Human Metapneumovirus	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Human Rhinovirus/Enterovirus	188 (36.8)	37 (50.7)	40 (53.3)	33 (44.0)	58 (32.0)	20 (18.7)
Influenza A	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza A H1	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza A H1-2009	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza A H3	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza B	2 (0.4)	0 (0.0)	0 (0.0)	1 (1.3)	1 (0.6)	0 (0.0)
Parainfluenza Virus 1	1 (0.2)	0 (0.0)	1 (1.3)	0 (0.0)	0 (0.0)	0 (0.0)
Parainfluenza Virus 2	13 (2.5)	3 (4.1)	4 (5.3)	3 (4.0)	2 (1.1)	1 (0.9)
Parainfluenza Virus 3	5 (1.0)	2 (2.7)	1 (1.3)	1 (1.3)	1 (0.6)	0 (0.0)
Parainfluenza Virus 4	8 (1.6)	1 (1.4)	4 (5.3)	2 (2.7)	1 (0.6)	0 (0.0)
RSV A	8 (1.6)	5 (6.8)	3 (4.0)	0 (0.0)	0 (0.0)	0 (0.0)
RSV B	9 (1.8)	3 (4.1)	4 (5.3)	0 (0.0)	2 (1.1)	0 (0.0)
Chlamydia pneumoniae	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Mycoplasma pneumoniae	4 (0.8)	0 (0.0)	1 (1.3)	2 (2.7)	1 (0.6)	0 (0.0)

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2014)
Organism	All Sites
(N=1951)
n (%)	Site 1
(N=165)
n (%)	Site 2
(N=248)
n (%)	Site 3
(N=350)
n (%)	Site 4
(N=892)
n (%)	Site 5
(N=296)
n (%)
Adenovirus	72 (3.7)	4 (2.4)	8 (3.2)	28 (8.0)	23 (2.6)	9 (3.0)
Coronavirus	102 (5.2)	8 (4.8)	11 (4.4)	32 (9.1)	29 (3.3)	22 (7.4)
Human Metapneumovirus	113 (5.8)	10 (6.1)	23 (9.3)	27 (7.7)	30 (3.4)	23 (7.8)
Human Rhinovirus/Enterovirus	388 (19.9)	27 (16.4)	33 (13.3)	61 (17.4)	185 (20.7)	82 (27.7)
Influenza A	110 (5.6)	5 (3.0)	21 (8.5)	48 (13.7)	19 (2.1)	17 (5.7)
Influenza A H1	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza A H1-2009	76 (3.9)	3 (1.8)	22 (8.9)	31 (8.9)	5 (0.6)	15 (5.1)
Influenza A H3	34 (1.7)	2 (1.2)	0 (0.0)	18 (5.1)	12 (1.3)	2 (0.7)
Influenza B	62 (3.2)	9 (5.5)	9 (3.6)	9 (2.6)	19 (2.1)	16 (5.4)
Parainfluenza Virus 1	24 (1.2)	0 (0.0)	0 (0.0)	5 (1.4)	2 (0.2)	17 (5.7)
Parainfluenza Virus 2	10 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	10 (1.1)	0 (0.0)
Parainfluenza Virus 3	99 (5.1)	13 (7.9)	3 (1.2)	28 (8.0)	41 (4.6)	14 (4.7)
Parainfluenza Virus 4	7 (0.4)	0 (0.0)	0 (0.0)	1 (0.3)	4 (0.4)	2 (0.7)
RSV A	28 (1.4)	4 (2.4)	6 (2.4)	7 (2.0)	4 (0.4)	7 (2.4)
RSV B	83 (4.3)	6 (3.6)	15 (6.0)	24 (6.9)	15 (1.7)	23 (7.8)
Chlamydia pneumoniae	3 (0.2)	0 (0.0)	0 (0.0)	1 (0.3)	2 (0.2)	0 (0.0)
Mycoplasma pneumoniae	5 (0.3)	1 (0.6)	0 (0.0)	3 (0.9)	0 (0.0)	1 (0.3)

Table 3: Expected Value (As Determined by ePlex RP Panel) Summary By Sample Collection Site in the Prospective Clinical Evaluation (Phase 1: March 2013 – August

Table 4: Expected Value (As Determined by ePlex RP Panel) Summary By Sample Collection Site in the Prospective Clinical Evaluation (Phase 2: September 2016 – Octobe 2016)

October 2010)
	All Sites	Site 5	Site 6	Site 7	Site 8
Organism	(N=511)	(N=49)	(N=101)	(N=161)	(N=200)
	n (%)	n (%)	n (%)	n (%)	n (%)
Adenovirus	10 (2.0)	2 (4.1)	3 (3.0)	3 (1.9)	2 (1.0)
Coronavirus	8 (1.6)	0 (0.0)	2 (2.0)	4 (2.5)	2 (1.0)
Human Metapneumovirus	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Human Rhinovirus/Enterovirus	188 (36.8)	24 (49.0)	49 (48.5)	62 (38.5)	53 (26.5)
Influenza A	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza A H1	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza A H1-2009	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza A H3	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Influenza B	2 (0.4)	1 (2.0)	0 (0.0)	0 (0.0)	1 (0.5)
Parainfluenza Virus 1	1 (0.2)	0 (0.0)	0 (0.0)	1 (0.6)	0 (0.0)
Parainfluenza Virus 2	13 (2.5)	2 (4.1)	4 (4.0)	3 (1.9)	4 (2.0)
Parainfluenza Virus 3	5 (1.0)	2 (4.1)	2 (2.0)	0 (0.0)	1 (0.5)
Parainfluenza Virus 4	8 (1.6)	1 (2.0)	1 (1.0)	4 (2.5)	2 (1.0)
RSV A	8 (1.6)	0 (0.0)	8 (7.9)	0 (0.0)	0 (0.0)
RSV B	9 (1.8)	1 (2.0)	4 (4.0)	0 (0.0)	4 (2.0)
Chlamydia pneumoniae	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Mycoplasma pneumoniae	4 (0.8)	0 (0.0)	3 (3.0)	0 (0.0)	1 (0.5)

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Summary of Performance Data

Clinical performance

Comparator Method

The performance of the ePlex RP Panel was compared to an FDA-cleared multiplexed molecular respiratory pathogen panel and analytically validated PCR tests with bi-directional sequencing for confirmation of RSV subtypes.. Details of the comparator method are described in Table 5.

Target	Comparator Method
Adenovirus	FDA-cleared multiplexed molecular respiratory pathogen
panel
Coronavirus
Human Metapneumovirus
Human Rhinovirus/Enterovirus
Influenza A
Influenza A H1
Influenza A H1-2009
Influenza A H3
Influenza B
Parainfluenza Virus 1
Parainfluenza Virus 2
Parainfluenza Virus 3
Parainfluenza Virus 4
Respiratory Syncytial Virus A	FDA-cleared multiplexed molecular respiratory pathogen
Respiratory Syncytial Virus B	FDA-cleared multiplexed molecular respiratory pathogen
panel followed by a PCR test with bi-directional sequencing
confirmation
Chlamydia pneumoniae	FDA-cleared multiplexed molecular respiratory pathogen
panel
Mycoplasma pneumoniae

Table 5: Comparator Methods Used to Assess ePlex RP Panel Clinical Performance

Prospective Clinical Samples

Clinical performance was evaluated in clinical nasopharyngeal swab samples in VTM prospectively-collected at 8 clinical sites in 2 phases. From March 2013 through August 2014, 2218 samples were prospectively-collected and frozen; from September 2016 through October

13

2016, 514 samples were prospectively-collected and tested fresh (never frozen). A total of 2732 samples were collected across the 2 phases. Prior to the start of investigational testing, 263 samples were withdrawn (251 had sample handling deviations, 9 were tested outside of protocol timelines, 2 had insufficient volume, and 1 had incomplete documentation). Of the 2469 prospectively-collected samples eligible for testing, 2462 were evaluable. Samples with final, valid results and a valid comparator result were considered evaluable. Seven prospectivelycollected samples were not evaluable because they did not have final, valid ePlex RP Panel results and were excluded from performance evaluations. Demographic information for prospectively-collected samples is described in Table 6. Subjects enrolled in this study were from a diverse demographic distribution and represent the intended patient population.

Table 6: Subject Demographic Data for Prospectively-Collected Samples by Collection Site (N=2462)

| | All Sites
N=2462
n (%) | Site 1
N=165
n (%) | Site 2
N=248
n (%) | Site 3
N=350
n (%) | Site 4
N=892
n (%) | Site 5
N=345
n (%) | Site 6
N=101
n (%) | Site 7
N=161
n (%) | Site 8
N=200
n (%) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
|-------------|------------------------------|--------------------------|--------------------------|--------------------------|--------------------------|--------------------------|--------------------------|--------------------------|--------------------------|-----|------------|-----------|----------|-----------|------------|-----------|-----------|---------|-----------|-------|------------|----------|----------|-----------|----------|------------|-----------|----------|----------|--------|------------|----------|---------|-----------|----------|------------|-----------|-----------|----------|---------|------------|-----------|------------|-----------|------------|-----------|---------|-----------|-----------|------|------------|-----------|-----------|-----------|------------|----------|---------|-----------|-----------|
| Sex | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Male | 1247 (50.6) | 96 (58.2) | 118 (47.6) | 186 (53.1) | 450 (50.4) | 188 (54.5) | 43 (42.6) | 84 (52.2) | 82 (41.0) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Female | 1215 (49.4) | 69 (41.8) | 130 (52.4) | 164 (46.9) | 442 (49.6) | 157 (45.5) | 58 (57.4) | 77 (47.8) | 118 (59.0) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Age (years) | | | | | | | | | | 0-1 | 388 (15.8) | 17 (10.3) | 21 (8.5) | 74 (21.1) | 164 (18.4) | 45 (13.0) | 28 (27.7) | 3 (1.9) | 36 (18.0) | > 1-5 | 325 (13.2) | 12 (7.3) | 22 (8.9) | 62 (17.7) | 64 (7.2) | 100 (29.0) | 39 (38.6) | 16 (9.9) | 10 (5.0) | > 5-21 | 321 (13.0) | 15 (9.1) | 6 (2.4) | 38 (10.9) | 82 (9.2) | 116 (33.6) | 34 (33.7) | 18 (11.2) | 12 (6.0) | > 21-65 | 926 (37.6) | 87 (52.7) | 131 (52.8) | 98 (28.0) | 385 (43.2) | 55 (15.9) | 0 (0.0) | 92 (57.1) | 78 (39.0) | > 65 | 502 (20.4) | 34 (20.6) | 68 (27.4) | 78 (22.3) | 197 (22.1) | 29 (8.4) | 0 (0.0) | 32 (19.9) | 64 (32.0) |
| Age (years) | | | | | | | | | | 0-1 | 388 (15.8) | 17 (10.3) | 21 (8.5) | 74 (21.1) | 164 (18.4) | 45 (13.0) | 28 (27.7) | 3 (1.9) | 36 (18.0) | > 1-5 | 325 (13.2) | 12 (7.3) | 22 (8.9) | 62 (17.7) | 64 (7.2) | 100 (29.0) | 39 (38.6) | 16 (9.9) | 10 (5.0) | > 5-21 | 321 (13.0) | 15 (9.1) | 6 (2.4) | 38 (10.9) | 82 (9.2) | 116 (33.6) | 34 (33.7) | 18 (11.2) | 12 (6.0) | > 21-65 | 926 (37.6) | 87 (52.7) | 131 (52.8) | 98 (28.0) | 385 (43.2) | 55 (15.9) | 0 (0.0) | 92 (57.1) | 78 (39.0) | > 65 | 502 (20.4) | 34 (20.6) | 68 (27.4) | 78 (22.3) | 197 (22.1) | 29 (8.4) | 0 (0.0) | 32 (19.9) | 64 (32.0) |
| Age (years) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| 0-1 | 388 (15.8) | 17 (10.3) | 21 (8.5) | 74 (21.1) | 164 (18.4) | 45 (13.0) | 28 (27.7) | 3 (1.9) | 36 (18.0) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| > 1-5 | 325 (13.2) | 12 (7.3) | 22 (8.9) | 62 (17.7) | 64 (7.2) | 100 (29.0) | 39 (38.6) | 16 (9.9) | 10 (5.0) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| > 5-21 | 321 (13.0) | 15 (9.1) | 6 (2.4) | 38 (10.9) | 82 (9.2) | 116 (33.6) | 34 (33.7) | 18 (11.2) | 12 (6.0) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| > 21-65 | 926 (37.6) | 87 (52.7) | 131 (52.8) | 98 (28.0) | 385 (43.2) | 55 (15.9) | 0 (0.0) | 92 (57.1) | 78 (39.0) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| > 65 | 502 (20.4) | 34 (20.6) | 68 (27.4) | 78 (22.3) | 197 (22.1) | 29 (8.4) | 0 (0.0) | 32 (19.9) | 64 (32.0) | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |

Prospective Clinical Performance

Positive percent agreement (PPA) was calculated by dividing the number of true positive (TP) results by the sum of TP and false negative (FN) results, while negative percent agreement (NPA) was calculated by dividing the number of true negative (TN) results by the sum of TN and false positive (FP) results. A TP result was one where the detected ePlex RP Panel result matched the detected comparator method result, while a TN result was one where a negative ePlex RP Panel result matched a negative comparator method result. The two-sided 95% confidence interval was also calculated.

14

A total of 2462 prospectively-collected samples (511 tested fresh and 1951 tested after previously frozen) were evaluated for 17 ePlex RP Panel organisms. PPA and NPA results are summarized by target in Tables 7 and 8 below.

Organism	Prevalence	TP/TP+FN	PPA (95% CI)	TN/TN+FP	NPA (95% CI)
Adenovirus	1.6%	6/8a	75.0 (40.9-92.9)	499/503a	99.2 (98.0-99.7)
Coronavirus	1.4%	7/7	100 (64.6-100)	503/504	99.8 (98.9-100)
Human Metapneumovirus	0.0%	0/0	---	511/511	100 (99.3-100)
Human Rhinovirus/Enterovirus	35.8%	176/183b	96.2 (92.3-98.1)	316/328b	96.3 (93.7-97.9)
Influenza A	0.0%	0/0	---	511/511	100 (99.3-100)
Influenza A H1	0.0%	0/0	---	511/511	100 (99.3-100)
Influenza A H1-2009	0.0%	0/0	---	511/511	100 (99.3-100)
Influenza A H3	0.0%	0/0	---	511/511	100 (99.3-100)
Influenza B	0.2%	1/1	100 (20.7-100)	509/510	99.8 (98.9-100)
Parainfluenza Virus 1	0.2%	1/1	100 (20.7-100)	510/510	100 (99.3-100)
Parainfluenza Virus 2	2.5%	12/13	92.3 (66.7-98.6)	497/498	99.8 (98.9-100)
Parainfluenza Virus 3	1.0%	5/5	100 (56.6-100)	506/506	100 (99.2-100)
Parainfluenza Virus 4	0.6%	3/3	100 (43.9-100)	503/508c	99.0 (97.7-99.6)
RSV A	1.8%	8/9	88.9 (56.5-98.0)	501/501	100 (99.2-100)
RSV B	2.0%	9/10	90.0 (59.6-98.2)	500/500	100 (99.2-100)
Chlamydia pneumoniae	0.0%	0/0	---	511/511	100 (99.3-100)
Mycoplasma pneumoniae	0.6%	3/3	100 (43.9-100)	507/508d	99.8 (98.9-100)

Table 7: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) in the ePlex RP Panel Clinical Study (Fresh)

ª Adenovirus was not detected in 2 of 2 FN samples and detected in 4 of 4 FP samples using PCR/sequencing.

b Human rhinovirus/enterovirus was not detected in 1 of 7 FN samples using of 12 FP samples using PCR/sequencing.

& Parainfluenza virus 4 was detected in 3 of 5 FP samples using PCR/sequencing.

d M. pneumoniae was detected in the 1 FP sample using PCR/sequencing.

15

| Organism | Prevalence | TP/TP+FN | Positive % Agreement
PPA (95% CI) | TN/TN+FP | Negative % Agreement
NPA (95% CI) |
|------------------------------|------------|----------|--------------------------------------|------------|--------------------------------------|
| Adenovirus | 2.7% | 48/53a | 90.6 (79.7-95.9) | 1874/1898a | 98.7 (98.1-99.1) |
| Coronavirus | 5.6% | 89/110b | 80.9 (72.6-87.2) | 1828/1841b | 99.3 (98.8-99.6) |
| Human Metapneumovirus | 5.8% | 107/113c | 94.7 (88.9-97.5) | 1832/1838c | 99.7 (99.3-99.9) |
| Human Rhinovirus/Enterovirus | 17.2% | 317/336d | 94.3 (91.3-96.4) | 1544/1615d | 95.6 (94.5-96.5) |
| Influenza Ae | 5.7% | 106/111f | 95.5 (89.9-98.1) | 1836/1840f | 99.8 (99.4-99.9) |
| Influenza A H1 | 0.0% | 0/0 | --- | 1951/1951 | 100 (99.8-100) |
| Influenza A H1-2009 | 3.6% | 70/71 | 98.6 (92.4-99.8) | 1874/1880g | 99.7 (99.3-99.9) |
| Influenza A H3 | 1.9% | 34/37h | 91.9 (78.7-97.2) | 1914/1914 | 100 (99.8-100) |
| Influenza B | 3.3% | 58/65i | 89.2 (79.4-94.7) | 1882/1886i | 99.8 (99.5-99.9) |
| Parainfluenza Virus 1 | 1.2% | 23/24 | 95.8 (79.8-99.3) | 1926/1927 | 99.9 (99.7-100) |
| Parainfluenza Virus 2 | 0.5% | 9/9 | 100 (70.1-100) | 1941/1942 | 99.9 (99.7-100) |
| Parainfluenza Virus 3 | 5.3% | 94/104j | 90.4 (83.2-94.7) | 1842/1847j | 99.7 (99.4-99.9) |
| Parainfluenza Virus 4 | 0.3% | 5/5 | 100 (56.6-100) | 1944/1946 | 99.9 (99.6-100) |
| RSV A | 1.6% | 27/31 | 87.1 (71.1-94.9) | 1917/1918 | 99.9 (99.7-100) |
| RSV B | 4.4% | 81/86 | 94.2 (87.1-97.5) | 1861/1863k | 99.9 (99.6-100) |
| Chlamydia pneumoniae | 0.3% | 2/5l | 40.0 (11.8-76.9) | 1945/1946l | 99.9 (99.7-100) |
| Mycoplasma pneumoniae | 0.3% | 4/5m | 80.0 (37.6-96.4) | 1945/1946 | 99.9 (99.7-100) |

Table 8: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) in the ePlex RP Panel Clinical Study (After Previously Frozen)

ే Adenovirus was not detected in 1 of 5 FN samples and detected in 9 of 24 FP samples using PCR/sequencing.

b Coronavirus was not detected in 2 of 21 FN samples and detected in 3 of 13 FP samples using PCR/sequencing.

° Human Metapneumovirus was not detected in 1 of 6 FN samples and detected in 4 of 6 FP samples using PCR/sequencing.

9 Human rhinovirus/enterovirus was not detected in 33 of 71 FP samples using PCR/sequencing.

ື Influenza A comparator results contain 71 samples with A H3, and 3 samples with A H3, and 3 samples with no subtype detected.

် Influenza A was not detected in 1 of 3 FN samples were not tested by PCR/sequencing) and detected in 1 of 4 FP samples using PCR/sequencing.

8 Influenza A H1-2009 was detected in 4 of 6 FP samples using PCR/sequencing.

" Influenza A H3 was not detected in 1 of 3 FN samples using PCR/sequencing.

් Influenza B was not detected in 3 of 7 FN samples and detected in 2 of 4 FP samples using PCR/sequencing.

1 Parainfluenza virus 3 was not detected in 3 of 10 FN samples and detected in 4 of 5 FP samples using PCR/sequencing.

RSV B was detected in 1 of 2 FP samples using PCR/sequencing.

¹ C. pneumoniae was not detected in 1 of 3 FN samples and detected in the 1 FP sample using PCR/sequencing.

10 M. pneumoniae was not detected in the 1 FN sample using PCR/sequencing.

16

Retrospective Clinical Samples

To supplement the number of positives for targets that were not sufficiently represented in the prospective collection, additional nasopharyngeal swab in VTM samples were retrospectively collected from 6 sites. A total of 535 nasopharyngeal swab samples that had previously tested positive for one or more of the target organisms during standard-of-care (SOC) testing were collected and stored frozen. Prior to the start of investigational testing, 11 samples were withdrawn due to noncompliance with the study protocol, and 52 samples were withdrawn because the organisms present had sufficient representation in other samples. In addition, the composition and integrity of the retrospective samples were confirmed with the same comparator method employed in the prospective clinical study (i.e., an FDA-cleared multiplexed respiratory pathogen panel). As the result of this confirmation testing using the comparator method, 26 additional samples were withdrawn because the original SOC testing positive results for the intended organisms were not confirmed when tested with the comparator method. Of the remaining 446 retrospectively-collected samples eligible for testing, all 446 were evaluable. Demographic information for retrospectively-collected samples is described in Table 9. Subjects enrolled in this study were from a diverse demographic distribution and represent the intended patient population.

