[Analysis of the relationship between intestinal segmented filamentous bacteria and rotavirus infection in children].

Objective: To investigate the association between intestinal colonization of segmented filamentous bacteria (SFB) and the risk of rotavirus infection, and the possible mechanisms by which SFB resist rotavirus infection. Methods: This case-control study enrolled 50 children aged 0 to 5 years who present to the outpatient Department of Children's Hospital, Zhejiang University School of Medicine with diarrhea and positive stool tests for rotavirus. The children were divided into rotavirus enteritis group and control group consisting of 55 children with non-gastrointestinal and non-infectious surgical diseases.The age and sex composition of the two groups was matched. The DNA of the fecal flora was extracted and SFB was detected by real-time fluorescence quantitative PCR analysis. The children in the rotavirus enteritis group and the control group were subgrouped by age and sex to analyze the differences in SFB positivity rates between different groups, and further compare and analyze the differences in SFB positivity rates between these two groups of children in the ≤2 years old subgroup and the >2-5 years old subgroup. Neutralization test was performed with p3340 protein and rotavirus to determine the relationship between rotavirus infection rate and p3340 concentration in Vero cells. χ2 test or Fisher's exact probability method was used for comparison between the two groups. Results: There were 50 children in the rotavirus enteritis group with an age of (1.7±0.9) years, and 55 children in the control group with an age of (1.8±1.1) years. The positive rate of SFB in children with rotavirus enteritis showed a declining trend across ages groups, with the highest rate of 10/14 in the ≤1 year old group, followed by 67% (14/21) in the >1-2 years old group, 9/15 in the >2-5 years old group, and there was no statistically significant difference (P=0.867). The positive rate of SFB in the control group was 12/15 in the ≤1 year old group, 95% (19/20) in the >1-2 years old group, 50% (10/20) in the >2-5 years old group, with statistical significance (P=0.004). The positive rate of SFB in children with rotavirus enteritis was 74% (20/27) in males and 56% (13/23) in females (χ2=1.71, P=0.192). In the control group, it was 79% (22/28) in males and 70% (19/27) in females (χ2=0.49, P=0.485). The positive rate of SFB was 66% (33/50) in the rotavirus enteritis group and 75% (41/55) in the control group, with no statistically significant (χ2=0.56, P=0.454). In the children ≤2 years old, the SFB positivity rate was 69% (24/35) in the rotavirus enteritis group and 89% (31/35) in the control group, with a statistically significant difference (χ2=4.16, P=0.041). However, in the children >2-5 years old, no statistically significant difference was observed, with the positive rate of SFB being 9/15 in the rotavirus enteritis group and 50% (10/20) in the control group (P=0.734). Pearson correlation analysis revealed a negative correlation between rotavirus infection and SFB positivity (r=-0.87,P<0.001). As the concentration of the p3340 specific protein increased, the luminescence intensity of the luciferase in the Vero cells, which were suitable for cultivating rotavirus, exhibited a decreasing trend (F=4.17, P=0.001). Conclusions: SFB colonization in infants less than 2 years old is associated with a reduced risk of rotavirus infection. Cloning of specific SFB functional protein p3340 neutralizes rotavirus infection of Vero cells, and this mechanism of targeting rotavirus infection differs from the common antiviral mechanism.

[The association between Helicobacter pylori virulence factor genotypes and gastroduodenal diseases in children].

