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

Tedim, Ana P.; Almansa, Raquel; Domínguez‐Gil, Marta; González-Rivera, Milagros; Micheloud, Dariela; Ryan, Pablo; Méndez, Raúl; Blanca‐López, Natalia; Pérez-García, Felipe; Bustamante, Elena; Gómez, José Manuel Naranjo; Doncel, Cristina; Trapiello, Wysali; Kelvin, Alyson A.; Booth, Ryan; Ostadgavahi, Ali Toloue; Oneizat, Ruth; Puertas, Carolina; Barbé, Ferrán; Ferrer, Ricard; Menéndez, Rosário; Bermejo-Martín, Jesús F.; Eirós, José María; Kelvin, David J.; Torres, Antoni

doi:10.1111/eci.13501

Cited by 21 publications

(20 citation statements)

References 14 publications

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“…Our finding that measurement using qPCR and ddPCR are highly correlated is consistent with other studies in nasopharyngeal swabs (Falzone et al 2020, Liu et al 2020), plasma (Tedim et al 2021), wastewater from aircraft and cruise ships (Ahmed et al 2020), and raw influent (Cieselski et al 2021). However, we observed that the correlation became weaker at the lower end of the concentration range we tested, spreading out at lower concentrations in a “broom-shaped” pattern consistent with studies comparing measurements of bacterial targets (Cao et al 2015).…”

Section: Discussionsupporting

confidence: 91%

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

Steele

Zimmer‐Faust

Griffith

et al. 2021

Preprint

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The rapid emergence of wastewater based surveillance has led to a wide array of SARS-CoV-2 RNA quantification methodologies being employed. Here we compare methods to store samples, inactivate viruses, capture/concentrate viruses, and extract/measure viral RNA from primary influent into wastewater facilities. We found that heat inactivation of the viruses led to a 1-3 log10 decrease compared to chemical inactivation. Freezing influent prior to concentration caused a 1-4 log10 decrease compared to processing fresh samples, but viral capture by membrane adsorption prior to freezing was robust to freeze-thaw variability. Concentration vs. direct extraction, and PCR platform also affected outcome, but by a smaller amount. The choice of nucleocapsid gene target had nearly no effect. Pepper mild-mottle virus was much less sensitive to these methodological differences than was SARS-CoV-2, which challenges its use as a population-level control among studies using different methods. Better characterizing the variability associated with different methodologies, in particular the impact of methods on sensitivity, will aid decision makers in following the effects of vaccination campaigns, early detection of future outbreaks, and potentially monitoring the appearance of SARS-CoV-2 variants in the population.

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Section: Discussionsupporting

confidence: 91%

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

Steele

Zimmer‐Faust

Griffith

et al. 2021

Preprint

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“…In recent years, digital PCR (dPCR) has emerged as an attractive alternative for environmental applications as it offers absolute quantification without the need for a standard curve, and it may be more sensitive and less prone to inhibition (Cao et al, 2015). As applied to the detection of SARS-CoV-2, RT-dPCR has been reported to be superior to RT-qPCR for clinical specimens, including nasopharyngeal samples (Falzone et al, 2020;Liu et al, 2020;Suo et al, 2020) and plasma from infected patients (Tedim et al, 2021), as it was found to be more sensitive and accurate.…”

Section: Pcr Platformsmentioning

confidence: 99%

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Ahmed¹,

Simpson²,

Bertsch³

et al. 2021

Preprint

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Wastewater surveillance for pathogens using the reverse transcription-polymerase chain reaction (RT-PCR) is an effective, resource-efficient tool for gathering additional community-level public health information, including the incidence and/or prevalence and trends of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater may provide an early-warning signal of COVID-19 infections in a community. The capacity of the world’s environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is rapidly increasing. However, there are no standardized protocols nor harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can lead to false-positive and -negative errors in the surveillance of SARS-CoV-2, culminating in recommendations and strategies that can be implemented to identify and mitigate these errors. Recommendations include, stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly during a low incidence of SARS-CoV-2 in wastewater. Corrective and confirmatory actions must be in place for inconclusive and/or potentially significant results (e.g., initial onset or reemergence of COVID-19 in a community). It will also be prudent to perform inter-laboratory comparisons to ensure results are reliable and interpretable for ongoing and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance was demonstrated during this global crisis. In the future, wastewater will play an important role in the surveillance of a range of other communicable diseases.

