← Product Code [OCC](/productcode/OCC) · K163636

# ePlex Respiratory Pathogen Panel (K163636)

_Genmark Diagnostics, Incorporated · OCC · Jun 9, 2017 · Microbiology · SESE_

**Canonical URL:** https://fda-staging.innolitics.com/device/K163636

## Device Facts

- **Applicant:** Genmark Diagnostics, Incorporated
- **Product Code:** [OCC](/productcode/OCC.md)
- **Decision Date:** Jun 9, 2017
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.3980
- **Device Class:** Class 2
- **Review Panel:** Microbiology
- **Attributes:** Real-World Evidence, Pediatric

## Real-World Evidence

| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
| --- | --- | --- | --- | --- | --- |
| K163636 · Jun 9, 2017 | ePlex Respiratory Pathogen Panel | Genmark Diagnostics, Incorporated | Retrospective clinical nasopharyngeal swab specimens collected during standard-of-care testing | Retrospective clinical specimens were used to supplement the prospective clinical study to provide sufficient positive samples for low-prevalence organisms. | Retrospective clinical study; Standard-of-care specimens; Clinical performance evaluation |

### Clinical Evidence

| Study Design | Population | Comparator | Key Endpoints |
| --- | --- | --- | --- |
| Retrospective Clinical Study; Retrospective collection of residual clinical specimens | Patients presenting with signs/symptoms of respiratory infection; Sample Size: 446 evaluable specimens; Number of Sites: 6 | FDA-cleared multiplexed respiratory pathogen panel | Positive and negative percent agreement |

## 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 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.

## Device Story

The ePlex RP Panel is an automated, multiplexed, qualitative in vitro diagnostic test for respiratory pathogens. It processes nasopharyngeal swab samples on the ePlex Instrument using a single-use cartridge. The device automates nucleic acid extraction, amplification (RT-PCR), and detection. It utilizes electrowetting (digital microfluidics) to manipulate droplets on a PCB and eSensor technology for electrochemical detection via voltammetry, avoiding optical/fluorescent methods. The system identifies 15 viral and 2 bacterial targets. It is intended for use in clinical laboratories by trained personnel. The instrument provides automated interpretation and reporting; raw data is inaccessible to users. Results aid clinicians in diagnosing respiratory infections, though they must be interpreted alongside clinical and epidemiological data. The device benefits patients by providing rapid, simultaneous identification of multiple pathogens, potentially guiding appropriate treatment and infection control decisions.

## Clinical Evidence

Prospective clinical study (N=2462) and retrospective study (N=446) compared ePlex RP Panel to FDA-cleared multiplexed respiratory panel. Mock clinical study (N=326) evaluated low-prevalence targets. Overall PPA/NPA were high across targets. Bench testing confirmed analytical sensitivity (LoD), reactivity, specificity, and stability.

## Technological Characteristics

Single-use cartridge; magnetic solid-phase extraction; PCR/RT-PCR amplification; eSensor electrochemical detection (ferrocene-labeled probes); electrowetting (digital microfluidics) on PCB; automated instrument-based processing; room temperature reagent storage.

## Regulatory Identification

A respiratory viral panel multiplex nucleic acid assay is a qualitative in vitro diagnostic device intended to simultaneously detect and identify multiple viral nucleic acids extracted from human respiratory specimens or viral culture. The detection and identification of a specific viral nucleic acid from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection when used in conjunction with other clinical and laboratory findings. The device is intended for detection and identification of a combination of the following viruses:(1) Influenza A and Influenza B; (2) Influenza A subtype H1 and Influenza A subtype H3; (3) Respiratory Syncytial Virus subtype A and Respiratory Syncytial Virus subtype B; (4) Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus; (5) Human Metapneumovirus; (6) Rhinovirus; and (7) Adenovirus.

## Special Controls

*Classification.* Class II (special controls). The special controls are:(1) FDA's guidance document entitled “Class II Special Controls Guidance Document: Respiratory Viral Panel Multiplex Nucleic Acid Assay;”
(2) For a device that detects and identifies Human Metapneumovirus, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Human Metapneumovirus (hMPV) Using Nucleic Acid Assays;” and
(3) For a device that detects and differentiates Influenza A subtype H1 and subtype H3, FDA's guidance document entitled “Class II Special Controls Guidance Document: Testing for Detection and Differentiation of Influenza A Virus Subtypes Using Multiplex Nucleic Acid Assays.” See § 866.1(e) for the availability of these guidance documents.

## Predicate Devices

- FilmArray® Respiratory Panel (RP) ([K160068](/device/K160068.md))

## Submission Summary (Full Text)

> This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com.
>
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY

A. 510(k) Number:

K163636

B. Purpose for Submission:

New device

C. Measurand:

Adenovirus, Coronavirus, Human Metapneumovirus (hMPV), Human Rhinovirus/Enterovirus (HRV/HEV), Influenza A, Influenza A H1 (Flu A/H1), Influenza A H1-2009 (Flu A/H1-2009), Influenza A H3 (Flu A/H3), Influenza B (Flu B), Parainfluenza virus 1 (PIV 1), Parainfluenza virus 2 (PIV 2), Parainfluenza virus 3 (PIV 3), Parainfluenza virus 4 (PIV 4), Respiratory Syncytial Virus A (RSV A), Respiratory Syncytial Virus B (RSV B), Chlamydia pneumoniae, and Mycoplasma pneumoniae nucleic acids target sequences.

D. Type of Test:

A multiplexed nucleic acid test intended for use with the GenMark ePlex instrument for the simultaneous qualitative in vitro detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) collected in viral transport media and obtained from individuals suspected of respiratory tract infections.

E. Applicant:

GenMark Diagnostics, Incorporated

F. Proprietary and Established Names:

ePlex® Respiratory Pathogen (RP) Panel

Common Name: ePlex RP® Panel

G. Regulatory Information:

|  Product Code | Classification | Regulation Section | Panel  |
| --- | --- | --- | --- |
|  OCC | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83)  |
|  OEM | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83)  |

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|  OEP | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83)  |
| --- | --- | --- | --- |
|  OOU | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83)  |
|  OTG | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83)  |
|  OZE | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83)  |
|  OZX | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83)  |
|  OZY | Class II | 21 CFR 866.3980 Respiratory Viral Panel Multiplex Nucleic Acid Assay | Microbiology (83)  |
|  OQW | Class II | 21 CFR 866.3332 Reagents for Detection of Specific Novel Influenza A Viruses | Microbiology (83)  |
|  NSU | Class II | 21 CFR 862.2570 Instrumentation for Clinical Multiplex Test Systems | Clinical Chemistry (75)  |

### H. Intended Use:

1. Intended 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,

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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.

2. Indication for use:
Same as Intended Use
3. Special conditions for use statement(s):
For prescription use only
4. Special instrument requirements:
ePlex instrument

# I. Device Description:

The ePlex Respiratory Pathogen (RP) Panel is based on the principles of competitive nucleic acid hybridization using a sandwich assay format, wherein a single-stranded target binds concurrently to a sequence-specific solution-phase signal probe and a solid-

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phase electrode-bound capture probe. The test employs nucleic acid extraction, target amplification via polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR), and hybridization of target DNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate single-stranded DNA suitable for hybridization.

Nucleic acid extraction from biological specimens occurs within the cartridge via cell lysis, nucleic acid capture onto magnetic beads, and release for amplification. The nucleic acid extraction is processed through microfluidic liquid handling. Once the nucleic acid targets are captured and inhibitors are washed away, the magnetic particles are delivered to the electrowetting environment on the printed circuit board (PCB) and the targets are eluted from the particles and amplified.

During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working gold electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to specific target sequence / region adjacent to the capture probe. Simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each working electrode on the array contains specific capture probes, and sequential analysis of each electrode allows detection of multiple analyte targets.

A summary of the ePlex RP Panel nucleic acid targets is presented in Table 1 below.

Table 1: A summary of the ePlex RP Targets

|  Organism | Target Gene(s)  |
| --- | --- |
|  Adenovirus | Hexon protein (L3) and Penton protein (L2)  |
|  Coronaviruses (229E, HKU1, NL63, and OC43) | Nucleoprotein (N)  |
|  Human Metapneumovirus | Nucleocapsid (N)  |
|  Influenza A | Matrix protein (M)  |
|  Influenza A H1 | Hemagglutinin (HA)  |
|  Influenza A 2009 H1N1 | Hemagglutinin (HA)  |
|  Influenza A/H3 | Hemagglutinin (HA)  |
|  Influenza B | RNA polymerase subunit PB1  |
|  Parainfluenza 1 | Hemagglutinin-neuraminidase (HN)  |
|  Parainfluenza 2 | Hemagglutinin-neuraminidase (HN)  |
|  Parainfluenza 3 | Hemagglutinin-neuraminidase (HN)  |
|  Parainfluenza 4 | Hemagglutinin-neuraminidase (HN)  |
|  Human Rhinovirus/Enterovirus | 5'- Untranslated Region (UTR)  |
|  RSV A | Nucleocapsid (N)  |
|  RSV B | Nucleocapsid (N)  |
|  Chlamydia pneumoniae | Major Outer Membrane Protein A (OmpA)  |
|  Mycoplasma pneumonia | Cytadhesin (P1)  |

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### Materials Provided:

Each ePlex Respiratory Pathogen Panel kit contains sufficient reagents to test 12 samples (EA001012):

• ePlex Respiratory Pathogen Panel Cartridge (12)
• Sample Delivery Device – RP Panel; 200 μl (12)

### Materials Needed but Not Provided:

#### Equipment

• GenMark ePlex instrument and software
• Pipettes calibrated to deliver 200 μl
- Vortex mixer
- Printer (Optional)

#### Consumables

• Pipette tips, aerosol resistant, RNase/DNase-free
• Disposable, powder free gloves
• 10% bleach for decontamination of appropriate surfaces
• 70% ethanol or isopropyl alcohol

### Interpretation of Results

Results interpretation of the ePlex Respiratory Pathogen Panel is performed by the ePlex instrument, and the interpretation of results on the ePlex RP Panel Detection Report for each targeted analyte is summarized in Table 2 below.

