key: cord-0891046-u2mk6jij
authors: Bruneau, Thomas; Wack, Maxime; Poulet, Geoffroy; Robillard, Nicolas; Philippe, Aurélien; Laurent-Puig, Pierre; Bélec, Laurent; Hadjadj, Jérôme; Xiao, Wenjin; Kallberg, Julia-Linnea; Kernéis, Solen; Diehl, Jean-Luc; Terrier, Benjamin; Smadja, David; Taly, Valerie; Veyer, David; Péré, Hélène
title: Circulating ubiquitous RNA, a highly predictive and prognostic biomarker in hospitalized COVID-19 patients
date: 2021-12-11
journal: Clin Infect Dis
DOI: 10.1093/cid/ciab997
sha: 73ec3f88a2fd80ada14140d89c0c39f73fd50474
doc_id: 891046
cord_uid: u2mk6jij

BACKGROUND: Approximately 15-30% of hospitalized COVID-19 patients develop acute respiratory distress syndrome, systemic tissue injury, and/or multi-organ failure leading to death in around 45% of cases. There is a clear need for biomarkers which quantify tissue injury, predict clinical outcomes and guide the clinical management of hospitalized COVID-19 patients. METHODS: We herein report the quantification by droplet-based digital PCR (ddPCR) of the SARS-CoV-2 RNAemia and the plasmatic release of a ubiquitous human intracellular marker, the ribonuclease P (RNase P) in order to evaluate tissue injury and cell lysis in the plasma of 139 COVID-19 hospitalized patients at admission. RESULTS: We confirmed that SARS-CoV-2 RNAemia was associated with clinical severity of COVID-19 patients. In addition, we showed that plasmatic RNase P RNAemia at admission was also highly correlated with disease severity (P<0.001) and invasive mechanical ventilation status (P<0.001) but not with pulmonary severity. Altogether, these results indicate a consequent cell lysis process in severe and critical patients but not systematically due to lung cell death. Finally, the plasmatic RNase P RNA value was also significantly associated with overall survival. CONCLUSION: Viral and ubiquitous blood biomarkers monitored by ddPCR could be useful for the clinical monitoring and the management of hospitalized COVID-19 patients. Moreover, these results could pave the way for new and more personalized circulating biomarkers in COVID-19, and more generally in infectious diseases, specific from each patient organ injury profile.

Coronavirus disease 2019 (COVID-19) is a global public health problem that has already caused more than 3 million deaths worldwide. A wide spectrum of disease severity was rapidly described ranging from asymptomatic or mild diseases to respiratory failure and multiple organ dysfunction syndromes, or failure requiring intensive care management of patients and leading to a high mortality rate. In severe cases, clinical observations rapidly described a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the onset of first symptoms [1] [2] [3] . Clinical deterioration is typically dominated by worsening of respiratory symptoms, which are potentially concomitant with severe systemic organ failure, including cardiovascular, renal and/or liver injuries [4] [5] [6] [7] [8] . Evidence tended to demonstrate that the second phase of COVID-19 was associated with a cytokine storm contributing to the development of acute respiratory distress syndrome (ARDS), systemic tissue injury, and multi-organ failure observed in severe cases of COVID-19 [9] . Approximately 5% of patients infected with SARS-CoV-2 require intensive care and admission for severe lung damage [7, 10] and 15-30% of patients hospitalized with COVID-19 develop ARDS [11, 12] leading to death in around 45% of cases [13] . Therefore, biomarkers that can quantify tissue injury, analyze disease pathogenesis, predict clinical outcomes, and guide the clinical management of hospitalized COVID-19 patients are clearly needed.

In recent years, the democratization of ultra-sensitive technologies, such as droplet-based digital PCR (ddPCR), has fostered the development of circulating markers, making them suitable for several clinical applications. Recently, we and others provided evidence that highly sensitive quantification of SARS-CoV-2 RNAaemia by ddPCR in peripheral blood could be a reliable marker of disease severity and that it could be used as a potential predictive biomarker of clinical worsening in COVID-19 patient follow-up in the second A c c e p t e d M a n u s c r i p t 5 phase of COVID-19 pathology [14, 15] . Besides SARS-CoV-2 RNAaemia, plasmatic release of ubiquitous human intracellular markers could be an accurate biomarker to evaluate tissue injury and cell lysis induced by COVID-19. Thus, in addition, we monitored the plasmatic release of the intracytoplasmic ribonuclease P (RNase P), targeting its H1 RNA catalytic part [16] in order to evaluate tissue injury and cell lysis induced by COVID-19 in this global study bringing together two cohorts of comparable COVID-19 patients hospitalized for respiratory deterioration during the first wave in Paris, France.

