Redondovirius-associated Periodontitis in People with Poor Oral Hygiene

doi:10.21203/rs.3.rs-5003719/v1

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Research Article

Redondovirius-associated Periodontitis in People with Poor Oral Hygiene

https://doi.org/10.21203/rs.3.rs-5003719/v1

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Objectives

This study aimed to investigate the prevalence of recently emerged members of the Redondoviridae family (ReDoVs) in individuals with periodontitis and their association with underlying diseases.

Materials and Methods

This case-control study involved 230 participants, 115 with chronic periodontitis and 115 healthy controls from October 2023 to May 2024. Demographic, health-related, and behavioral data were collected. Gingival samples were analyzed for ReDoVs using polymerase chain reaction (PCR), followed by sequencing and phylogenetic analysis.

Results

ReDoVs were detected in 51.30% of the periodontitis case group and 21.74% of the control group (p < 0.0001). ReDoV presence was associated with older age, lack of teeth brushing habits, and smoking history (p < 0.05). Also, a higher occurrence of underlying diseases was observed among participants with periodontitis (24.35%) compared to controls (4.35%). An association was observed between ReDoVs and diabetes (OR = 9.18, 95% CI: 1.48 to 56.98, p = 0.02).

Conclusions

ReDoVs and periodontitis were associated with adults with poor dental hygiene and those with diabetes. This supports a possible multifactorial and complex interaction between the presence of ReDoVs, periodontitis, and diabetes. We further update the message of our previous study by highlighting that poor oral hygiene increases the risk of ReDoV presence.

Clinical Relevance:

ReDoVs were significantly higher in individuals with periodontitis, with a strong correlation between ReDoVs and periodontitis, especially in diabetic patients. This could lead to improved periodontal treatments focusing on bacterial factors and addressing viral contributions, potentially reducing the burden of periodontal disease and its associated complications.

Redondoviridae

Periodontitis

Diabetes

Oral hygiene

Redondoviruses (ReDoVs), which are part of the larger group of eukaryotic circular rep-encoding single-stranded (CRESS) DNA viruses with small genomes and diverse host ranges, have received much attention due to their prevalence in human biological samples and potential link to human health conditions. These viruses are distinguished by their tiny circular, single-stranded DNA genomes, roughly 3,000 nucleotides long. Abbas et al. and subsequent studies have emphasized the broad distribution and possible adverse interactions of ReDoVs in humans, notably in respiratory and oral cavities [1, 2].

Recent studies show that ReDoVs are widely distributed in human populations, with varied prevalence rates in the respiratory tract, oral cavity, lungs, nasopharynx, and gastrointestinal system. Abbas et al. observed a 3.8% prevalence of ReDoV in the oral cavity, which is of great importance given the oral cavity's significance as a gateway for numerous infections [1]. The discovery of two major species, Vientovirus and Brisavirus, within this family further complicates their epidemiology because each may interact differently with the human host, impacting illness consequences across distinct body areas [1]. Once taken together, this distribution is crucial for understanding the ecological niches these viruses inhabit and the dynamics of their interactions with humans. Investigations like those reported in [3] and [4] demonstrate these viruses in populations ranging from healthy persons to those suffering from acute or chronic illnesses, implying a multifaceted role that may transcend basic pathogenicity [3, 4].

ReDoVs' clinical consequences are not known. Critical observations include increased viral loads in people with worse clinical outcomes, raising whether these viruses indicate illness severity or contribute to disease pathogenesis. This uncertainty highlights the need for more focused studies to better understand their function in human health and disease processes [5]. While their existence corresponds with periodontitis and other important disorders, causation linkages have yet to be shown clearly. Research into their function in inflammatory illnesses suggests that ReDoVs may affect disease processes, perhaps aggravating or altering immune responses in ways that might complicate patient outcomes in diseases such as COVID-19 and chronic respiratory problems [6, 7].

