Clinical Performance Of Fluorescence- Based Methods For .
Original ResearchCariologyClinical performance of fluorescencebased methods for detection of occlusalcaries lesions in primary teethLaura Regina Antunes PONTES(a)Tatiane Fernandes NOVAES(b)Bruna Lorena Pereira MORO(a)Mariana Minatel BRAGA(a)Fausto Medeiros MENDES(a)(a)Universidade de São Paulo – USP, School ofDentistry, Department of Orthodontics andPediatric Dentistry, São Paulo, SP, Brazil.(b)Universidade Cruzeiro do Sul, School ofDentistry, São Paulo, SP, Brazil.Declaration of Interest: The authors certifythat they have no commercial or associativeinterest that represents a conflict of interest inconnection with the manuscript.Corresponding Author:Fausto Medeiros MendesE-mail: R-2017.vol31.0091Submitted: May 11, 2017Accepted for publication: Aug 28, 2017Last revision: Sep 25, 2017Abstract: We aimed to investigate the performance of fluorescencebased methods (FBMs), compared to visual inspection after histologicalvalidation, in detecting and assessing the activity status of occlusal cariouslesions in primary teeth. One examiner evaluated 50 primary molarsclose to exfoliation in 24 children. Teeth were assessed using quantitativelight-induced fluorescence (QLF) and pen-type laser fluorescence(LFpen). After exfoliation, histological validation was performed. Teethwere cut and sections were evaluated for lesion depth and activity status(after utilization of a pH indicator) under a stereomicroscope. Parametersrelated to the performance of the methods in detecting caries lesions attwo thresholds (initial and dentin lesions) were calculated. Regardingthe activity status, lesions were classified into sound inactive or active,and the area under the ROC curve and the diagnostic odds ratio valuesof the methods were calculated and compared. Evaluation of redfluorescence using QLF presented higher sensitivity but lower specificitythan visual inspection in detecting dentin caries lesions. However, QLFconsidering different parameters and LFpen had similar performanceto that obtained with visual inspection. Regarding activity assessment,all FBMs and visual inspection also presented similar performance.In conclusion, FBMs did not prove advantageous for the detection andactivity assessment of occlusal caries lesions in primary molars whencompared to visual inspection.Keywords: Dental Caries; Fluorescence; Diagnosis, Oral; Dental CariesActivity Tests; Tooth, Deciduous.IntroductionAlthough visual inspection is the most widely used method for thedetection and activity assessment of caries lesions,1,2 it is subjective3 and itcould be influenced by the examiners’ experience.4 An attempt to overcomethese limitations is the creation of detailed visual indices, such as theInternational Caries Detection and Assessment System (ICDAS).5 Actually,the use of detailed and validated indices improves the performance ofvisual inspection for the detection of carious lesions.2 Another advantageof visual inspection is that it permits the evaluation of activity status ofcarious lesions through validated additional criteria.6 However, evenwhen well-described criteria are used, the method is examiner-dependent.Braz. Oral Res. 2017;31:e911
Clinical performance of f luorescence-based methods for detection of occlusal caries lesions in primary teethTherefore, objective and quantitative tools, suchas fluorescence-based methods (FBMs), have beenincreasingly researched for these purposes.3,7,8One of these FBMs is diode laser fluorescence (LF),the first method launched in the market to detectocclusal carious lesions.7,9 After that, a pen-typelaser fluorescence (LFpen) device was introducedfor the detection of carious lesions on both occlusaland approximal surfaces.8 This device is based on adiode laser emitting a red light with a wavelengthof approximately 655 nm, at which the dental tissueemits fluorescence, indicated on a numerical scale from0 to 99. Higher values indicate more severe cariouslesions.9 Even though these methods have been goodat detecting occlusal and approximal caries lesions,8they have not outperformed visual inspection.8 10Another FBM is quantitative light-inducedfluorescence (QLF), in which a blue light is emitted,capturing the back-scattered fluorescence in thegreen and red spectra.11 Loss of green fluorescence isobserved in initial enamel carious lesions, and the QLFdevice translates this fluorescence into a quantitativevalue. Therefore, this method is appropriate fordetecting the early signs of carious lesions and formonitoring the