A total of 76 blinded dry nasal swab samples were tested in one investigational site to evaluate the clinical performance of the CovClear™ SARS-Cov-2 Rapid Antigen Test strip. Nasal swab specimens were collected from patients with COVID-19 like symptoms during the 2020 COVID-19 season. All the Nasal swabs were added to the CovClear™ vial (either dry or in buffer). All the Nasal swab specimens were confirmed as positive or negative and validated with Ct value by the FDA EUA RT-PCR as a comparator method. The samples were randomized, blinded and tested using the instructions provided by the Instruction for Use.
All the study samples were random and assigned a study ID prior to testing. The expected results of the sample were completely blinded to the operators. 11% of the positive samples had Ct values over 30.
Analytical Sensitivity: Limit of Detection (LoD)
The LoD for direct swab was established using heat-inactivated SARS-CoV-2 isolate Hong Kong/VM20001061/2020). The strain was spiked into 0.5 saline solution prepared in accordance with BAM R66. The estimated LoD found from the initial two- fold serial dilution test was confirmed by testing 20 replicates. The confirmed LoD for direct swab was 7.12 x 103 TCID50/ml.
The specimen stability was established using heat-inactivated SARS-CoV-2 isolate Hong Kong/VM20001061/2020. The strain was spiked into 0.5% saline solution at 3x the LoD (2.16 x 104 TCID50/ml). Samples were then Incubated at room temperature for 0, 2 6, and 24 hours respectively prior to testing. All samples tested produced no qualitative impact on test line signal intensity as compared to the 0-hour condition, demonstrating that the CovClear™ COVID-19 Antigen Test performance was not affected by sample Instability for up to 24 hours at room temperature.
Analytical Specificity: Cross Reactivity and Microbial Interference
The potential cross-reactivity (exclusivity) of a panel of common organisms was evaluated with SARS-CoV-2 negative samples using the CovClear™ COVID-19 Antigen Test. Potential microbial interference was evaluated with samples containing heat-inactivated SARS-CoV-2 isolate isolate Hong Kong/VM20001061/2020 at approximately 3x LoD. A total of 7 bacteria were tested at a target at approximately 1 x 106 cfu/ml. The 13 viruses were tested at concentrations between 9.87 x 104 and 1 x 105 pfu. All negative samples gave negative results at the concentrations of the potentially cross-reactive common organisms tested showing no cross-reactivity with CovClear™ COVID-19 Antigen assay. All samples with SARS-CoV-2 strain tested positive showing no microbial interference at the concentrations of the potentially interfering common organisms tested.
SARS-coronavirus was not tested as part of this study. Additional testing may be required to determine if this pathogen will generate cross-reactivity at the CovClear test line.
To estimate the likelihood of cross-reactivity with SARS-CoV-2 of organisms that were not available for wet testing, in silico analysis using the Basic Local Alignment Search Tool (BLAST) managed by the National Center for Biotechnology Information (NCBI) was used to assess the degree of protein sequence homology.
- The homology between SARS-CoV-2 nucleocapsid protein and human coronavirus HKU1 nucleocapsid protein is relatively low, at 36.7% across 86.4% of sequences, but cross- reactivity cannot be ruled out.
- The homology between SARS-CoV-2 nucleocapsid protein and Mycobacterium tuberculosis total protein (3,991 proteins) is relatively low, homology-based cross-reactivity can be ruled out.
- The homology between SARS-CoV-2 nucleocapsid protein and Pneumocystis jirovecii total protein (3,745 proteins) is relatively low, homology-based cross-reactivity can be ruled out.
- The homology between SARS-CoV-2 nucleocapsid protein and human coronavirus 229E nucleocapsid protein is relatively low, but cross- reactivity cannot be ruled out. However, a result of the cross-reactivity wet study showed that CovClear™ COVID-19 Antigen had no cross-reactivity against human coronavirus 229E.
- No homologous protein was detected as a result of in silico assay with the proteins of Mycoplasma pneumoniae and the nucleocapsid protein (NP) of SARS-CoV-2. So, cross-reactivity of CovClear™ COVID-19 Antigen against Mycoplasma pneumoniae can be ruled out.
Endogenous Interfering Substances Effect
To assess substances with the potential to interfere with the performance of the CovClear™ COVID-19 Antigen Test, positive and negative samples were tested with the addition of potentially interfering substances. The SARS-CoV-2 target concentration in the positive samples was approximately 2x LoD. All samples tested produced no qualitative impact to test line signal intensity, demonstrating that the CovClear™ COVID-19 Antigen Test performance was not affected by any of the 14 potentially interfering substances listed in the table below at the concentrations tested.
The interfering effects of biotin concentrations ranging between 625 ng/mL and 10 µg/mL were tested in a separate study. Biotin concentrations up to 1.25 µg/ml did not lead to false results. Biotin concentrations =2.5 µg/ml can cause false-negative COVID-19 results with the CovClear™ COVID-19 Antigen Test.
High-dose Hook Effect
The CovClear™ COVID-19 Antigen Test was tested up to 1.15 x 105 TCID50/ml of heat-inactivated SARS- CoV-2 strain and no high-dose hook effect was observed.
For questions, or to report a problem, please call Empowered Diagnostics at 954-354-2768.
Test system problems may also be reported to the FDA using the MedWatch reporting system (phone: 1-800-FDA-1088; fax: 1-800-FDA-1078: or http://www.fda.gov/medwatch).