The oral cavity has a diverse microbiome composed of bacteria, fungi, and viruses that play critical roles in oral disease and health. Periodontal disease, a major worldwide health concern, is caused mostly by bacterial infections in tooth plaque. However, current research revealed a complicated interplay between diverse microbial species, including viruses, contributing to the etiology of periodontal diseases. Periodontal disease, a common chronic disease characterized by gum inflammation and tooth loss, has been the primary research focus on the complex interplay between microbial dysbiosis and immunological responses. ReDoVs have been discovered in the oral cavity, where their interactions with the microbial flora and the host immune system may affect the course and severity of periodontal disease. Studies have revealed that these viruses might enhance the inflammatory response in the gums, thus accelerating overall tissue damage and disease development [8, 9].

Despite growing knowledge of ReDoVs, there are gaps in the direct relationship between these viruses and periodontal disease outcomes. Most investigations have been correlational rather than definitive, highlighting the need for further targeted study into the processes by which ReDoVs impact periodontal disease [1]. The current work fills this gap by applying molecular methods to measure Redondovirus prevalence and strain diversity in gum samples from patients with periodontal disease, intending to shed light on their involvement in the etiopathogenesis of this disease [10, 11].

This study aimed to look at the incidence and association of ReDoVs in gum samples from referrals with periodontal complaints. By establishing their incidence and maybe identifying specific viral strains associated with disease severity, we want to shed light on the viral contributions to periodontal disease. This study's assumptions include the anticipation of a strong correlation between ReDoV prevalence and periodontal disease severity, which might open the door for new diagnostic and treatment strategies [6]. Since we have already demonstrated a possible association between ReDoVs and dental complications, clarifying the role of ReDoVs in periodontal disease may lead to a paradigm shift in how periodontal disease is diagnosed and treated, emphasising the need to consider viral components in preventive and therapeutic strategies [12, 13].

Study design, sample size, and data collection

This case-control study was designed to follow our recent report on the association between ReDoVs and dental complications that were orally interviewed patients referred for COVID-19 screening [14]. In this regard, a collaboration was formed between the Department of Virology, Iran University of Medical Sciences, Tehran, Iran, and the Department of Endodontics, Clinical Research Development Unit, School of Dentistry, Alborz University of Medical Sciences, Karaj, Iran. Accordingly, 230 referrals to the School of Dentistry from October 2023 to May 2024 were asked to participate in the study. The participants gave their informed consent and were orally informed of the study. In this regard, participants could leave the study at their request, and their information would not be included in the investigation. A group of randomly selected referrals who were referred for routine checks to the School of Dentistry, Alborz University of Medical Sciences, Karaj, Iran, with recognisable periodontal problems, formed the case groups. An unmatched control group comprised of the participants, students, and staff from both institutes diagnosed with healthy oral cavities with no sign of periodontitis was selected as the control group. The study comprised 230 participants, 115 in the case with chronic periodontitis and 115 in the control group.

It was considered not to be biased when selecting participants based on their lifestyles, hygiene habits, and any underlying disease variables. Data were collected through a structured questionnaire and clinical examinations. Participants in this study were assessed based on demographical (age, sex, occupation, marital state, and intravenous drug users [IVDU]), hygiene habits or health-related variables (dental floss usage, teeth brushing, smoking, and periodontitis severity), and medical history of underlying diseases.

Inclusion and exclusion criteria

According to the staging criteria for periodontitis [15], the case group comprised those with chronic periodontitis. The participants were aged 18–65 years (n = 260). Individuals suffering from systemic diseases, including renal or liver disorders, cardiovascular disease, diabetes mellitus, human immunodeficiency virus (HIV) infection or malignancies, and pregnant women were excluded. In addition, individuals who reported a history of periodontal treatment, smoking (> 15 cigarettes/day), or antibiotics treatment (during the previous 6 months) were also excluded. Clinical examination was performed for all participants, and the periodontal health was evaluated, including the pocket depth (PD), bleeding on probing (BOP), plaque index (PLI), gingival index (GI), and clinical attachment loss (CAL). Furthermore, the extent of alveolar bone resorption was determined radiographically using an orthopantomogram. Accordingly, the case group comprised participants with BOP (+), mean PD ≥ 6 mm, mean CAL ≥ 3 mm, and alveolar bone resorption. Additionally, the control group was formed by participants without signs of gingivitis, BOP, and CAL, with mean PD ≤ 4 mm.

