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Periodontitis is one of the world's most common chronic human diseases and has a significant impact on oral health. Recent evidence has revealed a link between periodontitis and certain severe systemic conditions. Moreover, periodontal patients remain so for life, even following successful therapy, requiring ongoing supportive care to prevent the disease's recurrence. The first challenge in treating the condition is ensuring a timely and accurate diagnosis since the loss of periodontal bone and soft tissue is progressive and largely irreversible. Although current clinical and radiographic parameters are the best available for identifying and monitoring the disease, the scientific community has a particular interest in finding quantifiable biomarkers in oral fluids that can improve early detection rates of periodontitis and evaluations of its severity. It is widely accepted that periodontitis is associated with polymicrobial dysbiosis and a chronic inflammatory immune response in the host. This response causes the generation of mediators like cytokines. Higher concentrations of cytokines are involved in inflammation and disease progression, acting as a network of biological redundancy. Most of the cytokines investigated concerning the periodontitis pathogenesis are proinflammatory. Of all of them, interleukin (IL) 1beta has been studied the most, followed by tumor necrosis factor (TNF) alpha and IL6. In contrast, only a few papers have evaluated antiinflammatory cytokines, with the most researched being IL4 and IL10. Several systemic reviews have concluded that the specific cytokines present in patients with periodontitis have a distinctive profile, which may indicate their possible discriminatory potential. In this chapter, the focus is on analyzing studies that investigate the accuracy of diagnoses of periodontitis based on the cytokines present in gingival crevicular fluid and saliva. The findings of our research group are also described.

In the literature, 16S rRNA gene sequencing is the most widely used technology for studying the periodontal microbiota. However, there is no evidence on how methodological aspects such as primer coverage, detection of matching amplicons (MAs), and clustering into operational taxonomic units (OTUs) could influence the results obtained for the oral niche. Furthermore, the comparison of 16S sequencing-based studies on periodontal microbiota is controversial due to significant methodological differences. Therefore, meta-omics analyses would favour the accuracy of phylogenetic data associated with different periodontal conditions. In the present Thesis, we analysed in silico 1) the coverage of primers employed in sequencing-based studies of the mouth microbiota using oral-specific databases containing bacterial and archaeal 16S rRNA gene sequences; 2) the number of 16S rRNA genes in the complete genomes of bacterial and archaeal species inhabiting the human mouth, and how the use of different primers would affect the detection of MAs from different taxa; and 3) the performance of different primers to detect distinct oral species with 16S rRNA gene amplicon similarity ≥97%, identifying the taxa that may be erroneously grouped into the same OTU. 2023-07-22

This in silico investigation aimed to: 1) evaluate a set of primer pairs with high coverage, including those most commonly used in the literature, to find the different oral species with 16S rRNA gene amplicon similarity/identity (ASI) values ≥97%; and 2) identify oral species that may be erroneously clustered in the same operational taxonomic unit (OTU) and ascertain whether they belong to distinct genera or other higher taxonomic ranks.Thirty-nine primer pairs were employed to obtain amplicon sequence variants (ASVs) from the complete genomes of 186 bacterial and 135 archaeal species. For each primer, ASVs without mismatches were aligned using BLASTN and their similarity values were obtained. Finally, we selected ASVs from different species with an ASI value ≥97% that were covered 100% by the query sequences. For each primer, the percentage of species-level coverage with no ASI≥97% (SC-NASI≥97%) was calculated.Based on the SC-NASI≥97% values, the best primer pairs were OP_F053-KP_R020 for bacteria (65.05%), KP_F018-KP_R002 for archaea (51.11%), and OP_F114-KP_R031 for bacteria and archaea together (52.02%). Eighty percent of the oral-bacteria and oralarchaea species shared an ASI≥97% with at least one other taxa, including Campylobacter, Rothia, Streptococcus, and Tannerella, which played conflicting roles in the oral microbiota. Moreover, around a quarter and a third of these two-by-two similarity relationships were between species from different bacteria and archaea genera, respectively. Furthermore, even taxa from distinct families, orders, and classes could be grouped in the same cluster.Consequently, irrespective of the primer pair used, OTUs constructed with a 97% similarity provide an inaccurate description of oral-bacterial and oral-archaeal species, greatly affecting microbial diversity parameters. As a result, clustering by OTUs impacts the credibility of the associations between some oral species and certain health and disease conditions. This limits significantly the comparability of the microbial diversity findings reported in oral microbiome literature.

