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We read with great interest the recent article by Yeoh et al, demonstrating an altered stool microbiome composition in patients with COVID-19 compared with controls, with greater dysbiosis correlating with elevated inflammatory markers.1 Additionally, dysbiosis was seen after disease resolution.1
To our knowledge, gut microbiome studies in young children with COVID-19 have not been reported. Critically, the developing gut microbiome of very young children differs from adults and establishes immune and inflammatory pathways.2 3 Moreover, children with COVID-19 can subsequently develop autoimmune and autoinflammatory diseases including Multisystem Inflammatory Syndrome in Children (MIS-C)4 5, which may in part be microbiome mediated, given recent findings by Yeoh et al.1 It is difficult to study this in young children, as many with SARS-CoV-2 infection are asymptomatic and rarely tested.6
To address this, knowing that SARS-CoV-2 can be detected in stool,7 we used an established study collecting longitudinal stool samples from before and throughout the pandemic to investigate the prevalence and associated microbiome changes of SARS-CoV-2 in very young children. We ran the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel assay on 769 serial stool samples from 595 children aged 0–24 months collected from February 2020 to February 2021. The prevalence of SARS-CoV-2 in faeces was 1.7% (13 samples from 13 separate children) with prevalence at <2 days and 2, 6, 12 and 24 months of 0% (0/1), 0% (0/21), 2.6% (4/156), 2.0% (7/357) and 0.9%,(2/234), respectively. Prevalence by month is shown in online supplemental figure 1A, with the first positive sample detected 31 days before the first reported case of COVID-19 regionally. No samples were positive in controls collected prior to the pandemic in 2019 (n=97 samples from 66 individuals). Of 13 positive children, 12 were asymptomatic with no personal or family history of SARS-CoV-2 (table 1A). Of 13 children, 1 was symptomatic with COVID-19 diagnosed 21 days before stool was collected. Hispanic ethnicity was associated with stool positivity (61.5% in positive samples vs 23.4% in negative samples, p=0.006 (χ2), table 1A). This study may underestimate prevalence rates as stool positivity may be lower than respiratory samples.
Supplemental material
(A) Characteristics of overall cohort and (B) characteristics of matched cohort for microbiome analysis
We successfully sequenced the SARS-CoV-2 genome from all positive samples (full methods in online supplemental data), with variant identification achieved for five samples (online supplemental figure 1B). We performed V4 16S rRNA gene sequencing on samples using DADA2 and the SILVA database for microbiome taxonomic profiling. We compared microbiomes using a 1:2 case–control match, controlled for ethnicity, age, delivery mode, gestational age, gender and recent antibiotic use (table 1B). Differential species abundance testing was performed using DESeq2 contrasting the SARS-CoV-2 positive and control samples. We found a significantly different relative abundance of taxa (adjusted p<0.05) between positive and control samples (all significantly different taxa at a species level shown in figure 1). Notably, we found a decreased abundance of Bifidobacterium bifidum and Akkermansia muciniphila in positive samples, both of which are linked to protection against inflammation.8 9 Bifidobacterium are also pioneering colonisers of the gut microbiota and have immunomodulatory properties.10 Bifidobacterium bifidum was found to be inversely correlated with disease severity in adults.1 While Yeoh et al 1 saw differences in beta diversity, our microbiome changes may be less robust compared with those symptomatic patients, with no differences seen in alpha or beta diversity. Detection of changes may also be limited by sample size.
Supplemental material
Significantly differentially abundant species between SARS-CoV-2 positive infants and controls identified using DESeq2. Negative log2 fold changes indicate a lower abundance of these species in SARS-CoV-2 positive samples relative to controls.
We show that microbiome changes are detectable even in asymptomatic infants infected with SARS-CoV-2. Of relevance, there is a decrease in anti-inflammatory taxa, similar to that seen in symptomatic adults. The impact of this on the developing microbiome, and subsequent immune and inflammatory responses is unknown, but deserves further exploration given the risk of development of autoimmune and autoinflammatory conditions in children with COVID-19.
Ethics statements
Patient consent for publication
Ethics approval
This study has been approved by the WCG institutional review board (IRB# 20120204). Written informed consent was obtained from all participants/guardians prior to collecting stool samples. Participants gave informed consent to participate in the study before taking part.
Supplementary materials
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Supplementary Data
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Footnotes
Twitter @TheDBStern, @DrSuchiHourigan
Contributors LN, SL, GM and SKH conceptualised and designed the study. LN, JM, SL and SH designed data collection instruments and collected and interpreted subject’s clinical data. SH conducted epidemiological analyses. DS and PS conducted microbiome analysis. LM conducted laboratory design and analysis. KB and WH conducted viral genome analysis. LN and SH drafted the initial manuscript. All authors reviewed and revised the manuscript. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.
Funding This work was supported in part by a National Institute of Child Health and Human Development K23 award (No. K23HD099240, Hourigan) and by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under the Intramural Research Programme (Hourigan) and BCBB Support Services Contract HHSN316201300006W/HHSN27200002 to MSC (Subramanian, Stern). This work was also supported by an Inova Health System Seed Grant (Hourigan, Nashed). Inova expresses its appreciation to the Fairfax County in VA, which has supported Inova’s research projects with annual funding from its Contributory Fund (Fund 10030, Maxwell).
Disclaimer The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.