← Product Code [QWR](/submissions/MI/subpart-d%E2%80%94serological-reagents/QWR) · K221925 # ID NOW COVID-19 2.0 (K221925) _Abbott Diagnostics Scarborough, Inc. · QWR · Aug 10, 2023 · Microbiology · SESE_ **Canonical URL:** https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QWR/K221925 ## Device Facts - **Applicant:** Abbott Diagnostics Scarborough, Inc. - **Product Code:** [QWR](/submissions/MI/subpart-d%E2%80%94serological-reagents/QWR.md) - **Decision Date:** Aug 10, 2023 - **Decision:** SESE - **Submission Type:** Dual Track - **Regulation:** 21 CFR 866.3982 - **Device Class:** Class 2 - **Review Panel:** Microbiology ## Indications for Use ID NOW COVID-19 2.0 performed on the ID NOW Instrument is a rapid molecular in vitro diagnostic test utilizing an isothermal nucleic acid amplification technology (NAAT) intended for the qualitative detection of nucleic acid from SARS-CoV-2 in direct anterior nasal (nasal) or nasopharyngeal swabs from individuals with signs and symptoms of respiratory tract infection. ID NOW COVID-19 2.0 performed on the ID NOW Instrument is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical, epidemiologic, and laboratory findings. SARS-CoV-2 RNA is generally detectable in nasal and nasopharyngeal swab specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA. Positive results do not preclude co-infection with bacteria or other viruses and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. A negative test result is presumptive, and it is recommended these results be confirmed by another molecular SARS-CoV-2 assay. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. This test is intended for prescription use only and can be used in Point-of-Care settings. ## Device Story Rapid, instrument-based isothermal nucleic acid amplification test (NAAT) for SARS-CoV-2 detection. Input: direct anterior nasal or nasopharyngeal swab. Process: sample added to Sample Receiver/Transfer Cartridge, inserted into ID NOW Instrument; instrument performs viral lysis, isothermal amplification, and fluorescence detection using molecular beacons. Output: qualitative result displayed on instrument, stored in on-board archive, and printable via USB. Used in point-of-care settings by healthcare professionals. Automated reporting aids clinical diagnosis when combined with other clinical/epidemiologic findings. Benefits: rapid results (<10 minutes) for acute phase infection detection; minimizes contamination risk via confined reaction environment. ## Clinical Evidence Prospective multi-center study (21 US sites, 914 specimens). Compared ID NOW COVID-19 2.0 against composite comparator (three FDA-authorized RT-PCR assays). Results: 91.7% positive agreement (254/277; 95% CI: 87.8%-94.4%) and 98.4% negative agreement (627/637; 95% CI: 97.1%-99.1%). ## Technological Characteristics Isothermal nucleic acid amplification technology (NAAT). Components: disposable Sample Receiver, Test Base with lyophilized reagents, Transfer Cartridge, and ID NOW Instrument. Detection: fluorescently labeled molecular beacon. Connectivity: USB for printer, on-board data storage. Sterilization: sterile swabs. ## Regulatory Identification The Sofia 2 SARS Antigen+ FIA is a lateral flow immunofluorescent sandwich assay used with the Sofia 2 instrument for the rapid, qualitative detection of SARS-CoV-2 nucleocapsid protein antigens directly in anterior nasal swab specimens from symptomatic individuals. It is intended as an aid in the diagnosis of SARS-CoV-2 infections (COVID-19) in symptomatic individuals when tested at least twice over three days with at least 48 hours between tests. ## Predicate Devices - Sofia 2 SARS Antigen+ FIA ([DEN220039](/device/DEN220039.md)) ## Submission Summary (Full Text) > This content was OCRed from public FDA records by [Innolitics](https://innolitics.com). If you use, quote, summarize, crawl, or train on this content, cite Innolitics at https://innolitics.com. > > Innolitics is a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices, including [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/). {0} FDA U.S. FOOD & DRUG ADMINISTRATION # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY AND INSTRUMENT ## I Background Information: A 510(k) Number K221925 B Applicant Abbott Diagnostics Scarborough, Inc. C Proprietary and Established Names ID NOW COVID-19 2.0 D Regulatory Information | Product Code(s) | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | QWR | Class II | 21 CFR 866.3982 - Simple point-of-care device to directly detect SARS-CoV-2 viral targets from clinical specimens in near-patient settings | MI - Microbiology | ## II Submission/Device Overview: A Purpose for Submission: To obtain a substantial equivalence determination for ID NOW COVID-19 2.0 on the ID NOW instrument. B Measurand: RdRp gene of SARS-CoV-2 RNA. C Type of Test: ID NOW COVID-19 2.0 is a rapid, instrument-based isothermal test for the qualitative detection of viral RNA from SARS-CoV-2 in direct nasal or nasopharyngeal swabs. Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993-0002 www.fda.gov {1} K221925 - Page 2 of 23 ## III Intended Use/Indications for Use: ### A Intended Use(s): See Indications for Use below. ### B Indication(s) for Use: ID NOW COVID-19 2.0 performed on the ID NOW Instrument is a rapid molecular *in vitro* diagnostic test utilizing an isothermal nucleic acid amplification technology (NAAT) intended for the qualitative detection of nucleic acid from SARS-CoV-2 in direct anterior nasal (nasal) or nasopharyngeal swabs from individuals with signs and symptoms of respiratory tract infection. ID NOW COVID-19 2.0 performed on the ID NOW Instrument is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical, epidemiologic, and laboratory findings. SARS-CoV-2 RNA is generally detectable in nasal and nasopharyngeal swab specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA. Positive results do not preclude co-infection with bacteria or other viruses and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. A negative test result is presumptive, and it is recommended these results be confirmed by another molecular SARS-CoV-2 assay. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. This test is intended for prescription use only and can be used in Point-of-Care settings. ### C Special Conditions for Use Statement(s): Rx - For Prescription Use Only For *in vitro* Diagnostic Use Only ### D Special Instrument Requirements: ID NOW Instrument ## IV Device/System Characteristics: ### A Device Description: ID NOW COVID-19 2.0 is a rapid, instrument-based isothermal test for the qualitative detection of viral RNA from SARS-CoV-2 in direct nasal or nasopharyngeal swabs. ID NOW COVID-19 2.0 System utilizes isothermal nucleic acid amplification technology and is comprised of: - Sample Receiver – single use, disposable containing the elution buffer - Test Base – single use, disposable comprising two sealed reaction tubes, each containing a lyophilized pellet - Transfer Cartridge – single use, disposable for transfer of the eluted sample to the Test Base - Patient Swabs – sterile anterior nasal swabs (foam) for anterior nasal swab collection and for use as a Negative Control Swab {2} - Positive Control Swab – single use, to ensure that test reagents are working properly and that the test is correctly performed, and - ID NOW Instrument. The reaction tubes in the Test Base contain lyophilized reagents required for amplification of the target nucleic acid and an internal control. ID NOW COVID-19 2.0 utilizes a pair of templates (similar to primers) for the specific amplification of RNA from SARS-CoV-2 and a fluorescently labeled molecular beacon designed to specifically identify the amplified nucleic acid targets. ID NOW COVID-19 2.0 is performed within the confinement of the Test Base, and no other part of the ID NOW Instrument has contact with the sample during the amplification process. To perform the assay, the Sample Receiver and Test