Evaluation of 16S rRNA primer sets for characterisation of microbiota in paediatric patients with autism spectrum disorder.

intestinal microbiota is becoming a significant marker that reflects differences between health and disease status also in terms of gut-brain axis communication. Studies show that children with autism spectrum disorder (ASD) often have a mix of gut microbes that is distinct from the neurotypical children. Various assays are being used for microbiota investigation and were considered to be universal. However, newer studies showed that protocol for preparing DNA sequencing libraries is a key factor influencing results of microbiota investigation. The choice of DNA amplification primers seems to be the crucial for the outcome of analysis. In our study, we have tested 3 primer sets to investigate differences in outcome of sequencing analysis of microbiota in children with ASD. We found out that primers detected different portion of bacteria in samples especially at phylum level; significantly higher abundance of Bacteroides and lower Firmicutes were detected using 515f/806r compared to 27f/1492r and 27f*/1495f primers. So, the question is whether a gold standard of Firmicutes/Bacteroidetes ratio is a valuable and reliable universal marker, since two primer sets towards 16S rRNA can provide opposite information. Moreover, significantly higher relative abundance of Proteobacteria was detected using 27f/1492r. The beta diversity of sample groups differed remarkably and so the number of observed bacterial genera.

The effect of saliva stimulation on the secretion of cortisol during stress and physiological conditions.

OBJECTIVES: Many studies use stimulated saliva for the assessment of cortisol. However, it is not yet clear how stimulation affects the flow of specific markers. The aim was to assess whether stimulation of salivation affects the physiological flow of cortisol during a stressing day as compared to an ordinary day. The second aim was to show how the normalising factor affects the outcome of the study. METHODS: Stimulated saliva was taken from 42 children at 8:00 a.m. and 12:00 a.m. on two separate days one month apart. During the first day, the children were exposed to stress situation, while the second day was considered a control day. The concentration of cortisol was analysed using ELISA. RESULTS: The highest level of cortisol was observed in the morning of the stress day (p 0.99). CONCLUSION: Based on our results, the examination of the cortisol diurnal rhythm is not reliable in stimulated saliva. Moreover, the effect of saliva stimulation has to be taken into account for every marker individually (Fig. 2, Ref. 22).

Specificity of gut microbiota in children with autism spectrum disorder in Slovakia and its correlation with astrocytes activity marker and specific behavioural patterns.

Recent research suggests the involvement of bidirectional gut-brain axis in autism spectrum disorder (ASD). The aim of this study was to establish the role of changed gut microbiota in behavioural and gastrointestinal manifestations, but also in astrocyte activation in children with ASD. Distinct faecal microbiota in children with ASD was found to be more heterogeneous compared to that in neurotypical children. Different bacterial abundance and correlation with behavioural and GI manifestations revealed several bacterial genera possibly important for ASD. Microbial-neuronal cross talk could be accomplished through S100B, released by glial cells, activated by low grade inflammation. More complex studies are required to understand ASD pathogenesis.

Fecal calprotectin levels correlate with main domains of the autism diagnostic interview-revised (ADI-R) in a sample of individuals with autism spectrum disorders from Slovakia.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by impaired social interaction and communication, as well as repetitive behavior and restricted interests. There is convincing evidence that the intestinal inflammation is involved in etiology of ASD. Increased levels of inflammatory markers were shown to be associated with more aberrant behaviors and communication of subjects with ASD. Calprotectin in the feces is produced by activated neutrophils and epithelial cells of the gut mucosa, and its levels reflect local inflammation of the gastrointestinal tract. Concentration of fecal calprotectin was determined by ELISA method in 87 individuals with ASD and 51 controls, of that 29 siblings of children with ASD and 22 non-related controls. In non-relatives significantly lower values of fecal calprotectin were observed than in both subjects with ASD and their siblings. In the group with ASD significant correlations of fecal calprotectin with all domains of the ADI-R diagnostic tool were found: qualitative abnormalities in reciprocal social interaction and communication, restrictive and repetitive patterns of behavior. Results suggest that low grade intestinal inflammation may be one of factors implicated in the pathophysiology of ASD.