key: cord-1029500-pqq52qk0
authors: Eiros, Rocío; Barreiro-Pérez, Manuel; Martín-García, Ana; Almeida, Julia; Villacorta, Eduardo; Pérez-Pons, Alba; Merchán, Soraya; Torres-Valle, Alba; Sánchez-Pablo, Clara; González-Calle, David; Pérez-Escurza, Oihane; Toranzo, Inés; Díaz-Peláez, Elena; Fuentes-Herrero, Blanca; Macías-Álvarez, Laura; Oliva-Ariza, Guillermo; Lecrevisse, Quentin; Fluxa, Rafael; Bravo-Grande, José L; Orfao, Alberto; Sánchez, Pedro L
title: Pericardial and myocardial involvement after SARS-CoV-2 infection: a cross-sectional descriptive study in health care workers
date: 2021-11-05
journal: Rev Esp Cardiol (Engl Ed)
DOI: 10.1016/j.rec.2021.11.001
sha: ca56b870e81542c78de03cdda8a0a38ce8fe0c28
doc_id: 1029500
cord_uid: pqq52qk0

Introduction and objectives: The cardiac sequelae of SARS-CoV-2 infection are still poorly documented. We conducted a cross-sectional study in health care workers to report evidence of pericardial and myocardial involvement after SARS-CoV-2 infection. Methods: We studied 139 health care workers with confirmed past SARS-CoV-2 infection. Participants underwent clinical assessment, electrocardiography, and laboratory tests, including immune cell profiling and cardiac magnetic resonance (CMR). Clinically suspected pericarditis was diagnosed when classic criteria were present and clinically suspected myocarditis was based on the combination of at least 2 CMR criteria. Results: Median age was 52 (41-57) years, 71.9% were women, and 16.5% were previously hospitalized for COVID-19 pneumonia. On examination (10.4 [9.3-11.0] weeks after infection-like symptoms), participants showed hemodynamic stability. Chest pain, dyspnea or palpitations were present in 41.7% participants, electrocardiographic abnormalities in 49.6%, NT-proBNP elevation in 7.9%, troponin in 0.7%, and CMR abnormalities in 60.4%. A total of 30.9% participants met criteria for either pericarditis and/or myocarditis: isolated pericarditis was diagnosed in 5.8%, myopericarditis in 7.9%, and isolated myocarditis in 17.3%. Most participants (73.2%) showed altered immune cell counts in blood, particularly decreased eosinophil (27.3%; P < .001) and increased cytotoxic T cell numbers (17.3%; P < .001). Clinically suspected pericarditis was associated (P < .005) with particularly elevated cytotoxic T cells and decreased eosinophil counts, while participants diagnosed with clinically suspected myopericarditis or myocarditis had lower (P < .05) neutrophil counts, natural killer-cells, and plasma cells. Conclusions: Pericardial and myocardial involvement with clinical stability are frequent after SARS-CoV-2 infection and are associated with specific immune cell profiles.

The novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is currently causing a sustained COVID-19 pandemic, with the risk of causing long-term cardiac sequelae in the infected population. 1 The fear of SARS-CoV-2 causing greater myocardial damage than other conventional viruses is based on its mechanism of infecting human cells by binding to the transmembrane angiotensin converting-enzyme 2-which is mainly expressed by cells in alveoli and myocardial tissue-the rise in troponin levels observed in COVID-19 patients hospitalized with pneumonia and its association with increased mortality, and the probably reduced innate antiviral defenses against a novel virus. 2 Pericarditis and myocarditis after conventional viral infections both stem from an inadequate or excessive immune response driven by T and B cell-mediated mechanisms. 3, 4 If there is an inadequate response, continued viral replication in the perimyocardium protracts inflammation by attracting killer T cells and the concomitant production of chemokines and cytokines. In contrast, molecular mimicry can result in the production of autoantibodies against cardiac proteins, leading to a cardio-specific autoimmune response that causes sustained inflammation, effusion, or cardiac remodeling. However, the specific immune profiles that occur after SARS-CoV-2 infection, particularly in patients showing cardiac sequelae remain unknown. 5 The present study was designed to search for evidence of pericardial and myocardial involvement after past SARS-CoV-2 infection comprehensively studied by clinical assessment, laboratory tests, electrocardiography and cardiac magnetic resonance (CMR) imaging.

Additionally, participants underwent an in-depth characterization of the immune cell compartments in blood and the virus-specific humoral immune response. As health care workers have been the group most affected by SARS-CoV-2 in Spain but have also been subject to more testing than the rest of the population, we decided to conduct the study in this singular cohort.

This cross-sectional study consecutively recruited 142 health care workers with laboratory confirmed SARS-CoV-2 infection in the University Hospital of Salamanca, and who volunteered for the study. Among them, 106 health care workers tested positive for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction (RT-PCR) from nasopharyngeal swab between March 13 and April 25, 2020; and 36 health care workers were diagnosed after testing positive for anti-SARS-CoV-2-immunoglobulin G (IgG) antibodies by ELISA between April 10 and May 22, 2020, as part of a major hospital campaign. The purpose of this second group was to also provide data from participants with past SARS-CoV-2 infection, who are more likely to have mild symptoms of viral infection, and because population-based SARS-CoV-2 seroprevalence studies are becoming more established. 6, 7 Study enrolment began on May 25 and finished on June 12, 2020.

Institutional approval (2020/05/490) for the study was provided by the University Hospital of Salamanca Ethics Committee, and all participants provided written informed consent. The study is registered with ClinicalTrials.gov NCT04413071. The responsibility for the study design, data collection and data interpretation lay solely with the study investigators. An internal adjudication monitoring board reviewed all cardiac study findings and adjudicated study outcomes. The authors had full access to all the data and elaborated all materials to submit for publication.

All participants underwent clinical evaluation, electrocardiography, laboratory tests and CMR imaging at the same visit. After obtaining written informed consent, trained interviewers used a structured questionnaire to collect baseline data in face-to-face interviews. A cardiologist Page 8 of 41 J o u r n a l P r e -p r o o f 8 took a complete medical history, performed a physical examination and reviewed the completeness of the questionnaire in a separate room, where an electrocardiogram was performed, and blood samples were drawn immediately before the CMR. Electrocardiograms were interpreted in consensus by 2 experienced readers, who were blinded to participant identification, clinical history, symptoms, physical examination, and other findings.

CMR was performed using a clinical 1.5 whole-body magnetic resonance scanner in the cardiac imaging laboratory of the University Hospital of Salamanca. 8 The imaging acquisition protocol is described in detail in Methods of the supplementary data. CMR images were globally and regionally analyzed using dedicated software, in consensus by 2 experienced readers, who were blinded in a similar manner to the electrocardiogram protocol. T2 and T1-based markers of myocardial inflammation were analyzed in each of the 16 segments of the 17-segment model of the American Heart Association (the true apex was excluded), where only positive segment concordances from the different T2 and T1-based markers were considered. Because myocarditis was diagnosed according to these T2 and T1-based CMR markers and an adequate selection of normal reference values is fundamental, we used a population-based control CMR imaging group from 50 sex-and aged-matched individuals without cardiac disease. 9 The prevalences of cardiovascular risk factors (hypertension, diabetes mellitus, dislipemia, current smoking) in the control cohort were similar to those in the study population.

Immunophenotypic analysis of (> 250) immune cell populations was performed in peripheral blood samples collected in K3-ethylenediaminetetra-acetic acid (EDTA, 10 mL/sample, and stained with the EuroFlow lymphocyte screening tube and the cluster of differentiation 4 T cell (TCD4), natural killer (NK)/TCD8, beta-lactoglobulin hydrolysates (BIgH) and monocyte-derived dendritic cell (MoDC) immune monitoring tubes by flow cytometry (FACSCANTO II and LSR-Fortessa, respectively; Becton/Dickinson Biosciences, United States) using a dual-platform assay previously described in detail. 10 Reference values for the individual immune cell subsets investigated in blood by flow cytometry were defined based on a population-based control 9 group of 463 age-matched adults (median age 52 [IQR 47-61] years) studied prior to the SARS-CoV-2 pandemic. Anti-SARS-CoV-2-IgM (AnshLabs, Webster, United States), IgG and IgA (Mikrogen Diagnostik, Neuried, Germany) antibody levels were measured in parallel in plasma from the same blood samples using commercially available in vitro diagnostic medical device approved (semiquantitative) ELISA kits, strictly as instructed by the manufacturers.

Study outcome measures were the prevalence of clinical pericarditis and of myocarditis, and the characterization of the immune cell compartments in blood, and the virus-specific humoral immune response. Clinically suspected pericarditis was diagnosed if at least 2 of the following criteria were present, following current guidelines 3 : pericarditic chest pain, pericardial rub on auscultation, widespread ST-elevation or PR segment depression on electrocardiogram, and evidence of pericardial effusion on CMR. Elevation of inflammation markers, C-reactive protein, and evidence of pericardial inflammation on CMR were used as additional supporting findings. The diagnosis of clinically suspected myocarditis was based on CMR criteria 11 ; we considered as main CMR criteria positive edema-sensitive T2-based markers (T2-weighted images or T2-mapping) or positive T1-based tissue characterization markers (abnormal T1relaxation time or extracellular volume or late gadolinium enhancement), and as supportive CMR criteria either pericardial effusion, or evidence of pericardial inflammation on CMR, or systolic left ventricular wall motion abnormalities. Considering that participants were being examined after the acute phase of SARS-CoV-2 infection, in this study clinically suspected myocarditis was defined as the presence of a combination of at least 2 T2 or T1-based CMR main criteria or the presence of combination of only 1 T2 or T1-based main criterion with 1 additional CMR supportive criterion.

As we were aware that pericarditis and myocarditis occur together in clinical practice, we therefore defined as clinically suspected myopericarditis those cases of pericarditis with associated myocarditis on CMR but without left ventricular wall motion abnormalities, and as clinically suspected perimyocarditis those cases where left ventricular wall motion abnormalities were present. 12

Descriptive statistics were used to summarized the data. Results are presented as the proportion (%) of valid cases for categorical variables and as the median [IQR] for continuous variables. Differences between groups were analyzed by the Fisher exact test for categorical variables and by the nonparametric Mann-Whitney or Kruskal-Wallis tests for continuous data.

Comparisons between immune cell counts in the blood of patients and controls were adjusted Cardiac-specific and inflammatory biomarkers were within the normal range in most participants (table 2) . CMR abnormalities were observed in 84 (60.4%) participants (table 3 and tables 4 and 5 of the supplementary data, Figure 2 ). Two (1.4%) participants showed increased myocardial T2-relaxation time, 5 (3.6%) edema on T2-weighted images, 40 (28.8%) increased native myocardial T1-relaxation time, 27 (19.4%) increased T1-extracellular volume, 10 (7.2%) T1-late gadolinium enhancement, 42 (30.2%) pericardial effusion, 1 (0.7%) a pericardial thickness of 3 mm and 7 (5.0%) systolic left ventricular wall motion abnormalities, global or regional.

A total of 43 (30.9%) participants fulfilled the criteria for either clinically suspected pericarditis or myocarditis. Clinically suspected isolated pericarditis was diagnosed in 8 (5.8%) participants, isolated myocarditis in 24 (17.3%), and myopericarditis in 11 (7.9%). These were no cases of perimyocarditis. Descriptions of criteria combinations are provided in figure 3 and baseline and examination characteristics for each diagnostic group are detailed in table 1 and table 2 Overall, no major differences were observed among participants with or without pericardial and myocardial involvement in the frequency and levels of anti-SARS-CoV-2-IgM, IgG and IgA antibodies in plasma (figure 5C). Importantly, overlapping immune profiles were detected between participants diagnosed by RT-PCR compared with those diagnosed by serology.

cohort of SARS-CoV-2-positive health care workers. In one of the largest cohort of participants with CMR imaging assessment reported so far, we demonstrate that pericardial and myocardial involvement is prevalent after SARS-CoV-2 infection in association with an altered immune response.

We decided to carry out a study in health care workers as this sector has been disproportionally infected in Spain, which provided us with the opportunity to study the prevalence of clinically suspected pericarditis and myocarditis in SARS-CoV-2-infected cases that were confirmed by positive RT-PCR or positive serology. In addition, because the proportion of female health care workers is high in Spain, our study does not underrepresent women who constituted more than two thirds of recruited participants. Unlike other observational studies suggesting that myocarditis may be slightly more prevalent in men than in women, 14 of the patients and late gadolinium enhancement in 31%. 16 Observations similar to a prospective study in 100 patients recovered from COVID-19 pneumoniae were the presence of myocardial edema in 60% and late gadolinium enhancement in 32%. 17 Our observations, performed mostly in nonhospitalized participants (83.5%) and also including participants At present, there is much interest in the long-term sequelae of COVID-19. It is intriguing that clinically suspected pericardial and myocardial manifestations were observed long after SARS-CoV-2 infection (more than 10 weeks after initial viral prodrome at infection) as well as in some currently asymptomatic participants (9 cases; 1 out of every 5 final clinically suspected cases of pericarditis, myopericarditis or myocarditis diagnoses). These long-term manifestations may be due to an inadequate innate and adaptative immune response with very limited data on the longer-term immunological consequences of past SARS-CoV-2 infection, 5 and no study has specifically focused on the settings of pericarditis and clinically suspected myocarditis. In this study, in-depth investigation of the distribution of major and minor populations of immune cells in blood showed a high frequency of overall altered immune profiles.

