TYPE Brief Research Report PUBLISHED 18 January 2023 DOI 10.3389/fimmu.2022.1105343 C-reactive protein isoforms OPEN ACCESS as prognostic markers of EDITED BY Yi Wu, Xi’an Jiaotong University, China COVID-19 severity REVIEWED BY Karlheinz Peter, Blanca Molins 1*, Marc Figueras-Roca 1,2, Oliver Valero 3, Baker Heart and Diabetes Institute, Australia Vı́ctor Llorenç 1,2, Sara Romero-Vázquez 1, Oriol Sibila 4, Christopher Sjöwall, Linköping University, Sweden Alfredo Adán 1,2, Carolina Garcı́a-Vidal 5 and Alex Soriano 5,6 Lawrence Albert Potempa, 1 Group of Ocular Inflammation: Clinical and Experimental Studies, Institut d’Investigacions Roosevelt University, United Sates Biomèdiques Agustı´ Pi i Sunyer (IDIBAPS), Barcelona, Spain, 2 Institut Clı´nic d’Oftalmologia (ICOF), *CORRESPONDENCE Hospital Clı´nic, Barcelona, Spain, 3 Statistical Department, Universitat Autònoma de Barcelona, Blanca Molins Barcelona, Spain, 4 Respiratory Department, Hospital Clinic of Barcelona-IDIBAPS, CIBERES, University of Barcelona, Barcelona, Spain, 5 Department of Infectious Diseases, Hospital Clinic of bmolins@recerca.clinic.cat Barcelona-IDIBAPS, University of Barcelona, Barcelona, Spain, 6 CIBERINF, Barcelona, Spain SPECIALTY SECTION This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology C-reactive protein (CRP), an active regulator of the innate immune system, has RECEIVED 22 November 2022 been related to COVID-19 severity. CRP is a dynamic protein undergoing ACCEPTED 26 December 2022 conformational changes upon activation in inflammatory microenvironments PUBLISHED 18 January 2023 between pentameric and monomeric isoforms. Although pentameric CRP is CITATION the circulating isoform routinely tested for clinical purposes, monomeric CRP Molins B, Figueras-Roca M, Valero O, shows more proinflammatory properties. Therefore, we aimed to determine Llorenç V, Romero-Vázquez S, Sibila O, Adán A, Garcı´a-Vidal C and the potential of monomeric CRP in serum as a biomarker of disease severity in Soriano A (2023) C-reactive protein COVID-19 patients (admission to intensive care unit [ICU] and/or in-hospital isoforms as prognostic markers of COVID-19 severity. mortality). We retrospectively determined clinical and biological features as Front. Immunol. 13:1105343. well as pentameric and monomeric CRP levels in a cohort of 97 COVID-19 doi: 10.3389/fimmu.2022.1105343 patients within 72h of hospital admission. Patients with severe disease had COPYRIGHT higher levels of both pentameric and monomeric CRP. However, multivariate © 2023 Molins, Figueras-Roca, Valero, Llorenç, Romero-Vázquez, Sibila, Adán, analysis showed increased mCRP but not pCRP to be independently associated Garcı´a-Vidal and Soriano. This is an to disease severity. Notably, mCRP levels higher than 4000 ng/mL (OR: 4.551, open-access article distributed under 95% CI: 1.329-15.58), together with number of co-morbidities, low lymphocyte the terms of the Creative Commons Attribution License (CC BY). The use, count, and procalcitonin levels were independent predictors of disease severity distribution or reproduction in other in the multivariate model. Our results show the potential of mCRP levels as a forums is permitted, provided the marker of clinical severity in COVID-19 disease. original author(s) and the copyright owner(s) are credited and that the original publication in this journal is KEYWORDS cited, in accordance with accepted academic practice. No use, C-reactive protein, COVID-19, isoforms, inflammation, prognosis distribution or reproduction is permitted which does not comply with these terms. 