Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description focuses on the electrochemical detection and analysis of hybridization signals based on predefined signal boundaries, which is a standard method for multiplex molecular diagnostics and does not indicate the use of AI/ML for data analysis or interpretation. The performance studies describe traditional analytical and clinical validation methods.

Therapeutic

No.

The device is an in vitro diagnostic test designed to detect and identify viral nucleic acids, aiding in the diagnosis of respiratory viral infections. It does not provide treatment or directly apply a therapeutic effect to the patient.

Diagnostic

Yes

Explanation: The device is explicitly described as "a qualitative nucleic acid multiplex in vitro diagnostic test intended for use... for the simultaneous detection and identification of multiple respiratory viral nucleic acids" and "aids in the diagnosis of respiratory viral infection".

SaMD (Software as a Medical Device)

No

The device is an in vitro diagnostic test system that includes hardware components (eSensor XT-8 instrument, eSensor XT-8 Cartridge) in addition to software for data analysis and reporting.

IVD (In Vitro Diagnostic)

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

The intended use statement explicitly states: "The eSensor® Respiratory Viral Panel (RVP) is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on the eSensor XT-8™ system for the simultaneous detection and identification of multiple respiratory viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals exhibiting signs and symptoms of respiratory infection."

The device description also details how the test is performed on patient samples (nasopharyngeal swabs) to detect viral nucleic acids, which is the core function of an in vitro diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The eSensor® Respiratory Viral Panel (RVP) is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on the eSensor XT-8™ system for the simultaneous detection and identification of multiple respiratory viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals exhibiting signs and symptoms of respiratory infection.

The following virus types and subtypes are identified using the eSensor RVP: Influenza A, Influenza A H1 Seasonal Subtype, Influenza A H3 Seasonal Subtype; Influenza A 2009 H1N1 subtype, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Human Metapneumovirus, Human Rhinovirus, Adenovirus species B/E, and Adenovirus species C.

The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection if used in conjunction with other clinical and epidemiological information.

Negative results do not preclude respiratory viral infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Positive results do not rule out bacterial infection, or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence and radiography) and clinical presentation must be taken into consideration in the final diagnosis of respiratory viral infection.

Performance characteristics for Influenza A were established during the 2010/2011 influenza season when Influenza A 2009 H1N1 and H3N2 were the predominant Influenza A viruses in circulation. When other Influenza A viruses emerge, performance characteristics may vary.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

For prescription use only.

Product codes

OCC, OEM, OOU, OEP, OQW, NSU, OUL, JJH

Device Description

The eSensor RVP is a multiplex microarray-based genotyping test system. It is based on the principles of competitive DNA hybridization using a sandwich assay format, wherein a singlestranded target binds concurrently to sequence-specific solution-phase signal probe and solidphase electrode-bound capture probe. The test employs reverse transcription polymerase chain reaction amplification, exonuclease digestion and hybridization of target DNA/RNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate singlestranded DNA suitable for hybridization. Hybridization occurs in the eSensor XT-8 Cartridge (described below) where the single-stranded target DNA is mixed with a hybridization solution containing labeled signal probes.

During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to the target adjacent to the capture probe. When inserted into the eSensor XT-8 instrument (described below), simultaneous hybridization of target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each pair of working electrodes on the array contains a different capture probe, and sequential analysis of each electrode allows detection of multiple viral targets.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Nasopharyngeal Swab (NPS)

Indicated Patient Age Range

Not Found

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

Prospective Clinical Study:

Sample Size: 1037 evaluable patient samples (out of 1182 collected).
Data Source: Nasopharyngeal (NP) swab specimens, prospectively collected during the 2010/11 influenza season at three North American clinical laboratories (Cleveland, Ohio; Providence, RI; and Albuquerque, NM).
Annotation Protocol:
- For Influenza A, Influenza B, RSV, Parainfluenza Viruses (PIV1, PIV2, PIV3), and adenovirus: Viral culture followed by DFA identification testing was used as the comparator method.
- For Influenza A H3, Influenza A 2009 H1N1, RSVA, RSVB, ADVB/E and ADVC: Subtyped by an independently developed qRT-PCR assay or qPCR assay followed by bidirectional sequencing.
- For HRV and HMPV: Evaluated using two independently developed and validated gRT-PCR assays followed by bidirectional sequencing.
- "True" positive definitions were based on positive results from the comparator method and, for subtyping, bidirectional sequencing with quality acceptance criteria matching GenBank database sequences.

Testing of Preselected Archived Samples (Retrospective):

Sample Size: 320 banked samples (including negative samples).
Data Source: Banked samples previously characterized as positive for Influenza A H1, Parainfluenza Virus 1, Parainfluenza Virus 2, Adenovirus B/E, and Adenovirus C. Collected from various sites across the United States or from the Centers for Disease Control and Prevention (CDC).
Annotation Protocol: Upon arrival at GenMark, banked samples were blinded and intermixed with negative samples before being sent for testing. Testing was performed identically to prospectively-collected patient specimens. With the exception of Flu A H1 samples (verified by CDC), these banked samples were sent to Beckman Coulter for comparator testing.

Summary of Performance Studies

1. Non-Clinical Performance Data:
- Limit of Detection (LoD): Verified for each viral target using serially diluted viral reference strains in M5 and M4 media. Detection rate of 95% or in at least 19 of 20 extraction replicates.
- Analytical Reactivity (Inclusivity): Evaluated performance with multiple viral target strains representing temporal and geographic diversity. Strains diluted to 3X LoD in M5 transport media and extracted in triplicate.
- Supplemental Analytical Inclusivity Testing of Other Influenza Subtypes: Tested additional live isolates or purified genomic RNA of Influenza A strains.
- Simulated Reactivity Information for Influenza Strains of Human, Swine, and Avian Origin: In silico analysis performed for strains not available for testing (e.g., Flu A H5 and Flu A H7 strains) by aligning primer sequences to GenBank® sequences.
- Reproducibility (Multisite): Evaluated Lot-to-lot, extraction, site/instrument-to-site/instrument, day-to-day, and operator/run-to-operator/run variability. Performed at three sites with a panel of samples at Moderate Positive (3x LoD), Low Positive (1x LoD), and Negative concentrations. Each analyte/concentration tested 108 times.
- Testing of Dual Infection Samples (Clinically Relevant Co-Infections): Determined capability to detect dual co-infections using representative viral cultures at 1x LoD and 10,000x LoD.
- Interference (Interfering Substances): Evaluated potentially interfering substances (e.g., blood, nasal secretions, medications, and microorganisms) by testing them individually with viral mixes at 3X LoD. No inhibition observed at tested concentrations for most substances. FluMist at 1% (v/v) showed inhibition for hMPV detection but not at 0.5% (v/v).
- Cross-Reactivity:
- Within Panel: Evaluated each viral target at high concentrations (10,000x, 1000x, 100x LoD) against other RVP viral targets. Not observed.
- With Other Respiratory Viruses Not Targeted: Assessed against 5 low-frequency respiratory viruses and 17 additional viruses.
- With Bacteria and Fungus: Tested against a panel of bacterial and fungal strains at 10^8 CFU/mL. Not observed.
- Carryover/Cross-Contamination: Tested using alternating high concentration positive and negative samples in a checkerboard pattern. No carryover/cross-contamination observed.

2. Clinical Performance Data:
- Prospective Clinical Study:
- Sample Size: 1037 evaluable nasopharyngeal (NP) swab specimens.
- Study Type: Prospective observational study.
- Key Results: See Key Metrics table below for Sensitivity and Specificity.
- Mixed Co-infections: eSensor RVP detected 128 mixed infections (18.4% of total positive specimens). 114 double, 11 triple, and 3 with four or more analytes. 95 samples contained one or more analytes that the reference/comparator method failed to detect.
- Testing of Preselected Archived Samples:
- Sample Size: 320 retrospective banked samples.
- Study Type: Independent validation using archived samples.
- Key Results: See Key Metrics table below for PPA and NPA.
- eSensor RVP Performance in Fresh vs. Frozen Clinical Specimen:
- Sample Size: 128 aliquots of ADV C at 3x LoD and 1x LoD.
- Study Type: Comparative study.
- Key Results: Positive percent agreement between fresh and frozen aliquots was 100% (95% CI 89.3% - 100%).

Key Metrics

Clinical Performance in Prospective Samples (N=1037):

Virus (Analyte)	Sensitivity	95% CI	Specificity	95% CI
Influenza A¹	96.4%	91.7% - 98.8%	94.8%	93.1% - 96.1%
Influenza A H1*	NA	NA	100.0%	99.6% - 100.0%
Influenza A H3	100.0%	95.1% - 100.0%	97.4%	96.2% - 98.3%
Influenza A 2009 H1N1	100.0%	92.7% - 100.0%	98.5%	97.5% - 99.1%
Influenza B	92.8%	83.9% - 97.6%	98.1%	97.1% - 98.9%
Parainfluenza Virus 1*	100.0%	39.8% - 100.0%	99.9%	99.5% - 100.0%
Parainfluenza Virus 2*	83.3%	35.9% - 99.6%	99.8%	99.3% - 100.0%
Parainfluenza Virus 3	94.1%	85.6% - 98.4%	97.7%	96.6% - 98.6%
Respiratory Syncytial Virus A	100.0%	94.7% - 100.0%	94.7%	93.1% - 96.0%
Respiratory Syncytial Virus B	100.0%	87.7% - 100.0%	95.9%	94.5% - 97.0%
Adenovirus B/E*	100.0%	75.3% - 100.0%	99.1%	98.3% - 99.5%
Adenovirus C*	100.0%	54.1% - 100.0%	96.6%	95.3% - 97.5%
Human Metapneumovirus	100.0%	93.5% - 100.0%	99.8%	99.3% - 100.0%
Human Rhinovirus	89.2%	83.0% - 93.7%	96.1%	94.6% - 97.3%

Performance in Retrospective Clinical Specimens (N=320):

Virus	Positive Percent Agreement	95% CI	Negative Percent Agreement	95% CI
Influenza A H1	96.7%	82.8% - 99.9%	100.0%	98.7% - 100.0%
Parainfluenza Virus 1	100.0%	86.3% - 100.0%	98.0%	95.6% - 99.3%
Parainfluenza Virus 2	100.0%	86.8% - 100.0%	96.6%	93.8% - 98.4%
Adenovirus B/E	100.0%	86.3% - 100.0%	98.3%	96.1% - 99.4%
Adenovirus C	100.0%	80.6% - 100.0%	88.8%	84.8% - 91.9%

Predicate Device(s)

Luminex® xTag™ RVP, 510(k) Number K081483, eSensor® Warfarin Sensitivity Test, 510(k) No. K073720

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

Not Found

Regulation Number and Section

§ 862.2310 Clinical sample concentrator.

(a)
Identification. A clinical sample concentrator is a device intended to concentrate (by dialysis, evaporation, etc.) serum, urine, cerebrospinal fluid, and other body fluids before the fluids are analyzed.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to the limitations in § 862.9.

Summary

0

K113731

.

510(k) Summary

510(k) Summary
Submitter Name:	GenMark Diagnostics
Address:	5964 La Place Court
Carlsbad, CA 92008
Contact:	Joel Centeno
Vice President, Quality, Regulatory, and Clinical Affairs
Phone:	1 (760) 448-4304
Fax:	1 (760) 683-6821
E-mail:	joel.centeno@genmarkdx.com
Date Prepared:	December 16, 2011
Device Trade Name:	eSensor® Respiratory Viral Panel (RVP)
Device Common Name:	Respiratory Viral Panel Multiplex Nucleic Acid Assay System
Measurand:	Viral RNA/DNA of the following targets:
Target	Abrv.	Target	Abrv.

Influenza A	Flu A	Human Rhinovirus	HRV
Influenza A H1	Flu A H1	Human Metapneumovirus	hMPV
Influenza A H3	Flu A H3	Adenovirus B/E	ADV B/E
Influenza A 2009 H1N1	2009 H1N1	Adenovirus C	ADV C
Influenza B	Flu B	Parainfluenza Virus 1	PIV 1
Respiratory Syncytial Virus A	RSV A	Parainfluenza Virus 2	PIV 2
Respiratory Syncytial Virus B	RSV B	Parainfluenza Virus 3	PIV 3

Sample Type:	Nasopharyngeal Swab (NPS)
Technology:	Polymerase Chain Reaction (PCR)
Device Panel:	OIVD Division of Immunology and Microbiology
Classification Name:	Respiratory Viral Panel Multiplex Nucleic Acid Assay, 866.3980
Instrumentation for clinical multiplex test systems, 862.2570
Classification Code:	OCC: Respiratory viral panel multiplex nucleic acid assay
OEM: Human Metapneumovirus (hMPV) RNA assay system
OEP: Influenza A virus subtype differentiation nucleic acid assay
OOU: Parainfluenza Multiplex Nucleic Acid Assay
NSU: Instrumentation for clinical multiplex test systems
Predicate Device(s):	Luminex® xTag™ RVP, 510(k) Number K081483
Classification Code: OCC, OEM, OEP
Regulation No. 866.3980
eSensor® Warfarin Sensitivity Test, 510(k) No. K073720
Classification Code (applicable): NSU Instrumentation for
Clinical Multiplex Test Systems

1

Intended Use:

The eSensor® Respiratory Viral Panel (RVP) is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on the eSensor XT-8™ system for the simultaneous detection and identification of multiple respiratory viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals exhibiting signs and symptoms of respiratory infection.

The following virus types and subtypes are identified using the eSensor RVP: Influenza A, Influenza A H1 Seasonal Subtype, Influenza A H3 Seasonal Subtype; Influenza A 2009 H1N1 subtype, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Human Metapneumovirus, Human Rhinovirus, Adenovirus species B/E, and Adenovirus species C.

The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection if used in conjunction with other clinical and epidemiological information.

Negative results do not preclude respiratory viral infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Positive results do not rule out bacterial infection, or co-infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence and radiography) and clinical presentation must be taken into consideration in the final diagnosis of respiratory viral infection.

Performance characteristics for Influenza A were established during the 2010/2011 influenza season when Influenza A 2009 H1N1 and H3N2 were the predominant Influenza A viruses in circulation. When other Influenza A viruses emerge, performance characteristics may vary.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

For prescription use only.

Indication for Use: Same as Intended Use

Device Description:

The eSensor RVP is a multiplex microarray-based genotyping test system. It is based on the principles of competitive DNA hybridization using a sandwich assay format, wherein a singlestranded target binds concurrently to sequence-specific solution-phase signal probe and solidphase electrode-bound capture probe. The test employs reverse transcription polymerase chain reaction amplification, exonuclease digestion and hybridization of target DNA/RNA. In the process, the double-stranded PCR amplicons are digested with exonuclease to generate singlestranded DNA suitable for hybridization. Hybridization occurs in the eSensor XT-8 Cartridge (described below) where the single-stranded target DNA is mixed with a hybridization solution containing labeled signal probes.

During hybridization, the single-stranded target DNA binds to a complementary, single-stranded capture probe immobilized on the working electrode surface. Single-stranded signal probes (labeled with electrochemically active ferrocenes) bind to the target adjacent to the capture probe. When inserted into the eSensor XT-8 instrument (described below), simultaneous hybridization of

2

target to signal probes and capture probe is detected by alternating current voltammetry (ACV). Each pair of working electrodes on the array contains a different capture probe, and sequential analysis of each electrode allows detection of multiple viral targets.

Box	Component	Packaging & Quantity	Storage
eSensor*
Respiratory Viral
Panel Cartridges	eSensor® RVP Cartridges	6 foil bags with 8 cartridges
each in each cartridge pack	10-25 °C
	eSensor® Respiratory Viral Panel
Product Insert	1 copy	Dry place
(retain for reference)
eSensor*
Respiratory Viral
Panel Amplification
Reagents	RVP Enzyme Mix	2 vials with 40 µL each	-15 to -30 °C
	RVP PCR Mix	2 vials with 1000 µL each	(in a designated pre-
PCR location)
	MS2 Internal Control	2 vials with 300 µL each
eSensor*
Respiratory Viral
Panel Detection
Reagents	RVP Signal Buffer	2 vials with 2200 µL each
	Exonuclease	2 vials with 145 µL each.	-15 to -30 °C
(in a designated post-
PCR location)
	Buffer-1	2 vials with 350 µL each
	Buffer-2	2 vials with 700 µL each

Table 1: Reagents supplied with the kit:

The Assay Cartridge (eSensor XT-8 Cartridge)

The eSensor XT-8 cartridge device consists of a printed circuit board (PCB) with a multi-layer laminate and a plastic cover that forms a hybridization chamber. The cartridge is fitted with a pump and check valves that circulate the hybridization when inserted into the eSensor XT-8 instrument. The PCB chip consists of an array of 72 gold-plated working electrodes, a silver reference electrode, and two gold-plated auxiliary electrodes. Each working electrode has a connector contact pad on the opposite side of the chip for electrical connection to the eSensor XT-8 instrument. Each electrode is modified with a multi-component; self-assembled monolayer that includes oligonucleotide capture probes specific for each polymorphic site on the test panel and insulator molecules. The cartridge also contains an electrically erasable programmable readonly memory component (EEPROM) that stores information related to the cartridge (e.g., assay identifier, cartridge lot number, and expiration date).

