PrimerEvalPy: a tool for in-silico evaluation of primers for targeting the microbiome.

BACKGROUND: The selection of primer pairs in sequencing-based research can greatly influence the results, highlighting the need for a tool capable of analysing their performance in-silico prior to the sequencing process. We therefore propose PrimerEvalPy, a Python-based package designed to test the performance of any primer or primer pair against any sequencing database. The package calculates a coverage metric and returns the amplicon sequences found, along with information such as their average start and end positions. It also allows the analysis of coverage for different taxonomic levels. RESULTS: As a case study, PrimerEvalPy was used to test the most commonly used primers in the literature against two oral 16S rRNA gene databases containing bacteria and archaea. The results showed that the most commonly used primer pairs in the oral cavity did not match those with the highest coverage. The best performing primer pairs were found for the detection of oral bacteria and archaea. CONCLUSIONS: This demonstrates the importance of a coverage analysis tool such as PrimerEvalPy to find the best primer pairs for specific niches. The software is available under the MIT licence at https://gitlab.citius.usc.es/lara.vazquez/PrimerEvalPy .

A systematic overview of dental methods for age assessment in living individuals: from traditional to artificial intelligence-based approaches.

Dental radiographies have been used for many decades for estimating the chronological age, with a view to forensic identification, migration flow control, or assessment of dental development, among others. This study aims to analyse the current application of chronological age estimation methods from dental X-ray images in the last 6 years, involving a search for works in the Scopus and PubMed databases. Exclusion criteria were applied to discard off-topic studies and experiments which are not compliant with a minimum quality standard. The studies were grouped according to the applied methodology, the estimation target, and the age cohort used to evaluate the estimation performance. A set of performance metrics was used to ensure good comparability between the different proposed methodologies. A total of 613 unique studies were retrieved, of which 286 were selected according to the inclusion criteria. Notable tendencies to overestimation and underestimation were observed in some manual approaches for numeric age estimation, being especially notable in the case of Demirjian (overestimation) and Cameriere (underestimation). On the other hand, the automatic approaches based on deep learning techniques are scarcer, with only 17 studies published in this regard, but they showed a more balanced behaviour, with no tendency to overestimation or underestimation. From the analysis of the results, it can be concluded that traditional methods have been evaluated in a wide variety of population samples, ensuring good applicability in different ethnicities. On the other hand, fully automated methods were a turning point in terms of performance, cost, and adaptability to new populations.

Biomarkers for diagnosis of stage III, grade C with molar incisor pattern periodontitis in children and young adults: a systematic review and meta-analysis.

AIM: To explore the existing salivary, gingival crevicular fluid (GCF), blood, and serum biomarkers associated with grade C molar-incisor pattern (C/MIP) periodontitis in systemically healthy children and young adults. MATERIALS AND METHODS: Cross-sectional, case-control, and cohort studies on stage III grade C periodontitis or former equivalent diagnosis with analysis of molecular biomarkers in saliva, GCF, blood, or serum were retrieved from six databases and screened based on the eligibility criteria. The risk of bias in included studies was evaluated. Meta-analysis was planned for biomarkers assessed using the same detection methods and sample type in at least two papers. RESULTS: Out of 5621 studies identified at initial screening, 28 papers were included in the qualitative analysis of which 2 were eligible for meta-analysis for IgG in serum samples. Eighty-seven biomarkers were assessed with the majority being higher in cases than in controls. Only the meta-analysis of total serum IgG with low heterogeneity value revealed a significant increase in its levels in C/MIPs compared to controls (standardised mean difference: 1.08; 95% CI: 0.76, 1.40). CONCLUSION: There is a paucity of data on biomarkers associated with molar-incisor pattern periodontitis. Although serum IgG levels are raised, other more specific biomarkers in saliva, GCF, and blood/serum may be promising but require further investigation.

Update on the Role of Cytokines as Oral Biomarkers in the Diagnosis of Periodontitis.

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.

Accuracy of single molecular biomarkers in saliva for the diagnosis of periodontitis: A systematic review and meta-analysis.

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.

Diagnostic accuracy of IL1ß in saliva: The development of predictive models for estimating the probability of the occurrence of periodontitis in non-smokers and smokers.

AIM: To obtain salivary interleukin (IL) 1ß-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. IL1ß 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, IL1ß 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 IL1ß 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 IL1ß remains acceptable for differentiating between untreated and treated periodontitis.

