Analysis of the association between salivary proteins and oral mucositis in patients with head and neck cancer undergoing IMRT: a longitudinal study.

INTRODUCTION: This longitudinal study assessed the association between salivary protein composition and the clinical onset/severity of oral mucositis (OM) in patients with head and neck tumours treated with intensity-modulated-radiotherapy (IMRT). METHODS: Saliva samples/clinical data were obtained from 40 head and neck cancer patients treated at Guy's Hospital before -IMRT(T0) and after-IMRT (T1 = 6 m, T2 = 12 m) (ethics approval/consent). Salivary flow rate, total protein concentration, and secretion rate were determined from saliva samples and compared with pre-treatment values. OM was assessed, total/specific salivary proteins, including mucin 5B and 7, IgA, cystatin-S, albumin, and α-amylase, were quantified. RESULTS: 95% patients experienced OM during IMRT, with 33 subjects reaching grade 2&3. At T1, there was a significant reduction in salivary flow rate, total protein secretion rate, α-amylase and cystatin-S compared to baseline. Remarkably IMRT did not significantly alter mucin 5B and 7, or the IgA secretion rate at any time point. At T1, all the analyzed proteins were associated with the OM outcomes. In addition, there was a significant inverse correlation between IgA concentration at T0 and the severity of OM during IMRT. CONCLUSION: This study revealed significant associations between several salivary proteins and OM in patients with head and neck cancer undergoing IMRT. Further longitudinal studies are needed to confirm these results. CLINICAL SIGNIFICANCE: The study contributes to the understanding of certain salivary proteins association with OM. This could be the first step towards identifying potential salivary markers that could offer perspectives for personalized medicine approaches to improve their quality of life (QoL). RESEARCH QUESTION: What is the association between salivary proteins and the occurrence and severity of OM in head and neck cancer patients? AIM: To assess the association between salivary protein composition with the clinical onset/severity of oral mucositis (OM) in head and neck cancer patients treated with intensity modulated radiotherapy. NULL HYPOTHESIS: There is no association between salivary proteins and onset/severity of OM in HNC patients.

The impact of an enhanced infection control protocol on molar root canal treatment outcome - a randomized clinical trial.

AIM: To evaluate the effect of an enhanced infection control protocol on root canal treatment outcomes and on microbial load within root canals after chemomechanical preparation. METHODOLOGY: A total of 144 molar teeth from 139 healthy patients receiving primary root canal treatment were block randomized to a standard protocol (StP) or an enhanced infection control protocol (EnP). Both treatment arms adhered to current best practice recommendations, while the EnP comprised additional steps that included replacing rubber dams, gloves, files, all instruments and surface barriers at the time of canal filling to reduce the chances of iatrogenic contamination. Patients and radiographic examiners were blinded to the protocol used. Intracanal microbial samples were taken at baseline (S1) and after completion of chemomechanical preparation (S2). Microbial 16S rDNA copy numbers were enumerated by quantitative polymerase chain reaction (qPCR). Cone beam computed tomography (CBCT) scans were taken before treatment and at one-year follow-up. The outcome was assessed clinically and radiographically using CBCT by logistic regression modelling. RESULTS: At one-year follow-up, 115 teeth were analysed (54 in StP and 61 in EnP). The percentage of favourable outcomes assessed by CBCT was 85.2% in the EnP and 66.7% in the StP. The odds of 12-month success was three times higher in the EnP group compared with the StP group (OR=2.89; p=0.022, CI: 1.17 - 7.15). The median bacterial reads were reduced from 8.1×103 in S1 samples to 3.5×103 in the StP group and from 8.6×103 to 1.3×103 in the EnP group. The enhanced protocol significantly reduced bacterial counts in pre-canal filling samples when compared to the standard protocol (p=0.009). CONCLUSIONS: The implementation of a facile, enhanced infection control protocol in primary root canal treatment resulted in less detectable bacterial DNA before canal filling and significantly more successful outcomes at one year.

Schneiderian membrane thickness variation following endodontic procedures: a retrospective cone beam computed tomography study.

