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BRIEF COMMUNICATION
Year : 2017  |  Volume : 35  |  Issue : 4  |  Page : 600-603
 

Molecular identification and detection of virulent factors in Helicobacter pylori from gastric biopsy samples of patients attended at Assam Medical College and Hospital, Dibrugarh, Assam, India


1 Department of Microbiology, Assam Medical College and Hospital, Dibrugarh, Assam, India
2 Department of Surgery, Assam Medical College and Hospital, Dibrugarh, Assam, India

Date of Web Publication1-Feb-2018

Correspondence Address:
Dr. Lahari Saikia
Department of Microbiology, Assam Medical College and Hospital, Dibrugarh - 786 002, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_232

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 ~ Abstract 

The present study aimed to detect the major virulence determinants of Helicobacter pylori, the gastric bacteria by polymerase chain reaction from genomic DNA of 314 gastric biopsies from dyspeptic patients in 2015-2016 through upper gastrointestinal endoscopy. In 153 cases out of 314, the high prevalence of oipA gene followed by cagA-vacA s1 m1 combined genotypes was found in mostly gastritis/duodenitis patients followed by the peptic ulcer and normal patients. Therefore, the clinical significance of the virulence markers of H. pylori associated with the severe forms of gastroduodenal diseases is still a matter of controversy since the endoscopically normal patients were found to harbour the virulent genes.


Keywords: Helicobacter pylori, peptic ulcer disease, polymerase chain reaction, virulent genes


How to cite this article:
Sarma A, Saikia L, Bhuyan RK, Hussain E. Molecular identification and detection of virulent factors in Helicobacter pylori from gastric biopsy samples of patients attended at Assam Medical College and Hospital, Dibrugarh, Assam, India. Indian J Med Microbiol 2017;35:600-3

How to cite this URL:
Sarma A, Saikia L, Bhuyan RK, Hussain E. Molecular identification and detection of virulent factors in Helicobacter pylori from gastric biopsy samples of patients attended at Assam Medical College and Hospital, Dibrugarh, Assam, India. Indian J Med Microbiol [serial online] 2017 [cited 2019 Dec 8];35:600-3. Available from: http://www.ijmm.org/text.asp?2017/35/4/600/224429



 ~ Introduction Top


Helicobacter pylori, a previously obscure organism, has now been associated with many diseases involving gastroduodenal tissue such as chronic gastritis, gastroduodenal ulcer disease and gastric carcinoma and infects 50% of the human population which might remain initially asymptomatic.[1],[2] There are increasing evidence that H. pylori pathogenesis and their diverse outcome of infection may be attributed to variability of genetic markers which may be clinically important.[2],[3]

The cytotoxin-associated gene (cagA), present in about 60% of H. pylori strains, is a marker for the cag region, a pathogenicity island consisting of several genes which induce morphological alterations as well as interleukin-8 (IL-8) secretion in the gastric epithelial cells.[4] The vacA gene, with two variable regions-the signal region (s) and middle region (m), encodes a protein toxin which induces the formation of vacuoles in primary gastric epithelial cells, thus causing injury to the gastric epithelium.[2] Several studies have suggested that the presence of these genes is associated with more severe forms of the gastroduodenal disease than any other clinical outcome. The induced by contact with epithelium, (iceA) gene has been considered as a marker for the peptic ulcer disease.[5] The outer membrane protein oipA is an inflammation-related gene which is associated with IL-8 secretion by gastric epithelial cells.[6] The blood group antigen binding adhesin (babA) and the sialic acid-binding adhesion (sabA) mediates bacterial adherence to human gastric epithelial cells.

The present study has been undertaken to evaluate the occurrence and spectrum of virulence factors of H. pylori in dyspeptic patients by polymerase chain reaction (PCR) from Assam, Northeast India.


