Sage Journals: Discover world-class research

Abstract

Objective

This study aimed to identify Helicobacter pylori virulence factors and examine their associations with clinical outcomes in Thai patients. Moreover, the association between these genotypes and gastric mucosa morphological patterns was investigated.

Methods

This retrospective study enrolled patients who underwent esophagogastroduodenoscopy at Suranaree University of Technology Hospital. The presence of the cagA and vacA genes was investigated by real-time polymerase chain reaction.

Results

The H. pylori-specific genes ureA and 16S rRNA were detected in all 698 gastric biopsy specimens. In total, 567 (81.23%) patients with H. pylori infection were positive for the cagA gene, 443 (63.46%) were positive for the vacA gene, and 370 (53.0%) were positive for both. The cagA genotype was significantly more common in patients with chronic gastritis and peptic ulcers (78.99% and 79.41%, respectively) than the vacA gene (51.48% and 55.88%, respectively) and combined genotypes (32.34% and 47.05%, respectively). Moreover, the cagA genotype was significantly more common in patients with type 4 or 5 gastric mucosa patterns (69.49% and 76.31%, respectively).

Conclusions

The cagA genotype is the main cause of serious inflammation of the gastric mucosa. The cagA gene is possibly an important factor explaining gastroduodenal disease outcomes in Thai patients with H. pylori infection.

Keywords

Helicobacter pylori cagA vacA gastroduodenal disease chronic gastritis peptic ulcer inflammation gastric mucosa

Introduction

Helicobacter pylori is a gram-negative bacterium that can colonize the human stomach. Infection by this bacterium is observed in approximately 25% of the population in developed countries, 70% to 90% of people in developing countries, and more than 50% of people in East Asia, particularly in Japan and China.^1–3 In Thailand, the prevalence of H. pylori infection is high in the northeastern region.⁴ Chronic infection by H. pylori is related to a wide variety of gastric mucosa damage ranging from chronic gastritis and gastric ulcer disease to gastric cancer and mucosa-associated lymphoid tissue (MALT) lymphoma.^5–7 The varieties of clinical outcomes of H. pylori infection are more likely attributable to variations in the virulence of individual strains. Several virulence factors have been described thus far. However, maculating cytotoxin A (vacA) and cytotoxin-associated antigen (cagA) play major roles in the clinical symptoms of H. pylori infection.^8,9 The cagA gene was proposed as a marker for the pathogenic island genome and it is an oncoprotein.¹⁰ There is strong evidence that the cagA gene suppresses phagocytosis, reduces antimicrobial peptide levels, blocks the effector T-cell response, and induces tolerogenic dendritic cells.¹¹ A meta-analysis of a case–control study revealed that cagA-positive strains increase the risk that gastric precancerous lesions develop into gastric cancer.¹² Another virulence factor, vacA, was reported to be important in the induction of vacuoles and apoptosis in the gastric epithelium.⁹ The vacA structure consists of two important regions: the signal region (s1 and s2) and the mid-region (m1 and m2).¹³ Several studies suggested that cagA and vacA are useful markers of most virulent strains of H. pylori related to severe gastroduodenal disease.^14–16 Although efforts in the past decade have been exerted to determine the association between the development of a specific disease and differences in the genotypes of H. pylori strains, the reports have not been conclusive. Therefore, the discovery of a marker for identifying patients at higher risk of gastroduodenal disease would be helpful for clinical practice. This study examined the association between the presence of the cagA and vacA genes of H. pylori and gastroduodenal diseases. Moreover, the association between these genotypes and the morphological patterns of the gastric mucosa was also investigated.

Materials and methods

Patients and specimens

Specimens were received from patients with H. pylori infection-associated diseases who underwent esophagogastroduodenoscopy (EGD) at Suranaree University of Technology Hospital (Nakhon Ratchasima, Thailand) from July 2021 to June 2022. The EGD procedures were conducted using an EVIS EXERA III (CV-190) endoscope (Olympus Corp., Tokyo, Japan). The whole stomach was reviewed by conventional gastrointestinal endoscopy. Then, gastric biopsy specimens were obtained using the site-specific biopsy method.¹⁷ Biopsy samples from the antrum and corpus were obtained for the rapid urease test, and only test-positive samples were used to extract DNA for real-time polymerase chain reaction (PCR). Biopsy samples for histological examinations were subsequently selected by the pathologist. Giemsa staining (Thermo Fisher Scientific, Waltham, MA, USA) and hematoxylin and eosin (H&E, Thermo Fisher Scientific) staining were applied for H. pylori detection. All specimens were stored at −20°C until further evaluation by molecular techniques. Patients signed a written informed consent form prior to enrollment. The study methods were approved by the Ethics Committee for Research Involving Human Subjects, Suranaree University of Technology (approval number: EC63-39). Moreover, this study followed relevant STROBE guidelines,¹⁸ and all patient details were de-identified.