BACKGROUND: To assess the change of the Schneider membrane thickness measured by CBCT before and after root canal treatment, retreatment and pulp capping procedures. METHODS: This retrospective study was conducted on CBCT scans of a patient population of Guy's Hospital NHS Foundation Trust, London. Three groups of patients were studied: Group 1 consisted of patients referred for primary endodontic treatment; Group 2 for endodontic retreatment; Group 3 for indirect pulp capping procedures (serving as a control group). Follow up scans were carried out 1 year after treatment. Measurements were carried out on CBCT scans and data were analysed statistically by Wilcoxon Signed Rank Test. Linear regression was used to assess predictive parameters for membrane thickness. RESULTS: A statistically significant reduction of the Schneider membrane thickness was observed one year after endodontic treatment and retreatment (P < 0.05) but no significant reduction was observed after pulp capping procedures. Linear regression showed that age and gender were significant predictors influencing the Schneider membrane thickness. CONCLUSIONS: Within the limitations of this retrospective study, following root canal treatment and re-treatment a Schneiderian membrane thickness reduction occurred at 1-year follow-up. The removal of odontogenic infection following endodontic treatment may help reducing the thickness of the Schneider membrane.

Combined systems approaches reveal highly plastic responses to antimicrobial peptide challenge in Escherichia coli.

Obtaining an in-depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. We investigated whether a whole organism view of antimicrobial peptide (AMP) challenge on Escherichia coli would provide a suitably sophisticated bacterial perspective on AMP mechanism of action. Selecting structurally and physically related AMPs but with expected differences in bactericidal strategy, we monitored changes in bacterial metabolomes, morphological features and gene expression following AMP challenge at sub-lethal concentrations. For each technique, the vast majority of changes were specific to each AMP, with such a plastic response indicating E. coli is highly capable of discriminating between specific antibiotic challenges. Analysis of the ontological profiles generated from the transcriptomic analyses suggests this approach can accurately predict the antibacterial mode of action, providing a fresh, novel perspective for previous functional and biophysical studies.

A colorimetric-based amplification system for proteinases including MMP2 and ADAM8.

We have developed a new amplification system for proteinases that is sensitive, simple, and inexpensive to run, exemplified by a horseradish peroxidase (HRP)-conjugated, dual MMP2 (matrix metalloproteinase 2) and ADAM8 (a disintegrin and metalloproteinase 8) peptide substrate assay presented herein. The HRP-conjugated substrate is attached to beads through a 6× histidine tag and then incubated with the target enzyme, cleaving the HRP reporter. This product is subsequently removed from the unreacted bound portions of the substrate by magnetic deposition of the beads. The amount of product is then quantified using a standard HRP color development assay employing 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2). This HRP amplification system represents a new approach to proteinase assays and could be applied to other enzymes, such as lipases, esterases, and kinases, as long as the unreacted substrate can be physically separated from the product and catalysis by the enzyme to be quantified is not impaired dramatically by steric hindrance from the HRP entity.

Assessment of Bacterial Load and Post-Endodontic Pain after One-Visit Root Canal Treatment Using Two Types of Endodontic Access Openings: A Randomized Controlled Clinical Trial.

The need for controlling bacteria and pain during root canal therapy is undeniable. This clinical trial aimed to assess whether there is a difference in colony-forming unit (CFU) reduction after instrumentation and post-endodontic pain after root canal treatment (RCT) using a traditional endodontic cavity (TEC) versus a conservative endodontic cavity (CEC). This clinical study was conducted on 89 patients designated for a single-visit RCT. Patients were allocated randomly (TEC n = 45 and CEC n = 44). The access opening was gained accordingly in each group by a single operator. A pre-instrumentation sample of root canal dentin was collected using an endodontic file; the second sample was collected similarly, right after shaping and cleaning the root canal. The CFU was calculated based on the samples collected. The pain level was recorded preoperatively and at 1, 7, and 21 days postoperatively utilizing a visual analog scale (VAS). There were no statistically significant differences in the CFU reduction between the TEC and CEC groups (p > 0.05). Additionally, there were no statistically significant differences found in postoperative pain levels between the TEC and CEC at 1, 7, and 21 days (p > 0.05). Despite the limitations of this study, both the CEC and TEC demonstrate a decrease in bacteria within the root canals and alleviate postoperative pain with no difference between them.

