Detection of fecal SARS-CoV-2 RNA in a prospective cohort of children with multisystem inflammatory syndrome (MIS-C)

Multisystem inflammatory syndrome related to SARS-CoV-2 infection (MIS-C) is a rare severe illness affecting paediatric patients. No data are available in literature about the presence of SARS-CoV-2 RNA in faces in patients with MIS-C. The SARS-CoV-2 positivity was evaluated in fecal samples obtained in a prospective cohort of paediatric patients admitted to our tertiary Hospital and diagnosed with MIS-C or COVID-19 between April 15, 2020, and February 29, 2021. The real-time RT-PCR was performed using a validated kit. The assay measures 3 target genes: SARS-CoV-2 gene E, gene N, gene ORF1ab. Overall, 16/63 (25%) fecal samples revealed the presence of SARS -CoV-2 RNA. No differences were detected about time from presumptive viral exposure and the time of stool collection (14 vs 8 days, p> 0,05) as well as about the presence of gastrointestinal symptoms (p>0.05) between patients with positive (+) stools for SARS-CoV-2 RNA and patients tested negative (-). Among children with MIS-C, stools were collected 27.5 days as median (95% CI 14-34) after presumed contact and the positivity rate was 12.5% (4/32). According to our data, we could suggest contact precautions with all patients with MIS-C. Further sample collection is ongoing to verify our preliminary data.


Introduction
Multisystem inflammatory syndrome related to SARS-CoV-2 infection (MIS-C) is a rare but severe illness affecting paediatric patients firstly described in April 2020, approximately one month after the first COVID-19 outbreak [1,2]. MIS-C usually develops after the infection rather than during the acute stage of the disease, suggesting a dysregulated immune response (i.e. continuous activation of adaptive immune responses driven by persisting antigen presentation) as the primary pathogenic mechanism [3]. Only one-third of reported cases, indeed, present a positive RT-PCR for SARS-CoV-2 on nasal swab; on the contrary, in most cases the serology results positive, suggesting a past infection [4,5].
MIS-C clinical presentation, characterized by multi-organ involvement, commonly involves gastrointestinal signs and symptoms [6][7][8]. To the best of our knowledge, no data are present in literature regarding the detection of SARS-CoV-2 RNA in fecal samples of patients with MIS-C, even if biospecimen collection methods for children were recently proposed [9].
The present prospective study aimed to detect SARS-CoV-2 RNA in fecal samples of patients who met CDC case definition criteria for MIS-C, admitted to our tertiary care paediatric hospital in the North-West of Italy between April 2020 and February 2021.

Materials and Methods
All patients admitted to Regina Margherita Children's Hospital (Turin, Italy) and diagnosed with MIS-C between April 15, 2020, and February 29, 2021, were prospectively enrolled in the study. Children admitted in the same period for COVID-19 or followed up in outpatients' service after hospitalization for COVID-19 were included as control group. Written informed consent was obtained from the parents of patients. Approval was obtained from Institutional Ethical Committee (Città della Salute e della Scienza di Torino, protocol number 00564/2020).
Fecal samples obtained after admission were stored at -80°C, immediately after collection, until the extraction and analysis were performed in the lab (Hygiene Section, Public Health and Pediatric Department, University of Turin). Extraction of total RNA from fecal samples was performed with a commercialized kit (Power Microbiome RNA kit, QIAGEN). The extracted RNA was quantified using a NanoQuant Plate (TECAN Trading AG, Switzerland), which allows quantification using a spectrophotometer read at 260 nm. The spectrophotometer used was the TECAN Infinite 200 PRO, and the software was i-Control (version 1.11.10). The extracted RNA concentration was meanly 17.6 ± 25.6 µg/mg stool. Samples were stored at -80°C until molecular analysis was performed.
The real-time RT-PCR was performed using the Novel Coronavirus (2019-nCoV) Real-Time Multiplex RT-PCR kit from LifeRiver Ltd., and CFX Instruments (Bio-Rad, Hercules, CA, USA) following the suggested thermal protocol included in the kit instructions and according to literature data [10]. The assay measures simultaneously 3 target genes: SARS-CoV-2 gene E, gene N, gene ORF1ab according to international validated testing protocols [10,11].
For each patient enrolled in the study, demographic data, clinical presentation and time from SARS-CoV-2 infection to fecal sample collection were registered (Table 1A). The time from the presumptive contact with the virus was defined as follow: a) when available, we considered the date of the first positive SARS-CoV-2 nasopharyngeal swab with the addiction of 4 days as typical incubation period [12]; b) for patients with MIS-C without previous positive swab and ascertained intrafamilial exposure to SARS-CoV-2, we considered the date of the first positive SARS-CoV-2 swab of familial contacts; c) for patients with MIS-C without positive swab and untested intrafamilial exposure, we considered 15 days as the minimum time to produce specific antibodies from exposure.
The statistical analysis was performed using IBM SPSS statistics package 27.0. A descriptive analysis of the variables was conducted. The data were reported as absolute numbers and percentages for categorical variables while as medians and confidence intervals -95% higher and 5% lower -for continuous variables. Moreover, the subjects were divided into two groups based on the diagnosis: COVID-19 or MIS-C. Differences between COVID-19 and MIS-C children were assessed using χ 2 test with Fisher's correction for categorical variables and Median test for independent samples for continuous variables. The Spearman rank-order correlation coefficient was also determined to assess the relationships between variables. A p-value p<0.05 was considered significant for all analyses.

