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BRIEF COMMUNICATION
Year : 2018  |  Volume : 36  |  Issue : 4  |  Page : 582-586
 

Clinical relevance of cagA and vacA and association with mucosal findings in Helicobacter pylori-infected individuals from Chennai, South India


1 Department of Digestive Health and Disease, Kilpauk Medical College, Chennai, Tamil Nadu, India
2 ICMR - National Institute of Epidemiology, Chennai, Tamil Nadu, India

Date of Web Publication18-Mar-2019

Correspondence Address:
Dr. Anand Vadivel
Department of Digestive Health and Disease, Kilpauk Medical College, Chennai - 600 010, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_406

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 ~ Abstract 

Helicobacter pylori is associated with a spectrum of severe gastrointestinal conditions. In this study, an attempt was made to correlate endoscopic mucosal patterns with H. pylori infection and examine the pathogenic potential of the strains. Among the 147 dyspeptic individuals studied, 42.2% were H. pylori infected. Association of H. pylori with type 3 and 4 mucosal patterns (P = 0.001) and intestinal metaplasia (P = 0.012) was seen. vacA was associated with histological (P = 0.014) and endoscopy findings (P = 0.009). Association of mucosal patterns with H. pylori infection could be useful for clinicians to decide on the need for eradication therapy.


Keywords: Helicobacter pylori, cagA, vacA, glmM, dyspepsia, mucosal pattern


How to cite this article:
Vadivel A, Kumar C P, Muthukumaran K, Ramkumar G, Balamurali R, Meena RL, Venkatasubramanian S, Solomon T R, Ganesh P, Kumar S J. Clinical relevance of cagA and vacA and association with mucosal findings in Helicobacter pylori-infected individuals from Chennai, South India. Indian J Med Microbiol 2018;36:582-6

How to cite this URL:
Vadivel A, Kumar C P, Muthukumaran K, Ramkumar G, Balamurali R, Meena RL, Venkatasubramanian S, Solomon T R, Ganesh P, Kumar S J. Clinical relevance of cagA and vacA and association with mucosal findings in Helicobacter pylori-infected individuals from Chennai, South India. Indian J Med Microbiol [serial online] 2018 [cited 2019 Aug 25];36:582-6. Available from: http://www.ijmm.org/text.asp?2018/36/4/582/254397



 ~ Introduction Top


Helicobacter pylori is an important human pathogen associated with severe gastrointestinal (GI) conditions, namely chronic gastritis, peptic ulcer disease (PUD) and gastric malignancies such as distal gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma.[1] Classified as a Class I carcinogen (WHO 1994), this Gram-negative microaerophilic bacterium chronically infects about half of the world population.[2] The prevalence varies from as low as 10% in the developed western nations to higher than 80% in developing countries like India where most infections occur in childhood with the prevalence rate exceeding 50% at 10 years of age and 80% in adulthood.[3],[4]

PUD is the most commonly recognised manifestation of H. pylori in India with high ratio of duodenal ulcer (DU) disease to gastric ulcers.[5] The point prevalence of active PUD has been reported to be 3% with a lifetime prevalence of 9%.[3]

Gastric cancer is the most serious outcome of H. pylori infection. In India especially in southern India with high prevalence of DU, the age-adjusted incidence rate of gastric cancer ranges between 2 and 57 per 100,000.[3]

There is increasing evidence for associating genetic variability of H. pylori with clinical outcomes. Vacuolating cytotoxin 'vacA' and cytotoxin-associated gene 'cagA' are the major virulence determinants of H. pylori. The vacA gene is virtually present in all H. pylori whereas cagA is reportedly seen in about 50%–60% strains.[6] The presence of cagA is associated with duodenal ulceration, gastric mucosal atrophy and gastric cancer. The sequence polymorphism in the signal peptide (s region) and middle region (m region) of the vacA gene has revealed two allelic forms m1, m2 and s1, s2, respectively. The s1/m1 strains produce high levels of toxin whereas s2/m2 do not produce any toxin. The s1/m2 strains reportedly elaborate low to moderate levels of toxin.[6]

Previous studies from India have reported predominance of vacA s1 and m1 alleles in Eastern India (Kolkata) whereas vacA m2 allele was more common in South India (Chennai).[7],[8] Overall, very few studies have been carried out on this important human pathogen in India.

