ORIGINAL RESEARCH

https://doi.org/10.5005/jp-journals-10015-2169
World Journal of Dentistry
Volume 14 | Issue 1 | Year 2023

Evaluation of Salivary Galectin-3 Level and its Potential Role in increasing the Severity of COVID-19 Infection in Patients with Periodontitis

Nimisha Mithradas¹, Uma Sudhakar², Vijayalakshmi Rajaram³, Shankar Ram V⁴, Navina Ravindran⁵, Devika Warrier V⁶, Sherine L Asirvatham⁷, Parthibhan Saket⁸

^1,2,4Department of Periodontics, Thai Moogambigai dental College and Hospital, Chennai, Tamil Nadu, India

^3,5–7Department of Periodontics, Meenakshi Ammal Dental College & Hospital, Chennai, Tamil Nadu, India

⁸Department of Periodontics, Adhiparasakthi Dental College and Hospital, Tamil Nadu, India

Corresponding Author: Nimisha Mithradas, Department of Periodontics, Thai Moogambigai Dental College and Hospital Chennai, Tamil Nadu, India, Phone: +91 9445006918 e-mail: drnimishamithradas1983@gmail.com

Received on: 01 November 2022; Accepted on: 02 December 2022; Published on: 25 March 2023

ABSTRACT

Aim: To evaluate and compare the salivary galectin-3 (Gal-3) level in chronic periodontitis patients, coronavirus disease 2019 (COVID-19) patients, and patients with COVID-19 + periodontitis and thus determining the risk of periodontitis in increasing the severity of COVID-19 infection.

Materials and methods: For the present study, a total of 77 participants were recruited to the study with 20 healthy controls (group I), 20 patients with chronic generalized periodontitis (group II), 19 COVID-19 patients (group III), and 18 with COVID-19 and periodontitis (group IV). Demographic characteristics and periodontal clinical parameters like plaque index (PI), probing pocket depth (PPD), and clinical attachment level (CAL) were recorded for all patients. Saliva samples were collected and Gal-3 levels were assessed using enzyme-linked immunosorbent assay (ELISA) kit. One-way analysis of variance (ANOVA) analysis and Tukey’s honest significant difference post hoc tests were carried out for data analysis.

Results: Group IV patients had a higher concentration of salivary Gal-3 (15.50 ng/mL) than that of group III (10.247 ng/mL) and group II (12.340 ng/mL), and the mean difference in Gal-3 level was statistically significant with the p-value 0.000. The mean PPD, CAL, and PI were significantly high in groups II and IV compared to groups I and II with a p-value of 0.000.

Conclusion: The result of the present study showed that patients with periodontitis and periodontitis + COVID-19 presented significant higher salivary Gal-3 levels in comparison with COVID-19 patients and healthy subjects. Thus periodontitis can be a risk factor in increasing the severity of COVID-19 infection.

Clinical significance: This study was carried out to evaluate whether periodontitis is a risk factor for increasing the severity of COVID-19 infection. This study also highlights the importance of maintaining good oral hygiene and periodontal health in preventing COVID-19 severity.

How to cite this article: Mithradas N, Sudhakar U, Rajaram V, et al. Evaluation of Salivary Galectin-3 Level and its Potential Role in increasing the Severity of COVID-19 Infection in Patients with Periodontitis. World J Dent 2023;14(1):3-8.

Source of support: Nil

Conflict of interest: None

Keywords: Acute phase proteins, Coronavirus disease 2019, Galectin-3, Periodontitis, Severe acute respiratory syndrome coronavirus 2.

INTRODUCTION

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV2) virus from the Coronaviridae family can infect patients of all age-groups. The disease condition which emerged in Wuhan, China, mainly spread through airborne droplets and aerosols, and subsequently had a global outbreak.¹^,² Even though, in most cases the disease resulted in mild symptoms, some cases showed increased mortality due to the presence of comorbidities resulting in the progression of the disease state to severe pneumonia and multiorgan failure. Although it is emphasized that the risk factors, such as age, sex, and comorbidities, increase the risk of complications and mortality, there is still a large proportion of patients with no identified risk factors who endure from severe COVID-19 related adversities and complications.³

