High Prevalence of Apical Periodontitis in Patients With Inflammatory Bowel Disease: An Age- and Gender- matched Case-control Study

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Inflammatory Bowel Diseases (2019): 27 June DOI: 10.1093/ibd/izz128 Copyright: © The Author 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is a pre-copyedited, author-produced version of an article accepted for publication in Inflammatory Bowel Diseases following peer review. The version of record Poyato-Borrego M, Segura-Sampedro JJ, Martín-González J, Torres-Domínguez Y, Velasco-Ortega E, Segura-Egea JJ. High Prevalence of Apical Periodontitis in Patients With Inflammatory Bowel Disease: An Ageand Gendermatched Case-control Study. Inflamm Bowel Dis. 2020 Jan 6;26(2):273-279. doi: 10.1093/ibd/izz128. PMID: 31247107. is available online at:https://academic.oup.com/ibdjournal/articleabstract/26/2/273/5524347?redirectedFrom=fulltext, doi.org/10.1093/ibd/izz128.” Inflammatory Bowel Diseases High Prevalence of Apical Periodontitis in Patients with Inflammatory Bowel Disease: An Ageand GenderMatched Case-Control Study --Manuscript Draft-- Manuscript Number: IBD-D-19-00308R2 Article Type: Original Research Articles - Clinical Section/Category: Epidemiology Keywords: apical periodontitis; root canal treatment; Inflammatory Bowel Disease; Crohn's disease; ulcerative colitis. Corresponding Author: Juan J Segura-Egea, MD, DDS, PhD Universidad de Sevilla Facultad de Odontología SPAIN First Author: Manuel Poyato-Borrego, MD Order of Authors: Manuel Poyato-Borrego, MD Juan J. Segura-Sampedro, MD, PhD Jenifer Martín-González, DDS, PhD Yolanda Torres-Domínguez, MD Juan J Segura-Egea, MD, DDS, PhD Eugenio Velasco-Ortega, MD, DDS, PhD Manuscript Region of Origin: SPAIN Abstract: Background: Deep carious lesion causes pulpitis, pulpal necrosis and, finally, apical periodontitis (AP). The elective treatment for AP is root canal treatment (RCT), becoming the tooth into a root-filled tooth (RFT). Several studies have linked AP and RFT to systemic diseases. Likewise, previous studies have also found an association between inflammatory bowel disease (IBD) and periodontal disease. The aim of this study was to analyze the prevalence of AP and RCT in patients with IBD and in control subjects. Methods: An ageand gendermatched case-control study design was used. The study group (SG) included 54 IBD patients (28 with Crohn´s disease, 26 with ulcerative colitis). Another 54 healthy subjects, without IBD and ageand gendermatched, were included in the control group (CG). The radiographic records were analyzed and AP was diagnosed as radiolucent periapical lesions (RPLs), using the periapical index score (PAI). Student’s t test, 2 test and multivariate logistic regression were used in the statistical analysis. Results: The presence of one or more tooth with RPL was found in 19 patients (35.2%) in the SG and in 9 subjects (16.7%) in the CG (p = 0.03). No differences were found between the two groups neither in the number of teeth with AP nor in the number of RFT (p > 0.05). However, multivariate logistic regression analysis adjusting for number of teeth and number of RFT showed that subjects with IBD have RPL with higher likelihood than control subjects (OR = 5.7; C.I. 95% = 1.7 – 19.1; p = 0.0048). Conclusions: Patients with ulcerative colitis or Crohn´s disease have higher prevalence of apical periodontitis. An oral health protocol should be established to address the higher prevalence of inflammatory oral processes. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation High Prevalence of Apical Periodontitis in Patients with Inflammatory Bowel Disease: An Ageand GenderMatched Case-Control Study Manuel Poyato-Borrego, MD1, Juan J. Segura-Sampedro, MD, PhD2, Jenifer Martín-González, DDS, PhD3, Yolanda Torres-Domínguez, MD4, Eugenio Velasco-Ortega, MD, DDS, PhD3*, Juan J. Segura-Egea, MD, DDS, PhD3*, 1Internal Medicine Unit. Hospital San Juan de Dios del Aljarafe. Sevilla, Spain. 2General & Digestive Surgery Unit. Hospital Universitario Son Espases. School of Medicine, University of Balearic Islands. Health Research Institute of Balearic Islands. Palma de Mallorca, Spain. 3Department of Stomatology, School of Dentistry, University of Sevilla, C/ Avicena s/n, 41009-Sevilla, Spain. 4Digestive Unit. Hospital San Juan de Dios del Aljarafe. Sevilla, Spain. Correspondence*: Prof. Juan J. Segura-Egea and Prof. Eugenio Velasco Ortega Facultad de Odontología, C/ Avicena s/n 41009-Sevilla (SPAIN) E-mail: [email protected]; [email protected] Phone number: 0034 954 481146. SOURCES AND SUPPORT The authors deny any conflicts of interest. SUMMARY Results of this ageand gendermatched case-control study show that the prevalence of apical periodontitis, diagnosed as radiolucent periapical lesions, is higher in patients with inflammatory bowel disease than in healthy control subjects (OR = 5.71; p = 0.0048). Main Document 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 ABSTRACT Background: Deep carious lesion causes pulpitis, pulpal necrosis and, finally, apical periodontitis (AP). The elective treatment for AP is root canal treatment (RCT), becoming the tooth into a root-filled tooth (RFT). Several studies have linked AP and RFT to systemic diseases. Likewise, previous studies have also found an association between inflammatory bowel disease (IBD) and periodontal disease. The aim of this study was to analyze