The present report is based on the study conducted at a public health gastroenterology referral facility in Chennai to examine mucosal patterns in H. pylori-associated gastritis. The authors also attempted to study the association of H. pylori infection with the clinical findings and H. pylori virulence markers cagA and vacA.


 ~ Materials and Methods Top


Patients

A total of 147 dyspeptic individuals attending the Department of Digestive Health and Diseases at the Government Peripheral Hospital, Chennai attached to the Government Kilpauk Medical College and Hospital (KMCH), Chennai between December 2012 and January 2014 were enrolled in the cross-sectional study. Patients with uninvestigated dyspepsia, active gastric or DU and gastric MALT-lymphoma were included whereas individuals with structural (hiatus hernia, gastroesophageal reflux disease) and metabolic causes for dyspepsia (uraemia, ischaemic heart disease, biliary colic and pancreatitis), recent use of proton pump inhibitor, antibiotics and bismuth-containing compounds were not included. The study was conducted with approval from the institute ethics committee of KMCH, and informed consent was obtained from all participants.

Endoscopic examination and biopsy sampling

On upper GI endoscopy (Oesophago-Gastro Duodenoscopy [OGD]), the mucosal pattern observed was categorised as type 1 (cleft-like appearance extending along the longitudinal axis of gastric body), type 2 (red dots arranged uniformly), type 3 (mosaic pattern without a focal area of hyperaemia) and type 4 (mosaic pattern with a focal area of hyperaemia).[9],[10]

Six biopsy samples (antrum/corpus) from suspected sites were collected during upper GI endoscopy. A portion of the specimen was inoculated into a tube for rapid urease test (RUT) and in formalin for histopathological examination (HPE). The remaining portion was kept in sterile TE buffer for DNA extraction. Samples for molecular analyses were transported in cold chain to the laboratory at National Institute of Epidemiology, Chennai within 6–8 h of collection. HPE by H and E staining was carried out in the Department of Pathology, KMCH and scored for gastritis and gastritis activity, from mild to severe, based on the level of infiltrating lymphocytes and neutrophils, respectively.

DNA extraction and polymerase chain reaction analysis

Biopsy specimens were homogenised, and genomic DNA was extracted using the commercial HiPurA™ Mammalian Genomic DNA purification kit (HiMedia, India), according to the manufacturer's instructions. Extracted DNA was stored at −20°C until further analysis. To ascertain the presence of H. pylori DNA, H. pylori-specific phosphoglucosamine mutase gene, glmM (formerly designated as ureC) was carried out. Further, amplification of the virulence genes cagA and vacA'm' alleles were done as described previously.[8],[11]

A case of H. pylori infection was diagnosed if any one of the polymerase chain reactions (glmM/CagA/vacA) is positive or if H. pylori was discernable on HPE.

Statistical analysis

Continuous and categorical data were analysed using Student's t-test and Chi-square/Fisher's exact tests, respectively. P < 0.05 was considered significant. Microsoft Excel was used for data entry. Descriptive data analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, Illinois, USA).


