European Journal of Cardiovascular Medicine

Myocarditis is an inflammatory disease of the heart characterized by inflammatory infiltrates and myocardial injury without an ischemic cause.(1) The most commonly identifiable cause of myocarditis is viral.(2,3) Esfandiarei and McManus1 proposed that the pathophysiology of viral myocarditis is a combination of direct cell injury and T-lymphocyte–mediated cytotoxicity, which can be augmented by the cytokine storm syndrome. Interleukin 6 (IL-6) seems to be the central mediator of cytokine storm, in which it orchestrates the proinflammatory responses from immune cells, including the T lymphocytes.(4) This process causes T-lymphocyte activation and a further release of inflammatory cytokines, which stimulate more T lymphocytes, leading to a positive feedback loop of immune activation and myocardial damage.

Some argued that up to 7% of COVID-19–related deaths were attributable to myocarditis.(5)

Gross changes in viral myocarditis are cardiac hypertrophy may present with associated ventricular dilation and the affected myocardium may appear pale, sometimes with hemorrhagic foci. Histologically in Lymphocytic myocarditis, a focal or diffuse lymphohistiocytic infiltrate (mostly T cell type) are seen within myocardial fibers, myocyte necrosis is typically present and presence of interstitial fibrosis of varying degree. Presence of edema should not be used as a criterion for diagnosis of myocarditis.

There are only a few studies on lymphocytic myocarditis and association with SARS-CoV-2 infection using IHC markers reported so far from India.

Myocarditis is an inflammatory disease of the myocardium diagnosed by established histological (Dallas criteria) , immunological, and immunohistochemical criteria (>14 leucocytes/mm2 including up to 4 monocytes/mm2 with the presence of CD3 positive T-lymphocytes >7 cells/mm2).(6) According to Dallas criteria, myocarditis is diagnosed in the setting of an "inflammatory infiltrate of the myocardium with necrosis and/or degeneration of adjacent myocytes, not typical of ischemic damage associated with coronary artery disease".(7)

In an autopsy based study in Germany, DianaLindner et aldocumented SARS-CoV-2 in the cardiac tissue by in situ hybridization in 24 of 39 patients (61.5%) in the absence of increased infiltration of mononuclear cells into the myocardium and those patients were not having fulminant myocarditis clinically.(8)

OBJECTIVES

1. To determine the presence of SARS- COV-2 infection with the help of IHC marker in diagnosed cases of lymphocytic myocarditis.

2. To characterize the cellular infiltrate of lymphocytic myocarditis using IHC markers

Study Design: Descriptive record based study Study Subjects All autopsy cases reported as lymphocytic myocarditis in the Department of Pathology, Government Medical College, Manjeri from 1st April 2020 to 31st March 2021, irrespective of the cause of death. The study includes 37 cases, reported as lymphocytic myocarditis. In the year 2019 there were only 4 cases of lymphocytic myocarditis. Since there was an increase in the number of lymphocytic myocarditis in the Covid 19 pandemic, we tried to find out whether it is related to Covid 19 infection or not. Inclusion Criteria The autopsy specimens of cardiac tissue with features of lymphocytic myocarditis were included. Exclusion Criteria Autopsy specimens of cardiac tissue with histologic features of ischemic heart disease, tissues with autolytic changes and tissues not adequate for IHC studies were excluded. Data Collection Tool To retrieve and review the slides filed in the histopathology lab reported as Lymphocytic myocarditis and perform IHC markers-antibodies to CD3, СD68, SARS-CoV-2, in the paraffin embedded tissue blocks. Analysis Entry in Excel sheet and analysis by SPSS. Univariables are to be expressed in mean and percentage. Methods All sequential autopsies that met the histological criteria given in the operational definition were included. We retrieved and reviewed the slides filed in the histopathology lab reported as Lymphocytic myocarditis and performed Immunohistochemistry (IHC) using the IHC markers- antibodies to CD3, СD68, SARS-CoV-2, in the paraffin embedded tissue blocks of cardiac tissue. Information provided included the presence of myocarditis and other patterns of inflammation and injury identified on haematoxylin and eosin- (H&E) stained slides of the heart. The degree of coronary artery stenosis and the presence of disrupted plaques were also provided. The assessment of the myocardium by light microscopy using Labomed LX 500 and evaluation of immunohistochemical stains for CD68, CD3 and SARS–COV / SARS–COV -2 (COVID 19) spike antibody (1:100 to 1: 500; GeneTex International corporation North America) were done in all selected cases. We standardised the covid antibody by obtaining posivity in cases of autopsy of lung tissue which were already diagnosed as covid positive by RT PCR (real-time reverse transcriptase polymerase chain reaction assays). For immunohistochemical stains, the number of cells staining in the × 400 high-power field with the most inflammation were counted. All immunohistochemical stains were performed by manual method. RESULTS In this study, a total of 37 subjects were included and all cases satisfied our defining criteria. The subjects had an age range from 6 years old to 72 years, with a mean age of 33.13 years (Majority of subjects belonged to the age group of 11 to 20 years. Of all the study subjects, 24 were males (64.9%) 13 (35.1%) were females. Figure 1: Age distribution of cases in the study group Figure 2: Sex distribution of cases in the study group Figure 3: Histology subtype of myocarditis based on H and E sections Of the total 37 cases, the histology results by studying the H and E stained sections showed the above results. Most of the cases satisfied the definition of lymphocytic myocarditis, the number of such cases were 27 (73%) followed by Mild lymphocytic myocarditis in 5 cases (13.5%), the 3 (8.1%) cases of Borderline lymphocytic myocarditis and one case of each of florid and acute lymphocytic myocarditis Immunohistochemistry Findings The myocardial cells were were positive for by SARS–COV / SARS–COV -2 (COVID19) spike antibody by IHC in one case of florid and one case of acute lymphocytic myocarditis. All 37 cases were negative for IHC markers CD 68 and CD 3. Immunohistochemistry marker Test result ( number of cases ) Positive Negative Monoclonal antibody CD 68 0 37 Monoclonal antibody CD 20 0 37 Monoclonal antibody CD 3 0 37 SARS–COV / SARS–COV -2 (COVID19) spike antibody 2 35 Table 1: Immunohistochemistry findings of IHC markers in the study group Image 1: Lyphocytic myocarditis with myocardial edema