Base are inserted into the ID NOW Instrument. All steps of the assay process are timed by the instrument; no timing by the test operator is required. When prompted by the instrument, the swab sample is added to the Sample Receiver and mixed by swirling motion for 10 seconds, then discarded. The Transfer Cartridge is pressed onto the Sample Receiver and the sample is then transferred via the Transfer Cartridge to the Test Base, resuspending the lyophilized pellet contained within the Test Base and initiating target amplification. No further operator intervention is required. The subsequent heating, mixing, and detection by fluorescence is performed by the instrument, with results automatically reported. Results are displayed by the ID NOW Instrument and are also stored in an on-board archive and are assigned to a sample ID that has been entered into the ID NOW Instrument by the operator either manually or using barcode scanner. Data can be retrieved and downloaded by the operator at any time after testing. An external Universal Printer can be attached via USB to the ID NOW Instrument to print test results. ## B Principle of Operation: ID NOW COVID-19 2.0 performed on the ID NOW Instrument utilizes an isothermal nucleic acid amplification technology NEAR (Nicking Enzyme Amplification Reaction). Individual reactions for SARS-CoV-2, provided as lyophilized pellets contained within the Test Base specifically amplify and detect unique target regions within the RNA genomes of the virus. ID NOW COVID-19 2.0 employs the use of fluorescently labeled molecular beacon probes for sensitive and specific real-time detection. ID NOW COVID-19 2.0 technology is isothermal and does not require a heat denaturation step to generate a single strand target for amplification. Instead a thermostable strand-displacing DNA polymerase, a thermostable nicking endonuclease, and two oligonucleotides (typically known as primers, but are referred to as templates in this technology) are utilized. Reverse transcriptase is also present in the reaction. The products of the ID NOW COVID-19 2.0 amplification are two complementary oligonucleotides 30-40 nucleotides in length. A product of this size is of sufficient length to be highly unique amongst genomes. The exact length of the products depends on the specific placement of the templates relative to one another and relative to their complementary sequences within the target genome. ID NOW COVID-19 2.0 specificity is achieved through two levels of sequence-specific interrogation, template annealing to complementary target sequence at an elevated temperature to drive target-specific amplification, and product detection as the molecular beacon anneals to the ID NOW COVID-19 2.0 product in a sequence-specific fashion. K221925 - Page 3 of 23 {3} A nasal or nasopharyngeal swab is tested directly and is added to a lysis buffer that provides access to the nucleic acid target. ## C Instrument Description Information: 1. Instrument Name: ID NOW Instrument. 2. Specimen Identification: Anterior nasal or nasopharyngeal swab. 3. Specimen Sampling and Handling: ID NOW COVID-19 2.0 is intended for testing a swab directly without elution in viral transport media. Please refer to the ID NOW COVID-19 2.0 Package Insert for anterior nasal swab and nasopharyngeal swab specimen collection procedures. 4. Calibration: ID NOW Instrument is factory calibrated and does not require any further calibration and verification at user site. However, if the instrument was transported or moved, a performance check using ID NOW Instrument Positive and Negative Controls is recommended to ensure proper functionality of the instrument. 5. Quality Control: ID NOW COVID-19 2.0 has built-in procedural control (Internal Control). An Internal Control (IC) is provided in the second tube as a lyophilized reaction and provides verification that the assay reagents are functioning properly and have not been inactivated or inhibited during a test. An RNA oligonucleotide is provided as the IC target. This RNA oligonucleotide contains 5' and 3' ends that are complementary to the target template set's recognition regions but with a spacer region (the region between the two recognition regions) that differs from the target's spacer region. The RNA oligonucleotide is 40 nucleotides in length, present at 100,000 copies per test. Detection of the IC occurs via a molecular beacon that specifically detects the amplified product generated from the IC RNA oligonucleotide. The result of the IC is displayed on the screen and is automatically stored in the instrument with each test result. This can be reviewed later by selecting Review Memory on the instrument. ID NOW COVID-19 2.0 External Controls are designed for use with the ID NOW COVID-19 2.0. The Positive Control swab is coated with inactivated SARS-CoV-2 virus dried onto a swab. This control confirms that the sample elution and lysis and workflow were performed correctly, and the ID NOW Instrument and kit components are performing as expected. A blank patient swab contained within the test kit can be used as the Negative Control Swab. It is recommended to run a Quality Control test using swabs once with each new shipment of test kits received and once for each untrained operator. If additional Positive or Negative Control Swabs are required, ID NOW COVID-19 2.0 Control Swab Kit can be purchased separately. ID NOW COVID-19 2.0 Control Swab Kit contains the same Positive and Negative Control Swabs that are provided in the ID NOW COVID-19 2.0 kit. K221925 - Page 4 of 23 {4} V Substantial Equivalence Information: A Predicate Device Name(s): Sofia 2 SARS Antigen+ FIA, Sofia 2 SARS Antigen+ FIA Control Swab Set B Predicate 510(k) Number(s): DEN220039 C Comparison with Predicate(s): | Device & Predicate Device(s): | K221925 | DEN220039 | | --- | --- | --- | | Device Trade Name | ID NOW COVID-19 2.0 | Sofia 2 SARS Antigen+ FIA | | Product Code | QWR | QVF | | Regulation Number/Name | Same | 21 CFR 866.3982 - Simple point-of-care device to directly detect SARS-CoV-2 viral targets from clinical specimens in near-patient settings | | General Device Characteristic Similarities | | | | Intended Use/Indications For Use | ID NOW COVID-19 2.0 performed on the ID NOW Instrument is a rapid molecular in vitro diagnostic test utilizing an isothermal nucleic acid amplification technology (NAAT) intended for the qualitative detection of nucleic acid from SARS-CoV-2 in direct anterior nasal (nasal) or nasopharyngeal swabs from individuals with signs and symptoms of respiratory tract infection. ID NOW COVID-19 2.0 performed on the ID NOW Instrument is intended for use as an aid in the diagnosis of COVID-19 if used in conjunction with other clinical, epidemiologic, and laboratory findings. SARS-CoV-2 RNA is generally detectable in nasal and nasopharyngeal swab | The Sofia 2 SARS Antigen+ FIA is a lateral flow immunofluorescent sandwich assay that is used with the Sofia 2 instrument for the rapid, qualitative detection of SARS-CoV-2 nucleocapsid protein antigens directly in anterior nasal swab specimens from individuals with signs and symptoms of upper respiratory infection (i.e., symptomatic) when testing is started within 6 days of symptom onset. The test is intended for use as an aid in the diagnosis of SARS-CoV-2 infections (COVID-19) in symptomatic individuals when tested at least twice over three days with at least 48 hours between tests. The test does not differentiate between SARS-CoV and SARS-CoV-2. A negative test result is presumptive, and it is recommended these results | K221925 - Page 5 of 23 {5} K221925 - Page 6 of 23 | | specimens during the acute phase of infection. Positive results are indicative of the presence of SARS-CoV-2 RNA. Positive results do not preclude co-infection with bacteria or other viruses and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. A negative test result is presumptive, and it is recommended these results be confirmed by another molecular SARS-CoV-2 assay. Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis for diagnosis, treatment, or other patient management decisions. This test is intended for prescription use only and can be used in Point-of-Care settings. | be confirmed by a molecular SARS-CoV-2 assay. Negative results do not preclude SARS-CoV-2 infections and should not be used as the sole basis for treatment or other patient management decisions. Positive results do not rule out co-infection with other respiratory pathogens. Performance characteristics for SARS-CoV-2 were established during the 2021-2022 SARS-CoV-2 pandemic when SARS-CoV-2 Omicron was the predominant SARS-CoV-2 variant in circulation. When other SARS-CoV-2 virus variant are emerging, performance characteristics may vary. This test is intended for prescription use only and can be used in Point-of-Care settings. | | --- | --- | --- | | Analyte Target | Same | SARS-CoV-2 | | Control | Same | Internal and External Controls | | Result Interpretation | Same | Automated | | Assay Result | Same | Qualitative | | Intended Environment for Use | Same | Professional use, in a medical laboratory or point-of-care | | Prescription Use Only | Same | Yes | | General Device Characteristic Differences | | | | Technology | Isothermal nucleic acid amplification for detecting the presence/absence of viral RNA in clinical specimens | Lateral flow immunofluorescent sandwich assay for detecting the presence/absence of the nucleocapsid protein antigen in clinical specimens | | SARS-CoV-2 Target Type | RdRp gene | Nucleocapsid protein | | Specimen Type | Direct anterior nasal or | Direct anterior nasal swabs | {6} | | nasopharyngeal swabs | | | --- | --- | --- | | Instrumentation | ID NOW Instrument | Sofia Q, Sofia 2, Sofia | | Automated Assay | Automated sample preparation, amplification, detection and result interpretation | Automated test interpretation and report generation | | Time to Result | ≤ 12 minutes | About 15 minutes | VI Standards/Guidance Documents Referenced: - Format for Traditional and Abbreviated 510(k)s, September 13, 2019. - The 510(k) Program: Evaluating Substantial Equivalence in Premarket Notifications (510(k)), July 28, 2014. - Recommendations for Clinical Laboratory Improvement Amendments of 1988 (CLIA) Waiver Applications for Manufacturers of In Vitro Diagnostic Devices, February 26, 2020 - Recommendations for Dual 510(k) and CLIA Waiver by Application Studies, February 26, 2020. - Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices, May 11, 2005. - Policy for Evaluating Impact of Viral Mutations on COVID-19 Tests, Guidance for Test Developers and FDA Staff, February 2021. - Medical device software - Software life cycle processes (IEC 62304:2006). - Clinical and Laboratory Standards Institute (CLSI) document, EP12-A2, User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline - Second Edition. - CLSI EP17-A2 - FDA’s Convenience Interim Regulatory Guidance document - Special Controls related to 21 CFR 866.3982. VII Performance Characteristics (if/when applicable): A Analytical Performance: 1. Reproducibility and Samples Near the Cut-Off Study: A reproducibility and samples near the cut-off study of the ID NOW COVID-19 2.0 was conducted by nine operators at three sites over five different days using panels of four SARS-CoV-2 samples contrived in clinical matrix diluent. All of the operators were representative of the intended users at CLIA waived sites having no training or hands-on experience in conducting laboratory testing. At each site, three operators tested eight samples (two replicates of each of the four Sample Panel Members) with three lots of Test Bases and three lots of Sample Receiver/Transfer Cartridges on each testing day. Samples were tested in random order. Each operator conducted testing for a minimum of five days. Testing days spanned a 10-day time period. No operator tested on five consecutive days. If a sample had an initial invalid result, the operator retested the sample once with the same lot and instrument. Each operator tested one positive and one negative control swab on each K221925 - Page 7 of 23 {7} instrument used for the study, on each day of testing prior to performing the study testing. External control swabs were tested once per day on each instrument. The percent agreement with the expected results is shown in the Tables 1-3 below. Table 1: Reproducibility Study – Overall Agreement with Expected Results Across All Sites, Operators, and Lots | Sample Type | Site | | | All Sites | | | | --- | --- | --- | --- | --- | --- | --- | | | | Site 1 | Site 2 | Site 3 | %Agreement (Count) | 95% CI | | 1.16x LoD | Percent Agreement | 97.8% | 94.4% | 96.7% | 96.3% (260/270) | 93.3%, 98.0% | | | Count | 88/90 | 85/90 | 87/90 | | | | 1.74x LoD | Percent Agreement | 98.9% | 96.6% | 98.9% | 98.1% (263/268) | 95.7%, 99.2% | | | Count | 89/90 | 86/89² | 88/89² | | | | 0.0235x LoD (High Negative) | Percent Agreement | 87.8% | 90.9% | 90.0% | 89.6% (240/268) | 85.3%, 92.7% | | | Count | 79/90 | 80/88² | 81/90 | | | | Virus Free Negative¹ | Percent Agreement | 100.0% | 100.0% | 98.9% | 99.6% (267/268) | 97.9%, 99.9% | | | Count | 90/90 | 89/89² | 88/89² | | | | Positive Control | Percent Agreement | 100% | 100% | 100% | 100% (137/137) | 97.3% - 100.0% | | | Count | 45/45 | 46/46 | 46/46 | | | | Negative Control | Percent Agreement | 100% | 100% | 100% | 100% (137/137) | 97.3% - 100.0% | | | Count | 45/45 | 46/46 | 46/46 | | | ¹Percent Agreement correlates to the percent of negative results. ²Sample(s) excluded due to protocol deviation. Table 2: Reproducibility Study -Percent Agreement with Expected Results by Operator | Sample Type | Site 1 | | | Site 2 | | | Site 3 | | | All Sites | | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | | Operator 1 % Agreement (Count) | Operator 2 % Agreement (Count) | Operator 3 % Agreement (Count) | Operator 1 % Agreement (Count) | Operator 2 % Agreement (Count) | Operator 3 % Agreement (Count) | Operator 1 % Agreement (Count) | Operator 2 % Agreement (Count) | Operator 3 % Agreement (Count) | All Operators % Agreement (Count) | 95% CI | | 1.16x LoD | 100% (30/30) | 93.3% (28/30) | 100% (30/30) | 93.3% (28/30) | 93.3% (28/30) | 96.7% (29/30) | 96.7% (29/30) | 96.7% (29/30) | 96.7% (29/30) | 96.3% (260/270) | 98.5% - 100.0% | | 1.74x LoD | 100% (30/30) | 96.7% (29/30) | 100% (30/30) | 93.3% (28/30) | 96.6% (28/29) | 100% (30/30) | 96.7% (29/30) | 100% (30/30) | 100% (30/30) | 98.1% (263/268)¹ | 95.7% - 99.2% | | 0.0235x LoD (High Negative) | 83.3% (25/30) | 90.0% (27/30) | 90.0% (27/30) | 89.3% (25/28) | 86.7% (26/30) | 96.7% (29/30) | 86.7% (26/30) | 93.3% (28/30) | 90.0% (27/30) | 89.6% (240/268)¹ | 85.3% - 92.7% | | Virus Free Negative | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (30/30) | 100% (29/29) | 100% (30/30) | 96.7% (29/30) | 100% (29/29) | 99.6% (267/268)¹ | 97.9% - 99.9% | ¹Sample(s) excluded due to protocol deviation. Table 3: Reproducibility Study - Percent Agreement with Expected Results by Lot | Sample Type | Lot 1 % Agreement (Count) | Lot 2 % Agreement (Count) | Lot 3 % Agreement (Count) | % Agreement (Count) | 95% CI | | --- | --- | --- | --- | --- | --- | K221925 - Page 8 of 23 {8} | 1.16x LoD | 96.7% (87/90) | 93.3% (84/90) | 98.9% (89/90) | 96.3% (260/270) | 93.3% - 98.0% | | --- | --- | --- | --- | --- | --- | | 1.74x LoD | 100% (89/89) | 95.5% (85/89) | 98.9% (89/90) | 98.1% (263/268)¹ | 95.7 – 99.2% | | 0.0235x LoD (High Negative) | 88.9% (80/90) | 92.0% (81/88) | 87.8% (79/90) | 89.6% (240/268)¹ | 85.3% - 92.7% | | Virus Free Negative | 100% (89/89) | 100% (89/89) | 98.9% (89/90) | 99.6% (267/268)¹ | 97.9% - 99.9% | ¹Sample(s) excluded due to protocol deviation. The data generated in this study demonstrate that ID NOW COVID-19 2.0 is reproducible when testing is conducted by intended users (i.e., untrained operators) in CLIA Waived settings. There were no significant differences observed within run (replicates tested by one operator), between run (five different days), between sites (three sites), or between operators (nine operators), or lots. 2. Linearity: Not applicable; this is a qualitative assay. 