GenMarkDx ePlex Respiratory Pathogen Panel 2 Instruction Manual

Instruction Manual for GenMarkDx models including: ePlexRP2, ePlex, Respiratory Pathogen Panel 2, PI1114-F

ePlex® Respiratory Pathogen Panel 2 Package Insert For Use Under the Emergency Use Authorization Only For in vitro Diagnostic Use Only For Prescription Use Only Designed For the Patient, Optimized For the Lab® GenMark Diagnostics, Inc. 5964 La Place Court Carlsbad, CA 92008 USA +1 760 448 4300 PI1114 F ePlex Respiratory Pathogen Panel 2 TABLE OF CONTENTS Intended Use........................................................................................................................ 4 Summary and Explanation of Test............................................................................................ 5 Summary of Detected Organisms ............................................................................................ 6 Principles of Technology......................................................................................................... 8 Materials Provided................................................................................................................. 9 Reagent Storage, Stability, and Handling .................................................................................. 9 Materials Not Provided ........................................................................................................... 9 Equipment.................................................................................................................................................................. 9 Consumables............................................................................................................................................................. 9 Warnings and Precautions.....................................................................................................10 General .................................................................................................................................................................... 10 Safety ....................................................................................................................................................................... 10 Laboratory................................................................................................................................................................ 11 Specimen Collection, Handling, and Storage ............................................................................11 Procedure ...........................................................................................................................12 Procedural Notes..................................................................................................................................................... 12 Detailed Procedure.................................................................................................................................................. 12 Quality Control.....................................................................................................................13 Internal Controls...................................................................................................................................................... 13 External Controls..................................................................................................................................................... 14 Results ...............................................................................................................................14 Influenza A Results ................................................................................................................................................. 14 Test Reports........................................................................................................................15 Detection Report...................................................................................................................................................... 15 External Control Report .......................................................................................................................................... 16 Summary Report..................................................................................................................................................... 16 Limitations of the Procedure...................................................................................................16 Conditions of Authorization for the Laboratory ...................................................................................................... 18 Performance Characteristics ..................................................................................................19 ePlex RP and RP2 Panels...................................................................................................................................... 19 Clinical performance.............................................................................................................19 Expected values...................................................................................................................19 Clinical performance.............................................................................................................21 Clin ical Perfo rman ce o f th e ePlex RP2 Pan el an d SARS-Co V-2 ......................................................................... 21 RP2 Clinical Study ePlex Instrument Performance............................................................................................... 23 Clinical Performance of the ePlex RP Panel ......................................................................................................... 23 Comparator Method ................................................................................................................................................ 23 Prospective Clinical Samples ................................................................................................................................. 23 Prospective Clinical Performance .......................................................................................................................... 24 Retrospective Clinical Samples.............................................................................................................................. 25 Retrospective Clinical Performance....................................................................................................................... 26 Contrived Sample Performance............................................................................................................................. 27 Clinical and Contrived Sample Performance by Target........................................................................................ 27 Co-detections in Prospective Clinical Samples ..................................................................................................... 31 Clinical Study ePlex RP Panel Instrument Performance...................................................................................... 34 Analytical Performance Characteristics....................................................................................34 Limit of Detection for SARS-CoV-2 ........................................................................................................................ 34 Limit o f Detection fo r All o th er RP2 Pan el Targ ets................................................................................................ 34 Analytical Reactivity (Inclusivity)............................................................................................................................. 36 Reactivity of SARS-CoV-2 Assays ......................................................................................................................... 36 Pred icted (in silico ) Reactivity (In clusivity) Results fo r SARS-Co V-2 ................................................................... 36 Inclusivity of All Other RP2 Targets........................................................................................................................ 36 Sup p lemen tal An alytical Reactivity (In clusivity) fo r In fluen za A ........................................................................... 39 Analytical Specificity (Cross-Reactivity and Exclusivity)....................................................................................... 44 Cross-Reactivity of the SARS-CoV-2 Assays........................................................................................................ 44 In silico Analysis of the ePlex RP2 Panel SARS-CoV-2 Assays .......................................................................... 45 Cross-Reactivity and Exclusivity of Other RP2 Panel Targets ............................................................................. 45 Reproducibility......................................................................................................................................................... 48 PI1114 F For Use Under Emergency Use Authorization Only. Page 2 ePlex Respiratory Pathogen Panel 2 Samples with Co-Detected Organisms.................................................................................................................. 52 Co-Detection of SARS-CoV-2 with Other Organisms ........................................................................................... 52 Samp les with Co -Detected Org anisms on th e RP2 Pan el.................................................................................... 52 Sample Matrix Equivalency .................................................................................................................................... 53 Interfering Substances............................................................................................................................................ 53 Carryover and Cross-contamination ...................................................................................................................... 55 Troubleshooting ...................................................................................................................55 Technical Support................................................................................................................................................... 56 Glossary of Symbols.............................................................................................................56 Ref erences..........................................................................................................................57 Trademarks.........................................................................................................................59 Patent Information................................................................................................................59 PI1114 F For Use Under Emergency Use Authorization Only. Page 3 ePlex Respiratory Pathogen Panel 2 INTENDED USE The ePlex® Respiratory Pathogen Panel 2 (ePlex RP2 Panel) is a multiplexed nucleic acid in vitro diagnostic test intended for use on the ePlex Instrument for the simultaneous qualitative detection and dif ferentiation of nucleic acids from multiple respiratory viral and bacterial organisms, including nucleic acid f rom Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), in nasopharyngeal swabs (NPS) eluted in viral transport media obtained from individuals suspected of respiratory viral infection consistent with COVID-19 by their healthcare provider. Clinical signs and symptoms of respiratory viral inf ection due to SARS-CoV-2 and the targeted respiratory viral and bacterial organisms can be similar. Testing is limited to laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. § 263a, that meet requirements to perform moderate or high complexity tests. The ePlex RP2 Panel is intended for the detection and differentiation of nucleic acid from SARS-CoV-2 and the f ollowing virus types, subtypes, and bacteria: adenovirus, coronavirus (229E, HKU1, NL63, OC43), SARS-CoV-2, human metapneumovirus, human rhinovirus/enterovirus, influenza A, influenza A H1, inf luenza A H1-2009, influenza A H3, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainf luenza virus 3, parainfluenza virus 4, respiratory syncytial virus (RSV) A, respiratory syncytial virus (RSV) B, Chlamydia pneumoniae, and Mycoplasma pneumoniae. SARS-CoV-2 RNA and nucleic acids from the other respiratory viral and bacterial organisms identified by this test are generally detectable in NPS specimens during the acute phase of infection. The detection and identification of specific viral and bacterial nucleic acids from individuals exhibiting signs and/or symptoms of respiratory infection aids in the diagnosis of respiratory infection when used in conjunction with other clinical and epidemiological information. The results of this test should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. Positive results are indicative of active infection with the identified respiratory pathogen but do not rule out infection or co-infection with non-panel organisms. The agent detected by the ePlex RP2 Panel may not be the definite cause of disease. Laboratories within the United States and its territories are required to report all results for SARS-CoV-2 to the appropriate public health authorities. Negative results for SARS-CoV-2 and other organisms on the ePlex RP2 Panel may be due to infection with pathogens that are not detected by this test, or lower respiratory tract infection that may not be detected by a nasopharyngeal swab specimen. Negative results do not preclude infection with SARSCoV-2 or other organisms on the ePlex RP2 Panel and should not be used as the sole basis for patient management decisions. Negative results must be combined with clinical observations, patient history, and epidemiological information. Negative results for other organisms detected by the test may require additional laboratory testing (e.g., bacterial and viral culture, immunofluorescence and radiography) when evaluating a patient with possible respiratory tract infection. Testing with the ePlex RP2 Panel is intended for use by qualified laboratory personnel who have been trained and are proficient in performing testing on the ePlex system. The ePlex RP2 Panel is only for use under the Food and Drug Administration's Emergency Use Authorization. Due to the genetic similarity between human rhinovirus and enterovirus, the ePlex RP2 Panel cannot reliably differentiate them. If differentiation is required, an ePlex RP2 Panel positive human PI1114 F For Use Under Emergency Use Authorization Only. Page 4 ePlex Respiratory Pathogen Panel 2 rhinovirus/enterovirus result should be followed-up using an alternative method (e.g., cell culture or sequence analysis). Perf ormance characteristics for influenza A were established when influenza A H1-2009 and A H3 were the predominant influenza A viruses in circulation. Performance of detecting influenza A may vary if other inf luenza A strains are circulating or a novel influenza A virus emerges. If infection with a novel influenza A virus is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate infection control precautions for novel virulent influenza viruses and sent to state or local health departments for testing. Viral culture should not be attempted in these cases unless a BSL-3+ facility is available to receive and culture specimens. SUMMARY AND EXPLANATION OF TEST The ePlex RP2 Panel is an automated qualitative nucleic acid multiplex in vitro diagnostic test for simultaneous detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (NPS) collected in viral transport media (VTM). The test is able to detect 16 respiratory viral targets and 2 bacterial targets as summarized in Table 1. This test is performed on ePlex instrument. Respiratory viruses and bacteria are responsible for a wide range of respiratory tract infections including the common cold, influenza, and croup, and represent the most common cause of acute illness. Disease severity can be especially high in the young, the immunocompromised, and elderly patients. Respiratory inf ections cause more doctor visits and absences from school and work than any other illness.1 Inf luenza viruses have a peak season in the winter months in the northern hemisphere and the severity of the flu season varies each year based on the particular strain or strains that are in circulation and how effective the vaccine is for that year.2 Globally, seasonal influenza results in about 3-5 million severe cases and 250,000 500,000 deaths annually.3 In late 2019, a novel coronavirus was identified in Wuhan, China. The disease caused by this novel coronavirus was initially called "2019 novel coronavirus" or "2019nCoV" and was later renamed Coronavirus Disease 2019, or COVID-19.4 As of July, 2020, cases have been identified in 188 countries around the world with over 16 million cases and 655,000 deaths.5 Inf luenza-like illness is a nonspecific respiratory illness characterized by fever, fatigue, cough, and other symptoms. The majority of influenza-like illnesses are not caused by influenza but by other viruses (e.g., rhinovirus, respiratory syncytial virus, adenovirus, and parainfluenza virus).6 Less common causes of inf luenza-like illness include bacteria such as Chlamydia pneumoniae and Mycoplasma pneumoniae.6 Table 1: Targets Detected by the ePlex RP2 Panel Target Adenovirus (A-F) Coronavirus (229E, HKU1, NL63, OC43) SARS-CoV-2 Human Metapneumovirus Classification (Genome Type) Adenovirus (DNA) Coronavirus (RNA) Coronavirus (RNA) Paramyxovirus (RNA) Seasonal Prevalence* Late winter to early summer7 Winter, spring9 Unknown4 Winter10 Most Commonly Infected Demographic All ages, immunocompromised8 All ages9 Not established4 Children, elderly, immunocompromised11 PI1114 F For Use Under Emergency Use Authorization Only. Page 5 ePlex Respiratory Pathogen Panel 2 Target Classification (Genome Type) Human Rhinovirus/ Enterovirus Picornavirus (RNA) Inf luenza A Inf luenza A H1 Inf luenza A H1-2009 Inf luenza A H3 Orthomyxovirus (RNA) Inf luenza B Parainf luenza Virus 1 Parainf luenza Virus 2 Parainf luenza Virus 3 Paramyxovirus (RNA) Parainf luenza Virus 4 Respiratory Syncytial Virus A Respiratory Syncytial Virus B Paramyxovirus (RNA) Chlamydia pneumoniae Bacterium (DNA) Mycoplasma pneumoniae Bacterium (DNA) * Based on northern hemisphere seasons Seasonal Prevalence* Fall, spring12/ Summer13 Winter3 Fall15 Fall, early winter15 Spring, summer15 Fall, early winter15 Winter17, 18 No peak season19 Late summer, f all20 Most Commonly Infected Demographic All ages, immunocompromised12, 13, 14 All ages3 All ages16 Inf ants, children, older adults17, 18 All ages, most common in children19 Children, young adults21 SUMMARY OF DETECTED ORGANISMS Adenovirus: Adenoviruses are non-enveloped DNA viruses that include seven human species (A - G) and more than 60 serotypes.22 Adenovirus species B, C, and E are frequently associated with upper respiratory infections; infections are common in children, and outbreaks often occur in crowded environments, such as military barracks.8 There is no vaccine available to the general public, but the introduction of a live, oral vaccine to the US military in 2011 has reduced the incidence of adenovirus outbreaks in this population.8, 23 Adenovirus infections generally cause mild illness but can result in severe disease in infants or in immunocompromised patients, particularly in hematopoietic stem cell transplant recipients.8, 22 In addition to respiratory infections, adenovirus can also cause gastroenteritis, conjunctivitis, and cystitis.8, 22 Adenovirus species A, D, and F are not typically associated with respiratory inf ections. Coronavirus: Human coronaviruses usually cause mild to moderate upper respiratory infections but can cause significant disease in the elderly, young children, and immunocompromised individuals.24, 25 Inf ection with coronaviruses 229E, HKU1, NL63, and OC43 is common worldwide. SARS-CoV-2: In late 2019, a novel coronavirus was identified in Wuhan, China. The disease caused by this novel coronavirus was initially called "2019 novel coronavirus" or "2019-nCoV" and was later renamed Coronavirus Disease 2019, or COVID-19.4 This novel coronavirus was named Severe Acute Respiratory Syndrome Coronavirus, or SARS-CoV-2 due to genetic similarity to the coronavirus responsible for an outbreak in 2003.26 As of July 2020, cases have been identified in 188 countries around the world with over 16 million cases and 655,000 deaths.5 Human Metapneumovirus: Human metapneumovirus is a member of the paramyxovirus family and is closely related to RSV.11 Metapneumovirus has been identified as an important respiratory pathogen in young children and is the second most common virus identified in pediatric respiratory tract infections.11 PI1114 F For Use Under Emergency Use Authorization