Positivization time of a COVID-19 rapid antigen self-test predicts SARS-CoV-2 viral load: a proof of concept

Abstract

Objectives This proof of concept study was aimed to validate the hypothesis that the time of positivization of SARS-CoV-2 self-performed rapid diagnostic tests (RDTs) may reflect the actual viral load in the specimen. Methods A SARS-CoV-2 positive sample with high viral load was diluted and concomitantly assayed with molecular assay (Xpert Xpress SARS-CoV-2) and RDT (COVID-VIRO ALL IN RDT). The (mean cycle threshold; Ct) values and RDT positivization times of these dilutions were plotted and interpolated by calculating the best fit. The parameters of this equation were then used for converting the positivization times into RDT-estimated SARS-CoV-2 Ct values in routine patient samples. Results The best fit between measured and RDT-estimated Ct values could be achieved with a 2-degree polynomial curve. The RDT-estimated Ct values exhibited high correlation (r=0.996) and excellent Deming fit (y=1.01 × x − 0.18) with measured Ct values. In 30 consecutive patients with positive RDT test, the correlation between RDT positivization time and measured Ct value was r=0.522 (p=0.003). The correlation of RDT-estimated and measured Ct values slightly improved to 0.577 (Deming fit: y=0.44 × x + 11.08), displaying a negligible bias (1.0; 95% CI, −0.2 to 2.2; p=0.105). Concordance of RDT-estimated and measured Ct values at the <20 cut-off was 80%, with 0.84 sensitivity and 0.73 specificity. Conclusions This proof of concept study demonstrates the potential feasibility of using RDTs for garnering information on viral load in patients with acute SARS-CoV-2 infection.

Quantification of SARS‐CoV‐2 viral load in patients using time‐to‐results in antigen lateral flow tests

“…Salvagno et al. also examined LFT predicted and measured Ct values in unknown clinical samples and found a correlation coefficient of 0.577, a lower value than we found with both the BN ( r = 0.93) or FF ( r = 0.97) assays 10 . We also found that high viral load samples could be diluted and accurately measured in the two antigen tests, which not only increases the upper range of viral concentrations that can be measured, but additionally allows a longer time frame between applying the samples and the test turning positive.…”

“…Salvagno et al also examined LFT predicted and measured Ct values in unknown clinical samples and found a correlation coefficient of 0.577, a lower value than we found with both the BN (r = 0.93) or FF (r = 0.97) assays. 10 We also found that high viral load samples could be diluted and accurately measured in the two antigen tests, which not only increases the upper range of viral concentrations that can be measured, but additionally allows a longer time frame between applying the samples and the test turning positive. This latter factor may increase the accuracy of the timing of when the test turns positive, because we found that measurements at both ends of the working range of the viral dose-response versus time-to-positivity curves are less precise than those in the middle part of the doseresponse curve (data not shown).…”

“…Between our results with two home tests and those of others, who measured time-to-positivity in eight other visual read LFTs versus actual or surrogates of viral load, it is likely that most, if not all, of the COVID-19 LFTs will behave similarly. [7][8][9][10]19 Accurate quantification requires first establishing a standard curve with known concentrations of the virus with each LFT before using it to estimate the viral load in a sample. Ideally this would be performed using artificial mucus spiked with the protein or virus rather than UTM as used in this study.…”

“…Additionally, two groups reported that the time-to-positive SARS-CoV-2 antigen result was inversely correlated with the Ct values. [8][9][10] In order to determine whether the utilization of timeto-positivity after application of nasal swab samples in two popular home SARS-CoV-2 antigen tests could be used to quantify actual viral load, we investigated the dose-response relationship between time-to-positive result by visual assessment in the antigen tests with purified nucleocapsid protein (NP), chemically inactivated WA1 and Omicron XBB.1.5 viral strains, and Ct values with dilutions of the viruses and in clinical samples. We also examined the feasibility of using time-to-positive result to predict the Ct values in clinical samples from patients with COVID-19.…”

“…demonstrated that measuring time‐to‐positive test results in six LFTs was shorter for a high viral concentration (2.5 × 10 5 TCID 50 /mL) than with a lower viral concentration (2.5 × 10 4 TCID 50 /mL). Additionally, two groups reported that the time‐to‐positive SARS‐CoV‐2 antigen result was inversely correlated with the Ct values 8–10 …”