SARS-CoV-2 in the abdomen or pelvis: SAFE SURGERY study

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As the global community recovers from the SARS-CoV-2 pandemic, the ability to return to normal clinical practice to relieve large clinical backlogs is critical. However, concerns over the risk of SARS-CoV-2 transmission to healthcare staff via surgery remain1. The angiotensin-converting enzyme (ACE) 2 protein receptor, through which SARS-CoV-2 infects cells, is expressed by cells across multiple organs2. Small single-centre studies3,4 have reported the presence of the virus in the surgical plume or peritoneal fluid, whereas others5–8 have not. In an emergency surgery setting, concern has been raised where there has been a breach in the gastrointestinal tract, which may provide an additional route for the virus to reach the peritoneal cavity9,10. To investigate this, a clinical study (SAFE SURGERY) was designed to assess the presence of SARS-CoV-2 in the abdominal cavity and organs of infected patients. SAFE SURGERY operated as three substudies. Substudy 1 was a multicentre, prospective study of peritoneal, rectal, and vaginal swabs from patients undergoing emergency abdominal or pelvic surgery who tested positive for SARS-CoV-2. Substudy 2 was a multicentre, retrospective analysis of archived surgical tissues from patients who tested positive for SARS-CoV-2, and who had previously undergone emergency abdominal or pelvic surgery. Substudy 3 comprised a multicentre analysis of post-mortem tissues from patients who had died from SARS-CoV-2 infection. Ethical approval for substudies 1 and 2 was obtained from the South Oxford B Research Ethics Committee (20/SC/0261). For substudy 3, ethical approval for sample collection was obtained from the Newcastle and North Tyneside Research Ethics Committee (REC: 19/NE/0336). Patients were recruited from five centres across the UK (Chester, Liverpool, Nottingham, Birmingham, Sandwell) between 23 July 2020 and 30 March 2021. Patients were eligible if they were undergoing emergency surgery following a recent diagnosis of SARS-CoV-2 infection (PCR-positive or chest X-ray suggestive of infection). All patients had swabs taken from the pelvis and paracolic gutters either via a laparotomy wound or a laparoscopic port, along with repeat nasopharyngeal and luminal (rectal, colonic or vaginal) swabs. Samples were placed in viral transport medium and sent to the University of Birmingham within 24 h. Samples were inactivated by heating to 65°C for 30 min in a temperature-mapped oven, and RNA was extracted using a Qiagen RNeasy® (Manchester, UK) kit with a modified protocol. SARS-CoV-2 RNA was measured by reverse transcriptase–PCR using the CERTEST ViaSure SARS-CoV-2 real-time PCR kit against the ORF1ab and N1 genes of the SARS-CoV-2 genome, as described previously11. Local study teams identified patients who had undergone emergency surgery or had died after a positive respiratory PCR swab. Retrospective samples were identified and relevant tissue blocks were sent to Blackpool Victoria Hospital for pathological examination. Haematoxylin and eosin-stained slides were produced for each tissue block to look for the presence of mesothelium-lined fatty tissue (indicative of the peritonealized surface) within the block. Patients were included if they had received a positive PCR swab, either in life or at post-mortem examination, and if COVID-19 was noted as the cause of death. Tissue samples were obtained from multiple organs and fixed for 72 h before processing and paraffin embedding. Wide-bore tissue cores were taken from each patient and used to construct tissue microarrays. For substudies 2 and 3, routine immunohistochemical (IHC) methods were used to stain for SARS-CoV-2 nucleocapsid protein (antibody clone ER81L, 1 : 200 dilution; Cell Signalling Technology) and ACE-2 receptor (antibody clone E-11, 1 : 100 dilution; Santa Cruz Biotechnology) using the Ventana Benchmark XT platform. Placental tissue from a patient known to be SARS-CoV-2-positive (Fig. S1A) and normal small bowel mucosa were used as positive controls for nucleocapsid and ACE-2 (Fig. S1B) respectively. Ten patients were recruited; 9 had positive nasopharyngeal swabs and 1 had clinical suspicion of infection based on a positive chest X-ray. Three of these patients underwent obstetric/gynaecological procedures and 7 had gastrointestinal surgery (Table 1). Repeat nasopharyngeal swabs were positive in 9 patients. Peritoneal swabbing was performed in all 10 patients; swabs were taken from the vagina in 1 patient, and from the colonic lumen in 1 patient. A total of 23 peritoneal, vaginal, and colonic swabs were taken, and all were negative for SARS-CoV-2 by PCR. Clinical and surgical characteristics of patients in substudy 1, and peritoneal swab results CXR, chest X-ray. Clinical and surgical characteristics of patients in substudy 1, and peritoneal swab results CXR, chest X-ray. Specimens from 21 patients with a positive PCR test were analysed for SARS-CoV-2 and ACE-2 receptor expression in mesothelial fatty cells. All 21 patients had undergone emergency gastrointestinal surgery, and 8 had a perforation of the gastrointestinal tract at the time of surgery (Table 2). All samples were negative for SAR-CoV-2. One large bowel specimen was focally positive for ACE-2 receptor expression which was seen faintly in adipose cells only adjacent to the mesothelial cells (Fig. 1a). Luminal expression of the ACE-2 receptor was noted in large and small bowel samples, as expected (Fig. S1C). Examples of immunohistochemical staining a Immunohistochemical (IHC) staining showing faint angiotensin-converting enzyme (ACE) 2 receptor expression in large bowel from a patient in substudy 2 (ACE-2 antibody, Santa Cruz Biotechnology; ×100 magnification); b positive SARS-CoV-2 staining in lung from post-mortem sample of a patient who died from acute COVID-19 infection (SARS-CoV-2 antibody, Cell Signalling Technology; ×100 magnification); c IHC staining of endothelial SARS-CoV-2 expression in bowel mucosa of a patient in substudy 3 (SARS-CoV-2 antibody, Cell Signalling Technology; ×100 magnification, haematoxylin counterstain). Clinical and surgical characteristics of patients in substudy 2, and summary of immunohistochemical analysis of SARS-CoV-2 and angiotensin-converting enzyme 2 receptor expression *Values in parentheses are number of patients with perforation. No virus or receptor expression was observed on peritoneal surfaces, other than weak focal expression in adjacent adipose cells from a single patient only. Clinical and surgical characteristics of patients in substudy 2, and summary of immunohistochemical analysis of SARS-CoV-2 and angiotensin-converting enzyme 2 receptor expression *Values in parentheses are number of patients with perforation. No virus or receptor expression was observed on peritoneal surfaces, other than weak focal expression in adjacent adipose cells from a single patient only. Samples were collected from 8 individuals with PCR-confirmed SAR-CoV-2. The serous membranes overlying abdominal and thoracic organs were examined for the presence of SARS-CoV-2 virus or ACE-2 receptor. None of the serous membranes examined by IHC showed evidence of virus protein or receptor expression (Table 3). Proportion of retrospective post-mortem samples in substudy 3 showing immunohistochemical detection of SARS-CoV-2 virus protein and angiotensin-converting enzyme 2 expression in post-mortem tissues from multiple organs in patients who died from COVID-19 No virus or angiotensin-converting enzyme (ACE) 2 receptor expression was detected in serous membranes. Positive virus staining was observed in half of the lung samples examined and a large bowel blood vessel. Positive ACE-2 receptor staining was detected in the lumen of control bowel tissue, as expected. Proportion of retrospective post-mortem samples in substudy 3 showing immunohistochemical detection of SARS-CoV-2 virus protein and angiotensin-converting enzyme 2 expression in post-mortem tissues from multiple organs in patient

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