Chronic biopsy proven post‐COVID myoendocarditis with SARS‐Cov‐2 persistence and high level of antiheart antibodies

Abstract Purpose To study the clinical signs and mechanisms (viral and autoimmune) of myoendocarditis in the long‐term period after COronaVIrus Disease 2019 (COVID‐19). Methods Fourteen patients (nine male, 50.1 ± 10.2 y.o.) with biopsy proven post‐COVID myocarditis were observed. The diagnosis of COVID‐19 was confirmed by IgG seroconversion. The average time of admission after COVID‐19 was 5.5 [2; 10] months. An endomyocardial biopsy (EMB) of the right ventricle was obtained. The biopsy analysis included polymerase chain reaction diagnosis of viral infection, morphological, immunohistochemical (IHC) examination with antibodies to CD3, CD45, CD68, CD20, SARS‐Cov‐2 spike, and nucleocapsid antigens. Coronary atherosclerosis was ruled out in all patients over 40 years. Results The new cardiac symptoms (congestive heart failure 3–4 New York Heart Association class with severe right ventricular involvement, various rhythm, and conduction disturbances) appeared 1–5 months following COVID‐19. Magnetic resonance imaging showed disseminated or focal subepicardial and intramyocardial late gadolinium enhancement, hyperemia, edema, and increased myocardial native T1 relaxation time. Antiheart antibodies levels were increased 3–4 times in 92.9% of patients. The mean left ventricular (LV) ejection fraction (EF) was 28% (24.5; 37.8). Active lymphocytic myocarditis was diagnosed in 12 patients, eosinophilic myocarditis in two patients. SARS‐Cov‐2 RNA was detected in 12 cases (85.7%), in association with parvovirus B19 DNA—in one. Three patients had also endocarditis (infective and nonbacterial, with parietal thrombosis). As a result of steroid and chronic heart failure therapy, the EF increased to 47% (37.5; 52.5). Conclusions COVID‐19 can lead to long‐term severe post‐COVID myoendocarditis, that is characterized by prolonged persistence of coronavirus in cardiomyocytes, endothelium, and macrophages (up to 18 months) in combination with high immune activity. Corticosteroids and anticoagulants should be considered as a treatment option of post‐COVID myoendocarditis.


| INTRODUCTION
The COronaVIrus Disease 2019 (COVID-19) pandemic has been going on for almost 2 years and largely influences routine clinical practice. The possibility of acute coronavirus-induced myocarditis was demonstrated in a series of studies. The first detection of coronavirus particles by electron microscopy in myocardial biopsy of a patient with COVID-19 and cardiogenic shock has been described, 1 as well as identification of RNA virus in myocardium of patients with myocarditis. 2 We have published a description of pancarditis in autopsy findings in patients with COVID-19 3 ; a little later, SARS-Cov-2 RNA was detected in the myocardium of all patients. However, descriptions refer only to cases of acute coronavirus myocarditis.
The maximum time since infection, in which RNA-positive myocarditis has been detected, is only 4 weeks. 4 The terms "post-COVID," "longterm," and "chronic" are not yet used in relation to coronavirus myocarditis. However, the ability of SARS-Cov-2 to induce prolonged, sustained over time, general and specific symptoms is accepted. This has given rise to the term "post-COVID syndrome." Inflammatory myocardial injury could be a component of this syndrome, which requires specific investigation using an endomyocardial biopsy (EMB).

| Purpose
To study the clinical signs and mechanisms (viral and autoimmune) of myoendocarditis in the long-term period after acute COVID-19.

| Comorbidities
The mean body mass index was 28.3 ± 3.5 kg/m 2 . Seven patients had a history of arterial hypertension, which was well controlled. One patient had a history of smoking, one-atopic bronchial asthma, onediabetes mellitus, and one-bicuspid aortic valve without significant valvular dysfunction. After COVID-19, all patients experienced prominent cardiac symptoms. The mean time to clinic admission after COVID-19 was 5.5 [2; 10] months, ranging from 2 to 9 months; the time to onset of symptoms after acute coronavirus infection was 1-5 months. In two cases, symptoms of myocarditis appeared not after COVID-19, but only after subsequent vaccination.  on the publicly available data on gene DNA and mRNA sequences in the NCBI database, Primer-BLAST software was used. The evaluation of antiheart antibodies (AHA) titers by indirect immunofluorescence method, echocardiography (EchoCG), 24-h ECG monitoring, coronary angiography, and cardiac MRI (n = 6) were also performed.

| Methods
Statistical analysis was performed using IBM SPSS statistics v.22.

| Ethical approval
The investigation is conform to the principles outlined in the Declaration of Helsinki. All patients signed an informed consent to participate in this study, which was approved by the local ethics committee of Sechenov University.

| RESULTS
3.1 | The general clinical characteristics of the patients are presented in Table 1 All patients were admitted for class 3-4 by New York Heart Association (NYHA) CHF. In 11 patients, it was biventricular (with peripheral edema, hepatomegaly, effusion in the cavities). We observed the moderate dilatation of all heart chambers and decreased LV ejection fraction (EF) up to 20%-30% ( Figure 1A,B). High pulmonary hypertension and signs of pulmonary embolism were not found, which ruled out the secondary cause of myocardial dysfunction.

| Results of morphological and IHC myocardial studies
A correlation of the clinical data of the patients with the myocardial morphological studies is presented in Table 2.
The diagnosis of active myocarditis was confirmed in all cases.
The evidence of activity included signs of cardiomyocytes' death (necrosis, lysis) and severe dystrophy, as well as interstitial edema in all patients, Figure 2A

| Results of virological (PCR and IHC) testing of myocardium
Real-time PCR analysis of myocardium detected no adenovirus or herpetic virus genome. Parvovirus B19 DNA was found in one patient (see Table 2). SARS-Cov-2 RNA was detected in 12 from 14 patients.
The maximal time after COVID-19, when the virus was detected in the myocardium of a patient with active myocarditis, was 18 months (

| Endocarditis in patients with post-COVID myocarditis
Concomitant endocarditis was diagnosed in four patients and manifested with two forms.
1. IE was diagnosed in a 39 y.o. patient with severe myocarditis (see Table 2) and a bicuspid aortic valve. The manifestations of IE included prolonged febrile fever, weight loss over 20 kg, progression of aortic stenosis and regurgitation, vegetations on the valve leaflets ( Figure 1C), splenomegaly, severe increasing in inflammatory markers with negative blood culture. Progressive CHF with a persistent decrease of LV EF up to 25% was observed. EMB revealed an active lymphocytic myocarditis.
Biopsy material culture and PCR analysis did not detect any bacteria.
There was no clinical suspicion of IE or parietal thrombosis (including MRI). Anticoagulant therapy was administered. EMB revealed signs of lymphocytic endocarditis with endocardial thickening and sclerosis, thrombotic masses. As well we observed a parietal thrombosis without endocarditis.
A common feature of patients with a combination of myo-and endocarditis was the detection of SARS-Cov-2 RNA in the myocardium and a low titer of antibodies to endothelium antigens in the blood, which may be considered as a consequence of escape of these antibodies to the endothelium as part of immune complexes. Other AHA titres were significantly elevated.  Table 2). In five This is primarily due to AНA production, which we detected in 73.5%

| Treatment approaches for decompensated post-COVID myocarditis
of inpatients with coronavirus pneumonia in acute COVID-19. 8 In the present study, high AHA titers were detected in 93% of patients;

DATA AVAILABILITY STATEMENT
All data generated or analyzed during this study are included in this article.