1 Introduction Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was declared in March 2020 a global pandemic by the World Health Organization with more than 4 million related deaths since then (1). COVID-19 is asymptomatic or mild in most cases. Yet, 10% of patients present severe Frontiers in Immunology 01 frontiersin.org Molins et al. 10.3389/fimmu.2022.1105343 disease due to pneumonia, acute respiratory distress syndrome 2 Methods (ARDS), systemic inflammation, thrombosis, and multiorgan failure, causing death in 1-3% of patients. Indeed, some patients 2.1 Study design, patients, and with mild symptoms may present a sudden progression to severe data collection disease (2). Noteworthy, although hospitalization for COVID-19 is We performed an observational retrospective study of 97 needed only in a minority of patients and disease management patients admitted to the hospital (Hospital Clinic of Barcelona, has majorly improved since the first outbreak in March 2020, the Spain) for > 48h with confirmed acute SARS-CoV-2 infection by highly infectious nature of SARS-CoV-2 driving to high rRT-PCR performed on nasopharyngeal and throat swabs incidence spikes in short periods of time is still posing a threat between March 1st and September 30th 2020. Included to health care systems worldwide. Thus, predicting individual patients had serum samples preserved in the COVIDBANK prognosis is crucial to provide a personalized treatment to within 72h of admission for further determination of reduce mortality. Age, male sex, hypertension, the presence of circulating levels of mCRP. The COVIDBANK is a certain co-morbidities, and several blood biomarkers have been biorepository of biological samples from SARS-CoV-2 patients consistently associated with worse prognosis (3, 4). treated in Hospital Clinic of Barcelona with the aim of providing C-reactive protein (CRP), an acute phase reactant and an samples of quality for SARS-COV-2-related scientific research. active regulator of the innate immune system, is increased in The Institutional Ethics Committee of Hospital Clinic of COVID-19 patients and has been associated to disease severity Barcelona approved the study (HCB/2020/0874) and patients (5, 6). Among its multiple functions, CRP activates the classical gave their informed consent to the COVIDBANK. pathway and inactivates the alternative pathway of the Data were retrospectively obtained for all patients included complement system (7). CRP is mainly synthesized in the liver in the study from the electronic health records. Variables upon interleukin- (IL-) 6 induction and its levels can increase up included were age, sex, and co-morbidities (chronic heart to 100-fold in response to several forms of tissue damage, disease, diabetes mellitus, chronic kidney disease, infection and inflammation. In plasma, CRP circulates as a hypertension, solid malignant neoplasm, chronic respiratory disk-shaped pentamer (also known as pentameric CRP, pCRP) disease, haematologic disease, hepatopathy, solid organ composed of five 23 kDa non-covalently bound subunits (8). transplant, HIV). Biochemical variables included blood cell Nevertheless, low pH, oxidative stress and bioactive lipids from count (eosinophils, basophils, lymphocytes), creatinine, D- activated cells can dissociate pCRP into its monomeric subunits dimer, ferritin, lactate dehydrogenase (LDH), procalcitonin, (9, 10) by means of phospholipase A2 activation and subsequent troponin, and pCRP at admission. The composite outcome lysophosphatidylcholine cell surface exposure (11). This variable included the need of intensive care unit (ICU) conformation of CRP, named monomeric CRP (mCRP), admission and/or in-hospital mortality at any time during in- shows different biological functions and antigenicity- hospital stay. In addition, mCRP levels were determined from expressing neoepitopes than pCRP representing the tissue- preserved serum samples of the COVIDBANK. associated insoluble form of CRP. Although there is no standardized method, circulating levels of mCRP in serum can be measured by means of customized ELISA (12). Unlike pCRP, 2.2 Serum mCRP determination mCRP presents a prothrombotic and proinflammatory phenotype (13–16). Given that most reports on the role of Serum mCRP was detected with the ELISA protocol CRP in systemic diseases are based on the pentameric described by Zhang et al. with some modifications (12, 17). conformation, additional research on the specific implications Briefly, mouse anti-human CRP mAb CRP-8 (Sigma-Aldrich, of mCRP over pCRP should be addressed. C1688) was immobilized as capture antibody at 1:1000 in Considering the hyperinflammatory nature of COVID-19 coating buffer (10 mM sodium carbonate/bicarbonate, pH 9.6) complications and the fact that mCRP dissociates