Sample collection and genome extraction

A skilled dentist gently took The samples from the gum with a sterile brush. Samples were immediately transferred into a vial containing viral transport media (VTM) and frozen at a temperature of ˗80°C for DNA extraction.

The genomic viral DNA was extracted from the samples retrieved from participants using the QIAamp® DNA Mini kit (Qiagen GmbH, Germany), according to the manufactured protocol. The quality of the extracted DNA was determined by NanoDrop (Thermo Scientific, Wilmington, MA) as described before [16].

Detection of the ReDoV by polymerase chain reaction

The presence of the ReDoV(s) genome was examined by our sensitive in-house polymerase chain reaction (PCR) assay, as described before [14]. Briefly, the assay was done in a 25 µL reaction containing 10 pmol of primers, 2.5 µl of 10X Ex Taq buffer (Mg2 + free), 1.0 U of Ex Taq DNA polymerase (TaKaRa Biotechnology, Dalian Co., Ltd., Shiga, Japan), 2.0 µl of MgCl2 (25 mM), 2.0 µl dNTPs Mixture (25 mM each), and 5 µl of extracted viral DNA as template.

The thermal cycles were comprised of an initial denaturation at 95°C for 4 minutes, 35 cycles at 95°C for 30 seconds, at 56°C for 30 seconds, and at 72°C for 35 seconds, followed by a final extension at 72°C for 5 minutes. The PCR products of the specimens, negative and positive controls, and size marker (100 bp) were electrophoresed on 1.8% Agarose gel, stained using gel red, and visualized by an ultraviolet transilluminator.

Sequencing and Phylogenic analysis

10 positive samples were randomly selected and purified using the high-pure PCR product purification kit (Roche Diagnostic, Mannheim, Germany), according to the manufactured protocol. The purified samples were sequenced bi-directionally by the chain termination method using the ABI 3730 XL sequencer.

The taxonomical data of ReDoVs are classified in different oral-, lung-, and human respiratory-associated, as well as unassigned ReDoVs’ sequences [14]. Here, we have updated the ReDoVs’ genomes to see if there were any more entries from the NCBI Taxonomy database. A multiple sequence alignment (MSA) was performed with MAFFT v7 [17, 18] using the retrieved data from NCBI’s Taxonomy database, our previously published ReDoVs sequencing data from the referrals for COVID-19 screenings, and data from the present study. Accordingly, MSA was performed with the G-INS-1 strategy to establish an accurate guide tree as described before [19]. The statistical significance of the phylogenetic tree was evaluated using the bootstrap method (1,000 replicates). A closely related Anellovirus genome was also used as the outgroup to root the tree. A Newick graph-theoretical trees file format was saved to reconstruct the radial phylogram using the Interactive Tree Of Life (iTOL; https://itol.embl.de/) online tool [20].

Statistical analysis

Statistical analysis was performed by MS-Excel XLSTAT v2019 software package, as reported before [21, 22]. The normality of the quantitative variable (age) was tested using the Kolmogorov-Smirnov test. The statistical differences between categorical variables were assessed using the Contingency table, and the strength of the test was approved by Fisher's exact test. A p-value ≤ 0.05 with an alpha value of 0.05 and a 95% confidence interval (CI 95%) was considered statistically significant.

Ethical considerations

The present study was approved ethically by the Ethics Committee, School of Medicine, Iran University of Medical Sciences, Tehran, Iran, by the Second Declaration of Helsinki, using the ethical code of IR.IUMS.FMD.REC.1402.351.

Report of the retrieved data

Based on the specialist’s clinical examinations, 115 individuals referred to the Department of Endodontics, Clinical Research Development Unit, School of Dentistry, Alborz University of Medical Sciences, Karaj, Iran, recognized with periodontitis, were randomly selected and participated in this study. A further control group was comprised of 115 participants who were. It was checked to have no confounding variable bias toward the parameters regarding lifestyle, hygiene habits, and underlying diseases. The mean age of the participants was 37.11 ± 13.48 years old (minimum of 10 years old and maximum of 71 years old). Also, the mean age of case and control groups was 41.05 ± 13.75 and 33.17 ± 13.48, respectively. The groups were not age-matched (test value = 4.43, p-value < 0.0001). The demographical reports of the participants are demonstrated in Table 1.

