Abstract

Background: Prolong infection with Helicobacter pylori may lead to chronic inflammation of gastroduodenal mucosa which in turns develops into severe diseases like peptic ulcer and gastric carcinoma. Bacterial virulence factor Cytotoxin Associated gene (cagA) is found to be responsible for developing such severe diseases in different countries. So this study was conducted to assess the relationship between occurrence of several gastroduodenal diseases and the presence of H. pylori cagA gene in Bangladeshi patients. Methods: Endoscopic gastroduodenal biopsy sample of 113 dyspeptic patients from different districts of Bangladesh were studied. H. pylori infection was detected by Rapid urease test, PCR of ureC gene and histological staining (Geimsa). Gastroduodenal disease was diagnosed by histopathological examination and cagA gene was detected by PCR. Result: H. pylori infection was identified among 48% (54/113) patients. Fifty seven percent of H. pylori infected patients were found to be cagA gene positive. cagA gene is significantly associated with Duodenal ulcer (p= .024) and Gastric carcimoma (p < .001). However, a further larger study is required to confirm this finding.

1. Introduction

Helicobacter pylori is a curved Gram negative bacteria that is colonizes in the gastric mucosa of about half of the world’s population where it causes a variety of clinical outcomes ranging from asymptomatic carriage to gastritis, peptic ulcers, and cancer ¹. However, less than 20% of infected patients present with clinical symptoms suggesting that, the disease severity is dependent on interactions between the host and the environment, and bacterial virulence ².

Many putative virulence factors have been identified in H. pylori that contribute to its pathogenesis. The 128-kDa cytotoxin-associated gene encoded antigen A (cagA) and vacuolating cytotoxin antigen gene (vacA) are known as the most important ones ^{3, 4}.

The cagA gene is a strain-specific H pylori gene and has been widely recognized as a marker for strains that confer increased risk for peptic ulcer disease ⁵ and gastric cancer ⁶. The cagA gene is present downstream of a 40-kb cluster of virulence genes known as the cag pathogenicity island (cag- PAI). These virulence genes encode a type IV secretion system that forms a syringe-like structure to translocate the cagA protein into the gastric epithelial cells. The cag-PAI has also been implicated in the induction of IL-8 in cultured gastric cells ⁷. This property contributes to the virulence capability of the cag positive strains by enhancing their proinflammatory power.

In contrast to the cagA gene, nearly all H. pylori strains around the world has possesed the vacA gene ⁸. A strong correlation has been found between the presence of the cagA gene with expression of the vacuolating cytotoxin activity ⁹. Furthermore, it has been found that most strains possessing cagA also possess the more virulent vacuolating form of VacA ¹⁰.

A number of recent studies have suggested that, infection with cag PAI-positive strains of H. pylori may significantly increase the risk of developing severe gastric mucosal inflammation, duodenal ulceration and gastric cancer and its precursor lesions ^{11, 12}.

Although, serological methods to detect specific antibodies to the H. pylori cagA protein would be more suitable ¹³ than genotype identification as it needs endoscopic gastroduodenal biopsy samples, Genotype identification is more accurate procedure as subjects infected with H. pylori strains containing the cagA gene do not always induce serum cagA antibody ¹⁴. Moreover, it has been suggested that host immunological responses to H. pylori may vary in different populations ¹⁵.

H. pylori has a global distribution but geographical differences in the prevalence of cagA status among H. pylori isolates have been reported ¹⁶. Even within country, the prevalence rate may vary between distinct geographic region ¹⁷. Moreover no study was done to determine the association between cagA gene and gastroduodenal diseases in our country.

So the aim of this study was to determine the association between the presence of cagA genotype in H. pylori and the severity of different gastroduodenal diseases in a group of Bangladeshi patients.

2. Methods

The study was conducted in Department of Microbiology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka.

The present study included 113 Bangladeshi patients who underwent upper Gastrointestinal endoscopic examination at outpatient department of Department of Gastroenterology of Bangabandhu Sheikh Mujib Medical University (BSMMU), Shahbagh, Dhaka, and Dhaka Medical college Hospital, Dhaka between March, 2015 to February 2016. Out of 113 patients 65 patients were endoscopically reported as gastritis followed by 18 duodenal ulcer, 17 gastric ulcer and 13 gastric carcinoma. Patient aged from 18 to onward presenting with symptoms of dyspepsia more than 1 month were included in the study. Patients who received H. pylori eradication treatment in the previous 2 months ¹⁸, Elderly individuals who had age more than 65 years, had severe medical or surgical illnesses or had used proton pump inhibitors, nonsteroidal anti-inflammatory drugs, colloidal bismuth compounds, or antibiotics within 4 weeks of enrollment were excluded from the study ¹⁹.