Periodontitis is a chronic inflammatory disease, multifactorial in origin, that is associated with dysbiotic dental plaque biofilms and characterized by progressive destruction of the tooth-supporting structures.⁴ Periodontitis not only leads to tooth loss and mobility, with the consequent impairment of the patient’s quality of life, but it also affects general health. Severe periodontitis has been associated with a range of systemic diseases.⁵ Due to this increased systemic risk, various analyses have proposed a possible link between periodontitis and increased severity of COVID-19.⁶^,⁷ The probable mechanisms which could expound the association between periodontitis and the COVID-19 disease could be due to the direct contact of the virus with the periodontal tissues, because of high expression of angiotensin-converting enzyme 2 (ACE2), CD147, transmembrane serine protease 2 (TMPRSS2), and furin which are expressed in oral epithelial cells.⁸^,⁹ The proteases produced by periodontopathic bacteria could cleave the S protein of the virus, thereby favoring SARS-CoV-2 infection.¹⁰ Thus, the presence of periodontopathic bacteria could increase the risk of SARS-CoV-2 infection. The similar overexpression of several cytokines, a COVID-19 “cytokine storm,” as seen in periodontitis with elevated serum levels of interleukin (IL)—1 β, IL-6, IL-7, IL-10, IL-17, IL-2, IL-8, IL-9, granulocyte-macrophage colony-stimulating factor, interferon γ, and tumor necrosis factor (TNF) α also increases the severity of COVID-19 infection.¹¹^-¹³ Another proposed immunological link between periodontitis and COVID-19 is through an acute phase protein, Gal-3.¹⁴ Gal-3, with a molecular weight of 30 kD is a member of β-galactoside binding chimera-type proteins, it is highly expressed in all human tissue, epithelial cells, endothelial cells, and immune cells like macrophages, activated T and B cells, dendritic cells, monocytes, and mast cells.¹⁵ Gal-3 plays a crucial role in many biological functions like homeostasis of immune cells, cell growth, fibrosis, inflammation, apoptosis, angiogenesis, premessenger ribonucleic acid addition, differentiation, and host defense.¹⁶ Recent studies have shown that Gal-3 is increasingly recognized as a potentially important diagnostic or prognostic biomarker in severe COVID-19 patients.¹⁷^,¹⁸ This increase in viral infection is due to the recruitment of more neutrophils, thereby enhancing airway inflammation.¹⁸ The host inflammatory responses is also increased by the release of several cytokines like IL-6 and TNF-α, which are some of the major cytokines present in severe COVID-19 patients.¹⁹ In addition to this, Gal-3 has a morphology that is almost identical to the spike protein of SARS-CoV-2, which eases the entry of the virus into the host cells.²⁰

Recent evidences suggested that increased Gal-3 level is associated with the severity of periodontitis and initial periodontal treatment lead to a decrease in the level. Thereby proving that Gal-3 can be considered a potential biomarker for the evaluation of gingival inflammation and the presence of periodontal disease.²¹

Currently no clinical data are available to explain the role of a biomarker in establishing the association of periodontitis and COVID-19 infection severity. Therefore, this present study evaluated and compared the salivary Gal-3 levels in chronic generalized periodontitis, COVID-19 positive individuals, and COVID-19 positive-patients with periodontitis, thereby determining the possibility of periodontitis being a risk factor for developing severe COVID-19 infection.

MATERIALS AND METHODS

A total of 80 individuals aged between 25 and 50 years who had at least 20 teeth were included in the study. The study was in compliance with the Helsinki Declaration on medical research reviewed in 2013. The study protocol was approved from Institutional Ethical Committee of Dr MGR University (Dr MGRDU/TMDCH/2021-22/625). Every participant was briefed about the study, and written informed consent was obtained in their own language.

Adequate sample size was achieved by performing a power sample analysis. To achieve an 80% power level and confidence interval of 95%, atleast 15 participants/group could be required for a total number of 75 patients.

After the initial screening phase, out of 80 patients, two patients refused to participate in the study during periodontal clinical examination and one patient was absent for the study and thus, a total of 77 patients were included for the study (Flowchart 1).

Flowchart 1: Flowchart of study

The enrolled patients were allotted into four groups. Group I included 20 healthy patients with PPD of <3 mm in all sites, no signs of inflammation, and clinical attachment loss. Group II included 20 patients with stage II–III grade B periodontitis with atleast 30–40% sites having PPD ≥ 4 mm, interdental CAL of ≥4 mm, radiographic bone loss extending to middle third of root interproximally in atleast two or more nonadjacent teeth.²² Both healthy and periodontitis patients were recruited from the outpatient ward of Department of Periodontics, Thai Moogambigai Dental College and Hospital (TMDCH), Chennai, Tamil Nadu, India.