DNA extraction

The extracted genomic DNA from gastric biopsies was obtained using a QIAamp DNA mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s guidelines. The concentration of the extracted DNA was investigated using a DS-11þ spectrophotometer (Denovix Inc., Wilmington, DE, USA), and the extracted DNA was stored at −20°C.

Real-time PCR

To investigate H. pylori infection, the presence of the 16S rRNA and ureA genes was examined. The H. pylori-positive cases were used to assess the presence of the cagA and vacA genes using real-time PCR. The primers for 16S rRNA, ureA, cagA, and vacA (Integrated DNA Technologies Inc., Coralville, IA, USA) are presented in Table 1. The extracted DNA from biopsy specimens was applied as templates for PCR. Real-time PCR was conducted according to the manufacturer’s instructions in a final volume of 20 µl consisting of the cDNA template, 5 pmol of each primer, and 2 × SYBR Green PCR Master Mix (Roche AG, Basel, Switzerland) using a Light Cycler VR 480 Instrument (Roche). The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 1 minute. The data were evaluated using Light Cycler 480 software, version 1.5 (Roche).

Table 1.

Primer sequences used for real-time polymerase chain reaction.

Gene	Forward	Reverse
cagA	5′-GAGTCATAATGGCATAGAACCTGAA-3′	5′-TTGTGCAAGAAATTCCATGAAA-3′
vacA	5′-CTCCAGAAGGCACACCAATAA-3′	5′-TGGCTTCCACTTCCCCATTAA-3′
ureA	5′-CGTGGCAAGCATGATCCAT-3′	5′-GGGTATGCACGGTTACGAGTTT-3′
16S	5′-GGAGTACGGTCGCAAGATTAAA-3′	5′-CTAGCGGATTCTCTCAATGTCAA-3′

Evaluation and classification of gastric mucosa morphological patterns

All endoscopic investigations were recorded, and images of the examination site were captured. The gastric mucosa morphological patterns were evaluated by three endoscopists as described by Anagnostopoulos et al.¹⁹ with some modifications. The gastric mucosa patterns were classified into five types as follows: type 1, normal gastric mucosa indicated by a honeycomb-type subepithelial capillary network pattern with a regular arrangement of collecting venules; type 2, mild chronic gastritis indicated by cone-shaped gastric pits; type 3, moderate-to-severe chronic gastritis indicated by rod-shaped gastric pits with sulci; type 4, ground-glass appearance; and type 5, dark brown patches with irregular borders and bluish margins. Types 1 and 2 indicate normal-to-mild inflammation and morphological features, and types 3 to 5 indicate moderate-to-severe inflammation with atypical morphological features.²⁰ Before reviewing the slides, the endoscopists were blinded to the results of H. pylori infection.

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, NY, USA). Comparisons between groups were performed using the χ² and Pearson correlation tests. P < 0.05 was considered statistically significant.

Results

H. pylori virulence factors and clinical outcomes

The 16S rRNA and ureA genes were detected in all 698 (100%) gastric biopsy cases. In total, 567 (81.23%) H. pylori-positive patients were positive for the cagA gene, 443 (63.46%) were positive for the vacA gene, and 370 (53.0%) were positive for both genes. Among infected patients, 538 (77.07%) had chronic gastritis, 103 (14.75%) had duodenitis, 34 (4.87%) had peptic ulcers, 13 (1.86%) had duodenal ulcers, and 10 (1.43%) had gastric cancer. The cagA genotype was significantly more common in patients with chronic gastritis and peptic ulcer (78.99% and 79.41%, respectively; P = 0.024 and 0.038, respectively), whereas the presence of vacA and the dual positive genotype had no significant association with clinical outcomes (Table 2).

Table 2.

Prevalence of Helicobacter pylori virulence factors in gastroduodenal diseases. The virulence genes and each clinical outcome were compared with those in chronic gastritis.

	Gastroduodenal diseases
Virulence genes	Chronic gastritis	Duodenitis	Peptic ulcer	Duodenal ulcer	Gastric cancer
cagA	425 (78.99%)*	64 (62.13%)	27 (79.41%)*	7 (53.84%)	6 (60.0%)
vacA	277 (51.48%)	57 (55.33%)	19 (55.88%)	6 (46.15%)	4 (40.0%)
cagA/vacA	174 (32.34%)	45 (43.68%)	16 (47.05%)	4 (30.76%)	3 (30.0%)

*, P < 0.05.

H. pylori virulence factors and the severity of chronic gastritis

The 698 patients included 441 (81.97%) patients with the type 2 or 3 pattern, 59 (10.96%) patients with a type 4 pattern, and 38 (7.06%) patients with a type 5 pattern. cagA was significantly more common in patients with the type 4 (69.49%; P = 0.041) and type 5 (76.31%; P = 0.035) patterns. However, no significant associations were established between the vacA gene or combination genotypes and gastric mucosa pathological patterns (Table 3).

Table 3.

Prevalence of Helicobacter pylori virulence factors according to the severity of chronic gastritis. The virulence genes and each gastric mucosa morphological pattern type were compared with those in gastric mucosa morphological pattern types 2–3.

	Gastric mucosa morphological pattern type
Virulence genes	Types 2–3	Type 4	Type 5
cagA	277 (62.81%)	41 (69.49%)*	29 (76.31%)*
vacA	239 (51.19%)	22 (37.28%)	17 (44.73%)
cagA/vacA	173 (39.22%)	18 (30.50%)	13 (34.21%)

*, P < 0.05.

Discussion

H. pylori virulence factors and the inflammatory response of the host have been reported to be involved in gastric lesion development induced by the bacterium. Persistent infection by H. pylori causes the development of severe complications such as chronic gastritis, peptic ulcer, duodenal ulcer, gastric MALT lymphoma, and gastric cancer.^5–7 Many studies have examined the effect of H. pylori virulence genes on clinical symptoms, whereas data about the effects of virulence genes on gastrointestinal diseases remain limited. Two main pathogenic factors have been identified in H. pylori, namely the cagA and vacA genes.^8,9 The presence of these two virulence genes has been associated with a more aggressive phenotype of H. pylori strains, and these genes have been linked to severe disease outcomes.^14–16 This study determined the prevalence of the cagA and vacA genes and combination genotypes in Thai patients with H. pylori-associated gastroduodenal diseases. Our study demonstrated that the presence of the cagA gene was associated with chronic gastritis and peptic ulcers. Meanwhile, the vacA gene and the combination genotype were not associated with any clinical outcomes. Moreover, this is the first report to evaluate the association between H. pylori virulence factors and gastric mucosa morphological patterns in gastroduodenal diseases. We revealed that the cagA gene was associated with type 4 and type 5 gastric mucosa patterns. These findings suggest that the cagA gene is an influential marker for chronic gastritis and peptic ulcer development, and it might be involved in the severity of the disease.

The prevalence of the cagA gene varies in different geographical regions. In Western countries, the prevalence of the cagA gene is 60% to 70%,²¹ but the gene was found in more than 80% to 90% of patients in East Asian countries.²² It has been demonstrated that most H. pylori strains in Thailand carry the cagA gene.²³ Our results are supportive of previous findings that Thai strains predominantly carried the cagA genotype. In addition, the prevalence of cagA-positive strains in this study was similar to that reported in the northeastern region of Thailand²⁴ and in other Asian countries, in which the prevalence rate of cagA-positive strains exceeded 80%. Moreover, this study revealed that H. pylori strains harboring the cagA gene are associated with chronic gastritis and peptic ulcers. The results were consistent with a previous report of Brazilian clinical isolates that found a significant association between cagA and peptic ulcers.²⁵ Therefore, the cagA gene might be an influential marker for identifying patients at higher risk of gastritis and peptic ulcer development.