 ~ Results Top


Age and gender

Among the 147 patients included in the present study, 105 (71%) were males and 42 (29%) were females. Fifty per cent (74/147) of patients were in the age group of 21–39 years. RUT was positive in 46% (68/147) of individuals. Sixty-two patients (42.2%) were categorised as H. pylori infected [Table 1].
Table 1: Demographic and clinical characteristics of study participants (n=147)

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Analysis of age group-wise positivity showed that age group 40–59 years had significantly lower proportion of H. pylori infection (P = 0.035) [Table 2]. There was no significant difference in the proportion of H. pylori positivity in other age groups. Similarly, there was no gender-specific association among the study participants. There was also no significant difference in RUT positivity across age groups or gender.
Table 2: Characteristics of Helicobacter pylori-infected individuals

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Clinical features and co-morbidities

The most common presenting symptom was abdominal bloating (27%; 39/147) followed by epigastric burning (24%; 36/147) [Table 1]. History of diabetes mellitus was noted in 28% (41/147) of participants and 17% (25/147) had hypertension. The percentage of individuals who self-reported alcohol consumption and smoking were 26% (38/147) and 22% (33/147), respectively. A history of drug intake was obtained in 15% (22/147) individuals, and 5% (8/147) reported that they occasionally skipped meals. There was no significant association of H. pylori infection with any clinical feature and co-morbidities such as diabetes mellitus and hypertension [Table 2].

Endoscopic findings

Endoscopic examination revealed normal mucosal study in 31% (41/147) of patients. In 69% (101/147) of patients, abnormal findings were seen, with 31% (45/147) having either type 3 or 4 mucosal pattern and 22% (32/147) having type 1 or 2 pattern [Figure 1]. DU was observed in 17 patients (12%) and 7 individuals had gastric ulcer (5%).
Figure 1: Mucosal patterns on upper gastrointestinal endoscopy

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There was significant association of H. pylori infection with type 3 mucosal pattern (P = 0.007) [Table 2]. The proportion of H. pylori infected with type 1 pattern was lower than seen among uninfected individuals, and this difference was significant (P = 0.008).

Histopathological examination

HPE showed 56% (83/147) patients to have chronic non-specific gastritis (CNSG). The proportion of CNSG was significantly lower among H. pylori-infected individuals (P = 0.002). Erosive and nodular gastritis was seen in 16% (24/147) and 10% (14/147) of cases. Precancerous lesions such as atrophic gastritis and intestinal metaplasia were seen in 4% 6/147) and 12% (17/147) cases. Analysis revealed significant association of H. pylori infection with intestinal metaplasia (P = 0.012) [Table 2]. Only in 3 cases, H. pylori bacilli were discernible.

Molecular studies

glmM gene was amplified in 95.2% cases (59/62) with H. pylori infection [Figure 2]. vacA and cagA genes were amplified in 85.5% and 59.7% of H. pylori-infected cases. Among the vacA positive cases, 50.9% (27/53) had m2 allele whereas in 37.7% (20/53) cases, m1 allele was seen. In 11.3% cases, both m1 and m2 alleles were detected. Both cagA and vacA were present in 51.6% (31/62) of the infected individuals.
Figure 2: (a) Amplification of Helicobacter pylori-specific glmM gene (294 bp) M-100 bp ladder, Lane 1- Negative control; Lane 2 to 4 gastric biopsy samples (b) amplification of cagA gene (394 bp) M = 100bp ladder, Lane 1 - Negative control; Lane 2–4 gastric biopsy samples (c) Amplification of vacA gene (Allele m1 [401 bp] and Allele m2 [476bp]) M = 100bp ladder, Lane 1 - Negative control; Lane 2 and 3 - Allele m2; Lane 4 to 5 - Allele m1 and Lane 6 - Allele m1 + m2 coinfection

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There was association of vacA positivity with histological (P = 0.014) as well as with OGD findings (P = 0.009). Among patients with type 3 and 4 mucosal patterns, 53.3% (24/45) were vacA positive. Such an association was not seen in the case of cagA positivity.


 ~ Discussion Top


H. pylori is one of the most frequent and persistent bacterial infections affecting nearly half of the world's population. The associated clinical conditions range from mild CNSG to severe conditions such as atrophic gastritis, ulcers and eventually gastric carcinoma. Hitherto, very few studies have been conducted in South India on H. pylori especially examining the association with mucosal patterns and virulence determinants such as cagA and vacA subtypes. The present study was an attempt by the authors to correlate endoscopic mucosal patterns with H. pylori infection status and also examine the pathogenic potential of H. pylori strains circulating in this region.

