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We had observed increased apoptosis in two appendixes in children who developed acute appendicitis after COVID-19 infection. We wondered if this was a chance finding or was causal. To investigate whether increased apoptosis was indicative of COVID-19 infection and if the appendix was involved in COVID-19, we evaluated H&E-stained slides of 12 consecutive cases of acute appendicitis in children (6 with past/current COVID-19, 6 negative on PCR). SARS-CoV-2 immunohistochemistry was performed on sections on the Leica BOND-III platform using Severe Acute Respiratory Syndrome (SARS) nucleocapsid mouse monoclonal antibody at 1:100 dilution (catalogue #MA1-7404; Thermo Fisher Scientific) following antigen retrieval, as previously described.1 To determine apoptosis, we used immunofluorescence-based labelling with rabbit polyclonal caspase-3 (1:500 dilution; ab4051, Abcam UK) and visualised the localised protein using a laser scanning confocal microscope (DMi8, Leica Microsystems, Germany), as described previously.2 Positivity was graded based on intensity and number as 0 to 3. Apoptotic cells were semiquantitatively assessed and graded from 1 to 3+ (at 20×): 0: no immunofluorescence-positive cells; 1+: less than or equal to 5 cells/high-power-field; 2+: between 6 and 10 cells/high-power-field; 3+: more than 10 cells/high-power-field.
There were three boys and three girls in the COVID-19 group (one toddler, five children (early to middle childhood); cases 1–6 in table 1). One patient was positive for SARS-CoV-2 by PCR, while the other five were negative by PCR but reactive to COVID-19 IgG antibodies in serum, indicating prior infection. In the COVID-19-negative arm, there were six children (one early childhood, one adolescent, four middle childhood; cases 7–12 in …
Handling editor Runjan Chetty.
Contributors SAP wrote the initial draft. PW offered comments to improve the draft. SAP, TC and AM evaluated the histology slides. BB and PAA evaluated the immunofluorescence. SC and TRS offered clinical inputs. All authors have contributed to the manuscript and have seen the final version and approved of it.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests TC and PW are associated with Arkana Labs, USA, where the immunostains were done.
Provenance and peer review Not commissioned; internally peer reviewed.