key: cord-0746258-rp2apmmg
authors: Veyer, David; Kernéis, Solen; Poulet, Geoffroy; Wack, Maxime; Robillard, Nicolas; Taly, Valérie; L’Honneur, Anne-Sophie; Rozenberg, Flore; Laurent-Puig, Pierre; Bélec, Laurent; Hadjadj, Jérôme; Terrier, Benjamin; Péré, Hélène
title: Highly sensitive quantification of plasma SARS-CoV-2 RNA shelds light on its potential clinical value
date: 2020-08-17
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1196
sha: d651e854a520e4bb29a60ab7b9f9324c0bf59d2d
doc_id: 746258
cord_uid: rp2apmmg

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a global public health problem that has already caused more than 662,000 deaths worldwide. Although the clinical manifestations of COVID-19 are dominated by respiratory symptoms, some patients present other severe damage such as cardiovascular, renal and liver injury or/and multiple organ failure, suggesting a spread of the SARS-CoV-2 in blood. Recent ultrasensitive polymerase chain reaction (PCR) technology now allows absolute quantification of nucleic acids in plasma. We herein intended to use the droplet-based digital PCR technology to obtain sensitive detection and precise quantification of plasma SARS-CoV-2 viral load (SARS-CoV-2 RNAaemia) in hospitalized COVID-19 patients. METHODS: Fifty-eight consecutive COVID-19 patients with pneumonia 8 to 12 days after onset of symptoms and 12 healthy controls were analyzed. Disease severity was categorized as mild-to-moderate in 17 patients, severe in 16 patients and critical in 26 patients. Plasma SARS-CoV-2 RNAaemia was quantified by droplet digital Crystal Digital PCR™ next-generation technology (Stilla Technologies, Villejuif, France). RESULTS: Overall, SARS-CoV-2 RNAaemia was detected in 43 (74.1%) patients. Prevalence of positive SARS-CoV-2 RNAaemia correlated with disease severity, ranging from 53% in mild-to-moderate patients to 88% in critically ill patients (p=0.036). Levels of SARS-CoV-2 RNAaemia were associated with severity (p=0.035). Among nine patients who experienced clinical deterioration during follow-up, eight had positive SARS-CoV-2 RNAaemia at baseline while only one critical patient with undetectable SARS-CoV-2 RNAaemia at the time of analysis died at day 27. CONCLUSION: SARS-CoV-2 RNAaemia measured by droplet-based digital PCR constitutes a promising prognosis biomarker in COVID-19 patients

Coronavirus disease 2019 (COVID-19) 1 is a global public health problem that has already caused more than 662,000 deaths worldwide. Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) 2 , responsible for the development of COVID-19, was first isolated and sequenced in early January 2020 3 . Although most patients present mild-to-moderate disease, 5 to 10% progress to severe or critical disease 4 , including pneumonia and acute respiratory failure. Based on data from patients with laboratory-confirmed COVID-19 from mainland China, admission to intensive care unit (ICU), invasive mechanical ventilation or death occurred in 5.0%, 2.3% and 1.4% of cases, respectively 1 . In severe cases, clinical observations typically describe a two-step disease progression, starting with a mild-tomoderate presentation followed by a secondary respiratory worsening 9 to 12 days after onset of first symptoms [4] [5] [6] . Clinical deterioration is dominated by worsening of respiratory symptoms, potentially concomitant with severe systemic damage including cardiovascular, renal and liver injury and/or multiple organ failure 4, 7 . In addition, coagulopathy has been reported in severe COVID-19 cases 8, 9 . These systemic clinical features strongly suggest the spread of SARS-CoV-2 within extra-pulmonary sites via the blood flow.

Detection of circulating SARS-CoV-2 RNA by conventional real-time reverse transcriptionpolymerase chain reaction assay (RT-PCR) which is used for routine SARS-CoV-2 molecular diagnosis from respiratory tract samples was previously reported in COVID-19 patients in few studies 4, 10, 11 . As there is no certainty as whether this blood RNA belongs to entire infectious viral particles or come from infected-cell lysis, "SARS-CoV-2 RNAaemia" was preferred to "viraemia" when referring to this circulating SARS-CoV-2 RNA. In their retrospective series of 57 COVID-19 patients, Chen W. et al. found only 6 patients with positive SARS-CoV-2 RNAaemia by RT-PCR and suggested an association between A c c e p t e d M a n u s c r i p t 5 detectable serum SARS-CoV-2 RNA with disease severity 11 . However, conventional RT-PCR for SARS-CoV-2 RNA may lack sensitivity in patients showing low viremia.

Furthermore, in the absence of adequate quantitative standards availability, RT-PCR does not allow precise quantification of low SARS-CoV-2 viral loads. Ultrasensitive PCR technology is now available for absolute quantification of nucleic acids in plasma, such as droplet-based digital PCR (ddPCR), a more sensitive technique for virus detection than RT-PCR 12-14 . ddPCR was primarily developed for cancer research to detect and quantify mutated circulating tumoral DNA among cell-free DNA in liquid biopsies by optimizing sensitivity and absolute quantification compared with classical q(RT-)PCR. This innovative method is based on the realization of thousands of single-molecule PCRs in parallel in independent compartment, here droplets of an induced emulsion with nucleic acid extracts. Therefore, every single PCR is performed separately and consequently avoids the bias seen in conventional PCR. Every single droplet containing targeted DNA or RNA will become fluorescent after (RT-)PCR and is detected as a positive droplet by laser excitation. In that way, droplet-based digital PCR allows the generation of quantitative and accurate data without standard curves. As the raw data fits the Poisson distribution, it allows absolute quantification of the amplified target. Besides higher sensitivity over qPCR, ddPCR also presents many advantages for the analysis of biological samples that are complex in nature.