Most of the patients in the present study were in the age group of 21–39 years, and it was in this group that the H. pylori positivity was the highest. Relatively lower positivity was observed in the 40–59 years age group. A recent study from Odisha in Eastern India also showed higher positivity among adolescent and young adults (15–30 years) followed by adults in the age group of 40–60 years.[12] A study from Thailand reported the older age group of 41–60 years to be mostly infected.[13] No gender-specific association with H. pylori infection was observed in the present study, and this was similar to the observation in a recent study from Kerala in southern India.[14] However, higher positivity among males (P = 0.001) was recently reported from Odisha.[12]

In the present study, there was significant association of H. pylori positivity with types 3 and 4 mucosal patterns. In contrast, there was lower proportion of positivity among individuals with types 1 and 2 mucosal patterns. These findings are in concordance with previous reports that described type 1 and 2 patterns to be predictive of H. pylori negative status and types 3 and 4 patterns to correspond to H. pylori positive status. Thus far, no other Indian report has attempted to associate mucosal patterns with H. pylori status.

H. pylori infection has been associated with the incidence of dysplasia or carcinoma.[14] Intestinal metaplasia and gastric dysplasia are the main precancerous lesions of the stomach. Although no cases of gastric carcinoma were encountered in the present study, there was a significant association between H. pylori infection and intestinal metaplasia. The proportion of H. pylori positives among cases of CNSG was significantly lower compared to H. pylori uninfected individuals. Unlike other Indian reports that had documented significant association of H. pylori infection with duodenal/gastric ulcers, no such association was observed in this study.

The high prevalence of vacA among the H. pylori-infected cases was similar to the previous reports from this region and other parts of India.[8],[15] The predominance of m2 subtype in this study was similar to previous reports from South India.[8],[16] The presence of multiple genotypes (m1 + m2) is indicative of multiple strains in the same individual as only a single m type is seen in one H. pylori strain. The presence of multiple strains due to either coinfection or superinfection is reportedly higher in countries with high H. pylori prevalence.[6] Multiple m types were not observed in the previous study from Chennai that had analysed the m types. m1 strains are reportedly more common in northern and eastern parts of India.[8],[16] vacA was significantly associated with intestinal metaplasia (P = 0.008) and type 3 and 4 mucosal pattern (P = 0.003) and significantly lower in patients with type 1 and 2 mucosal pattern (0.006).

The proportion of strains with cagA in the present study was lesser than from the previous report from this region and other parts of India. Comparable rates have been reported from another study from North India but are more common in reports from Europe and America.[13] cagA has been previously associated with gastric cancer, but with limited sample size, the present study was not powered to study such associations, and moreover, no cases of gastric cancer were encountered. cagA was found to be significantly associated with type 3 and 4 mucosal pattern (P = 0.019).

A key limitation in the present study was that only in three cases, H. pylori bacilli were discernable. This could be attributed to the presence of low bacterial load coupled with poor quality of the stain used that would have rendered visualising the bacteria difficult. Further in this study, a lot of emphasis was laid on submitting substantial portion of biopsy material for molecular studies. This also resulted in lesser material getting submitted for HPE. The present study examined only individuals who visited the study hospital seeking treatment for dyspepsia, and hence, the findings of the study should not be generalised to explain H. pylori infection in the community settings.