3. Analytical Reactivity: a) In silico Inclusivity Analysis An alignment was performed with the oligonucleotide primer and probe sequences of the ID NOW COVID-19 2.0 with all publicly available SARS-CoV-2 genomic sequences submitted to NCBI (National Center for Biotechnology Information) GenBank and GISAID (Global Initiative on Sharing All Influenza Data) databases between December 1, 2019 and December 3-4, 2021. A total of 431,147 high quality SARS-CoV-2 sequences (<1% unknown or unidentified nucleotides) plus a reference genome were available from NCBI GenBank, and 4,252,920 from GISAID databases. Both datasets contained sequences obtained from human hosts only. 217,267 sequences were present in both databases. To avoid redundancy only the GISAID copies of the duplicated sequences were retained for analysis bringing the total number of high quality human SARS-CoV-2 sequences available from both databases to 4,466,800. Of the total number of sequences analyzed, 3,274 sequences contained at least 1 ambiguous or unidentified nucleotide within the target region, bringing the total number of isolates suitable for inclusivity analysis down to 4,463,526. From this analysis 99.58% of the sequences provided 100% homology to the ID NOW COVID-19 2.0 primer and probe sequences. Only 18,702 out of 4,463,526 SARS-CoV-2 isolates, or 0.42%, contained single nucleotide polymorphisms (SNPs) within the assay primer or probe annealing region. Of these 18,702 isolates: 18,609 isolates carried a single mismatch, 36 isolates carried 2 mismatches and 57 isolates carried >2 mismatches. Based on mismatch location, the vast majority of the 18,702 isolates containing a mismatch are expected to have no impact or low impact on target detection. An additional alignment was performed with the oligonucleotide primer and probe sequences of the ID NOW COVID-19 2.0 with all publicly available SARS-CoV-2 genomic sequences collected within the United States and submitted to the GISAID database between October 17, 2022 and April 17, 2023. The dataset contained sequences obtained from human hosts only and totaled 382,309 sequences. ID NOW COVID-19 2.0 provided 100% sequence homology across 99.51% of the 382,309 sequences. For the 0.49% of sequences which had mismatches present, K221925 - Page 9 of 23 {9} 0.40% of the sequences had mismatches within the assay annealing region for Template 2 while 0.41% occurred within the molecular beacon. Only 0.05% of the 389,309 sequences had mismatches within Template 1. Despite not having 100% homology for all of the sequences analyzed, there were no mismatch scenarios in any of the assay annealing regions that occurred at >1% frequency. ## b) Inclusivity Wet-Testing The inclusivity in detection of SARS-CoV-2 variants by ID NOW COVID-19 2.0 was further evaluated through empirical evaluation of particular strains. Swab specimens were prepared using SARS-CoV-2 strains diluted in nasal specimen matrix diluent to approximately 1-3x LoD. Contrived swab specimens were prepared by coating 50 microliters of virus dilution onto each swab. A concentration level was considered "reactive/positive" in this study if all five replicates generated a positive result. If 5/5 COVID-19 positive results were not obtained across all three device lots at the concentration tested, the isolate was tested at increasing concentrations until 5/5 positive results were obtained. All daily control testing generated expected results on each day of testing. Each result in the Table 4 below represents 100% agreement for all replicates tested. | SARS-CoV-2 Strains | Detected Concentrations (Copies/reaction) | Detected Concentrations (Copies/swab) | | --- | --- | --- | | Hong Kong/VM200001061/2020 | 60 | 1,500 | | Italy-INMI1 | 60 | 1,500 | | SARS-CoV-2-USA- WA1/2020 | 58.3 | 1,457.5 | | P.2 (Zeta) | 26 | 650 | | P.1 (Gamma) | 61.1 | 1,527.5 | | B.1.1.7 (Alpha) | 45.9 | 1,147.5 | | B.1.429 (Epsilon) | 18.7 | 467.5 | | B.1.1.318 | 28.8 | 720 | | WA1-wt | 41 | 1,025 | | B.1.351 (Beta) | 23 | 575 | | B.1.1.7 (Alpha) | 100.2 | 2,505 | | B.1.617.1 (Kappa) | 19 | 475 | | B.1.617.1 (Kappa) | 40.5 | 1,012.50 | | B.1.617.2 (Delta) | 22.4 | 560 | | B.1.617.2 (Delta) | 20.7 | 517.5 | | B.1.1.529 (Omicron) | 60 | 1,500 | | BA.2.12.1 (Omicron) | 60 | 1,500 | | BA.4.6 (Omicron) | 60 | 1,500 | | BA.5.1 (Omicron) | 60 | 1,500 | | BA.5.2 (Omicron)* | 80 | 2,000 | | BE.1 (Omicron) | 60 | 1,500 | | BF.5 (Omicron)* | 100 | 2,500 | | BF.7 (Omicron)* | 80 | 2,000 | | BA.4.1 (Omicron) | 60 | 1,500 | | BQ.1 (Omicron) | 60 | 1,500 | | BQ.1.1 (Omicron) | 60 | 1,500 | | XBB.1 (Omicron)* | 80 | 2,000 | | XBB.6 (Omicron) | 60 | 1,500 | * Detected concentration greater than 3x LoD (60 copies/reaction). K221925 - Page 10 of 23 {10} # 4. Analytical Specificity/Interference: # a) Wet-Testing To determine the analytical specificity of ID NOW COVID-19 2.0, 38 commensal and pathogenic microorganisms (24 viruses, 12 bacteria, and 2 yeasts) that may be present in the nasal cavity or nasopharynx were tested. All of the microorganisms were diluted to a clinically relevant concentrations ranging from $10^{6}$ to $10^{7}$ cells/mL, IFU/mL, or CFU/mL (bacteria), $10^{5}$ to $10^{8}$ TCID $_{50}$ /mL, copies/mL, GE/mL or IU/mL (viruses), and $10^{6}$ to $10^{7}$ cells/mL or CFU/mL (yeast) (Table 5). Each microorganism was tested in replicates of five. On each day of testing, one positive control swab and one UTM inoculated swab serving as the negative control were tested first. All daily control testing generated expected results on each day of testing. Expected negative results were obtained. None of the bacteria, viruses or yeast tested cross-reacted in ID NOW COVID-19 2.0 at the concentrations tested. A summary of the cross-reactivity evaluation is presented in the Table 5 below. Table 5: Analytical Specificity Study Results | Organism/Virus | Source/ID | Catalog Number | Test Concentration | Cross-Reactivity Detected | | --- | --- | --- | --- | --- | | Adenovirus 1 | ATCC | VR-1 | 2.0 x 10^6 TCID50/mL | None | | Adenovirus 7 | ATCC | VR-7 | 2.0 x 10^6 TCID50/mL | None | | Human Coronavirus 229E | ATCC | VR-740 | 1.0 x 10^5 TCID50/mL | None | | Human Coronavirus HKU1† | ATCC | VR-3262SD | 1.0 x 10^8 copies/mL | None | | Human Coronavirus NL63 | Zeptometrix | 0810228CF | 1.0 x 10^5 TCID50/mL | None | | Human Coronavirus OC43 | ATCC | VR-1558 | 1.0 x 10^5 TCID50/mL | None | | SARS-coronavirus | ATCC | VR-3280SD | 1.0 x 10^5 cp/mL | None | | MERS-coronavirus* | BEI | NR-50171 | 1.0 x 10^5 GE/mL | None | | Enterovirus 70 | ATCC | VR-836 | 4.2 x 10^5 TCID50/mL | None | | Human Echovirus 7 | ATCC | VR-37 | 2.0 x 10^6 TCID50/mL | None | | hMPV | Zeptometrix | 0810161CF | 2.0 x 10^6 U/mL | None | | Human Parainfluenza virus 1 | ATCC | VR-94 | 3.64 x 10^5 TCID50/mL | None | | Human Parainfluenza virus 2 | ATCC | VR-92 | 2.0 x 10^6 TCID50/mL | None | | Human Parainfluenza virus 3 | ATCC | VR-93 | 2.0 x 10^6 TCID50/mL | None | | Human Parainfluenza virus 4a | Zeptometrix | 0810060CF | 1.0 x 10^5 TCID50/mL | None | K221925 - Page 11 of 23 {11} | Organism/Virus | Source/ID | Catalog Number | Test Concentration | Cross-Reactivity Detected | | --- | --- | --- | --- | --- | | Influenza A H1N1 | Virapur | A/California/7/09 | 2.0 x 10^{6} IU/mL | None | | Influenza A H3N2 | Virapur | A/Texas/50/2012 | 2.0 x 10^{6} IU/mL | None | | Influenza B Victoria Lineage | Virapur | B/Malaysia/2506/04 | 2.0 x 10^{6} IU/mL | None | | Influenza B Yamagata Lineage | Virapur | B/Wisconsin/1/2010 | 2.0 x 10^{6} IU/mL | None | | Mumps virus | Zeptometrix | 0810079CF | 1.0 x 10^{5} TCID_{50}/mL | None | | RSV A | Virapur | A2 | 2.0 x 10^{6} IU/mL | None | | RSV B | Virapur | B1 | 2.0 x 10^{6} IU/mL | None | | Rhinovirus 1 | ATCC | VR-1559 | 2.0 x 10^{6} TCID_{50}/mL | None | | Rhinovirus 2 | ATCC | VR-482 | 2.0 x 10^{6} TCID_{50}/mL | None | | Bordetella pertussis** | ATCC | 9797 | 2.0 x 10^{7} cells/mL | None | | Chlamydia pneumoniae | ATCC | VR-2282 | 1.0 x 10^{6} IFU/mL | None | | Haemophilus influenzae | Zeptometrix | 0801679 | 1.0 x 10^{6} CFU/mL | None | | Legionella pneumophila** | ATCC | 33152 | 2.0 x 10^{7} cells/mL | None | | Mycobacterium tuberculosis | Zeptometrix | 0801660 | 1.0 x 10^{6} CFU/mL | None | | Mycoplasma pneumoniae | ATCC | 15531-TTR | 2.0 x 10^{7} CFU/mL | None | | Pseudomonas aeruginosa*** | ATCC | 15442 | 1.0 x 10^{6} CFU/mL | None | | Staphylococcus aureus** | ATCC | 25923 | 2.0 x 10^{7} CFU/mL | None | | Staphylococcus epidermidis | Zeptometrix | 0804276 | 1.0 x 10^{6} CFU/mL | None | | Streptococcus salivarius*** | ATCC | BAA-1024 | 1.0 x 10^{6} CFU/mL | None | | Streptococcus pneumoniae | Zeptometrix | 0801439 | 1.0 x 10^{6} CFU/mL | None | | Streptococcus pyogenes | Zeptometrix | 0801512 | 1.0 x 10^{6} CFU/mL | None | | Candida albicans** | ATCC | 60193 | 2.0 x 10^{7} cells/mL | None | | Pneumocystis jirovecii (PJP) †‡ | Zeptometrix | 0801698 | 1.0 x 10^{6} CFU/mL | None | *Active organism could not be sourced; therefore, inactivated organisms were evaluated. ** The reported concentration aligns with the assigned internal lot number. A Certificate of Analysis could not be identified for these organisms. ***Cultured internally from ATCC strain on ATCC recommended culture media. Quantified by the counting of colony forming units from serially diluted bacterial stocks. † Active or inactivated organism could not be sourced; therefore, RNA was evaluated. ‡ Active or inactivated organism could not be sourced; therefore, a recombinant yeast was evaluated. b) In Silico Analysis In silico analysis was performed to identify overlaps between the ID NOW COVID-19 2.0 target nucleic acid sequence and the genomes of a defined set of respiratory tract microorganisms. Basic local alignments were downloaded from the NCBI nucleotide sequence database (accessed on December 10, 2021). The Basic Local Alignment Search K221925 - Page 12 of 23 {12} Tool (BLAST) analysis was performed to compare the recognition regions of the ID NOW COVID-19 2.0 templates and molecular beacons to all GenBank sequence records for high priority pathogens from the coronavirus genetic family and from prevalent disease agents and normal or pathogenic flora GenBank entries of non-human host viruses were excluded from the analysis. The top hit GenBank accession record for each Taxonomy ID is reported in Table 6 below. Table 6: Cross Reactivity Top Hits for each Taxonomy ID with Percent Identity Shown | Organism | Tax ID | Forward Template | Probe | Reverse Template | | --- | --- | --- | --- | --- | | Human coronavirus 229E | 11137 | 75% | 45.83% | 50% | | Human coronavirus OC43 | 31631 | 68.75% | 50% | 61.11% | | Human coronavirus HKU1 | 290028 | 56.25% | 50% | 50% | | Human coronavirus NL63 | 277944 | 75% | 58.33% | no hits | | SARS-coronavirus | 694009 | 93.75% | 79.17% | 83.33% | | MERS-coronavirus | 1335626 | 81.25% | 45.83% | 55.56% | | Human adenovirus 1 | 10533 | no hits | no hits | no hits | | Human adenovirus 2 | 10515 | no hits | no hits | no hits | | Human adenovirus 3 | 45659 | no hits | no hits | no hits | | Human adenovirus 4 | 28280 | no hits | no hits | no hits | | Human adenovirus 5 | 28285 | no hits | no hits | no hits | | Human adenovirus 7 | 10519 | no hits | no hits | no hits | | Human adenovirus 11 | 10541 | no hits | no hits | no hits | | Human adenovirus 14 | 10521 | no hits | no hits | no hits | | Human adenovirus 31 | 10529 | no hits | no hits | no hits | | Cytomegalovirus | 10358 | no hits | no hits | no hits | | Echovirus E6 | 12062 | no hits | no hits | no hits | | Echovirus E7 | 46018 | no hits | no hits | no hits | | Echovirus E9 | 12060 | no hits | no hits | no hits | | Echovirus E11 | 12078 | no hits | no hits | no hits | | Epstein Barr virus | 10376 | no hits | no hits | no hits | | Human Metapneumovirus (hMPV) | 162145 | no hits | no hits | no hits | | Influenza A | 11320 | no hits | no hits | no hits | | Influenza B | 11520 | no hits | no hits | no hits | | Measles virus | 351680 | no hits | no hits | no hits | | Mumps virus | 2560602 | no hits | no hits | no hits | | Parainfluenza Type 1 | 12730 | no hits | no hits | no hits | | Parainfluenza Type 2 | 2560525 | no hits | no hits | no hits | | Parainfluenza Type 3 | 11216 | no hits | no hits | no hits | | Parainfluenza Type 4a or 4b | 2560526 | no hits | no hits | no hits | | RSV A | 208893 | no hits | no hits | no hits | | RSV B | 208895 | no hits | no hits | no hits | | Rhinovirus: | 12059 | N/A | N/A | N/A | | Coxsackievirus B4 | 12073 | no hits | no hits | no hits | | Human rhinovirus B35 | 167329 | no hits | no hits | no hits | | Enterovirus 70 (VR-836) | 31915 | no hits | no hits | no hits | | Rest of rhinovirus spp. | 12059 | 87.50% | no hits | 72.22% | K221925 - Page 13 of 23 {13} | Bordetella pertussis | 520 | no hits | no hits | no hits | | --- | --- | --- | --- | --- | | Bordetella bronchiseptica | 518 | no hits | no hits | no hits | | Candida albicans | 5476 | 81.25% | 62.50% | no hits | | Chlamydia pneumoniae | 83558 | no hits | no hits | no hits | | Chlamydia trachomatis | 813 | 81.25% | no hits | no hits | | Corynebacterium diphtheriae | 1717 | 87.50% | no hits | 72.22% | | Escherichia coli | 562 | 87.50% | 70.83% | 88.89% | | Haemophilus influenzae | 727 | no hits | 70.83% | no hits | | Klebsiella pneumoniae | 573 | 81.25% | 70.83% | 72.22% | | Lactobacillus plantarum | 1590 | no hits | 58.33% | no hits | | Legionella pneumophila | 446 | 81.25% | 62.50% | 77.78% | | Moraxella catarrhalis | 480 | no hits | 58.33% | no hits | | Mycobacterium tuberculosis | 1773 | no hits | no hits | no hits | | Mycoplasma pneumoniae | 2104 | no hits | no hits | no hits | | Neisseria gonorrhoeae | 485 | 81.25% | no hits | no hits | | Neisseria meningitidis | 487 | no hits | 58.33% | no hits | | Neisseria mucosa | 488 | no hits | no hits | no hits | | Pneumocystis jirovecii (PJP) | 42068 | 87.50% | 62.50% | no hits | | Proteus mirabilis | 584 | 81.25% | 75.00% | no hits | | Proteus vulgaris | 585 | 81.25% | 66.67% | 72.22% | | Pseudomonas aeruginosa | 287 | no hits | 70.83% | 72.22% | | Staphylococcus aureus | 1280 | no hits | 83.33% | 88.89% | | Staphylococcus epidermidis | 1282 | no hits | 83.33% | no hits | | Streptococcus pneumoniae | 1313 | no hits | 58.33% | no hits | | Streptococcus pyogenes | 1314 | no hits | 70.83% | no hits | | Streptococcus salivarius | 1304 | no hits | 62.50% | no hits | Fifteen (15) organisms were identified with >80% homology to at least one template and/or molecular beacon and were examined further to evaluate the risk of cross-reactivity in silico. None of these organisms are expected to negatively impact test performance due to reasons including, (i) homology to a single template or probe, (ii) where both templates show homology, they anneal in genome locations extremely far from one another, or (iii) nucleotide mismatches present at or near the very 3'-ends of the template/target organism homologous sequences, which will significantly reduce the ability of the template to anneal and extend. Overall, none of the evaluated microorganisms are predicted/expected to cross-react with ID NOW COVID-19 2.0. ## c) Microbial Interference A microbial interference study was performed to determine if ID NOW COVID-19 2.0 can detect SARS-CoV-2 virus near the limit of detection (LoD) in the presence of non-SARS-CoV-2 viruses, bacteria, and yeasts. Vendor provided stocks of SARS-CoV-2 were diluted in Negative Nasal Swab Matrix Diluent (NSMD) to 1.74x LoD for RSV A, RSV B, Flu A/California, Flu A/Texas, Flu B/Wisconsin, and Flu B/Malaysia; all others were tested with SARS-CoV-2 virus diluted in NSMD to 3x LoD. Contrived SARS-CoV-2 positive swab samples were prepared by coating 50 microliters of virus dilution onto each swab. The