Only. Page 6 ePlex Respiratory Pathogen Panel 2 Illness is more severe in children who are immunocompromised or have underlying conditions, such as HIV or cardiac disease; it can also cause more severe disease in immunocompromised adults, especially those with COPD (chronic obstructive pulmonary disease), asthma, cancer, or in transplant patients.26 Human Rhinovirus and Enterovirus: Rhinovirus and enterovirus are closely related RNA viruses in the Picornaviridae family.13, 14 There are more than 100 different serotypes that all share high sequence homology.25 Rhinovirus causes up to 80% of all cases of the common cold worldwide and is more common in children than adults. It is the cause of a significant number of mild upper respiratory tract inf ections throughout the year, especially during the spring and fall seasons.12, 29 Most infections are mild, but rhinovirus has been associated with severe infections in at-risk populations including young children, the elderly, immunocompromised patients, and those with asthma.12, 13 There are 62 non-polio enteroviruses that can cause disease in humans.14 Enterovirus primarily infects the gastrointestinal tract but can also cause respiratory illness, which is generally mild, like the common cold, but can result in serious complications, especially in infants.14 A 2014 outbreak of enterovirus D68 (EV-D68) resulted in severe respiratory infections, some of which were fatal.30 Influenza virus: There are three types of influenza viruses: A, B, and C.3 In the northern hemisphere, inf luenza A and B circulate during the winter months causing seasonal epidemics most years; influenza C inf ections are less common and not believed to cause epidemics.3, 31 Both influenza A and B mutate, and the impact of influenza varies from year to year depending on the severity of the changes and ef f ectiveness of influenza vaccines.32 The two most common influenza A subtypes infecting humans are H1N1 (including the 2009 Pandemic H1N1 variant) and H3N2, and prevalence varies annually.33 Other rare inf luenza A subtypes also known to infect humans, such as H5N1 (avian influenza) and H3N2v, can cause severe illness and, in some cases, death.33 Inf luenza is easily spread from person to person and those most at risk for complications from infection include infants and children, the elderly, and anyone who is immunocompromised or who has co-morbidities such as heart or lung disease.34 Influenza A 2009 H1N1: During the 2009 - 2010 inf luenza season, a new strain of influenza A, now known as 2009 H1N1 became the dominant circulating virus, accounting for approximately 95% of reported influenza infections.31 This strain replaced the H1N1 virus that was previously circulating in humans and is common in both Europe and the U.S. 31, 33 Parainfluenza Virus: The parainfluenza viruses are members of the paramyxovirus family that commonly cause respiratory infections in children.35 Prevalence of parainfluenza viruses is seasonal and varies by type; most infections are mild and self-limited, but parainfluenza virus can cause life threatening pneumonia in immunocompromised patients, such as those with cystic fibrosis or transplant recipients.36 Respiratory Syncytial Virus: RSV is the most common cause of pediatric viral respiratory infections.11 Inf ection with RSV can occur at any age, and those most at risk for complications and more severe disease are the very young, especially premature infants, the elderly, and anyone with a weakened immune system.37 There are two types of respiratory syncytial virus, RSV A and B. Inf ections with RSV A are thought to be more severe than infections with RSV B.17, 38 Chlamydia pneumoniae (formerly known as Chlamydophila pneumoniae): Chlamydia pneumoniae is a common cause of upper respiratory infections including atypical pneumonia.39 C. pneumoniae is transmitted person-to-person by respiratory secretions and outbreaks are common in close contact settings.19 Inf ection severity can be mild or result in more severe disease, particularly in high risk populations such as people with heart or lung disease, diabetes, and the elderly.19, 40 The true prevalence PI1114 F For Use Under Emergency Use Authorization Only. Page 7 ePlex Respiratory Pathogen Panel 2 of C. pneumoniae infections is unknown, but the use of molecular diagnostics has improved detection of this organism, as it is difficult to identify using traditional laboratory methods.40 Mycoplasma pneumoniae: Mycoplasma pneumoniae is a bacterium lacking a cell wall and is a major cause of respiratory disease.21 M. pneumoniae is transmitted person-to-person by respiratory droplets and is a common cause of atypical, or walking pneumonia.41 M. pneumoniae is frequently undiagnosed but is estimated to be involved in up to 30% of respiratory infections.120 Inf ection often results in mild illness such as tracheobronchitis, or a chest cold, and is most prevalent in young adults and school-aged children.22, 41 Outbreaks of M. pneumoniae occur mostly in crowded environments, like schools, college dormitories, military barracks, and nursing homes.41 PRINCIPLES OF TECHNOLOGY The True Sample-to-Answer Solution ePlex instrument automates all aspects of nucleic acid testing including extraction, amplification, and detection, combining electrowetting and GenMark's eSensor® technology in a single-use cartridge. eSensor technology is based on the principles of competitive DNA hybridization and electrochemical detection, which is highly specific and is not based on fluorescent or optical detection. Electrowetting, or digital microfluidics, uses electrical fields to directly manipulate discrete droplets on the surf ace of a hydrophobically coated printed circuit board (PCB). Sample and reagents are moved in a programmable fashion in the ePlex cartridge to complete all portions of the sample processing from nucleic acid extraction to detection. A sample is loaded onto the ePlex cartridge and nucleic acids are extracted and purified from the specimen via magnetic solid phase extraction. For RNA targets, a reverse transcription step is performed to generate complementary DNA from the RNA, followed by PCR to amplify the targets. Exonuclease digestion creates single-stranded DNA in preparation for eSensor detection. The target DNA is mixed with ferrocene-labeled signal probes that are complementary to the specific targets on the panel. Target DNA hybridizes to its complementary signal probe and capture probes, which are bound to gold-plated electrodes, as shown below in Figure 1. The presence of each target is determined by voltammetry which generates specific electrical signals from the ferrocene-labeled signal probe. PI1114 F For Use Under Emergency Use Authorization Only. Page 8 ePlex Respiratory Pathogen Panel 2 Figure 1: Hybridization complex. Target-specific capture probes are bound to the gold electrodes in the eSensor microarray on the ePlex cartridge. The amplified target DNA hybridizes to the capture probe and to a complementary ferrocene-labeled signal probe. Electrochemical analysis determines the presence or absence of targets using voltammetry. MATERIALS PROVIDED Table 2: The True Sample-to-Answer Solution® ePlex Respiratory Pathogen Panel 2 Kit Contents Product Item number Components (quantity) Storage ePlex Respiratory Pathogen Panel 2 EA001222 ePlex Respiratory Pathogen Panel 2 Cartridge (12) 28 ºC The ePlex RP2 Panel reagents are shipped at room temperature; upon receipt, reagents should be stored at 2-8 °C. Saf ety Data Sheets (SDS) for all reagents provided in this kit may be obtained at https://www.genmarkdx.com/support/safety-data-sheets-sds/. For paper copies, please contact GenMark Customer Service at CustomerService@genmarkdx.com. REAGENT STORAGE, STABILITY, AND HANDLING · Store the ePlex RP2 Panel kit components at 28 ºC. · Do not use RP Panel kit components beyond the expiration date. · Do not open a cartridge pouch until you are ready to perform testing. MATERIALS NOT PROVIDED Equipment · GenMark ePlex instrument and Software · Pipettes calibrated to deliver 200 µL · Vortex mixer · Printer (optional) - See ePlex Operator Manual for compatibility guidelines Consumables · Pipette tips, aerosol resistant, RNase/DNase-free · Disposable, powder free gloves PI1114 F For Use Under Emergency Use Authorization Only. Page 9 ePlex Respiratory Pathogen Panel 2 · 10% bleach for decontamination of appropriate surfaces · 70% ethanol or isopropyl alcohol WARNINGS AND PRECAUTIONS General · For use under Emergency Use Authorization Only. · For in vitro diagnostic use only. · This test has not been FDA cleared or approved. · This test has been authorized by FDA under an EUA for use by authorized laboratories. · This test has been authorized only for the simultaneous qualitative detection and differentiation of nucleic acids from multiple respiratory viral and bacterial organisms, including nucleic acid from Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2). · This test is only authorized for the duration of the declaration that circumstances exist justifying the authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of COVID-19 under Section 564(b)(1) of the Federal Food, Drug, and Cosmetic Act, 21 U.S.C. § 360bbb-3(b)(1), unless the authorization is terminated or revoked sooner. · A trained healthcare professional should carefully interpret the results from the ePlex RP2 Panel in conjunction with a patient's signs, symptoms, and results from other diagnostic tests. · Positive results do not rule out co-infection with other viruses or bacteria. 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 diagnosis of respiratory infection. · Laboratories within the United States and its territories are required to report all results for SARSCoV-2 to the appropriate public health authorities. · Do not reuse ePlex RP2 Panel kit components. · Do not use reagents beyond the expiration date printed on the labeling. · Do not use a reagent that is damaged. · Follow the procedure as described in this package insert. Read all instructions before starting the test. Any deviation from the procedures and guidelines may affect optimal test performance. · All human-sourced materials should be considered potentially infectious and should be handled with universal precautions. · The use of sterile, disposable, nuclease-free pipette tips is recommended. Use only supplied or specified required consumables to ensure optimal test performance. Safety · Handle all specimens and waste materials as if they were capable of transmitting infectious agents in accordance with Universal Precautions. Observe safety guidelines such as those outlined in CDC/NIH Biosafety in Microbiological and Biomedical Laboratories, CLSI Document M29 Protection of Laboratory Workers from Occupationally Acquired Infections, or other appropriate guidelines. · Do not eat, smoke, drink, apply cosmetics, or handle contact lenses in areas where reagents or human specimens are handled. · Follow national biological safety procedures for handling biological samples. (e.g., do not pipette by mouth, wear appropriate protective clothing and eye protection). · If inf ection 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 Inf luenza viruses and sent to 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. · Dispose materials used in this test, including reagents, specimens, and used vials, in accordance with all f ederal, state, and local regulations. PI1114 F For Use Under Emergency Use Authorization Only. Page 10 ePlex Respiratory Pathogen Panel 2 · Do not stick fingers or other objects inside the ePlex instrument bays. · Wash hands thoroughly with soap and water after handling reagents. Launder contaminated clothing prior to re-use. · Do not puncture or pierce reagent blisters on the ePlex cartridge. Reagents may cause irritation to skin, eyes, and respiratory tract. Harmful if swallowed or inhaled. Contains oxidizing liquids. · The ePlex RP2 Panel cartridge contains chemicals that are classified as hazardous. Review the Saf ety Data Sheet (SDS) bef ore use, and in cases of exposure, ref er to the SDS f or more inf ormation. · Observe safety guidelines such as wearing proper protective equipment including laboratory coats, gowns, gloves, eye protection, and a biological safety cabinet as outlined in Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition https://www.cdc.gov/labs/BMBL.html. · If inf ection with SARS-CoV-2 is suspected based on current clinical and epidemiological screening criteria recommended by public health authorities, specimens should be collected with appropriate inf ection control precautions. · Thoroughly decontaminate the lab and all equipment with 10% bleach followed by 70% ethanol or isopropyl alcohol (or equivalent) prior to processing a specimen. · Immediately clean up any spill containing potentially infectious material with a 0.5-1% (w/v) sodium hypochlorite (20% v/v bleach). · Perf ormance characteristics have been determined with nasopharyngeal swab samples from human patients with signs and symptoms of respiratory infection. · Specimens should be processed in a Class II (or higher) biological safety cabinet. · To mitigate the risk of sample-to-sample contamination, change gloves af ter dispensing sample into the cartridge. · Contamination of the sample may occur if the sample is loaded in an area where PCR amplicons f or respiratory pathogens are generated. Avoid loading sample in areas that are potentially contaminated with PCR amplicon. Laboratory · Contamination of the sample may occur if laboratory personnel processing the sample are inf ected with common respiratory pathogens. To avoid this, specimens should be processed in biosafety cabinets. If a biosafety cabinet is not used, a splash shield or face mask should be used when processing samples. · A biosafety cabinet that is used for viral or bacterial culture should not be used for sample preparation. · Samples and cartridges should be handled and/or tested one at a time. To mitigate the risk of sample-to-sample contamination, change gloves after dispensing sample into the cartridge. · Thoroughly decontaminate the lab and all equipment with 10% bleach followed by 70% ethanol or isopropyl alcohol (or equivalent) prior to processing a specimen. · Contamination of the sample may occur if the sample is loaded in an area where PCR amplicons f or respiratory pathogens are generated. Avoid loading sample in areas that are potentially contaminated with PCR amplicon. SPECIMEN COLLECTION, HANDLING, AND STORAGE Nasopharyngeal Swab Collection Nasopharyngeal swab specimen collection should be performed according to standard technique and placed in viral transport media. Minimum Sample Volume 200 µL nasopharyngeal swab specimen in viral transport media is required f or testing. PI1114 F For Use Under Emergency Use Authorization Only. Page 11 ePlex Respiratory Pathogen Panel 2 Transport and Storage Clinical specimens can be stored at room temperature (1530 ºC) f or up to 12 hours or ref rigerated at 2-8 ºC f or up to 10 days after collection in viral transport media. Specimens can also be stored at -20 ºC or -80 ºC f or 12 months with up to 2 freeze/thaw cycles. Inadequate or inappropriate specimen collection, storage, and transport are likely to yield false test results. Training in specimen collection is highly recommended due to the importance of specimen quality. CLSI MM13-A may be referenced as an appropriate resource. PROCEDURE Procedural Notes · All f rozen samples should be thawed completely before testing. · Samples should be nasopharyngeal swabs in viral transport media. · Cartridge can be used immediately upon removal from 2-8 °C storage. There is no need to equilibrate to room temperature before use. · Once cartridge is removed from foil pouch, it should be used within 2 hours. Do not open the cartridge pouch until the sample is ready to be tested. · Once the sample is loaded into the ePlex RP2 Panel cartridge, the sample should be tested as soon as possible or within 2 hours. · Do not re-use cartridges. · Use a new, sterile pipette tip for loading each sample. · Do not insert a wet cartridge into the ePlex instrument. If the cartridge or sample has leaked, dispose of cartridge in accordance with all federal, state, and local regulations. · Samples should be transferred into the ePlex RP2 Panel cartridge in an amplicon-free, clean environment. · Samples, consumables, and lab areas should be protected from aerosol or direct contamination with amplicon. Decontaminate laboratory areas and affected equipment with 10% bleach f ollowed by 70% ethanol or isopropyl alcohol (or equivalent). · Samples and cartridges should be handled and/or tested one at a time. To mitigate the risk of sample-to-sample contamination, change gloves after dispensing sample into the cartridge. · Specimens should be processed in biosafety cabinets. If a biosafety cabinet is not used, a splash shield or face mask should be used when processing samples. · Dispose materials used in this test, including reagents, specimens, and used vials, in accordance with all regulations. Detailed Procedure 1. Decontaminate the clean area used for setting up the ePlex RP2 Panel with 10% bleach followed by 70% ethanol or isopropyl alcohol (or equivalent). 2. Remove one RP2 Panel cartridge pouch from kit packaging. 3. Open the RP2 Panel cartridge pouch. 4. Write the accession ID or place a barcode label with accession ID on the RP2 Panel cartridge. 5. Vortex the sample for 3-5 seconds. 6. Use a calibrated pipette to aspirate 200 µL of sample and dispense into the sample loading port of the ePlex RP2 Panel cartridge. 7. Close the sample loading port by sliding the cap over the port and firmly pushing down on the cap to securely seal the sample delivery port. NOTE: Bubbles can be present when closing the cap. 8. Scan the RP2 Panel cartridge using the barcode reader provided with the ePlex instrument. NOTE: If an accession ID barcode label is not used, manually enter accession ID with the onscreen keyboard and scan the cartridge barcode when prompted by the ePlex System. PI1114 F For Use Under Emergency Use Authorization Only. Page 12 ePlex Respiratory Pathogen Panel 2 NOTE: The barcode scanner will read both the accession ID barcode (if placed on the cartridge by the operator) and the 2D barcode printed on the cartridge label; however, the barcode scanner will only beep once to indicate that both barcodes have been read. 9. Insert the RP2 Panel cartridge into any available bay, indicated by a flashing, white LED light. The test will begin automatically when the cartridge has been inserted into the bay and the pre- run check (cartridge initialization) is completed, indicated by a blue LED light. QUALITY CONTROL Internal Controls Each cartridge includes internal controls that monitor performance of each step of the testing process. A DNA control verifies extraction, amplification and detection of DNA targets, and RNA controls verify amplification and detection of RNA targets. Each amplification reaction on the cartridge has at least one internal control and in each reaction either the internal control or a target must generate signal above the defined threshold for a valid test result. Internal control results are interpreted by the ePlex software and displayed on ePlex RP Panel Reports as Internal Control with a result of PASS, FAIL, N/A or INVALID. Table 3 includes details on the interpretation of Internal Control results. Internal Control Result PASS FAIL N/A INVALID Table 3: Internal Control Results Explanation Action The internal control or a target from each amplification reaction has generated signal above the threshold. All results are displayed on the RP2 Panel Detection Report. The test was completed and internal controls were successful, Test is valid, report results. indicating valid results were generated. Neither the internal control nor any target in at least one amplification reaction generates signal above the threshold. No results are displayed on the RP2 Panel Detection Report. The test was completed but at least one internal control was not detected, indicating that results are not valid. Test is not valid, repeat the test using a new cartridge. The internal control in every amplification reaction does not generate signal above the threshold, but a target in every amplification reaction does generate signal above the th res h o l d. The test was completed and internal controls were not successful, however detection of signal above the threshold for a target in every amplification reaction indicates valid results were generated. An error has occurred during processing that prevents analysis of signal data. The test has not successfully completed and results for this test are not valid. This is often due to an instrument or software error. All results are displayed on the RP2 Panel Detection Report. Test is valid, report results. No results are displayed on the RP2 Panel Detection Report. Test is not valid, repeat the test using a new cartridge. PI1114 F For Use Under Emergency Use Authorization Only. Page 13 ePlex