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

Patients and Methods

We present a retrospective case series of paediatric patients diagnosed clinically and histopathologically with chilblains during the COVID-19 pandemic. Approval from the institutional ethics committee and board was obtained. Standard informed consents were obtained for recording images and for skin biopsies in all patients.

The patients were seen in the emergency department and then referred to the dermatology department of a children's hospital over a period of 4 weeks (April to May 2020), shortly after the peak of incidence of COVID-19 in Madrid. Six of the patients (cases 1–6) were included in a previous report focused on the clinical and epidemiological features of 22 patients with similar lesions.[1]

Seven biopsy specimens from seven patients with skin lesions clinically diagnosed as COVID-19-related chilblains were examined. All of the biopsies were taken from lesions located on the feet or toes. Clinical parameters were recorded including age, sex, personal history of previous diseases, exposure to potentially infected household contacts, skin symptoms, location of lesions, systemic symptoms, therapies administered and follow-up. Laboratory analyses performed were also recorded, as well as SARS-CoV-2 PCR from nasopharyngeal and oropharyngeal swabs.

All of the biopsies were assessed on multiple serial sections stained with haematoxylin and eosin, Mowry's colloid iron and periodic acid–Schiff. The following histological features were recorded: vacuolar changes of the basal layer, exocytosis of lymphocytes, necrotic keratinocytes, erosion and ulceration of the epidermis, parakeratosis, spongiosis, oedema of the superficial dermis, perivascular superficial inflammatory infiltrate, perivascular deep inflammatory infiltrate, perieccrine lymphoid cell infiltrate, lymphocytic lobular panniculitis, fibrinoid material within the vessel walls, thrombi in papillary dermal vessels, thrombi in reticular dermis and subcutis vessels, lymphocytic infiltration of dermal vessels, purpura, dermal mucin, and basal membrane thickening. In each case, these features were scored as absent (−), mild/focal (+), moderate (++) or marked/extensive (+++).

Immunohistochemical stains with antibodies against CD3, CD4, CD8, CD20, CD30 and CD61 (Dako, Glostrup, Denmark) were performed in all of the samples. For SARS-CoV and SARS-CoV-2 immunostains the tissue was cut from paraffin blocks in 3-mm sections using a microtome and stretched on a water bath at 40 °C, mounted on glass slides and incubated at 60 °C for 1 h. The slides were deparaffinized in xylene and hydrated in ethanol. Antigen retrieval was performed with 10 mmol L−1 citrate buffer (pH 6.0) in a PT Link (Agilent Technologies, Santa Clara, CA, USA) at 95 °C for 20 min. We used a monoclonal antibody (1A9, dilution 1 : 200; GeneTex Inc., Irvine, CA, USA) against the spike protein of SARS-CoV and SARS-CoV-2. The staining was visualized with the chromogenic substrate 3-amino-9-ethylcarbazole [Dako EnVision+ System – HRP (AEC); Agilent Technologies] for 4 min, counterstained with Mayer's haematoxylin, and mounted with commercially ready-to-use Dako Ultramount Aqueous Permanent Mounting Medium. The staining was carried out with a Dako Autostainer Link 48 (Agilent Technologies). Negative controls (tonsils from patients without COVID-19) were included together with each sample. The antibody was previously optimized using sections of lungs from autopsies of patients with COVID-19 as positive controls and different inflammatory skin conditions as negative controls.

We evaluated the relative proportion of T and B cells, the proportion of helper to cytotoxic T cells and the presence of activated lymphocytes. The presence and location of the virus were recorded as present (+) or absent (−). CD61 was examined to highlight the presence (+) or absence (−) of intravascular platelet thrombi. Transmission electron microscopy examination was performed in case 2.

All of the morphological, histochemical and immunohistochemical parameters were assessed by two of the authors (I.C. and A.T.) and SARS-CoV- and SARS-CoV-2-stained slides were seen by four (I.C., A.T., L.R. and C.S.).

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