Indian Journal of Pathology and Microbiology

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 61  |  Issue : 1  |  Page : 66--69

CagA and VacA genes of Helicobacter pylori and their clinical relevance


Lavanya Jeyamani1, Jayalakshmi Jayarajan2, Venkatakrishnan Leelakrishnan2, Mukundan Swaminathan2,  
1 Department of Microbiology, Karpagam Faculty of Medical Sciences and Research, Coimbatore, Tamil Nadu, India
2 Department of Microbiology and Gastroenterology, PSG Institute of Medical Science and Research, Coimbatore, Tamil Nadu, India

Correspondence Address:
Lavanya Jeyamani
197/13, Asiad Colony, 7th Avenue, Annanagar West, Chennai - 600 101, Tamil Nadu
India

Abstract

Context: Helicobacter pylori is associated with the development of a variety of gastroduodenal diseases which varies with ethnicity and the type of strains that infect the population. Aims: This study aims to evaluate the prevalence of H. pylori cagA and vacA genotypes in our region and to determine their relationship to the severity of the lesions that they cause. Settings and Design: This study was an observational cross-sectional study. Subjects and Methods: DNA was extracted from 165 gastric biopsies from patients evaluated for dyspepsia. PCR was used to detect cagA and vacA (s1, s2, m1, m2) genes of H. pylori. Statistical analysis of associations was performed between endoscopy findings and virulence genes. Statistical Analysis Used: Pearson Chi-square test and Fischer's exact test. Results: The prevalence of H. pylori infection was 37% and the dominant genotypes was vacA s1 cagA-positive strain (54.1%) in this study. The vacAs1 subtype was found in all patients with peptic ulcer disease (PUD). The entire normal study group had VacA s2 variant only. This clearly shows that vacA s1 is a significant virulence marker and patients harboring s1 strains are more prone to develop ulcers (P = 0.007). There was a significant association of cagA with s1 strain rather than s2. Variation in VacA m genotype did not seem to have any association with disease status. There was a statistically significant association between the presence of cagA gene and PUD rather than the nonulcer dyspepsia (P = 0.027). Conclusion: The predominant genotype in our population was cagA positive vacA s1, which was found to be significantly associated with patients with gastric diseases, especially PUD. VacA s1 can serve as a single best virulence marker of the disease manifestation.



How to cite this article:
Jeyamani L, Jayarajan J, Leelakrishnan V, Swaminathan M. CagA and VacA genes of Helicobacter pylori and their clinical relevance.Indian J Pathol Microbiol 2018;61:66-69


How to cite this URL:
Jeyamani L, Jayarajan J, Leelakrishnan V, Swaminathan M. CagA and VacA genes of Helicobacter pylori and their clinical relevance. Indian J Pathol Microbiol [serial online] 2018 [cited 2022 Jan 17 ];61:66-69
Available from: https://www.ijpmonline.org/text.asp?2018/61/1/66/228163


Full Text



 Introduction



Ever since the discovery of Helicobacter pylori, the scientific facts associated with it have undergone a drastic change over a period of time. The role of this bacterium either as a pathogen or a commensal still remains centered around important factors – lifestyle of the patient, food habits in the region, and virulence of the strain.[1],[2]H. pylori has two important cytotoxin – vacA and cagA. VacA is a polymorphic gene showing variance in s and m region – s1/s2 and m1/m2. Among this, s2 is not known to be associated with virulence. It is widely studied in the western countries that the presence cagA and VacA s1 genes contribute to severe manifestation of H. pylori infection, whereas in Asian population, studied so far, cagA bore no significance. One exception was Japanese population where it was associated with gastric cancer.[2] There is a wide variation in the prevalence of these virulence factors and their association with clinical manifestations in different ethnic groups.[3],[4],[5],[6],[7],[8] In this scenario, very few studies have been carried out on virulence factors in Indian population. Hence, the current study was undertaken to evaluate the prevalence of cagA and vacA genotypes of H. pylori and their relationship to the severity of the gastric lesions produced in patients in Coimbatore region (Western Tamil Nadu, India).