The eSensor XT-8 Instrument

The eSensor XT-8 instrument was previously cleared for IVD use by the FDA under K073720 and K090901.

The eSensor XT-8 is a clinical multiplex instrument that has a modular design consisting of a base module and one, two, or three cartridge-processing towers containing 8, 16, or 24 cartridge slots, respectively. The cartridge slots operate independently of each other. Any number of cartridges can be loaded at one time, and the remaining slots are available for use while the instrument is running.

The base module controls each processing tower, provides power, and stores and analyzes data. The instrument is designed to be operated solely with the touch screen interface. Entering patient accession numbers and reagent lot numbers can be performed by the bar code scanner or the touch screen.

Each processing tower consists of eight cartridge modules, each containing a cartridge connector, a precision-controlled heater, an air pump, and electronics. The air pumps drive the pump and valve system in the cartridge, eliminating fluid contact between the instrument and the cartridge. The pneumatic pumping enables recirculation of the hybridization solution allowing the target DNA and the signal probes to hybridize with the complementary capture probes on the electrodes. The pump in the cartridge is connected to a pneumatic source from the eSensor XT-8 instrument and provides unidirectional pumping of the hybridization mixture through the channel

3

during hybridization. Using this process to circulate the hybridization solution minimizes the unstirred boundary laver at the electrode surface and continuously replenishes the volume above the electrode that has been depleted of complementary targets and signal probes.

The XT-8 instrument provides electrochemical detection of bound signal probes by ACV and subsequent data analysis and test report generating functions. All hybridization, ACV scanning and analysis parameters are defined by a scanning protocol loaded into the XT-8 Software, and then specified for use by the EEPROM on each cartridge.

Principle of eSensor Technology: eSensor technology uses a solid-phase electrochemical method for determining the presence of one or more of a defined panel of virus target sequences. Purified DNA/RNA is isolated from the patient specimen according to defined laboratory procedures and the extracted nucleic acid is reverse transcribed and/or amplified using virus specific primers with an RT-PCR enzyme mix. The amplified DNA is converted to single-stranded DNA via exonuclease digestion and is then combined with a signal buffer containing ferrocenelabeled signal probes that are specific for the different viral targets. The mixture of amplified sample and signal buffer is loaded onto a cartridge containing single-stranded oligonucleotide capture probes bound to gold-plated electrodes. The cartridge is inserted into the XT-8 instrument where the single-stranded targets hybridize to the complementary sequences of the capture probes and signal probes, as shown in Figure 1. The presence of each target is determined by voltammetry, which generates specific electrical signals from the ferrocene-labeled signal probe.

The eSensor RVP provides a qualitative result based upon the presence (Positive) or absence (Target Not Detected) of the viruses contained in the panel along with the internal MS2 control. Positive and negative results are determined based on the electrical signals generated being either above or below specified signal boundaries, respectively.

Image /page/3/Picture/4 description: The image shows a schematic of a DNA biosensor. The biosensor consists of a gold electrode with a self-assembled monolayer on top. A capture probe is attached to the monolayer, which binds to the target DNA. A signal probe is then used to detect the presence of the target DNA. The 5' and 3' ends of the DNA strands are labeled.

Figure 1: Hybridization complex formed at the surface of each electrode. Different, target specific, capture probes are covalently attached to the gold electrodes in the eSensor microarray. The amplified viral target DNA hybridizes to the capture probe and to a complementary ferrocene label signal probe. The ferrocene label is detected at the electrode surface using voltammetry.

Substantial Equivalence Discussion: The eSensor Respiratory Viral Panel (RVP) uses the similar fundamental scientific technologies and has the same intended use as that of the predicate device, the Luminex® xTag® RVP and eSensor XT-8 Instrument. The eSensor XT-8 Instrument described in K078720 (eSensor Warfarin Sensitivity Test) is the identical instrument with a unique Assay Analysis Module (AAM) necessary to support the RVP IVD.

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Element	Table 2: Substantial Equivalence Predicate Comparison:
	GenMark eSensor RVP	Luminex xTag RVP
	Subject Device	K081483
Intended Use	The eSensor® Respiratory Viral Panel (RVP) is a qualitative nucleic acid multiplex in vitro diagnostic test intended for use on the eSensor XT-8TM system for the simultaneous detection and identification of multiple respiratory viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals exhibiting signs and symptoms of respiratory infection.	The xTAG RVP (Respiratory Viral Panel) is a qualitative nucleic acid multiplex test intended for the simultaneous detection and identification of multiple respiratory virus nucleic acids in nasopharyngeal swabs from individuals suspected of respiratory tract infections.
	The following virus types and subtypes are identified using the eSensor RVP: Influenza A, Influenza A H1 Seasonal Subtype, Influenza A H3 Seasonal Subtype, Influenza A 2009 H1N1 subtype, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Human Metapneumovirus, Human Rhinovirus, Adenovirus species B/E, and Adenovirus species C.	The following virus types and subtypes are identified using RVP: Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza 1, Parainfluenza 2, and Parainfluenza 3 virus, Human Metapneumovirus, Rhinovirus, and Adenovirus.
	The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection if used in conjunction with other clinical and epidemiological information.	The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory infection aids in the diagnosis of respiratory viral infection if used in conjunction with other clinical and laboratory findings. It is recommended that specimens found to be negative after examination using RVP be confirmed by cell culture. Negative results do not preclude respiratory viral infection and should not be used as the sole basis for diagnosis, treatment or other management decisions.
	Negative results do not preclude respiratory viral infection and should not be used as the	Positive results do not rule out bacterial infection or co-infection with other organisms. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence, and radiography) and clinical presentation must be taken into consideration in order to obtain the final
Element	GenMark eSensor RVP	Luminex xTag RVP
	Subject Device	K081483
	sole basis for diagnosis, treatment or other
patient management decisions. Positive
results do not rule out bacterial infection, or
co-infection with other viruses. The agent
detected may not be the definite cause of
disease. The use of additional laboratory
testing (e.g. bacterial and viral culture,
immunofluorescence and radiography) and
clinical presentation must be taken into
consideration in the final diagnosis of
respiratory viral infection.	diagnosis of respiratory infection.
	Performance characteristics for Influenza A
were established during the 2010/2011
influenza season when Influenza A 2009
H1N1 and H3N2 were the predominant
Influenza A viruses in circulation. When
other Influenza A viruses emerge,
performance characteristics may vary.	Due to seasonal prevalence, performance
characteristics for Influenza A/H1 were established
primarily with retrospective specimens.
	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.	The RVP assay cannot adequately detect
Adenovirus species C, or serotypes 7a and 41.
The RVP primers for detection of rhinovirus cross-
react with enterovirus. A rhinovirus reactive result
should be confirmed by an alternate method (e.g.
cell culture).
	For prescription use only.	Performance characteristics for Influenza A Virus
were established when Influenza A/H3 and A/H1
were the predominant Influenza A viruses in
circulation. When other Influenza A viruses are
emerging, performance characteristics may vary. If
infections 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 a state or local health department for
testing. Viral culture should not be attempted in
these cases unless a BSL 3+ facility is available to
receive and culture specimens.
		6
Element	GenMark eSensor RVP	Luminex xTag RVP
	Subject Device	K081483
Organisms Detected	Influenza A, Influenza A H1 Seasonal
Subtype, Influenza A H3 Seasonal Subtype,
Influenza A 2009 H1N1 strain, Influenza B,
Respiratory Syncytial Virus subtype A,
Respiratory Syncytial Virus subtype B,
Parainfluenza 1, Parainfluenza 2,
Parainfluenza 3, Human Metapneumovirus,
Human Rhinovirus, Adenovirus B/E and
Adenovirus C	Influenza A, Influenza A subtype H1, Influenza A
subtype H3, Influenza B, Respiratory Syncytial
Virus subtype A, Respiratory Syncytial Virus
subtype B, Parainfluenza 1, Parainfluenza 2, and
Parainfluenza 3 virus, Human Metapneumovirus,
Rhinovirus, and Adenovirus
Specimen type	Same	Nasopharyngeal swabs (NPS)
Sample preparation	Same	Up front sample processing is required to extract
nucleic acid
Assay technology	Same	PCR-based system for detecting viral nucleic acids
in clinical specimens targeting unique regions of
virus RNA/DNA
Detection
technology	Solid phase electrochemical	Spectrofluorometry
Instrument	1. Same

eSensor XT-8 | 1. EasyMag® extraction system
Luminex® 100/200™ instrument |
| Software | Application software and embedded
firmware (controls hardware functions) on
XT-8 in addition to Assay Analysis Module
(AAM) for RVP-IVD | IS or XPONENT software; xTAG Data Analysis
Software RVP (US) |
| Time to results | Approximately 6 hours | Approximately 8 hours |
| Results | Same | Qualitative |
| Test interpretation | Automated test interpretation and report
generation. User can access the raw signals
generated by the assay. | Semi-automated test interpretation. User must
review all "no call" results to determine cause and
retesting strategy. |
| Controls | Same | Internal control added to each sample. External
control processed with each batch of samples. |

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control control control control controllers.

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and the comments of the comments of the comments of the comments of

and the comments of the country of

and the control control of the control of

7

NON-CLINICAL PERFORMANCE DATA

Limit of Detection

Limit of Detection (LoD)

The Limit of Detection (LoD) was identified and verified for each viral target of the eSensor RVP using samples prepared from regrown and re-titered viral reference strains as detailed in Table 3. Initial estimations involved serially diluting each viral strain in M5 media. The dilutions prepared from each target were extracted in triplicate and the assay was performed once for each extract. Verification of the LoD was performed by meeting 95% detection or in at least 19 of the 20 extraction replicates on the selected dilution of each culture. Once the LoD was verified for each viral target in M5. verification of the LoD was also performed with the M4 media. Each viral target was diluted in M4 media down to the LoD concentration. The LoD dilution of each culture was extracted 20 times and the eSensor RVP assay was performed on each extract. The final results summary with the verified LoD concentration in TCID55/mL for both M5 and M4 media is shown in Table 3.

| Viral Target | Strain | Starting
TCID50/mL | LoD Spiked
Conc.
(TCID50/mL) | %
Positive |
|--------------------|------------------------|-----------------------|------------------------------------|---------------|
| Flu A | H1N1
Brisbane/59/07 | 4.17 x 105 | 4.17 x 10-1 | 100% |
| Flu A H1 | H1N1
Brisbane/59/07 | 4.17 x 105 | 4.17 x 10-1 | 97.5% |
| Flu A | H3N2 | 1.58 x 108 | 1.58 x 103 | 100% |
| Flu A H3 | H3N2 | 1.58 x 108 | 1.58 x 103 | 100% |
| Flu A 2009
H1N1 | NY/2009 | 1.05 x 106 | 1.05 x 10-1 | 95% |
| Flu B | Florida/02/06 | 3.16 x 106 | 3.16 x 10-1 | 100% |
| hMPV | B2 | 4.17 x 105 | 4.17 x 100 | 100% |
| HRV | 3 | 1.58 x 104 | 1.58 x 10-3 | 97.5% |
| PIV1 | C35 | 2.81 x 104 | 2.81 x 10-2 | 100% |
| PIV2 | Greer | 2.81 x 106 | 2.81 x 100 | 100% |
| PIV3 | C 243 | 2.81 x 107 | 2.81 x 101 | 100% |
| RSV A | A2 | 2.81 x 105 | 2.81 x 100 | 97.5% |
| RSV B | 9320 | 1.58 x 105 | 1.58 x 100 | 100% |
| ADV B/E | Type 4 | 1.58 x 106 | 1.58 x 101 | 100% |
| ADV C | Type 1 | 8.89 x 107 | 8.89 x 101 | 100% |

Table 3: LoD Results Summary

Analytical Reactivity (Inclusivity)

Analytical Inclusivity Testing

The performance of the eSensor RVP with multiple viral target strains representing temporal and geographic diversity for each claimed viral target was evaluated. Each viral target strain was diluted in M5 transport media to a titer of 3X LoD for the corresponding viral target and extracted in triplicate using the bioMerieux NucliSENS easyMAG System. Following extraction, each

8

replicate was tested once using the eSensor RVP. In the case that a viral target strain is not detected at 3X LoD, 1000-fold serial dilutions were made from the viral stock and then each dilution was extracted in triplicate and tested using the eSensor RVP.

In cases where unexpected False Negative results were obtained, in silico analysis was performed. Table 4 shows the results.

| Target | Strain Detected
(TCID50/ml) | LoD Multiple
Detected |
|--------------------|--------------- | Flu A H1 | A/New
Caledonia/20/1999 | 4.2 | | A/Brisbane/59/07 | | FM/1/47H1 | | A/Denver/1/57 | | A/Solomon
Islands/3/2006 | 1.26 | | A/Taiwan/42/06 | | A/NWS/33 H1- 3000x* |
| | A/PR/8/34 H1- not detected* |
| | A/Mal/302/54 H1- 15172x* |
| | A/Fort (H1N1) | 5.5 H1-13x* |
| | A/Aichi/2/68 H3N2 | Flu A H3 | A/Brisbane/10/07 | | A/Victoria/3/75 | | A/Port Chalmers/1/73 | | A/Wisconsin/67/05 | | A/Hong Kong/8/68 | | A/Perth/16/2009 | | Alice A/England/42/72 | 4743 | | MRC-2 Strain | 4743 | | A/Nanchang/933/95 | Flu A 2009
H1N1 | A/NY/02/2009 | | A/New Jersey/8/76 | | A/California/7/2009 | Concentration
---------------------|------------------------------------------|---------------------------------|
| 10x |
| 1.26 | 3x |
| 1.26 | 3x |
| 1.26 | 3x |
| 3x |
| 1.26 | 3x |
| 1260 | Flu A- 3x;
| 1.26 | Flu A- 3x;
| 6372 | Flu A- 3x
Monmouth/1/1947
| Flu A- 3x
| 4743 | 3x |
| 4743 | 3x |
| 4743 | 3x |
| 4743 | 3x |
| 4743 | 3x |
| 4743 | 3x |
| 4743 | 3x |
(vaccine)
| 3x |
Recombinant
| 3x |
| 4743 | 3x |
| 0.3 | 3x |
| 0.3 | 3x |
| 0.3 | 3x |

			Table 4: Analytical Reactivity (Inclusivity) Results
--	--	--	------------------------------------------------------	--

9

| Target | Strain | Concentration
Detected
(TCID50/ml) | LoD Multiple
Detected |
|------------------------------------------------------|-------------------------------|------------------------------------------|-------------------------------------|
| | A/Swine NY/01/2009 | 0.3 | 3x |
| | A/Swine NY/03/2009 | 0.3 | 3x |
| | A/Mexico/4108/09 | 0.3 | 3x |
| | A/Virginia/ATCC1/2009 | 0.3 | 3x |
| | A/Virginia/ATCC2/2009 | 0.6 | Flu A- 3x
2009 H1N1- 6x** |
| | A/Virginia/ATCC3/2009 | 2.7 | Flu A- 3x
2009 H1N1-
27x** |
| | A/Iowa/15/30 | 100 | Flu A- 3x;
2009 H1N1-
1000x** |
| | B/Florida/02/06
(Yamagata) | 1 | 3x |
| | B/Malaysia/2506/04 | 1 | 3x |
| Flu B
(Victoria
lineage except
where noted) | B/Lee/40 | 1 | 3x |
| | B/Allen/45 | 1 | 3x |
| | B/GL/1739/54 | 1 | 3x |
| | B/Taiwan/2/62 | 1 | 3x |
| | B/Hong Kong/5/72 | 1 | 3x |
| | B/Maryland/1/59 | 1 | 3x |
| RSV A | A2 | 8.4 | 3x |
| | Long | 8.4 | 3x |
| | 9320 | 4.8 | 3x |
| RSV B | WV/14617/85 | 4.8 | 3x |
| | Wash/18537/62 | 4.8 | 3x |
| PIV1 | C35 | 0.084 | 3x |
| | Type 1 | 0.084 | 3x |
| PIV2 | Greer | 8.4 | 3x |
| | Type 2 | 8.4 | 3x |
| PIV3 | C-243 | 84 | 3x |
| | Type 3 | 84 | 3x |
| hMPV | IA3-2002 G, A1 | 12.6 | 3x |
| | IA14-2003 G, A2 | 12.6 | 3x |
| | Peru2-2002 G, B1 | 12.6 | 3x |
| Target | Strain | Concentration
Detected
(TCID50/ml) | LoD Multiple
Detected |
| | 1A | 0.9 | 450x# |
| | A2 | 0.9 | 569x# |
| | A7 | 0.005 | 3x |
| | A16 | 0.005 | 3x |
| HRV A | 18 | Detected## | N/A |
| | A34 | 0.005 | 3x |
| | A57 | 0.005 | 3x |
| | A77 | 0.005 | 3x |
| | 277G | 0.2 | 100x# |
| | B3 | 0.1 | 80x |
| | B14 | 0.02 | 14x |
| | B17 | 0.4 | 253x |
| HRV B | B42 | 0.005 | 3x |
| | FO2-2547 | 0.2 | 89x# |
| | B83 | 0.2 | 127x |
| | 84 | Detected## | N/A |
| HRV C | C$ | Detected## | N/A |
| | Type 3 | 0.3 | 3x |
| | Type 7A | 0.3 | 3x |
| ADV B | Type 11 (lot 306523) | 0.3 | 3x |
| | De Wit Type 14 | 0.3 | 3x |
| | Ch.79 Type 16 | 0.3 | 3x |
| | Type 21 (lot 307610) | 0.3 | 3x |
| | Compton Type 34 | 0.3 | 3x |
| | Holden Type 35 | 0.3 | 3x |
| | Wan Type 50 | 0.3 | 3x |
| | Type 1 | 267 | 3x |
| | Type 2 | 533 | 6x |
| ADV C | Type 5 | 533 | 6x |
| | Type 6 | 533 | 6x |
| ADV E | Type 4 | 47 | 3x |

:

September 19.