Respiratory Pathogen Panel 2 External Controls Positive and negative external controls should be tested with each new lot of reagents or monthly, whichever occurs first. Viral transport medium can be used as the negative control. Previously characterized positive samples or viral transport medium spiked with well characterized organisms can be used as the external positive control. External controls should be run in accordance with laboratory protocols and accrediting organizations, as applicable. RESULTS Table 4: Interpretation of Results on the ePlex RP2 Panel Detection Report Target Result Explanation Action Target Detected The test was completed successfully, and the target has generated signal above its defined threshold, and the Internal Control was reported as PASS. All results are displayed on the RP2 Panel Detection Report. Test is valid, report results. All results are displayed on the RP2 Panel Detection Report. Multiple Targets Detected Target Not Detected In v al i d The test was completed successfully, and multiple targets have generated signal above their defined threshold, and the Internal Control was reported as PASS. The test was completed successfully, and the target did not generate signal above its defined threshold, and the Internal Control was reported as PASS. The test has not successfully completed, and results for this test are not valid. This is often due to an instrument or software error or failure of an internal c o n tro l . Test is valid, report results. Detection of more than 3 pathogens may indicate contamination. Re-test of the sample is recommended to confirm results. All results are displayed on the RP2 Panel Detection Report. Test is valid, report results. No results are displayed on the RP2 Panel Detection Report. Test is not valid, repeat test. Influenza A Results The ePlex RP2 Panel detects Inf luenza A and the H1, H1-2009, and H3 subtypes using unique assays for each. Interpretation of results for Inf luenza A are described in Table 5. Results for Influenza A and Subtypes Influenza A detected, at least one subtype (H1, H1-2009, or H3) reported as detected. Table 5: Results for Influenza A Explanation Results on Report This is an expected result. Result reported as influenza A and influenza A subtype d etec ted . Recommended Action No n e PI1114 F For Use Under Emergency Use Authorization Only. Page 14 ePlex Respiratory Pathogen Panel 2 Results for Influenza A and Subtypes Explanation Results on Report Recommended Action Re-test to confirm result. Influenza A detected, all subtypes (H1, H1-2009, and H3) reported as not d etec ted Low virus titers can result in detection of influenza A matrix without a subtype. Detection of influenza A matrix without a subtype can also indicate the presence of a novel strain. Result reported as influenza A detected. No Influenza A subtype detected. Influenza A detected and more than one subtype (H1, H1-2009, or H3) reported as detected. Sample is co-infected with multiple influenza subtypes. Infection with multiple subtypes of influenza are possible but rare. A live intranasal multivalent influenza virus vaccine may cause false positive results for influenza A, A/H1, A/H3, A/H1-2009, and/or influenza B. Result reported as influenza A and multiple subtypes d etec ted . If the original result is confirmed, contact the appropriate public health authorities for additional tes ti n g . If the re-test provides a different result, test the sample a third time to ensure the accuracy of the result. Re-test to confirm result. If the re-test result confirms the original result, it is recommended that the sample be further investigated using a different FDA-cleared influenza A subtyping assay. Contamination has o c c urred . Re-test to confirm result. Influenza A not detected, at least one subtype (H1, H1-2009, or H3) reported as detected. Low virus titers can result in detection of influenza A subtype without the influenza A matrix. Detection of influenza A subtype without the influenza A matrix can also indicate the presence of a novel strain. Influenza A (subtype) detected. Re-testing of this sample to confirm Influenza A (subtype) is recommended. Refer to package insert for additional information. If the re-test result confirms the original result, the influenza A subtype is considered positive. It is recommended that the sample be further investigated using a different FDA-cleared influenza A subtyping assay and/or sending the residual sample to local public health laboratory for further testing. TEST REPORTS There are several different reports that are available on the ePlex instrument. Results are provided in a printable format, may be viewed electronically, or may be exported for additional analysis. Reports can be customized with account specific information such as the address, logo, and institution specific footers on each report. For more information on ePlex reports, refer to the ePlex Operator Manual. Detection Report The RP2 Panel Detection Report includes the results for each individual sample run on the ePlex instrument. PI1114 F For Use Under Emergency Use Authorization Only. Page 15 ePlex Respiratory Pathogen Panel 2 The Summary section indicates the overall test result and lists all detected targets in that sample. The Results section includes a list of all targets on the panel with an individual result for each. Results for each target are reported as Detected, Not Detected, or Invalid (displayed as a red x); results for the Internal Control are reported as PASS, FAIL, INVALID, or N/A. External Control Report The RP2 Panel External Control Report is generated for an external control that has been pre-defined in the ePlex RP2 Panel software. For more information on defining external controls on ePlex RP2 Panel, ref er to the ePlex Operator Manual. The Summary section indicates the overall result (Pass or Fail status) and lists all detected targets for that external control. The Results section includes a list of all panel targets with the result, expected result, and Pass/Fail status for each. Results are reported as Detected, Not Detected, or Invalid (displayed as a red x). A target is reported as Pass if the actual result matches the expected result (as defined for that control); a target is reported as Fail if the actual result does not match the expected result. If the actual results f or each target match the expected result for each target (all targets reported as Pass), the overall result f or the external control is reported as Pass in the Summary section. If the actual result for any target does not match the expected result, the overall result for the external control is reported as Fail in the Summary section. Summary Report The Summary Report allows the operator to use defined searchable criteria to create customized reports, using specified targets, dates, range of dates, sample, external control, test bay, or operator. For more inf ormation on creating Summary Reports, refer to the ePlex Operator Manual. LIMITATIONS OF THE PROCEDURE · This product can be used only with the GenMark ePlex instrument. · At high titers, cross-reactivity with SARS-CoV-1 was observed with the ePlex RP2 Panel. · Due to the genetic similarity between human rhinovirus and enterovirus, this test cannot reliably dif ferentiate them. An ePlex RP2 Panel Rhinovirus/Enterovirus positive result should be followedup using an alternate method (e.g. cell culture or sequence analysis) if differentiation between the viruses is required. · This test is a qualitative test and does not provide a quantitative value of detected organism present. · The perf ormance of the test has been evaluated for use with human sample material only. · This test has not been validated for testing samples other than nasopharyngeal swab samples in viral transport media. · The perf ormance of this test has not been established for immunocompromised individuals. · The perf ormance of this test has not been established for patients without signs and symptoms of respiratory infection. · Results f rom this test must be correlated with the clinical history, epidemiological data, and other data available to the clinician evaluating the patient. · The ef f ect of antibiotic treatment on test performance has not been evaluated. · The perf ormance of this test has not been established for screening of blood or blood products. PI1114 F For Use Under Emergency Use Authorization Only. Page 16 ePlex Respiratory Pathogen Panel 2 · Targets (viral and bacterial nucleic acids) may persist in vivo, independent of viral or bacterial viability. Detection of target(s) does not imply that the corresponding virus(es) or bacteria are inf ectious or are the causative agents for clinical symptoms. · The detection of viral or bacterial nucleic acid is dependent upon proper specimen collection, handling, transportation, storage, and preparation. Failure to observe proper procedures in any one of these steps can lead to incorrect results. There is a risk of false positive or f alse negative values resulting from improperly collected, transported, or handled samples. · There is a risk of false negative values due to the presence of sequence variants in the viral or bacterial targets of the test, the presence of inhibitors, technical error, sample mix-up, or an inf ection caused by an organism not detected by the panel. Test results may be affected by concurrent antibacterial or antiviral therapy or levels of bacteria or virus in the sample that are below the limit of detection for the test. A result of No Targets Detected on the ePlex RP2 Panel should not be used as the sole basis for diagnosis, treatment or other patient management decisions. · A result of No Targets Detected on the ePlex RP2 Panel in the setting of a respiratory illness may be due to infection with pathogens that are not detected by this test or lower respiratory tract inf ection that is not detected by a nasopharyngeal swab sample. · There is a risk of false positive results due to contamination of the sample with target organisms, their nucleic acids, or amplicons. Particular attention should be given to the Laboratory precautions noted under the Warnings and Precautions section. · There is a risk of false positive results due to non-specific amplification and cross-reactivity with organisms found in the respiratory tract. Erroneous results due to cross-reactivity with organisms that were not specifically evaluated or new variant sequences that emerge are possible. · If f our or more organisms are detected in a sample, retesting is recommended to confirm polymicrobial result. · The ePlex RP2 Panel influenza A subtyping reagents target the influenza A hemagglutinin gene only. The ePlex RP2 Panel does not detect or differentiate the influenza A neuraminidase gene. · The perf ormance of this test has not been established for monitoring treatment of infection with any of the panel organisms. · Positive and negative predictive values are highly dependent on prevalence. False negative test results are more likely during peak activity when prevalence of disease is high. False positive test results are more likely during periods when prevalence is moderate to low. · Clinical performance was established when influenza A H3 and influenza A H1-2009 were the predominant influenza A viruses in circulation. When other influenza A viruses emerge, perf ormance may vary. · Due to the small number of positive samples collected for Chlamydia pneumoniae during the prospective and retrospective clinical studies, performance characteristics for Chlamydia pneumoniae were established primarily with contrived clinical specimens. Performance characteristics for Influenza A H1 were established using contrived clinical specimens only. · Clinical evaluation indicates a lower sensitivity for the detection of coronavirus OC43. If infection with coronavirus OC43 is suspected, negative samples should be confirmed using an alternative method. · The ef f ect of interfering substances has only been evaluated for those listed in this package insert. Interference due to substances other than those described in the "Interfering Substances" section can lead to erroneous results. · At concentrations greater than 1% weight/volume in the sample, tobramycin was found to inhibit assay performance. · Minimum Essential Media (MEM) may be inhibitory and negatively impact the performance of the ePlex RP2 Panel. · Diluents from external quality controls or proficiency testing materials that include the following substances have been shown to interfere with the performance of the ePlex RP2 Panel: human plasma proteins and 941 L media with methanol. PI1114 F For Use Under Emergency Use Authorization Only. Page 17 ePlex Respiratory Pathogen Panel 2 · The perf ormance of this test has not been specifically evaluated for specimens collected from individuals who recently received influenza vaccine. Recent administration of a live intranasal inf luenza virus vaccine may cause false positive results for influenza A, H1, H3, H1-2009, and/or inf luenza B. · The ePlex RP2 Panel cannot differentiate variant viruses, such as H3N2v, from seasonal inf luenza A viruses. If variant virus infection is suspected, clinicians should contact their state or local health department to arrange specimen transport and request a timely diagnosis at a state public health laboratory. Conditions of Authorization for the Laboratory The ePlex RP2 Panel Letter of Authorization, along with the authorized Fact Sheet for Healthcare Providers, the authorized Fact Sheet for Patients, and authorized labeling are available on the FDA website: https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-useauthorizations-medical-devices/in-vitro-diagnostics-euas. However, to assist clinical laboratories using the ePlex RP2 Panel, the relevant Conditions of Authorization are listed below: A. Authorized laboratories1 using the ePlex RP2 Panel will include with test result reports, all authorized Fact Sheets. Under exigent circumstances, other appropriate methods for disseminating these Fact Sheets may be used, which may include mass media. B. Authorized laboratories using the ePlex RP2 Panel will use the ePlex RP2 Panel as outlined in the Instructions for Use. Deviations from the authorized procedures, including the authorized instruments, authorized extraction methods, authorized clinical specimen types, authorized control materials, authorized other ancillary reagents and authorized materials required to use the ePlex RP2 Panel are not permitted. C. Authorized laboratories that receive the ePlex RP2 Panel will notify the relevant public health authorities of their intent to run the ePlex RP2 Panel prior to initiating testing. D. Authorized laboratories using the ePlex RP2 Panel will have a process in place for reporting test results to healthcare providers and relevant public health authorities, as appropriate. E. Authorized laboratories will collect information on the performance of the ePlex RP2 Panel and report to DMD/OHT7-OIR/OPEQ/CDRH (via email: CDRH-EUA-Reporting@fda.hhs.gov) and GenMark Diagnostics, Inc. (via email: technicalsupport@genmarkdx.com) any suspected occurrence of false positive or false negative results and significant deviations from the established performance characteristics of the ePlex RP2 Panel Test of which they become aware. F. All laboratory personnel using the ePlex RP2 Panel must be appropriately trained in RT-PCR techniques and use appropriate laboratory and personal protective equipment when handling this kit and use the ePlex RP2 Panel in accordance with the labeling. G. GenMark Diagnostics, Inc., authorized distributors, and authorized laboratories using the ePlex RP2 Panel will ensure that any records associated with this EUA are maintained until otherwise notif ied by FDA. Such records will be made available to FDA for inspection upon request. 1 The letter of authorization refers to, "laboratories certified under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet requirements to perform moderate or high complexity tests" as "authorized laboratories." PI1114 F For Use Under Emergency Use Authorization Only. Page 18 ePlex Respiratory Pathogen Panel 2 PERFORMANCE CHARACTERISTICS ePlex RP and RP2 Panels The ePlex RP2 Panel was developed by incorporating the reagents required to detect the SARS-CoV-2 targets from the ePlex SARS-CoV-2 Test into the existing ePlex Respiratory Pathogen Panel (RP Panel). The assays for detection of SARS-CoV-2 were added into PCR pools that contain additional targets. The targets that are now co-amplified with SARS-CoV-2 are influenza A, influenza A H1, influenza A H1-2009, inf luenza A H3, influenza B, and adenovirus; assays for all other targets were unchanged. Studies were conducted to demonstrate that the performance characteristics of the RP Panel were not affected by the addition of the SARS-CoV-2 assays. Additional studies to support the addition of SARS-CoV-2 are included in the sections below. The original studies from the RP Panel are still relevant for the RP2 Panel. CLINICAL PERFORMANCE EXPECTED VALUES A prospective, multicenter clinical study was conducted to evaluate the clinical performance of the ePlex RP Panel in nasopharyngeal swab samples. 2462 nasopharyngeal swab samples were prospectively collected at 8 collection sites in 2 phases from patients of all ages and genders presenting with signs and/or symptoms of respiratory infection. In the first phase from March 2013 through August 2014, 1951 samples were prospectively collected and frozen; from September 2016 through October 2016, 511 samples were prospectively collected and tested fresh (never frozen). The expected values of individual analytes based on ePlex RP Panel results in prospective samples for each phase are summarized in Tables 6-9. NOTE: Expected values for SARS-CoV-2 have not been determined. Table 6: Expected Value (As Determined by ePlex RP Panel) Summary By Age Group in the Prospective Clinical Evaluation (Phase 1: March 2013 August 2014) Organism All Ages (N=1951) n (%) Age 0-1 (N=315) n (%) Age >1-5 (N=250) n (%) Age >5-21 (N=246) n (%) Age >21-65 (N=745) n (%) Age >65 (N=395) n (%) Adenovirus Coronavirus (229E, HKU1, NL63, OC43) 72 (3.7) 102 (5.2) 31 (9.8) 19 (6.0) 24 (9.6) 18 (7.2) 7 (2.8) 16 (6.5) 7 (0.9) 32 (4.3) 3 (0.8) 17 (4.3) Human Metapneumovirus Human Rhinovirus/Enterovirus Influenza A Influenza A H1 Influenza A H1-2009 Influenza A H3 Influenza B Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Parainfluenza Virus 4 RSV A RSV B 113 (5.8) 388 (19.9) 110 (5.6) 0 (0.0) 76 (3.9) 34 (1.7) 62 (3.2) 24 (1.2) 10 (0.5) 99 (5.1) 7 (0.4) 28 (1.4) 83 (4.3) 22 (7.0) 113 (35.9) 6 (1.9) 0 (0.0) 4 (1.3) 1 (0.3) 4 (1.3) 4 (1.3) 4 (1.3) 31 (9.8) 3 (1.0) 13 (4.1) 33 (10.5) 28 (11.2) 94 (37.6) 18 (7.2) 0 (0.0) 13 (5.2) 5 (2.0) 9 (3.6) 12 (4.8) 4 (1.6) 20 (8.0) 2 (0.8) 6 (2.4) 19 (7.6) 6 (2.4) 58 (23.6) 20 (8.1) 0 (0.0) 14 (5.7) 6 (2.4) 10 (4.1) 4 (1.6) 0 (0.0) 3 (1.2) 1 (0.4) 3 (1.2) 6 (2.4) 31 (4.2) 87 (11.7) 49 (6.6) 0 (0.0) 37 (5.0) 12 (1.6) 24 (3.2) 3 (0.4) 2 (0.3) 27 (3.6) 1 (0.1) 2 (0.3) 15 (2.0) 26 (6.6) 36 (9.1) 17 (4.3) 0 (0.0) 8 (2.0) 10 (2.5) 15 (3.8) 1 (0.3) 0 (0.0) 18 (4.6) 0 (0.0) 4 (1.0) 10 (2.5) PI1114 F For Use Under Emergency Use Authorization Only. Page 19 ePlex Respiratory Pathogen Panel 2 Organism Chlamydia pneumoniae Mycoplasma pneumoniae All Ages (N=1951) n (%) 3 (0.2) 5 (0.3) Age 0-1 (N=315) n (%) 0 (0.0) 1 (0.3) Age >1-5 (N=250) n (%) 0 (0.0) 1 (0.4) Age >5-21 (N=246) n (%) 1 (0.4) 2 (0.8) Age >21-65 (N=745) n (%) 1 (0.1) 1 (0.1) Age >65 (N=395) n (%) 1 (0.3) 0 (0.0) Table 7: Expected Value (As Determined by ePlex RP Panel) Summary By Age Group in the Prospective Clinical Evaluation (Phase 2: September 2016 October 2016) Organism All Ages (N=511) n (%) Age 0-1 (N=73) n (%) Age >1-5 (N=75) n (%) Age >5-21 (N=75) n (%) Age >21-65 (N=181) n (%) Age >65 (N=107) n (%) Adenovirus 10 (2.0) 3 (4.1) 4 (5.3) 1 (1.3) 1 (0.6) 1 (0.9) Coronavirus (229E, HKU1, NL63, OC43) 8 (1.6) 2 (2.7) 0 (0.0) 1 (1.3) 4 (2.2) 1 (0.9) Human Metapneumovirus Human Rhinovirus/Enterovirus 0 (0.0) 188 (36.8) 0 (0.0) 37 (50.7) 0 (0.0) 40 (53.3) 0 (0.0) 33 (44.0) 0 (0.0) 58 (32.0) 0 (0.0) 20 (18.7) Influenza A 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza A H1 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza A H1-2009 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza A H3 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza B 2 (0.4) 0 (0.0) 0 (0.0) 1 (1.3) 1 (0.6) 0 (0.0) Parainfluenza Virus 1 1 (0.2) 0 (0.0) 1 (1.3) 0 (0.0) 0 (0.0) 0 (0.0) Parainfluenza Virus 2 13 (2.5) 3 (4.1) 4 (5.3) 3 (4.0) 2 (1.1) 1 (0.9) Parainfluenza Virus 3 5 (1.0) 2 (2.7) 1 (1.3) 1 (1.3) 1 (0.6) 0 (0.0) Parainfluenza Virus 4 8 (1.6) 1 (1.4) 4 (5.3) 2 (2.7) 1 (0.6) 0 (0.0) RSV A 8 (1.6) 5 (6.8) 3 (4.0) 0 (0.0) 0 (0.0) 0 (0.0) RSV B 9 (1.8) 3 (4.1) 4 (5.3) 0 (0.0) 2 (1.1) 0 (0.0) Chlamydia pneumoniae 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Mycoplasma pneumoniae 4 (0.8) 0 (0.0) 1 (1.3) 2 (2.7) 1 (0.6) 0 (0.0) Table 8: Expected Value (As Determined by ePlex RP