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

Affiliation

Tedim AP(1)(2), Almansa R(1)(2)(3), Domínguez-Gil M(4), González-Rivera M(5), Micheloud D(6), Ryan P(7), Méndez R(8), Blanca-López N(7), Pérez-García F(9), Bustamante E(10), Gómez JM(11), Doncel C(1)(2)(3), Trapiello W(12), Kelvin AA(13)(14), Booth R(13)(14), Ostadgavahi AT(13)(14), Oneizat R(4), Puertas C(5), Barbé F(3)(15), Ferrer R(3)(16), Menéndez R(3)(8), Bermejo-Martin JF(#)(1)(2)(3), Eiros JM(4), Kelvin DJ(#)(13)(14), Torres A(#)(3)(17).
Author information:
(1)Group for Biomedical Research in Sepsis
(BioSepsis), Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.
(2)Hospital Universitario Río Hortega, Valladolid, Spain.
(3)Centro de Investigación Biomédica en Red en Enfermedades Respiratorias
(CIBERES), Instituto de Salud Carlos III, Av. de Monforte de Lemos, Madrid, Spain.
(4)Microbiology Service, Hospital Universitario Rio Hortega, Valladolid, Spain.
(5)Department of Laboratory Medicine, Hospital General Universitario Gregorio Marañón, Madrid, Spain.
(6)Emergency Department, Hospital General Universitario Gregorio Marañón, Madrid, Spain.
(7)Hospital Universitario Infanta Leonor, Madrid, Spain.
(8)Pulmonology Service, Hospital Universitario y Politécnico de La Fe, Avinguda de Fernando Abril Martorell, Spain.
(9)Servicio de Microbiología Clínica, Hospital Universitario Príncipe de Asturias, Madrid, Spain.
(10)Intensive Care Unit, Hospital Clínico Universitario de Valladolid. Av. Ramón y Cajal, Valladolid, Spain.
(11)Intensive Care Unit. Hospital General Universitario Gregorio Marañón, Madrid, Spain.
(12)Clinical Analysis Service. Hospital, Clínico Universitario de Valladolid, Av. Ramón y Cajal, Valladolid, Spain.
(13)Department of Microbiology and Immunology, Faculty of Medicine, Canadian Center for Vaccinology CCfV, Dalhousie University, Halifax, Nova Scotia, Canada.
(14)Laboratory of Immunity, Shantou University Medical College, Jinping, Shantou, China.
(15)Respiratory Department, Institut Ricerca Biomedica de Lleida, Av. Alcalde Rovira Roure, Lleida, Spain.
(16)Intensive Care Department, SODIR Research Group, Vall d'Hebron Hospital Universitari, Vall d'Hebron Institut de Recerca, Barcelona, Spain.
(17)Department of Pulmonology, Institut D investigacions August Pi I Sunyer
(IDIBAPS), Hospital Clinic de Barcelona, Universidad de Barcelona, Barcelona, Spain.
(#)Contributed equally

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.