The rate of vacA genotype positivity in this study was 63%, which corresponded to the findings of a cross-sectional study in northeastern Thailand.²⁴ Conflicting results have been obtained in studies on the association between the vacA gene and gastroduodenal disease in different geographical regions. Our study revealed no association between vacA positivity and gastroduodenal disease, in accordance with previous findings,^26–28 suggesting that the vacA genotype is not a good marker of clinical outcomes in H. pylori infection. However, other studies recorded an association between gastroduodenal disease (e.g., atrophic gastritis, peptic ulcer) and the vacA gene in H. pylori-infected patients.²⁹ Although the presence of both genes is often observed simultaneously, which increases the pathogenicity of the strains, we found that combination genotypes did not affect the clinical outcomes of H. pylori-infected patients in Thailand. One important reason for the difference might be geographical differences in H. pylori strains. Several studies demonstrated that cagA-positive H. pylori strains are involved in gastroduodenal disease severity.^8,30 Our results also indicated that H. pylori strains harboring the cagA gene were associated with inflammatory changes in the gastric mucosa, suggesting that the cagA gene could be useful in indicating severe disease in H. pylori-infected patients. Although this study included a large number of subjects, the number of patients in some groups (peptic ulcer, duodenal ulcer, and gastric cancer) remained small, and larger numbers of cases are needed.

Conclusions

The cagA gene is the major virulence factor present in H. pylori, and it is associated with severe gastroduodenal pathology that includes both chronic gastritis and peptic ulcer disease. The presence of this gene can be used as an indicator of the severity of the disease and risk of progression of gastric mucosa damage in Thai patients.

Footnotes

Author contributions

Conception and design: T. Tongtawee, W. Wattanawongdon

Acquisition of data: T. Tongtawee, W. Wattanawongdon, T.S. Bartpho

Analysis and interpretation of data: T. Tongtawee, W. Wattanawongdon, T.S. Bartpho

Writing and revision of the manuscript: T. Tongtawee, W. Wattanawongdon

Declaration of conflicting interests

The authors declare no conflict of interest regarding the publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by Suranaree University of Technology, Thailand Science Research and Innovation, and the National Science, Research and Innovation Fund.

ORCID iD

Taweesak Tongtawee

References

Ghose

Perez-Perez

Dominguez-Bello

, et al. East Asian genotypes of Helicobacter pylori strains in Amerindians provide evidence for its ancient human carriage. Proc Natl Acad Sci U S A 2002; 99: 15107–15111.

Ghotaslou

Milani

Akhi

, et al. Diversity of Helicobacter pylori cagA and vacA genes and its Relationship with clinical outcomes in Azerbaijan, Iran. Adv Pharm Bull 2013; 3: 57–62.

Siegel

Naishadham

Jemal

Cancer statistics. CA Cancer J Clin 2012; 62: 10–29.

Shoosanglertwijit

Kamrat

Werawatganon

, et al. Real-world data of Helicobacter pylori prevalence, eradication regimens, and antibiotic resistance in Thailand, 2013–2018. JGH Open 2020; 4: 49–53.

Park

Kim

, et al. Relevance of vacA genotypes of Helicobacter pylori to cagA status and its clinical outcome. Korean J Intern Med 2001; 16: 8–13.

Atherton

JC.

The pathogenesis of Helicobacter Pylori-induced gastro-duodenal disease. Annu Rev Pathol Mech Dis 2006; 1: 69–96.

Fock

Ang

TL.

Epidemiology of Helicobacter Pylori and gastric cancer in Asia. J Gastroenterol Hepatol 2010; 25: 479–486.

Blaser

Perez-Perez

Kleanthous

, et al. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res 1995; 55: 2111–2115.

Atherton

JC.

The clinical relevance of strain types of Helicobacter pylori. Gut 1997; 40: 701–703.

10.

Odenbreit

Puls

Sedlmaier

, et al. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 2000; 287: 1497–1500.

11.

Chang

Yeh

Sheu

BS.

The impacts of H. pylori virulence factors on the development of gastroduodenal diseases. J Biomed Sci 2018; 25: 68.

12.

Matos

De Sousa

Marcos-Pinto

, et al. Helicobacter pylori CagA and VacA genotypes and gastric phenotype: a meta-analysis. Eur J Gastroenterol Hepatol 2013; 25: 1431–1441.

13.

Andreson

Loivukene

Sillakivi

, et al. Association of cagA and vacA genotypes of Helicobacter pylori with gastric diseases in Estonia. J Clin Microbiol 2022; 40: 298–300.

14.

