Use of Deep-Amplicon Sequencing (DAS), Real-Time PCR and In Situ Hybridization to Detect H. pylori and Other Pathogenic Helicobacter Species in Feces from Children.

BACKGROUND: Detecting Helicobacter pylori in fecal samples is easier and more comfortable than invasive techniques, especially in children. Thus, the objective of the present work was to detect H. pylori in feces from children by molecular methods as an alternative for diagnostic and epidemiological studies. METHODS: Forty-five fecal samples were taken from pediatric patients who presented symptoms compatible with H. pylori infection. HpSA test, culture, real-time quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), direct viable count associated with FISH (DVC-FISH), and Illumina-based deep-amplicon sequencing (DAS) were applied. RESULTS: No H. pylori colonies were isolated from the samples. qPCR analysis detected H. pylori in the feces of 24.4% of the patients. In comparison, DVC-FISH analysis showed the presence of viable H. pylori cells in 53.3% of the samples, 37% of which carried 23S rRNA mutations that confer resistance to clarithromycin. After DAS, H. pylori-specific 16S rDNA sequences were detected in 26 samples. In addition, DNA from H. hepaticus was identified in 10 samples, and H. pullorum DNA was detected in one sample. CONCLUSION: The results of this study show the presence of H. pylori, H. hepaticus, and H. pullorum in children's stools, demonstrating the coexistence of more than one Helicobacter species in the same patient. The DVC-FISH method showed the presence of viable, potentially infective H. pylori cells in a high percentage of the children's stools. These results support the idea that fecal-oral transmission is probably a common route for H. pylori and suggest possible fecal-oral transmission of other pathogenic Helicobacter species.

Retinol-binding protein 4 levels are associated with measures of liver and renal function and oxidant/antioxidant status in obese children.

Serum retinol-binding protein 4 (RBP4) has been proposed as a metabolic risk factor in obesity. We found that RBP4 levels also were associated with liver enzymes and cystatin C. Oxidant stress is a significant feature in obese children with greater values of RBP4 that can mediate the development of comorbidities.

Vitamin D status is linked to biomarkers of oxidative stress, inflammation, and endothelial activation in obese children.

OBJECTIVE: To examine vitamin D, parathyroid hormone, and serum calcium-phosphorus levels relationships to biomarkers of oxidative/nitrosative stress, inflammation, and endothelial activation, potential contributors for vascular complications in obese children. STUDY DESIGN: Cross-sectional clinical study of 66 obese Caucasian children aged 7 to 14 years. Cardiovascular risk factors were assessed. Malondialdehyde and myeloperoxidase as measures of oxidative stress, and plasma nitrite+nitrate, urinary nitrate, and 3-nitrotyrosine as markers of nitrosative stress were measured. Adipocytokines, inflammatory molecules (high-sensitivity C-reactive protein, interleukin-6, and tumor necrosis factor-α), endothelial activation molecules (soluble intercellular adhesion molecule-1, soluble vascular cell adhesion molecule 1 [sVCAM-1]), E-selectin, and vascular endothelial growth factor were also investigated. Serum 25-hydroxy-cholecalciferol [25(OH)D], intact parathormone, and calcium-phosphorus levels were determined in these children and in a comparison group of 39 non-obese children. RESULTS: Obese children had a significantly lower 25(OH)D level (P = .002) and a higher intact parathormone (P = .011) than non-obese children. Phosphorus and the calcium-phosphorus product were also significantly higher (P < .0001). Insufficient serum concentrations of 25(OH)D (<20 ng/mL) were detected in 5% of normal children and in 30% of the obese children. In the obese children with vitamin D insufficiency, malondialdehyde, myeloperoxidase, 3-nitrotyrosine, interleukin-6, and sVCAM-1 were substantially elevated. A partial correlation analysis showed an inverse relationship of 25(OH)D levels with 3-nitrotyrosine (r = -0.424, P = .001), and sVCAM-1 (r = -0.272, P = .032). CONCLUSIONS: Insufficient 25(OH)D levels were detected in severely obese children with increased markers of oxidative/nitrosative stress, inflammation, and endothelial activation.

Oxidative markers in children with severe obesity following low-calorie diets supplemented with mandarin juice.

AIM: To evaluate the effect of supplementing a hypocaloric diet with mandarin juice, a food with a high content of antioxidants (vitamin C, flavonoids and carotenoids), on biomarkers of oxidant/antioxidant status of severe obese children. METHODS: Forty obese children were randomized into two groups pair-wise in a 4-week controlled intervention study. Both groups followed a hypocaloric diet. One group received additionally a supplementation of 500mL of 100% mandarin juice daily. Clinical data, anthropometry, dietary intake and fasting blood samples were collected at baseline and after the intervention. Lipid peroxidation was assessed by circulating levels of malondialdehyde, and protein oxidation was determined by the concentration of plasma carbonyl groups. The antioxidant defence was evaluated by red cell-reduced glutathione and plasma levels of α-tocopherol and vitamin C. RESULTS: The supplemented group experienced a decrease in the levels of malondialdehyde (-9.6%, p =0.014) and carbonyl groups (-36.1%, p =0.006) and an increase in antioxidants (α-tocopherol +16.1%, p=0.006, glutathione +36.1%, p < 0.0001, and vitamin C + 94.6%, p < 0.0001). CONCLUSION: The mandarin juice consumption with a reduced calorie diet positively affects the antioxidant defence and produces a decrease in biomarkers of oxidative stress in obese children.

Is obesity associated with oxidative stress in children?

OBJECTIVE: We evaluated the presence of oxidative stress in obese children without co-morbidities. METHODS: The study population included 68 children (30 girls, 38 boys), between 6 and 14 years of age. The levels of markers of oxidative damage (malondialdehyde [MDA], and plasma carbonyl groups [CG]) and measures of antioxidant defense, such as the enzyme glutathione peroxidase (GPx) and low molecular scavengers (erythrocyte-reduced glutathione [GSH], alpha-tocopherol and beta-carotene) were determined. Children were categorized in groups by the standard deviation score of body mass index (SDS-BMI). Twenty children were non-obese (SDS-BMI< or =1.33), and the 48 obese children (SDS-BMI> or =2) were further divided into two groups: SDS-BMI> or =3 (22 children) and > or =2 SDS-BMI<3 (26 children). RESULTS: The levels of MDA and CG were significantly higher (p<0.05) in children with SDS-BMI> or =3. The GPx activity was increased, while the GSH concentration was lower in obese children compared with non-obese children (p<0.01). There were no differences in serum alpha-tocopherol and beta-carotene levels between groups. MDA was the sole marker of oxidative damage that was positively correlated with SDS-BMI, (r=0.35, p=0.015), and negatively with high-density lipoprotein cholesterol (HDL-C) (r=- 0.32, p=0.027). GPx was inversely related to total cholesterol (r=- 0.34, p=0.019). In multiple regression analysis, we confirmed that SDS-BMI and HDL-C were determinants of MDA. CONCLUSIONS: Severe childhood obesity is associated with oxidative stress. Thus, providing foods with high antioxidant capacity in addition to a hypocaloric diet is crucial for the treatment of obese children.