Declining levels of serum chemokine (C-X-C motif) ligand 10 over time are associated with new onset of psoriatic arthritis in patients with psoriasis: a new biomarker?

BACKGROUND: We previously found that serum levels of chemokine (C-X-C motif) ligand 10 (CXCL10) decreased after the onset of psoriatic arthritis (PsA). OBJECTIVES: We measured CXCL10 levels over time in patients with psoriasis who developed PsA to determine whether the drop in CXCL10 was specific to these patients and further assess its association with PsA development. METHODS: Prospectively followed patients with psoriasis without arthritis [cutaneous psoriasis (PsC)] were assessed yearly by rheumatologists for the presence of PsA. Patients with PsC who developed PsA (converters) were matched to those that did not develop PsA (nonconverters) based on psoriasis duration and the interval between follow-up visits. The duration between baseline and the first visit postconversion in converters was used to assign a pseudoconversion date in nonconverters. Linear mixed-effects models were used to model the expression of CXCL10 over time. RESULTS: CXCL10 significantly declined over time in converters prior to PsA development with a significant difference in the trend over time between converters (n = 29) and nonconverters (n = 52; P < 0·001). CXCL10 continued to decline after PsA onset in a subset of converters. There was a significant difference in the trend of CXCL10 levels between converters (n = 24) and nonconverters (n = 16; P = 0·01) preconversion/pseudoconversion. This difference remained postconversion (P = 0·006) and was not different from the preconversion period (P = 0·75). CONCLUSIONS: A large difference in CXCL10 was identified in patients with PsC that are destined to develop PsA over time. This exploratory analysis supports the association of CXCL10 with PsA development in patients with PsC and warrants further study of the predictive ability of this chemokine. What is already known about this topic? Chemokine (C-X-C motif) ligand 10 (CXCL10) is elevated in psoriatic affected tissues and serum and/or plasma. Patients with psoriasis that develop psoriatic arthritis (PsA) have elevated CXCL10 levels at baseline and these levels drop after arthritis onset. What does this study add? By monitoring levels of CXCL10 in serum over multiple visits in patients with psoriasis that develop PsA as well as those that do not develop PsA, an association was identified between CXCL10 and PsA development. What is the translational message? CXCL10 is a strong candidate for use by physicians for the detection of patients with psoriasis that are at risk of developing PsA. Linked Comment: Kirby and Fitzgerald. Br J Dermatol 2020; 183:805-806.

Validation of an Oral Disease Severity Score (ODSS) tool for use in oral mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid (MMP) is a rare autoimmune bullous disease predominantly affecting the oral mucosa. Optimal management relies upon thorough clinical assessment and documentation at each visit. OBJECTIVES: The primary aim of this study was to validate the Oral Disease Severity Score (ODSS) for the assessment of oral involvement in MMP. We also compared its inter- and intraobserver reliability with those of the oral parts of the Mucous Membrane Pemphigoid Disease Area Index (MMPDAI), Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) and Physician's Global Assessment (PGA). METHODS: Fifteen patients with mild-to-moderately severe oral MMP were scored for disease severity by 10 oral medicine clinicians from four U.K. centres using the ODSS, the oral sections of MMPDAI and ABSIS, and PGA. Two clinicians rescored all patients after 2 h. RESULTS: In terms of reliability, the interobserver ODSS total score intraclass correlation coefficient (ICC) was 0·97, MMPDAI activity 0·59 and damage 0·15, ABSIS total 0·84, and PGA 0·72. The intraobserver ICCs (two observers) for ODSS total were 0·97 and 0·93; for MMPDAI activity 0·93 and 0·70 and damage 0·93 and 0·79; for ABSIS total 0·99 and 0·94; and for PGA 0·92 and 0·94. Convergent validity between ODSS and MMPDAI was good (correlation coefficient 0·88). The mean ± SD time for completion of ODSS was 93 ± 31 s, with MMPDAI 102 ± 24 s and ABSIS involvement 71 ± 18 s. The PGA took < 5 s. CONCLUSIONS: This study has validated the ODSS for the assessment of oral MMP. It has shown superior interobserver agreement over MMPDAI, ABSIS and PGA, and superior intraobserver reliability to MMPDAI. It is quick and easy to perform. What's already known about this topic? There are no validated scoring methodologies for oral mucous membrane pemphigoid (MMP). Proposed disease activity scoring tools for MMP include the Mucous Membrane Disease Area Index (MMPDAI) and the Autoimmune Bullous Skin Disorder Intensity Score (ABSIS). The Oral Disease Severity Score (ODSS) has been validated for use in oral pemphigus vulgaris (PV). It has been shown to be reliable and sensitive in both lichen planus (LP) and MMP. What does this study add? The ODSS has been shown to be a thorough, sensitive and reproducible, yet quick scoring tool for the assessment of oral involvement in MMP. Its versatility for use in oral PV, MMP and LP is an added advantage over other scoring methodologies. What are the clinical implications of this work? We propose that the ODSS be used as a clinical scoring tool for monitoring activity in oral MMP in clinical practice as well as for use in multicentre studies.

An Oral Disease Severity Score validated for use in oral pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is a rare autoimmune bullous disease, which can present with recalcitrant oral mucosal lesions. Optimal management of PV relies upon careful clinical assessment and documentation. OBJECTIVES: The primary aim of this study was to validate the Oral Disease Severity Score (ODSS) for the assessment of oral involvement in PV. A secondary aim was to compare its inter- and intraobserver variability and ease of use with the Physician's Global Assessment (PGA) and the oral scoring methods used in the Autoimmune Bullous Skin Disorder Intensity Score (ABSIS) and the Pemphigus Disease Area Index (PDAI). METHODS: Fifteen patients with mild-to-moderately severe oral PV were scored for disease severity by 10 oral medicine clinicians using the ODSS, the PGA and the oral sections of ABSIS and PDAI. Two clinicians rescored all patients after a minimum 2-h interval. RESULTS: Interobserver reliability was assessed using an intraclass correlation coefficient (ICC). For the ODSS total score the ICC was 0·83, for PDAI (oral total activity) 0·79, ABSIS (oral total) 0·71 and PGA 0·7. Intraobserver agreement between initial scoring and rescoring of the same patient by two clinicians demonstrated an ICC for each of 0·97 and 0·96 for ODSS total score; 0·99 and 0·82 for PDAI oral activity; 0·86 and 0·45 for ABSIS total; and 0·99 and 0·64 for PGA. Convergent validity was good, with a correlation coefficient > 0·5 (P < 0·001). The mean ± SD times taken to complete each scoring method were ODSS 76 ± 37 s, PDAI 117 ± 16 s and ABSIS 75 ± 19 s. CONCLUSIONS: This study has validated the ODSS for the assessment of oral PV. It has shown superior inter- and intraobserver reliability to PDAI, ABSIS and PGA and is quick to perform.

Human Disease/Clinical Medical Sciences in Dentistry: Current state and future directions of undergraduate teaching in the UK and Ireland.

In March 2017, a group of teachers of human disease/clinical medical science (HD/CMSD) representing the majority of schools from around the UK and Republic of Ireland met to discuss the current state of teaching of human disease and also to discuss how the delivery of this theme might evolve to inform improved healthcare. This study outlines how the original teaching in medicine and surgery to dental undergraduate students has developed into the theme of HD/CMSD reflecting changing needs as well as guidance from the regulators, and how different dental schools have developed their approaches to reach their current state. Each school was also asked to share a strengths, weakness, opportunities and threats (SWOT) analysis of their programme and to outline how they thought their HD/CMSD programme may develop. The school representatives who coordinate the delivery and assessment of HD/CMSD in the undergraduate curriculum have extensive insight in this area and are well-placed to shape the HD/CMSD development for the future.

Group O RBCs: where is universal donor blood being used.

BACKGROUND: There have been recurrent shortages of group O blood due to insufficient inventory and use of group O blood in ABO non-identical recipients. We performed a 12-year retrospective study to determine utilization of group O Rh-positive and Rh-negative red blood cells (RBCs) by recipient ABO group. Reasons for transfusing group O blood to ABO non-identical recipients were also assessed. METHODS: Utilization data from all group O Rh-positive and Rh-negative RBCs transfused at three academic hospitals between April 2002 and March 2014 were included. Data were extracted from Transfusion Registry for Utilization Surveillance and Tracking, a comprehensive database with inventory information on all blood products received at the hospitals. Extracted data included product type, ABO and Rh, final disposition (transfused, wasted, outdated), and demographic and clinical data on all patients admitted to hospital. Descriptive statistics were performed using sas 9.3. RESULTS: There were 314 968 RBC transfusions: 151 645 (48·1%) were group O, of which 138 136 (91·1%) RBC units were transfused to group O individuals. ABO non-identical recipients received 13 509 group O RBCs (8·9%). The percentage of group O RBCs transfused to ABO non-identical recipients by fiscal year varied from 7·8% to 11·1% with a steady increase from 2011 to 2013. Reasons for this included: trauma, outdating, outpatient usage and shortages. CONCLUSION: The practice of transfusing O RBCs to non-O individuals has been increasing. Specific hospital and blood supplier policies could be targeted to change practice, leading to a more sustainable group O red blood cell supply.

Transfusion-related alloimmunization in children: epidemiology and effects of chemotherapy.

BACKGROUND & OBJECTIVES: Alloimmunization rates following red blood cell (RBC) transfusion in paediatric oncology are not known. This study aimed to: (1) describe frequency and specificity of alloantibodies in paediatric oncology patients after RBC transfusions; (2) determine the effect of chemotherapy on alloimmunization rate. MATERIALS & METHODS: Retrospective cohort study of paediatric patients at a tertiary care hospital is evaluated by two groups: control group, paediatric patients without cancer; study group, paediatric oncology patients who received chemotherapy. Alloimmunization was defined as clinically significant IgG alloantibody formation against RBC antigens. RESULTS: A total of 1273 children were evaluated including 324 in study group, 909 controls, and 40 haemoglobinopathy patients. Overall, frequency of alloimmunization was 1·5%: 0·3% (95% CI: 0, 1·90) in study group; 1·3% (95% CI: 0·73, 2·32) in control group and 15% in haemoglobinopathies. The association between chemotherapy and alloimmunization was not significant; P value = 0·20 Fisher's exact test, OR 0·23 (95% CI: 0·03, 1·79). CONCLUSION: This is the first study exploring RBC alloimmunization in paediatric patients by diagnosis. Alloimmunization frequency was low. It was not possible to determine an association between chemotherapy and alloimmunization due to the low event rate.

Multimodal optical characterisation of collagen photodegradation by femtosecond infrared laser ablation.

Collagen is a structural component of the human body, as a connective tissue it can become altered as a result of pathophysiological conditions. Although the collagen degradation mechanism is not fully understood, it plays an important role in ageing, disease progression and applications in therapeutic laser treatments. To fully understand the mechanism of collagen alteration, in our study photo-disruptive effects were induced in collagen I matrix by point-irradiation with a femtosecond Ti-sapphire laser under controlled laser ablation settings. This was followed by multi-modal imaging of the irradiated and surrounding areas to analyse the degradation mechanism. Our multi-modal methodology was based on second harmonic generation (SHG), scanning electron microscope (SEM), autofluorescence (AF) average intensities and the average fluorescence lifetime. This allowed us to quantitatively characterise the degraded area into four distinct zones: (1) depolymerised zone in the laser focal spot as indicated by the loss of SHG signal, (2) enhanced crosslinking zone in the inner boundary of the laser induced cavity as represented by the high fluorescence ring, (3) reduced crosslinking zone formed the outer boundary of the cavity as marked by the increased SHG signal and (4) native collagen. These identified distinct zones were in good agreement with the expected photochemical changes shown using Raman spectroscopy. In addition, imaging using polarisation-resolved SHG (p-SHG) revealed both a high degree of fibre re-orientation and a SHG change in tensor ratios around the irradiation spot. Our multi-modal optical imaging approach can provide a new methodology for defining distinct zones that can be used in a clinical setting to determine suitable thresholds for applying safe laser treatments without affecting the surrounding tissues. Furthermore this technique can be extended to address challenges observed in collagen based tissue engineering and used as a minimally invasive diagnostic tool to characterise diseased and non-diseased collagen rich tissues.

Microbiochemical analysis of carious dentine using Raman and fluorescence spectroscopy.

The aim of this study was to evaluate and correlate objectively the microspectroscopically derived biochemical components of sound, infected and affected carious dentine with their microhardness and autofluorescence (AF) characteristics. Over 3 million high-resolution Raman spectra from 8 extracted human carious teeth were recorded using Raman spectrometer with parallel spectrum acquisition. Green AF signals across each carious lesion from all samples were acquired with a similar spatial resolution using confocal fluorescence microscopy. The Knoop microhardness (KHN) from a total of 233 co-localized areas was recorded from the same samples and allocated subjectively into the three zones. Cluster analysis of the Raman data, performed using in-house software, produced five independent spectral components representing mineral content, protein content, porphyrin fluorescence (PF), putative infected dentine signal (IDS) and affected dentine signal (ADS). The distributions of the 5 Raman components and the AF signal were matched across all samples and their average values were calculated for each corresponding KHN area. The infected dentine was defined significantly by the KHN, AF and by the relative contribution of the mineral, PF and IDS clusters. Protein cluster was not statistically related to the KHN or AF. A delineation between affected and sound dentine was observed using the KHN, AF, PF and ADS parameters. This study concludes that micro-Raman spectroscopy can provide a non-invasive and objective evaluation of different carious dentine zones. Being able to detect and assess clinically the caries-affected dentine during minimally invasive operative caries management is important to control the risk of unnecessary tissue removal.

Impact on agriculture of the mount st. Helens eruptions.

Ash from Mount St. Helens has fallen over a diverse agricultural area, with deposits of up to 30 kilograms per square meter. Crop losses in eastern Washington are estimated at about $100 million in 1980-about 7 percent of the normal crop value in the affected area and less than was expected initially. Production of wheat, potatoes, and apples will be normal or above normal because the favorable conditions for growth of these crops since the ashfall helped offset the losses. Alfalfa hay was severely lodged under the weight of the ash, but ash-contaminated hay is apparently nontoxic when eaten by livestock. The ash as an abrasive is lethal to certain insects, such as bees and grasshoppers, but populations are recovering. The ash has increased crop production costs by necessitating machinery repairs and increased tillage. On soil, the ash reduces water infiltration, increases surface albedo, and may continue to affect water runoff, erosion, evaporation, and soil temperature even when tilled into the soil. Ash on plant leaves reduced photosynthesis by up to 90 percent. Most plants have tended to shed the ash. With the possible exception of sulfur, the elements in the ash are either unavailable or present in very low concentrations; and no significant contribution to the nutrient status of soils is expected.

Goftte: A R package for assessing goodness-of-fit in proportional (sub) distributions hazards regression models.

BACKGROUND AND OBJECTIVE: In this paper, we introduce a new R package goftte for goodness-of-fit assessment based on cumulative sums of model residuals useful for checking key assumptions in the Cox regression and Fine and Gray regression models. METHODS: Monte-Carlo methods are used to approximate the null distribution of cumulative sums of model residuals. To limit the computational burden, the main routines used to approximate the null distributions are implemented in a parallel C++ programming environment. Numerical studies are carried out to evaluate the empirical type I error rates of the different testing procedures. The package and the documentation are available to users from CRAN R repositories. RESULTS: Results from simulation studies suggested that all statistical tests implemented in goftte yielded excellent control of the type I error rate even with modest sample sizes with high censoring rates. CONCLUSIONS: As compared to other R packages goftte provides new useful method for testing functionals, such as Anderson-Darling type test statistics for checking assumptions about proportional (sub-) distribution hazards. Approximations for the null distributions of test statistics have been validated through simulation experiments. Future releases will provide similar tools for checking model assumptions in multiplicative intensity models for recurrent data. The package may help to spread the use of recent advocated goodness-of-fit techniques in semiparametric regression for time-to-event data.

Single wavelength micro-endoscopy in non-surgical vascular lesion diagnosis & characterization.

Malignant and dysplastic epithelial lesions have often been reported to excite vascular responses by histopathological characterization. Little is reported concerning in vivo real-time imaging of vascular patterns and flow in health or disease but the development of miniature imaging instrumentation has now allowed such developments. We describe the application of a selective wavelength (540 nm) epi-illumination Hopkins pattern endoscopic imaging system to image vascular tissues and capillary blood flow in vivo. The contrast mechanism in such imaging was characterized, haemoglobin acting as a chromatic transmission filter despite endoscopy being a non-invasive and therefore principally reflection mode imaging system. In vivo adrenergic vascular responses, capillary flow rate variations over time and variations in normal capillary architecture around the oral cavity were recorded; demonstrating that simple imaging systems can be used for non-surgical diagnosis and characterization of vascular lesions, tumours and treatment responses.

Reproductive health and public health ethics.

Individuals' reproductive choices are private matters, but sexual conduct and pregnancy impose significant public health burdens. Ethical principles of public health are distinguishable from principles applied in modern bioethics. Bioethical principles have been developed at the clinical or microethical level, affecting relations among individuals, whereas pubic health ethics applies at the population-based or macroethical level. Resolution of issues, for instance of consent to healthcare interventions and preservation of privacy, is different in public health practice from in clinical medicine. Public health aspects of human reproduction concern reduction of maternal mortality and morbidity, particularly in resource-poor countries, and the contribution to high rates of each of unsafe abortion, most prevalent where abortion laws are restrictive. Further aspects of public health ethics concern limited access to contraceptive services, the spread of sexually transmitted infections (STIs), including HIV, causes of infertility, especially due to STIs, and responses to each of these concerns.

Acquiring human embryos for stem-cell research.

Human tissue engineering and regenerative medicine may be considerably advanced by embryonic stem-cell research and cell line development, to provide preventive means, cures and treatment strategies for a range of debilitating conditions and injuries. Research may result in embryos from which stem-cells are derived losing viability, which offends some religious convictions. The different status religions and laws may attribute to embryos serves different purposes and results from different approaches. Neither need depend on, nor impose itself on, the other. Embryos surplus to IVF patients' needs may be donated to research with appropriate consent. In some circumstances, it may be ethical to ask patients to make their fresh embryos available for research. Prohibitions against deliberately creating embryos for research purposes are common, but not universally adopted, and are being challenged. Women who donate ova require information about risks, which for women considering donation for research may not be balanced by compensating benefits.

Respecting adolescents' confidentiality and reproductive and sexual choices.

Adolescents, defined as between 10 and 19 years old, present a growing challenge to reproductive health. Adolescent sexual intercourse contributes to worldwide burdens of unplanned pregnancy, abortion, spread of sexually transmitted infections (STIs), including HIV, and maternal mortality and morbidity. A barrier to contraceptive care and termination of adolescent pregnancy is the belief that in law minors intellectually mature enough to give consent also require consent of, or at least prior information to, their parental guardians. Adolescents may avoid parental disclosure by forgoing desirable reproductive health care. Recent judicial decisions, however, give effect to internationally established human rights to confidentiality, for instance under the Convention on the Rights of the Child, which apply without a minimum age. These judgments contribute to modern legal recognition that sufficiently mature adolescents can decide not only to request care for contraception, abortion and STIs, but also whether and when their parents should be informed.

Achieving transparency in implementing abortion laws.

National and international courts and tribunals are increasingly ruling that although states may aim to deter unlawful abortion by criminal penalties, they bear a parallel duty to inform physicians and patients of when abortion is lawful. The fear is that women are unjustly denied safe medical procedures to which they are legally entitled, because without such information physicians are deterred from involvement. With particular attention to the European Court of Human Rights, the UN Human Rights Committee, the Constitutional Court of Colombia, the Northern Ireland Court of Appeal, and the US Supreme Court, decisions are explained that show the responsibility of states to make rights to legal abortion transparent. Litigants are persuading judges to apply rights to reproductive health and human rights to require states' explanations of when abortion is lawful, and governments are increasingly inspired to publicize regulations or guidelines on when abortion will attract neither police nor prosecutors' scrutiny.

Statistical methods for incomplete data: Some results on model misspecification.

Inverse probability weighted estimating equations and multiple imputation are two of the most studied frameworks for dealing with incomplete data in clinical and epidemiological research. We examine the limiting behaviour of estimators arising from inverse probability weighted estimating equations, augmented inverse probability weighted estimating equations and multiple imputation when the requisite auxiliary models are misspecified. We compute limiting values for settings involving binary responses and covariates and illustrate the effects of model misspecification using simulations based on data from a breast cancer clinical trial. We demonstrate that, even when both auxiliary models are misspecified, the asymptotic biases of double-robust augmented inverse probability weighted estimators are often smaller than the asymptotic biases of estimators arising from complete-case analyses, inverse probability weighting or multiple imputation. We further demonstrate that use of inverse probability weighting or multiple imputation with slightly misspecified auxiliary models can actually result in greater asymptotic bias than the use of naïve, complete case analyses. These asymptotic results are shown to be consistent with empirical results from simulation studies.

In-vivo imaging of the microvasculature of the soft tissue margins of osteonecrotic jaw lesions.

Introduction Given the increasing incidence of medication-related jaw osteonecrosis, and recognition of the mucosal blood supply's importance, we have developed a non-invasive Real Time Optical Vascular Imaging (RTOVI) instrument. Imaging the red blood cells within the sub-mucosal capillary networks demonstrates the microcirculatory anatomy. We report a small trial, demonstrating the technique's viability, examining mucosal microcirculatory changes adjacent to osteonecrotic lesions.Aims Imaging the microvasculature of soft tissue margins of patients' exposed necrotic bone lesions in situ was intended to provide unique observational as well as quantitative data, using an image analysis routine, based on ImageJ software. Our interest was to evaluate whether this could offer valuable information for complex wound margin management.Methods Four osteoradionecrosis and four medication-related osteonecrosis patients (M:F 1:1 mean 68.25 years) were enrolled under the NRES Ethics 11/LON/0354 and KCL Research Ethics Committee (REC) BDM/14/15-14 approvals. Microvascular images from mucosal margins of exposed mandibular osteonecrosis lesions were compared with equivalent images from both uninvolved contralateral mucosa and similar mucosal sites in four healthy subjects.Results We demonstrated narrow hypo-vascularised oedematous lesion margins surrounded by a concentric inflammatory band and normal mucosa beyond. Parameters reporting individual capillary shape, via mean percentage of occupancy per capillary per field of view and capillary loop aspect ratio, differed significantly between groups (ANOVA, p = 0.0002 and p = 0.04 respectively). Values reporting capillary number and area showed expected changes but did not reach statistical significance.Conclusion This pilot study demonstrated the feasibility of mucosal microvascular imaging in assessing the microvascular changes found in the soft tissues at the margins of osteonecrotic lesions, with potential to inform therapeutic interventions and clinical decisions to continue or modify regime strategies at the earliest opportunity. Given the increasing incidence of medication-related jaw osteonecrosis, and the recognition of the importance of mucosal blood supply, we developed a non-invasive instrument demonstrating microcirculation anatomy by imaging transiting red blood cells.