Background: Sequencing has been widely used to study the composition of the oral microbiome present in various health conditions. The extent of the coverage of the 16S rRNA gene primers employed for this purpose has not, however, been evaluated in silico using oral-specific databases. This paper analyses these primers using two databases containing 16S rRNA sequences from bacteria and archaea found in the human mouth and describes some of the best primers for each domain. Results: A total of 369 distinct individual primers were identified from sequencing studies of the oral microbiome and other ecosystems. These were evaluated against a database reported in the literature of 16S rRNA sequences obtained from oral bacteria, which was modified by our group, and a self-created oral-archaea database . Both databases contained the genomic variants detected for each included species. Primers were evaluated at the variant and species levels, and those with a species coverage (SC) ≥75.00% were selected for the pair analyses. All possible combinations of the forward and reverse primers were identified, with the resulting 4638 primer pairs also evaluated using the two databases. The best bacteria-specific pairs targeted the 3-4, 4-7 and 3-7 16S rRNA gene regions, with SC levels of 97.14-98.83%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-5 and 3-6, with SC estimates of 95.88%. Finally, the best pairs for detecting both domains targeted regions 4-5, 3-5 and 5-9, and produced SC values of 94.54-95.71% and 96.91-99.48% for bacteria and archaea, respectively. Conclusions: Given the three amplicon length categories (100-300, 301-600 and >600 bps), the primer pairs with the best coverage values for detecting oral bacteria were: KP_F048-OP_R043 (region 3-4; primer pair position for Escherichia coli J01859.1: 342-529), KP_F051-OP_R030 (4-7; 514-1079), and KP_F048-OP_R030 (3-7; 342-1079). For detecting oral archaea, these were: OP_F066-KP_R013 (5-6; 784-undefined), KP_F020-KP_R013 (3-6; 518-undefined) and OP_F114-KP_R013 (3-6; 340-undefined). Lastly, for detecting both domains jointly they were KP_F020-KP_R032 (4-5; 518-801), OP_F114-KP_R031 (3-5; 340-801) and OP_F066-OP_R121 (5-9; 784-1405). The primer pairs with the best coverage identified herein are not among those described most widely in the oral microbiome literature.

The present study used 16S rRNA gene amplicon sequencing to assess the impact on salivary microbiome of different grades of dental and periodontal disease and the combination of both (hereinafter referred to as oral disease), in terms of bacterial diversity, co-occurrence network patterns and predictive models. Our scale of overall oral health was used to produce a convenience sample of 81 patients from 270 who were initially recruited. Saliva samples were collected from each participant. Sequencing was performed in Illumina MiSeq with 2 × 300 bp reads, while the raw reads were processed according to the Mothur pipeline. The statistical analysis of the 16S rDNA sequencing data at the species level was conducted using the phyloseq, DESeq2, Microbiome, SpiecEasi, igraph, MixOmics packages. The simultaneous presence of dental and periodontal pathology has a potentiating effect on the richness and diversity of the salivary microbiota. The structure of the bacterial community in oral health differs from that present in dental, periodontal or oral disease, especially in high grades. Supragingival dental parameters influence the microbiota’s abundance more than subgingival periodontal parameters, with the former making a greater contribution to the impact that oral health has on the salivary microbiome. The possible keystone OTUs are different in the oral health and disease, and even these vary between dental and periodontal disease: half of them belongs to the core microbiome and are independent of the abundance parameters. The salivary microbiome, involving a considerable number of OTUs, shows an excellent discriminatory potential for distinguishing different grades of dental, periodontal or oral disease; considering the number of predictive OTUs, the best model is that which predicts the combined dental and periodontal status This investigation was supported by the Instituto de Salud Carlos III (General Division of Evaluation and Research Promotion, Madrid, Spain) and co-financed by FEDER (“A way of making Europe”) under grant ISCIII/PI17/01722, and the CESPU under grants MVOS2016 and MVOS-PT-IINFACTS-2019 SI

Very few 16S rRNA-based studies have conducted a simultaneous analysis to identify the impact of various dental and periodontal parameters and determine which of them have the greatest repercussion for the salivary microbiota. Consequently, this study used 16S rRNA gene amplicon sequencing to assess the impact on salivary microbiome of different grades of dental, periodontal and global oral disease. Our global oral health scale was used to produce a convenience sample of 81 patients from 270 who were initially recruited. These subjects were assigned the following grades: 47 had a periodontal grade (PG) of 0 and dental grades (DGs) between 0-3, and 46 had a DG of 0 and PGs between 0-3. Saliva samples were collected from each participant. Sequencing was performed in Illumina MiSeq with 2 x 300 bp reads, while the raw reads were processed according to the Mothur pipeline. The statistical analysis of the 16S rDNA sequencing data at the species level was conducted using the Phyloseq, DESeq2 and Microbiome packages. The impact on the salivary microbiota of the different DGs, PGs and global oral grades (GGs) was investigated in relation to: 1) indicators of alpha diversity and the structure of the bacterial community; and 2) the composition of the core microbiome and the results of differential abundance tests. The simultaneous presence of dental and periodontal pathology has a potentiating effect on the richness and diversity of the salivary microbiota. The structure of the bacterial community in oral health differs from that present in dental, periodontal or global oral disease, especially in high grades. The non-specific microbiome core contains a greater number of more abundant species than the specific core of a particular dental or periodontal condition (health or pathology). The number of taxa in the salivary microbiota with differential abundances between the DGs, PGs or GGs represents, at most, a quarter of the bacterial community and are mainly non-core species. Supragingival dental parameters influence the microbiota`s abundance more than subgingival periodontal parameters, with the former making a greater contribution to the impact that global oral health has on salivary microbiome.

