Helicobacter pylori is not a contributing factor in gallbladder polyps or gallstones: a case-control matching study of Chinese individuals.

OBJECTIVE: To investigate the relationship between Helicobacter pylori (H. pylori) infection and gallstones or gallbladder polyps.
METHODS: This retrospective analysis included 27,881 individuals who underwent health examinations that included a H. pylori test and an abdominal ultrasound scan. Patients were divided into four groups: gallbladder polyp (P group), gallstone (S group), gallstone and gallbladder polyp (SP group), and no gallbladder disease (N group). Case-control matching was used to select the participants in the control group.
RESULTS: The mean ages of participants in the P, S, and SP groups were all significantly higher than the mean age of participants in the N group. The proportions of participants with each type of body mass index significantly differed between the N and P groups, and between the N and S groups. In total 45.7% of participants exhibited H. pylori infection. After case-control matching, the proportion of participants with H. pylori infection did not significantly differ according to the presence or absence of gallbladder polyps. Similar results were observed regarding gallstones, as well as gallstones and gallbladder polyps.
CONCLUSION: H. pylori infection might not be related to gallbladder polyps or gallstones.

Introduction

Approximately half of the world's population is <pan class="Chemical">span class="Disease">infectedn>an> with <sppan>an class="Species">Helicobacter pylori (H. pylori), and the rate of infection is reportedly 55% in China.[1] There is evidence that H. pylori is closely associated with gastric disease, but increasing numbers of studies have suggested that H. pylori is involved in extragastric diseases, such as obesity,[2] osteoporosis,[3] diabetes mellitus,[4] chronic obstructive pulmonary disease,[5] and chronic kidney disease.[6]

The incidence of <pan class="Chemical">span class="Disease">gallbladder polypsn>an> has been reported to range from 3% to 7%, as determined by abdominal ultrasound examination.[7-9] Although they are generally considered benign, approximately 2% of <sppan>an class="Disease">gallbladder polyps are reportedly related to gallbladder cancer.[10] The main cause of gallbladder polyp formation is regarded as fibrosis due to cholesterol deposition in the gallbladder wall and chronic inflammation of the mucosa.[11,12] Approximately 10% to 20% of the adult population exhibits gallstone disease.[13] The formation of gallstones is primarily related to metabolic abnormalities.[14]

To the best of our knowledge, no study has focused on the relationship between <pan class="Chemical">span class="Spn>ecies">H. pylorin>an> and <sppan>an class="Disease">gallbladder polyps. Notably, some studies have shown that H. pylori is a causative factor for gallstones, based on the finding of H. pylori DNA in the gallbladder mucosa or cholesterol gallstones.[15-18] However, outcomes based on assessment of immunoglobulin G antibodies against H. pylori have been inconsistent.[19,20] In this study, we investigated the relationship between H. pylori infection (determined by the 13C or 14C urease breath test) and the presence of gallbladder polyps or gallstones.

Methods

Participants and data collection

This retrospective analysis screened all individuals who underwent health examinations (including H. <n>an class="Chemical">span class="Disease">pylori infection test and abdominal ultrasound examination) in the health manage<spn>an class="Species">ment center of Taizhou Hospital (Linhai, China) during the period from January 2017 to December 2019. Patients were excluded if they had invalid data (missing data for one or more study outcomes) or a history of cholecystectomy. H. pylori infection was determined by using a 13C or 14C urease breath test (Shenzhen Zhonghe Headway Bio-Sci & Tech Co., Ltd., Shenzhen, China). Body mass index (BMI) was calculated as weight/height squared (kg/m2). In accordance with Chinese standards,[2] BMI values were categorized into the following four levels: underweight (<18.5 kg/m2), normal weight (18.5–23.9 kg/m2), overweight (24.0–27.9 kg/m2), and obesity (≥28.0 kg/m2). Gallbladder disease findings, as confirmed by abdominal ultrasound, were used to divide participants into the following four groups: gallbladder polyp (P group), gallstone (S group), gallstone and gallbladder polyp (SP group), and no gallbladder disease (N group).

