Comparison Between Radiographs, White and Fluorescent Images in the Diagnosis and Treatment Decisions for Occlusal Caries: An Ex Vivo Study

ABSTRACT Objective: To compare the agreement of images in white light (WL), fluorescence (FL), and digital radiographs (DR), on the diagnosis and treatment decisions for occlusal caries lesions against a micro-CT gold standard. Material and Methods: Ten extracted third molars, with enamel and/or dentin caries (ICDAS 2-4), were included. Occlusal surface images were acquired with an intraoral camera (SoproLife®) in WL and FL modes. DR was obtained using an intraoral X-ray and a semi-direct digital system. A total of 780 images were needed, organized in a template, to be later examined by twenty-six dentists invited to compose the study. The Generalized Estimation Equations model was used to compare the proportions of the correct answers between the three methods and the gold standard. When significant, Bonferroni post-hoc test was used to identify differences (α=5%). Results: Most of the examiners were specialists (76.9%) with 14.5 years of experience. All diagnostic methods were similar and showed low agreement (DR 12.7%, WL 16.5%, and FL 16.5%) compared with gold standard caries diagnostic scores. Regarding treatment decisions, mean agreement for all diagnostic methods was higher (43.2%; p<0.001), and among all methods, WL (48.1%) and FL (51.2%) modes performed better than DR (30.4%, p<0.001). Conclusion: SoproLife® images could help clinicians to propose rational, minimally invasive treatments for occlusal caries lesions.


Introduction
Diagnosing the occlusal surface is still a challenge for clinicians in practice, but the current accuracy of visual examination in detecting the full extent of the lesions can be considered good if a well-established index is used by trained examiners [1].However, results from another recent systematic review on the subject performed by the same authors pointed out that studies on the accuracy of the visual method for caries detection should consider clinically relevant outcomes [2] since a "correct" diagnosis made by clinicians is not necessarily translated into a direct benefit for patients in terms of more conservative treatment approaches [3].
The gap between an accurate diagnosis and a rational and minimally invasive operative treatment decision should be bridged if patient-centered outcomes are being put into the spotlight of evidence-based dentistry.
Clinical guidelines still recommend intraoral radiographs as an auxiliary method to the visual clinical examination in detecting caries lesions [4,5] with the claim that solely; its use may result in failure to detect some lesions.Therefore, the combination of both visual and radiographic methods would provide greater diagnostic sensibility while keeping good sensitivity [6].However, although a digital radiographic examination is easy to perform and provides images in a few seconds, it has strict indications because of health risk hazards associated with ionizing radiation [7].In this context, it is important to validate and implement diagnostic methods that do not use ionizing radiation sources to minimize exposure to patients and professionals [8,9].Moreover, as mentioned, desirable clinical diagnostic aids should also improve treatment decisions towards more rational and minimally invasive approaches, resulting in a clear benefit for patients [2].
SoproLife ® intraoral camera (Acteon Group Ltd., La Ciotat, France) is a light-induced fluorescence caries diagnostic system based on the auto-fluorescence of dental tissues when illuminated by a wavelength of 450 nanometers [10].It offers two lighting modes: white and fluorescent (blue color).The fluorescent mode is indicated for caries diagnosis, with the colors blue and acid green indicating sound enamel and dentin, while the colors bright red and black green indicate the presence and activity of the lesion, active and inactive, respectively [11].Caries activity assessments are essential to propose rational treatment plans for occlusal caries lesions because, for example, arrested caries generally do not need operative treatment [12].
To measure the sensitivity, specificity, and accuracy of digital diagnostic alternatives for caries, a micro-CT technique has been proposed as a gold standard to validate caries severity under laboratory conditions [13,14], especially because histological methods have low accuracy in detecting the real depth of dentin lesions [15].Thus, the aim of the present ex vivo study was to compare the agreement on caries diagnosis and treatment decisions of occlusal surfaces images of permanent carious teeth obtained by SoproLife ® , in white light and fluorescence mode, and digital radiographs, against a micro-CT gold standard.
The hypothesis tested was that all caries diagnostic methods result in similar agreement when used for detecting/diagnosing lesions or when used to help define treatment decisions.