DenTiUS Plaque, a Web-Based Application for the Quantification of Bacterial Plaque: Development and Usability Study.

BACKGROUND: In the dentistry field, the analysis of dental plaque is vital because it is the main etiological factor in the 2 most prevalent oral diseases: caries and periodontitis. In most of the papers published in the dental literature, the quantification of dental plaque is carried out using traditional, non-automated, and time-consuming indices. Therefore, the development of an automated plaque quantification tool would be of great value to clinicians and researchers. OBJECTIVE: This study aimed to develop a web-based tool called DenTiUS and various clinical indices to evaluate dental plaque levels using image analysis techniques. METHODS: The tool was executed as a web-based application to facilitate its use by researchers. Expert users are free to define experiments, including images from either a single patient (to observe an individual plaque growth pattern) or several patients (to perform a group characterization) at a particular moment or over time. A novel approach for detecting visible plaque has been developed as well as a new concept known as nonvisible plaque. This new term implies the classification of the remaining dental area into 3 subregions according to the risk of accumulating plaque in the near future. New metrics have also been created to describe visible and nonvisible plaque levels. RESULTS: The system generates results tables of the quantitative analysis with absolute averages obtained in each image (indices about visible plaque) and relative measurements (indices about visible and nonvisible plaque) relating to the reference moment. The clinical indices that can be calculated are the following: plaque index of an area per intensity (API index, a value between 0 and 100), area growth index (growth rate of plaque per unit of time in hours; percentage area/hour), and area time index (the time in days needed to achieve a plaque area of 100% concerning the initial area at the same moment). Images and graphics can be obtained for a moment from a patient in addition to a full report presenting all the processing data. Dentistry experts evaluated the DenTiUS Plaque software through a usability test, with the best-scoring questions those related to the workflow efficiency, value of the online help, attractiveness of the user interface, and overall satisfaction. CONCLUSIONS: The DenTiUS Plaque software allows automatic, reliable, and repeatable quantification of dental plaque levels, providing information about area, intensity, and growth pattern. Dentistry experts recognized that this software is suitable for quantification of dental plaque levels. Consequently, its application in the analysis of plaque evolution patterns associated with different oral conditions, as well as to evaluate the effectiveness of various oral hygiene measures, can represent an improvement in the clinical setting and the methodological quality of research studies.

Accuracy of single molecular biomarkers in gingival crevicular fluid for the diagnosis of periodontitis: A systematic review and meta-analysis.

AIM: To analyse, by means of a meta-analytical approach, the diagnostic accuracy of molecular biomarkers in gingival crevicular fluid (GCF) for the detection of periodontitis in systemically healthy subjects. MATERIAL AND METHODS: Studies on GCF molecular biomarkers providing a binary classification table (or sensitivity and specificity values and group sample sizes) in individuals with clinically diagnosed periodontitis were considered eligible. The search was performed using six electronic databases. The methodological quality of studies was assessed through the tool Quality Assessment of Diagnostic Studies. Meta-analyses were performed using the Hierarchical Summary Receiver Operating Characteristic, which adjusts classification data using random effects logistic regression. RESULTS: The included papers identified 36 potential biomarkers for the detection of periodontitis and for four of them meta-analyses were performed. The median sensitivity and specificity were for MMP8, 76.7% and 92.0%; for elastase, 74.6% and 81.1%; for cathepsin, 72.8% and 67.3%, respectively. The worst estimates of sensitivity and specificity were for trypsin (71.3% and 66.1%, respectively). CONCLUSIONS: MMP8 showed good sensitivity and excellent specificity, which resulted in this biomarker being clinically the most useful or effective for the diagnosis of periodontitis in systemically healthy subjects, regardless of smoking condition.

Evaluation of a new oral health scale of infectious potential based on the salivary microbiota.

