Knowledge and opinions of French dental students related to caries risk assessment and dental sealants (preventive and therapeutic).

OBJECTIVES: A national questionnaire study was performed to document knowledge and opinions of French dental students (FDSs) about minimal intervention (MI) in dentistry especially caries risk assessment (CRA) and dental sealants (DSs). MATERIALS AND METHODS: A questionnaire was administered to the fifth-year dental FDSs (n = 1370) from the 16 French dental schools. Descriptive and statistical analyses were performed. RESULTS: The response rate was 84.5%. A large majority of respondents (87.8%) linked MI with minimally invasive dentistry and 77.4% considered MI as a concept based on prevention. About 80% stated they use CRA in clinical practice, mostly without any specific form. If 80.4% of the respondents would base their treatment plans on CRA, only 55.1% would regularly plan preventive regimens according to individual risk level. However, while 96.6% declared they perform preventive DSs, only 44.3% considered therapeutic sealants as a routine treatment. Although 75.1% of FDSs stated that they had sufficient learning and training related to CRA, 55.9% thought that they need further education about preventive and therapeutic DSs. CONCLUSION: Although FDSs seem to be aware of the importance of CRA and preventive strategies, this study shows the need to harmonize the teaching in cariology according to the latest European recommendations. CLINICAL RELEVANCE: A national questionnaire study showed variability towards knowledge and opinions of FDSs related to MI in cariology. This may impact care provisions in their future professional life showing the urgent need to harmonize the teaching of MI in cariology in France.

Culture of salivary methanogens assisted by chemically produced hydrogen.

Methanogen cultures require hydrogen produced by fermentative bacteria such as Bacteroides thetaiotaomicron (biological method). We developed an alternative method for hydrogen production using iron filings and acetic acid with the aim of cultivating methanogens more efficiently and more quickly (chemical method). We developed this new method with a reference strain of Methanobrevibacter oralis, compared the method to the biological reference method with a reference strain of Methanobrevibacter smithii and finally applied the method to 50 saliva samples. Methanogen colonies counted using ImageJ software were identified using epifluorescence optical microscopy, real-time PCR and PCR sequencing. For cultures containing pure strains of M. oralis and M. smithii, colonies appeared three days postinoculation with the chemical method versus nine days with the biological method. The average number of M. smithii colonies was significantly higher with the chemical method than with the biological method. There was no difference in the delay of observation of the first colonies in the saliva samples between the two methods. However, the average number of colonies was significantly higher with the biological method than with the chemical method at six days and nine days postinoculation (Student's test, p = 0.005 and p = 0.04, respectively). The chemical method made it possible to isolate four strains of M. oralis and three strains of M. smithii from the 50 saliva samples. Establishing the chemical method will ease the routine isolation and culture of methanogens.

Performance of Fluorescence-based Systems in Early Caries Detection: A Public Health Issue.

AIM: Modern clinical caries management involves early stage caries diagnosis and should fit with dental health policy. The objective of this study was to achieve early caries detection in enamel and dentine with a laser-based system (DIAGNOdent™ pen) first and secondary with a new fluorescence intra-oral camera (Soprolife®). A visual inspection with a loupe was used as control. MATERIALS AND METHODS: Following the consolidated standards of reporting trials recommendations, 628 occlusal fissures were included for analysis. RESULTS: The sensitivity and specificity of both devices varied depending on the cutoff threshold of the caries score, and the ROC curve showed higher values for the Soprolife® than for DIAGNOdent™ pen. The values of the area under the curve decreased from 0.81 (Soprolife® in daylight) to 0.79 (Soprolife® in fluorescent mode) and 0.67 for DIAGNOdent™ pen. DIAGNOdent™ pen reproducibility (intra and inter-investigator) showed a wide dispersion, with many values scattered beyond the confidence limits (±2 SD), and the weighted kappa coefficient, which was quite low (0.58), confirmed this tendency. CONCLUSION: Caries prevalence in terms of public health policy is of interest and caries detection increased significantly when using an fluorescence-based intra-oral camera. CLINICAL SIGNIFICANCE: The clinical significance of these findings is that fluorescence could help improve caries diagnosis, reduce clinical misinterpretations, and finally benefit the patients. How to cite this article: Terrer E, Slimani A, Giraudeau N, et al. Performance of Fluorescence-based Systems in Early Caries Detection: A Public Health Issue. J Contemp Dent Pract 2019;20(10):1126-1132.

