Correlation Analysis of Helicobacter pylori Infection and Digestive Tract Symptoms in Children and Related Factors of Infection

Background: To analyze the correlation between Helicobacter pylori infection and digestive tract symptoms in children and other related factors, and to explore the risk factors of H. pylori infection in children and the expression of inflammatory factors in H. pylori-positive and H. pylori-negative children. Methods: Overall, 234 children with H. pylori test in Xuzhou Children’s Hospital, Xuzhou Medical University (Xuzhou, China) were enrolled. Among them, 73 children were H. pylori-positive and 161 were H. pylori-negative. The expression levels of cytokines interleukin-8 (IL-8), interleukin-18 (IL-18) and interferon-γ (IFN-γ) in H. pylori-positive and H. pylori-negative children were determined by ELISA. The correlation between H. pylori-positive and general data, digestive tract symptoms, other clinical symptoms, living habits, eating habits, family history and other related factors was statistically analyzed. Multivariate Logistic regression analysis was used to analyze the independent risk factors of H. pylori infection in children. Results: Family monthly income, inattentive eating, sharing toothbrushes and cups, gnawing fingers, eating fried food, drinking raw water, eating smoked and pickled food, father suffering from gastropathy and mother suffering from gastropathy were independent risk factors for H. pylori infection in children. The most common digestive tract symptoms of children with H. pylori infection were abdominal pain, accompanied by one or more clinical symptoms. The expression levels of IL-8, IL-18 and IFN-γ in H. pylori-positive children were significantly higher than those in H. pylori-negative children. Conclusion: Prevention of H. pylori infection in children is helpful for healthy growth of children, and cytokines IL-8, IL-18, IFN-γ have the potential to be used as biomarkers for diagnosis of H. pylori-positive children.


Introduction
Helicobacter pylori (H. pylori) is a gram-negative bacterium that can live in human stomach and is also one of the most common pathogens in the world. Once infected, it may cause functional dyspepsia, peptic ulcer and even gastric cancer (1)(2)(3). According to epidemiological statistics, the infection rate of H. pylori is 18.9-87.7%. In 2015, there were about 4.4 billion H. pylori -infected patients in the world, with 780,000 cancer cases caused by H. pylori (4,5). H. pylori infection mainly lies in childhood, but symptoms begin to appear in adulthood (6). Research on the treatment scheme of H. pylori different resistance has become the mainstream treatment choice (7,8).
Therefore, we decided to explore the relevant factors that might affect children's H. pylori infection, to explore the independent risk factors of children's H. pylori infection, and to provide valuable prevention suggestions for children's H. pylori infection. Interleukin-8 (IL-8) is a kind of pro-inflammatory chemotactic cytokine with various cell sources, which works on recruiting leukocytes or neutrophils to carry out anti-infection or tissue repair (9,10). The expression of polymorphic IL-8 gene is closely related to H. pylori infection and may affect the occurrence of peptic ulcer symptoms in H. pylori-positive patients (11). Interleukin-18 (IL-18) is an important cytokine that plays an important role in innate and adaptive immune function and participates in the key innate immune defense process of intracellular infection (12,13). IL-18 can protect mouse models from H. pyloriinduced asthma and inflammatory bowel disease (14). IL-18 can play a pro-inflammatory role by producing IL-8 from immune cell population, and can also establish a link between immune response and interferon-γ (IFN-γ) by driving polarization of helper T cells and inducing antiinfection natural killer cells (15,16). IFN-γ, a key cytokine produced by inflammatory cells to regulate the development of immune system and related functions, works in immune and tumor monitoring (17,18). The increase of IFN-γ level plays an important regulatory role in triggering the anti-inflammatory response of helper T cells and preventing H. pylori bacterial infection (19). In this study, we investigated IL-8, IL-18 and IFN-γ by detecting their expression levels in H. pylori-positive children and H. pylori-negative children.