Tooth loss and depression in Chilean participants of the National Health Survey 2016-2017: Oral and social functions mediation analysis.

BACKGROUND: Previous studies have indicated the association between poor oral health and depression in adults. This study evaluated oral and social functions contribution to the association between tooth loss and depressive symptoms in Chilean individuals. METHODS: We used data from the Chilean National Health Survey. The number of remaining teeth (≤19 versus ≥20 teeth) and anterior tooth losses were the exposure variables. Outcome was depression, measured through a self-report question and with the Composite International Diagnostic Interview - Short Form (CIDI SF). Mediating variables were determined by five questions, including problems regarding "speaking", "pain and suffering", "eating", "daily activities", and "social relationships". We performed logistic regression models adjusted by multiple confounders variables. Finally, we calculated indirect, direct effect, total effect, and the proportion mediated (PM). RESULTS: We included 5383 participants. The self-reported depression and suspected depression prevalence were 22,1 % and 14,0 % respectively. The total effect of fewer remaining teeth (≤19) on self-reported depression was 1.21 (95 % CI 1.02-1.44), and 1.09 (95 % CI 0.90-1.33) for suspected depression. All five variables of oral and social functions significantly mediated the association between tooth loss and depression. Feeling uncomfortable when speaking or eating discomfort were the most significant mediators. LIMITATIONS: The mediation analysis should be interpreted with caution due to the cross-sectional design. CONCLUSIONS: Deterioration of oral and social functions was a significant mediator in the association between tooth loss and depression, in particular feeling uncomfortable when speaking or eating. This mechanism should be considered in interventions to improve mental health.

A self-etch bonding system with potential to eliminate selective etching and resist proteolytic degradation.

OBJECTIVES: Bonded restorations using self-etch (SE) systems exhibit a limited lifespan due to their susceptibility to hydrolytic, enzymatic or fatigue degradation and poor performance on enamel. This study was conducted to develop and assess the performance of a two-step SE system using a functional monomer bis[2-(methacryloyloxy)ethyl]phosphate (BMEP) and demonstrate a strategy to enhance stability of bonded resin composite restorations to both enamel and dentine. METHODS: A two-step SE system was formulated with a primer containing BMEP, with an adhesive with or without BMEP, and compared to a commercial 10-MDP-containing system, ClearfilTM SE Bond 2 (CFSE). The systems were evaluated on enamel for surface roughness and microshear bond strength (µSBS) and on dentine for microtensile bond strength (µTBS), nanoleakage, MMP inhibition and cyclic flexural fatigue. RESULTS: Whilst all bonding systems resulted in statistically similar µSBS, BMEP-based primers yielded greater enamel surface roughness than the CFSE primer. The BMEP-free adhesives resulted in statistically similar or higher µTBS and lower nanoleakage compared to CFSE. In situ zymography revealed minimal to no MMP activity within the hybrid layer of BMEP-based systems. The BMEP-free adhesive exhibited flexural strength and fatigue resistance statistically similar to CFSE. CONCLUSIONS: Incorporation of BMEP in the primer led to satisfactory bond strengths with both enamel and dentine, potentially eliminating the need for selective enamel etching. Combined with an adhesive formulation that is solvent-free and hydrophobic, and confining the acidic functional monomer in the primer resulted in minimal interfacial leakage, and resistance to proteolytic degradation and the cyclic nature of chewing. CLINICAL SIGNIFICANCE: The SE bonding system containing BMEP combines the potent etching of phosphoric acid with the therapeutic function of the phosphate-based monomer in creating a homogenous hybrid layer with protection against endogenous proteolytic enzymes. This strategy may overcome current challenges that arise during selective enamel etching.

Assessing the Iatrogenic Contribution to Contamination During Root Canal Treatment.

INTRODUCTION: Asepsis in endodontics aims to control all potential sources of infection. Inadvertent introduction of bacteria into the root canal system may occur when the aseptic chain is breached during treatment. Therefore, measures are taken to prevent such microbial access and establish an aseptic environment. This study aimed to assess potential bacterial contamination and the potential risk of iatrogenic introduction from 7 sites comprising surfaces, instruments, and files acquired during the treatment of 30 vital, pulpitic teeth. METHODS: Bacterial samples were collected from access burs, files, endodontic rulers, rubber dam surfaces, gloves, and instruments. Genomic DNA was extracted and quantified by quantitative polymerase chain reaction. Bacterial types were determined using next-generation sequencing. RESULTS: High frequencies of contamination and microbial numbers were encountered in all sample types examined.Thirty-eight percent of the initial files introduced into the root canal had significant levels of bacteria at the point of obturation, including endodontic pathogens. Around half of the rubber dam surfaces were contaminated with substantial bacterial loads at the time of obturation, and bacteria were also detected in 20%-30% of gloves, instruments, and rulers before obturation. Next-generation sequencing revealed the predominant oral or endodontic origin of these contaminants, with the following genera identified: Streptococcus, Rothia, Granulicatella, Cutibacterium, Corynebacterium, Peptostreptococcus, and Fusobacterium. Together, these findings highlight the potential risk of reintroducing endodontically relevant bacteria during treatment. CONCLUSIONS: Gloves, rubber dams, instruments, and files acquire bacterial contamination during treatment at high frequencies and loads. This highlights the potential risk of iatrogenic contamination at the clinically vulnerable point of canal obturation. Measures to address these may improve clinical outcomes.

Impact of an Enhanced Infection Control Protocol on the Microbial Community Profile in Molar Root Canal Treatment: An in Vivo NGS Molecular Study.

INTRODUCTION: Recent findings demonstrated that 1-year cone-beam computed tomography-based outcomes of molar root canal treatment were improved through an enhanced infection protocol (EnP), when compared with a current best-practice standard infection control protocol (StP). The EnP comprised measures to reduce iatrogenic contamination from direct and indirect contact surfaces, including the replacement of the rubber dams, gloves, files, all instruments, and surface barriers before root canal obturation. The aim of this study was to investigate the effect of such an EnP on resident microbiome present after chemomechanical instrumentation and the protocol ability in reducing iatrogenic contamination in molar teeth during root canal treatment. METHODS: Molar teeth were block-randomized to receive treatment under EnP or StP. To compare the differential effect of the protocol on the identity of bacteria present, 150 matched DNA extracts from 75 molar teeth samples (StP, n = 39; EnP, n = 36) were evaluated. Samples were taken before (S1) and after (S2) chemomechanical preparation and were subjected to next-generation sequencing of the V3-V4 region of the 16S rRNA gene before bioinformatical identification using the HOMD oral microbiome database and downstream taxonomic processing, providing measures of richness and diversity of bacteria and significant bacterial taxa during chemomechanical instrumentation and the effect of the 2 treatment groups. RESULTS: Eighty-eight microbial taxa were significantly more abundant in StP S2 samples, including endodontically relevant contaminants taxa as Actinomyces, Cutibacterium, and Haemophilus. The S2 samples demonstrated fewer residual bacterial species in the EnP group, with 26.8 observed species compared with 38.3 in the StP. Reduced diversity and richness measures were noted in the EnP pre-obturation samples compared with the StP in OTU, Chao1, and ACE indices (P ≤ .05). Differential microbial identities between S1 and S2 samples and protocols demonstrated that the previously observed increased effectiveness of the EnP protocol was likely to prevent recontamination or de novo contamination of the root canal space during treatment. CONCLUSIONS: The implemented EnP resulted in a specific reduction of microbial taxa often associated with recontamination or iatrogenic contamination, suggesting the basis for improved infection control measures during root canal treatment.

Methacrylic monomer derived from cardanol incorporated in dental adhesive as a polymerizable collagen crosslinker.

OBJECTIVES: The aim of this study was to evaluate the influence on MMP inhibition, dentin adhesion and physicochemical properties of an adhesive system incorporated with polymerizable collagen crosslinker monomer derived from cardanol. METHODS: The intermediary cardanol epoxy (CNE) was synthesized through cardanol epoxidation, followed by synthesis of cardanol methacrylate through methacrylic acid solvent-free esterification. Zymographic analysis was performed to evaluate the substances' ability to inhibit gelatinolytic enzymes. Collagen crosslinkers were added into adhesives systems according to the following groups: Ybond Universal® (Control), Ybond® + 2 % proanthocyanidin (PAC), Ybond® + 2 % unsaturated cardanol (Cardanol) and Ybond® + 2 % cardanol methacrylate (CNMA). Degree of conversion (DC) of the adhesives was assessed by FT-IR. Disk-shaped specimens were prepared for water sorption (WS) and solubility (SL) tests. Human third molars were sectioned to expose medium dentin and restored according to the different adhesives used (n = 5). Then, the specimens were cut into 1 mm2 sticks to evaluate, after 24 h and 6-month aging, microtensile bond strength (µTBS) and nanoleakage by scanning electron microscopy. Data were analysed with ANOVA and Tukey's post-test (α = 0.05). RESULTS: CNMA and PAC completely inhibited all forms of gelatinolytic enzymes. Cardanol achieved a significantly lowest DC, while the other groups did not differ from each other (p > 0.05). PAC achieved significantly higher water sorption, while CNMA solubility was significantly lower when compared to the other adhesives (p < 0.05). PAC provided a statistically higher 24 h and 6-month aging bond strength. Intermediary similar µTBS were presented by control and CNMA (p = 0.108). All adhesives applied attained significantly reduced bond strength after aging (p < 0.05). Interfaces created using CNMA were almost devoid of silver deposits initially, however all groups showed large amounts of silver deposits on resin-dentin interface subjected to water aging. SIGNIFICANCE: Although CNMA was effective in inhibiting gelatinolytic enzymes, when incorporated into a universal adhesive it could not promote less degradation of the adhesive interface after water aging. Since it is a hydrophobic monomer, CNMA did not interact well with dentin collagen, however it reduced the solubility of the adhesive system besides not interfering in its polymerization.

Tumor necrosis factor-alpha (TNF-alpha) regulates shedding of TNF-alpha receptor 1 by the metalloprotease-disintegrin ADAM8: evidence for a protease-regulated feedback loop in neuroprotection.

Tumor necrosis factor alpha (TNF-alpha) is a potent cytokine in neurodegenerative disorders, but its precise role in particular brain disorders is ambiguous. In motor neuron (MN) disease of the mouse, exemplified by the model wobbler (WR), TNF-alpha causes upregulation of the metalloprotease-disintegrin ADAM8 (A8) in affected brain regions, spinal cord, and brainstem. The functional role of A8 during MN degeneration in the wobbler CNS was investigated by crossing WR with A8-deficient mice: a severely aggravated neuropathology was observed for A8-deficient WR compared with WR A8(+/-) mice, judged by drastically reduced survival [7 vs 81% survival at postnatal day 50 (P50)], accelerated force loss in the forelimbs, and terminal akinesis. In vitro protease assays using soluble A8 indicated specific cleavage of a TNF-alpha receptor 1 (p55 TNF-R1) but not a TNF-R2 peptide. Cleavage of TNF-R1 was confirmed in situ, because levels of soluble TNF-R1 were increased in spinal cords of standard WR compared with wild-type mice but not in A8-deficient WR mice. In isolated primary neurons and microglia, TNF-alpha-induced TNF-R1 shedding was dependent on the A8 gene dosage. Furthermore, exogenous TNF-alpha showed higher toxicity for cultured neurons from A8-deficient than for those from wild-type mice, demonstrating that TNF-R1 shedding by A8 is neuroprotective. Our results indicate an essential role for ADAM8 in modulating TNF-alpha signaling in CNS diseases: a feedback loop integrating TNF-alpha, ADAM8, and TNF-R1 shedding as a plausible mechanism for TNF-alpha mediated neuroprotection in situ and a rationale for therapeutic intervention.

Characterization of Root Canal Microbiota in Teeth Diagnosed with Irreversible Pulpitis.

INTRODUCTION: Previous studies have shown that in teeth presenting with symptoms of irreversible pulpitis (IP), bacteria and their by-products driving inflammation are confined mainly within the coronal pulpal tissue. The present study aimed to determine the presence and identity of bacteria within pulps presenting with clinical symptoms of IP using molecular methods. METHODS: Samples were obtained from 30 adult patients presenting to the dental emergency department with signs and symptoms of IP. After meticulous surface decontamination, the pulp space was accessed, and clinical samples were collected from inflamed pulp tissue using sterile paper points. Genomic DNA was extracted from the clinical samples, and quantification of bacteria was performed using quantitative polymerase chain reaction targeting the conserved 16S ribosomal RNA (rRNA) gene. To characterize the microbial composition, the V3-V5 hypervariable regions of the 16S rRNA gene were amplified and subjected to next-generation sequencing on the MiSeq platform (Illumina, San Diego, CA). RESULTS: Of the 30 teeth that presented with IP, half of the intracanal samples had a substantial bacterial load (16S rRNA copies) within the IP vital pulp as determined by quantitative polymerase chain reaction. Next-generation sequencing microbial identification was successful in 7 intracanal samples and yielded 187 bacterial operational taxonomic units within the IP samples. The most abundant genera observed among the vital cases were Veillonella (16%), Streptococcus (13%), Corynebacterium (10%), Cutibacterium (9.3%), and Porphyromonas (5.7%). CONCLUSIONS: The current study highlighted the evidence of vital teeth diagnosed as IP harboring considerable bacterial loads and composed of genera reflective of established endodontic pathology and thus may offer insights into the initial events preceding pulpal necrosis.

Clinical Evaluation of Pathognomonic Salivary Protease Fingerprinting for Oral Disease Diagnosis.

Dental decay (Caries) and periodontal disease are globally prevalent diseases with significant clinical need for improved diagnosis. As mediators of dental disease-specific extracellular matrix degradation, proteases are promising analytes. We hypothesized that dysregulation of active proteases can be functionally linked to oral disease status and may be used for diagnosis. To address this, we examined a total of 52 patients with varying oral disease states, including healthy controls. Whole mouth saliva samples and caries biopsies were collected and subjected to analysis. Overall proteolytic and substrate specific activities were assessed using five multiplexed, fluorogenic peptides. Peptide cleavage was further described by inhibitors targeting matrix metalloproteases (MMPs) and cysteine, serine, calpain proteases (CSC). Proteolytic fingerprints, supported by supervised machine-learning analysis, were delineated by total proteolytic activity (PepE) and substrate preference combined with inhibition profiles. Caries and peridontitis showed increased enzymatic activities of MMPs with common (PepA) and divergent substrate cleavage patterns (PepE), suggesting different MMP contribution in particular disease states. Overall, sensitivity and specificity values of 84.6% and 90.0%, respectively, were attained. Thus, a combined analysis of protease derived individual and arrayed substrate cleavage rates in conjunction with inhibitor profiles may represent a sensitive and specific tool for oral disease detection.

More than the Eye Can See: Shedding New Light on SARS-CoV-2 Lateral Flow Device-Based Immunoassays.

Containing the global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has been an unprecedented challenge due to high horizontal transmissivity and asymptomatic carriage rates. Lateral flow device (LFD) immunoassays were introduced in late 2020 to detect SARS-CoV-2 infection in asymptomatic or presymptomatic individuals rapidly. While LFD technologies have been used for over 60 years, their widespread use as a public health tool during a pandemic is unprecedented. By the end of 2020, data from studies into the efficacy of the LFDs emerged and showed these point-of-care devices to have very high specificity (ability to identify true negatives) but inadequate sensitivity with high false-negative rates. The low sensitivity (<50%) shown in several studies is a critical public health concern, as asymptomatic or presymptomatic carriers may wrongly be assumed to be noninfectious, posing a significant risk of further spread in the community. Here, we show that the direct visual readout of SARS-CoV-2 LFDs is an inadequate approach to discriminate a potentially infective viral concentration in a biosample. We quantified significant immobilized antigen-antibody-labeled conjugate complexes within the LFDs visually scored as negative using high-sensitivity synchrotron X-ray fluorescence imaging. Correlating quantitative X-ray fluorescence measurements and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) determined numbers of viral copies, we identified that negatively scored samples could contain up to 100 PFU (equivalent here to ∼10 000 RNA copies/test). The study demonstrates where the shortcomings arise in many of the current direct-readout SARS-CoV-2 LFDs, namely, being a deficiency in the readout as opposed to the potential level of detection of the test, which is orders of magnitude higher. The present findings are of importance both to public health monitoring during the Coronavirus Disease 2019 (COVID-19) pandemic and to the rapid refinement of these tools for immediate and future applications.

Amoebae in Chronic, Polymicrobial Endodontic Infections Are Associated with Altered Microbial Communities of Increased Virulence.

BACKGROUND: Infections of the root canal space involve polymicrobial biofilms and lead to chronic, low grade inflammatory responses arising from the seeding of microbes and by-products. Acute exacerbation and/or disseminating infections occur when established microbial communities undergo sudden changes in phenotypic behaviour. METHODS: Within clinical endodontic infections, we assessedcategorical determinants comprising, and changing microbial composition of, chronic polymicrobial infections and their association with amoebae. After standardised assessment, primary or secondary infections underwent sampling and DNA processing, targeting bacteria, fungi and amoebae, including 16S high-throughput sequencing. After taxonomic assignment, community composition was correlated with clinical signs and symptoms. Diversity and abundance analyses were carried out in relation to the presence of non-bacterial amplicons. RESULTS: Clinical specimens revealed two distinct community clusters, where specific changes correlated with clinical signs. An association between the compositions of microbiomes was found between these groups and the presence of Entamoeba gingivalis in 44% of cases. When amoebae were present in endodontic infections, we demonstrate changes in microbial community structure that mirror those observed in treatment-resistant or recurrent infections. CONCLUSIONS: Amoeba are present in endodontic infections at a high prevalence, and may promote increased virulence by enrichment for phagocytosis-resistant bacteria.

Rapid Chairside Microbial Detection Predicts Endodontic Treatment Outcome.

BACKGROUND: The aim of this longitudinal, one-year cohort study was to explore the hypothesis that fluorescence sampling of the root canal space prior to obturation could predict the outcome of root canal treatment (RCT). METHODS: Sixty-five teeth underwent primary RCT and were followed up clinically and radiographically. The outcome was determined radiographically with periapical radiographs (PR) and cone beam computed tomography (CBCT) scans. RESULTS: Success at 12 months was predictable based on the fluorescence score. When the fluorescence score (defined as the percentage of signal over total signal including background) was lower than 67, there was a 4.5 times (Odds ratio (OR) = 0.028; 95% confidence interval (CI): 0.003, 0.291, p = 0.001) greater chance of success (90% overall). When the readings were above this threshold, the success rate was 20%. CONCLUSION: A chairside sampling method is able to predict the outcome of RCT, through the use of paper point sampling and fluorescence staining. This has reduced the prevalence of persistent infections by guiding the optimum time for obturation. ClinicalTrials.gov trial NCT03660163.

The airways microbiome of individuals with asthma treated with high and low doses of inhaled corticosteroids.