Table 2: Interpretation of Results on the ePlex RP Panel Detection Report

|  Target Result | Explanation | Action  |
| --- | --- | --- |
|  Target Detected | The test was completed successfully, and the target has generated signal above its defined threshold, and the Internal Control was reported as PASS. | All results are displayed on the RP Panel Detection Report.Test is valid, report results.  |
|  Multiple Targets Detected | The test was completed successfully, and multiple targets have generated signals above its defined threshold, and the Internal Control was reported as PASS. | All results are displayed on the RP Panel Detection Report.Test is valid, report results.Detection of more than 3 pathogens may indicate contamination. Re-test of the sample is recommended to confirm results.  |
|  Not Detected | The test was completed successfully, and the target did not generate signal above its defined threshold, and the Internal Control was reported as PASS. | All results are displayed on the RP Panel Detection Report.Test is valid, report results.  |
|  Invalid | The test has not successfully completed, and results for this test are not valid. This is often due to an instrument or software error or failure of an internal control. | No results are displayed on the RP Panel Detection Report.Test is not valid, repeat test.  |

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# Influenza A Results

The ePlex RP Panel detects Influenza A and the A/H1, A/H1-2009, and A/H3 subtypes using unique assays for each. Interpretation of results for Influenza A are described in Table 3 below.

Table 3: Possible Assay Results for Influenza A and the Corresponding Interpretation

|  Results for Influenza A and Subtypes | Explanation | Results on Report | Recommended Action  |
| --- | --- | --- | --- |
|  Influenza A detected, at least one subtype (A/H1, A/H1-2009, or A/H3) reported as detected. | This is an expected result. | Result reported as influenza A and influenza A subtype detected. | None  |
|  Influenza A detected, all subtypes (A/H1, A/H1-2009, and A/H3) reported as not detected | Low virus titers can result in detection of influenza A matrix without a subtype. Detection of influenza A matrix without a subtype can also indicate the presence of a novel strain. | Result reported as influenza A detected (no subtype detected) | Re-test to confirm result. If the re-test provides a different result, test the sample a third time to ensure the accuracy of the result. If the re-test provides the same result, then the function of the ePlex RP Panel cartridge should be verified by testing with appropriate external positive control materials (known positive samples for influenza A H1, influenza A H3 and influenza A H1-2009), and a negative control should also be run to test for potential PCR-product contamination. If the ePlex RP Panel accurately identifies the external and negative controls, contact the appropriate public health authorities for confirmatory testing.  |
|  Influenza A detected and more than one subtype (A/H1, A/H1-2009, or A/H3) reported as detected. | Sample is co-infected with multiple influenza subtypes. Infections with multiple subtypes of influenza are possible but rare. A live intranasal multivalent influenza virus vaccine may cause | Result reported as influenza A and multiple subtypes detected. | Re-test to confirm result. If the re-test result confirms the original result, it is recommended that the sample be further investigated using a different FDA-cleared influenza A subtyping assay.  |

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|  Results for Influenza A and Subtypes | Explanation | Results on Report | Recommended Action  |
| --- | --- | --- | --- |
|   | false positive results for influenza A, A/H1, A/H3, A/H1-2009, and/or influenza B. Contamination has occurred. |  |   |
|  Influenza A not detected, at least one subtype (A/H1, A/H1-2009, or A/H3) reported as detected. | Low virus titers can result in detection of influenza A subtype without the influenza A matrix. Detection of influenza A subtype without the influenza A matrix can also indicate the presence of a novel strain. | Influenza A (subtype) detected. Re-testing of this sample to confirm influenza A (subtype) is recommended. Refer to package insert for additional information. | Re-test to confirm result. If the re-test result confirms the original result, the influenza A subtype is considered positive. It is recommended that the sample be further investigated using a different FDA-cleared influenza A subtyping assay and/or sending the residual sample to local public health laboratory for further testing.  |

### ePlex RP Panel Test Reports

There are several different reports that are available on the ePlex instrument. Results are provided in a printable format, may be viewed electronically, or may be exported for additional analysis. Reports can be customized with account specific information such as the address, logo, and institution specific footers on each report.

#### Detection Report

The ePlex RP Panel Detection Report includes the results for each individual sample run on the ePlex instrument. The Summary section indicates the overall test result and lists all detected analytes in that sample. The Results section includes a list of all analytes on the panel with an individual result for each. Results for each analyte are reported as Detected, Not Detected, or Invalid (displayed as a red x); results for the Internal Control are reported as PASS, FAIL, INVALID, or N/A.

#### External Control Report

The ePlex RP Panel External Control Report is generated for an external control that has been pre-defined in the ePlex RP Panel software. The Summary section indicates the overall result (Pass or Fail status) and lists all detected analytes for that external control. The Results section includes a list of all panel analytes with the result, expected result, and Pass/Fail status for each. Results are reported as Detected, Not Detected, or Invalid (displayed as a red x). An analyte is reported as Pass if the actual result matches the expected result (as defined for that control); an analyte is reported as Fail if the actual result does not match the expected result. If the actual results for each analyte match the

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expected results (all analytes reported as Pass), the overall result for the external control is reported as Pass in the Summary section. If the actual result for any analyte does not match the expected result, the overall result for the external control is reported as Fail in the Summary section.

# Summary Report

The Summary Report allows the operator to use defined searchable criteria to create customized reports, using specified analytes, dates, range of dates, sample, external control, test bay, or operator.

# J. Substantial Equivalence Information:

1. Predicate device name(s):

FilmArray® Respiratory Panel (RP)

2. Predicate K number(s):

K160068

3. Comparison with predicate(s):

|  Similarities or Differences  |   |   |
| --- | --- | --- |
|  Element | ePlex Respiratory Pathogen (RP) Panel (K163636) | FilmArray Respiratory Panel (RP) (K160068)  |
|  Specimen Types | NPS (in transport media) | Same  |
|  Organisms Detected | Adenovirus, Coronavirus, Human Metapneumovirus, Influenza A, Influenza A H1, Influenza A H3, Influenza A H1-2009, Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Human Rhinovirus/Enterovirus, Respiratory Syncytial Virus A, , Respiratory Syncytial Virus B, Chlamydia pneumoniae, and Mycoplasma pneumoniae | Same, except that it also detects and differentiates Coronavirus 229E, Coronavirus HKU1, Coronavirus NL63, and Coronavirus OC43; it also detects Bordetella pertussis; it does not differentiate Respiratory Syncytial Virus A and Respiratory Syncytial Virus B  |
|  Analyte | RNA/DNA | Same  |
|  Technological Principles | Multiplex nucleic acid amplification test | Same  |
|  Instrumentation | ePlex Instrument | FilmArray 2.0 or FilmArray® Torch  |

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|  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.  |
| --- | --- | --- |
|  Reagent Storage | Room temperature | Same  |
|  Test Interpretation | Automated test interpretation and report generation. User cannot access raw data. | Same  |
|  Controls | Eight internal controls 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.  |

### K. Standard/Guidance Documents Referenced (if applicable):

None

### L. Test Principle:

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, as shown below in Figure 1.

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![img-0.jpeg](img-0.jpeg)

Figure 1: Hybridization complex. Target-specific capture probes are bound to the gold electrodes in the eSensor microarray on the ePlex cartridge. The amplified target DNA hybridizes to the capture probe and to a complementary ferrocene-labeled signal probe. Electrochemical analysis determines the presence or absence of targets using voltammetry.

The presence of each target is determined by voltammetry which generates specific electrical signals from the ferrocene-labeled signal.

### M. Performance Characteristics (if/when applicable):

#### 1. Analytical performance:

##### a. Reproducibility Study :

A multisite reproducibility study of the ePlex RP Panel was conducted at three testing sites (two external sites and one internal site) across major potential sources of variability, such as site-to-site, lot-to-lot, day-to-day, and operator-to-operator. One ePlex instrument per study site with either three or four towers was employed in this reproducibility study. Two operators performed testing at each site on six days (five nonconsecutive days) with three unique lots of ePlex RP Panel cartridges. A reproducibility study panel consisting of three panel members with six organisms that represent seven RP Panel targets (multi-spiked \( ^{1} \) ) at three concentrations (moderate positive - 3x LoD, low positive - 1x LoD, and negative) was tested in triplicate. The six representative organisms in the reproducibility study panel included adenovirus, coronavirus OC43 (CoV-OC43), human metapneumovirus,

\( ^{1} \)  Due to the design of ePlex RP Panel, where each of the representative targets are located in separate PCR reactions/lanes (except for CoV-OC43 and RSV A), nucleic acid amplification of each target is conducted in a reaction that is physically separated from the other targets. Therefore, pooling of multiple analyte targets in one specimen (i.e., the multi-spiking approach) most likely does not perform differently than a test sample containing a single analyte target. Analytical study results also demonstrated that the same analytical sensitivity was achieved for CoV-OC43 and RSV A regardless of whether they were tested separately/independently or together within the same sample mix.

