OraQuick Ebola Rapid Antigen Test

{0}------------------------------------------------ # EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR ORAQUICK EBOLA RAPID ANTIGEN TEST DECISION SUMMARY # A. De Novo Number: DEN190025 # B. Purpose for Submission: De Novo request for evaluation of automatic class III designation for the OraQuick Ebola Rapid Antigen Test. # C. Measurand: Ebolavirus Antigens # D. Type of Test: Lateral flow immunoassay # E. Applicant: OraSure Technology, Inc. # F. Proprietary and Established Names: OraQuick Ebola Rapid Antigen Test # G. Regulatory Information: - 1. Regulation section: 21 CFR 866.4002 - 2. Classification: Class II - 3. Product code(s): QID {1}------------------------------------------------ # 4. Panel: Microbiology (83) # H. Intended Use: - 1. Intended use(s): The OraQuick Ebola Rapid Antigen Test is an in vitro diagnostic single-use immunoassay for the qualitative detection of antigens within the Ebolavirus genus but does not differentiate between these viruses. Testing with the OraQuick Ebola Rapid Antigen Test must only be performed when public health authorities have determined the need for this test. Testing for Ebola Virus Disease (EVD) must be performed in accordance with current guidelines provided by the appropriate public health authorities that address appropriate biosafety conditions, interpretation of test results, and coordination of testing, results and patient management with public health authorities. The OraQuick Ebola Rapid Antigen Test is intended for use with specimens from: - individuals with epidemiological risk factors with signs and symptoms of EVD or - . recently deceased individuals with epidemiological risk factors who are suspected to have died of EVD. EVD is a nationally notifiable condition and must be reported to public health authorities in accordance with local, state, and federal regulations. The OraQuick Ebola Rapid Antigen Test is intended for use with venipuncture whole blood and fingerstick whole blood specimens as an aid in diagnosis of EVD in patients suspected of and with signs or symptoms consistent with EVD who have epidemiological risk factor(s) for Ebolavirus exposure (e.g., contact with a known or suspected case, travel to a geographic location at a time when Ebolavirus transmission was known or suspected to have occurred). Performance of the device with Ebolavirus positive fingerstick whole blood was established in a non-human primate model. The OraQuick Ebola Rapid Antigen Test is intended for use with cadaveric oral fluid collected from recently deceased individuals with epidemiological risk factors who are suspected to have died of EVD. Cadaveric oral fluid should be collected directly with the device or collected with oral swabs in viral transport media. The OraQuick Ebola Rapid Antigen Test is intended as an aid in the determination of EVD as the cause of death to inform decisions on safe handling of cadavers to prevent disease transmission. The OraQuick Ebola Rapid Antigen Test results are presumptive, definitive identification of EVD requires performing additional testing and confirmation procedures in consultation with public health and/or other authorities to whom reporting is required. Negative results were observed in individuals with low levels of circulating virus, therefore negative results do not preclude infection with viruses within the Ebolavirus genus. {2}------------------------------------------------ The level of Ebolavirus antigens that would be present in EVD clinical specimens from individuals with early systemic infection is unknown. Test performance of the OraQuick Ebola Rapid Antigen Test is associated with the level of Ebolavirus antigens in the patient; therefore, the test is not intended for use in an asymptomatic population for massscreening purposes (e.g., as the sole means of EVD control at airports or bordercrossings) or for testing of individuals at risk of exposure without observable signs of infection. The OraQuick Ebola Rapid Antigen Test is intended for use by experienced personnel who have documented device specific training offered by OraSure Technologies Inc., training in the correct use of recommended personal protective equipment (PPE) and expertise in infectious disease diagnostic testing, including the safe handling of clinical specimens potentially containing Ebolavirus. The test is intended for use by laboratory professionals or healthcare workers who have demonstrated availability of biosafety equipment, access to patient containment facilities, and established procedures (e.g., SOP) for coordinating testing, results and patient management with public health authorities consistent with state, local and federal recommendations and guidelines. - 2. Indication(s) for use: Same as intended use. - 3. Special conditions for use statement(s): For prescription use only. For in vitro diagnostic use only. - 4. Special instrument requirements: Not applicable. # I. Device Description: The OraQuick Ebola Rapid Antigen Test is a manually performed, visually read immunoassay for the qualitative detection of Ebola virus in human venipuncture whole blood, fingerstick whole blood and cadaveric oral fluid. The OraQuick Ebola Rapid Antigen Test is comprised of both a single-use test device and a vial containing a pre-measured amount of a buffered developer solution. The single-use test devices consist of a plastic housing that protects the assay test strip with the reagents. The assay strip can be viewed through the test device result window. The test result window has indicated zones for the Ebolavirus specific Test Line and the Control Line of the test. The test consists of a sealed pouch with two separate compartments for each of the following two components: - 1. OraQuick Ebola Rapid Antigen Test {3}------------------------------------------------ # 2. Developer Vial The OraQuick Ebola Rapid Antigen Test further contains the following materials for the sample processing: - 1. Test Stand - 2. Micropipettes capable of pipetting 20ul of sample The OraQuick Ebola Rapid Antigen Test kit must be used with the OraQuick Ebola Rapid Antigen Test Kit Controls and a Visual Reference Panel (available separately). The OraQuick Ebola Rapid Antigen Test Kit Controls contain: - 1. One Ebola positive control vial (orange capped) - 2. One Ebola negative control vial (white capped) The positive control contains 0.25mL of Ebola recombinant VP40 equivalent to a moderate positive result and is diluted in a defibrinated pool of normal human plasma negative for Ebolavirus. The negative control contains 0.25 mL of defibrinated pool of normal human plasma negative for Ebolavirus. The controls do not contain infectious Ebolavirus material. The OraQuick Ebola Visual Reference Panel (VRP) is intended to assist new operators in becoming proficient at reading specimens with antigen levels near the limit of detection of the device. The devices in the VRP do not contain any infectious material. The VRP contains three Quick Ebola Rapid Antigen Test devices that have been designed to represent reading intensities representative of - 1. A test result near the limit of detection (LoD) - 2. A low positive test result - 3. A negative test result Additional materials required but not provided include 1. Timer # J. Standard/Guidance Document Referenced (if