LGCmain
Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 2056
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 3  |  Page : 535-544
Correlation of Helicobacter pylori virulence genotype & severity of mucosal inflammation in gastric biopsies from two geographically diverse regions in India


1 Department of Pathology, All India Institute of Medical Sciences, New Delhi, India
2 Centre for Medical Biotechnology, Amity Institute of Biotechnology, Amity University, Noida, Uttar Pradesh, India
3 Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, India
4 Department of Gastroenterology and Human Nutrition, All India Institute of Medical Sciences, New Delhi, India

Click here for correspondence address and email

Date of Submission06-Jun-2021
Date of Decision09-Jun-2021
Date of Acceptance11-Jun-2021
Date of Web Publication21-Jul-2022
 

   Abstract 


Background: H. pylori-associated gastritis in patients from the high-altitude area of Ladakh showed severe gastritis, mucosal nodularity, atrophy, and cancer in comparison to those from North India. This study served to analyze if differences in the H. pylori virulence genotypes decide the extent of gastric mucosal inflammation. Methods: Fifty gastric biopsies each from patients with H. pylori-associated gastritis from Ladakh and a tertiary care center in North India were included. The presence of H. pylori strain was confirmed with Warthin starry stain and polymerase chain amplification of the H. pylori-specific 16S rRNA. The cagA, vacA s1, s2, and m1, m2 alleles, and dupA virulence genotypes were studied in all archival samples, followed by their histological correlations. Results: cagA (P 0.009) and vacAs1 m1 (P 0.009) genes were distinctly more in H. pylori strains colonizing the biopsies of North Indian patients. In contrast, the cagA -ve vacAs2 m2 strains were significantly more in H. pylori strain colonizing the biopsies from Ladakhi patients. dupA genotype was almost similarly present in strains from both regions. Among these, only cagA and dupA virulence genes were associated with severe mucosal neutrophilic activity and deep infiltration of H. pylori strains in North Indian patients. Conclusions: Differences in virulence genotypes of H. pylori in gastric biopsies from North Indian and Ladakhi patients were found not significant in deciding the severity of H. pylori-associated gastritis.

Keywords: Gastric biopsy, genotype, Helicobacter pylori, histology, Ladakh, North India

How to cite this article:
Sharma N, Das P, Das R, Mahant S, Kalaivani M, Yadav R, Rajeshwari M, Kedia S, Makharia GK, Saraya A, Gupta SD, Ahuja V. Correlation of Helicobacter pylori virulence genotype & severity of mucosal inflammation in gastric biopsies from two geographically diverse regions in India. Indian J Pathol Microbiol 2022;65:535-44

How to cite this URL:
Sharma N, Das P, Das R, Mahant S, Kalaivani M, Yadav R, Rajeshwari M, Kedia S, Makharia GK, Saraya A, Gupta SD, Ahuja V. Correlation of Helicobacter pylori virulence genotype & severity of mucosal inflammation in gastric biopsies from two geographically diverse regions in India. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Aug 15];65:535-44. Available from: https://www.ijpmonline.org/text.asp?2022/65/3/535/351603





   Introduction Top


The diverse socio-economic and geographical landscape, and the ethnic differences of its resident population have shaped the mélange of disease outcomes across India. Whereas the prevalence of Helicobacter pylori–induced gastritis varies between 60% and 70% in rural India, in high-altitude areas of Ladakh (situated nearly 14,070 feet above sea level), a prevalence of almost 90% has been reported. The most recognized manifestation of H. pylori infection in India is peptic ulcer disease, particularly duodenal ulcers.[1],[2] A severe form of pangastritis, gastric mucosal nodularity, and the presence of mucosal atrophy were reported in patients from Ladakh.[3],[4],[5],[6],[7] Whereas the etiological organism is the same, differences of disease manifestations in gastric mucosa between these two regions can be either due to 1) differences in host genotypes, 2) genetic diversity of the colonizing H. pylori strains, or 3) other environmental factors that affect the infection course, like dietary practices or cultural habits, etc.[8],[9],[10],[11]