Table 1

Demographical information of the total participants
Variable\Statistic	Nbr. of observations	Nbr. of missing values	Categories	Frequency per category	Rel. frequency per category (%)
Sex	230	0	Female	137	59.57
			Male	93	40.43
Dental floss usage	230	0	No	124	53.91
			Sometimes	19	8.26
			Yes	87	37.83
Age category	230	0	1–10	3.00	1.30
			11–19	7.00	3.04
			20–29	66.00	28.70
			30–39	70.00	30.43
			40–49	40.00	17.39
			50–65	36.00	15.65
			> 65	8.00	3.48
Occupation	230	0	Employed	214.00	93.04
			Retired	11.00	4.78
			Unemployed	5.00	2.17
Marital state	230	0	Married	130.00	56.52
			Single	97.00	42.17
			Widow	3.00	1.30
Teeth brushing	230	0	> Once	59.00	25.65
			None	40.00	17.39
			Once	131.00	56.96
Smoking history	230	0	No	199.00	86.52
			Yes	31.00	13.48
IVDU^£ history	230	0	No	229.00	99.57
			Yes	1.00	0.43
^£ Intravenous drug users

Further comparison of demographical findings between the case and control groups is depicted in Fig. 1. In terms of age groups, most of the individuals in the case group were between the ages of 30–39 (26.96%) and 40–49 (25.22%), whereas the majority of the control group fell within the 20–29 (43.48%) and 30–39 (33.91%) age categories. Both groups had a high employment rate, with 90.43% of cases and 95.65% of controls employed. There was a significant difference in marital status between the two groups, with a larger percentage of married individuals in the case group (73.91%) compared to the control group (39.13%). In contrast, the control group had a higher proportion of singles (60.00%). The data on teeth brushing habits revealed that 57.39% of the case group brushed their teeth once a day, while 31.30% did not brush at all. On the other hand, 56.52% of the control group brushed once daily, with only 3.48% not brushing at all. In terms of smoking history, it was found that 74.78% of the case group did not smoke, while the control group had a higher percentage of non-smokers at 98.26%. A minimal history of IVDU was reported by 99.13% of the case group and 100% of the control group, with no history of IVDU (see Fig. 1).

The analysis of hygiene habits or health-related variables and medical history of underlying diseases

Underlying diseases that the participants reported included Anemia, asthma, hyperlipidemia, hypertension, diabetes, gastric surgery, Hashimoto's thyroiditis, cardiovascular disease, hyperthyroidism, hypothyroidism, rheumatism, thalassemia minor, and alopecia totalis (Table 2). Accordingly, 33/230 (14.35%) of participants reported underlying diseases. Among the 115 participants in the case group, 24.35% reported having an underlying disease. In contrast, the control group, with 115 participants, reported a lower prevalence of underlying disease (4.35%). This indicates a higher occurrence of underlying diseases among participants with periodontitis compared to the control group.

Table 2

Data regarding the reported underlying diseases or complications by the participants (n = 230)
Variable\Statistic	Nbr. of observations	Nbr. of missing values	Categories	Frequency per category	Rel. frequency per category (%)
Anemia	230	0	No	226.00	98.26
			Yes	4.00	1.74
Asthma	230	0	No	229.00	99.57
			Yes	1.00	0.43
Hyperlipidemia	230	0	No	228.00	99.13
			Yes	2.00	0.87
Hypertension	230	0	No	225.00	97.83
			Yes	5.00	2.17
Diabetes	230	0	No	224.00	97.39
			Yes	6.00	2.61
Gastric surgery	230	0	No	228.00	99.13
			Yes	2.00	0.87
Hashimoto's thyroiditis	230	0	No	229.00	99.57
			Yes	1.00	0.43
Cardiovascular disease	230	0	No	226.00	98.26
			Yes	4.00	1.74
Hyperthyroidism	230	0	No	225.00	97.83
			Yes	5.00	2.17
Hypothyroidism	230	0	No	222.00	96.52
			Yes	8.00	3.48
Rheumatism	230	0	No	228.00	99.13
			Yes	2.00	0.87
Thalassemia minor	230	0	No	229.00	99.57
			Yes	1.00	0.43
Alopecia totalis	230	0	No	229.00	99.57
			Yes	1.00	0.43

