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-Martin; José María Eiros; David J. Kelvin; Antoni Torres

doi:10.1111/eci.13501

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 FerrerRosario Menéndez, Jesús F. Bermejo-Martin, José María Eiros, David J. Kelvin, Antoni Torres

Medicine

Research output: Contribution to journal › Article › peer-review

21 Citations (Scopus)

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.

Original language	English
Article number	e13501
Journal	European Journal of Clinical Investigation
Volume	51
Issue number	6
DOIs	https://doi.org/10.1111/eci.13501
Publication status	Published - Jun 2021

Bibliographical note

Funding Information:
This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID‐19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li‐Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the ‘Subvenciones de concesión directa para proyectos y programas de investigación del virus SARS‐CoV2, causante del COVID‐19’, FONDO ‐ COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Inflammation. The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript. APT was supported by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co‐financed by the European Development Regional Fund (A Way to Achieve Europe program). This study has also been funded by a Research Grant 2020 from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) APT.

Funding Information:
Funding This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID-19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li-Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the ‘Subvenciones de concesión directa para proyectos y programas de investigación del virus SARS-CoV2, causante del COVID-19’, FONDO - COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Inflammation. The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript. APT was supported by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (A Way to Achieve Europe program). This study has also been funded by a Research Grant 2020 from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) APT. We thank the nurse teams of the participant Clinical Services for their collaboration in sample collection.

Publisher Copyright:
© 2021 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd

ASJC Scopus Subject Areas

Biochemistry
Clinical Biochemistry

PubMed: MeSH publication types

Comparative Study
Journal Article

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10.1111/eci.13501

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Tedim, A. P., Almansa, R., Domínguez-Gil, M., González-Rivera, M., Micheloud, D., Ryan, P., Méndez, R., Blanca-López, N., Pérez-García, F., Bustamante, E., Gómez, J. M., Doncel, C., Trapiello, W., Kelvin, A. A., Booth, R., Ostadgavahi, A. T., Oneizat, R., Puertas, C., Barbé, F., ... Torres, A. (2021). Comparison of real-time and droplet digital PCR to detect and quantify SARS-CoV-2 RNA in plasma. European Journal of Clinical Investigation, 51(6), Article e13501. https://doi.org/10.1111/eci.13501

Tedim, AP, Almansa, R, Domínguez-Gil, M, González-Rivera, M, Micheloud, D, Ryan, P, Méndez, R, Blanca-López, N, Pérez-García, F, Bustamante, E, Gómez, JM, Doncel, C, Trapiello, W, Kelvin, AA, Booth, R, Ostadgavahi, AT, Oneizat, R, Puertas, C, Barbé, F, Ferrer, R, Menéndez, R, Bermejo-Martin, JF, Eiros, JM, Kelvin, DJ & Torres, A 2021, 'Comparison of real-time and droplet digital PCR to detect and quantify SARS-CoV-2 RNA in plasma', European Journal of Clinical Investigation, vol. 51, no. 6, e13501. https://doi.org/10.1111/eci.13501

@article{61564cf648ae4ffa869aa757af3af120,

title = "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{\texttrademark} 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.",

author = "Tedim, {Ana P.} and Raquel Almansa and Marta Dom{\'i}nguez-Gil and Milagros Gonz{\'a}lez-Rivera and Dariela Micheloud and Pablo Ryan and Ra{\'u}l M{\'e}ndez and Natalia Blanca-L{\'o}pez and Felipe P{\'e}rez-Garc{\'i}a and Elena Bustamante and G{\'o}mez, {Jos{\'e} Manuel} and Cristina Doncel and Wysali Trapiello and Kelvin, {Alyson A.} and Ryan Booth and Ostadgavahi, {Ali Toloue} and Ruth Oneizat and Carolina Puertas and Ferr{\'a}n Barb{\'e} and Ricard Ferrer and Rosario Men{\'e}ndez and Bermejo-Martin, {Jes{\'u}s F.} and Eiros, {Jos{\'e} Mar{\'i}a} and Kelvin, {David J.} and Antoni Torres",