from pCRP in for 18h at 4°C. This commercially available monoclonal proinflammatory microenvironments and it is also the main antibody has been reported to specifically recognize mCRP, active proinflammatory isoform of CRP, we hypothesized that but not pCRP (18). After washing three times for 2 min each mCRP could be considered as a more specific prognostic marker with TBS, non-specific binding was blocked with filtered 1% of inflammatory progression and severity than pCRP in BSA-TBS for 1 hour at 37°C. Samples were diluted 1:100 in COVID-19. Thus, we aimed to evaluate mCRP and pCRP blocking buffer and added into wells for 1h at 37°C. Then, levels in COVID-19 patients at hospital admission to washing step was repeated and samples were incubated with determine their prognostic value to progression to more severe sheep anti-human CRP polyclonal antibody (MBS223280, disease forms. MyBioSource) at 1:5000 in blocking buffer for 1h at room Frontiers in Immunology 02 frontiersin.org Molins et al. 10.3389/fimmu.2022.1105343 temperature (RT), before incubation with a HRP-labelled 3 Results donkey anti-sheep IgG (Abcam) at 1:10000 in blocking buffer for 30 min at RT. Signalling was detected with VersaMax Microplate Reader and the OD value of each sample was Study population consisted of 97 patients admitted to calculated as OD 450 –OD 570 nm. A standard curve was Hospital Clı́nic of Barcelona between March and September prepared by serial dilutions of mCRP (0-50 ng/mL) obtained 2020 for > 48h with confirmed acute SARS-CoV-2 infection. The by urea-chelation of pCRP (Calbiochem) (16) in blocking buffer mean age was 60 ± 17 years and 59 (61%) were males. The most (1% BSA-TBS) in the presence of reference diluted sera (1:100). common co-morbidities were hypertension (50,5%), diabetes The concentration of mCRP in the reference sera was below mellitus (28.9%), chronic heart disease (23.7%), chronic detection level and therefore undetectable following 1:100 respiratory disease (21.6%), solid malignant neoplasm (15.5%), dilution. Controls using purified pCRP at a concentration of and chronic kidney disease (15.5%). Other minor co-morbidities 100 ng/mL, which would be equivalent to 10 µg/mL after 1:100 included chronic liver disease, hematologic disease, solid organ dilution, only generated background signal, showing specificity transplant, and HIV infection. A total of 27 (27.8%) patients for mCRP. were admitted to the ICU and the in-hospital mortality rate was 25.8% (Table 1). Although the majority of patients that died were previously admitted to ICU (15/25, 60%), some died in 2.3 Statistical analysis normal ward (10/25, 40%). Severe disease was defined as ICU admission and/or in-hospital mortality. Categorical variables were described as absolute (N) and Patients with severe disease (37/97) were older (68.76 ± 14.8 relative (%) frequencies and Chi square or Fisher’s test was used vs. 55.02 ± 16.1 years, P<0.001) and had more co-morbidities for comparisons. Quantitative variables following a normal (2.43 [0.00-5.00] vs. 1.20 [0.00-5.00], P<0.001). Patients with distribution were represented as mean ± standard deviation higher levels at admission of creatinine (1.17 [0.24-6.49] vs. 0.85 and differences between groups were determined with [0.50-4.05] mg/dL, P=0.012), D-dimer (1200 [400-28400] vs. Student’s t-test. Variables with a non-normal distribution were 450 [200-6000] ng/mL, P<0.001), procalcitonin (0.18 [0.03- expressed as median and minimum and maximum value and the 15.03] vs. 0.08 [0.00-1.58] ng/mL, P<0.001), troponin (11.65 Mann-Whitney test was employed to determine significant [0.00-304] vs. 3.25 [0-803] ng/L, P<0.001), pCRP (11.61 [1.65- differences. Linear relationships were analysed by 33.50] vs. 5.63 [0.00-33.34] µg/mL, P=0.013), and mCRP (3551.3 determination of Spearman correlation coefficient. Statistical [30-9806] vs. 206 [30-8086], ng/mL P<0.001) and lower assumptions were made based on significance level below 0.05. lymphocyte count (0.60 [0.10-2.90] vs. 1.15 [0.30-2.20] 109/L, To determine the variables associated with disease severity P<0.001) were also more likely to develop severe disease. (need for ICU admission and/or in-hospital mortality), a Chronic kidney and heart disease, hypertension, and solid multivariable logistic regression model was performed adding malignant neoplasm were also associated to disease severity the variables with a P value lower than 0.2 in the bivariate (Table 1). As shown in Figure 1, patients that required ICU analysis. Variables with more than 10% of missing values were admission and/or died had higher levels of both monomeric excluded. Quantitative variables added to the multivariate (*P<0.001) and pentameric CRP (*P=0.013). mCRP significantly analysis were dichotomized as follows: age (>70), pCRP (>10 correlated with pCRP (r=0.377, P<0.001), although the µg/mL), mCRP (>4000 ng/mL), creatinine (>1.1 mg/dL in correlation coefficient was only intermediate (Figure 1C). women and >1.3 mg/dL in men), D-dimer (>500 ng/mL), When analysing the ratio log_mCRP/pCRP no association lymphocytes (<0.004 x109/L), procalcitonin (>0.5 ng/mL), with disease severity was found. In fact, when determining the ferritin (>306 ng/mL in women and >336 ng/mL in men), correlation of log_mCRP/pCRP with established severity troponin (>50 ng/mL). Cut-off values of pCRP, creatinine, D- markers only a statistically significant correlation was observed dimer, lymphocyte count, procalcitonin, and ferritin were for creatinine (r=0.241, P=0.019) and troponin (r=0.264, chosen based on their abnormal levels associated to pathology. P=0.015), but not for procalcitonin (r=-0.007, P=0.94), The final model was reached through backward stepwise lymphocyte count (r=-0.173, P=0.096), ferritin (r=-0.157, removal of variables with p-value higher than 0.1 and using P=0.139), and D-dimer (r=0.133, P=0.208). Instead, mCRP Wald tests to demonstrate that each model was better than its significantly correlated with more markers of severity previous iteration. Odds ratio (OR) with 95% confidence interval including procalcitonin (r=0.278, P=0.007), lymphocyte count (CI) were calculated. To assess the predictive ability of each (r=-0.335, P=0.001), creatinine (r=0.205, P=0.044), troponin model, we calculated the area under the receiver operating (r=0.275, P=0.01), and D-dimer (r=0.329, P=0.001) but not characteristic (ROC) curve with its respective 95% confidence with ferritin (r=0.087, P=0.410). Nevertheless, in most cases interval (95% CI) and determined the cut-off value to maximize correlation coefficient was weak (r<0.3). sensitivity and specificity. Statistical analyses were performed Quantitative variables were then dichotomized and added to using SAS v9.4 software (SAS Institute Inc., Cary, NC, USA). the multivariate analysis: number of pathologies, high levels of Frontiers in Immunology 03 frontiersin.org Molins et al. 10.3389/fimmu.2022.1105343 TABLE 1 Clinical and analytical features of COVID-19 patients upon admission and significant risk factors for disease severity (ICU admission and/or in-hospital mortality) on bivariate analysis. Variable Total N=97 Non-severe disease N=60 Severe disease N=37 P-value Age, mean (SD), years 60.3 (16.9) 55.02 (16.1) 68.76 (14.8) *<0.001 Male, N (%) 59 (60.2) 36 (61.0) 23 (39.0) 0.911 Pre-existing comorbidities, N (%) Chronic heart disease 23 (23.7) 7 (30.4) 16 (69.6) *<0.001 Diabetes mellitus 28 (28.9) 15 (53.6) 13 (46.4) 0.285 Chronic kidney disease 15 (15.5) 3 (20.0) 12 (80.0) *<0.001 Hypertension 49 (50.5) 24 (49.0) 25 (51.0) *0.008 Solid malignant neoplasm 15 (15.5) 5 (33.3) 10 (66.7) *0.013 Chronic respiratory disease 21 (21.6) 15 (71.4) 6 (28.6) 0.308 Other pathologies 3 (33.3) 6 (66.7) 0.064 Haematologic disease 2 (2.1) 1 (50.0) 1 (50.0) 1.000 Chronic liver disease 3 (3.1) 0 (0) 3 (100) 0.053 Solid organ transplant 3 (3.1) 1 (33.3) 2 (66.7) 0.556 HIV 3 (3.1) 1 (33.3) 2 (66.7) 0.556 Number of pathologies, median (min-max) 1.67 (0.00-5.00) 1.20 (0.00-5.00) 2.43 (0.00-5.00) *<0.001 Analytical variables, median (min-max) Basophils, 109/L 0.00 (0.00-0.10) 0.00 (0.00-0.10) 0.00 (0.00-0.10) 0.838 Eosinophils, 109/L 0.00 (0.00-0.50) 0.00 (0.00-0.40) 0.00 (0.00-0.50) 0.150 Lymphocytes, 109/L 1.00 (0.10-2.90) 1.15 (0.30-2.20) 0.60 (0.10-2.90) *<0.001 Creatinine, mg/dL 0.87 (0.24-6.49) 0.85 (0.50-4.05) 1.17 (0.24-6.49) *0.012 D-dimer, ng/mL 650 (200-28400) 450 (200-6000) 1200 (400-28400) *<0.001 Ferritin, ng/mL 596 (12-6126) 487 (12-4309) 740 (21-6126) 0.127 LDH, U/L 302 (148-971) 298 (148-675) 332 (168-971) 0.178 Procalcitonin, ng/mL 0.12 (0.00-15.03) 0.08 (0.00-1.58) 0.18 (0.03-15.03) *<0.001 Troponin, ng/L 7.20 (0.00-803) 3.25 (0.00-803) 11.65 (0.00-304) *<0.001 pCRP, µg/mL 6.43 (0.00-33.50) 5.63 (0.00-33.34) 11.61 (1.65-33.50) *0.013 mCRP, ng/mL 1860 (30-9805) 206.0 (30-8086) 3551.3 (30-9806) *<0.001 Log, mCRP/pCRP 5.44 (-0.11-7.82) 4.75 (-0.11-7.82) 5.64 (0.54-7.27) 0.216 *Statistically significant. mCRP (>4000 ng/mL) and procalcitonin (>0.5 ng/mL), and low 4 Discussion lymphocyte count (<0.004 x109/L) were independently associated to disease severity. Lymphocyte count (OR: 4.615, 95% CI: 1.493- Since the first outbreak in early 2020 huge labour has been 14.26), procalcitonin (OR: 18.199, 95% CI: 2.235-148.17), and devoted to understand COVID-19 pathophysiology and to mCRP (OR: 4.551, 95% CI: 1.329-15.58) were retained in the identify prognosis factors for disease severity worldwide. The model as independent predictors of severity (Table 2). The area wide range of signs, symptoms and clinic severity urge to a under the ROC curve (AUC) was 0.869 (95% CI: 0.794-0.945, and personalized approach for disease management. In this regard, the best cut-off had a 86.1% sensitivity and 75.0% specificity) certain blood biomarkers such as IL-6, ferritin, D-dimer, and showing a good ability to predict in-hospital mortality (Figure 2). CRP may be able to anticipate the development of the cytokine Frontiers in Immunology 04 frontiersin.org Molins et al. 10.3389/fimmu.2022.1105343 between circulating (pCRP) and monomeric (mCRP) tissue- b a s e d i s of o r m s . N o t a b l y , m CR P m a n i f e s t s p o t e n t A proinflammatory effects and activates platelets (16), leukocytes (15), and endothelial cells (21). Moreover, mCRP deposition has been localized in inflamed tissues, thus suggesting an active role in the progression of several inflammatory disorders including Alzheimer’disease (22), cardiovascular disease (23), and age- related macular degeneration (24). Although mCRP could represent a more accurate marker of inflammation, pentameric pCRP is the CRP isoform determined for clinical purposes. Currently, there are no commercially available assays to determine serum or plasma levels of mCRP, likely due to the insoluble nature of the monomeric subunit. However, several reports have developed customized ELISA assays to determine circulating mCRP using specific antibodies against mCRP. Some B authors have generated their own antibodies against mCRP (25– 27), others have used mCRP antibodies (clone 8C10) developed by Potempa et al. (28), while others have used the commercially available clone CRP-8 that has been described to specifically recognize mCRP (12, 29). In our study, we followed the protocol described by Zhang et al., and used the monoclonal antibody CRP-8 to quantify circulating mCRP in our cohort as this protocol avoided cross-reactivity with pCRP. Alternatively, flow cytometry has also been used by some authors to determine circulating mCRP (30, 31). The precise nature of the mCRP detected by ELISA is unclear as both highly denaturated or globular mCRP forms C may be present in serum samples. Given the reduced solubility of mCRP it is conceivable that detected mCRP can be also bound to microparticles. Alternatively, circulating mCRP can be found in a globular form with binding properties similar to the pentameric form as recently described by Williams et al. (29). Indeed, further research regarding the structure and function of physiological circulating mCRP is warranted. No standardized method for circulating mCRP determination has been established yet and different techniques and protocols may result in different mCRP values, which currently limit the FIGURE 1 translational application of mCRP in the clinical setting. Yet, few mCRP and pCRP levels in COVID-19 patients. Box-and whisker plots of mCRP (A) and pCRP (B) levels according to disease TABLE 2 Significant risk factors for disease severity (ICU admission severity (ICU-admission and/or in-hospital mortality). Median and/or in-hospital mortality) on multivariate regression analysis. values are highlighted by solid lines. Statistical analysis was conducted using Mann-Whitney test (*P < 0.05, **P < 0.001). Variable OR 95% CI P- value Correlation between mCRP and pCRP (C). Number of pathologies *0.036 1 vs. 0 10.660 1.448-78.48 storm leading to COVID-19 complications (19, 20). Here, we 2 vs. 0 12.135 1.787-82.41 provide insights on the potential of the conformational isoforms 2 vs. 1 1.138 0.344-3.77 of CRP to predict disease severity, defined as ICU admission and/or in-hospital mortality. Our results show that high levels of mCRP >4000 ng/mL 4.551 1.329-15.58 *0.016 circulating mCRP in COVID-19 patients at hospital admission Lymphocytes <0.004x109/L 4.615 1.493-14.26 *0.008 are independently associated to disease severity. Procalcitonin >0.5 ng/mL 18.199 2.235-148.17 *0.007 CRP is a dynamic protein subjected to conformational *Statistically significant. changes upon activation in inflammatory microenvironments Frontiers in Immunology 05 frontiersin.org Molins et al. 10.3389/fimmu.2022.1105343 Whether mCRP plays a role in the progression of COVID-19 or is merely a marker remains unclear. A recent study showed that mCRP can bind to the SARS-CoV-2 spike receptor binding- domain (RBD) and competes with the binding of the spike RBD to angiotensin-converting enzyme 2 (ACE2) receptor (32). Increased levels of CRP induced by SARS-CoV-2 infection may eventually lead to unrestrained inflammation. Indeed, CRP apheresis can reduce the immune response in COVID-19 patients, as showed in a case series report of seven patients (33). Targeting mCRP in COVID-19 patients could represent a novel therapeutic target. Besides CRP apheresis, approaches aimed at FIGURE 2 ROC curve of the multivariate model. inhibiting either CRP dissociation into mCRP (34, 35) or antibodies against mCRP (36) could represent attractive alternatives to prevent the cytokine storm and studies have determined circulating mCRP levels in several hyperinflammatory status associated to COVID-19. Yet, diseases including skin related autoimmune disorders (0-120 ng/ caution should be taken when targeting CRP as the effects of mL) (12), Adult-Onset Still-s disease (0-1000 ng/mL) (25), CRP isoforms may differ depending on the infection stage; in systemic lupus erythematosus (1-7 ng/mL), antineutrophil early phases of infection CRP acts as a soluble pattern cytoplasmic antibody-associated vasculitis (5-20 ng/mL) (28), recognition receptor targeting necrotic or damaged tissue, atherosclerosis (0-50 ng/mL) (27), and chronic pulmonary while in late phases of infection, unrestrained levels of CRP obstructive disease (600-1000 ng/mL) (26). In these studies, may cause hype rin flammation with its subsequent mCRP levels were lower than those for hospitalized COVID-19 complications. mCRP is mainly generated within inflamed patients from our study. As a matter of fact, the study of Karlsson tissues and has a short circulating half-life (21), thus et al. also included a control group of healthy subjects with a mean suggesting that circulating mCRP might not play a direct role level of mCRP below 10 ng/mL (28). Although differences may be in autoimmune induction. Yet, it may act as an indicator of local attributed to the method used to determine mCRP it is mCRP accumulation and subsequent inflammation. conceivable that COVID-19 patients at hospital admission may Immunostaining of mCRP in damaged infected tissue could present increased levels of mCRP compared to other pathologies. shed light on the role of mCRP in COVID-19 pathophysiology. Because mCRP dissociates from pCRP, it is also conceivable that In summary, despite certain intrinsic limitations including high levels of pCRP, as the ones of our cohort, lead to higher levels retrospective and single centre nature together with limited of mCRP. Indeed, mCRP significantly correlated with pCRP sample size, our study shows for the first time the role of although the correlation coefficient was not particularly strong. circulating mCRP as a prognostic factor of disease severity in Given the wide range of COVID-19 clinical presentation, COVID-19 which