Multiphoton Microscopy for Caries Detection with ICDAS Classification.

Dentin carious lesion is a dynamic process that involves demineralization and collagen denaturation. Collagen type I is the major protein in dentin and it has been investigated based on its optical properties. Multiphoton microscopy (MPM) is a nonlinear imaging technique that reveals the caries process using the collagen two-photon excitation fluorescence (2PEF) and its second-harmonic generation (SHG). Combining the histological and the International Caries Detection and Assessment System (ICDAS) classifications with nonlinear optical spectroscopy (NLOS), 2PEF and SHG intensities of enamel and dentin were highly altered during the caries process. It has been proven that the ratio SHG/2PEF is a relevant indicator of the organic matrix denaturation [Terrer et al.: J Dent Res 2016; 96: 574-579]. In the present study, a series of measurable signals is made to detect early stages of carious lesion according to the ICDAS classification and to explore the relationship between these measures and the ICDAS scale. Comparison of the efficiency of nonlinear optical signals for caries detection with the ICDAS classification is essential to evaluate their potential for clinical application. In our study, the use of the NLOS measured by MPM allowed us to monitor a quantitative parameter (SHG/2PEF ratio) according to the dentin carious lesion state (ICDAS and histological examination). Three coherent new groups were defined (ICDAS 0/1; ICDAS 2/3; ICDAS 4/5/6), where the carious process can be clearly described with a statistically significant decrease of the SHG/2PEF ratio.

Preventive management of carious lesions: from non-invasive to micro-invasive operative interventions.

Understanding that dental carious lesions occur as a result of the action of micro-organisms in the dental plaque biofilm, where demineralisation on the tooth surface is the first sign of the disease, such incipient lesions can be treated using preventive, non-operative and minimally invasive operative dentistry. If the caries process is left unmanaged, the lesions progress towards cavitation, leading to more invasive treatments. This article discusses the principles of preventive, non-invasive and micro-invasive treatments of early carious lesions, outlining the clinical situations where these therapies can be applied.

Candidatus Methanosphaera massiliense sp. nov., a methanogenic archaeal species found in a human fecal sample and prevalent in pigs and red kangaroos.

Methanosphaera stadtmanae was the sole Methanosphaera representative to be cultured and detected by molecular methods in the human gut microbiota, further associated with digestive and respiratory diseases, leaving unknown the actual diversity of human-associated Methanosphaera species. Here, a novel Methanosphaera species, Candidatus Methanosphaera massiliense (Ca. M. massiliense) sp. nov. was isolated by culture using a hydrogen- and carbon dioxide-free medium from one human feces sample. Ca. M. massiliense is a non-motile, 850 nm Gram-positive coccus autofluorescent at 420 nm. Whole-genome sequencing yielded a 29.7% GC content, gapless 1,785,773 bp genome sequence with an 84.5% coding ratio, encoding for alcohol and aldehyde dehydrogenases promoting the growth of Ca. M. massiliense without hydrogen. Screening additional mammal and human feces using a specific genome sequence-derived DNA-polymerase RT-PCR system yielded a prevalence of 22% in pigs, 12% in red kangaroos, and no detection in 149 other human samples. This study, extending the diversity of Methanosphaera in human microbiota, questions the zoonotic sources of Ca. M. massiliense and possible transfer between hosts.IMPORTANCEMethanogens are constant inhabitants in the human gut microbiota in which Methanosphaera stadtmanae was the only cultivated Methanosphaera representative. We grew Candidatus Methanosphaera massiliense sp. nov. from one human feces sample in a novel culture medium under a nitrogen atmosphere. Systematic research for methanogens in human and animal fecal samples detected Ca. M. massiliense in pig and red kangaroo feces, raising the possibility of its zoonotic acquisition. Host specificity, source of acquisition, and adaptation of methanogens should be further investigated.