.

:

.

:

.

10

in silico analysis revealed little homology between the strain sequence and the H1
primer sequences. The degree of mis-match to the H1 subtype primer sequences
resulted subtyping result.

:

11

** In silico analysis revealed little homology between the strain sequence and the 2009 H1N1 primer sequences. The degree of mis-match to the 2009 H1N1 subtype primer sequences resulted in decreased reactivity to these influenza strains for the 2009 H1N1 subtyping result.
- HRV strain 3, used for LoD determination, had a TCIDs /ml of 0.0016. HRV strains 1A, FO2-2547, 277G were detected at a higher LoD multiple to the reference strain, respectively with their corresponding TCIDsoml values of 0.9, 0.2, and 0.2. In silico analysis revealed mis-matches between the capture probe sequence and the HRV strains (2, 3 and 1 mis-match(es) respectively).

** No concentration available since it was an extracted RNA sample. § Only one test done for HRV C due to limited sample availability

Supplemental Analytical Inclusivity Testing of Other Influenza Subtypes

Additional analytical inclusivity testing was carried out with either live isolates or purified genomic RNA of Influenza A strains.

Subtype	Host	Strain	Testing Conc.	RVP Result
Flu A H1N2	Human	A/NWS/34 (HA) x A/Rockefeller Institute/5/57 (NA),
(H1N2), Reassortant NWS-F, RNA	0.74 ng	Flu A H1
Flu A H2N2	Avian	A/Japan/305/57, RNA	1.625 ng	Flu A
Flu A H5N3		A/Korea/426/68 (HA, NA) x A/Puerto Rico/8/34, RNA	3.12 ng	Flu A
Flu A H5N3	Avian	A/duck/Singapore/645/97, Wild Type	1.26 TCID50/ml	Flu A
Flu A H10N7		A/chicken/Germany/N/49	1.26 TCID50/ml	Flu A

Table 5: Additional Analytical Reactivity (Inclusivity) Results

Simulated Reactivity Information for Influenza Strains of Human, Swine, and Avian Origin

For any strains that were not available for testing with the eSensor RVP, such as Flu A H5 and Flu A H7 strains in silico analysis was performed. The eSensor RVP primers for Flu A, Flu A H1, Flu A 2009 H1N1 and Flu A H3 were aligned to the GenBank® sequences of the reactivity strains. Exclusivity was predicted based on the number and location of mismatches between assay primers and available strain sequences.

Simulated reactivity of the eSensor RVP with influenza strains was generated using a bioinformatics approach. Assay primer sequences, signal probes and capture probes were aligned with GenBank sequences corresponding to the appropriate gene targets and reactivity were predicated based on the number and location of mismatches in the targeted region shown in Table 6.

Table 6: Supplemental Reactivity of eSensor RVP Influenza A with
Human. Swine, and Avian Influenza Strains

| Subtype | Host | Strain | GenBank
ID | Simulated
RVP
Reactivity
Result |
|--------------|-------|---------------------------------------------------------|----------------------------|------------------------------------------|
| H2N2 | Human | A/Albany/20/1957(H2N2) | CY022014 | Flu A |
| | | A/chicken/New York/13828-3/1995(H2N2) | CY014822 | Flu A |
| | Avian | A/Japan/305/1957(H2N2) | CY014977 | Flu A |
| | | A/Korea/426/1968(H2N2) | CY031596 | Flu A |
| H4N6 | Avian | A/blue-winged teal/Minnesota/Sg-00043/2007(H4N6) | CY063978 | Flu A |
| H5N1 | | A/peregrine falcon/Aomori/7/2011 | AB629716 | Flu A |
| Subtype | Host | Strain | GenBank
ID | Simulated
RVP
Reactivity
Result |
| H5N1 | Avian | A/chicken/West Bengal/239022/2010 | CY061305 | Flu A |
| | | A/chicken/West Bengal/193936/2009 | GU272009 | Flu A |
| | | A/chicken/Hunan/1/2009 | HM172150 | Flu A |
| | | A/chicken/Hunan/8/2008 | GU182162 | Flu A |
| | | A/chicken/West Bengal/106181/2008 | GU083632 | Flu A |
| | | A/chicken/Primorsky/85/2008 | FJ654298 | Flu A |
| | | A/chicken/West Bengal/82613/2008 | GU083648 | Flu A |
| | | A/duck/France/080036/2008 | CY046185 | Flu A |
| | | A/duck/Vietnam/G12/2008 | AB593450 | Flu A |
| | | A/chicken/Thailand/PC-340/2008 | EU620664 | Flu A |
| | | A/great egret/Hong Kong/807/2008 | CY036240 | Flu A |
| | | A/rook/Rostov-on-Don/26/2007(H5N1) | EU814504 | Flu A |
| | | A/turkey/VA/505477-18/2007(H5N1) | GU186510 | Flu A |
| | | A/chicken/Bangladesh/1151-10/2010(H5N1) | HQ156766 | Flu A |
| | | A/Bangladesh/3233/2011 | CY088772 | Flu A |
| | Human | A/Cambodia/R0405050/2007(H5N1) | HQ200572 | Flu A |
| | | A/Cambodia/S1211394/2008 | HQ200597 | Flu A |
| | | A/Hong Kong/486/97(H5N1) | AF255368 | Flu A |
| | Swine | A/swine/East Java/UT6010/2007(H5N1) | HM440124 | Flu A |
| H5N2 | | A/duck/Pennsylvania/10218/1984(H5N2) | AB286120 | Flu A |
| | | A/American black duck/Illinois/08OS2688/2008 | CY079453 | Flu A |
| | | A/American green-winged teal/California/HKWF609/2007 | CY033447 | Flu A |
| | | A/Canada goose/New York/475813-2/2007 | GQ923358 | Flu A |
| | Avian | A/blue-winged teal/Saskatchewan/22542/2007 | CY047705 | Flu A |
| | | A/chicken/Taiwan/A703-1/2008 | AB507267 | Flu A |
| | | A/duck/France/080032/2008 | CY046177 | Flu A |
| | | A/duck/New York/481172/2007 | GQ117202 | Flu A |
| | | A/gadwall/Altai/1202/2007 | CY049759 | Flu A |
| H5N3 | | A/mallard/Louisiana/476670-4/2007 | GQ923390 | Flu A |
| | | A/waterfowl/Colorado/476466-2/2007 | GQ923374 | Flu A |
| | Avian | A/duck/Singapore/F119/3/1997(H5N3) | GU052803 | Flu A |
| H6N1 | Avian | A/duck/PA/486/1969(H6N1) | EU743287 | Flu A |
| H6N2
H7N2 | | A/mallard/Czech Republic/15902-17K/2009(H6N2) | HQ244433 | Flu A |
| | Avian | A/chicken/Hebei/1/2002 | AY724263 | Flu A |
| | | A/chicken/PA/149092-1/02 | AY241609 | Flu A |
| | | A/chicken/NJ/294508-12/2004 | EU743254 | Flu A |
| | | A/chicken/New York/23165-6/2005 | CY031077 | Flu A |
| Subtype | Host | Strain | GenBank ID | Simulated RVP Reactivity Result |
| | | A/muscovy duck/New York/87493-3/2005 | CY034791 | Flu A |
| | | A/mallard/Netherlands/29/2006 | CY043833 | Flu A |
| | | A/northern shoveler/California/JN1447/2007 | CY076873 | Flu A |
| H7N2 | Human | A/New York/107/2003(H7N2) | EU587373 | Flu A |
| H7N3 | | A/Canada/rv504/2004(H7N3) | CY015007 | Flu A |
| | | A/American green-winged teal/Mississippi/09OS046/2009 | CY079309 | Flu A |
| | Avian | A/chicken/Germany/R28/03 | AJ619676 | Flu A |
| | | A/chicken/Netherlands/1/03 | AY340091 | Flu A |
| H7N7 | | A/mallard/California/HKWF1971/2007 | CY033383 | Flu A |
| | | A/mallard/Korea/GH171/2007 | FJ959087 | Flu A |
| | | A/mute swan/Hungary/5973/2007 | GQ240816 | Flu A |
| | | A/northern shoveler/Mississippi/ 09OS643/2009 | CY079413 | Flu A |
| | | Human | A/Netherlands/219/03(H7N7) | AY340089 |
| | H9N2 | | A/Hong Kong/1073/99(H9N2) | AJ278647 |
| | Avian | A/turkey/Wisconsin/1/1966(H9N2) | CY014664 | Flu A |
| H11N9 | | A/duck/Memphis/546/1974(H11N9) | GQ257441 | Flu A |
| | Swine | A/swine/Wisconsin/1/1971 (H1N1) | CY022414 | Flu A |
| H1N1 | | A/swine/Wisconsin/1/1971 (H1N1) | CY022417 | Flu A H1 |
| | | A/California/UR06-0393/2007(H1N1) | CY026540 | Flu A |
| | | A/California/UR06-0393/2007(H1N1) | CY026539 | Flu A H1 |
| | Human | A/New York/297/2003(H1N2) | CY002665 | Flu A |
| H1N2 | | A/New York/297/2003(H1N2) | CY002664 | Flu A H1 |
| H1N1 (2009) | | A/Aalborg/INS133/2009(H1N1) | CY063607 | Flu A |
| | | A/Aalborg/INS133/2009(H1N1) | CY063606 | 2009 H1N1 |
| | Swine | A/swine/Hong Kong/NS857/2001 (H1N2) | GQ229350 | Flu A |
| H1N2 | | A/swine/Hong Kong/NS857/2001 (H1N2) | GQ229348 | Flu A H1 |
| | | A/swine/Sweden/1021/2009(H1N2) | GQ495135 | Flu A |
| | | A/swine/Sweden/1021/2009(H1N2) | GQ495132 | Flu A H1 |
| | Avian | A/blue-winged teal/ALB/452/1983(H3N1) | CY004635 | Flu A |
| H3N1 | | A/blue-winged teal/ALB/452/1983(H3N1) | CY005940 | Flu A H3 |
| H3N2 | | A/swine/NY/A01104005/2011(H3N2)* | N/A* | Flu A |
| | Swine | A/swine/NY/A01104005/2011(H3N2)* | JN940422 | Flu A H3 |
| | | A/Maine/06/2011(H3N2) | JN866181 | Flu A |
| | | A/Maine/06/2011(H3N2) | JN866186 | Flu A H3 |
| | | A/Indiana/08/2011(H3N2) | JN655558 | Flu A |
| | | A/Indiana/08/2011(H3N2) | JN638733 | Flu A H3 |
| | | CY044581 | | Flu A |
| Subtype | Host | Strain | GenBank ID | Simulated RVP Reactivity Result |
| | | A/American black duck/North Carolina/675-075/2004(H3N2) | GU051136 | Flu A |
| | | | GU051135 | Flu A H3 |
| H3N5 | | A/mallard/Netherlands/2/1999(H3N5) | CY060264 | Flu A |
| | | | CY060261 | Flu A H3 |
| H3N6 | Avian | A/American black duck/New Brunswick/25182/2007(H3N6) | CY047697 | Flu A |
| | | | CY047696 | Flu A H3 |
| H3N7 | | A/northern shoveler/California/HKWF1367/2007(H3N7) | CY033375 | Flu A |
| | | | CY033372 | Flu A H3 |
| H3N8 | | A/American black duck/Washington/699/1978(H3N8) | GU052300 | Flu A |
| | | | GU052299 | Flu A H3 |

12

.

:

.

13

and the country of the country of the country

14

Influenza A H3N2v (swine-origin). No sequence available but literature from CDC states that strains the conserved Matrix Protein (14) sequence from 2009 H1N1. Therefore Flu A should also be able to be detected.

Reproducibility

Multisite Reproducibility

Multisite reproducibility of the eSensor RVP was performed to evaluate the major sources of variability, such as lot-to-lot, extraction, site/instrument-to-site/instrument, day-to-day and operator/run-to-operator/run. Reproducibility testing occurred at three sites, utilizing a panel of samples containing viral material from culture derived isolates in media, simulating NPS specimen. Each of the selected panel targets was prepared at concentrations representing the following three viral load levels: Moderate Positive (100% positive, 3x LoD), Low Positive (95% positive, 1x LoD), and Negative (100% negative). Each simulated sample was divided into aliquots, blinded, and stored frozen (-70 °C) prior to testing. Each site received three lots of RVP Cartridges/Reagents, a set of samples for two operators and one XT-8 instrument. All samples were independently extracted using the bioMérieux easyMAG extraction system. Every analyte at each concentration was tested a total of 108 times (two operators at three sites, each testing three replicates on six testing days). Each lot of RVP Cartridges/Reagents was used in 36 tests per analyte/concentration. Summary results for each tested analyte are summarized below.

| Flu A
Concentration | Site | # Positive | # Negative | % Agreement
with Expected
Results | 95% CI | Mean
(nA) | Std Dev | % CV |
|--------------------------------------|-----------|------------|------------|-----------------------------------------|------------|--------------|---------|------|
| MOD POS
(3x LoD)
1.3 TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 243.0 | 23.6 | 9.7 |
| | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 246.4 | 29.7 | 12.0 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 235.0 | 32.6 | 13.9 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 241.5 | 29.0 | 12.0 |
| LOW POS
(1x LoD)
0.4 TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 248.3 | 28.6 | 11.5 |
| | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 244.7 | 26.4 | 10.8 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 232.8 | 23.2 | 10.0 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 242.0 | 26.7 | 11.1 |

Table 7: Summary of Influenza A Calls (H3N2)

15

| Flu A
Concentration | Site | # Positive | # Negative | % Agreement
with Expected
Results | 95% CI | Mean
(nA) | Std Dev | % CV |
|------------------------|-----------|------------|------------|-----------------------------------------|-----------------|--------------|---------|------|
| Negative | Site 1 | 3/288 | 285/288 | 99.0% | 97.0%-
99.8% | 1.2 | 2.4 | N/A |
| | Site 2 | 1/288 | 287/288 | 99.7% | 98.1%-100% | 1.0 | 2.2 | N/A |
| | Site 3 | 2/288 | 286/288 | 99.3% | 97.5%-
99.9% | 1.0 | 0.9 | N/A |
| | All Sites | 6/864 | 858/864 | 99.3% | 98.5%-
99.7% | 1.1 | 1.9 | N/A |

Table 8: Summary of Influenza A H3 Calls

| Flu A H3
Concentration | Site | # Positive | # Negative | % Agreement
with Expected
Results | 95% CI | Mean
(nA) | Std Dev | % CV |
|--------------------------------------------------------|-----------|------------|------------|-----------------------------------------|------------|--------------|---------|------|
| MOD POS
(3x LoD)
$4.7 \times 10^3$
TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 86.5 | 23.9 | 27.7 |
| | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 77.4 | 26.7 | 34.5 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 86.0 | 30.5 | 35.4 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 83.3 | 27.3 | 32.7 |
| LOW POS
(1x LoD),
$1.6 \times 10^3$
TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 81.5 | 26.1 | 32.0 |
| | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 68.2 | 29.6 | 43.4 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 84.9 | 21.3 | 25.1 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 78.2 | 26.6 | 34.1 |
| Negative | Site 1 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.4 | 0.3 | N/A |
| | Site 2 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.3 | 0.3 | N/A |
| | Site 3 | 1/288 | 287/288 | 99.7% | 98.1%-100% | 0.4 | 0.4 | N/A |
| | All Sites | 1/864 | 863/864 | 99.9% | 99.4%-100% | 0.4 | 0.4 | N/A |

Table 9: Summary of Adenovirus B/E Calls

| ADV B
Concentration | Site | # Positive | # Negative | % Agreement
with Expected
Results | 95% CI | Mean
(nA) | Std Dev | % CV |
|---------------------------------------|-----------|------------|------------|-----------------------------------------|------------|--------------|---------|------|
| | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 109.1 | 10.9 | 10.0 |
| MOD POS
(3x LoD)
47.4 TCID50/ml | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 102.6 | 11.5 | 11.2 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 102.2 | 14.5 | 14.1 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 104.7 | 12.7 | 12.1 |
| | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 92.7 | 11.4 | 12.3 |
| LOW POS
(1x LoD)
15.8 TCID50/ml | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 89.9 | 10.9 | 12.1 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 84.5 | 16.9 | 20.0 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 89.1 | 13.7 | 15.3 |
| | Site 1 | 1/288 | 287/288 | 99.7% | 98.1%-100% | 1.6 | 6.0 | N/A |
| Negative | Site 2 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 1.2 | 0.4 | N/A |
| | Site 3 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 1.2 | 0.4 | N/A |
| | All Sites | 1/864 | 863/864 | 99.9% | 99.4%-100% | 1.3 | 3.5 | N/A |

.