Patients reporting to the communicable disease ward of the institution (Sri Lalithambigai Medical College and Hospital, Chennai, Tamil Nadu, India) from the period of January–May 2022 were recruited to the study after they undergo real-time reverse-transcriptase-polymerase chain reaction (rRT-PCR) (according to World Health Organization interim guidelines, 2020) for diagnosis of COVID-19.²³ Patients with mild to moderate COVID-19 disease that turned positive were included for the study. Patients’ contact details were obtained from the record maintenance section of institution and they were contacted over the telephone concerning their readiness to take part in the study.

Coronavirus disease 2019 (COVID-19) positive patients who were willing to participate in the study were scheduled within 1–2 weeks for periodontal examination after their rRt-PCR turned negative and became disease free. On clinical examination, periodontal parameters like PPD (pocket probing depth), CAL, and PI was measured. PPD and CAL were recorded in all six sites of a tooth using a University of North Carolina-15 periodontal probe. PI index was measured in four surfaces of the teeth (mesial, buccal, distal, and lingual) and scores were obtained as per Silliness and Loe PI score criteria. Patients with PPD and CAL of <4–5 mm, PI < 2.2 were included in group III and those with PPD ≥ 6 mm, CAL ≥ 5 mm, and PI ≥ 2 in group IV. A single investigator who was blind to the categorization of the study participants had measured all clinical parameters.

Coronavirus disease 2019 (COVID-19) associated complications, such as existence of COVID-19 pneumonia, intensive care unit (ICU) admission, and any need of hospital-assisted ventilation were excluded. Smokers, patients with any systemic diseases, any histories of periodontal treatment done or intake of any systemic antibiotics, immunosuppressant in past 6 months, and pregnant or lactating women were also excluded.

Saliva Collection and Gal-3 Analysis

About 2–3 hours prior to saliva collection, patients were asked to abstain from drinking or eating. Patients were then instructed to collect unstipulated saliva in the floor of their mouth and then expectorate into a graduated container (0.5–1 mL). Saliva samples were transported to lab and stored at −800°C.

The level of salivary Gal-3 was measured by ELISA using a commercial kit [Human Gal-3 (LGALS3) ELISA Kit, Abkinne].

Human Gal-3 (LGALS3) ELISA kit employed a two-site sandwich ELISA to quantitate LGALS3 in samples. An antibody specific for LGALS3 had been precoated onto a microplate. Standards and samples were pipetted into the wells, and any LGALS3 present was bound by the immobilized antibody. After removing any unbound substances, an horseradish peroxidase (HRP)—conjugated human LGALS3 detection antibody was added to the wells. Following a wash to remove any unbound HRP reagent, a chromogen solution was added to the wells, and color developed in proportion to the amount of LGALS3 bound in the initial step. The color development was stopped and the intensity of the color was measured. Optical density was read at 450 nm using a microplate reader within 15 minutes. Salivary gal-3 levels were obtained from an ELISA reader. The minimum detectable dose of human LGALS3 was <0.50 ng/mL and the detection range was between 5–80 ng/mL.

STATISTICAL ANALYSIS

The Statistical Package for the Social Sciences (IBM Corporation, Chicago, Illinois, United States of America) software program version 16 was used to perform statistical analysis. One-way ANOVA was used to compare the mean values of demographic and clinical parameters. Tukey’s honest significant difference post hoc tests were carried out for multiple comparisons between groups I and II, I and III, I and IV, II and III, II and IV, and III and IV.

RESULTS

A total of 77 patients with similar gender distribution of 42 males (56.0%) and 38 females (45.0%) were selected for the study. The result of this study showed that the mean age of group IV (50.13) was higher in comparison with other groups. The mean PPD, CAL, and PI was significantly high in groups II and IV compared to groups I and III, and on comparison of clinical parameters among the groups, the mean values showed a statistically significant difference with a p-value of 0.000 (Table 1). However, on multiple comparisons of PPD and CAL between groups II and IV did not show a statistically significant value (0.550 and 0.800, respectively), as depicted in Table 2. But on comparison of PI index score between groups II and IV a statistically significant value was obtained (p-value 0.000). The mean salivary Gal-3 level in group II patients (12.340 ng/mL) was more in comparison to group III (10.247 ng/mL) and group I (6.987 ng/mL). Group IV patients had a higher concentration of salivary Gal-3 (15.50 ng/mL) than that of group III and II and the mean difference in Gal-3 level was statistically significant with the p-value 0.000 (Table 3). But no statistically significant difference was observed on the comparison of salivary Gal-3 level between group II and III (p-value of 0.287) (Table 4). Thus, mean salivary Gal-3 level was higher in COVID-19 positive individuals with periodontitis compared to COVID-19 patients and periodontitis patients.