The prevalence of the underlying illnesses is presented in Fig. 2 in both case and control groups. In this regard, anemia was found in 3.48% of individuals in the case group, whereas the control group did not report it. 0.87% of the individuals in the case group had asthma, whereas none of the control group members reported having asthma. Hyperlipidemia was reported in 1.74% of individuals in the case group, while it was not present in the control group. 4.35% of the case group had hypertension, while none of the control group had it. 5.22% of individuals in the case group were found to have diabetes, while none of the individuals in the control group were reported to have the condition. A history of gastric surgery was reported by 1.74% of the case group, whereas none was reported by the control group. Hashimoto's thyroiditis was not seen in the case group, whereas it was reported by 0.87% of the control group. Cardiovascular disease had the same prevalence of 1.74% in both groups. 4.35% of the case group had hyperthyroidism, while none of the control group had the illness. 6.09% of the case group had hypothyroidism, compared to just 0.87% of the control group who had the same condition. Both rheumatism and thalassemia minor were found in 0.87% of the individuals in the case group, while none were present in the control group. There were no reports of alopecia totalis in the case group, while it was found in 0.87% of the control group.

ReDoVs prevalence in the participants

Here, the presence of ReDoV’s genome was investigated in 230 participants. The results showed that 63.48% (146 participants) tested negative for ReDoV, while 36.52% (84 participants) tested positive. Furthermore, comparing ReDoVs status between case and control groups revealed significant differences. Among the case group, 59 (51.30%) were ReDoV-positive. In contrast, 25 (21.74%) participants in the control group had ReDoV-positive results. The test of independence between groups and ReDoV status yielded a significant difference between the groups (chi-square value of 21.68 > the critical value of 3.84, df(1), p-value < 0.0001). This indicates a significant association between the case group and ReDoV genome presence. Fisher's exact test confirmed the significance, showing that the number of ReDoV-negative cases in the control group and ReDoV-positive cases in the case group were significant. The cell-specific chi-square values indicated that the observed associations were considerable, further supporting the conclusion of an important link between group classification (case/control) and ReDoV status.

To calculate the odds ratio (OR) for the prevalence of ReDoVs in the case and control groups, Fisher's exact test was performed to determine the statistical significance of the observed association between group classification (case/control) and ReDoV status. The test was conducted with the alternative hypothesis that the odds ratio is not equal to 1. The Monte Carlo method with 5000 simulations was used to ensure robust p-value estimation, and the significance level (alpha) was set at 0.05. Standard bootstrap intervals with 100 samples were also used to validate the results. The OR for ReDoV prevalence in the case group compared to the control group was 0.26, indicating no significant impact of periodontitis on the detection of ReDoVs. This is supported by a log odds ratio of -1.33 with a standard deviation of 0.29 and a 95% confidence interval ranging from − 1.90 to -0.76. Several association coefficients were calculated (see Table 3).

Table 3

The coefficient analysis of the odds for detecting ReDoVs genome in case and control groups
Coefficient	Value	Standard deviation	Lower bound 95%	Upper bound 95%
Goodman and Kruskal Gamma	-0.58	0.10	-0.77	-0.39
Kendall's tau	-0.31	0.06	-0.43	-0.19
Stuart's tau	-0.30	0.06	-0.41	-0.18
Somers' D (R/C)	-0.30	0.06	-0.41	-0.18
Somers' D (C/R)	-0.32	0.06	-0.44	-0.19
Theil's U (R/C)	0.07	0.03	0.01	0.13
Theil's U (C/R)	0.07	0.03	0.01	0.13
Theil's U (Symmetric)	0.07	0.03	0.01	0.13
Log(Odds ratio)	-1.33	0.29	-1.90	-0.76
Odds ratio	0.26		0.15	0.47

The association of ReDoV status and demographic and behavioral oral hygiene factors