 Subjects and Methods



This observational cross-sectional study was conducted during the period from May 2014 to June 2015 in the Department of Microbiology, after obtaining institutional Human ethical committee's approval. Gastric biopsy samples were collected from 165 patients with gastroduodenal diseases by upper gastrointestinal endoscopy, after obtaining their written informed consent to participate in the study. Patients who had received antibiotics within the past 1 month period and antisecretory drugs within the past 2 weeks before endoscopy were excluded. Endoscopic findings were noted down. One biopsy tissue was obtained aseptically preferably from the site of lesion or gastric antrum. The sample was transported to the molecular laboratory in sterile freshly prepared 70% ethanol and stored under –80°C for further processing. DNA extraction was done using QIAmp DNA MINI extraction kit as per the manufacturers' instruction (QIAGEN, Germany).

The presence of H. pylori in the tissue was identified by conventional PCR for glmM gene (housekeeping gene). All the positive samples were further tested for the presence of cagA and variance of vacA genes by PCR for individual genes. The respective primers and amplicon sizes are mentioned in [Table 1].[9],[10],[11],[12],[13] A single strain harbors any one of the variants in each gene, for example, VacA s1/m1, VacA s1/m2, VacA s2/m1, or VacA s2/m2. All PCRs were performed in AB StepOne-Thermocycler.{Table 1}

Single reaction mixture (25 μl) contained 4 μl of DNA suspension, 12.5 μL of Mastermix (Sigma-Aldrich, Mumbai), and 1 μl of each primer depending on the gene amplified [Sigma-Aldrich, Mumbai] (1 μM of each primer for glmM gene or 25 pmol of each primer for VacA genes or 10pmol of each primer for cagA gene). The remaining volume was adjusted with PCR grade water. Positive and negative controls were set up with each batch of tests run. H. pylori ATCC 700392 (strain 26695) was used as positive control for glmM, cagA, vacA s1 m1 the genes. Negative controls were run with PCR grade sterile water in place of template. PCR condition for glmM gene was denaturation at 95°C for 10 min, 35 cycles of 95°C for 30 s, annealing temperature 60°C for 45 s and extension at 72°C for 1 min and a final elongation at 72°C for 5 min.[9] PCR condition for cagA And variants of vacA genes were denaturation at 94°C for 3 min, 35 cycles of the following condition –94°C for 1 min, annealing temperature of 55°C for 1 min and extension at 72°C for 1 min, and a final elongation at 72°C for 10 min.[10],[11],[12],[13] The amplified product is visualized by use of agarose gel electrophoresis. Images of the gel were captured by gel doc and interpreted.

Statistical analysis was performed using SPSS software (Statistical Product and Services Solutions, version 17, SPSS Inc., Chicago, IL, USA) to analyze data. Association between gastric lesions and genotypes was tested independently using Pearson Chi-square test and Fischer's exact test. All Chi-square test and Fischer's exact test results with P < 0.05 were considered statistically significant.

 Results



Among the dyspeptic patients, 107 (65%) were male and 58 (35%) were female. [Figure 1] shows the distribution of cases according to their endoscopy finding. Out of 165 patients included in this study, 86% (142) had significant endoscopic findings – gastritis, gastroduodenitis, peptic ulcer, etc., Out of 165 patients, 61 (37%) had H. pylori infection confirmed by glmM PCR. On analysis of virulence genes, cagA positivity was 57.4% in our study. [Table 2] shows the percentage of cagA positivity among different pathological conditions. There was a statistically significant association between the presence of cagA gene and peptic ulcer disease (PUD) rather than the nonulcer dyspepsia (P = 0.027, odds ratio was 6 with 95% confidence interval [CI], range 1.19–30.1) as shown in [Table 3]. Although cagA positivity was high among gastric ulcer (90.9%) and gastroduodenitis (83.3%) patients compared to patients with normal endoscopic findings, statistical analysis by Fisher's exact test found no significance (P = 0.642, odds ratio 2.15 with 95% CI, range 0.33–13.92) attached to the cagA status and disease manifestation.{Figure 1}{Table 2}{Table 3}

Among 61 strains distribution of vacA s and m polymorphs were as follows – 44 vacA s1 and 17 vacA s2, 31 vacA m1 and 30 vacA m2. [Table 4] shows that all 44 vacA s1 subtype identified were found only in patients with pathological findings, whereas the s2 subtype was found in both normal as well as diseased patients.{Table 4}

In the light of clinical manifestations, among the 35 patients who had gastritis s1 m1 and s1 m2 subtypes predominated with 34.3% each. Gastric ulcer and gastroduodenitis patients shared a similar picture where all the strains were either s1 m1 or s1 m2 subtypes with no vacA s2 subtype. All 3 duodenal ulcer patients had harbored only vacA s1 m1 subtype. Statistical analysis was done using Pearson Chi-square test, and there was a significant association between the vacA subtypes and the above-mentioned endoscopy findings (P = 0.007).