17

| | All Sites
N=446 | Site 1
N=1 | Site 2
N=1 | Site 3
N=129 | Site 4
N=18 | Site 5
N=131 | Site 6
N=166 |
|-------------|--------------------|---------------|---------------|-----------------|----------------|-----------------|-----------------|
| | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) | n (%) |
| Sex | | | | | | | |
| Male | 232 (52.0) | 0 (0.0) | 1 (100) | 76 (58.9) | 11 (61.1) | 68 (51.9) | 76 (45.8) |
| Female | 214 (48.0) | 1 (100) | 0 (0.0) | 53 (41.1) | 7 (38.9) | 63 (48.1) | 90 (54.2) |
| Age (years) | | | | | | | |
| 0 - 1 | 122 (27.4) | 0 (0.0) | 0 (0.0) | 24 (18.6) | 5 (27.8) | 56 (42.7) | 37 (22.3) |
| > 1 - 5 | 107 (24.0) | 0 (0.0) | 1 (100) | 51 (39.5) | 3 (16.7) | 16 (12.2) | 36 (21.7) |
| > 5 - 21 | 59 (13.2) | 0 (0.0) | 0 (0.0) | 9 (7.0) | 2 (11.1) | 19 (14.5) | 29 (17.5) |
| > 21 - 65 | 99 (22.2) | 1 (100) | 0 (0.0) | 11 (8.5) | 8 (44.4) | 31 (23.7) | 48 (28.9) |
| > 65 | 59 (13.2) | 0 (0.0) | 0 (0.0) | 34 (26.4) | 0 (0.0) | 9 (6.9) | 16 (9.6) |

Table 9: Subject Demographic Data for Retrospectively-Collected Samples by Collection Site (N=446)

Retrospective Clinical Performance

A total of 446 retrospectively-collected samples were evaluated for 17 ePlex RP Panel organisms. The following specimens with the original positive SOC results for the unintended organisms that were not confirmed by the comparator method were excluded from the performance calculation for the respective organism: 1 coronavirus positive specimen, 3 human rhinovirus/enterovirus positive specimens, 1 influenza A positive specimen, 1 influenza A H3 positive specimen. 1 parainfluenza virus positive specimen. In addition, 5 unintended RSV positive specimens by the comparator method were not confirmed by PCR/sequencing with regard to determining RSV subtypes and therefore were excluded from the performance calculations for RSV A and RSV B. PPA and NPA results are summarized by target in Table 10 below.

		Positive % Agreement	Negative % Agreement
Organism	TP/TP+FN	PPA (95% CI)	TN/TN+FP	NPA (95% CI)
Adenovirus	55/56a	98.2 (90.6-99.7)	386/390a	99.0 (97.4-99.6)
Coronavirus	121/138b	87.7 (81.2-92.2)	307/307	100 (98.8-100)
Human Metapneumovirus	5/7	71.4 (35.9-91.8)	439/439	100 (99.1-100)
Human Rhinovirus/Enterovirus	37/41	90.2 (77.5-96.1)	384/402	95.5 (93.0-97.1)
Influenza Ac	75/82d	91.5 (83.4-95.8)	363/363	100 (99.0-100)
Influenza A H1	0/0	---	446/446	100 (99.1-100)
Influenza A H1-2009	27/31e	87.1 (71.1-94.9)	415/415	100 (99.1-100)
Influenza A H3	45/51f	88.2 (76.6-94.5)	394/394	100 (99.0-100)
Influenza B	1/1	100 (20.7-100)	445/445	100 (99.1-100)
Parainfluenza Virus 1	43/48g	89.6 (77.8-95.5)	396/397	99.7 (98.6-100)

Table 10: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of the ePlex RP Panel With Comparator Methods (Retrospective Collection)

18

		Positive % Agreement		Negative % Agreement
Organism	TP/TP+FN	PPA (95% CI)	TN/TN+FP	NPA (95% CI)
Parainfluenza Virus 2	46/51	90.2 (79.0-95.7)	395/395	100 (99.0-100)
Parainfluenza Virus 3	2/2	100 (34.2-100)	444/444	100 (99.1-100)
Parainfluenza Virus 4	18/20	90.0 (69.9-97.2)	426/426	100 (99.1-100)
RSV A	25/27	92.6 (76.6-97.9)	414/414	100 (99.1-100)
RSV B	21/22	95.5 (78.2-99.2)	419/419	100 (99.1-100)
Chlamydia pneumoniae	1/1	100 (20.7-100)	445/445	100 (99.1-100)
Mycoplasma pneumoniae	7/7	100 (64.6-100)	439/439	100 (99.1-100)

4 Adenovirus was not detected in the 1 FN sample and detected in 2 of 4 FP samples using PCR/sequencing.

Coronavirus was not detected in 2 of 16 FN samples using (1 sample was not tested by PCR sequencing).

6 Influenza A comparator results contain 31 samples with A H1-2009 and 51 samples with A H3 detected.

4 Influenza A was not detected in 3 of 7 FN samples using PCR/sequencing.

e Influenza A H1-2009 was not detected in 2 of 4 FN samples using PCR/sequencing.

1 Influenza A H3 was not detected in 1 of 6 FN samples using PCR/sequencing.

8 Parainfluenza virus 1 was not detected in 2 of 5 FN samples using PCR/sequencing.

Contrived Sample Performance

There were 327 contrived samples created and tested to supplement the low prevalence targets on the RP Panel; 104 contained one or more low prevalence organisms and 223 were negative for the contrived organisms. All 327 contrived samples were tested with the ePlex RP Panel and 326 were evaluable. PPA and NPA results are summarized for these low prevalence organisms in Table 11 below.

Table 11: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of the ePlex RP Panel With Comparator Method (Contrived Samples)

Organism	Positive % Agreement		Negative % Agreement
	TP/TP+FN	PPA (95% CI)	TN/TN+FP	NPA (95% CI)
Chlamydia pneumoniae	52/52	100 (93.1-100)	274/274	100 (98.6-100)
Influenza A H1	51/51	100 (93.0-100)	275/275	100 (98.6-100)

Co-Detections in Prospective Clinical Samples

The ePlex RP Panel identified a total of 135 prospective samples with multiple organisms detected, or 5.5% of all prospectively-collected samples. Of these, 118 (4.8%) had two organisms, 14 (0.6%) had three organisms, and 3 (0.1%) had four organisms detected. Of the 135 co-detected samples, 58 included 1 or more organisms that had not been detected by the comparator method(s). Results are summarized in Tables 12 and 13.

19

| Distinct Co-Detection Combinations Detected by
the ePlex RP Panel | | | | Of Co-detections
(% of samples) | Number of
Discrepant
Co-detections | Discrepant
Organism(s) a |
|----------------------------------------------------------------------|------ | Organism
1 | Organism
2 | Organism
3 | Organism
4 | | | ADV | CoV | ADV | CoV | ADV (1), Flu B (1),
HRV/EV (1) |
| ADV | Flu AH3 | ADV | Flu B | ADV | FluA09H1 (1) |
| ADV | FluA09H1 | ADV | FluA09H1 | ADV | HMPV | ADV | HMPV | ADV | HRV/EV | ADV | HRV/EV | ADV | HRV/EV | ADV | HRV/EV | ADV | HRV/EV | ADV | PIV 2 | ADV | PIV 3 | ADV | PIV 4 | ADV | RSV B | CPneum | HRV/EV | CoV | FluA09H1 | CoV | HMPV | CoV | HMPV | CoV | HRV/EV | CoV | HRV/EV | CoV | PIV 1 | CoV | RSV A | CoV | RSV B | Flu A (unk) | HRV/EV | Flu AH3 | HRV/EV | Flu AH3 | RSV B | Flu B | HRV/EV | Flu B | HRV/EV | Flu B | PIV 3 | FluA09H1 | HMPV | FluA09H1 | HRV/EV | HMPV | HRV/EV | HMPV | HRV/EV | HMPV | PIV 3 | HRV/EV | PIV 1 | HRV/EV | PIV 2 | HRV/EV | PIV 3 | HRV/EV | PIV 4 | Distinct Co-Detection Combinations Detected by
the ePlex RP Panel | | | | Of Co-detections
(% of samples) | Number of
Discrepant
Co-detections | Discrepant
Organism(s) a |
| Organism
1 | Organism
2 | Organism
3 | Organism
4 | | | HRV/EV | RSV A | HRV/EV | RSV B | PIV 1 | PIV 4 | PIV 3 | RSV B | RSV A | RSV B | Total Number of Co-Detections | | Total Number with 2 Organisms Detected | | Total Number with 3 Organisms Detected | | Total Number with 4 Organisms Detected | Total Number
---------|---------------|---------------|----------------------------------------------------|------------------------------------------|-------------------------------------------------------|
| |
| | | 2 (0.08%) | 0 | |
| HRV/EV | | 2 (0.08%) | 1 | ADV (1) |
| Flu A (unk) | Flu B | HRV/EV | 1 (0.04%) | 1 | ADV (1), Flu A (unk)
| | | 1 (0.04%) | 0 | |
| HRV/EV | RSV B | 1 (0.04%) | 1 | ADV (1), Flu B (1) |
| | | 1 (0.04%) | 1 | ADV (1), FluA09H1
| HRV/EV | | 1 (0.04%) | 0 | |
| PIV 3 | | 1 (0.04%) | 1 | PIV 3 (1) |
| | | 3 (0.12%) | 2 | ADV (2) |
| HRV/EV | RSV A | 1 (0.04%) | 1 | RSV A (1) |
| | | 18 (0.73%) | 7 | ADV (6), HRV/EV (1) |
| Mpneum | | 1 (0.04%) | 0 | |
| PIV 1 | | 1 (0.04%) | 1 | PIV 1 (1) |
| PIV 4 | | 1 (0.04%) | 1 | ADV (1), PIV 4 (1) |
| RSV B | | 1 (0.04%) | 0 | |
| | | 2 (0.08%) | 1 | ADV (1) |
| | | 2 (0.08%) | 1 | ADV (1) |
| | | 1 (0.04%) | 1 | ADV (1) |
| | | 2 (0.08%) | 2 | ADV (2) |
| | | 1 (0.04%) | 0 | |
| | | 1 (0.04%) | 0 | |
| | | 4 (0.16%) | 0 | |
| HRV/EV | | 2 (0.08%) | 0 | |
| | | 12 (0.49%) | 4 | CoV (1), HRV/EV (4) |
| RSV B | | 1 (0.04%) | 1 | CoV (1) |
| | | 1 (0.04%) | 0 | |
| | | 3 (0.12%) | 0 | |
| | | 3 (0.12%) | 2 | CoV (2) |
| | | 1 (0.04%) | 1 | Flu A (unk) (1) |
| | | 2 (0.08%) | 1 | HRV/EV (1) |
| | | 1 (0.04%) | 0 | |
| | | 4 (0.16%) | 2 | HRV/EV (2) |
| RSV B | | 1 (0.04%) | 0 | |
| | | 1 (0.04%) | 0 | |
| HRV/EV | | 1 (0.04%) | 1 | HRV/EV (1) |
| | | 2 (0.08%) | 1 | HRV/EV (1) |
| | | 5 (0.20%) | 1 | HRV/EV (1) |
| RSV B | | 1 (0.04%) | 1 | HRV/EV (1) |
| | | 1 (0.04%) | 0 | |
| | | 3 (0.12%) | 0 | |
| | | 7 (0.28%) | 3 | HRV/EV (1), PIV 2 (2) |
| | | 11 (0.45%) | 5 | HRV/EV (5) |
| | | 4 (0.16%) | 4 | PIV 4 (4) |
Total Number
| |
| | | 5 (0.20%) | 0 | |
| | | 11 (0.45%) | 6 | HRV/EV (6) |
| | | 1 (0.04%) | 1 | PIV 4 (1) |
| | | 1 (0.04%) | 0 | |
| | | 1 (0.04%) | 1 | RSV B (1) |
| | | 135 (5.5%) | 57 | 64/290b |
| | | 118 (4.8%) | 47 | 49/236 |
| | | 14 (0.6%) | 7 | 8/42 |
| | | 3 (0.1%) | 3 | 7/12 |

Table 12: Distinct Co-Detection Combinations Detected by the

20

Note: ADV= adenovirus, CoV= coronavirus, HMPV= human metapneumovirus, HRV/EV= human rhinovirus/enterovirus, Flu= Influenza, (unk)= unknown subtype, PIV= parainfluenza, RSV= respiratory syncytial virus, Cpneumoniae, Mpneumoniae

4 A discrepant organism is defined as one that was detected by the ePlex RP Panel but not by the comparator method(s).

b 64/64 discrepant organisms were investigated using PCR/sequencing; the discrepant organism was detected in 20/64 cases:

-In 8/18 samples, adenovirus was detected by PCR/sequencing.