note = "Funding Information: This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID‐19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li‐Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the {\textquoteleft}Subvenciones de concesi{\'o}n directa para proyectos y programas de investigaci{\'o}n del virus SARS‐CoV2, causante del COVID‐19{\textquoteright}, FONDO ‐ COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Inflammation. The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript. APT was supported by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co‐financed by the European Development Regional Fund (A Way to Achieve Europe program). This study has also been funded by a Research Grant 2020 from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) APT. Funding Information: Funding This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID-19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li-Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the {\textquoteleft}Subvenciones de concesi{\'o}n directa para proyectos y programas de investigaci{\'o}n del virus SARS-CoV2, causante del COVID-19{\textquoteright}, FONDO - COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Inflammation. The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript. APT was supported by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (A Way to Achieve Europe program). This study has also been funded by a Research Grant 2020 from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) APT. We thank the nurse teams of the participant Clinical Services for their collaboration in sample collection. Publisher Copyright: {\textcopyright} 2021 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd",

year = "2021",

month = jun,

doi = "10.1111/eci.13501",

language = "English",

volume = "51",

journal = "European Journal of Clinical Investigation",

issn = "0014-2972",

publisher = "Wiley-Blackwell",

number = "6",

}

TY - JOUR

T1 - Comparison of real-time and droplet digital PCR to detect and quantify SARS-CoV-2 RNA in plasma

AU - Tedim, Ana P.

AU - Almansa, Raquel

AU - Domínguez-Gil, Marta

AU - González-Rivera, Milagros

AU - Micheloud, Dariela

AU - Ryan, Pablo

AU - Méndez, Raúl

AU - Blanca-López, Natalia

AU - Pérez-García, Felipe

AU - Bustamante, Elena

AU - Gómez, José Manuel

AU - Doncel, Cristina

AU - Trapiello, Wysali

AU - Kelvin, Alyson A.

AU - Booth, Ryan

AU - Ostadgavahi, Ali Toloue

AU - Oneizat, Ruth

AU - Puertas, Carolina

AU - Barbé, Ferrán

AU - Ferrer, Ricard

AU - Menéndez, Rosario

AU - Bermejo-Martin, Jesús F.

AU - Eiros, José María

AU - Kelvin, David J.

AU - Torres, Antoni

N1 - Funding Information: This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID‐19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li‐Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the ‘Subvenciones de concesión directa para proyectos y programas de investigación del virus SARS‐CoV2, causante del COVID‐19’, FONDO ‐ COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Inflammation. The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript. APT was supported by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co‐financed by the European Development Regional Fund (A Way to Achieve Europe program). This study has also been funded by a Research Grant 2020 from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) APT. Funding Information: Funding This work was supported by awards from the Canadian Institutes of Health Research, the Canadian 2019 Novel Coronavirus (COVID-19) Rapid Research Funding initiative (CIHR OV2 – 170357), Research Nova Scotia (DJK), Atlantic Genome/Genome Canada (DJK), Li-Ka Shing Foundation (DJK), Dalhousie Medical Research Foundation (DJK), the ‘Subvenciones de concesión directa para proyectos y programas de investigación del virus SARS-CoV2, causante del COVID-19’, FONDO - COVID19, Instituto de Salud Carlos III (COV20/00110, CIBERES, 06/06/0028), (AT). DJK is a recipient of the Canada Research Chair in Translational Vaccinology and Inflammation. The funding sources did not play any role neither in the design of the study and collection, not in the analysis, in the interpretation of data or in writing the manuscript. APT was supported by the Sara Borrell Research Grant CD018/0123 funded by Instituto de Salud Carlos III and co-financed by the European Development Regional Fund (A Way to Achieve Europe program). This study has also been funded by a Research Grant 2020 from the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) APT. We thank the nurse teams of the participant Clinical Services for their collaboration in sample collection. Publisher Copyright: © 2021 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd

PY - 2021/6

Y1 - 2021/6

N2 - 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.

AB - 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.

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U2 - 10.1111/eci.13501

DO - 10.1111/eci.13501

M3 - Article

C2 - 33512013

AN - SCOPUS:85100583142

SN - 0014-2972

VL - 51

JO - European Journal of Clinical Investigation

JF - European Journal of Clinical Investigation

IS - 6

M1 - e13501

ER -

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

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