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The effect of vitamin D deficiency on eradication rates of Helicobacter pylori infection

Background/Aim Many studies have investigated risk factors other than antibiotic resistance linked to Helicobacter pylori (H. pylori) eradication failure. The aim of this study was to study the effect of serum levels of 25‐hydroxy‐vitamin D (25[OH]D) on eradication rates of H. pylori infection. Methods This study included 150 patients diagnosed with H. pylori gastritis using magnifying narrow‐band imaging endoscopy supported by stool antigen test. Serum 25‐OH vitamin D levels were measured via the Enzyme‐Linked Immune Sorbent assay (ELISA) method before starting eradication therapy of H. pylori infection. All patients were treated with clarithromycin‐based triple therapy for 14 days. H. pylori eradication was determined via a stool antigen test performed 4 weeks after the end of therapy. According to the serum level of 25‐OH vitamin D levels, the patients were divided into two groups: group I (sufficient) had a vitamin D level of ≥20 ng/mL, while group II (deficient) had a vitamin D level of <20 ng/mL. Results Our results revealed that eradication was successful in 105 (70%) patients and failed in 45 (30%) patients. The mean 25[OH]D level was significantly lower in the eradication failure group compared to the successful treatment group (14.7 ± 4.5 vs 27.41 ± 7.1; P < 0.001). Furthermore, there were significantly more patients with deficient 25[OH]D levels in the failed treatment group, 30 (66.6%), compared to the successful group, 10 (9.5%) (P < 0.001). Conclusions Our results demonstrated that 25‐OH vitamin D deficiency may be considered a risk factor related to eradication failure of H. pylori infection. In addition, a further randomized trial to evaluate the effect of vitamin D supplementation in H. pylori eradication is mandatory.


Introduction
Infection by Helicobacter pylori has been established as a major cause of chronic gastritis. It affects 50% of the world's population, and it is important in the pathogenesis of other gastrointestinal diseases such as peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma. 1 Up to 76-95% of gastric cancers and 90% of duodenal ulcers are associated with H. pylori infection. 2 The National Institute of Health Consensus Development Conference concluded that patients with H. pylori infection should receive antimicrobial therapy as the risk of ulcer recurrence and associated complications do not diminish unless H. pylori infection is cured. 3 Although triple therapy with a proton pump inhibitor (PPI), clarithromycin, and amoxicillin or metronidazole has been used as the first-line treatment for H. pylori infections, the American College of Gastroenterology suggested that the cure rates were 70-85% in 2007. 4 In addition, recent systematic review showed that the cure rates of sequential and standard triple therapy were 84.1 and 75.1%, respectively. 5 The most important factor affecting H. pylori cure rates is the antibiotic resistance of H. pylori strains. The number of H. pylori strains that are resistant to antibiotics is increasing. 6 In addition, the successful treatment of H. pylori infection depends on host genetic factors such as cytochrome P450 2C19 (CYP2C19), interleukin 1B (IL-1B), and multidrug-resistant transporter-1 (MDR1). 7 Host immunity also plays an important role against an infectious disease such as H. pylori infection. Vitamin D is an important secosteroid hormone with pleiotropic effects. While its role in the regulation of calcium and bone homeostasis is well established, recently, there is increasing recognition that vitamin D has immunomodulatory, anti-inflammatory and antifibrotic properties and plays an important role in the regulation of cell proliferation and differentiation. 8 Recent studies have demonstrated that vitamin D regulates the expression of specific endogenous antimicrobial peptides (AMPs) in immune cells; this action leads to a potential role for vitamin D in modulating the immune response to various infectious diseases. In the vitamin D deficiency state, the infected macrophage is unable to produce sufficient 1,25-(OH) 2 D to upregulate the production of AMP cathelicidin. 9 Although this antimicrobial mechanism of vitamin D has been demonstrated only in macrophages infected with mycobacterium tuberculosis, it is also well known that cathelicidin has broad-spectrum activity against a wide variety of other pathogens, including Gram-negative and Gram-positive bacteria, viruses, and fungi. 10 Evidence exists that vitamin D has a potential antimicrobial activity, and its deficiency has deleterious effects on general well-being and longevity. Vitamin D may reduce the risk of infection through multiple mechanisms; Vitamin D boosts innate immunity by modulating the production of AMPs and cytokine response. 11 Moreover, Vitamin D helps in boosting the activity of monocytes and macrophages, thereby contributing to a potent systemic antimicrobial effect. A vitamin D replete state appears to benefit most infections. 12

Materials and methods
This study was conducted at gastrointestinal endoscopy unit, Internal Medicine Department of El-Hussein University Hospital, AL-Azhar University, Cairo, Egypt, between March 2018 and June 2018. In this study, 150 sequential Egyptian patients with dyspeptic symptoms for at least 1 month were consecutively enrolled. For each patient, full medical history along with complete physical examination was conducted. Informed consent was obtained from all patients. Diagnostic esophagogastroduodenoscopy using magnifying narrow-band imaging (ME-NBI) was performed for all patients afterward; if the patient had endoscopic findings suggesting H. pylori chronic gastritis, a stool antigen test for the detection of H. pylori infection was conducted to support endoscopic diagnosis. Serum 25-OH vitamin D levels were measured using the ELISA method before beginning eradication therapy for the H. pylori infection. According to the serum level of 25-OH vitamin D levels, the patients were divided into two groups: group I (sufficient) had a vitamin D level of ≥20 ng/mL, while group II (deficient) had a vitamin D level of <20 ng/mL. All patients were treated with clarithromycin-based triple therapy for 14 days. H. pylori eradication was determined via a stool antigen test performed 4 weeks after the end of therapy. Endoscopic evaluation. The instruments used in this study were a videoendoscope and an electronic endoscopic system (Olympus EVIS Lucera Ellit CV-290; Olympus Medical Systems, Tokyo, Japan) equipped with three imaging modes: highresolution white light endoscopy (WLE), autofluorescence imaging (AFI), and narrow-band imaging (NBI). Mode switch from WLE to NBI or AFI was controlled by buttons on the control head. Combining magnifying endoscopy and the NBI system can lead to visualizing microscopic mucosal structures and their capillary patterns more clearly. All patients were offered conscious sedation with intravenous midazolam (2.5-5 mg) and/or propofol (40-200 mg) and then underwent endoscopic assessment for the presence or absence of regular arrangement of collecting venules (RAC) as well as mucosal and vascular patterns of the gastric body. All of the endoscopic examinations were performed by one senior endoscopist with more than 10 years of experience in diagnostic and therapeutic endoscopy. The entire endoscopic procedure was recorded on a digital video disc (DVD). NBI endoscopic diagnosis was performed on two occasions. In the first occasion, diagnosis was made during real-time endoscopy, while in the second occasion, it was made on the basis of the pictures and videos obtained, which was then confirmed. Using ME-NBI, the normal gastric body mucosal pattern was determined by small, round pits surrounded by subepithelial capillary networks creating a honeycombing pit pattern, with the presence of regular arrangement of collecting venules (RAC-positive) ( Fig. 1), while diagnosis of H. pylori chronic gastritis was made according to the presence of an obviously enlarged oval or prolonged pit with increased density of irregular vessels as well as the presence of a well-demarcated oval or tubulovillous pit with clearly visible coiled or wavy subepithelial capillary networks along with the absence of regular arrangement of collecting venules (RAC-negative) (Fig. 2).
Treatment protocol. All patients were treated with clarithromycin-based triple therapy (clarithromycin 500 mg, amoxicillin 1000 mg, and esomeperazol 20 mg) twice daily for 14 days. Statistical analysis. The data obtained from the history, clinical examination, and investigations were tabulated and statistically analyzed using the GraphPad Prism program version 6. Mean standard deviations (SDs) were used to identify the data related to the continuous variables, and categorical variables were provided as percentages. The comparison of the variables with normal distribution was tested with an unpaired t-test, and the comparison of the variables without normal distribution was tested with a Mann-Whitney U test. The categorical variables were compared with Pearson's χ 2 test, and a P-value < 0.05 was considered statistically significant.

Results
The present study included 150 patients diagnosed with H. pylori chronic gastritis by ME-NBI plus stool antigen test, who were divided into two groups according to the vitamin D level measured before starting eradication therapy for H. pylori infection: Group I, 110 (73.3%) patients with sufficient vitamin D level (≥20 ng/mL), and Group II, 40 (26.6%) patients with deficient  vitamin D levels (<20 ng/mL). There was no significant differences between the two groups regarding age or gender distribution (P = 0.747 and P = 0.507, respectively) ( We found 30 (66.6%) patients in the failed treatment group and 10 (9.5%) patients in the successful treatment group to be vitamin 25(OH)D-deficient. Therefore, vitamin 25(OH)D deficiency was significantly higher in the failed treatment group compared to the successful treatment group (P < 0.001) ( Table 2 and Fig. 3).