-In 1/4 samples, coronavirus was detected by PCR/sequencing.

-In 7/25 samples, human rhinovirus/enterovirus was detected by PCR/sequencing.

-In 1/1 sample, influenza A H1-2009 was detected by PCR/sequencing.

-In 1/1 sample, parainfluenza virus 3 was detected by PCR/sequencing.

-In 2/6 samples, parainfluenza virus 4 was detected by PCR/sequencing.

Table 13: Additional Co-Detection Combinations Detected by the Comparator Method in the
Prospective Clinical Samples

| Distinct Co-Detection Combinations Detected
by the Comparator Method | | | Total Number
Of Co-detections
(% of samples) | Number of
Discrepant
Co-detections | Discrepant
Organism(s) a,b |
|-------------------------------------------------------------------------|------------|------------|----------------------------------------------------|------------------------------------------|-------------------------------|
| Organism 1 | Organism 2 | Organism 3 | | | |
| ADV | CoV | | 1 (0.04%) | 1 | ADV (1), CoV (1) |
| ADV | HRV/EV | | 4 (0.16%) | 4 | ADV (4) |
| ADV | HRV/EV | PIV 3 | 1 (0.04%) | 1 | HRV/EV (1), PIV 3 (1) |
| ADV | HRV/EV | RSV A | 1 (0.04%) | 1 | ADV (1) |
| CPneum | HRV/EV | | 1 (0.04%) | 1 | CPneum (1) |
| CPneum | PIV 3 | | 1 (0.04%) | 1 | CPneum (1) |
| CoV | FluA09H1 | | 2 (0.08%) | 2 | CoV (2) |
| CoV | HMPV | | 1 (0.04%) | 1 | CoV (1) |
| CoV | HRV/EV | | 6 (0.24%) | 6 | CoV (4), HRV/EV (2) |
| CoV | PIV 3 | | 1 (0.04%) | 1 | CoV (1) |
| CoV | RSV B | | 3 (0.12%) | 3 | CoV (2), RSV B (1) |
| Flu AH3 | HRV/EV | PIV 3 | 1 (0.04%) | 1 | Flu AH3 (1), PIV 3 (1) |
| Flu AH3 | PIV 3 | | 1 (0.04%) | 1 | PIV 3 (1) |
| FluA09H1 | HMPV | HRV/EV | 1 (0.04%) | 1 | HMPV (1), HRV/EV (1) |
| HMPV | HRV/EV | | 1 (0.04%) | 1 | HRV/EV (1) |
| HRV/EV | PIV 1 | | 1 (0.04%) | 1 | HRV/EV (1) |
| HRV/EV | PIV 3 | | 2 (0.08%) | 2 | HRV/EV (2) |
| HRV/EV | PIV 3 | RSV B | 1 (0.04%) | 1 | PIV 3 (1) |
| HRV/EV | RSV A | | 2 (0.08%) | 2 | RSV A (2) |

ª A discrepant organism is defined as one that was detected by the comparator method(s) but not by the ePlex RP Panel.

b 36/36 discrepant organisms were investigated using the discrepant organism was not detected in 10/36 cases:

-In 2/6 samples, adenovirus was not detected by PCR/sequencing.

-In 1/2 samples, Chlamydia pneumoniae was not detected by PCR/sequencing.

-In 1/11 samples, coronavirus was not detected by PCR/sequencing.

-In 5/8 samples, human rhinovirus/enterovirus was not detected by PCR/sequencing.

-In 1/1 sample, influenza A H3 was not detected by PCR/sequencing.

21

Clinical Study ePlex Instrument Performance

A total of 3281 samples (including prospective, retrospective, and contrived samples) were initially tested in the clinical evaluations and 3127/3281 = 95.3% (95% CI: 94.5%-96.0%) generated valid results on the first attempt. After re-test, 8 samples had invalid results; final validity rate was 3273/3281 = 99.8% (95% CI: 99.5%-99.9%).

Selected Analytical Studies

Limit of Detection

The limit of detection (LoD), or analytical sensitivity was identified and verified for each viral and bacterial target on the ePlex RP Panel using quantified reference strains/isolates. Serial dilutions were prepared in a natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples) with one or more organisms per series, and at least 20 replicates per target were tested in the study. The limit of detection was defined as the lowest concentration at which each target is detected at least 95% of the time. The confirmed LoD for each ePlex RP Panel organism is shown in Table 14.

Target	Strain	LoD Concentration
Adenovirus	Type 1 (C)	1 x 103 TCID50/mL
	Type 4 (E)	2 x 100 TCID50/mL
	Type 7 (B)	2 x 100 TCID50/mL
Coronavirus 229E	229E	1 x 100 TCID50/mL
Coronavirus HKU1	HKU1a	5 x 104 copies/mL
Coronavirus NL63	NL63	7.5 x 100 TCID50/mL
Coronavirus OC43	OC43	5 x 102 TCID50/mL
Human Metapneumovirus	A1 IA3-2002	2 x 10-1 TCID50/mL
	A2 IA14-2003	2 x 103 TCID50/mL
	B1 Peru2-2002	2 x 102 TCID50/mL
	B2 Peru1-2002	2.25 x 102 TCID50/mL
Human Rhinovirus/Enterovirus	Enterovirus Type 68 (2007)	1 x 100 TCID50/mL
	Rhinovirus 1A	1.5 x 100 TCID50/mL
	Rhinovirus B14	1 x 100 TCID50/mL
	Rhinovirus C a	1 x 105 copies/mL
Influenza A	H1N1 Brisbane/59/07	3 x 10-1 TCID50/mL
Influenza A H1	H1N1 Brisbane/59/07	3 x 10-1 TCID50/mL
Influenza A H1-2009	NY/01/2009	1 x 10-1 TCID50/mL

--	--	--	------------------------------------

22

Target	Strain	LoD Concentration
	A/Perth/16/2009	1 x 101 TCID50/mL
	A/Texas/50/2012	1 x 100 TCID50/mL
Influenza A H3	A/Victoria/361/2011	5 x 10-1 TCID50/mL
	H3N2 Brisbane/10/07	5 x 101 TCID50/mL
Influenza B	Florida/02/06	1 x 10-1 TCID50/mL
	B/Brisbane/60/2008	1 x 100 TCID50/mL
Influenza B (Victoria Lineage)	B/Montana/5/2012	1 x 100 TCID50/mL
	B/Nevada/03/2011	1 x 100 TCID50/mL
	B/Massachusetts/02/2012	1 x 102 TCID50/mL
Influenza B (Yamagata Lineage)	B/Texas/06/2011	1 x 10-1 TCID50/mL
	B/Wisconsin/01/2010	1 x 100 TCID50/mL
Parainfluenza Virus 1	Clinical Isolate	4 x 10-1 TCID50/mL
Parainfluenza Virus 2	Clinical Isolate	5 x 101 TCID50/mL
Parainfluenza Virus 3	Clinical Isolate	5 x 100 TCID50/mL
Parainfluenza Virus 4	4a	3 x 101 TCID50/mL
Respiratory Syncytial Virus A	2006 Isolate	1.5 x 100 TCID50/mL
Respiratory Syncytial Virus B	CH93(18)-18	2 x 10-1 TCID50/mL
Chlamydia pneumoniae	AR-39	3 x 102 TCID50/mL
Mycoplasma pneumoniae	FH strain of Eaton Agent [NCTC 10119]	3 x 102 CCU/mL

ª Clinical samples confirmed positive for coronavirus C by bi-directional sequencing and quantified by real-time RT-PCR were used for determination of LoD.

Analytical Reactivity (Inclusivity)

A panel of 101 strains/isolates representing the genetic, temporal, and geographic diversity of each target on the ePlex RP Panel was evaluated to demonstrate analytical reactivity. Each strain/isolate was tested in triplicate at 3x LoD in natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples); if the organism was not detected at this concentration, testing of higher concentrations was performed. Additional in silico analysis was also performed on a subset of ePlex RP Panel organisms.

All of the 101 strains/isolates tested for inclusivity were detected by the ePlex RP Panel. Results of analytical reactivity are shown in Tables 15-25.

Adenovirus Species	Serotype	Concentration	Multiple of LoD Detected
A	Type 31	3 x 103 TCID50/mL	3x
B	Type 3	6 x 100 TCID50/mL	3x
	Type 11	6 x 100 TCID50/mL	3x
	De Wit Type 14	6 x 100 TCID50/mL	3x

Table 15: Analytical Reactivity (Inclusivity) Results for Adenovirus

23

Adenovirus Species	Serotype	Concentration	Multiple of LoD Detected
	Ch.79 Type 16	2 x 102 TCID50/mL	100xa
	Type 21	6 x 100 TCID50/mL	3x
	Compton Type 34	6 x 100 TCID50/mL	3x
	Holden Type 35	6 x 100 TCID50/mL	3x
	Wan Type 50	2 x 101 TCID50/mL	10xb
C	Type 2	3 x 103 TCID50/mL	3x
	Type 5	3 x 103 TCID50/mL	3x
	Type 6	3 x 103 TCID50/mL	3x
D	Type 26	3 x 103 TCID50/mL	3x
	Type 37	3 x 103 TCID50/mL	3x
F	Type 40 Dugan	3 x 103 TCID50/mL	3x
	Type 41/ Strain Tak	3 x 103 TCID50/mL	3x

ª In silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic material

present in the culture of this or the reference strain (TCID30 value is based only on infectious virus particles).