Objective: To investigate the association between Helicobacter pylori (Hp) virulence factor genotypes and the degree and activity of gastric mucosa pathological changes in pediatric gastroduodenal diseases. Methods: This retrospective cohort study was conducted from May 2020 to October 2020. The frozen strains of Hp, which were cultured with the gastric mucosa of 68 children with gastroscopy confirmed gastroduodenal diseases who visited the children's hospital of Zhejiang University School of Medicine from April 2012 to December 2014, were resuscitated. After extracting DNA from these Hp strains, PCR amplification and agarose gel electrophoresis were performed to determine the detection rate of cytotoxin-associated protein A (cagA),vacuolating cytotoxin A (vacA)(s1a、s1b/s2,m1/m2), outer inflammatory protein A (oipA),blood group antigen binding adhesin (babA),duodenal ulcer promoting protein A (dupA) genes; oipA genes were sequenced to determine the gene status. The patients were divided into different groups according to the findings of gastroscopy and gastric mucosa pathology. The detection rates of various virulence factor genotypes among different groups were compared using χ2 tests or Fisher's exact tests. Results: The 68 Hp strains all completed genetic testing. According to the diagnostic findings of gastroscopy, the 68 cases were divided into 47 cases of superficial gastritis and 21 cases of peptic ulcer. Regarding the pathological changes of gastric mucosa, 8 cases were mild, and 60 cases were moderate and severe according to the degree of inflammation; 61 cases were active and 7 cases inactive according to the activity of inflammation. The overall detection rates of cagA, vacA, vacA s1/m2, functional oipA, babA2, and dupA virulence factor genes were 100% (68/68), 100% (68/68), 94% (64/68), 99% (67/68), 82% (56/68), and 71% (48/68), respectively. In the superficial gastritis group, their detection rates were 100% (47/47), 100% (47/47), 96% (45/47), 98% (46/47), 81% (38/47), and 70% (33/47), respectively; in the peptic ulcer group, their detection rates were 100% (21/21), 100% (21/21), 90% (19/21), 100% (21/21), 86% (18/21), and 71% (15/21), respectively. There was no statistically significant difference between the two groups (all P>0.05). In the mild gastric mucosa inflammation group, the detection rates of the above six genotypes were 8/8, 8/8, 8/8, 7/8, 7/8, and 5/8, respectively; and in the moderate to severe inflammation groups, the detection rates were 100% (60/60), 100% (60/60), 93% (56/60), 100% (60/60), 82% (49/60), and 72% (43/60), respectively, with no statistically significant difference between the two groups (all P>0.05). In the active inflammation group, the detection rate of six genotypes were 100% (61/61), 100% (61/61), 93% (57/61), 98% (60/61), 82% (50/61), and 72% (44/61), respectively; and in the inactive inflammation group, they were 7/7, 7/7, 7/7, 7/7, 6/7, and 4/7, respectively. Again, there was no statistically significant difference between the two groups (all P>0.05). There was no statistically significant difference in the detection rate of combinations of 4 or 5 virulence factor genes among the different groups (all P>0.05). Conclusions: CagA, vacA, vacA s1/m2, functional oipA, babA2, and dupA genes are not associated with superficial gastritis and peptic ulcer in children, or with the degree and activity of gastric mucosa pathological inflammation. Different gene combinations of cagA, vacA, oipA, babA2, and dupA have no significant effects on predicting the clinical outcome of Hp infection in children.

[The relationship between genetic polymorphism of CYP2C19 and the efficacy of Helicobacter pylori eradication therapy in children].

Objective: To investigate the relationship between genetic polymorphisms of cytochrome P450 2C19 (CYP2C19) and the efficacy of Helicobacter pylori (Hp) eradication therapy in children. Methods: The retrospective cohort study was conducted on 125 children with gastroscopy and positive rapid urease test (RUT) from September 2016 to December 2018 who presented to the Children's Hospital of Zhejiang University School of Medicine due to gastrointestinal symptoms including nausea, vomiting, abdominal pain, bloating, acid reflux, heartburn, chest pain, vomiting blood and melena. Hp culture and drug susceptibility test were carried out with gastric antrum mucosa before treatment. All the patients completed 2 weeks of standardized Hp eradication therapy and had 13C urea breath test 1 month after that, which was used to evaluate the curative effect. The DNA of gastric mucosa after RUT was analyzed and CYP2C19 gene polymorphism was detected. Children were grouped according to metabolic type. Combined with the results of Hp culture and drug susceptibility, the relationship between CYP2C19 gene polymorphism and the efficacy of Hp eradicative treatment was analyzed in children. Chi square test was used for row and column variables, and Fisher exact test was used for comparison between groups. Results: One hundred and twenty five children were enrolled in the study, of whom 76 were males and 49 females. The genetic polymorphism of CYP2C19 in these children found poor metabolizer (PM) of 30.4% (38/125), intermediate metabolizer (IM) of 20.8% (26/125), normal metabolizer (NM) of 47.2% (59/125), rapid metabolizer (RM) of 1.6% (2/125), and ultrarapid metabolizer (UM) of 0. There were statistically significant in positive rate of Hp culture among these groups (χ2=124.00, P<0.001). In addition, the successful rates of Hp eradication in PM, IM, NM and RM genotypes were 84.2% (32/38), 53.8% (14/26), 67.8% (40/59), and 0, respectively, with significant differences (χ2=11.35, P=0.010); those in IM genotype was significantly lower than that in PM genotype (P=0.011). With the same standard triple Hp eradicative regimen, the successful rate of Hp eradication for IM type was 8/19, which was lower than that of PM (80.0%, 24/30) and NM type (77.3%, 34/44) (P=0.007 and 0.007, respectively). There was a significant difference in the efficacy of Hp eradication treatment among different genotypes (χ2=9.72, P=0.008). According to the clarithromycin susceptibility result, the successful rate of Hp eradication treatment for IM genotype was 4/15 in the sensitive group and 4/4 in the drug-resistant group (χ2=6.97, P=0.018). Conclusions: The genetic polymorphism of CYP2C19 in children is closely related to the efficacy of Hp eradication treatment. PM has a higher successful rate of eradication treatment than the other genotypes.

