Supplementary appendix

This appendix formed part of the original submission. We post it as supplied by the
authors.

Supplement to: Varga Z, Flammer AJ, Steiger P, et al. Endothelial cell infection and
endotheliitis in COVID-19. Lancet 2020; published online April 17. http://dx.doi.org/
10.1016/S0140-6736(20)30937-5.
 Supplementary Appendix

Table of Contents

1. Detailed case histories of case 1, 2 and 3

2. Detailed methods of pathology sampling
3. Supplementary figure
Case 1: A 71 years old kidney transplanted male, admitted on March 20th, 2020 with
dyspnea, fever, tachycardia, hypotension, and confusion and tested positive for
COVID-19. Notably, the patient had pre-existing coronary artery disease and arterial
hypertension. He rapidly required mechanical ventilation because of severe hypoxic
failure, renal replacement therapy and norepinephrine and vasopressin for septic
shock. Cardiac biomarkers (NTproBNP: 10’456ng/l, Troponin T: 51ng/l), inflammation
parameters (CRP: 232mg/l) and D-dimers (2.42mg/l) were elevated. Radiologic
findings included bilateral infiltration and ground glass opacities with consolidations in
the right lung. Echocardiography showed preserved left ventricular ejection fraction,
but a severely enlarged left atrium (59ml/m2) indicating longstanding diastolic
dysfunction. Treatment included hydroxychloroquine and empiric antibiotic treatment
with piperacillin/tazobactam and levofloxacin as well as anticoagulation with
unfractionated heparin (UFH). After one week, the patient could not be weaned,
neither from vasoactive drugs, nor from renal replacement therapy and ventilation. He
developed abdominal distension and impaired passage (reflux of > 500ml). A CT-scan
revealed pneumatosis intestinalis of the jejunum and air within the portal vein as signs
of mesenteric ischemia. In accordance to the patient’s presumed will, palliative care
was provided and the patient died on day 8 after admission with clinical symptoms of
sepsis.
Autopsy demonstrated mesenteric ischemia complicated by severe diffuse alveolar
damage in the lungs. Postmortem analysis of kidney transplants by electron
microscopy revealed viral inclusion structures in endothelial cells of the transplanted
kidney (Fig. 1A and Fig 1B). Histologically, there was a prominent endotheliitis with
recruitment of inflammatory cells, as well as an unusual high amount of apoptotic
bodies in many organs, especially in the pulmonary vessels (Fig. 1D) but also in small
bowel and heart (Supplementary figure). In addition, a strong accumulation of
mononuclear cells was found in the lung, leading to congestion of most small lung
vessels.

Case 2: A 58 years old women with preexisting diabetes mellitus type 2, arterial
hypertension and obesity had cough, fever and dyspnea for 3 days at home. She was
directly admitted to the intensive care unit on March 18th 2020 because of very rapidly
progressive respiratory failure due to severe acute respiratory distress syndrome
(ARDS). On admission, cardiac (CK: 1387 U/l, myoglobin: 1033ug/l, troponin T: 29ng/)
and inflammation biomarkers (CRP: 342ng/l, PCT: 3.43ng/l) were elevated, D-dimer
was 0.83mg/l. Echocardiography showed normal sized left ventricle with concentric
remodeling and normal ejection fraction (EF 65%) without regional wall motion
abnormalities. Treatment included hydroxychloroquine and empiric antibiotic
treatment, as well as thrombo-prophylaxis with UHF. Within the first week after
admission, the patient developed multi-organ failure requiring ventilation and renal
replacement therapy. On day 16, mesenteric ischemia developed and the necrotic
small intestine had to be removed. Liver failure, circularity failure and signs of cardiac
ischemia including new inferior ST-segment elevation in the ECG were noted and
echocardiography demonstrated new inferior akinesia suggestive for an acute right
coronary artery occlusion. Right heart failure occurred and the patient died.
Autopsy revealed histology signs of ARDS and lymphocytic endotheliitis in lung, heart,
kidney and liver with massive centrilobular and parenchyma necrosis. In the ICU a
myocarditis had been suspected. Histology showed an acute posterior myocardial
infarction, but no signs of viral lymphocytic myocarditis. Histology of the resected small
intestine showed mucosal ischemic necrosis as well as prominent endotheliitis and
many apoptotic bodies of the submucosal vessels with only scattered fibrin thrombi,
almost identical to case 3.

Case 3: A 69 years old male with preexisting arterial hypertension but otherwise
healthy. He developed cough, fever and dyspnea on March 11th 2020. On March
20th he was admitted to an external hospital and was tested positive for COVID-19 on
the same day. After developing respiratory failure, the patient was intubated on March
28th and was urgently transferred to our hospital for further intensive care treatment.
On admission, the patient developed diffuse bilateral infiltrates suggestive for COVID-
19 induced ARDS. Cardiac biomarkers were normal or only mildly elevated (CK: 22U/l,
myoglobin: <21ug/l, Troponin: 17ng/l), inflammatory parameters were elevated (CRP:
286mg/l, PCT 0.23ug/l and Il-6: 289ng/l) as well as D-dimers (8.8g/l). The patient
needed vasopressor support (norepinephrine) and developed atrial
fibrillation. Echocardiography showed a dilated left ventricle with severely reduced
ejection fraction (25%) and diffuse hypokinesia as well as moderate mitral
regurgitation. Due to his COVID infection, the patient was treated with
hydroxychloroquine and prophylactic piperacillin/tazobactam. Anticoagulation with
UFH was given from the beginning of intensive care treatment. Unfortunately, 10 days
after being tested positive and after two days in intensive care unit, mesenteric
ischemia developed and small intestine resection (300cm) had to be performed. The
patient is still alive.
Histology of the small intestine resection demonstrated ischemic mucosal necrosis
and prominent endotheliitis of the submucosal vessels along with a large amount of
apoptotic bodies (Fig. 1C).

Detailed methods of pathology sampling

Informed consent for autopsy was available in both deceased patients. Patients
underwent a complete autopsy in a dedicated room for infectious diseases, including
histological assessment of each organ. Z.A. and M.H. reviewed macroscopic and
microscopic findings. Formalin fixed paraffin embedded tissues were analyzed. For
histology (hematoxylin and eosin) and immunohistochemistry (Caspase-3), sections
of 3-4 micrometer thickness were used. All sections were evaluated on light
microscope
Caspase-3 protein was assessed by the polyclonal Cleaved Caspase-3 antibody (Cell
Signaling Asp 175; dilution 1.:300). The whole staining procedure (including
pretreatment and staining) was conducted with the Ventana Benchmark semi-
automated staining system using Ventana reagents for the entire procedure (including
iVIEW DAB detection kit and the signal was enhanced using the amplification kit).
Electron microscopy of the kidney was performed using standard protocols for renal
biopsy processing at the electron microscopy unit of the Department of Pathology and
Molecular Pathology, University Hospital Zürich. Electron microscopy tissue was
processed and embedded in a plastic, hard media, and semi-thick sections were
stained with toluidine blue to identify the specific area to be cut for thin sections to be
placed on a grid for electron microscopy examination.

Supplementary figure
Post-mortem findings in myocardial tissue: Presence of vascular changes without
lymphocytic myocarditis. Star: accumulation of intravascular mononuclear cells. Inset:
Minimal heart endotheliitis with scattered lymphocytes beneath the endothelium
(arrows). Hematoxylin-eosin staining Original magnification, X400