:

.

16

| hMPV
Concentration | Site | # Positive | # Negative | % Agreement
with Expected
Results | 95% CI | Mean
(nA) | Std Dev | % CV |
|-------------------------------------|-----------|------------|------------|-----------------------------------------|-------------|--------------|---------|------|
| MOD POS
(3x LoD)
13 TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 91.2 | 26.1 | 28.6 |
| MOD POS
(3x LoD)
13 TCID50/ml | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 92.5 | 37.1 | 40.1 |
| MOD POS
(3x LoD)
13 TCID50/ml | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 100.4 | 22.7 | 22.6 |
| MOD POS
(3x LoD)
13 TCID50/ml | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 94.7 | 29.3 | 30.9 |
| LOW POS
(1x LoD)
4 TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 56.4 | 30.0 | 53.2 |
| LOW POS
(1x LoD)
4 TCID50/ml | Site 2 | 35/36 | 1/36 | 97.2% | 85.5%-99.9% | 51.0 | 31.2 | 61.2 |
| LOW POS
(1x LoD)
4 TCID50/ml | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 63.8 | 28.1 | 44.0 |
| LOW POS
(1x LoD)
4 TCID50/ml | All Sites | 107/108 | 1/108 | 99.1% | 94.9%-100% | 57.1 | 30.0 | 52.5 |
| Negative | Site 1 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.1 | 0.0 | N/A |
| Negative | Site 2 | 8/288 | 280/288 | 97.2% | 94.6%-98.8% | 0.7 | 4.1 | N/A |
| Negative | Site 3 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.1 | 0.1 | N/A |
| Negative | All Sites | 8/864 | 856/864 | 99.1% | 98.2%-99.6% | 0.3 | 2.4 | N/A |

Table 10: Summary of hMPV Calls

Table 11: Summary of PIV 3 Calls

・

·

.

| PIV3
Concentration | Site | # Positive | # Negative | % Agreement
with Expected
Results | 95% CI | Mean
(nA) | Std Dev | % CV |
|-------------------------------------|-----------|------------|------------|-----------------------------------------|------------|--------------|---------|------|
| MOD POS
(3x LoD)
84 TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 178.1 | 27.4 | 15.4 |
| | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 193.8 | 29.8 | 15.4 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 160.8 | 27.4 | 17.0 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 177.6 | 31.1 | 17.5 |
| LOW POS
(1x LoD)
28 TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 139.0 | 34.8 | 25.1 |
| | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 162.4 | 27.9 | 17.2 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 126.9 | 38.9 | 30.7 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 142.8 | 37.0 | 25.9 |
| Negative | Site 1 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.2 | 0.1 | N/A |
| | Site 2 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.2 | 0.1 | N/A |
| | Site 3 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.2 | 0.1 | N/A |
| | All Sites | 0/864 | 864/864 | 100.0% | 99.6%-100% | 0.2 | 0.1 | N/A |

Table 12: Summary of RSV A Calls

| RSV A Concentration | Site | # Positive | # Negative | % Agreement
with Expected
Results | 95% CI | Mean
(nA) | Std Dev | % CV |
|--------------------------------------|-----------|------------|------------|-----------------------------------------|-------------|--------------|---------|------|
| MOD POS
(3x LoD)
8.4 TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 166.3 | 19.2 | 11.5 |
| | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 156.7 | 31.7 | 20.2 |
| | Site 3 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 156.4 | 22.9 | 14.7 |
| | All Sites | 108/108 | 0/108 | 100.0% | 96.6%-100% | 159.8 | 25.3 | 15.8 |
| LOW POS
(1x LoD)
2.8 TCID50/ml | Site 1 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 146.6 | 22.7 | 15.5 |
| | Site 2 | 36/36 | 0/36 | 100.0% | 90.3%-100% | 124.6 | 41.0 | 32.9 |
| | Site 3 | 35/36 | 1/36 | 97.2% | 85.5%-99.9% | 128.2 | 33.4 | 26.1 |
| | All Sites | 107/108 | 1/108 | 99.1% | 94.9%-100% | 133.1 | 34.3 | 25.8 |
| Negative | Site 1 | 4/288 | 284/288 | 98.6% | 96.5%-99.6% | 0.7 | 4.0 | N/A |
| | Site 2 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.2 | 0.1 | N/A |

17

| RSV A
Concentration | Site | # Positive | # Negative | % Agreement
with Expected
Results | 95% CI | Mean
(nA) | Std Dev | % CV |
|------------------------|-----------|------------|------------|-----------------------------------------|-------------|--------------|---------|------|
| | Site 3 | 0/288 | 288/288 | 100.0% | 98.7%-100% | 0.2 | 0.2 | N/A |
| | All Sites | 4/864 | 860/864 | 99.5% | 98.8%-99.9% | 0.4 | 2.3 | N/A |

Testing of Dual Infection Samples

Clinically Relevant Co-Infections

An internal co-infection study was performed to determine the capability of the eSensor RVP to detect clinically relevant dual co-infections in patient samples. Nine clinically relevant coinfections were evaluated in this study. Dual co-infections were prepared by using the representative viral cultures at two different concentrations - Virus A at 1x LoD and Virus B at 10,000x LoD, as well as Virus A at 10,000x LoD and Virus B at 1x LoD. Relevant medical literature was sourced for selection of viral mix composition of common or expected co-infections.

The table below summarizes the TCIDsoml and LoD multiple detected in each viral co-infection.

| Viral Co-
Infection

(Virus 1/Virus 2)	Virus 1 Detected		Virus 2 Detected
	LoD
Multiple	TCID50/ml	LoD
Multiple	TCID50/ml
H3-RSV	1x LoD	$1.58 x 10^3$	10,000x LoD	$2.81 x 10^4$
RSV-H3	3x LoD	$8.43 x 10^0$	10,000x LoD	$1.58 x 10^7$
H3-FLUB	1x LoD	$1.58 x 10^3$	10,000x LoD	$3.16 x 10^3$
FLUB-H3	1x LoD	$3.16 x 10^{-1}$	10,000x LoD	$1.58 x 10^7$
H1N1-HRV	3x LoD	$3.15 x 10^{-1}$	10,000x LoD	$1.58 x 10^7$
HRV-H1N1	1x LoD	$1.58 x 10^{-3}$	10,000x LoD	$1.05 x 10^3$
H1N1-PIV3	1x LoD	$1.05 x 10^{-1}$	10,000x LoD	$2.81 x 10^5$
PIV3-H1N1	1x LoD	$2.81 x 10^1$	10,000x LoD	$1.05 x 10^3$
H1N1-RSV	1x LoD	$1.05 x 10^{-1}$	10,000x LoD	$2.81 x 10^4$
RSV-H1N1	3x LoD	$8.43 x 10^0$	10,000x LoD	$1.05 x 10^3$
RSV-ADV	1x LoD	$2.81 x 10^0$	10,000x LoD	$8.89 x 10^{-2}$
ADV-RSV	1x LoD	$8.89 x 10^{-2}$	10,000x LoD	$2.81 x 10^4$
HMPV-RSV	1x LoD	$4.17 x 10^0$	10,000x LoD	$2.81 x 10^4$
RSV-HMPV	1x LoD	$2.81 x 10^0$	10,000x LoD	$4.17 x 10^4$
HMPV-ADV	1x LoD	$4.17 x 10^0$	10,000x LoD	$8.89 x 10^{-2}$
ADV-HMPV	1x LoD	$8.89 x 10^{-2}$	10,000x LoD	$4.17 x 10^4$
HRV-RSV	3x LoD	$4.74 x 10^{-3}$	10,000x LoD	$2.81 x 10^4$
RSV-HRV	1x LoD	$2.81 x 10^0$	10,000x LoD	$1.58 x 10^7$

Table 13: Dual Infection Reproducibility Summary Results

18

Interference

Interfering Substances

Potentially interfering substances were selected based on the fact that they could pre-exist in the specimen (e.g. blood, nasal secretions or mucus, and throat medications used to relieve congestion, nasal dryness, irritation, or asthma and allergy symptoms) as well as those that could be introduced during specimen collection and preparation. Each potentially interfering substance was tested individually with the exception of Luffa opperculata. Galphimia glauca. Histaminum hydrochloricum, and Sulfur, which were tested together as Zicam® Allergy Relief Nasal spray and Oxymetazoline and Menthol, which were tested together as Afrin® No Drip Severe Congestion nasal spray, thereby bringing the total to 21 potentially interfering test combinations. Viral samples representative of the 14 viral targets on the eSensor RVP were obtained from commercially available cultured cell lines as indicated in Table 14. Seven viral mixes were made, each containing unique viral targets. Viral mixes were added to each potentially interfering substance resulting in a final testing concentration of 3X LoD for each analyte. Each was extracted in triplicate with each extract tested once with the eSensor RVP. Twenty-four (24) potentially interfering substances were tested in this study with 21 combinations. Additionally, nine potentially interfering microorganisms (viral and bacterial) were also tested in the same manner as described above. The microorganisms and their testing concentrations are listed in Table 14. All substances and microorganisms tested for interference were shown to be compatible with the eSensor RVP. No potentially interfering substance or microorganism was shown to inhibit the eSensor RVP at all tested concentrations .

| Potentially Interfering
Substance | Active
Ingredient | Substance
Form | Tested
Concentration |
|---------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------|-------------------|-------------------------|
| Sample Matrix | Control for no interfering substance | Liquid | N/A |
| Viral transport medium | Becton Dickinson VTM | Liquid | N/A |
| Blood (human) | Blood | Liquid | 2% v/v |
| | Human gDNA | 50 ng/rxn | 50 ng/rxn |
| Throat lozenges, oral anesthetic
and analgesic | Benzocaine | Dry | 30% w/v |
| | Menthol* | Nasal Spray | 1% v/v |
| Mucin: bovine submaxillary gland,
type I-S | Purified mucin protein | Dry | 1% w/v |
| | Phenylephrine (Neo-Synephrine) | Dry | 1.5% v/v |
| Nasal sprays
or drops | Oxymetazoline* (also contains Benzalkonium
Chloride, Menthol, Eucalyptol, Camphor, benzyl
alcohol and phosphate buffers) | Nasal Spray | 1% v/v |
| | Sodium chloride | Dry | 0.8% w/v |
| Antibacterial, systemic | Tobramycin | Dry | 5% w/v |
| Antibiotic, nasal ointment | Mupirocin | Dry | 2% w/v |
| | Beclomethasone | Dry | 1.5% w/v |
| | Dexamethasone | Dry | 1.5% w/v |
| Nasal corticosteroids | Flunisolide | Dry | 1.5% w/v |
| | Triamcinolone | Dry | 1.5% w/v |
| | Budesonide (Pulmicort) | Dry | 1.5% w/v |
| | Fluticasone (Flonase) | Dry | 3% w/v |
| Nasal gel | Luffa opperculata** | Nasal Gel | 1% v/v |
| | Sulfur** | Nasal Gel | 1% v/v |

Table 14: Potentially Interfering Substances

19

| Potentially Interfering
Substance | Active
Ingredient | Substance
Form | Tested
Concentration |
|--------------------------------------|------------------------------------------|-------------------|-------------------------|
| Homeopathic allergy relief medicine | Galphimia glauca** | Nasal Gel | 1% v/v |
| Homeopathic allergy relief medicine | Histaminum hydrochloricum** | Nasal Gel | 1% v/v |
| FluMist | Live intranasal influenza virus vaccine" | Liquid | 0.5%-1% v/v |
| Anti-viral drugs | Zanamivir (Relenza®) | Dry | 550 ng/ml |
| Anti-viral drugs | Oseltamivir (Tamiflu") | Dry | 142 ng/ml |
| Virus | Cytomegalovirus | Culture | 1 x 105 PFU/ml |
| Virus | Enterovirus 71 | Culture | 1 x 105 PFU/ml |
| Bacteria | Streptococcus pneumoniae | Culture | |
| Bacteria | Bordetella pertussis | Culture | |
| Bacteria | Haemophilus influenza | Culture | |
| Bacteria | Mycoplasma pneumoniae | Culture | 1 x 106 CFU/ml |
| Bacteria | Staphylococcus aureus | Culture | |
| Bacteria | Neisseria meningitidis | Culture | |
| Bacteria | Corynebacterium diptheriae | Culture | |

*Tested together (Afrin No Drip Severe Congestion nasal spray)

** Tested together (Zicam Allergy Relief)

FluMist vaccine: Addition of FluMist Live Intranasal Influenza Vaccine to the transport media control resulted in positive calls for Flu A, Flu A 2009 H1N1 and Flu B. This was due to the live attenuated influenza virus present in the vaccine.

4 Testing of FluMist at 1% (v/v) resulting in an inhibition in the detection of hMPV . FluMist did not inhibit the detection of hMPV when tested at 0.5% (v/v).

Cross-Reactivity

Cross-Reactivity Evaluation for Viruses Detected by the eSensor RVP

Cross-reactivity of each viral target (14 viral targets) was evaluated at high concentrations with the eSensor RVP by making three serial dilutions of viral reference strains with viral transport media (Remel M5) at 10,000x, 1000x and 100x the LoD. The titer of each virus dilution and corresponding LoD values were determined and provided in the table below. Cross-reactivity was not observed with any of the RVP viral targets at the concentrations tested. Table 15 summarizes the cross-reactivity results.

| Viral Target | Strain | LoD
Concentration
(TCID50/mL) | Highest Test
Concentration
(TCID50/mL) | Highest Multiple
of LoD Tested | Cross-Reactivity
Results |
|-----------------|---------------------|-------------------------------------|----------------------------------------------|-----------------------------------|-----------------------------|
| Flu A | H1N1 Brisbane/59/07 | 4.17 x 10-1 | 4.17 x 103 | 10,000x | Not Observed |
| Flu A H1 | H1N1 Brisbane/59/07 | 4.17 x 10-1 | 4.17 x 103 | 10,000x | Not Observed |
| Flu A | H3N2 | 1.58 x 103 | 1.58 x 107 | 10,000x | Not Observed |
| Flu A H3 | H3N2 | 1.58 x 103 | 1.58 x 107 | 10,000x | Not Observed |
| Flu A 2009 H1N1 | NY/2009 | 1.05 x 10-1 | 1.05 x 103 | 10,000x | Not Observed |
| Flu B | Florida/02/06 | 3.16 x 10-1 | 3.16 x 103 | 10,000x | Not Observed |
| hMPV | B2 | 4.17 x 100 | 4.17 x 104 | 10,000x | Not Observed |
| HRV | 3 | 1.58 x 10-3 | 1.58 x 101 | 10,000x | Not Observed |
| PIV1 | C35 | 2.81 x 10-2 | 2.81 x 102 | 10,000x | Not Observed |

Table 15: Within Panel Cross-Reactivity Final Results

20

| Viral Target | Strain | LoD
Concentration
(TCID50/mL) | Highest Test
Concentration
(TCID5o/mL) | Highest Multiple
of LoD Tested | Cross-Reactivity
Results |
|--------------|--------|-------------------------------------|----------------------------------------------|-----------------------------------|-----------------------------|
| PIV2 | Greer | 2.81 x 100 | 2.81 x 104 | 10,000x | Not Observed |
| PIV3 | C 243 | 2.81 x 101 | 2.81 x 105 | 10,000x | Not Observed |
| RSV A | A2 | 2.81 x 100 | 2.81 x 104 | 10,000x | Not Observed |
| RSV B | 9320 | 1.58 x 100 | 1.58 x 104 | 10,000x | Not Observed |
| ADV B/E | Type 7 | 8.89 x 10-2 | 1.58 x 105 | 10,000x | Not Observed |
| ADV B/E | Type 4 | 1.58 x 101 | | | |
| ADV C | Type 1 | 8.89 x 101 | 8.89 x 105 | 10,000x | Not Observed |

Cross-Reactivity with Other Respiratory Viruses Not Targeted by the eSensor RVP Cross-reactivity with 5 respiratory viruses known to circulate with Jow frequency in the general population was assessed. All viral strains were diluted in M5 transport media to a titer of 100 PFU/mL and extracted using the bioMérieux easyMAG extraction method in triplicate. Following extraction, each replicate was tested once in the RVP assay.