following panel of non-SARS-CoV-2 viruses, bacteria, and yeast were tested in five replicates at the concentration provided in Table 7 below. K221925 - Page 14 of 23 {14} All daily control testing generated expected results. Expected positive results were obtained for all co-infection specimens tested. Each result in the table represents 100% agreement for all replicates tested unless otherwise noted. Table 7: Microbial Interference Study Results | Specimen ID | Testing Concentration | %Agreement (Count) | | --- | --- | --- | | Adenovirus 1 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Adenovirus 7 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Human Coronavirus 229E | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Human Coronavirus NL63 | 1.17 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Human Coronavirus OC43 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Human Coronavirus HKU1 | 1.0 x 10^{8} copies/mL | 100% (5/5) | | SARS-Coronavirus** | 2.0 x 10^{5} copies/mL | 100% (5/5) | | MERS-Coronavirus | 1.0 x 10^{5} GE/mL | 100% (5/5) | | Enterovirus 70 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Human Echovirus 7 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | hMPV | 1.0 x 10^{5} U/mL | 100% (5/5) | | Human Parainfluenza Virus 1** | 2.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Human Parainfluenza Virus 2 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Human Parainfluenza Virus 3 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Human Parainfluenza Virus 4a | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Influenza A H1N1/California/7/2009* | 1.0 x 10^{5} IU/mL | 100% (5/5) | | Influenza A H3N2/Texas/50/2012* | 1.0 x 10^{5} IU/mL | 100% (5/5) | | Influenza B Yamagata/Wisconsin/1/2010* | 1.0 x 10^{5} IU/mL | 100% (5/5) | | Influenza B Victoria/Malaysia/2506/04* | 1.0 x 10^{5} IU/mL | 100% (5/5) | | Mumps Virus | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | RSV A* | 1.0 x 10^{5} IU/mL | 100% (5/5) | | RSV B* | 1.0 x 10^{5} IU/mL | 100% (5/5) | | Rhinovirus 1 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Rhinovirus 2 | 1.0 x 10^{5} TCID_{50}/mL | 100% (5/5) | | Bordetella pertussis | 1.0 x 10^{6} CFU/mL | 100% (5/5) | | Chlamydia pneumoniae | 1.0 x 10^{6} IFU/mL | 100% (5/5) | | Haemophilus influenzae | 1.0 x 10^{6} CFU/mL | 100% (5/5) | | Legionella pneumophila | 1.0 x 10^{6} cells/mL | 100% (5/5) | | Mycobacterium tuberculosis | 1.0 x 10^{6} CFU/mL | 100% (5/5) | | Mycoplasma pneumoniae* | 1.0 x 10^{6} CFU/mL | 100% (5/5) | | Pseumonoas aeruginosa | 1.0 x 10^{6} CFU/mL | 100% (5/5) | K221925 - Page 15 of 23 {15} | Staphylococcus aureus | 1.0 x 10^6 CFU/mL | 100% (5/5) | | --- | --- | --- | | Staphylococcus epidermidis | 1.0 x 10^6 CFU/mL | 100% (5/5) | | Streptococcus salivarius | 1.0 x 10^6 CFU/mL | 100% (5/5) | | Streptococcus pneumoniae | 1.0 x 10^6 CFU/mL | 100% (5/5) | | Streptococcus pyogenes | 1.0 x 10^6 CFU/mL | 100% (5/5) | | Candida albicans | 1.0 x 10^6 cells/mL | 100% (5/5) | | Pneumocystis jirovecii (PJP) | 1.0 x 10^6 CFU/mL | 100% (5/5) | *RSV A, RSV B, Influenza A (California/7/2009 and Texas/50/2012), Influenza B (Wisconsin/1/2010 and Malaysia/2506/04) and M. pneumoniae - SARS-CoV-2 were tested at 34.8 copies/reaction (1.74x LoD) rather than 60 copies/reaction. **Due to a calculation error Coronavirus HKU1 was tested at $1.0 \times 10^{8}$ copies/mL, SARS-Coronavirus was tested at $2.0 \times 10^{5}$ copies/mL, and Parainfluenza Virus 1 was tested at $2.0 \times 10^{5}$ TCID $_{50}$ /mL. # d) Endogenous/Exogenous Interference Substances An interfering substances study was performed to evaluate if substances naturally present or artificially introduced into the nasal cavity or nasopharynx do not interfere with ID NOW COVID-19 2.0 test performance. Each interfering substance was tested at the concentration listed below (Table 8) in the presence of SARS-CoV-2 at $3\mathrm{x}$ LoD. Negative specimens were prepared by combining the interfering substance with clinical matrix diluent. All specimens were tested in five replicates on the ID NOW COVID-19 2.0 according to the test procedure. All daily controls generated the expected results on each day of testing. Expected results were obtained for all interfering substances tested, except for Mucin (2% w/v) and Zicam Rapid Melt Tab (20% w/v Zincum gluconium, Zincum aceticum). Table 8 below presents the concentration at which no interference was seen for each substance tested. Table 8: Interfering Substances Study Results | Type | Active Ingredient | Concentration | SARS-CoV-2 Positive %Agreement (Count) | SARS-CoV-2 Negative %Agreement (Count) | | --- | --- | --- | --- | --- | | Endogenous | Mucin1 | 1% w/v | 100% (5/5) | 100% (5/5) | | | Whole Blood | 1% v/v | 100% (5/5) | 100% (5/5) | | | Leukocytes | 1 x 10^6 cells/mL | 100% (5/5) | 100% (5/5) | | | Post nasal lavage discharge | 1% v/v | 100% (5/5) | 100% (5/5) | | OTC Nasal Spray 1 | Phenylephrine | 20% v/v | 100% (5/5) | 100% (5/5) | | OTC Nasal Spray 2 | Oxymetazoline | 20% v/v | 100% (5/5) | 100% (5/5) | | OTC Nasal Spray 3 | Cromolyn sodium | 20% v/v | 100% (5/5) | 100% (5/5) | | OTC Nasal Mist | Sodium chloride with preservatives | 20% v/v | 100% (5/5) | 100% (5/5) | | OTC Homeopathic Nasal Spray 1 | Alkalol | 20% v/v | 100% (5/5) | 100% (5/5) | | OTC Homeopathic Nasal Gel | Galphimia glauca, Histaminum hydrochloricum, Luffa opperculata, Sulfur | 20% v/v | 100% (5/5) | 100% (5/5) | | OTC Homeopathic Nasal Spray 3 | Fluticasone furoate | 20% v/v | 100% (5/5) | 100% (5/5) | K221925 - Page 16 of 23 {16} | OTC Homeopathic Nasal Spray 4 | Fluticasone propionate | 20% v/v | 100% (5/5) | 100% (5/5) | | --- | --- | --- | --- | --- | | OTC Homeopathic Oral Tablet | Zincum gluconium, Zincum aceticum² | 10% w/v | 100% (5/5) | 100% (5/5) | | Sore Throat Spray | Phenol | 20% v/v | 100% (5/5) | 100% (5/5) | | Corticosteroid | Beclomethasone | 0.068 mg/mL | 100% (5/5) | 100% (5/5) | | | Dexamethasone | 0.48 mg/mL | 100% (5/5) | 100% (5/5) | | | Flunisolide | 0.04 mg/mL | 100% (5/5) | 100% (5/5) | | | Triamcinolone | 0.04 mg/mL | 100% (5/5) | 100% (5/5) | | | Budesonide | 0.051 mg/mL | 100% (5/5) | 100% (5/5) | | | Mometasone | 0.04 mg/mL | 100% (5/5) | 100% (5/5) | | Antiviral | Zanamivir | 0.284 mg/mL | 100% (5/5) | 100% (5/5) | | Antibiotic, Nasal Ointment | Mupirocin | 4.3 mg/mL | 100% (5/5) | 100% (5/5) | | Antibacterial, Systemic | Tobramycin | 1.44 mg/mL | 100% (5/5) | 100% (5/5) | | Throat Lozenge | Benzocaine, Menthol | 0.63 mg/mL | 100% (5/5) | 100% (5/5) | | Toothpaste | Fluoride | 1% w/v | 100% (5/5) | 100% (5/5) | | Tobacco | Tobacco | 0.1% w/v | 100% (5/5) | 100% (5/5) | | Nicotine | Nicotine | 0.1% w/v | 100% (5/5) | 100% (5/5) | | Oral Rinse | Eucalyptol, Menthol, Methyl Salicylate, Thymol | 10% v/v | 100% (5/5) | 100% (5/5) | ¹Mucin at 2% w/v in the absence of SARS-CoV-2 yielded 1/5 invalid results and therefore was tested at a lower concentration. ²Zincum gluconium, Zincum aceticum at 20% w/v in the presence of SARS-CoV-2 yielded 1/5 invalid results and therefore was tested at a lower concentration. 5. **Assay Reportable Range:** Not applicable; this is a qualitative assay. 