the prevalence of AP and RCT in patients with IBD and in control subjects. Methods: An ageand gendermatched case-control study design was used. The study group (SG) included 54 IBD patients (28 with Crohn´s disease, 26 with ulcerative colitis). Another 54 healthy subjects, without IBD and ageand gendermatched, were included in the control group (CG). The radiographic records were analyzed and AP was diagnosed as radiolucent periapical lesions (RPLs), using the periapical index score (PAI). Student’s t test, 2 test and multivariate logistic regression were used in the statistical analysis. Results: The presence of one or more tooth with RPL was found in 19 patients (35.2%) in the SG and in 9 subjects (16.7%) in the CG (p = 0.03). No differences were found between the two groups neither in the number of teeth with AP nor in the number of RFT (p > 0.05). However, multivariate logistic regression analysis adjusting for number of teeth and number of RFT showed that subjects with IBD have RPL with higher likelihood than control subjects (OR = 5.7; C.I. 95% = 1.7 – 19.1; p = 0.0048). Conclusions: Patients with ulcerative colitis or Crohn´s disease have higher prevalence of apical periodontitis. An oral health protocol should be established to address the higher prevalence of inflammatory oral processes. Key words: apical periodontitis, root canal treatment, inflammatory bowel disease, Crohn’s disease, ulcerative colitis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 INTRODUCTION Under the name Inflammatory Bowel Diseases (IBD), two recurrent and chronic inflammatory processes of the gastrointestinal tract are included: Crohn's disease (CD) and ulcerative colitis (UC). Both diseases are characterized by diffuse inflammation of the intestinal mucosa, but EC can affect any segment of the gastrointestinal tract, from the mouth to the anus. Terminal ileum is the most frequent site affected. UC affects the large bowel, being the distal colon the most affected region1. Between 4% and 16% of patients with IBD present oral manifestations, including mucosal edema, linear ulceration, angular cheilitis and granulomatous gingivitis2, also presenting a higher prevalence of caries3,4 and periodontal disease5,6. Apical periodontitis (AP) occurs as a sequel of tooth decay, once caries lesion reaches the dental pulp causing irreversible pulpitis and pulp necrosis. The leakage of polymicrobial and antigenic content of the root canal through the apical foramen triggers the inflammatory response of the periapical tissues7. Chronic periapical inflammation is characterized by the presence on the radiograph of a radiolucent image around the apex of the affected tooth resulting from inflammatory resorption of alveolar bone8. The prevalence of AP is high all over the world9,10 and the elective treatment for teeth with AP in order to achieve satisfactory periapical wound healing is root canal treatment (RCT)7. Several studies have found an association between AP and systemic diseases in whose aetiology the pro-inflammatory status of the patient intervenes, such as diabetes mellitus11,12 and cardiovascular desease13,14. Moreover, a previous study has shown that women with IBD had a higher prevalence of AP and patients with IBD had larger periapical lesions than healthy subjects15. However, the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 available scientific evidence is inconsistent. The aim of this ageand gendermatched case-control study was to analyze the prevalence of apical periodontitis, assessed as radiolucent periapical lesions (RPL), and the frequency of root filled teeth (RFT) in healthy control patients and patients with IBD. The null hypothesis was there are no significant differences in the prevalence of endodontic variables between control subjects and IBD patients. MATERIALS AND METHODS Patients’ selection Participants were recruited among patients with IBD receiving treatment at the San Juan de Dios Hospital (Sevilla, Spain) between the years 2017 and 2018. Subjects reporting a history of CD and UC, diagnosed according to the international investigational protocols16, and following the Montreal classification of IBD17, were asked to voluntarily participate. Inclusion criteria were as follows: patients older than 18 years, having at least 8 remaining teeth, who agreed a radiological examination. Exclusion criteria encompassed patients younger than 18 years old, having less than 8 remaining teeth, or who did not agree a radiological examination. Consecutive patients were invited to participate at each clinical site. Only two patients refused to participate. Once the minimum sample size (n = 49) was widely surpassed (n = 54), the recruitment was terminated. A total of 54 patients, 31 men and 23 women (43.1 ± 14.0 years) that agreed and met the inclusion/exclusion criteria constituted the “study group”. Table 1 shows the diagnoses and clinical characteristics of the patients with IBD included in the study group, according to the Montreal classification16, and table 2 shows the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 distribution of patients according to treatment (corticosteroids, immunosuppression and biologic medication). An additional 54 subjects, 31 men and 23 women (43.1 ± 13.8 years), that agreed and met the same inclusion/exclusion criteria, reporting no history of IBD or any clinical sign of ongoing systemic diseases, were matched for age and