 ~ Conclusions Top


The association of type 3 and 4 mucosal patterns with H. pylori infection could be useful to inform the clinician to decide on the need for eradication therapy especially in settings without adequate laboratory support. This could also help circumvent unnecessary use of antibiotics in an era of escalating levels of antimicrobial resistance. The high prevalence of virulence determinants including multiple vacA subtypes is suggestive of the circulation of highly virulent H. pylori strains in the community. Further studies involving larger sample size, geographical representativeness and examining various other virulence genes is required to gather more insights into the clinico-epidemiological and molecular aspects of H. pylori infections in relation to population and geography in India.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
Breurec S, Michel R, Seck A, Brisse S, Côme D, Dieye FB, et al. Clinical relevance of cagA and vacA gene polymorphisms in Helicobacter pylori isolates from Senegalese patients. Clin Microbiol Infect 2012;18:153-9.  Back to cited text no. 1
    
2.
International Agency for Research on Cancer. Working group on the evaluation of carcinogenic risks to humans. Helicobacter pylori. In: Schistosomes, liver flukes, and Helicobacter pylori: Views and Expert Opinions of an IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon: International Agency for Research on Cancer; 1994. p. 177-240.  Back to cited text no. 2
    
3.
Thirumurthi S, Graham DY. Helicobacter pylori infection in India from a western perspective. Indian J Med Res 2012;136:549-62.  Back to cited text no. 3
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Misra RN, Bhagat M, Ahmed N. Helicobacter pylori in dyspepsia – Antibiotic sensitivity and virulence patterns. Med J Armed Forces India 2006;62:22-6.  Back to cited text no. 4
    
5.
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. 5
    
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van Doorn LJ, Figueiredo C, Rossau R, Jannes G, van Asbroek M, Sousa JC, et al. Typing of Helicobacter pylori vacA Gene and Detection of cagA Gene by PCR and Reverse Hybridization. J Clin Microbiol 1998;36:1271-6.  Back to cited text no. 6
    
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Mukhopadhyay AK, Kersulyte D, Jeong JY, 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. 7
    
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Udhayakumar G, Senthilkumar C, Jayanthi V, Devaraj N, Devaraj H. Helicobacter pylori detection and genotyping in gastric biopsy specimens from Chennai patients (India). Can J Microbiol 2009;55:126-32.  Back to cited text no. 8
    
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Yan SL, Wu ST, Chen CH, Hung YH, Yang TH, Pang VS, et al. Mucosal patterns of Helicobacter pylori-related gastritis without atrophy in the gastric corpus using standard endoscopy. World J Gastroenterol 2010;16:496-500.  Back to cited text no. 9
    
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Cho JH, Chang YW, Jang JY, Shim JJ, Lee CK, Dong SH, et al. Close observation of gastric mucosal pattern by standard endoscopy can predict Helicobacter pylori infection status. J Gastroenterol Hepatol 2013;28:279-84.  Back to cited text no. 10
    
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Kansau I, Raymond J, Bingen E, Courcoux P, Kalach N, Bergeret M, et al. Genotyping of Helicobacter pylori isolates by sequencing of PCR products and comparison with the RAPD technique. Res Microbiol 1996;147:661-9.  Back to cited text no. 11
    
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Satpathi P, Satpathi S, Mohanty S, Mishra SK, Behera PK, Maity AB, et al. Helicobacter pylori infection in dyspeptic patients in an industrial belt of India. Trop Doct 2017;47:2-6.  Back to cited text no. 12
    
13.
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. 13
    
14.
Adlekha S, Chadha T, Krishnan P, Sumangala B. Prevalence of Helicobacter pylori infection among patients undergoing upper gastrointestinal endoscopy in a medical college hospital in Kerala, India. Ann Med Health Sci Res 2013;3:559-63.  Back to cited text no. 14
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Mishra KK, Srivastava S, Dwivedi PP, Prasad KN, Ayyagari A. Genotypes of Helicobacter pylori isolated from various acid peptic diseases in and around Lucknow. Curr Sci 2002;83:749-55.  Back to cited text no. 15
    
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Kumar S, Kumar A, Dixit VK. Genetic diversity in strains of Helicobacter pylori from India and their relatedness to strains from other parts of the world. Infect Genet Evol 2011;11:242-7.  Back to cited text no. 16
    


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