BACKGROUND: Inhaled corticosteroids (ICS) are the mainstay of asthma treatment, but evidence suggests a link between ICS usage and increased rates of respiratory infections. We assessed the composition of the asthmatic airways microbiome in asthma patients taking low and high dose ICS and the stability of the microbiome over a 2 week period. METHODS: We prospectively recruited 55 individuals with asthma. Of these, 22 were on low-dose ICS and 33 on high-dose ICS (16 on budesonide, 17 on fluticasone propionate). Sputum from each subject underwent DNA extraction, amplification and 16S rRNA gene sequencing of the bacterial component of the microbiome. 19 subjects returned for further sputum induction after 24 h and 2 weeks. RESULTS: A total of 5,615,037 sequencing reads revealed 167 bacterial taxa in the asthmatic airway samples, with the most abundant being Streptococcus spp. No significant differences in sputum bacterial load or overall community composition were seen between the low- and high-dose ICS groups. However, Streptococcus spp. showed significantly higher relative abundance in subjects taking low-dose ICS (p = 0.002). Haemophilus parainfluenzae was significantly more abundant in subjects on high-dose fluticasone propionate than those on high-dose budesonide (p = 0.047). There were no statistically significant changes in microbiota composition over a 2-week period. DISCUSSION: Whilst no significant differences were observed between the low- and high-dose ICS groups, increased abundance of the potential pathogen H. parainfluenzae was observed in patients taking high-dose fluticasone propionate compared to those taking high-dose budesonide. The microbiota were stable over fourteen days, providing novel evidence of the established community of bacteria in the asthmatic airways. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT02671773.

Assessing the diagnostic importance of nonviable bacterial cells in respiratory infections.

Identification of bacteria in clinical samples is fundamental to combating infections. Modern molecular genetic approaches exploit nucleic acids signals from clinical samples. However, DNA-derived signals can originate from nonviable bacterial cells and, therefore, generate data that could be misinterpreted. Terminal restriction fragment length polymorphism profiling of cystic fibrosis sputum samples was combined with propidium monoazide (PMA) photo-induced cross-linking. PMA is highly membrane impermeant and is excluded from viable bacteria but readily penetrates dead cells. Exposure to a light source renders DNA in permeable cells incapable of contributing to polymerase chain reaction. PMA treatment was shown to effectively prevent dead bacteria, spiked into sputum samples, from contributing to profiles. Comparison of treated and untreated clinical samples indicated that dead bacterial cells significantly bias untreated profiles. These findings highlight the significant contribution that nonviable bacteria can make to DNA-based diagnostic analysis of clinical samples while providing a simple and effective means of avoiding such bias.

Bacterial Contamination of Endodontic Materials before and after Clinical Storage.

INTRODUCTION: The aim of this study was to evaluate the bacterial contamination in endodontic consumables (gutta-percha points, rubber dams, paper mixing pads, caulking agents, and endodontic instrument sponges [EISs]) before and after clinical use and storage. METHODS: Materials were randomly sampled in triplicates at 3 time points (t0, at package opening; t1, at 7 days; and t2, at 14 days) during their clinical usage. The gutta-percha points and caulking agent (25 mg) were added to 1 mL phosphate-buffered saline (PBS). The rubber dam, paper mixing pad, and EIS were added to 25 mL PBS. After vortexing, centrifuging, and removing the supernatant, the pellet was resuspended in 1 mL PBS, plated on fastidious anaerobic agar, and incubated aerobically and anaerobically. The grown colonies were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The total bacterial load was calculated in the remaining volume (800 µL) from each sample by quantitative polymerase chain reaction after DNA extraction. RESULTS: All tested materials showed a varied number of contaminated samples at the 3 time points (except EIS at t0) using MALDI-TOF MS. The most isolated genera were Propionibacterium (42%) and Staphylococcus (32%). By using non-culture-based approaches, all tested materials at the 3 time points (except gutta-percha at t0 and the caulking agent at t0, t1, and t2) carried bacterial DNA. CONCLUSIONS: The majority of the tested materials harbored bacteria in their samples before and after clinical storage. Nosocomial infection derived from commonly used consumables could have an impact on the outcome of endodontic treatment.