Panel) Summary By Sample Collection Site in the Prospective Clinical Evaluation (Phase 1: March 2013 August 2014) Organism All Sites (N=1951) n (%) Site 1 (N=165) n (%) Site 2 (N=248) n (%) Site 3 (N=350) n (%) Site 4 (N=892) n (%) Site 5 (N=296) n (%) Adenovirus Coronavirus (229E, HKU1, NL63, OC43) Human Metapneumovirus 72 (3.7) 102 (5.2) 113 (5.8) 4 (2.4) 8 (4.8) 10 (6.1) 8 (3.2) 11 (4.4) 23 (9.3) 28 (8.0) 32 (9.1) 27 (7.7) 23 (2.6) 29 (3.3) 30 (3.4) 9 (3.0) 22 (7.4) 23 (7.8) Human Rhinovirus/Enterovirus 388 (19.9) 27 (16.4) 33 (13.3) 61 (17.4) 185 (20.7) 82 (27.7) Influenza A 110 (5.6) 5 (3.0) 21 (8.5) 48 (13.7) 19 (2.1) 17 (5.7) Influenza A H1 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza A H1-2009 76 (3.9) 3 (1.8) 22 (8.9) 31 (8.9) 5 (0.6) 15 (5.1) Influenza A H3 34 (1.7) 2 (1.2) 0 (0.0) 18 (5.1) 12 (1.3) 2 (0.7) Influenza B 62 (3.2) 9 (5.5) 9 (3.6) 9 (2.6) 19 (2.1) 16 (5.4) Parainfluenza Virus 1 24 (1.2) 0 (0.0) 0 (0.0) 5 (1.4) 2 (0.2) 17 (5.7) Parainfluenza Virus 2 10 (0.5) 0 (0.0) 0 (0.0) 0 (0.0) 10 (1.1) 0 (0.0) Parainfluenza Virus 3 99 (5.1) 13 (7.9) 3 (1.2) 28 (8.0) 41 (4.6) 14 (4.7) Parainfluenza Virus 4 7 (0.4) 0 (0.0) 0 (0.0) 1 (0.3) 4 (0.4) 2 (0.7) PI1114 F For Use Under Emergency Use Authorization Only. Page 20 ePlex Respiratory Pathogen Panel 2 Organism RSV A RSV B Chlamydia pneumoniae Mycoplasma pneumoniae All Sites (N=1951) n (%) 28 (1.4) 83 (4.3) 3 (0.2) 5 (0.3) Site 1 (N=165) n (%) 4 (2.4) 6 (3.6) 0 (0.0) 1 (0.6) Site 2 (N=248) n (%) 6 (2.4) 15 (6.0) 0 (0.0) 0 (0.0) Site 3 (N=350) n (%) 7 (2.0) 24 (6.9) 1 (0.3) 3 (0.9) Site 4 (N=892) n (%) 4 (0.4) 15 (1.7) 2 (0.2) 0 (0.0) Site 5 (N=296) n (%) 7 (2.4) 23 (7.8) 0 (0.0) 1 (0.3) Table 9: Expected Value (As Determined by ePlex RP Panel) Summary By Sample Collection Site in the Prospective Clinical Evaluation (Phase 2: September 2016 October 2016) Organism Adenovirus All Sites (N=511) n (%) 10 (2.0) Site 5 (N=49) n (%) 2 (4.1) Site 6 (N=101) n (%) 3 (3.0) Site 7 (N=161) n (%) 3 (1.9) Site 8 (N=200) n (%) 2 (1.0) Coronavirus (229E, HKU1, NL63, OC43) 8 (1.6) 0 (0.0) 2 (2.0) 4 (2.5) 2 (1.0) Human Metapneumovirus 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Human Rhinovirus/Enterovirus 188 (36.8) 24 (49.0) 49 (48.5) 62 (38.5) 53 (26.5) Influenza A 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza A H1 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza A H1-2009 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza A H3 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Influenza B 2 (0.4) 1 (2.0) 0 (0.0) 0 (0.0) 1 (0.5) Parainfluenza Virus 1 1 (0.2) 0 (0.0) 0 (0.0) 1 (0.6) 0 (0.0) Parainfluenza Virus 2 13 (2.5) 2 (4.1) 4 (4.0) 3 (1.9) 4 (2.0) Parainfluenza Virus 3 5 (1.0) 2 (4.1) 2 (2.0) 0 (0.0) 1 (0.5) Parainfluenza Virus 4 8 (1.6) 1 (2.0) 1 (1.0) 4 (2.5) 2 (1.0) RSV A 8 (1.6) 0 (0.0) 8 (7.9) 0 (0.0) 0 (0.0) RSV B 9 (1.8) 1 (2.0) 4 (4.0) 0 (0.0) 4 (2.0) Chlamydia pneumoniae 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Mycoplasma pneumoniae 4 (0.8) 0 (0.0) 3 (3.0) 0 (0.0) 1 (0.5) CLINICAL PERFORMANCE Clinical Performance of the ePlex RP2 Panel and SARS-CoV-2 Perf ormance characteristics of the ePlex RP2 Panel for SARS-CoV-2 detection were established using previously frozen clinical specimens (nasopharyngeal swab (NPS) samples) collected from U.S. patients. In the f irst arm of the study, a total of 189 samples, 174 NPS samples (60 known SARS-CoV-2 positive and 114 f rom the initial RP Panel clinical study) and 15 contrived samples were tested with the ePlex RP2 Panel in the clinical evaluation study. Samples with final, valid results and a comparator result were considered evaluable. Four samples (1 known SARS-CoV-2 positive, 3 from the initial RP Panel clinical study) were not evaluable because they did not have final, valid ePlex RP2 Panel results and were excluded from analysis. The comparator methods for the SARS-CoV-2 target were COVID-19 molecular diagnostic tests that received FDA Emergency Use Authorization (EUA). Only the 60 SARS-CoV-2 known positive NPS samples were tested with these methods. There was no comparator method for the SARS-CoV-2 target in the remaining 114 NPS samples from the initial clinical study. These samples were presumed SARSCoV-2 negative based on their collection prior to 2017. The comparator method for the other RP2 Panel PI1114 F For Use Under Emergency Use Authorization Only. Page 21 ePlex Respiratory Pathogen Panel 2 targets was the ePlex RP Panel. Only the 114 NPS samples from the initial RP Panel clinical study were tested with this method. Positive percent agreement (PPA) was calculated by dividing the number of true positive (TP) results by the sum of TP and false negative (FN) results, while negative percent agreement (NPA) was calculated by dividing the number of true negative (TN) results by the sum of TN and false positive (FP) results. A TP result was one where the detected ePlex RP2 Panel result matched the detected comparator method result, while a TN result was one where a negative ePlex RP2 Panel result matched a presumed negative result. Since archived negative samples were not tested by the comparator method, false positives are calculated based on a presumed negative clinical truth. The two-sided 95% confidence interval was also calculated. Results are shown in Table 10 below. Table 10. Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for SARS-CoV-2 in the ePlex RP2 Panel Clinical Study ePlex RP2 Test Comparator Result Positive Negative Total Positive 59 0 59 Negative 0 111 111 Total 59 111 170 PPA: 59/59 100% (95% CI: 93.9-100) NPA: 111/111 100% (95% CI: 96.7-100 In the second arm of the study, testing was done to evaluate the performance of ePlex RP2 Panel targets co-amplified with the SARS-CoV-2 assays (assays for SARS-CoV-2 were incorporated into PCR pools that also include influenza A, influenza A H1, influenza A H1-2009, influenza A H3, influenza B, and adenovirus; assays for all other targets were unchanged). Samples were tested with the ePlex RP2 Panel and the original ePlex Respiratory Pathogen Panel, or to the known contrived organism. For the Inf luenza A H1 target only, contrived samples were evaluated. Results are shown in Tables 11-12 below. Table 11: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of the ePlex RP2 Panel with the ePlex RP Panel in Nasopharyngeal Swab Samples Organism Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Adenovirus Influenza A 25/25 56/57b 100 (86.7-100) 98.2 (90.7-99.7) 83/86a 54/54 96.5 (90.2-98.8) 100 (93.4-100) Influenza A H1 Influenza A H1-2009 0/0 26/27c --96.3 (81.7-99.3) 111/111 84/84 100 (96.7-100) 100 (95.6-100) Influenza A H3 29/30b 96.7 (83.3-99.4) 81/81 100 (95.5-100) Influenza B 28/29d 96.6 (82.8-99.4) 82/82 100 (95.5-100) a 3 FP Adenovirus results were detected by the ePlex RP Panel (IUO) in the original clinical study. b 1 FN Influenza A H3 result was not detected by the RP Panel (IUO) in the original clinical study. c 1 FN Influenza A 2009 H1N1 result was detected by the RP Panel (IUO) in the original clinical study. d 1 FN Influenza B result was detected by the RP Panel (IUO) in the original clinical study. Table 12: Positive Percent Agreement (PPA) for the ePlex RP2 Panel with the ePlex RP Panel with Contrived Samples Organism Positive % Agreement TP/TP+FN PPA (95% CI) Influenza A 15/15 100 (79.6-100) Influenza A H1 15/15 100 (79.6-100) PI1114 F For Use Under Emergency Use Authorization Only. Page 22 ePlex Respiratory Pathogen Panel 2 RP2 Clinical Study ePlex Instrument Performance A total of 189 samples (174 NPS and 15 contrived) were initially tested with the ePlex RP2 Panel and 183/189 = 96.8% (95% CI: 93.2% - 98.5%) generated valid results on the first attempt. Two samples were re-tested and both had valid results. The remaining invalid samples did not have sufficient volume for repeat testing. Clinical Performance of the ePlex RP Panel Comparator Method The perf ormance of the ePlex RP Panel was compared to an FDA-cleared multiplexed molecular respiratory pathogen panel and analytically validated PCR tests with bi-directional sequencing for conf irmation of RSV subtypes. Details of the comparator method are described in Table 13. Table 13: Comparator Methods Used to Assess ePlex RP Panel Clinical Performance Target Comparator Method Adenovirus Coronavirus (229E, HKU1, NL63, OC43) Human Metapneumovirus Human Rhinovirus/Enterovirus Influenza A Influenza A H1 Influenza A H1-2009 Influenza A H3 Influenza B Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Parainfluenza Virus 4 Respiratory Syncytial Virus A Respiratory Syncytial Virus B Chlamydia pneumoniae Mycoplasma pneumoniae FDA-cleared multiplexed molecular respiratory pathogen panel FDA-cleared multiplexed molecular respiratory pathogen panel followed by a PCR test with bi-directional sequencing confirmation FDA-cleared multiplexed molecular respiratory pathogen panel Prospective Clinical Samples Clinical performance was evaluated in clinical nasopharyngeal swab samples in VTM prospectively collected at 8 clinical sites in 2 phases. From March 2013 through August 2014, 2218 samples were prospectively collected and frozen; from September 2016 through October 2016, 514 samples were prospectively collected and tested fresh (never frozen). A total of 2732 samples were collected across the 2 phases. Prior to the start of investigational testing, 263 samples were withdrawn (251 had sample handling deviations, 9 were tested outside of protocol timelines, 2 had insufficient volume, and 1 had incomplete documentation). Of the 2469 prospectively collected samples eligible for testing, 2462 were evaluable. Samples with final, valid results and a valid comparator result were considered evaluable. Seven prospectively collected samples were not evaluable because they did not have final, valid ePlex RP Panel results and were excluded from performance evaluations. Demographic information for prospectively collected samples is described in Table 14. Subjects enrolled in this study were f rom a diverse demographic distribution and represent the intended patient population. PI1114 F For Use Under Emergency Use Authorization Only. Page 23 ePlex Respiratory Pathogen Panel 2 Table 14: Subject Demographic Data for Prospectively Collected Samples by Collection Site (N=2462) All Sites N=2462 n (%) Site 1 N=165 n (%) Site 2 N=248 n (%) Site 3 N=350 n (%) Site 4 N=892 n (%) Site 5 N=345 n (%) Site 6 N=101 n (%) Site 7 N=161 n (%) Sex Male Female 1247 (50.6) 1215 (49.4) Age (years) 01 > 15 > 521 > 2165 388 (15.8) 325 (13.2) 321 (13.0) 926 (37.6) > 65 502 (20.4) 96 (58.2) 69 (41.8) 17 (10.3) 12 (7.3) 15 (9.1) 87 (52.7) 34 (20.6) 118 (47.6) 130 (52.4) 186 (53.1) 164 (46.9) 450 (50.4) 442 (49.6) 188 (54.5) 157 (45.5) 43 (42.6) 58 (57.4) 21 (8.5) 22 (8.9) 6 (2.4) 131 (52.8) 68 (27.4) 74 (21.1) 62 (17.7) 38 (10.9) 98 (28.0) 78 (22.3) 164 (18.4) 64 (7.2) 82 (9.2) 385 (43.2) 197 (22.1) 45 (13.0) 100 (29.0) 116 (33.6) 55 (15.9) 29 (8.4) 28 (27.7) 39 (38.6) 34 (33.7) 0 (0.0) 0 (0.0) 84 (52.2) 77 (47.8) 3 (1.9) 16 (9.9) 18 (11.2) 92 (57.1) 32 (19.9) Site 8 N=200 n (%) 82 (41.0) 118 (59.0) 36 (18.0) 10 (5.0) 12 (6.0) 78 (39.0) 64 (32.0) Prospective Clinical Performance Positive percent agreement (PPA) was calculated by dividing the number of true positive (TP) results by the sum of TP and false negative (FN) results, while negative percent agreement (NPA) was calculated by dividing the number of true negative (TN) results by the sum of TN and false positive (FP) results. A TP result was one where the detected ePlex RP Panel result matched the detected comparator method result, while a TN result was one where a negative ePlex RP Panel result matched a negative comparator method result. The two-sided 95% confidence interval was also calculated. A total of 2462 prospectively collected samples (511 tested fresh and 1951 tested after previously frozen) were evaluated for 17 ePlex RP Panel organisms. PPA and NPA results are summarized by target in Tables 15 and 16 below. Table 15: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) in the ePlex RP Panel Clinical Study (Fresh) Organism Positive % Agreement Negative % Agreement Prevalence TP/TP+FN PPA (95% CI) TN/TN+FP NPA (95% CI) Adenovirus 1.6% 6/8a Coronavirus (229E, HKU1, NL63, OC43) 1.4% 7/7 75.0 (40.9-92.9) 499/503a 100 (64.6-100) 503/504 99.2 (98.0-99.7) 99.8 (98.9-100) Human Metapneumovirus 0.0% Human Rhinovirus/Enterovirus 35.8% 0/0 176/183b --- 511/511 96.2 (92.3-98.1) 316/328b 100 (99.3-100) 96.3 (93.7-97.9) Influenza A 0.0% 0/0 --- 511/511 100 (99.3-100) Influenza A H1 0.0% 0/0 --- 511/511 100 (99.3-100) Influenza A H1-2009 0.0% 0/0 --- 511/511 100 (99.3-100) Influenza A H3 0.0% 0/0 --- 511/511 100 (99.3-100) Influenza B 0.2% 1/1 100 (20.7-100) 509/510 99.8 (98.9-100) Parainfluenza Virus 1 0.2% 1/1 100 (20.7-100) 510/510 100 (99.3-100) Parainfluenza Virus 2 2.5% 12/13 92.3 (66.7-98.6) 497/498 99.8 (98.9-100) Parainfluenza Virus 3 Parainfluenza Virus 4 1.0% 5/5 0.6% 3/3 100 (56.6-100) 506/506 100 (43.9-100) 503/508c 100 (99.2-100) 99.0 (97.7-99.6) RSV A 1.8% 8/9 88.9 (56.5-98.0) 501/501 100 (99.2-100) RSV B 2.0% 9/10 90.0 (59.6-98.2) 500/500 100 (99.2-100) PI1114 F For Use Under Emergency Use Authorization Only. Page 24 ePlex Respiratory Pathogen Panel 2 Organism Positive % Agreement Negative % Agreement Prevalence TP/TP+FN PPA (95% CI) TN/TN+FP NPA (95% CI) Chlamydia pneumoniae 0.0% 0/0 --- 511/511 100 (99.3-100) Mycoplasma pneumoniae 0.6% 3/3 100 (43.9-100) 507/508d 99.8 (98.9-100) a Adenovirus was not detected in 2 of 2 FN samples and detected in 4 of 4 FP samples using PCR/sequencing. b Human rhinovirus/enterovirus was not detected in 1 of 7 FN samples and detected in 9 of 12 FP samples using PCR/sequencing. c Parainfluenza virus 4 was detected in 3 of 5 FP samples using PCR/sequencing. d M. pneumoniae was detected in the 1 FP sample using PCR/sequencing. Table 16: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) in the ePlex RP Panel Clinical Study (After Previously Frozen) Organism Prevalence Adenovirus Coronavirus (229E, HKU1, NL63, OC43) 2.7% 5.6% Human Metapneumovirus 5.8% Human Rhinovirus/Enterovirus 17.2% Influenza Ae 5.7% Influenza A H1 0.0% Influenza A H1-2009 3.6% Influenza A H3 1.9% Influenza B 3.3% Parainfluenza Virus 1 1.2% Positive % Agreement TP/TP+FN PPA (95% CI) 48/53a 90.6 (79.7-95.9) Negative % Agreement TN/TN+FP NPA (95% CI) 1874/1898a 98.7 (98.1-99.1) 89/110b 80.9 (72.6-87.2) 1828/1841b 99.3 (98.8-99.6) 107/113c 317/336d 106/111f 0/0 70/71 34/37h 58/65i 23/24 94.7 (88.9-97.5) 94.3 (91.3-96.4) 95.5 (89.9-98.1) --98.6 (92.4-99.8) 91.9 (78.7-97.2) 89.2 (79.4-94.7) 95.8 (79.8-99.3) 1832/1838c 1544/1615d 1836/1840f 1951/1951 1874/1880g 1914/1914 1882/1886i 1926/1927 99.7 (99.3-99.9) 95.6 (94.5-96.5) 99.8 (99.4-99.9) 100 (99.8-100) 99.7 (99.3-99.9) 100 (99.8-100) 99.8 (99.5-99.9) 99.9 (99.7-100) Parainfluenza Virus 2 Parainfluenza Virus 3 0.5% 5.3% 9/9 94/104j 100 (70.1-100) 1941/1942 90.4 (83.2-94.7) 1842/1847j 99.9 (99.7-100) 99.7 (99.4-99.9) Parainfluenza Virus 4 0.3% 5/5 100 (56.6-100) 1944/1946 99.9 (99.6-100) RSV A 1.6% 27/31 87.1 (71.1-94.9) 1917/1918 99.9 (99.7-100) RSV B Chlamydia pneumoniae 4.4% 0.3% 81/86 2/5l 94.2 (87.1-97.5) 1861/1863k 40.0 (11.8-76.9) 1945/1946l 99.9 (99.6-100) 99.9 (99.7-100) Mycoplasma pneumoniae 0.3% 4/5m 80.0 (37.6-96.4) 1945/1946 99.9 (99.7-100) a Adenovirus was not detected in 1 of 5 FN samples and detected in 9 of 24 FP samples using PCR/sequencing. b Coronavirus was not detected in 2 of 21 FN samples and detected in 3 of 13 FP samples using PCR/sequencing. c Human Metapneumovirus was not detected in 1 of 6 FN samples and detected in 4 of 6 FP samples using PCR/sequencing. d Human rhinovirus/enterovirus was not detected in 6 of 19 FN samples and detected in 33 of 71 FP samples using PCR/sequencing. e Influenza A comparator results contain 71 samples with A H1-2009, 37 samples with A H3, and 3 samples with no subtype detected. f Influenza A was not detected in 1 of 3 FN samples (2 samples were not tested by PCR/sequencing) and detected in 1 of 4 FP samples using PCR/sequencing. g Influenza A H1-2009 was detected in 4 of 6 FP samples using PCR/sequencing. h Influenza A H3 was not detected in 1 of 3 FN samples using PCR/sequencing. i Influenza B was not detected in 3 of 7 FN samples and detected in 2 of 4 FP samples using PCR/sequencing. j Parainfluenza virus 3 was not detected in 3 of 10 FN samples and detected in 4 of 5 FP samples using PCR/sequencing. k RSV B was detected in 1 of 2 FP samples using PCR/sequencing. l C. pneumoniae was not detected in 1 of 3 FN samples and detected in the 1 FP sample using PCR/sequencing. m M. pneumoniae was not detected in the 1 FN sample using PCR/sequencing. Retrospective Clinical Samples To supplement the number of positives for targets that were not sufficiently represented in the prospective collection, additional nasopharyngeal swab in VTM samples were retrospectively collected from 6 sites. A PI1114 F For Use Under Emergency Use Authorization Only. Page 25 ePlex Respiratory Pathogen Panel 2 total of 535 nasopharyngeal swab samples that had previously tested positive for one or more of the target organisms during standard-of-care (SOC) testing were collected and stored frozen. Prior to the start of investigational testing, 11 samples were withdrawn due to noncompliance with the study protocol, and 52 samples were withdrawn because the organisms present had sufficient representation in other samples. In addition, the composition and integrity of the retrospective samples were confirmed with the same comparator method employed in the prospective clinical study (i.e., an FDA-cleared multiplexed respiratory pathogen panel). As the result of this confirmation testing using the comparator method, 26 additional samples were withdrawn because the original SOC testing positive results for the intended organisms were not confirmed when tested with the comparator method. Of the remaining 446 retrospectively collected samples eligible for testing, all 446 were evaluable. Demographic information for retrospectively collected samples is described in Table 17. Subjects enrolled in this study were f rom a diverse demographic distribution and represent the intended patient population. Sex Male Female Age (years) 0 1 > 15 > 5 21 > 21 65 > 65 Table 17: Subject Demographic Data for Retrospectively Collected Samples by Collection Site (N=446) All Sites N=446 n (%) Site 1 N=1 n (%) Site 2 N=1 n (%) Site 3 N=129 n (%) Site 4 N=18 n (%) Site 5 N=131 n (%) 232 (52.0) 214 (48.0) 0 (0.0) 1 (100) 1 (100) 0 (0.0) 76 (58.9) 53 (41.1) 11 (61.1) 7 (38.9) 68 (51.9) 63 (48.1) 122 (27.4) 107 (24.0) 59 (13.2) 99 (22.2) 59 (13.2) 0 (0.0) 0 (0.0) 0 (0.0) 1 (100) 0 (0.0) 0 (0.0) 1 (100) 0 (0.0) 0 (0.0) 0 (0.0) 24 (18.6) 51 (39.5) 9 (7.0) 11 (8.5) 34 (26.4) 5 (27.8) 3 (16.7) 2 (11.1) 8 (44.4) 0 (0.0) 56 (42.7) 16 (12.2) 19 (14.5) 31 (23.7) 9 (6.9) Site 6 N=166 n (%) 76 (45.8) 90 (54.2) 37 (22.3) 36 (21.7) 29 (17.5) 48 (28.9) 16 (9.6) Retrospective Clinical Performance A total of 446 retrospectively collected samples were evaluated for 17 ePlex RP Panel organisms. The f ollowing specimens with the original positive SOC results for the unintended organisms that were not conf irmed by the comparator method were excluded from the performance calculation for the respective organism: 1 coronavirus positive specimen, 3 human rhinovirus/enterovirus positive specimens, 1 inf luenza A positive specimen, 1 influenza A H3 positive specimen, 1 parainfluenza virus positive specimen. In addition, 5 unintended RSV positive specimens by the comparator method were not conf irmed by PCR/sequencing with regard to determining RSV subtypes and therefore were excluded f rom the performance calculations for RSV A and RSV B. PPA and NPA results are summarized by target in Table 18 below. Table 18: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of the ePlex RP Panel with Comparator Methods (Retrospective Collection) Organism Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Ad en o v i rus 55/56a 98.2 (90.6-99.7) 386/390a 99.0 (97.4-99.6) Coronavirus (229E, HKU1, NL63, OC43) 121/138b 87.7 (81.2-92.2) 307/307 100 (98.8-100) Human Metapneumovirus 5/7 71.4 (35.9-91.8) 439/439 100 (99.1-100) Human Rhinovirus/Enterovirus 37/41 90.2 (77.5-96.1) 384/402 95.5 (93.0-97.1) PI1114 F For Use Under Emergency Use Authorization Only. Page 26 ePlex Respiratory Pathogen Panel 2 Organism Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Influenza Ac 75/82d 91.5 (83.4-95.8) 363/363 100 (99.0-100) Influenza A H1 0/0 --- 446/446 100 (99.1-100) Influenza A H1-2009 Influenza A H3 27/31e 45/51f 87.1 (71.1-94.9) 88.2 (76.6-94.5) 415/415 394/394 100 (99.1-100) 100 (99.0-100) Influenza B Parainfluenza Virus 1 1/1 43/48g 100 (20.7-100) 89.6 (77.8-95.5) 445/445 396/397 100 (99.1-100) 99.7 (98.6-100) Parainfluenza Virus 2 46/51 90.2 (79.0-95.7) 395/395 100 (99.0-100) Parainfluenza Virus 3 2/2 100 (34.2-100) 444/444 100 (99.1-100) Parainfluenza Virus 4 18/20 90.0 (69.9-97.2) 426/426 100 (99.1-100) RSV A 25/27 92.6 (76.6-97.9) 414/414 100 (99.1-100) RSV B 21/22 95.5 (78.2-99.2) 419/419 