Respiratory dysfunction three months after severe COVID‐19 is associated with gut microbiota alterations

  1. Beate Vestad
  2. Thor Ueland
  3. Tøri Vigeland Lerum
  4. Tuva Børresdatter Dahl
  5. Kristian Holm
  6. Andreas Barratt‐Due
  7. Trine Kåsine
  8. Anne Ma Dyrhol‐Riise
  9. Birgitte Stiksrud
  10. Kristian Tonby
  11. Hedda Hoel
  12. Inge Christoffer Olsen
  13. Katerina Nezvalova Henriksen
  14. Anders Tveita
  15. Ravinea Manotheepan
  16. Mette Haugli
  17. Ragnhild Eiken
  18. Åse Berg
  19. Bente Halvorsen
  20. Tove Lekva
  21. Trine Ranheim
  22. Annika Elisabeth Michelsen
  23. Anders Benjamin Kildal
  24. Asgeir Johannessen
  25. Lars Thoresen
  26. Hilde Skudal
  27. Bård Reiakvam Kittang
  28. Roy Bjørkholt Olsen
  29. Carl Magnus Ystrøm
  30. Nina Vibeche Skei
  31. Raisa Hannula
  32. Saad Aballi
  33. Reidar Kvåle
  34. Ole Henning Skjønsberg
  35. Pål Aukrust
  36. Johannes Roksund Hov
  37. Marius Trøseid
  38. the NOR‐Solidarity study group

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Abstract

Background

Although coronavirus disease 2019 (COVID‐19) is primarily a respiratory infection, mounting evidence suggests that the gastrointestinal tract is involved in the disease, with gut barrier dysfunction and gut microbiota alterations being related to disease severity. Whether these alterations persist and are related to long‐term respiratory dysfunction remains unknown.

Methods

Plasma was collected during hospital admission and after 3 months from the NOR‐Solidarity trial ( n = 181) and analyzed for markers of gut barrier dysfunction and inflammation. At the 3‐month follow‐up, pulmonary function was assessed by measuring the diffusing capacity of the lungs for carbon monoxide (DL CO ). Rectal swabs for gut microbiota analyses were collected ( n = 97) and analyzed by sequencing the 16S rRNA gene.

Results

Gut microbiota diversity was reduced in COVID‐19 patients with respiratory dysfunction, defined as DL CO below the lower limit of normal 3 months after hospitalization. These patients also had an altered global gut microbiota composition, with reduced relative abundance of 20 bacterial taxa and increased abundance of five taxa, including Veillonella , potentially linked to fibrosis. During hospitalization, increased plasma levels of lipopolysaccharide‐binding protein (LBP) were strongly associated with respiratory failure, defined as pO 2 /fiO 2 (P/F ratio) <26.6 kPa. LBP levels remained elevated during and after hospitalization and were associated with low‐grade inflammation and respiratory dysfunction after 3 months.

Conclusion

Respiratory dysfunction after COVID‐19 is associated with altered gut microbiota and persistently elevated LBP levels. Our results should be regarded as hypothesis generating, pointing to a potential gut–lung axis that should be further investigated in relation to long‐term pulmonary dysfunction and long COVID.

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  1. Version published to 10.1111/joim.13458
  2. ScreenIT

    SciScore for 10.1101/2021.07.13.21260412: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsField Sample Permit: The study was approved by the Committee for Medical Research Ethics Region South East Norway (118684) and by the Norwegian Medicines Agency (20/04950-23) and registered in ClinicalTrials.gov (NCT04321616).
    IRB: The study was approved by the Committee for Medical Research Ethics Region South East Norway (118684) and by the Norwegian Medicines Agency (20/04950-23) and registered in ClinicalTrials.gov (NCT04321616).
    Consent: All participants gave informed consent prior to inclusion, either by themselves or a legally authorised representative.
    Sex as a biological variablenot detected.
    RandomizationStudy design and participants: NOR-Solidarity is a multicentre, open label, adaptive randomised clinical trial evaluating the effect of antiviral drugs on hospitalized COVID-19 patients admitted to 23 Norwegian hospitals(12).
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Plasma levels of LBP, intestinal fatty acid binding protein (IFABP), CCL25 and regenerating islet-derived protein 3α (REG-3α) were measured in duplicate by enzyme immunoassays (EIA) using commercially available antibodies (R&D Systems, Minneapolis, MN) in a 384 format using a combination of a SELMA (Jena, Germany) pipetting robot and a BioTek (Winooski, VT) dispenser/washer.
    binding protein (IFABP)
    suggested: None
    CCL25
    suggested: None
    Software and Algorithms
    SentencesResources
    Quality control and quantification of pooled libraries were performed using Agilent Bioanalyzer (Agilent Technologies, USA) and Kapa Library Quantification Kit (Kapa Biosystems, London, UK).
    Agilent Bioanalyzer
    suggested: None
    Sequence processing and bioinformatics: Paired-end reads containing Illumina Universal Adapters or PhiX were discarded using bbduk version 38.90 (BBTools, https://jgi.doe.gov/data-and-tools/bbtools/) (parameters adaptor filter: k=23 hdist=1 tbo cf=TRUE ftm=5. parameters phix filter: k=31 hdist=1) and the remaining reads were demultiplexed using cutadapt version 3.3(19) (parameters:-e 0.1 --no-indels --overlap 12 --discard-untrimmed --action none).
    https://jgi.doe.gov/data-and-tools/bbtools/
    suggested: (Bestus Bioinformaticus Tools, RRID:SCR_016968)
    Taxonomic classification of ASVs was done based on RESCRIPt(23) in QIIME2 using a naïve Bayes classifier trained on the V3-V4 region of a preclustered version (99% sequence similarity) of the Silva database version 138(24)
    Silva
    suggested: (SILVA, RRID:SCR_006423)
    Differential abundance testing with LEfSe(26) was done on the rarefied dataset, while for differential abundance testing with ALDEx2(27), a prevalence-filtered (25% most prevalent) version of the non-rarefied dataset was used.
    ALDEx2
    suggested: (ALDEx2, RRID:SCR_003364)
    SPSS release 26.0.0.1 and 27.0.0.0 were used for statistical analysis.
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    The present study has some limitations. Due to logistic constraints of launching a randomized trial during the first wave of the pandemic, the gut microbiota samples were only collected at the three-month follow-up. We therefore cannot relate the long-term microbiota alterations to potential microbiota alterations during hospitalization. Also, the analysis of microbiota composition was only performed in one cohort, and without a validation panel these must be considered explorative. Moreover, pulmonary function tests beyond P/F-ratio were only performed at three-month follow-up, eliminating the possibility for baseline comparisons. The study also has obvious strengths, including standardized data capture in a randomized trial with longitudinal biobanking, as well as comprehensive long-term follow-up with blood tests, microbiota sampling and assessment of pulmonary function by DLCO. Also, to our knowledge, our study is the first to link pulmonary function to gut microbiota alterations in COVID-19. In conclusion, the decreased microbial diversity and compositional gut microbiota alterations in patients with persistent respiratory dysfunction, as well as the association of persistently raised LBP levels with these clinical features, point to a potential gut-lung axis in COVID-19. These observations could be related to not only acute respiratory failure during hospitalization, but also to long-term COVID-19 morbidity. Our findings warrant further research on the potential role of...

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04321616RecruitingThe Efficacy of Different Anti-viral Drugs in COVID 19 Infec…

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2021.07.13.21260412v1 on medRxiv