Indeed, performing every single PCR in an isolated droplet provides higher resistance to a variety of inhibitors that could be contained within biological samples and consequently significantly increases reproducibility of the obtained data and comparability of the results between different laboratories 15 . The increased tolerance of ddPCR to PCR inhibitory molecules makes it an attractive alternative to qPCR for medical applications including viral diagnostics 16 . Recently, ddPCR was used to quantify SARS-CoV-2 RNA in lower respiratory A c c e p t e d M a n u s c r i p t 6 tract samples of COVID-19 patients, and showed higher sensitivity than RT-PCR, especially with low viral loads 17 .

In the present study, ddPCR was used to precisely quantify circulating SARS-CoV-2 RNA in the blood from COVID-19 patients hospitalized with different clinical severity.

COVID-19 patients admitted to the Cochin Hôpital, Paris, France, at the time of respiratory deterioration and between days 8 and 12, were included between March 19, 2020 and April 3, 2020, in the setting of the local RADIPEM biological samples collection derived from samples collected in routine care. The present work focused on SARS-CoV-2 RNAaemia is part of another study primarily designed to explore innate inflammatory responses in hospitalized COVID-19 patients 18 , and used the same inclusion criteria. Our study is an ancillary study focused on circulating SARS-CoV-2 RNA from another more global study regarding Inclusion criteria for COVID-19 inpatients were: age between 18 A c c e p t e d M a n u s c r i p t 7 days after disease onset was related to early Chinese clinical observations reporting a twostep disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening few days after onset of first symptoms in severe cases. The mean interval between the onset of illness and the admission to ICU was of 9-12 days in these early reports [4] [5] [6] 18 .

Healthy controls were RT-PCR SARS-CoV-2 negative asymptomatic adults, matched with cases on age and sex.

The clinical severity of COVID-19 was described according to the adaptation of the Sixth Computations were performed using the R software, and the ordinal package for the ordinal regression model. P values < 0.05 were considered significant.

Role of the funding source. The funder of the study had no role in study design, data collection, data analysis, data interpretation, or report writing. The corresponding author had full access to all the data in the study and shared with all co-authors the final decision to submit the paper for publication.

A c c e p t e d M a n u s c r i p t 10

Fifty-eight COVID-19 patients and 12 healthy controls were analyzed. Demographic and clinical characteristics of the patients are shown in Table 1 Proportion of positive SARS-CoV-2 RNAaemia was significantly different between patients from different clinical classes, from 53% in mild-to-moderate patients to 88% in critically ill patients (p=0.036) ( Table 1) .

Overall, SARS-CoV-2 RNAaemia were associated with COVID-19 severity (p=0.035, Kruskal-Wallis), as depicted in Figure 1 , with median SARS-CoV-2 RNAaemia of 89, 279

and 301 cp/mL, in mild-to-moderate, severe and critically ill patient groups, respectively.

Raw plasma SARS-CoV-2 RNA value for each patient in the three different clinical classes were detailed in supplementary table 1.

In multivariate analysis adjusted on age, sex and comorbidities, SARS-CoV-2 RNAaemia was strongly associated with the clinical class (adjusted hazard ratio 2.2, 95% CI 1.39-3.9;

p=0.0018) ( Table 2) .

All healthy controls showed negative SARS-CoV-2 RNAaemia with no detection of both specific SARS-CoV-2 N and ORF-1 genes.

A c c e p t e d M a n u s c r i p t 12

Clinical deterioration was observed in nine COVID-19 patients during follow-up:

introduction of high-flow nasal oxygen therapy (n=3), introduction of mechanical ventilation (n=3), or death (n=3).

Eight of the nine COVID-19 patients with clinical deterioration had positive SARS-CoV-2

RNAaemia at baseline, while only one critical patient with undetectable SARS-CoV-2

RNAaemia at the time of analysis died at day 27 (Figure 2) . Of note, the patient with the highest SARS-CoV-2 RNAaemia (65,476 cp/ml) died from COVID-19 one day after plasma sampling.

In the present study, SARS-CoV- within biological samples 15 . This increased tolerance of ddPCR to PCR inhibitory molecules makes it an attractive alternative to quantitative PCR or RT-PCR for medical applications including viral diagnostics 16 and cancer research 13 . Finally, digital RT-PCR was described as presenting high potentiality over conventional real time RT-qPCR 21 . In the present study, SARS-CoV-2 RNAaemia assessed by ddPCR was detected in roughly 75% of hospitalized COVID-19 patients, and ddPCR was highly specific for the detection of SARS-COV-2

RNAaemia. Compared to recent studies reporting positive blood SARS-CoV-2 viral load by RT-PCR in less than a half of hospitalized patients for COVID-19 11, 22 , our results indicate that ddPCR is more efficient and more precise to detect and quantify SARS-CoV-2

RNAaemia as compared to classical RT-PCR.

In one previous study, SARS-CoV-2 RNAaemia indirectly assessed with the cycle threshold of RT-PCR was correlated with the COVID-19 severity 11 . We show here that precise quantification of SARS-CoV-2 RNAaemia by ddPCR confirms these previous observations. In our study, detectable SARS-CoV-2 RNAaemia was also more frequently 

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We thank the patients, the nurses and clinical staff who are providing care for the patients. We would like to acknowledge and thank Stilla Technologies for their prompt support with this project. 

A c c e p t e d M a n u s c r i p t 17 Myers Squibb, AstraZeneca, GlaxoSmithKline, Vifor Pharma, Terumo BCT, and Lilly, outside the submitted work. V.T. reports being the cofounder of Emulseo, and receiving grants from Biomnis, outside submitted work. In addition, V.T. reports patent EP20305571.