[The effect of Helicobacter pylori infection on duodenal bulbar microbiota in children with duodenal ulcer].

Objective: To investigate the characteristics of duodenal bulbar microbiota in children with duodenal ulcer and Helicobacter pylori (Hp) infection. Methods: This prospective cohort study enrolled 23 children with duodenal ulcers diagnosed by gastroscopy who were admitted to the Children's Hospital of Zhejiang University School of Medicine due to abdominal pain, abdominal distension, and vomiting from January 2018 to August 2018. They were divided into Hp-positive and Hp-negative groups according to the presence or absence of Hp infection. Duodenal bulbar mucosa was sampled to detect the bacterial DNA by high-throughput sequencing. The statistical difference in α diversity and ß diversity, and the relative abundance in taxonomic level between the two groups were compared. Microbial functions were predicted using the software PICRUSt. T-test, Rank sum test or χ2 test were used for comparison between the two groups. Results: A total of 23 children diagnosed with duodenal ulcer were enrolled in this study, including 15 cases with Hp infection ((11.2±3.3) years of age, 11 males and 4 females) and 8 cases without Hp infection ((10.1±4.4) years of age, 6 males and 2 females). Compared with Hp-negative group, the Hp-positive group had higher Helicobacter abundance (0.551% (0.258%, 5.368%) vs. 0.143% (0.039%, 0.762%), Z=2.00, P=0.045) and lower abundance of Fusobacterium, Streptococcus and unclassified- Comamonadaceae (0.010% (0.001%, 0.031%) vs. 0.049% (0.011%, 0.310%), Z=-2.24, P=0.025; 0.031% (0.015%, 0.092%) vs. 0.118% (0.046%, 0.410%), Z=-2.10, P=0.036; 0.046% (0.036%, 0.062%) vs. 0.110% (0.045%, 0.176%), Z=-2.01, P=0.045). Linear discriminant analysis (LDA) effect sized showed that at the genus level, only Helicobacter was significantly enriched in Hp-positive group (LDA=4.89, P=0.045), while Streptococcus and Fusobacterium significantly enriched in Hp-negative group (LDA=3.28, 3.11;P=0.036,0.025, respectively). PICRUSt microbial function prediction showed that the expression of oxidative phosphorylation and disease-related pathways (pathways in cancer, renal cell carcinoma, amoebiasis, type 1 diabetes mellitus) in Hp-positive group were significantly higher than that in Hp-negative group (all P<0.05), while the expression of pathways such as energy metabolism and phosphotransferase system pathways were significantly lower than that in Hp-negative group (all P<0.05). Conclusion: In children with Hp-infected duodenal ulcers, the mucosal microbiota of the duodenal bulb is altered, characterized by an increased abundance of Helicobacter and a decreased abundance of Clostridium and Streptococcus, and possibly alters the biological function of the commensal microbiota through specific metabolic pathways.

[Analysis of clinical characteristics of 126 children with recurrent intussusception].

