Excellent option for mass testing during the SARS-CoV-2 pandemic: painless self-collection and direct RT-qPCR

Abstract

The early identification of asymptomatic yet infectious cases is vital to curb the 2019 coronavirus (COVID-19) pandemic and to control the disease in the post-pandemic era. In this paper, we propose a fast, inexpensive and high-throughput approach using painless nasal-swab self-collection followed by direct RT-qPCR for the sensitive PCR detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This approach was validated in a large prospective cohort study of 1038 subjects, analysed simultaneously using (1) nasopharyngeal swabs obtained with the assistance of healthcare personnel and analysed by classic two-step RT-qPCR on RNA isolates and (2) nasal swabs obtained by self-collection and analysed with direct RT-qPCR. Of these subjects, 28.6% tested positive for SARS-CoV-2 using nasopharyngeal swab sampling. Our direct RT-qPCR approach for self-collected nasal swabs performed well with results similar to those of the two-step RT-qPCR on RNA isolates, achieving 0.99 positive and 0.98 negative predictive values (cycle threshold [Ct] < 37). Our research also reports on grey-zone viraemia, including samples with near-cut-off Ct values (Ct ≥ 37). In all investigated subjects (n = 20) with grey-zone viraemia, the ultra-small viral load disappeared within hours or days with no symptoms. Overall, this study underscores the importance of painless nasal-swab self-collection and direct RT-qPCR for mass testing during the SARS-CoV-2 pandemic and in the post-pandemic era.

Keywords: COVID-19; Mass molecular testing; Nasal mid-turbinate swab; PCR diagnostics; Post-pandemic era; Self-collection.

Fig. 1

a Study design: comparison of nasal-swab self-collection followed by direct RT-qPCR (left panel) vs nasopharyngeal-swab  healthcare personnel-assisted sampling with two-step RT-qPCR (RNA isolation followed by PCR; right panel). b Nylon-flocked swab tips tested for self-collected nasal swabs. (1) FLOQSwabs MFS-98000KQ (iClean), (2) MFS-97000KQ (iClean) and 3) 520CS01 (COPAN Diagnostics Inc.)

Fig. 2

Detection of a control human RNase P (RP) gene (a) and virus-specific N1/N2 genes* (b) in self-collected nasal swabs by direct RT-qPCR; the specimen was heat inactivated before the PCR analysis. To avoid false-negative results in the RT-qPCR, the human RP gene had to be investigated to control for proper specimen collection and amplification reaction inhibition. Positive (red line) and negative (black line) controls from direct RT-qPCR (DIOS-RT-qPCR Kit) were included in each run. *The RT-qPCR setup, primers and probe sequences have been reported previously [15]

Fig. 3

Inhibition of SARS-CoV-2 virus detection by direct RT-qPCR tested with titrated copies of viable virus culture either spiked immediately into COVID media (orange lines) or spiked into COVID media with nasal swabs from COVID-19-negative patients (blue lines). The live SARS-CoV-2 virus culture was spiked at the following concentrations: 32, 16, 8, 4, 2 and 0 copies/reaction (which translates into 1143, 571, 286, 143 and 0 copies/ml). The lowest concentrations were analysed in triplicate. The data are presented as a amplification curves for particular SARS-CoV-2 concentrations and b the corresponding Ct values. Ct: cycle threshold; SD: standard deviation; Delta Ct: the difference between the COVID medium alone and the COVID medium with negative nasal swabs for the same viral load. *A Ct difference of around 3.3 cycles corresponds to every 1log10 copies/ml change in viral load detection (2^3.3 = 9.48)

Fig. 4

Comparison of unnormalised Ct values calculated for two-step RT-qPCR and direct RT-qPCR detection of the SARS-CoV-2 virus in all samples found to be positive by both nasopharyngeal and nasal swabs. a The linear regression analysis for the Ct detected for paired samples in both SARS-CoV-2 RT-qPCR methods. The x-axis shows the Ct values obtained by direct RT-qPCR, and the y-axis shows the values for the paired samples analysed by two-step RT-qPCR. b The distance between the Ct values from the two-step RT-qPCR (blue dot) and paired direct RT-qPCR (orange dot) represents the rate of Ct discordance between the techniques for an individual study subject