Several of the immune cell alterations identified mimic abnormalities reported during active infection for the general population with COVID-19, including decreased eosinophil and NK cell counts. 23 The overall pattern in this study emerges as a unique SASR-CoV-2-associated immune profile. For example, while decreased eosinophil counts in blood have been reported among participants infected with influenza, 24 no association has yet been reported with increased counts of cytotoxic (CD4 -CD8 -/lo ) T cells and plasmablasts in blood, which have been identified among HIV-infected participants in the absence of eosinopenia. 25 More detailed analysis of the altered immune profiles among the different groups of participants showed that those with clinically suspected myopericarditis or myocarditis had closer to normal lymphocyte counts, but reduced numbers in blood of circulating eosinophils and NK cells. Such a unique profile mimics what has been described recently during the acute phase of SARS-CoV-2 infection, suggesting an ongoing cytotoxic response with increased tissue migration or death by apoptosis of specific subsets of cytotoxic cells. These findings suggest that a less pronounced (potentially insufficient) or a delayed humoral response may occur in these participants, which may lead to decreased neutralization, opsonization and/or clearance of the virus locally at the perimyocardium; local viral persistence would favor an increased tissue-homing (or early death) of eosinophils, immunomodulatory and intermediate monocytes, in addition to cytotoxic (effector) cells. Thus, similar to influenza, 26 although SARS-CoV-2 pneumoniae is the most widely recognized complication, the coronavirus could trigger pericarditis or myocarditis as part of the host immune response rather than viral-mediated myocarditis per se. In this sense, cases of cardiac involvement are beginning to be described after the second dose of COVID-19 vaccine. 27

The major limitation of this study is that clinically suspected myocarditis was not confirmed via endomyocardial biopsy. CMR T1 and T2 measures, although significant, were small between the participants with SARS-CoV-2 infection and the control group. The study analysis was limited to health care workers and therefore has limited external generalizability to other nonhealth care settings. However, the strength of this study is the addition of nonhospitalized participants, as well as the inclusion of participants diagnosed with past SARS-CoV-2 infection through serology, who also had a high prevalence of pericardial and myocardial involvement.

This study shows that clinically suspected pericarditis and myocarditis are frequent in health care workers after SARS-CoV-2 infection, as well as in some currently asymptomatic individuals; in addition, we provide evidence for an altered immune cell distribution in blood which affects cells involved in both the innate (eg, eosinophils, monocytes and NK cells) and 

-There is increasing evidence of cardiac sequelae after SARS-CoV-2 infection. -Clinical pericarditis and myocarditis are associated with specific immune cell profiles, paving the way for a better understanding of the immune mechanisms involved. In this regard, pericardial and myocardial involvement is beginning to be described after COVID-19 vaccination. Table 4 Distribution of subsets of myeloid and lymphoid immune cells in blood Healthy donors (n = 463)

All participants (N = 139)

Pandemic: A Global Perspective

Drives Development of COVID-19

ESC Guidelines for the diagnosis and management of pericardial diseases: The Task Force for the Diagnosis and Management of Pericardial Diseases of the European Society of Cardiology (ESC)Endorsed by: The European Association for Cardio-Thoracic Surgery (EACTS)

Immunology of COVID-19: Current State of the Science

Seroprevalence of anti-SARS-CoV-2 IgG antibodies in Geneva, Switzerland (SEROCoV-POP): a population-based study

Prevalence of SARS-CoV-2 in Spain (ENE-COVID): a nationwide, population-based seroepidemiological study

First Magnetic Resonance Managed by a Cardiology Department in the Spanish Public Healthcare System. Experience and Difficulties of an Innovative Model

Rationale and design of a population-based study to identify structural heart disease abnormalities: a spatial and machine learning analysis

Age Distribution of Multiple Functionally Relevant Subsets of CD4+ T Cells in Human Blood Using a Standardized and Validated 14-Color EuroFlow Immune Monitoring Tube

Cardiovascular Magnetic Resonance in Nonischemic Myocardial Inflammation: Expert Recommendations

Myopericarditis: Etiology, management, and prognosis

EuroFlow Lymphoid Screening Tube (LST) data base for automated identification of blood lymphocyte subsets

National Trends, Gender, Management, and Outcomes of Patients Hospitalized for Myocarditis

Sex differences in immune responses that underlie COVID-19 disease outcomes

Cardiac involvement in recovered COVID-19 patients identified by magnetic resonance imaging

Outcomes of Cardiovascular Magnetic Resonance Imaging in Patients Recently Recovered From Coronavirus Disease 2019 (COVID-19)

Patterns of myocardial injury in recovered troponinpositive COVID-19 patients assessed by cardiovascular magnetic resonance

The hidden burden of influenza: A review of the extra-pulmonary complications of influenza infection

Pathological features of COVID-19-associated myocardial injury: a multicentre cardiovascular pathology study

COVID-19-associated Non-Occlusive Fibrin Microthrombi in the Heart

High Prevalence of Pericardial Involvement in College Student Athletes Recovering From COVID-19

Dysregulation of immune response in patients with COVID-19 in Wuhan, China

Predictors of H1N1 influenza in the emergency department: proposition for a modified H1N1 case definition

Eosinophilia in patients infected with human immunodeficiency virus

Immune Mechanisms in Cardiovascular Diseases Associated With Viral Infection

Acute myocarditis after administration of the BNT162b2 vaccine against COVID-19