**1. Introduction**

The introduction Down syndrome (DS) is a congenital genetic disease, caused by the presence of an extra chromosome in par 21 [1], being the most common human aneuploidy among living births, with an estimated prevalence of one case per 800 births [2]. Its phenotypic expression, however, may be associated with significant genetic complexity [3]. Subjects with DS present a characteristic phenotype and intellectual disability [4], together with several systemic diseases [5], such as congenital heart disease and thyroid dysfunction, as well as a number of oral diseases [6,7]. Furthermore, there is compromise in their innate immune response [8] and DS subjects suffer more frequently from oral infections, when compared with the general population [9,10].

**Citation:** Cuenca, M.; Marín, M.J.; Nóvoa, L.; O'Connor, A.; Sánchez, M.C.; Blanco, J.; Limeres, J.; Sanz, M.; Diz, P.; Herrera, D. Periodontal Condition and Subgingival Microbiota Characterization in Subjects with Down Syndrome. *Appl. Sci.* **2021**, *11*, 778. https://doi.org/ 10.3390/app11020778

Received: 6 December 2020 Accepted: 11 January 2021 Published: 15 January 2021

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Early onset forms of periodontitis and late onset of caries lesions represent the most frequent oral conditions associated with DS subjects [9], which may become further aggravated by their motor disability and manual dexterity compromise, that limits their performance in oral hygiene practices [11]. Several observational studies have reported high prevalence of gingivitis and periodontitis (ranging from 58–96%) at young ages (less than 35) in DS subjects [12]. In Brazil, in a study sample of 93 DS patients aged 6–20 years, only 9% showed a healthy periodontium and 33% had periodontitis [13]; in another study including 64 DS subjects (mean age 23.8 years), 28.1% presented gingivitis and 71.9% periodontitis [14]. In The Netherlands, in a study group conformed by 182 DS subjects, 36.6% were diagnosed of periodontitis [15]. In addition, SD periodontitis patients have shown more severity, when compared with controls [16], and a higher impact in their quality of life [13]. In fact, in the latest classification of periodontal and peri-implant diseases and conditions [17], periodontitis in DS patients was included within the category "periodontitis as a manifestation of a systemic disease," in which the systemic condition has a major impact in the onset and course of periodontitis, as clearly occurs in DS subjects [18].

Furthermore, some authors have suggested a specific microbial profile associated to DS subjects with periodontitis [19,20], raising the hypothesis that a distinctive pathogenic microbiota combined with an impaired host-response [21], may result in an earlier dysbiosis and onset of periodontitis [22]. However, the evidence supporting this hypothesis is very scarce and the reported results are highly heterogeneous [19,20,23]. In light of this limited information, we have designed this observational study aimed to evaluate the subgingival microbiota of DS subjects with different periodontal health status (periodontal health, gingivitis or periodontitis), by means of cultural and molecular microbiological methods.

## **2. Materials and Methods**

#### *2.1. Study Population*

A cross-sectional observational study was designed in a Spanish Caucasian population diagnosed of DS. This investigation followed the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) criteria for reporting [24] and was approved by the Research Ethics Committee (EC) of Santiago-Lugo, Spain (Registration Code: 2018/510). All subjects and, when appropriate, their legal guardians, confirmed in writing their agreement to participate and signed the EC-approved informed consent, once they were informed on the objectives and processes associated with this investigation.

Screening among DS subjects regularly attending seven educational or occupational therapy centers in the region of Galicia, in the North-West of Spain, was carried out between September 2017 and November 2018.

At this screening visit, information on their age, gender, presence of co-morbidities and use of current medications was collected. Study subjects were consecutively included if DS was genetically confirmed and did not have any of the following exclusion criteria: under 18 years of age, comorbidities that could influence the periodontal condition (e.g., diabetes), presence of harmful habits (e.g., smoking), having received antimicrobial therapy in the previous month (e.g., systemic antimicrobials and/or oral antiseptics), insufficient degree of collaboration for performing clinical assessment and microbiological sampling.

Upon inclusion in the study, subjects had an intraoral examination where the following periodontal variables were recorded in four sites at the six reference teeth [25] or when absent, at the adjacent tooth: plaque index (PlI) [26], probing depth (PD) (using a PCP UNC15 periodontal probe, applying a force of 20–25 g), bleeding on probing (BOP) [27], gingival recession (REC) (distance from the gingival margin to the cemento-enamel junction) and clinical attachment level (CAL). All measurements were recorded by a trained and calibrated clinical investigator (N.L.). Calibration was achieved during several sessions of repeated measurements, until the clinical investigator demonstrated an intra-class correlation coefficient (ICC) higher than 0.75 in all the periodontal variables evaluated. The intra-class correlation coefficient values for intra-examiner calibration were 0.87 (95% confidence interval = 0.75–0.98). Clinical assessments were not conducted in dental clinics/chairs so, together with the specific condition of the subjects, neither full-mouth clinical evaluations or radiographical assessments were feasible.

Following a modification of the case definitions proposed by the Classification of Periodontal and Peri-Implant Diseases and Conditions [17], the study group was categorized as:

