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SARS‐CoV‐2 N‐antigenemia in critically ill adult COVID‐19 patients: Frequency and association with inflammatory and tissue‐damage biomarkers

Abstract The current study aimed at characterizing the dynamics of SARS‐CoV‐2 nucleocapsid (N) antigenemia in a cohort of critically ill adult COVID‐19 patients and assessing its potential association with plasma levels of biomarkers of clinical severity and mortality. Seventy‐three consecutive critically ill COVID‐19 patients (median age, 65 years) were recruited. Serial plasma (n = 340) specimens were collected. A lateral flow immunochromatography assay and reverse‐transcription polymerase chain reaction (RT‐PCR) were used for SARS‐CoV‐2 N protein detection and RNA quantitation and in plasma, respectively. Serum levels of inflammatory and tissue‐damage biomarkers in paired specimens were measured. SARS‐CoV‐RNA N‐antigenemia and viral RNAemia were documented in 40.1% and 35.6% of patients, respectively at a median of 9 days since symptoms onset. The level of agreement between the qualitative results returned by the N‐antigenemia assay and plasma RT‐PCR was moderate (k = 0.57; p < 0.0001). A trend towards higher SARS‐CoV‐2 RNA loads was seen in plasma specimens testing positive for N‐antigenemia assay than in those yielding negative results (p = 0.083). SARS‐CoV‐2 RNA load in tracheal aspirates was significantly higher (p < 0.001) in the presence of concomitant N‐antigenemia than in its absence. Significantly higher serum levels of ferritin, lactose dehydrogenase, C‐reactive protein, and D‐dimer were quantified in paired plasma SARS‐CoV‐2 N‐positive specimens than in those testing negative. Occurrence of SARS‐CoV‐2 N‐antigenemia was not associated with increased mortality in univariate logistic regression analysis (odds ratio, 1.29; 95% confidence interval, 0.49‐3.34; p = 0.59). In conclusion, SARS‐CoV‐2 N‐antigenemia detection is relatively common in ICU patients and appears to associate with increased serum levels of inflammation and tissue‐damage markers. Whether this virological parameter may behave as a biomarker of poor clinical outcome awaits further investigations.

since symptoms onset. The level of agreement between the qualitative results returned by the N-antigenemia assay and plasma RT-PCR was moderate (k = 0.57; p < 0.0001).
A trend towards higher SARS-CoV-2 RNA loads was seen in plasma specimens testing positive for N-antigenemia assay than in those yielding negative results (p = 0.083).
SARS-CoV-2 RNA load in tracheal aspirates was significantly higher (p < 0.001) in the presence of concomitant N-antigenemia than in its absence. Significantly higher serum levels of ferritin, lactose dehydrogenase, C-reactive protein, and D-dimer were quantified in paired plasma SARS-CoV-2 N-positive specimens than in those testing negative. Occurrence of SARS-CoV-2 N-antigenemia was not associated with increased mortality in univariate logistic regression analysis (odds ratio, 1.29; 95% confidence interval, 0.49-3.34; p = 0.59). In conclusion, SARS-CoV-2 N-antigenemia detection is relatively common in ICU patients and appears to associate with increased serum levels of inflammation and tissue-damage markers. Whether this virological parameter may behave as a biomarker of poor clinical outcome awaits further investigations.

| INTRODUCTION
Severe COVID-19 is a multisystem disease involving the lower respiratory tract (LRT) and extra-pulmonary organs such as the liver, kidney, spleen, and central nervous system. 1 Following infection, SARS-CoV-2 initially replicates in the upper respiratory tract (URT) before reaching the LRT 2,3 where it may cause severe damage, by virtue of its own cytopathogenicity and notably by inducing a persistent, dysregulated proinflammatory state. 4,5 SARS-CoV-2 may access the systemic compartment early after infection. In fact, depending upon clinical severity, SARS-CoV-2 RNAemia can be detected in up to 88% of COVID-19 patients within the first week after symptoms onset and has been associated with ICU admission, need for invasive mechanical ventilation, multiple organ failure, and mortality rate. 6 Likewise, SARS-CoV-2 nucleocapsid (N) antigenemia, which has also been found in a large percentage of COVID-19 patients, [7][8][9] has been associated with higher ICU admission rates and overall mortality. 7,9 The current study aimed at further characterizing the dynamics of SARS-CoV-2 N-antigenemia in a cohort of critically ill adult COVID-19 patients and assessing its potential association with plasma levels of biomarkers of clinical severity and mortality. Studies of this nature may contribute to clarifying the pathogenesis of SARS-CoV-2 infection, as well as precisely identifying virological factors modulating COVID-19 prognosis.

| Patients and specimens
In this prospective observational study, 73 consecutive critically ill COVID-19 patients (51 males and 22 females; median age, 65 years; range, 21-80 years) were enrolled between October 2020 and February 2021 (Table 1). According to the Centers for Disease Control and Prevention (CDC) (https://www.covid19treatmentguidelines.nih. gov/management/critical-care), critical illness was defined by the presence of respiratory failure, septic shock, and/or multiple organ dysfunction. Plasma specimens were scheduled to be collected at least once a week from ICU admission, were obtained by centrifugation of whole blood ethylenediaminetetraacetic acid tubes, cryopreserved at −80°C and retrieved for analyses within 1 month after collection.
Nonpreviously thawed specimens were used for analyses. Medical history and laboratory data were prospectively recorded. The current study was approved by the Ethics Committee of Hospital Clínico Universitario INCLIVA (May, 2020).