We herein report the quantification of SARS-CoV-2 RNAemia and circulating RNase The Cox proportional hazards model was used to evaluate the risk of death at inclusion between patients with low and high plasmatic RNase P concentration.

Patients' clinical outcomes are presented using Kaplan-Meier curves.

Computations were performed using the R software, and the survival package for the Cox proportional hazards model. P values < 0.05 were considered statistically significant

Global and per-cohort demographic and clinical characteristics of the patients are shown in Table 1 . Mean age was 58 years (SD = 14), and 78% were male. Patients in both cohorts were comparable, except for patients from Cochin-Port Royal Hospital cohort who were slightly younger (mean age 54±13 vs. 62±14 years) and presented less comorbidities (hypertension 30% vs 53%, chronic renal failure 3% vs 15%) at inclusion. The degree of severity of COVID-19 was categorized as mild-to-moderate in 37 (27%) patients, severe in 35 (25%) and critical in 67 (48%).

SARS-CoV-2 RNAemia by ddPCR was significantly correlated with clinical severity (respectively at a median of 25 (101) copy/ml ; 36 (330) copy/ml in severe patients and 113 (528) copy/ml in critical patients) in hospitalized patients at admission (Figure 1A; P=0.021). Plasma SARS-CoV-2 RNAemia was also correlated with mechanical ventilation status, with a higher concentration in IMV (median of 113 (528) copy/ml) than in nonventilated COVID-19 patients (median of 36 (199) copy/ml) (Figure 1B; P=0.012) . No A c c e p t e d M a n u s c r i p t 9 correlation was found between SARS-CoV-2 RNAemia and pulmonary severity objected by CT-scan at admission (Figure 1C ; Kruskal-Wallis test, P =0.47).

Plasmatic RNase P concentration was highly correlated with clinical severity classes (Figure   2A ; P < 0.001 on log values) and the invasive mechanical ventilation status (Figure 2B ; P < 0.001), with median plasma RNase P concentration of 14345 copy/mL (IQR=27500 copy/mL) in non-IMV patients and 103482 (192500) copy/mL in IMV patients. Median plasma RNase P concentration in the control group of no disease patients (n = 18) was 3053 (1051) copy/mL. No correlation was found between RNase P RNAemia and pulmonary severity objected by CT-scan at admission (Figure 2C ; P=0.53). (Figure 3) , whereas SARS-CoV-2 RNAemia value did not predict mortality in our study (data not shown). Interestingly, the median of delay between elevated plasma RNase P RNA concentration (>4.63log copy/mL) and death was of 4 days. Intubated patients without deadly outcome despite a concentration of RNAse P superior to 4.63log cp/mL (n=31) were finally extubated. Among the 16 A c c e p t e d M a n u s c r i p t 10 moderate and severe patients with a concentration of RNAse P superior to 4.63log cp/mL, 50% of them presented a clinical deterioration, such as intensive care unit transfer during their hospitalization but with a final favorable clinical outcome.

We measured SARS-CoV-2 RNAemia and plasma RNase P RNA concentrations at admission in a cohort of 139 COVID-19 patients referred at the time of disease worsening.

SARS-CoV-2 RNAemia was detectable in most hospitalized patients. These results confirmed previous data on the correlation between viral RNAemia and clinical severity,

showing higher viral loads in severe and even more in critical patients compared to the mildto-moderate patients [14, 15, 17, 18] .

We also observed that RNase P RNA concentration, an ubiquitous and aspecific human intracellular RNA marker, was also highly correlated with disease severity and invasive mechanical ventilation status in hospitalized COVID-19 patients, indicating a consequent cell lysis process in severe and critical patients. Fisher test P=0.12; supp. Table 1 b) . Therefore, RNase P RNA alone appears to be an interesting biomarker to determine COVID19 severity. Finally, even if additional experiments are needed to confirm our data, circulating RNase P appears as a highly predictive and prognostic marker in COVID19.