100 (99.1-100) Chlamydia pneumoniae 1/1 100 (20.7-100) 445/445 100 (99.1-100) Mycoplasma pneumoniae 7/7 100 (64.6-100) 439/439 100 (99.1-100) a Adenovirus was not detected in the 1 FN sample and detected in 2 of 4 FP samples using PCR/sequencing. b Coronavirus was not detected in 2 of 16 FN samples using PCR/sequencing (1 sample was not tested by PCR/sequencing). c Influenza A comparator results contain 31 samples with A H1-2009 and 51 samples with A H3 detected. d Influenza A was not detected in 3 of 7 FN samples using PCR/sequencing. e Influenza A H1-2009 was not detected in 2 of 4 FN samples using PCR/sequencing. f Influenza A H3 was not detected in 1 of 6 FN samples using PCR/sequencing. g Parainfluenza virus 1 was not detected in 2 of 5 FN samples using PCR/sequencing. Contrived Sample Performance There were 327 contrived samples created and tested to supplement the low prevalence targets on the RP Panel; 104 contained one or more low prevalence organisms and 223 were negative for the contrived organisms. All 327 contrived samples were tested with the ePlex RP Panel and 326 were evaluable. PPA and NPA results are summarized for these low prevalence organisms in Table 19 below. Table 19: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) of the ePlex RP Panel with Comparator Method (Contrived Samples) Organism Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Chlamydia pneumoniae 52/52 100 (93.1-100) 274/274 100 (98.6-100) Influenza A H1 51/51 100 (93.0-100) 275/275 100 (98.6-100) Clinical and Contrived Sample Performance by Target Tables 20-36 below include the clinical performance by pathogen of prospective samples tested fresh (shown in Table 15), prospective samples tested after previously freezing (shown in Table 16), retrospective samples (shown in Table 18), and contrived samples (shown in Table 1). PI1114 F For Use Under Emergency Use Authorization Only. Page 27 ePlex Respiratory Pathogen Panel 2 Table 20: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Adenovirus in the ePlex RP Panel Clinical Study Adenovirus Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Fresh 6/8a 75.0 (40.9-92.9) 499/503a 99.2 (98.0-99.7) Prospectively Collected Samples Frozen Total 48/53b 54/61 90.6 (79.7-95.9) 88.5 (78.2-94.3) 1874/1898b 2373/2401 98.7 (98.1-99.1) 98.8 (98.3-99.2) Retrospectively Collected Samples 55/56c 98.2 (90.6-99.7) 386/390c 99.0 (97.4-99.6) a Adenovirus was not detected in 2 of 2 FN samples and detected in 4 of 4 FP samples using PCR/sequencing. b Adenovirus was not detected in 1 of 5 FN samples and detected in 9 of 24 FP samples using PCR/sequencing. c Adenovirus was not detected in the 1 FN sample and detected in 2 of 4 FP samples using PCR/sequencing. Table 21: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Coronavirus (229E, HKU1, NL63, OC43) in the ePlex RP Panel Clinical Study Coronavirus (229E, HKU1, NL63, OC43) Sample Positive % Agreement Type TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Fresh Prospectively Collected Samplesa Frozen 7/7 89/110b 100 (64.6-100) 80.9 (72.6-87.2) 503/504 1828/1841b 99.8 (98.9-100) 99.3 (98.8-99.6) Total 96/117 82.1 (74.1-88.0) 2331/2345 99.4 (99.0-99.6) Retrospectively Collected Samplesc 121/138d 87.7 (81.2-92.2) 307/307 100 (98.8-100) a 20 FN prospectively collected frozen samples were repeat tested with the comparator method and 12 had coronavirus detected. Of these 12 samples, 11 were repeat tested with the ePlex RP Panel and 3 had coronavirus detected. b Coronavirus was not detected in 2 of 21 FN samples and detected in 3 of 13 FP samples using PCR/sequencing. c 10 FN retrospectively collected samples were repeat tested with the comparator method and all 10 had coronavirus detected. Of these 10 samples, 9 were repeat tested with the ePlex RP Panel and 5 had coronavirus detected. d Coronavirus was not detected in 2 of 16 FN samples using PCR/sequencing (1 sample was not tested by PCR/sequencing). Table 22: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Human Metapneumovirus in the ePlex RP Panel Clinical Study Human Metapneumovirus Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Prospectively Collected Samples Fresh Frozen 0/0 107/113a --- 511/511 94.7 (88.9-97.5) 1832/1838a 100 (99.3-100) 99.7 (99.3-99.9) Total 107/113 94.7 (88.9-97.5) 2343/2349 99.7 (99.4-99.9) Retrospectively Collected Samples 5/7 71.4 (35.9-91.8) 439/439 100 (99.1-100) a Human Metapneumovirus was not detected in 1 of 6 FN samples and detected in 4 of 6 FP samples using PCR/sequencing. Table 23: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Human Rhinovirus/Enterovirus in the ePlex RP Panel Clinical Study Human Rhinovirus/Enterovirus Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Prospectively Collected Samples Fresh Frozen 176/183a 317/336b 96.2 (92.3-98.1) 316/328a 94.3 (91.3-96.4) 1544/1615b 96.3 (93.7-97.9) 95.6 (94.5-96.5) Total 493/519 95.0 (92.8-96.6) 1860/1943 95.7 (94.7-96.5) Retrospectively Collected Samples 37/41 90.2 (77.5-96.1) 384/402 95.5 (93.0-97.1) a Human rhinovirus/enterovirus was not detected in 1 of 7 FN samples and detected in 9 of 12 FP samples using PCR/sequencing. b Human rhinovirus/enterovirus was not detected in 6 of 19 FN samples and detected in 33 of 71 FP samples using PCR/sequencing. PI1114 F For Use Under Emergency Use Authorization Only. Page 28 ePlex Respiratory Pathogen Panel 2 Table 24: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Influenza A in the ePlex RP Panel Clinical Study Influenza A Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Fresh 0/0 --- 511/511 100 (99.3-100) Prospectively Collected Samplesa Frozen 106/111b 95.5 (89.9-98.1) 1836/1840b 99.8 (99.4-99.9) Total 106/111 95.5 (89.9-98.1) 2347/2351 99.8 (99.6-99.9) Retrospectively Collected Samplesc 75/82d 91.5 (83.4-95.8) 363/363 100 (99.0-100) a Influenza A comparator results contain 71 samples with A H1-2009, 37 samples with A H3, and 3 samples with no subtype detected. b Influenza A was not detected in 1 of 3 FN samples (2 samples were not tested by PCR/sequencing) and detected in 1 of 4 FP samples using PCR/sequencing. c Influenza A comparator results contain 31 samples with A H1-2009 and 51 samples with A H3 detected. d Influenza A was not detected in 3 of 7 FN samples using PCR/sequencing. Table 25: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Influenza A H1 in the ePlex RP Panel Clinical Study Influenza A H1 Prospectively Collected Samples Retrospectively Collected Samples Contrived Samples Sample Type Fresh Frozen Total Positive % Agreement TP/TP+FN PPA (95% CI) 0/0 0/0 0/0 0/0 51/51 --------100 (93.0-100) Negative % Agreement TN/TN+FP NPA (95% CI) 511/511 1951/1951 2462/2462 446/446 275/275 100 (99.3-100) 100 (99.8-100) 100 (99.8-100) 100 (99.1-100) 100 (98.6-100) Table 26: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Influenza A H1-2009 in the ePlex RP Panel Clinical Study Influenza A H1-2009 Sample Positive % Agreement Type TP/TP+FN PPA (95% CI) Fresh 0/0 --- Prospectively Collected Samples Frozen 70/71 98.6 (92.4-99.8) Total 70/71 98.6 (92.4-99.8) Retrospectively Collected Samples 27/31b 87.1 (71.1-94.9) a Influenza A H1-2009 was detected in 4 of 6 FP samples using PCR/sequencing. b Influenza A H1-2009 was not detected in 2 of 4 FN samples using PCR/sequencing. Negative % Agreement TN/TN+FP NPA (95% CI) 511/511 1874/1880a 2385/2391 415/415 100 (99.3-100) 99.7 (99.3-99.9) 99.7 (99.5-99.9) 100 (99.1-100) Table 27: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Influenza A H3 in the ePlex RP Panel Clinical Study Influenza A H3 Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Fresh 0/0 --- Prospectively Collected Samples Frozen 34/37a 91.9 (78.7-97.2) Total 34/37 91.9 (78.7-97.2) Retrospectively Collected Samples 45/51b 88.2 (76.6-94.5) a Influenza A H3 was not detected in 1 of 3 FN samples using PCR/sequencing. b Influenza A H3 was not detected in 1 of 6 FN samples using PCR/sequencing. Negative % Agreement TN/TN+FP NPA (95% CI) 511/511 100 (99.3-100) 1914/1914 100 (99.8-100) 2425/2425 100 (99.8-100) 394/394 100 (99.0-100) PI1114 F For Use Under Emergency Use Authorization Only. Page 29 ePlex Respiratory Pathogen Panel 2 Table 28: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Influenza B in the ePlex RP Panel Clinical Study Influenza B Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Prospectively Collected Samples Fresh Frozen Total 1/1 58/65a 59/66 100 (20.7-100) 89.2 (79.4-94.7) 89.4 (79.7-94.8) 509/510 1882/1886a 2391/2396 99.8 (98.9-100) 99.8 (99.5-99.9) 99.8 (99.5-99.9) Retrospectively-Collected Samples 1/1 100 (20.7-100) 445/445 100 (99.1-100) a Influenza B was not detected in 3 of 7 FN samples and detected in 2 of 4 FP samples using PCR/sequencing. Table 29: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Parainfluenza Virus 1 in the ePlex RP Panel Clinical Study Parainfluenza Virus 1 Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Fresh 1/1 100 (20.7-100) Prospectively Collected Samples Frozen 23/24 95.8 (79.8-99.3) Total 24/25 96.0 (80.5-99.3) Retrospectively Collected Samples 43/48a 89.6 (77.8-95.5) a Parainfluenza virus 1 was not detected in 2 of 5 FN samples using PCR/sequencing. Negative % Agreement TN/TN+FP NPA (95% CI) 510/510 1926/1927 2436/2437 396/397 100 (99.3-100) 99.9 (99.7-100) 100 (99.8-100) 99.7 (98.6-100) Table 30: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Parainfluenza Virus 2 in the ePlex RP Panel Clinical Study Parainfluenza Virus 2 Prospectively Collected Samples Retrospectively Collected Samples Sample Type Fresh Frozen Total Positive % Agreement TP/TP+FN PPA (95% CI) 12/13 9/9 21/22 46/51 92.3 (66.7-98.6) 100 (70.1-100) 95.5 (78.2-99.2) 90.2 (79.0-95.7) Negative % Agreement TN/TN+FP NPA (95% CI) 497/498 1941/1942 2438/2440 395/395 99.8 (98.9-100) 99.9 (99.7-100) 99.9 (99.7-100) 100 (99.0-100) Table 31: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Parainfluenza Virus 3 in the ePlex RP Panel Clinical Study Parainfluenza Virus 3 Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Negative % Agreement TN/TN+FP NPA (95% CI) Fresh 5/5 100 (56.6-100) 506/506 100 (99.2-100) Prospectively Collected Samples Frozen 94/104a 90.4 (83.2-94.7) 1842/1847a 99.7 (99.4-99.9) Total 99/109 90.8 (83.9-94.9) 2348/2353 99.8 (99.5-99.9) Retrospectively Collected Samples 2/2 100 (34.2-100) 444/444 100 (99.1-100) a Parainfluenza virus 3 was not detected in 3 of 10 FN samples and detected in 4 of 5 FP samples using PCR/sequencing. Table 32: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Parainfluenza Virus 4 in the ePlex RP Panel Clinical Study Parainfluenza Virus 4 Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Fresh 3/3 100 (43.9-100) Prospectively Collected Samples Frozen 5/5 100 (56.6-100) Total 8/8 100 (67.6-100) Retrospectively Collected Samples 18/20 90.0 (69.9-97.2) a Parainfluenza virus 4 was detected in 3 of 5 FP samples using PCR/sequencing. Negative % Agreement TN/TN+FP 503/508a 1944/1946 2447/2454 426/426 NPA (95% CI) 99.0 (97.7-99.6) 99.9 (99.6-100) 99.7 (99.4-99.9) 100 (99.1-100) PI1114 F For Use Under Emergency Use Authorization Only. Page 30 ePlex Respiratory Pathogen Panel 2 Table 33: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Respiratory Syncytial Virus A in the ePlex RP Panel Clinical Study Respiratory Syncytial Virus A Prospectively Collected Samples Retrospectively Collected Samples Sample Type Fresh Frozen Total Positive % Agreement TP/TP+ FN PPA (95% CI) 8/9 88.9 (56.5-98.0) 27/31 87.1 (71.1-94.9) 35/40 87.5 (73.9-94.5) 25/27 92.6 (76.6-97.9) Negative % Agreement TN/TN+FP NPA (95% CI) 501/501 1917/1918 2418/2419 414/414 100 (99.2-100) 99.9 (99.7-100) 100 (99.8-100) 100 (99.1-100) Table 34: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Respiratory Syncytial Virus B in the ePlex RP Panel Clinical Study Respiratory Syncytial Virus B Sample Type Positive % Agreement TP/TP+ FN PPA (95% CI) Fresh 9/10 90.0 (59.6-98.2) Prospectively Collected Samples Frozen 81/86 94.2 (87.1-97.5) Total 90/96 93.8 (87.0-97.1) Retrospectively Collected Samples 21/22 a RSV B was detected in 1 of 2 FP samples using PCR/sequencing. 95.5 (78.2-99.2) Negative % Agreement TN/TN+FP NPA (95% CI) 500/500 1861/1863a 2361/2363 419/419 100 (99.2-100) 99.9 (99.6-100) 99.9 (99.7-100) 100 (99.1-100) Table 35: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Chlamydia pneumoniae in the ePlex RP Panel Clinical Study Chlamydia pneumoniae Sample Positive % Agreement Negative % Agreement Type TP/TP+FN PPA (95% CI) TN/TN+FP NPA (95% CI) Prospectively Collected Samples Fresh 0/0 Frozen 2/5a --- 511/511 40.0 (11.8-76.9) 1945/1946a 100 (99.3-100) 99.9 (99.7-100) Total 2/5 40.0 (11.8-76.9) 2456/2457 100 (99.8-100) Retrospectively Collected Samples 1/1 100 (20.7-100) 445/445 100 (99.1-100) Contrived Samples 52/52 100 (93.1-100) 274/274 100 (98.6-100) a C. pneumoniae was not detected in 1 of 3 FN samples and detected in the 1 FP sample using PCR/sequencing. Table 36: Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) for Mycoplasma pneumoniae in the ePlex RP Panel Clinical Study Mycoplasma pneumoniae Sample Type Positive % Agreement TP/TP+FN PPA (95% CI) Prospectively Collected Samples Fresh 3/3 Frozen 4/5b 100 (43.9-100) 80.0 (37.6-96.4) Total 7/8 87.5 (52.9-97.8) Retrospectively Collected Samples 7/7 100 (64.6-100) a M. pneumoniae was detected in the 1 FP sample using PCR/sequencing. b M. pneumoniae was not detected in the 1 FN sample using PCR/sequencing. Negative % Agreement TN/TN+FP 507/508a 1945/1946 2452/2454 439/439 NPA (95% CI) 99.8 (98.9-100) 99.9 (99.7-100) 99.9 (99.7-100) 100 (99.1-100) Co-detections in Prospective Clinical Samples The ePlex RP Panel identified a total of 135 prospective samples with multiple organisms detected, or 5.5% of all prospectively collected samples. Of these, 118 (4.8%) had two organisms, 14 (0.6%) had three organisms, and 3 (0.1%) had four organisms detected. Of the 135 co-detected samples, 58 included 1 or more organisms that had not been detected by the comparator method(s). Results are summarized in Table 37 and 38. PI1114 F For Use Under Emergency Use Authorization Only. Page 31 ePlex Respiratory Pathogen Panel 2 Table 37: Distinct Co-Detection Combinations Detected by the ePlex RP Panel in the Prospective Clinical Samples Distinct Co-Detection Combinations Detected by the ePlex RP Panel Organism 1 Organism 2 Organism 3 Organism 4 Total Number Of Co-detections (% of samples) Number of Discrepant Co-detections Discrepant Organism(s) a ADV CoV 2 (0.08%) 0 ADV CoV HRV/EV 2 (0.08%) 1 ADV (1) ADV Flu A (unk) Flu B HRV/EV 1 (0.04%) 1 ADV Flu AH3 1 (0.04%) 0 ADV (1), Flu A (unk) (1), Flu B (1), HRV/EV (1) ADV Flu B HRV/EV RSV B 1 (0.04%) 1 ADV FluA09H1 1 (0.04%) 1 ADV (1), Flu B (1) ADV (1), FluA09H1 (1) ADV FluA09H1 HRV/EV 1 (0.04%) 0 ADV FluA09H1 PIV 3 1 (0.04%) 1 PIV 3 (1) ADV HMPV 3 (0.12%) 2 ADV HMPV HRV/EV RSV A 1 (0.04%) 1 ADV (2) RSV A (1) ADV ADV HRV/EV HRV/EV Mpneum 18 (0.73%) 7 1 (0.04%) 0 ADV (6), HRV/EV (1) ADV HRV/EV PIV 1 1 (0.04%) 1 PIV 1 (1) ADV HRV/EV PIV 4 1 (0.04%) 1 ADV (1), PIV 4 (1) ADV ADV HRV/EV PIV 2 RSV B 1 (0.04%) 0 2 (0.08%) 1 ADV (1) ADV ADV PIV 3 PIV 4 2 (0.08%) 1 1 (0.04%) 1 ADV (1) ADV (1) ADV RSV B 2 (0.08%) 2 ADV (2) CPneum HRV/EV 1 (0.04%) 0 CoV FluA09H1 CoV HMPV 1 (0.04%) 0 4 (0.16%) 0 CoV HMPV HRV/EV CoV HRV/EV 2 (0.08%) 0 12 (0.49%) 4 CoV (1), HRV/EV (4) CoV HRV/EV RSV B 1 (0.04%) 1 CoV (1) CoV PIV 1 1 (0.04%) 0 CoV RSV A CoV RSV B 3 (0.12%) 0 3 (0.12%) 2 CoV (2) Flu A (unk) Flu AH3 HRV/EV HRV/EV 1 (0.04%) 1 2 (0.08%) 1 Flu A (unk) (1) HRV/EV (1) Flu AH3 RSV B 1 (0.04%) 0 Flu B HRV/EV 4 (0.16%) 2 HRV/EV (2) Flu B Flu B HRV/EV PIV 3 RSV B 1 (0.04%) 0 1 (0.04%) 0 FluA09H1 FluA09H1 HMPV HRV/EV HRV/EV 1 (0.04%) 1 2 (0.08%) 1 HRV/EV (1) HRV/EV (1) HMPV HRV/EV 5 (0.20%) 1 HRV/EV (1) HMPV HRV/EV RSV B 1 (0.04%) 1 HRV/EV (1) HMPV HRV/EV PIV 3 PIV 1 1 (0.04%) 0 3 (0.12%) 0 HRV/EV PIV 2 7 (0.28%) 3 HRV/EV (1), PIV 2 (2) PI1114 F For Use Under Emergency Use Authorization Only. Page 32 ePlex Respiratory Pathogen Panel 2 Distinct Co-Detection Combinations Detected by the ePlex RP Panel Organism 1 Organism 2 Organism 3 Organism 4 Total Number Of Co-detections (% of samples) Number of Discrepant Co-detections Discrepant Organism(s) a HRV/EV PIV 3 11 (0.45%) 5 HRV/EV (5) HRV/EV PIV 4 4 (0.16%) 4 PIV 4 (4) HRV/EV RSV A 5 (0.20%) 0 HRV/EV PIV 1 RSV B PIV 4 11 (0.45%) 6 1 (0.04%) 1 HRV/EV (6) PIV 4 (1) PIV 3 RSV B 1 (0.04%) 0 RSV A RSV B 1 (0.04%) 1 RSV B (1) Total Number of Co-Detections 135 (5.5%) 57 64/290b Total Number with 2 Organisms Detected 118 (4.8%) 47 49/236 Total Number with 3 Organisms Detected 14 (0.6%) 7 8/42 Total Number with 4 Organisms Detected 3 (0.1%) 3 7/12 Note: ADV= adenovirus, CoV= coronavirus (229E, HKU1, NL63, OC43), HMPV= human metapneumovirus, HRV/EV= human rhinovirus/enterovirus, Flu= Influenza, (unk)= unknown subtype, PIV= parainfluenza, RSV= respiratory syncytial virus, Cpneum= C. pneumoniae, Mpneum= M. pneumoniae a A discrepant organism is defined as one that was detected by the ePlex RP Panel but not by the comparator method(s). b 64/64 discrepant organisms were investigated using PCR/sequencing; the discrepant organism was detected in 20/64 cases: -In 8/18 samples, adenovirus was detected by PCR/sequencing. -In 1/4 samples, coronavirus was detected by PCR/sequencing. -In 7/25 samples, human rhinovirus/enterovirus was detected by PCR/sequencing. -In 1/1 sample, influenza A H1-2009 was detected by PCR/sequencing. -In 1/1 sample, parainfluenza virus 3 was detected by PCR/sequencing. -In 2/6 samples, parainfluenza virus 4 was detected by PCR/sequencing. Table 38: Additional Co-Detection Combinations Detected by the Comparator Method in the Prospective Clinical Samples Distinct Co-Detection Combinations Detected by the Comparator Method Organism 1 Organism 2 Organism 3 Total Number Of Co-detections (% of samples) Number of Discrepant Co-detections Discrepant Organism(s) a,b ADV CoV 1 (0.04%) 1 ADV (1), CoV (1) ADV HRV/EV 4 (0.16%) 4 ADV (4) ADV HRV/EV PIV 3 1 (0.04%) 1 HRV/EV (1), PIV 3 (1) ADV HRV/EV RSV A 1 (0.04%) 1 ADV (1) CPneum HRV/EV 1 (0.04%) 1 CPneum (1) CPneum PIV 3 1 (0.04%) 1 CPneum (1) CoV FluA09H1 2 (0.08%) 2 CoV (2) CoV HMPV 1 (0.04%) 1 CoV (1) CoV HRV/EV 6 (0.24%) 6 CoV (4), HRV/EV (2) CoV PIV 3 1 (0.04%) 1 CoV (1) CoV RSV B 3 (0.12%) 3 CoV (2), RSV B (1) Flu AH3 HRV/EV PIV 3 1 (0.04%) 1 Flu AH3 (1), PIV 3 (1) Flu AH3 PIV 3 1 (0.04%) 1 PIV 3 (1) FluA09H1 HMPV HRV/EV 1 (0.04%) 1 HMPV (1), HRV/EV (1) HMPV HRV/EV 1 (0.04%) 1 HRV/EV (1) HRV/EV PIV 1 1 (0.04%) 1 HRV/EV (1) HRV/EV PIV 3 2 (0.08%) 2 HRV/EV (2) HRV/EV PIV 3 RSV B 1 (0.04%) 1 PIV 3 (1) HRV/EV RSV A 2 (0.08%) 2 RSV A (2) a A discrepant organism is defined as one that was detected by the comparator method(s) but not by the ePlex RP Panel. b 36/36 discrepant organisms were investigated using PCR/sequencing; the discrepant organism was not detected in 10/36 cases: -In 2/6 samples, adenovirus was not detected by PCR/sequencing. -In 1/2 samples, Chlamydia pneumoniae was not detected by PCR/sequencing. PI1114 F For Use Under Emergency Use Authorization Only. Page 33 ePlex Respiratory Pathogen Panel 2 -In 1/11 samples, coronavirus was not detected by PCR/sequencing. -In 5/8 samples, human rhinovirus/enterovirus was not detected by PCR/sequencing. -In 1/1 sample, influenza A H3 was not detected by PCR/sequencing. Clinical Study ePlex RP Panel Instrument Performance A total of 3281 samples (including prospective, retrospective, and contrived samples) were initially tested in the clinical evaluations and 3127/3281 = 95.3% (95% CI: 94.5%-96.0%) generated valid results on the f irst attempt. After re-test, 8 samples had invalid results; final validity rate was 3273/3281 = 99.8% (95% CI: 99.5%-99.9%). ANALYTICAL PERFORMANCE CHARACTERISTICS Limit of Detection for SARS-CoV-2 The limit of detection (LoD), or analytical sensitivity was identified and verified for SARS-CoV-2 using commercially available heat inactivated quantified virus. Serial dilutions were prepared in a natural clinical matrix (pooled, negative nasopharyngeal swab in VTM) and at least 20 replicates per concentration were tested in the study. The limit of detection was defined as the lowest concentration at which SARS-CoV-2 is detected at least 95% of the time. The confirmed LoD for detection of SARS-CoV2 is shown in Table 39. Table 39: SARS-CoV-2 LoD Results Summary Target Strain LoD Concentration SARS-Co V-2 USA-WA1/2020 1 x 10-2 TCID50/mLa a The LoD concentration for detection of SARS-CoV-2 was determined to be 0.01 TCID50/mL, which corresponds to 250 genomic copies per milliliter, as determined by digital droplet PCR. FDA SARS-CoV-2 Reference Panel Testing The evaluation of sensitivity and MERS-CoV cross-reactivity was performed using reference material (T1), blinded samples and a standard protocol provided by the FDA. The study included a range finding study and a confirmatory study for LoD. Blinded sample testing was used to establish specificity and to corroborate the LoD. The ePlex RP2 Panel showed no cross-reactivity with MERS-CoV at the highest concentration supplied. The LoD of the ePlex RP2 Panel using the FDA SARS-CoV-2 Reference