Methanobrevibacter massiliense and Pyramidobacter piscolens Co-Culture Illustrates Transkingdom Symbiosis.

Among oral microbiota methanogens, Methanobrevibacter massiliense (M. massiliense) has remained less studied than the well-characterised and cultivated methanogens Methanobrevibacter oralis and Methanobrevibacter smithii. M. massiliense has been associated with different oral pathologies and was co-isolated with the Synergistetes bacterium Pyramidobacter piscolens (P. piscolens) in one case of severe periodontitis. Here, reporting on two additional necrotic pulp cases yielded the opportunity to characterise two co-cultivated M. massiliense isolates, both with P. piscolens, as non-motile, 1-2-µm-long and 0.6-0.8-µm-wide Gram-positive coccobacilli which were autofluorescent at 420 nm. The two whole genome sequences featured a 31.3% GC content, gapless 1,834,388-base-pair chromosome exhibiting an 85.9% coding ratio, encoding a formate dehydrogenase promoting M. massiliense growth without hydrogen in GG medium. These data pave the way to understanding a symbiotic, transkingdom association with P. piscolens and its role in oral pathologies.

In vitro investigation of fluorescence of carious dentin observed with a Soprolife® camera.

OBJECTIVES: Our aim was to determine the origin of the red fluorescence of carious dentine observed with the Soprolife® camera. METHODS: We conducted in vitro studies to evaluate the origin of the red fluorescence using acids and matrix metalloproteinase (MMP) to mimic caries and methylglycoxal (MGO) to evaluate the effect of glycation reactions on the red fluorescence. In every step of these models, we detected the changes of dentin photonic response with Soprolife® in daylight mode and in treatment mode. A Raman spectroscopy analysis was performed to determine the variations of the dentin organic during the in vitro caries processes. Raman microscopy was performed to identify change in the collagen matrix of dentine. RESULTS: The red fluorescence observed in carious dentine using a Soprolife® camera corresponds to the brownish color observed using daylight. Demineralization using nitric acid induces a loss of the green fluorescence of dentine. The red fluorescence of carious dentine is resistant to acid treatment. Immersion of demineralized dentine in MGO induces a change of color from white to orange-red. This indicates that the Maillard reaction contributes to lesion coloration. Immersion of demineralized dentine in an MMP-1 solution followed by MGO treatment results in a similar red fluorescence. Raman microspectroscopy analysis reveals accumulation of AGE's product in red-colored dentine. CONCLUSIONS: Our results provide important information on the origin of the fluorescence variation of dentine observed with the Soprolife® camera. We demonstrate that the red fluorescence of carious dentine is linked to the accumulation of Advanced Glycation End products (AGE). CLINICAL RELEVANCE: The study provides a new biological basis for the red fluorescence of carious dentine and reinforces the importance of the Soprolife® camera in caries diagnostics.

Culturing the Human Oral Microbiota, Updating Methodologies and Cultivation Techniques.

Recent years have been marked by a paradigm shift in the study of the human microbiota, with a re-emergence of culture-dependent approaches. Numerous studies have been devoted to the human microbiota, while studies on the oral microbiota still remain limited. Indeed, various techniques described in the literature may enable an exhaustive study of the microbial composition of a complex ecosystem. In this article, we report different methodologies and culture media described in the literature that can be applied to study the oral microbiota by culture. We report on specific methodologies for targeted culture and specific culture techniques and selection methodologies for cultivating members of the three kingdoms of life commonly found in the human oral cavity, namely, eukaryota, bacteria and archaea. This bibliographic review aims to bring together the various techniques described in the literature, enabling a comprehensive study of the oral microbiota in order to demonstrate its involvement in oral health and diseases.

Culturing clinical Methanobrevibacter smithii using GG medium in a minimal anaerobe atmosphere.

