**4. Discussion**

In the present study, a large cohort of 124 subjects with DS showed significantly higher PCR counts of *T. forsythia,* culture counts of *E. corrodens* and total anaerobic counts, when comparing periodontitis with periodontal health or gingivitis. Subjects with periodontitis were also significantly older and the prevalence of hypothyroidism and levothyroxine intake significantly lower, as compared with the periodontal health and gingivitis groups.

Studies that have previously assessed the subgingival microbiota in DS subjects, have included a limited number of subjects and applied culture-independent techniques [19,20,23,37]. In the present study, a combination of culture and molecular techniques were selected, since they can be considered as complementary methodologies [32,38]. Using these techniques, higher counts and frequencies of detection of relevant periodontal pathogens, such as *P. gingivalis, A. actinomycetemcomitans, P. intermedia, T. forsythia* and *E. corrodens*, were identified in periodontitis, when compared with gingivitis and periodontally healthy DS subjects, as it has been previously reported in periodontitis patients without DS [39]. These quantitative and qualitative differences in the subgingival microbiota of periodontitis subjects may, in part, explain the early onset and progression of periodontitis in DS subjects, as it has been highlighted in previous studies [20,37]. However, in the present study, statistically significant differences among groups were only observed for culture counts of *E. corrodens* and total anaerobic counts, between periodontal health and periodontitis patients, and for qPCR counts of *T. forsythia,* when comparing periodontitis with periodontal health or gingivitis patients. This microbiological profile is also in agreement with the results obtained using Next Generation Sequencing (NGS), reported in a subset of this study population, which showed significantly higher levels of the genera *Tannerella*, *Porphyromonas* and *Aggregatibacter* in periodontitis, as compared with the periodontal health group [36].

In the present study, when using qPCR, *T. forsythia* was the most prevalent bacterial species in periodontitis patients (67.9%) but also in the other two groups. A similar trend has been reported in previous studies: Martinez-Martinez et al. [20] reported that *T. forsythia* was the most prevalent species in periodontitis DS patients (95.5%), being significantly higher when compared with periodontal health; Amano et al. [37] reported a high frequency of detection for *T. forsythia* (89.7%) in periodontitis DS patients, also significantly higher than in gingivitis patients; and *T. forsythia* was the only bacterial species with significantly higher presence and counts in DS subjects, when compared with healthy controls and individuals with cerebral palsy [23].

For *A. actinomycetemcomitans,* low frequencies of detection in the three study groups were reported in the present study. Much higher frequencies were detected in previous studies [20,37]. These differences could be explained by geographical variability, since a low prevalence of this bacterium has been reported in Spain [40,41] or due to technical aspects, such as the use of different probe-primers in the PCR technique or to differences in the patient selection process.

Higher counts and frequencies, for *T. forsythia* and *A. actinomycetemcomitans*, were reported when using qPCR, compared with culture. Similar results have been found in previous comparative studies [42]. These differences could be explained by the ability of qPCR to detect DNA of both viable and non-viable bacteria and to the lower detection limits of qPCR [32,38]. In addition, detection for *T. forsythia* by culturing is challenging [43,44], resulting in higher sensitivity and lower specificity when comparing qPCR technique *versus* culture [32,43,45]. However, the overall microbial tendency of an increase in prevalence for the three main target bacterial species from periodontal health to gingivitis and to periodontitis, was similar irrespective of the applied microbiological diagnostic technique.