**Table 1:** Comparison of mean clinical parameters among groups
	Groups	N	Mean ± standard deviation	F value	p-value
Age	Group I	20	39.47 ± 4.291	30.939	0.000
	Group II	20	48.20 ± 7.389
	Group III	19	46.07 ± 7.186
	Group IV	18	50.13 ± 5.630
PPD	Group I	20	2.20 ± 0.414	143.121	0.000
	Group II	20	7.20 ± 0.941
	Group III	19	5.47 ± 0.743
	Group IV	18	8.00 ± 0.756
CAL	Group I	20	2.20 ± 0.414	147.387	0.000
	Group II	20	7.73 ± 0.961
	Group III	19	4.73 ± 0.961
	Group IV	18	8.60 ± 0.828
PI	Group I	20	0.580 ± 0.2042	77.557	0.000
	Group II	20	2.233 ± 0.3773
	Group III	19	1.480 ± 0.3649
	Group IV	18	2.673 ± 0.2738

CAL, clinical attachment level; PPD, probing pocket depth; PI, plaque index; p-value, probability value; p-value significant at 0.05

**Table 2:** Multiple comparison of mean clinical parameters between the groups
		Groups	Mean difference	p-value
PPD	Group I	Group II	−4.267^*	0.000
		Group III	−5.000^*	0.000
		Group IV	−5.800^*	0.000
	Group II	Group III	−1.533^*	0.000
	Group II	Group IV	−0.733	0.550
	Group III	Group IV	−0.800^*	0.028
CAL	Group I	Group II	−4.533^*	0.000
		Group III	−5.533^*	0.000
		Group IV	−6.400^*	0.000
	Group II	Group III	−1.867^*	0.000
	Group II	Group IV	−1.000^*	0.800
	Group III	Group IV	−0.867^*	0.029
PI	Group I	Group II	−1.1000^*	0.000
		Group III	−1.6533^*	0.000
		Group IV	−2.0933^*	0.000
	Group II	Group III	−0.5533^*	0.000
	Group II	Group IV	−0.9933^*	0.000
	Group III	Group IV	−0.4400^*	0.007

CAL, clinical attachment level; PPD, probing pocket depth; PI, plaque index; p-value, probability value; p-value significant at 0.05; *The mean difference is significant at the 0.05 level

**Table 3:** Comparison of mean values of salivary Gal-3 (ng/mL) level concentration in various groups
Groups	N	Mean ± standard deviation	F value	p-value
Group I	20	6.987 ± 0.6174	63.914	0.00
Group II	20	12.340 ± 1.1096
Group III	19	10.247 ± 1.2906
Group IV	18	15.500 ± 2.7240

**Table 4:** Multiple comparisons of salivary Gal-3 level (ng/mL) concentration between the groups
Group	Groups	Mean difference (I−J)	Significance
Group I	Group II	−3.2600^*	0.000
	Group III	−4.3533^*	0.000
	Group IV	−8.5133^*	0.000
Group II	Group III	−1.0933	0.287
Group II	Group IV	−5.2533^*	0.000
Group III	Group IV	−4.1600^*	0.000*

*, the mean difference is significant at the 0.05 level

DISCUSSION

The present study results showed that periodontitis could be a risk factor for increasing the severity of COVID-19 infection. In the last 2 years during pandemic, COVID-19 disease had become great challenge to society, particularly in the field of health care. Individuals with any comorbidities, such as cardiovascular diseases, diabetes, renal diseases, and respiratory diseases, can increase the severity of COVID-19. Periodontitis an inflammatory, chronic condition acts as a crucial factor in not only determining the oral health status but also acts as a trivial contributing factor in the pathophysiology of various systemic conditions. The association between the presence of periodontal disease and the progressive course of respiratory illnesses has been justified through various studies.²⁴

Persual of literature has shown several speculative mechanisms that describe the severity of COVID-19 infection with the presence of periodontitis.²⁵ The linkage mechanism could be by direct aspiration of periodontopathic pathogen to lungs by providing a favorable environment due to oral dysbiosis created by increased plaque formation and thereby increasing COVID-19 complications.²⁶ Takahashi et al. proposed that periodontal bacteria cause this aggravation of COVID-19 infection by increasing the expression of ACE2, a receptor for SARS-CoV-2 and the proteases produced by bacteria, cleaves the S-protein of the virus, and enable its entry. Thus, oral cavity becomes a potential reservoir for the virus.²⁷ This is further in credence to the recent study by Gupta et al., where SARS-CoV-2 virus was detected from the gingival crevicular fluid (GCF) of patients rendering that poor oral hygiene could possibly result in increasing the viral load.²⁸ Periodontitis and COVID-19 complications share some common inflammatory signals resulting from the release of proinflammatory cytokines like ILs 1β, 6, and 17; TNF-α; and C-reactive protein (CRP). Unrelenting periodontal disease leads to hyperresponsiveness of host and release of proinflammatory cytokines, which enter the systemic circulation and thereby alter the respiratory epithelium. Apart from this, viral replication in host cells leads to activation of the nucleotide-binding domain-like receptor protein 3 inflammasome, which may also result in the release of proinflammatory cytokines creating a vicious cycle called a cytokine release syndrome. Altered clotting cascades with an increase in D-dimer levels, increased prothrombin time, and decreased fibrinogen levels are commonly associated with COVID-19 patients. Worsening of these parameters, especially with elevated D-dimer levels, is an investigation of severe COVID-19 infections. Patients with moderate to advanced periodontitis had shown to have elevated blood parameters like D-Dimer, hemoglobin A1C, lymphocytes, and white blood cells, which are relevant to the course of COVID-19, and thereby, likely to be more prone to exacerbations of COVID-19 than patients with mild periodontitis.²⁹ These findings are in par with a case–control study done by Marouf et al., which investigated a significant association of periodontitis with COVID-19 complications like hospital-assisted ventilation, ICU admission, and death. It is well-documented that there is a marked increase of systemic inflammatory markers like CRP in the serum of periodontitis patients.²⁹ Another acute phase protein is Gal-3, a proinflammatory protein involved in T-cell mediated inflammation. Gal-3 exaggerates the expression of proinflammatory mediators, and thereby plays a crucial role in the evolution of the periodontal disease. This is consistent with the findings of a recent study by Hendek et al. that showed an increase of GCF Gal-3 in periodontitis patients, as it plays a key role in the homeostasis of immune cells and also acts as a modulator for cell signaling and cell surface functioning in various inflammatory conditions. The initial periodontal treatment led to a decrease in GCF Gal-3 level and thus became a potential biomarker for evaluating the severity of periodontitis.²¹ During active inflammation, Gal-3 is released from fibroblasts and macrophages systemically and becomes a marker of coronary heart disease (CHD), cardiovascular fibrosis, and heart failure by increasing endothelial dysfunction. Gal-3 acts as a chemokine and plays a key role in the phagocytosis of microorganisms by stimulating monocyte and macrophage migration. Isola et al. suggested that there is an increase in Gal-3 levels in periodontitis patients and periodontitis with CHD compared to patients with CHD, thereby establishing as a predictive marker of periodontitis and CHD.³⁰ Current literature indicates, Gal-3 as a potential prognostic biomarker in predicting the severity of COVID-19 infection as SARS-COV-2 spike protein and Gal-3 share the same morphology, thus facilitating the ingress of the virus into host cells.¹⁷

The viral host interaction can be interfered with by targeting Gal-3 due to its functional similarity to the N-Acetylneuraminic acid binding domain on the spike protein of SARS-CoV-2. Also, it has been suggested that Gal-3 inhibitors can suppress the release of proinflammatory markers like IL-1 and IL-6 and increase the level of IL-10, thus resulting in the potential decrease of viral load.³¹ There are numerous studies to correlate the association of periodontal disease and COVID-19 infection. But this study is one among the first study done to assess the periodontal clinical parameters and potential role of a biomarker Gal-3, in the saliva of COVID-19 positive patients, thereby suggesting a role in the severity of periodontal disease and COVID-19 infection. The PI score was significantly high in COVID-19 patients in this study which is in accordance with a study by Larvin et al., which concluded that the risk of mortality was high in COVID-19 patients who had bleeding gums.³²

However, the study had a few limitations. The patients were not examined clinically during the active phase of COVID-19 infection because of the risk of transmission of the disease. Periodontal destruction is a slow process, and it was speculated that participants had periodontitis prior to developing COVID-19 infections. This is in consistent with the study by Anand et al. in 2021.³³

CONCLUSION

An abundance of evidence suggested the association between the severity of periodontitis and COVID-19 infection, which require further exploration. This association could be caused by Gal-3-mediated increased viral attachment and increased immune response with inflammation. Poor oral hygiene with increased plaque formation and gingival bleeding is more often seen in COVID-19 patients. In this perspective, this study was done to emphasize the significance of maintaining periodontal disease under control. However larger sample size with more clinical trials are required to evaluate the periodontal status in patients with COVID-19 infection and to establish a relationship with periodontal disease and COVID-19.

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