Whereas the residents of Ladakh are mostly Tibetan-Buddhists (about 70%) and ancestrally related to the Baltistan area of Pakistan and the rest to the Jammu and Kashmir (about 30%),[12],[13] in comparison, the inhabitants of north India are mostly Indo-Aryans.[14],[15] Being a mountain desert, fruits and vegetables are sparingly available in Ladakh, and plentiful salt is commonly used as a food preservative. The milk, butter, and salt used as essential ingredients in local tea by the Ladakh inhabitants have been suspected to create a favorable milieu for H. pylori-like organisms.[3] However, these sociocultural-ethnic differences may not be enough to justify the differences in gastric H. pylori colonization and severity in gastritis in patients from these two regions.[16] Hence, the virulence genotyping of the colonizing H. pylori strains should be analyzed in these patient populations. Among the H. pylori virulence genes, whereas cagA causes epithelial tight junction damage and induces inflammation by forming a bacterial type IV secretion system produced by the cag pathogenicity island (cagPAI),[17] the vacA genotype forms large vacuoles in epithelial cell endosomal membrane and inhibits antigen presentation. Among the vacA genes, the vacA s1m1 genotype has been associated with significant mucosal inflammation and damage.[18] The dupA gene on the other hand appears to act at the next level by polarizing the inflammatory cell infiltrate towards a Th1 phenotype.[19] Some earlier studies have indicated differences in H. pylori genotypes between inhabitants of Ladakh and the rest of India. Whereas H. pylori strains found in most Indians are clustered genetically around the European strains, the Ladakhi strains comprise a distinct sub-population of hpAsia2 within the European variability.[20] Taking clues from this available information, we hypothesized that the distinctness of virulence genotypes of H. pylori strains between the Ladakhi patients and North Indians is one of the essential determinants of difference in the severity of the gastric mucosal inflammation in patients from North India and Ladakh, and analyzed this aspect in this study.


   Materials and Methods Top


Subjects: A total of 100 archival gastric biopsies comprising 50 biopsies each from patients residing in lower plains of North India and high-altitude areas of Ladakh, with evidence of H. pylori-associated chronic active gastritis, were selected. The biopsies selected were not age-matched. The patients underwent esophagogastroduodenoscopy for various conditions detailed in the result section. Multiple site biopsies were collected as per the updated Sydney system recommendations.[21] The biopsies collected in our institution were fixed in 10% neutral buffered formalin for 4 h, processed routinely, and embedded in paraffin. However, biopsies collected at our institute's outreach camp in Ladakh by the visiting gastroenterologists were stored in 10% buffered formalin and processed 5–8 days after collection. Paraffin sections from the blocks were stained with Hematoxylin and Eosin, Warthin Starry, and Giemsa stains and were reviewed by two pathologists. Histological parameters were examined, and sub-graded as per the updated Sydney system recommendations.[21] The archival tissue samples were used for genomic DNA extraction.

Histological analyses

Chronic mononuclear cell infiltrate: Mononuclear lymphocytes and plasma cells assessed in lamina propria away from lymphoid aggregates and graded as minimal, moderate, and marked infiltrate as per the updated Sydney reporting system.[21]

Neutrophilic activity: The presence of ≥1 polymorph in the lamina propria or neutrophilic infiltrate in the mucosal epithelium was taken as a feature of activity, and the same was also graded.[21]

Mucosal atrophy and metaplasia: Loss of gastric mucosal glands with lamina propria fibrosis, and goblet cell or pseudopyloric metaplasia were looked for and graded.[21]

Lymphoid follicle: The presence of lymphoid collection, with or without germinal centers in the mucosa away from muscularis mucosae was noted down.

Deep infiltration pattern of H pylori: Apart from the histological parameters mentioned above, as suggested by Ito T, et al.,[22] we also examined the deep infiltration pattern of the H. pylori defined as the identification of spiral bacilli into the deeper part of the foveolae or mucus glands and lamina propria. This was taken as one of the histological markers of disease severity.[22]

Histological confirmation of the presence of H pylori like strains in the gastric biopsies

The gastric biopsies of patients from Ladakh and North India included were selected after confirming the presence of H pylori-like organisms by Warthin Starry silver stain.

DNA Isolation from FFPE samples

Genomic DNA was extracted using the Purelink Genomic DNA Mini Kit provided by Invitrogen (CA, USA) using the manufacturer's protocol. Briefly, four 10 μm thick sections cut from the formalin-fixed, paraffin-embedded (FFPE) blocks were scrapped, and the extract was collected in a sterile microcentrifuge tube. Subsequent deparaffinization, washing, and tissue digestion were performed by using PureLink® Genomic Digestion Buffer and proteinase K. The genomic DNA was eluted by using 100% ethanol, using micro-spin columns.

The following two methods were undertaken to check the quality of the eluted DNA:

  1. Nanodrop spectrophotometer: The collected DNA was then quantified using a Nanodrop spectrophotometer (Thermo, USA), and the concentration and 260/280 (acceptable range 1.8-2) and 260/230 ratios (acceptable range 1.8) were determined.
  2. Agarose gel electrophoresis was also performed on the genomic DNA samples, using the housekeeping gene primer BTK (Bruton Tyrosine Kinase), exon 9 to check the integrity of eluted DNA [Table 1].
Table 1: Primer sequences, their product lengths, and their references used in this study

Click here to view


Further confirmation of H pylori strains on gastric biopsies by polymerase chain reaction using H pylori-specific HP1 and HP2 primers

