ESENSOR RESPIRATORY VIRAL PANEL

{0}------------------------------------------------ K113731 . # 510(k) Summary | 510(k) Summary | | | | |---------------------|---------------------------------------------------------------------------|--------|-------| | Submitter Name: | GenMark Diagnostics | | | | Address: | 5964 La Place Court<br>Carlsbad, CA 92008 | | | | Contact: | Joel Centeno<br>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<br>Instrumentation for clinical multiplex test systems, 862.2570 | | Classification Code: | OCC: Respiratory viral panel multiplex nucleic acid assay<br>OEM: Human Metapneumovirus (hMPV) RNA assay system<br>OEP: Influenza A virus subtype differentiation nucleic acid assay<br>OOU: Parainfluenza Multiplex Nucleic Acid Assay<br>NSU: Instrumentation for clinical multiplex test systems | | Predicate Device(s): | Luminex® xTag™ RVP, 510(k) Number K081483<br>Classification Code: OCC, OEM, OEP<br>Regulation No. 866.3980<br>eSensor® Warfarin Sensitivity Test, 510(k) No. K073720<br>Classification Code (applicable): NSU Instrumentation for<br>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*<br>Respiratory Viral<br>Panel Cartridges | eSensor® RVP Cartridges | 6 foil bags with 8 cartridges<br>each in each cartridge pack | 10-25 °C | | | eSensor® Respiratory Viral Panel<br>Product Insert | 1 copy | Dry place<br>(retain for reference) | | eSensor*<br>Respiratory Viral<br>Panel Amplification<br>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-<br>PCR location) | | | MS2 Internal Control | 2 vials with 300 µL each | | | eSensor*<br>Respiratory Viral<br>Panel Detection<br>Reagents | RVP Signal Buffer | 2 vials with 2200 µL each | | | | Exonuclease | 2 vials with 145 µL each. | -15 to -30 °C<br>(in a designated post-<br>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. {4}------------------------------------------------ | 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 | | | <b>Subject Device</b> | K081483 | | | sole basis for diagnosis, treatment or other<br>patient management decisions. Positive<br>results do not rule out bacterial infection, or<br>co-infection with other viruses. The agent<br>detected may not be the definite cause of<br>disease. The use of additional laboratory<br>testing (e.g. bacterial and viral culture,<br>immunofluorescence and radiography) and<br>clinical presentation must be taken into<br>consideration in the final diagnosis of<br>respiratory viral infection. | diagnosis of respiratory infection. | | | Performance characteristics for Influenza A<br>were established during the 2010/2011<br>influenza season when Influenza A 2009<br>H1N1 and H3N2 were the predominant<br>Influenza A viruses in circulation. When<br>other Influenza A viruses emerge,<br>performance characteristics may vary. | Due to seasonal prevalence, performance<br>characteristics for Influenza A/H1 were established<br>primarily with retrospective specimens. | | | If infection with a novel Influenza A virus is<br>suspected based on current clinical and<br>epidemiological screening criteria<br>recommended by public health authorities,<br>specimens should be collected with<br>appropriate infection control precautions for<br>novel virulent influenza viruses and sent to<br>state or local health departments for testing.<br>Viral culture should not be attempted in<br>these cases unless a BSL 3+ facility is<br>available to receive and culture specimens. | The RVP assay cannot adequately detect<br>Adenovirus species C, or serotypes 7a and 41.<br>The RVP primers for detection of rhinovirus cross-<br>react with enterovirus. A rhinovirus reactive result<br>should be confirmed by an alternate method (e.g.<br>cell culture). | | | For prescription use only. | Performance characteristics for Influenza A Virus<br>were established when Influenza A/H3 and A/H1<br>were the predominant Influenza A viruses in<br>circulation. When other Influenza A viruses are<br>emerging, performance characteristics may vary. If<br>infections with a novel Influenza A virus is<br>suspected based on current clinical and<br>epidemiological screening criteria recommended by<br>public health authorities, specimens should be<br>collected with appropriate infection control<br>precautions for novel virulent Influenza viruses and<br>sent to a state or local health department for<br>testing. Viral culture should not be attempted in<br>these cases unless a BSL 3+ facility is available to<br>receive and culture specimens. | | | | 6 | | Element | GenMark eSensor RVP | Luminex xTag RVP | | | Subject Device | K081483 | | Organisms Detected | Influenza A, Influenza A H1 Seasonal<br>Subtype, Influenza A H3 Seasonal Subtype,<br>Influenza A 2009 