Pathological Evidence for SARS-CoV-2 as a Cause of Myocarditis

JACC Review Topic of the Week

Rika Kawakami, MD; Atsushi Sakamoto, MD; Kenji Kawai, MD; Andrea Gianatti, MD; Dario Pellegrini, MD; Ahmed Nasr, MD; Bob Kutys, PA; Liang Guo, PHD; Anne Cornelissen, MD; Masayuki Mori, MD; Yu Sato, MD; Irene Pescetelli, MD; Matteo Brivio, MD; Maria Romero, MD; Giulio Guagliumi, MD; Renu Virmani, MD; Aloke V. Finn, MD

Disclosures

J Am Coll Cardiol. 2021;77(3):314-325. 

In This Article

CVpath Experience

In a series of 384 autopsy cases (239 cardiac, 51 noncardiac, and 94 non-natural deaths), inflammatory infiltrates were found in the heart in 18% of all cases, with multifocal infiltrates in 9%. Incidental infiltrates were most frequent in natural noncardiac deaths (31%), but were also seen in drug-related deaths (20%) and cardiac deaths (16%), and were least frequent in traumatic deaths (16%).[21] We previously reported autopsy findings in the hearts of patients dying during viral infection (in this case during the AIDS epidemic) and showed that focal mild inflammatory infiltrates not meeting the definition of myocarditis in the heart were not uncommon (31%).[20] We also found focal mild mononuclear inflammatory infiltrates in 10 of 24 (42%) hearts in sudden traumatic deaths. Because these foci were not associated with myocyte necrosis, they were not diagnosed as myocarditis.

We have examined the hearts of 15 subjects dying with COVID-19 infection from the Lombardy region outbreak (Papa Giovanni Hospital, Bergamo, Italy) and 1 case from the Medical Examiner in Baltimore, Maryland. The hearts of 15 unselected patients dying from COVID-19 at Ospedale Papa Giovanni XXIII (Bergamo, Italy) and undergoing autopsy were collected and sent to CVPath Institute (Gaithersburg, Maryland) for detailed pathological analysis. The study protocol was reviewed and approved by the ethical committee at Ospedale Papa Giovanni XXIII Bergamo (2020-0056) and by CVPath Institute IRB (RP0112), and was registered at ClinicalTrials.gov (NCT04367792). The specimens were anonymized before shipping, and the examining pathologist (R.V., M.R., R.K.) was blinded to the clinical details. All tissue specimens were fixed in 10% buffered formalin for at least 72 to 96 h prior to shipment. Whole hearts and lungs (either paraffin blocks or tissues), were sent to CVPath Institute for pathological examination.

As shown in Table 2, the average age of the subjects was 70 years, and 69% were men. In total, 4 subjects had a history of prior cardiovascular intervention and 4 had other relevant past medical histories. The average length of hospitalization until death was 6 days. A complete autopsy with detailed cardiac examination (histological sections examined from all 4 walls, at 2 levels of the heart) was conducted. Overall, none of the cases met the criteria for myocarditis, although 3 had evidence of either microvascular or epicardial thrombosis in the setting of acute myocardial infarction, and these cases all had neutrophilic infiltration. Inflammatory cell infiltration was found in the epicardium in 10 of 16 cases (63%), consisting mostly of lymphocytes, and myocardial mononuclear inflammatory cell infiltration was found in 5 of 16 (31%) cases (Table 3).

Total RNA was extracted from myocardial tissue (1 sample from each chamber of the heart) from each of the 16 cases. Concentration and quality of RNA samples were measured. Quantitative reverse transcriptase PCR was performed using specifically designed primers for SARS-CoV-2 (N1 and N2 from CDC EUA essay). Virus copy number was quantified based on the reverse transcriptase PCR results with standard control containing the complete nucleocapsid gene from SARS-CoV-2. RNase P was used as a control. The detection of SARS-CoV-2 RNA was determined by the amplification at Ct ≤40.[33] In only 2 cases was the virus detected in the heart by PCR and both were in the atria (1 left atrium and 1 right atrium) and not in the ventricles, and neither was accompanied by inflammation, although in most of the 16 cases the virus was detected in the lung (Table 3).

We also evaluated the nature of the inflammatory cells found in the myocardium in cases of (noninfectious) traumatic (control) versus COVID-19 deaths. We examined histological sections from the left ventricles from 5 randomly selected cases of control death from our CVPath registry and from 5 randomly selected cases of subjects dying of COVID-19 (see Table 2 for list of cases selected). We stained left ventricular myocardial sections using antibodies against CD3 (T-cell marker) and CD68 (macrophage). Overall, there was no difference in the total number of T cells and macrophages in the 2 groups (Figure 1). However, the cell counts in COVID-19 hearts were higher than what others have used to diagnose myocarditis. We believe because these cells are scattered and not accompanied by myocyte necrosis, these cases do not fulfill the criteria of myocarditis. Moreover, CD3 cells were significantly more frequent in control cases than in COVID-19 cases, whereas macrophages were more frequent in COVID-19 than in control cases, but we did not determine their exact location (i.e., interstitium vs. intravascular). Figure 1 contrasts these results with a typical case of mild myocarditis from the CVPath biobank, which showed greater CD3 abundance than macrophages, but both are typically more frequently observed in myocarditis than in control deaths and COVID-19 cases.

Figure 1.

Myocardial Inflammatory Cell Infiltrates in Cases of Traumatic Death, COVID-19, and Myocarditis
Representative low and high power of hematoxylin and eosin images, and immunostains for CD3 and CD68 from cases of traumatic death (control) (A to D and P), COVID-19 (E to H), and myocarditis (I to L). Bar graphs represent total inflammatory (i.e., CD3 and CD68) cell counts in control subjects and COVID-19 cases (n = 5 each) (M, N, and O). A photomicrograph of single-cell necrosis with CD3 and CD68 staining is shown in P. CD3 = cluster of differentiation 3; CD68 = cluster of differentiation 68; COVID-19 = coronavirus disease-2019; SARS-CoV-2 = severe acute respiratory syndrome-coronavirus-2.

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