Role of Helicobacter Species in Chinese Patients with Inflammatory Bowel Disease

Abstract

Based on 16S rRNA gene PCR, no significant difference was observed in rates of detection of Helicobacter species in intestinal biopsy specimens from 160 Chinese inflammatory bowel disease (IBD) patients (10%) and 80 controls (6.3%). By using a [¹³C]urea breath test, the H. pylori infection rate in 208 Chinese IBD patients (19.7%) was found to be significantly lower than that in 416 controls (48.8%).

TEXT

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), is a chronic relapsing idiopathic inflammatory disorder of the gastrointestinal tract with unknown etiology (20). While it is well accepted that microorganisms play an essential role in the development of IBD, the specific microorganisms involved in the disease remain unclear. Although a number of recent studies in Western countries have reported increased prevalence of enterohepatic Helicobacter species in the intestinal tracts of IBD patients, the role of these organisms remains controversial (1–3, 5, 7, 10, 12–14, 18, 21). To date, such studies have not been conducted in China.

Studies showing lower prevalence rates of gastric Helicobacter pylori infection in IBD patients than controls have suggested that gastric H. pylori infection may protect against IBD development (11). This concept seems to be supported by the finding that the prevalence of IBD has been increasing in China over the last 2 decades (19) while the prevalence of H. pylori infection has been decreasing. However, studies investigating H. pylori prevalence in Chinese IBD patients are limited, due partly to the low prevalence of IBD among the Chinese population (19).

This study used two separate arms to investigate Helicobacter in Chinese IBD patients. In one arm, we used ribosomal PCR to broadly investigate the prevalence of Helicobacter species in the intestinal mucosae of IBD patients. In a separate arm, we used the [¹³C]urea breath test (UBT) to compare the prevalence rates of gastric H. pylori infection between IBD patients and the general population.

Using a Helicobacter-specific 16S rRNA gene PCR, we determined whether Helicobacter species were present in intestinal biopsy specimens from 240 patients (88 CD patients, 72 UC patients, and 80 controls) undergoing colonoscopy at the endoscopy center of the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People's Republic of China, between September 2008 and November 2010. According to recent consensus guidelines, diagnosis of IBD was based on the combination of clinical presentation and endoscopic, histological, and radiological findings (4). Patients who had received antibiotics 3 months prior to colonoscopy or had a history of previous H. pylori eradication therapy were excluded. Controls were randomly recruited from among patients who underwent colonoscopy for various reasons, such as hematochezia, polyp follow-up, constipation, and cancer screening, but had no mucosal lesions found upon colonoscopy and no abnormal histopathological findings detected by microscopy. In the IBD group, two biopsy specimens per patient were collected, one each from endoscopically involved and noninvolved areas. In the control group, two biopsy specimens were collected from locations on the normal intestinal mucosa matched with the IBD group. Biopsy specimens were used for DNA extraction and histological staining. DNA samples were extracted from intestinal biopsy specimens (about 5 mg per biopsy sample) by using the QIAamp DNeasy tissue kit (Qiagen, Hilden, Germany). Two different Helicobacter genus-specific primer sets, pair 1 (H276f and H676r) and pair 2 (C97 and C98) (14, 21), were used to amplify a fragment of approximately 400 bp within the 16S rRNA gene that is conserved among members of the Helicobacter genus. Positive (H. pylori DNA) and negative (water) controls were included in all PCR runs. All 16S rRNA gene PCR-positive samples were sequenced.

A second cohort was recruited to investigate the gastric H. pylori status as evaluated by UBT. A total of 208 IBD patients and 416 age- and sex-matched healthy controls participated in a regular check-up program in the hospital over the same period. Details of previous treatment history (i.e., use of sulfasalazine, 5-aminosalicylic acid, corticosteroids, antibiotics, and immunosuppressants) were obtained from medical records and verified by face-to-face interview. Patients were excluded if they had current or past use of proton pump inhibitor therapy or histamine blockers 1 month prior to study entry, if they had used antibiotics 3 months prior to study entry, or if they had a history of H. pylori eradication therapy. A positive history of antibiotic usage was defined as accumulated treatment duration of more than 1 week (17). The study protocol was approved by the human ethics committee of the First Affiliated Hospital, Sun Yat-sen University. All patients provided written informed consent.

The demographic and clinical characteristics of 240 patients who underwent colonoscopy are presented in Table 1. Based on the two Helicobacter genus-specific PCRs, eight CD patients (9.1%), eight UC patients (11.1%), and five controls (6.3%) were positive for Helicobacter spp. Subsequent sequencing of the PCR products showed all to be 100% similar to H. pylori. Fifteen PCR-positive patients (71.4%) had known gastric H. pylori infection, while the status of H. pylori infection for the remaining six patients was unknown. Examination of biopsy tissues stained with hematoxylin and eosin or with modified Giemsa stain showed no evidence of Helicobacter species in intestinal tissue from any group.

Table 1.