Aim To obtain salivary interleukin (IL) 1β-based models to predict the probability of the occurrence of periodontitis, differentiating by smoking habit. Materials/methods A total of 141 participants were recruited, 62 periodontally healthy controls and 79 subjects affected by periodontitis. Fifty of the diseased patients were given non-surgical periodontal treatment and showed significant clinical improvement in 2 months. IL1β was measured in the salivary samples using the Luminex instrument. Binary logistic regression models were obtained to differentiate untreated periodontitis from periodontal health (first modelling) and untreated periodontitis from treated periodontitis (second modelling), distinguishing between non-smokers and smokers. The area under the curve (AUC) and classification measures were calculated. Results In the first modelling, IL1β presented AUC values of 0.830 for non-smokers and 0.689 for smokers (accuracy = 77.6% and 70.7%, respectively). In the second, the predictive models revealed AUC values of 0.671 for non-smokers and 0.708 for smokers (accuracy = 70.0% and 75.0%, respectively). Conclusion Salivary IL1β has an excellent diagnostic capability when it comes to distinguishing systemically healthy patients with untreated periodontitis from those who are periodontally healthy, although this discriminatory potential is reduced in smokers. The diagnostic capacity of salivary IL1β remains acceptable for differentiating between untreated and treated periodontitis.

Aim To analyse, using a meta-analytical approach, the diagnostic accuracy of single molecular biomarkers in saliva for the detection of periodontitis in systemically healthy subjects. Materials and methods Articles on molecular biomarkers in saliva providing a binary contingency table (or sensitivity and specificity values and group sample sizes) in individuals with clinically diagnosed periodontitis were considered eligible. Searches for candidate articles were conducted in six electronic databases. The methodological quality was assessed through the tool Quality Assessment of Diagnostic Studies. Meta-analyses were performed using the Hierarchical Summary Receiver Operating Characteristic model. Results Meta-analysis was possible for 5 of the 32 biomarkers studied. The highest values of sensitivity for the diagnosis of periodontitis were obtained for IL1beta (78.7%), followed by MMP8 (72.5%), IL6 and haemoglobin (72.0% for both molecules); the lowest sensitivity value was for MMP9 (70.3%). In terms of specificity estimates, MMP9 had the best result (81.5%), followed by IL1beta (78.0%) and haemoglobin (75.2%); MMP8 had the lowest specificity (70.5%). Conclusions MMP8, MMP9, IL1beta, IL6 and Hb were salivary biomarkers with good capability to detect periodontitis in systemically healthy subjects. MMP8 and IL1beta are the most researched biomarkers in the field, both showing clinically fair effectiveness for the diagnosis of periodontitis.

The objective of the present study was to determine cytokine thresholds derived from predictive models for the diagnosis of chronic periodontitis, differentiating by smoking status. Seventy-five periodontally healthy controls and 75 subjects affected by chronic periodontitis were recruited. Sixteen mediators were measured in gingival crevicular fluid (GCF) using multiplexed bead immunoassays. The models were obtained using binary logistic regression, distinguishing between non-smokers and smokers. The area under the curve (AUC) and numerous classification measures were obtained. Model curves were constructed graphically and the cytokine thresholds calculated for the values of maximum accuracy (ACC). There were three cytokine-based models and three cytokine ratio-based models, which presented with a bias-corrected AUC > 0.91 and > 0.83, respectively. These models were (cytokine thresholds in pg/ml for the median ACC using bootstrapping for smokers and non-smokers): IL1alpha (46099 and 65644); IL1beta (4732 and 5827); IL17A (11.03 and 17.13); IL1alpha/IL2 (4210 and 7118); IL1beta/IL2 (260 and 628); and IL17A/IL2 (0.810 and 1.919). IL1alpha, IL1beta and IL17A, and their ratios with IL2, are excellent diagnostic biomarkers in GCF for distinguishing periodontitis patients from periodontally healthy individuals. Cytokine thresholds in GCF with diagnostic potential are defined, showing that smokers have lower threshold values than non-smokers. This work was supported by the Instituto de Salud Carlos III (General Division of Evaluation and Research Promotion, Madrid, Spain) and co-financed by FEDER (“A way of making Europe”) under Grant ISCIII/PI17/01722, and the Consellería de Cultura, Educación e Ordenación Universitaria da Xunta de Galicia (Spain) under Grant ED431B 2017/029 and A. Regueira-Iglesias support ED481A-2017 SI