OBJECTIVES: The objective of this study is to analyse the correlation of our own design of oral health scale (grades 0 and 1-better oral health vs. grades 2 and 3-poorer oral health) with the salivary microbiota. PATIENTS AND METHODS: The oral health scale we elaborated was evaluated in 100 adults (25 patients from each global oral health grade). Saliva samples collected from these patients were analysed using microbiological culture techniques, determining the presence/absence and the concentrations of some odontopathogens and periodontopathogens. RESULTS: In comparison with the global oral health grades 0-1, the grades 2-3 presented significantly higher values for the presence of odontopathogens (78 vs. 38 %; Streptococcus mutans, Lactobacillus spp. and Actinomyces spp.) and periodontopathogens (100 vs. 90 %; Aggregatibacter actinomycetemcomitans, Campylobacter spp., Fusobacterium spp. and Prevotella gingivalis). In comparison with the grades 0-1, the grades 2-3 presented significantly higher values for the concentrations (CFU/mL log10) of facultative anaerobes, strict anaerobes, odontopathogens (S. mutans, Lactobacillus spp. and Actinomyces spp.) and periodontopathogens (A. actinomycetemcomitans, Capnocytophaga spp., Campylobacter spp. and Fusobacterium spp.). CONCLUSION: Our new global oral health scale shows a positive correlation with the detection and quantification of certain odontopathogens and periodontopathogens present in the saliva, confirming their possible infectious potential. CLINICAL RELEVANCE: Our own design of oral health scale could be particularly useful for the epidemiological study of different populations, the evaluation of the influence of oral health on the development of certain systemic diseases as well as the analysis of inter- and intra-individual variability of the oral microbiota in relation to the different grades of the oral health scale.

In situ antimicrobial activity on oral biofilm: essential oils vs. 0.2 % chlorhexidine.

OBJECTIVES: This study aims to evaluate the in situ antibacterial activity of a mouthwash containing essential oils (M-EO) on undisturbed de novo plaque-like biofilm (PL-biofilm) up to 7 h after its application. PATIENTS AND METHODS: An appliance was designed to hold six glass disks on the buccal sides of the lower teeth, allowing PL-biofilm growth. Fifteen healthy volunteers wore the appliance for 48 h and then performed a M-EO. Disks were removed after 30 s and at 1, 3, 5, and 7 h later. After a washout period, the same procedure was repeated with a M-WATER and a M-0.2 % chlorhexidine. After PL-biofilm vital staining, samples were analyzed using a confocal laser scanning microscope. RESULTS: At 30 s after M-EO, the levels of bacterial vitality were 1.18 %, significantly lower than that of the basal sample (p < 0.001). After 7 h, the antibacterial effect of essential oils was still patent with a 47.86 % difference in bacterial vitality compared to the basal sample (p < 0.001). CONCLUSION: A single M-EO presents high antibacterial immediate activity and penetration capacity in situ and a substantivity which lasts for at least 7 h after its application over de novo biofilm. These results were better than those observed with 0.2 % chlorhexidine under the same conditions. CLINICAL RELEVANCE: A single M-EO is an effective measure against the de novo biofilm, presenting a good alternative to clorhexidine such as a preoperative rinse, in periodontal procedures or post-treatment applications.

The accuracy of estimating chronological age from Demirjian and Nolla methods in a Portuguese and Spanish sample.

BACKGROUND: Age determination has great importance in many clinical decisions, being commonly used in odontopediatrics, orthodontics, pediatrics, and forensic medicine. The Nolla and Demirjian et al. methods have been used for these purposes. However, estimating chronological age by means of the dental mineralization stage is not a straightforward analysis, and it is fundamental to test the validity of these methods and their applicability to populations. In this article we intend to compare the accuracy of estimating chronological age from dental age measured with the Nolla and Demirjian methods in a Portuguese and Spanish sample, considering the variables of sex and age-group. METHODS: The sample was composed of 821 orthopantomographs of healthy Portuguese (n = 270) and Spanish (n = 551) subjects from 4 to 34 years old. For the Nolla and Demirjian methods, seven mandibular left teeth were examined, staged according to the dental maturity scale of each method. We obtained a good index of inter-rater agreement, a good internal consistency for the teeth assessment, and a good temporal consistency. RESULTS: Dental age was calculated for each method. The Demirjian et al. method tends to overestimate the real age of participants and the Nolla method tends to underestimate it. The accuracy of both methods varied between the sexes and age groups. Both methods were found to be more precise with males. As the age-group increases, the predictive capacities of both methods diminish. The Nolla method was more accurate than the Demirjian method in early and late childhood for both sexes. Neither method could predict chronological age in adults. CONCLUSIONS: We can estimate chronological age for early and late childhood, through the Nolla and Demirjian methods, with the former showing greater predictive capacities than the latter. The Demirjian method tends to overestimate age and the Nolla method tends to underestimate it, leading to the importance of forming regression equations adapted to the population studied. Nolla and Demirjian formulas adapted to our sample were created as a function of sex and age-group.

Relationship between the oral cavity and cardiovascular diseases and metabolic syndrome.

The components of the human body are closely interdependent; as a result, disease conditions in some organs or components can influence the development of disease in other body locations. The effect of oral health upon health in general has been investigated for decades by many epidemiological studies. In this context, there appears to be a clear relationship between deficient oral hygiene and different systemic disorders such as cardiovascular disease and metabolic syndrome. The precise relationship between them is the subject of ongoing research, and a variety of theories have been proposed, though most of them postulate the mediation of an inflammatory response. This association between the oral cavity and disease in general requires further study, and health professionals should be made aware of the importance of adopting measures destined to promote correct oral health. The present study conducts a Medline search with the purpose of offering an update on the relationship between oral diseases and cardiovascular diseases, together with an evaluation of the bidirectional relationship between metabolic syndrome and periodontal disease. Most authors effectively describe a moderate association between the oral cavity and cardiovascular diseases, though they also report a lack of scientific evidence that oral alterations constitute an independent cause of cardiovascular diseases, or that their adequate treatment can contribute to prevent such diseases. In the case of metabolic syndrome, obesity and particularly diabetes mellitus may be associated to an increased susceptibility to periodontitis. However, it is not clear whether periodontal treatment is able to improve the systemic conditions of these patients.

Oral Health Scales: design of an oral health scale of infectious potential.

OBJECTIVES: In this paper we propose a new Global Oral Health Scale that will allow the infectious potential of the oral cavity, clinically manifest as local and focal infections, to be condensed into a single parameter. STUDY DESIGN: Based on a number of oral health scales previously designed by our group, we designed a final version that incorporates dental and periodontal variables (some of them evaluated using corroborated objective indices) that reflect the presence of caries and periodontal disease. RESULTS: The application of the proposed oral health scale requires the examination of 6 sites per tooth (mesio-buccal, medio-buccal, disto-buccal, disto-lingual, medio-lingual and mesio-lingual). The following variables are analysed: number of tooth surfaces with supragingival plaque, determined using the O'Leary index; number of teeth with caries and the severity of the caries; number of tooth surfaces with gingival inflammation, determined using the Ainamo and Bay index; and number of tooth surfaces with pockets ≥ 4 mm and severity of the pockets. These variables are then grouped into 2 categories, dental and periodontal. The final grades of dental and periodontal health correspond to the grades assigned to a least 2 of the 3 variables analysed in each of these categories. The category (dental or periodontal) with the highest grade is the one that determines the grade of the Global Oral Health Scale. CONCLUSION: This scale could be particularly useful for the epidemiological studies comparing different populations and for analysis of the influence of distinct degrees of oral health on the development of certain systemic diseases.

Evaluation of an oral health scale of infectious potential using a telematic survey of visual diagnosis.

OBJECTIVE: To compare the results of a subjective estimation of oral health through review of a set of intraoral photographs with those of an objective oral health scale of infectious potential. METHOD: The pool of patients was made up of 100 adults. Using an infectious-potential scale based on dental and periodontal variables, we assigned 1 of the 4 grades of the scale (range, 0 to 3; 0 corresponds to an excellent oral health status and 3 to the poorest oral health status) to each subject. A total of 20 representative subjects were selected from the pool of patients, 5 subjects for each one of the grades of the scale, and a standardized photographic record was made. One thousand dentists practicing in Spain were sent the survey by e-mail and 174 completed forms were received. We then calculated the concordance of the oral health status indicated by the respondents after visualising the photographs on comparison with the results of the oral health scale of infectious potential; concordance was termed correct grade allocation (CGA). RESULTS: The majority of respondents (69.1%) achieved a CGA in 8 to 12 cases and none achieved more than 15 CGAs. The poorest CGA rates were found with grades 1 and 2, with a mean of 1.74 ± 1.09 and 1.87 ± 1.18, respectively, out of a maximum of 5. The concordance in terms of CGA was high for grade 0 (70.5%), very low for grade 1 (10.8%), low for grade 2 (37.3%), and moderate for grade 3 (42.6%). CONCLUSION: In comparison with visual examination of the oral cavity, the use of objective scale that establishes a reliable diagnosis of oral health in terms of infectious potential was found to be advantageous.

Critical review of 16S rRNA gene sequencing workflow in microbiome studies: From primer selection to advanced data analysis.

The multi-batch reanalysis approach of jointly reevaluating gene/genome sequences from different works has gained particular relevance in the literature in recent years. The large amount of 16S ribosomal ribonucleic acid (rRNA) gene sequence data stored in public repositories and information in taxonomic databases of the same gene far exceeds that related to complete genomes. This review is intended to guide researchers new to studying microbiota, particularly the oral microbiota, using 16S rRNA gene sequencing and those who want to expand and update their knowledge to optimise their decision-making and improve their research results. First, we describe the advantages and disadvantages of using the 16S rRNA gene as a phylogenetic marker and the latest findings on the impact of primer pair selection on diversity and taxonomic assignment outcomes in oral microbiome studies. Strategies for primer selection based on these results are introduced. Second, we identified the key factors to consider in selecting the sequencing technology and platform. The process and particularities of the main steps for processing 16S rRNA gene-derived data are described in detail to enable researchers to choose the most appropriate bioinformatics pipeline and analysis methods based on the available evidence. We then produce an overview of the different types of advanced analyses, both the most widely used in the literature and the most recent approaches. Several indices, metrics and software for studying microbial communities are included, highlighting their advantages and disadvantages. Considering the principles of clinical metagenomics, we conclude that future research should focus on rigorous analytical approaches, such as developing predictive models to identify microbiome-based biomarkers to classify health and disease states. Finally, we address the batch effect concept and the microbiome-specific methods for accounting for or correcting them.

Impact of 16S rRNA Gene Redundancy and Primer Pair Selection on the Quantification and Classification of Oral Microbiota in Next-Generation Sequencing.

This study aimed to evaluate the number of 16S rRNA genes in the complete genomes of the bacterial and archaeal species inhabiting the human mouth and to assess how the use of different primer pairs would affect the detection and classification of redundant amplicons and matching amplicons (MAs) from different taxa. A total of 518 oral-bacterial and 191 oral-archaeal complete genomes were downloaded from the NCBI database, and their complete 16S rRNA genes were extracted. The numbers of genes and variants per genome were calculated. Next, 39 primer pairs were used to search for matches in the genomes and obtain amplicons. For each primer, we calculated the number of gene amplicons, variants, genomes, and species detected and the percentage of coverage at the species level with no MAs (SC-NMA). The results showed that 94.09% of oral bacteria and 52.59% of oral archaea had more than one intragenomic 16S rRNA gene. From 1.29% to 46.70% of bacterial species and from 4.65% to 38.89% of archaea detected by the primers had MAs. The best primers were the following (SC-NMA; region; position for Escherichia coli [GenBank version no. J01859.1]): KP_F048-OP_R030 for bacteria (93.55%; V3 to V7; 342 to 1079), KP_F018-KP_R063 for archaea (89.63%; V3 to V9; undefined to 1506), and OP_F114-OP_R121 for both domains (92.52%; V3 to V9; 340 to 1405). In addition to 16S rRNA gene redundancy, the presence of MAs must be controlled to ensure an accurate interpretation of microbial diversity data. The SC-NMA is a more useful parameter than the conventional coverage percentage for selecting the best primer pairs. The pairs used the most in the oral microbiome literature were not among the best performers. IMPORTANCE Hundreds of publications have studied the oral microbiome through 16S rRNA gene sequencing. However, none have assessed the number of 16S rRNA genes in the genomes of oral microbes, or how the use of primer pairs targeting different regions affects the detection of MAs from different taxa. Here, we found that almost all oral bacteria and more than half of oral archaea have more than one intragenomic 16S rRNA gene. The performance of the primer pairs in not detecting MAs increases as the length of the amplicon augments. As none of those most employed in the oral literature were among the best performers, we selected a series of primers to detect bacteria and/or archaea based on their percentage of species detected without MAs. The intragenomic 16S rRNA gene redundancy and the presence of MAs between distinct taxa need to be considered to ensure an accurate interpretation of microbial diversity data.

In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea.

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 98.83-97.14%; meanwhile, the optimum archaea-specific primer pairs amplified regions 5-6, 3-6, 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 95.71-94.54% and 99.48-96.91% for bacteria and archaea, respectively. CONCLUSIONS: Given the three amplicon length categories (100-300, 301-600, and >600 base pairs), the primer pairs with the best coverage values for detecting oral bacteria were as follows: 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 as follows: 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. Video Abstract.

Periodontal health status and bacteraemia from daily oral activities: systematic review/meta-analysis.

