Adherence to dietary treatment and clinical factors associated with anti-transglutaminase antibodies in celiac disease during the follow-up.

Clinical manifestations and mucosa atrophy at the time of the diagnosis, antibody concentrations during the follow up, and demographic characteristics of the study, are shown in Table 1 . The mean age of the patients included in the study was 24.2 years, with no significant differences between sexes. A total of 254 patients (60.2% women and 39.8% men) were studied. Among them, 212 (83.5%) had a confirmed diagnosis of CD (59.9% were women with a mean age of 25.3 years and 40.1% men with a mean age of 22.7 years). Women showed a greater tendency to suffer diarrhoea and abdominal pain (p-value Chi-square test = 0.009 and 0.015, respectively). 75.6% of the sample registered a correct therapeutic follow-up, which was stronger for the older group, which registered a higher frequency of abdominal pain and loss of weight (p-value from ANOVA F-test = 0.002 and 0.046, respectively). The youngest group reported the highest frequency of vomiting (p-value from ANOVA F- test <0.001). Participants aged between 12-18 years had the lowest degree of therapeutic follow-up (22.9%), being also the group with the highest number of extra-digestive disorders (mean (SD) = 4.1 (1.3)). Table 1 Descriptive variables based on age and sex. Table 1 Variable Total Sex Age Men Women p-value a 0–11 years 12–18 years >18 years p-value a Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Mean (SD) Ab (U/mL) – 1 st measure 147.8 (118.4) 138.55 (119.0) 153.9 (117.9) 0.311 181.9 (125.1) † 136.2 (112.6) † 131.1 (112.8) † 0.009 ∗ Ab (U/mL) – 2 nd measure 91.8 (92.3) 93.2 (99.8) 90.8 (87.3) 0.842 133.7 (106.3) † 92.7 (90.4) † 64.1 (71.5) † <0.001 ∗ Ab (U/mL) – 3 rd measure 44.1 (52.0) 42.6 (58.0) 45.1 (47.8) 0.712 47.5 (62.8) 42.8 (60.1) 42.5 (38.9) 0.789 Ab (U/mL) – 4 th measure 23.8 (26.7) 21.4 (22.5) 25.5 (29.1) 0.229 20.3 (21.2) † 17.8 (16.6) † 28.9 (32.3) † 0.013 ∗ Age (years) 24.2 (19.3) 22.7 (18.4) 25.3 (19.9) 0.283 7.0 (2.8) 14.2 (2.1) 40.3 (17.0) Digestive alterations (N) 3.7 (1.2) 3.5 (1.2) 3.7 (1.2) 0.202 3.7 (1.0) 3.5 (1.3) 3.7 (1.3) 0.384 Extradigestive alterations (N) 3.7 (1.3) 3.7 (1.3) 3.8 (1.3) 0.433 3.9 (1.4) 4.1 (1.3) 3.5 (1.2) 0.014 ∗ Associated pathologies (N) 2.6 (0.9) 2.5 (0.8) 2.7 (1.0) 0.200 2.7 (1.1) 2.6 (0.8) 2.6 (0.9) 0.709 N (%) N (%) N (%) p-value b N (%) N (%) N (%) p-value b Sex 254 (100%) 101 (39.8%) 153 (60.2%)  Men 30 (29.7%) 24 (23.8%) 47 (46.5%) 0.862  Women 48 (31.4%) 32 (20.9%) 73 (47.7%) CD diagnosis  Confirmed 212 (83.5%) 85 (40.1%) 127 (59.9%) 0.131 64 (30.2%) 45 (21.2%) 103 (48.6%) 0.539  Not confirmed 24 (9.4%) 6 (25%) 18 (75%) 7 (29.2%) 5 (20.8%) 12 (50%)  Suspicion 18 (7.1%) 10 (55.6%) 8 (44.4%) 7 (38.9%) 6 (33.3%) 5 (27.8%) Therapeutic follow-up  No 62 (24.4%) 19 (30.6%) 43 (69.4%) 0.092 9 (14.5%) 12 (19.4%) 41 (66.1%) <0.001 ∗  Yes 192 (75.6%) 82 (42.7%) 110 (57.3%) 69 (35.9%) 44 (22.9%) 79 (41.1%) HLA diagnosis  Negative 12 (4.7%) 1 (8.3%) 11 (91.7%) 0.023 ∗ 2 (16.7%) 1 (8.3%) 9 (75%) 0.140  Positive 242 (95.3%) 100 (41.3%) 142 (58.7%) 76 (31.4%) 55 (22.7%) 111 (45.9%) Diagnostic EMA  Negative 6 (2.4%) 2 (33.3%) 4 (66.7%) 0.745 0 (0%) 0 (0%) 6 (100%) 0.032  Positive 248 (97.6%) 99 (39.9%) 149 (60.1%) 78 (31.5%) 56 (22.6%) 114 (46%) Ferritin  Increased 2 (0.8%) 1 (50%) 1 (50) 0.766 0 (0%) 0 (0%) 2 (100%) 0.324  Deficit 252 (99.2%) 100 (39.7%) 152 (60.3%) 78 (31%) 56 (22.2%) 118 (46.8%) Iron  Increased 5 (2%) 2 (40%) 3 (60%) 0.991 2 (40%) 1 (20%) 2 (40%) 0.900  Deficit 249 (98%) 99 (39.8%) 150 (60.2%) 76 (30.5%) 55 (22.1%) 118 (47.4%) Diarrhoea  No 90 (35.4%) 26 (28.9%) 64 (71.1%) 0.009 ∗ 28 (31.1%) 21 (23.3%) 41 (45.6%) 0.907  Yes 164 (64.6%) 75 (45.7%) 89 (54.3%) 50 (30.5%) 35 (21.3%) 79 (48.2%) Abdominal pain  No 100 (39.4%) 49 (49%) 51 (51%) 0.015 ∗ 43 (43%) 21 (21%) 36 (36%) 0.002 ∗  Yes 154 (60.6%) 52 (33.8%) 102 (66.2%) 35 (22.7%) 35 (22.7%) 84 (54.5%) Weight loss  No 189 (74.4%) 81 (42.9%) 108 (57.1%) 0.086 59 (31.2%) 48 (25.4%) 82 (43.4%) 0.046 ∗  Yes 65 (25.6%) 20 (30.8%) 45 (69.2%) 19 (29.2%) 8 (12.3%) 38 (58.5%) Vomits  No 155 (61%) 60 (38.7%) 95 (61.3%) 0.668 30 (19.4%) 24 (15.5%) 101 (65.2%) <0.001 ∗  Yes 99 (39%) 41 (41.4%) 58 (58.6%) 48 (48.5%) 32 (32.3%) 19 (19.2%) Dietary non-compliance  No 138 (54.3%) 50 (36.2%) 88 (63.8%) 0.210 44 (31.9%) 34 (24.6%) 60 (43.5%) 0.375  Yes 116 (45.7%) 51 (44%) 65 (56%) 34 (29.3%) 22 (19%) 60 (51.7%) Marsh diagnosis  0 22 (8.7%) 12 (54.5%) 10 (45.5%) 0.673 2 (9.1%) 1 (4.5%) 19 (86.4%) <0.001 ∗  1 5 (2%) 1 (20%) 4 (80%) 1 (20%) 1 (20%) 3 (60%)  2 1 (0.4%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 0 (0%)  3a 54 (21.3%) 23 (42.6%) 31 (57.4%) 5 (9.3%) 13 (24.1%) 36 (66.7%)  3b 68 (26.8%) 25 (36.8%) 43 (63.2%) 13 (19.1%) 13 (19.1%) 42 (61.8%)  3c 61 (24%) 23 (37.7%) 38 (62.3%) 23 (37.7%) 22 (36.1%) 16 (26.2%)  No biopsy ‡ 43 (16.9%) 17 (39.5%) 26 (60.5%) 34 (79.1%) 5 (11.6%) 4 (9.3%) Ab: antibodies TGA-IgA. SD: standard deviation. HLA: Human leukocyte antigen. EMA: anti-endomysial antibody. a p-value from ANOVA F- test. b p-value from Chi-square test. ∗ significant ANOVA/Chi-square test (p value < 0.05). † significant Tukey test (p value < 0.05). ‡ Patients under 2 years had no biopsy following the ESPGHAN criteria [ 43 ]. Descriptive variables based on age and sex. Ab: antibodies TGA-IgA. SD: standard deviation. HLA: Human leukocyte antigen. EMA: anti-endomysial antibody. p-value from ANOVA F- test. p-value from Chi-square test. significant ANOVA/Chi-square test (p value < 0.05). significant Tukey test (p value < 0.05). Patients under 2 years had no biopsy following the ESPGHAN criteria [ 43 ]. From the total of participants, 45.7% registered dietary transgressions at some point of the treatment, without observing significant differences regarding sex or age. At the time of diagnosis, most of the adults presented a mild-moderate villous atrophy degree (66.7% type 3a and 61.8% type 3b). In contrast, children under 12 years of age showed a tendency to present a severe atrophy of the intestinal villi (see Table 1 ). The mean of diseases associated with CD was 2.6 (0.9), with no differences between sexes or age ranges, highlighting: 1) thyroid disorders (36.6%), which included: autoimmune thyroiditis, hypo and hyperthyroidism (both confirmed and suspected by altered levels of Thyroid-Stimulating Hormone (TSH) or free T3 and T4 thyroxins); 2) diabetes mellitus (11.0%), including type I and II; 3) irritable colon (9.4%); 4) obesity (8.3%), considering a Body Mass Index (BMI) ≥30 kg/m 2 ; and Attention Deficit Hyperactivity Disorder (ADHD) (6.3%) ( Table 2 ). Table 2 Frequencies of potentially related diseases to CD and known associated prevalence. Table 2 Diseases Study frequencies N (%) Associated prevalence according to bibliography Thyroid disorder 93 (36.6%) 30.3% in adults [ 44 ] 26.2% in children [ 45 ] Diabetes mellitus 28 (11%) 1–12% [ 46 , 47 ] Irritable colon 24 (9.4%) 0–11.4% [ 5 , 6 ] Obesity 21 (8.3%) 0–6% [ 48 ] Attention deficit hyperactivity disorder 16 (6.3%) 6–11% [ 49 ] Sterility 7 (2.8%) 2.1–4,1% [ 50 ] Colon polyps 7 (2.8%) 20% [ 51 ] Cancer 7 (2.8%) Associated to all cancers with high prevalence variability [ 52 ] Uterine fibroid 7 (2.8%) – Dermatitis herpetiformis 6 (2.4%) 13% [ 53 ] Down syndrome 4 (1.6%) 3–12% [ 50 , 54 ] Alopecia areata 4 (1.6%) 1–2% [ 55 ] Endometriosis 4 (1.6%) 2.5% [ 56 ] Erosive duodenitis 3 (1.2%) 2–42% [ 57 ] Raynaud's disease 3 (1.2%) 0.33–1.34% [ 58 , 59 ] Ulcerative colitis 3 (1.2%) 1,3% [ 49 ] Crohn's disease 3 (1.2%) 1,3% [ 49 ] Polyendocrine syndromes 3 (1.2%) 10–30% [ 60 ] Sjögren syndrome 2 (0.8%) 2–15% [ 54 , 61 ] Asperger 2 (0.8%) 0.95–2.62% [ 62 , 63 ] Polycystic ovaries 2 (0.8%) Associated in only 1 study [ 64 ] Perthes 1 (0.4%) – Frequencies of potentially related diseases to CD and known associated prevalence. Figure 1 shows the Kernel diagram with the TGA-IgA antibody concentrations in each of the four measurements versus the reference value proposed as a positive diagnosis of CD (10 IU/mL) [ 20 ]. Results also show that the mean of serum TGA-IgA antibody decreased progressively between the first and fourth measure (p-value from ANOVA F- test <0.001, p-value Tukey t-test <0.05 for every pair measured), being the steepest decrease among patients aged 0–18 years (see Table 1 ). Figure 1 Kernel diagram with TGA-IgA antibodies concentrations at each measure. Reference concentration for positive diagnose (>10 U/mL). Figure 1 Kernel diagram with TGA-IgA antibodies concentrations at each measure. Reference concentration for positive diagnose (>10 U/mL). The multivariate mixed model ( Table 3 ) showed a better fit (lower AIC score and higher determination coefficient R-square) for the direct association between TGA-IgA antibodies concentrations logarithm and the number of digestive disorders as well as the reported transgressions (ß = 0.146, p-value = 0.115 and ß = -0.530, p-value = 0.017, respectively). The model also showed an inverse relationship between antibodies' concentrations and the number of extra-digestive disorders (ß = - 0.164, p-value = 0.056). Sensitivity analysis did not report significant changes (see Table 3 ). Table 3 Output from the fitted mixed multivariate model before backward procedure and sensitivity analyses. Table 3 Covariates Total sample (n = 254) Excluding non-confirmed cases (n = 183) β[CI 95%] p-value β[CI 95%] p-value N of digestive disorders 0.146 [−0.035, 0.327] 0.115 0.175 [−0.020, 0.370] 0.081 N of extra-digestive disorders −0.164 [−0.331, 0.004] 0.056 −0.274 [−0.478, −0.070] 0.009 ∗ Transgressions (reference 'no') 0.530 [0.095, 0.965] 0.017 ∗ 0.670 [0.162, 1.178] 0.010 ∗ Adjustment variables Age (years) 0.006 [−0.005, 0.017] 0.299 0.008 [−0.005, 0.021] 0.236 Sex (reference 'male') 0.319 [−0.124, 0.762] 0.159 0.173 [−0.353, 0.700] 0.519 2nd measure time (reference 1st) 1.661 [1.146, 2.176] <0.001 ∗ 1.960 [1.351, 2.569] <0.001 ∗ 3rd measure time (reference 1st) −2.402 [−2.916, −1.887] <0.001 ∗ −2.250 [−2.859, −1.641] <0.001 ∗ 4th measure time (reference 1st) −3.010 [−3.615, −2.585] <0.001 ∗ −2.908 [−3.518, −2.299] <0.001 ∗ N: number. CI: Confidence interval. ∗ Significant p-value < 0.05. Output from the fitted mixed multivariate model before backward procedure and sensitivity analyses. N: number. CI: Confidence interval. Significant p-value < 0.05.

From 2007 to 2017, a prospective observational study was carried out in 254 participants, who were recruited from patients of the Hospital San Agustín (Linares, Jaén), which is responsible for a population area of 129,233 inhabitants (2018) in southern Spain. Only patients with IgA negative tissue transglutaminase antibody serology (<10 U/mL) were excluded from the study. From the total sample (mean age [95% CI] = 24.3 ± [23.1–25.5] years), 212 were registered with a confirmatory diagnosis of CD, 18 were kept with a diagnosis of suspected CD (with symptomatology and positive serology but pending CD confirmation), and 24 were considered silent patients (positive serology but without symptomatology) [ 13 ]. The protocol of this study was approved by the Ethics Committee of the San Agustín Hospital. Study participants were informed of the objectives and the scope of the study and signed an informed consent for their participation. Criteria for patient's inclusion in the study followed the below protocol: positive serological test result (IgA tissue anti-transglutaminase antibodies ≥10 U/mL), determination of HLA DQ2/DQ8 haplotypes and confirmation by duodenal biopsy (evaluated by Marsh-Oberhuber classification [ 14 ]). TGA-IgA antibodies were determined by Fluorescent Enzyme Immunoassay (EliA Celikey® IgA Well), in the ImmunoCAP 250 autoanalyzer from Phadia Thermofisher®. The HLA DQ2/DQ8 haplotypes were determined using the GENVINSET HLA CELIAC® kit [ 15 ]. Briefly, preparation of the duodenal biopsy is performed using a formalin fixation (10% buffered formalin), paraffin inclusion and hematoxylin-eosin staining (it was used immunohistochemical staining to evaluate Marsh I and II stages) [ 16 ]. Serial biopsies prepared according to suggested were reviewed by a pathologist. Also, antibodies TGA-IgA, total IgA immunoglobulin and antibodies TGA-IgG in patients with selective déficit of IgA, were determined by an external laboratory. According to hospital protocol, first measurement of antibodies is recommended at 3–6 months after the introduction of dietary treatment, with successive measurements every 6 months until the patient's antibody level is normalized [ 16 ]. For every patient included in the study, at least 4 measurements were ensured in order to determine the existence of eating transgressions. The determination of non-compliance to GFD, based on this periodic evaluation of antibodies, was developed as follows: 1) 'inconclusive', when there were less than 3 TGA-IgA antibody values per patient (excluded from the study); 2) 'without transgression', when the progression was clearly descending and evolving towards normal values; 3) 