6. **Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):** a) **Assay Controls** Please refer to the Instrument Description Information (Section C.5) above for assay controls. b) **Sample Stability and Swab Equivalency** A Sample Stability and Swab Equivalency Study was performed to evaluate the specimen storage and swab type recommendations stated in the product insert. Negative Nasal Swab Matrix Diluent (NSMD) was prepared with pooled negative anterior nasal swabs eluted in UTM. On each day of testing, one positive control swab and one blank swab serving as the negative control were tested on each instrument used. Swab specimens were prepared using inactivated SARS-CoV-2 diluted NSMD to approximately 1.74x LoD. Each of the 15 swab types (Table 9) was tested in ten replicates on the ID NOW COVID-19 2.0 according to the test procedure at Time 0 and after 2 hours of storage at room temperature (15-30°C). All daily control testing generated expected results. The results are presented in Table 9 below. K221925 - Page 17 of 23 {17} Table 9: Sample Stability and Swab Equivalency | Swab Type | ID NOW COVID-19 2.0 Results | | | | | --- | --- | --- | --- | --- | | | Time 0 | | 2 Hours | | | | Clinical Matrix Diluent % Agreement (Count) | SARS-CoV-2 %Agreement (Count) | Clinical Matrix Diluent % Agreement (Count) | SARS-CoV-2 % Agreement (Count) | | Nasal Swabs | | | | | | Puritan Regular Foam Tip* | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | Puritan Regular Rayon Tip | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | Puritan HydraFlock Flock Swabs-Standard Tip | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | Puritan PurFlock Ultra Flocked Swabs-Standard Tip | 0% (0/10) (7/10 negatives, 3/10 invalids) | 50% (5/10) (2/10 negatives, 3/10 invalids) | 10% (1/10) (2/10 negatives, 7/10 invalids) | 0% (0/10) (4/10 negatives, 6/10 invalids) | | Copan Standard Rayon Tip Swab | 100% (10/10) | 100% (10/10) | 100% (10/10) | 80% (8/10) (2/10 negatives) | | Copan Standard Flocked Swab | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | MRC Technologies Foam Tipped Applicator | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | Foamtec Int'l CleanFOAM Diagnostics | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | FA Polyurethane Foam Swab | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | JCF Polyurethane Foam Swab | 100% (10/10) | 100% (10/10) | 100% (10/10) | 90% (9/10) (1/10 negative) | | Nasopharyngeal Swabs | | | | | | Puritan Small Foam Tip | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | Puritan Mini Rayon Tip | 100% (10/10) | 100% (10/10) | 100% (10/10) | 60% (6/10) (4/10 negatives) | | Puritan HydraFlock Flock Swabs-Mini Tip | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | | Puritan PurFlock Ultra Flocked Swabs-Mini Tip | 0% (0/10) (5/10 negatives) (5/10 invalids) | 0% (0/10) (10/10 invalids) | 0% (0/10) (3/10 negatives) (7/10 invalids) | 0% (0/10) (2/10 negatives) (8/10 invalids) | | Copan Mini-Tip Flocked Swab | 100% (10/10) | 100% (10/10) | 100% (10/10) | 100% (10/10) | *Swab provided in the ID NOW COVID-19 2.0 kits Based on the results of the study the following swabs are not suitable for use with the ID NOW COVID-19 Assay: Puritan Mini Rayon Tip, Puritan PurFlock Ultra Flocked Swabs-Standard Tip, Puritan PurFlock Ultra Flocked Swabs-Mini Tip, Copan Standard Rayon Tip Swab, and JCF Polyurethane Foam. 7. Detection Limit: a) LoD in Nasal Swab Matrix Negative Nasal Swab Matrix Diluent (NSMD) was prepared with pooled negative AN swabs eluted in Universal Transport Media (UTM). On each day of testing, one positive control swab and one blank swab serving as the negative control were tested on each instrument used for that day's testing. Heat inactivated SARS-CoV-2 (USA_WA1/2020) was diluted in NSMD. Swab samples were prepared by dispensing $50~\mu \mathrm{L}$ of the testing dilution into a conical tube. A sterile foam-tipped swab was inserted into the tube and swirled to absorb the testing dilution onto the swab head. To determine the LoD, serial limiting dilutions were tested in seven replicates per dilution. Testing was performed over three days, for a total of K221925 - Page 18 of 23 {18} 21 replicates at each dilution level for each device lot. The results of this testing are provided in Table 10. Table 10: Preliminary LoD Results | SARS-CoV-2 Concentration (Copies/Reaction) | ID NOW COVID-19 2.0 Results | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | Lot 1 # Detected | Lot 1 % Detected | Lot 2 # Detected | Lot 2 % Detected | Lot 3 # Detected | Lot 3 % Detected | All Lots % Agreement (Count) | | 20 | 21/21 | 100% | 21/21 | 100% | 21/21 | 100% | 100 % (63/63) | | 10 | 19/21 | 90% | 19/21 | 90% | 18/21 | 86% | 88.9% (56/63) | | 5 | 15/21 | 71% | 9/21 | 43% | 14/21 | 67% | 60.3% (38/63) | | 2.5 | 8/21 | 38% | 10/21 | 48% | 7/21 | 33% | 39.7% (25/63) | | 1.25 | 3/21 | 14% | 4/21 | 19% | 8/21 | 38% | 23.8% (15/63) | | 0.625 | 3/21 | 14% | 2/21 | 10% | 4/21 | 19% | 14.3% (9/63) | | 0.3125 | 3/21 | 14% | 1/21 | 5% | 2/21 | 10% | 9.5% (6/63) | The data generated in Preliminary LoD testing was used for Probit Regression analysis using statistical software JMP v14.3 (SAS). An LoD point estimate was determined on each device lot tested. The highest calculated LoD value from all three lots (i.e., worst case) was considered the predicted LoD. Based on this analysis, the LoD was predicted to be 117 copies/mL or 11.7 copies/reaction. Confirmation of the LoD was performed by testing 20 swabs prepared with 11.6 copies/reaction. Confirmation testing of this level generated $\geq 85\%$ positive results across the three device lots. Due to the results being below the expected $95\%$ positive, the next highest level tested during study execution to generate $\geq 95\%$ positive results across all three device lots, 20 copies/reaction, was determined to be the assay LoD. The results from the confirmation of the LoD are provided in Table 11. Table 11: Confirmation of the LoD | ID NOW COVID-19 2.0 Lot (20 copies/reaction) | ID NOW COVID-19 2.0 Results | | | --- | --- | --- | | | # Detected | % Detection | | Lot 1 | 17/20 | 85% | | Lot 2 | 19/20 | 95% | | Lot 3 | 20/20 | 100% | The LoD for the ID NOW COVID-19 2.0 for nasal swabs in NSDM was determined to be 20 copies/reaction or 500 copies/swab. The LoD is summarized in Table 12 below. K221925 - Page 19 of 23 {19} Table 12: LoD in Nasal Swab Matrix | Specimen Type | LoD (copies/μL) | LoD (copies/swab)* | LoD (copies/reaction) | | --- | --- | --- | --- | | Inactivated SARS-CoV-2 in NSMD | 10 | 500 | 20 | *Copies/Swab = Virus concentration per reaction (copies/reaction) / 100 (μL/reaction) x 2500 (μL Elution Buffer per Sample Receiver) x 1 (Sample Receiver/Swab) b) LoD in Nasopharyngeal Swab Matrix Negative Nasopharyngeal Swab Matrix Diluent (NPMD) was prepared with pooled negative NP swabs eluted in UTM. On each day of testing, one positive control swab and one blank swab serving as the negative control were tested on each instrument used. Heat inactivated SARS-CoV-2 (USA_WA1/2020) was diluted in NPMD. For each of the three lots of the ID NOW COVID-19 2.0 devices, the testing solutions were prepared at 20 copies/reaction of SARS-CoV-2 in NPMD. Swab samples were prepared by dispensing 10 μL of the testing dilution into a conical tube. A sterile foam-tipped nasopharyngeal swab was inserted into the tube and swirled to absorb the testing dilution onto the swab head. A total of 60 replicates were tested according to the assay procedure. The LoD for ID NOW COVID-19 2.0 in NP swabs in NPMD was confirmed at 20 copies/reaction or 500 copies/swab. The results are provided in the Table 13. Table 13: LoD Confirmation in Nasopharyngeal in Swab Matrix | SARS-CoV-2 Concentration (Copies/Reaction) | ID NOW COVID-19 2.0 Results | | | | | | | | --- | --- | --- | --- | --- | --- | --- | --- | | | Lot 1 # Detected | Lot 1 % Detected | Lot 2 # Detected | Lot 2 % Detected | Lot 3 # Detected | Lot 3 % Detected | All Lots % Agreement (Count) | | 20 | 58/60* | 96.7% | 59/60 | 98.3% | 60/60 | 100% | 98.3% (177/180)** | *Includes retest of one of the invalid results. **Does not include the invalid that was retested per the approved protocol. 8. Assay Cut-Off: ID NOW COVID-19 2.0 consists of two fluorescence channels, ROX for the Internal Control (IC) channel and FAM for the COVID-19 assay channel. The fluorescent signal of each channel is sampled and processed using a full amplification cycle to produce the final assay result. The assay terminates once a determinate assay result is obtained, when configured to do so by the instrument configuration file. During the acquisition of its fluorescent signal, each channel is first individually assessed using the decision algorithm and assigned a result as “Asserted”, “Not Asserted”, or “Indeterminate”. The channel results are then combined to arrive at the assay result as “Positive”, “Negative”, or “Invalid” based on assay decision logic. 