applicable): EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline—Third Edition EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline-Second Edition EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition {4}------------------------------------------------ # K. Test Principle: The OraQuick Ebola Rapid Antigen Test utilizes a proprietary lateral flow immunoassay procedure. The assay test strip, which can be viewed through the test device result window, is comprised of a series of components: the blocker pad, the conjugate pad, the nitrocellulose membrane, and finally the absorbent pad. The performance of the assay occurs by hydration and transport of reagents as they interact with the specimen across the strip via chromatographic lateral flow. The conjugate pad contains salts, buffers, and a signal generating reagent consisting of Ebola antibodies conjugated to colloidal gold. Ebolavirus antigens in the sample are captured by Ebola antibodies at the Test (T) Zone, which become immobilized on the nitrocellulose membrane and visualized by colloidal gold labeled with Ebola antibodies. The Control (C) Zone immobilized onto the nitrocellulose membrane is visualized by colloidal gold ensuring component elution, reagent activity, and adequate device performance. A fingerstick or venipuncture whole blood specimen is collected using a plastic micropipette or calibrated lab pipette both of which transfer 20 ul of blood to the device, followed by the insertion of the device into the developer vial. Cadaveric oral fluid specimens can be collected in two ways: - 1. Swabbing the gum line or the soft pallet tissue in the back of the throat with the flat pad of the device and then insert the device directly into the developer solution, or - 2. Swabbing the gum line or the soft pallet tissue in the back of the throat with a recommended swab that will then be inserted into recommended viral transport media The following viral transport systems are recommended and were validated for use with the device: Σ- Virocult VTM Collection (K082472) and BD Universal Viral Transport Medium (K042970). When using recommended viral transport media, 20 ul specimen are collected from the transport media tube using a calibrated lab pipette and transferred to the device through the sample port. The device is then inserted into the developer solution. For all sample types, the developer solution facilitates the capillary flow of the specimen into the device and onto the assay strip. As the specimen flows through the device, antigens from the specimen are bound by the Ebola antibody labeled gold colorimetric reagent present on the assay strip. If the specimen contains Ebola antigens, the resulting labeled complexes bind to the Test (T) Zone resulting in a red to purple line. If the specimen does not contain Ebolavirus, no labeled complexes bind at the Test Zone and no line is observed. The intensity of the line color is not proportional to the amount of virus present in the specimen. The remaining colloidal gold is transported and bound to the Control (C) Zone. This procedural control serves to demonstrate that the fluid has migrated adequately through the device. A red to purple line will appear at the Control (C) Zone during the performance of all valid tests whether or not the sample is positive or negative for Ebolavirus (refer to the Test Result and Interpretation of Test Result section in the package insert). Positive results may be interpreted as soon as red to purple lines are visible at the Test (T) Zone and Control (C) Zone. Negative results must be read 30 minutes after inserting the device into the Developer Vial. {5}------------------------------------------------ The OraQuick Ebola Rapid Antigen Test must be used with the external OraQuick Ebola Rapid Antigen Test Kit Controls and the Visual Reference Panel (available separately) according to the Instructions for Use. # L. Performance Characteristics (if/when applicable): Notes: - . The term 'Ebolavirus' refers to the genus Ebolavirus. The term 'Ebola virus' specifically refers to the species Ebola Zaire, one member of the genus Ebolavirus. Where this specific reference is not desired the general term Ebolavirus is used. - In the absence of sufficient amounts of cadaveric oral fluid analytical device ● validation studies were performed with oral fluid from living individuals. Contrived positive samples were derived by adding live or inactivated Ebolavirus or recombinant VP40 antigen to oral fluid from living individuals. - . Generally, the term contrived is used in this document for all non-natural samples. There are two ways Ebolavirus samples have been contrived for the studies described in this document: (1) negative matrix plus recombinant VP40 antigen and (2) negative matrix plus a live or inactivated Ebolavirus preparation. In both instances negative matrix is natural negative clinical matrix sourced from humans. No artificial matrices have been used in this de novo. The way samples were contrived is described for each study individually. # 1. Analytical performance: ### a. Precision/Reproducibility: A reproducibility study was performed at three (3) testing sites, two external and one internal, with three (3) operators per site. Each of the three (3) operators per site, tested each of three panel members twice daily with each of three lots over the course of 5 days. Panel members derived from recombinant Ebolavirus VP40 protein (rVP40-Ag) added into negative matrix at each of three different reactivity levels. i.e., negative, low positive (2x LoD) and moderate positive (5x LoD). Sample testing was performed blinded. Three sub-studies were performed for each of three matrices: Direct Collect Oral Fluid, Viral Transport Media (VTM), and Whole Blood. In the absence of large enough amounts of cadaveric oral fluid, this study was performed with oral fluid from living individuals as a surrogate for cadaveric oral fluid. Panel members for each matrix were formulated based on the respective LoD in the specific matrix. Combined results were analyzed per lot, operator and site. Results across operators, runs, lots and days are shown below per site and overall in Table 1 (Whole Blood), Table 2 (direct collected Oral Fluid) and Table 3 (VTM collected Oral Fluid). {6}------------------------------------------------ | | Negative | | Low Positive | | Moderate Positive | | |------------------------|----------|-----------------------|--------------|------------------------|-------------------|-------------------------| | | n/N | %CI | n/N | %CI | n/N | %/CI | | Site 1 | (b) (4) | | | | | | | Site 2 | | | | | | | | Site 3 | | | | | | | | Overall<br>Concordance | (b) (4) | 99.6%<br>(98.9, 99.9) | (b) (4) | 99.9%<br>(99.3, 100.0) | (b) (4) | 100.0%<br>(99.5, 100.0) | Table 1: Reproducibility of Functional Test Results – Summary Whole Blood # Table 2: Reproducibility of Functional Test Results - Summary Oral Fluid (Direct Collection) | | Negative | | Low Positive | | Moderate Positive | | |------------------------|----------|-----------------------|--------------|-----------------------|-------------------|-----------------------| | | n/N | %CI | n/N | %CI | n/N | %/CI | | Site 1 | (b) (4) | | | | | | | Site 2 | | | | | | | | Site 3 | | | | | | | | Overall<br>Concordance | (b) (4) | 98.1%<br>(97.0, 99.0) | (b) (4) | 95.2%<br>(93.5, 96.5) | (b) (4) | 