'with transgression' when the positivity of the abnormal level of antibodies persisted, or when the medical specialist, based on the interview with the patient, stated in the medical history that there had been transgressions. Antibodies concentrations depend on gluten, so a persistently positive serology or a lack of decrease in serum levels after one year of GFD indicates poor dietary compliance or unintentional exposure to gluten [ 17 ]. The covariates introduced in this study were: age (years), sex (man, woman), year of CD diagnosis, presence (yes/no) of digestive disturbances: 1) classic pattern (diarrhea, steatorrhea, weight loss); 2) non-classic pattern with unspecific digestive symptoms, actually more frequent (abdominal pain, vomiting, epigastralgia, constipation, bloating, dyspepsia, loss of appetite), presence (yes/no) of extra-digestive disorders derived from in much of micronutrient malabsorption (muscle, respiratory, skin, liver, neurology, anemia, allergies, osteopenia/osteoporosis, oral thrush), determination of the level of TGA-IgA antibodies, presence (yes/no) of food transgressions, presence (yes/no) of diseases associated with CD, detected in the patients included in this study (diabetes mellitus, thyroid disorder, neurological and psychiatric disorders such as attention deficit disorder and hyperactivity, dermatitis herpetiformis, irritable colon, Crohn's disease, ulcerative colitis, erosive duodenitis, alopecia areata, infertility, cancer, Down's syndromes, Asperger's, Sjögren and poliendocrine, obesity, Raynaud's disease, and results of biochemical tests associated with CD (iron (μg/dL) and ferritin (ng/mL)). Mean (M) and standard deviation (SD) were calculated for antibodies TGA-IgA concentrations according to sex and the age-range of the study population. The Pearson Chi-square test and the ANOVA F-test were applied to the TGA-IgA concentrations to compare the mean of sex and age range across categories. Probable association between TGA-IgA repeated measures, as the response variable, and predictive covariates, was assessed by using mixed multivariate models. Predictors for the response variable were adjusted according to the following procedure: (1) A basal model was built following a backward elimination procedure considering the complete 254 participants: starting from model including all covariates related to the TGA-IgA concentrations at p-value 0.10. The model with lower AIC [ 18 ] and higher determination coefficient R 2 was selected. Sex, age and time of each measure (from first to fourth time point) were included for adjustment purposes in all models, regardless of their statistical significance [ 17 ]. The predictors effect was considered statistically significant if the p-value < 0.05, although marginally significant effects (p-value <0.1) were also considered. Sensitivity analyses were carried out to evaluate the robustness of the multivariate model by excluding all non-confirmed cases using Marsh scale (<level 3). The validity of the regression models was tested by residual analyses. No influential data were identified by graphical representation. Collinearity diagnostics were conducted on the final models. Analyses were conducted with the statistical software R, version 3.5.1 [ 19 ].