In silico analysis revealed that lower sensitivity may be

Table 16: Analytical Reactivity (Inclusivity) Results for Human Metapneumovirus

| Metapneumovirus

Subtype	Strain	Concentration	Multiple of LoD Detected
Human metapneumovirus	Peru6-2003 G, B2	6.75 x $10^2$ TCID50/mL	3x

Rhinovirus/Enterovirus	Strain	Concentration	Multiple of LoD Detected
Human Rhinovirus	Type A2	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	Type A7	$1.5 \times 10^{1} \text{ TCID}_{50}/\text{mL}$	10xa
	Type A16	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	Type A18	$1.5 \times 10^{2} \text{ TCID}_{50}/\text{mL}$	100xa
	Type A34	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	Type A57	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	Type A77	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	277G	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	Type B3	$1.5 \times 10^{1} \text{ TCID}_{50}/\text{mL}$	10xa
	Type B17	$1.5 \times 10^{1} \text{ TCID}_{50}/\text{mL}$	10xa
	Type B42	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	Type B83	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	Type B84	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	FO2-2547	$4.5 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
Enterovirus	Type 71	$3 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
Coxsackievirus	A9	$3 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	A10	$3 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	A21	$3 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x
	A24	$3 \times 10^{0} \text{ TCID}_{50}/\text{mL}$	3x

Table 17: Analytical Reactivity (Inclusivity) Results for Human Rhinovirus/Enterovirus

24

Rhinovirus/Enterovirus	Strain	Concentration	Multiple of LoD Detected
	B2	1 x 102 TCID50/mL	100xa
	B3	3 x 100 TCID50/mL	3x
	B4	3 x 100 TCID50/mL	3x
	B5	1 x 101 TCID50/mL	10xa
Echovirus	9	3 x 100 TCID50/mL	3x
	E6	1 x 101 TCID50/mL	10xb
	25	1 x 101 TCID50/mL	10xa
	30	3 x 100 TCID50/mL	3x
Poliovirus	1	1 x 102 TCID50/mL	100xa

^a In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes.

" In silico analysis revealed that lower sensilt of mismatches in the assay primers andro probes.
^ In silico analysis revealed god homology to primers and probes. Lower sens present in the culture of this or the reference strain (TCID50 value is based only on infectious virus particles).

Table 18: Analytical Reactivity (Inclusivity) Results for Influenza A

Note: Due to different assays for influenza A matrix and influenza A subtypes on the ePlex RP Panel, if different LoDs are observed for inclusivity for a Flu A matrix vs. a subtype, the differences are noted in the Multiple of LoD Detected column.

| Influenza A

Subtype	Strain	Concentration	Multiple of LoD Detected
Influenza A H1	A/FM/1/47	3 x 100 TCID50/mL	10x (Influenza A matrix)a
10000x H1 subtypeb
	A/New Caledonia/20/1999	9 x 10-1 TCID50/mL	3x
	A/New Jersey/8/76	9 x 10-1 TCID50/mL	3x
H1 subtype not detectedc
	A/NWS/33	3 x 100 TCID50/mL	10x (Influenza A matrix)a
H1 subtype not detectedd
	A/PR/8/34	9 x 10-1 TCID50/mL	3x (Influenza A matrix)
H1 subtype not detectede
	A/Solomon Islands/3/2006	9 x 10-1 TCID50/mL	3x
	A/Taiwan/42/06	9 x 100 TCID50/mL	30xf
	A/Hong Kong/8/68
	A/Port Chalmers/1/73
Influenza A H3	A/Nanchang/933/95	1.5 x 102 TCID50/mL	3x
	A/Victoria/3/75
	A/Wisconsin/67/05

Influenza A
2009 H1N1	A/California/7/2009	1 x 100 TCID50/mL	10xg
	A/Mexico/4108/09	3 x 10-1 TCID50/mL	3x
	A/NY/02/2009	1 x 100 TCID50/mL	10xh
	A/Swine NY/03/2009	3 x 10-1 TCID50/mL	3x
	A/Swine/Iowa/15/30	3 x 10-1 TCID50/mL	3x (Influenza A matrix)
100,000x (H1-2009 subtype)i
	A/Virginia/ATCC1/2009	1 x 100 TCID50/mL	10xj
	A/Virginia/ATCC2/2009	1 x 101 TCID50/mL	100xj
	A/Virginia/ATCC3/2009	1 x 102 TCID50/mL	1,000xj

ª In silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic naterial present in the culture of this or the reference strain (TCIDso value is based only on infectious virus particles).

In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes.

25

C H1-2009 subtype was detected in this seasonal influenza A H1 strain at 30x LoD.

4 In silico analysis revealed little homology between this non-contemporary strain sequence and the H1 signal probe sequences.

· In silico analysis revealed little homology between this non-contemporary influenza strain sequence and the H1 primer sequences. f For Influenza A matrix, in silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect

estimation of genetic material present in the reference strain (TCIDs, value is based only on infectious virus particles). For H1 subtype, in silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes.

6 For Influenza A matrix, in silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and or probes. For H1 subtype, in silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of 111 saterial present in the culture of this or the reference strain (TCD23) value is based only on infectious vinus particles).
genetic material present in the culture of thi

For Influenza A matrix, in silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic material present in the culture of this or the reference strain (TCID3) value is based only on infectious virus particles). For H1-2009 subtype, in silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. 1 In silico analysis revealed little homology between the H1 or H1-2009 primer, signal probe and capture probe sequences.

1 No sequence data was available to investigate lower sensitivity of the influenza A 2009 HIN1 A/Virginia/ATCC1/2009,

A/Virginia/ATCC2/2009 and A/Virginia/ATTC3/2009 strains.

Table 19: Analytical Reactivity (Inclusivity) Results for Influenza A Strains Titered with Methods Different From the Reference Strain

Influenza A Subtype	Strain	Concentration Detected
Influenza A H1	A/Denver/1/57	1.6 x 102 CEID50/mL (Influenza A matrix)
1.6 x 108 CEID50/mL (H1 subtype)
	A/Mal/302/54	1.58 x 102 CEID50/mL (Influenza A matrix)
1.58 x 105 CEID50/mL (H1 subtype)
	A/Aichi/2/68 H3N2	1.58 x 103 CEID50/mL
Influenza A H3	Alice (vaccine) A/England/42/72	5 x 100 EID50/mL (Influenza A matrix)
5 x 101 EID50/mL (H3 subtype)
	MRC-2 Recombinant Strain	8.89 x 102 CEID50/mL (Influenza A matrix)
8.89 x 103 CEID50/mL (H3 subtype)
Influenza A H1N1	A/Washington/24/2012 (A/H1 pdm09)	3.16 x 103 EID50/mL (Influenza A matrix)
3.16 x 102 EID50/mL (H1-2009 subtype)
Influenza A H1N2	Kilbourne F63: A/NWS/34 (HA) x
A/Rockefeller Institute/5/57 (NA),
Reassortant NWS-F- Matrix	8.89 x 101 CEID50/mL (Influenza A matrix)
No subtype detecteda
Influenza A H5N8	A/Gyrfalcon/Washington/41088-
6/2014 BPL	1.58 x 103 EID50/mL (Influenza A matrix)
No subtype detectedb
Influenza A H5N2	A/Northern
Pintail/Washington/40964/2014 BPL	2.51 x 103 EID50/mL (Influenza A matrix)
No subtype detectedb
Influenza A H7N9	A/ANHUI/1/2013	7.94 x 103 EID50/mL (Influenza A matrix)
No subtype detectedc
Influenza A H3N2v	A/Indiana/21/2012	2.51 x 104 EID50/mL (Influenza A matrix and H3
subtype)

ª In silico analysis revealed little homology between this non-contemporary strain sequence and the H1 Signal Probe sequences.

b Detection of the H5 Subtype not expected

• Detection of the H7 Subtype not expected

NOTE: CEID50/mL= Chick Embryo Infectious Dose; EID50/mL= Egg Infectious Dose

26

Supplemental Analytical Reactivity (Inclusivity) for Influenza A

For human, avian, and swine influenza strains not available for testing on the ePlex RP Panel, in silico analysis was performed. Bioinformatics analysis was used to predict a result based on the number and location of mismatches in the primers, capture probes, and signal probes found in the ePlex RP Panel relative to an alignment of GenBank sequences.