Panel was observed to be 1.8x105 NDU/mL. The results of the FDA SARS-CoV-2 Reference Panel testing are summarized in Table 40. Table 40: FDA SARS-CoV-2 Reference Panel Testing Summary FDA Reference Material Specimen Type ePlex RP2 Panel LoD Cross-Reactivity SARS-Co V-2 NPS 1.8 x 105 NDU/mLa N/A MERS-Co V NPS N/A ND a The sample matrix used for the FDA reference panel, Minimum Essential Media (MEM), is not a media typically used for collection of respiratory specimens; this material was not tested as part of the interfering substances study (shown in Table 67). Additional characterization testing indicated that the diluent used for the FDA SARS-CoV-2 Reference Panel (Minimal Essential Media; MEM) may interfere with the performance of the ePlex RP2 Panel. NDU/mL: RNA NAAT detectable units/mL N/A: Not applicable ND: Not detected PI1114 F For Use Under Emergency Use Authorization Only. Page 34 ePlex Respiratory Pathogen Panel 2 Limit of Detection for All other RP2 Panel Targets The limit of detection (LoD), or analytical sensitivity was identified and verified for each viral and bacterial target on the ePlex RP2 Panel using quantified reference strains/isolates. Serial dilutions were prepared in a natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples) with one or more organisms per series, and at least 20 replicates per target were tested in the study. The limit of detection was def ined as the lowest concentration at which each target is detected at least 95% of the time. The conf irmed LoD for each ePlex RP2 Panel organism is shown in Table 41. Table 41: LoD Results Summary Target Strain Ad en o v i rus Coronavirus 229E Coronavirus HKU1 Coronavirus NL63 Coronavirus OC43 Human Metapneumovirus Human Rhinovirus/Enterovirus Influenza A Influenza A H1 Influenza A H1-2009 Influenza A H3 Influenza B (Victoria Lineage) Influenza B (Yamagata Lineage) Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Type 1 (C) Type 4 (E) Type 7 (B) 229E HKU1a NL63 OC43 A1 IA3-2002 Type Bb B1 Peru2-2002 B2 Peru1-2002 Enterovirus Type 68 (2007) Rhinovirus 1A Rhinovirus B14 Rhinovirus Ca H1N1 Brisbane/59/07 H1N1 Brisbane/59/07 NY/01/2009 A/Perth /16/2009 A/Texas/50/2012 A/Vi c to ri a/361/2011 H3N2 Brisbane/10/07 B/Bri s ban e/60/2008 B/Mo n tan a/5/2012 B/Nev ad a/03/2011 Fl o ri d a/02/06 B/Mas s ac h us etts /02/2012 B/Texas/06/2011 B/W i s c o n s i n/01/2010 Clinical Isolate Clinical Isolate Clinical Isolate LoD Concentration 1 x 103 TCID50/mL 2 x 100 TCID50/mL 2 x 100 TCID50/mL 1 x 100 TCID50/mL 5 x 104 copies/mL 7.5 x 100 TCID50/mL 5 x 102 TCID50/mL 2 x 10-1 TCID50/mL 2 x 103 TCID50/mL 2 x 102 TCID50/mL 2.25 x 102 TCID50/mL 1 x 100 TCID50/mL 1.5 x 100 TCID50/mL 1 x 100 TCID50/mL 1 x 105 copies/mL 3 x 10-1 TCID50/mL 3 x 10-1 TCID50/mL 1 x 10-1 TCID50/mL 1 x 101 TCID50/mL 1 x 100 TCID50/mL 5 x 10-1 TCID50/mL 5 x 101 TCID50/mL 1 x 100 TCID50/mL 1 x 100 TCID50/mL 1 x 100 TCID50/mL 1 x 10-1 TCID50/mL 1 x 102 TCID50/mL 1 x 10-1 TCID50/mL 1 x 100 TCID50/mL 4 x 10-1 TCID50/mL 5 x 101 TCID50/mL 5 x 100 TCID50/mL PI1114 F For Use Under Emergency Use Authorization Only. Page 35 ePlex Respiratory Pathogen Panel 2 Target Strain LoD Concentration Parainfluenza Virus 4 4a 3 x 101 TCID50/mL Respiratory Syncytial Virus A 2006 Isolate 1.5 x 100 TCID50/mL Respiratory Syncytial Virus B CH93(18)-18 2 x 10-1 TCID50/mL Chlamydia pneumoniae AR-39 3 x 102 TCID50/mL Mycoplasma pneumoniae FH strain of Eaton Agent [NCTC 10119] 3 x 102 CCU/mL a Clinical samples confirmed positive for coronavirus HKU1 and human rhinovirus C by bi-directional sequencing and quantified by real-time RT-PCR were used for determination of LoD. b The human metapneumovirus strain tested was originally identified as A2 IA14-2003. Subsequent vendor communications identified an error and corrected the strain identification as type B. Analytical Reactivity (Inclusivity) Reactivity of SARS-CoV-2 Assays Inclusivity was evaluated using RNA for SARS-CoV-2 (Hong Kong/VM20001061/2020) at 750 copies/mL. Three replicates were tested and all replicates were detected as expected as shown in Table 42. Table 42: Analytical Reactivity (Inclusivity) Results for SARS-CoV-2 Target Test Material SARS-CoV-2 Hong Kong/VM20001061/2020 (BEI Resource Isolated RNA) Concentration 750 copies/mL Percent Detected (positive replicates / total) 100% (3/3) Predicted (in silico) Reactivity (Inclusivity) Results for SARS-CoV-2 In silico analysis of >118,000 sequences from GISAID was conducted to assess the ability of the ePlex RP2 Panel to detect the most recent COVID-19 strains (analysis was conducted on October 5, 2020). The results of these analyses show that the sequences are 99% identical. Inclusivity of All Other RP2 Targets Inclusivity of all other RP2 Panel targets was evaluated using a panel of strains/isolates representing the genetic, temporal, and geographic diversity of each target on the panel to demonstrate analytical reactivity. Each strain/isolate was tested in triplicate at 3x LoD in natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples); if the organism was not detected at this concentration, testing of higher concentrations was performed. Additional in silico analysis was also performed on a subset of ePlex RP2 Panel organisms. All of the strains/isolates tested for inclusivity were detected by the ePlex RP2 Panel. Results of analytical reactivity are shown in Tables 43-53. Table 43: Analytical Reactivity (Inclusivity) Results for Adenovirus Adenovirus Species Serotype Concentration Multiple of LoD Detected A Type 31 3 x 103 TCID50/mL 3x Type 3 6 x 100 TCID50/mL 3x B Type 11 6 x 100 TCID50/mL 3x De Wit Type 14 6 x 100 TCID50/mL 3x PI1114 F For Use Under Emergency Use Authorization Only. Page 36 ePlex Respiratory Pathogen Panel 2 Adenovirus Species Serotype Concentration Multiple of LoD Detected Ch.79 Type 16 2 x 102 TCID50/mL 100xa Type 21 6 x 100 TCID50/mL 3x Compton Type 34 6 x 100 TCID50/mL 3x Holden Type 35 6 x 100 TCID50/mL 3x Wan Type 50 2 x 101 TCID50/mL 10xb Type 2 3 x 103 TCID50/mL 3x C Type 5 3 x 103 TCID50/mL 3x Type 6 3 x 103 TCID50/mL 3x Type 26 3 x 103 TCID50/mL 3x D Type 37 3 x 103 TCID50/mL 3x Type 40 Dugan 3 x 103 TCID50/mL 3x F Type 41/ Strain Tak 3 x 103 TCID50/mL 3x a In silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic material present in the culture of this or the reference strain (TCID50 value is based only on infectious virus particles). b In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. Table 44: Analytical Reactivity (Inclusivity) Results for Human Metapneumovirus Metapneumovirus Subtype Human Metapneumovirus Strain Concentration Peru6-2003 G, B2 6.75 x 102 TCID50/mL Multiple of LoD Detected 3x Table 45: Analytical Reactivity (Inclusivity) Results for Human Rhinovirus/Enterovirus Rhinovirus/Enterovirus Human Rhinovirus En tero v i rus Co x s ac k i ev i rus Strain Type A2 Type A7 Type A16 Type A18 Type A34 Type A57 Type A77 277G Type B3 Type B17 Type B42 Type B83 Type B84 FO2-2547 Type 71 A9 A10 A21 A24 B2 Concentration 4.5 x 100 TCID50/mL 1.5 x 101 TCID50/mL 4.5 x 100 TCID50/mL 1.5 x 102 TCID50/mL 4.5 x 100 TCID50/mL 4.5 x 100 TCID50/mL 4.5 x 100 TCID50/mL 4.5 x 100 TCID50/mL 1.5 x 101 TCID50/mL 1.5 x 101 TCID50/mL 4.5 x 100 TCID50/mL 4.5 x 100 TCID50/mL 4.5 x 100 TCID50/mL 4.5 x 100 TCID50/mL 3 x 100 TCID50/mL 3 x 100 TCID50/mL 3 x 100 TCID50/mL 3 x 100 TCID50/mL 3 x 100 TCID50/mL 1 x 102 TCID50/mL Multiple of LoD Detected 3x 10xa 3x 100xa 3x 3x 3x 3x 10xa 10xa 3x 3x 3x 3x 3x 3x 3x 3x 3x 100xa PI1114 F For Use Under Emergency Use Authorization Only. Page 37 ePlex Respiratory Pathogen Panel 2 Rhinovirus/Enterovirus Strain Concentration Multiple of LoD Detected B3 3 x 100 TCID50/mL 3x B4 3 x 100 TCID50/mL 3x B5 1 x 101 TCID50/mL 10xa 9 3 x 100 TCID50/mL 3x E6 Ec h o v i rus 25 1 x 101 TCID50/mL 1 x 101 TCID50/mL 10xb 10xa 30 3 x 100 TCID50/mL 3x Po l i o v i rus 1 1 x 102 TCID50/mL 100xa a In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. b In silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic material present in the culture of this or the reference strain (TCID50 value is based only on infectious virus particles). Table 46: Analytical Reactivity (Inclusivity) Results for Influenza A Note: Due to different assays for influenza A matrix and influenza A subtypes on the ePlex RP Panel, if dif ferent LoDs are observed for inclusivity for an Inf luenza A matrix vs. HA subtype, the differences are noted in the Multiple of LoD Detected column. Influenza A Subtype Strain A/FM/1/47 Concentration 3 x 100 TCID50/mL Multiple of LoD Detected 10x (Influenza A matrix)a 10000x H1 subtypeb A/New Caledonia/20/1999 9 x 10-1 TCID50/mL 3x A/New Jersey/8/76 9 x 10-1 TCID50/mL 3x H1 subtype not detectedc Influenza A H1 A/NWS/33 3 x 100 TCID50/mL 10x (Influenza A matrix)a H1 subtype not detectedd A/PR/8/34 9 x 10-1 TCID50/mL 3x (Influenza A matrix) H1 subtype not detectede A/Solomon Islands/3/2006 9 x 100 TCID50/mL 30x A/Tai wan /42/06 9 x 100 TCID50/mL 30xf A/Hong Kong/8/68 A/Port Chalmers/1/73 Influenza A H3 A/Nan c h an g /933/95 A/Vi c to ri a/3/75 1.5 x 102 TCID50/mL 3x A/W i s c o n s i n/67/05 A/Cal i fo rn i a/7/2009 1 x 100 TCID50/mL 10xg A/Mex i c o /4108/09 3 x 10-1 TCID50/mL 3x Influenza A 2009 H1N1 A/NY/02/2009 A/Swine NY/03/2009 A/Swi n e/Io wa/15/30 A/Vi rg i n i a/ATCC1/2009 1 x 100 TCID50/mL 3 x 10-1 TCID50/mL 3 x 10-1 TCID50/mL 1 x 100 TCID50/mL 10xh 3x 3x (Influenza A matrix) 100,000x (H1-2009 subtype)i 10xj A/Vi rg i n i a/ATCC2/2009 1 x 101 TCID50/mL 100xj A/Vi rg i n i a/ATCC3/2009 1 x 102 TCID50/mL 1,000xj a In silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic material present in the culture of this or the reference strain (TCID50 value is based only on infectious virus particles). b In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. PI1114 F For Use Under Emergency Use Authorization Only. Page 38 ePlex Respiratory Pathogen Panel 2 c H1-2009 subtype was detected in this seasonal influenza A H1 strain at 30x LoD. d In silico analysis revealed little homology between this non-contemporary strain sequence and the H1 signal probe/capture probe sequences. e In silico analysis revealed little homology between this non-contemporary influenza strain sequence and the H1 primer sequences. f For Influenza A matrix, in silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic material present in the culture of this or the reference strain (TCID50 value is based only on infectious virus particles). For H1 subtype, in silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. g For Influenza A matrix, in silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. For H1 subtype, in silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic material present in the culture of this or the reference strain (TCID50 value is based only on infectious virus particles). h For Influenza A matrix, in silico analysis revealed good homology to primers and probes. Lower sensitivity is likely the result of incorrect estimation of genetic material present in the culture of this or the reference strain (TCID50 value is based only on infectious virus particles). For H1-2009 subtype, in silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. i In silico analysis revealed little homology between the strain sequence and the H1 or H1-2009 primer, signal probe and capture probe sequences. j No sequence data was available to investigate lower sensitivity of the influenza A 2009 H1N1 A/Virginia/ATCC1/2009, A/Virginia/ATCC2/2009 and A/Virginia/ATTC3/2009 strains. Table 47: Analytical Reactivity (Inclusivity) Results for Influenza A Strains Titered with Methods Different from the Reference Strain Influenza A Subty p e Strain Concentration Detected Influenza A H1 A/Den v er/1/57 A/Mal/302/54 1.6 x 102 CEID50/mL (Influenza A matrix) 1.6 x 108 CEID50/mL (H1 subtype) 1.58 x 102 CEID50/mL (Influenza A matrix) 1.58 x 105 CEID50/mL (H1 subtype) A/Aichi/2/68 H3N2 1.58 x 103 CEID50/mL Influenza A H3 Influenza A H1N1 Alice (vaccine) A/England/42/72 MRC-2 Recombinant Strain A/Washington/24/2012 (A/H1 pdm09) 5 x 100 EID50/mL (Influenza A matrix) 5 x 101 EID50/mL (H3 subtype) 8.89 x 102 CEID50/mL (Influenza A matrix) 8.89 x 103 CEID50/mL (H3 subtype) 3.16 x 103 EID50/mL (Influenza A matrix) 3.16 x 102 EID50/mL (H1-2009 subtype) Influenza A H1N2 Kilbourne F63: A/NWS/34 (HA) x A/Rockefeller Institute/5/57 (NA), Reassortant NWS-F- Matrix 8.89 x 101 CEID50/mL (Influenza A matrix) No subtype detecteda Influenza A H5N8 Influenza A H5N2 A/Gy rfal c o n /W as h ing ton/410886/2014 BPL A/No rth ern Pintail/Washington/40964/2014 BPL 1.58 x 103 EID50/mL (Influenza A matrix) No subtype detectedb 2.51 x 103 EID50/mL (Influenza A matrix) No subtype detectedb Influenza A H7N9 A/ANHUI/1/2013 7.94 x 103 EID50/mL (Influenza A matrix) No subtype detectedc Influenza A H3N2v A/Indiana/21/2012 2.51 x 104 EID50/mL (Influenza A matrix and H3 s ubty p e) a In silico analysis revealed little homology between this non-contemporary strain sequence and the H1 Signal Probe/Capture Probe sequences. b Detection of the H5 Subtype not expected c Detection of the H7 Subtype not expected NOTE: CEID50/mL= Chick Embryo Infectious Dose; EID50/mL= Egg Infectious Dose Supplemental Analytical Reactivity (Inclusivity) for Influenza A For human, avian, and swine influenza strains not available for testing on the ePlex RP Panel, in silico analysis was performed. Bioinformatics analysis was used to predict a result based on the number and location of mismatches in the primers, capture probes, and signal probes found in the ePlex RP Panel relative to an alignment of GenBank sequences. PI1114 F For Use Under Emergency Use Authorization Only. Page 39 ePlex Respiratory Pathogen Panel 2 Table 48: Predicted (in silico) Reactivity (Inclusivity) Results for Influenza A Influenza A Subtype Host H2N2 Human Strain A/Al ban y /20/1957(H2N2) Kilbourne F38: A/Korea/426/68 (HA, NA) x A/Puerto Rico/8/34 A/chicken/New York/13828-3/1995(H2N2) GenBank ID CY022014 CY037296 CY014822 Predicted ePlex Result Influenza A Influenza A Influenza A Avian A/J ap an /305/1957(H2N2) CY014977 Influenza A A/Ko rea/426/1968(H2N2) CY031596 Influenza A H4N6 A/Blue-winged teal/Minnesota/Sg00043/2007(H4N6) A/Peregrine falcon/Aomori/7/2011 CY063978 AB629716 Influenza A Influenza A A/Chicken/West Bengal/239022/2010 CY061305 Influenza A A/Chicken/West Bengal/193936/2009 GU272009 Influenza A A/Ch i c k en /Hun an /1/2009 HM172150 Influenza A A/Ch i c k en /Hun an /8/2008 GU182162 Influenza A A/Chicken/West Bengal/106181/2008 GU083632 Influenza A Avian A/Ch i c k en /Pri mo rs k y/85/2008 A/Chicken/West Bengal/82613/2008 FJ654298 GU083648 Influenza A Influenza A A/Duc k /Fran c e/080036/2008 CY046185 Influenza A H5N1 A/Duc k /Vi etn am/G12/2008 A/Ch i c k en /Th ai l and /PC-340/2008 AB593450 EU620664 Influenza A Influenza A A/Great egret/Hong Kong/807/2008 CY036240 Influenza A A/Ro o k /Ro s to v -on-Don /26/2007(H5N1) EU814504 Influenza A A/Turkey/VA/505477-18/2007(H5N1) GU186510 Influenza A A/Ch i c k en /Ban g l ad es h/1151-10/2010(H5N1) HQ156766 Influenza A A/Ban g l ad es h /3233/2011 CY088772 Influenza A Human A/Cambo d i a/R0405050/2007(H5N1) A/Cambo d i a/S1211394/2008 HQ200572 HQ200597 Influenza A Influenza A A/Hong Kong/486/97(H5N1) AF255368 Influenza A Swine A/Swine/East Java/UT6010/2007(H5N1) HM440124 Influenza A A/Duc k /Pen n s y l v an i a/10218/1984(H5N2) AB286120 Influenza A A/American black duck/Illinois/08OS2688/2008 A/American green-winged teal /Cal i fo rn i a/HKW F609/2007 CY079453 CY033447 Influenza A Influenza A A/Canada goose/New York/475813-2/2007 GQ923358 Influenza A H5N2 Avian A/Blue-winged teal/Saskatchewan/22542/2007 A/Ch i c k en /Tai wan /A703-1/2008 CY047705 AB507267 Influenza A Influenza A A/Duc k /Fran c e/080032/2008 CY046177 Influenza A A/Duck/New York/481172/2007 GQ117202 Influenza A A/Gad wal l /Al tai /1202/2007 CY049759 Influenza A A/Mal l ard /Lo ui s i an a/476670-4/2007 GQ923390 Influenza A A/W aterfo wl /Co l o rad o/476466-2/2007 GQ923374 Influenza A H5N3 H6N1 Avian A/Duc k /Si n g ap o re/F119/3/1997(H5N3) A/Duck/PA/486/1969(H6N1) GU052803 EU743287 Influenza A Influenza A PI1114 F For Use Under Emergency Use Authorization Only. Page 40 ePlex Respiratory Pathogen Panel 2 Influenza A Subtype H6N2 Host H7N2 Avian H7N3 Human H7N7 Avian H7N9 Human Human Avian H9N2 H10N7 H11N9 H1N1 Human Avian Swi n e H1N2 H1N1 (2009) Human H1N2 Swi n e Strain A/Mallard/Czech Republic/1590217K/2009(H6N2) A/Ch i c k en /Hebei /1/2002 A/Ch i c k en /PA/149092-1/02 A/Ch i c k en /NJ /294508-12/2004 A/Chicken/New York/23165-6/2005 A/Muscovy duck/New York/23165-13/2005 A/Muscovy duck/New York/87493-3/2005 A/Mal l ard /Neth erl an d s /29/2006 A/Northern shoveler/California/JN1447/2007 A/New York/107/2003(H7N2) A/Can ad a/rv 504/2004(H7N3) A/American green-winged teal /Mi s s i s s i p pi /09OS046/2009 A/Ch i c k en /German y /R28/03 A/Ch i c k en /Neth erl an ds /1/03 A/Mal l ard /Cal i fo rn i a/HKW F1971/2007 A/Mal l ard /Ko rea/GH171/2007 A/Mute swan/Hungary/5973/2007 A/Northern shoveler/Mississippi/ 09OS643/2009 A/Neth erl an d s /219/03(H7N7) A/Sh an g h ai /1/2013(H7N9) A/No rth ern s h o v el er/Mi s s is s ipp i/11OS145/2011(H7N9) A/Ruddy turnstone/Delaware Bay/220/1995(H7N9) A/Turk ey /Mi n n es o ta/1/1988(H7N9) A/Blue-winged teal/Ohio/566/2006(H7N9) A/Hong Kong/1073/99(H9N2) A/Turk ey /W i s c o n s in/1/1966(H9N2) A/c h i c k en /German y /N/1949(H10N7) A/Duc k /Memp h i s /546/1974(H11N9) A/Swi n e/W i s c o n s in /1/1971(H1N1) A/Cal i fo rn i a/UR06-0393/2007(H1N1) A/New York/297/2003(H1N2) A/Aal bo rg /INS133/2009(H1N1) A/South Carolina/02/2010(H1N1) A/Swine/Hong Kong/NS857/2001(H1N2) A/Swi n e/Swed en /1021/2009(H1N2) GenBank ID HQ244433 AY724263 AY241609 EU743254 CY031077 CY033226 CY034791 CY043833 CY076873 EU587373 CY015007 CY079309 AJ619676 AY340091 CY033383 FJ959087 GQ240816 CY079413 AY340089 EPI439493 CY133650 Predicted ePlex Result Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A CY127254 CY014787 CY024819 AJ278647 CY014664 GQ176135 GQ257441 CY022414 CY026540 CY026539 CY002664 CY002665 CY063606 CY063607 KC781370 KC781372 GQ229350 GQ495135 Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A H1 Influenza A H1 Influenza A H12009 Influenza A H12009 Influenza A Influenza A PI1114 F For Use Under Emergency Use Authorization Only. Page 41 ePlex Respiratory Pathogen Panel 2 Influenza A Subtype H3N1 Host Avian Strain A/Blue-winged teal/ALB/452/1983(H3N1) A/Io wa/07/2011(H3N2) A/Io wa/08/2011(H3N2) A/Io wa/09/2011(H3N2) A/In d i an a/08/2011(H3N2) H3N2v Human A/Mai n e/06/2011(H3N2) A/Mai n e/07/2011(H3N2) A/Pen n s y l v an i a/09/2011(H3N2) A/Pen n s y l v an i a/11/2011(H3N2) A/Pen n s y l v an i a/10/2011(H3N2) A/West Virginia/06/2011(H3N2) A/West Virginia/07/2011(H3N2) A/In d i an a/10/2011(H3N2) H3N5 H3N6 H3N7 H3N8 Swi n e Avian A/Bo s to n /38/2008(H3N2) A/s wi n e/NY/A01104005/2011(H3N2v ) A/Mai n e/06/2011(H3N2) A/In d i an a/08/2011(H3N2) A/American black duck/North Carolina/675075/2004(H3N2) A/Mal l ard /Neth erl an d s /2/1999(H3N5) A/American black duck/New Brun s wi c k /25182/2007(H3N6) A/No rth ern s h o v el er/Cal i forni a/HKW F1367/2007(H3N7) A/American black d uc k /W as h i ng ton/699/1978(H3N8) GenBank ID CY004635 JQ070760 JQ290177 JQ070768 JQ290167 JQ070776 JQ290183 JQ070800 JQ070795 JN866181 JN866186 JN992746 JN655534 JN655540 JN655550 JQ290159 JQ290164 JQ348839 KJ942592 JQ070787 CY044580 CY044581 JN940422 JN866181 JN866186 JN655558 JN638733 GU051135 GU051136 CY060261 CY060264 CY047696 CY047697 CY033372 CY033375 GU052300 GU052299 Predicted ePlex Result Influenza A Influenza A H3 Influenza A H3 Influenza A H3 Influenza A H3 Influenza A H3 Influenza A Influenza A Influenza A Influenza A Influenza A H3 Influenza A Influenza A H3 Influenza A H3 Influenza A H3 Influenza A H3 Influenza A H3 Influenza A H3 Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A Influenza A H3 PI1114 F For Use Under Emergency Use Authorization Only. Page 42 ePlex Respiratory Pathogen Panel 2 Table 49: Analytical Reactivity (Inclusivity) Results for Influenza B Influenza B Subtype Strain Concentration Multiple of LoD Detected B/Lee/40 3 x 10-1 TCID50/mL 3x Influenza B (Yamagata Lineage) B/Al l en /45 B/Mary l an d /1/59 1 x 100 TCID50/mL 1 x 101 TCID50/mL 10xa 100xa B/Tai wan /2/62 1 x 101 TCID50/mL 100xa Influenza B (Victoria Lineage) B/Hong Kong/5/72 B/Malaysia/2506/04 1 x 101 TCID50/mL 3 x 10-1 TCID50/mL 100xb 3x Influenza B (Lineage unknown) B/GL/1739/54 3 x 10-1 TCID50/mL 3x a No sequence data available. Lower sensitivity may be a result of mismatches in the assay primers and/or probes. In addition, the reduced sensitivity may be the result of incorrect estimation of genetic material present in the culture of this or the reference strain (TCID50/mL value is based only on infectious virus particles). b In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. Table 50: Analytical Reactivity (Inclusivity) Results for Parainfluenza Virus Parainfluenza Subtype Strain Concentration Multiple of LoD Detected Parainfluenza Virus 1 C35 1.2 x 100 TCID50/mL 3x Parainfluenza Virus 2 Greer 1.5 x 102 TCID50/mL 