Table 16: Cross-Reactivity Results of Rare Respiratory Virus

Organism	Source	Test Concentrations	Cross-Reactivity Results
Parainfluenza Virus 4	Zeptometrix	2.92 x 105 PFU/mL	Not Observed
Coronavirus OC43*	Zeptometrix	5.96 x 104 PFU/mL	Not Observed
Coronavirus 229E	Zeptometrix	1.36 x 105 PFU/mL	Not Observed
Coronavirus NL63**	Zeptometrix	9.89 x 104 PFU/mL	Not Observed
Coronavirus HKU1	Clinical Isolate	N/A§	Not Observed

*OC43 had one replicate fail the IC control at high (10) concentration.

**NL63 was tested at the highest concentration available - 9.89 x 10° PFU/mL.

3 The Coronavirus HKU1 sample was a clinical isolate identified during the method comparison study. The method used was qualitative so no copy information was available.

Cross-Reactivity with 17 additional viruses that are not targets of the eSensor RVP were also assessed (Table 17.)_All viral strains were diluted in M5 transport media to a titer of 10° PFU/mL and extracted using the bioMérieux easyMAG extraction method in triplicate reactions.

Table 17: Cross-Reactivity Results of with other Viruses
- A B - A - A - A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

| Organism | Source | Test Concentrations | Cross-Reactivity
Results |
|--------------------|-----------------------|------------------------|--------------------------------------|
| Adenovirus 18 (A) | Zeptometrix VPL-030 | 2.37 x $10^{5}$ PFU/mL | Not Observed |
| Adenovirus 9 (D) | Zeptometrix VPL-030 | 4.63 x $10^{5}$ PFU/mL | ADV C False Positive* |
| Adenovirus 41 (F) | Zeptometrix VPL-030 | 8.05 x $10^{5}$ PFU/mL | ADV C False Positive* |
| Enterovirus 71 | Zeptometrix 0810047CF | 2.92 x $10^{5}$ PFU/mL | Not Observed |
| Coxsackievirus A10 | Zeptometrix 0810106CF | 1.72 x $10^{5}$ PFU/mL | Not Observed |
| Coxsackievirus A9 | Zeptometrix 0810017CF | 2.21 x $10^{5}$ PFU/mL | Not Observed |
| Echovirus E6 | Zeptometrix 0810076CF | 7.16 x $10^{5}$ PFU/mL | Not Observed |
| Coxsackievirus B2 | ATCC VR-29 | 6.22 x $10^{6}$ PFU/mL | Not Observed |
| Coxsackievirus B3 | Zeptometrix 0810074CF | 1.06 x $10^{5}$ PFU/mL | Not Observed |
| Coxsackievirus B4 | Zeptometrix 0810075CF | 8.04 x $10^{6}$ PFU/mL | 2/3 Not Observed
1 HRV Positive** |

21

| Organism | Source | Test Concentrations | Cross-Reactivity
Results |
|---------------------------------|------------------------|-------------------------|-----------------------------|
| Coxsackievirus B5 | Zeptometrix 081019CF | 7.16 x $10^7$ PFU/mL | Not Observed |
| Echovirus 9 | Zeptometrix 081007CF | 1.41 x $10^5$ PFU/mL | Not Observed |
| Echovirus 25 | Zeptometrix VPL-030 | 1.93 x $10^5$ PFU/mL | Not Observed |
| Echovirus 30 | Zeptometrix 0810078CF | 9.89 x $10^4$ PFU/mL | Not Observed |
| Coxsackievirus A21 | Zeptometrix 0810018CF | 2.92 x $10^5$ PFU/mL | Not Observed |
| Coxsackievirus A24 | ATCC VR-583 | 7.00 x $10^5$ PFU/mL | Not Observed |
| Enterovirus 68 | ATCC VR-561 | 1.40 x $10^5$ PFU/mL | Not Observed |
| Poliovirus | ATCC VR-193 | 1.11 x $10^5$ PFU/mL, | HRV False Positive |
| Bocavirus | Clinical Isolate | N/A | Not Observed |
| Herpesvirus 1: Herpes Simplex | Zeptometrix 0810005CF | 1.01 x $10^5$ PFU/mL | Not Observed |
| Herpesvirus 3: Varicella Zoster | Zeptometrix 081 0026CF | 2.35 x $10^6$ copies/mL | Not Observed |
| Herpesvirus 4: Epstein Barr | Zeptometrix 0810008CF | 1.06 x $10^5$ PFU/mL | Not Observed |
| Herpesvirus 5: Cytomegalovirus | Zeptometrix 0810003CF | 6.68 x $10^5$ PFU/mL | Not Observed |
| Measles | Zeptometrix | 1.37 x $10^5$ PFU/mL | Not Observed |
| Mumps | Zeptometrix 0810079CF | 1.93 x $10^5$ PFU/mL | Not Observed |

*ADV C cross-reactive signal was also obtained from Adenovirus 9 (D) and Adenovirus 41 (F) when it was

diluted 1000 fold from the initial testing concentration. Due to the genetic similarity between Adenovirus C, D,

and F, the eSensor RVP cannot reliably differentiate them. A positive eSensor RVP Adenovirus species C result should be followed-up using an alternative method (e.g., sequence analysis) if definitive Adenovirus speciation

is needed.

One replicate of Coxsackievirus B4 at high concentration (8.04 x 10° PFU/mL ) generated a HRV positive result which was silghtly above the assay threshold. None of the other thirteen (13) enterovinus analytes tested at similar high concentrations generated a positive call for HRV.

5 Due to the genetic similarity between human rhinovirus and poliovirus, the eSensor RVP cannot reliably

differentiate them. If a polio infection is suspected, a positive eSensor RVP human rhinovirus (HRV) result

should be confirmed using an alternate method (e.g., cell culture).

Quantification of the viral RNA contained in the Herpesvirus-3 (Varicella Zoster Virus) sample was performed using realtime RT-PCR and provided in copies/mL

Cross-Reactivity with Bacteria and Fungus

Bacterial and fungal strains were tested for cross-reactivity with the eSensor RVP and were diluted in M5 transport media to a titer of 108 CFU/mL. These organisms were extracted in triplicate with the bioMérieux easyMAG system.

Following extraction, each replicate was tested once using the eSensor RVP as shown in Table 18.

Organism	Source	Test Concentrations	Cross-Reactivity Results
Acinetobacter baumanii	Zeptometrix 0801597	$5.2 x 10^8$ CFU/mL	Not Observed
Bordetella parapertussis	Zeptometrix 0801461	$9.8 x 10^8$ CFU/mL	Not Observed
Bordetella pertussis	Zeptometrix 0801459	$5.8 x 10^8$ CFU/mL	Not Observed
Burkholderia cepacia	Zeptometrix BacT-050	$2.3 x 10^8$ CFU/mL	Not Observed
Candida albicans	Zeptometrix 0801504	$1.0 x 10^8$ CFU/mL	Not Observed
Candida glabrata	Zeptometrix 0801535	$9.73 x 10^8$ CFU/mL	Not Observed
Chlamydophila pneumoniae DNA	ABI 08-942-250	$1.4 x 10^7$ copies/mL	Not Observed
Corynebacterium diphtheriae	Zeptometrix BacT-050	$3.58 x 10^8$ CFU/mL	Not Observed

Table 18: Cross-Reactivity Results of with Bacteria and Fungus

22

| Organism | Source | Test Concentrations | Cross-Reactivity
Results |
|-----------------------------------|----------------------|---------------------|-----------------------------|
| Escherichia coli | Zeptometrix 0801624 | 1.5 x 106 CFU/mL | Not Observed |
| Haemophilus influenzae | Zeptometrix 0801680 | 2.6 x 106 CFU/mL | Not Observed |
| Klebsiella pneumoniae | Zeptometrix 0801506 | 1.07 x 106 CFU/mL | Not Observed |
| Lactobacillus acidophilus | Zeptometrix 0801540 | 2.12 x 106 CFU/mL | Not Observed |
| Lactobacillus planarum | Zeptometrix 0801507 | 1.75 x 106 CFU/mL | Not Observed |
| Legionella pneumophila | Zeptometrix 0801645 | 2.6 x 106 CFU/mL | Not Observed |
| Moraxella catarrhalis | Zeptometrix 0801509 | 3.9 x 106 CFU/mL | Not Observed |
| Mycobacterium tuberculosis | Zeptometrix 0801660 | 2.2 x 106 CFU/mL | Not Observed |
| Mycoplasma pneumoniae | Zeptometrix 0801579 | 2.47 x 106 CCU/mL | Not Observed |
| Neisseria meningiditis | Zeptometrix 0801511 | 3.37 x 106 CFU/mL | Not Observed |
| Neisseria sicca | Zeptometrix 0801754 | 3.37 x 106 CFU/mL | Not Observed |
| Porphyromonas gingivalis | Zeptometrix BacT-050 | 3.55x106 CFU/mL | Not Observed |
| Proteus vulgaris | Zeptometrix BacT-050 | 1.0 x 106 CFU/mL | Not Observed |
| Pseudomonas aeruginosa | Zeptometrix 0801519 | 1.05 x 106 CFU/mL | Not Observed |
| Serratia marcescens | Zeptometrix 0801723 | 6.1 x 106 CFU/mL | Not Observed |
| Staphylococcus aureus (COL) | Zeptometrix 0801638 | 8.4 x 106 CFU/mL | Not Observed |
| Staphylococcus aureus (MSSA) | Zeptometrix 0801675 | 1.2 x 106 CFU/mL | Not Observed |
| Staphylococcus epidermidis (MSSE) | Zeptometrix 0801689 | 2.2 x 106 CFU/mL | Not Observed |
| Staphylococcus epidermidis (MRSE) | Zeptometrix 0801651 | 6.2 x 106 CFU/mL | Not Observed |
| Staphylococcus haemolyticus | Zeptometrix 0801591 | 2.16 x 106 CFU/mL | Not Observed |
| Streptococcus agalactiae | Zeptometrix 0801545 | 2.2 x 106 CFU/mL | Not Observed |
| Streptococcus dysgalactiae | Zeptometrix 0801516 | 6.46 x 106 CFU/mL | Not Observed |
| Streptococcus mitis | Zeptometrix 0801695 | 2.43 x 106 CFU/mL | Not Observed |
| Streptococcus pneumoniae | Zeptometrix 0801439 | 2.8 x 106 CFU/mL | Not Observed |
| Streptococcus pyrogenes | Zeptometrix 0801512 | 1.55 x 106 CFU/mL | Not Observed |
| Streptococcus salivarius | Zeptometrix BacT-050 | 6.53 x 106 CFU/mL | Not Observed |

Carryover/Cross-Contamination

The carryover/cross-contamination study challenged the extraction, RT-PCR, and detection portions of the assay within and between runs and operators tested over the course of five testing days. A representative strain of Parainfluenza Virus 3 was obtained as a commercially available cultured cell line. Positive Parainfluenza Virus 3 samples were prepared at a concentration of 1.00 x 10 TCIDgomL (3559x LoD) while negative samples were un-inoculated Remel M5 transport media. All samples were extracted using the bioMérieux easyMAG System. Five sets of alternating high concentration positive and negative samples were extracted and tested in a checkerboard pattern. Each set of samples contained 24 tests (12 positive and 12 negative). Total number of tests for the duration of the study was 120 samples (60 positive and 60 negative).

No carryover/cross-contamination was observed in the eSensor RVP, as 100% of the PIV 3 negative samples were reported as 'Target Not Detected'.

23

CLINICAL PERFORMANCE DATA

Clinical Performance

Expected Values

A prospective clinical study testing nasopharyngeal (NP) swab specimens was conducted during the 2010/11 influenza season at three North American clinical laboratories. The expected values of individual analytes based on eSensor RVP results in prospective samples are summarized in Tables 19 and 20. The expected values of mixed co-infections based on eSensor RVP results in prospective samples are summarized in Tables 21 and 22.

| Virus (Analyte) | Age 0-1
(N = 270) | Age >1-5
(N = 136) | Age >5-21
(N = 127) | Age >21-65
(N = 333) | Age >65
(N = 171) | All Ages
(N = 1037) |
|-------------------------------|----------------------|-----------------------|------------------------|-------------------------|----------------------|------------------------|
| | N (%) | N (%) | N (%) | N (%) | N (%) | N (%) |
| Influenza A (Un-Subtypable) | 2 (0.7) | 0 (0.0) | 2 (1.6) | 5 (1.5) | 1 (0.6) | 10 (1.0) |
| Influenza A (Total) | 25 (9.3) | 22 (16.2) | 17 (13.4) | 84 (25.2) | 31 (18.1) | 179 (17.3) |
| Influenza A H3 | 12 (4.8) | 15 (11.0) | 7 (5.5) | 43 (12.9) | 22 (12.9) | 99 (9.5) |
| Influenza A 2009 H1N1 | 10 (3.7) | 8 (5.9) | 6 (4.7) | 33 (9.9) | 7 (4.1) | 64 (6.2) |
| Influenza B | 10 (3.7) | 17 (12.5) | 33 (26.0) | 15 (4.5) | 7 (4.1) | 82 (7.9) |
| Human Metapneumovirus | 18 (6.7) | 11 (8.1) | 3 (2.4) | 15 (4.5) | 10 (5.9) | 57 (5.5) |
| Human Rhinovirus | 82 (30.4) | 27 (19.9) | 21 (16.6) | 26 (7.8) | 11 (6.4) | 167 (16.1) |
| Parainfluenza Virus 1 | 3 (1.1) | 0 (0.0) | 1 (0.8) | 0 (0.0) | 1 (0.6) | 5 (0.5) |
| Parainfluenza Virus 2 | 1 (0.4) | 1 (0.7) | 0 (0.0) | 3 (0.9) | 2 (1.2) | 7 (0.7) |
| Parainfluenza Virus 3 | 43 (15.9) | 15 (11.0) | 5 (3.9) | 18 (5.4) | 5 (2.9) | 86 (8.3) |
| Respiratory Syncytial Virus A | 69 (25.6) | 21 (15.4) | 8 (6.3) | 11(3.3) | 10 (5.8) | 119 (11.4) |
| Respiratory Syncytial Virus B | 28 (10.4) | 17 (12.5) | 4 (3.2) | 14 (4.2) | 6 (3.5) | 69 (6.7) |
| Adenovirus B/E | 6 (2.2) | 8 (5.9) | 3 (1.6) | 5 (1.8) | 0 (0.0) | 22 (2.1) |
| Adenovirus C | 21 (7.7) | 4 (2.9) | 1 (0.8) | 9 (2.7) | 6 (3.5) | 41 (3.9) |

Table 19: Expected Value (As Determined by eSensor RVP) Summary by Age Group in the Prospective Clinical Evaluation