arrestment of these lesions afternon-operative treatments, mainly on free smoothsurfaces.3,8 However, studies to investigate the accuracyof QLF in detecting occlusal carious lesions havebeen unsatisfactory for permanent teeth,12,13 and noprevious studies were conducted with primary teeth.Moreover, the accuracy of QLF in detecting occlusalcaries lesions using histological evaluation has notbeen investigated as reference standard.Furthermore, QLF is capable of detecting redfluorescence emitted by caries lesions, which isprobably related to bacterial metabolic by-products.14,15Thus, assessment of red fluorescence could be anadjunctive method for caries activity assessment, sinceactive lesions are more infected than inactive ones.16,17To the best of our knowledge, however, no previousstudy was carried out to evaluate the accuracy ofQLF in detecting occlusal caries lesions, nor for theassessment of caries activity status in primary teeth.Therefore, the objective of this clinical study withhistological validation of the whole sample was toevaluate the accuracy of QLF and other methods in2Braz. Oral Res. 2017;31:e91detecting severity and assessing the activity status ofocclusal carious lesions in primary teeth, comparingFBMs with the visual inspection performed with ICDAS.The working hypothesis is that some of these FBMsare better at detecting the severity or assessing theactivity status of caries lesions than visual inspection.MethodologyEthical concerns and samplingThe present study was approved by the localResearch Ethics Committee (process number 203/2008).Guardians of the children who participated in thestudy signed an informed consent form prior tothe study. This study used a sample from anotherrecently published study.18 Children participatingin this previous study who had at least one primarymolar close to exfoliation were selected to participatein the present study.For the first study,18 children who sought dentaltreatment in our dental school were randomly selectedand invited to participate in the study throughenrolment forms with no details on their oral healthstatus. In the first study, 113 children aged 4 to 14 yearswere examined.18 Children who verbally agreed totheir participation and whose parents signed theconsent form were screened to check whether theyhad at least one primary molar close to exfoliation.Moreover, those eligible primary molars shouldnot have enamel defects, restorations, sealants,or frankly cavitated lesions. Then, one suspect site(non-evident caries lesions) of each tooth included waschosen and marked on a drawing to indicate furtherevaluations. Therefore, we included apparently soundocclusal surfaces, surfaces with probable non-cavitatedcaries lesions, or microcavitated lesions, which werenon-obvious in the first evaluation. This choice wasmade by an independent examiner who did notparticipate in the subsequent assessments (LRAP).More than one tooth per children could be selected.Clinical examinationPrior to the examinations, the participants werepositioned in a dental unit under operating light andsubjected to cleaning procedures using a rotating bristlebrush and water. No prophylactic pastes were used to
Pontes LRA, Novaes TF, Moro BLP, Braga MM, Mendes FMavoid possible influence on measurements made byFBMs. Then, one trained examiner (TFN) carried outall examinations using the different methods.The teeth were first assessed through visualinspection. The examination was performed incleaned teeth, under illumination, with the aid ofplane mouth mirrors and WHO periodontal probes.The drawing with the suspect site was shown to theexaminer to guide the evaluation. The teeth wereexamined wet and were then dried for 5 s using a3-in-1 syringe. The examiner evaluated the pre-selectedsite for the presence of lesions using the ICDAS.19For activity assessment, the criteria described inthe reference manual of the ICDAS,19 which is basedon a previously described and validated system,20,21were used. The examiner was instructed to classifythe site according to the ICDAS scores, and in thepresence of any type of carious lesion (scores from1 to 6), the lesion was classified as active or inactive.The ICDAS and additional criteria used for activityassessment are described in Table 1.Fluorescence-based methodsAfter the visual inspection, the LFpen methodwas used. This method was carried out using theDIAGNOdent pen (Kavo, Biberach, Germany),according to the manufacturer’s instructions. Tip 2,appropriate for occlusal surfaces, was used. Thedevice was first calibrated