The presence of H. pylori strains was also confirmed by using HP1 and HP2 primers (amplicon size 109 bp) demonstrated specific for 16S rRNA of H. pylori strains, especially using the FFPE tissue.[23] In this mentioned study, the authors confirmed the H. pylori strains by direct sequencing of the ssDNA of PCR product and found >97.3% homology with the H. pylori isolates with negligible cross-reactivity, in comparison to the Universal 16S rRNA sequence.[23]

Polymerase chain reaction for detection of bacterial virulence genes

Singleplex PCR was performed using a total 20 μL reaction mix, containing 1 × PCR buffer, 2.5 mM MgCl2, 200 μM dNTP, 200 nM of each forward and reverse primers in separate tubes specific for H. pylori cag A, dupA, vacA s1/s2, and vacA m1/m2 virulence genes [Table 1], 1U of Taq polymerase, and 5 μL of samples of DNA. These primer sequences were picked up from the literature where these sequences worked well.[24],[25],[27],[28] The following PCR conditions were followed: 95°C for 3 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and final denaturation of 72°C for 5 min in an Agilent 8800, USA, PCR machine. The PCR products were resolved using 2% Agarose- Tris-Borate-EDTA (TBE) gel, stained using ethidium bromide (EtBr) staining, and visualized with a Gel Doc machine (Genie transilluminator). The band sizes on the gel plate were compared to a 100 bp DNA ladder. Positive controls were run by using the DNA isolates of known H. pylori strain cultures, and a negative reaction was constituted using nuclease-free water. The interpretation of gel images was performed by two independent groups comprising two contributors each, and the results were finally discussed and reviewed.

Statistical analysis

Descriptive and inferential statistical analysis has been carried out in the present study. Results on continuous measurements were presented on mean ± SD (Min-Max), and results on categorical measurements were presented in number (%). Significance was assessed at a 5% level of significance. Chi-square/Fisher Exact tests were used to find the significance of study parameters on a categorical scale between the two or more groups, the nonparametric setting for qualitative data analysis.


   Results Top


Basic clinical profile of the patients included: Esophagogastroduodenoscopy from patients residing in the North Indian plain with a mean age of 40 years were performed for the following indications: dyspepsia (22%), epigastric pain (24%), vomiting (14%), jaundice, upper GI bleed, and small bowel diarrhea in 8% patients each, dysphagia in 6%, and gastric distension, carcinoma stomach, chronic liver disease, and cirrhosis in 2% patients each. The mean age of Ladakhi patients was 38.4 years and had the following indications for esophagogastroduodenoscopy: epigastric pain (96.5%), heartburn (38.1%), regurgitation (32.1%), and pain radiating to the back (48.8%). On endoscopy, the Ladakhi patients showed antral gastritis in 70.7%, pangastritis in 9.5%, and nodular gastritis in 18.3%. on the other hand, endoscopic examination in North Indian patients showed antral erythema in 82.3% of patients, pangastritis in 5.4% of the patients, and the features of gastroduodenitis in 85.1% of patients.

Histological changes observed: Out of the total 100 gastric biopsies included from patients with H. pylori-associated gastritis, H pylori-like organisms identified in following densities in the biopsies from North Indian patients- minimal: 6 (12%), moderate: 17 (34%), and marked: 27 (54%). And out of the 50 gastric biopsies examined from Ladakhi patients with H pylori-associated gastritis, 12 (24%) showed minimal, 17 (34%) moderate, and 21 (42%) marked the presence of organisms [Figure 1]. The presence of H. pylori strains in these biopsies was confirmed with PCR assay using HP1 and HP2 primers (amplicon size 109 bp) specific for the 16S rRNA of H. pylori strain and all these biopsies. Whereas the density of H. pylori in gastric biopsies was significantly higher in the presence of mucosal lymphoid aggregates and neutrophilic activity in Ladakhi patients (P 0.02 and < 0.001, respectively), a similar correlation was not found in North Indians (P 0.91 and 0.24, respectively). Whereas the density of mucosal chronic mononuclear cell infiltrates correlated with the grade of mucosal neutrophil activity in both the patient groups (both P < 0.001), no such correlation was observed with the presence of mucosal lymphoid aggregates. Mild and moderate mucosal atrophy was noted in 24% and 2% biopsies, respectively in North Indians, and 38% and 6% in Ladakhi patients (P 0.21). Goblet cell metaplasia was not identified in either of the groups. Whereas the deep infiltration pattern of H. pylori was identified in 36 (72%) North Indian patients, it was seen in 31 (62%) of biopsies from Ladakhi patients. The deep infiltration pattern of the H. pylori significantly correlated with the density of mucosal mononuclear cell infiltrate (P 0.006), lymphoid aggregates (P 0.02), neutrophilic activity (P 0.01), and overall Sydney scores (P 0.002) in Ladakhi patients; but not in the North Indian patients [Table 2].
Figure 1: Photomicrographs show moderate mononuclear cell infiltration in lamina propria including lymphocytes and plasma cell infiltrate in lamina propria (arrow) [a × 100]. Neutrophilic activity is seen in lamina propria and the gastric pits (arrows) [b × 200]. Gastric biopsy shows surface mucosal ulceration and acute inflammatory exudate (arrow) [c × 100]. Warthin Starry silver stain showing minimal [d × 400] and marked [e × 400] colonization by H. pylori in gastric foveolar mucosa (arrows). A gastric antral biopsy shows a deep infiltration pattern of H. pylori in the deeper part of the foveolae (arrows), antral mucus glands (arrows), and lamina propria [f × 100]