9. Carry-Over: A study was performed to evaluate carry-over/contamination risk when alternating testing between high positive and negative specimens on the ID NOW COVID-19 2.0. NSMD was prepared with pooled negative AN swabs eluted in UTM. Heat inactivated SARS-CoV-2 virus was diluted in NSMD. On each day of testing, one positive control swab and one blank swab serving as the negative control were tested on each instrument. A positive swab was tested first, followed by a negative swab on each ID NOW Instrument. One round of testing consisted of one positive swab test followed by one negative swab test. This procedure was repeated for a total of 15 rounds per instrument (15 positive and 15 negative) over the course of two days using a total K221925 - Page 20 of 23 {20} of two instruments. All positive and negative daily controls generated the expected results on each day of testing. A summary of the results is provided in Table 14 below. Table 14: Results Summary Carry-over/Contamination Study | ID NOW Instrument | Testing Day | Specimen Tested | Replicates Tested | % Agreement (Count) | | --- | --- | --- | --- | --- | | 1 | 1 | SARS-CoV-2 Positive Swab | 7 | 100% (7/7) | | | | Negative Swab | 7 | 100% (7/7) | | | 2 | SARS-CoV-2 Positive Swab | 8 | 100% (8/8) | | | | Negative Swab | 8 | 100% (8/8) | | 2 | 1 | SARS-CoV-2 Positive Swab | 7 | 100% (7/7) | | | | Negative Swab | 7 | 100% (7/7) | | | 2 | SARS-CoV-2 Positive Swab | 8 | 100% (8/8) | | | | Negative Swab | 8 | 100% (8/8) | B Comparison Studies: 1. Method Comparison with Predicate Device: Not applicable. Please refer to the Clinical Studies Section of this document. 2. Matrix Comparison: Refer to the Limit of Detection (LoD) studies performed in anterior nasal (AN) and nasopharyngeal (NPS) swab matrices. C Clinical Studies: The clinical performance of the ID NOW COVID-19 2.0 was established in a multi-center, prospective clinical study conducted at 21 U.S. sites in 2020/2021. A total of 60 different operators, across the sites tested subjects with ID NOW COVID-19 2.0. The study sites and the test operators used in this clinical study were representative of the CLIA waived setting. To be enrolled in the study, patients had to be presenting at the study centers showing signs and symptoms of upper respiratory infection. Two nasal (NS) or nasopharyngeal (NPS) swabs were collected from each patient and tested using ID NOW COVID-19 2.0 at all study sites. Three FDA Emergency Use Authorized Real-Time Polymerase Chain Reaction (RT-PCR) assays for the detection of SARS-CoV-2 were utilized in a composite comparator method to establish a composite comparator result for each sampel. At all sites, one nasal or nasopharyngeal swab was tested directly with the ID NOW COVID-19 2.0 according to product instructions and the other swab was eluted in UTM. All sites shipped the UTM sample to a central testing laboratory for RT-PCR testing with the comparator assays. The demographics of study participants are presented in Table 15 below. The Positive Percent Agreement (PPA) and Negative Percent Agreement of ID NOW COVID-19 2.0 with composite comparator are presented in Table 16 below. K221925 - Page 21 of 23 {21} Table 15: Subject Demographics | Age | Subjects (N = 914) | | --- | --- | | N | 914 | | Mean (SD) | 42 (17.3) | | Median | 40 | | Min-Max | 1.0, 87 | | Age Category (years) | | | ≤ 5 | 3 (0.3%) | | 6 – 21 | 111 (12.1%) | | 22 – 59 | 644 (70.5%) | | ≥60 | 156 (17.1%) | | Gender | | | Female | 914 | | Male | 42 (17.3) | Table 16: Performance of ID NOW COVID-19 2.0 against Composite Comparator (Nasal and Nasopharyngeal Swabs Combined) | | Composite Comparator Result | | | | --- | --- | --- | --- | | ID NOW COVID-19 2.0 | POSITIVE | NEGATIVE | Total | | Positive | 254 | 10 | 264 | | Negative | 23 | 627 | 650 | | Total | 277 | 637 | 914 | | PPA (95% CI) | 91.7% (87.8% - 94.4%) | | | | NPA (95% CI) | 98.4% (97.1% - 99.1%) | | | D Clinical Cut-Off: Not applicable. E Expected Values/Reference Range: Not applicable. F Other Supportive Instrument Performance Characteristics Data: ID NOW COVID-19 2.0 and ID NOW Influenza A & B 2 Sequential Workflow Study: Sequential Workflow utilizes one patient specimen to run both an ID NOW COVID-19 2.0 assay and an ID NOW Influenza A & B 2 assay by reusing the Sample Receiver. In this workflow, ID NOW COVID-19 2.0 assay must be run BEFORE the ID NOW Influenza A & B 2 assay. A study was conducted to evaluate the impact of a sequential workflow on ID NOW COVID-19 2.0 and on the ID NOW Influenza A & B 2 test performance. For this study, individual swab samples were prepared by spiking NSMD with viruses at the following concentrations: SARS- K221925 - Page 22 of 23 {22} CoV-2 (inactivated) at 1.74x LoD, influenza A at ~0.57x LoD, and influenza B at ~0.61x LoD. NSMD was prepared with pooled negative AN swabs eluted in UTM. Negative test samples consisted of swabs with unspiked NSMD. The sequential workflow consisted of first testing a single anterior nasal swab sample with ID NOW COVID-19 2.0 and, after obtaining the test result, followed by testing the same sample with an ID NOW Influenza A&B 2 test. In this study, the same Sample Receiver containing the test sample was used to run both tests. ID NOW COVID-19 2.0 and ID NOW Influenza A & B 2 assays both utilize the same Elution Buffer (contained within the Sample Receiver) and both assays are intended for use with an anterior nasal or nasopharyngeal swab specimen. On each day of testing, one COVID-19 Positive Control Swab, one Influenza A & B Positive Control Swab and two Negative Control Swabs were tested on each instrument. Each sample was tested in five replicates following the sequential workflow: swab was run as a patient specimen with ID NOW COVID-19 2.0. Then, using the same Sample Receiver and the same ID NOW instrument, an ID NOW Influenza A & B 2 test was run. All tests generated expected results. A summary of results is provided in the Table 17 below. Table 17: Sequential Workflow Study | | Sample Type | Assay | ID NOW COVID-19 2.0 Result % Agreement (Count) | ID NOW Influenza A&B 2 Result | | | --- | --- | --- | --- | --- | --- | | | | | | Flu A % Agreement (Count) | Flu B % Agreement (Count) | | Sequential workflow on the same instrument: ID NOW COVID-19 2.0 first, followed by ID NOW Influenza A&B 2 | SARS-CoV-2 and Flu A & B Positive Swab | ID NOW COVID-19 2.0 | 100% (5/5) | N/A | N/A | | | | ID NOW Influenza A & B 2 | N/A | 100% (5/5) | 100% (5/5) | | | Negative Swab | ID NOW COVID-19 2.0 | 100% (0/5) | N/A | N/A | | | | ID NOW Influenza A & B 2 | N/A | 100% (0/5) | 100% (0/5) | | | SARS-CoV-2 negative and Flu A & B Positive Swab | ID NOW COVID-19 2.0 | 100% (0/5) | N/A | N/A | | | | ID NOW Influenza A & B 2 | N/A | 100% (5/5) | 100% (5/5) | VIII Proposed Labeling: The labeling supports the finding of substantial equivalence for this device. IX Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision. K221925 - Page 23 of 23 --- **Source:** [https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QWR/K221925](https://fda.innolitics.com/submissions/MI/subpart-d%E2%80%94serological-reagents/QWR/K221925) **Published by [Innolitics](https://innolitics.com)** — a medical-device software consultancy. We help companies design, build, and clear FDA-regulated software and AI/ML devices. If you're preparing [a 510(k)](https://innolitics.com/services/510ks/), [a De Novo](https://innolitics.com/services/regulatory/), [a SaMD](https://innolitics.com/services/end-to-end-samd/), [an AI/ML medical device](https://innolitics.com/services/medical-imaging-ai-development/), or [an FDA regulatory strategy](https://innolitics.com/services/regulatory/), [get in touch](https://innolitics.com/contact). **Cite:** Innolitics at https://innolitics.com