Table 20: Predicted (in silico) Reactivity (Inclusivity) Results for Influenza A
Influenza
A Subtype	Host	Strain	GenBank ID	Predicted ePlex
Result
H2N2	Human	A/Albany/20/1957(H2N2)	CY022014	Influenza A
		Kilbourne F38: A/Korea/426/68 (HA, NA) x
A/Puerto Rico/8/34	CY037296	Influenza A
	Avian	A/chicken/New York/13828-3/1995(H2N2)	CY014822	Influenza A
	Avian	A/Japan/305/1957(H2N2)	CY014977	Influenza A
		A/Korea/426/1968(H2N2)	CY031596	Influenza A
H4N6		A/Blue-winged teal/Minnesota/Sg-
00043/2007(H4N6)	CY063978	Influenza A
		A/Peregrine falcon/Aomori/7/2011	AB629716	Influenza A
		A/Chicken/West Bengal/239022/2010	CY061305	Influenza A
		A/Chicken/West Bengal/193936/2009	GU272009	Influenza A
		A/Chicken/Hunan/1/2009	HM172150	Influenza A
		A/Chicken/Hunan/8/2008	GU182162	Influenza A
H5N1	Avian	A/Chicken/West Bengal/106181/2008	GU083632	Influenza A
		A/Chicken/Primorsky/85/2008	FJ654298	Influenza A
		A/Chicken/West Bengal/82613/2008	GU083648	Influenza A
		A/Duck/France/080036/2008	CY046185	Influenza A
		A/Duck/Vietnam/G12/2008	AB593450	Influenza A
		A/Chicken/Thailand/PC-340/2008	EU620664	Influenza A
		A/Great egret/Hong Kong/807/2008	CY036240	Influenza A
		A/Rook/Rostov-on-Don/26/2007(H5N1)	EU814504	Influenza A
		A/Turkey/VA/505477-18/2007(H5N1)	GU186510	Influenza A
	Human	A/Chicken/Bangladesh/1151-10/2010(H5N1)	HQ156766	Influenza A
		A/Bangladesh/3233/2011	CY088772	Influenza A
		A/Cambodia/R0405050/2007(H5N1)	HQ200572	Influenza A
		A/Cambodia/S1211394/2008	HQ200597	Influenza A
		A/Hong Kong/486/97(H5N1)	AF255368	Influenza A
	Swine	A/Swine/East Java/UT6010/2007(H5N1)	HM440124	Influenza A
		A/Duck/Pennsylvania/10218/1984(H5N2)	AB286120	Influenza A
H5N2		A/American black duck/Illinois/08OS2688/2008	CY079453	Influenza A
	Avian	A/American green-winged
teal/California/HKWF609/2007	CY033447	Influenza A
		A/Canada goose/New York/475813-2/2007	GQ923358	Influenza A
Influenza
A Subtype	Host	Strain	GenBank ID	Predicted ePlex
Result
		A/Blue-winged teal/Saskatchewan/22542/2007	CY047705	Influenza A
		A/Chicken/Taiwan/A703-1/2008	AB507267	Influenza A
		A/Duck/France/080032/2008	CY046177	Influenza A
		A/Duck/New York/481172/2007	GQ117202	Influenza A
		A/Gadwall/Altai/1202/2007	CY049759	Influenza A
		A/Mallard/Louisiana/476670-4/2007	GQ923390	Influenza A
		A/Waterfowl/Colorado/476466-2/2007	GQ923374	Influenza A
H5N3		A/Duck/Singapore/F119/3/1997(H5N3)	GU052803	Influenza A
H6N1	Avian	A/Duck/PA/486/1969(H6N1)	EU743287	Influenza A
H6N2		A/Mallard/Czech Republic/15902-
17K/2009(H6N2)	HQ244433	Influenza A
		A/Chicken/Hebei/1/2002	AY724263	Influenza A
		A/Chicken/PA/149092-1/02	AY241609	Influenza A
		A/Chicken/NJ/294508-12/2004	EU743254	Influenza A
		A/Chicken/New York/23165-6/2005	CY031077	Influenza A
H7N2	Avian	A/Muscovy duck/New York/23165-13/2005	CY033226	Influenza A
		A/Muscovy duck/New York/87493-3/2005	CY034791	Influenza A
		A/Mallard/Netherlands/29/2006	CY043833	Influenza A
		A/Northern shoveler/California/JN1447/2007	CY076873	Influenza A
		A/New York/107/2003(H7N2)	EU587373	Influenza A
H7N3	Human	A/Canada/rv504/2004(H7N3)	CY015007	Influenza A
	Avian	A/American green-winged
teal/Mississippi/09OS046/2009	CY079309	Influenza A
		A/Chicken/Germany/R28/03	AJ619676	Influenza A
		A/Chicken/Netherlands/1/03	AY340091	Influenza A
H7N7		A/Mallard/California/HKWF1971/2007	CY033383	Influenza A
		A/Mallard/Korea/GH171/2007	FJ959087	Influenza A
		A/Mute swan/Hungary/5973/2007	GQ240816	Influenza A
		A/Northern shoveler/Mississippi/09OS643/2009	CY079413	Influenza A
	Human	A/Netherlands/219/03(H7N7)	AY340089	Influenza A
	Human	A/Shanghai/1/2013(H7N9)	EPI439493	Influenza A
H7N9	Avian	A/Northern
shoveler/Mississippi/11OS145/2011(H7N9)	CY133650	Influenza A
		A/Ruddy turnstone/Delaware
Bay/220/1995(H7N9)	CY127254	Influenza A
		A/Turkey/Minnesota/1/1988(H7N9)	CY014787	Influenza A
		A/Blue-winged teal/Ohio/566/2006(H7N9)	CY024819	Influenza A
	Human	A/Hong Kong/1073/99(H9N2)	AJ278647	Influenza A
H9N2		A/Turkey/Wisconsin/1/1966(H9N2)	CY014664	Influenza A
H10N7	Avian	A/chicken/Germany/N/1949(H10N7)	GQ176135	Influenza A
H11N9		A/Duck/Memphis/546/1974(H11N9)	GQ257441	Influenza A
	Swine	A/Swine/Wisconsin/1/1971(H1N1)	CY022414	Influenza A
H1N1	Human	A/California/UR06-0393/2007(H1N1)	CY026540	Influenza A H1
Influenza
A Subtype	Host	Strain	GenBank ID	Predicted ePlex
Result
H1N2		A/New York/297/2003(H1N2)	CY026539
CY002664
CY002665	Influenza A H1
H1N1
(2009)		A/Aalborg/INS133/2009(H1N1)	CY063606
CY063607	Influenza A H1-
2009
		A/South Carolina/02/2010(H1N1)	KC781370
KC781372	Influenza A H1-
2009
	H1N2	Swine	A/Swine/Hong Kong/NS857/2001(H1N2)	GQ229350
H1N2	Swine	A/Swine/Sweden/1021/2009(H1N2)	GQ495135	Influenza A
H3N1	Avian	A/Blue-winged teal/ALB/452/1983(H3N1)	CY004635	Influenza A
H3N2v	Human	A/Iowa/07/2011(H3N2)	JQ070760
JQ290177	Influenza A H3
		A/Iowa/08/2011(H3N2)	JQ070768
JQ290167	Influenza A H3
		A/Iowa/09/2011(H3N2)	JQ070776
JQ290183	Influenza A H3
		A/Indiana/08/2011(H3N2)	JQ070800
JQ070795	Influenza A H3
		A/Maine/06/2011(H3N2)	JN866181
JN866186	Influenza A H3
		A/Maine/07/2011(H3N2)	JN992746	Influenza A
		A/Pennsylvania/09/2011(H3N2)	JN655534	Influenza A
		A/Pennsylvania/11/2011(H3N2)	JN655540	Influenza A
		A/Pennsylvania/10/2011(H3N2)	JN655550	Influenza A
		A/West Virginia/06/2011(H3N2)	JQ290159
JQ290164	Influenza A H3
		A/West Virginia/07/2011(H3N2)	JQ348839	Influenza A
		A/Indiana/10/2011(H3N2)	KJ942592
JQ070787	Influenza A H3
	Swine	A/Boston/38/2008(H3N2)	CY044580
CY044581	Influenza A H3
		A/swine/NY/A01104005/2011(H3N2v)	JN940422	Influenza A H3
		A/Maine/06/2011(H3N2)	JN866181
JN866186	Influenza A H3
		A/Indiana/08/2011(H3N2)	JN655558
JN638733	Influenza A H3
		Avian	A/American black duck/North Carolina/675-
075/2004(H3N2)	GU051135
GU051136
H3N5	A/Mallard/Netherlands/2/1999(H3N5)		CY060261
CY060264	Influenza A
H3N6	A/American black duck/New		CY047696	Influenza A
Influenza
A Subtype	Host	Strain	GenBank ID	Predicted ePlex
Result
		Brunswick/25182/2007(H3N6)	CY047697	Influenza A
H3N7		A/Northern
shoveler/California/HKWF1367/2007(H3N7)	CY033372
CY033375	Influenza A
H3N8		A/American black
duck/Washington/699/1978(H3N8)	GU052300
GU052299	Influenza A H3

Table 20: Predicted (in silico) Reactivity (Inclusivity) Results for Influ

27

28

29

Table 21: Analytical Reactivity (Inclusivity) Results for Influenza B

| Influenza B Subtype | Strain | Concentration | Multiple of LoD
Detected |
|-----------------------------------|--------------------|-------------------------|-----------------------------|
| Influenza B
(Yamagata Lineage) | B/Lee/40 | 3 x $10^{-1}$ TCID50/mL | 3x |
| | B/Allen/45 | 1 x $10^{0}$ TCID50/mL | 10xa |
| | B/Maryland/1/59 | 1 x $10^{1}$ TCID50/mL | 100xa |
| | B/Taiwan/2/62 | 1 x $10^{1}$ TCID50/mL | 100xa |
| Influenza B
(Victoria Lineage) | B/Hong Kong/5/72 | 1 x $10^{1}$ TCID50/mL | 100xb |
| | B/Malaysia/2506/04 | 3 x $10^{-1}$ TCID50/mL | 3x |
| Influenza B
(Lineage unknown) | B/GL/1739/54 | 3 x $10^{-1}$ TCID50/mL | 3x |

ª No sequence data available. Lower sensitivity may be a result of mismatches in the assay primers and/or probes. In addition, the reduced sensitivity may be the result of incorrect estimation of genetic material present in the reference strain (TCD3) value is based only on infectious virus particles).

b In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes.

Table 22: Analytical Reactivity (Inclusivity) Results for Parainfluenza Virus
Parainfluenza Subtype	Strain	Concentration	Multiple of LoD
Detected
Parainfluenza Virus 1	C35	1.2 x 100 TCID50/mL	3x
Parainfluenza Virus 2	Greer	1.5 x 102 TCID50/mL	3x
Parainfluenza Virus 3	C-243	5 x 101 TCID50/mL	10xa
Parainfluenza Virus 4	4b	9 x 101 TCID50/mL	3x

ª In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes.

Table 23: Analytical Reactivity (Inclusivity) Results for Respiratory Syncytial Virus

| RSV Subtype | Strain | Concentration | Multiple of LoD
Detected |
|----------------------------------|---------------|---------------------|-----------------------------|
| Respiratory Syncytial Virus
A | A2 | 4.5 x 100 TCID50/mL | 3x |
| | Long | 4.5 x 100 TCID50/mL | 3x |
| Respiratory Syncytial Virus
B | 9320 | 6 x 10-1 TCID50/mL | 3x |
| | Wash/18537/62 | 6 x 10-1 TCID50/mL | 3x |
| | WV/14617/85 | 6 x 10-1 TCID50/mL | 3x |

Table 24: Analytical Reactivity (Inclusivity) Results for Chlamydia pneumoniae

| | Strain | Concentration | Multiple of LoD
Detected |
|----------------------|------------------|-------------------|-----------------------------|
| Chlamydia pneumoniae | CWL-029 | $9 x 10^2$ CFU/mL | 3x |
| | TWAR strain 2043 | $9 x 10^2$ CFU/mL | 3x |

30

| | Strain | Concentration | Multiple of LoD
Detected |
|-----------------------|-----------|----------------|-----------------------------|
| Mycoplasma pneumoniae | [Bru] | 9 x 102 CCU/mL | 3x |
| | M129-B170 | 9 x 102 CCU/mL | 3x |
| | M129-B7 | 9 x 102 CCU/mL | 3x |
| | [M52] | 9 x 102 CCU/mL | 3x |
| | [Mac] | 9 x 102 CCU/mL | 3x |
| | Mutant 22 | 3 x 104 CCU/mL | 100xa |
| | PI 1428 | 3 x 104 CCU/mL | 100xb |

Table 25: Analytical Reactivity (Inclusivity) Results for Mycoplasma pneumoniae

No sequence data available. Lower sensitivity may be a result of mismatches in the assay primers and/or probes. In addition, the reduced sensitivity may be the result of incorrect estimation of genetic material present in the culture of this or the reference strain (CCU/ml value is based only on live bacteria).

b In silico analysis revealed good homology to primers and probes. The result of incorrect estimation of genetic material present in the culture of this or the reference strain (CCU/ml value is based only on live bacteria).

Analytical Specificity (Cross-Reactivity and Exclusivity)

Cross-reactivity of each viral and bacterial target on the ePlex RP Panel was evaluated at high concentrations (1 x 105 TCID50/mL or >1 x 105 EID50/mL for viruses, 1 x 106 CFU/mL or CCU/mL for bacterial isolates, or 1 x 106 copies/mL for in vitro transcripts) of quantified strains/isolates diluted in viral transport media. In vitro transcript for coronavirus HKU1 was diluted in PBS. Table 26 summarizes the results of the on-panel viral and bacterial strains/isolates tested. No cross-reactivity was observed between any of the on-panel viruses or bacteria.