Click here to view
Table 2: Correlation of chronic inflammation, lymphoid follicle, neutrophilic activity, and total Sydney score with deep infiltration pattern of H pylori strains in gastric biopsies of patients from North India and Ladakh

Click here to view


Virulence-associated genotyping of H. pylori strains

Detection of cagA genotype

Amplification of the cagA region (350-bp amplicon) was noted in 21 (42%) H. pylori-positive biopsies from Ladakhi patients, in comparison to 34 (68%) biopsies from North India patients. North Indian strains had more cagA virulence genotype than the Ladakhi strains, and this difference was statistically significant (P = 0.009) [Figure 2] and [Flow chart].
Figure 2: Agarose gel electrophoresis (2%) images showing protein bands of HP gene (109 bp), cagA gene (350bp), vacA s1 gene (259bp), vacA s2 gene (259bp), vacA m1 gene (567bp), vacA m2 (642 bp), and dupA gene (306 bp) in patient samples as compared to 100 bp and 50 bp ladders (Invitrogen, USA)

Click here to view



Detection of vacA s1/s2 and m1/m2 alleles

The vacA s and m bands were identified as follows: s1 (amplicon size 259 bps), s2 (amplicon size 286 bps), m1 (amplicon size 567 bps), m2 (amplicon size 642 bps), respectively [Figure 2]. All gastric biopsies from North Indian and Ladakhi strains which showed cagA positivity (n-34 and n-21, respectively), also showed a vacA s1m1 genotype (Flow chart). The cagA negative H. pylori-positive biopsy samples from North Indian and Ladakhi patients (n-16 and 29, respectively) showed a vacA s2m2 genotype. The cagA + vacA s1m1 genotype was commoner in H. pylori-positive biopsy samples from North Indian patients than in Ladakhi patients (P 0.009).

Detection of DupA genotype

A total of 22 out of 50 H. pylori-positive biopsy samples (44%) from North Indian patients and 20 out of 50 biopsy samples from Ladakhi patients (40%) showed dupA genotype, respectively (Flow chart). However, this difference was not statistically significant (P = 0.68). All dupA +ve strains in North India patients were also cagA vacAs1m1 +ve. Four out of the 20 dupA +ve H pylori strains in patients from Ladakh had cagA -ve vacA s2m2 +ve genotype (Flow chart).

H. pylori virulence genotype and gastric mucosal histological changes

When individual virulence genotypes were correlated with mucosal histological changes, cagA genotype in North Indian patients was associated with the severe neutrophilic mucosal activity (P 0.02), whereas in Ladakhi patients vacA s2m2 genotype was associated with more severe neutrophilic activities (P 0.02) [Table 3]. The dupA genotype in North Indian patients also was associated with deeper mucosal infiltration by the bacilli, though there was no significant difference in the dupA genotype in the North Indian and Ladakhi patients. It seems that the CagA VacAs1 m1 +ve dupA +ve strains in North Indian patients have more neutrophilic activity and deep infiltration pattern of H pylori. However, when the overall virulence genotypes were considered together, there were no definite mucosal histological differences between the cagA+ vacA s1m1 genotype versus cagA -ve vac s2m2 genotypes both in North Indians and in Ladakhi patients [Table 4]. No association of H pylori genotypes with mucosal atrophy was noted.
Table 3: Correlation of individual H pylori virulence genotype and histological changes of gastric mucosa in patients from Ladakh and North India