3x Parainfluenza Virus 3 C-243 5 x 101 TCID50/mL 10xa Parainfluenza Virus 4 4b 9 x 101 TCID50/mL 3x a In silico analysis revealed that lower sensitivity may be a result of mismatches in the assay primers and/or probes. Table 51: Analytical Reactivity (Inclusivity) Results for Respiratory Syncytial Virus RSV Subtype Respiratory Syncytial Virus A Respiratory Syncytial Virus B Strain A2 Lo n g 9320 W as h /18537/62 WV/14617/85 Concentration 4.5 x 100 TCID50/mL 4.5 x 100 TCID50/mL 6 x 10-1 TCID50/mL 6 x 10-1 TCID50/mL 6 x 10-1 TCID50/mL Multiple of LoD Detected 3x 3x 3x 3x 3x Table 52: Analytical Reactivity (Inclusivity) Results for Chlamydia pneumoniae Chlamydia pneumoniae Strain CWL-029 TWAR strain 2043 Concentration 9 x 102 CFU/mL 9 x 102 CFU/mL Multiple of LoD Detected 3x 3x PI1114 F For Use Under Emergency Use Authorization Only. Page 43 ePlex Respiratory Pathogen Panel 2 Table 53: Analytical Reactivity (Inclusivity) Results for Mycoplasma pneumoniae Strain [Bru] Concentration 9 x 102 CCU/mL Multiple of LoD Detected 3x M129-B170 9 x 102 CCU/mL 3x M129-B7 9 x 102 CCU/mL 3x Mycoplasma pneumoniae [M52] [Mac] 9 x 102 CCU/mL 3x 9 x 102 CCU/mL 3x Mutant 22 3 x 104 CCU/mL 100xa PI 1428 3 x 104 CCU/mL 100xb a No sequence data available. Lower sensitivity may be a result of mismatches in the assay primers and/or probes. In addition, the reduced sensitivity may be the result of incorrect estimation of genetic material present in the culture of this or the reference strain (CCU/ml value is based only on live bacteria). b In silico analysis revealed good homology to primers and probes. The reduced sensitivity is likely the result of incorrect estimation of genetic material present in the culture of this or the reference strain (CCU/ml value is based only on live bacteria). Analytical Specificity (Cross-Reactivity and Exclusivity) Cross-Reactivity of the SARS-CoV-2 Assays Cross-reactivity of the SARS-CoV-2 assays was evaluated using both in silico analysis and by testing quantif ied analytes for organisms likely to be found in circulation and other pathogens in the same genetic f amily. Synthetic constructs were used for analytes where high-titer cultures were not available (SARSCoV-1, MERS-CoV, and Coronavirus HKU1). A pool of two to four analytes were tested in triplicate. Viral analytes were diluted to testing concentrations ranging from 1x104 - 1x106 TCID50/mL. Bacterial and f ungal analytes were diluted to a testing concentration of 1x108 CFU/mL. Synthetic constructs were tested at a concentration of 1x106 copies/mL. A summary of the results of cross-reactivity testing are shown in Table 54 below. At high titers, cross-reactivity with SARS-CoV-1 was observed with the ePlex RP2 Panel. Table 54: Cross-Reactivity of SARS-CoV-2 Assays with On and Off-Panel Organisms Virus/Bacteria Adenovirus C Co ro n av i rus Co ro n av i rus Co ro n av i rus Co ro n av i rus Co ro n av i rus Co ro n av i rus Echovirus T En tero v i rus Influenza A Influenza B Metap n eumo v i rus Parai n fl uen za Parai n fl uen za Parai n fl uen za Parai n fl uen za Strain 1 229E HKU1a NL63 OC43 MERS-Co Va SARS-Co V-1a 30 68 H1N1/NY01/2009 Yamag ata B2 Peru1-2002 1 2 3 4a Concentration 1 x 104 TCID50/mL 1 x 104 TCID50/mL 5 x 104 TCID50/mL 1 x 104 TCID50/mL 1 x 106 TCID50/mL 1 x 104 copies/µL 1 x 106 copies/µL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL Cross-Reactivity Not observed Not observed Not observed Not observed Not observed Not observed Observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed PI1114 F For Use Under Emergency Use Authorization Only. Page 44 ePlex Respiratory Pathogen Panel 2 Virus/Bacteria Respiratory Syncytial Virus A Rh i n o v i rus Bordetella pertussis Candida albicans Corynebacterium diphtheriae Haemophilus influenzae Legionella pneumophila Mycobacterium tuberculosis Moraxella catarrhalis Neisseria meningitidis Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus epidermidis Staphylococcus salivarius Streptococcus pneumoniae Streptococcus pyogenes Pooled Nasal Swab a in vitro transcript Strain 2006 B14 ATCC53894 ATCC24433 ATCC53281 ATCC43065 ATCC35096 H37Rv ATCC23246 NCTC10026 ATCC BAA-1744 ATCC25923 ATCC700567 ATCC25975 ATCC49136 ATCC49399 Human Clinical Sample Concentration 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL 1 x 108 CFU/mL N/A Cross-Reactivity Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed In silico Analysis of the ePlex RP2 Panel SARS-CoV-2 Assays In silico analysis was performed for the gene regions targeted by the ePlex RP2 Panel to evaluate cross-reactivity. GenMark conducted a primer BLAST® search of the NCBI database against all bacteria, negative-stranded RNA viruses (negarnavariota), picornaviruses, adenoviruses, common human coronaviruses, MERS, Candida albicans, and Pneumocystis. The BLAST searches did not identify any cross-reactivity with the exception of SARS coronavirus, which is in the same subgenus (Sarbecovirus) as SARS-CoV-2. Cross-Reactivity and Exclusivity of Other RP2 Panel Targets The design of the ePlex RP2 Panel incorporates assays for the detection of SARS-CoV-2 without af f ecting the original designs of the ePlex RP Panel assays. The original RP Panel targets impacted by the addition of the SARS-CoV-2 assays (influenza A, influenza A H1, influenza A H1-2009, influenza A H3, inf luenza B, and adenovirus) were tested and no cross-reactivity was observed. Theref ore, the established cross-reactivity claims of the ePlex RP Panel are applicable to the ePlex RP2 Panel. Cross-reactivity of each viral and bacterial target on the ePlex RP Panel was evaluated at high concentrations (1 x 104 1 x 106 TCID50/mL f or viruses, 1 x 108 CFU/mL for bacterial and fungal isolates, or 1 x 104 1 x 106 copies/mL for in vitro transcripts) of quantified strains/isolates diluted in viral transport media. In vitro transcript for coronavirus HKU1 was diluted in PBS. Table 55 summarizes the results of the on-panel viral and bacterial strains/isolates tested. No cross-reactivity was observed between any of the on-panel viruses or bacteria. PI1114 F For Use Under Emergency Use Authorization Only. Page 45 ePlex Respiratory Pathogen Panel 2 Table 55: Cross-reactivity with ePlex RP Panel Target Organisms Target Adenovirus A Adenovirus B Adenovirus C Adenovirus D Adenovirus E Adenovirus F Co ro n av i rus Co ro n av i rus Co ro n av i rus Co ro n av i rus En tero v i rus Human metapneumovirus Human rhinovirus Influenza A Influenza A H1 Influenza A H1-2009 Influenza A H3 Influenza A H3N2va Strain Type 31 Type 7A Type 1 Type 9 Type 4 Type 41 229E HKU1 in vitro transcript NL63 OC43 Type 68 2007 isolate B1 1A A/Bri s ban e/59/07 A/Bri s ban e/59/07 A/NY/01/2009 A/Bri s ban e/10/07 A/Indiana/21/2012 Concentration 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 106 copies/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 2.51 x 105 EID50/mL Cross-Reactivity Results Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Influenza A H5N2b A/Northern Pintail Washington/40964/14BPL 2.51 x 105 EID50/mL Not observed Influenza A H5N8c A/Gyrfalcon/Washington /410886/2014 BPL 1.58 x 105 EID50/mL Not observed Influenza A H7N9d Influenza B Parainfluenza Virus 1 Parainfluenza Virus 2 Parainfluenza Virus 3 Parainfluenza Virus 4 RSV A RSV B Chlamydia pneumoniae A/ANHUI/1/2013 B/Fl o ri d a/02/06 C35 Type 2 Type 3 Type 4a 2006 Isolate CH93(18)-18 AR-39 7.94 x 105 EID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 106 CFU/mL Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Not observed Mycoplasma pneumoniae FH strain of Eaton Agent [NCTC 10119] a Influenza A H3N2v detected as Influenza A, Influenza A H3 b Influenza A H5N2 detected as Influenza A c Influenza A H5N8 detected as Influenza A d Influenza A H7N9 detected as Influenza A 1 x 106 CCU/mL Not observed Cross-reactivity of viruses, bacteria, and fungi that are not targets on the ePlex RP Panel was evaluated at high concentrations (1 x 105 TCID50/mL or copies/mL for viruses, 1 x 106 CFU/mL for bacterial and yeast isolates, or 1 x 106 copies/mL for plasmid DNA or genomic RNA) by diluting quantified strains in viral transport media. Plasmid for bocavirus and genomic RNA for MERS coronavirus (MERS-CoV) were PI1114 F For Use Under Emergency Use Authorization Only. Page 46 ePlex Respiratory Pathogen Panel 2 diluted in PBS. Table 56 summarizes the results of the strains tested. No cross-reactivity was observed between any of the off-panel viruses, bacteria or fungi with the ePlex RP Panel targets. Table 56: Cross-reactivity with Organisms Not Detected by the ePlex RP Panel (Exclusivity) Target Acinetobacter baumanii Strain ATCC® 19606 Concentration 1 x 106 CFU/mL Cross-Reactivity Results Not observed Bordetella pertussis 18323 [NCTC 10739] 1 x 106 CFU/mL Not observed Bordetella parapertussis ATCC 15311 1 x 106 CFU/mL Not observed Burkholderia cepacia ATCC 25416 1 x 106 CFU/mL Not observed Candida albicans ATCC 10231 1 x 106 CFU/mL Not observed Candida glabrata ATCC 15126 1 x 106 CFU/mL Not observed MERS Coronavirus (MERS-CoV) EMC/2012a 1 x 105 copies/mL Not observed Corynebacterium diphtheriae ATCC 13812 1 x 106 CFU/mL Not observed Cy to meg al o v i rus Epstein Barr Virus Escherichia coli AD 169 Strain B95-8 ATCC 10279 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 106 CFU/mL Not observed Not observed Not observed Haemophilus influenzae ATCC 43065 1 x 106 CFU/mL Not observed Herpes Simplex Virus Human bocavirus Isolate 2 Bocavirus plasmidb 1 x 105 TCID50/mL 1 x 106 copies/mL Not observed Not observed Klebsiella pneumoniae ATCC 51504 1 x 106 CFU/mL Not observed Lactobacillus acidophilus ATCC 314 1 x 106 CFU/mL Not observed Lactobacillus plantarum ATCC 8014 1 x 106 CFU/mL Not observed Legionella pneumophila Ph i l ad el p h ia-1 1 x 106 CFU/mL Not observed Measles N/A 1 x 105 TCID50/mL Not observed Moraxella catarrhalis ATCC 23246 1 x 106 CFU/mL Not observed Mump s Mycobacterium tuberculosis Isolate 2 ATCC 25177 1 x 105 TCID50/mL 1 x 106 CFU/mL Not observed Not observed Neisseria meningiditis ATCC 13077 1 x 106 CFU/mL Not observed Neisseria sicca ATCC 29193 1 x 106 CFU/mL Not observed Porphyromonas gingivalis ATCC 33277 1 x 106 CFU/mL Not observed Proteus vulgaris ATCC 33420 1 x 106 CFU/mL Not observed Pseudomonas aeruginosa ATCC 15442 1 x 106 CFU/mL Not observed Serratia marcescens ATCC 13880 1 x 106 CFU/mL Not observed Staphylococcus aureus (MRSA) NRS384 1 x 106 CFU/mL Not observed Staphylococcus aureus (MSSA) ATCC 25923 1 x 106 CFU/mL Not observed Staphylococcus epidermidis (MRSE) ATCC 35983 1 x 106 CFU/mL Not observed Staphylococcus epidermidis (MSSE) ATCC 49134 1 x 106 CFU/mL Not observed Staphylococcus haemolyticus ATCC 29970 1 x 106 CFU/mL Not observed Streptococcus agalactiae ATCC 12401 1 x 106 CFU/mL Not observed Streptococcus dysgalactiae ATCC 35666 1 x 106 CFU/mL Not observed Streptococcus mitis ATCC 15914 1 x 106 CFU/mL Not observed Streptococcus pneumoniae ATCC 49619 1 x 106 CFU/mL Not observed Streptococcus pyogenes ATCC 12384 1 x 106 CFU/mL Not observed Streptococcus salivarius ATCC 13419 1 x 106 CFU/mL Not observed PI1114 F For Use Under Emergency Use Authorization Only. Page 47 ePlex Respiratory Pathogen Panel 2 Target Strain Varicella Zoster Virus 82 a Extracted genomic RNA b Plasmid does not contain full length viral genome. Concentration 8.9 x 103 TCID50/mL Cross-Reactivity Results Not observed Reproducibility A multisite reproducibility study of the ePlex RP Panel was performed to evaluate agreement with expected results across major sources of variability, such as site-to-site, lot-to-lot, day-to-day, and operator-to-operator. Testing occurred at 3 sites (2 external, 1 internal) on one ePlex instrument per site with either 3 or 4 towers. Two operators performed testing at each site on 6 days (5 nonconsecutive days) with 3 unique lots of RP Panel cartridges. A reproducibility panel consisting of 3 panel members with 6 organisms (representing 7 RP Panel targets) at 3 concentrations (moderate positive- 3x LoD, low positive- 1x LoD, and negative) was tested in triplicate. The 6 organisms tested included adenovirus, coronavirus (229E, HKU1, NL63, OC43) , human metapneumovirus, influenza A H3, parainfluenza virus 1, and RSV A; organisms were diluted in natural clinical matrix (pooled, negative nasopharyngeal swab samples). Negative samples consisted of natural clinical matrix only. Each simulated sample was divided into aliquots and stored frozen (-70 °C) prior to testing. Each operator tested 9 samples (3 member reproducibility panel in triplicate) each day; each panel member was tested 108 times (3 replicates x 3 sites x 2 operators x 3 lots x 2 days of testing/operator/lot) for a maximum of 324 tests. Af ter completion of initial and repeat testing for invalid results, 1 low positive sample tested at Site 3 had an invalid result and was excluded from reproducibility performance analyses. Percent agreement (95% CI) with expected results was 100% for all 7 targets for the moderate positive and negative panel, and 100% for 6 of 7 low positive panel targets (coronavirus, human metapneumovirus, influenza A, influenza A H3, parainfluenza 1, and RSV A); percent agreement was 91.6% f or adenovirus. Summary results for the 7 ePlex RP Panel targets that correspond to the 6 organisms in the reproducibility panel are provided in Tables 57-63. Summary results for the 10 ePlex RP Panel targets that did not have organisms included in the reproducibility panel are provided in Table 64. Table 57: Percent Agreement for Adenovirus Adenovirus Concentration Agreement with Expected Results Site Agreed / N % 95% CI Moderate Positive 3x LoD 6 x 100 TCID50/mL 1 36/36 100 2 36/36 100 3 36/36 100 All 108/108 100 (90.4-100) (90.4-100) (90.4-100) (96.6-100) Low Positive 1x LoD 2 x 100 TCID50/mL 1 36/36 100 2 34/36 94.4 3 28/35 80.0 All 98/107 91.6 (90.4-100) (81.9-98.5) (64.1-90.0) (84.8-95.5) 1 36/36 100 (90.4-100) Neg ati v e 2 36/36 100 3 36/36 100 (90.4-100) (90.4-100) CI=Confidence Interval All 108/108 100 (96.6-100) PI1114 F For Use Under Emergency Use Authorization Only. Page 48 ePlex Respiratory Pathogen Panel 2 Table 58: Percent Agreement for Coronavirus (229E, HKU1, NL63, OC43) Coronavirus (229E, HKU1, NL63, OC43) Concentration Site Agreement with Expected Results Agreed / N % 95% CI Moderate Positive 3x LoD 1.5 x 103 TCID50/mL Low Positive 1x LoD 5 x 102 TCID50/mL 1 36/36 100 2 36/36 100 3 36/36 100 All 108/108 100 1 36/36 100 2 36/36 100 3 35/35 100 All 107/107 100 (90.4-100) (90.4-100) (90.4-100) (96.6-100) (90.4-100) (90.4-100) (90.1-100) (96.5-100) Neg ati v e 1 36/36 100 2 36/36 100 3 36/36 100 (90.4-100) (90.4-100) (90.4-100) All 108/108 100 (96.6-100) Table 59: Percent Agreement for Human Metapneumovirus (hMPV) hMPV Concentration Agreement with Expected Results Site Agreed / N % 95% CI Moderate Positive 3x LoD 6.75 x 102 TCID50/mL Low Positive 1x LoD 2.25 x 102 TCID50/mL 1 36/36 100 2 36/36 100 3 36/36 100 All 108/108 100 1 36/36 100 2 36/36 100 3 35/35 100 All 107/107 100 (90.4-100) (90.4-100) (90.4-100) (96.6-100) (90.4-100) (90.4-100) (90.1-100) (96.5-100) Neg ati v e 1 36/36 100 2 36/36 100 3 36/36 100 (90.4-100) (90.4-100) (90.4-100) All 108/108 100 (96.6-100) Influenza A Concentration Moderate Positive 3x LoD 1.5 x 102 TCID50/mL Low Positive 1x LoD 5 x 101 TCID50/mL Table 60: Percent Agreement for Influenza A Agreement with Expected Results Site Agreed / N % 1 36/36 100 2 36/36 100 3 36/36 100 All 108/108 100 1 36/36 100 2 36/36 100 3 35/35 100 All 107/107 100 95% CI (90.4-100) (90.4-100) (90.4-100) (96.6-100) (90.4-100) (90.4-100) (90.1-100) (96.5-100) PI1114 F For Use Under Emergency Use Authorization Only. Page 49 ePlex Respiratory Pathogen Panel 2 Influenza A Concentration Neg ati v e Agreement with Expected Results Site Agreed / N % 1 36/36 100 2 36/36 100 3 36/36 100 All 108/108 100 95% CI (90.4-100) (90.4-100) (90.4-100) (96.6-100) Influenza A H3 Concentration Moderate Positive 3x LoD 1.5 x 102 TCID50/mL Low Positive 1x LoD 5 x 101 TCID50/mL Neg ati v e Table 61: Percent Agreement for Influenza A H3 Agreement with Expected Results Site Agreed / N % 1 36/36 100 2 36/36 100 3 36/36 100 All 108/108 100 1 36/36 100 2 36/36 100 3 35/35 100 All 107/107 100 1 36/36 100 2 36/36 100 3 36/36 100 All 108/108 100 95% CI (90.4-100) (90.4-100) (90.4-100) (96.6-100) (90.4-100) (90.4-100) (90.1-100) (96.5-100) (90.4-100) (90.4-100) (90.4-100) (96.6-100) PIV 1 Concentration Moderate Positive 3x LoD 1.2 x 100 TCID50/mL Low Positive 1x LoD 4 x 10-1 TCID50/mL Neg ati v e Table 62: Percent Agreement for Parainfluenza Virus (PIV) 1 Agreement with Expected Results Site Agreed / N % 95% CI 1 36/36 100 (90.4-100) 2 36/36 100 (90.4-100) 3 36/36 100 (90.4-100) All 108/108 100 (96.6-100) 1 36/36 100 (90.4-100) 2 36/36 100 (90.4-100) 3 35/35 100 (90.1-100) All 107/107 100 (96.5-100) 1 36/36 100 (90.4-100) 2 36/36 100 (90.4-100) 3 36/36 100 (90.4-100) All 108/108 100 (96.6-100) Table 63: Percent Agreement for Respiratory Syncytial Virus (RSV) A RSV A Concentration Agreement with Expected Results Site Agreed / N % 95% CI Moderate Positive 3x LoD 4.5 x 100 TCID50/mL 1 36/36 100 2 36/36 100 3 36/36 100 (90.4-100) (90.4-100) (90.4-100) PI1114 F For Use Under Emergency Use Authorization Only. Page 50 ePlex Respiratory Pathogen Panel 2 RSV A Concentration Low Positive 1x LoD 1.5 x 100 TCID50/mL Neg ati v e Agreement with Expected Results Site Agreed / N % All 108/108 100 1 36/36 100 2 36/36 100 3 35/35 100 All 107/107 100 1 36/36 100 2 36/36 100 3 36/36 100 All 108/108 100 95% CI (96.6-100) (90.4-100) (90.4-100) (90.1-100) (96.5-100) (90.4-100) (90.4-100) (90.4-100) (96.6-100) Table 64: Negative Percent Agreement with Organisms Not Included in the Reproducibility Panel Target Agreement with Expected Negative Results Site Agreed / N % 95% CI 1 108/108 100 (96.6-100) 2 108/108 100 Human Rhinovirus/Enterovirus 3 104/107 97.2 (96.6-100) (92.1-99.0) All 320/323 99.1 1 108/108 100 (97.3-99.7) (96.6-100) Influenza A H1 2 108/108 100 3 107/107 100 (96.6-100) (96.5-100) All 323/323 100 (98.8-100) 1 108/108 100 (96.6-100) Influenza A H1-2009 2 108/108 100 3 107/107 100 (96.6-100) (96.5-100) All 323/323 100 (98.8-100) 1 108/108 100 (96.6-100) Influenza B 2 108/108 100 3 107/107 100 (96.6-100) (96.5-100) All 323/323 100 (98.8-100) 1 108/108 100 (96.6-100) Parainfluenza Virus 2 2 108/108 100 3 107/107 100 (96.6-100) (96.5-100) All 323/323 100 (98.8-100) 1 108/108 100 (96.6-100) Parainfluenza Virus 3 2 108/108 100 3 106/107 99.1 (96.6-100) (94.9-99.8) All 322/323 99.7 (98.3-99.9) 1 108/108 100 (96.6-100) Parainfluenza Virus 4 2 108/108 100 3 107/107 100 (96.6-100) (96.5-100) All 323/323 100 (98.8-100) PI1114 F For Use Under Emergency Use Authorization Only. Page 51 ePlex Respiratory Pathogen Panel 2 Target Respiratory Syncytial Virus B Chlamydia pneumoniae Mycoplasma pneumoniae Agreement with Expected Negative Results Site Agreed / N % 95% CI 1 108/108 100 (96.6-100) 2 108/108 100 (96.6-100) 3 107/107 100 (96.5-100) All 323/323 100 (98.8-100) 1 108/108 100 (96.6-100) 2 108/108 100 (96.6-100) 3 107/107 100 (96.5-100) All 323/323 100 (98.8-100) 1 108/108 100 (96.6-100) 2 107/108 99.1 (94.9-99.8) 3 106/107 99.1 (94.9-99.8) All 321/323 99.4 (97.8-99.8) Samples with Co-Detected Organisms Co-Detection of SARS-CoV-2 with Other Organisms Detection of SARS-CoV-2 in the presence of another clinically relevant organism was evaluated using a natural clinical matrix (pooled, negative nasopharyngeal swab samples) spiked with SARS-CoV-2 and a second organism co-amplified in the same PCR reaction. In this study, SARS-CoV-2 was tested at a low concentration (3x LoD) in combination with the second organism at a high concentration (1 x 106 copies/mL). SARS-CoV-2 was also tested a high concentration (2.5 x 106 copies/mL) in combination with the second organism at low concentration (3x LoD). Table 65 contains the results of co-detection testing which demonstrated that there is no competitive inhibition when SARS-CoV-2 is co-amplified at low concentrations in the presence of the indicated organisms at high concentrations or when SARS-CoV-2 at high concentration is co-amplified with the indicated organism at low concentration. Table 65: Detection of Co-detections Organism 1 SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 Influenza A H1-2009 Adenovirus Influenza B High Titer 2.5 x 106 copies/mL 2.5 x 106 copies/mL 2.5 x 106 copies /mL 1 x 106 copies /mL 1 x 106 copies /mL 1 x 106 copies /mL Organism 2 Influenza A H1-2009 Adenovirus Influenza B SARS-CoV-2 SARS-CoV-2 SARS-CoV-2 Multiple of LoD 3x 3x 3x 3x 3x 3x % Positive of Organism 2 100% 100% 100% 100% 100% 100% Samples with Co-Detected Organisms on the RP2 Panel Detection of more than one clinically relevant viral organism in a sample was evaluated with the ePlex RP Panel using a natural clinical matrix (pooled, negative nasopharyngeal swab samples) spiked with two RP Panel organisms: one organism at a low concentration (1-3x LoD) and the second organism at a high concentration (1 x 105 TCID50/mL). Table 66 contains the results of co-detection testing which demonstrated the ability of the ePlex RP Panel to detect 