using the ceramic standardprovided by the manufacturer, and also on a soundsmooth surface of the tooth under evaluation. Thisvalue was electronically subtracted from thoseobtained from the examined site. After drying thetooth for 3 s with a 3-in-1 syringe, the tip was placedvertically and then rotated around the verticalaxis. The examiner made two assessments on eachsuspicious site and recorded the mean value.After that, the same examiner performed theevaluations with another FBM. The assessmentswere made with the QLF device (QLF InspektorPro; Inspektor Research Systems, Amsterdam,Netherlands), and the images were analyzed usinga software program (Inspektor Pro Software, InspektorResearch Systems, Amsterdam, Netherlands). Theimages were taken from the entire occlusal surfaceafter drying with cotton pellets. Then, the markedarea corresponding to the suspect site was manuallyselected and the images were assessed by the software.A sound region of the surface was defined as referencefor the measurements. We recorded three differentparameters provided by the QLF system:a. Red fluorescence of the lesion ( R%): percentageof ratio of red fluorescence compared with ratioof sound tissue;b. Green fluorescence of the lesion ( F%):percentage of green fluorescence loss onthe suspicious site compared with greenfluorescence from sound tooth tissue;Table 1. International Caries Detection and Assessment System (ICDAS)5 and additional criteria used in the caries activity assessment.19,20,21ScoresICDASClinical features of active carious lesionClinical features of inactive caries lesion0No or slight change in enamel translucencyafter prolonged air drying (5 s)--1First visual change in enamel (seen onlyafter prolonged air drying or restricted towithin the confines of a pit or fissure2Distinct visual changes in enamelLocalized enamel breakdown inopaque or discoloured enamel (withoutvisual signs of dentinal involvement)Surface of enamel is whitish, brownish orblack. Enamel may be shiny and feels hardand smooth when the tip of the probe ismoved gently across the surface.3Surface of enamel is whitish/yellowishopaque with loss of luster; feels roughwhen the tip of the probe is moved gentlyacross the surface. Lesion is in a plaquestagnation area.4Underlying dark shadow from dentineProbably active-5Distinct cavity with visible dentin6Extensive distinct cavity with visible dentine(involving more than half of the surface)Cavity feels soft or leathery on gentlyprobing the dentine.Cavity may be shiny and feels hard ongently probing the dentine.Braz. Oral Res. 2017;31:e913
Clinical performance of f luorescence-based methods for detection of occlusal caries lesions in primary teethc. Green fluorescence loss integrated over thelesion area ( Q%.mm2): percentage of greenfluorescence loss in the lesion integrated overthe lesion area.Reference standardSubsequently, teeth exfoliated or they wereextracted (for orthodontic purposes) within 3 weeksafter the end of the clinical recordings. The teeth werethen stored at -20 C for up to 1 month.22 Thereafter,the teeth were sectioned from the sites marked on thedrawing using a 0.3-mm-thick diamond saw mountedin a cutting machine (Isomet 5000 Linear PrecisionSaw, Buehler, Lake Bluff, USA). The histologicalevaluation after sectioning was the reference standardof the present study. After that, the sections werefirst evaluated under a stereomicroscope at X8–20magnification and reflected light (Leica DM 750, LeicaMicrosystems, Heidelberg, Germany). Immediatelyafterwards, two different examiners (LRAP and FMM),unaware of the results obtained with the methods,evaluated the sections for lesion depth. The lesionswere classified according to a 5-point scale: D0 nocaries lesions, D1 caries lesion limited to the outerhalf of the enamel, D2 caries lesion reaching theinner half of the enamel; D3 outer half dentin carieslesion and, D4 dentin caries lesion reaching theinner half of the dentin.Immediately thereafter, a 0.1% methyl red solution(Aldrich, Milwaukee, USA) was dripped onto thesections. After 1 min, the excess dye was removed with afilter paper and the sections were evaluated as describedpreviously. The examiners then classified the lesionsinto inactive, when the sections were stained yellow,or active, when the lesions appeared red or reddish.22, 23After some training using images from other studies, theexaminers performed the evaluations independently,and in case of disagreements, a consensus was reachedafter a discussion between them.Statistical analysisRegarding histological