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influenza A H3, parainfluenza virus 1, and RSV A; organisms were diluted in natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples). Negative samples consisted of the same natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples) only. Each contrived sample was divided into aliquots and stored frozen (-70 °C) prior to testing. Each operator tested nine samples (i.e., three 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 minimum of 324 tests.

Summary results for the ePlex RP Panel reproducibility study are provided in Table 4 below.

Table 4: Reproducibility of ePlex RP Panel Results

|  Analyte | Concentration Tested | Expected Result | Agreement with Expected Result  |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  Site 1 | Site 2 | Site 3^{a} | All Sites (95% CI)  |
|  **Viruses**  |   |   |   |   |   |   |
|  Adenovirus | Moderate Positive (3× LoD) (6.0E+00 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|   |  Low Positive (1× LoD) (2.0E+00 TCID_{50}/mL) | Detected | 36/36 100% | 34/36 94.4% | 28/35 80.0% | 98/107 91.6% (84.8%-95.5%)  |
|   |  None (no analyte) | Not Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|  Coronavirus | Moderate Positive (3× LoD) (1.5E+03 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|   |  Low Positive (1× LoD) (5.0E+02 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 35/35 100% | 107/107 100% (96.5%-100%)  |
|   |  None (no analyte) | Not Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|  Human Metapneumovirus | Moderate Positive (3× LoD) (6.8E+02 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|   |  Low Positive (1× LoD) (2.3E+02 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 35/35 100% | 107/107 100% (96.5%-100%)  |

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|  Analyte | Concentration Tested | Expected Result | Agreement with Expected Result  |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  Site 1 | Site 2 | Site 3^{a} | All Sites (95% CI)  |
|   | None (no analyte) | Not Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|  **Human Rhinovirus/Enterovirus** | None (no analyte) | Not Detected | 108/108 100% | 108/108 100% | 104/107 97.2% | 320/323 99.1% (97.3%-99.7%)  |
|  **Influenza A** | Moderate Positive (3× LoD) (1.5E+02 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|   |  Low Positive (1× LoD) (5.0E+01 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 35/35 100% | 107/107 100% (96.5%-100%)  |
|   |  None (no analyte) | Not Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|  **Influenza A H1-2009** | None (no analyte) | Not Detected | 108/108 100% | 108/108 100% | 107/107 100% | 323/323 100% (98.8%-100%)  |
|  **Influenza A H1** | None (no analyte) | Not Detected | 108/108 100% | 108/108 100% | 107/107 100% | 323/323 100% (98.8%-100%)  |
|  **Influenza A H3** | Moderate Positive (3× LoD) (1.5E+02 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|   |  Low Positive (1× LoD) (5.0E+01 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 35/35 100% | 107/107 100% (96.5%-100%)  |
|   |  None (no analyte) | Not Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|  **Influenza B** | None (no analyte) | Not Detected | 108/108 100% | 108/108 100% | 107/107 100% | 323/323 100% (98.8%-100%)  |
|  **Parainfluenza Virus 1** | Moderate Positive (3× LoD) (1.2E+00 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 36/36 100% | 108/108 100% (96.6%-100%)  |
|   |  Low Positive (1× LoD) (4.0E-01 TCID_{50}/mL) | Detected | 36/36 100% | 36/36 100% | 35/35 100% | 107/107 100% (96.5%-100%)  |

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|  Analyte | Concentration Tested | Expected Result | Agreement with Expected Result  |   |   |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |   |   |  Site 1 | Site 2 | \( Site\ 3^a \) | All Sites (95% CI)  |
|   | None(no analyte) | Not Detected | 36/36100% | 36/36100% | 36/36100% | 108/108100%(96.6%-100%)  |
|  Parainfluenza Virus 2 | None(no analyte) | Not Detected | 108/108100% | 108/108100% | 107/107100% | 323/323100%(98.8%-100%)  |
|  Parainfluenza Virus 3 | None(no analyte) | Not Detected | 108/108100% | 108/108100% | 106/10799.1% | 322/32399.7%(98.3%-99.9%)  |
|  Parainfluenza Virus 4 | None(no analyte) | Not Detected | 108/108100% | 108/108100% | 107/107100% | 323/323100%(98.8%-100%)  |
|  Respiratory Syncytial Virus A | Moderate Positive (3× LoD)(4.5E+00 TCID50/mL) | Detected | 36/36100% | 36/36100% | 36/36100% | 108/108100%(96.6%-100%)  |
|   |  Low Positive (1× LoD)(1.5E+00 TCID50/mL) | Detected | 36/36100% | 36/36100% | 35/35100% | 107/107100%(96.5%-100%)  |
|   |  None(no analyte) | Not Detected | 36/36100% | 36/36100% | 36/36100% | 108/108100%(96.6%-100%)  |
|  Respiratory Syncytial Virus B | None(no analyte) | Not Detected | 108/108100% | 108/108100% | 107/107100% | 323/323100%(98.8%-100%)  |
|  Bacteria  |   |   |   |   |   |   |
|  Chlamydia pneumoniae | None(no analyte) | Not Detected | 108/108100% | 108/108100% | 107/107100% | 323/323100%(98.8%-100%)  |
|  Mycoplasma pneumoniae | None(no analyte) | Not Detected | 108/108100% | 107/10899.1% | 106/10799.1% | 321/32399.4%(97.8%-99.8%)  |

\( ^{a} \)  1/324 reproducibility panel samples had initial and re-tested invalid results (tested at Site 3) and was excluded from this analysis.

Over the course of the reproducibility study, a total of three different ePlex instruments and 66 different ePlex bays (one 4-tower ePlex instrument each at Site 1 and Site 2, and one 3-tower ePlex instrument at Site 3, each tower has six bays) were used by six different operators at three different sites. All bays were utilized at least once during the conduct of this study.

In total, 334 runs were attempted with one pre-flight check failure. Of the 333 runs that initiated, all runs completed and valid results were obtained from 323 out of the 333 runs that were initiated (323/333, 97.0%).

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Of the 324 reproducibility panel samples initially tested, 315 (97.2%; 95% CI: 94.8% - 98.5%) had valid results and nine (2.8%; 95% CI: 1.5% - 5.2%) had invalid results. Initial validity rates were similar across the testing sites: 96.3% at Site 1, 98.1% at Site 2, 97.2% at Site 3. Initially invalid samples were re-tested and eight had valid results. The final validity rate was 99.7% (95% CI: 98.3%-99.9%).

b. Linearity/assay reportable range:

Not applicable, qualitative assay

c. Traceability, Stability, Expected values (controls, calibrators, or methods):

### Assay Controls

#### Internal Controls

Each ePlex RP Panel cartridge includes internal controls that monitor performance of each step of the testing process. A DNA control verifies extraction, amplification and detection of DNA targets, and RNA controls verify amplification and detection of RNA targets. Each amplification reaction on the cartridge has at least one internal control and in each reaction either the internal control or a target must generate signal above the defined threshold for a valid test result. Internal control results are interpreted by the ePlex software and displayed on ePlex RP Panel Reports as Internal Control with a result of PASS, FAIL, N/A, or INVALID. Table 5 below includes detailed interpretation of Internal Control results.

Table 5: Internal Control Results

|  Internal Control Result | Explanation | Action  |
| --- | --- | --- |
|  PASS | The internal control or a target from each amplification reaction has generated signal above the threshold.The test was completed and internal controls were successful, indicating valid results were generated. | All results are displayed on the RP Panel Detection Report.Test is valid, report results.  |
|  FAIL | Neither the internal control nor any target in at least one amplification reaction generates signal above the threshold.The test was completed but at least one internal control was not detected, indicating that results are not valid. | No results are displayed on the RP Panel Detection Report.Test is not valid, repeat the test using a new cartridge.  |

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|  Internal Control Result | Explanation | Action  |
| --- | --- | --- |
|  N/A | The internal control in every amplification reaction does not generate signal above the threshold, but a target in every amplification reaction does generate signal above the threshold. The test was completed and internal controls were not successful, however detection of signal above the threshold for a target in every amplification reaction indicates valid results were generated. | All results are displayed on the RP Panel Detection Report. Test is valid, report results.  |
|  INVALID | An error has occurred during processing that prevents analysis of signal data. The test has not successfully completed and results for this test are not valid. This is often due to an instrument or software error. | No results are displayed on the RP Panel Detection Report. Test is not valid, repeat the test using a new cartridge.  |

# External Controls

External controls are not provided with the ePlex RP Panel. However, during the prospective clinical evaluation of the ePlex RP Panel, each testing site was provided with positive and negative external control samples. There were four external positive controls (A, B, C, D) and one external negative control. The positive controls together represent all the targets on the ePlex RP Panel. On each day of testing with the ePlex RP Panel, each site tested at least one positive and one negative control. Sites rotated through the four positive controls throughout the study.