The study populations were categorized into 2 groups based on the case definition used in this study: H. pylori positive patients (n = 54) & H. pylori negative patients (n = 59). Patients were considered as H. pylori positive when positive results were obtained in at least two of the three tested methods (Rapid urease test, histology for H. pylori and PCR for ureC gene) and considered as negative when the results of all diagnostic tests were negative. Gastroduodenal diseases was diagnosed by endoscopic and histopathological examinations and established in accordance with the Sydney System Classification.

From each patient three pieces of gastric tissue were taken from nonlesional mucosa of the lesser curvature side of the antrum and three pieces from midbody for H. pylori detection (Pentax Video-Endoscopy EG/3485). Additional biopsies were taken from margins of malignant looking ulcers or proliferative growths for histopathological examination to confirm the diagnosis. Two specimen, each from the antrum and body were fixed in 10% buffered formalin and send to the Pathology Department of BSMMU and DMC hospital for histopathological examination. One specimen each from the body and antrum were examined for the presence of H. pylori by rapid urease test and one specimen from the antrum and body were preserved in 1.5 ml microcentrifuge tube containing 1 ml phosphate buffer solution for detection of H. pylori ureC gene and cagA gene by PCR. All biopsy samples were stored at -20°C until DNA extraction from the samples were performed.

DNA from gastric tissues was extracted by using the QlAamp (QIAGENÒ) DNA Mini Kit according to the manufecturer’s instructions. For confirming the presence of H. pylori DNA in tissue, the ureC gene was identified by PCR using the following primers ²⁰. (Figure 1)

H. pylori cagA gene was detected by using following primers designed by Yamaoka et al. (1997) ²¹ (Figure 2).

The PCR was performed as previously reported in another study ²². The DNA were denatured at 94°C for 4 minutes, followed by 35 cycles at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds with a final extension at 72°C for 7 minutes.

This work was ethically approved by Institutional Review Board, BSMMU; process number BSMMU/2015/10008. Written, informed consent was obtained from all patients.

The sample size was calculated by using the following formula. A 95% confidence interval was used. The seroprevalence of H. pylori infection in Bangladesh was 92% and acceptable error was 5%. The statistical analysis was based on the creation and categorization of variables and was performed by using SPSS version 20.0 (Statistical Program for the Social Sciences Inc., Chicago, USA). The Chi-square test or Fisher’s exact test were used to compare between proportions. Values of p < 0.05 were considered statistically significant.

3. Results

The Out of 113 patients, majority (65) were endoscopically reported as gastritis followed by 18 duodenal ulcer, 17 gastric ulcer and 13 gastric carcinoma. The study populations were between the ages 18- 65 years, with mean age 39.4±12.8 years. Majority of gastritis patients (15, 23%) were in age group between 21-30 years whereas 35.3% (6/17) of gastric ulcers and 46.2 % (6/13) of gastric carcinoma patients were in 41-50 years of age group. Thirty three percent (6/18) patients of duodenal ulcer were in age group between 60-65 years (Table 1). 72 (63.7%) of the study population were male and 41 (36.3%) were female with male female ratio of being 2:1. The relation between endoscopic findings and histopathologial findings are shown in Table 2. Endoscopic findings of the study population were significantly associated with histopathological findings (p<.001) (Table 2). In the present study 47.8% (54/113) were defined as H. pylori positive cases and 52.2 % (59/113) were considered as H. Pylori negative cases (Table 3).

The rate of H. pylori infection in different histopathologically confirmed gastroduodenal diseases are demonstrated in Table-4. Gastric carcinoma is significantly associated with H. pylori infection (p=.046). Table-5 has revealed the frequency of positive cagA gene in different gastroduodenal diseases among H. pylori positive cases. Out of 54 H. pylori positive patients, 31 (57.4%) were cagA gene positive.

The cagA gene is significantly associated with duodenal ulcer cases (p=.024) and gastric carcinoma (p=<.001).