General information
Overall, 234 children tested for H. pylori in Xuzhou Children's Hospital, Xuzhou Medical University, China from Feb 2017 to Feb 2019 were enrolled, including 118 boys and 116 girls, aged 3-15 yr, with an average age of (8.91±3.25) yr, 80 cases at preschool age of 3-6 yr old, 88 cases at school age of 7-12 yr old, 66 cases at puberty of 13-16 yr old, 159 cases from cities and towns, 75 cases from countryside, 73 cases of H. pyloripositive and 161 cases of H. pylori-negative. This study was approved by the Ethics Committee of Xuzhou Children's Hospital,Xuzhou Medical University, and their families were informed and they all signed a fully informed consent.

Inclusion and exclusion criteria
Inclusion criteria were as follows: those who informed and willing to cooperate with this study; patients without other types of infection; patients with complete clinical data; those who had not received H. pylori infection treatment; those who had not undergone surgery for nearly a month. Exclusion criteria were as follows: Bismuth, proton pump inhibitors, H2 receptor blockers, antibiotics and other drugs were ingested in the past four weeks; patients comorbid with serious dysfunction of heart, liver, spleen, lung, kidney, etc.; those with communication difficulties; those who were unwilling to sign a fully informed consent.

Diagnostic criteria for H. pylori infection
H. pylori was positive in gastric mucosa tissue culture, and there was a large range of H. pylori in gastric mucosa tissue section staining, which was consistent with one diagnosis of H. pylori infection. There was a small range of H. pylori in gastric mucosa tissue section staining. As a result, 13C urea breath test were positive, serum H. pylori-IgG was positive or stool H. pylori antigen was positive, and rapid urease test were positive, which was in accordance with the second diagnosis of H. pylori infection.

Observation indicators
Through examining pathological data and designing questionnaires, relevant factors were statistically recorded to carry out correlation analysis and risk factor analysis of H. pylori-positive, including general data such as gender, age, place of residence, character, family population, family monthly income. Within six months, whether there were digestive tract symptoms such as reflux, abdominal pain, epigastric pain, nausea and vomiting, hematochezia and peptic ulcer, other clinical symptoms such as recurrent urticaria, anemia, malnutrition, anaphylactoid purpura, anorexia and halitosis, living habits such as inattentive eating, sharing tableware, toothbrushes and cups, keeping pets and gnawing fingers often, eating habits such as eating snacks or fried food, drinking raw water, eating smoked and pickled food, and eating garlic often, family history such as father suffering from gastropathy, mother suffering from gastropathy, and diners suffering from gastropathy.

Detection methods
Gastric mucosa tissue of subjects was taken out by gastroscopy, incubated with RPMI 1640 medium of 10%FBS at 37 ℃ for one day, ground evenly, centrifuged at 18,000 r/min for 25 min, and 1 ml of the absorbed supernatant was stored in a freezer at -35 ℃. Serum was taken out from the freezer, placed in a refrigerator at 4 ℃ for dissolution, and then placed at room temperature for complete dissolution. The expression levels of IL-8, IL-18 and IFN-γ in gastric mucosa tissue were detected by ELISA (20), and the detection was carried out in strict accordance with the specifications of human IL-8 ELISA kit, human IL-18 ELISA kit and human IFN-γ ELISA kit (Shanghai Zhenyu Biotechnology Co., Ltd., Shanghai, China, E-EL-H0048km, E-EL-H0253km, E-EL-H0108km). Three kinds of wells were set up: sample, standard and blank wells. Altogether 50 μL of sample to be tested was added to the sample well and 50 μL of standard was added to the standard well, nothing in the blank well. The sample well and the standard well were each added with 100 μL of horseradish peroxidase labeled detection antibody, sealed and incubated at 37 ℃ for 60 min. The liquid was poured out, spin-dried and washed 5 times repeatedly. Substrates A and B were fully mixed in a volume of 1:1, 100 μL of substrate mixed solution was added to all wells, the plates were sealed, incubation was carried out at 37 ℃ for 15 min, and 50 μL of termination solution was added to each well. The absorbance (OD value) at 450 nm of each well was read by an enzyme-labeled analyzer (China Shanghai Chenlian Biotechnology Development Co., Ltd., MB530), and the concentrations of IL-8, IL-18 and IFN-γ were calculated.

