Ethical statement
This monocentric study was performed in a “hors Jardé” law legislative context according to the French legislation for the protection of the people during health care management (https://www.legifrance.gouv.fr/codes/article_lc/LEGIARTI000045629992/2022-04-22/), on existing medical data in the file, not justifying the signed consent of the holders of parental authority for the children included. All experimental protocols were approved by Bichat-Claude Bernard hospital, Assistance Publique- Hôpitaux de Paris (AP-HP), authorities.
No informed consent was required for this study but a non-objection of the patient to participate to medical research. Information about the possibility of using patients' medical data unless they express their opposition is present in the medical reports. An information note presenting the objectives of the research is sent to patients to inform them that the clinical data from their medical file will be used in the context of this research. Patients can then oppose the use of their data. In case of refusal of data collection, the patient will not be included. The data processing implemented as part of this research will be recorded in the AP-HP register, placed under the responsibility of the AP-HP Data Protection Officer, in accordance with the provisions of the General Data Protection Regulation.
Study Population
This prospective study was conducted from 01 March 2020 to 03 May 2020 during the first wave of the SARS-CoV-2 pandemic in France. Outpatients and hospitalised patients receiving care in Bichat-Claude Bernard hospital, who has provided at least one stool sample for microbiological investigations, were included regardless of clinical context. For patients suspected of being infected with COVID-19, a SARS-CoV-2 molecular diagnostic test was performed on a respiratory sample on admission. If the respiratory sample was negative but clinical suspicion remained high (clinical presentation and history associated with radiographic detection of ground glass opacities), SARS-CoV-2 molecular detection on stool samples and/or SARS-CoV-2 serology in the month following the acute infectious episode were performed to confirm COVID-19 (COVID+ group). Patients with SARS-CoV-2 RNA detected in the stool sample were assigned to the excretory group as opposed to patients with undetectable faecal viral shedding (non-excretory group). Patients with no suspicion of COVID-19 (atypical clinical presentation and history without typical pulmonary radiography) with or without negative results for SARS-CoV-2 infection (PCR on respiratory samples and/or stools and/or serology) were retrospectively and prospectively included, according to the same previous criteria, from 01 February 2020 to 03 May 2020, to constitute a negative control group for gastro-intestinal co-infections (COVID- group). Nosocomial COVID-19 infection (i.e. proven SARS-CoV-2 infection acquired during hospital stay) and onset of digestive symptoms later than 30 days after hospital care were excluded. For each included patient, epidemiological (age, sex, place of residence, travel history), treatment (background and anti-infectious treatment), clinical (underlying disease, disease history, type and duration of clinical signs, clinical course and outcomes), radiological (extent of pulmonary ground-glass opacities), and biological data (sampling, microbiological, haematological and biochemical investigations) were collected from medical charts.
The aetiology of the digestive symptoms was determined by assessing the current medical treatment, underlying disease, disease history, type and duration of gastrointestinal symptoms and microbiological results. Where possible, the onset and duration of digestive and respiratory symptoms, as well as the interval between onset of symptoms and start of medical care, the delay between digestive and respiratory symptoms, length of hospital stay, respiratory and stool sampling (i.e. time between onset of clinical symptoms and biological sampling) as well as delay between digestive and respiratory sampling were extracted from patient record information. ICU treatment was taken into account from at least day 1 of hospitalisation in the unit. Fever was defined as body temperature over 38°C. Diarrhoea was defined as a modification of stool consistency associated with an increased frequency of bowel movements. Diarrhoea was considered chronic if it persisted for at least 30 days.
Virological investigations
The RealStar® SARS-CoV-2 RT-PCR Kit 1.0 assay, targeting the SARS-CoV-2 N and E genes was used for viral detection. RNA extraction was performed with the MagNA Pure LC 2.0 System (Roche) according to manufacturer’s protocol. For each sample, 200 µL of nasopharyngeal (NP) sample or faeces suspension was diluted in 2 mL of lysis buffer and eluted in 50 µL. 10 µL of extracted RNA was used to perform the real-time RT-PCR with the ABI Prism® 7500 SDS (Applied Biosystems). A heterologous amplification system (Internal Control), included in the kit, was used to identify possible RT-PCR inhibition. In case of inhibition, NP and faeces samples were retested under the same conditions at a 1:10 dilution in PBS. SARS-CoV-2 anti-N antibody detection was performed using Abbott Architect SARS-CoV-2 immunoglobulin G (IgG) (Abbott, Maidenhead, United Kingdom) and expressed as an index (cutoff: 0.49).
Microbiological investigation on stool samples
A syndromic approach using QIAstat-Dx® gastrointestinal Panel (Qiagen®), which targets 14 bacteria (Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Campylobacter spp. (C. jejuni, C. upsaliensis, C. coli), Salmonella spp., Clostridioides difficile (tcdA/tcdb), Yersinia enterocolitica, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), E. coli producers of shigatoxins (STEC) (enterohaemorrhagic E. coli/serotype O157 : H7), enteroinvasive E. coli (EIEC)/Shigella spp, Plesiomonas shigelloides), 4 viruses (human Adenovirus F40/F41, Norovirus (group 1 and 2), Rotavirus A, Astrovirus and Sapovirus (group 1, 2, 4, 5)) and 4 parasites (Entamoeba histolytica, Cryptosporidium spp, Giardia intestinalis and Cyclospora cayetanensis) was performed on stool samples according to manufacturer’s instructions. In case of positive results for C. difficile, detection of the free toxins A and B was performed by membrane enzyme immunoassay (CDIFF QUIK CHEK COMPLETE®, Alère®).
Trained microscopists also performed microscopic parasitological examinations on the same fresh sample after iodine-stained wet mount, flotation and biphasic concentration. In addition, stool smears stained by modified Ziehl-Neelsen were examined. Microsporidia PCR (Bio-evolution®) and a 4-plex protozoa PCR targeting E. histolytica, Cryptosporidium spp, G. intestinalis and D. fragilis (Amplidiag®, Mobidiag®) were also systematically performed according to manufacturer’s instructions. DNA was extracted from stool on EZ1® Advanced XL (Qiagen®) using EZ1® DNA tissue kit and the EZ1 bacteria card from PBS-suspensions of stool samples pretreated with proteinase K.
Gastrointestinal infection aetiology was considered positive if the patient presented digestive symptoms without other evident aetiology, together with the detection of a high burden of known pathogenic microorganisms (positive microscopic examination and/or Ct < 30 on QIAstat-Dx® and/or Ct < 35 on Amplidiag®). Otherwise, patients were considered carriers of the detected microorganisms.
Statistical analysis
Quantitative parametric variables were represented by mean and standard deviation (SD) and by median and interquartile range [25th percentile – 75th percentile] for non-parametric variables. Categorical variables were evaluated according to size and frequency. Fisher’s exact test was used to compare categorical variables between groups. Kruskall-Wallis tests were used to compare quantitative variables between groups when appropriated. For all tests, a difference was considered significant when p<0.05. All reported p values are two-tailed. Statistical analyses were performed using STATA, version 12 (Stata corp®, College station, Texas, USA).
Data availability
Authors confirm to editorial Scientific reports board that all materials and raw datas necessary for manuscript reviewing and comprehension will be available from the text as supplemental data or from the corresponding author when necessary.