The association between ReDoV status and demographic and behavioral oral hygiene variables was investigated using chi-square and Fisher's exact test (Fig. 3). The contingency table for sex and ReDoV status showed no significant association (chi-square = 1.97, p-value = 0.16). In contrast, age category and ReDoV status were significantly associated (chi-square = 20.02, p-value < 0.0001), suggesting a strong link between age and ReDoV prevalence. No significant association was found for occupation with ReDoV status (chi-square = 2.16, p-value = 0.34). Marital status and ReDoV status also showed no significant association (chi-square = 5.58, p-value = 0.06). However, teeth brushing habits were significantly associated with ReDoV status (chi-square = 19.78, p-value < 0.0001), with the "none" and "once" categories being linked considerably to positive ReDoV status. Smoking history was significantly associated with ReDoV status (chi-square = 5.19, p-value = 0.02), where smokers were more likely to be ReDoV-positive (odds ratio = 2.39, 95% CI: 1.12 to 5.09). Lastly, no significant association was found between IVDU history and ReDoV status (chi-square = 1.75, p-value = 0.19).

The association between ReDoV status and underlying diseases

No significant association was found between the presence or absence of the ReDoVs genome and underlying diseases reported by the participants. Further analysis included a contingency table comparing individuals with and without diabetes against their ReDoV status was performed. It was observed that among the 230 individuals, 145 individuals without diabetes tested negative for ReDoV, while 79 individuals without diabetes tested positive for ReDoV. In contrast, only 1 individual with diabetes tested negative for ReDoV, whereas 5 individuals with diabetes tested positive for ReDoV (chi-square value of 5.82 > critical value of 3.84, df(1), p-value = 0.02). Fisher's exact test further confirmed this significance, with diabetic participants being more likely to be ReDoV-positive. In support of this finding, the calculated odds ratio was 9.18 (95% CI: 1.48 to 56.98), indicating that diabetic participants had significantly higher odds of being ReDoV-positive than non-diabetic participants (Table 4).

Table 4

The coefficient analysis of the odds for detecting ReDoVs genome participants with diabetes
Coefficient	Value	Standard deviation	Lower bound 95%	Upper bound 95%
Goodman and Kruskal Gamma	0.80	0.20	0.42	1.19
Kendall's tau	0.16	0.06	0.04	0.28
Stuart's tau	0.05	0.02	0.00	0.10
Somers' D (R/C)	0.05	0.03	0.00	0.11
Somers' D (C/R)	0.48	0.16	0.18	0.79
Theil's U (R/C)	0.10	0.07	-0.04	0.25
Theil's U (C/R)	0.02	0.02	-0.01	0.05
Theil's U (Symmetric)	0.03	0.03	-0.02	0.08
Log(Odds ratio)	2.22	0.93	0.39	4.04
Odds ratio	9.18		1.48	56.98

Phylogenic analysis

Randomly, ten samples were sequenced and submitted to GenBank with the accession numbers PP930330 to PP930339. Furthermore, data from the previous study was also retrieved with the accession numbers PP319424 to PP319438. As illustrated in Fig. 4, several clades (I to X) could be established, with few with consistent organ associations. Accordingly, clades II and V comprised the genomic data of ReDoVs with mostly oral cavity distribution. Also, clades XI and X were composed of respiratory- (lung) associated ReDoVs. Other clades represented a mixture of different organ-associated or unidentified ReDoV genomes.

The primary study by Abbas et al. (2019) reported a high prevalence of ReDoVs in periodontal tissues and suggested that these viruses might play a role in the pathogenesis of periodontitis [7]. This study was also designed to confirm the findings of our previous study on interviewed referrals for COVID-19 screening who have reported oral/dental complications during the time of their testing [14]. In the preceding research, due to the type of study, we could only manage to interview the referrals for any dental or gum issues. During the interview, the participants acknowledged their gum or dental complications; whether their problems were due to periodontitis was unclear. Nevertheless, consistent with our previous finding using the developed in-house PCR method, it was observed that individuals with moderate to chronic periodontitis represent a higher presence of the ReDoVs genome (51.30%) than the control group (21.74%). This is also aligned with a report that identified a correlation between ReDoV and chronic periodontitis [23]. The case and control groups were carefully selected to avoid potential confounding bias toward factors that may present any conflict regarding lifestyle, oral hygiene factors, and underlying diseases. In this regard, the control group comprised students or clinical staff with no recognizable oral, gum, or dental issues. However, we could not find a similar significant OR as the (0.26 versus 13.03) supporting the finding of our last study [14]. This indicates that an accurate diagnosis of periodontitis is essential for selecting a case group. Still, the prevalence of the ReDoVs was significantly higher among cases than in the control group.

