Comparative Study of the Myocardium of Patients from Four COVID-19 Waves

Abstract

Background: Few studies have compared COVID-19 patients from different waves. This study aims to conduct a clinical and morphological analysis of patients who died from COVID-19 during four waves.

Methods: The study involved 276 patients who died from COVID-19 during four waves, including 77 patients in the first wave, 119 patients in the second wave, and 78 patients in the third wave. We performed a histological examination of myocardium samples from autopsies and additionally analyzed the samples by PCR. We conducted immunohistochemistry of the myocardium for 21 samples using antibodies against CD3, CD45, CD8, CD68, CD34, Ang1, VWF, VEGF, HLA-DR, MHC1, C1q, enteroviral VP1, and SARS-CoV-2 spike protein. We also did immunofluorescent staining of three myocardial specimens using VP1/SARS-CoV-2 antibody cocktails. Further, we ran RT-ddPCR analysis for 14 RNA samples extracted from paraffin-embedded myocardium. Electron microscopic studies of the myocardium were also performed for two samples from the fourth wave.

Results: Among the 276 cases, active myocarditis was diagnosed in 5% (15/276). Of these cases, 86% of samples expressed VP1, and individual cells contained SARS-CoV-2 spike protein in 22%. Immunofluorescence confirmed the co-localization of VP1 and SARS-CoV-2 spike proteins. ddPCR did not confidently detect SARS-CoV-2 RNA in the myocardium in any myocarditis cases. However, the myocardium sample from wave IV detected a sub-threshold signal of SARS-CoV-2 by qPCR, but myocarditis in this patient was not confirmed. Electron microscopy showed several single particles similar to SARS-CoV-2 virions on the surface of the endothelium of myocardial vessels. A comparison of the cardiovascular complication incidence between three waves revealed that the incidence of hemorrhage (48 vs. 24 vs. 17%), myocardial necrosis (18 vs. 11 vs. 4%), blood clots in the intramural arteries (12 vs. 7 vs. 0%), and myocarditis (19 vs. 1 vs. 6%) decreased over time, and CD8-T-killers appeared. Immunohistochemistry confirmed the presence of endotheliitis in all 21 studied cases.

Conclusions: This study compared myocardial damage in patients who died during three COVID-19 waves and showed a decrease in the incidence of endotheliitis complications (thrombosis, hemorrhage, necrosis) and myocarditis over time. However, the connection between myocarditis and SARS-CoV-2 infection remains unproven.

Keywords: COVID-19 waves; immunofluorescent myocardial study; immunohistochemical; myocarditis; polymerase chain reaction; ultrastructural myocardial study.

Figure 1

Large polymorphic cells in the myocardium of patient with coronavirus infection. (a)—perivascular large polymorphic cells with nuclear hypertrophy; H&E; (b)—CD34 expression in large polymorphic cells, (c)—CD68 expression in large polymorphic perivascular cells, (d)—VEGF expression in large polymorphic, activated endotheliocytes; ×200.

Figure 2

Immunohistochemical study of the myocardium of patients with active myocarditis. (a)—active lymphocytic myocarditis; H&E, ×100; (b)—CD45 expression on lymphocytes of a large infiltrate in the myocardium; (c,d)—expression of CD3, CD8 on infiltrate lymphocytes (respectively); (e–h)—expression of CD34, VEGF, Ang2, and VWF (respectively) on activated endotheliocytes and Ang2 in cardiomyocytes; (i,j)—expression of MHC1 and HLA-DR (respectively) on vessels and infiltrate cells; ×200.

Figure 3

Immunohistochemical study of the myocardium of patients with myocarditis. (a–c)—SARS-CoV-2 Spike protein expression in vascular endothelium, macrophages, and rare cardiomyocytes; (d)—VP1 expression of enteroviruses in myocardial vessels; (a)—×100, (b–d)—×200.

Figure 4

RT-ddPCR analysis of RNA. Single non-reproducible SARS-CoV-2-positive droplets have been identified using FFPE samples of myocardial tissue from patients with myocarditis (marked with a red oval). (a) Results of ddPCR for Act-B transcript, isolated from FFPE samples. Multiple positive droplets confirm suitability of RNA for ddPCR analysis; (b) Results of ddPCR for Sars-Cov-2 specific RNA (gene N). Single non-reproducible positive droplets have been identified (marked with a red oval).

Figure 5

Immunofluorescent study of the myocardium with a cocktail of antibodies against SARS-CoV-2 Spike protein/enterovirus VP1 protein. Green represents fluorescence of CoV-2 Spike protein, red represents fluorescence of enterovirus VP1, yellow or orange represent fluorescence of colocalization of viral antigens. Nuclei were counterstained with DAPI. (a–d)—×200. (e–h)—×630.

Figure 6

Electron microscopy examination of myocardial vessels. On the apical surface of endotheliocytes, membrane particles morphologically similar to SARS-CoV-2 virions are found (arrows). Legends: e—endotheliocytes, bm—basal membrane, ec—erythrocytes.

Figure 7

Changes in the myocardium during coronavirus infection. (a)—large polymorphic cells in the myocardium; ×200; (b)—non-coronary ischemic necrosis; ×100, (c)—hemorrhagic myocardial infarction; ×100; (d)—thrombi in intramural arteries, myocardial necrosis; ×100; (e)—focal plasma impregnation of the intima of the intramural arteries; ×100; (f)—active lymphocytic myocarditis; ×100; H&E.

Figure 8

Columns of different colors demonstrate the severity of the feature in the wave.