Table 26: Cross-Reactivity with ePlex RP Panel Target Organisms
Target	Strain	Concentration	Cross-Reactivity
Results
Adenovirus A	Type 31	1 x 105 TCID50/mL	Not observed
Adenovirus B	Type 7A	1 x 105 TCID50/mL	Not observed
Adenovirus C	Type 1	1 x 105 TCID50/mL	Not observed
Adenovirus D	Type 9	1 x 105 TCID50/mL	Not observed
Adenovirus E	Type 4	1 x 105 TCID50/mL	Not observed
Adenovirus F	Type 41	1 x 105 TCID50/mL	Not observed
Coronavirus	229E	1 x 105 TCID50/mL	Not observed
Coronavirus	HKU1 in vitro transcript	1 x 106 copies/mL	Not observed
Coronavirus	NL63	1 x 105 TCID50/mL	Not observed
Coronavirus	OC43	1 x 105 TCID50/mL	Not observed
Enterovirus	Type 68 2007 isolate	1 x 105 TCID50/mL	Not observed
Human metapneumovirus	B1	1 x 105 TCID50/mL	Not observed
Human rhinovirus	1A	1 x 105 TCID50/mL	Not observed

Table 26: Cross-reactivity with ePlex RP Panel Target Organisms

31

| Target | Strain | Concentration | Cross-Reactivity
Results |
|-----------------------|----------------------------------------------|---------------------|-----------------------------|
| Influenza A | A/Brisbane/59/07 | 1 x 105 TCID50/mL | Not observed |
| Influenza A H1 | A/Brisbane/59/07 | 1 x 105 TCID50/mL | Not observed |
| Influenza A H1-2009 | A/NY/01/2009 | 1 x 105 TCID50/mL | Not observed |
| Influenza A H3 | A/Brisbane/10/07 | 1 x 105 TCID50/mL | Not observed |
| Influenza A H3N2va | A/Indiana/21/2012 | 2.51 x 105 EID50/mL | Not observed |
| Influenza A H5N2b | A/Northern Pintail
Washington/40964/14BPL | 2.51 x 105 EID50/mL | Not observed |
| Influenza A H5N8c | A/Gyrfalcon/Washington
/410886/2014 BPL | 1.58 x 105 EID50/mL | Not observed |
| Influenza A H7N9d | A/ANHUI/1/2013 | 7.94 x 105 EID50/mL | Not observed |
| Influenza B | B/Florida/02/06 | 1 x 105 TCID50/mL | Not observed |
| Parainfluenza Virus 1 | C35 | 1 x 105 TCID50/mL | Not observed |
| Parainfluenza Virus 2 | Type 2 | 1 x 105 TCID50/mL | Not observed |
| Parainfluenza Virus 3 | Type 3 | 1 x 105 TCID50/mL | Not observed |
| Parainfluenza Virus 4 | Type 4a | 1 x 105 TCID50/mL | Not observed |
| RSV A | 2006 Isolate | 1 x 105 TCID50/mL | Not observed |
| RSV B | CH93(18)-18 | 1 x 105 TCID50/mL | Not observed |
| Chlamydia pneumoniae | AR-39 | 1 x 106 CFU/mL | Not observed |
| Mycoplasma pneumoniae | FH strain of Eaton Agent
[NCTC 10119] | 1 x 106 CCU/mL | Not observed |

ª Influenza A H3N2v detected as Influenza A, Influenza A H3

b Influenza A H5N2 detected as Influenza A

° Influenza A H5N8 detected as Influenza A

ª Influenza A H7N9 detected as Influenza A

Cross-reactivity of viruses, bacteria, and fungi that are not targets on the ePlex RP Panel was evaluated at high concentrations (1 x 105 TCID50/mL or copies/mL for viruses, 1 x 10° CFU/mL for bacterial and yeast isolates, or 1 x 10 copies/mL for plasmid DNA or genomic RNA) by diluting quantified strains/isolates in viral transport media. Plasmid for bocavirus and genomic RNA for MERS coronavirus (MERS-CoV) were diluted in PBS. Table 27 summarizes the results of the strains tested. No cross-reactivity was observed between any of the off-panel viruses, bacteria or fungi with the ePlex RP Panel targets.

Table 27: Cross-reactivity with Organisms Not Detected by the ePlex RP Panel (Exclusivity)

| Target | Strain | Concentration | Cross-Reactivity
Results |
|--------------------------------------|--------------------|---------------------|-----------------------------|
| Acinetobacter baumanii | ATCC® 19606 | 1 x 106 CFU/mL | Not observed |
| Bordetella pertussis | 18323 [NCTC 10739] | 1 x 106 CFU/mL | Not observed |
| Bordetella parapertussis | ATCC 15311 | 1 x 106 CFU/mL | Not observed |
| Burkholderia cepacia | ATCC 25416 | 1 x 106 CFU/mL | Not observed |
| Candida albicans | ATCC 10231 | 1 x 106 CFU/mL | Not observed |
| Target | Strain | Concentration | Cross-Reactivity
Results |
| Candida glabrata | ATCC 15126 | 1 x 106 CFU/mL | Not observed |
| MERS Coronavirus (MERS-CoV) | EMC/2012a | 1 x 105 copies/mL | Not observed |
| Corynebacterium diphtheriae | ATCC 13812 | 1 x 106 CFU/mL | Not observed |
| Cytomegalovirus | AD 169 | 1 x 105 TCID50/mL | Not observed |
| Epstein Barr Virus | Strain B95-8 | 1 x 105 TCID50/mL | Not observed |
| Escherichia coli | ATCC 10279 | 1 x 106 CFU/mL | Not observed |
| Haemophilus influenzae | ATCC 43065 | 1 x 106 CFU/mL | Not observed |
| Herpes Simplex Virus | Isolate 2 | 1 x 105 TCID50/mL | Not observed |
| Human bocavirus | Bocavirus plasmidb | 1 x 106 copies/mL | Not observed |
| Klebsiella pneumoniae | ATCC 51504 | 1 x 106 CFU/mL | Not observed |
| Lactobacillus acidophilus | ATCC 314 | 1 x 106 CFU/mL | Not observed |
| Lactobacillus plantarum | ATCC 8014 | 1 x 106 CFU/mL | Not observed |
| Legionella pneumophila | Philadelphia-1 | 1 x 106 CFU/mL | Not observed |
| Measles | N/A | 1 x 105 TCID50/mL | Not observed |
| Moraxella catarrhalis | ATCC 23246 | 1 x 106 CFU/mL | Not observed |
| Mumps | Isolate 2 | 1 x 105 TCID50/mL | Not observed |
| Mycobacterium tuberculosis | ATCC 25177 | 1 x 106 CFU/mL | Not observed |
| Neisseria meningiditis | ATCC 13077 | 1 x 106 CFU/mL | Not observed |
| Neisseria sicca | ATCC 29193 | 1 x 106 CFU/mL | Not observed |
| Porphyromonas gingivalis | ATCC 33277 | 1 x 106 CFU/mL | Not observed |
| Proteus vulgaris | ATCC 33420 | 1 x 106 CFU/mL | Not observed |
| Pseudomonas aeruginosa | ATCC 15442 | 1 x 106 CFU/mL | Not observed |
| Serratia marcescens | ATCC 13880 | 1 x 106 CFU/mL | Not observed |
| Staphylococcus aureus (MRSA) | NRS384 | 1 x 106 CFU/mL | Not observed |
| Staphylococcus aureus (MSSA) | ATCC 25923 | 1 x 106 CFU/mL | Not observed |
| Staphylococcus epidermidis
(MRSE) | ATCC 35983 | 1 x 106 CFU/mL | Not observed |
| Staphylococcus epidermidis
(MSSE) | ATCC 49134 | 1 x 106 CFU/mL | Not observed |
| Staphylococcus haemolyticus | ATCC 29970 | 1 x 106 CFU/mL | Not observed |
| Streptococcus agalactiae | ATCC 12401 | 1 x 106 CFU/mL | Not observed |
| Streptococcus dysgalactiae | ATCC 35666 | 1 x 106 CFU/mL | Not observed |
| Streptococcus mitis | ATCC 15914 | 1 x 106 CFU/mL | Not observed |
| Streptococcus pneumoniae | ATCC 49619 | 1 x 106 CFU/mL | Not observed |
| Streptococcus pyogenes | ATCC 12384 | 1 x 106 CFU/mL | Not observed |
| Streptococcus salivarius | ATCC 13419 | 1 x 106 CFU/mL | Not observed |
| Varicella Zoster Virus | 82 | 8.9 x 103 TCID50/mL | Not observed |

32

^a Extracted genomic RNA

ª Extracted genomic RNA
b Plasmid does not contain full length viral genome.

33

Reproducibility

A multisite reproducibility study of the ePlex RP Panel was performed to evaluate agreement with expected results across major sources of variability, such as site-to-site, lot-to-lot, day-today, and operator-to-operator. Testing occurred at 3 sites (2 external, 1 internal) on one ePlex instrument per site with either 3 or 4 towers. Two operators performed testing at each site on 6 days (5 nonconsecutive days) with 3 unique lots of RP Panel cartridges. A reproducibility panel consisting of 3 panel members with 6 organisms (representing 7 RP Panel targets) at 3 concentrations (moderate positive- 3x LoD, low positive- 1x LoD, and negative) was tested in triplicate. The 6 organisms tested included adenovirus, coronavirus, human metapneumovirus, influenza A H3, parainfluenza virus 1, and RSV A; organisms were diluted in natural clinical matrix (pooled, negative nasopharyngeal swab samples). Negative samples consisted of natural clinical matrix only. Each simulated sample was divided into aliquots and stored frozen (-70 °C) prior to testing. Each operator tested 9 samples (3 member reproducibility panel in triplicate) each day; each panel member was tested 108 times (3 replicates x 3 sites x 2 operators x 3 lots x 2 days of testing/operator/lot) for a maximum of 324 tests. After completion of initial and repeat testing for invalid results, 1 low positive sample tested at Site 3 had an invalid result and was excluded from these analyses.

Percent agreement (95% CI) with expected results was 100% for all 7 targets for the moderate positive and negative panel, and 100% for 6 of 7 low positive panel targets (coronavirus, human metapneumovirus, influenza A, influenza A H3, parainfluenza 1, and RSV A); percent agreement was 91.6% for adenovirus. Summary results for the 7 ePlex RP Panel targets that correspond to the 6 organisms in the reproducibility panel are provided in Tables 28-34. Summary results for the 10 ePlex RP Panel targets that did not have organisms included in the reproducibility panel are provided in Table 35.