Ethical Aspects and Study Design
This study was approved by the institution's Research Ethics Committee (#3.442.162).This was an in vitro accuracy study with a cross-sectional design, which followed the recommendations of the Standard for Reporting of Diagnostic Accuracy (STARD) steering committee [16].

Sample Power
Prior to the beginning of this study, a power calculation was performed using WinPepi v.11.65 [17] to estimate the sample size needed to compare the proportion of correct answers given by examiners using three diagnostic methods.For each individual evaluating 30 images of the same teeth (10 images per diagnostic method group), a total of 690 images of teeth would be needed to disclose significant results and thus, 23 individuals would have to be recruited for this study.This calculation would detect a 15% difference between the proportions of the methodologies as significant, with the estimated hit ratio of the image without fluorescence of 84.9% [18] with a power of 90% and a significance level of 5%.Approximately an extra 10% of images were added to this calculation to prevent possible losses, resulting in a total of 780 images of the same teeth, and thus, 26 examiners were needed for the present study.

Tooth Specimens
Initially, 20 third molars presenting enamel and/or dentin caries lesions on the occlusal surface ranging from ICDAS 2-4 [19], previously detected by an experienced and calibrated examiner (MMA), were selected from a biorepository organized for this specific study.Tooth specimens presenting enamel defects other than carious lesions (hypomineralization, hypoplasia, etc) were excluded from the sample.The selected teeth were kept in saline, to avoid dehydration for a maximum of three months before the start of the study [20].Sound, restored or teeth presenting changes not related to carious involvement were not considered for the study.
Ten teeth were randomly drawn from this selection (page: https://sorteador.com.br/) and included in the sample (3 teeth with ICDAS 2, two with ICDAS 3 and five with ICDAS 4).After selection, the specimens were cleaned with ultrasonic tips to remove calculus and debris and were subsequently incorporated by the roots using utility wax on a plastic base to facilitate manipulation.Each specimen was placed in approximal contact with two other sound molars, one on each side, in order to simulate the position of a second permanent molar in the oral cavity, as shown in Figure 1.The 10 tooth specimens sets were kept in a closed container under 100% humidity conditions during the study.The examiners were instructed to score the images of the occlusal surface of each selected tooth in each set (middle tooth) acquired with each method (DR, WL and FL) using a modified dental caries scores (Table 1).These scores included just five simple progressive categories, from sound surfaces to dentin cavitation.After that, and for each tooth specimen image, the examiners should also score one best treatment option for each examined tooth (no specific treatment, preventive treatment, non-operative and operative treatment) based on the most recent and comprehensive guidelines [21] (Table 1).

Selection of Examiners
From a universe of thirty dentists working in a public clinical setting, twenty-six with routine experience of diagnosing carious lesions, but without a specific academic background in the field of Cariology were approached by convenience and were invited to participate in the study.They all provided their written consent before enrolling in the study procedures.Sociodemographic data were collected, such as gender, educational level (specialist, master, and Ph.D.), age and time since graduation.They were individually provided with a 10-minute explanation of how to score the tooth images using the proposed scores (Table 1) and the interpretation of the fluorescent colors.

Gold Standard for Dental Caries and Treatment Options
After the acquisition of the occlusal surface images and radiographs, the teeth were scanned in a highenergy 1173 micro-CT (Bruker Belgium SA, Kontich, Belgium) using the following acquisition parameters: 100kV, 80μA, 6.47μm pixel size, 1mm thick Al filter, 1s exposure, 0.5° rotation step at 360°, and 20 lines random movements.After this, the acquired projections were reconstructed using the NRecon software (version 1.7.0.4,NRecon, Bruker Corporation, Billerica, MA, USA) using standardized parameters: ring artifact correction (10), beam hardening correction (52%), and standardized contrast limits between 0 and 0.05.After reconstruction, the teeth were aligned with the occlusal surface parallel to the ground using a dedicated software (Data Viewer, Bruker Corporation, Billerica, MA, USA) and the slice with the deepest part of the lesion was detected.This slice was classified according to the dental caries scores presented in Table 1 by an experienced operator (LPS), and defined as the gold standard for caries presence in this study (Figure 2).Possible gold standard for treatment options were defined based on the summary of the best available evidence [21].Table 2 shows corresponding matched dental caries and treatment option scores.