This study has observed differences in digestive and extra-digestive clinical manifestations according to sex and age. Also, TGA-IgA antibodies concentrations were directly associated with the number of digestive disorders manifested by the patient and the record of dietary non-compliance, and inversely related to the number of extra-digestive disorders. Differences in digestive and extra-digestive symptoms were observed. According to sex and age, a higher level of villus atrophy in children under 18 years of age and a progressive decrease in the level of TGA-IgA antibodies, were directly associated with presence of digestive disorders, registry of dietary transgressions and inversely related to the presence of extra-digestive disorders. The female trend in the frequency of diarrhoea and abdominal pain had been previously observed in other studies [ 21 ]. However, in another study, also in Minnesota, it was observed that the prevalence of abdominal pain and diarrhoea was higher in men [ 22 ]. Possible causes of atypical manifestations, such as abdominal pain, could be based on the superposition of other functional disorders such as irritable bowel syndrome, where CD is 3–4 times more prevalent, or an undiagnosed intolerance to food components [ 23 ]. In our study, we observed that patients >18 years were associated with a higher frequency for abdominal pain and weight loss. A recent pilot study in adult patients with seronegative CD, showed weight loss and abdominal pain as a characteristic form of presentation of the illness [ 24 ]. There are other studies in celiac adults they also include weight loss and abdominal pain among its main symptoms [ 25 ]. Another study carried out in the Netherlands in 412 celiac adults, reported the following results in order of prevalence: diarrhea, fatigue, weight loss and abdominal pain [ 26 ], similar to those obtained in the present study. On the other hand, children under 18 years of age were more frequently associated with vomiting in our study, which is consistent with research carried out in 16 countries in the Mediterranean area on 749 celiac children, divided by age groups, which it related vomiting as the most prevalent symptom in the age group between 6-10 years [ 27 ]. However, in a multicenter study carried out in Spanish children, divided by age groups (0–2, > 2–6, > 6–15 years), it was associated with other symptoms such as bloating (6 years) [ 28 ]. Regarding non-classical or atypical extra-intestinal alterations, our study related them to the group of 12–18 years. This fact is also supported by other studies. In this sense, it is worth mentioning a study developed in the United States, which observed that the mean age at the time of diagnosis for patients with classic CD was 8.09 years with symptoms such as diarrhoea and developmental delay. However, the mean age for children with non-classical CD was 10.19 years and it was associated with extra-intestinal manifestations [ 29 ]. Another study carried out in 165 children (2–18 years old) from New York, showed a mean age of diagnosis for CD of 10.7 years, most of them associated with non-classic manifestations such as abdominal pain and constipation. The authors pointed out that this increase in the presentation of CD in its non-classical form could be due to changes in eating habits and a greater consumption of processed food products that contribute to a greater exposure to the gliadin antigen [ 30 ]. It is well known that the antibody titter decreases with age [ 31 , 32 ]. A proposed mechanism for this phenomenon is based in the theory of the sequestration of TGA-IgA antibodies over time by the intestinal mucosa [ 32 ]. The results of the present study indicate a higher antibody titter at younger ages, although they reach similar values to the group of adults in the fourth serological measure. This result was also observed in a study conducted in northern Spain (97 children and 227 adults), in which the correlation of TGA-IgA antibodies and the degree of villous atrophy was evaluated to avoid duodenal biopsy. Adults showed less severe histopathology and lower antibody titers than children [ 20 ]. The results of the present study also highlight that those under 18 years were associated with the highest degree of intestinal villous atrophy (3c-severe lesion according to the Marsh-Oberhuber classification in duodenal biopsy), compared to those over 18 years of age, associated with a degree of injury 3a-mild and 3b-moderate. This association has also been observed in previous studies. In a multicenter study developed on 974 celiac Spanish children under the age of 15, 46.4% showed a level of atrophy 3b, followed by level 3c in 28.4% of cases, associated with a high prevalence of classic forms (digestive symptoms) [ 28 ]. Another Canadian study of 140 celiac children under 17 years of age found that 42.8% of patients had severe villous atrophy or Marsh 3c [ 33 ]. The most outstanding result associating the level of TGA-IgA antibodies directly with the number of digestive disorders and dietary non-compliance, as well as inversely with extra-digestive disorders is a study of 445 celiac patients in Finland which correlated the highest TGA-IgA antibody titre with the greatest deterioration of the digestive mucosa, and