99.4%<br>(98.6, 99.8) | # Table 3: Reproducibility of Functional Test Results - Summary Oral Fluid (VTM Collection) | | Negative | | Low Positive | | Moderate Positive | | |------------------------|----------|------------------------|--------------|------------------------|-------------------|-------------------------| | | n/N | %CI | n/N | %CI | n/N | %/CI | | Site 1 | (b) (4) | | | | | | | Site 2 | | | | | | | | Site 3 | | | | | | | | Overall<br>Concordance | (b) (4) | 99.9%<br>(99.3, 100.0) | (b) (4) | 99.8%<br>(99.1, 100.0) | (b) (4) | 100.0%<br>(99.5, 100.0) | - b. Linearity/assay reportable range: Not applicable {7}------------------------------------------------ - c. Traceability, Stability, Expected values (controls, calibrators, or methods): Controls Traceability: N/A Expected values: To monitor the assay performance, reagent performance, and procedural errors, positive and negative external controls must be run in accordance with the guidelines or requirements of local, state, and/or federal regulations or accrediting organizations. External Controls are provided separate from the OraQuick Ebola Rapid Antigen Test Kit devices in the OraOuick Ebola Rapid Antigen Test Kit Control kit. The Ebola positive control consists of recombinant Ebola VP40 diluted into a defibrinated pool of Ebolavirus negative normal human plasma. The Ebola negative control vial consists of defibrinated Ebolavirus negative normal human plasma. The positive control is expected to give a positive signal, the negative control is expected to provide a negative signal. #### Stability: Storage conditions for the control kit were evaluated in multipl (4) real-time stability ted (b) eplicates studies each of which tested one control kit lot. The main studie per time point for Shelf Life and open vial stability. Open vial stability is sassessed based on kits that had undergone(b) (4) month Shelf Life. All visual observations were graded as (b) (4) by comparing each test result to an Ebola Attribute Chart that consists of devices for which the colored Test Lines represent teading scores of 0 (b) (4) The targeted average level for the Test Line of the positive control is individual test replicates may have Test Lines of(b) (4) The targeted average level for the Test Line of the negative control i he data of each of each of the fours studies supports an expiration dating of 12 months for closed vials and 8 weeks for open vial when stored at 5℃ ± 3℃. In addition (b) (4) was evaluated as a storage condition. (b) (4) is not a recommended storage temperature due to (b) (4) (b) (4) Calibrators This device does not contain calibrators. {8}------------------------------------------------ ### Reagents Traceability: N/A Expected values: N/A Stability: #### Shelf Life (Realtime): Multiple real-time and accelerated stability studies were performed for the OraQuick Ebola Rapid Antigen Test kits. In the main study, multiple lots of the pouched OraQuick Ebola Rapid Antigen Test kits are subjected to real-time stability testing at both (b) (4) (b) (4) . The test panel consisted of the Ebola positive control from the associated control kit, a negative control (Ebolavirus negative whole blood) and an additional Panel Member (PM1). Starting at(b) (4) an additional panel member at 4x LoD of recombinant VP40 diluted into whole blood was added. Whole blood as a testing matrix was included in the assessment because it was identified as the limiting factor to correct shelf life assignment. Testing in clinical matrix curtails the risk that shelf life is over assigned. All test panel members were evaluated at each time point in replicates of each per lot. All visual observations were graded as (b) (4) by comparing each test result to an internal Ebola Attribute Chart that consists of devices for which the colored Test Lines represent reading scores of(b) (4) While assay output (signal strength) decrease at higher temperatures, all lots met the acceptance criteria over the entire course of the study at both 2-8°C and 0°C when tested with the positive control and PM1. With testing of the whole blood panel member at 4x LoD one lot met acceptance criteria up to month 11. one lot up to month 15 and the third lot up to 10 months. Based on the combined data of three lots upon consideration of the 4x LoD whole blood test panel member the stability assessment for the pouched OraQuick Ebola Rapid Antigen Test kit supports a shelf life of(b) (4) when stored at (b) (4) #### Shelf Life (Accelerated): The OraQuick Ebola Rapid Antigen product had been subjected also to accelerated stability testing performed a(b) (4) Based on the low positive test sample used in the study the accelerated stability study supports a time limited storage at elevated temperatures for up to(b) (4) {9}------------------------------------------------ # Shipping Stability: One of the lots subjected to reagent stability assessment underwent additional assessment for shipping stability. The shipping stability study evaluated exposure to cvcling temperature extremes of frozen (i.e., -18°C), tropical wet (i.e., 40°C with 80% Relative Humidity [RH]) and ambient temperature (b) (4) with(b) (4) RH). The data support shipping at ambient temperature with a transit time of up to 12 days. # Visual Reference Panel Traceability: N/A Expected values: The OraQuick Ebola Visual Reference Panel is intended to assist new operators in becoming proficient at reading specimens with antigen levels near the limit of detection of the device. All new operators must be able to correctly interpret all devices provided within the OraQuick Ebola Visual Reference Panel prior to using the OraQuick Ebola Rapid Antigen Test since the failure to read at low intensities can result in the inability to detect low positive patient samples near the limit of detection of the OraQuick Ebola Rapid Antigen Test and may consequently result in false negative results. The OraQuick Ebola Visual Reference Panel is comprised of OraQuick Ebola Rapid Antigen Test devices that have been designed to represent reading intensities of limit of detection, low positive, and negative test results. Stability: # Shelf Life (Realtime): Shelf life of the Visual Reference Panel (VRP) was assessed using (b) (4)lots of each of the devices contained in the VRP, i.e., Negative (Reading Level 0), LoD (Target Reading Level 1+) and Low Positive (Target Reading Level 3+). The following conditions were tested in the real time stability study: - Pouched devices at (b) (4) . - Pouched devices at (b) (4) . - Un-pouched devices at (b) (4) . - Un-pouched devices at (b) (4) . - Un-pouched devices (b) (4) . All three lots of VRP met all stability acceptance criteria for the Test and Control lines up to and including the month 13 time point for all conditions. There was no longitudinal degradation in signal intensity throughout the course of the study. This {10}------------------------------------------------ study supports a pouched and un-pouched shelf life of 12 months when stored at 15°C to 30°C including normal in-use benchtop conditions during a standard 8-hour work shift. # Shelf Life (Accelerated): An accelerated stability study was performed in which VRP panel members were tested at (b) (4) for each of the following conditions: - Pouched . - Un-pouched . - Benchtop condition . In the accelerated stability study open, closed and benchtop conditions all three VRP lots demonstrated no degradation in signal intensity after being subjected to (b) (4) Accelerated stability testing supports temporary temperature deviations during the shelf life. # Sample Stability - # Whole Blood Sample Stability: Individual whole blood samples were collected in K3-EDTA and tested with the OraQuick Ebola Rapid Antigen Test after various storage conditions. Each unique whole blood sample was divided in half. One half was left untreated (control), to the other half a dilution of gamma irradiated Ebola virus (Strain E. Zaire Mavinga, NR-31807) was added that produced a low positive sample at 2.5 x Limit of Detection (LoD). The following test time points were assessed: - Time 0 (immediate testing) - - (b) C storage testing conducted at hours (b) (4) - °C storage testing conducted at hours(b) (4) and at days(b) (4) - Three (3) freeze/thaw cycles at (b) (4) l with testing conducted at 24, 48 and 72 hours; evaluation of the frozen storage and freeze/thaw was included to support the used of clinical whole blood and fingerstick specimens that were a mix of fresh and frozen whole blood samples. Aliquots were evaluated for correct identification by the OraQuick Ebola Rapid Antigen Test after blinding and randomization. Results are listed below in Table 4 for refrigerated and incubated storage and in Table 5 for frozen storage and freeze/thaw cycles. {11}------------------------------------------------ | Time (Days) | % Correctly Identified at Indicated Storage Temperature | | | | |-------------|---------------------------------------------------------|----------|--------------|----------| | | 2-8°C | | 30°C | | | | Low Positive | Negative | Low Positive | Negative | | (b) (4) | | | | | Table 4. Whole Blood Sample Stability All samples were correctly identified throug (4) ours of testing. The whole blood sample stability study supports a sample stab for 24 hours up to 30℃. In the initial round of fresh/frozen testing 2 samples were missed after the first freeze/thaw cycle even though aliquots of the same sample were correctly detected after two and three freeze/thaw cycles. An investigation was performed that concluded that samples had likely been tested before fully equilibrating to room temperature. Additional 30 samples were subjected to one freeze thaw cycle only. Generally, samples after a 2 and 3 freeze-thaw cycle exhibited no loss of analyte signal due to the freezing process and the study therefore supports the use of frozen whole blood specimens with up to three freeze/thaw cycles. | Freeze/Thaw<br>Cycles | % Correctly Identified at Indicated Storage Temperature | | | | | |-----------------------|---------------------------------------------------------|----------|--|--|--| | | Low Positive | Negative | | | | | | (D) (4) | | | | | | 2 | | | | | | | | | | | | | Table 5: Whole Blood Freeze/Thaw Sample Stability # Oral Fluid Sample Stability - Direct Collection Method: For oral fluid samples directly collected with the test device through swabbing the gumline of an individual 10 samples each were tested that were negative (untreated) and low positive at 2.5x LoD. The low positive sample was prepared by spiking a pool of saliva with gamma irradiated virus at 2.5x LoD. Testing was performed by pipetting 70 ul of prepared negative and low positive solution directly onto the flat pad. The devices were then stored as follows to validate immediate and delayed insertion of the devices into the developer vial provided with the kit; samples were read randomized and blinded: {12}------------------------------------------------ - Refrigerated (2-8℃)(b) (4) । All negative and low positive samples were correctly identified upon immediate testing (b) (4) Upon storage of the sample loaded devices at (b) (4) the agreement for the low positive samples was 100% for all timepoints at 30, 60, and 90 minutes. The negative samples yielded expected results with(b) (4) concordance. Upon storage of the sample loaded devices at 2-8°C one low positive sample each at (b) (4) This study supports storage delayed insertion of the test device after the device with the oral fluid sample was stored at 2-8°C for up to (b) (4) # Oral Fluid Sample Stability - Viral Transport Media (VTM) Collection: (b) (4) {13}------------------------------------------------ This study supports sample stability for oral fluid in both the BD VTM and the 2-Virocult VTM for up to 48 hours from -70℃ to 40℃ including three freeze/thaw cycles. # d. Detection limit: Multiple studies were conducted to establish the limit of detection (LoD) of the OraQuick Ebola Rapid Antigen Test using gamma irradiated Ebola Zaire virus and recombinant VP40 Antigen. Considering feasibility in the context of a (b) (4) analyte the testing procedure had to be slightly modified for some of the analytical studies; however, each analytical and clinical study that relied on contrived samples is supported by an LoD study specific for the material and procedure used. Inactivation: To support the use of inactivated material in the LoD and other analytical studies parallel testing of live and inactivated Ebola Zaire material was performed as follows: Ebola Zaire virus stock (strain Mayinga) was diluted in PBS at a concentration of (b) (4) /mL and then diluted down to (b) (4) dilution series. Dilutions were aliquoted and frozen. For each of the dilutions inactivation was performed on one of the frozen aliquots using gamma irradiation at 5 x 106 RAD. Aliquots of each dilution of live Ebola Zaire virus and inactivated Ebola Zaire virus were tested with the OraQuick Ebola Rapid Antigen Test according to Package Insert instructions and were also subjected to nucleic acid extraction and VP40-PCR testing. Results as summarized in Table 6 below demonstrate that the reading score of the OraQuick Ebola Rapid Antigen Test is not impacted by the inactivation process as no difference between live and inactivated Ebola Zaire virus material was observed. Table 6: PCR and OraQuick Ebola Antigen Test results for parallel testing of live and inactivated Ebola Zaire Virus | | | Live | | Inactive | | |----------------------|-----------|-------------------------|----------|-------------------------|----------| | Dilution of<br>virus | Replicate | OraQuick<br>Visual Read | Ct value | OraQuick<br>Visual Read | Ct value | | | (b) | | | (4) | | {14}------------------------------------------------ Limit of Detection (LoD) in Whole Blood: To determine a tentative LoD, serial dilutions of inactivated Ebola Zaire virus (strain Mayinga) were made in a pool of Ebolavirus negative venous whole blood. Each dilution was tested in 10 or more replicates. The dilutions were randomized with Ebolavirus negative whole blood and blinded. Three operators each tested a subset of the randomized and blinded samples using the OraQuick Ebola Rapid Antigen Test. A Probit analysis was then used to determine a tentative LoD. defined as the concentration calculated to vield positive results 95% of the time. To confirm the tentative LoD, a panel of 20 samples at the tentative LoD (1.2 x 106 TCID50/mL), 20 negative samples, and 20 moderate positive samples (7.81 x 101 TCID50/mL) were prepared in negative venous whole blood, randomized and tested by three blinded operators. Table 7 summarizes the number of positive (LoD and Moderate Positive test devices) and