Table 20: Expected Value (As Determined by eSensor RVP) Summary by Site in the Prospective Clinical Evaluation

| Virus (Analyte) | Site 1
(N = 245) | Site 2
(N = 533) | Site 3
(N = 259) | All Sites
(N = 1037) |
|-----------------------------|----------------------|----------------------|---------------------|-------------------------|
| | N (%) | N (%) | N (%) | N (%) |
| Influenza A (Un-Subtypable) | 0 (0.0) | 8 (1.5) | 2 (0.8) | 10 (1.0) |
| Influenza A (Total) | 58 (23.7) | 89 (16.7) | 32 (12.4) | 179 (17.3) |
| Influenza A H3 | 32 (13.1) | 54 (10.1) | 13 (5.0) | 99 (9.5) |

24

| Virus (Analyte) | Site 1
(N = 245) | Site 2
(N = 533) | Site 3
(N = 259) | All Sites
(N = 1037) |
|-------------------------------|---------------------|---------------------|---------------------|-------------------------|
| | N (%) | N (%) | N (%) | N (%) |
| Influenza A 2009 H1N1 | 19 (7.8) | 28 (5.3) | 17 (6.6) | 64 (6.2) |
| Influenza B | 4 (1.6) | 59 (11.1) | 19 (7.3) | 82 (7.9) |
| Human Metapneumovirus | 23 (9.4) | 25 (4.7) | 9 (3.5) | 57 (5.5) |
| Human Rhinovirus | 44 (18.0) | 99 (18.6) | 24 (9.3) | 167 (16.1) |
| Parainfluenza Virus 1 | 0 (0.0) | 4 (0.8) | 1 (0.4) | 5 (0.5) |
| Parainfluenza Virus 2 | 1 (0.4) | 6 (1.1) | 0 (0.0) | 7 (0.7) |
| Parainfluenza Virus 3 | 3 (1.2) | 68 (12.8) | 15 (5.8) | 86 (8.3) |
| Respiratory Syncytial Virus A | 17 (6.9) | 85 (15.9) | 17 (6.6) | 119 (11.4) |
| Respiratory Syncytial Virus B | 15 (6.1) | 41 (7.7) | 13 (5.0) | 69 (6.7) |
| Adenovirus B/E | 0 (0.0) | 14 (2.6) | 8 (3.1) | 22 (2.1) |
| Adenovirus C | 16 (6.5) | 19 (3.6) | 6 (2.3) | 41 (3.9) |

Table 21: Expected Value (Co-infections as Determined by eSensor RVP) Summary by Age
Group in the Prospective Clinical Evaluation

and the comments of the comments of the comments of

| Co-Infection | Age 0-1
(N = 270) | Age >1-5
(N = 136) | Age >5-21
(N = 127) | Age >21-65
(N = 333) | Age >65
(N = 171) | All Ages
(N = 1037) |
|--------------------------------------|----------------------|------------------------|------------------------|-------------------------|----------------------|------------------------|
| | N | N | N | N | N | N (%) |
| ADV B/E + Flu B | 0 | 0 | 0 | 2 | 0 | 2 (0.2) |
| ADV B/E + HRV | 0 | 2 | 0 | 0 | 0 | 2 (0.2) |
| ADV B/E + PIV3 | 3 | 0 | 0 | 0 | 0 | 3 (0.3) |
| ADV B/E + RSV A | 1 | 1 | 0 | 0 | 0 | 2 (0.2) |
| ADV B/E + RSV B | 0 | 1 | 0 | 0 | 0 | 1 (0.1) |
| ADV B/E + HMPV + HRV + RSV A + RSV B | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| ADV C + Flu B | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| ADV C + HMPV | 3 | 0 | 0 | 0 | 0 | 3 (0.3) |
| ADV C + HRV | 3 | 1 | 0 | 1 | 1 | 6 (0.6 |
| ADV C + PIV3 | 0 | 0 | 0 | 1 | 0 | 1 (0.1) |
| ADV C + RSV A | 2 | 2 | 0 | 0 | 0 | 4 (0.4) |
| ADV C + RSV B | 1 | 0 | 0 | 1 | 1 | 3 (0.3) |
| ADV C + HRV + PIV3 | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| ADV C + HRV + RSV A | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| Flu A + ADV B/E | 0 | 0 | 1 | 0 | 0 | 1 (0.1) |
| Flu A + ADV C | 1 | 1 | 0 | 2 | 2 | 6 (0.6) |
| Flu A + Flu B | 0 | 0 | 1 | 1 | 0 | 2 (0.2) |
| Flu A + HMPV | 0 | 0 | 0 | 1 | 1 | 2 (0.2) |
| Flu A + HRV | 3 | 0 | 0 | 0 | 1 | 4 (0.4) |
| Flu A + PIV2 | 0 | 0 | 0 | 0 | 1 | 1 (0.1) |
| Flu A + PIV3 | 2 | 0 | 0 | 0 | 0 | 2 (0.2) |

.

and the comments of the comments of the comments of

25

| Co-Infection | Age 0-1
(N = 270) | Age >1-5
(N = 136) | Age >5-21
(N = 127) | Age >21-65
(N = 333) | Age >65
(N = 171) | All Ages
(N = 1037) |
|-----------------------------|----------------------|-----------------------|------------------------|-------------------------|----------------------|------------------------|
| | N | N | N | N | N | N (%) |
| Flu A + RSV A | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| Flu A + RSV B | 0 | 1 | 0 | 1 | 0 | 2 (0.2) |
| Flu A + HRV + PIV3 | 2 | 0 | 0 | 0 | 0 | 2 (0.2) |
| Flu A + RSV A + RSV B | 2 | 0 | 0 | 0 | 0 | 2 (0.2) |
| Flu A + ADV C + HRV + RSV A | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| Flu A + ADV C + HRV + PIV3 | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| Flu B + HRV | 1 | 0 | 1 | 1 | 1 | 4 (0.4) |
| Flu B + PIV3 | 0 | 2 | 0 | 0 | 1 | 3 (0.3) |
| Flu B + RSV A | 2 | 0 | 2 | 0 | 1 | 5 (0.5) |
| Flu B + RSV B | 0 | 1 | 0 | 0 | 0 | 1 (0.1) |
| Flu B + HRV + PIV2 | 0 | 1 | 0 | 0 | 0 | 1 (0.1) |
| Flu B + HRV + RSV A | 2 | 0 | 0 | 0 | 0 | 2 (0.2) |
| HMPV + HRV | 4 | 1 | 0 | 0 | 0 | 5 (0.5) |
| HMPV + PIV3 | 0 | 0 | 0 | 1 | 0 | 1 (0.1) |
| HMPV + RSV B | 0 | 0 | 0 | 1 | 0 | 1 (0.1) |
| HRV + PIV1 | 2 | 0 | 0 | 0 | 0 | 2 (0.2) |
| HRV + PIV2 | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| HRV + PIV3 | 9 | 0 | 1 | 1 | 0 | 11 (1.1) |
| HRV + RSV A | 11 | 3 | 1 | 1 | 0 | 16 (1.6) |
| HRV + RSV B | 6 | 2 | 0 | 0 | 0 | 8 (0.8) |
| HRV + PIV3 + RSV A | 1 | 0 | 0 | 0 | 0 | 1 (0.1) |
| HRV + PIV3 + RSV B | 0 | 1 | 0 | 0 | 0 | 1 (0.1) |
| PIV3 + RSV A | 1 | 3 | 0 | 2 | 0 | 6 (0.6) |
| PIV3 + RSV B | 0 | 0 | 0 | 1 | 0 | 1 (0.1) |

Table 22: Expected Value (Co-infections as Determined by eSensor RVP) Summary by Site in the Prospective Clinical Evaluation

| Co-Infection | Site 1
(N = 245)
N | Site 2
(N = 533)
N | Site 3
(N = 259)
N | All Sites
(N = 1037)
N (%) | | | | | |
|-----------------------------------------|--------------------------|--------------------------|--------------------------|----------------------------------|--------------------------|---|--------------------------|---------|----------------------------------|
| ADV B/E + Flu B | 0 | 0 | 2 | 2 (0.2) | | | | | |
| ADV B/E + HRV | 0 | 2 | 0 | 2 (0.2) | | | | | |
| ADV B/E + PIV3 | 0 | 2 | 1 | 3 (0.3) | | | | | |
| ADV B/E + RSV A | 0 | 1 | 1 | 2 (0.2) | | | | | |
| ADV B/E + RSV B | 0 | 1 | 0 | 1 (0.1) | | | | | |
| ADV B/E + HMPV + HRV + RSV A +
RSV B | 0 | 0 | 1 | 1 (0.1) | | | | | |
| ADV C + Flu B | 0 | 1 | 0 | 1 (0.1) | | | | | |
| ADV C + HMPV | Co-Infection | 1 | Site 1
(N = 245)
N | 2 | Site 2
(N = 533)
N | 0 | Site 3
(N = 259)
N | 3 (0.3) | All Sites
(N = 1037)
N (%) |
| ADV C + HRV | 2 | 3 | 1 | 6 (0.6) | | | | | |
| ADV C + PIV3 | 0 | 1 | 0 | 1 (0.1) | | | | | |
| ADV C + RSV A | 1 | 3 | 0 | 4 (0.4) | | | | | |
| ADV C + RSV B | 3 | 0 | 0 | 3 (0.3) | | | | | |
| ADV C + HRV + PIV3 | 0 | 1 | 0 | 1 (0.1) | | | | | |
| ADV C + HRV + RSV A | 0 | 1 | 0 | 1 (0.1) | | | | | |
| Flu A + ADV B/E | 0 | 1 | 0 | 1 (0.1) | | | | | |
| Flu A + ADV C | 3 | 2 | 1 | 6 (0.6) | | | | | |
| Flu A + Flu B | 0 | 1 | 1 | 2 (0.2) | | | | | |
| Flu A + HMPV | 1 | 0 | 1 | 2 (0.2) | | | | | |
| Flu A + HRV | 2 | 2 | 0 | 4 (0.4) | | | | | |
| Flu A + PIV2 | 1 | 0 | 0 | 1 (0.1) | | | | | |
| Flu A + PIV3 | 0 | 2 | 0 | 2 (0.2) | | | | | |
| Flu A + RSV A | 0 | 1 | 0 | 1 (0.1) | | | | | |
| Flu A + RSV B | 0 | 1 | 1 | 2 (0.2) | | | | | |
| Flu A + HRV + PIV3 | 0 | 2 | 0 | 2 (0.2) | | | | | |
| Flu A + RSV A + RSV B | 0 | 2 | 0 | 2 (0.2) | | | | | |
| Flu A + ADV C + HRV + RSV A | 0 | 1 | 0 | 1 (0.1) | | | | | |
| Flu A + ADV C + HRV + PIV3 | 0 | 1 | 0 | 1 (0.1) | | | | | |
| Flu B + HRV | 1 | 3 | 0 | 4 (0.4) | | | | | |
| Flu B + PIV3 | 0 | 2 | 1 | 3 (0.3) | | | | | |
| Flu B + RSV A | 0 | 2 | 3 | 5 (0.5) | | | | | |
| Flu B + RSV B | 0 | 1 | 0 | 1 (0.1) | | | | | |
| Flu B + HRV + PIV2 | 0 | 1 | 0 | 1 (0.1) | | | | | |
| Flu B + HRV + RSV A | 0 | 2 | 0 | 2 (0.2) | | | | | |
| HMPV + HRV | 2 | 2 | 1 | 5 (0.5) | | | | | |
| HMPV + PIV3 | 0 | 0 | 1 | 1 (0.1) | | | | | |
| HMPV + RSV B | 1 | 0 | 0 | 1 (0.1) | | | | | |
| HRV + PIV1 | 0 | 2 | 0 | 2 (0.2) | | | | | |
| HRV + PIV2 | 0 | 1 | 0 | 1 (0.1) | | | | | |
| HRV + PIV3 | 0 | 11 | 0 | 11 (1.1) | | | | | |
| HRV + RSV A | 3 | 12 | 1 | 16 (1.6) | | | | | |
| HRV + RSV B | 1 | 6 | 1 | 8 (0.8) | | | | | |
| HRV + PIV3 + RSV A | 0 | 1 | 0 | 1 (0.1) | | | | | |
| HRV + PIV3 + RSV B | 0 | 1 | 0 | 1 (0.1) | | | | | |
| PIV3 + RSV A | 2 | 4 | 0 | 6 (0.6) | | | | | |
| PIV3 + RSV B | 0 | 0 | 1 | 1 (0.1) | | | | | |

.

r ·

26

.

:

27

Prospective Clinical Study

All clinical specimens in the prospective clinical study were nasopharyngeal (NP) swab specimens, prospectively collected and tested during the 2010/11 influenza season at three North American clinical laboratories. Clinical laboratories were located in Cleveland. Ohio: Providence, RI; and Albuquerque, NM. Demographic details for patient population are summarized in Table 23. Study sites enrolled subjects from diverse demographic groups; about 40% of the specimens were obtained from patients enrolled at a hospital. The remaining specimens were collected from outpatients and patients in an emergency department. A total of 1182 patient samples were collected prospectively across the three clinical sites from January 2011. Out of these patient samples, 1037 were evaluable. A total of 145 samples were excluded for the following reasons: samples not tested within 5 days of specimen collection (72/145), operator and/or easyMAG mechanical errors (62/145), samples not retested (11/145). Out of the 1037 samples collected, an even split of patients were male and female. Approximately one quarter of the samples came from children under the age of 1, and patients aged 21-65 contributed the largest share of the samples.

| Demographic | Site 1
N = 245 (%) | Site 2
N = 533 (%) | Site 3
N = 259 (%) | All Sites
N = 1037 (%) |
|----------------------|-----------------------|-----------------------|-----------------------|---------------------------|
| SEX | | | | |
| Male | 105 (42.9) | 296 (55.5) | 117 (45.2) | 518 (50.0) |
| Female | 140 (57.1) | 237 (44.5) | 142 (54.8) | 519 (50.0) |
| AGE (yrs) | | | | |
| 0 - 1 | 46 (18.8) | 197 (37.0) | 27 (10.4) | 270 (26.0) |
| > 1 - 5 | 20 (8.2) | 94 (17.6) | 22 (8.4) | 136 (13.1) |
| > 5 - 21 | 19 (7.8) | 82 (15.4) | 26 (10.0) | 127 (12.2) |
| > 21 - 65 | 97 (39.6) | 106 (19.9) | 130 (50.2) | 333 (32.1) |
| > 65 | 63 (25.7) | 54 (10.1) | 54 (20.8) | 171 (16.5) |
| SUBJECT STATUS | | | | |
| Outpatient | 7 (2.9) | 219 (41.1) | 90 (34.7) | 316 (30.5) |
| Hospitalized | 131 (53.5) | 162 (30.4) | 114 (44.0) | 407 (39.2) |
| Emergency Department | 107 (43.7) | 152 (28.5) | 55 (21.2) | 314 (30.3) |

Table 23: General Demographic Data for Prospectively Collected Specimens (N=1037)

A total of 1037 specimens were evaluated for all 14 RVP panel viruses with the prospectively collected samples, the performance for each respiratory virus was described by the clinical sensitivity and specificity. Sensitivity for a respiratory virus is the ability of the test to obtain positive results for this respiratory virus in the samples with positive results obtained by the comparator method for the particular virus. Specificity for a respiratory virus is the ability of the test to obtain negative results for this respiratory virus in the samples with negative results obtained by the comparator method for this respiratory virus. Depending on the comparator method used for a particular virus, performance is described as sensitivity/specificity or Positive Percent Agreement (PPA)/Negative Percent Agreement (NPA).

28

The performance of the RVP assay was compared to the established gold standard reference method of viral culture for most viral targets. For respiratory viruses in which culture was not available, a composite (multi-lest) reference method (a predetermined algorithm that combined the results of a few tests) was used as the comparator method. As seen in Table 24, viral culture followed by DFA identification testing was used as the comparator method for Influenza A, Influenza B, RSV, Parainfluenza Viruses (PIV1, PIV2, PIV3), and adenovirus. Since viral culture cannot determine the subtype for influenza A. RSVs, and adenoviruses, these viruses were subtyped by an independently developed qRT-PCR assay or qPCR assay followed by bidirectional sequencing to determine the subtypes (Influenza A H3, Influenza A 2009 H1N1, RSVA, RSVB, ADVB/E and ADVC). HRV and HMPV were evaluated using two independently developed and validated gRT-PCR assays followed by bidirectional sequencing.

