Comparison of real-time and droplet digital PCR to detect and quantify SARS-CoV-2 RNA in plasma

Abstract

Background: The presence of SARS-CoV-2 RNA in plasma has been linked to disease severity and mortality. We compared RT-qPCR to droplet digital PCR (ddPCR) to detect SARS-CoV-2 RNA in plasma from COVID-19 patients (mild, moderate, and critical disease).

Methods: The presence/concentration of SARS-CoV-2 RNA in plasma was compared in three groups of COVID-19 patients (30 outpatients, 30 ward patients and 30 ICU patients) using both RT-qPCR and ddPCR. Plasma was obtained in the first 24h following admission, and RNA was extracted using eMAG. ddPCR was performed using Bio-Rad SARS-CoV-2 detection kit, and RT-qPCR was performed using GeneFinder™ COVID-19 Plus RealAmp Kit. Statistical analysis was performed using Statistical Package for the Social Science.

Results: SARS-CoV-2 RNA was detected, using ddPCR and RT-qPCR, in 91% and 87% of ICU patients, 27% and 23% of ward patients and 3% and 3% of outpatients. The concordance of the results obtained by both methods was excellent (Cohen's kappa index = 0.953). RT-qPCR was able to detect 34/36 (94.4%) patients positive for viral RNA in plasma by ddPCR. Viral RNA load was higher in ICU patients compared with the other groups (P < .001), by both ddPCR and RT-qPCR. AUC analysis revealed Ct values (RT-qPCR) and viral RNA load values (ddPCR) can similarly differentiate between patients admitted to wards and to the ICU (AUC of 0.90 and 0.89, respectively).

Conclusion: Both methods yielded similar prevalence of RNAemia between groups, with ICU patients showing the highest (>85%). RT-qPCR was as useful as ddPCR to detect and quantify SARS-CoV-2 RNAemia in plasma.

Keywords: COVID-19; RNAemia; RT-qPCR; SARS-CoV-2; ddPCR; viral RNA load.