Click here to view



   Discussion Top


All over the world, approximately, 50%–60% of the population is colonized by H. pylori, a designated class I carcinogen for gastric cancer, which is the second leading cause of cancer-related death worldwide.[29] Developed nations of Asia such as Korea, China, and Japan have a high prevalence of H pylori-associated gastric carcinomas.[30] Paradoxically, studies from Asian countries such as Thailand, India, Pakistan, Bangladesh, Iran, Saudi Arabia, Israel, Malaysia, and Africa have reported a high prevalence of H. pylori infection, with a low incidence of gastric cancer.[8],[30],[31],[32],[33],[34] Whereas the prevalence of H. pylori infection is about 56%–60% in North Indian patients with dyspepsia,[35] a prevalence of up to 93% was noted in patients residing in the high altitude of Ladakh. Earlier, we had reported the finding of severe gastric inflammation, nodularity, and atrophy associated with H. pylori infection in patients from Ladakh.[36] Pandey R et al.[37] demonstrated that p53 (Arg/Arg) and (Pro/Arg) variants in the p53 codon 72 were higher (40.59% and 33.66%) in the healthy population, as compared to p53 Pro/Pro variant (25.74%) in gastric cancer patients with H. pylori infection. Incidentally, a high prevalence of p53 (Arg/Arg) variants in the North Indian patients was hypothesized to impart more stress-bearing capabilities leading to low carcinogenesis. Also, various other host-associated genes were found to have an impact on the carcinogenic potential of H. pylori infection, including the CYP2E1 (96 bp insertion), GSTM1 null genotype, and GSTT1 non-null genotype in smokers, and an inverse correlation of H. pylori infection with the expression of microsatellite marker proteins.[31] However, the direct role of these host-associated genetic alterations in carcinogenesis was not proven. Hence, there is a need to look into other environmental determinants, regional food, and cultural practices, and diversity of virulence genotypes of H. pylori strains identified among the different geographical regions, age of acquiring infection, etc., which may be responsible for diverse mucosal histological changes and disease outcomes. We chose to investigate the association of virulence genes of H pylori-associated chronic active gastritis from the patients residing in the North Indian plains and compared with those living in the high-altitude areas of Ladakh, as the mucosal histological changes were reported more severe in the latter.[38] Though we found that the cagA+ vacA s1m1 + genotype was more prevalent in North Indian strains than in the H. pylori strains from Ladakh, there was no conclusive correlation of H. pylori genotypes with the severity of gastric mucosal changes.

In contrast to our expectation, the cagA and vacs1m1 genotype was noted more in North Indians, than those from Ladakh. Whether the difference in the time of exposure to formalin in biopsies from North India (4 h) and Ladakh (5–8 days) was a reason for these findings, needs further evaluation, but unlikely to be the cause. In general, the cagA+ vacA s1m1 or cagA-ve vacA s2m2 genotypes did not show significant differences of mucosal inflammatory pathology in this study, except for an isolated correlation of cagA in North Indian patients and vacA s2 m2 genotype in Ladakhi patients with mucosal neutrophilic activity and dupA gene with deep infiltration pattern of H. pylori in the North India patients [Table 4]. These findings seem to suggest that the severity of gastric mucosal inflammation in H. pylori infection may be influenced by other local factors such as food habits, cultural practices, and age of infection in presence of an inflammogenic bacterial virulence genotype. Though the investigation of these environmental, dietary, or host factors was beyond the scope of this study, it is interesting to note that despite frequent H. pylori infection in Indian children, especially those living in low socioeconomic conditions, they mostly remain asymptomatic throughout their childhood and only a small fraction of them develop peptic ulcer disease when they are young adults and gastric cancer is extremely rare.[39] In our previous study on H. pylori-associated gastritis patients from Ladakh, about 75%–77% of patients were found to consume daily hot salted-butter tea and salted meat; whether high salt consumption has any influence on gastric mucosal pathology needs further investigation.[36] We also did not evaluate variability in the conserved regions of cagPAI in our patients. A highly conserved cagPAI among the Japanese isolates was found to be associated with high gastric complications and in Indian patients, the cagPAI was found to be least conserved.[40]
Table 4: Correlation of overall H pylori virulence genotype and histological changes of gastric mucosa

Click here to view


In contrast to our finding of cagA+ vacA s1m1 prevalent genotype in North Indian patients than in Ladakhis, Romero-Gallo J, et al. reported 95% cagA+ seropositivity and culture growth in Tibetan orphans in Ladakh.[13] In comparison to the Tibetan orphans included in this study, our study population also comprised local Buddhists and Muslims from the region.[13] In a previous study on genotyping of 10 H pylori strains from Ladakhi patients, Kauser F et al.[41] found that the vacA s1m1 genotype is very rare in Ladakh, whereas vacA s2 genotype was identified in 60% strains and vacA m1 and m2 genotypes were identified in equal frequencies. This study results support our findings although the two cannot be compared. Our study is based on the analysis of FFPE samples. On the other hand, the study by Kauser et al.[41] involved the analysis of DNA extracted from cultured isolates of H pylori. Nevertheless, the two studies do concur that the pathogenic vac As1 m1 subtype does not seem to be common in the Ladakhi population studied. The H. pylori virulence genotype in Ladakh showed a mosaic pattern with a close resemblance to the European-American strains than in the eastern Asian strains.[3] Mukhopadhyay A K. et al.[26] from the city of Kolkata in Eastern India also found that 80%– 90% of their patient's strains are cag A + vacAs1+, independent of the disease severity, like our findings. The cagA and vacA s1m1 genes were identified in Japanese and South Korean isolates with a high prevalence of gastric cancer.[30] However, this divergent data indicates that only H. pylori virulence genotype is not enough to produce severe mucosal inflammation. In a previous study from this group on 166 North Indian patients, including 96 patients with duodenal ulcer and 70 patients with functional dyspepsia, dupA gene was identified in 37.5% of H. pylori strains isolated from the duodenal ulcer patients and in 22.8% of the patients with dyspepsia. A total of 97% of these strains from duodenal ulcer patients and 81% of patients with functional dyspepsia also showed cagA positivity,[42] like the index study where all dupA +ve strains from North Indian patients (44%) also had cagA vacAs1 m1 genotype. These strains in our patients also showed denser neutrophilic activity in gastric mucosa and a deep infiltration pattern of H. pylori in gastric pits and lamina propria. However, we did not identify significant differences of dupA genotype in H. pylori strains from North Indian and Ladakhi patients. Comparative studies on the prevalence of dupA genotype in H. pylori strains from North Indian and Ladakhi patients are not available.

