key: cord-0706854-w1ddmozu
authors: Veyrenche, Nicolas; Pisoni, Amandine; Debiesse, Ségolène; Bollore, Karine; Bedin, Anne-Sophie; Makinson, Alain; Niel, Clémence; Alcocer-Cordellat, Carmen; Mondain, Anne-Marie; Le Moing, Vincent; Van de Perre, Philippe; Tuaillon, Edouard
title: SARS-CoV-2 nucleocapsid urine antigen in hospitalized patients with Covid-19
date: 2022-03-01
journal: J Infect Dis
DOI: 10.1093/infdis/jiac073
sha: e0777364b396db86904dd02b7e6501da4e3ecd95
doc_id: 706854
cord_uid: w1ddmozu

BACKGROUND: SARS-CoV-2 nucleocapsid antigen (N-Ag) can be detected in the blood of patients with Covid-19. We used a highly sensitive and specific assay to explore the presence of N-Ag in urine during the course of Covid-19, and explore its relationship with the severity of the disease. METHODS: We studied urine and blood N-Ag using highly sensitive immunoassay in 82 patients with a SARS-CoV-2 infection proven by PCR. RESULTS: In the first and second weeks of Covid-19, hospitalized patients tested positive for urinary N-Ag (81.25% and 71.79%, respectively), and blood N-Ag (93.75% and 94.87%, respectively). High urinary N-Ag levels were associated with the absence of SARS-CoV-2 nucleocapsid antibodies, admission in intensive care units, high C-reactive protein levels, lymphopenia, eosinopenia, and high lactate dehydrogenase. A higher accuracy was observed for urine N-Ag as a predictor of severe Covid-19 compared to blood N-Ag. CONCLUSIONS: Our study demonstrate that N-Ag is present in the urine of patients hospitalized in the early phase of Covid-19. As a direct marker of SARS-CoV-2, urinary N-Ag reflects the dissemination of viral compounds in the body. Urine N-Ag may be a useful marker for disease severity of SARS-CoV-2 infections.

The SARS-CoV-2 pandemic has upset the world and challenged medical knowledge about viral respiratory infections. One of the major characteristics of SARS-CoV-2 infection is the diversity of clinical expression, with a wide range of symptoms reported in infected persons [1] . While severe forms constitute only a small share of Covid-19 cases, they put major pressure on health systems and are responsible for the direct excess mortality associated with the pandemic.

Although knowledge on the natural history of Covid-19 has improved considerably over the last 18 months, the exact mechanisms of impairment in the innate and adaptive immune system leading to severe forms of Covid-19 remain to be elucidated. According to our current understanding of the pathophysiology, different phases of virus and host interactions characterize SARS-CoV-2 infection [2] [3] . Following the incubation period, a high viral replication triggers the innate immune response. A strong inflammatory syndrome and the onset of the adaptive immune response characterize the second phase of Covid-19 before recovery or worsening. Severe forms of Covid-19 are associated with an excessive release of cytokines, known as a "cytokine storm", occurring generally during the second phase of the disease [4] . The administration of corticosteroids reduces death and time to recovery [5, 6] , and interleukin-6 antagonist Tocilizumab also have obtained encouraging results [7] [8] [9] .

On the virus side of the host-virus interplay, the contribution to disease severity of a high SARS-CoV-2 load measured in respiratory samples remains uncertain. The kinetics of SARS-CoV-2 RNA in the upper respiratory tract during the course of Covid-19 is well established, with high concentrations observed during the initial phase, followed by a rapid decrease in the second week after the onset of symptoms, and a low or undetectable level of RNA later [7] . The viral loads of asymptomatic or mild forms of SARS-CoV-2 infections are similar to severe forms [10] . Studies suggest that SARS-CoV-2 RNA decreases faster in mild/asymptomatic infections and in young subjects compared to severe forms and elderly subjects [11, 12] . The presence of SARS-CoV-2 components also has been reported outside the respiratory tract, such as in blood, stool and saliva. When detected, the plasma SARS-CoV-2 RNA level is generally low. The detection of SARS-CoV-2 is also found in extrapulmonary organs of immunocompromised patients, including heart, kidney, liver, and spleen [13] . However, no case of SARS-CoV-2 transmission via transfusion has been described [14] . On the other hand, the presence of the SARS-CoV-2 nucleocapsid has been reported in the blood of Covid-A c c e p t e d M a n u s c r i p t 4 19 patients. Circulating antigen (Ag) is detectable in almost all hospitalized patients in the early phase of the disease [15, 16] . A high level of nucleocapsid antigenemia (N-Ag) could be associated with severe forms of Covid-19 and the presence of circulating SARS-CoV-2 RNA [17, 18] .