Methanobrevibacter smithii (M. smithii), the most prevalent and abundant gut methanogen, detoxifies hydrogen into methane and is, therefore, of paramount importance for the equilibrium of the gut microbiota. The isolation by culture of M. smithii has routinely relied upon hydrogen­carbon dioxide-enriched, oxygen-deprived atmospheres. In this study, we developed a medium referred to as "GG", which allowed for M. smithii growth and isolation by culture in an oxygen-deprived atmosphere, with no supply of either hydrogen or carbon dioxide, making it easier to detect M. smithii by culture in clinical microbiology laboratories.

Molecular structural analysis of carious lesions using micro-Raman spectroscopy.

In clinical situations carious dentine tissues can be discriminated by most caries fluorescence detection tools, including a new fluorescence intra-oral camera. The objectives of this study were: (i) to analyze the Raman spectra of sound, carious, and demineralized dentine, (ii) to compare this spectral analysis with the fluorescence variation observed when using a fluorescence camera, and (iii) to evaluate the involvement of the Maillard reaction in the fluorescence variations. The first positive hypothesis tested was that the fluorescence of carious dentine obtained using a fluorescence camera and the Raman spectra variation were closely related. The second was that the variation of fluorescence could be linked with the Maillard reaction. Sound dentine, sound dentine demineralized in aqueous nitric acid solution, carious soft dentine, sound dentine demineralized in lactic acid solution, sound dentine demineralized in aqueous nitric acid solution and immersed in methylglycoxal solution, and sound dentine demineralized in aqueous nitric acid solution and immersed in methylglycoxal and glucose solutions, were studied using micro-Raman spectroscopy. Modifications in the band ratio of amide, phosphate, and carbonate were observed in the decayed and demineralized groups compared with the sound dentine group. The results indicate that a close relationship exists between the Maillard reaction and fluorescence variation.

Knowledge and Opinions of French Dental Students in Operative Dentistry - Management of Deep Carious Lesions.

PURPOSE: A questionnaire survey was recently undertaken among French dental students (FDSs) to investigate their practices, knowledge and opinions in various domains of minimal intervention (MI) in cariology. The present work focuses on management of deep carious lesions (DCLs). MATERIALS AND METHODS: The questionnaire was administered (Spring 2018) to all the fifth-year students of the 16 French dental schools. Descriptive analyses were performed. RESULTS: Among 1370 FDSs (response rate: 84.5%), hardness was the most commonly reported criterion for assessing the endpoint of carious tissue removal (53.9%), followed by firm dentin (40.0%). Regarding FDSs' opinion of leaving carious dentine under a restoration, 41.9% of the respondents agreed that carious tissues should always be removed completely. For an asymptomatic tooth with DCLs and exposed pulp, direct pulp capping was mainly chosen (93.9%). In a clinical case correctly diagnosed as a reversible pulpitis by 79.7% of respondents, nearly half of FDSs chose a one-step complete excavation (48.3%) followed by selective excavation (25.1%), then two-step complete excavation (20.9%) and a minority (5.7%) opted for pulpal therapy (biopulpotomy or endodontic treatment). CONCLUSION: The present results suggest an inadequate dissemination of MI concepts among FDSs towards DCL management. The present results show the need for a harmonisation and a reinforcement of teaching evidence-based MI according to the latest European recommendations.

Knowledge and Opinions of French Dental Students Toward Occlusal and Proximal Restorative Thresholds.

