Study shows that nucleic acid amplification tests more accurate in detecting SARS-CoV-2 than rapid antigen

By Sreetama Dutt M.Sc.Nov 30 2021Reviewed by Benedette Cuffari, M.Sc.

The currently available tests for detecting antigen load in patients suspected of being infected with the severe acute respiratory syndrome coronavirus (SARS-CoV-2) include the rapid antigen and reverse-transcriptase polymerase chain reaction (RT-PCR) tests. Several studies have compared the clinical sensitivity of molecular assays and rapid antigen tests on nasopharyngeal or nasal swab samples to detect SARS-CoV-2 levels.

Study: Analytical sensitivity and effectiveness of different SARS-CoV-2 testing options. Image Credit: anyaivanova / Shutterstock.com

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

However, a major limitation in these studies has been the necessity to collect separate swabs for detecting SARS-CoV-2 ribonucleic acid (RNA) and antigen, respectively, wherein these individuals were usually tested because of (mild) symptoms and already had higher viral loads. The available data from these studies that directly compared the analytical sensitivity of different nucleic acid amplification technology (NAT) systems and lateral flow devices for rapid detection of SARS-CoV-2 antigens using serial dilutions of swab fluid with a known viral load have been very limited.

About the study

Dutch researchers recently published a study on the preprint server medRxiv*, wherein they used reference preparations from pooled swab samples in Viral Transport Medium (VTM) to compare the analytical sensitivity of different SARS-CoV-2 assays. To this end, the researchers used betapropiolactone to inactivate these samples and measured the viral load before and after inactivation.

Since rapid antigen tests are known to miss a considerable proportion of infected individuals, the researchers also evaluated two NAT methods that were developed for fast detection of SARS-CoV-2 infection. These methods include a loop-mediated isothermal amplification (LAMP) assay and a simple amplification-based assay (SAMBA). The researchers then compared the sensitivity of these tests with the RT-PCR and transcription-mediated amplification (TMA) assays.

Study findings

In the current study, the researchers prepared SARS-CoV-2 working standards and reference panels from a pool of swab fluid samples before and after inactivation by beta-propiolactone. They further quantified viral load in NAT detectable RNA copies/mL using limiting dilution analysis.

The 50% lower limits of detection (LOD) values were then estimated by probit analysis as compared to LODs of rapid antigen tests on 1.5 fold dilutions of the native material.  The researchers used four NAT tests including the Roche cobas PCR, Hologic Aptima TMA 6.6, DRW SAMBA, and Molgen LAMP 23. The sensitivity of these assays was then compared with Fluorecare antigen, Abbott Panbio antigen, and Roche antigen tests.

The four NAT assays were able to detect early viremia 40-66 hours earlier than the 1,000 copies/mL infectivity threshold, whereas the three antigen tests would become positive 41-48 hours later. The Roche cobas assay for open reading frame a/b (ORFa/b) was the most sensitive with 50% and 95% LOD of 1.8 and 8.3 copies/mL, respectively, when used on single samples, and 11.1 and 49.8, respectively, when used on mini-pools of six samples.

The Aptima TMA assay was 3.6 fold less sensitive than the Roche cobas PCR with 50% and 95% LODs of 6.6 and 29.7 copies/mL, respectively. The LAMP assay was 12.4-fold less sensitive than the Roche assay with estimated 50% and 95% LODs of 23 and 102 copies/mL, respectively. The SAMBA II assay showed values for the 50% LOD and 95% LOD as 15 and 133 copies/mL, respectively.

The analytical sensitivity of the rapid antigen assays on 1.5-fold dilutions of the native standard dilutions was 28,000 to 56,000 fold lower than the Roche cobas PCR assay when comparing the lowest concentration giving indeterminate antigen reactivity with the 50% LOD in PCR. The LODs with rapid antigen test positive reactivity were 9,000 to 21,000-fold higher than the 95% LOD in the Roche cobas assay.

One 50% Tissue Culture Infectious Dose (TCID50)/mL of culture fluid was estimated to be equivalent to approximately 1,000 RNA copies/mL in the selected working standard. When assuming this level as start of contagiousness in a logarithmic, linear, ramp up viremia model with a 10-fold rise of viral load per day for the wild-type strain of SARS-CoV-2, the researchers estimated relative time points of first detectability of early infection by the different SARS-CoV-2 assays from the aforementioned LODs.

The analytical sensitivity data modeling was found to be compatible with clinical sensitivity data of rapid antigen tests. This confirmed that NAT assays were more reliable than antigen assays for identifying early infected asymptomatic individuals who are potentially infectious.

Implications

The analytical sensitivity of four different NAT assays was found to be better than three lateral flow devices for rapid SARS CoV-2 antigen detection on swab fluid samples before and after inactivation by beta-propiolactone. The researchers further estimated up to 56,000 fold differences in detection limits between several SARS-CoV-2 detection methods on a pool of swab fluid samples before inactivation.

Taken together, NAT assays could offer a new and more effective approach to detect SARS-CoV-2 in asymptomatic individuals and help in preventing the spread of COVID-19.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources