Iron deficiency anemia in H.pylori pediatric patients and the role of IL-1β

Introduction

The incidence of IDA and H. pylori infection are believed to be associated, indicating a substantial improvement in IDA with successful H. pylori eradication. One meta-analysis study revealed that combination therapy for H. pylori elimination with administration of iron was more potent in treating IDA.¹ While, other meta-analyses indicated different results, where the accrual of Hb levels following H. pylori eradication was not significant and IDA refractory events after H. pylori eradication were still found, especially in severe cases.²

The inflammatory process has a crucial impact in the occurrence of IDA with H. pylori infection. Gastric mucous membrane layer inflammation is influenced by Interleukin (IL)-1β. The surge of IL-1β production in the antrum and corpus then stimulates parietal cells to inhibit gastric acid secretion and increase the transcription of other proinflammatory cytokines such as interferon-α, IL-6, and IL-8.³ Multi center studies (Brazil, Chile and England) in adolescents show that H. pylori infection can lead to decreased iron absorption in children by inducing IL-1B increase.⁴

Only few H. pylori-infected children experience IDA. One of the pro-inflammatory cytokines, particularly IL-1β, increased significantly in this infection and is associated with impaired iron absorption. Therefore, the correlation between IL-1β levels and the incidence of IDA in H. pylori infection needs a further study.

Methods

This research was conducted in the period between January 2022 to January 2023. The subjects for this study were children who experienced recurrent abdominal pain, nausea and vomiting. The selections of subjects begin with determining the target population, the accessible population, the method of selection, sample size, as well as determining the inclusion and exclusion criteria. The study has received the necessary approval from the University Sumatera Utara Ethics Commission (No: 1185/KEP/USU/2021).

Measurement of serum IL-1β levels was carried out using the ELISA method. The examination uses a quantitative sandwich enzyme immuno-assay technique. Previously, a monoclonal antibody specific for IL-1β was coated on the microplate. Standard, sample, control and conjugate were introduced into the well with a pipette and the presence of IL-1β would be paired by immobilized antibody with a monoclonal enzyme-linked antibody specific for IL-1β. The color intensity formed would be proportional to the amount of bounded IL1B.

Endoscopic examination was necessary to support the occurrence of Helicobacter Pylori infection showing a hyperemic appearance of the gastrointestinal mucosa, which is also supported by CLO examination. Preparation for the CLO examination by screening the patient who was not taking antibiotics, PPI (proton pump inhibitor) and bismuth 2 weeks prior the procedure.

Study subjects had to meet the following inclusion criteria: pediatric patients aged 2-18 years who experienced recurrent abdominal pain, nausea and vomiting; the patient’s parents/guardians’ writtenconsent to be included as a research subject; recently not getting antibiotics, bismuth, H2 antagonists, proton pump inhibitors, and any immunomodulators in the last four weeks; patients who have not taken iron supplements in the last 3 months; in good nutritional status and no history of gastrointestinal surgery. The exclusion criteria were patients with malignancy, immunosuppression and worm infections.

To analyze the correlation between subject characteristics and the presence of H. pylori infection, the Chi square test (if the nominal data is 2 values), the Kruskall Wallis test (the nominal data is more than 2 values), the independent T test (for interval/ratio data) were used. The Mann Whitney test was used to determine the correlation between IL-1β laboratory characteristics and the presence of H. pylori infection. Chi square test was a bivariate test used to find the presence of H. pylori infection and IDA. For IL-1β levels, the cut-off value was sought and plotted onto the ROC curve to gain sensitivity, specificity, positive and negative predictive values.

Results

The population study were 52 children who experienced recurrent abdominal pain, nausea and vomiting and consisting of 26 H. pylori (+) and 26 H. pylori (-) patients. All children involved in this study met the inclusion criteria. Table 1 contained complete subject characteristic.

Table 2 shows the analysis results of the corellation between H. pylori infection and IDA.

Of the 26 children with H. pylori (+), there were 23 (88.5%) with IDA. Meanwhile, out of 26 children without H. pylori, only 2 (7.7%) had IDA. The analysis using the Chi Square test demonstrated a significant correlation between H. pylori infection and iron deficiency anemia (p<0.001). The Prevalence Ratio value obtained was 11.5 (95% CI 3.015 – 43.864) meaning that H. pylori-infected (+) children would be 11.5 times more probable to have iron deficiency anemia than children with H. pylori (-). The analysis with ROC curve (Figure 1) demonstrated that the AUC area of IL-1β levels in predicting IDA H. pylori-infected children was 87% with p=0.041 and 95% CI 65.7%-100%. This shows that IL-1β levels can be used to predict IDA in H. pylori (+) children.

Figure 1. ROC curve of IL-1β ability in predicting IDA in children with H. pylori (+).

As seen on the line chart in Figure 2, the cut-off value of IL-1β levels in predicting IDA in H. pylori (+) children is 1.3 pg/mL. Table 3 displays the accuracy values which include sensitivity, specificity, positive predictive value (PPV), negative predictive values (NPV) and the accuracy of IL-1β levels in predicting IDA in H. pylori (+) children.

Figure 2. Line graph of the sensitivity and specificity of IL-1β in predicting IDA in children with H. pylori (+).

The sensitivity value of IL-1β levels in predicting IDA was 87%, the specificity value was 66.7%, the positive predictive value (PPV) was 95.2%, the negative predictive values (NPV) was 40% and the accuracy level was 84.6%.

Discussion

Most studies reveal that persistent H. pylori infection in the mucosa of the gaster can cause iron deficiency anemia or IDA, especially in the population of children and adolescents. The cause of H. pylori infection-associated IDA is still indefinite, where in general the majority of pediatric patients with IDA associated with H. pylori infection do not display any signs of blood loss as a result of gastrointestinal hemorrhagic lesions. In general, in developing countries with low socioeconomic levels, it is often difficult to maintain an adequate iron balance where intake of iron-rich foods with high iron bioavailability from animals is rare or nonexistent. Lack of vitamin C intake combine with frequent consumption of tea and a diet high in phytates and polyphenols are risk factors for IDA.⁵ Iron deficiency in children will also affect learning ability, lack of ability memory, lack of concentration, failure in education and affect children’s cognitive and motor development.^6,7 Our study is the first study of the association between IL-1β and the incidence of IDA in children with abdominal pain due to H. pylori. This study showed a corellation between H. pylori infection and IDA (p<0.001) 95% CI 3.015-43.865) (Table 2).

Recurrent abdominal pain (RAP) is a frequent incident in children, characterized by symptoms of 3 or more episodes of pain over a 3-month period that interferes with normal daily activities.^8,9 The occurrence of RAP caused by H. pylori is inconclusive, despite the fact that several studies have reported that CagA+ strains might be causing recurrent abdominal pain in H. pylori-infected children. Cytotoxin-Associated Gene A (CagA) is one of the primary virulence factors in H. pylori infection.¹⁰ This study shows that abdominal pain in the H. pylori (+) infection group is more dominant than H. pylori (-) around 65.4% (Table 1). Another possible cause of abdominal pain in children with H. pylori (+) is the gastric mucosa inflammation. Interleukin-1β cooperates with other inflammatory cytokines to stimulate neutrophils in the mucosa of the gaster, causing inflammation.

Inflammation that occurs in H. pylori infection depends on bacterial virulence factors and the immune response of the host. The occurrence of H. pylori infection results in inflammation that persists in the gastric mucosa and is chronic, the host immune system, both innate and adaptive immunity plays a crucial role in the pathogenesis of gastroduodenal disorders in response to the inflammatory process against infection by H. pylori. This situation is mediated by a series of pro (IL-1β, IL-6 and IL-8) and anti (IL-1RN and IL-10) inflammatory cytokines.¹¹

Interleukin 1β (IL-1β) is involved in various cellular activities, such as the inflammatory response and gastric acid secretion. The inflammatory response of H. pylori infection help activaties neutrophils and lymphocytes which cause epithelial damage by cytokines release. The correlation between cytokines with its nflammation degree and neutrophil activity is positive, where the cytokine level is proportional to the degree of inflammation and also the neutrophil activity.¹²

Interleukin-1β is a proinflammatory cytokine that acts as an acute phase response and is associated with disease onset. These cytokines can be detected in various medical conditions, including H. pylori infection. During inflammation and infection IL-1β is released and causes inflammatory response which affects chronic diseases as well as H. pylori infection.¹³ This study found significant differences in Interleukin-1β levels in H .pylori (+) compared to H. pylori (-) with p<0.001. This proves that IL-1β is crucial in the severity of H. pylori infection and has clinical aspects for H. pylori-infected patients’ management to prevent gastric malignancy in the future.

Our study showed the correlation between H. pylori infection and IDA (p<0.001) 95%CI 3.015-43.865). This can be assessed from the ROC curve analysis (Figure 1) where the AUC area obtained from IL-1β levels can predict IDA in H. pylori (+) children that is equal to 87% with p=0.041 and 95% CI 65.7%-100%.

In the study, we found that the cut-off value of IL-1β levels for predicting IDA in children with H. pylori (+) was 1.3 pg/mL with a sensitivity value of 87% and a specificity value of 66.7% (Figure 2 and Table 3). The cut off value of 1.3 pg/mL is important because this value portrays the accuracy of IL-1B in predicting IDA in infection caused by H. pylori.

The pathophysiology infection caused by H. pylori initiates an inflammatory response through a process of mediator release, such as cytokines which interacts between immunocompetent/hematopoietic cells and between the immune system/neuroendocrine system. Interleukin-1β (IL-1β) induces the release of other interleukins such as IL-8 and the combination of the two cytokines affects neutrophil activation in the gastric mucosa of H. pylori-infected patients.¹⁴

In addition, IL-1β can reduce gastric acidity by suppressing gastric acid secretion, through regulation of H+/K+ATPase expression and suppressing gastrin release, which in turn suppresses Sonic Hedgehog gene, causing gastric atrophy.¹⁵ If the above situation does not get an immediate treatment, it will eventually lead to stomach cancer. This decrease in gastric secretion can also cause disturbances of iron metabolism, where gastric acid is important for iron absorption by converting ferric iron into ferrous iron which is more easily absorbed.

Conclusions

This research found a significant correlation between Helicobacter pylori infection and iron deficiency anemia, even though the mechanism is not fully comprehensive. H. pylori-infected children had significantly different IL-1β levels compared to H. pylori (-), demonstrating that IL-1β is crucial in determining how severe an infection is and that treatment of infection with IL-1β could help prevent future gastric cancer.