Van Doorn LJ et al.[43] detected 611 vacA strains across different countries of Europe, America, and East Asia. In most of the published studies, vacA s1/cagA-positive H. pylori genotype was associated with peptic ulcer disease (P < 0.001); hence, it was considered that the vacA s1/cagA has a certain potential to cause clinically significant disease. However, it is not yet clear from our study that despite having a low prevalence of cagA+ vacA s1m1 genotype in patients from Ladakh, why the gastric mucosal pathologies are more severe, in comparison to North Indian patients. Hence, we now hypothesize that apart from H. pylori or host genotypes other psychosocial, environmental, and dietary factors may have a role in the disease outcome and need to be studied.

This study adds to the existing knowledge that the virulence genotype of H. pylori strains is not only the sole factor that determines the severity of gastric mucosal inflammation in patients from high altitude areas of Ladakh and North India. However, we included only 50 gastric biopsies each from these two geographically divergent regions. Sequencing of the virulence genes could have given more comprehensive information; however, we chose to investigate a commoner virulence gene panel in this study based on our previous observations.

To conclude, the prevalence of cagA1+ vacA s1m1+ H. pylori virulence genotypes is higher in gastric biopsies from North Indian patients than in Ladakhi patients. The North Indian cagA1+ vacA s1m1+ H. pylori strains are also dupA+. Despite having a distinct H pylori genotype in patients from both geographically divergent regions, the mucosal inflammatory changes are not remarkably different. Hence, other possible influencing factors as local environmental and dietary practices should be systematically be investigated.

Acknowledgments

We express our sincere acknowledgment to Dr. Asish K. Mukhopadhyay from the National Institute of Cholera and Enteric Disease, Kolkata for providing us H. pylori strain-expressing dupA gene, which we used as a positive control.

Ethics approval

This is a retrospective study, the protocol was approved by the Institute's Ethical Clearance Committee (Memorandum no. IECPG – 208/24.02.2016, RT – 6/29.0.6.2016 dated 2nd July 2016).

Consent to Participate

As per the institute's protocol, before taking the endoscopic biopsies, informed consent was taken from each patent for biopsy procedure, use of leftover biopsy material for further research, academic activities, and publications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Abraham P, Bhatia SJ. Position paper on Helicobacter pylori in India. Indian Society of Gastroenterology. Indian J Gastroenterol 1997;16(Suppl 1):S29-33.  Back to cited text no. 1
    
2.
Graham DY, Asaka M. Eradication of gastric cancer and more efficient gastric cancer surveillance in Japan: Two peas in a pod. J Gastroenterol 2010;45:1–8.  Back to cited text no. 2
    
3.
Ramirez-Ramos A, Gilman RH, Spira W, Recavarren S, Watanabe J, Leon-Barua R, et al. Ecology of Helicobacter pylori in Peru: Infection rates in coastal, high altitude, and jungle communities. Gut 1992;33:604–5.  Back to cited text no. 3
    
4.
Recavarren-Arce S, Ramirez-Ramos A, Gilman RH, Chinga-Alayo E, Watanabe-Yamamoto J, Rodriguez-Ulloa C, et al. Severe gastritis in the Peruvian Andes. Histopathology 2005;46:374–9.  Back to cited text no. 4
    
5.
Fox JG, Dangler CA, Taylor NS, King A, Koh TJ, Wang TC. High-salt diet induces gastric epithelial hyperplasia and parietal cell loss and enhances Helicobacter pylori colonization in C57BL/6 mice. Cancer Res 1999;59:4823–8.  Back to cited text no. 5
    