negative results obtained for the inactivated virus positive and negative whole blood samples. Table 7: Summary of the confirmatory LoD study for the OraQuick Ebola Rapid Antigen Test with Whole Blood and inactivated Ebola Zaire virus (Mayinga) | Whole<br>Dilution | Concentration<br>[TCID50/mL] | Total<br>Number<br>of Tests | Ct Value of<br>inactivated viral<br>stock at indicated<br>concentration | Number of<br>Positive<br>Results | Number of<br>Negative<br>Results | |-------------------|------------------------------|-----------------------------|-------------------------------------------------------------------------|----------------------------------|----------------------------------| | LoD | 1.64 x 106 | 20 | (b) (4) | 19 | 1 | | (b) (4) | | | | | | The same procedure (i.e., initial range finding study followed by a confirmatory study with 20 replicates) was followed for determining an LoD with recombinant VP40 antigen and a second Ebola Zaire virus stock from BEI Resources (NR-31807) in support of analytical studies that used these additional materials. Table 8 below provides a summary of the LoD of the OraQuick Ebola Rapid Antigen Test with the different materials used in the analytical and contrived clinical studies. {15}------------------------------------------------ | | Ebola Zaire (Mayinga)<br>[TCID50/mL whole<br>blood]1 | rVP40-Ag<br>[ng/mL whole<br>blood]1 | E. Zaire Mayinga<br>(NR-31807)]<br>[Dilution]1 | |-----|------------------------------------------------------|-------------------------------------|------------------------------------------------| | LoD | 1.64 x 106 | 53ng/mL | (b) (4) | Table 8: LoD of the OraQuick Ebola Rapid Antigen Test for Whole Blood when used with specified test materials. 1 LoD when 20 ul of this dilution in whole blood is applied to the sample port of the test device. Limit of Detection (LoD) in Oral Fluid: A similar study, designed as the whole blood LoD study (above), was performed; i.e., an initial range finding study with at least 10 replicates per dilution was followed by a confirmatory LoD study that tested 20 replicates each of samples that were negative. moderate positive and concentrated at the tentative LoD. In support of the analytical studies that used contrived samples the LoD was established for inactivated virus as well as for recombinant VP40 antigen. Both collection methods. i.e., (1) direct collection of oral fluid and (2) collection of oral fluid with a flocked swab/VTM (see section K. Test Principle) were validated for their respective LoD. For the flocked swab/VTM method two swab/VTM systems were validated, 2- Virocult VTM and the BD Universal Transport Medium. Oral fluid with each of the methods was obtained from 5 or more individuals using either the flat pad of the OraQuick device or the flocked swab of the respective VTM systems. After collection, 5uL of the inactivated virus solutions or of the recombinant VP40 antigen (rVP40-Ag) solutions were pipetted on either the collection pad for the direct collection method) or onto the flocked swab (for the VTM methods). Negative samples were collected the same way but 5ul of Ebolavirus negative diluent was added. Collection and spiking were performed by an unblinded operator per a randomization schedule. Each of three blinded operators then tested and read a subset of the devices 30 minutes after placement of the device in the developer solution. Results of the confirmatory study are summarized in Table 9 below for inactivated Ebola Zaire Mayinga and in Table 10 for rVP40-Ag. Note that the Ebola Zaire Mayinga (BEI Resources, NR-31807) used in the oral fluid studies was a different viral stock from the one used for the whole blood LoD study above: the BEI material was not provided with a titered concentration prior to inactivation and as such no concentration can be listed for the dilutions. Table 9: Summary of the confirmatory LoD study for the OraQuick Ebola Rapid Antigen Test with Oral Fluid using gamma-irradiated E. Zaire Mayinga (BEI, NR-31807) | Sample | Dilution of<br>Viral Stock<br>NR-31807 | Total<br>Number of<br>Tests | Number of<br>Positive<br>Results | Number of<br>Negative<br>Results | |---------|----------------------------------------|-----------------------------|----------------------------------|----------------------------------| | (b) (4) | | | | | {16}------------------------------------------------ | Direct | (b) (4) | |-----------------------------------------------------------------------|---------| | Collection | | | OraQuick | | | Σ- Virocult | | | VTM | | | Collections | | | BD Universal | | | Transport | | | Medium | | | LR: Low positive based on range finding study | | | MR: Moderate positive based on range finding study | | | N/A: Not applicable; no inactivated virus was added onto the devices. | | Table 10: Summary of the confirmatory LoD study for the OraQuick Ebola Rapid Antigen Test with Oral Fluid and rVP40-Ag | | Sample | Concentration<br>of added<br>rVP40-Ag<br>Solution<br>(µg/mL) | Total<br>Number of<br>Tests | Number of<br>Positive<br>Results | Number of<br>Negative<br>Results | |-------------------------------------|----------|--------------------------------------------------------------|-----------------------------|----------------------------------|----------------------------------| | Direct<br>Collection<br>OraQuick | LoD | (b) (4) | 20 | 19 | 1 | | | Moderate | | 20 | 20 | 0 | | | Negative | | 20 | 0 | 20 | | Σ- Virocult<br>VTM<br>Collections | LoD | | 20 | 20 | 0 | | | Moderate | | 20 | 19 | 1 | | | Negative | | 20 | 0 | 20 | | BD Universal<br>Transport<br>Medium | LoD | | 20 | 20 | 0 | | | Moderate | | 20 | 20 | 0 | | | Negative | | 20 | 0 | 20 | Table 11 below provides an overview over the oral fluid LoD obtained with all different oral fluid sample collection/processing methods and Ebola virus materials used in the analytical and contrived clinical studies and/or claimed per the intended use of the device. {17}------------------------------------------------ | Collector | LoD<br>[Amount of<br>rAg per Test]1 | LoD<br>[Concentration<br>of rVP40-Ag in<br>Oral Fluid1] | LoD<br>[Dilution of<br>gamma-irradiated<br>E. Zaire Mayinga | |----------------------------------------|-------------------------------------|---------------------------------------------------------|-------------------------------------------------------------| | Direct Collection<br>OraQuick | 0.53 ng/test | 7.6 ng/mL | (b) (4) | | Σ-Virocult<br>Transport System | 3.20 ng/test | 3200 ng/mL | (b) (4) | | BD Universal Viral<br>Transport Medium | 0.22 ng/test | 465 ng/mL | | Table 11: LoD of the OraQuick Ebola Rapid Antigen Test for Oral Fluid when used with different collection methods and test materials. 1 LOD concentration in oral fluid was calculated using the average volumes of oral fluid that are absorbed by each of the swabs/devices and the volume of VTM (i.e., 70 ul for the OraQuick Ebola Rapid Antigen Test flat pad, 70 ul for the BD swab diluted into 3 mL of VTM and 50 ul for the 2-Virocult swab diluted in 1 mL VTM) and the subsequent volume of 20ul of the VTM solution that was transferred to the device. 