In this study, we used a highly sensitive and specific nucleocapsid-Ag assay to: i) explore the presence of N-Ag in the urine of hospitalized patients, ii) study the kinetics of N-Ag concentration in urine and blood during infection, iii) assess the relationship between N-Ag concentrations in urine and blood, and iv) compare urine and blood N-Ag levels in moderate versus severe forms of Covid-19.

Design of the study. Plasma, urine and nasopharyngeal samples were collected from 82 SARS- [19] .

According to manufacturer's instructions, a signal ratio « Sample/Positive control% » (S/P%) ≥ 40% was considered positive, 30% < S/P% < 40% was considered suspicious and S/P% ≤ 30 was considered negative.

A c c e p t e d M a n u s c r i p t 5 N-antigen detection. N-SARS-CoV-2 antigen levels in urine and plasma were determined with a CE-IVD ELISA microplate assay, COV-QUANTO® (AAZ-LMB, Boulogne-Billancourt, France). The cut-off value was defined by the manufacturer's instructions: samples with antigen N concentration ≥ 2.97 pg/mL were considered positive. Assay reproducibility was established by the manufacturer using three batches: CV is 9.01% at 6.49pg/mL, and 4.8% at 168.02 pg/mL. M a n u s c r i p t 7

Levels of urine and plasma N-Ag were analyzed according to abnormalities in biological markers associated with Covid-19 severity (Figure 6, supplementary Figure 3 ). Higher urine and blood N-Ag levels were measured among patients with a C-reactive protein (CRP) over 100 mg/L. The differences remained significant when N-Ag levels in urine and blood were analyzed according to N-IgG serological status. Higher urine and blood N-Ag levels also were measured among patients with lymphopenia and low eosinophil counts. Urine N-Ag levels were higher when the Lactate dehydrogenase (LDH) level was elevated. We did not observe an association between N-Ag levels and low platelet counts (<200/µL), abnormal troponin levels (>60 mg/mL), high D-dimer levels (> 1200 mg/mL), or the Glomerular Filtration Rate (GFR) based on the Modification of Diet in Renal Disease (MDRD) study equation (< 60 ml/min/1.73m2). Urine N-Ag levels were lower in patients with elevated alanine aminotransferase (ALT) concentrations.

In this study, we assess N-Ag in urine of patients hospitalized for a Covid-19 infection confirmed by PCR. Our results demonstrate that N-Ag is present in urine of patients hospitalized for a SARS-CoV-2 infection. Urinary N-Ag concentrations decreased progressively after the onset of symptoms following a low decay during the first and second weeks, and a sharp decrease during the third week. The presence of circulating antibodies against nucleocapsid was associated with a lower level of urine N-Ag, but the SARS-CoV-2 nucleocapsid remained detectable in the urine samples of most of the patients seropositive for N-IgG.

Covid-19 severity since high concentrations of virus in nasopharyngeal and saliva specimens are observed in asymptomatic, mild and severe forms of SARS-CoV-2 infections [6] . By contrast, blood SARS-CoV-2 RNA is more frequently detectable and found at higher levels in severe forms of Covid-19 [8, 9] . SARS-CoV-2 viremia is associated with disease severity, patient outcome and inflammatory biomarkers [20] . Systemic clinical manifestations suggest that SARS-CoV-2 also can infect different organs through the bloodstream, such as endothelial cells [21] , gastrointestinal cells and angiotensin A c c e p t e d M a n u s c r i p t 8 converting enzyme 2 receptor (ACE2) positive distal tubule cells [22] . Furthermore, the administration of convalescent plasma therapy and monoclonal antibodies (mAb) against the Spike protein help improve Covid-19 recovery, although studies shown conflicting results [23] [24] [25] [26] [27] [28] . These observations suggest that SARS-CoV-2 replication and plasma viremia may contribute to the severity of Covid-19.

The first reports on Covid-19 infrequently detected circulating SARS-CoV-2 RNA [29] . Recent studies have inconsistently detected SARS-CoV-2 RNA, and generally with a low viral load [30, 31] . In contrast, in the study of Le Hingrat et al., N-antigenemia appeared to be a sensitive marker of SARS-CoV-2 infection in hospitalized patients, able to provide a surrogate test to molecular approaches [15] .

Dandan S. et al. confirmed this observation using a digital enzyme-linked immunosorbent method [17] . In line with these studies, we observed that the N-Ag levels in our population of hospitalized patients most of the time were over 100 pg/mL during the first week after the onset of symptoms, and remained largely over the lower limit of quantification of the assay during the second week. Given the much better analytical sensitivity of PCR methods compared to antigen immunoassays, these findings are surprising. SARS-CoV-2 nucleocapsid antigen may be released in the bloodstream or circulate after destruction of the virus particle or produced in excess.