The use of different microbiological technologies makes difficult to interpret the results from different studies, although common trends can be identified. In the present study, in which two complementary techniques were used, qPCR yielded higher counts for the three main target bacterial species, and higher prevalence for *T. forsythia* and *A. actinomycetemcomitans*, than culturing techniques, but the same tendency was observed with both methods, that is, an increase in prevalence/counts for the three main target bacterial species from health to gingivitis and to periodontitis. Both techniques are restricted in terms of target bacterial species, in this case three for qPCR and ten for culture. Other techniques may have a wider target such a checkerboard DNA-DNA hybridization: in the study by Sakellari et al. [23], 14 species were targeted and *T. forsythia* was identified as a relevant pathogen in all age cohorts, while *P. gingivalis, C. rectus, P. intermedia* and *A. actinomycetemcomitans* were relevant (among others) in the older cohorts; conversely, in the study by Khocht et al. [19], 40 species were targeted and *Treponema socrankii* showed significantly higher levels in DS subjects with periodontitis, as compared to individuals without periodontitis. These restrictions in the number of target species can be solved today using Next Generation Sequencing approaches, as shown in a publication based on subset of samples of the present study, but only comparing periodontitis and healthy periodontal subjects [36]: significant differences were observed at different taxonomical levels, with periodontitis patients demonstrating not only higher levels of periodontal pathogens, including *Tannerella, Treponema, Porphyromonas* and *Aggregatibacter* but also of new putative pathogens, such as species of *Peptostreptococcus, Filifactor, Fretibacterium* and *Desulfobulbus*; subjects with periodontal health showed more frequently species of *Veillonella, Neisseria, Gamella* and *Granulicatella*.

Different case definitions have been used to assess the periodontal status of DS subjects. For example, López-Pérez et al. [16] defined four categories as sound (CAL, 0 to 1 mm), mild periodontitis (CAL, 2 to 3 mm), moderate periodontitis (CAL, 4 to 5 mm) and severe periodontitis (CAL, 6 mm or more); while Khocht et al. [19] defined periodontitis as 5% or more teeth with CAL of 5 mm or more. In the present study, a more solid reference, the one proposed by the 2018 Classification of Periodontal and Peri-Implant Diseases and Conditions [17,28,29], was applied, with some modifications for the optimal use of the registered variables. The significant differences in PD, CAL and BOP among the studied categories were expected, since those variables were part of the case definition. For PlI, closely associated with BOP, it should be highlighted the large magnitude of the differences between periodontal health (around 45%), when compared with gingivitis and periodontitis (over 70%). The influence of this finding in the overall results deserve a careful analysis and the influence of a poor plaque control in the onset of gingivitis and periodontitis should be considered. In light of the possible impact of the limited skills in supragingival biofilm control in DS individuals, this factor should be taken into consideration [11].

In the present study, patients with periodontitis presented a significantly lower prevalence of hypothyroidism (10%) and the use of substitute medication (levothyroxine), compared with periodontal health and gingivitis subjects (42–44%). Hypothyroidism is the most common pathological hormone deficiency and, among the known risk factors for developing hypothyroidism, DS has been listed, usually as an autoimmune thyroiditis [46], which is consistent with the higher prevalence of autoimmune diseases in DS. Thus, the estimated prevalence of thyroid disorders in DS subjects reaches 40% in some series [47] and thanks to systematic screening, usually hypothyroidism is detected in subclinical stages in DS individuals [48], being subclinical hypothyroidism the most common detected thyroid abnormality in DS subjects [49]. Since the rate of conversion to overt hypothyroidism has been reported to be low in follow up studies [50], treatment of subclinical hypothyroidism is only advised by most authors in case of conversion to overt hypothyroidism [51].

Since the endocrine system can modulate the immune system in a bidirectional manner [52] and, in a recent scoping review, on 29 selected articles, a positive relationship between hypothyroidism and periodontitis was found [53], thus the findings of the present study are totally unexpected. One possible explanation is that periodontitis patients are actually suffering subclinical hypothyroidism and/or they have not yet been properly diagnosed [54], and, this were true, the surveillance of hypothyroid hormone function can be further supported in DS individuals. However, only speculations can be made at this point, since the previous hypothesis cannot be tested now.