PURPOSE: To investigate the practices, knowledge and opinions of French dental students (FDSs) in various domains of minimal intervention (MI) in cariology. MATERIALS AND METHODS: A cross-sectional, questionnaire-based study was conducted in spring 2018 among all fifth-year French dental students (FDSs) from the 16 French dental schools. The present article focuses on restorative management. Statistical analyses (descriptive, chi-squared) were performed. RESULTS: The response rate was 84.5%. Overall, 97.4% of respondents would have operatively intervened for proximal and 83% for occlusal carious lesions, respectively, while non-or micro-invasive intervention would have been possible. Interestingly, 15% would completely open the occlusal fissures. For both occlusal and proximal lesions requiring a restoration, composite resin was indicated by over 95% of the respondents. In a clinical case, 51.6% of FDSs who rightly diagnosed an enamel carious lesion would operatively intervene. When FDSs could not diagnose the type of carious lesions, a high proportion of invasive actions were also reported (40%). FDSs who read scientific articles were more likely to consider the high importance of not filling sound teeth unnecessarily (p = 0.033). CONCLUSION: FDSs do not have sufficient awareness of MI guidelines regarding occlusal and proximal restorative thresholds. Efforts are required in dental schools to teach FDSs to postpone invasive/restorative strategies to later stages of carious progression. There is a need to strengthen prevention techniques and non-invasive options in the teaching of MI in cariology.

Probing structural transitions in the intrinsically disordered C-terminal domain of the measles virus nucleoprotein by vibrational spectroscopy of cyanylated cysteines.

Four single-cysteine variants of the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL)) were cyanylated at cysteine and their infrared spectra in the C triple bond N stretching region were recorded both in the absence and in the presence of one of the physiological partners of N(TAIL), namely the C-terminal X domain (XD) of the viral phosphoprotein. Consistent with previous studies showing that XD triggers a disorder-to-order transition within N(TAIL), the C triple bond N stretching bands of the infrared probe were found to be significantly affected by XD, with this effect being position-dependent. When the cyanylated cysteine side chain is solvent-exposed throughout the structural transition, its changing linewidth reflects a local gain of structure. When the probe becomes partially buried due to binding, its frequency reports on the mean hydrophobicity of the microenvironment surrounding the labeled side chain of the bound form. The probe moiety is small compared to other common covalently attached spectroscopic probes, thereby minimizing possible steric hindrance/perturbation at the binding interface. These results show for the first time to our knowledge the suitability of site-specific cysteine mutagenesis followed by cyanylation and infrared spectroscopy to document structural transitions occurring within intrinsically disordered regions, with regions involved in binding and folding being identifiable at the residue level.

A new concept in restorative dentistry: LIFEDT-light-induced fluorescence evaluator for diagnosis and treatment: part 2 - treatment of dentinal caries.

AIM: A new and innovative therapeutic concept using a light-induced fluorescence evaluator for diagnosis and treatment (LIFEDT) of dental caries based on the imaging and autofluorescence of dental tissues is proposed. The aims of this series of in vivo experiments are to compare and analyze the brightness variations of sound dentin and active and arrested carious dentin illuminated with an intraoral LED camera and to determine if this new device could be helpful in daily practice to discriminate between caries and sound dentin. METHODS AND MATERIALS: A new intraoral LED camera that emits visible blue light was used in this in vivo study to illuminate and photograph 15 teeth at high magnification. The magnified images were examined using the free Image J V version 1.41 software. Four standardized rectangular areas were drawn on each picture that included both healthy and pathologic areas to analyze variations in brightness using a brightness formula: L = 0.299 Red + 0.587 Green + 0.114 Blue. RESULTS: Statistically significant differences in the brightness were found between active and arrested caries processes in an area of infected dentin designated Z2. Within the limitations of this in vivo study, the images created with the intraoral LED camera revealed significant variations in fluorescence between sound dentin and active and arrested caries processes. CONCLUSIONS: The LIFEDT concept provides a therapeutic concept based on these findings of variations in fluorescence between healthy and pathologic tissue. CLINICAL SIGNIFICANCE: This concept defines a pragmatic clinical and therapeutic approach for treating active and arrested carious lesions based on the interpretation of variations of a fluorescence signal and applying the LIFEDT concept to the treatment of dentin carious lesions.

Carious lesion detection technologies: factual clinical approaches.

Minimal intervention dentistry is now accepted as the contemporary approach for caries management. The development of adjunctive technologies to assist early lesion detection has led to widespread marketing of various devices over the past two decades. A thorough understanding of the clinical relevance and limitations of such devices is required to make valid interpretation of their results. This paper will discuss the most common commercially available carious lesion detection technologies in order to give dental practitioners clear information about the devices' scientific principles, advantages and limitations within a clinical context.