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“…An overall of 3651 samples, from 2825 patients and 145 controls, were extracted from the 39 included studies. The studies were conducted worldwide; 16 in Asia [ 12 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ], 14 in Europe [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ], and 9 in North America [ 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ]. A minority of studies (8) included control samples [ 21 , 28 , 29 , 33 , 38 , 47 , 54 , 56 ].…”

Section: Resultsmentioning

confidence: 99%

“…Although most eligible studies of this systematic review evaluated the diagnostic performance of ddPCR compared to RT-qPCR, we also extracted data from 10 studies assessing the prognostic performance of ddPCR and their main findings are summarized in Table 2 . Four of them showed that ddPCR can accurately quantify SARS-CoV-2 RNA levels, especially in immunocompromised patients and hospitalized patients with severe COVID-19 infections [ 23 , 37 , 46 , 49 ]. Furthermore, a study conducted by Ram-Mohan et al showed that ddPCR, compared to traditional RT-qPCR, was superior for detecting and quantifying SARS-CoV-2 RNAemia in hospitalized patients over a course of 30 days [ 52 ].…”

Section: Resultsmentioning

confidence: 99%

“…One study reported elevated SARS-CoV-2 RNA concentration in plasma was associated with severe illness in COVID-19 patients [ 49 ]. Furthermore, a higher viral load in the plasma was associated with increased mortality or ICU admission [ 46 , 49 ]. ddPCR was also accurate in detecting and quantifying SARS-CoV-2 RNA plasma levels in immunocompromised and ICU patients, a finding with potential clinical significance [ 23 , 37 , 46 , 49 ].…”

Section: Discussionmentioning

confidence: 99%

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Diagnostic, Prognostic, and Therapeutic Value of Droplet Digital PCR (ddPCR) in COVID-19 Patients: A Systematic Review

Ishak

AlRawashdeh

Esagian

et al. 2021

JCM

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Accurate detection of SARS-CoV-2, the pathogen causing the global pandemic of COVID-19, is essential for disease surveillance and control. Quantitative reverse transcription PCR (RT-qPCR) is considered the reference standard test for the diagnosis of SARS-CoV-2 by the World Health Organization and Centers for Disease Control and Prevention. However, its limitations are a prompt for a more accurate assay to detect SARS-CoV-2, quantify its levels, and assess the prognosis. This article aimed to systematically review the literature and assess the diagnostic performance of droplet digital PCR (ddPCR), also to evaluate its potential role in prognosis and management of COVID-19 patients. PubMed and Scopus databases were searched to identify relevant articles published until 13 July 2021. An additional PubMed search was performed on 21 October 2021. Data from the 39 eligible studies were extracted and an overall 3651 samples from 2825 patients and 145 controls were used for our qualitative analysis. Most studies reported ddPCR was more accurate than RT-qPCR in detecting and quantifying SARS-CoV-2 levels, especially in patients with low viral loads. ddPCR was also found highly effective in quantifying SARS-CoV-2 RNAemia levels in hospitalized patients, monitoring their disease course, and predicting their response to therapy. These findings suggest ddPCR could serve as a complement or alternative SARS-CoV-2 tool with emerging diagnostic, prognostic, and therapeutic value, especially in hospital settings. Additional research is still needed to standardize its laboratory protocols, also to accurately assess its role in monitoring COVID-19 therapy response and in identifying SARS-CoV-2 emerging variants.

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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).

Cited by 21 publications

References 14 publications

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

Sources of variability in methods for processing, storing, and concentrating SARS-CoV-2 in influent from urban wastewater treatment plants

Minimizing Errors in RT-PCR Detection and Quantification of SARS-CoV-2 RNA for Wastewater Surveillance

Diagnostic, Prognostic, and Therapeutic Value of Droplet Digital PCR (ddPCR) in COVID-19 Patients: A Systematic Review

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