• appendix
• immunohistochemistry
• apoptosis

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 …