Table 6: External Controls Utilized in the Clinical Evaluations

|  External Controls | Expected Calls  |
| --- | --- |
|  Positive Control A | Adenovirus, Coronavirus, Influenza A H3, PIV 4, RSV A, and C. pneumoniae  |
|  Positive Control B | Adenovirus, Coronavirus, Human Metapneumovirus, Influenza A H1-2009, PIV 2, and RSV B  |
|  Positive Control C | Adenovirus, Coronavirus, Influenza A H1, PIV 1, and PIV  |
|  Positive Control D | Coronavirus, Human Rhinovirus/Enterovirus, Influenza B, and M. pneumoniae  |
|  Negative Control | Negative (Not Detected)  |

Over 104 days of investigational testing across five testing sites, there were 209 external controls tested with valid results: 27 Positive Control A, 24 Positive Control B, 27 Positive Control C, 25 Positive Control D, and 106 Negative Control. Of these, 203/209 had the expected results. The remaining six controls had the expected results except as follows:

- two Negative Controls each had one false positive (FP) result: one for Adenovirus, one for Coronavirus

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- one Negative Control had four FP results: Coronavirus, Human Rhinovirus/Enterovirus, Influenza B, and Mycoplasma pneumoniae. It is likely the site inadvertently tested a Positive Control D sample and not a Negative Control since these four organisms are the expected results for Positive Control D
- two Positive Controls (A and B) each had a FP result for Human Rhinovirus/Enterovirus
- one Positive Control A had a false negative (FN) result for PIV 4.

The sponsor is also recommending the following in the product package insert: "Positive and negative external controls should be tested with each new lot of reagents or monthly, whichever occurs first. Viral transport medium can be used as the negative control. Previously characterized positive samples or viral transport medium spiked with well characterized organisms can be used as the external positive control. External controls should be run in accordance with laboratory protocols and accrediting organizations, as applicable."

### Specimen Stability

The ePlex RP Panel package insert claims that clinical specimens can be stored at room temperature (15–30°C) for up to 12 hours or refrigerated at 4°C for up to 10 days after collection in transport media. Specimens can also be stored at -20°C or -80°C for six months with up to two freeze/thaw cycles.

These clinical specimen stability claims are supported by analytical study which investigated the effect of different specimen storage conditions on specimen integrity (as determined by performance on the ePlex RP Panel).

A representative panel of six analytes (Table 7) was spiked into a pooled nasopharyngeal (NP) sample to approximately 1x LoD concentrations. It is noted that the six-analyte test sample generates seven results for the ePlex RP Panel.

Table 7: Representative Analytes Contained in Pooled NP Sample for Analytical Studies

|  Analyte Strain | Strain | Expected Positive Result on the ePlex RP Panel  |
| --- | --- | --- |
|  Influenza A H3 | H3N2 Brisbane/10/07 | Flu A and Flu A H3  |
|  RSV A | 2006 Isolate | RSV-A  |
|  Parainfluenza Virus 1 | Clinical Isolate | PIV-1  |
|  Human Metapneumovirus | B2 Peru1-2002 | hMPV  |
|  Coronavirus | OC43 | Coronavirus  |
|  Adenovirus B | Type 7 | Adenovirus  |

Baseline condition (T0) was established with 20 replicates. Each subsequent time point/test condition was assessed with 10 replicates unless otherwise noted. The overall study design (identifying all of the test conditions assessed in the study) is summarized in Table 8 below.

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**Table 8: Study Design – Specimen Storage and Testing Time points**

|  Storage Condition | Time Points  |   |   |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  *Ambient Storage Conditions*  |   |   |   |   |   |   |   |   |   |   |   |   |
|  15°C – 30°C | T0 | 4h | 8h | 12h | 24h |  |  |  |  |  |  |   |
|  *Refrigerated Storage Conditions*  |   |   |   |   |   |   |   |   |   |   |   |   |
|  2°C - 8°C | T0 | 1d | 3d | 5d | 7d | 10d | 14d |  |  |  |  |   |
|  *Frozen Storage Conditions*  |   |   |   |   |   |   |   |   |   |   |   |   |
|  -20°C & -80°C | T0 | 1w | 2w | 1m | 3m | 6m | 12m | 18m | 24m | 30m | 36m | 42m  |

Note: Shaded boxes indicate future time points that have not yet been tested.

The specimen stability study acceptance criteria were the following:

- Performance at each time point will be considered acceptable if the positivity rate for each analyte (either from the first 10 replicates or from the final 20 replicates in case of additional testing) is equivalent to the positivity rate for the corresponding analyte at time point T0 (baseline time point). “Equivalent” is defined as positivity rates that differ by 5% or less.
- If a time point did not meet the positivity acceptance criteria, the next time point was evaluated. If the subsequent time point met the acceptance criteria, the study was continued. If two consecutive time points failed, the specimen stability was determined to be the last time point when the acceptance criteria were met.

The specimen stability assessment data are summarized in Table 9 to Table 13 below.

**Table 9: Ambient Storage Conditions (Positivity and Mean Signal)**

|  Target | T0 |   | 4 Hrs |   | 8 Hrs |   | 12 Hrs |   | 24 Hrs  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA  |
|  Flu A H3 | 100 | 279.2 | 100 | 318.3 | 100 | 306.1 | 100 | 309.0 | 100 | 158.9  |
|  Flu A | 100 | 852.2 | 100 | 872.2 | 100 | 659.9 | 100 | 651.2 | 100 | 576.0  |
|  RSV-A | 100 | 619.0 | 100 | 669.6 | 100 | 567.6 | 100 | 604.4 | 95 | 500.5  |
|  hMPV | 100 | 421.9 | 100 | 448.6 | 100 | 373.5 | 100 | 404.1 | 85 | 259.8  |
|  PIV-1 | 100 | 619.5 | 100 | 645.3 | 100 | 629.6 | 100 | 661.8 | 95 | 388.2  |
|  Adv B | 100 | 376.1 | 100 | 405.9 | 100 | 299.4 | 100 | 361.6 | 95 | 296.7  |
|  CoV | 100 | 776.0 | 100 | 820.7 | 100 | 745.8 | 100 | 790.6 | 100 | 776.8  |

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Table10: Refrigerated Storage Conditions (Positivity)

|  Target | % Positivity  |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |  T0 | 1 Day | 3 Days | 5 Days | 7 Days | 10 days | 14 days  |
|  Flu A H3 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  Flu A | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  RSV-A | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  hMPV | 100 | 100 | 100 | 100 | 100 | 100 | 95  |
|  PIV-1 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  Adv B | 100 | 100 | 100 | 100 | 100 | 100 | 90  |
|  CoV | 100 | 100 | 100 | 100 | 100 | 100 | 100  |

Table 11: Refrigerated Storage Conditions (Mean Signal)

|  Target | Mean Signal (nA)  |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |  T0 | 1 Day | 3 Days | 5 Days | 7 Days | 10 days | 14 days  |
|  Flu A H3 | 330.6 | 159.6 | 270.5 | 269.1 | 322.0 | 351.0 | 174.6  |
|  Flu A | 725.4 | 691.8 | 719.3 | 586.1 | 844.0 | 762.7 | 546.7  |
|  RSV-A | 589.3 | 522.0 | 589.9 | 577.8 | 600.2 | 588.5 | 462.8  |
|  hMPV | 404.0 | 361.4 | 347.9 | 328.8 | 391.7 | 352.4 | 245.3  |
|  PIV-1 | 475.6 | 613.1 | 453.8 | 576.9 | 413.7 | 558.9 | 411.2  |
|  Adv B | 328.7 | 277.5 | 319.1 | 248.2 | 341.4 | 280.2 | 187.1  |
|  CoV | 767.1 | 669.9 | 752.2 | 696.8 | 815.8 | 802.1 | 531.0  |

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**Table 12: Frozen Storage Conditions (Positivity)**

|  Target | T0 | 1 Week |   | 2 Weeks |   | 1 Month |   | 3 Months |   | 6 Months  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  -20°C | -80°C | -20°C | -80°C | -20°C | -80°C | -20°C | -80°C | -20°C | -80°C  |
|  Flu A H3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  Flu A | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  RSV-A | 100 | 100 | 95* | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  hMPV | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  PIV-1 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  Adv B | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  CoV | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 95  |

* When the first 10 replicates were tested at the one week time point, RSV-A was missed in 1/10 replicates. Ten additional replicates were tested and the combined percent positivity for RSV-A was 95%.