also with the highest presence of digestive symptoms [ 32 ]. In a prospective study in 170 Hungarian patients an increase in the level of TGA-IgA antibodies was shown in CD patients with more severe enteropathy (Marsh 3b and 3c) and a greater degree of gastrointestinal clinical presentation. Furthermore, the study associated a higher degree of recovery in the level of antibodies in children after the GFD [ 34 ]. On the other hand, a study conducted in the Netherlands in 116 celiac children showed that those with TGA-IgA antibodies ≥100 U/mL had a higher prevalence of extra-intestinal symptoms than children with levels of TGA-IgA antibodies <100 U/mL, which manifested more gastrointestinal symptoms. The authors suggested that those patients with high antibody titers, extra-digestive manifestations and more severe digestive injuries, were related to a more advanced or generalized disease [ 35 ]. However, other studies do not link the level of antibodies with age or the symptoms of CD. For example, a study carried out in 59 celiac children from Saudi Arabia studied the clinical presentation, serology and the degree of atrophy of the disease, noting that although the children had the highest degree of villous atrophy, it was not related to TGA-IgA antibody levels [ 36 ]. In another wider study conducted in the Netherlands which included 4,442 children for screening of CD TGA-IgA antibodies, there was also no relationship between gastrointestinal symptoms, the degree of enteropathy and the level of TGA-IgA antibodies [ 37 ]. On the other hand, celiac patients are more susceptible to develop other diseases associated with CD, possibly by sharing the same genetic component, which show extra-digestive symptoms. The most common are type 1 diabetes mellitus, autoimmune thyroiditis, and inflammatory bowel disease. These comorbidities present an heterogeneous immune response which can be confound the diagnosis of CD based on the antibody titer [ 38 , 39 ]. Little is known about the dynamics of antibody reduction after GFD and the effect of non-compliance. After treatment, around 80% of patients offer negative serologies at 6–12 months and 90% at 5 years [ 17 ]. Non-compliance with GFD, has been significantly related to increased serum TGA-IgA antibody level [ 17 ] (45.7% of patients committed dietary non-compliance during the present study). The relationship between the level of TGA-IgA and dietary non-compliance has been well established in previous studies [ 40 , 41 ]. An Italian study in 204 patients, which divided the sample into two age groups, reported eating transgressions in 26.5% of the sample, with the serum level of TGA-IgA antibodies increased in children (13 years old) [ 40 ]. Another Italian study in 2,245 celiac patients, developed a follow-up strategy with annual serologies (TGA-IgA antibodies) and periodic clinical visits, observing that a third of the patients presented fluctuating serologies due to unstable adherence to GFD [ 41 ]. There are limitations in the present study: (1) a bias derived from the study design is the lack of knowledge about the presence of previous diseases associated with CD before the diagnosis; (2) not known is if the patient starts the gluten-free diet right after the CD confirmed diagnosis; a precise period of time has been studied including patients with a previous EC diagnosis and new diagnosed; (3) a relatively small sample size, although the follow-up period was long and the low prevalence of CD in southern Europe (260–1000 cases/100,000) should be considered [ 42 ]; (4) the lack of inclusion in the study of other serum antibodies such as antiendomysium or antigliadin, although tissue antitransglutaminase was chosen for its sensitivity and specificity in diagnosing and monitoring the disease. On the contrary, this study strengths are: (1) obtention of the data directly from the medical records and not by means of questionnaires, which avoids biases both from the interviewer and patient's suggestion; (2) the statistical analysis, which also includes the performance of sensitivity analysis and confounding assessment; and (3) the study's prospective design, which made it possible to obtain detailed information concerning biological and clinical characteristics that may affect antibodies concentrations outcomes. In conclusion, clinical manifestations on CD showed differences in digestive and extra-digestive symptoms according to sex and age. Women showed a greater tendency to suffer from diarrhea and abdominal pain, those over 18 years were associated with a higher frequency of abdominal pain and weight loss, and those under 18 years were associated with the presence of vomiting, as well as a higher level of villous atrophy. TGA-IgA antibodies levels were directly associated with the number of digestive disorders manifested by the patient and the record of dietary non-compliance, and inversely related to the number of extra-digestive disorders. The less monitored group and with more extradigestive disorders reported were those between 12 and 18 years old. Therefore, it is necessary to develop strategies in clinical practice aimed at this population group and continuous monitoring should be implemented to improve life quality and reduce complications that may arise in the long term.