gender, constituting the “control group”. Controls were recruited from patients of the same city and health district, seeking for the first time routine dental care (not emergency care) at the Dental Clinic of the School of Dentistry between the years 2016 and 2018. Radiographic examination Radiographic periapical status was diagnosed on the basis of examination of digital panoramic radiographs of the jaws. Two trained radiographic technicians, with over ten years of experience, took the panoramic radiographs using a digital ortho-pantomograph machine (Promax®, Planmeca, class 1, type B, 80 KHz, Planmeca, Helsinki, Finland). Radiographic evaluation The periapical status was assessed using the “Periapical Index” (PAI) score18, as described previously14,19(table 3). A score greater than 2 (PAI ≥ 3) was considered to be a sign of periapical pathology. The worst score of all roots was taken to represent the PAI score for multi-rooted teeth. Teeth were categorized as root-filled teeth if they had been filled with a radiopaque material in the root canal(s). The following information was recorded on a structured form for each subject: (a) number of teeth present; (b) number and location of teeth having identifiable 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 radiolucent periapical lesions, (c) number and location of root-filled teeth, and (d) number and location of root-filled teeth having identifiable radiolucent periapical lesions. Observers’ calibration Three blinded observers with extensive clinical experience in endodontics examined the radiographs. Before evaluation, the observers participated in a calibration course for PAI system, which consisted of 100 radiographic images of teeth, some root-filled and some not, kindly provided by Dr. Ørstavik. Each tooth was assigned to 1 of the PAI scores by using visual references (also provided by Dr. Ørstavik) for the 5 categories within the scale18. After scoring the teeth, the results were compared to a “gold standard atlas”, and a Cohen Kappa was calculated (0.76 – 0.84). Intra-observer reproducibility was evaluated for each examiner. Every observer scored the panoramic radiographs of 20 patients (10 of each group, randomly selected). Then, one month after this first examination, the observer was recalibrated in the PAI system and repeated the scoring of the radiographs of the same 20 patients. The intra-observer agreement test on PAI scores on the 20 patients produced a Cohen’s Kappa ranging 0.86 - 0.93. Finally, intra-observers reproducibility was also determined comparing the PAI scores on the 20 radiographs provided by each observer. The agreement test produced a Cohen’s Kappa ranging 0.81 - 0.90. The Cohen’s Kappa for interobservers variability ranged 0.76 - 0.88. The consensus radiographic standard was the simultaneous interpretation by the three examiners of the panoramic radiograph of each patient20,21. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Statistical analysis The prevalence of AP and RCT was evaluated on the total number of individuals and the total number of teeth. The minimal sample size (n = 49) was calculated using the sample size calculator software of the National Center for Advancing Translational Sciences (NIH, UK) (http://www.sample-size.net/sample-sizeproportions/)22 for the comparison of proportions in two independent samples, with continuity correction. They were taken into account a two-sided significance level of 5% ( = 0.05, Z = 1.960), and 80% power ( = 0.20, Z = 0.842) to detect a hypothesized difference between the proportion of the two groups of 30 points (prevalence of AP reported previously in Spain  40%9, hypothesized prevalence of AP in the study group = 70%). Raw data were entered into Excel (Microsoft Corporation, Redmond, WA). All analyses were done in an SPSS environment (Version 11; SPSS, Inc, Chicago, IL). The Student t test, 2 test, and logistic regression analysis were used to determine the significance of differences between groups. Data are reported as mean ± standard deviation. According to the established significance level, a p value ≤ 0.05 was considered statistically significant. ETHICAL CONSIDERATIONS The protocol of this ageand gendermatched case-control study (number 1500N-16) was approved by the Ethical Committee of the University Hospitals of Sevilla (Spain). 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Factors involved in the T helper type 1 and type 2 cell commitment and osteoclast regulation in inflammatory apical diseases. Oral Microbiol Immunol. 2009;24(1):25-31. doi:10.1111/j.1399-302X.2008.00469.x 41. Stashenko P, Wang C-Y, Tani-Ishii N, Yu SM. Pathogenesis of induced rat periapical lesions. Oral Surgery, Oral Med Oral Pathol. 