In a study by Zhang et al. comprised of 240 individuals, including 120 with chronic periodontitis and 120 healthy controls, ReDoVs were found in 71.67% of the periodontitis group and 51.67% of the control group (20% difference with an OR of 2.53) [23]. This indicates the high rate of ReDoVs in individuals with periodontitis, supporting the idea of ReDoV involvement in human dental diseases. In this regard, demographic and behavioral factors, such as age, oral hygiene practices, and smoking history, were significant determinants of ReDoV prevalence in both Zhang et al. and our study. We have also found that dental floss is significantly associated with a lower incidence of periodontitis and less frequency of the ReDoV genome in those who frequently use dental floss. This further supports the complex multifactorial impact of ReDoV on dental health, which could involve other regulatory factors such as recently reported circular RNAs (circRNAs) in the setting of periodontitis [13]. Additionally, ReDoVs may exploit E. gingivalis as a host in the oral cavity, potentially influencing the pathogenicity of periodontal disease and making the ReDoV pathogenicity even more complex [24, 25]. We suggest a possibly higher incidence rate of ReDoVs in those with poor oral hygiene and less dental floss use. This is due to the increased population of fungal infections, especially E. gingivalis, in the setting of periodontitis, which hosts ReDoVs. This interaction may be a contributing factor in some renal complications like diabetes, which are discussed below.

Periodontitis is identified as an inflammatory condition affecting the periodontium, causing tissue damage and loss of alveolar bone [26]. It is common among adults, leading to a considerable impact on public health [27]. Periodontitis is also associated with local immune system alteration and systemic diseases [28, 29]. This supports the establishment of viral infection in the gastrointestinal and respiratory tract axes. As a result, the occurrence of underlying diseases was higher among participants with periodontitis (24.35%) compared to controls (4.35%). Significantly, there was an association between diabetes and ReDoV status (odds ratio = 9.18, 95% CI: 1.48 to 56.98). Periodontitis and diabetes are interconnected through a bidirectional relationship where each condition exacerbates the other, primarily through systemic inflammation [30]. This chronic inflammatory state likely establishes a favorable environment for the persistence and proliferation of ReDoVs. The elevated cytokines and other inflammatory mediators in periodontitis could facilitate viral activity and impact the host immune response, contributing to higher ReDoV prevalence in individuals with periodontitis. Furthermore, the advantageous effects of periodontal treatment on glycaemic control in diabetic patients are demonstrated by reductions in HbA1c levels and systemic benefits of managing periodontal health [31]. This suggests that effective periodontal treatment and taking care of oral hygiene could also potentially reduce ReDoV load, potentially thoroughly reducing the population of E. gingivalis and improving overall health outcomes. The presented finding aligns with the increasing evidence that the health of the gums has a significant effect on overall health, including viral infections.

A phylogenetic analysis was performed as a routine investigation of the genetic distance between the detected genomes of the viruses. We have performed a highly sensitive and accurate progressive MSA using data provided by the NCBI’s Taxonomy database and our sequencing results. To cluster the ReDoVs as a group, the tree was rooted with a closely related member of Annelovirus. There are currently a few categories, including the human gut, lung, oral, respiratory-associated Brisaviruses or Vientovirues, and unclassified and non-human ReDoVs. These groups lack enough members to accurately and robustly distinguish them. Also, it is poorly understood that lung-associated ReDoVs could not be associated with oral or respiratory ReDoVs and vice versa. Additionally, as a limitation of our study, we have only partially sequenced the positive samples, which might influence the genetic distance evaluation as some entries are used to establish the phylogenetic tree.