Marta Miró: Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper. Manuel Alonso-Garrido: Analyzed and interpreted the data; Wrote the paper. Manuel Lozano, Lara Manyes: Conceived and designed the experiments. Juanjo Peiró: Analyzed and interpreted the data. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors do not have permission to share data. The authors declare no conflict of interest. No additional information is available for this paper.

The only effective treatment for celiac disease (CD) is a strict gluten free diet (GFD). However, the reported celiac adult compliance varies between 17-45% [ 1 ]. Among other factors, it depends on the age of the patient or the age at the time of diagnosis, especially during the puberty and adolescence, where there is a greater autonomy in dietary decisions, making it hard to comply with the GFD [ 2 ]. The occasional ingestion of gluten does not usually produce any symptoms, although it does affect the intestinal mucosa causing inflammation and increasing the risk of long-term complications [ 3 ]. In clinical practice, CD is monitored through TGA-IgA antibody levels, since the normalization of serum levels in successive periodic measurements indicates good response and adherence to treatment. A dietary interview, along with each antibody measurement, is recommended in order to detect non-compliance with the GFD [ 4 ]. Once gluten is removed from the diet, levels of TGA-IgA antibodies should stabilize in the next 6–12 months and damage to the digestive mucosa should be reversed in around 6–24 months [ 5 ]. CD is an inflammatory, chronic, and multisystem disorder mediated by the immune system, with a prevalence of 1–3%; It is triggered by the ingestion of water-insoluble proteins (gluten) contained in cereals such as wheat, barley and rye, in genetically predisposed individuals [ 6 ]. A delay in the diagnosis of CD increases the risk of developing other immune disorders due to the duration of exposure to gluten, so there is evidence of a gluten-related autoimmunity in genetically predisposed people, which may develop autoimmune disorders involving other organs than the intestine; Thus, CD has been associated with endocrine manifestations (autoimmune thyroiditis, Addison, type I diabetes mellitus), liver (autoimmune hepatitis, primary biliary cirrhosis), dermal (herpetiform dermatitis) and connective tissue (Sjögren's syndrome), among other immune diseases [ 7 , 8 ]. Furthermore, previous studies reported that prevalence of concomitant autoimmune disease increases with age at the diagnosis of celiac disease [ 9 ]. Delay in diagnosis CD could also be associated with the atypical clinical presentation profile [ 10 ]. Currently, the atypical CD form is the most common with non-digestive symptoms. The increase in severity in these symptoms not related to CD has been associated with an heterogeneous immune response which is able to interfere with the diagnosis [ 11 ]. Therefore, early detection of CD is necessary in order to reduce the risk due to continuous gluten exposure, especially in patients with extraintestinal manifestations which can lead to a late diagnosis of the disease, unlike patients with classical CD whose typical gastrointestinal symptoms allow an early diagnosis [ 12 ]. The aim of this study is to evaluate the factors association with the evolution of TGA-IgA antibodies and with dietary non-compliance and diseases related with CD.