Our data strongly support the use of this cell free RNA (cfRNA) quantification by ddPCR as a prognostic tool for early detection and monitoring of cell and tissue injury associated with COVID-19. Ordinarily, circulating endogenous RNA is considered to be extremely fragile and not sufficiently stable to represent a marker for monitoring, as compared to circulating DNA. However, in our study, the massive release of this endogenous marker seems to counteract the intrinsic weakness of RNA properties making it a marker of choice to quantify and monitor in parallel of the viral RNAemia the degree of cell lysis and systemic viral invasion simultaneously. Another hypothesis would be that this particular RNA, as a subunit of a protein complex, may be more protected from degradation and therefore more easily detected and quantitated than classical RNA.

Solely based on CT-scan imaging, a lytic and an inflammatory process cannot be distinguished. However, a high plasma level of RNase P RNA may more likely indicate a lytic process rather than an inflammatory one. In the case of a more frequent lytic pulmonary process in COVID-19, our data can also explain and predict the longer mean stay in ICU observed in critical COVID-19 patients (15 days) compared to critical seasonal influenza infected patients (8 days) [19] that could correlate with the highest degree of pulmonary cell lysis in COVID-19 patients and thus a longer time to recover functional lung cells.

Distinction between pulmonary lytic and inflammatory lesions could be of great interest for the clinical management of COVID-19 patients, especially in a therapeutic perspective, considering that anti-inflammatory treatments would be more efficient in the context of A c c e p t e d M a n u s c r i p t 12 inflammatory process and maybe not in the context of lytic process. Finally, the delay between elevated RNase P (>4.63 log copy/mL) and death of patients of 4 days (from 0 to 28 days) also asks the question of refining timing therapeutics early to patients with elevated RNase P at admission. Obviously, such considerations could be applied to other pulmonary infectious pathologies.

The lack of correlation between RNase P RNA concentration and pulmonary severity estimated by the percentage of lung damage on CT-scan illustrates that radiologic lesions may not systematically reflect lytic process but both lytic and inflammatory lesions. This lack of correlation could also be explained by the fact that RNase P RNA level is not lung-specific and could therefore reflect extra-pulmonary tissue lysis. Therefore, we investigated the 9 patients (6 critical; 2 severe and 1 moderate, Figure 2C ) with plasmatic RNase P concentration above 4.63 log copy/mL in the two less severe pulmonary groups (<10% and 10-25%). Very interestingly, in 6 of these 9 specific patients for whom other biological information were available, we found signal for other organ injury such as kidney, liver or heart with respectively, elevated blood creatinine, ASAT/ALAT or troponin levels. Finally, a possible hypothesis is that endogenous RNA release in plasma comes directly from infected cells lysis where SARS-CoV-2 replication occurs. However, we found 12 critical patients, with plasmatic RNase P levels above 4.63 log copy/mL but without any plasmatic SARS-CoV-2 RNA detection concurrently. In such patients, cfRNA plasmatic release could reflect the destruction of non-infected cells probably due to immunopathological mechanisms. As COVID-19 has been described as a systemic disease with multi-organ involvement, with regard to our preliminary results, we need further tissue-specific molecular markers to understand and specify the origin of observed cell lysis. Profiling organ-specific methylation markers within circulating cell-free DNA (cfDNA) to trace its origin and to quantify tissuespecific injury due to COVID-19 is possible. We aim to develop and validate such tissue-A c c e p t e d M a n u s c r i p t 13 specific biomarkers in further studies to determine the origin of hospitalized COVID-19 patients' complications. In this way, Cheng et al. recently reported a blood test to quantify cell-, tissue-, and organ-specific injury due to COVID-19 [20] . The authors assessed the utility of this test to identify subjects with severe disease and report an evidence of injury to the lung and liver and the involvement of red blood cell progenitors associated with severe COVID-19. In their study, the concentration of cfDNA correlated with the WHO ordinal scale for disease progression and was significantly increased in patients requiring intubation. M a n u s c r i p t 

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