34

| Adenovirus

Concentration		Agreement with Expected Results
	Site	Agreed / N	%	95% CI
Moderate Positive
3x LoD
6 x $10^0$ TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Low Positive
1x LoD
2 x $10^0$ TCID50/mL	1	36/36	100	(90.4-100)
	2	34/36	94.4	(81.9-98.5)
	3	28/35	80.0	(64.1-90.0)
	All	98/107	91.6	(84.8-95.5)
Negative	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)

Table 28: Percent Agreement for Adenovirus

CI=Confidence Interval

Table 29: Percent Agreement for Coronavirus

| Coronavirus

Concentration	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Moderate Positive
3x LoD
$1.5 x 10^3$ TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Low Positive
1x LoD
$5 x 10^2$ TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	35/35	100	(90.1-100)
	All	107/107	100	(96.5-100)
Negative	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)

35

| hMPV

Concentration	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Moderate Positive
3x LoD
6.75 x 102 TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Low Positive
1x LoD
2.25 x 102 TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	35/35	100	(90.1-100)
	All	107/107	100	(96.5-100)
Negative	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)

Table 30: Percent Agreement for Human Metapneumovirus (hMPV)

Table 31: Percent Agreement for Influenza A

| Influenza A

Concentration	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Moderate Positive
3x LoD
1.5 x 10² TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Low Positive
1x LoD
5 x 10¹ TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	35/35	100	(90.1-100)
	All	107/107	100	(96.5-100)
Negative	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)

36

| Influenza A H3

Concentration	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Moderate Positive
3x LoD
$1.5 x 10^2$ TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Low Positive
1x LoD
$5 x 10^1$ TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	35/35	100	(90.1-100)
	All	107/107	100	(96.5-100)
Negative	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)

Table 32: Percent Agreement for Influenza A H3

Table 33: Percent Agreement for Parainfluenza Virus (PIV) 1

| PIV 1

Concentration	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Moderate Positive
3x LoD
$1.2 x 10^{0} TCID_{50}/mL$	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Low Positive
1x LoD
$4 x 10^{-1} TCID_{50}/mL$	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	35/35	100	(90.1-100)
	All	107/107	100	(96.5-100)
Negative	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)

37

| RSV A

Concentration	Site	Agreement with Expected Results
		Agreed / N	%	95% CI
Moderate Positive
3x LoD
$4.5 x 10^0$ TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)
Low Positive
1x LoD
$1.5 x 10^0$ TCID50/mL	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	35/35	100	(90.1-100)
	All	107/107	100	(96.5-100)
Negative	1	36/36	100	(90.4-100)
	2	36/36	100	(90.4-100)
	3	36/36	100	(90.4-100)
	All	108/108	100	(96.6-100)

Table 34: Percent Agreement for Respiratory Syncytial Virus (RSV) A

Table 35: Negative Percent Agreement for Targets With Organisms Not Included in the Reproducibility Panel

Target	Site	Agreement with Expected Negative Results
		Agreed / N	%	95% CI
Human Rhinovirus/Enterovirus	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	104/107	97.2	(92.1-99.0)
	All	320/323	99.1	(97.3-99.7)
Influenza A H1	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	107/107	100	(96.5-100)
	All	323/323	100	(98.8-100)
Influenza A H1-2009	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	107/107	100	(96.5-100)
	All	323/323	100	(98.8-100)
Influenza B	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	107/107	100	(96.5-100)
	All	323/323	100	(98.8-100)
Parainfluenza Virus 2	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	107/107	100	(96.5-100)
	All	323/323	100	(98.8-100)

38

Target	Site	Agreement with Expected Negative Results
		Agreed / N	%	95% CI
Parainfluenza Virus 3	l	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	106/107	99.1	(94.9-99.8)
	All	322/323	99.7	(98.3-99.9)
Parainfluenza Virus 4	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	107/107	100	(96.5-100)
	All	323/323	100	(98.8-100)
Respiratory Syncytial Virus B	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	107/107	100	(96.5-100)
	All	323/323	100	(98.8-100)
Chlamydia pneumoniae	1	108/108	100	(96.6-100)
	2	108/108	100	(96.6-100)
	3	107/107	100	(96.5-100)
	All	323/323	100	(98.8-100)
Mycoplasma pneumoniae	1	108/108	100	(96.6-100)
	2	107/108	99.1	(94.9-99.8)
	3	106/107	99.1	(94.9-99.8)
	All	321/323	99.4	(97.8-99.8)

39

Samples with Co-Detected Organisms

Detection of more than one clinically relevant viral and/or bacterial organism in a sample was evaluated with the ePlex RP Panel using a natural clinical matrix (pooled, negative nasopharyngeal swab samples) spiked with two RP Panel organisms: one organism at a low concentration (1-3x LoD) and the second organism at a high concentration (1 x 10 TCIDsymL). Table 36 contains the results of co-detection testing which demonstrated the ability of the ePlex RP Panel to detect 2 organisms in a sample at both high and low concentrations as indicated in the table.

Organism 1	High Titer	Organism 2	Low Titer
Influenza A H3	$1 x 10^5$
TCID50/mL	Adenovirus B	$2 x 10^0$
TCID50/mL	1x
Adenovirus	$1 x 10^5$
TCID50/mL	Influenza A H3	$5 x 10^1$ TCID50/mL	1x
Influenza A H3	$1 x 10^5$
TCID50/mL	RSV A	$1.5 x 10^0$
TCID50/mL	1x
RSV A	$1 x 10^5$
TCID50/mL	Influenza A H3	$5 x 10^1$
TCID50/mL	1x
Influenza A H1-2009	$1 x 10^5$
TCID50/mL	RSV B	$6 x 10^{-1}$ TCID50/mL	3x
RSV B	$1 x 10^5$
TCID50/mL	Influenza A H1-2009	$1 x 10^{-1}$
TCID50/mL	1x
Influenza A H1-2009	$1 x 10^5$
TCID50/mL	Rhinovirus	$1.5 x 10^0$
TCID50/mL	1x
Rhinovirus	$1 x 10^5$
TCID50/mL	Influenza A H1-2009	$3 x 10^{-1}$
TCID50/mL	3x
Influenza A H1-2009	$1 x 10^5$
TCID50/mL	Parainfluenza Virus 3	$5 x 10^0$
TCID50/mL	1x
Parainfluenza Virus 3	$1 x 10^5$
TCID50/mL	Influenza A H1-2009	$1 x 10^{-1}$
TCID50/mL	1x
Rhinovirus	$1 x 10^5$
TCID50/mL	RSV A	$1.5 x 10^0$
TCID50/mL	1x
RSV A	$1 x 10^5$
TCID50/mL	Rhinovirus	$1.5 x 10^0$
TCID50/mL	1x
Coronavirus	$1 x 10^5$
TCID50/mL	RSV A	$1.5 x 10^0$
TCID50/mL	1x
RSV A	$1 x 10^5$
TCID50/mL	Coronavirus	$7.5 x 10^0$
TCID50/mL	1x
Human
Metapneumovirus	$1 x 10^5$
TCID50/mL	Adenovirus	$2 x 10^0$ TCID50/mL	1x
Adenovirus	$1 x 10^5$
TCID50/mL	Human
Metapneumovirus	$2.25 x 10^2$
TCID50/mL	1x
Adenovirus	$1 x 10^5$
TCID50/mL	RSV A	$1.5 x 10^0$
TCID50/mL	1x
RSV A	$1 x 10^5$
TCID50/mL	Adenovirus	$2 x 10^0$
TCID50/mL	1x

Table 36: Detection of Co-detections

40

Sample Matrix Equivalency

All analytical studies that utilized viral and bacterial cultures close to LoD were performed by spiking the viral and bacterial cultures into a pool of natural negative NPS in VTM as sample matrix. For analytical studies that used viral and bacterial cultures at a concentration which was at least 10x LoD or higher, the viral and bacterial cultures were spiked into MicroTest™ M5® transport media from Remel instead of negative pooled NPS for ease of use. A sample matrix equivalency study was performed to demonstrate equivalency of natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples) with viral transport media for targets spiked at a concentration of approximately 10x LoD. Quantified, representative viral and bacterial strains were diluted in a natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples) and in viral transport media. All samples were tested in duplicate. There was no difference observed in detection of targets in natural clinical matrix vs. viral transport media.

Interfering Substances

Substances commonly found in respiratory samples, substances that could be introduced during specimen collection, or medications commonly used to treat congestion, allergies, or asthma symptoms that could potentially interfere with the ePlex RP Panel were individually evaluated. To simulate clinical samples, quantified representative viral and bacterial strains were diluted to 1x LoD in a natural clinical matrix (pooled, negative nasopharyngeal swab specimens) and tested in triplicate for negative and positive interference. Natural clinical matrix (pooled, negative nasopharyngeal swab samples) with no organisms added was used as a control. All substances and organisms tested for interference were shown to be compatible with the ePlex RP Panel. No potentially interfering substances were found to inhibit the ePlex RP Panel at the concentrations tested in Table 37.

41

Potentially Interfering Substance	Active Ingredient	Testing Concentration
Control Sample Matrixa	Becton Dickinson UVT	N/A
Transport Mediuma	Copan eSwab (Liquid Amies media)	N/A
	MicroTest M4	N/A
	MicroTest M4-RT	N/A
Viral Transport Mediuma	MicroTest M5	N/A
	MicroTest M6	N/A
	Copan Minitip in UVT	N/A
Flocked Swabs	Copan Regular Tip in UVT	N/A
Blood (human)	Blood	2% v/v
	Human gDNA	50 ng/rxn
Throat lozenges, oral anesthetic
and analgesic	Benzocaine, menthol	26% w/v
Mucin	Purified mucin protein	1% w/v
Nasal sprays or drops	Phenylephrine HCl (Neo-Synephrine®)	1.5% v/v
	Oxymetazoline HCl (Afrin®)	1% v/v
	Sodium chloride	0.8% w/v
Antibacterial, systemic	Tobramycin b	1% w/v
Antibiotic, nasal ointment	Mupirocin	2% w/v
Nasal corticosteroids	Beclomethasone	1.5% w/v
	Dexamethasone	1.5% w/v
	Flunisolide	1.5% w/v
	Budesonide (Rhinocort®)	0.9% v/v
	Triamcinolone (Nasacort®)	1.5% v/v
	Fluticasone (Flonase®)	1.5% v/v
ZICAM® Allergy Relief Nasal Gel	Luffa opperculata
	Sulfur	1% v/v
	Galphimia glauca
	Histaminum hydrochloricum
Anti-viral drugs	Zanamivir	550 ng/mL
	Oseltamivir	142 ng/mL
Virus	Cytomegalovirus	1 x 105 TCID50/mL
Bacteria	Streptococcus pneumoniae	1 x 106 CFU/mL
	Bordetella parapertussis
	Haemophilus influenza
	Staphylococcus aureus
	Neisseria meningitides
	Corynebacterium diptheriae

Table 37: List of Substances for Testing

ª Testing of media was done by adding a negative NPS collected in the specified media and diluting in the natural clinical matrix.

" room of the active of tading a negall of in the sample, tobramycin was found to inhibit assay parformance.

42

Carryover and Cross-contamination

The carryover/cross-contamination rate of the ePlex RP Panel and ePlex instrument was tested in a checkerboard approach by running high positive and negative samples interspersed in all bays of a four-tower ePlex instrument (24 bays total) over 5 separate runs on 5 separate days. Quantified parainfluenza virus 3 was prepared in viral transport media at a high concentration (1 x 105 TCIDso/mL, 20,000x LoD) to simulate a clinically relevant high positive and was tested as a representative target organism. Transport media was used to represent negative samples. On each round of testing, 24 ePlex RP Panel cartridges were evaluated. 100% of parainfluenza 3positive samples generated a result of Detected and 100% of parainfluenza 3-negative samples generated a parainfluenza 3 result of No Target Detected, indicating no carryover or crosscontamination was observed between bays or within bays with the ePlex RP Panel when testing consecutively or in adjacent bays.