The results of the present study demonstrated a high prevalence of the ReDoVs genome in adult individuals with periodontitis who are less concerned about dental hygiene. Furthermore, the substantial association between the presence of ReDoVs in participants with diabetes increases the possibility of a multifactorial contribution of the viral infection of tooth and renal diseases. This could be future research on the clinical association between ReDoVs and renal diseases in those with periodontitis. Moreover, future research should focus on elucidating the molecular mechanisms by which ReDoVs interact with host cells and contribute to periodontal disease pathogenesis.

The study's findings demonstrated a significant association between the presence of ReDoVs and chronic periodontitis, with a higher prevalence of the virus in individuals with periodontal disease compared to healthy controls. The strong correlation between ReDoVs and diabetes suggests a multifactorial contribution of these viruses to periodontal pathology, particularly in diabetic patients. Phylogenetic analysis identified distinct clades with a mixture of ReDoVs with different origins. The significant association between ReDoVs and poor oral hygiene practices, such as infrequent tooth brushing, smoking, and using less dental floss, especially among adult participants, further emphasizes the accumulation of oral pathogens like E. gingivalis and increased risk of ReDoVs presence. Effective periodontal treatment has been shown to improve glycemic control in diabetic patients, suggesting that addressing periodontal infections could have broader health benefits, including reducing the viral load of ReDoVs.

Acknowledgments

We thank the Iran University of Medical Sciences, Tehran, Iran, for their financial support of this work. We also appreciate the support of the students and staff of the Department of Virology, Iran University of Medical Sciences, Tehran, Iran, and the Department of Endodontics, Clinical Research Development Unit, School of Dentistry, Alborz University of Medical Sciences, Karaj, Iran, for making this cooperation happen. All authors gave their final approval and agreed to be accountable for all aspects of the work.

Statements and Declarations

Ethics approval

This study was approved by the Ethics Committee of Iran University of Medical Sciences, Tehran, Iran (Ethics Code: IR.IUMS.REC.1402.530) in 2024. All participants provided written informed consent prior to enrolment in the study. This research was conducted ethically following the World Medical Association Declaration of Helsinki.

Consent for publication

Not applicable

Declaration of conflicting interest

The author(s) declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article.

Funding statement

This work was supported by the Iran University of Medical Sciences, Tehran, Iran [grant number 85162].

Data availability

The datasets generated and/or analyzed during the current study are available in the GenBank (https://www.ncbi.nlm.nih.gov/nucleotide/) repository under the accession numbers PP930330 to PP930339.

Author contribution

AM: Contributed to conceptualization, data curation, formal analysis, investigation, methodology, resources, software, validation, visualization, and writing—original draft, and review and editing.

ZD: Contributed to data curation, resources, software, and validation.

ZM: Contributed to investigation, validation, and writing—original draft.

KK: Contributed to data curation, investigation, and validation.

NE: Contributed to investigation and validation.

SJK: Contributed to formal analysis, methodology, software, and writing—original draft, and review and editing.

TD: Contributed to resources and writing—review and editing.

AS: Contributed to resources.

RB: Contributed to resources and writing—review and editing.

FBS: Contributed to conceptualization, funding acquisition, methodology, project administration, supervision, validation, and writing—original draft, review, and editing.

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No competing interests reported.

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Redondovirius-associated Periodontitis in People with Poor Oral Hygiene

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Abstract

Objectives

Materials and Methods

Results

Conclusions

Clinical Relevance:

Figures

Introduction

Methodology

Study design, sample size, and data collection

Inclusion and exclusion criteria

Sample collection and genome extraction

Detection of the ReDoV by polymerase chain reaction

Sequencing and Phylogenic analysis

Statistical analysis

Ethical considerations

Results

Report of the retrieved data

The analysis of hygiene habits or health-related variables and medical history of underlying diseases

ReDoVs prevalence in the participants

The association of ReDoV status and demographic and behavioral oral hygiene factors

The association between ReDoV status and underlying diseases

Phylogenic analysis

Discussion

Conclusion

Declarations

Acknowledgments

Statements and Declarations

Ethics approval

Consent for publication

Declaration of conflicting interest

Funding statement

Data availability

Author contribution

References

Additional Declarations

Status:

Version 1