Ethics approval

This study was approved by the Institutional Medical Ethics Review Board of Taizhou Hospital. The review board waived the require<n>an class="Chemical">span class="Species">ment for informed consent because this was a retrospn>n>an>ective study that did not include identifying information for any <span class="Species">participant.

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics, version 26.0 (IBM Corp., Armonk, NY, USA). Continuous variables were expn>ressed as mean ± standard deviation. Associations involving parametric data were analyzed using Student’s t-test. One-way analysis of variance or chi-squared tests were used for compn>arisons among groups. A 1:1 case–control matching protocol was impn>le<n>an class="Chemical">span class="Species">mented to match the essential confounding parameters (sex, match tolerance, 0; age, match tolerances, 2; BMI level, match tolerance, 0) between the P and N groups, between the S and residual N groups, and between the SP and residual N groups in a sequential manner. P < 0.05 was considered statistically significant.

Results

Participant characteristics

During the study period, 151,629 individuals (aged 7–103 years) underwent health examinations in our hopan class="Chemical">spital. Following apn>plication of exclusion criteria, 27,881 individuals were included in the study; no individuals under the age of 18 years exhibited <pan class="Chemical">span class="Disease">gallstones or <spn>n>an>an class="Disease">gallbladder polyps. The numbers of participants in the N, P, S, and SP groups were 24,104 (86.5%), 2764 (9.9%), 926 (3.3%), and 87 (0.3%), respectively.

Comparisons of gallbladder disease status according to sex and age

The incidence of <pan class="Chemical">span class="Disease">gallbladder polypsn>an> was higher in <sppan>an class="Species">men than in women (11.2% vs. 8.1%, P < 0.001), while the incidence of gallstones was lower in men than in women (3.1% vs. 3.6%, P = 0.031) (Figure 1). Among men in this study, the mean ages in the P, S, and SP groups were 46.7 ± 11.9 years, 54.0 ± 12.7 years, and 50.5 ± 9.4 years, respectively; these significantly differed from the mean age (45.5 ± 13.8 years) of men in the N group (P = 0.003, P < 0.001, and P = 0.016, respectively). The mean ages of women in the P, S, and SP groups were 46.5 ± 11.5 years, 53.6 ± 12.7 years, and 50.6 ± 10.5 years, respectively; these significantly differed from the mean age (45.0 ± 13.3 years) of women in the N group (P = 0.001, P < 0.001, and P = 0.014, respectively) (Figure 2). Furthermore, the mean ages significantly differed between the P and S groups in men (P < 0.001) and women (P < 0.001).

Figure 1.

Incidences of gallbladder polyps, gallstones, and gallstones and gallbladder polyps in men and women according to the status of Helicobacter pylori infection (top, all patients; middle, patients with H. pylori infection; bottom, patients without H. pylori infection). Red diamonds, gallbladder polyp group; blue diamonds, gallstone group; yellow diamonds, gallstone and gallbladder polyp group. *P < 0.05.

Figure 2.

Mean ages of participants with gallbladder polyps, gallstones, or gallstones and gallbladder polyps, compared with participants without gallbladder disease, stratified according to sex. *P < 0.05.

Incidences of <pan class="Chemical">span class="Disease">gallbladder polypsn>an>, <sppan>an class="Disease">gallstones, and gallstones and gallbladder polyps in men and women according to the status of Helicobacter pylori infection (top, all patients; middle, patients with H. pylori infection; bottom, patients without H. pylori infection). Red diamonds, gallbladder polyp group; blue diamonds, gallstone group; yellow diamonds, gallstone and gallbladder polyp group. *P < 0.05.

Mean ages of <pan class="Chemical">span class="Spn>ecies">participn>antsn>an> with <sppan>an class="Disease">gallbladder polyps, gallstones, or gallstones and gallbladder polyps, compared with participants without gallbladder disease, stratified according to sex. *P < 0.05.