 ~ Materials and Methods Top


A total of 314 adult patients, undergoing upper gastrointestinal endoscopy for dyspeptic symptoms were enrolled in the study. Patients were grouped into on the basis of age and gender. Of these, 115 patients had gastritis/duodenitis, 47 had peptic ulcer disease (PUD), 92 had non-ulcer dyspepsia, 20 had gastric carcinoma and the remaining 40 was categorised into 'others', which included gastroesophageal reflux disease, stomach and oesophageal varices, oesophageal growth. Two biopsy samples from the antrum of the stomach were obtained in 0.6 ml brain heart infusion broth (Difco Laboratories, Detroit, MI, USA) with 15% glycerol and transported to the microbiology laboratory. All cases were included after obtaining written consent from them or their guardians (in case of minors). The present study was approved by the institutional ethics committee (human) of our institution before initiation of the work and was carried out according to the guidelines of the committee. Genomic DNA was extracted by the QIAamp DNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The extracted DNA was kept at −20°C until used for PCR.

Polymerase chain reaction amplification of Helicobacter pylori virulent genes (CagA, VacA, IceA, OipA, BabA and SabA)

H. pylori was detected form the biopsy samples by PCR of ureA gene and 16S rRNA gene. PCR primers of all genes were designed on the basis of published sequences.[7],[8],[9],[10] After DNA extraction, amplification of the virulent genes was carried out in a final volume of 20 μl containing PCR buffer, 200 μM (each) deoxynucleotides, 1.5 mM MgCl2, 2U Tag polymerase (all from Sigma Chemicals Co., St. Louis, MO, USA), 10–50 ng of genomic DNA and 10 μM of corresponding primers. The reaction mixtures were amplified as follows: initial denaturation at 96°C for 2 min, followed by 30 cycles of denaturation −30 s at 94°C, 40 s of annealing (55°C for vacA m1, m2, s1, cagA PCR, 45°C for iceA PCR, 49°C for oipA and 57°C babA and sabA PCR) and 1 min of extension at 72°C. Final extension was continued for 5 min at 72°C.

For all PCR assays, genomic DNA from H. pylori strain 26695 was used as positive control. Agarose gel electrophoresis was performed on the amplified PCR products.

Statistical analysis

Analysis of data was performed using Fisher's exact test or the Chi-square test. Epi-Info software (version 7.1.3, Atlanta, Georgia, US) was used and the value of P < 0.05 was considered to be statistically significant.


 ~ Results Top


The study results showed that the age groups of 30–60 years and the male population of our study region showed an increased rate of H. pylori infection than any other group. However, there was no significant association between the age and the sex of the patients and the presence of H. pylori (P > 0.05).

Out of 153 cases studied, gastritis/duodenitis was present in 57 (37.25%) cases, followed by 30 (19.61%) peptic ulcer cases, 37 cases (24.18%) had non-ulcer dyspepsia and nine cases (5.88%) had gastric cancer. Those positive by ureA PCR (153 cases) were subjected to PCR of virulent genes and statistical analysis.

Helicobacter pylori virulent genes detected in biopsy samples in different types of clinical lesions

In the present study, high prevalence (80.3%) of H. pylori oipA gene was noted mostly in H. pylori positive chronic gastritis and duodenitis patients, followed by cagA (66.6%) and vacA s1 m1 allelic variant (60.7%). VacA s1 m1 gene was present in 93 patients, whereas s1 m2 gene was present in only 9 patients out of 153 [Figure 1]. VacA genotypic combination s2 m1 and s2 m2 was not found in our study. Eighty-three cases (54.2%) were found to be positive for both cagA and vacA genotypes whereas 67 (43.8%) cases were positive for combined cagA, vacA, iceA and oipA genotypes.
Figure 1: Amplification of Helicobacter pylori virulent genes: (a) VacA m1, m2, s1, CagA- Lane 2- positive control. Lanes 3,5,6 and 8- VacA s1m1 CagA genotype at 259 bp, 567 bp and 350 bp, respectively. Lane 7- VacA s1m2 genotype at 259 bp and 642 bp, respectively (b) BabA and SabA- Lane 1 and 2- amplification of SabA gene at 622 bp. Lane 4- multiplex polymerase chain reaction of BabA and SabA genes at 833 bp and 622 bp, respectively. (c) IceA 1, A2- Lane 1- IceA 2 at 229 bp. Lane 2- IceA 1 at 247 bp (d) OipA- Lanes 2-5- OipA genotype at 401 bp. DNA ladder uded-100 bp