AIM: The aim of this study was to investigate the robustness of the observations on the influence of oral hygiene, gingival and periodontal status on the development of bacteraemia from everyday oral activities (B-EOA), analysing its prevalence, duration, magnitude and bacterial diversity. MATERIAL AND METHODS: This systematic review/meta-analysis complies with PRISMA reporting guidelines. MEDLINE-PubMed, the Cochrane Library and Embase were explored for detecting studies on B-EOA. RESULTS: There were 290 potentially eligible articles, of which 12 article on B-EOA fulfilled the inclusion criteria and were processed for data extraction (seven on toothbrushing, one on dental flossing and four on chewing). Evaluating the influence of plaque and gingival indices on the prevalence of bacteraemia following toothbrushing, the pooled odds ratios were 2.61 [95% confidence interval (CI) = 1.45-4.69] and 2.77 (95% CI = 1.50-5.11), respectively. None of five studies on bacteraemia following dental flossing and chewing revealed a statistically significant association between oral hygiene, gingival or periodontal status and the development of bacteraemia. CONCLUSIONS: Meta-analysis showed that plaque accumulation and gingival inflammation scores significantly increased the prevalence of bacteraemia following toothbrushing. However, systematic review showed no relationship between oral hygiene, gingival and periodontal status and the development of B-chewing, and there is no evidence that gingival and periodontal health status affects B-flossing.

Predictors for tumor recurrence after primary definitive surgery for oral cancer.

PURPOSE: The purpose of this study was to identify significant predictors for oral squamous cell carcinoma recurrence. PATIENTS AND METHODS: This Ambispective cohort study was performed in consecutive metastasis-free patients treated for oral squamous cell carcinoma with curative intent from 1998 through 2003. Variables included gender, age, tumor site, macroscopic pattern of the lesion, coexisting disorders (diabetes, hepatic and heart disorders, other tumors/diseases), degree of differentiation, and pathologic TNM stage. Tumor recurrence was considered the dependent variable (outcome). The distribution of recurrences was assessed with χ(2) test. Survival times were estimated by Kaplan-Meier curves and differences were examined with log-rank test. Multiple Cox regression study was also performed. The significance level chosen for all tests was P < .05. RESULTS: One hundred eighteen patients entered the study. Tumor recurrence was 44.9% during the follow-up period (10% local, 29.7% regional, and 5% distant). The mean period that had elapsed before recurrence was 15 months (1.5 to 81.8), with most recurrences (66%) during the first year after treatment (84.9% before 2 years). Multivariate Cox regression analysis indicated the presence of a coexisting disorder (P = .022) as the most relevant prognostic factor for relapse, because patients with associated diseases had a 2.43-fold risk of recurrence. Tumor stage (P = .037), degree of differentiation (P = .042), and macroscopic pattern of the lesion (P = .022) were also identified as prognostic factors for relapse. CONCLUSIONS: The risk profile for oral cancer recurrence includes patients younger than 60 years with coexisting diseases whose primary tumor occurred as an ulcerated lesion, and diagnosed at an advanced stage with a poorly differentiated tumor.

Simulation for training in sinus floor elevation: new surgical bench model.

OBJECTIVES: to describe a bench model (workshop of abilities) for sinus floor elevation (SFE) training that simulates the surgical environment and to assess its effectiveness in terms of trainees' perception. STUDY DESIGN: thirty-six randomly selected postgraduate students entered this cross-sectional pilot study and asked to fill in an anonymous, self-applied, 12-item questionnaire about a SFE workshop that included a study guide containing the workshop' s details, supervised practice on a simulated surgical environment, and assessment by means of specific check-lists. RESULTS: Thirtiy-six fresh sheep heads were prepared to allow access to the buccal vestible. Using the facial tuber, third premolar and a 3D-CT study as landmarks for trepanation, the sinus membrane was lifted, the space filled with ceramic material and closed with a resorbable membrane. The participants agreed on their ability to perform SFE in a simulated situation (median score= 4.5; range 2-5) and felt capable to teach the technique to other clinicians or to undertake the procedure for a patient under supervision of an expert surgeon (median= 4; range 1-5 ). There were no differences on their perceived ability to undertake the technique on a model or on a real patient under supervision of an expert surgeon (p=0.36). CONCLUSIONS: Clinical abilities workshops for SFE teaching are an essential educational tool but supervised clinical practice should always precede autonomous SFE on real patients. Simulation procedures (workshop of abilities) are perceived by the partakers as useful for the surgical practice. However, more studies are needed to validate the procedure and to address cognitive and communication skills, that are clearly integral parts of surgical performance.