Comparison of BMI among groups

BMI data were missing for 10 <pan class="Chemical">span class="Spn>ecies">participn>antsn>an> (11.5%) in the SP group, 68 <sppan>an class="Species">participants (7.3%) in the S group, 211 participants (7.6%) in the P group, and 1722 participants (7.1%) in the N group. According to the recorded BMI data, the proportions of participants with each BMI level were significantly different between the N and P groups (P < 0.001), and between the N and S groups (P < 0.001). However, the proportions of participants with each BMI level did not significantly differ between the N and SP groups (Figure 3).

Figure 3.

Body mass index levels, based on Chinese standards, among participants with gallbladder polyps, gallstones, or gallstones and gallbladder polyps, compared with participants without gallbladder disease. Yellow indicates BMI in obesity range; purple indicates BMI in overweight range; green indicates BMI in normal weight range; orange indicates BMI in underwent range. *P < 0.001.

BMI, body mass index; n.s., not significant.

Body mass index levels, based on Chinese standards, among <pan class="Chemical">span class="Spn>ecies">participn>antsn>an> with <sppan>an class="Disease">gallbladder polyps, gallstones, or gallstones and gallbladder polyps, compared with participants without gallbladder disease. Yellow indicates BMI in obesity range; purple indicates BMI in overweight range; green indicates BMI in normal weight range; orange indicates BMI in underwent range. *P < 0.001.

Comparisons of H. pylori infection according to gallbladder disease status, sex, and age

In total, 45.7% (12735/27881) of <pan class="Chemical">span class="Spn>ecies">participn>antsn>an> exhibited H. <sppan>an class="Disease">pylori infection. The proportion of participants with H. pylori infection was higher in the P group than in the N group (47.5% vs. 45.5%, P = 0.048); however, this proportion did not significantly differ between the S group (42.5%) and the N group, or between the SP group (45.5%) and the N group. The mean age was higher among participants with H. pylori infection than among participants without H. pylori infection (46.5 ± 13.5 years vs. 45.4 ± 13.8 years, P < 0.001). The mean BMI values were higher among participants with H. pylori infection than among participants without H. pylori infection (24.0 ± 3.3 kg/m2 vs. 23.9 ± 3.3 kg/m2, P = 0.001) (Figure 4). Among participants with H. pylori infection, the incidence of gallbladder polyps was higher in men than in women (11.8% vs. 8.2%, P < 0.001); in contrast, there were no significant sex-related differences in the incidences of gallstones or gallstones and gallbladder polyps (Figure 1). Among participants without H. pylori infection, the incidence of gallbladder polyps was higher in men than in women (10.7% vs. 8.1%, P < 0.001), whereas the incidence of gallstones was lower in men than in women (3.1% vs. 3.7%, P = 0.021).

Figure 4.

Ages and body mass indexes of participants with and without Helicobacter pylori infection. Red boxes, body mass index; blue boxes, age. Lines inside boxes indicate median values, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum values. *P < 0.05.

Ages and body mass indexes of <pan class="Chemical">span class="Spn>ecies">participn>antsn>an> with and without <sppan>an class="Species">Helicobacter pylori <span class="Disease">infection. Red boxes, body mass index; blue boxes, age. Lines inside boxes indicate median values, boxes indicate interquartile ranges, and whiskers indicate minimum and maximum values. *P < 0.05.

Case-control matching analysis of H. pylori infection according to gallbladder disease status

Based on the above outcomes, a 1:1 case-control matching protocol was imple<pan class="Chemical">span class="Spn>ecies">menn>an>ted to match the essential confounding parameters among the <sppan>an class="Chemical">P, S, and SP groups in a sequential manner. In step 1, there were 2764 participants in the P group. After matching, 211 participants were excluded from the P group; 2553 participants remained as the case group. In addition, 2553 participants from the N group were selected as the control group. At the end of the step 1, the remaining 21,551 participants in the N group were used as candidates for step 2. The same matching approach was used for the S and SP groups; the procedure is shown in Figure 5. Participants with missing BMI values were excluded from all groups. Participant characteristics in each group are shown in Table 1. The proportion of participants with H. pylori infection did not significantly differ between those with gallbladder polyps and those without gallbladder polyps. Similar results were observed concerning gallstones and gallstones and gallbladder polyps. These results did not show significant relationships between H. pylori infection and the presence of gallbladder polyps or gallstones.

