Comparison of real‐time and droplet digital PCR to detect and quantify SARS‐CoV‐2 RNA in plasma
Ana P. Tedim, Raquel Almansa, Marta Domínguez‐Gil, Milagros González‐Rivera, Dariela Micheloud, Pablo Ryan, Raúl Méndez, Natalia Blanca‐López, Felipe Pérez‐García, Elena Bustamante, José Manuel Gómez, Cristina Doncel, Wysali Trapiello, Alyson A. Kelvin, Ryan Booth, Ali Toloue Ostadgavahi, Ruth Oneizat, Carolina Puertas, Ferrán Barbé, Ricard Ferrer, Rosario Menéndez, Jesús F. Bermejo-Martín, José María Eirós Bouza, David J. Kelvin, Antoní Torres
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.