examinations, interexaminerreproducibility before the consensus between theexaminers was evaluated using weighted kappa forboth lesion depth and activity assessment.4Braz. Oral Res. 2017;31:e91As to lesion depth, receiver operating characteristics(ROC) analyses were carried out to evaluate thevalidity of the FBMs for detecting carious lesions attwo different thresholds: all lesions (D0 sound vs. D1,D2, D3 and D4 carious), and dentin carious lesions(D0, D1 and D2 sound vs. D3 and D4 carious).The best cutoff points were determined (highest sumof specificity and sensitivity rates) for each methodat both thresholds, as well as the areas under theROC curves (Az).With these cutoff points, sensitivity, specificity, andaccuracy (percentage of right diagnosis, consideringsound and carious surfaces) were calculated at boththresholds. The same parameters were also calculatedfor visual inspection considering lesions higher thanICDAS score 0 as cutoff point at all lesion thresholdsand higher than score 2 for the dentin lesion threshold.The values obtained for each method were comparedby McNemar’s test.With regard to activity assessment, the performanceof FBMs was calculated in terms of diagnostic oddsratio (DOR) using logistic regression and Az values,considering the values obtained for each method.By using the best cutoff point calculated in the ROCanalysis, results were dichotomized into sound inactivevs. active, and DOR and accuracy were calculated. Thecluster nature of the data (more than 1 tooth per child)was taken into account. The level of significance was setat 5%, and MedCalc 13.1.2.0 (MedCalc software bvba,Ostend, Belgium) and Stata 13.0 (StataCorp LP, CollegeStation, USA) were used for the analyses.ResultsOur sample included 50 occlusal primary teethfrom 24 children (12 male and 12 female) aged 8 to 12years (mean 10.2; standard deviation 1.1 years).From the 50 teeth, histological examinationrevealed that only four teeth were sound (D0). Thedistribution of the caries lesions in terms of lesiondepth is shown in Table 2. As to the reproducibility ofthe histological depth evaluation obtained before theconsensus, the weighted kappa value was 0.915 (95%confidence interval – 95%CI 0.824 to 1.000). From46 teeth with lesions, 16 with enamel lesions and 9with dentin caries lesions were classified as active
Pontes LRA, Novaes TF, Moro BLP, Braga MM, Mendes FMafter the utilization of methyl red (a total of 25 activecarious lesions) and 20 enamel lesions and 1 dentincarious lesion were classified as inactive (21 inactivecaries lesions). Interexaminer reliability in assessingthe activity status after the use of the dye yieldeda weighted kappa of 0.800 (95%CI 0.634 to 0.966).Regarding the presence of caries lesions, at D1threshold, visual inspection presented higher sensitivity,but with no statistically significant differences comparedTable 2. Distribution among scores obtained with theInternational Caries Detection and Assessment System (ICDAS)and caries lesions activity status and the lesions depth evaluatedthrough histological activeActiveLesion 5101412248250Total(8.0%) (24.0%) (48.0%) (16.0%) (4.0%) (100.0%)D0: sound; D1: initial enamel caries lesions; D2: advanced enamelcaries lesions; D3: initial dentin caries lesions; D4: advanced dentincaries lesions111with the sensitivities obtained with LFpen and QLFconsidering R and Q. The F obtained with QLFpresented statistically significant lower sensitivity(Table 3). Both specificity and Az yielded similarresults. However, regarding accuracy, visual inspectionshowed statistically significant and higher values thanthe R and F obtained with QLF (Table 3).At the dentin threshold, although visual inspectionand LFpen methods presented lower values, therewere no statistically significant differences amongthe methods. On the other hand, specificity wassignificantly different from that obtained with QLFconsidering F. The other parameters obtained withQLF ( R and Q) and LFpen presented intermediatespecificity values, with no statistical differences(Table 3). Concerning accuracy and Az, all methodsshowed similar values (Table 3).When we compared the methods regardingcaries activity assessment, there were no statisticallysignificant differences among them. In the analysesmade considering the values obtained with FBMs(quantitative variables), Az values ranged from0.67 to 0.77 (Table 4). When we dichotomized thevariables considering the best cutoff points calculatedthrough ROC analysis, accuracy was around 0.72 forall methods. DOR ranged from 6.43 (obtained