2 LoD when 5 ul of this dilution is applied to the swab or directly to the flatpad of the OraQuick test device. #### e. Analytical specificity: #### Reactivity (Inclusivity) Analytical reactivity of the OraQuick Ebola Rapid Antigen Test was evaluated for additional strains of the Ebolavirus (Table 12). Testing of three (3) replicates was performed using negative venous whole blood as the testing sample matrix. The OraQuick Ebola Rapid Antigen Test also reacts with E. Sudan and E. Bundibugyo in addition to reacting with E. Zaire. All Ebolavirus strains were provided as inactivated viruses. Each viral strain was tested in three (3) replicates when added at a 1:2 and/or 1:10 dilution in whole blood at the concentrations listed in Table 12 below. The OraQuick Ebola Rapid Antigen Test is positive with Ebola Sudan (strains Boneface [1976] and Gulu [2000]) and with Ebola Bundibugyo (strain Uganda [2007]) but is negative with Ebola Ivory Coast [1994] and Ebola Reston [1989/90]. {18}------------------------------------------------ | Ebolavirus Strain | Concentration Tested | Positive<br>Replicates | Reactivity<br>(Positive (P)/<br>Negative (N)) | |--------------------------------------------|-------------------------|------------------------|-----------------------------------------------| | Ebola Zaire Mayinga<br>ZZXDK901/812094/VSP | 1.5 x 106 TCID50/mL | | Positive as per LoD study | | Taï Forest | Unknown (1:10 dilution) | 0/3 | N | | (Ivory Coast, 11/27/94) | Unknown (1:2 dilution) | 0/3 | N | | Taï Forest | 1.36 x 107 VP/mL1 | 0/3 | N | | (Ivory Coast) | 7.5 x 107 VP/mL1 | | | | Reston | 3.16 x 106 pfu/mL | 0/3 | N | | (119876 Pennsylvania) | 1.58 x 107 pfu/mL | 0/3 | N | | Reston (aka H28) | 5.83 x 105 PFU/mL | 0/3 | N | | Sudan | 1 x 105 pfu/mL | 0/3 | N | | (Boneface) | 5 x 105 pfu/mL | 3/3 | P | | Sudan | 5.6 x 104 pfu/mL | 2/3 | P | | (200011676 Gulu) | 2.8 x 105 pfu/mL | 3/3 | P | | | 3.25 x 105 PFU/mL1 | 0/3 | N | | Sudan | 5.95 x 105 PFU/mL 1 | 0/3 | N | | (200011676 Gulu) | 1.19 x 106 PFU/mL 1 | 3/3 | P | | | 1.79 x 106 PFU/mL 1 | 3/4 | P | | Bundibugyo | 3.98 x 104 pfu/mL | 2/3 | P | | (200706291 Uganda<br>prototype) | 1.99 x 105 pfu/mL | 3/3 | P | | | 3.73 x 104 PFU/mL 1 | 0/3 | N | | Bundibugyo | 6.83 x 104 PFU/mL 1 | 1/3 | N | | (Uganda) | 1.37 x 105 PFU/mL 1 | 3/3 | P | | | 2.05 x 105 PFU/mL 1 | 3/3 | P | Table 12: Inclusivity with inactivated virus of different Ebolavirus species # Cross reactivity: Cross reactivity of the OraQuick Ebola Rapid Antigen Test was initially evaluated by testing additional viral, bacterial, and parasitic pathogens according to the same protocol and study design that was outlined under reactivity (above). Testing was performed with live organisms unless otherwise indicated. In the study, three (3) replicates were tested with the pathogens when added to venous whole blood at the concentrations listed in Table 13 below. None of the tested organisms produced false positive results in the OraQuick Ebola Rapid Antigen Test at the concentration tested. Potential cross reactivity with Dengue Virus was not assessed. HIV-2 cross reactivity was evaluated with HIV-2 positive samples from the Ivory Coast. {19}------------------------------------------------ Table 13: Cross reactivity | Virus/Bacteria/Parasite | Type/Strain | Concentration Tested | Reactivity | |--------------------------------------|-------------------------------------------------------------------|-------------------------------------------------------|------------| | Marburg1 | RAVN | Log 5.53 TCID50/mL | None | | | Musoke | 3.16 x106 PFU/mL | None | | | | 3.73 x 106 PFU/mL | None | | | 200501379 Angola prototype | 1.44 x 106 PFU/mL | None | | | VOEGE | 3.6 x106 PFU/mL | None | | Crimean Congo<br>Hemorrhagic Fever1 | OMAN199809166 #811466 | 5.6 x104 TCID50/mL | None | | Lassa1 | Josiah | 3.16 x 105 PFU/mL | None | | | | 1.88 x 105 PFU/mL | None | | | Macenta (aka Z-136) | 1.19 x 107 PFU/mL | None | | | Pinneo | 2.00 x 106 PFU/mL | None | | Rift Valley Fever1 | ZH-501 | 3 x106 PFU/mL | None | | | | 5.43 x 106 PFU/mL | None | | Yellow Fever1 | Vaccine Strain #806588 | Unknown | None | | | Asibi | 1.88 x 106 PFU/mL | None | | Chikungunya virus | ATCC VR-64 | 3.0 x 108 LD50/mL | None | | Dengue virus | Serotype 1, strain WP74 | 6.20 x 104 PFU/mL | None | | | Serotype 2, strain 16803 | 7.18 x 105 PFU/mL | None | | | Serotype 3, strain CH53489 | 1.52 x 104 PFU/mL | None | | | Serotype 4, strain 34150 | 2.12 x 105 PFU/mL | None | | Influenza A | A/Wisconsin/10/1998 | 2.3 x 105 TCID50/mL | None | | Influenza B | B/Florida/04/06 | 4.6 x 105 TCID50/mL | None | | Rotavirus | ATCC VR-899 | 6.4 x 106 TCID50/mL | None | | Adenovirus | Type 5 ATCC VR-5 | 2.0 x 105 TCID50/mL | None | | RSV | ATCC VR-26 | 9.0 x 105 TCID50/mL | None | | Enterovirus | Enterovirus 71 ATCC VR-<br>1432 | 5.1 x 105 TCID50/mL | None | | Salmonella | <i>S. enterica</i> ATCC 10708 | 3.8 x 107 CFU/mL | None | | Salmonella typhi | <i>S. typhi</i> ATCC 6539 | 5.4 x 105 CFU/mL | None | | Shigella | <i>S. dysenteriae</i> ATCC 9361 | 4.0 x 106 CFU/mL | None | | Pseudomonas<br>aeruginosa | <i>P. aeruginosa</i> ATCC 15442 | 3.4 x 108 CFU/mL | None | | Vibrio Cholera | <i>V. cholera</i> ATCC 39050 | 6.7 x 105 CFU/mL | None | | Streptococcus<br>pneumonia | <i>S. pneumonia</i> ATCC 6303 | 2.1 x 108 CFU/mL | None | | Hemophilus influenza<br>(meningitis) | <i>H. influenzae</i> ATCC 33930 | 3.0 x 107 CFU/mL | None | | Leptospira genus | <i>L. biflexa</i> ATCC 23582 | ~ 9.1 x 104 CFU/mL | None | | Neisseria meningitides | <i>N. meningitides</i> ATCC 13090 | 3.5 x 106 CFU/mL | None | | Yersinia enterocolitica | <i>Y. enterocolitica</i> ATCC 23715 | 7.0 x 106 CFU/mL | None | | Plasmodium falciparum<br>(malaria) | <i>P. falciparum</i> ATCC 30932 | 0.26% parasitemia | None | | Virus/Bacteria/Parasite | Type/Strain | Concentration<br>Tested | Reactivity | | Plasmodium viva<br>(malaria) | <i>P. vivax</i> ATCC 30151 | 6.8 x 105 cells/mL | None | | Trypanosoma | <i>T. cruzi</i> ATCC 30013 | 3.1 x 107 CFU/mL | None | | Rickettsia | <i>R. africae</i> (protein) BEI NR-<br>42992 | 11.6 mg/mL | None | | Bacteroides fragilis | VPI 2553 [EN-2; NCTC 9343]<br>ATCC 25285 | 1.0 x 108 CFU/mL | None | | Klebsiella pneumoniae | [CIP 104216, NCIB 10341]<br>ATCC 4352 | 5.8 x 107 CFU/mL | None | | Enterococcus faecium | NCTC 7171 [DSM 20477,<br>JCM 8727, NCDO 942] ATCC<br>19434 | 7.6 x 107 CFU/mL | None | | Escherichia coli | AMC 198 ATCC 11229 | 4.5 x 107 CFU/mL | None | | Vesicular Stomatitis<br>Virus | Rhabdovirus Indiana Lab [V-<br>520-001-522]<br>ATCC VR-158 | 7.9 x 106 TCID50/mL | None | | Human<br>Immunodeficiency<br>Virus-1 | Clinical Sample<br>ZeptoMetrix 022117B1D | 5.1 x 106 TCID50/mL | None | | Hepatitis A Virus | Clinical Sample<br>University of Ottawa<br>021916B1C-1D | 4.5 x 107 TCID50/mL | None | | Cytomegalovirus | Herpesviridae AD-169 ATCC<br>VR-1788 | 2.9 x 105 TCID50/mL | None | | Epstein-Barr Virus | Herpesviridae,<br>Lymphocryptovirus P-3 ATCC<br>VR-602 | 1.44 x 105 copies/mL2 | None | | Hepatitis B Virus | Clinical Sample<br>DLS13-01459 | 1.2 x 107 IU/mL | None | | West Nile Virus | Flaviviridae, Flavivirus<br>B 956 [V-554-001-522]<br>ATCC VR-1267 | 2.3 x 108<br>TCID50/mL | None | | Hepatitis C Virus | Clinical Sample<br>DLS14-08008 | 4.7 x 106 IU/mL | None | | Mumps Virus | Paramyxoviridae, Rubulavirus<br>Jones<br>ATCC VR-1438 | 7.1 x 105 TCID50/mL | None | | Measles | Paramyxoviridae,<br>Morbillivirus Edmonston<br>ATCC VR-24 | 1.5 x 106 TCID50/mL | None | | Rubella | Togaviridae, Rubivirus M33<br>ATCC VR-315 | 1.5 x 105 TCID50/mL | None | | Streptococcus<br>pneumoniae | Slovakia 14-10 [29055]<br>ATCC 700677 | 4.6 x 104 CFU/mL | None | | Virus/Bacteria/Parasite | Type/Strain | Concentration<br>Tested | Reactivity | | Borrelia hermsii3 | HS1 Serotype 26<br>ATCC BAA-2821<br>Lot 6020 | 1:11 Dilution of<br>purchased<br>unquantified culture | None | | Yersinia<br>pseudotuberculosis3 | IP2666 BEI NR-4371<br>Lot 57852108 | 1:11 Dilution of<br>purchased<br>unquantified culture | None | | Rickettsia australis3 | JC BEI NR-10454 | 8.1 x 105 TCID50/mL | None | {20}------------------------------------------------ {21}------------------------------------------------ 1 = Organisms and strains marked with an asterisk were tested inactivated 2 = determined by digital droplet PCR in plasma 3 = Due to the unavailability of Yersinia pestis, Borrelia recurrentis, Rickettsia prowazekii, and Rickettsia typhi, surrogate organisms Borrelia hermsii, Yersinia pseudotuberculosis, and Rickettsia australis were utilized. # Interference: Exogenous and endogenous interfering substances were evaluated for interference by adding the potential interfering substances to Ebolavirus negative matrix and to rVP40 antigen containing matrix (positive). Recombinant VP40 antigen was spiked at 2 x LoD; some substances were repeated at 4 x LoD (see footnotes in the tables below). Endogenous interferents testing for Bilirubin, Hemoglobin, Protein and HAMA was completed in venous whole blood. Three whole blood samples were each tested in two replicates; for Cholesterol 4 whole blood samples were each tested in 3 replicates; for HAMA; Rheumatoid Factor (RF) testing was performed on 3 serum samples, that were each tested in two dilutions with 2 replicates. Dilutions for RF were made in serum. The concentrations of RF that were tested ranged from 525 IU/mL to 11,900 IU/mL. For testing oral fluid specific potential interferents, mucin and leukocytes were added into Ebolavirus negative and rVP40 antigen containing positive expectorated oral fluid from living individuals. Additionally, subjects used toothpaste prior to direct oral fluid collections with the test device. Results of the interference testing are summarized in Table 14 and Table 15 below for endogenous (in venous whole blood and oral fluid as indicated) and exogenous (tested in venous whole blood only) interfering substances, respectively. {22}------------------------------------------------ Table 14: Testing of potentially interfering endogenous substances specific to whole blood and oral fluid | Interfering<br>Substance | Target Testing<br>Concentration | n | rVP40 Ag Negative<br>Samples | | rVP40-Ag Positive<br>Samples | | |----------------------------------------------------|---------------------------------|---------|------------------------------|--------------|------------------------------|---------------| | <b>whole blood-specific potential interferents</b> | | | | | | | | Bilirubin | 25 mg/dL | 6 | 0 | 6 (100%) | 6 (100%) | 0 | | Hemoglobin | 20 g/dL | 6 | 0 | 6 (100%) | 6 (100%) | 0 | | Protein | 5 g/dL | 6 | 0 | 6 (100%) | 6 (100%) | 0 | | HAMA | 2464 ng/mL | 6 | 0 | 6 (100%) | 6 (100%) | 0 | | Rheumatoid<br>Factor | 11,900 IU/mL | 6 | 6 (100%) | 0 | 6 (100%) | 0 | | Rheumatoid<br>Factor<br>(KB82487)2 | 2000 IU/mL | 6 | 5 (83%) | 1 (17%) | 6 (100%) | 0 | | | 2920 IU/mL | 2 | 2 (100%) | 0 | 2 (100%) | 0 | | Rheumatoid<br>Factor | 1460 IU/mL | 4 | 4 (100%) | 0 | 4 (100%) | 0 | | Rheumatoid<br>Factor | 1050 IU/mL | 2 | 0 | 2 (100%) | 2 (100%) | 0 | | Cholesterol | 13 mmol/L | 12 | 0 | 12 (100%) | 12<br>(100%) | 0 | | Antinuclear<br>Antibodies | 1:160 titer | 12 | 0 | 12 (100%) | 11<br>(91.7%) | 1 (8.3%)1 | | Antinuclear<br>Antibodies | 1:120 titer | 3 | | | 3 (100%) | 0 | | Antinuclear<br>Antibodies | 1:160 titer at 4.0 x<br>LoD | 12 | | | 12<br>(100%) | 0 | | <b>oral fluid-specific potential interferents</b> | | | | | | | | Toothpaste | N/A | | | | | | | Mucin | 20 mg/mL | (b) (4) | | | | | | Mucin | 15 mg/mL | | | | | | | Leukocytes | 6.12 x 109 cells / L | | | | | | | Interfering<br>Substance | Target Testing<br>Concentration | n | rVP40-Ag Negative<br>Samples | | rVP40-Ag Positive<br>Samples | | | | | | Positive | Negative | Positive | Negative | | Acetylsalicylic<br>Acid | 3.62 mmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Salicylic Acid | 4.34 mmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Sulfamethoxazole | 1.58 mmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Ribavirin | 11.04 µg/mL | 12 | 0 | 12<br>(100%) | 11<br>(91.7%) | 1<br>(8.3%)1 | | | 5.52 µg/mL | 3 | NT | NT | 3<br>(100%) | 0 | | | 11.04 µg/mL at<br>4.0 x LoD | 12 | NT | NT | 12<br>(100%) | 0 | | Emtricitabine | 7.5 µg/mL | 12 | 0 | 12<br>(100%) | 11<br>(91.7%) | 1<br>(8.3%)1 | | | 3.75 µg/mL | 3 | NT | NT | 3<br>(100%) | 0 | | | 7.5 µg/mL at<br>4.0 x LoD | 12 | NT | NT | 12<br>(100%) | 0 | | Efavirenz | 16.6 µg/mL | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Ritonavir | 33.6 µg/mL | 12 | 0 | 12<br>(100%) | 10<br>(83.3%) | 2<br>(16.7%)1 | | | 16.8 µg/mL | 6 | NT | NT | 6<br>(100%) | 0 | | | 33.6 µg/mL at<br>4.0 x LoD | 12 | NT | NT | 12<br>(100%) | 0 | | Elvitegravir | 5.61 µg/mL | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Chloroquine | 17.95 µg/mL | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Atovaquone /<br>Proguanil | 89.1/2.8 µg/mL | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Ibuprofen | 2425 µmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Acetaminophen | 1324 µmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Interfering | Target Testing<br>Concentration | n | rVP40-Ag Negative<br>Samples | | rVP40-Ag Positive<br>Samples | | | Substance | | | Positive | Negative | Positive | Negative | | Quinine | 148 µmol/L | 12 | 0 | 12<br>(100%) | 11<br>(91.7%) | 1<br>(8.3%)1 | | | 74 µmol/L or<br>24 µg/mL | 3 | NT | NT | 3<br>(100%) | 0 | | | 148 µmol/L at<br>4.0 x LoD | 12 | NT | NT | 12<br>(100%) | 0 | | Rifampin | 78.1 µmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Amoxicillin | 206 µmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Tetracycline | 34 µmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Erythromycin | 81.6 µmol/L | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | | Biotin | 3.6 µg/mL | 12 | 0 | 12<br>(100%) | 12<br>(100%) | 0 | of failed replicates in the entirety of this study is consistent with the statistical failure rate of a low positive sample near the LoD. Therefore, those interfering substances that produced occasional false negative results in the low positive rVP40-Ag containing samples (2x LoD) were repeated in the presence of slightly higher rVP40-Ag concentration (4x LoD) to distinguish random statistical distribution of measurements on a low positive sample from true interference of the test substance. 