Association between periodontitis and COVID-19 infection: a two-sample Mendelian randomization study

Introduction

The coronavirus disease 2019 (COVID-19) worldwide pandemic stemming from outbreaks of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection triggers grim conditions such as pneumonia and respiratory failure and has been related with many deaths (Group et al., 2021; Guan et al., 2020). By November 2, 2021, >240 million cases had been confirmed, and total deaths have numbered >5.01 million globally. Given the high incidence and severity, there is an urgent need to identify risk factors related with higher susceptibility to COVID-19.

One of the most prevalent chronic inflammatory illnesses, periodontitis has a prevalence of 20–50%. Severe periodontitis is the sixth most prevalent illness worldwide (Collaborators, 2018). It plays a key function in stimulating inflammatory cytokine release (Cardoso, Reis & Manzanares-Cespedes, 2018). Therefore, if causal, periodontitis can increase the global burden of COVID-19, and interventions targeting oral inflammation will play an important role in preventing COVID-19 and its complications. Recent observational studies have found that periodontitis and its severity, gingival bleeding, and plaque accumulation (Anand et al., 2021; Gupta et al., 2021b; Marouf et al., 2021; Vieira, 2021) might be related with higher risk for severe COVID-19 (i.e., hospitalization, mechanical ventilation, intubation, mortality). Moreover, the periodontium inflammation level has been related to SARS-CoV-2 infection (Andrade et al., 2021; Marouf et al., 2021). In addition, metagenomic analyses of COVID-19 patients have repeatedly revealed uncharacteristically elevated bacterial reads of Prevotella intermedia, a periodontal pathogen (Zhu et al., 2020). However, a recent study of 13,253 UK Biobank participants who had undergone a COVID-19 test yielded inadequate proof to connect periodontal disease with greater COVID-19 risk (Larvin et al., 2020): participants with painful gums, gums that bled, and loose teeth did not show increased COVID-19 infection, admission to hospital, or mortality risk compared to the control group. Given the global outbreak of COVID-19 and its hazards, determining whether periodontitis causes COVID-19 and targeting prevention strategies are imperative.

Mendelian randomization (MR) is the method for causal inferences in non-experimental situations with the advantage of overcoming the limitations of both observational and randomized controlled trials (RCTs) studies. It uses germline genetic variants as instrumental variable (IV) to proxy for environmentally modifiable exposures within observational epidemiological studies and is considered as analogous to RCT due to the fact that the alleles of a genetic variant are distributed randomly at meiosis (Mendel’s second law of independent assortment) and individuals are then “randomized” by nature (Jansen, Lieb & Schunkert, 2016; Lawlor et al., 2008). Observational studies may be subject to confounding factors and reverse causality, which poses challenges to causal inference. Many confounding factors might exist in these studies because periodontitis is a comorbidity with many other systemic diseases (Hajishengallis & Chavakis, 2021), which have been related to COVID-19 outcomes (Ramos-Casals, Brito-Zeron & Mariette, 2021). Therefore, it is difficult to exclude the interference of confounding factors in these studies. Further, a reverse causal association is also possible due to the coexistence of excessive inflammatory states (Hajishengallis, 2015; Mangalmurti & Hunter, 2020). Mendelian randomization (MR) can overcome confounding bias because genetic alleles are randomized to individuals at conception, thereby breaking the link with most confounders. Moreover, as genetic alleles are always assigned before disease onset, reverse causality does not affect them. And although RCT had a pivotal role in establishing causal associations, a rigorously designed, well-executed RCT is sometimes too time-consuming and expensive to be unrealistic, which makes MR design more prominent and practical especially when Genome-Wide Association Study (GWAS) summary data are publicly available. Therefore, together with vigorous observational analyses, MR analyses can yield corresponding insights to improve the assessment of possibly causal associations. Determining causal relations between COVID-19 and periodontitis might facilitate the alleviation of their influence on disease risk.

Considering the recent evidence that periodontitis patients may be more susceptible to SARS-CoV-2 infection due to a higher inflammatory burden and the infection status of periodontal pathogens, we hypothesized that periodontitis and its related phenotypes, including periodontal pathogens and gingival inflammation, are causally associated with COVID-19 susceptibility. Therefore, a two-sample MR analysis was performed to assess the causal relationship of periodontitis and its associated phenotypes with COVID-19 using OpenGWAS data (https://gwas.mrcieu.ac.uk/).

Materials & Methods

This study is reported as per the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline. No additional ethics approval was required for this study because the data are public available de-identified summary-level data. The Mendelian randomization unbiased causal associations and assumptions of this study were showed in Fig. 1. The study pipeline is illustrated on Fig. 2.