**Table 13: Frozen Storage Conditions (Mean Signal)**

|  Target | T0 | 1 Week |   | 2 Weeks |   | 1 Month |   | 3 Months |   | 6 Months  |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  -20°C | -80°C | -20°C | -80°C | -20°C | -80°C | -20°C | -80°C | -20°C | -80°C  |
|  Flu A H3 | 330.6 | 260.7 | 281.3 | 282.6 | 209.6 | 249.5 | 301.9 | 286.2 | 274.0 | 308.4 | 349.0  |
|  Flu A | 725.4 | 901.0 | 665.5 | 595.7 | 626.0 | 621.6 | 723.5 | 574.4 | 640.2 | 714.7 | 708.1  |
|  RSV-A | 589.3 | 587.7 | 518.8 | 497.5 | 516.0 | 525.9 | 561.7 | 458.9 | 515.7 | 523.2 | 567.2  |
|  hMPV | 404.0 | 395.2 | 384.1 | 345.2 | 369.0 | 298.8 | 318.0 | 251.7 | 254.0 | 267.2 | 298.0  |
|  PIV-1 | 475.6 | 422.0 | 465.6 | 577.1 | 567.7 | 464.4 | 555.1 | 422.8 | 470.1 | 484.7 | 478.9  |
|  Adv B | 328.7 | 349.6 | 324.1 | 303.2 | 296.2 | 272.5 | 273.1 | 234.4 | 226.0 | 237.7 | 265.5  |
|  CoV | 767.1 | 802.9 | 987.1 | 666.5 | 291.6 | 621.1 | 677.2 | 476.3 | 515.1 | 473.2 | 487.2  |

The data from the specimen stability studies supported the following specimen stability claims:

- storage under ambient conditions (15–30°C) for up to 12 hours in VTM

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• storage under refrigerated conditions (4°C) for up to 10 days in VTM
• storage under frozen conditions (-20°C and -80°C) for up to six months

### Fresh vs. Frozen Study

An analytical study was conducted to demonstrate clinical specimen stability after subjecting samples to one or two freeze-thaw cycles. A representative panel of six analytes (Table 7 above) was spiked into pooled negative NPS in VTM to approximately 1x LoD concentrations and tested fresh after one and after two freeze-thaw cycles at 10 replicates each.

The specimen freeze-thaw study results are summarized in Table 14 below.

Table 14: Freeze-Thaw Study (Positivity and Mean Signal)

|  Target | T_{0} |   | T_{1} |   | T_{2}  |   |
| --- | --- | --- | --- | --- | --- | --- |
|   |  % Pos | Mean nA | % Pos | Mean nA | % Pos | Mean nA  |
|  Flu A H3 | 100 | 229.6 | 100 | 355.3 | 100 | 379.1  |
|  Flu A | 100 | 790.6 | 100 | 720.7 | 100 | 714.0  |
|  RSV-A | 100 | 622.7 | 100 | 588.6 | 100 | 593.9  |
|  hMPV | 100 | 424.6 | 100 | 389.5 | 100 | 410.1  |
|  PIV-1 | 100 | 585.6 | 100 | 385.4 | 100 | 355.1  |
|  Adv B | 100 | 365.2 | 100 | 351.9 | 100 | 361.4  |
|  CoV | 100 | 816.4 | 100 | 703.4 | 100 | 685.1  |

The data from the specimen freeze-thaw study supported the following specimen stability claim:

• specimens in VTM are stable after two freeze-thaw cycles

### In-Cartridge Sample Stability Study

An analytical study was carried out to demonstrate that the cartridge can be stored at room temperature (15–30°C) for two hours after the sample has been loaded prior to running the cartridge in an ePlex bay.

A representative panel of six analytes (Table 7 above) was spiked into pooled negative NPS as matrix. Each target mix was tested at a final concentration of approximately 1x LoD. Forty (40) cartridges were loaded following package insert

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instructions. Twenty (20) of these cartridges were run within 10 minutes of loading; the other 20 cartridges were kept for two hours at room temperature and then run following package insert instructions.

The acceptance criteria for this analytical study were the following:

- The sample is stable at room temperature after it has been loaded into the cartridge for up to 120 minutes prior to running the cartridge. Stability is defined as equal positivity rates for each target in each of the tested conditions that will be compared to each other. Equivalent positivity rate is defined as positivity rates that differ by 5% or less.

The in-cartridge sample stability study results are summarized in Table 15 and Table 16 below.

**Table 15: In-Cartridge Sample Stability Study Comparison of Positivity Rate at 10 and 120 Minutes**

|  Target | % Positive  |   |
| --- | --- | --- |
|   |  Sample loaded after 10 min | Sample loaded after 120 min  |
|  Flu A H3 | 100% (20/20) | 95% (19/20)  |
|  Flu A | 100% (20/20) | 100% (20/20)  |
|  RSV-A | 100% (20/20) | 100% (20/20)  |
|  hMPV | 100% (20/20) | 95% (19/20)  |
|  PIV-1 | 100% (20/20) | 100% (20/20)  |
|  Adv B | 100% (20/20) | 100% (20/20)  |
|  CoV | 100% (20/20) | 95% (19/20)  |

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Table 16: In-Cartridge Sample Stability Study Comparison of Mean and Mean minus 2 SD at 10 and 120 Minutes

|  Target | Cutoff (nA) | Sample loaded after 10 min |   | Sample loaded after 120 min  |   |
| --- | --- | --- | --- | --- | --- |
|   |   |  mean signal (nA) | mean signal minus 2SD (nA) | mean signal (nA) | mean signal minus 2SD (nA)  |
|  Flu A H3 | 5 | 314.8 | 181.2 | 225.0 | 10.1  |
|  Flu A | 20 | 712.9 | 420.9 | 688.6 | 568.9  |
|  RSV-A | 10 | 588.5 | 535.6 | 580.1 | 438.7  |
|  hMPV | 5 | 413.0 | 313.7 | 353.8 | 167.7  |
|  PIV-1 | 10 | 542.5 | 333.3 | 561.0 | 369.1  |
|  Adv B | 10 | 339.6 | 254.9 | 382.7 | 235.3  |
|  CoV | 10 | 774.8 | 558.8 | 605.5 | 270.8  |

The study results demonstrated that the cartridge can be stored at room temperature (15–30°C) for two hours after the sample has been loaded prior to running the cartridge in an ePlex bay.

### Simulated vs. Natural NPS in VTM Specimen Study

An analytical study was conducted to verify sample matrix equivalency between Remel M5 VTM (simulated matrix) and pooled negative nasopharyngeal swabs (NPS) collected in commercially available VTM (natural matrix) for the ePlex RP Panel for targets that are tested at 10x LoD and above.

A representative panel of six analytes (Table 7 above) was spiked either into Remel M5 VTM or pooled negative NPS at a final concentration of approximately 10x LoD. A negative control was run in duplicate for VTM and the NPS pool. Twenty (20) cartridge runs of spiked NPS matrix and 20 cartridge runs of spiked VTM were performed, as were two negative controls with each set of 20 runs (i.e., 44 cartridge runs were performed).

The acceptance criteria for this analytical study were the following:

- The assay will be determined to not be impacted by sample matrix if the positivity rate for each target is equivalent to the positivity rate for the corresponding target in the simulated matrix control. Equivalent is defined as positivity rates that differ by 5% or less.

The sample matrix equivalence study results are summarized in Table 17 below.

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Table 17: Sample Matrix Equivalence Study Positivity rate and mean signals for all targets in VTM and NPS

|  Target | Remel M5 VTM |   |   |   | NPS in VTM |   |   |   | Cut off in nA | % difference in positivity rate  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  % positive | Mean (nA) | SD (nA) | Mean nA- | % positive | Mean (nA) | SD (nA) | Mean nA-2SD  |   |   |
|  Flu A H3 | 100% (20/20) | 308.9 | 125.5 | 57.9 | 100% (20/20) | 317.8 | 97.8 | 122.2 | 5 | 0%  |
|  Flu A | 100% (20/20) | 724.5 | 93.7 | 537.1 | 100% (20/20) | 763.0 | 112.6 | 537.8 | 20 | 0%  |
|  RSV-A | 100% (20/20) | 631.3 | 98.6 | 434.1 | 100% (20/20) | 623.7 | 79.0 | 465.7 | 10 | 0%  |
|  hMPV | 100% (20/20) | 466.2 | 77.6 | 311 | 100% (20/20) | 474.1 | 71.7 | 330.8 | 5 | 0%  |
|  PIV-1 | 100% (20/20) | 484.5 | 133.5 | 217.5 | 100% (20/20) | 502.9 | 138.3 | 226.3 | 10 | 0%  |
|  Adv B | 100% (20/20) | 371.0 | 47.2 | 276.6 | 100% (20/20) | 354.3 | 52.4 | 249.5 | 10 | 0%  |
|  CoV | 100% (20/20) | 790.3 | 90.2 | 609.9 | 100% (20/20) | 736.2 | 106.7 | 522.8 | 10 | 0%  |

The results from the sample matrix equivalency study demonstrated the equivalency between Remel M5 VTM and natural nasopharyngeal swabs (NPS) collected in Remel M5 VTM for targets at approximately 10x LoD.

### d. Detection limit:

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 verification. 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 18 below.