Statistical Analysis
Categorical variables related to demographic data of the examiners were represented by absolute and relative frequencies.The variable "age" and "time since graduation" were represented by mean, standard deviation (SD) and amplitude.The Generalized Estimation Equations model (GEE Model) [22,23] was used to compare the proportions of the correct answers between the three methods evaluated by the same examiner.
This analysis was chosen because it considers related samples.The model was composed by an independent work correlation matrix, and a robust estimator covariance matrix.When significant, the Bonferroni post-hoc test was used to identify the different categories.The level of significance adopted was 5%.All analyses were performed in SPSS software, version 25.0 (IBM Corporation, Armonk, NY, USA).

Results
Most of the examiners were female, (n=21; 80.8%), with an average age of 38.4 years.The most prevalent education level was that of specialist (n=20; 76.9%), with a mean of 14.5 years of experience, as shown in Table 3.  Table 4 shows a comparison of the proportions of correct answers (as agreed with the gold standard scores) given by the examiners on the diagnosis of caries at the occlusal surface of the specimens.In general, no difference was found among the three methods (p=0.415), with a general low agreement (mean 15.3%) between the assigned caries scores and the gold standard for caries diagnosis.However, in tooth 6 (ICDAS 4), the evaluators responded better to the images in WL (50% correct answers) compared to DR (15.4%) and FL (19.2%) while in tooth 8 (ICDAS 4), DR underperformed compared to FL, while this was better than WL.As tooth 4 (ICDAS 3) resulted in no correct answers for DR and FL, it was not possible to estimate the p-value.Table 5 shows the comparison of the proportions of correct answers given by the evaluators in relation to treatment decisions proposed for the specimens.The mean percentage of correct answers among the methods was higher for WL (48.1%) and LF (51.2%) compared to DR (30.4%) (p<0.001).The total proportion of correct answers was different among the methods also in teeth 4, 6 and 8 (p<0.05).For tooth 4 (ICDAS 3), the examiners responded better to the images in WL (88.5%) and FL (80.8%) compared to DR (11.5%).For tooth 6 (ICDAS 4), the evaluators responded better to the images in WL (84.6%) compared to FL (53.8%), but without difference from DR (57.7%).As for tooth 8 (ICDAS 4), the evaluators responded better to the images in FL (88.5%) compared to DR (34.6%) and WL (50%).The remaining teeth did not show a more assertive method.