Discussion
In the present study, we have evaluated the possible association between vitamin D levels and H. pylori infection. Our study primarily investigates the relationship between H. pylori eradication rates and low vitamin D levels. In this study, we found the H. pylori eradication rates to be significantly lower in patients with low vitamin D levels. In addition, the number of patients with deficient vitamin D levels was significantly higher in the eradication failure group compared to the successful group. A potential pathogenic mechanism explaining the observed association between vitamin D status and eradication rates is the impairment of the vitamin D signal immune function, which may lead to inadequate immune response. Vitamin D deficiency may be a risk factor associated with H. pylori infection treatment failure and may lead to a need for supplementation of vitamin D before H. pylori eradication therapy. The link between vitamin D deficiency and susceptibility to infection has been suggested for longer than a century, with the early observation that children with nutritional rickets were more likely to experience infections of the respiratory system, leading to the coining of the phrase "rachitic lung". 14 Recently, one of the most interesting reports on vitamin D by Guo et al. 15 has shown that vitamin D demonstrates an antimicrobial effect against H. pylori. Guo et al. 15 found that vitamin D plays an important role in gastric mucosa homeostasis and host protection from H. pylori infection. Besides its effect on bone metabolism, vitamin D may reduce inflammatory markers such as CRP, TNF-α, IL-6, and IL-18, and the level of anti-inflammatory cytokine IL-10 may increase. 16 Vitamin D is also known to regulate the expression of AMPs cathelicidin and β-defensin, which kill the bacteria. Although the effect of cathelicidin has been demonstrated only in macrophages infected with Mycobacterium tuberculosis, antibacterial action against Gram-negative and Gram-positive bacteria has also been  The mean of vitamin 25(OH) D level was significantly lower in the eradication failure group compared to the successful treatment group (P < 0.0001). Vitamin 25(OH) D deficiency was significantly higher in the failed treatment group compared to the successful treatment group (P < 0.001). reported. 17 In a vitamin D-deficient state, the infected macrophage is unable to produce sufficient 1,25-(OH)D2 to upregulate the production of cathelicidin and β-defensin, thus rendering them unable to kill the H. pylori strains. More recently, epidemiological studies have demonstrated strong associations between seasonal variations in vitamin D levels and the incidence of various infectious diseases, including septic shock. 18  patterns, microvessels, cell morphology, and even microbes. NBI is a novel endoscopic technique that may enhance the accuracy of diagnosis by using narrow-bandwidth filters in a red-greenblue (R/G/B) sequential illumination system. 19 In the present study, we diagnose H. pylori gastritis by using magnifying NBI supported by a stool antigen test for H. pylori infection as the diagnostic gold standard. NBI diagnosis was based on the examination of micro surface structures of the gastric corpus, including both the gastric pit pattern and vascular pattern. Diagnosis of H. pylori gastritis was made according to the presence of obviously enlarged oval or prolonged pit with increased density of irregular vessels as well the presence of well-demarcated oval or tubulovillous pit with clearly visible coiled or wavy subepithelial capillary networks along with the absence of regular arrangement of collecting venules. Magnifying endoscopy using NBI has two distinct applications: the analysis of the surface architecture of the epithelium (pit pattern) and the analysis of the vascular network. 20 According to Yagi's classification, the normal body mucosa shows collecting venules and true capillaries that form a network surrounding gastric pits with a pinhole-like appearance (Z0 pattern), while the other three types (Z1, Z2, and Z3) corresponded to the H. pylori-positive mucosa. The Z0 pattern had 93.8% sensitivity and 96.2% specificity for predicting normal gastric mucosa without H. pylori infection. 21 Similarly, Nakagawa's classification divided the morphology of collecting venules into three patterns: regular (R), irregular (I), and obscured (O). The sensitivity and specificity of R pattern gastric mucosa as an indicator of the absence of H. pylori infection were 63.9 and 100%, respectively 22 . Anagnostopoulos et al. 23 tested the feasibility of magnifying endoscopy in a Western population, and the gastric body was categorized into four types: type 1, regular arrangement of the majority of the studies to evaluate gastric mucosal patterns has been conducted by Japanese investigators collecting venules and regular, round pits; type 2, regular, round pits, but loss of collecting venules; type 3, loss of normal collecting venules, with enlarged white pits surrounded by erythema; and type 4, loss of normal round pits, with irregular arrangement of collecting venules. The sensitivity and specificity of types 2 and 3 patterns for predicting H. pylori infection were 100 and 92.7%, respectively. The type 4 pattern corresponded to atrophic gastritis with a sensitivity of 90% and a specificity of 96%. A prospective study of 129 patients performed in Turkey confirmed that high-resolution magnifying endoscopy is superior to standard endoscopy for the diagnosis of H. pylori-associated gastritis. 24 In the present study, we combined both ME-NBI to diagnose H. pylori chronic gastritis supported with a stool antigen test as a gold standard to increase the sensitivity and specificity for the detection of H. pylori infection. The stool antigen test is a sensitive and specific noninvasive test for the diagnosis of H. pylori infection. It is inexpensive 25

Conclusions
Our results demonstrated that 25-OH vitamin D deficiency may be considered a risk factor related to the eradication failure of H. pylori infection. In addition, a further randomized trial to evaluate the effect of vitamin D supplementation in H. pylori eradication is mandatory.