1994;78(4):494502. doi:10.1016/0030-4220(94)90044-2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Table 1. Distribution of patients in the experimental group (IBD) according to the type of disease (UC, ulcerative colitis; CD, Crohn's disease), location, and behavior pattern, according to the Montreal classification16. IBD (n = 54) Number (%) Crohn`s disease (n = 28) Location L1: Ileal 16 (57.1%) L2: Colonic 2 ( 7.1%) L3: Ileocolonic 8 (28.6%) L4: Ileal and upper disease 2 ( 7.1%) Behavior pattern B1: Stricturing 15 (53.6%) B2: Penetrating 4 (14.3%) B3: Non constricturing/non penetrating 9 (32.1%) Age at diagnosis A1: < 16 years 4 (14.3%) A2: 16-40 years 20 (71.4%) A3: > 40 years 4 (14.3%) Ulcerative colitis (n = 26) Location E1: Rectum and sigmoid colon 6 (23.1%) E2: Left side of colon 12 (46.2%) E3: All of colon 8 (30.8%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Table 2. Distribution of patients in the experimental group (IBD) according to the treatment (steroids, immunosuppression and biologic medication). Ulcerative Crohn´s Total Treatment Colitis (n = 26) Disease (n = 28) (n = 54) n (%) n (%) n (%) Corticosteroids 3 (11.5) 3 (10.7) 6 (11.1) 5-aminosalicylic acid Mesalazine 26 (100) 18 (64.3) 44 (81.5) Immunomodulators Methotrexate 3 (11.5) 2 (7.1) 5 (9.3) Azathioprine 4 (15.4) 14 (50.0) 18 (33.3) Cyclosporine 1 (3.8) 1 (1.9) Biologic agents Anti-TNF Infliximab 2 (7.7) 2 (3.7) Adalimumab 1 (3.8) 1 (1.9) Golimumab 1 (3.6) 1 (1.9) Others Vedolizumab 1 (3.6) 1 (1.9) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Table 3. Periapical Index (PAI) (Ørstavik et al. 1986)18. Score Criteria 1 Normal periapical structures 2 Small changes in bone structure 3 Changes in bone structure with some mineral loss 4 Periodontitis with well defined radiolucent area 5 Severe periodontitis with exacerbating features 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Table 4. Distribution of analyzed variables among patients with IBD (study group) and normal subjects (control group). Study group Control group Total n = 54 (50%) n = 54 (50%) n = 108 (100%) p value Age/Years t test Mean ± SD 43.1 ± 14.0 43.1 ± 13.8 43.1 ± 13.8 > .05 Gender Male 31 (57.4%) 31 (57.4%) 62 (57.4%) 2 test Female 23 (42.6%) 23 (42.6%) 46 (42.63%) > .05 No. of teeth Mean ± SD 24.9 ± 3.9 25.5 ± 5.5 25.2 ± 4.7 t test Median 26 27 27 > .05 Teeth with AP Any 19 (35.2%) 9 (16.7%) 28 (25.9%) 2 test None 35 (64.8%) 45 (83.3%) 80 (74.1%) = .03 No. of Teeth with AP Mean ± SD 0.5 ± 0.8 0.4 ± 0.8 0.5 ± 0.8 t test Median 0 0 0 > .05 RFT Any 29 (53.7%) 22 (40.7%) 51 (47.2%) 2 test None 25 (46.3%) 32 (59.3%) 57 (52.7%) > .05 No. of RFT Mean ± SD 0.8 ± 1.0 1.1 ± 1.8 1.0 ± 1.5 t test Median 1 0 0 > .05 RFT-AP Any 14 (48.3%) 8 (36.4%) 22 (43.1%) 2 test None 15 (51.7%) 14 (63.6%) 29 (56.9%) > .05 No. of RFT-AP Mean ± SD 0.3 ± 0.5 0.3 ± 0.7 0.3 ± 0.6 t test Median 0 0 0 > .05 Smoking Yes 7 (13.0%) 11 (20.4%) 17 (15.7%) 2 test No 47 (87.0%) 43 (79.6%) 91 (84.3%) > .05 Abbreviations: RFT: root-filled teeth. AP: apical periodontitis. RFT-AP: root-filled teeth with apical periodontitis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 distribution of patients according to treatment (corticosteroids, immunosuppression and biologic medication). An additional 54 subjects, 31 men and 23 women (43.1 ± 13.8 years), that agreed and met the same inclusion/exclusion criteria, reporting no history of IBD or any clinical sign of ongoing systemic diseases, were matched for age and gender, constituting the “control group”. Controls were recruited from patients of the same city and health district, seeking for the first time routine dental care (not emergency care) at the Dental Clinic of the School of Dentistry between the years 2016 and 2018. Radiographic examination Radiographic periapical status was diagnosed on the basis of examination of digital panoramic radiographs of the jaws. Two trained radiographic technicians, with over ten years of experience, took the panoramic radiographs using a digital ortho-pantomograph machine (Promax®, Planmeca, class 1, type B, 80 KHz, Planmeca, Helsinki, Finland). Radiographic evaluation The periapical status was assessed using the “Periapical Index” (PAI) score18, as described previously14,19(table 3). A score greater than 2 (PAI ≥ 3) was considered to be a sign of periapical pathology. The worst score of all roots was taken to represent the PAI score for multi-rooted teeth. Teeth were categorized as root-filled teeth if they had been filled with a radiopaque material in the root canal(s). The following information was recorded on a structured form for each subject: (a) number of teeth present; (b) number and location of teeth having identifiable radiolucent periapical lesions, (c) number and location of root-filled teeth, and (d) number and location of root-filled teeth having identifiable radiolucent periapical lesions. Observers’ calibration Three blinded observers with extensive clinical experience in endodontics examined the radiographs. Before evaluation, the observers participated in a calibration course for PAI system, which consisted of 100 radiographic images of teeth, some root-filled and some not, kindly provided by Dr. Ørstavik. Each tooth was assigned to 1 of the PAI scores by using visual references (also provided by Dr. Ørstavik) for the 5 categories within the scale18. After scoring the teeth, the results were compared to a “gold standard atlas”, and a Cohen Kappa was calculated (0.76 – 0.84). Intra-observer reproducibility was evaluated for each examiner. Every observer scored the panoramic radiographs of 20 patients (10 of each group, randomly selected). Then, one month after this first examination, the observer was recalibrated in the PAI system and repeated the scoring of the radiographs of the same 20 patients. The intra-observer agreement test on PAI scores on the 20 patients produced a Cohen’s Kappa ranging 0.86 - 0.93. Finally, intra-observers reproducibility was also determined comparing the PAI scores on the 20 radiographs provided by each observer. The agreement test produced a Cohen’s Kappa ranging 0.81 - 0.90. The Cohen’s Kappa for interobservers