Statistical analysis
The data were processed by GraphPad Prism 6 (GraphPad Software, San Diego, USA). The counting data were expressed by the number of cases/percentage [n(%)], and chi-square test was used for comparison of counting data between groups. The measurement data were expressed by mean±SD, and the comparison of measurement data between the two groups was conducted by t test with independent samples. P<0.05 was considered to be statistically significant.

General information
In the general data of the study subjects, the correlation between H. pylori infection and gender was not significant, but the correlation between H. pylori infection and age, place of residence, personality, family population and monthly family income was significant (P<0.05) ( Table 1).

Digestive tract symptoms in children with H. pylori infection
Digestive tract symptoms of children with H. pylori infection include reflux, abdominal pain, epigastric pain, nausea and vomiting, hematochezia and peptic ulcer. Among them, abdominal pain accounted for the highest percentage (47.95%), followed by reflux (32.88%), nausea, vomiting and hematochezia accounting for the lowest percentage (2.74%) ( Table 2).

Other clinical symptoms of children with H. pylori infection
Children with H. pylori infection were also accompanied by recurrent urticaria, anemia, malnutrition, allergic purpura, anorexia, halitosis and other clinical symptoms. Anorexia accounted for the highest percentage (17.81%), followed by hal-itosis (15.07%), and malnutrition (4.11%) with the lowest percentage. It should be noted that some H. pylori children were accompanied by various clinical symptoms (Table 3).

Correlation between infection and children's living habits
H. pylori-infected children had significant correlation with inattentive eating, sharing tableware, sharing toothbrushes and cups, keeping pets and gnawing fingers (P<0.05) ( Table 4).

Correlation between H. pylori infection and children's eating habits
H. pylori infection was significantly correlated with eating habits such as eating snacks, or fried food, drinking raw water, eating smoked and pickled foods, and eating garlic often (P<0.05), but not with eating vegetables raw (Table 5).

Correlation between H. pylori infection and children's family history
H. pylori infection had a significant correlation with the family history of children such as father suffering from gastropathy, mother suffering from gastropathy, and diners suffering from gastropathy (P<0.05) ( Table 6).

Discussion
Among the relevant symptoms of H. pylori infection in children, the typical digestive tract symptom is abdominal pain, and other common clinical symptoms are anorexia, halitosis, malnutrition, etc. Studies have shown that anorexia caused by H. pylori infection is related to activa-tion of hypothalamic cavitation cytotoxin, while halitosis caused by H. pylori infection may be related to increase of H2S content caused by H. pylori decay (21,22). In the research on the relationship between H. pylori infection and digestive tract diseases, H. pylori might cause gastritis, peptic ulcer, malignant lymphoma and even diseases outside the digestive tract, eventually leading to malnutrition in children, manifested by short stature, which suggested that preventing H. pylori infection was important for children's development (23). Therefore, it is of great significance for the improvement of children's quality of life and healthy growth to find out the relevant risk factors of H. pylori infection and propose preventive measures with reference significance.   (31). In our study, the expression levels of cytokines IL-8, IL-18 and IFNγ in gastric mucosa tissues in children with H. pyloripositive were significantly higher than those in negative controls, suggesting that they might become biomarkers for diagnosing H. pylori-positive, but the specific diagnostic value would not be analyzed for the time being in this study.
Although this study confirmed that healthy living and eating habits, improving family economic level had certain preventive effects on children's H. pylori infection, and IL-8, IL-18, IFN-γ were highly expressed in children's H. pylori-positive, there was still some room for improvement. First of all, we could expand the sample, extract relevant data from the database for large sample analysis, and improve the reliability of the research. Second, we could supplement the diagnostic value of the above cytokines for H. pyloripositive. Further, basic experiments could be conducted to explore the specific regulatory mechanism of the above cytokines on H. pylori infection. We need to perfect these points and further supplement them in future research.

Conclusion
Prevention of H. pylori infection in children is helpful for healthy growth of children, and cytokines IL-8, IL-18, IFN-γ have the potential to be used as biomarkers for diagnosis of H. pyloripositive children.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.