Virus (Analyte)	Comparator Method	Subtyping
Influenza A
Influenza A H1
Influenza A H3
Influenza A 2009 H1N1
RSV A	Viral culture followed by DFA identification1	qRT-PCR + Bidirectional
Sequencing
RSV B
Adenovirus B/E
Adenovirus C
Influenza B
PIV 1	Viral culture followed by DFA identification2	N/A
PIV 2
PIV 3
Human Metapneumovirus	2 qRT-PCR (2 methods) with Bidirectional
Sequencing3	N/A
Human Rhinovirus

Validated Performance of the eSensor RVP assay detecting Influenza A, RSV or ADV respectively was

Table 24: Comparator Methods used to assess RVP performance

compared to viral culture followed by fluorescent antibody identification. "True" Influenza A, RSV or ADV positives respectively, were considered as any sample that tested positive for Influenza A, RSV or ADV respectively, by viral culture followed by DFA testing. True positive samples were subtyped using one analytically validated gRT-PCR assay with bi-directional sequence confirmation. The comparator assays were designed to amplify a different sequence from that amplified by the eSensor RVP assay(s). None of the comparator PCR assays overlapped any RVP amplicon sequence even if the same gene was targeted. "True" Influenza A H1, H3, or 2009 H1N1 positives, respectively, were considered as any sample that tested positive for Influenza A by viral culture, and had bi-directional sequencing pre-defined quality acceptance criteria that matched Influenza A/H1, A/H3, or A/2009 H1 sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), respectively, with acceptable Evalues. "True" RSV A or RSV B positively, were considered as any sample that tested positive for Influenza A by viral culture, and had bi-directional sequencing data meeting pre-defined quality acceptance criteria that matched RSV A or RSV B sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), respectively, with acceptable E-values. "True" ADV C or ADV B/E positives, respectively, were considered as any sample that tested positive for Influenza A by viral culture, and had bi-directional sequencing data meeting pre-defined quality acceptance criteria that matched ADV C or ADV B/E sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih.gov), respectively, with acceptable E-values. Performance of the eSensor RVP assay detecting Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2 and Parainfluenza Virus 3 respectively was compared to viral culture followed by fluorescent antibody identification. "True" Influenza B, Parainfluenza Virus 1, Parainfluenza Virus 2 or Parainfluenza Virus 3 positives, respectively, were considered as any sample that tested positive for Influenza Virus

Parainfluenza Virus 2, or Parainfluenza Virus 3. respectively, by viral culture followed by DFA testing. 3 Performance of the eSensor RVP assay detecting Human Rhinovirus or Human Metapneumovirus, respectively, was compared to a predetermined algorithm that used composite comparator methods. The methods consist of two analytically validated PCR assays followed by bi-directional sequencing. "True" Human

29

Rhinovirus or Human Metapneumovirus positively, were considered as any sample that had bidirectional sequencing data meeting pre-defined quality acceptance criteria that matched Human Rhinovirus or Human Metapneumovirus sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank database (www.ncbi.nlm.nih. gov), respectively, with acceptable E-values.

Sensitivity or positive percent agreement (PPA) was calculated by dividing the number of true positive (TP) results by the sum of true positive and false negative (FN) results, while specificity or negative percent agreement (NPV) was calculated by dividing the number of true negative (TN) results by the sum of true negative and false positive (FP) results. A TP result was one where the positive RVP result matched the positive reference/comparator result, while a TN result was one whereby a negative RVP result matched a negative reference/comparator result. The two-sided 95% confidence interval was also calculated. The results are summarized below.

Virus (Analyte)	TP/(TP+FN)	Sensitivity		Specificity
		Percent	95% CI	TN/(TN+FP)	Percent	95% CI
Influenza A¹	132/137ª	96.4%	91.7% - 98.8%	850/897ᵇ	94.8%	93.1% - 96.1%
Influenza A H1*	0/0	NA	NA	1027/1027	100.0%	99.6% - 100.0%
Influenza A H3	74/74	100.0%	95.1% - 100.0%	927/952ᶜ	97.4%	96.2% - 98.3%
Influenza A 2009 H1N1	49/49	100.0%	92.7% - 100.0%	956/971ᵈ	98.5%	97.5% - 99.1%
Influenza B	64/69ᵉ	92.8%	83.9% - 97.6%	947/965ᶠ	98.1%	97.1% - 98.9%
Parainfluenza Virus 1*	4/4	100.0%	39.8% - 100.0%	1029/1030ʲ	99.9%	99.5% - 100.0%
Parainfluenza Virus 2*	5/6ⁱ	83.3%	35.9% - 99.6%	1026/1028ᵏ	99.8%	99.3% - 100.0%
Parainfluenza Virus 3	64/68ˡ	94.1%	85.6% - 98.4%	944/966ᵐ	97.7%	96.6% - 98.6%
Respiratory Syncytial Virus A	68/68	100.0%	94.7% - 100.0%	905/956ⁿ	94.7%	93.1% - 96.0%
Respiratory Syncytial Virus B	28/28	100.0%	87.7% - 100.0%	955/996ᵒ	95.9%	94.5% - 97.0%
Adenovirus B/E*	13/13	100.0%	75.3% - 100.0%	1012/1021ᵖ	99.1%	98.3% - 99.5%
Adenovirus C*	6/6	100.0%	54.1% - 100.0%	993/1028q	96.6%	95.3% - 97.5%
Virus (Analyte)	TP/(TP+FN)	PPA		NPA
		Percent	95% CI	TN/(TN+FP)	Percent	95% CI
Human Metapneumovirus	55/55	100.0%	93.5% - 100.0%	979/981ʳ	99.8%	99.3% - 100.0%
Human Rhinovirus	132/148	89.2%	83.0% - 93.7%	853/888ˢ	96.1%	94.6% - 97.3%

Table 25: Performance in Prospective Clinical Specimens (N=1037)

*These viral targets were supplemented with retrospective samples as shown below.

1 Influenza A results contain 14 Flu A samples without a positive subtype and 123 samples with either Influenza A H3 or 2009 H1N1 positive results.

4 Flu A was not detected in all 5 RVP False Negative samples using independently developed and validated aPCR assays.

b Flu A viruses were confirmed positive in 35/47 RVP False Positive samples using bidirectional sequencing.

° Flu A H3 viruses were confirmed positive in 22/25 RVP False Positive samples using bidirectional sequencing.

9 Flu A 2009 H1N1 viruses were confirmed positive in 14/15 RVP False Positive samples using bidirectional sequencing.

8 Flu B was not detected in 4/5 RVP False Negative samples using bidirectional sequencing.

' Flu B was confirmed positive in 11/18 RVP False Positive samples using bidirectional sequencing.

0 hMPV was confirmed positive in 1/2 RVP False Positive samples using bidirectional sequencing.

" HRV was confirmed positive in 7/35 RVP False Positive samples using bidirectional sequencing.

| PIV 1 was not detected in this RVP False Positive sample by bidirectional sequencing.

PIV 2 was not detected in this RVP False Negative sample using independently developed and validated gPCR assays.

30

PIV 2 virus was confirmed positive in 0/2 RVP False Positive samples by bidirectional sequencing. ' PIV 3 was not detected in 4/4 RVP False Negative samples using independently developed and validated

qPCR assays.
™ PIV 3 virus was confirmed positive in 10/22 RVP False Positive samples using bidirectional sequencing.

" RSV A were confirmed positive in 43/51 RVP False Positive samples using bidirectional sequencing.

° RSV B was confirmed positive in 35/41 RVP False Positive samples using bidirectional sequencing.

º ADV B/E was confirmed positive in 8/9 RVP False Positive samples using bidirectional sequencing.

9 ADV C was confirmed positive in 16/35 False Positive samples using bidirectional sequencing.

The eSensor RVP system detected a total of 128 mixed infections in the prospective clinical evaluation (1037 tested and analyzed specimens). This represents 18.4% of the total positive specimens (128/696). One hundred fourteen (114/128; 89.1%) were double infections, eleven (11/128, 8.6%) were triple infections, and three (3/128; 2.3%) samples with four or more RVP analytes were identified. Ninety five of the 128 samples contained one or more analytes that the reference/comparator method failed to detect.

| Analyte
1 | Analyte
2 | Analyte
3 | Analyte
4 | Analyte
5 | Total Number
of
Co-infections | Number of
Discrepant
Co-infections | Discrepant
Analyte(s) |
|---------------------------------------------------------------|--------------------------------------|--------------|--------------|--------------|-------------------------------------|------------------------------------------|--------------------------------|
| ADV B/E | Flu B | | | | 2 | 2 | ADV B (2), Flu B (1) |
| ADV B/E | HRV | | | | 2 | 0 | |
| ADV B/E | PIV3 | | | | 3 | 3 | ADV B (3) |
| ADV B/E | RSV A | | | | 2 | 2 | ADV B (1), RSV A (2) |
| ADV B/E | RSV B | | | | 1 | 1 | RSV B (1) |
| ADV B/E | HMPV | HRV | RSV A | RSV B | 1 | 1 | RSV A (1), RSV B (1) |
| ADV C | Flu B | | | | 1 | 1 | ADV C (1) |
| ADV C | HMPV | | | | 3 | 3 | ADV C (3) |
| ADV C | HRV | | | | 6 | 4 | ADV C (4), HRV (1) |
| ADV C | PIV3 | | | | 1 | 1 | ADV C (1) |
| ADV C | RSV A | | | | 4 | 4 | ADV C (3), RSV A (2)' |
| ADV C | RSV B | | | | 3 | 3 | ADV C (3), RSV B (2) |
| ADV C | HRV | PIV3 | | | 1 | 1 | ADV C (1) |
| ADV C | HRV | RSV A | | | 1 | 0 | |
| Flu A | ADV B/E | | | | 1 | 1 | Flu A |
| Flu A | ADV C | | | | 6 | 6 | ADV C (6) |
| Flu A | Flu B | | | | 2 | 2 | Flu A (2), HRV (1) |
| Flu A | HMPV | | | | 2 | 2 | H1N1 (1), H3 (1), HMPV (1) . |
| Flu A | HRV | | | | 4 | 2 | H1N1 (1), HRV (2) |
| Flu A | PIV2 | | | | 1 | 1 | PIV2 (1) |
| Flu A | PIV3 | | | | 2 | 2 | Flu A (1), PIV3 (2) |
| Flu A | RSV A | | | | 1 | 1 | RSV A (1) |
| Distinct Co-infection Combinations Detected
by eSensor RVP | | | | | Total Number
of
Co-infections | Number of
Discrepant
Co-infections | Discrepant
Analyte(s) |
| Analyte
1 | Analyte
2 | Analyte
3 | Analyte
4 | Analyte
5 | | | |
| Flu A | RSV B | | | | 2 | 2 | RSV B (2) |
| Flu A | HRV | PIV3 | | | 2 | 1 | H1N1 (1) |
| Flu A | RSV A | RSV B | | | 2 | 2 | RSV A (2), RSV B (2) |
| Flu A | ADV C | HRV | RSV A | | 1 | 1 | ADV C (1), HRV (1) |
| Flu A | ADV C | HRV | PIV3 | | 1 | 1 | ADV C (1), Flu A (1), PIV3 (1) |
| Flu B | HRV | | | | 4 | 2 | Flu B (1), HRV (1) |
| Flu B | PIV3 | | | | 3 | 3 | Flu B (2), PIV3 (2) |
| Flu B | RSV A | | | | 5 | 5 | Flu B (2), RSV A (5) |
| Flu B | RSV B | | | | 1 | 1 | RSV B (1) |
| Flu B | HRV | PIV2 | | | 1 | 1 | HRV (1), PIV2 (1) |
| Flu B | HRV | RSV A | | | 2 | 1 | RSV A (1) |
| HMPV | HRV | | | | 5 | 1 | HMPV (1) |
| HMPV | PIV3 | | | | 1 | 0 | |
| HMPV | RSV B | | | | 1 | 1 | RSV B (1) |
| HRV | PIV1 | | | | 2 | 1 | PIV1 (1) |
| HRV | PIV2 | | | | 1 | 1 | HRV (1) |
| HRV | PIV3 | | | | 11 | 4 | HRV (4), PIV3 (2) |
| HRV | RSV A | | | | 16 | 9 | HRV (5), RSV A (6) |
| HRV | RSV B | | | | 8 | 6 | HRV (1), RSV B (5) |
| HRV | PIV3 | RSV A | | | 1 | 1 | RSV A (1) |
| HRV | PIV3 | RSV B | | | 1 | 1 | RSV B (1) |
| PIV3 | RSV A | | | | 6 | 6 | PIV3 (4), RSV A (5) |
| PIV3 | RSV B | | | | 1 | 1 | PIV3 (1), RSV B (1) |
| | Total Number of Co-infections | | | | 128 | 95 | 117/278 |
| | Total Number of Double Infections | | | | 114 | 85 | 99/232 |
| | Total Number of Triple Infections | | | | 11 | 8 | 11/33 |
| | Total Number of Quadruple Infections | | | | 2 | 2 | 5/8 |
| | Total Number of Quintuple Infections | | | | 1 | 1 | 2/5 |

Table 26: Distinct Co-infection Combinations Detected by the eSensor RVP Assay in the Prospective Clinical Trial

31

*A discrepant co-infection or discrepant analyte was defined as one that was detected by RVP but not the reference/comparator methods.

'117/117 discrepant analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 58/117 cases.

16/6 discrepant ADV B/E analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 5/6 cases

°24/24 discrepant ADV C analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 11/24 cases

for discrepant Flu B analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 3/6 cases

4/4 discrepant Flu A 2009 H1N1 analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 4/4 cases

32

° // discrepant Flu A H3 analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 1/1 cases

'2/2 discrepant HMPV analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 1/2 cases

919/19 discrepant HRV analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 3/19 cases

"12/12 discrepant PIV3 analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 3/12 cases

'27/27 discrepant RSV A analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 17/27 cases

17/17 discrepant RSV B analytes were investigated using an alternate method; bi-directional sequence analysis identified the analyte in question in 11/17 cases

Table 27: Additional Co-Infection Combinations Detected by Reference/Comparator Methods, But Not by the eSensor RVP Assay in the Prospective Clinical Trial

| Distinct Co-Infection
Combinations* | | Total
Number of
Co-Infections | Number of
Discrepant
Co-infections | Discrepant
Analyte(s) |
|----------------------------------------|-----------|-------------------------------------|------------------------------------------|--------------------------|
| Analyte 1 | Analyte 2 | | | |
| Flu B | HRV | 6 | 3 | Flu B (2), HRV (3) |
| Flu B | RSV B | 1 | 1 | Flu B (1), RSV B (1) |
| HRV | PIV3 | 13 | 3 | HRV (3), PIV3 (3) |

*This table includes only co-infections that were detected by the reference/comparator method but not by RVP; the remaining co-infections detected by the reference/comparator method are already represented in Table above.

| Organism
Combinations | Number
of
Samples | % of
Samples
Analyzed
(N=1037) | Organism
Combinations | Number
of
Samples | % of
Samples
Analyzed
(N=1037) |
|-----------------------------------------|-------------------------|-----------------------------------------|-----------------------------|-------------------------|-----------------------------------------|
| ADV B/E + Flu B | 2 | 0.2 | Flu A + HRV + PIV3 | 2 | 0.2 |
| ADV B/E + HRV | 2 | 0.2 | Flu A + RSV A + RSV B | 2 | 0.2 |
| ADV B/E + PIV3 | 3 | 0.3 | Flu A + ADV C + HRV + PIV3 | 1 | 0.1 |
| ADV B/E + RSV A | 2 | 0.2 | Flu A + ADV C + HRV + RSV A | 1 | 0.1 |
| ADV B/E + RSV B | 1 | 0.1 | Flu B + HRV | 4 | 0.4 |
| ADV B/E + HMPV + HRV + RSV A +
RSV B | 1 | 0.1 | Flu B + PIV3 | 3 | 0.3 |
| ADV C + Flu B | 1 | 0.1 | Flu B + RSV A | 5 | 0.5 |
| ADV C + HMPV | 3 | 0.3 | Flu B + RSV B | 1 | 0.1 |
| ADV C + HRV | 6 | 0.6 | Flu B + HRV + PIV2 | 1 | 0.1 |
| ADV C + PIV3 | 1 | 0.1 | Flu B + HRV + RSV A | 2 | 0.2 |
| ADV C + RSV A | 4 | 0.4 | HMPV + HRV | 5 | 0.5 |
| ADV C + RSV B | 3 | 0.3 | HMPV + PIV3 | 1 | 0.1 |
| ADV C + HRV + PIV3 | 1 | 0.1 | HMPV + RSV B | 1 | 0.1 |
| ADV C + HRV + RSV A | 1 | 0.1 | HRV + PIV1 | 2 | 0.2 |

Table 28: Mixed Infections Detected by eSensor RVP in Prospective Samples

33

| Organism
Combinations | Number
of
Samples | % of
Samples
Analyzed
(N=1037) | Organism
Combinations | Number
of
Samples | % of
Samples
Analyzed
(N=1037) |
|--------------------------|-------------------------|-----------------------------------------|--------------------------|-------------------------|-----------------------------------------|
| Flu A + ADV B/E | 1 | 0.1 | HRV + PIV2 | 1 | 0.1 |
| Flu A + ADV C | 6 | 0.6 | HRV + PIV3 | 11 | 1.1 |
| Flu A + Flu B | 2 | 0.2 | HRV + RSV A | 16 | 1.6 |
| Flu A + HMPV | 2 | 0.2 | HRV + RSV B | 8 | 0.8 |
| Flu A + HRV | 4 | 0.4 | HRV + PIV3 + RSV A | 1 | 0.1 |
| Flu A + PIV2 | 1 | 0.1 | HRV + PIV3 + RSV B | 1 | 0.1 |
| Flu A + PIV3 | 2 | 0.2 | PIV3 + RSV A | 6 | 0.6 |
| Flu A + RSV A | 1 | 0.1 | PIV3 + RSV B | 1 | 0.1 |
| Flu A + RSV B | 2 | 0.2 | Total Mixed Infections | 128 | 12.3 |

93% (963/1037) of the evaluable prospective clinical specimens vielded valid results on the first attempt. Invalid results or no results were obtained for the remaining 74 specimens (45 of which generated results on the first run, but required retesting due to a negative control failure caused by operator error). Data generated from the retests was used in the final analysis. All 74 specimens yielded valid results after a single retest when tested according the retest recommendations.