4. Discussion

In the present study 47.8% was diagnosed as H. pylori positive cases but the prevalence of H. pylori in Bangladesh is reported to be 42% by 2 years of age with a rapid increase to 67% by 10 years of age ²³. On the other hand, Rahman et al. (2009) ²⁴ has found H. pylori positive in 53.3% cases by RUT and in 43.4% cases by histology. In another study about 92% adult population in Bangladesh was seropositive for H. pylori ²⁵. The present study has found lower H. pylori detection rate in comparison to a study done by Hanif et al. (2010) ²⁶ in Pakistan who found 68% H. pylori positive by RUT, 62% by PCR for ureC gene and 64% by histological staining. On the other hand, 91% of the studied dyspeptic patients were positive for H. pylori in an Egyptian study by Amer et al. ²⁷ Prevalence of H. pylori infection varies between and within countries depending on socioeconomic factors, different demographic distribution of the organism among various regions, previous antibiotic consumption ^{28, 29} and the method of detection of infection because gastroduodenal biopsy based tests may give false negative results due to sampling error ³⁰

Among the histopathology confirmed intestinal metaplasia patients, 75% were H. pylori positive cases but In the current study, 39.3% of the gastritis and 58.8% of the duodenal ulcer patients were infected with H. pylori. This findings correlate with the findings reported by Helaly et al. (2009) ³¹ in Egypt who found 41.1% of the gastritis patients and 54.5% of duodenal ulcer patients to be H. pylori positive. In spite of high prevalence rate of H. pylori infection among dyspeptic patients, a low incidence of gastric adenocarcinoma has been found in the present study as reported by Miwa et al, 2002 ³².

In the present study, H. pylori infection was predominant among the gastric carcinoma patients (p=.046). Yamagata et al. (2000) ³³ in Japan observed similar findings among the age-adjusted gastric cancer patients. Only a minority of infected patients develops severe diseases and variations in clinical outcome may be due to the considerable genetic diversity of the H. pylori strains that cause infection, and host factors.

The prevalence of cagA positive H. pylori varies among different geographic regions. The primary geographic influence has an important role in the adaptation of organism to the environment and climatc conditions ³⁴. In this study, out of 54 H. pylori positive cases 31(57.4%) were cagA gene positive. This finding correlates with the findings of several studies, where cagA positivity rate was reported 61% in China ³⁵, 61.6% in Tunissia ³⁶ and 65.9% in Brazil ³⁷. Almost similar finding was reported by Rahman et al. ³⁸ (2003) in Bangladesh who found 68.4% cagA gene positive among 57 culture positive H. pylori cases. H. pylori cagA positive strains are more virulent causing higher level of gastric mucosal inflammation in gastritis and gastric cancer ³⁹. In the present study, in relation to different gastroduodenal diseases cagA gene was positive in 70% of duodenal ulcer and 41.7% of gastritis patients. It has been reported that, cagA was present among 75% of the strains from the patients with duodenal ulcer disease and 55% of strains from patients with gastritis in Bangladeshi patients ³⁸. The cagA was significantly associated with duodenal ulcer cases (p =.024) in this study. This finding is in agreement with studies done by Nomura et al. (2002) ⁵ and Yamoka et al. (1999) ⁴⁰ in Japan who found strong association between cagA gene and peptic ulcer diseases but in contrast, no association was found between cagA status and duodenal ulcer in Singapore ⁴¹. The large variation in the H. pylori genome that amplifies the cagA gene from H. pylori isolated in one country failed to detect cagA from another country ⁴². Moreover, there may be several distinct form of cagA gene with an uneven geographical distribution that may provide a marker for difference in virulence among cagA positive H. pylori strain. This may be the reason for difference in cagA status in different countries. Moreover, selection criteria of patients and large diverse study groups with respect to genotypes and clinical symptoms are also considered as important factor for variation in results in different regions ⁴³

A high frequency of cagA-positive strains was observed in intestinal metaplasia and gastric dysplasia patients (Table 5), indicating that a statistical association could be reached by increasing the number of patients in future studies.

In this study, 87.5% (7/8) of the gastric carcinoma patients were infected with cagA gene positive strains. Zohu et al (2004) ⁴⁴ reported 100% of gastric carcinoma patients were cagA positive. A significant association is observed between cagA status and gastric carcinoma (p <.001) in the present study. It has been suggested that, the persons infected with cagA positive H. pylori are at considerably increased risk of gastric cancer than with uninfected subjects ⁴⁵.

5. Conclusion

Our present study demonstrated that, the cagA gene is significantly associated with duodenal ulcer disease and gastric carcinoma in Bangladeshi patients. One of the major limitations of our study was that we could not compare our result with serological test as anti cagA antibody cannot be detected due to limitation of time and budget. Besides H. pylori colonizer was not identified as asymptomatic patients were not included in the study. Though our study provides a significant association between infections by cagA gene-positive strains of H. pylori and severe clinical outcomes, but future multicentre studies on a large scale with a full characterization of Bangladeshi H pylori isolates are required to confirm our findings and overcome above mentioned limitations.

Acknowledgements

We are very grateful to Scientific Officer Md. Yunus Ali & all the laboratory technologists of Department of Microbiology, BSMMU.

References