Patients' characteristics
Overall, 63 patients (28 females and 35 males, aged 1 months to 15 years) were prospectively enrolled in the study (Table 1A).
Thirty-two children fulfilled CDC case definition criteria for MIS-C [13,14]. Among 31 patients with COVID-19, 24 were enrolled during the acute stage of the disease, and 7 at the follow-up in outpatients' clinic. Clinical and demographic characteristics of patients are reported in Table 1A.
Data regarding IgG against SARS-CoV-2 were available for 28/32 patients with MIS-C, as 4 children were enrolled before the development of validated methods; 27/28 (96%) children had a positive serology, confirming past infection. All patients with COVID-19 displayed at least one positive Real-Time Polymerase Chain Reaction (RT-PCR) nasal swab for SARS-CoV-2.
No differences were detected about time from presumptive viral exposure and the time of stool collection (14 vs 8 days, p> 0,05) as well as about the presence of gastrointestinal symptoms (p>0.05) between patients with positive (+) stools for SARS-CoV-2 RNA and patients tested negative (-). In particular, the positivity rate of stools was comparable in patients with or without gastrointestinal clinical involvement, 37.5% (6/35) vs 35% (10/28), p>0.05.
Multiple studies have reported gastrointestinal symptoms in children with COVID-19, with diarrhoea, nausea, vomiting, anorexia and abdominal pain being described as the main symptoms [6,28]. Children, compared to adults, have a higher ACE-2 expression in the gut [15] and present a higher ACE expression inside the intestine than in the respiratory tract [24]; these data may explain both the higher proportion of gastrointestinal symptoms and the higher percentage of positive faeces in the paediatric than in the adult form of COVID-19 [15].
MIS-C clinical presentation, characterized by multi-organ involvement, commonly includes gastrointestinal signs and symptoms. According to literature data [6][7][8], in our cohort diarrhoea, nausea, vomiting or abdominal pain were more frequent in patients with MIS-C compared to children with COVID-19 (respectively more than 75% in the first group vs less than 30% in the second one); this difference resulted statistically significant (p< 0.000).
To the best of our knowledge, data regarding the presence of the virus in the gut of patients with MIS-C are lacking. In our prospective cohort of pedaitric patients with MIS-C, more than 10% of patients tested positive (+) for SARS-CoV-2 RNA in faeces. The rate of patients with acute COVID-19 and fecal SARS-CoV-2 positivity (39%) was consistent with previously reported data [24], confirming the reliability of our methods.
Despite the limits deriving from the small number of patients with MIS-C tested positive (+) for SARS-CoV-2 fecal RNA, some preliminary conclusions could be drawn. First, it should be noted that there is no difference in the occurrence rate of gastrointestinal symptoms between patients with MIS-C with a positive (+) or a negative (-) result for SARS-CoV-2-RNA in the stools. Whether the virus titres in fecal fomites are of sufficient concentration and infectivity for subsequent transmission remains unknown, even in patients with acute COVID-19 [19,[25][26][27][28][29]. Nevertheless, due to the impossibility of clinically differentiating in our cohort children with positive (+) stools (>10%) from the others with negative (-) stools, we suggest contact precautions during hospitalization to be taken in all patients with MIS-C.
In addition, the wide range of days observed between presumed primary viral infection and detection of viral RNA in faeces (72, 43, 36 and 27 days) does not permit to determine precisely how long these precautions should be applied in children with MIS-C.
Further sample collection is ongoing to verify our preliminary data on a larger cohort.
To further complete the study, gut microbiota analysis on DNA extracted from fecal samples of our patients with MIS-C will be performed in the next months. In adults, microbiota modulation linked to SARS-CoV-2-host interaction has been proven to promote inflammation (through reduction of short-chain fatty acid producers and increase of pathogenic microorganisms) [30,31]. The quite long pre-symptomatic phase in MIS-C suggests an additional trigger that likely occurs 2-4 weeks after the initial infection; in this context, microbiota might represent a crucial key to explain the mechanism of disease onset. As a consequence, complete microbiota analysis could be an innovative approach to better understand MIS-C pathogenesis and identify potential early biomarkers of susceptibility to this emerging paediatric disease. Author Contributions: EP and DT and UR gave substantial contribution to conception and design, drafted the article, reviewed and critically revised the manuscript; DT and EF made stool sample processing and extraction and bio-molecular analysis; DT made statistical analysis and bioinformatic; AC, EF, GP, FP and MD contributed to conception and design, collected data, and revised the manuscript; all authors approved the final version of the manuscript and agree to be accountable for all aspects of the work.
Funding: This research received no external funding.
Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Città della Salute e della Scienza di Torino, protocol number 00564/2020.
Informed Consent Statement: Written informed consent has been obtained from the parents of the patients to publish this paper.