Demographic and clinical features of IBD patients and controls who underwent intestinal biopsies

Characteristic	Value for:			P value
	CD patients	UC patients	Controls
No. of subjects	88	72	80
Gender (no. of males/females)	62/26	46/26	51/29	>0.05
Mean age ± SD (yr)	29.2 ± 12.4	42.3 ± 14.1	35.1 ± 14.6	<0.05
Mean disease duration ± SD (mos)	52.9 ± 33.9	52.3 ± 49.1		>0.05
Drug history
No. treated with sulfasalazine or 5-aminosalicylic	56	60
No. treated with corticosteroid	42	21
No. treated with immunosuppressant	44	14
No. treated with no medication	17	12

The demographic and clinical characteristics of IBD patients and controls who underwent the UBT are presented in Table 2. The prevalence of H. pylori infection in IBD patients was 19.7% (18.3% for CD patients and 21.2% for UC patients) and was significantly lower than that in the age- and sex-matched controls (48.8%) (χ² = 49.267; P < 0.001) (Table 2). Patients with UC had a higher H. pylori infection rate than patients with CD, although the difference was not statistically significant (χ² = 0.273; P = 0.601). The H. pylori infection rate in IBD patients who had previously used metronidazole or ciprofloxacin was significantly lower (8.7%) than the rate in those who had not (22.8%) (χ² = 4.529; P = 0.033). However, the prevalence of H. pylori infection in nonusers of metronidazole or ciprofloxacin (22.8%) remained significantly lower than that in age- and sex-matched controls (48.5%) (χ² = 29.551; P < 0.001). Intake of other drugs (i.e., sulfasalazine, 5-aminosalicylic acid, corticosteroids, and immunosuppressants) had no significant influence on the H. pylori infection rate (P > 0.05).

Table 2.

Demographic and clinical characteristics of IBD patients and controls undergoing the [¹³C]urea breath test

Characteristic	Value for:			P value
	CD patients	UC patients	Controls
No. of subjects	104	104	416
Gender (no. of males/females)	69/35	60/44	258/158	>0.05
Mean age ± SD (yr)	31.0 ± 12.2	40.9 ± 13.6	36.0 ± 13.8	<0.05
Mean disease duration ± SD (mos)	59.6 ± 58.4	48.3 ± 45.8		0.124
No. of smokers/nonsmokers	10/94	7/97	35/381	0.749
Prevalence of H. pylori infection (%)	18.3	21.2	48.8	<0.001

In this study, which consisted of the largest number of IBD patients included in a study to date, we employed for the first time two pairs of highly sensitive and specific primers to detect Helicobacter species in Chinese IBD patients and controls (14, 21). In our study, no significant difference in the prevalence of Helicobacter species DNA in the intestinal mucosa was found between patients with IBD and controls. The sequences of all positive samples were found to be identical to the H. pylori 16S rRNA gene, which may reflect H. pylori gastric infection given that 15 of the PCR-positive patients were known to have H. pylori gastric infection. Currently, the data on whether the presence of Helicobacter species is increased in patients with IBD are inconclusive. While some studies have found an increased prevalence rate (3, 10, 12, 14, 18, 21), other studies have found otherwise (1, 2, 5, 7, 13). Discrepancies among studies may be explained by geographical and genetic differences among countries and populations and/or differences in disease stage, the length of time since initial diagnosis, and previous antibiotic or other treatments received. Further studies including newly diagnosed patients, without any history of intervention, are required to confirm this result.

We found a significantly lower prevalence of gastric H. pylori infection in both Chinese UC and CD patients than in matched controls, consistent with the result of a recent meta-analysis (11). Our study also indicated that a history of treatment with metronidazole or ciprofloxacin might, to some extent, lower the gastric H. pylori infection rate, as these antibiotics have the potential to eradicate H. pylori. However, after adjusting for the impact of metronidazole or ciprofloxacin, we still found a significantly lower prevalence of H. pylori infection in IBD patients than in matched controls. Usage of other drugs such as sulfasalazine, 5-aminosalicylic acid, steroids, and azathioprine did not influence the prevalence of H. pylori infection, in agreement with the results of most previous studies (11, 16, 17). In support of this finding, a recent study conducted on a small scale in eastern China also reported a lower prevalence of gastric H. pylori infection in Chinese IBD patients than in controls (15). The researchers in that study also found significantly increased prevalence of gastric H. pylori infection in UC patients compared to that in CD patients, while such a significant increase was absent in our study. Although the design of that study was similar to our study design, results from the previous study are questionable in several ways. First, it involved a small number of subjects. Second, it did not clearly address the criteria for diagnosing IBD and it did not clearly define exclusion and inclusion criteria in relation to treatment intervention, both of which are factors that could lead to false-negative results. Furthermore, only serological testing (for IgG antibodies) was used to diagnose H. pylori infection, which may result in false-positive findings, given the low specificity of serological testing (11). In contrast, our study involved a large number of patients and used consensus diagnostic criteria, clear inclusion/exclusion criteria, and UBT for diagnosis of H. pylori infection. Overall, results from our study provide a more accurate prevalence of gastric H. pylori infection among IBD patients.

The decline in the prevalence of H. pylori infection accompanied by a simultaneous rise in the occurrence of IBD in the 20th century had led to the hypothesis of the protective role of H. pylori against IBD (9). Our results are in concordance with this hypothesis. However, the mechanism behind this hypothesis is unknown. It was reported previously that gastric infection with H. pylori alters the dendritic cell-polarized Th17/Treg balance toward a Treg-biased response, which may influence the immunological response in the intestine (8). Another study reporting that prior H. pylori infection influences distal mucosal immune responses, as indicated by increased interleukin-10 expression measured in the mesenteric lymph nodes of mice infected with H. pylori, also confirmed that H. pylori infection in the stomach alters the immunological environment of the lower gastrointestinal tract, providing mechanistic support for the epidemiological observation of a negative association between H. pylori status and the risk of IBD (6). Further studies investigating the effect of H. pylori eradication on the risk of development of IBD are warranted to justify the beneficial role of H. pylori for IBD prevention.

In conclusion, Helicobacter species are not found to play an important role in the etiology of IBD in Chinese adult patients. The lower rate of gastric H. pylori infection found in our study in Chinese patients with IBD than in the controls suggests that gastric H. pylori infection may protect against the development of IBD. Future studies addressing this protective mechanism will be of great interest.