Rapid ex vivo examination of Mohs specimens using optical coherence tomography.

BACKGROUND: Mohs micrographic surgery (MMS) is an effective treatment for certain non-melanoma skin cancers. Optical coherence tomography (OCT) is a biomedical imaging modality that permits high-resolution imaging of the epidermis and dermis with the potential to detect both healthy tissue and tumour. OCT may also provide a means of detecting and differentiating between the various histological subtypes of basal cell carcinomas (BCC) in vivo. OBJECTIVE: The aim of this prospective ex vivo study was to evaluate the efficacy of OCT in recognising healthy and pathological margins of excised BCC lesions and detecting different BCC subtypes. METHODS: Seventy-three subjects with biopsy-proven BCCs on the facial region undergoing MMS were recruited. Narrow clinically healthy margins of the skin surrounding the tumour were included in the excisional biopsy. Biopsies were scanned with the OCT instrument immediately ex vivo and processed to obtain horizontal Mohs frozen sections and compared with their corresponding OCT images. RESULTS: Histopathological analysis of 280 margins showed 232 tumour free margins and 48 tumour-involved margins. OCT showed very good sensitivity (81.2%) and specificity (94.3%) in detecting healthy from tumour-involved margins. OCT accuracy was 93.4%, and the intra- and inter-observer reliability was substantial (Kappa value ranged between 0.63-0.76). CONCLUSION: This study shows the accuracy of ex vivo OCT in identifying the margin status of BCCs of the head and neck region. Moreover, this modality has demonstrated good capability in distinguishing different BCC subtypes and the potential for in vivo in situ diagnostics.

Rapid Bacterial Detection during Endodontic Treatment.

Bacteria present in the root canal (RC) space following an RC treatment (RCT) can lead to persistent infections, resulting in treatment failure and the need for reintervention or extraction. Currently, there are no standardized methods in use to clinically detect bacterial presence within RC spaces. The use of paper point sampling and fluorescence staining was shown to be a rapid method, able to detect residual bacteria following treatment. The study demonstrated that Calcein acetoxymethyl (AM) proved to be a suitable dye for detecting vital bacteria within mature endodontic biofilms, with an improved sensitivity over colony-forming unit counting in a stressed biofilm model. Furthermore, in a clinical trial with primary RCTs, 53 infected teeth were sampled in vivo, and increased detection of vital cells was found when compared with colony-forming unit counting, highlighting the sensitivity of the technique in detecting low cell numbers. By combining fluorescent staining and microspectroscopy with software-based spectral analysis, successful detection of vital cells from RCs was possible after 5 min of Calcein AM incubation. Application of this technology during RCT has the potential to reduce persistent infections through vital cell detection and additional treatment. Furthermore, this technique could be applied to antimicrobial research and disinfection control in clinical settings ( ClinicalTrials.gov NCT03055975).

Conflict of interest: legal and ethical aspects.

Conflicts of interest arise when those who owe conscientious duties to others appear to have personal interests that might tempt them to subordinate those duties to their self-interest. Conflicts of interest are distinguishable from conflicts of commitment, which arise when individuals bear two or more mutually exclusive duties to others. If persons in conflicts of interest favor their self-interest, they may violate binding legal duties such as fiduciary duties. Conflicts arise when, for instance, medical practitioners refer their patients to other practitioners in exchange for payments (fee splitting), and when they gain secondary incomes such as from testing patients' samples in laboratories in which they hold ownership or investment interests. Financial dealings with commercial sponsors may place researchers in apparent conflicts of interest with research subjects. When conflicts of interest are unavoidable, they may be resolved by appropriate disclosure.