Autism diagnosed with autism (X ?= 8 ± 2

Autism is a serious and complex disorder first described in 1943 by Kanner based on brain development (1). The main characteristics of autism are related to socialization issues, communication deficit, repetitive and stereotyped behavior and cognitive inflexibility. There is no biochemical marker that allows diagnosing autism, therefore, it is identified based on the patient´s clinic and behavior (2).In relation to the severity of autism, it has been classified in grades depending on dimensions within the spectrum. For instance, grade 1 refers to a mild deficit and grade 3 the most severe level of nonverbal communication skills (3).There are some clinical conditions present in autism, such as gastrointestinal problems, disorders in neurological development affecting brain function, high prevalence of epilepsy in up to 46% of patients and sleep disorders, such as insomnia or apnea (4). Due to such disability to digest certain proteins, Defeat Autism Now (DNA) protocol is indicated in patients with autism. This is a biological and nutritional treatment proposition published for the first time in 1996 (5). The main goal of this treatment is to help the organism to detox. In this protocol the diet must be free of gluten, casein, additives and refined sugar (6,7), elements associated to dental caries. Oral health of these patients has also specific characteristics. Autistic children prefer soft foods and this makes them more susceptible to dental caries. Additionally, behavior disorders usually render oral hygiene and dental attention more difficult in this type of patients (8). Despite these conditions which favor the development of caries, it is reported that autistic children do not evidence a higher index, considering all their predisposing factors. Therefore, there is a growing interest on the diagnosis through the saliva in relation to the potential benefits to prevent dental caries (9). The objective of this study was to evaluate salivary factors related to caries in autistic patients.Materials and MethodsPopulation of study68 male patients were selected. 34 children had been diagnosed with autism (X ?= 8 ± 2 years) and other 34 boys were included as healthy controls (X ?= 8 ± 2 years). Autistic patients were selected from 2 schools for patients with autism at the city of Caracas, and healthy patients were selected from private schools in the same city. All those patients with grade 1 and 2 diagnosed autism were included. Children with GI disorders, such as gastroesophageal reflux altering oral pH, patients consuming drug which might cause xerostomia or produce gingival hyperplasia, patients with concomitant syndromes, such as Down syndrome and patients with autism grade 3 were excluded. Ethical aspects Bioethical approval was granted by the Bioethics Committee of the Faculty of Dentistry of Universidad Central de Venezuela (Number 0421-2013). All parents were informed in detailed about the study and consent was signed for approval of participation. Clinical evaluationAll patients were evaluated using artificial light and a clinical dental mirror. The presence of caries, gingivitis and dental plaque were measured. In this procedure the number of affected teeth was analyzed through decayed, missing and filled teeth index (DMFT) under World Health Organization 1997 criteria (10). Also, the Simplified Oral Hygiene Index (IHO-S) was measured in six teeth’s surfaces with a score from 0 to 3 to determine the presence of debris (11). All the patient’s parents were asked about the diet of the children, if the follow or not a diet free of gluten, casein and salicylates. Recollection of saliva and pH measurement After the clinical exam a 5 cc of unstimulated whole saliva sample was collected using sterile disposable cups.  Patients were told not to eat or drink two hours before the sample was taken. pH was measured using a Hanna Piccolo manual pH meter, previously calibrated with pH 4 and 7 standards. Then, the sample was cryopreserved and taken to the laboratory and centrifuged at 16000 g for 10 minutes to remove unwanted debris.Calcium and phosphate assessment Calcium content in the saliva sample was estimated by the colorimetric method of Connerty & Briggs (12) using O-cresolphthalein complex. Absorbance was measured at 570 nm in any suitable spectrophotometer or colorimeter. Phosphate was determined by the Fiske and Subbarow (13) method which is based on the ready solubility of the reducible phosphomolybdic acid in isobutyl alcohol. It consists essentially of the reduction of phosphomolybdic acid to the blue complex by shaking the alcoholic extract with an acidified aqueous solution of stannous chloride. Finally, total proteins were estimated by Folin Ciocalteu method and absorbance was measured at 750 nm after 30 minutes (14).Polyacrylamide gel electrophoresis and zymography for protein evaluationSalivary proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) (10% separating gel, 5% stacking gel) according to the method of Laemmli (15). Zymography was performance for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. Gelatin embedded in a polyacrylamide gel was digested by active gelatinases run through the gel. After Coomassie staining, areas of degradation were visible as clear bands against a darkly stained background (16).IgA concentration measurement The determination of total salival IgA was performed by ELISA modified method and the absorbance was measured at 492 nm (17).ResultsCaries and IHO-S indexesIn the group of patients with autism, 79.41% (27 patients) were caries free. Contrary, 73.52% (25 patients) of control group were diagnosed with caries. Patients with autism exhibited less percentage of caries which was statistically significant (p?0.001). DMFT index in autistic patients was 1 ± 1 and controls 3 ± 2. Patients with autism showed a statistically significant less caries index (p?0.001). In terms of oral hygiene, children with autism showed more dental plaque than controls (64.70% vs 61.80%), however no statistically significant (p= 0.042). IHO-S index was 2.23 ± 0.83 in autistic children while it was 1.82 ± 0.60 in healthy children. Patients with autism exhibited a statistically significant (p=0.008) poorer oral hygiene. 

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