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Table 18: LoD Results Summary

|  Target | Strain | LoD Concentration  |
| --- | --- | --- |
|  Adenovirus | Type 1 (C) | 1.0E+03 TCID_{50}/mL  |
|   |  Type 4 (E) | 2.0E+00 TCID_{50}/mL  |
|   |  Type 7 (B) | 2.0E+00 TCID_{50}/mL  |
|  Coronavirus 229E | 229E | 1.0E+00 TCID_{50}/mL  |
|  Coronavirus HKU1 | HKU1^{a} | 5.0E+04 copies/mL  |
|  Coronavirus NL63 | NL63 | 7.5E+00 TCID_{50}/mL  |
|  Coronavirus OC43 | OC43 | 5.0E+02 TCID_{50}/mL  |
|  Human Metapneumovirus | A1 IA3-2002 | 2.0E-01 TCID_{50}/mL  |
|   |  A2 IA14-2003 | 2.0E+03 TCID_{50}/mL  |
|   |  B1 Peru2-2002 | 2.0E+02 TCID_{50}/mL  |
|   |  B2 Peru1-2002 | 2.3E+02 TCID_{50}/mL  |
|  Human Rhinovirus/Enterovirus | Enterovirus Type 68 (2007) | 1.0E+00 TCID_{50}/mL  |
|   |  Rhinovirus 1A | 1.5E+00 TCID_{50}/mL  |
|   |  Rhinovirus B14 | 1.0E+00 TCID_{50}/mL  |
|   |  Rhinovirus C^{a} | 1.0E+05 copies/mL  |
|  Influenza A | H1N1 Brisbane/59/07 | 3.0E-01 TCID_{50}/mL  |
|  Influenza A H1 | H1N1 Brisbane/59/07 | 3.0E-01 TCID_{50}/mL  |
|  Influenza A H1-2009 | NY/01/2009 | 1.0E-01 TCID_{50}/mL  |
|  Influenza A H3 | A/Perth/16/2009 | 1.0E+01 TCID_{50}/mL  |
|   |  A/Texas/50/2012 | 1.0E+00 TCID_{50}/mL  |
|   |  A/Victoria/361/2011 | 5.0E-01 TCID_{50}/mL  |
|   |  H3N2 Brisbane/10/07 | 5.0E+01 TCID_{50}/mL  |
|  Influenza B | Florida/02/06 | 1.0E-01 TCID_{50}/mL  |
|  Influenza B (Victoria Lineage) | B/Brisbane/60/2008 | 1.0E+00 TCID_{50}/mL  |
|   |  B/Montana/5/2012 | 1.0E+00 TCID_{50}/mL  |
|   |  B/Nevada/03/2011 | 1.0E+00 TCID_{50}/mL  |
|  Influenza B (Yamagata Lineage) | B/Massachusetts/02/2012 | 1.0E+02 TCID_{50}/mL  |
|   |  B/Texas/06/2011 | 1.0E-01 TCID_{50}/mL  |
|   |  B/Wisconsin/01/2010 | 1.0E+00 TCID_{50}/mL  |
|  Parainfluenza Virus 1 | Clinical Isolate | 4.0E-01 TCID_{50}/mL  |
|  Parainfluenza Virus 2 | Clinical Isolate | 5.0E+01 TCID_{50}/mL  |
|  Parainfluenza Virus 3 | Clinical Isolate | 5.0E+00 TCID_{50}/mL  |
|  Parainfluenza Virus 4 | 4a | 3.0E+01 TCID_{50}/mL  |
|  Respiratory Syncytial Virus A | 2006 Isolate | 1.5E+00 TCID_{50}/mL  |
|  Respiratory Syncytial Virus B | CH93(18)-18 | 2.0E-01 TCID_{50}/mL  |
|  *Chlamydia pneumoniae* | AR-39 | 3.0E+02 TCID_{50}/mL  |
|  *Mycoplasma pneumoniae* | FH strain of Eaton Agent [NCTC 10119] | 3.0E+02 CCU/mL  |

$^{a}$ Clinical samples confirmed positive for coronavirus HKU1 and human rhinovirus C by bi-directional sequencing and quantified by real-time RT-PCR were used for determination of LoD.

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#### e. Analytical Reactivity:

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.

Results of analytical reactivity study, including in silico analysis results, are shown in Tables 19 to Table 29.

Table 19: Analytical Reactivity (Inclusivity) Results for Adenovirus

|  Adenovirus Species | Serotype | Concentration | Multiple of LoD Detected  |
| --- | --- | --- | --- |
|  A | Type 31 | \( {3.0}\mathrm{E} + {03}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|  B | Type 3 | \( {6.0}\mathrm{E} + {00}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Type 11 | \( {6.0}\mathrm{E} + {00}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  De Wit Type 14 | \( {6.0}\mathrm{E} + {00}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Ch.79 Type 16 | \( {2.0}\mathrm{E} + {02}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | \( {100}{\mathrm{x}}^{\mathrm{a}} \)  |
|   |  Type 21 | \( {6.0}\mathrm{E} + {00}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Compton Type 34 | \( {6.0}\mathrm{E} + {00}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Holden Type 35 | \( {6.0}\mathrm{E} + {00}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Wan Type 50 | \( {2.0}\mathrm{E} + {01}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | \( {10}{\mathrm{x}}^{\mathrm{b}} \)  |
|  C | Type 2 | \( {3.0}\mathrm{E} + {03}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Type 5 | \( {3.0}\mathrm{E} + {03}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Type 6 | \( {3.0}\mathrm{E} + {03}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|  D | Type 26 | \( {3.0}\mathrm{E} + {03}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Type 37 | \( {3.0}\mathrm{E} + {03}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|  F | Type 40 Dugan | \( {3.0}\mathrm{E} + {03}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 3x  |
|   |  Type 41/ Strain Tak | \( {3.0}\mathrm{E} + {03}\mathrm{{TCID}}_{50}/\mathrm{{mL}} \) | 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 (TCID \( _{50} \)  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 20: Analytical Reactivity (Inclusivity) Results for Human Metapneumovirus

|  Metapneumovirus Subtype | Strain | Concentration | Multiple of LoD Detected  |
| --- | --- | --- | --- |
|  Human metapneumovirus | Peru6-2003 G, B2 | \( 6.8E+02 \) TCID\( _{50} \)/mL | 3x  |

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Table 21: Analytical Reactivity (Inclusivity) Results for Human Rhinovirus/Enterovirus

|  Rhinovirus/Enterovirus | Strain | Concentration | Multiple of LoD Detected  |
| --- | --- | --- | --- |
|  Human Rhinovirus | Type A2 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  Type A7 | \( 1.5E+01 TCID_{50}/mL \) | \( 10x^a \)  |
|   |  Type A16 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  Type A18 | \( 1.5E+02 TCID_{50}/mL \) | \( 100x^a \)  |
|   |  Type A34 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  Type A57 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  Type A77 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  277G | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  Type B3 | \( 1.5E+01 TCID_{50}/mL \) | \( 10x^a \)  |
|   |  Type B17 | \( 1.5E+01 TCID_{50}/mL \) | \( 10x^a \)  |
|   |  Type B42 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  Type B83 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  Type B84 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|   |  FO2-2547 | \( 4.5E+00 TCID_{50}/mL \) | 3x  |
|  Enterovirus | Type 71 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|  Coxsackievirus | A9 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|   |  A10 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|   |  A21 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|   |  A24 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|   |  B2 | \( 1.0E+02 TCID_{50}/mL \) | \( 100x^a \)  |
|   |  B3 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|   |  B4 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|   |  B5 | \( 1.0E+01 TCID_{50}/mL \) | \( 10x^a \)  |
|  Echovirus | 9 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|   |  E6 | \( 1.0E+01TCID_{50}/mL \) | \( 10x^b \)  |
|   |  25 | \( 1.0E+01TCID_{50}/mL \) | \( 10x^a \)  |
|   |  30 | \( 3.0E+00 TCID_{50}/mL \) | 3x  |
|  Poliovirus | 1 | \( 1.0E+02 TCID_{50}/mL \) | \( 100x^a \)  |

\( ^{a} \)  In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes.  
 \( ^{b} \)  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 (TCID \( _{50} \)  value is based only on infectious virus particles).

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Table 22: 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 | 3.0E+00 TCID\( _{50} \)/mL | 10x (Influenza A matrix)\( ^a \)10000x H1 subtype\( ^b \)  |
|   |  A/New Caledonia/20/1999 | 9.0E-01 TCID\( _{50} \)/mL | 3x  |
|   |  A/New Jersey/8/76 | 9.0E-01 TCID\( _{50} \)/mL | 3x (Influenza A matrix)H1 subtype not detected\( ^c \)  |
|   |  A/NWS/33 | 3.0E+00 TCID\( _{50} \)/mL | 10x (Influenza A matrix)\( ^a \)H1 subtype not detected\( ^d \)  |
|   |  A/PR/8/34 | 9.0E-01 TCID\( _{50} \)/mL | 3x (Influenza A matrix)H1 subtype not detected\( ^e \)  |
|   |  A/Solomon Islands/3/2006 | 9.0E-01 TCID\( _{50} \)/mL | 3x  |
|   |  A/Taiwan/42/06 | 9.0E+00 TCID\( _{50} \)/mL | 30x\( ^f \)  |
|  Influenza A H3 | A/Hong Kong/8/68 | 1.5E+02 TCID\( _{50} \)/mL | 3x  |
|   |  A/Port Chalmers/1/73  |   |   |
|   |  A/Nanchang/933/95  |   |   |
|   |  A/Victoria/3/75  |   |   |
|   |  A/Wisconsin/67/05  |   |   |
|  Influenza A 2009 H1N1 | A/California/7/2009 | 1.0E+00 TCID\( _{50} \)/mL | 10x\( ^g \)  |
|   |  A/Mexico/4108/09 | 3.0E-01 TCID\( _{50} \)/mL | 3x  |
|   |  A/NY/02/2009 | 1.0E+00 TCID\( _{50} \)/mL | 10x\( ^h \)  |
|   |  A/Swine NY/03/2009 | 3.0E-01 TCID\( _{50} \)/mL | 3x  |
|   |  A/Swine/Iowa/15/30 | 3.0E-01 TCID\( _{50} \)/mL | 3x (Influenza A matrix)100,000x (H1-2009 subtype)\( ^i \)  |
|   |  A/Virginia/ATCC1/2009 | 1.0E+00 TCID\( _{50} \)/mL | 10x\( ^j \)  |
|   |  A/Virginia/ATCC2/2009 | 1.0E+01 TCID\( _{50} \)/mL | 100x\( ^j \)  |
|   |  A/Virginia/ATCC3/2009 | 1.0E+02 TCID\( _{50} \)/mL | 1,000x\( ^j \)  |

\( ^{a} \)  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 (TCID \( _{50} \)  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.