6.
Gamboa-Dominguez A, Ubbelohde T, Saqui-Salces M, Romano-Mazzoti L, Cervantes M, Domínguez-Fonseca C, et al. Salt and stress synergize H. pylori-induced gastric lesions, cell proliferation, and p21 expression in Mongolian gerbils. Dig Dis Sci 2007;52:1517–26.  Back to cited text no. 6
    
7.
Tsugane S, Tei Y, Takahashi T, Watanabe S, Sugano K. Salty food intake and risk of Helicobacter pylori infection. Jpn J Cancer Res 1994;85:474–8.  Back to cited text no. 7
    
8.
Graham DY, Lu H, Yamaoka Y. African, Asian or Indian enigma, the East Asian Helicobacter pylori: Facts or medical myths. J Dig Dis 2009;10:77–84.  Back to cited text no. 8
    
9.
Graham DY. Helicobacter pylori infection in the pathogenesis of duodenal ulcer and gastric cancer: A model. Gastroenterology 1997;113:1983–91.  Back to cited text no. 9
    
10.
Tovey FI. Helicobacter pylori infection and upper gastrointestinal pathology in a British immigrant Indian community. Eur J Gastroenterol Hepatol 1997;9:647–8.  Back to cited text no. 10
    
11.
Dikshit RP, Mathur G, Mhatre S, Yeole BB. Epidemiological review of gastric cancer in India. Indian J Med Paediatr Oncol 2011;32:3–11.  Back to cited text no. 11
[PUBMED]  [Full text]  
12.
Thirumurthi S, Graham DY. Helicobacter pylori infection in India from a western perspective. Indian J Med Sci 2010;64:423-40.  Back to cited text no. 12
  [Full text]  
13.
Romero-Gallo J, Pérez-Pérez GI, Novick RP, Kamath P, Norbu T, Blaser MJ. Responses of endoscopy patients in Ladakh, India, to Helicobacter pylori whole-cell and CagA antigens. Clin Diagn Lab Immunol 2002;9:1313-7.  Back to cited text no. 13
    
14.
Basu A, Mukherjee N, Roy S, Sengupta S, Banerjee S, Chakraborty M, et al. Ethnic India: A genomic view, with special reference to peopling and structure. Genome Res 2003;13:2277-90.  Back to cited text no. 14
    
15.
Gautam P, Jha P, Kumar D, Tyagi S, Varma B, Dash D, et al. Spectrum of large copy number variations in 26 diverse Indian populations: Potential involvement in phenotypic diversity. Hum Genet 2012;131:131-43.  Back to cited text no. 15
    
16.
Backert S, Clyne M, Tegtmeyer N. Molecular mechanisms of gastric epithelial cell adhesion and injection of CagA by Helicobacter pylori. Cell Commun Signal 2011;9:28.  Back to cited text no. 16
    
17.
Amieva MR, El-Omar EM. Host-bacterial interactions in Helicobacter pylori infection. Gastroenterology 2008;134:306–23.  Back to cited text no. 17
    
18.
Molinari M, Salio M, Galli C, Norais N, Rappuoli R, Lanzavecchia A, et al. Selective inhibition of Ii-dependent antigen presentation by Helicobacter pylori toxin VacA. J Exp Med 1998;187:135–40.  Back to cited text no. 18
    
19.
Hussein NR, Argent RH, Marx CK, Patel SR, Robinson K, Atherton JC. Helicobacter pylori dupA is polymorphic, and its active form induces proinflammatory cytokine secretion by mononuclear cells. J Infect Dis 2010;202:261–9.  Back to cited text no. 19
    
20.
Devi SM, Ahmed I, Francalacci P, Hussain MA, Akhter Y, Alvi A, et al. Ancestral European roots of Helicobacter pylori in India. BMC Genomics 2007;8:184.  Back to cited text no. 20
    
21.
Dixon MF, Genta RM, Yardley JH, Correa P. Classification and grading of gastritis: The updated Sydney system. Am J Surg Pathol 1996;20:1161-81.  Back to cited text no. 21
    
22.
Ito T, Kobayashi D, Uchida K, Takemura T, Nagaoka S, Kobayashi I, et al. Helicobacter pylori invades the gastric mucosa and translocates to the gastric lymph nodes. Lab Invest 2008;88:664-81.  Back to cited text no. 22
    
23.
Ho SA, Hoyle JA, Lewis FA, Secker AD, Cross D, Mapstone NP, et al. Direct polymerase chain reaction test for detection of Helicobacter pylori in humans and animals. J Clin Microbiol 1991;29:2543–9.  Back to cited text no. 23
    
24.
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. 24
    
25.
Alam J, Maiti S, Ghosh P, De R, Chowdhury A, Das S, et al. Significant association of the dupA gene of Helicobacter pylori with duodenal ulcer development in a South-east Indian population. J Med Microbiol 2012;61:1295–302.  Back to cited text no. 25
    
