SARS-CoV-2 Endothelial Infection Causes COVID-19 Chilblains

Histopathological, Immunohistochemical and Ultrastructural Study of Seven Paediatric Cases

I. Colmenero; C. Santonja; M. Alonso-Riaño; L. Noguera-Morel; A. Hernández-Martín; D. Andina; T. Wiesner; J.L. Rodríguez-Peralto; L. Requena; A. Torrelo

Disclosures

The British Journal of Dermatology. 2020;183(4):729-737. 

In This Article

Results

Clinical Features

Our seven patients (four male, three female; 11–17 years old) had skin lesions on the toes and lateral aspects of their feet and heels that were consistent with chilblains (Figure 1). They were minimally painful or pruritic. The hands were also affected in one patient, who had associated lesions of erythema multiforme on the elbows and knees.[7] Of note, no patient had a history of rheumatic disease, lupus erythematosus, Raynaud phenomenon, acrocyanosis or previous chilblains, and two of them were on treatment with methylphenidate hydrochloride for more than 1 year at the same dose for attention deficit hyperactivity disorder. The lesions had been present for 4–30 days before the biopsy procedure (Table 1). In all patients, the lesions had a benign outcome, with no significant systemic complaints, and gradual spontaneous resolution was achieved in all of them after 8 weeks of follow-up.

Figure 1.

(a, b) Case 2 and (c, d) case 6. Clinical spectrum of perniotic acral ischaemic lesions.

SARS-CoV-2 PCR from nasopharyngeal and oropharyngeal swabs was negative in all cases tested (six of six). PCR tests were performed between 1 and 21 days (median 10) from the beginning of the skin lesions. Coagulation studies were normal in six patients tested. D-dimer levels in serum were measured in six cases and were minimally elevated in one case (900 ng mL−1; normal < 500 ng mL−1), but this abnormal result had no clinical significance, with the patient showing good health, no systemic symptoms and other coagulation tests within normal limits. Full blood count was normal in all tested cases.

Histopathology

The biopsies showed similar findings with variable intensity (Table 2). All showed a mild interface dermatitis featuring vacuolar degeneration of the basal epidermal layer. Exocytosis of lymphocytes was seen in three cases and scattered necrotic keratinocytes in four. Lymphocytic vasculitis was demonstrated in all biopsies. Lymphocytes infiltrated the wall of dermal venules and arterioles. Endotheliitis, defined as swollen endothelial cells separated from the underlying basement membrane by subendothelial lymphocytes, was frequently observed (Figure 2). Indirect features of vascular damage such as red cell extravasation and dermal oedema were present in all cases. Fibrinoid necrosis of vessels was seen in two biopsies and microthrombosis in four. These thrombi were noted in papillary dermal capillaries and also involved reticular dermis vessels (Figure 3). Transmural lymphocytic infiltration of a large subcutaneous vessel, not associated with fibrinoid necrosis or thrombosis, was noted in one case.

Figure 2.

Case 6, skin biopsy. (a) Acral skin showing superficial and deep perivascular inflammation extending into the subcutis and papillary dermal oedema [haematoxylin and eosin (H&E), original magnification × 20]. (b) Mild exocytosis and vacuolar degeneration (H&E, × 200). (c–f) Lymphocytic infiltration of vessel walls. Note the lifting of endothelium with underlying lymphocytes in (e) (arrow) and transmural inflammation of a large subcutaneous vessel in (f). H&E, original magnification (c, e) × 400; (d, f) × 200.

Figure 3.

Case 2, skin biopsy. (a) Acral skin showing superficial and deep perivascular inflammation extending into the subcutis [haematoxylin and eosin (H&E), original magnification × 20]. (b) Mild papillary dermal oedema, vacuolar degeneration of the basal layer and lymphocytic exocytosis together with prominent red cell extravasation (H&E, × 100). (c) Thrombi in superficial dermis vessels (H&E, × 200). (d–f) Lymphocytic vasculitis with thrombosis and fibrin deposition in vessel walls. H&E, original magnification (d) × 200, (e, f) × 400.

A superficial and deep angiocentric and eccrinotropic lymphocytic infiltrate was seen in all samples. Inflammation extended to the subcutaneous fat in the six biopsies where the subcutis was represented and involved mostly the fat lobule. The inflammatory infiltrate was predominantly composed of small lymphocytes. Large activated lymphocytes were noted in only two cases (cases 2 and 3) with more severe inflammation. Plasma cells were only focally seen in these two biopsies showing more dense inflammation.

Immunohistochemistry

The inflammatory infiltrate was predominantly composed of mature T cells (CD3+) with a predominance of helper T lymphocytes (CD4+) over cytotoxic T lymphocytes (CD8+). Only scattered mature B lymphocytes (CD20+) were seen, except for case 5, where aggregates of B cells were noted in the centre of dense nodular lymphoid nodules. Scattered CD30+ cells were observed in cases 2 and 3. CD61 highlighted the presence of small microthrombi in four cases, including ones where thrombi were not apparent on haematoxylin and eosin-stained sections (case 6) (Table 3).

Cytoplasmic granular positivity for SARS-CoV-2 spike protein was mainly demonstrated in endothelial cells of the capillary and postcapillary venules of the upper dermis, and also in epithelial cells of the secretory portion of eccrine units in all cases (Figure 4a–c).

Figure 4.

(a–c) Cases 4–6. Cytoplasmic granular positivity for SARS-CoV-2 spike protein in endothelial cells (immunohistochemistry, original magnification × 400). (d) Case 2. Ultrastructural image of an endothelial cell showing coronavirus-like particles consistent with SARS-CoV-2 (arrow), next to a mitochondrion for size comparison (electron microscopy, × 60 000).

Electron Microscopy

Ultrastructural examination revealed the presence of round membrane-bound structures within the cytoplasm of endothelial cells showing an electrolucent centre, and surrounded by tiny spikes, giving them a halo-like appearance. Their mean diameter was 92.26 nm (range 80.76–109.76) and the mean thickness of the spikes was 13.18 nm (range 12.36–13.88). Based on previous descriptions in the literature, these structures were interpreted as coronavirus-like particles[8–14] (Figure 4d). Tubuloreticular inclusions were also found within the endothelial cells, similarly to other descriptions of SARS-CoV-2 and SARS-CoV infections.[8,15]

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