withQLF, F) to 12.74 (with LFpen). However, there wereno significant differences since the 95%CI valuesoverlapped (Table 4).Table 3. Performance of methods for detecting occlusal caries lesions at different thresholds regarding the lesion depth.MethodsCut-off pointsSensitivitySpecificityAccuracyAzInitial enamel caries lesions (D1)Visual inspection 00.870 a0.500 a0.840 a0.766 aLFpen 40.848 a0.500 a0.820 a, b0.649 aQLFRed fluorescence ( R) 21.90.717 a, b0.750 a0.720 b, d0.668 aGreen fluorescence loss ( F) 9.30.565 b0.750 a0.580 c, d0.590 aaaa, bGreen fluorescence loss X lesion size ( Q) 2.10.8260.5000.8000.571 aDentin caries lesions (D3)Visual inspection 20.600 a0.950 a0.880 a0.871 aaa, baLFpen 300.6000.8250.6720.674 aQLFRed fluorescence ( R) 31.30.900 a0.825 a, b0.840 a0.870 aaa, baGreen fluorescence loss ( F) 16.70.7000.8750.8400.780 aabaGreen fluorescence loss X lesion size ( Q) 13.80.7000.7750.7600.745 aAz: area under receiver operating characteristics curve. LFpen: pen-type laser fluorescence method; QLF: quantitative light-induced fluorescencemethod. Different letters indicate statistically significant differences among figures within the same column at the same threshold (p 0.05).Braz. Oral Res. 2017;31:e915
Clinical performance of f luorescence-based methods for detection of occlusal caries lesions in primary teethTable 4. Performance of visual inspection and fluorescence-based methods in assessing activity of occlusal caries lesions in primary teethAnalysis considering quantitative valuesMethodsVisual inspectionLFpenDOR(95
Clinical performance of fluorescence-based methods for detection of occlusal caries lesions in primary teeth Abstract: We aimed to investigate the performance of fluorescence-based methods (FBMs), compared to visual inspection after histological validation, in detecting and assessing the activity status of occlusal carious lesions in primary teeth.
Specialized Fluorescence Techniques 171 Single Molecule Fluorescence 172 Fluorescence Correlation Spectroscopy 173 Forster Resonance Energy Transfer 173 Imaging and Super-Resolution Imaging (Con-ventional and Lifetime) 174 Instrumentation and Laser Based Fluorescence Techniques 175 Nonlinear Emission Processes in Fluorescence Spectroscopy 176
Practical fluorescence microscopy 37 4.1 Bright-field versus fluorescence microscopy 37 4.2 Epi-illumination fluorescence microscopy 37 4.3 Basic equipment and supplies for epi-illumination fluorescence . microscopy. This manual provides basic information on fluorescence microscopy
observation to a fluorescence-based experiment. This added dimension can provideinformation on the local environment, fluorescence lifetime and molecular mass. A variety of instruments are utilized in fluorescence polarization studies. These instruments are based on the design of existing fluorescence spectroscopy or microscopy techniques.
P.R. Selvin (2000) The renaissance of fluorescence resonance energy transfer. Nat Struct Biol.7:730-4. P.R. Selvin (1995) Fluorescence resonance energy transfer. Meth Enzymol246:300-334. J.R. Lakowicz (2006) Principles of Fluorescence Spectroscopy, 3rd edn. Springer. Olympus Resource Center: Fluorescence resonance energy transfer
fiber-based fluorescence imaging was mainly limited to epifluorescence and scanning confocal modalities. Two new classes of photonic crystal fiber facilitate ultrashort pulse delivery for fiber-optic two-photon fluorescence imaging. An upcoming generation of fluorescence imaging devices will be based on mic rofabricated device components.
one or more resonance fluorescence series of the Na2 (X l!:'u _B lllu) blue-green band system. Altogether, 19 different fluorescence progressions have been identified and assigned v, J quantum numbers. The absolute . I. INTRODUCTION Laser-induced fluorescence of moleculesl ,2 is a recent
Introduction - Optical resolution - Optical sectioning with a laser scanning confocal microscope - Confocal fluorescence imaging Stimulated emission depletion (STED) microscopy Fluorescence resonance energy transfer (FRET) Fluorescence lifetime imaging Two photon excitation microscopy Conclusion @Physics, IIT Guwahati Page 3
Nutrition and Food Science [CODE] SPECIMEN PAPER Assessment Unit A2 1 assessing. 21 Option A: Food Security and Sustainability or Option B: Food Safety and Quality. 22 Option A: Food Security and Sustainability Quality of written communication will be assessed in all questions. Section A Answer the one question in this section. 1 (a) Outline the arguments that could be used to convince .