Results

Mendelian randomization analyses for COVID-19

The causal relations between COVID-19 and periodontitis were estimated with IVW, weighted median, and MR-Egger regression methods (Table 2; Fig. 3). The two GWAS datasets for COVID-19 present the approximate results. IVW yielded proof of causal relationships between COVID-19 and periodontitis. Periodontitis was associated positively with COVID-19 risk (ebi-a-GCST010776: IVW odds ratio [OR] = 1.02 [95% confidence interval (CI), 1.00–1.05], P = 0.0171; ebi-a-GCST010777: IVW OR = 1.03 (95% CI [1.00–1.05]), P = 0.0397). The weighted median also revealed estimates that were directionally similar, while MR-Egger regression appeared insufficiently powered for exceeding standard thresholds of significance (ebi-a-GCST010776: weighted median OR = 1.03 (95% CI [1.00–1.06]), P = 0.0282; ebi-a-GCST010777: weighted median OR = 1.03 (95% CI [1.00–1.06]), P = 0.0295). The intercepts of MR-Egger indicated no proof for significant directional pleiotropy for both datasets (ebi-a-GCST010776: P = 0.7660; ebi-a-GCST010777: P = 0.6017; Table 2). Our findings indicate that there are no effects of directional pleiotropy between COVID-19 and periodontitis.

Table 2:

Mendelian randomization for periodontitis on COVID-19.

Methods	COVID-19 (hospitalized vs population) ebi-a-GCST010777			COVID-19 ebi-a-GCST010776
	N SNPs	OR Estimates (95%CI)	P-value	N SNPs	OR Estimates (95% CI)	P-value
Inverse variance weighted	5	1.03(1.00–1.05)	0.0397	5	1.02(1.00–1.05)	0.0171
Weighted median	5	1.03(1.00–1.06)	0.0295	5	1.03(1.00–1.06)	0.0282
MR Egger	5	1.05(0.89–1.24)	0.5808	5	1.06(0.93–1.21)	0.4181
MR Egger intercept		−0.0322	0.7660		−0.0468	0.6017

DOI: 10.7717/peerj.14595/table-2

Notes:

COVID

coronavirus disease 2019

ebi-a-GCST010777 and ebi-a-GCST010776

two largest COVID-19 GWAS data

SNP

the label of single-nucleotide polymorphism

N

sample size

OR

odds ratio

P

strength of evidence against the null hypothesis of no association between variant and outcome

We explored the causal associations between PCTs derived by principal component analysis and COVID-19 (Fig. 4). COVID-19 risk across the two datasets was slightly affected by the local inflammatory response (GCF IL-1β) (ebi-a-GCST010776: IVW OR = 1.02 (95% CI [1.01–1.03]), P < 0.001; ebi-a-GCST010777: IVW OR = 1.03 (95% CI [1.02–1.04]), P < 0.001), whereas PCT3 and PCT5 showed no statistically significant association with COVID-19.

Discussion

In the present study, the two-sample MR IVW method revealed a causal association between genetically predicted periodontitis and SARS-CoV-2 infection; that is, periodontitis increases COVID-19 susceptibility. We explored whether there is a causal association between the periodontitis-related phenotypes and COVID-19, and found that increased GCF IL-1β levels also increase COVID-19 susceptibility. The causal role of periodontitis in COVID-19 was verified in the two largest COVID-19 GWAS datasets.

Here, we present evidence that periodontitis and GCF IL-1β are COVID-19 risk factors. A newly published study has also explored the causal relationship between periodontitis and COVID-19 based on MR methods and reached the same conclusion that there is a causal association between periodontitis and COVID-19 (Wang et al., 2021). However, in this study, we further explored the causal effects of periodontitis-related phenotypes, including PCT3, PCT5 and inflammatory indicators IL-1β on COVID-19, and obtained the causal association of IL-1β in gingival crevicular fluid on COVID-19. In addition, previous epidemiological studies support our findings (Anand et al., 2021; Gupta et al., 2021b; Marouf et al., 2021; Vieira, 2021). It suggests that periodontitis and a high inflammatory burden are associated with higher COVID-19 infection, hospitalization rates and poorer prognosis, which our findings support. However, the results from a large-sample study are inconsistent with our findings (Larvin et al., 2020). In that study, the authors found that gum pain or bleeding was not related with COVID-19 risk and that hospitalization or mortality risk were not increased by loose teeth. We speculate that there are two major reasons for the inconsistent results. First, the oral manifestations, such as gum pain, gum bleeding, and tooth loss, were self-reported and not diagnosed; the inaccurate exposure information might have led to inconsistent results. Second, confounding factors, which that study did not adjust well, might have influenced the true correlation between periodontitis and COVID-19.