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High percentage of most of the virulent genes was noted in chronic gastritis and duodenitis patients followed by peptic ulcer patients. The iceA gene, prevalent in gastritis and peptic ulcer patients, was detected in 96 cases out of 153 with iceA1 allelic variant being the predominant gene (79 out of 153) followed by iceA2 ( 17 out of 153). Both sabA and babA genotypes were more prevalent in gastritis/duodenitis patients than peptic ulcer patients (38.5% and 18.9%, respectively) [Table 1] and [Figure 1].
Table 1: Status of Helicobacter pylori virulent genotypes obtained from 153 polymerase chain reaction positive cases with different clinical outcomes

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While establishing a relationship between H. pylori PUD and other clinical conditions with the presence of virulent genotypes, our study showed a statistically significant association between PUD and non-ulcer dyspepsia in patients positive for cagA (P = 0.0028), iceA (P = 0.001), babA (P = 0.0183) and sabA genes (P = 0.0204). PUD was also significantly associated with gastric carcinoma for patients positive for cagA (P = 0.014), vacA (P = 0.04) and oipA genes (P = 0.018).


 ~ Discussion and Conclusion Top


The study of H. pylori genotypes by PCR can be used as a marker for infections since the sensitivity of PCR is similar to that of culture.[11] Due to the fastidious nature of the bacteria, lengthy culture times along with low culture detection rate,[11] we decided to perform PCR from DNA of gastric biopsies for efficient determination of the distribution of virulent genotypes in our area of study.

The presence of combined genotypes in single biopsy samples strongly indicates that the patients might possess mixed infection with more than one H. pylori strain rather than single infection. These results correlate with the studies performed by Ben Mansour et al., where they showed colonisation of highly virulent H. pylori strains in single patients in the relation of presence of vacA m1/m2 and iceA 1/iceA 2 in the same biopsy.[9]

In this study, the vacA s1 m1 allele was found to be associated with the presence of cagA gene. The predominance of s1 m1 allele over s1 m2 might indicate that this variant produced high levels of vacA cytotoxic activity and that there is a high accumulation of vacA s1 m1 strain among the patients of our district since they might adapt well to the gastric environment more effectively than other genotypes. This result can be compared with analytical studies on vacA typing done by Ito et al. in Japan in which there was high prevalence of vacA s1 m1 strain in almost all cagA positive isolates due to their clustering with vacA s1 m1 allelic variant.[12]

An important aspect of the present study was that all the related H. pylori virulent factors were found to be more associated with chronic gastritis and duodenitis than PUD whereas there was no H. pylori infection in 12 cases of PUD and 49 cases of gastritis/duodenitis. A study by Bindayna et al. showed a high prevalence of cagA gene in duodenal ulcer patients.[3] Peek et al. showed that the iceA gene of H. pylori was more significantly associated with PUD and may be a precursor for severity of the disease.[5] In most H. pylori genotypic-related research, maximum incidence of virulent factors can be found mainly in PUD. However, this present finding is in contrast with the literature aspect of H. pylori that it is mainly associated with PUD and might indicate the beginning of an underlying infection which might progress into PUD and other gastric conditions.

However, in the present study, 16 patients who were endoscopically normal were found to harbour cagA gene and 11 patients were found to harbour both cagA and vacA gene. Since infection with H. pylori is often associated with the absence of clinical symptoms, the possible hypothesis for this is that these patients might be initially infected with H. pylori which may eventually progress into chronic gastritis or adenocarcinomas. Similar studies were conducted by Figura et al. where non-ulcer dyspeptic patients found to be infected with both cag positive and negative colonies which might play an important role in the development of intestinal metaplasia.[13]

A major limiting factor of this study is that the analysis of the findings of the current study was based only on upper GI endoscopy and not on histopathology. The endoscopically normal or non-ulcer dyspeptic patients were categorised into non-ulcer dyspepsia, or the endoscopically proven gastric or duodenal ulcer patients were categorised into PUD.