H1N1 strain, Influenza B,<br>Respiratory Syncytial Virus subtype A,<br>Respiratory Syncytial Virus subtype B,<br>Parainfluenza 1, Parainfluenza 2,<br>Parainfluenza 3, Human Metapneumovirus,<br>Human Rhinovirus, Adenovirus B/E and<br>Adenovirus C | Influenza A, Influenza A subtype H1, Influenza A<br>subtype H3, Influenza B, Respiratory Syncytial<br>Virus subtype A, Respiratory Syncytial Virus<br>subtype B, Parainfluenza 1, Parainfluenza 2, and<br>Parainfluenza 3 virus, Human Metapneumovirus,<br>Rhinovirus, and Adenovirus | | Specimen type | Same | Nasopharyngeal swabs (NPS) | | Sample preparation | Same | Up front sample processing is required to extract<br>nucleic acid | | Assay technology | Same | PCR-based system for detecting viral nucleic acids<br>in clinical specimens targeting unique regions of<br>virus RNA/DNA | | Detection<br>technology | Solid phase electrochemical | Spectrofluorometry | | Instrument | 1. Same<br>2. eSensor XT-8 | 1. EasyMag® extraction system<br>2. Luminex® 100/200™ instrument | | Software | Application software and embedded<br>firmware (controls hardware functions) on<br>XT-8 in addition to Assay Analysis Module<br>(AAM) for RVP-IVD | IS or XPONENT software; xTAG Data Analysis<br>Software RVP (US) | | Time to results | Approximately 6 hours | Approximately 8 hours | | Results | Same | Qualitative | | Test interpretation | Automated test interpretation and report<br>generation. User can access the raw signals<br>generated by the assay. | Semi-automated test interpretation. User must<br>review all "no call" results to determine cause and<br>retesting strategy. | | Controls | Same | Internal control added to each sample. External<br>control processed with each batch of samples. | : ഗ : . : . : ・ : . · {5}------------------------------------------------ : : control control control control controllers. . : {6}------------------------------------------------ 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<br>TCID50/mL | LoD Spiked<br>Conc.<br>(TCID50/mL) | %<br>Positive | |--------------------|------------------------|-----------------------|------------------------------------|---------------| | Flu A | H1N1<br>Brisbane/59/07 | 4.17 x 105 | 4.17 x 10-1 | 100% | | Flu A H1 | H1N1<br>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<br>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 | Concentration<br>Detected<br>(TCID50/ml) | LoD Multiple<br>Detected | |--------------------|------------------------------------|------------------------------------------|---------------------------------| | Flu A H1 | A/New<br>Caledonia/20/1999 | 4.2 | 10x | | | A/Brisbane/59/07 | 1.26 | 3x | | | FM/1/47H1 | 1.26 | 3x | | | A/Denver/1/57 | 1.26 | 3x | | | A/Solomon<br>Islands/3/2006 | 1.26 | 3x | | | A/Taiwan/42/06 | 1.26 | 3x | | | A/NWS/33 | 1260 | Flu A- 3x;<br>H1- 3000x* | | | A/PR/8/34 | 1.26 | Flu A- 3x;<br>H1- not detected* | | | A/Mal/302/54 | 6372 | Flu A- 3x<br>H1- 15172x* | | | A/Fort Monmouth/1/1947<br>(H1N1) | 5.5 | Flu A- 3x<br>H1-13x* | | | A/Aichi/2/68 H3N2 | 4743 | 3x | | Flu A H3 | A/Brisbane/10/07 | 4743 | 3x | | | A/Victoria/3/75 | 4743 | 3x | | | A/Port Chalmers/1/73 | 4743 | 3x | | | A/Wisconsin/67/05 | 4743 | 3x | | | A/Hong Kong/8/68 | 4743 | 3x | | | A/Perth/16/2009 | 4743 | 3x | | | Alice (vaccine)<br>A/England/42/72 | 4743 | 3x | | | MRC-2 Recombinant<br>Strain | 4743 | 3x | | | A/Nanchang/933/95 | 4743 | 3x | | Flu A 2009<br>H1N1 | A/NY/02/2009 | 0.3 | 3x | | | A/New Jersey/8/76 | 0.3 | 3x | | | A/California/7/2009 | 0.3 | 3x | | | | | Table 4: Analytical Reactivity (Inclusivity) Results | | |--|--|--|------------------------------------------------------|--| |--|--|--|------------------------------------------------------|--| {9}------------------------------------------------ | Target | Strain | Concentration<br>Detected<br>(TCID50/ml) | LoD Multiple<br>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<br>2009 H1N1- 6x** | | | A/Virginia/ATCC3/2009 | 2.7 | Flu A- 3x<br>2009 H1N1-<br>27x** | | | A/Iowa/15/30 | 100 | Flu A- 3x;<br>2009 H1N1-<br>1000x** | | | B/Florida/02/06<br>(Yamagata) | 1 | 3x | | | B/Malaysia/2506/04 | 1 | 3x | | Flu B<br>(Victoria<br>lineage except<br>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<br>Detected<br>(TCID50/ml) | LoD Multiple<br>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 | : 1. September 19. . . : . : . 10 {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),<br>(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<br>ID | Simulated<br>RVP<br>Reactivity<br>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<br>ID | Simulated<br>RVP<br>Reactivity<br>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/2254…