Detection of SARS-CoV-2 RNA in urine specimens has been reported in less than 5% of confirmed Covid-19 cases [32] . Using mass spectrometry, Mishra C et al. have reported detection of nucleocapsid-derived peptides in urine of a third of Covid-19 patients [33] . Our results using a highly sensitive immunoassay demonstrate the presence of N-antigen in urine in most of Covid-19 hospitalized patients during the first two weeks after onset of the symptoms. The kidney is among the most frequently affected extrapulmonary organs during SARS-CoV-2 infection, and varying degrees of renal damage have been reported in Covid-19 patients [34, 35] . Acute kidney disease was observed in a quarter of the patients included in our study. We did not observed any association between urine N-Ag levels and altered renal function. N-Ag in urine may originate from the blood and be excreted by the kidney, as suggested by the correlation between N-Ag concentrations in blood and urine. The SARS-CoV-2 nucleocapsid is a 46 kd protein. The glomerular permeability and filtration probably permit the excretion of this small-size protein. However, after seroconversion against the nucleocapsid, immune complexes also form, with a size that makes them unable to be filtered. Of note, although Covid-19 associated glomerular disease has been reported, this type of kidney injury A c c e p t e d M a n u s c r i p t 9 seems infrequent among acute kidney diseases associated to SARS-CoV-2 infection [36] . A local production of SARS-CoV-2 nucleocapsid may be another possible origin of the antigen detected in urine. Alongside hypoxia, circulating disorder and inflammation, SARS-CoV-2 infection may directly contribute to kidney injury. Cells expressing ACE2 are present in the tubules, and studies have shown that SARS-CoV-2 RNA, nucleocapsid and spike protein accumulate in tubules [22, 37] .

Both blood and urine N-Ag levels may reflect SARS-CoV-2 disseminated infection. We observed a moderate correlation between N-Ag in blood and urine, perhaps better if the couple of blood and urine were collected in the same time but the kinetic of this marker may be different in these two compartments [13] . The development of anti-nucleocapsid humoral response may induce the formation of immune complexes that interfere with N-Ag quantification in blood. Hence, after seroconversion N-Ag and N-IgG levels may represent only the unbound fraction available to be measured by the immunoassays. In urine, antibodies directed against nucleocapsid were only detected in one quarter of the patients who tested positive for circulating N-IgG, limiting the risk of underestimation of N-Ag levels by the immunoassay. In other words, N-Ag levels in urine may be more accurate since nucleocapsid Ag quantitation in urine is probably less impacted by the formation of immune Ag-Abs complexes compared to blood. N-Ag in urine may better reflect disseminated infection than nucleocapsid antigenemia, especially after seroconversion against SARS-CoV-2 nucleoprotein. Besides interfering with assay measurement, the presence of circulating antibodyantigen complexes may bind FC receptors activating monocytes/macrophages, and fuel the hyper inflammation observed in the second phase of Covid-19 [38] .

Risk factors related to age and comorbidities, alongside inflammation and cytopenia, are associated with the development of severe forms of Covid-19 requiring hospitalisation and intensive care. At present, however, the progression to a severe form of Covid-19 remains unpredictable. We observed higher concentrations of urine N-Ag in samples collected in patients hospitalised in ICU compared to medical wards. This result is in line with the study of Caceres P. et al., reporting that SARS-CoV-2 viral load in urine sediments was associated with higher mortality in hospitalized patients [6] .

Furthermore, in our study, urine and plasma N-Ag levels were associated with several early markers of Covid-19 severity, such as lymphopenia, low eosinophil count, and CRP levels. N-Ag level should be evaluated in a combination of markers to predict the risk of severe form of Covid-19. We observed lower levels of urine N-Ag in patients with abnormal ALT, which may be because liver injury is A c c e p t e d M a n u s c r i p t 10 delayed after the first week in the course of prolonged Covid-19 while the decay of N-Ag is already underway [39] .

Our study has several limitations. The population is not representative of SARS-CoV-2 infected individuals. All of the subjects had moderate or severe forms of Covid-19 and required oxygen, whereas a majority of SARS-CoV-2 infected individuals do not require hospitalization. In addition, patients requiring critical care are over-represented because their urine samples were frequently collected in routine care. We did not assess the value of N-Ag as a predictive marker of adverse evolution but only as a marker associated to severe Covid-19. Finally, N-Ag levels were analysed on a single urine sample while results on urine samples collected taken over a 24-hour period would be more accurate.

In conclusion, these results demonstrate that N-Ag is present in urine of patients hospitalized for 

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M a n u s c r i p t 11

The author would like to thank Grace Delobel for English language editing and review services.

This work was funded by the Montpellier University Hospital, Muse I-SITE Program Grant, University of Montpellier.

The authors declare that there are no conflict of interests or personal relationships that could have appeared to influence the work reported in this paper.