26.
Mukhopadhyay AK, Kersulyte D, Jeong J-Y, Datta S, Ito Y, Chowdhury A, et al. Distinctiveness of genotypes of helicobacter pylori in Calcutta. India. J Bacteriol 2000;182:3219–27.  Back to cited text no. 26
    
27.
Atherton JC, Cao P, Peek RM, Tummuru MK, Blaser MJ, Cover TL. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 1995;270:17771–7.  Back to cited text no. 27
    
28.
Atherton JC, Cover TL, Twells RJ, Morales MR, Hawkey CJ, Blaser MJ. Simple and accurate PCR-based system for typing vacuolating cytotoxin alleles of Helicobacter pylori. J Clin Microbiol 1999;37:2979–82.  Back to cited text no. 28
    
29.
Bhurgri Y, Pervez S, Kayani N, Haider S, Ahmed R, Usman A, et al. Rising incidence of gastric malignancies in Karachi, 1995- 2002. Asian Pac J Cancer Prev 2009;10:41–4.  Back to cited text no. 29
    
30.
Ishaq S, Nunn L. Helicobacter pylori and gastric cancer: A state-of-the-art review. Gastroenterol Hepatol Bed Bench 2015;8:S6-14.  Back to cited text no. 30
    
31.
Misra V, Pandey R, Misra SP, Dwivedi M. Helicobacter pylori and gastric cancer: Indian enigma. World J Gastroenterol 2014;20:1503-9.  Back to cited text no. 31
    
32.
Wroblewski LE, Peek RM, Wilson KT. Helicobacter pylori and gastric cancer: Factors that modulate disease risk. Clin Microbiol Rev 2010;23:713–39.  Back to cited text no. 32
    
33.
Goh KL. Epidemiology of Helicobacter pylori infection in Malaysia--Observations in a multiracial Asian population. Med J Malaysia 2009;64:187-92.  Back to cited text no. 33
    
34.
Hirai I, Sasaki T, Kimoto A, Yamamoto Y, Azuma T, Mahachai V, et al. Infection of less virulent Helicobacter pylori strains in asymptomatic healthy individuals in Thailand as a potential contributing factor to the Asian enigma. Microbes Infect 2010;12:227–30.  Back to cited text no. 34
    
35.
Singh V, Trikha B, Nain CK, Singh K, Vaiphei K. Epidemiology of Helicobacter pylori and peptic ulcer in India. J Gastroenterol Hepatol 2002;17:659–65.  Back to cited text no. 35
    
36.
Sharma PK, Suri TM, Venigalla PM, Garg SK, Mohammad G, Das P, et al. Atrophic gastritis with a high prevalence of Helicobacter pylori is a predominant feature in patients with dyspepsia in a high-altitude area. Trop Gastroenterol 2015;35:246-51.  Back to cited text no. 36
    
37.
Pandey R, Misra V, Misra SP, Dwivedi M, Misra A. Helicobacter pylori infection and a P53 codon 72 single nucleotide polymorphism: A reason for an unexplained Asian enigma. Asian Pac J Cancer Prev 2014;15:9171-6.  Back to cited text no. 37
    
38.
Covacci A, Telford JL, Del Giudice G, Parsonnet J, Rappuoli R. Helicobacter pylori virulence and genetic geography. Science 1999;284:1328–33.  Back to cited text no. 38
    
39.
Poddar U, Yachha SK. Helicobacter pylori in children: An Indian perspective. Indian Pediatr 2007;44:761-70.  Back to cited text no. 39
    
40.
Nomura A, Stemmermann GN, Chyou PH, Kato I, Perez-Perez GI, Blaser MJ. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med 1991;325:1132–6.  Back to cited text no. 40
    
41.
Kauser F, Hussain MA, Ahmed I, Ahmad N, Habeeb A, Khan AA, et al. Comparing genomes of helicobacter pylori strains from the high-altitude desert of Ladakh, India. J Clin Microbiol 2005;43:1538–45.  Back to cited text no. 41
    
42.
Arachchi HJ, Kalra V, Lal B, Bhatia V, Baba CS, Chakravarthy S, et al. Prevalence of duodenal ulcer-promoting gene (dupA) of Helicobacter pylori in patients with duodenal ulcer in North Indian population. Helicobacter 2007;12:591-7.  Back to cited text no. 42
    
43.
Van Doorn LJ, Figueiredo C, Mégraud F, Pena S, Midolo P, Queiroz DM, et al. Geographic distribution of vacA allelic types of Helicobacter pylori. Gastroenterology 1999;116:823–30.  Back to cited text no. 43
    

Top
Correspondence Address:
Prasenjit Das
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi – 110029
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_565_21

Rights and Permissions


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed594    
    Printed36    
    Emailed0    
    PDF Downloaded42    
    Comments [Add]    

Recommend this journal