Testing of Preselected Archived Samples

Banked samples previously characterized as positive for Influenza A H1, Parainfluenza Virus 1, Parainfluenza Virus 2, Adenovirus B/E, and Adenovirus C were used to supplement the performance studies for these analytes. These frozen banked samples were collected from various sites across the United States or from the Centers for Disease Control and Prevention (CDC). Upon arrival at GenMark, banked samples were blinded and intermixed with negative samples before being sent for testing, which was conducted by multiple sites involved in the prospective analysis of the patient samples. Testing of the banked samples was performed identically to prospectively-collected patient specimens. Results from the banked samples are presented separately from the prospectively collected specimens.

A total of 343 retrospective banked samples were collected for analysis. Out of this sample set, 11 samples were sent which didn't contain a banked viral target so these eleven samples were not tested further. Eight additional samples were excluded as they didn't contain a banked viral target as originally reported by the collection site and confirmed by comparator testing. Two samples reported errors on targets but were not retested as indicated. One sample was not sequenced. One sample had an internal control failure but was not retested as indicated. After these data were excluded, a total of 320 banked samples (including negative samples) for 5 viral targets were collected and analyzed.

With the exception of Flu A H1 samples, these banked samples were also sent to Beckman Coulter for comparator testing, and the results from the Beckman Coulter testing were compared to the results obtained by the eSensor RVP. Since the Flu A H1 samples came from the Centers for Disease Control and Prevention and were verified to be Flu A H1, these samples were not sent to Beckman Coulter for further testing. The results are summarized in Table 29.

34

	Positive Percent Agreement			Negative Percent Agreement
Virus	TP/(TP+FN)	Percent	95% CI	TN/(TN+FP)	Percent	95% CI
Influenza A H1	29/30	96.7%	82.8% - 99.9%	290/290	100%	98.7% - 100.0%
Parainfluenza Virus 1	25/25	100.0%	86.3% - 100.0%	289/295	98.0%	95.6% - 99.3%
Parainfluenza Virus 2	26/26	100.0%	86.8% - 100.0%	284/294	96.6%	93.8% - 98.4%
Adenovirus B/E	25/25	100.0%	86.3% - 100.0%	290/295	98.3%	96.1% - 99.4%
Adenovirus C	16/16	100.0%	80.6% - 100.0%	270/304	88.8%	84.8% - 91.9%

Table 29: Performance in Retrospective Clinical Specimens (N=320)

eSensor RVP Performance in Fresh vs. Frozen Clinical Specimen

Simulated viral specimens were prepared by spiking viral transport media (Remel M5) with two different concentrations of ADV C viral culture (3x LoD and 1x LoD). To evaluate the performance of frozen specimens, 128 aliguots of ADV C (64 replicates each at 3x and 1x LoD) were prepared. Sixty four aliquots (32 at each testing concentration) were tested immediately after preparation (fresh) while 64 aliquots were tested after undergoing two freeze/thaw cycles (frozen). Positive percent agreement between RVP results from fresh versus frozen aliquots for all concentrations tested was calculated. The positive percent agreement between RVP results from fresh versus frozen aliquots was 100% (95% confidence interval 89.3% - 100%).

Prospective 2X2 Performance Tables:

Influenza A
	Reference
eSensor RVP	Positive	Negative	Total
Positive	132	47a	179
Negative	5b	850	855
Total	137	897	1034
Sensitivity: 96.4% (95% CI: 91.7% - 98.8%)
Specificity: 94.8% (95% CI: 93.1% - 96.1%)

Table 30: Prospective Influenza A Results

a Influenza A virus was confirmed positive in 35/47 RVP False Positive samples using bidirectional sequencing. b Influenza A virus was not detected in all 5 RVP False Negative samples using independently developed and validated qPCR assays.

	Table 31: Prospective Influenza A H1 Results
--	----------------------------------------------	--	--	--

Influenza A H1
	Reference
eSensor RVP	Positive	Negative	Total
Positive	0	0	0
Negative	0	1027	1027
Total	0	1027	1027
Sensitivity: N/A
Specificity: 100.0% (95% CI: 99.6% - 100.0%)

35

Influenza A H3
eSensor RVP	Reference
	Positive	Negative	Total
Positive	74	25a	99
Negative	0	927	927
Total	74	952	1026
Sensitivity: 100.0% (95% CI: 95.1% - 100.0%)
Specificity: 97.4% (95% CI: 96.2% - 98.3%)

Table 32: Prospective Influenza A H3 Results

a Influenza A H3 virus was confirmed positive in 22/25 RVP False Positive samples using bidirectional sequencing.

Influenza A 2009 H1N1
eSensor RVP	Reference
	Positive	Negative	Total
Positive	49	15a	64
Negative	0	956	956
Total	49	971	1020
Sensitivity: 100.0% (95% CI: 92.7% - 100.0%)
Specificity: 98.5% (95% CI: 97.5% - 99.1%)

Table 33: Prospective Influenza A 2009 H1N1 Results

a Influenza A 2009 HINI virus was confirmed positive in 14/15 RVP False Positive samples using bidirectional sequencing.

Influenza B
eSensor RVP	Reference
	Positive	Negative	Total
Positive	64	18a	82
Negative	5b	947	952
Total	69	965	1034
Sensitivity: 92.8% (95% CI: 83.9% - 97.6%)
Specificity: 98.1% (95% CI: 97.1% - 98.9%)

Table 34: Prospective Influenza B Results

a Influenza B virus was confirmed positive in 11/18 RVP False Positive samples using bidirectional sequencing.

b Influenza B virus was not detected in 4/5 RVP False Negative samples using independently developed and validated qPCR assays.

36

Respiratory Syncytial Virus A
eSensor RVP	Reference
	Positive	Negative	Total
Positive	68	51a	119
Negative	0	905	905
Total	68	956	1024
Sensitivity: 100.0% (95% CI: 94.7% - 100.0%)
Specificity: 94.7% (95% CI: 93.1% - 96.0%)

Table 35: Prospective RSVA Results

a Respiratory Syncytial Virus type A was confirmed positive in 43/51 RVP False Positive samples using bidirectional sequencing.

Respiratory Syncytial Virus B
eSensor RVP	Reference	Positive	Negative	Total
Positive	28.	41a	69
Negative	0	955	955
Total	28	996	1024
Sensitivity: 100.0% (95% CI: 87.7% - 100.0%)
Specificity: 95.9% (95% CI: 94.5% - 97.0%)

Table 36: Prospective RSVB Results

a Respiratory Syncytial Virus type B was confirmed positive in 35/41 RVP False Positive samples using bidirectional sequencing.

Parainfluenza Virus 1
	Reference
eSensor RVP	Positive	Negative	Total
Positive	4	1a	5
Negative	0	1029	1029
Total	4	1030	1034
Sensitivity: 100.0% (95% CI: 39.8% - 100.0%)
Specificity: 99.9% (95% CI: 99.5% - 100.0%)

Table 37: Prospective PIV1 Results

a PIV 1 was not detected in this RVP False Positive sample by bidirectional sequencing.

37

Parainfluenza Virus 2
eSensor RVP	Reference
	Positive	Negative	Total
Positive	5	2a	7
Negative	1b	1026	1027
Total	6	1028	1034
Sensitivity: 83.3% (95% CI: 35.9% - 99.6%)
Specificity: 99.8% (95% CI: 99.3% - 100.0%)

Table 38: Prospective PIV2 Results

a Parainfluenza type 2 virus was confirmed positive in 0/2 RVP False Positive samples by bidirectional sequencing.

b Parainfluenza type 2 virus was not detected in this RVP False Negative sample using independently developed and validated qPCR assays.

Parainfluenza Virus 3
eSensor RVP	Reference
	Positive	Negative	Total
Positive	64	22a	86
Negative	4b	944	948
Total	68	966	1034
Sensitivity: 94.1% (95% CI: 85.6% - 98.4%)
Specificity: 97.7% (95% CI: 96.6% - 98.6%)

Table 39: Prospective PIV3 Results

a Parainfluenza type 3 virus was confirmed positive in 10/22 RVP False Positive samples using bidirectional sequencing. b Parainfluenza type 3 virus was not detected in 4/4 RVP False Negative samples using independently developed and validated qPCR assays.

Human Metapneumovirus
eSensor RVP	Reference
	Positive	Negative	Total
Positive	55	2a	57
Negative	0	979	979
Total	55	981	1036
Sensitivity: 100.0% (95% CI: 93.5% - 100.0%)
Specificity: 99.8% (95% CI: 99.3% - 100.0%)

Table 40: Prospective HMPV Results

a Human metapneumovirus was confirmed positive in 1/2 RVP False Positive samples using bidirectional sequencing.

38

Human Rhinovirus
eSensor RVP	Reference
	Positive	Negative	Total
Positive	132	35a	167
Negative	16	853	869
Total	148	888	1036
Sensitivity: 89.2% (95% CI: 83.0% - 93.7%)
Specificity: 96.1% (95% CI: 94.6% - 97.3%)

Table 41: Prospective HRV Results

a Human rhinovirus was confirmed positive in 7/35 RVP False Positive samples using bidirectional sequencing.

Adenovirus B/E
	Reference
eSensor RVP	Positive	Negative	Total
Positive	13	9a	22
Negative	0	1012	1012
Total	13	1021	1034
Sensitivity: 100.0% (95% CI: 75.3% - 100.0%)
Specificity: 99.1% (95% CI: 98.3% - 99.5%)

			Table 42: Prospective ADV B/E Results

a Adenovirus type B/E was confirmed positive in 8/9 RVP False Positive samples using bidirectional sequencing.

Table 43: Prospective ADV C Results
--------------------------------------	--

:

Adenovirus C
eSensor RVP	Reference
	Positive	Negative	Total
Positive	6	35a	41
Negative	0	993	993
Total	6	1028	1034
Sensitivity: 100.0% (95% CI: 54.1% - 100.0%)
Specificity: 96.6% (95% CI: 95.3% - 97.5%)

a Adenovirus type C was confirmed positive in 16/35 RVP False Positive samples using bidirectional sequencing.

39

Retrospective 2X2 Performance Tables:

Influenza A (Banked Samples)
eSensor RVP	Reference
	Positive	Negative	Total
Positive	29	0	29
Negative	1	290	291
Total	30	290	320
Positive Percent Agreement: 96.7% (95% CI: 82.8% - 99.9%)
Negative Percent Agreement: 100.0% (95% CI: 98.7% - 100.0%)

Table 44: Retrospective Influenza A H1 Results

r Table 45: Retrospective PIV1 Results

	PIV1 (Banked Samples)
eSensor-RVP	Reference	Positive	Negative	Total
Positive		25	6	31
Negative		0	289	289
Total		25	295	320
Positive Percent Agreement: 100.0% (95% CI: 86.3% - 100.0%)
Negative Percent Agreement: 98.0% (95% CI: 95.6% - 99.3%)

Table 46: Retrospective PIV2 Samples

PIV2 (Banked Samples)
eSensor RVP	Reference
	Positive	Negative	Total
Positive	26	10	36
Negative	0	284	284
Total	26	294	320
Positive Percent Agreement: 100.0% (95% CI: 86.8% - 100.0%)
Negative Percent Agreement: 96.6% (95% CI: 93.8% - 98.4%)

Table 47: Retrospective ADV B/E Results

ADV B/E (Banked Samples)
eSensor RVP	Reference Positive	Reference Negative	Total
Positive	25	5	30
Negative	0	290	290
Total	25	295	320
Positive Percent Agreement: 100.0% (95% CI: 86.3% - 100.0%)
Negative Percent Agreement: 98.3% (95% CI: 96.1% - 99.4%)

·

40

Table 48: Retrospective ADV C Results
ADV C (Banked Samples)
eSensor RVP	Reference
	Positive	Negative	Total
Positive	16	34	50
Negative	0	270	270
Total	16	304	320
Positive Percent Agreement: 100.0% (95% CI: 80.6% - 100.0%)
Negative Percent Agreement: 88.8% (95% CI: 84.8% - 91.9%)

. . . . .

.

Table 48: Retrospective ADV C Results

.

:

.

And Children Children

41

DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/41/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 image of an eagle or other bird with its wings spread.

Public Health Service

SEP

Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993

1 0 2012

GenMark Diagnostics, Inc.
c/o Joel Centeno
VP Regulatory, Quality, Clinical
5964 La Place Court
Carlsbad, CA 92008

c/o Joel Centeno
VP Regulatory, Quality, Clinical
5964 La Place Court
Carlsbad, CA 92008

Re: K113731 Trade/Device Name: eSensor® Respitory Viral Panel (RVP) Regulation Number: 21 CFR 866.3980 Regulation Name: Respitory viral panel multiplex nucleic acid assay Regulatory Class: Class II Product Code: OCC, OEM, OOU, OEP, OQW, NSU, OUL, JJH Dated: September 4, 2012 Received: September 6, 2012

Dear Mr. Centeno:

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.

If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. 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); and good manufacturing practice

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requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. 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/ReportalProblem/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 Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Soy aos

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Indications for Use Form

510(k) Number (if known): K113731

Device Name: eSensor® Respiratory Viral Panel (RVP)

Indications for Use:

The eSensor® Respiratory Viral Panel (RVP) is a qualitative nucleic acid multiplex In vitro diagnostic test intended for use on the eSensor XT-8TM system for the simulaneous detection of multiple respiratory viral nucleic acids in nasopharyngeal swabs (NPS) obtained from individuals exhibiting signs and symptoms of respiratory infection.

The following virus types and subtypes are identified using the eSensor RVP: Influenza A H1 Seasonal Subtype, Influenza A H3 Seasonal Subtype, Influenza A 2009 H1N1 sublype, Influenza B, Respiratory Syncytial Virus subtype A, Respiratory Syncytial Virus subtype B, Parainfluenza Virus 2, Parainfluenza Virus 3, Human Metapneumovirus, Human Rhinovirus, Adenovirus species B/E, and Adenovirus species C.

The detection and identification of specific viral nucleic acids from individuals exhibiting signs and symptoms of respiratory intection aids in the diagnosis of respiratory viral Infection if used in conjunction with other clinical and epidemiological information.

Negative results do not preclude respiratory viral infection and should not be used as the sole basis for diagnosis, treatment or other patient management decisions. Positive results do not nate out bacterial infection with other viruses. The agent detected may not be the definite cause of disease. The use of additional laboratory testing (e.g. bacterial and viral culture, immunofluorescence and radiography) and clinical presentation must be taken into consideration in the final diagnosis of respiratory viral infection.

Performance characteristics for Influenza A were established during the 2010/2011 influenza A 2009 H1N1 and H3N2 were the predominant Influenza A viruses in circulation. When other Influenza A viruses emerge performance characteristics may vary.

If infection with a novel Influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL 3+ facility is available to receive and culture specimens.

X
Prescription Use
(Part 21 CFR 801 Subpart D)
AND/OR
Over-The-Counter Use
(21 CFR 801 Subpart C

(21 CFR 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE OF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)

Tamara Feldblyum

Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) K113731

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