In the present study, patients with periodontitis were significantly older that those in periodontal health or gingivitis groups. This finding agrees with previous reports, that have pointed out that the prevalence of periodontal diseases in DS, as it occurs in the general population, increases with age [14,15]. It is also relevant to highlight that the prevalence of periodontitis in DS individual is relatively high at a young age, ranging 58–96% in subjects younger that 35 years old [12]. As for other periodontitis as a manifestation of systemic diseases, periodontitis in DS individual presents more frequently with earlier onset, faster and generalized and more severe progression, as compared with individuals without a systemic immune compromised [16,23]. A longitudinal study has also shown that periodontal pathogens can be frequently detected in DS subjects with periodontitis, for up to 6 months, despite periodontal treatment and frequent supportive periodontal care, suggesting a higher risk of progression and giving more relevance to supragingival biofilm control as performed by the subject [55]. The importance of poor oral hygiene has been also emphasized in a recent study, suggesting a larger impact of a poor oral hygiene and an impaired immune response in the development of periodontal diseases in DS subjects [56]. However, the possible relevance of specific periodontal pathogens should not be discarded, since a recent 24-month study has even reported an association of high counts *of P. gingivalis* with risk of disease progression [57], although the study population did not include DS patients.

The present study has clinically and microbiologically evaluated a large group of DS individuals. However, some limitations have to be acknowledged, being the main limitation that periodontal diagnosis was based on a partial-mouth clinical evaluation (Ramfjord teeth), with no radiographical evaluation, which may have underestimated disease levels. This approach was selected to ease the evaluation of the special patient population included in the study and knowing that partial-mouth recording systems may present adequate degrees of accuracy [58]. Additional limitations can also be listed: (i) in a cross-sectional design, causal associations cannot be established [59]; (ii) some confounding factors may have not been properly controlled, such as diet, life-style and habits, care givers support in oral hygiene; and (iii) differences in age and thyroid disfunction may have impacted both clinical and microbiological findings.

## **5. Conclusions**

In the present study, DS patients with periodontitis were characterized, as compared with those with periodontal health or gingivitis, by an older age, a lower frequency of thyroid dysfunction, deeper periodontal pockets and more attachment loss, higher prevalence of bleeding on probing and dental plaque accumulation and higher levels of anaerobic bacterial counts, *T. forsythia* and *E. corrodens*. It is, therefore, important, to promote actions for DS individuals, including surveillance of thyroid hormone function, improvement of oral hygiene measures and frequent evaluation of periodontal health, in order to make possible an early detection of disease (i.e., gingivitis) and, thus, providing adequate treatment or help in maintaining periodontal health, when it is still preserved.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2076-341 7/11/2/778/s1, Supplementary Table S1. Presumptive identification of bacterial species in culture. Supplementary Table S2. Demographic characteristics for the complete study population. Supplementary Table S3. Periodontal clinical outcomes, expressed as means and standard deviations (SD), for the complete study population and for each study group. Supplementary Table S4. Microbiological findings (counts and frequencies of detection), as evaluated by means of quantitative polymerase chain reaction, expressed as means and standard deviations (SD), for the complete study population and for each study group. Supplementary Table S5. Microbiological findings, as evaluated by means of culture, with counts expressed as means and standard deviations (SD) and frequencies of detection expressed as percentage, for the complete study population and for each study group. Supplementary Table S6. Microbiological findings (proportions), as evaluated by means of culture, expressed as means and standard deviations (SD), for the complete study population and for each study group.

**Author Contributions:** M.C., M.J.M., A.O., M.C.S., contributed to data acquisition (microbiological analyses), statistical analyses and critically revised the manuscript; L.N., contributed to conception, data acquisition (clinical evaluations) and critically revised the manuscript; J.B., J.L., critically revised the manuscript; M.S., D.H., P.D., contributed to conception, data analysis and interpretation, critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was co-funded by Xunta de Galicia under Ignicia Programme, Axencia Galega de Innovación, GAIN (12/08/2016; GRANT\_NUMBER: IN855A). The participation of M.C. Sánchez in the project occurred within the activities of the Extraordinary Chair of DENTAID in Periodontal Research, University Complutense of Madrid, Spain.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki of the World Medical Association (2008) and approved by the Research Ethics Committee of Santiago-Lugo, Spain (registration code: 2018/510).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved (and their legal guardians, where appropriate) in the study.

**Data Availability Statement:** Data available on request due to restrictions. The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy and ethical issues.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