should be confirmed in prospective severe disease was considered on either ICU admission and/or multicentric studies. It should be also pointed that our patient in-hospital death in order to search for high external validity cohort was recruited during the first outbreak of the pandemic, findings. Accordingly, our results showed that both mCRP and before proper treatments and vaccines were available, which may pCRP were significantly associated to disease severity in the differ with the phenotype of current COVID-19 patients as bivariate analysis. Interestingly, mCRP, but not pCRP, was disease is now better understood and treated. Determination independently associated to disease severity in the multivariate of mCRP levels in vaccinated COVID-19 patients would help to analysis. Because mCRP represents the proinflammatory better understand the role of mCRP in COVID-19. Further conformation of CRP we also aimed to determine whether the studies are warranted to elucidate the role of mCRP in the ratio mCRP/pCRP was associated to disease severity. different stages of SARS-COV-2 infection in order to identify Nevertheless, unlike mCRP, this variable was not associated to novel targets to prevent the eventual progression to disease severity. Notably, the multivariate model including cytokine storm. mCRP, number of pathologies, low lymphocyte count, and procalcitonin had an AUC of 0.869, with a 86.1% sensitivity and 75.0% specificity in the best cut-off showing a good ability to predict in-hospital mortality. Although our results suggest that Data availability statement mCRP could serve as a prognostic factor of disease severity in COVID-19, a standardized method for determining circulating The raw data supporting the conclusions of this article will mCRP should be established before its clinical application. be made available by the authors, without undue reservation. Frontiers in Immunology 06 frontiersin.org Molins et al. 10.3389/fimmu.2022.1105343 Ethics statement COVIDBANK of HCB-IDIBAPS. This work was financed by ad hoc patronage funds for research on COVID-19 from donations The studies involving human participants were reviewed and from citizens and organizations to the Hospital Clı́nic de approved by The Institutional Ethics Committee of Hospital Barcelona-Fundació Clı́nic per a la Recerca Biomèdica. Clinic of Barcelona. The patients/participants provided their written informed consent to participate in this study. Conflict of interest Author contributions AS has received honoraria for talks on behalf of Merck Sharp and Dohme, Pfizer, Novartis, Gilead, Menarini and Angelini, as BM, MF-R, CG-V, and AS contributed to the design of the well as grant support from Pfizer and Gilead. study and experiments. BM, OV, SR-V, MF-R, and VL The remaining authors declare that the research was performed the experiments, data capture, and analysis. BM, conducted in the absence of any commercial or financial MF-R, OS, AA, CG-V, and AS performed the data relationships that could be construed as a potential conflict interpretation. All authors contributed to the article and of interest. approved the submitted version. Publisher’s note Funding All claims expressed in this article are solely those of the This work was funded by the Ministry of Science and authors and do not necessarily represent those of their affiliated Innovation of Spain, ‘Instituto de Salud Carlos III,’ ‘Fondo de organizations, or those of the publisher, the editors and the Investigació n Sanitaria’ (grant numbers PI19/00265), and funds reviewers. Any product that may be evaluated in this article, or FEDER “Una manera de hacer Europa. We are indebted to the claim that may be made by its manufacturer, is not guaranteed Fundació Glòria Soler for its contribution and support to the or endorsed by the publisher. References 1. COVID-19 map - johns Hopkins coronavirus resource center. Available at: platelets localizes inflammation to atherosclerotic plaques. Circ Res (2009) https://coronavirus.jhu.edu/map.html (Accessed August 16, 2022). 105:128–37. doi: 10.1161/CIRCRESAHA.108.190611 2. Wu C, Chen X, Cai Y, Xia J, Zhou X, Xu S, et al. 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