2 organisms in a sample at both high and low concentrations as indicated in the table. PI1114 F For Use Under Emergency Use Authorization Only. Page 52 ePlex Respiratory Pathogen Panel 2 Organism 1 Influenza A H3 Adenovirus Influenza A H3 RSV A Influenza A H1-2009 RSV B Influenza A H1-2009 Rhinovirus Influenza A H1-2009 Parainfluenza Virus 3 Rhinovirus RSV A Coronavirus (229E, HKU1, NL63, OC43) RSV A Human Metapneumovirus Adenovirus Adenovirus RSV A Table 66: Detection of Co-detections High Titer 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL 1 x 105 TCID50/mL Organism 2 Adenovirus B Influenza A H3 RSV A Influenza A H3 RSV B Influenza A H1-2009 Rhinovirus Influenza A H1-2009 Parainfluenza Virus 3 Influenza A H1-2009 RSV A Rhinovirus RSV A Coronavirus (229E, HKU1, NL63, OC43) Adenovirus Human Metapneumovirus RSV A Adenovirus Low Titer 2 x 100 TCID50/mL 5 x 101 TCID50/mL 1.5 x 100 TCID50/mL 5 x 101 TCID50/mL 6 x 10-1 TCID50/mL 1x 10-1 TCID50/mL 1.5 x 100 TCID50/mL 3 x 10-1 TCID50/mL 5 x 100 TCID50/mL 1 x 10-1 TCID50/mL 1.5 x 100 TCID50/mL 1.5 x 100 TCID50/mL 1.5 x 100 TCID50/mL 7.5 x 100 TCID50/mL 2 x 100 TCID50/mL 2.25 x 102 TCID50/mL 1.5 x 100 TCID50/mL 2 x 100 TCID50/mL Multiple of LoD 1x 1x 1x 1x 3x 1x 1x 3x 1x 1x 1x 1x 1x 1x 1x 1x 1x 1x Sample Matrix Equivalency All analytical studies that utilized viral and bacterial cultures close to LoD were performed by spiking the viral and bacterial cultures into a pool of natural negative NPS in VTM as sample matrix. For analytical studies that used viral and bacterial cultures at a concentration which was at least 10x LoD or higher, the viral and bacterial cultures were spiked into MicroTestTM M5® transport media from Remel instead of negative pooled NPS for ease of use. A sample matrix equivalency study was performed to demonstrate equivalency between natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples) and viral transport media when spiked with targets at a concentration of approximately 10x LoD. Quantified, representative viral and bacterial strains were diluted in a natural clinical matrix (pooled, negative nasopharyngeal swab in VTM samples) and in viral transport media. All samples were tested in duplicate. There was no difference observed in detection of targets in natural clinical matrix vs. viral transport media. Interfering Substances Substances commonly found in respiratory samples, substances that could be introduced during specimen collection, or medications commonly used to treat congestion, allergies, or asthma symptoms that could potentially interfere with the ePlex RP Panel were individually evaluated. To simulate clinical samples, quantified representative viral and bacterial strains were diluted to 1x LoD in a natural clinical matrix (pooled, negative nasopharyngeal swab specimens) and tested in triplicate for negative and positive interference. Natural clinical matrix (pooled, negative nasopharyngeal swab samples) with no organisms added was used as a control. All substances and organisms tested for interference were PI1114 F For Use Under Emergency Use Authorization Only. Page 53 ePlex Respiratory Pathogen Panel 2 shown to be compatible with the ePlex RP Panel. No potentially interfering substances were found to inhibit the ePlex RP Panel at the concentrations tested in Table 67. Table 67: List of Substances for Testing Potentially Interfering Substance Active Ingredient Testing Concentration Control Sample Matrixa Transport Mediuma Becton Dickinson UVT N/A Copan eSwab (Liquid Amies media) N/A MicroTest M4 N/A MicroTest M4-RT N/A Viral Transport Mediuma MicroTest M5 N/A MicroTest M6 N/A Flocked Swabs Copan Minitip in UVT N/A Copan Regular Tip in UVT N/A Blood (human) Throat lozenges, oral anesthetic and analgesic Mucin Nasal sprays or drops Bl o o d Human gDNA Benzocaine, menthol Purified mucin protein Phenylephrine HCl (Neo-Synephrine®) Oxymetazoline HCl (Afrin®) 2% v/v 50 ng/rxn 26% w/v 1% w/v 1.5% v/v 1% v/v Antibacterial, systemic Antibiotic, nasal ointment Sodium chloride To bramy c i n b Mup i ro c i n 0.8% w/v 1% w/v 2% w/v Bec l o meth as o n e 1.5% w/v Dex ameth as o n e 1.5% w/v Nasal corticosteroids Fl un i s o l id e Budesonide (Rhinocort®) Triamcinolone (Nasacort®) Fluticasone (Flonase®) 1.5% w/v 0.9% v/v 1.5% v/v 1.5% v/v Luffa opperculata ZICAM® Allergy Relief Nasal Gel Sulfur Galphimia glauca 1% v/v Histaminum hydrochloricum Anti-viral drugs Virus Zan ami v i r Oseltamivir Cy to meg al o v i rus Streptococcus pneumoniae 550 ng/mL 142 ng/mL 1 x 105 TCID50/mL Bordetella parapertussis Bacteria Haemophilus influenza Staphylococcus aureus 1 x 106 CFU/mL Neisseria meningitides Corynebacterium diptheriae a Testing of media was done by adding a negative NPS collected in the specified media and diluting in the natural clinical matrix. b At concentrations greater than 1% weight/volume in the sample, tobramycin was found to inhibit assay performance. PI1114 F For Use Under Emergency Use Authorization Only. Page 54 ePlex Respiratory Pathogen Panel 2 Carryover and Cross-contamination The carryover/cross-contamination rate of the ePlex RP Panel and ePlex instrument was tested in a checkerboard approach by running high positive and negative samples interspersed in all bays of a f ourtower ePlex instrument (24 bays total) over 5 separate runs on 5 separate days. Quantified parainfluenza virus 3 was prepared in viral transport media at a high concentration (1 x 105 TCID50/mL, 20,000x LoD) to simulate a clinically relevant high positive and was tested as a representative target organism. Transport media was used to represent negative samples. On each round of testing, 24 ePlex RP Panel cartridges were evaluated. 100% of parainfluenza 3-positive samples generated a result of Detected and 100% of parainf luenza 3-negative samples generated a parainfluenza 3 result of No Target Detected, indicating no carryover or cross-contamination was observed between bays or within bays with the ePlex RP Panel when testing consecutively or in adjacent bays. TROUBLESHOOTING Table 68: Troubleshooting Table For a complete list of all ePlex error messages and a description of the messages, please refer to the ePlex Operator Manual. Error Test did not start Test did not finish Error Messages Description Re-test Recommendations Cartridge failure The cartridge initialization test failed Cartridge not present Bay heater failure Unknown error Bay main / fluid motor failure Bay over pressured Bay temperature out of range The system was unable to read the cartridge Cartridge inserted doesn't match the serial number of the cartridge scanned The system is not ready to accept the cartridge The system failed to prepare the cartridge for processing Bay heater failure Bay main / fluid motor failure Bay voltage failure Bay sub-system communication timeout Cartridge failure Bay over pressured Bay auto-calibration failure Bay temperature out of range The system was unable to eject the cartridge from the bay An error that occurs during prerun checks (cartridge initialization) of the cartridge upon insertion into the bay. Cartridge initialization occurs when the cartridge is first inserted into the bay and takes approximately 90 seconds. Upon completion of cartridge initialization, the cartridge cannot be restarted, but prior to this point, the cartridge can be restarted. To verify cartridge initialization has completed, examine the cartridge label upon removal from the bay. If the cartridge label has been pierced, the test has already started and cartridge cannot be reused. If the label has not been pierced, follow the recommendation as stated. This type of error occurs during the run, after pre-run checks (cartridge initialization) have finished, and prevents the cartridge from being processed to completion. 1. Remove cartridge from bay. a. Reset bay to clear the error b. Restart cartridge in any available bay 2. If the cartridge is not able to be run on the second try and again generates an error during cartridge initialization, this indicates an issue with the cartridge. This cartridge should be discarded following laboratory procedures and the sample should be repeated using a new cartridge. Bay(s) should be reset to clear the errors. Please contact GenMark Technical Support to alert them of the issue. If the bay remains in an error state (flashing red) after the cartridge has been removed, then the bay must be reset through the Bay Configuration menu before it can be used to run cartridges. Reagents have been consumed and the cartridge cannot be reused. Contact GenMark Technical Support and proceed with repeat testing of the sample using a new cartridge. If the bay remains in an error state (flashing red) after the cartridge has been removed, then the bay must be reset through the Bay Configuration menu before it can be used to run cartridges. PI1114 F For Use Under Emergency Use Authorization Only. Page 55 ePlex Respiratory Pathogen Panel 2 Error Invalid Error Messages Description Re-test Recommendations This is an error that results in no valid results being generated. A test report will be generated, but all targets and the internal control will be invalid. Reagents have been consumed and the cartridge cannot be reused. Contact GenMark Technical Support and proceed with repeat testing of the sample using a new cartridge. Technical Support GenMark Technical support is available 24 hours a day, 7 days a week to provide the highest level of customer support and satisfaction. GenMark Diagnostics, Inc. 5964 La Place Court Carlsbad, CA 92008 USA Phone: 1 800 eSensor (1 800 373 6767), Option 2 Email: technicalsupport@genmarkdx.com GLOSSARY OF SYMBOLS Symbol Description Batch Code Cauti o n Contains sufficient for <n> tests European Union Conformity In vitro diagnostic medical device Consult instructions for use Authorized representative in the European Community Man ufac turer Cartridge Lot Symbol Description Use by date YYYY-MM-DD Serial number Catalog number Biological risks Upper limit of temperature Lower limit of temperature Temperature range Irritant, dermal sensitizer, acute toxicity (harmful), narcotic effects, respiratory tract irritation O x i d i zers PI1114 F For Use Under Emergency Use Authorization Only. Page 56 ePlex Respiratory Pathogen Panel 2 Symbol Description Rx Only For prescription use only Symbol Description For Use Under the Emergency Use Authorization Only REFERENCES 1. Upper Respiratory Inf ection (URI or Common Cold). Johns Hopkins Medicine. Retrieved from http://www.hopkinsmedicine.org/healthlibrary/conditions/pediatrics/upper_respiratory_infection_ur i_or_common_cold_90,P02966/ (Date accessed: 3/22/2016). 2. Seasonal influenza, More Information. Centers for Disease Control and Prevention. Retrieved f rom http://www.cdc.gov/flu/about/qa/disease.htm (Date accessed: 6/10/2016). 3. Inf luenza (Seasonal). (2014). World Health Organization. Retrieved from http://www.who.int/mediacentre/factsheets/fs211/en/ (Date accessed: 3/22/2016). 4. Coronavirus Disease 2019 (COVID-19) Frequently Asked Questions. Centers for Disease Control and Prevention Retrieved from https://www.cdc.gov/coronavirus/2019ncov/faq.html#Basics (Date accessed 6/18/2020) 5. COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU). Retrieved from: https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467 b48e9ecf6 (Date accessed: 7/27/2020). 6. Mossad, S., Upper Respiratory Tract Infections. Cleveland Clinic Center for Continuing Education. Retrieved from http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/infectiousdisease/upper-respiratory-tract-infection/ (Date Published 8/2013). 7. Adenovirus Infections. University of Rochester Medical Center. Retrieved from https://www.urmc.rochester.edu/Encyclopedia/Content.aspx?ContentTypeID=90&ContentID=P02 508 (Date accessed: 3/22/2016). 8. Adenoviruses Clinical Overview. Centers for Disease Control and Prevention. Retrieved from http://www.cdc.gov/adenovirus/hcp/clinical-overview.html (Date accessed: 3/22/2016). 9. Gaunt, E.R. et al. (2010). Epidemiology and Clinical Presentations of the Four Human Coronaviruses 229E, HKU1, NL63, and OC43 Detected over 3 Years Using a Novel Multiplex Real-Time PCR Method. J. Clin. Microbiol. 48(8) 2940-2947. 10. Human Metapneumovirus Clinical Features. Centers for Disease Control and Prevention. http://www.cdc.gov/surveillance/nrevss/hmpv/clinical.html (Date accessed: 3/22/2016). 11. Auwaerter, P., Metapneumovirus. Johns Hopkins Antibiotics (ABX) Guide. Retrieved from http://www.hopkinsguides.com/hopkins/view/Johns_Hopkins_ABX_Guide/540614/all/Metapneum ovirus (Updated March 2013). 12. Anzueto, A., et al. (2003) Diagnosis and Treatment of Rhinovirus Respiratory Inf ections. Chest. 123(5) 1664-1672. 13. Auwaerter, P., Rhinovirus. Johns Hopkins Antibiotics (ABX) Guide. Retrieved from http://www.hopkinsguides.com/hopkins/view/Johns_Hopkins_ABX_Guide/540476/all/Rhinovirus? q=rhinvorirus&ti=0#0 (Updated February 2016). 14. Auwaerter, P., Enterovirus. Johns Hopkins Antibiotics (ABX) Guide. Retrieved from http://www.hopkinsguides.com/hopkins/view/Johns_Hopkins_ABX_Guide/540204/all/Enterovirus? q=enterovirus&ti=0#0 (Updated November 2014). 15. Henrickson, K.J. (2003). Parainfluenza viruses. Clin. Microbiol. Rev. 16(2):242-264. 16. Schomacker, H. et al., (2012) Pathogenesis of acute respiratory illness caused by human parainf luenza viruses. Curr Opin Virol. 2(3) 294-299. 17. Mahony, J.B. Detection of Respiratory Viruses by Molecular Methods. Clin. Microbiol. Rev. (2008). 21(4) 716-747. PI1114 F For Use Under Emergency Use Authorization Only. Page 57 ePlex Respiratory Pathogen Panel 2 18. Resch, B., et al., (2011). Epidemiology of Respiratory Syncytial Virus Infection in Preterm Infants. Open Microbiol J. 5(Suppl 2-M3) 135-143. 19. Atypical Pneumonia. Chlamydia pneumoniae Infection. Centers for Disease Control and Prevention. Retrieved from https://www.cdc.gov/pneumonia/atypical/cpneumoniae/index.html (Updated February 7, 2014). 20. Auwaerter, P., Mycoplasma Pneumoniae. Johns Hopkins Antibiotics (ABX) Guide. Retrieved f rom http://www.hopkinsguides.com/hopkins/view/Johns_Hopkins_ABX_Guide/540373/all/Mycoplasma %20pneumoniae (Date accessed: 3/28/2016). 21. Epidemiological update: Mycoplasma pneumoniae infections- recent increases reported in EU countries. (2012). European Centre for Disease Prevention and Control. Retreived from http://ecdc.europa.eu/en/press/news/_layouts/forms/News_DispForm.aspx?ID=340&List=8db728 6c-f e2d-476c-9133-18ff4cb1b568 (Date accessed: 3/28/2016). 22. Spacek, L. and Auwaerter, P., Adenovirus. Johns Hopkins Antibiotics (ABX) Guide. Retrieved f rom http://www.hopkinsguides.com/hopkins/view/Johns_Hopkins_ABX_Guide/540009/all/Adenovirus? q=adenovirus&ti=0#0 (Updated December 2014). 23. Technical Guidance, Naming the coronavirus disease (COVID-19) and the virus that causes it. World Health Organization. Retrieved from: https://www.who.int/emergencies/diseases/novelcoronavirus-2019/technical-guidance/naming-the-coronavirus-disease-(covid-2019)-and-thevirus-that-causesit#:~:text=ICTV%20announced%20%E2%80%9Csevere%20acute,two%20viruses%20are%20diff erent (Date accessed: 6/18/2020) 24. Friedman, J., (2012). Vaccination Program Appears to Reduce Respiratory Infections Among Recruits. Story number NNS120131-22. Retrieved from http://www.navy.mil/submit/display.asp?story_id=65070. 25. Coronavirus, About Coronavirus. Centers for Disease Control and Prevention. Retrieved from http://www.cdc.gov/coronavirus/about/index.html (Date accessed: 3/22/2016). 26. Coronavirus Inf ections. European Centre for Disease Prevention and Control. Retrieved from http://ecdc.europa.eu/en/healthtopics/coronavirus-infections/Pages/index.aspx (Date accessed: 3/24/2106). 27. Human Metapneumovirus Clinical Features, National Respiratory and Enteric Virus Surveillance System (NREVSS). Centers for Disease Control and Prevention. Retrieved from http://www.cdc.gov/surveillance/nrevss/hmpv/clinical.html (Date accessed: 3/22/2016). 28. Tapparel, C., et al., (2009). New Respiratory Enterovirus and Recombinant Rhinoviruses among Circulating Picornaviruses. Emerg Infect Dis Retrieved from http://wwwnc.cdc.gov/eid/article/15/5/08-1286 (Date accessed: 3/24/2016). 29. Jacobs, S., et al., (2013). Human Rhinoviruses. Clin Microbiol Rev 26(1) 135-162. 30. Enterovirus D68 detections in the USA, Canada and Europe, Second update 25 November 2014. European Centre for Disease Prevention and Control. Stockholm: ECDC; 2014. Retrieved from http://ecdc.europa.eu/en/publications/Publications/Enterovirus-68-detected-in-the-USA-CanadaEurope-second-update-25-November-2014.pdf (Date accessed: 3/22/2016). 31. Inf luenza, Types of Influenza Viruses. Centers for Disease Control and Prevention. Retrieved f rom http://www.cdc.gov/flu/about/viruses/types.htm (Date accessed: 3/22/2016). 32. Seasonal Influenza Factsheet for the General Public. European Centre for Disease Prevention and Control. Retrieved from http://ecdc.europa.eu/en/healthtopics/seasonal_influenza/basic_facts/Pages/factsheet_general_p ublic.aspx (Date accessed: 3/24/2016). 33. 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Retrieved from http://www.hopkinsguides.com/hopkins/view/Johns_Hopkins_ABX_Guide/540415/all/Parainfluenz a_virus?q=parainfluenza&ti=0#0 (Date accessed: 3/24/2016). 37. Respiratory Syncytial Virus Inf ection, Infection and Incidence. Centers for Disease Control and Prevention. Retrieved from http://www.cdc.gov/rsv/about/infection.html (Date accessed: 3/24/2016). 38. Walsh, E., et al., (1997). Severity of Respiratory Syncytial Virus Infection Is Related to Virus Strain. J Inf ect Dis 175(4) 814-820. 39. Bartlett, J., Chlamydophila pneumoniae. Johns Hopkins Antibiotics (ABX) Guide. Retrieved from http://www.hopkinsguides.com/hopkins/view/Johns_Hopkins_ABX_Guide/540117/all/Chlamydoph ila%20pneumoniae (Updated October 4, 2015). 40. Pneumonia. Centers for Disease Control and Prevention. Retrieved from http://www.cdc.gov/pneumonia/index.html (Updated February 25, 2015). 41. Mycoplasma pneumoniae Inf ection. Centers for Disease Control and Prevention. Retrieved from http://www.cdc.gov/pneumonia/atypical/mycoplasma/index.html (Date accessed: 3/28/2016). TRADEMARKS GenMark®, GenMark Dx®, eSensor®, ePlex®, Designed For the Patient, Optimized For the Lab® and The True Sample-to-Answer Solution® are registered trademarks of GenMark Diagnostics, Inc. BLAST® is a registered trademark of the National Library of Medicine KimwipesTM is a trademark of Kimberly-Clark Corporation. Flonase® is a registered trademark of GlaxoSmithKline, plc. Nasacort® is a registered trademark of Sanofi-Aventis Pharmaceuticals. Rhinocort® is a registered trademark of AstraZeneca AB. Af rin® is a registered trademark of Bayer. MicroTestTM M4®, M4RT®, M5®, and M6®, are registered trademarks of Thermo Fisher Scientific. Neo-Synephrine® is a registered trademark of Foundation Consumer Healthcare, LLC. Zicam® is a registered trademark of Matrixx Initiatives, Inc. ATCC® is a registered trademark of the American Type Culture Collection. PATENT INFORMATION ePlex® Respiratory Pathogen Panel and/or use thereof features technology claimed in one or more of the f ollowing United States and European patents owned or licensed by GenMark Diagnostics Inc. or its subsidiaries, with multiple additional foreign and domestic patents pending: U.S. Patent Nos. 7,172,897, 7,312,087, 7,534,331, 7,820,391, 8,486,247, 9,222,623, 9,410,663, 9,453,613, 9,498,778, 9,500,663, 9,598,722; 9,874,542, 9,957,553, 10,001,476, 10,391,489, 10,495,656, 10,352,983, 10,564,211, D881409, 10,669,592, D900330, 10,753,986, International Patent Nos. 1218541, 1246699, 60125713.8, 2220102, 602008031596.7, 1246699, 2278757, 60125713.8, 3548159, 9874542, 60017809.9, 1350568, 3548159, 2965817, 2889415, 3218725, 005250651-00001, 2889415, 1239806; and other international counterparts. Unless otherwise agreed to in writing, by using a cartridge, Recipient acknowledges that Recipient has read, accepts and agrees to be bound by and comply with the General Terms and Conditions of Sale available on GenMark's website which can be amended from time to time by GenMark without consent. If PI1114 F For Use Under Emergency Use Authorization Only. Page 59 ePlex Respiratory Pathogen Panel 2 Recipient does not accept and agree to be bound by the General Terms and Conditions of Sale, Recipient will immediately cease any further use of the cartridge. This product is subject to a limited license to use the product in the field of human in vitro diagnostics and research reasonably related thereto. Users are prohibited from using this product for other applications, including in the field of forensics (including human identification testing). Ef fective Date: July 2021 2021 GenMark Diagnostics, Inc. All rights reserved. Clinical Micro Sensors, Inc. dba GenMark Diagnostics, Inc. 5964 La Place Court, Carlsbad, CA 92008 +1 760 448 4300 www.genmarkdx.com PI1114 F For Use Under Emergency Use Authorization Only. Page 60

ePlex Respiratory Pathogen Panel 2 - Instructions for Use

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US FDA

References