ACE2 gene expression and inflammatory conditions in periodontal microenvironment of COVID-19 patients with and without diabetes evaluated by qPCR

  1. Boy M. Bachtiar
  2. Endang W. Bachtiar
  3. Hari Sunarto
  4. Yuniarti Soeroso
  5. Benso Sulijaya
  6. Citra Fragrantia Theodorea
  7. Irandi Putra Pratomo
  8. Yudhistira
  9. Ardiana Kusumaningrum
  10. Defi Efendi
  11. Efa Apriyanti
  12. Nastiti Rilo Utami
  13. Astri Deviana
  14. Anissa Dien Andriyani

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Abstract

Objective

Chronic periodontitis has been proposed to be linked to coronavirus disease (COVID-19) on the basis of its inflammation mechanism. We aimed to evaluate this association by investigating the expression of Angiotensin Converting Enzyme-2 (ACE2) in periodontal compartments, which contain dysbiosis-associated pathogenic bacteria, and how it can be directly or indirectly involved in exacerbating inflammation in periodontal tissue.

Material and Methods

This observational clinical study included 23 adult hospitalized patients admitted to Universitas Indonesia Hospital with PCR-confirmed COVID-19, while 6 non-COVID-19 participants come to periodontal clinic were included as control. Using real time-PCR (qPCR) and gingival crevicular fluids (GCF) samples from COVID-19 patients with and without diabetes and periodontitis, we assessed the mRNA expression of angiotensin-converting enzyme 2 (ACE2), IL-6, IL-8, complement C3, and LL-37 as well as the relative proportion of Porphyromonas gingivalis, Fusobacterium nucleatum , and Veillonella parvula to represent the dysbiosis condition in periodontal microenvironment. All analyses were performed to determine their relationship.

Results

ACE2 mRNA expression was detected in the GCF of periodontitis-COVID-19 patients with and without diabetes. However, only periodontitis-COVID-19 patients with diabetes showed a positive relationship between ACE2 expression and inflammatory conditions in the periodontal microenvironment. In addition, the interplay between pro-inflammatory cytokine (IL-6) and complement C3 could be used as a predictor of the severity of periodontal inflammation in COVID-19 patients with diabetes.

Conclusion

The study data show that the SARS-CoV-2 entry gene is expressed in the GCF of patients with COVID-19, and its expression correlates with inflammatory markers.

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  1. ScreenIT

    SciScore for 10.1101/2022.03.10.22271304: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Patients: Before starting this study, we obtained ethical approval from the Ethical Review Committee of Medical Ethical Committee of Rumah Sakit Universitas Indonesia (RSUI) and followed the principles of the Medical Research Involving Human Subject Act (ETHICAL APPROVAL Nomor: 0042/SKPE/KKO/2021/00).
    Field Sample Permit: All sampling methods were carried out in accordance with committee guidelines and regulations, and this study was carried out in accordance with the guidelines provided by the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement [21].
    Consent: After a briefing on the procedure, only participants who provided written consent were included in the study.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Statistical analyses were conducted using GraphPad PRISM 9.0 (GraphPad Software, San Diego, CA, USA).
    GraphPad PRISM
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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:
    Our study has intrinsic limitations. First, as an observational study, it was difficult to exclude all potential confounders. Therefore, the involvement of some variables may be underestimated in predicting the result. Second, the number of subjects involved was small, and we could not be sure if the altered microbial composition in the periodontal niche had already occurred in our COVID-19 patients when GCF samples were collected. Periodontitis is a common infectious disease that is triggered by oral bacteria. It is important to analyze certain quantities of specific pathogens in the oral microbiota of patients to initiate inflammation. In this study, we used the relative abundance of each targeted species as a proportion rather than the actual levels. Finally, although GCF reflects the serum immune response [11], the results of this study cannot be linked directly to suggest that the level of inflammation determinants in GCF may correspond to the systemic inflammation mechanism of COVID-19. Based on the results of this preliminary study, we are planning a forthcoming study in searching the oral microbiome and trying to correlate the viral load and the functional inference of oral bacterial composition, and how it relates to the host response. However, with these limitations, the current approach hints at the importance of the oral ecosystem in modulating clinical symptoms related to COVID-19 cases [60-63].

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    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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  2. Version published to 10.1101/2022.03.10.22271304v1 on medRxiv