SARS-CoV-2 RDT lot consistency
One lot of 2750 test cassettes were prepared from a total of 38 printed and assembled cards, with approximately 72 test strips produced from each card. Each test strip was assembled into a cassette and sealed into a foil pouch with a silica desiccant packet. To evaluate the intra-lot consistency, test cassettes assembled with strips collected from the beginning, middle, and end of each even-numbered card were collected. Recombinant nucleocapsid antigen at 2.5ng/mL in sample extraction buffer was chosen to target 2X the LOD signal on the RDS-2500 reader; 85mL was dispensed into the sample port of each cassette. After 15 minutes each cassette was visually inspected and run on an RDS-2500 reader and intensity values recorded for both test and control lines. Results were analyzed by one-way ANOVA in Minitab with a significance level of a = 0.05; test line intensity p = 0.800 and control line p = 0.270 demonstrated no significant difference in performance across the entire lot of test devices.
Linearity and limit of detection of the RDT device
Linearity and Limit of Detection (LOD) studies utilized the guidelines outlined by the U.S. Food and Drug Administration Coronavirus Disease 2019 (COVID-19) Emergency Use Authorizations for Medical Devices Antigen Template for Test Developers, October 26, 2020 version.14 Heat inactivated SARS-CoV-2 virus (BEI Resources, isolate USA0WA 1/2020) was diluted into a pool of 10 individual RT-qPCR SARS-CoV-2 negative nasal samples collected into saline.
Linearity was evaluated by testing serial dilutions of inactivated SARS-CoV-2 virus in pooled negative nasal swab samples and the intensity of the test line was evaluated by visual inspection (Table 1) as well as quantified using the RDS-2500 reader (Fig. 4).
Table 1
Linearity study summary. Visually interpreted test results of heat inactivated reference SARS-CoV-2 virus dilutions into pooled negative nasal swab sample (n = 2 per dilution). All samples with 1.79x104 viral copies or less per RDT cassette as well as the zero-virus negative control did not generate a visible test line and were interpreted as negative. All test cassettes have a visible red control line, indicating all tests were valid.
Viral copies / RDT | TCID50/mL | Test line result (+/-) | Control line result (+/-) |
2.86 x 105 | 1.6 x 103 | + | + |
1.43 x 105 | 8.0 x 102 | + | + |
7.17 x 104 | 4.0 x 102 | + | + |
3.59 x 104 | 2.0 x 102 | + | + |
1.79 x 104 | 1.0 x 102 | - | + |
8.96 x 103 | 50 | - | + |
0 | 0 | - | + |
Following visual inspection, RDT cassettes were read in the RDS-2500 reader to quantify line intensity. Test line signal counts were correlated to viral copy input. Figure 4 demonstrates a linear response between the amount of viral material applied to the cassette and the amount of specific SARS-CoV-2 signal detected at the test line. The correlation coefficient for the resultant best fit line demonstrated an R2 of 0.9684, indicating an acceptable dose-response for the N-antigen detection assay.
The FDA EUA guidance defines LOD as the lowest concentration at which 19 of 20 (95%) replicates are positive.14 Limit of Detection Robustness was evaluated by running n = 20 of the dilutions above, at, and below the presumed LOD of 3.59 x 104 viral copies per test cassette; results are depicted in Table 2. The LOD was verified using inactivated virus spiked into pooled negative nasal swab samples and found to be 3.59 x 104 genomic viral copies or 200 TCID50/mL per RDT cassette (Table 2).
Table 2
LOD robustness using heat-inactivated virus in pooled negative swab samples. SARS-CoV-2 RDT cassette replicates of both 7.17 x 104 viral copies/RDT and 3.59 x 104 viral copies/RDT had all 20/20 cassettes with positive test lines by visual inspection. For the 10 RDT cassettes to which 1.79 x 104 viral copies/RDT was applied, all 10/10 test lines were negative by visual inspection. The lowest level of virus where at least 95% of the replicates were detected was 3.59 x 104 viral copies/ RDT.
Viral copies / RDT | Positive test line | Positive control line |
1.79 x 104 | 0/10 | 10/10 |
3.59 x 104 | 20/20 | 20/20 |
7.17 x 104 | 20/20 | 20/20 |
To quantitatively assess the sensitivity of the SARS-CoV-2 antigen RDT, each of the fifty cassettes used in the determination of the assay LOD were read using the RDS-2500 reader. Figure 5 demonstrates the range of signals detected at the test line and the control line locations.
For all 50 cassettes tested, the control line intensity was strongly positive, indicating all tests were valid (B). Samples with more viral antigen present (≥ 3.59 x 104 viral copies/RDT) showed a control line signal approximately 5% lower than cassettes where less or no analyte was present, however this was not discernable by visual inspection.