Kuipers

Perez-Perez

Meuwissen

, et al. Helicobacter pylori and atrophic gastritis: importance of the cagA status. J Natl Cancer Inst 1995; 87: 1777–1780.

15.

Zhou

Zhang

, et al. Caga genotype and variants in Chinese Helicobacter pylori strains and relationship to gastroduodenal diseases. J Med Microbiol 2004; 53: 231–235.

16.

Memon

Hussein

Miendje

, et al. Vacuolating cytotoxin genotypes are strong markers of gastric cancer and duodenal ulcer-associated Helicobacter pylori strains: A matched case-control study. J Clin Microbiol 2014; 52: 2984–2989.

17.

Tongtawee

Dechsukhum

Leeanansaksiri

, et al. Improved detection of Helicobacter pylori infection and premalignant gastric mucosa using “site specific biopsy”: a randomized control clinical trial. Asian Pac J Cancer Prev 2015; 16: 8487–8490.

18.

Von Elm

Altman

Egger

, et al. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. Ann Intern Med 2007; 147: 573–577.

19.

Anagnostopoulos

Yao

Kaye

, et al. High-resolution magnification endoscopy can reliably identify normal gastric mucosa, Helicobacter pylori-associated gastritis, and gastric atrophy. Endoscopy 2007; 39: 202–207.

20.

Tongtawee

Kaewpitoon

, et al. Correlation between gastric mucosal morphologic patterns and histopathological severity of Helicobacter pylori associated gastritis using conventional narrow band imaging gastroscopy. Biomed Res Int 2015; 2015: 808505.

21.

Rudi

Kolb

Maiwald

, et al. Diversity of Helicobacter pylori vacA and cagA genes and relationship to VacA and CagA protein expression, cytotoxin production, and associated diseases. J Clin Microbiol 1998; 36: 944–948.

22.

Maeda

Yoshida

Ikenoue

, et al. Structure of cag pathogenicity island in Japanese Helicobacterpylori isolates. Gut 1999; 44: 336–341.

23.

Uchida

Miftahussurur

Pittayanon

, et al. Helicobacter pylori Infection in Thailand: A Nationwide Study of the CagA Phenotype. PLoS ONE 2015; 10: e0136775.

24.

Bartpho

Wattanawongdon

Tongtawee

, et al. Precancerous Gastric Lesions with Helicobacter pylori vacA+/babA2+/oipA+ Genotype Increase the Risk of Gastric Cancer. Biomed Res Int 2020; 2020: 7243029.

25.

Ribeiro

Godoy

Benvengo

, et al. Clinical relevance of the cagA, vacA and iceA genotypes of Helicobacter pylori in Brazilian clinical isolates. FEMS Immunol Med Microbiol 2003; 36: 181–185.

26.

Reza

Morteza

Mohammad

, et al. Diversity of Helicobacter Pylori cagA and vacA Genes and Its Relationship with Clinical Outcomes in Azerbaijan, Iran. Adv Pharm Bull 2013; 3: 57–62.

27.

Siavoshi

Malekzadeh

Daneshmand

, et al. Helicobacter pylori endemic and gastric disease. Dig Dis Sci 2005; 50: 2075–2080.

28.

Kamali-Sarvestani

Bazargani

Masoudian

, et al. Association of h pylori caga and vaca genotypes and il-8 gene polymorphisms with clinical outcome of infection in Iranian patients with gastrointestinal diseases. World J Gastroenterol 2006; 12: 5205–5210.

29.

Sugimoto

Zali

Yamaoka

The association of vacA genotypes and Helicobacter pylori -related gastroduodenal diseases in the Middle East. Eur J Clin Microbiol Infect Dis 2009; 28: 1227–1236.

30.

Jang

Jones

Olsen

, et al. Epidemiological link between gastric disease and polymorphisms in VacA and CagA. J Clin Microbiol 2010; 48: 559–567.

Relationship between Helicobacter pylori virulence genes and gastroduodenal disease

Abstract

Objective

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Patients and specimens

DNA extraction

Real-time PCR

Evaluation and classification of gastric mucosa morphological patterns

Statistical analysis

Results

H. pylori virulence factors and clinical outcomes

H. pylori virulence factors and the severity of chronic gastritis

Discussion

Conclusions

Footnotes

Author contributions

Declaration of conflicting interests

Funding

ORCID iD

References