In conclusion, the clinical significance of virulence markers of H. pylori such as cagA, vacA, iceA and oipA which are associated with severe forms of gastro-duodenal diseases is still a matter of controversy since the endoscopically normal patients without any clinical lesions were found to harbour the cagA and vacA genes.

Acknowledgement

The authors are highly grateful to the Multidisciplinary Research Unit (MRU) ICMR, Assam Medical College and Hospital, Dibrugarh, Assam, India for providing financial and infrastructure support for the study. The authors are highly grateful to Dr. R. K. Kotokey, Principal, Assam Medical College and Hospital, Dibrugarh, for allowing to conduct this study in the Multidisciplinary Research Unit (ICMR) of this institution. The authors would like to thank the endoscopy unit of the Department of Surgery, Assam Medical College and Hospital, Dibrugarh, Assam for providing the biopsy specimens.

Financial support and sponsorship

Financial and Infrastructure support from Multidisciplinary Research Unit (MRU) ICMR, Assam Medical College and Hospital, Dibrugarh, Assam, India.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clin Microbiol Rev 1997;10:720-41.  Back to cited text no. 1
    
2.
Kusters JG, van Vliet AH, Kuipers EJ. Pathogenesis of Helicobacter pylori infection. Clin Microbiol Rev 2006;19:449-90.  Back to cited text no. 2
    
3.
Bindayna KM, Al Baker WA, Botta GA. Detection of Helicobacter pylori cagA gene in gastric biopsies, clinical isolates and faeces. Indian J Med Microbiol 2006;24:195-200.  Back to cited text no. 3
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4.
Crabtree JE, Covacci A, Farmery SM, Xiang Z, Tompkins DS, Perry S, et al. Helicobacter pylori induced interleukin-8 expression in gastric epithelial cells is associated with cagA positive phenotype. J Clin Pathol 1995;48:41-5.  Back to cited text no. 4
    
5.
Peek RM Jr., Thompson SA, Donahue JP, Tham KT, Atherton JC, Blaser MJ, et al. Adherence to gastric epithelial cells induces expression of a Helicobacter pylori gene, iceA, that is associated with clinical outcome. Proc Assoc Am Physicians 1998;110:531-44.  Back to cited text no. 5
    
6.
Yamaoka Y, Ojo O, Fujimoto S, Odenbreit S, Haas R, Gutierrez O, et al. Helicobacter pylori outer membrane proteins and gastroduodenal disease. Gut 2006;55:775-81.  Back to cited text no. 6
    
7.
Chattopadhyay S, Patra R, Ramamurthy T, Chowdhury A, Santra A, Dhali GK, et al. Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens. J Clin Microbiol 2004;42:2821-4.  Back to cited text no. 7
    
8.
Chomvarin C, Namwat W, Chaicumpar K, Mairiang P, Sangchan A, Sripa B, et al. Prevalence of Helicobacter pylori vacA, cagA, cagE, iceA and babA2 genotypes in Thai dyspeptic patients. Int J Infect Dis 2008;12:30-6.  Back to cited text no. 8
    
9.
Ben Mansour K, Fendri C, Zribi M, Masmoudi A, Labbene M, Fillali A, et al. Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients. Ann Clin Microbiol Antimicrob 2010;9:10.  Back to cited text no. 9
    
10.
Goudarzi H, Rezaee H, Rafizadeh M, Taghavi A. Determination of the status of Helicobacter pylori SabA gene in relation to clinical findings. J Med Bacteriol 2012;1:3-8.  Back to cited text no. 10
    
11.
Mégraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 2007;20:280-322.  Back to cited text no. 11
    
12.
Ito Y, Azuma T, Ito S, Miyaji H, Hirai M, Yamazaki Y, et al. Analysis and typing of the vacA gene from cagA-positive strains of Helicobacter pylori isolated in Japan. J Clin Microbiol 1997;35:1710-4.  Back to cited text no. 12
    
13.
Figura N, Vindigni C, Covacci A, Presenti L, Burroni D, Vernillo R, et al. CagA positive and negative Helicobacter pylori strains are simultaneously present in the stomach of most patients with non-ulcer dyspepsia: Relevance to histological damage. Gut 1998;42:772-8.  Back to cited text no. 13
    


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