\( ^{c} \)  H1-2009 subtype was detected in this seasonal influenza A H1 strain at 30x LoD.

\( ^{d} \)  In silico analysis revealed little homology between this non-contemporary strain sequence and the H1 signal probe/capture probe sequences.

\( ^{e} \)  In silico analysis revealed little homology between this non-contemporary influenza strain sequence and the H1 primer sequences.

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 (TCID \( _{50} \) 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.

\( ^{8} \)  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 genetic material present in the culture of this or the reference strain (TCID \( _{50} \)  value is based only on infectious virus particles).

\( ^{b} \)  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

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(TCID \( _{50} \) 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 strain sequence and 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 H1N1

A/Virginia/ATCC1/2009, A/Virginia/ATCC2/2009 and A/Virginia/ATTC3/2009 strains.

Table 23: 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 | \( 1.6E+02 \text{ CEID}_{50}/\text{mL} \) (Influenza A matrix)\( 1.6E+08 \text{ CEID}_{50}/\text{mL} \) (H1 subtype)  |
|   |  A/Mal/302/54 | \( 1.6E+02 \text{ CEID}_{50}/\text{mL} \) (Influenza A matrix)\( 1.6E+05 \text{ CEID}_{50}/\text{mL} \) (H1 subtype)  |
|  Influenza A H3 | A/Aichi/2/68 H3N2 | \( 1.6E+03 \text{ CEID}_{50}/\text{mL} \)  |
|   |  Alice (vaccine) A/England/42/72 | \( 5.0E+00 \text{ EID}_{50}/\text{mL} \) (Influenza A matrix)\( 5.00E+01 \text{ EID}_{50}/\text{mL} \) (H3 subtype)  |
|   |  MRC-2 Recombinant Strain | \( 8.9E+02 \text{ CEID}_{50}/\text{mL} \) (Influenza A matrix)\( 8.9E+03 \text{ CEID}_{50}/\text{mL} \) (H3 subtype)  |
|  Influenza A H1N1 | A/Washington/24/2012 (A/H1 pdm09) | \( 3.2E+03 \text{ EID}_{50}/\text{mL} \) (Influenza A matrix)\( 3.2E+02 \text{ EID}_{50}/\text{mL} \) (H1-2009 subtype)  |
|  Influenza A H1N2 | Kilbourne F63: A/NWS/34 (HA) x A/Rockefeller Institute/5/57 (NA), Reassortant NWS-F- Matrix | \( 8.89 \times 10^{1} \text{ CEID}_{50}/\text{mL} \) (Influenza A matrix)No subtype detecteda  |
|  Influenza A H5N8 | A/Gyrfalcon/Washington/41088-6/2014 BPL | \( 1.6E+03 \text{ EID}_{50}/\text{mL} \) (Influenza A matrix)No subtype detectedb  |
|  Influenza A H5N2 | A/Northern Pintail/Washington/40964/2014 BPL | \( 2.5E+03 \text{ EID}_{50}/\text{mL} \) (Influenza A matrix)No subtype detectedb  |
|  Influenza A H7N9 | A/ANHUI/1/2013 | \( 7.9E+03 \text{ EID}_{50}/\text{mL} \) (Influenza A matrix)No subtype detectedc  |
|  Influenza A H3N2v | A/Indiana/21/2012 | \( 2.5E+04 \text{ EID}_{50}/\text{mL} \) (Influenza A matrix and H3 subtype)  |

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

\( ^{b} \)  Detection of the H5 Subtype not expected

\( ^{c} \)  Detection of the H7 Subtype not expected

NOTE: \(\mathrm{CEID}_{50} / \mathrm{mL} =\) Chick Embryo Infectious Dose; \(\mathrm{EID}_{50} / \mathrm{mL} =\) Egg Infectious Dose

For human, avian, and swine influenza A strains that were not available for wet 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. The in silico analysis results are summarized in Table 24 below.

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**Table 24: 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  |
|   |   |  A/Japan/305/1957(H2N2) | CY014977 | Influenza A  |
|   |   |  A/Korea/426/1968(H2N2) | CY031596 | Influenza A  |
|  H4N6 | Avian | A/Blue-winged teal/Minnesota/Sg-00043/2007(H4N6) | CY063978 | Influenza A  |
|  H5N1 |   | 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  |
|   |   |  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  |
|   |   |  A/Chicken/Bangladesh/1151-10/2010(H5N1) | HQ156766 | Influenza A  |
|   |  Human | 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  |
|  H5N2 | Avian | A/Duck/Pennsylvania/10218/1984(H5N2) | AB286120 | Influenza A  |
|   |   |  A/American black duck/Illinois/08OS2688/2008 | CY079453 | Influenza A  |
|   |   |  A/American green-winged teal/California/HKWF609/2007 | CY033447 | Influenza A  |
|   |   |  A/Canada goose/New York/475813-2/2007 | GQ923358 | Influenza A  |
|   |   |  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 | Avian | A/Duck/Singapore/F119/3/1997(H5N3) | GU052803 | Influenza A  |
|  H6N1 |   | A/Duck/PA/486/1969(H6N1) | EU743287 | Influenza A  |

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|  Influenza A Subtype | Host | Strain | GenBank ID | Predicted ePlex Result  |
| --- | --- | --- | --- | --- |
|  H6N2 |  | A/Mallard/Czech Republic/15902-17K/2009(H6N2) | HQ244433 | Influenza A  |
|  H7N2 | Avian | 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  |
|   |   |  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  |
|  H7N3 | Human | A/New York/107/2003(H7N2) | EU587373 | Influenza A  |
|   |   |  A/Canada/rv504/2004(H7N3) | CY015007 | Influenza A  |
|  H7N7 | 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  |
|   |   |  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  |
|  H7N9 | Human | A/Shanghai/1/2013(H7N9) | EPI439493 | Influenza A  |
|   |  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  |
|  H9N2 | Human | A/Hong Kong/1073/99(H9N2) | AJ278647 | Influenza A  |
|   |  Avian | A/Turkey/Wisconsin/1/1966(H9N2) | CY014664 | Influenza A  |
|  H10N7 |   | A/chicken/Germany/N/1949(H10N7) | GQ176135 | Influenza A  |
|  H11N9 |   | A/Duck/Memphis/546/1974(H11N9) | GQ257441 | Influenza A  |
|  H1N1 | Swine | A/Swine/Wisconsin/1/1971(H1N1) | CY022414 | Influenza A  |
|   |  Human | A/California/UR06-0393/2007(H1N1) | CY026540 | Influenza A H1  |
|   |   |  A/New York/297/2003(H1N2) | CY026539  |   |
|  H1N2 |   |   | CY002664 | Influenza A H1  |
|   |  H1N1 (2009) | A/Aalborg/INS133/2009(H1N1) | CY002665  |   |
|  A/Aalborg/INS133/2009(H1N1) |   |   | CY063606 | Influenza A H1-2009  |
|  A/South Carolina/02/2010(H1N1) |   | CY063607  |   |   |
|   |   |  A/South Carolina/02/2010(H1N1) | KC781370 | Influenza A H1-2009  |
|  H1N2 | Swine | A/Swine/Hong Kong/NS857/2001(H1N2) | KC781372  |   |
|   |   |  A/Swine/Sweden/1021/2009(H1N2) | GQ229350 | Influenza A  |
|   |  |  | GQ495135 | Influenza A  |

30

{30}

|  Influenza A Subtype | Host | Strain | GenBank ID | Predicted ePlex Result  |
| --- | --- | --- | --- | --- |
|  H3N1 | Avian | A/Blue-winged teal/ALB/452/1983(H3N1) | CY004635 | Influenza A  |
|  H3N2v | Human | A/Iowa/07/2011(H3N2) | JQ070760 | Influenza A H3  |
|   |   |   |  JQ290177  |   |
|   |   |  A/Iowa/08/2011(H3N2) | JQ070768 | Influenza A H3  |
|   |   |   |  JQ290167  |   |
|   |   |  A/Iowa/09/2011(H3N2) | JQ070776 | Influenza A H3  |
|   |   |   |  JQ290183  |   |
|   |   |  A/Indiana/08/2011(H3N2) | JQ070800 | Influenza A H3  |
|   |   |   |  JQ070795  |   |
|   |   |  A/Maine/06/2011(H3N2) | JN866181 | Influenza A H3  |
|   |   |   |  JN866186  |   |
|   |   |  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 | Influenza A H3  |
|   |   |   |  JQ290164  |   |
|   |   |  A/West Virginia/07/2011(H3N2) | JQ348839 | Influenza A  |
|   |   |  A/Indiana/10/2011(H3N2) | KJ942592 | Influenza A H3  |
|   |  JQ070787  |   |   |   |
|   |  A/Boston/38/2008(H3N2) | CY044580 | Influenza A H3  |   |
|   |   |  CY044581  |   |   |
|   |  Swine | A/swine/NY/A01104005/2011(H3N2v) | JN940422 | Influenza A H3  |
|   |   |  A/Maine/06/2011(H3N2) | JN866181 | Influenza A H3  |
|   |   |   |  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 | Influenza A  |
|   |   |   |  GU051136 | Influenza A  |
|   |   |  A/Mallard/Netherlands/2/1999(H3N5) | CY060261 | Influenza A  |
|  CY060264 |   |   | Influenza A  |   |
|  A/American black duck/New Brunswick/25182/2007(H3N6) |   | CY047696 | Influenza A  |   |
|   |   |  CY047697 | Influenza A  |   |
|  A/Northern shoveler/California/HKWF1367/2007(H3N7) |   | CY033372 | Influenza A  |   |
|   |   |  CY033375 | Influenza A  |   |
|  A/American black duck/Washington/699/1978(H3N8) |   | GU052300 | Influenza A H3  |   |
|   |   |  GU052299  |   |   |