0 (4 was a RF positive plasma and testing was performed in plasma not in whole blood Interference (i.e., false positive results) was observed when testing RF and high concentrations of Mucin (>15mg/mL). Titration indicated that this effect disappeared when concentrations fell to 1050 IU/mL for RF and to 15 mg/mL for mucin. This observation is noted in a limitation described in the package insert. {23}------------------------------------------------ Table 15: Testing of potentially interfering exogenous substances (commonly used drugs) in whole blood {24}------------------------------------------------ Considering the technology (i.e. manually performed and visually read test), the total number of failed replicates in this study is consistent with the statistical failure rate of a low positive sample near the LoD. Therefore, those interfering substances that produced occasional false negative results in the low positive rVP40-Ag containing samples (2x LoD) were repeated in the presence of slightly higher rVP40-Ag concentration (4x LoD) to distinguish random statistical distribution of measurements on a low positive sample from true interference of the test substance. > Occasional false negative results in the presence of rVP40-Ag were observed for some endogenous interferents (i.e., mucin and ANA) and some drugs (i.e., Ribavirin, Emtricitabine, Ritonavir and Quinine). However, considering the low spiking concentration of 2x LoD, the total number of rVP40 positive samples included in the study and that this test is a manually performed and visually read test, this observation was likely due to the random distribution of a very low positive sample. Therefore, testing was repeated at a slightly higher but still low positive concentration (i.e., 4x LoD). The additional testing confirmed that the observation of occasional false negative results with a very low positive sample were likely due to random statistical distribution of a low positive sample and not true interference of the substance. {25}------------------------------------------------ #### f. Sensitivity with the WHO Reference Reagent: There is currently no International Standard for Ebolavirus Antigens available. However, multiple candidate EBOV recombinant protein preparations were evaluated to serve as WHO International Reference Reagents (IRR) for monitoring the performance of Ebolavirus Antigen based tests to be used for presumptive diagnosis of EVD. This study used the 1st Reference Reagent 2016, lyophilized Ebola VP40 Antigen: Item# 2016.2302 (refer to Wilkinson et al. 2016, WHO collaborative study to assess the suitability of a WHO International Reference Panel for Ebola virus VP40 antigen; WHO/BS/2016.2302), obtained from the National Institute for Biological Standards and Control (NIBSC). Only 'Sample 3' of the WHO Reference Reagent was used; it is a low positive sample based on freeze-dried recombinant VP40 (full-length) expressed in E.coli. The VP40 sequence is based on a 2014 isolate of Ebola Zaire (i.e., Makona-Kissidougou-C15; GenBank KJ660346). This study was designed to test the sensitivity of the OraQuick Ebola Rapid Antigen Test with the Ebola Reference Reagent described above. Only 5 replicates per dilution were tested because of the limiting amount of material available. The study was performed with one lot of the OraQuick Ebola Rapid Antigen Test and one lot of the OraQuick Ebola Rapid Antigen Test Kit Controls. A total of three vials of the reference material were combined and diluted with the following dilution factors: 1:3, 1:6, 1:9, 1:12, 1:24 and 1:48. Dilutions were performed using the most challenging matrix, i.e., venous whole blood and dilutions and replicates were randomized for testing. Each dilution was tested in five replicates. Negative venous whole blood was tested in three replicates as a negative control. Results were graded visually as 0 to 5+ or >+5 by comparing each test result to an internal Ebola Attribute Chart that consists of devices for which the colored Test Lines represent reading scores of 0 to >+5. Results are summarized in Table 16 below. | | Test Replicate | | | | | Final<br>Result | |----------|----------------|---|---|---|---|-----------------| | Dilution | 1 | 2 | 3 | 4 | 5 | | | 1:3 | (b) (4) | | | | | | | 1:6 | | | | | | | | 1:9 | | | | | | | | 1:12 | | | | | | | | 1:24 | | | | | | | | 1:48 | | | | | | | Table 16: Testing of the WHO Reference Reagent The reconstituted Ebola rVP40-Ag resulted in high positive visual grades (> 4+) when tested undiluted with the OraQuick Ebola Rapid Antigen Test. The visual grade intensity decreased to low positive (≤ 3+) or negative when diluted 1:6 in whole blood. {26}------------------------------------------------ - g. High Dose Hook Effect study Performance of the OraQuick Ebola Rapid Antigen Test was evaluated utilizing high concentrations of Ebola specific rVP40-Ag. A single operator utilizing a single lot of reagents tested samples corresponding to rVP40-Ag concentrations of (4) ug/mL to 5+ at (b) ug/mL ug/mL. Line intensity increased from 2+ at (b) and remained at 5+ at the higher testing concentrations. There was no visible decline in band intensity when going from (b) (4) ug/mL analyte concentrations. - h. Assay cut-off: Not applicable - 2. Comparison studies: - a. Method comparison with predicate device: Not applicable - b. Matrix comparison: Not applicable - 3. Clinical studies: Clinical performance as outlined below was assessed in multiple studies using samples and results from routine testing performed as part of the response to the 2014 Ebola Zaire outbreak in West Africa. All natural clinical study samples were collected from patients of the intended use population, i.e., patients with signs and symptoms of EVD with epidemiological risk factors or individuals who recently died and were suspected to have died of EVD with epidemiological risk factors. Studies were performed according to the instructions for use of the OraQuick Ebola Rapid Antigen Test that was under emergency use authorization (EUA) at that time. Data collection for the West African studies was not based on a pre-defined study protocol developed by Orasure Technologies but was instead collected as part of the routine testing. Demographic study information was not available for these studies and patients and samples were not individually identifiable. Studies performed by OraSure, such as the U.S. based specificity studies were IRB approved and samples collected with informed consent where required. Study specific enrollment criteria and demographics for the specificity studies in a U.S. based population are provided for each of the studies in their respective sections below. Samples were tested, and results were read per instructions for use; i.e., positive sample results were read as soon as a signal appeared, negative sample results were read at 30 minutes (unless otherwise noted) and within a 3 minutes maximal time window. Clinical {27}------------------------------------------------ studies were performed upon use of the control kit and visual reference panel (VRP) as follows: # External Controls During the conduct of the clinical trial protocols, external control testing was performed at the following time points: - Each new operator: - । Each new lot of test kits; - -Each new shipment of test kits: - ្រី Test kit storage temperature was outside the range of 2°C - 40°C or 36°F -104°F: and - ା Testing area temperature was outside the range of 15°C - 40°C or 59°F -104°F - i For the new de novo specificity cohort controls were also run when testing was repeated for invalid results. # Visual Reference Panels Clinical sites and participating organizations were provided with visual reference panels to assist new operators in becoming proficient at reading specimens with antigen levels near the limit of detection of the device. The following table provides a summary of all studies that support clinical performance: | | Sen…