Discussion
The results of the present study confirm that imaging methods alone underperform in the diagnosis of occlusal caries, with a general low mean of correct hits (15.3%) based on the golden standard used.However, regarding treatment options, the image obtained by the intraoral camera in WL (48.1%) or FL (51.2%) resulted in statistically significant higher correct hits than DR alone in establishing an acceptable treatment plan.Thus, these imaging methods have aided the examiners in making rational treatment decisions for occlusal caries based on minimally invasive approaches.
The reasons for the low agreement among the diagnostic methods in relation to the diagnosis of caries are probably related to the definition of lesion severity provided by the golden standard used in the present study (microtomography).Although histological processing and analysis is still used as a gold standard for dental caries detection, it is fundamentally a destructive method because it requires sectioning of the sample for microscopic examination.Therefore, the micro-CT technique used as the golden standard in the present study has been recently proposed and validated to define caries severity under laboratory conditions [13,14,24,25], especially because histological methods have generally low accuracy in detecting the depth of dentin lesions [15].
Evaluation of tooth 4 (ICDAS 3) resulted in no correct answers for caries diagnosis for DR and FL.
This could be explained by the fact that a small dentin demineralization area, very near to the threshold between ICDAS 2 and 3, was detected by the micro-CT.This technique has, indeed an improved accuracy in detecting the actual depth of the lesion [24].However, in line with the general findings of this study, treatment decisions were much better scored using the WL (88.5%) and FL (80.8%) mode, compared to the DR (11.5%) mode (p<0.001).
Tooth 6 (ICDAS 4) resulted in a better agreement for caries diagnosis when WL was used and this was probably because the FL pushed the diagnosis towards the activity of the lesion (inactive -color black).
Another study has, in fact, not been able to confirm the validity of SoproLife ® in determining the actual lesion activity [20].Caries severity and activity are two different criteria and ideally, treatment options should include both.However, in tooth 6, the enamel cavitation evidenced by WL favored the correct treatment decision -operative treatment focused on the cavitation.
Tooth 8 (ICDAS 4) was favored by the FL mode also due to the lesion activity.In this tooth, the red color of the dentin cavitation area would have called the attention of the examiner towards more correct hits for caries diagnosis and treatment decisions.For the other teeth, no statistically significant differences were detected, but in general, FL and WL resulted in higher correct hits for treatment decisions in the present study.
Fluorescent caries diagnostic devices are considered attractive for their simplicity of use, compact size and absence of exposure to radiation [26].By combining the advantages of a fluorescent device with an intraoral camera, SoproLife ® proves to be a non-destructive and clinically applicable diagnostic method to aid in caries detection.In fact, compared to a gold standard visual examination based on ICDAS, sensitivity values of both SoproLife ® imaging modes range from 0.75-0.86while specificity values are around 0.81-0.89[27,28].
Indeed, SoproLife ® has an acceptable accuracy in detecting the presence (sensitivity) and absence (specificity) of lesions [29], but the gold standard caries detection used are based on clinically scored teeth.In the present study, however, no sensitivity/specificity values were calculated.We have rather employed an agreement evaluation using a laboratory and "true" gold standard for caries detection, as mentioned before.
Although the location where the images were presented was the same for all examiners, with the screen calibrated with the same amplitude and brightness, there were still concerns expressed by the examiners regarding the difficulty in detecting occlusal caries through digital images in view of the activity and depth of pigmented grooves.Although most professional evaluators were specialists (n=20; 76%) with experience in caries diagnosis, they reported a lack of intimacy with the digital fluorescent device and the practice of diagnosing and treatment decision for occlusal caries using digital images.A previous study reported that the ability to evaluate intraoral photographs on a screen at a convenient time and place was considered advantageous by a cohort of dentists with experience in epidemiological studies [30] and to facilitate distance diagnosis, such as remote and difficult to access areas [31].
The limitations of this study include the ex vivo conditions in which the evaluations were carried out, that may be different from the in vivo dynamic complex of caries initiation and arrestment.Indeed, it is known that caries activity detection can be facilitated in the clinical situation when characteristics such as biofilm accumulation and gingival inflammation can be used as surrogates to whether the lesion is active or inactive [32].However, it is still possible to detect caries activity by considering some lesion characteristics, such as luster, roughness and staining of the surface [33], and these characteristics were considered in the present study.Moreover, treatment decision gold standards in the present study were broader (more options) due to the nature of the minimally invasive approaches, what could have influenced higher agreement for treatment decisions compared to caries diagnosis.Finally, it is important to mention that the initial implementation costs generally associated with improved imaging detection methods may soon be worth its effectiveness in reducing the cost of care and preventing unnecessary operative treatments for the patient and the healthcare services.

Conclusion
The total proportion of correct answers was greater for all diagnostic methods relative to the treatment decisions compared to the current diagnosis of carious lesion in permanent molars.White-light or fluorescent SoproLife ® images may help clinicians to propose rational minimally invasive treatments for occlusal caries lesions.

Figure 1 .
Figure 1.Example of one sample assembled for photography and radiographic examination, simulating positions in the oral cavity.

Figure 2 .
Figure 2. DR, WL, and FL images of the selected teeth, their respective representative slices, gold standard classification of caries lesions and respective treatments.

Figure 2
Figure2illustrates the teeth included in the study, its respective DR, WL and FL images, and the corresponding micro-CT slice showing the deepest part of the caries lesion.For each tooth, the gold standard dental caries and treatment options scores are also detailed.