SARS-CoV-2, which causes COVID-19, has been detected in the periodontium (Fernandes Matuck et al., 2020). The positive causal correlation between periodontitis and susceptibility to COVID-19 may be connected to the augmented viral invasion. It is thought that periodontitis-induced epithelial mucosal ulcers may cause the loss of defenses against SARS-CoV-2. This renders periodontitis patients more vulnerable to infection by SARS-CoV-2 (Pfutzner, Lazzara & Jantz, 2020). Recently, it was hypothesized that the oral immune barrier breakdown, which can take place in periodontitis, may result in SARS-CoV-2 dissemination into the systemic circulation through its GCF and saliva oral reservoirs (Gupta et al., 2021a; Graham Lloyd-Jones et al., 2021). In addition, this association may be associated with the dysregulated inflammatory response. Cytokine storms caused by inflammation are important drivers of COVID-19 (Mangalmurti & Hunter, 2020). Periodontitis, one of the most common chronic inflammatory diseases, induces a dysregulated inflammatory response (Hajishengallis, 2015). Subgingival plaque biofilm interacts with host cells, leading to the release of proinflammatory cytokines and aggravating systemic inflammation (Pan, Wang & Chen, 2019). Therefore, it might cause overstimulation of the immune system, thereby aggravating SARS-CoV-2 infection severity because of the cytokine storm. Our findings that increased GCF IL-1β levels were associated with increased COVID-19 also support this assumption. GCF IL-1β might be a potential biomarker for predicting COVID-19 occurrence. However, exactly how IL-1β affects COVID-19 remains unclear, and the association between GCF IL-1β and SARS-CoV-2 infection warrants further exploration. Some studies have proposed that molecular factors in healthy oral mucosa might enable virus-host cell interactions. Gingival fibroblasts and the periodontal ligament contain angiotensin-converting enzyme 2 (ACE2), which has an affinity for SARS-CoV-2 and penetrates target cells easily (Madapusi Balaji et al., 2020). Therefore, ACE2-expressing cells may be vulnerable to infection.

It is worth mentioning that we found no causal association between PCT4 (Aa) and PCT5 (Pg) with COVID-19. Chakraborty et al. found that metagenomic analysis of patients with SARS-CoV-2 infection repeatedly yielded uncharacteristically high P. intermedia bacterial reads and failed to detect higher Aa and Pg reads (Zhu et al., 2020). This suggests that there might be no direct causal relationship between COVID-19 and these two periodontal pathogens. However, the specific correlation requires further exploration.

In the present two-sample MR study, we established a causal relationship between periodontitis and COVID-19 susceptibility, excluding the effects of confounders and reverse causality. The finding is important because we have established a link between periodontitis and COVID-19 risk from a high evidence level and broadened the understanding of COVID-19 risk. Therefore, periodontitis is a COVID-19 risk factor, which may contribute to the development of effective directives and strategies for controlling COVID-19 spread to vulnerable populations. Furthermore, we demonstrate for the first time that higher GCF IL-1β levels elevate COVID-19 risk, suggesting a novel mechanism through which SARS-CoV-2 might cause an individual onset of COVID-19 through the IL-1β-related pathways. Therefore, interventions targeting periodontal inflammation might also act in COVID-19 prevention. In addition, we demonstrate the result’s stability by using a causal relationship between periodontitis and COVID-19 in two databases.

However, our study has some limitations. The main limitation of our study is the insufficient power due to the relatively small sample size of GWAS for PD. Although the heritability of dental caries and periodontitis has been reported to be as high as 50%, GWAS with small sample sizes limits the ability to extract more significant instrumental variables, which results in the instrumental variables we used only explaining a small portion of phenotypic variation (Shungin et al., 2019). We partly verified the robustness of the results by applying multiple MR methods in parallel, while a larger sample size of GWAS for PD was still expected to validate the findings in our study. In addition, MR analysis relied on periodontitis exposure attributable to genetic predisposition, which is a lifetime exposure level and thus does not reflect the short-term effect of periodontitis and its components on COVID-19 risk. Therefore, the short-term exposure risk needs to be verified by further clinical studies. Third, due to limited data resources, our results are largely built on European populations and do not characterize the general inferences for other ethnic groups. While we assessed the causality of COVID-19 according to current data and numerous corresponding approaches, these results should be authenticated with added clinical resources and detailed investigation of the underlying causality mechanism.

Conclusions

The findings suggests that periodontitis and the higher GCF IL-1β levels is causally related to increase susceptibility of COVID-19. However, given the limitations of our study, the well-designed randomized controlled trials are needed to confirm its findings, which may represent a new non-pharmaceutical intervention for preventing COVID-19.

Supplemental Information