variability ranged 0.76 - 0.88. The consensus radiographic standard was the simultaneous interpretation by the three examiners of the panoramic radiograph of each patient20,21. Statistical analysis The prevalence of AP and RCT was evaluated on the total number of individuals and the total number of teeth. The minimal sample size (n = 49) was calculated using the sample size calculator software of the National Center for Advancing Translational Sciences (NIH, UK) (http://www.sample-size.net/sample-sizeproportions/)22 for the comparison of proportions in two independent samples, with continuity correction. They were taken into account a two-sided significance level of 5% ( = 0.05, Z = 1.960), and 80% power ( = 0.20, Z = 0.842) to detect a hypothesized difference between the proportion of the two groups of 30 points (prevalence of AP reported previously in Spain  40%9, hypothesized prevalence of AP in the study group = 70%). Raw data were entered into Excel (Microsoft Corporation, Redmond, WA). All analyses were done in an SPSS environment (Version 11; SPSS, Inc, Chicago, IL). The Student t test, 2 test, and logistic regression analysis were used to determine the significance of differences between groups. Data are reported as mean ± standard deviation. According to the established significance level, a p value ≤ 0.05 was considered statistically significant. ETHICAL CONSIDERATIONS The protocol of this ageand gendermatched case-control study (number 1500N-16) was approved by the Ethical Committee of the University Hospitals of Sevilla (Spain). Each subject signed a consent form after being advised of the nature of the study. RESULTS The distribution of analysed variables in the two groups is shown in table 4. The average number of teeth per patient was 24.9 ± 3.9 and 25.5 ± 5.5 teeth in the study and control groups, respectively (p > 0.05). In the study group, the average number of teeth with AP was 0.5 ± 0.8, whereas in the control group it was 0.4 ± 0.8 (p >0.05). The number of RFT was also similar in both groups, being 0.8 ± 1.0 in the SG and 1.1 ± 1.8 in the CG (p > 0.05). There were no significant differences in smoking between the study group and the control group (p > 0.05). Analysing the SG, no significant differences were observed between patients with UC and patients with CD in the number of teeth with AP, in the number of RFT, nor in the number of RFT with AP (p > 0.05). Taking the patient as a reference (table 5), in the SG group 19 patients (35.2%) had at least one tooth with radiolucent periapical lesion (RPL), whereas in the CG it was presented only 9 subjects (16.7%) (p = 0.03). The calculated odds ratio (OR) was 2.71 (C. I. 95% = 1.09 – 6.73; p = 0.03) (table 5). There were no differences between both groups in the number of patients with one or more RFT (OR = 1.69; C.I. 95% = 0.79 – 3.62; p = 0.18) nor in the number of patients with one or more RFT with RPL (OR = 1.63; C.I. 95% = 0.53 – 5.07; p = 0.39). To analyse which variables influenced the periapical status, multivariate logistic regressions were run with number of teeth, number of RFT, and the presence of IBD (0 = healthy control; 1 = IBD), taking as dependent variable and outcome “periapical status” (0 = no tooth with RPL; 1 = at least one tooth with RPL) (Table 6). In the multivariate analysis, including all the above factors as covariates, IBD status (OR = 5.7; C.I. 95% = 1.7 – 19.1; p = 0.0048) was highly significant, indicating that subjects with IBD have RPL with higher likelihood than control subjects. The number of RFT was also significantly associated to the presence of RPL (OR = 2.2; C.I. 95% = 1.5 – 3.2; p = 0.0001). DISCUSSION This ageand gendermatched case-control study aimed to investigate the prevalence of RPL and RFT in patients with IBD and control healthy subjects. The null hypothesis tested (i.e. there are no significant differences in the prevalence of endodontic variables between control subjects and IBD patients) has been partly refused. The results show a significant association between IBD and the presence of RPL. Specifically, IBD patients are 5.7 times more likely to present apical periodontitis compared to control healthy subjects (p = 0.005). On the contrary, the frequency of RFT was similar in IBD patients and control subjects (p > 0.05). The number of teeth and the number of root-filled teeth23 have been shown to be significantly associated to radiographically diagnosed periapical lesions. However, in the present study the number of teeth and the number of RFT were similar in IBD patients and control subjects. The recruitment method of the patients was similar to that used in previous studies10,11,15. It is possible that IBD patients had more concerns about their overall dental health and were more likely to participate in the study. This could result in a spurious association between IBD and periapical disease. However, we think this is unlikely as only two patients refused to participate. IBD was diagnosed according to the current criteria for the diagnosis of CD and UC 16,17. The low percentage of patients with ileocolonic disease and the high percentage of patients with isolated ileal disease is striking. However, previous studies conducted in Spain24 have found similar distribution of IBD subtypes. In relation to periapical status, the ‘periapical index’ (PAI) is a scoring system18 widely used in epidemiological and clinical studies in which the