31

{31}

Table 25: Analytical Reactivity (Inclusivity) Results for Influenza B

|  Influenza B Subtype | Strain | Concentration | Multiple of LoD Detected  |
| --- | --- | --- | --- |
|  Influenza B (Yamagata Lineage) | B/Lee/40 | 3.0E-01 TCID\( _{50} \)/mL | 3x  |
|   |  B/Allen/45 | 1.0E+00 TCID\( _{50} \)/mL | 10x\( ^a \)  |
|   |  B/Maryland/1/59 | 1.0E+01 TCID\( _{50} \)/mL | 100x\( ^a \)  |
|   |  B/Taiwan/2/62 | 1.0E+01 TCID\( _{50} \)/mL | 100x\( ^a \)  |
|  Influenza B (Victoria Lineage) | B/Hong Kong/5/72 | 1.0E+01 TCID\( _{50} \)/mL | 100x\( ^b \)  |
|   |  B/Malaysia/2506/04 | 3.0E-01 TCID\( _{50} \)/mL | 3x  |
|  Influenza B (Lineage unknown) | B/GL/1739/54 | 3.0E-01 TCID\( _{50} \)/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 culture of this or the reference strain (TCID \( _{50} \)  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 26: Analytical Reactivity (Inclusivity) Results for Parainfluenza Virus

|  Parainfluenza Subtype | Strain | Concentration | Multiple of LoD Detected  |
| --- | --- | --- | --- |
|  Parainfluenza Virus 1 | C35 | \( 1.2E+00 \text{ TCID}_{50}/\text{mL} \) | 3x  |
|  Parainfluenza Virus 2 | Greer | \( 1.5E+02 \text{ TCID}_{50}/\text{mL} \) | 3x  |
|  Parainfluenza Virus 3 | C-243 | \( 5.0E+01 \text{ TCID}_{50}/\text{mL} \) | \( 10x^a \)  |
|  Parainfluenza Virus 4 | 4b | \( 9.0E+01 \text{ TCID}_{50}/\text{mL} \) | 3x  |

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

Table 27: Analytical Reactivity (Inclusivity) Results for Respiratory Syncytial Virus

|  RSV Subtype | Strain | Concentration | Multiple of LoD Detected  |
| --- | --- | --- | --- |
|  Respiratory Syncytial Virus A | A2 | \( 4.5E+00 \text{ TCID}_{50}/\text{mL} \) | 3x  |
|   |  Long | \( 4.5E+00 \text{ TCID}_{50}/\text{mL} \) | 3x  |
|  Respiratory Syncytial Virus B | 9320 | \( 6.0E-01 \text{ TCID}_{50}/\text{mL} \) | 3x  |
|   |  Wash/18537/62 | \( 6.0E-01 \text{ TCID}_{50}/\text{mL} \) | 3x  |
|   |  WV/14617/85 | \( 6.0E-01 \text{ TCID}_{50}/\text{mL} \) | 3x  |

Table 28: Analytical Reactivity (Inclusivity) Results for Chlamydia pneumoniae

|   | Strain | Concentration | Multiple of LoD Detected  |
| --- | --- | --- | --- |
|  Chlamydia pneumoniae | CWL-029 | 9.0E+02 CFU/mL | 3x  |
|   |  TWAR strain 2043 | 9.0E+02 CFU/mL | 3x  |

32

{32}

Table 29: Analytical Reactivity (Inclusivity) Results for Mycoplasma pneumoniae

|   | Strain | Concentration | Multiple of LoD Detected  |
| --- | --- | --- | --- |
|  Mycoplasma pneumoniae | [Bru] | \( 9 \times 10^{2} \) CCU/mL | 3x  |
|   |  M129-B170 | \( 9 \times 10^{2} \) CCU/mL | 3x  |
|   |  M129-B7 | \( 9 \times 10^{2} \) CCU/mL | 3x  |
|   |  [M52] | \( 9 \times 10^{2} \) CCU/mL | 3x  |
|   |  [Mac] | \( 9 \times 10^{2} \) CCU/mL | 3x  |
|   |  Mutant 22 | \( 3 \times 10^{4} \) CCU/mL | \( 100x^a \)  |
|   |  PI 1428 | \( 3 \times 10^{4} \) CCU/mL | \( 100x^b \)  |

\( ^{a} \)  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 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 reduced sensitivity is likely 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).

All of the 101 strains/isolates wet tested for inclusivity were detected by the ePlex RP Panel.

f. Analytical Specificity/Cross-reactivity Evaluation:

### On-Panel Organisms Testing

Potential cross-reactivity of each viral and bacterial target on the ePlex RP Panel was evaluated at high concentrations (i.e., 1.0E+05 TCID \( _{50} \) /mL or >1.0E+05 EID \( _{50} \) /mL for viruses; 1.0E+06 CFU/mL or CCU/mL for bacterial isolates, or 1.0E+06 copies/mL for in vitro transcripts) of quantified strains/isolates diluted in VTM. In vitro transcripts for coronavirus HKU1 was diluted in PBS. Table 30 below summarizes the results of the on-panel viral and bacterial strains/isolates tested.

Table 30: Cross-reactivity Study with ePlex RP Panel Target Organisms

|  Target | Strain | Concentration | Cross-Reactivity Results  |
| --- | --- | --- | --- |
|  Adenovirus A | Type 31 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Adenovirus B | Type 7A | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Adenovirus C | Type 1 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Adenovirus D | Type 9 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Adenovirus E | Type 4 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Adenovirus F | Type 41 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Coronavirus | 229E | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Coronavirus | HKU1 in vitro transcript | \( 1.0E+06 \text{ copies/mL} \) | Not observed  |
|  Coronavirus | NL63 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Coronavirus | OC43 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Enterovirus | Type 68 2007 isolate | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |

33

{33}

|  Target | Strain | Concentration | Cross-Reactivity Results  |
| --- | --- | --- | --- |
|  Human metapneumovirus | B1 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Human rhinovirus | 1A | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Influenza A | A/Brisbane/59/07 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Influenza A H1 | A/Brisbane/59/07 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Influenza A H1-2009 | A/NY/01/2009 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Influenza A H3 | A/Brisbane/10/07 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Influenza A H3N2va | A/Indiana/21/2012 | \( 2.5E+05 \text{ EID}_{50}/\text{mL} \) | Not observed  |
|  Influenza A H5N2b | A/Northern Pintail Washington/40964/14BPL | \( 2.5E+05 \text{ EID}_{50}/\text{mL} \) | Not observed  |
|  Influenza A H5N8c | A/Gyrfalcon/Washington /410886/2014 BPL | \( 1.6E+05 \text{ EID}_{50}/\text{mL} \) | Not observed  |
|  Influenza A H7N9d | A/ANHUI/1/2013 | \( 7.9E+05 \text{ EID}_{50}/\text{mL} \) | Not observed  |
|  Influenza B | B/Florida/02/06 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Parainfluenza Virus 1 | C35 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Parainfluenza Virus 2 | Type 2 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Parainfluenza Virus 3 | Type 3 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Parainfluenza Virus 4 | Type 4a | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  RSV A | 2006 Isolate | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  RSV B | CH93(18)-18 | \( 1.0E+05 \text{ TCID}_{50}/\text{mL} \) | Not observed  |
|  Chlamydia pneumoniae | AR-39 | \( 1.0E+06 \text{ CFU}/\text{mL} \) | Not observed  |
|  Mycoplasma pneumoniae | FH strain of Eaton Agent [NCTC 10119] | \( 1.0E+06 \text{ CCU}/\text{mL} \) | Not observed  |

\( ^{a} \)  Influenza A H3N2v detected as Influenza A, Influenza A H3

\( ^{b} \)  Influenza A H5N2 detected as Influenza A

\( ^{c} \)  Influenza A H5N8 detected as Influenza A

\( ^{d} \)  Influenza A H7N9 detected as Influenza A

No cross-reactivity was observed between any of the on-panel viruses or bacteria.

### Off-Panel Organisms Testing

Potential cross-reactivity of viruses, bacteria, and fungi that are not targets on the ePlex RP Panel was evaluated at high concentrations (i.e., 1.0E+05 TCID \( _{50} \) /mL or copies/mL for viruses, 1.0E+06 CFU/mL for bacterial and yeast isolates, or 1.0E+06 copies/mL for plasmid DNA or genomic RNA) by diluting quantified strains/isolates in VTM. Plasmid for…

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**Source:** [https://fda-staging.innolitics.com/device/K163636](https://fda-staging.innolitics.com/device/K163636)

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