The reader can interpret signal outside of the actual band, which increases the perceived background signal (as shown by the overlapping error bars of the 1.79 x 104 viral genomic copies and the 3.59 x 104 viral copies in Fig. 5A). Visual images of representative RDTs are therefore shown in Fig. 6 to highlight the intensity level of the band for each level of inactivated virus-spiked negative nasal swab samples alongside a RT-qPCR positive swab sample. Weak positive blue test lines were visible in the “T” location of cassettes 1 and 2, both at and above 3.59 x 104 viral copies/RDT. No test line was visible on cassette 3, below 3.59 x 104 viral copies/RDT. For a comparison, the RT-qPCR positive swab sample demonstrates a clearly visible test line on cassette 4. Control lines were visible at location “C” for all cassettes 1–4.
A summary of other Emergency Use Authorization (EUA) issued antigen tests for SARS-CoV-2 collected in October of 2020 from the Food and Drug Administration (FDA) website demonstrated a range of LOD from 1.0 x 102 TCID50/mL – 4.5 x 105 TCID50/mL.15 Our LOD of 3.59 x 104 viral copies per test is equivalent to 2.0 x 102 TCID50/mL and therefore is among the most sensitive SARS-CoV-2 RDT devices. For comparison, the BinaxNOW COVID-19 Ag card has shown a limit of detection of about 4.04 x 104 to 8.06 x 104 copies per swab.16
Clinical Sample Performance Testing
Clinical sample performance testing as outlined by the FDA EUA recommendation is a minimum of 30 positive specimens and 30 negative specimens collected either retrospectively or prospectively. Tests should demonstrate a minimum sensitivity of ≥ 80% for sample types tested.14 Retrospective nasal samples collected into saline were mixed with 10X extraction buffer to mimic our intended sample-extraction buffer concentration and run on RDT cassettes; all samples were tested for SARS-CoV-2 by RT-qPCR using the validated ExoDx COVID-19 RT-qPCR Test (a CLIA-validated FDA EUA-authorized CDC protocol; this assay has an LOD of ≤ 0.85 copies/µL). Samples are considered positive by RT-qPCR if both the N1 and N2 assay are detected. Cycle threshold (Ct) levels are inversely proportional to the amount of viral nucleic acid in the sample. A dose-response curve of Ct values vs SARS-CoV-2 RNA copies using the clinically validated RT-qPCR assay is shown in Supplemental Fig. 1.
A total of 35 out of 40 nasal swab samples positive by RT-qPCR were shown positive by RDT, demonstrating 87.5% sensitivity, and 40 out of 40 patient nasal swab samples negative by RT-qPCR were negative by RDT, demonstrating 100% specificity. This performance from a preliminary evaluation is comparable to SARS-CoV-2 RDT devices that have received FDA EUA approval.15 However, it is important to note that the reported clinical sensitivity of these assays is dependent on the viral load of the patients in the clinical cohort. The viral load varies widely between patients and is dependent on when and how the sample was taken during the course of an infection, making it challenging to compare across different studies. The concordance between RDT and RT-qPCR testing of patient nasal swabs collected into saline is summarized in Table 3 and Fig. 7.
Table 3
Concordance of the SARS-COV-2 antigen RDT assay and a validated RT-qPCR assay. SARS-CoV-2 antigen RDT results achieved 87.5% sensitivity and 100% specificity in a clinical cohort of 40 negative samples and 40 positives that were confirmed by RT-qPCR.
RT-qPCR result | RDT test line result | RDT control line result |
negative | 0/40 | 40/40 |
positive | 35/40 | 40/40 |
SARS-CoV-2 RT-qPCR-positive samples that had Ct values of 25 or lower were universally positive in the SARS-CoV-2 antigen RDT. RT-qPCR positive samples that were not detected as positive by RDT had Ct values of 25.3, 25.5, 25.7, 26.5 and 33.5, respectively, for the N1 assay. This indicates that the LOD for the SARS-CoV-2 antigen RDT assay correlates with patient samples that have Ct values around 25 for this CLIA-validated FDA EUA-authorized CDC test protocol.
To further assess the SARS-CoV-2 antigen RDT kit components and determine if a higher dilution of the samples could be used (using the 1X extraction buffer instead of 10X), 10 additional samples were evaluated by combining 400mL of saline-collected swab directly into the kit sample tube containing 400mL of extraction buffer, pipetting up and down to mix, and adding 3 drops (approximately 85mL) to the sample port of the RDT cassette. As was observed previously, all known positive RT-qPCR samples with Ct values ≤ 25.0 resulted in a positive test line on the RDT; one sample at 26.5 Ct reported positive on RDT while one sample at 25.28 Ct was negative. Visual results are depicted in Table 4 and Fig. 8.
Table 4
Visual interpretation summary of test kit-based study. 400mL of RT-qPCR known positive SARS-CoV-2 saline swab samples added to sample extraction buffer tube and evaluated on SARS-CoV-2 antigen RDT results agree with previous cutoff for RT-qPCR positives on RDT around 25 Ct for the N1 assay.
RT-qPCR N1 Ct | Test line result (+/-) | Control line result (+/-) |
12.2 | + | + |
15.8 | + | + |
14.5 | + | + |
19.8 | + | + |
20.4 | + | + |
23.0 | + | + |
22.3 | + | + |
25.0 | + | + |
26.5 | + | + |
25.3 | - | + |