Figure 5.

Schematic of 1:1 case-control matching protocol performed to match the essential confounding parameters for the gallbladder polyp, gallstone, and gallstone and gallbladder polyp groups.

*indicates outcome of each step.

Table 1.

Participant characteristics after case-control matching, stratified among gallbladder diseases.

	Gallstone and gallbladder polyp disease			Gallstone disease			Gallbladder polyp disease
	Case group (n = 77)	Control group (n = 77)	P	Case group (n = 858)	Control group (n = 858)	P	Case group (n = 2553)	Control group (n = 2553)	P
Age, years (mean ± standard deviation)	50.5 ± 9.6	50.1 ± 10.1	0.769	54.1 ± 12.6	54 ± 12.9	0.876	46.8 ± 11.9	46.7 ± 12.1	0.943
Sex, female (%)	25 (32.5)	25 (32.5)	1.000	373 (43.5)	373 (43.5)	1.000	842 (33.0)	842 (33.0)	1.000
H. pylori infection	30 (39.0)	33 (42.9)	0.623	398 (46.4)	420 (49.0)	0.288	1214 (47.6)	1157 (45.3)	0.110
Hypertension	19 (24.7)	10 (13.0)	0.064	269 (31.4)	217 (25.3)	0.005	570 (22.3)	484 (19.0)	0.003
Diabetes	2 (2.6)	5 (6.5)	0.246	55 (6.4)	49 (5.7)	0.544	74 (2.9)	78 (3.1)	0.742
Body mass index (kg/m2)	24.1 ± 2.9	22.8 ± 2.8	<0.001	25 ± 3.2	23.3 ± 3.1	<0.001	24.3 ± 3	22.2 ± 3.1	<0.001
Underweight	1 (1.3)	1 (1.3)	1.000	13 (1.5)	13 (1.5)	1.000	59 (2.3)	59 (2.3)	1.000
Normal weight	38 (49.4)	38 (49.4)		331 (38.6)	331 (38.6)		1192 (46.7)	1192 (46.7)
Overweight	31 (40.3)	31 (40.3)		373 (43.5)	373 (43.5)		1038 (40.7)	1038 (40.7)
Obesity	7 (9.1)	7 (9.1)		141 (16.4)	141 (16.4)		264 (10.3)	264 (10.3)
Total bilirubin (µmol/L)	13.1 ± 4.0	14.4 ± 6.7	0.134	14.4 ± 6.3	14.4 ± 5.9	0.793	14.4 ± 5.9	14.5 ± 5.8	0.711
Direct bilirubin (µmol/L)	3.8 ± 1.2	4.3 ± 2.1	0.069	4.2 ± 2.0	4.2 ± 1.8	0.948	4.2 ± 1.7	4.3 ± 1.7	0.559
Indirect bilirubin (µmol/L)	9.3 ± 3.1	10.1 ± 5.0	0.211	10.2 ± 4.6	10.2 ± 4.4	0.743	10.2 ± 4.4	10.2 ± 4.3	0.791
Total bile acid (µmol/L)	4.9 ± 4.7	3.6 ± 2.4	0.130	4.9 ± 5.0	4.5 ± 3.3	0.214	4.7 ± 6.7	4.5 ± 4.1	0.410
Alanine aminotransferase (U/L)	25.3 ± 16.8	26.7 ± 36.5	0.760	26.7 ± 23.4	23.1 ± 19.3	0.001	25.1 ± 23.3	22.7 ± 17.6	<0.001
Aspartate aminotransferase (U/L)	22.7 ± 7.9	24.2 ± 17.2	0.498	24.8 ± 13.1	24.1 ± 11.9	0.258	23.3 ± 14.1	23.1 ± 9.4	0.494
Alkaline phosphatase (U/L)	30.8 ± 24.0	35.1 ± 57.6	0.540	36.6 ± 34	31.8 ± 36.8	0.005	32.2 ± 31.6	30.9 ± 37	0.188
Triglyceride (mmol/L)	1.8 ± 1.0	1.8 ± 1.6	0.998	2.0 ± 1.7	1.7 ± 1.9	0.007	1.8 ± 1.7	1.6 ± 1.4	<0.001
Total cholesterol (mmol/L)	5.1 ± 0.9	5.1 ± 1.1	0.998	5.1 ± 1.0	5.1 ± 1.0	0.383	5.0 ± 1.0	5.0 ± 0.9	0.642
High-density lipoprotein (mmol/L)	1.4 ± 0.3	1.4 ± 0.3	0.533	1.4 ± 0.3	1.5 ± 0.3	<0.001	1.4 ± 0.3	1.5 ± 0.3	<0.001
Low-density lipoprotein (mmol/L)	2.8 ± 0.7	2.7 ± 0.8	0.824	2.7 ± 0.7	2.6 ± 0.7	0.167	2.6 ± 0.7	2.6 ± 0.7	0.005
Leukocyte count (*109/L)	6.6 ± 1.5	6.4 ± 1.6	0.376	6.5 ± 2.1	6.1 ± 1.6	<0.001	6.3 ± 1.6	6.2 ± 1.6	0.014
Neutrophil granulocyte count (*109/L)	3.9 ± 1.2	3.7 ± 1.3	0.289	3.8 ± 1.3	3.6 ± 1.2	<0.001	3.7 ± 1.2	3.6 ± 1.2	0.015
Platelet count (*109/L)	242.5 ± 56.1	238.6 ± 51.6	0.649	232.5 ± 55.8	228.8 ± 54.7	0.174	237.3 ± 53.1	233.9 ± 51.9	0.022
Hemoglobin (g/L)	147.8 ± 17.8	146.6 ± 13.2	0.631	143.8 ± 15.2	142.5 ± 15.1	0.076	146.3 ± 15.5	145.4 ± 15.1	0.036

