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

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). 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. 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). 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.

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 ... -  《-》

Comparison of Different Reverse Transcriptase-Polymerase Chain Reaction-Based Methods for Wastewater Surveillance of SARS-CoV-2: Exploratory Study.

Länsivaara A ，Lehto KM ，Hyder R ，Janhonen ES ，Lipponen A ，Heikinheimo A ，Pitkänen T ，Oikarinen S ，WastPan Study Group ... -  《JMIR Public Health and Surveillance》

Analytical and Clinical Performance of Droplet Digital PCR in the Detection and Quantification of SARS-CoV-2.

Since the initial coronavirus disease outbreak in late 2019 (COVID-19), reverse-transcription real-time polymerase chain reaction (RT-qPCR) has become the gold standard test to detect severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). However, a more sensitive and accurate diagnostic tool was required. Therefore, droplet digital polymerase chain reaction (ddPCR) was suggested as an alternative method. Here, we evaluated the performance of ddPCR to detect SARS-CoV-2 and compared it to the performance of RT-qPCR. The analytical performances, including limit of blank and limit of detection, were established using positive and negative SARS-CoV-2 reference materials. A total of 366 RNA extracts (173 positive and 193 negative by RT-qPCR) were collected from four institutions and tested with a Bio-Rad SARS-CoV-2 ddPCR kit that detects the SARS-CoV-2 genome using primers for N1 and N2. Limit of blank was set at 0, and the limits of detection of N1 and N2 were 1.99 copies/μL and 5.18 copies/μL, respectively. Linearity was evaluated using serial dilution samples, which demonstrated good results (R2: 0.999, linear range: 5.88-6825.25 copies/μL for N1 and R2: 0.999, 5.53-5855.47 copies/μL for N2). The results of ddPCR and RT-qPCR revealed substantial agreement (Cohen's kappa: 0.639, p < 0.01). The 63 samples with positive ddPCR but negative RT-qPCR showed low copy numbers, and 55% of them had COVID-19-related symptoms. Droplet digital polymerase chain reaction demonstrated excellent sensitivity for SARS-Cov-2 detection and consistently agreed with the results from conventional RT-qPCR. Furthermore, ddPCR provided quantitative data that can be used to monitor changes in the viral load of patients with COVID-19.

Kim KB ，Choi H ，Lee GD ，Lee J ，Lee S ，Kim Y ，Cho SY ，Lee DG ，Kim M ... -  《-》

Viral RNA load in plasma is associated with critical illness and a dysregulated host response in COVID-19.

Bermejo-Martin JF ，González-Rivera M ，Almansa R ，Micheloud D ，Tedim AP ，Domínguez-Gil M ，Resino S ，Martín-Fernández M ，Ryan Murua P ，Pérez-García F ，Tamayo L ，Lopez-Izquierdo R ，Bustamante E ，Aldecoa C ，Gómez JM ，Rico-Feijoo J ，Orduña A ，Méndez R ，Fernández Natal I ，Megías G ，González-Estecha M ，Carriedo D ，Doncel C ，Jorge N ，Ortega A ，de la Fuente A ，Del Campo F ，Fernández-Ratero JA ，Trapiello W ，González-Jiménez P ，Ruiz G ，Kelvin AA ，Ostadgavahi AT ，Oneizat R ，Ruiz LM ，Miguéns I ，Gargallo E ，Muñoz I ，Pelegrin S ，Martín S ，García Olivares P ，Cedeño JA ，Ruiz Albi T ，Puertas C ，Berezo JÁ ，Renedo G ，Herrán R ，Bustamante-Munguira J ，Enríquez P ，Cicuendez R ，Blanco J ，Abadia J ，Gómez Barquero J ，Mamolar N ，Blanca-López N ，Valdivia LJ ，Fernández Caso B ，Mantecón MÁ ，Motos A ，Fernandez-Barat L ，Ferrer R ，Barbé F ，Torres A ，Menéndez R ，Eiros JM ，Kelvin DJ ... -  《CRITICAL CARE》

Evaluation of diagnostic performance of SARS-CoV-2 infection using digital droplet polymerase chain reaction in individuals with or without COVID-19 symptoms.

Kim Y ，Lee E ，Kim B ，Cho J ，Ryu SW ，Lee KA ... -  《-》