Co-occurrence of cagA and vacA analysis showed that 33 (75%) of the 44 vacAs1 isolates were cagA positive, while only 2 (11.8%) of the 17 vacAs2 isolates were cagA positive.

Among 30 nonulcer dyspepsia patients with vacA s1 subtype, 21 (70%) were cagA positive. Among 14 PUD patients with vacA s1 subtype, 12 (85.7%) were cagA positive. Difference in cagA status had no statistically significant association with the disease manifestation in vacA s1-positive patients. [Figure 2] shows the gel doc images of the genes studied.{Figure 2}

 Discussion



The prevalence of H. pylori infection among this study population was 37%. Although WHO states that the prevalence is 70%–90% among Asian population, it is known to vary widely across our country.[14]H. pylori infection was maximum in patients with duodenal ulcer (100%) and gastric ulcer (78.6%), followed by gastroduodenitis (54.5%). H. pylori infection had a significant association with PUD rather than nonulcer dyspepsia (P = 0.001, odds ratio 8 with 95% CI, range 2.2–29.8). On contrary to the studies from Eastern Asia,[15] all the 3 carcinoma stomach patients included in this study did not have H. pylori in their gastric mucosa. Only 21.7% of the study group with normal endoscopic finding had H. pylori in their gastric mucosa and all of them were vacA s2 strains, clearly showing that vacA s2 strains are far less virulent than s1 strains.

Studies show that H. pylori-induced gastroduodenal diseases varied in severity and manifestations depending on the strain virulence. In this study, we have analyzed cagA and vacA variant status of bacteria directly from the tissue biopsy.

Prevalence of vacAs1 variant (72%) was greater compared to s2 variant (28%). A statistically significant association was found between the presence of vacA s1 and disease manifestation (P< 0.01). Nonulcer dyspepsia patients showed a higher prevalence of vacA s1 compared to s2, and this association was significant. None of the vacA s1 strains was found in cases with normal endoscopic findings. All the strains from duodenal ulcer cases were vacAs1 m1 type whereas gastric ulcer patients had 63.6% and 36.4% of s1 m1 and s1 m2, respectively. VacAs1 subtype was found in all patients with PUD.

The entire normal study group had VacA s2 variant only. Research evidences show that vacA s2 strain is not an efficient toxin producer. None of the ulcer disease and gastroduodenitis patients had s2 variants. Twelve vacA s2 strains were found in patients with gastritis. This could be due to confounding factors which were not included in this study. This clearly shows that vacA s1 is a significant virulence marker for development of ulcers and s2 is a benign marker with a questionable need for eradication therapy.

On analysis of virulence genes, cagA positivity was 57.4% in our study. This is in accordance with the global data (60%–70%). There was a statistically significant association between presence of cagA gene and PUD rather than the nonulcer dyspepsia (P = 0.027). Although cagA positivity was high among patients with gastric ulcer (90.9%) and gastroduodenitis (83.3%) compared to those with normal endoscopic findings, statistical analysis by Fisher's exact test found no significance (P = 0.642) attached to the cagA status and disease manifestation.

When the co-occurrence of cagA and vacA subtypes was studied, we found that cagA was more often associated with s1 strain rather than s2. Percentage of vacA s1 cagA positive strain was 54.1 in Coimbatore region. On analysis, this co-occurrence of vacA s1 cagA positivity had no influence on the diseases. VacA m subtype was equally distributed among s1 variants and did not have any significance.

All infected patients were treated with anti-H. pylori therapy and clinically cured. However, long-term follow-up of the infected cases could not be carried out due to shorter time period of the study.

 Conclusion



These findings show that the vacA s1/s2 genotype has an important role in the clinical outcomes. Hence, this genotype can be used to identify patients who are at a higher risk for gastroduodenal disease and those who may not require treatment. Patients infected with the strain carrying vacA s1 genotype should be checked for eradication status on completion of antibiotic treatment, to prevent the development of PUD in future.

Acknowledgment

We gratefully acknowledge the financial support offered by Research committee, PSGIMSR. We are thankful to Head of the department of Microbiology for his support. We also thank the patients who have participated in this study.

Financial support and sponsorship

This study was financially supported by PSGIMSR through PSG Prime Funds.

Conflicts of interest

There are no conflicts of interest.

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