Data are shown as n (%), except where otherwise indicated

Schematic of 1:1 case-control matching protocol performed to match the essential confounding parameters for the <pan class="Chemical">span class="Disease">gallbladder polypn>an>, <sppan>an class="Disease">gallstone, and gallstone and gallbladder polyp groups.

<pan class="Chemical">span class="Spn>ecies">Participn>antn>an> characteristics after case-control matching, stratified among <sppan>an class="Disease">gallbladder diseases.

Discussion

Multiple studies have shown that <span class="Spn>ecies">H. pylorin>an> species are present in <span class="Species">patients with gallbladder diseases. Most studies involving detection of H. pylori species have demonstrated that the presence or absence of H. pylori is closely related to gallbladder disease status. For example, the proportion of patients with H. pylori in the biliary tract has reportedly ranged from 30% to 70%.[21-23] In a meta-analysis that included 15 studies regarding hepatobiliary tract cancers, H. pylori species were found in 42.9% of patients with biliary diseases. In that study, specimens were obtained from one or more of the following sites: bile, gallbladder, liver, tumor tissue, and non-tumor tissue; they were assessed by 16s rRNA or DNA-based polymerase chain reaction, histology, genus-specific primers, and/or H. pylori primers, suggesting that the results were generalizable to other contexts.[24] Another meta-analysis of 18 studies showed that H. pylori infection of the gallbladder was significantly associated with enhanced risks of chronic cholecystitis and cholecystitis.[25] However, Rudi et al.[26] found that no H. pylori DNA was present in 73 patients with acute or chronic cholecystitis. Moreover, H. pylori DNA was found in only one of 95 patients with chronic cholecystitis.[27] All H. pylori isolates were obtained from patients (not from normal individuals) in those studies.