presence of RPL is assessed to determine the prevalence of AP 10,25. In the last decades, the results of epidemiological studies suggest a link between AP and some systemic diseases, such as diabetes mellitus12, cardiovascular disease26, tobacco smoking19, osteoporosis, inherited coagulation disorders and others27. Although a causal relationship has not been established between AP and these diseases28, these findings have contributed to promote a greater attention to the oral health of these patients. In addition, they have led to investigate the possible association of other diseases with endodontic pathology. This has happened too in recent times with IBD15,29. Even though the etiology of IBD is not exactly known, it has been shown that both genetic and environmental factors play a role in its pathogenesis30. The characteristic intestinal inflammation would be produced by an inadequate immune response of the intestinal mucosa against the luminal antigens in a host with genetic susceptibility, resulting in an imbalance of pro-inflammatory and antiinflammatory factors31. Signs and symptoms of these diseases are abdominal pains, poor appetite, weight loss, diarrhea, and rectal bleeding, showing active episodes and asymptomatic intervals32. Several studies have analyzed the possible relationship between IBD and periodontal disease5,6. The pathogenesis of periodontal disease, similarly to that of IBD, involves a complex interplay between periodontopathogens and the host immune-inflammatory response, greatly influenced by genetic and environmental factors33. A systematic review by Papageorgiou et al. (2017)34, concluded that IBD patients had significantly higher risk of periodontal disease and worse oral health compared to control non-IBD patients. Although AP has an etiology, pathogenesis and clinical manifestations similar to that of periodontal disease27, very few studies have been carried out investigating the periapical and endodontic status of patients with IBD. A cross-sectional study analyzed the consumption of dental treatment among patients with IBD, finding that either CD or UC patients needed significantly higher number of procedures. CD patients required significantly more endodontic treatments, when compared to controls35. Recently, Piras et al. (2017)15 have reported that women with IBD had a significantly higher number of teeth with AP. IBD patients exhibited significantly higher PAI index score when compared with controls. Multivariate logistic regression analysis shows RPLs are more frequent in IBD patients after adjusting for age, gender, number of teeth, and number of root-filled teeth (OR = 5.7; C.I. 95% = 1.7 – 19.1; p = 0.005).These results agree with previous reports which found a worse oral health status in patients with IBD5,6,15. Therefore IBD could be considered an independent risk factor for the development of chronic inflammatory oral diseases such as periodontal disease and apical periodontitis. The percentage of patients with at least one RFT associated to RPLs was similar in both the study and control groups (p > 0.05). Although periapical radiolucent lesions associated to RFT may represent persistent chronic apical periodontitis or incomplete healed lesions after root canal treatment, they can also represent healing lesions, particularly if the time elapsed since treatment was less than 2 years36. A previous study has shown that periapical lesion of patients with IBD taking biologic medications, such as anti–TNFα, healed faster after nonsurgical endodontic treatment, compared to healthy control subjects29. The authors attribute this fact to a possible beneficial effect for the endodontic treatment of biological drugs29. Nevertheless, in the present study only three of the IBD patients were taken adalimubad or infliximab, two anti–TNFα medicaments. Some considerations should be made about the coherence and biological plausibility of this results regarding the mechanisms by which periapical or endodontic status could affect bowel diseases, and vice-versa. The dysbiosis of the gut microbiota, typical of IBD, not only could contribute to the development of intestinal disorder, but also to the extraintestinal inflammatory oral diseases 15. On the other hand, oral biofilms implicated in both periodontal and periapical diseases could influence the development of IBDs 37. It has been proposed that swallowing of oral dead bacteria could stimulate several pathogens in the gut (necrotrophy), creating new phenotypes by upregulation of bacterial virulence genes37,38. Another possible link between apical periodontitis and IBD could be the immune system. UC is classified as a TH2 type immune disease, characterized by an up-regulation of interleukin (IL)-5, whereas CD is classified as a TH1 type immune disease, showing high levels of interferon gamma (IFN-), IL-12, and tumor necrosis factor alpha (TNF-α)39. Both type of immune responses are implicated in the course of apical periodontitis. The progression of apical periodontitis and its characteristic bone destruction have been attributed to the earlier onset of Th1 response, with activation of osteoclasts by nuclear factor kappa B ligand (RANKL)40. On the contrary, periapical repair after endodontic treatment seems to be related to the later onset of Th2 response41. Thus, the genotype, the main determinant of the immune response of each person, could be the link that associates both pathologies. The