Study Design: Descriptive record based study

Study Subjects

All autopsy cases reported as lymphocytic myocarditis in the Department of Pathology, Government Medical College, Manjeri from 1st April 2020 to 31st March 2021, irrespective of the cause of death. The study includes 37 cases, reported as lymphocytic myocarditis. In the year 2019 there were only 4 cases of lymphocytic myocarditis. Since there was an increase in the number of lymphocytic myocarditis in the Covid 19 pandemic, we tried to find out whether it is related to Covid 19 infection or not.

Inclusion Criteria

The autopsy specimens of cardiac tissue with features of lymphocytic myocarditis were included.

Exclusion Criteria

Autopsy specimens of cardiac tissue with histologic features of ischemic heart disease, tissues with autolytic changes and tissues not adequate for IHC studies were excluded.

Data Collection Tool

To retrieve and review the slides filed in the histopathology lab reported as Lymphocytic myocarditis and perform IHC markers-antibodies to CD3, СD68, SARS-CoV-2, in the paraffin embedded tissue blocks.

Analysis

Entry in Excel sheet and analysis by SPSS. Univariables are to be expressed in mean and percentage.

Methods

All sequential autopsies that met the histological criteria given in the operational definition were included. We retrieved and reviewed the slides filed in the histopathology lab reported as Lymphocytic myocarditis and performed Immunohistochemistry (IHC) using the IHC markers- antibodies to CD3, СD68, SARS-CoV-2, in the paraffin embedded tissue blocks of cardiac tissue.

Information provided included the presence of myocarditis and other patterns of inflammation and injury identified on haematoxylin and eosin- (H&E) stained slides of the heart. The degree of coronary artery stenosis and the presence of disrupted plaques were also provided. The assessment of the myocardium by light microscopy using Labomed LX 500 and evaluation of immunohistochemical stains for CD68, CD3 and SARS–COV / SARS–COV -2 (COVID 19) spike antibody (1:100 to 1: 500; GeneTex International corporation North America) were done in all selected cases. We standardised the covid antibody by obtaining posivity in cases of autopsy of lung tissue which were already diagnosed as covid positive by RT PCR (real-time reverse transcriptase polymerase chain reaction assays).

For immunohistochemical stains, the number of cells staining in the × 400 high-power field with the most inflammation were counted. All immunohistochemical stains were performed by manual method.

RESULTS

In this study, a total of 37 subjects were included and all cases satisfied our defining criteria. The subjects had an age range from 6 years old to 72 years, with a mean age of 33.13 years (Majority of subjects belonged to the age group of 11 to 20 years. Of all the study subjects, 24 were males (64.9%) 13 (35.1%) were females.

Of the total 37 cases, the histology results by studying the H and E stained sections showed the above results. Most of the cases satisfied the definition of lymphocytic myocarditis, the number of such cases were 27 (73%) followed by Mild lymphocytic myocarditis in 5 cases (13.5%), the 3 (8.1%) cases of Borderline lymphocytic myocarditis and one case of each of florid and acute lymphocytic myocarditis

Immunohistochemistry Findings

The myocardial cells were were positive for by SARS–COV / SARS–COV -2 (COVID19) spike antibody by IHC in one case of florid and one case of acute lymphocytic myocarditis. All 37 cases were negative for IHC markers CD 68 and CD 3.

This autopsy-based study evaluated lymphocytic myocarditis during the COVID-19 pandemic to determine its association with SARS-CoV-2 infection. Although an increased number of myocarditis cases was observed during the study period, direct evidence of SARS-CoV-2 myocardial infection was identified in only two cases.

These findings are consistent with international autopsy studies showing low prevalence of true viral myocarditis despite frequent myocardial injury in COVID-19 deaths8-11. Increasing evidence suggests that myocardial injury in COVID-19 is often mediated by systemic inflammatory responses, cytokine storm, hypoxia, endothelial dysfunction, and thrombotic complications rather than direct viral cytopathic effects.(9-12)

Preliminary observations in the literature, together with the observations in this series, suggest that myocardial injury with or without depressed cardiac function in our study group may result from aetiologies other than viral myocarditis. Acute myocardial tissue injury and inflammatory infiltrate might be related to elevated cytokines, hypoxaemia, right ventricular strain, and thrombotic complications. Details of the cardiac pathology from autopsies of patients dying with COVID-19 are currently very limited. Despite the high mortality rate worldwide, only a few studies, comprising a small number of patients, have so far reported information concerning the cardiac pathology in these patients.(8,9,10,11) Limitation of our Study A value of >14 leukocytes/mm2 with the presence of T lymphocytes >7 cells/mm2 has been considered a cutoff to reach a diagnosis of myocarditis. We selected the cases purely based on hematoxyline and eosin stained sections without IHC and after a period of more than 10 days of viral infection the markers need not be positive always. Ethical Considerations The research protocol was submitted to obtain approval from Institutional Research Committee (IRC) and Institutional Ethics Committee, Government. Medical College, Manjeri. The details were collected from histopathology records after obtaining permission from the Head of the Department of Pathology. The financial assistance was given by SBMR. Conflict of Interest: Nill

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