| Detection of SARS-CoV-2 N protein in plasma
The CLINITEST Rapid COVID-19 Antigen Test (Siemens, Healthineers), a lateral flow immunochromatography (LFIC) device licensed for detection of SARS-CoV-2 nucleocapsid protein nasopharyngeal specimens or nasal swabs, was used on plasma specimens. The limit of detection (LOD) of the assay in plasma was determined by spiking a prepandemic plasma pool testing negative by SARS-CoV-2 reversetranscription polymerase chain reaction (RT-PCR) with 10, 25, 50, 100, and 150 pg/ml of a recombinant N protein (MT-25C19NC, Certest Biotec S.L.). The LOD was found to be at least 50 pg/ml ( Figure 1A). N-antigen line intensity was scored visually using a 3-level scale: 0, negative result; 1+, intensity of test band lower than control band, and 2+, intensity of test band equal or greater to control line (which roughly corresponded to less than 100 pg/ml and greater than or equal to 100 pg/ml, respectively) ( Figure 1A).      (Figure 2A). Moreover, as shown in Figure 3, specimens yielding strong reactivity on the N-antigen assay (2+) had significantly higher SARS-CoV-2 RNA loads (p = 0.002) than those yielding weak reactivity (1+).

| Detection of SARS-CoV-2 N-antigenemia
One plasma specimen yielding discordant results (positive RT-PCR and negative N-antigen assay) was subjected to N depletion as detailed above. As shown in Figure 1B, treatment of plasma with anti-N protein antibody reduced the test line intensity by 54% compared with that obtained with the rabbit isotype control. A total of 61 patients had one or more TA collected (total number, 165; median of 2 specimens/patient; range, 1-11) within the study period. As previously reported, 10 SARS-CoV-2 RNA load in TA ranged between 3.03 and 10.6 log 10 copies/ml (median, 6.5 log 10 copies/ml). SARS-CoV-2 RNA load in TA was significantly higher (p < 0.001) in the presence of concomitant N-antigenemia than in its absence ( Figure 2B).

| Relationship between n-antigenemia and plasma levels of inflammatory or tissue-damage biomarkers and absolute lymphocyte counts
Significantly higher serum levels of ferritin, LDH, CRP, and D-D were measured in paired SARS-CoV-2 N-positive specimens than in those testing negative (Table 2). In contrast, plasma levels of IL-6 were comparable across groups. Lymphocyte counts were significantly lower in the presence of SARS-CoV-2 N antigen in plasma than in its absence ( Table 2). Median serum levels of the above biomarkers tended to be higher when paired plasma specimens yielded stronger N-antigen reactivity (2+ vs. 1+), yet the difference was only significantly different for ferritin (Figure 4).

| DISCUSSION
Here, we investigated the dynamics of SARS-CoV-2 N-antigenemia in a relatively homogeneous cohort of ICU patients with severe pneumonia, most of whom underwent mechanical ventilation, by means of LFIC with an analytical sensitivity of around 50 pg/ml. In contrast to previous studies, serial specimens from patients rather than a single one drawn at hospital admission were analyzed. SARS-CoV-2 N-antigenemia was detected in 41.0% of patients and was cleared before day 33 after ICU admission, those exhibiting higher plasma viral RNA loads being more likely to test positive, as previously noticed, 8 and yielding stronger N-antigen line reactivities. This concurs with the figure (49%) reported in a previous study 9 also using LFIC (Panbio COVID-19 Ag Rapid Test Device from Abbott), but is much lower than the proportion found by Hingrat et al. 8 employing an ultrasensitive immunoassay (limit of detection of 2.8 pg/ml). We did not measure serum antibodies against SARS-CoV-2, which may impact the rate of N-antigenemia detection, 9 and precludes certainty concerning the comparability of our data with those reported in the other studies. 8,9 Interestingly, in line with a previous report, 8 N-antigenemia had a higher detection rate than viral RNAemia; as depletion experiments proved the true nature of the N protein detected in discordant specimens, we favor the idea that this phenomenon could be explained by the fact that N protein is likely less prone to degradation than RNA in cryopreserved-thawed specimens. Given that detection of SARS-CoV-2 RNA in blood has not been associated with the infectious virus, 11 we T A B L E 2 SARS-CoV-2 N-antigenemia and plasma level COVID-19 severity biomarkers A novel observation was that, as previously shown for SARS-CoV-2 RNAemia, 10 the presence of N-antigenemia was significantly associated with high viral loads in the lower respiratory tract.
Interestingly, neither treatment with remdesivir nor with tocilizumab appeared to have a major impact on the rate of SARS-CoV-2 Nantigenemia detection. This is in line with previous studies showing that neither of these drugs had a tangible effect on SARS-CoV-2 RNA load in the upper airways even when given early after symptoms onset. 12,13 We provide for the first time some SARS-CoV-2 N-antigenemia at hospital admission has been independently associated with disease progression in mixed cohorts of COVID-19 patients with severe disease 7 and independently associated with increased 30-day overall mortality in a series including only ICU patients. 9 Nevertheless, we found no evidence of an association between detection of N-antigenemia and mortality rate.
Reasons for this discrepancy are unclear, but may be linked to differences in baseline characteristics of patients and clinical and therapeutic management of COVID-19 across centers; It may also be related to different sample collection timing across the studies.

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The main limitations of the current study are the relatively small sample size and the use of an analytical semiquantitative method which may have underestimated the N-antigenemia detection rate.
Analysis of sequential specimens from patients could be considered a strength of the research.
In conclusion, SARS-CoV-2 N-antigenemia detection is relatively common in ICU patients and appears to associate with increased plasma levels of inflammation and tissue-damage markers. Whether this virological parameter may behave as a biomarker of poor clinical outcome awaits data from larger and well-powered studies.

ACKNOWLEDGMENTS
We are grateful to all personnel who work at Clinic University

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

DATA AVAILABILITY STATEMENT
The data presented in the manuscript have not been made available, but can be shared upon request.