present study has several limitations. Firstly, some relevant factors that influence the periapical status and the prevalence of root canal treatment has not been considered, such as educational level, socioeconomic status, diabetes, caries, quality of coronal restorations, quality of endodontic treatment, history of trauma23. These factors could be acting as confounding factors. Secondly, the quality of root canal filling and coronal restoration, which have not been considered when evaluating the presence of periapical radiolucencies. This has been proven to be associated to the prevalence of chronic apical periodontitis, and could act as confounding factors14,23. It could explain the wide OR confidence interval observed in the multivariate analysis. The control of confounding factors is difficult, particularly when any influence on apical periodontitis is likely to be multifactorial. CONCLUSION These results suggest that both types of IBD, ulcerative colitis and Crohn´s disease, are associated to higher prevalence of apical periodontitis. Prospective longitudinal studies are needed to clarify if there is a causal relationship between periapical status and IBD. Therefore an oral health protocol should be established in these patients to address the higher prevalence of inflammatory oral processes. ACKNOWLEDGEMENTS The authors deny any conflicts of interest. REFERENCES 1. Baumgart DC, Sandborn WJ. Crohn’s disease. Lancet. 2012;380(9853):1590-1605. 2. Kalmar JR. Crohn’s disease: orofacial considerations and disease pathogenesis. Periodontol 2000. 1994;6:101-115. http://www.ncbi.nlm.nih.gov/pubmed/9673174. Accessed March 31, 2019. 3. Brito F, Barros FC de, Zaltman C, et al. Prevalence of periodontitis and DMFT index in patients with Crohn’s disease and ulcerative colitis. J Clin Periodontol. 2008;35(6):555-560. doi:10.1111/j.1600-051X.2008.01231.x 4. Rooney TP. Dental caries prevalence in patients with Crohn’s disease. Oral Surgery, Oral Med Oral Pathol. 1984;57(6):623-624. doi:10.1016/00304220(84)90284-6 5. Koutsochristou V, Zellos A, Dimakou K, et al. Dental caries and periodontal disease in children and adolescents with inflammatory bowel disease: A case-control study. Inflamm Bowel Dis. 2015;21(8):1839-1846. doi:10.1097/MIB.0000000000000452 6. Vavricka SR, Manser CN, Hediger S, et al. Periodontitis and gingivitis in inflammatory bowel disease: a case-control study. Inflamm Bowel Dis. 2013;19(13):2768-2777. doi:10.1097/01.MIB.0000438356.84263.3b 7. Eriksen HM. Epidemiology of apical periodontitis. In: Orstavik D, Ford TP, eds. Essential Endodontology: Prevention and Treatment of Apical Periodontitis. Blackwell Science; 1998:179-191. 8. Liu S, Cheng Y, Xu W, Bian Z. Protective effects of follicle-stimulating hormone inhibitor on alveolar bone loss resulting from experimental periapical lesions in ovariectomized rats. J Endod. 2010;36(4):658-663. Table 2. Distribution of patients in the experimental group (IBD) according to the treatment (steroids, immunosuppression and biologic medication). Ulcerative Crohn´s Total Treatment Colitis (n = 26) Disease (n = 28) (n = 54) n (%) n (%) n (%) Corticosteroids 3 (11.5) 3 (10.7) 6 (11.1) 5-aminosalicylic acid Mesalazine 26 (100) 18 (64.3) 44 (81.5) Immunomodulators Methotrexate 3 (11.5) 2 (7.1) 5 (9.3) Azathioprine 4 (15.4) 14 (50.0) 18 (33.3) Cyclosporine 1 (3.8) 1 (1.9) Biologic agents Anti-TNF Infliximab 2 (7.7) 2 (3.7) Adalimumab 1 (3.8) 1 (1.9) Golimumab 1 (3.6) 1 (1.9) Others Vedolizumab 1 (3.6) 1 (1.9) Table 3. Periapical Index (PAI) (Ørstavik et al. 1986)18. Score Criteria 1 Normal periapical structures 2 Small changes in bone structure 3 Changes in bone structure with some mineral loss 4 Periodontitis with well defined radiolucent area 5 Severe periodontitis with exacerbating features Table 4. Distribution of analyzed variables among patients with IBD (study group) and normal subjects (control group). Study group Control group Total n = 54 (50%) n = 54 (50%) n = 108 (100%) p value Age/Years t test Mean ± SD 43.1 ± 14.0 43.1 ± 13.8 43.1 ± 13.8 > .05 Gender Male 31 (57.4%) 31 (57.4%) 62 (57.4%) 2 test Female 23 (42.6%) 23 (42.6%) 46 (42.63%) > .05 No. of teeth Mean ± SD 24.9 ± 3.9 25.5 ± 5.5 25.2 ± 4.7 t test Median 26 27 27 > .05 Teeth with AP Any 19 (35.2%) 9 (16.7%) 28 (25.9%) 2 test None 35 (64.8%) 45 (83.3%) 80 (74.1%) = .03 No. of Teeth with AP Mean ± SD 0.5 ± 0.8 0.4 ± 0.8 0.5 ± 0.8 t test Median 0 0 0 > .05 RFT Any 29 (53.7%) 22 (40.7%) 51 (47.2%) 2 test None 25 (46.3%) 32 (59.3%) 57 (52.7%) > .05 No. of RFT Mean ± SD 0.8 ± 1.0 1.1 ± 1.8 1.0 ± 1.5 t test Median 1 0 0 > .05 RFT-AP Any 14 (48.3%) 8 (36.4%) 22 (43.1%) 2 test None 15 (51.7%) 14 (63.6%) 29 (56.9%) > .05 No. of RFT-AP Mean ± SD 0.3 ± 0.5 0.3 ± 0.7 0.3 ± 0.6 t test Median 0 0 0 > .05 Smoking Yes 7 (13.0%) 11 (20.4%) 17 (15.7%) 2 test No 47 (87.0%) 43 (79.6%) 91 (84.3%) > .05 Abbreviations: RFT: root-filled teeth. AP: apical periodontitis. RFT-AP: root-filled teeth with apical periodontitis. Table 5. Estimation of odds ratio (OR) values, and their 95% confidence interval (C.I.), using 2 test, for the association between the prevalence of apical periodontitis (AP), root-filled teeth (RFT), and root-filled teeth with apical periodontitis (RFT-AP) in patients with IBD (study group, SG; n = 54) and normal subjects (control group; n = 54). AP (%) RFT (%) RFT-AP (%) Study group 19 (35.2) 29 (53.7) 14 (48.3) Control group 9 (16.7) 22 (40.7) 8 (36.4) Total 28 (25.9) 51 (47.2) 22 (43.1) OR SG 2.71 1.69 1.63 95% C.I. 1.09 – 6.73 0.79 – 3.62 0.53 – 5.07 p value 0.03 0.18 0.39 Each value represents the number of patients with at least 1 tooth with AP, 1 RFT or 1 RFT with AP. Abbreviations: IBD: inflammatory bowel disease. OR SG: odds ratio calculated for study group. Table 6. Multivariate logistic regression analyse of the influence of the independent variables number of teeth, number of root-filled teeth, and the presence of IBD (0 = absent, 1 = present), on the dependent variable “periapical status” (0 = no tooth with apical periodontitis, 1 = at least one tooth with apical periodontitis). Overall model fit: Chi Square = 33.4735; df = 3; p = 0.0000 RFT: root-filled teeth; IBD: inflammatory bowel disease status. Dependent variable B p Odds Ratio C. I. 95% Inf. Limit C. I. 95% Sup. Limit. No. Teeth -0.1442 0.0150 0.8657 0.7708 0.9724 No. RFT 0.7823 0.0001 2.1866 1.4835 3.2229 IBD 1.7403 0.0048 5.6992 1.7021 19.0836 IBD-D-19-00308R1 High Prevalence of Apical Periodontitis in Patients with Inflammatory Bowel Disease: An Ageand GenderMatched Case-Control Study Author's Point-by-Point Response to Reviewers In blue: answers to referees. In red: modifications and/or new paragraphs in the manuscript. Reviewers´ comments to authors and answers: Reviewer #1: The authors have addressed most of my critiques of the previous submission. I have one minor and one major suggestion. 1) First, I appreciate that consecutive patients were recruited from the various clinical sites with only two patients refusing to participate. I agree that it is unlikely then that patients in need of dental evaluation were more likely to be recruiting resulting in a spurious association with periapical abnormalities. However, the wording needs to be changed. I would simply say, "Consecutive patients were invited to participate at each clinical site. Only two patients refused to participate." In the discussion, "It is possible that IBD patients had more concerns about their overall dental health and were more likely to participate in the study. This could result in a spurious association between IBD and periapical abnormalities. However, we think this is unlikely as only two patients refused to participate." Authors: Thanks for your comments and suggestion. In Mat & Met we have changed the wording according to your suggestion, as follows: Consecutive patients were invited to participate at each clinical site. Only two patients refused to participate. In Discussion, the following paragraph has been removed: Taking into account the way in which the components of the study group were selected, it can be ruled out that the patients with IBD included in the study had a special concern for their oral health, which would bias the results by providing a spurious association with IBD. This paragraph was changed to the one you suggested, as follows: It is possible that IBD patients had more concerns about their overall dental health and were more likely to participate in the study. This could result in a spurious association between IBD and periapical disease. However, we think this is unlikely as only two patients refused to participate. 2) My major critique is that subanalyses assessing the impact of steroid and immune suppressant use is lacking. Presumably, controls were not treated with these therapies; thus, steroid and immune suppressant use cannot be added to the regression model. However, I would recommend that you do sub-analyses of the IBD patients to determine if periapical abnormalities were more common in patients treated with either therapy alone or both therapies. Authors: Thanks for your comments and suggestion. Indeed, given that the controls did not take corticosteroids or immune suppressants, this was not included in the logistic regression analysis. Nevertheless, coinciding with your suggestion, we had already done subanalyses to see if there were differences Supplementary Material For Review between patients in the study group according to their treatment, and to investigate whether radiolucent periapical lesions were more common in patients treated with corticosteroids alone or in those treated with corticosteroids and immune suppressant. However, probably because of the small size of the sample and the small number of patients taking both medications, no significant correlations were observed. Moreover, we believe that these results are not adequate to be published in the article, taking into account the small number of patients with IBD treated with corticosteroids (only 6) or both therapies (only 3). We attach the table of multivariate logistic regression analysis with the independent variables 1) number of teeth, 2) type of IBD (0=CD, 1=UC), and 3) type of medication (0=only corticosteroid, 1=corticosteroid + immune suppressants), and the dependent variable periapical status (0=no periapical lesion, 1= at least 1 periapical lesion): Overall Model Fit: Chi Square = 7.9314; df = 3; p = 0.0475 Variable Coeff. r p O.R. Low High 1 -0.2434 0.0173 0.7840 0.6416 0.9579 2 0.2693 0.6762 1.3091 0.3698 4.6338 3 -0.7459 0.5485 0.4743 0.0415 5.4213 Following your suggestion, we have decided to prepare a new study, including a greater sample of IBD patients, designed to assess the possible impact of steroid and immune suppressant in the periapical status. Reviewer #2: I accept the answers and corrections. Authors: Thanks for your comments.