Objective: To analyze and summarize the clinical features in children with recurrent intussusception. Methods: This retrospective cohort study collected the clinical data of 126 children with recurrent intussusception who were admitted to the Children's Hospital of Zhejiang University School of Medicine due to "abdominal pain, paroxysmal crying, vomiting, bloody stools" from January 1, 2015 to November 30, 2019. The clinical manifestations of recurrent intussusception between ≤3 years old group and >3 years old group were compared, the etiology and age characteristics of pathologic lead points (PLP) were analyzed, and the clinical characteristics of PLP group and non-PLP group were also compared. The χ2 test and Mann-Whitney U test were used to compare the differences between groups. Results: A total of 126 children with recurrent intussusception were included, of whom 76 were males and 50 were females, with the age of 2.9 (1.7, 5.1) years. The proportion of children aged more than 1 year was 87.3% (110/126), and 48.4% (61/126) more than 3 years. Clinical manifestations mostly lacked the typical triad of symptoms. The percentage of paroxysmal crying in ≤ 3 years old group was significantly higher than that in >3 years old group (52.3% (34/65) vs. 24.6% (15/61), χ2=10.17, P=0.001), while the percentage of abdominal pain was significantly lower than that in the >3 years old group (46.1% (30/65) vs. 75.4% (46/61), χ2=11.25, P=0.001). The rate of positive ultrasound examination was 17.5% (22/126), and 63.6% (14/22) of them were diagnosed. The positive rate of CT examination was 4/13, of which 2 cases were diagnosed. In this study, 37 children were diagnosed with PLP by colonoscopy, laparoscopy or laparotomy, and 89 children were found without PLP. The positive rate of PLP in >3 years old group was significantly higher than that in ≤3 years old group (37.7% (23/61) vs. 21.5% (14/65), χ2=3.96, P=0.046). Meckel's diverticulum and juvenile polyp were the main contributors of PLP in ≤3 years old group, accounting for 7/14 and 3/14 respectively, while lymphoma and juvenile polyp accounted for 34.8% (8/23) and 26.1% (6/23), respectively in >3 years old group. Compared with non-PLP group, PLP group had higher age (5.2 (1.6, 6.7) vs. 2.7 (1.8, 4.2) years, Z=-2.26, P=0.01). However, there were no significant differences in gender and recurrence frequency between the two groups (both P>0.05). Conclusions: Recurrent intussusception is more common in children more than 1 year old, and has a wide spectrum of non-specific clinical presentations. Imaging examinations can be used to identify PLP. The most recurrent intussusception is idiopathic, but the presence of PLP should be alerted for, such as Meckel's diverticulum, lymphoma and juvenile polyp. Colonoscopy sometimes is necessary, surgical exploration and treatment should be carried out in time.

[Characteristics of gastric mucosa microbiota in children with chronic gastritis and duodenal ulcer].

Objective: To investigate the differences of gastric mucosa microbiota between children with chronic gastritis and duodenal ulcer under the condition of Helicobacter pylori (Hp) infection. Methods: This prospective cohort study involved 57 children with Hp infection diagnosed by gastric endoscopy who were admitted to the Children's Hospital of Zhejiang University School of Medicine due to "abdominal pain, abdominal distension and vomiting" between January 2018 to August 2018. According to gastroscopy and pathological examination, the children were divided into chronic gastritis group and duodenal ulcer group. Gastric mucosa from Hp infected patients were sampled, and the flora DNA was analyzed by high-throughput sequencing. The statistical difference of α diversity, ß diversity between two groups were analyzed. The relative abundance of the two groups in each taxonomic level was analyzed statistically. T test, Rank sum test or χ2 test was used for comparison between the two groups. Results: A total of 57 children diagnosed with Hp infection were enrolled in this study, including 42 cases of chronic gastritis (the age was (9.3±2.8) years, 22 males and 20 females) and 15 cases of duodenal ulcer (the age was (11.1±3.3) years, 9 males and 6 females). Alpha diversity index Chao and ACE in Hp infected chronic gastritis group were significantly higher than those in Hp infected duodenal ulcer group (217±50 vs. 183±64, t=2.088, P=0.009;218±47 vs. 192±76, t=1.566, P=0.016, respectively). The Beta-diversity index such as nonmetric multidimensional scaling (NMDS) analysis were significantly different in the two groups (analysis of similarity R=0.304, P=0.028). Among the main bacteria genera, there were 6 genera with significant differences between the two groups, which were Prevotella (0.190% (0.008%-1.983%) vs. 0.021% (0.005%-2.398%), Z=-2.537, P=0.011), Alloprevotella (0.097% (0.010%-0.813%) vs. 0.015% (0.003%-0.576%), Z=-2.492, P=0.013), Haemophilus (0.109% (0.004%-0.985%) vs. 0.014% (0.004%-0.356%), Z=-2.900, P=0.004), Neisseria (0.074% (0.004%-0.999%) vs. 0.024% (0.003%-0.255%), Z=-2.718, P=0.007), Streptococcus (0.166% (0.008%-1.869%) vs. 0.045% (0.006%-0.879%), Z=-2.537, P=0.010), and an unclassified-Microbacteriaceae (0.214% (0.060%-1.762%) vs. 0.117% (0.010%-0.954%), Z=-2.120, P=0.034). Linear discriminant analysis (LDA) effect sized analysis showed that at the genus level, only Prevotella was significantly enriched in the duodenal ulcer group (LDA=2.90, P=0.010), while Streptococcus, Neisseria and Haemophilus were significantly enriched in the chronic gastritis group (LDA=2.83, 2.82, 2.69, P=0.011, 0.007, 0.004, respectively). Conclusions: The gastric mucosal microbiota in duodenal ulcer associated with Hp is significantly different from that in chronic gastritis. Hp may promote the occurrence of peptic ulcer together with gastric microbiota.

