Influence of serum vitamin D level on Helicobacter pylori eradication: A multi‐center, observational, prospective and cohort study

Objectives This study was designed to test whether serum vitamin D levels affected Helicobacter pylori (H. pylori) infection and eradication rates. Methods A multicenter observational prospective cohort study was conducted. A total of 496 H. pylori − positive (H. pylori +) and 257 H. pylori‐negative (H. pylori −) patients were enrolled from four hospitals in China. Baseline serum vitamin D levels were measured and a 13C‐urea breath test (UBT) was performed for all the participants. The H. pylori + patients were divided into two subgroups based on their serum vitamin D levels (<10 or ≥10 ng/mL). A second 13C‐UBT was performed between 4 and 8 weeks after 14‐day bismuth‐containing quadruple eradication therapies. Factors potentially affecting H. pylori eradication were determined using a questionnaire survey. Results Serum vitamin D levels were significantly lower in the H. pylori + group than in the H. pylori − group ([17.0 ± 6.9] ng/mL vs [19.2 ± 8.0] ng/mL, P = 0.000). H. pylori eradication rate significantly differed between patients with serum vitamin D levels of <10 ng/mL and ≥10 ng/mL (71.7% vs 87.3%, P = 0.005). A multivariate analysis showed that having serum vitamin D level ≥10 ng/mL was an independent risk factor for a successful H. pylori eradication (odds ratio 0.381, 95% confidence interval 0.183‐0.791, P = 0.010). Conclusions Serum vitamin D level may affect H. pylori infection and its eradication. Randomized controlled trials are needed to find out whether vitamin D supplements may increase the H. pylori eradication rate.


| INTRODUCTION
Gastric cancer (GC) generates a heavy disease burden in China.
Helicobacter pylori (H. pylori) infection is known to play a role in the pathogenesis in most patients with GC. 1 Accumulating evidence has shown that H. pylori infection causes almost 90% of non-cardiac GC. 2 Therefore, a successful eradication of H. pylori would significantly decrease the incidence of GC. However, both bacterial strains and host factors have been reported to impede the eradication of H. pylori infection, including the resistance to antibiotics, the virulence of the strains, and host-related genetic disorders. 3 Vitamin D regulates the calcium and phosphorus metabolism needed for bone formation, and the influence of vitamin D on H. pylori infection and eradication rates has recently been widely investigated. Several clinical studies have illustrated that vitamin D analogs may have anti-H. pylori antimicrobial effects. [4][5][6][7][8][9][10] Cytological research has also found that vitamin D 3 decomposition product 1 (VDP1) can lyse H. pylori bacterial cells by inducing the collapse of the cell membrane. 11,12 However, the correlation between vitamin D levels and H. pylori has not been fully illustrated, and studies on the impact of serum vitamin D levels on H. pylori eradication were mostly of small sample sizes. Therefore, we conducted a multicenter prospective cohort study with a relatively large sample size to determine whether serum vitamin D levels had an impact on H. pylori infection and eradication, and whether its low level was an independent risk factor affecting H. pylori eradication.

| Study population
We conducted a non-randomized, multicenter, observational and prospective cohort study with consecutively enrolled patients to estimate the influence of serum vitamin D levels on the rates of H. pylori infection and eradication. All patients were recruited between October 2017 and July 2018 from either one of the four centers: the Xijing Hospital (h) had refused H. pylori eradication treatment, or were allergic to any one of the drugs given in the eradication schemes; and (i) who used daily vitamin D supplements. In total, 496 patients who were tested positive (H. pylori + ) and the 257 who were negative (H. pylori − ) for H. pylori infection and who had had their serum vitamin D levels tested were enrolled in our study. The study was approved by the Ethics Com-

| Data collection
The following information were extracted from the patients who were H. pylori + using a questionnaire on the factors affecting H. pylori eradication, as follows: patients' age, sex, occupation, residential area, body mass index (BMI), marital status, educational level, family members, annual income, smoking, alcohol consumption, history of periodontal disease, hygiene of dining place, main source of drinking water, drinking of untreated water or not, 13 C-UBT results, diagnosis by gastroscopy, treatment of adequate dosage and duration, choice of proton pump inhibitors (PPIs), and serum vitamin D levels. Treatment of an adequate dosage and duration was defined as having received one of two different H. pylori eradication schemes for 14 days without forgetting to take the prescribed medication.

| Treatment and follow-up for H. pylori + patients
The H. pylori + patients were further divided into two subgroups based on their serum vitamin D levels (<10 or ≥10 ng/mL) and were report. 13 A cut-off value for serum vitamin D level was set at 10 ng/mL because a study from Turkey has reported that 10 ng/mL may be the cut-off value that affects H. pylori eradication. 7 Finally, a 13 C-UBT was repeated between 4 and 8 weeks after the treatment was completed.

| Statistical analysis
Statistical analyses were performed using SPSS version 22.0 software (IBM, Armonk, NY, USA). A double entry and verification method was used to record and enter the data collected from the patients. A one-sample Kolmogorov-Smirnov test was adopted to test the normality of continuous variables, which were expressed as medians and ranges for the variables with an abnormal distribution and mean ± standard deviation for variables with a normal distribution. Comparisons between the groups were performed using the Student's t-test, Wilcoxon signed-rank test, or the χ 2 test, when appropriate. A two-tailed P < 0.05 was considered statistically significant.
Binary logistic regression was applied to determine whether an independent risk factor of H. pylori eradication was the patient's serum vitamin D level. The degrees of this association were measured using the odds ratio (OR) and 95% confidence interval (CI).

| Patients' characteristics
As shown in Figure 1, there were 496 H. pylori + patients and 257 H. pylori − patients who underwent a 13 C-UBT and whose serum vitamin D levels were tested in our study. Of the H. pylori + patients, 81 (16.3%) out of 496 patients were lost to follow-up; H. pylori was successfully eradicated in 355 (eradication rate 85.5% [355/415]) .

| Comparison of serum vitamin D levels between H. pylori + and H. pylori − patients
We found that serum vitamin D levels were significantly lower in the H. pylori + group than in the H. pylori − group ([17.0 ± 6.9] ng/mL vs [19.2 ± 8.0] ng/mL, P = 0.000); while there were no significant differences in others factors such as age, sex, season of vitamin D detection, or occupation between the two groups, as shown in Table 1. This indicates that serum vitamin D levels may affect H. pylori infection.
3.3 | Differences in the H. pylori eradication rate between patients with serum vitamin D levels <10 and ≥10 ng/mL A second 13 C-UBT was performed between 4 and 8 weeks after 14-day bismuth-containing quadruple eradication therapy. Significant differences were seen between the serum vitamin D levels of <10 and ≥10 ng/mL groups in terms of their H. pylori eradication rates (71.7% vs 87.3%, P = 0.005; Table 2). This result suggests that having a lower serum level of vitamin D may be related to a lower eradication rate of H. pylori.

| Multivariate analyses of factors affecting the H. pylori eradication rate
Factors influencing the eradication rate of H. pylori were analyzed using a questionnaire with 20 factors. As shown in Table 3, univariate analysis showed that a treatment of adequate dosage and duration, alcohol consumption and serum levels of vitamin D differed significantly between the groups in which H. pylori was successfully eradicated and that failed, suggesting that these factors might affect the H. pylori eradication rate.