Currently, there is little evidence available on the development of predictive models for the diagnosis or prognosis of chronic periodontitis based on the qPCR quantification of subgingival pathobionts. Our objectives were to: (1) analyze and internally validate pathobiont-based models that could be used to distinguish different periodontal conditions at site-specific level within the same patient with chronic periodontitis; (2) develop nomograms derived from predictive models. Subgingival plaque samples were obtained from control and periodontal sites (probing pocket depth and clinical attachment loss 4 mm, respectively) from 40 patients with moderate-severe generalized chronic periodontitis. The samples were analyzed by qPCR using TaqMan probes and specific primers to determine the concentrations of Actinobacillus actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), Parvimonas micra (Pm), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythia (Tf), and Treponema denticola (Td). The pathobiont-based models were obtained using multivariate binary logistic regression. The best models were selected according to specified criteria. The discrimination was assessed using receiver operating characteristic curves and numerous classification measures were thus obtained. The nomograms were built based on the best predictive models. Eight bacterial cluster-based models showed an area under the curve (AUC) ≥0.760 and a sensitivity and specificity ≥75.0%. The PiTfFn cluster showed an AUC of 0.773 (sensitivity and specificity = 75.0%). When Pm and AaPm were incorporated in the TdPiTfFn cluster, we detected the two best predictive models with an AUC of 0.788 and 0.789, respectively (sensitivity and specificity = 77.5%). The TdPiTfAa cluster had an AUC of 0.785 (sensitivity and specificity = 75.0%). When Pm was incorporated in this cluster, a new predictive model appeared with better AUC and specificity values (0.787 and 80.0%, respectively). Distinct clusters formed by species with different etiopathogenic role (belonging to different Socransky’s complexes) had a good predictive accuracy for distinguishing a site with periodontal destruction in a periodontal patient. The predictive clusters with the lowest number of bacteria were PiTfFn and TdPiTfAa, while TdPiTfAaFnPm had the highest number. In all the developed nomograms, high concentrations of these clusters were associated with an increased probability of having a periodontal site in a patient with chronic periodontitis This work was supported by the EM2014/025 project from the Regional Ministry of Culture, Education and University (regional government of Galicia, Spain), which is integrated in the Regional Plan of Research, Innovation and Development 2011–2015; and grants PI13/02390-PI16/01163 awarded to MT within the State Plan for R+D+I 2013–2016 (National Plan for Scientific Research, Technological Development and Innovation 2008–2011) and co-financed by the ISCIII-Deputy General Directorate of evaluation and Promotion of Research-European Regional Development Fund “A way of Making Europe” and Instituto de Salud Carlos III FEDER SI

We present the case of a patient with an intracerebral hemorrhage as complication of an infectious endocarditis (EI) produced by Neisseria sicca on a prosthetic mitral valve. The patient was anticoagulated previously with Acenocumarol as prophylaxis of thrombosis of the prosthetic valve. He was diagnosed as having IE and later he presented neurological symptoms as consequence of several intracerebral hemorrhagic foci. We decided to continue the anticoagulation with sodium heparin followed of Bemiparina and no new hemorrhagic complications nor thrombosis of the valve were observed after three months of the event. We have not found in the literature any case where low molecular weight heparin has been used as method of anticoagulation in these cases beyond two weeks.

Presentamos el caso de un paciente con una hemorragia intracerebral como complicación de una endocarditis infecciosa (EI) producida por Neisseria sicca sobre una válvula mitral metálica. El paciente estaba previamente anticoagulado con Acenocumarol como profilaxis de trombosis de la válvula protésica. Fue diagnosticado de EI y posteriormente presentó síntomas neurológicos como consecuencia de varios focos hemorrágicos intracerebrales. Se decidió continuar la anticoagulación con heparina sódica inicialmente y posteriormente con Bemiparina, no observándose nuevas complicaciones hemorrágicas ni trombosis de la válvula a los tres meses del evento. No hemos encontrado en la literatura ningún caso donde la heparina de bajo peso molecular (HBPM) haya sido utilizada como método de anticoagulación en estos casos más allá de dos semanas.