A new concept in restorative dentistry: light-induced fluorescence evaluator for diagnosis and treatment. Part 1: Diagnosis and treatment of initial occlusal caries.

AIM: The objective of this in vivo experiment is to propose an innovative therapeutic concept using a light-induced fluorescence evaluator for diagnosis and treatment (LIFEDT) that is based on the imaging and autofluorescence of dental tissues. BACKGROUND: Processes with the aim of diagnosing carious lesions in the initial stage with optimum sensitivity and specificity employ a wide variety of technologies, but like the conventional diagnosis tools, they remain either inefficient or too subjective. TECHNIQUE: This experiment evaluated a fluorescence light-induced camera that illuminates tooth surfaces within an excitation radiation band of light with a wavelength of 450 nm and facilitates a high magnification image. CONCLUSION: An analysis of 50 occlusal grooves revealed three clinical forms of enamel caries: (1) enamel caries on the surface, (2) suspicious grooves with a positive autofluorescent red signal, and (3) suspicious grooves with a neutral fluorescent dark signal. Two decision-making diagrams were proposed in accordance with international recommendations for preventive dentistry, but modified as a result of the accurate information obtained with this new LIFEDT device. CLINICAL SIGNIFICANCE: The lighting of suspect occlusal grooves with the SoproLife camera enables observation of any variations in the optical properties to refine a caries diagnosis and facilitates more than a 50x magnification of occlusal groove anatomy to provide additional information on the carious potential of the tooth surface.

Tobacco Smoking Affects the Salivary Gram-Positive Bacterial Population.

The microbial communities of the oral fluid are in direct contact with tobacco smoke, which may thus affect these communities. Few culture-based studies have analyzed the effects of tobacco smoking on the oral fluid microbiota. Using bacterial culture we investigated whether tobacco smoking altered the microbial diversity of the oral fluid, focusing on aerobic and facultative anaerobic Gram-positive bacteria otherwise comprising of major pathogens. Among 90 oral fluid specimens collected in 19 tobacco-smokers and 71 controls, the diversity did not significantly differ with age and with sex. However, diversity was significantly lower in tobacco-smokers (nine different species) than in non-smokers (18 different species) with all the species cultured in tabocco-smokers being also cultured in non-smokers. We isolated the human pathogen Streptococcus australis for the first time from oral fluid. Tobacco smoking significantly alters the saliva Gram-positive bacterial microbiota, including pathogens with potential implication in the pathogenesis of tobacco-related diseases such as periodontitis and peri-implantitis.

Specific clones of Trichomonas tenax are associated with periodontitis.

Trichomonas tenax, an anaerobic protist difficult to cultivate with an unreliable molecular identification, has been suspected of involvement in periodontitis, a multifactorial inflammatory dental disease affecting the soft tissue and bone of periodontium. A cohort of 106 periodontitis patients classified by stages of severity and 85 healthy adult control patients was constituted. An efficient culture protocol, a new identification tool by real-time qPCR of T. tenax and a Multi-Locus Sequence Typing system (MLST) based on T. tenax NIH4 reference strain were created. Fifty-three strains of Trichomonas sp. were obtained from periodontal samples. 37/106 (34.90%) T. tenax from patients with periodontitis and 16/85 (18.80%°) T. tenax from control patients were detected by culture (p = 0.018). Sixty of the 191 samples were tested positive for T. tenax by qPCR, 24/85 (28%) controls and 36/106 (34%) periodontitis patients (p = 0.089). By combining both results, 45/106 (42.5%) patients were positive by culture and/or PCR, as compared to 24/85 (28.2%) controls (p = 0.042). A link was established between the carriage in patients of Trichomonas tenax and the severity of the disease. Genotyping demonstrates the presence of strain diversity with three major different clusters and a relation between disease strains and the periodontitis severity (p<0.05). More frequently detected in periodontal cases, T. tenax is likely to be related to the onset or/and evolution of periodontal diseases.