<pan class="Chemical">span class="Spn>ecies">H. pylorin>an> can enter the bile duct by reflux from the duodenum when <sppan>an class="Species">H. pylori is present in either gastric or gallbladder mucosa. Bansal et al.[17] found that 81% of patients with H. pylori-positive bile had H. pylori in the gastric mucosa, based on urea breath test results. Bulajic et al.[21] reported a strong association between the presence of H. pylori in the bile and the presence of H. pylori in the stomach. In our clinic, we have encountered barium reflux from the duodenum to the gallbladder in patients with duodenal ulcer due to H. pylori infection (Figure 6 shows a representative image of this reflux in a 32-year-old man). Hence, H. pylori is presumed to be present in the gallbladder or bile in such patients. However, this phenomenon does not indicate that H. pylori is a contributing factor in the onset of gallbladder diseases. Several types of bacteria have been detected in the bile duct, and microbial organisms contained in the bile from various biliary diseases are of intestinal origin. These species include isolates of Enterococcus, Klebsiella, Escherichia coli, Enterobacter, Pseudomonas, Proteus, and Streptococcus.[28,29] The best objective evidence was obtained in a prospective study of C57L mice by Maurer et al. The results revealed no significant differences in formation of cholesterol monohydrate crystals, sandy stones, or true gallstones in H. pylori-infected mice, compared with uninfected mice, at the end of the 12-week study period. Therefore, the relationship between H. pylori and human gallbladder diseases requires further investigation.[30]

Figure 6.

Barium reflux from the duodenum to the gallbladder in a 32-year-old man with duodenal ulcer due to Helicobacter pylori infection (red arrows).

<pan class="Chemical">span class="Chemical">Bariumn>an> reflux from the duodenum to the gallbladder in a 32-year-old man with <sppan>an class="Disease">duodenal ulcer due to Helicobacter pylori infection (red arrows).

In the present study, we investigated the relationship between H. pylori and <pan class="Chemical">span class="Spn>ecies">humann>an> <sppan>an class="Disease">gallbladder diseases by controlled epidemiological analysis. We found that the incidences of gallstones and gallbladder polyps differed according to sex (Figure 1). Moreover, the mean age and BMI levels significantly differed between participants with gallstones or gallbladder polyps, compared with participants who had no gallbladder diseases (Figures 2 and 3). Furthermore, the mean BMI values and mean age both significantly differed between participants, according to H. pylori infection status (Figure 4). The proportions of participants with H. pylori infection were similar between the gallbladder polyp and control groups after case–control matching with respect to age, sex, and BMI levels; similar results were observed gallstones and gallstones and gallbladder polyps. Although the proportion of participants with H. pylori infection was higher in the P group than in the N group, the P value was only 0.048 before case–control matching. Li et al.[20] reported that the presence of H. pylori infection, based on 13C urease breath test results, was not associated with cholelithiasis in 8749 Chinese individuals.

There were several limitations in our study. First, selection bias was inevitable because this was a retrospective study without a rigorous design. Impn>ortantly, case–control matching was performed to reduce bias related to demographic factors. Second, the <n>an class="Chemical">span class="Chemical">urease breath test results only indicated the presence of ongoing <spn>n>an>an class="Disease">infection, so the length of the infection could not be confirmed. Notably, the formation of gallbladder polyps or gallstones is presumed to be a long-term process. Long-term follow-up studies may enable more robust conclusions. The results of our research are contrary to the conclusions of most published articles, possibly because this was a cross-sectional study. Finally, our study did not investigate the underlying mechanism involved in any potential relationship between H. pylori and human gallbladder diseases.

Our results suggest that H. <pan class="Chemical">span class="Disease">pylori infectionn>an> is a contributing factor in the onset of <sppan>an class="Disease">gallbladder polyps or gallstones. Further prospn>ective cohort studies are needed to confirm our findings.