[Eosinophilic esophagitis in children: analysis of 22 cases].

Objective: Eosinophilic esophagitis (EoE) is a chronic immune-mediated esophageal disease.The current domestic reports of EoE in children is rare.The aim of this study was to analyze the clinical features, the diagnosis and treatment advance of EoE in children by case analysis and literature review. Method: Clinical data of 22 children with EoE from January, 2011 to December, 2015 in Children's Hospital, Zhejiang University School of Medicine were recorded, retrospective analysis was performed on clinical presentation, gastroendoscopy and histopathological examination features and the treatment. Result: (1) Clinical data: EoE can occur at any age in children (5 months to 13 years). The most common clinical manifestations of EoE are vomiting and abdominal pain, 45% (10/22) and 41%(9/22) respectively. (2) Endoscopy and pathological features of esophageal mucosa: 11 cases with coarse mucous membrane (50%), 6 cases with congestion or erosion of esophageal membrane (27%), 5 cases with longitudinal crack (23%), 3 cases with ring uplift (14%), 3 cases with granular uplift (14%), 3 cases with normal mucosa(14%). Histopathologic manifestation is eosinophil infiltration and the eosinophil counts were all more than or equal to 15/HP. (3) Laboratory results: 13 cases had increasing eosinophil counts and eosinophils proportion (62%). (4)Allergy history: among 22 cases, 7 patients had allergy history (32%). (5) Situation of treatment and remission: 16 cases had clinical remission by oral omeprazole; 2 cases had clinical remission by oral Omeprazole and Montelukast sodium; 1 case acquired remission by elimination diet; 1 case acquired remission by elimination diet and oral prednisone. 2 cases dropped out; Only 2 patients received gastroendoscopy re-examination after 3 months and revealed esophageal mucosal histologic complete recovery. Conclusion: The clinical symptoms of EoE in children varies.Esophageal mucosal features of gastroendoscopy examination in children with EoE were longitudinal crack, white exudates or plaques, paper mucosa, ring uplift and granular uplift.Most patients could achieve remission by using proton-pump inhibitors, only few children needed elimination diet and change formula, or even oral glucocorticoids.

Role of proximal gastric acid reflux in causation of respiratory symptoms in children with gastroesophageal reflux.

OBJECTIVES: The association of gastroesophageal reflux (GER) and respiratory disorders is well known but the mechanism is still unclear. This study aims to evaluate the presence and severity of proximal gastric acid reflux in children having GER with or without respiratory symptoms. METHODS: 24 hour esophageal pH monitoring with a dual pH probe placed in the proximal and distal esophagus was performed in 23 and 31 children having GER with or without respiratory symptoms, respectively. RESULTS: No significant difference in the parameters of pH monitoring in either proximal or distal esophagus was observed between GER patients with or without respiratory symptoms. The proportion of patients having proximal GER among those with respiratory symptoms was not significantly different from those without respiratory symptoms (P > 0.05). CONCLUSION: Proximal esophageal acid reflux does not seem to play a role in the development of persistent respiratory symptoms in children with GER. Distal esophageal acid reflux is the predominant form of reflux in children with GER regardless of the occurrence of respiratory symptoms.