Prognostic significance of genotyping Helicobacter pylori infection in patients in younger age groups with gastric cancer

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1 See Editorial, p Center for Liver Researh and Diagnostis, Dean College of Medial Sienes, Kanhanbagh, Hyderabad, Andhra Pradesh, India; 2 Department of Gastroenterology, Osmania General Hospital, Afzalgunj, Hyderabad, Andhra Pradesh, India; 3 Department of Pathology, Dean College of Medial Sienes, Kanhanbagh, Hyderabad, Andhra Pradesh, India Correspondene to: Dr A A Khan, Center for Liver Researh and Diagnostis, Dean College of Medial Sienes, Kanhanbagh, Hyderabad , Andhra Pradesh, India; aleem_a_khan@rediffmail.om SKT and GM ontributed equally to this work. Reeived 6 Otober 2007 Aepted 25 January 2008 Prognosti signifiane of genotyping Heliobater pylori infetion in patients in younger age groups with gastri aner S K Tiwari, 1 G Manoj, 1 G Vasanth Kumar, 1 G Sivaram, 1 S I Hassan, 2 B Prabhakar, 2 U Devi, 2 S Jalaluddin, 2 K Kumar, 2 S Ahmed, 1 Z Abid, 1,3 M A Habeeb, 1 A A Khan, 1 C M Habibullah 1 ABSTRACT Bakground: Malignant tumours of the stomah are ommon, but the inidene of stomah aner varies from ountry to ountry, probably a result of geneti, epigeneti and environmental fators. Stomah aner often ours in older people whose stomahs produe only small quantities of aid. Although infetion with Heliobater pylori has been proven beyond doubt in the aetiopathogenesis of various gastri disorders, not muh is known about the genotypes of H pylori infetion in early-onset gastri aner. Aim: To asertain the genotypes of H pylori in gastri aner. Methods: Ninety-two patients were separated into three groups on the basis of their endosopi findings: group 1, gastri aner; group 2, gastri uler; group 3, non-uler dyspepsia. Gastri biopsy speimens were obtained for ulture and DNA isolation; additional speimens were taken from subjets with gastri aner for histopathologial analysis. Amplifiation was performed using speifi oligonuleotide primers to obtain genotypi data. Four samples from eah group were randomly seleted for sequene analysis. Results: Genotypi analysis showed agt+ve/hrga+ve/ aga+ve/age+ve/vaas1+ve to be highly prevalent in 79% of ases of H pylori infetion. This genotype was found in 88% of subjets in group 1 and 78% in group 2. Intestinal-type adenoarinoma was found in 35 subjets (83%), 32 (9%) of whih harboured this genotype. Sequene analysis showed no signifiant strain-speifi variations. Conlusions: Certain genotypes of H pylori have higher preditive value for the development of intestinal-type arinoma at an early age. Genotyping of H pylori may well be a useful tool for sreening people at inreased risk of developing malignany. Gastri aner is the seond leading ause of aner-related deaths worldwide. From the data provided by the International Ageny for Researh on Caner, 1 malignany of the stomah is the most ommon aner in Asia, nearly two-thirds of whih ours in developing ountries. 2 Despite a dramati fall in the inidene and mortality of distal gastri aners in the Western world, the inidene of proximal tumours is inreasing, espeially in the male population. In ontrast, data from the Asian sub-ontinent reveal an inrease in distal tumours on a par with proximal tumours. The marked geographi variation, differenes in lifestyle, time trends and migratory effets assoiated with the Original artile inidene of gastri aner suggest that environmental and lifestyle fators are major ontributors to the aetiology of this disease. 3 Our understanding of gastri aner underwent a disernible shift with the disovery of Heliobater pylori. Infetion with H pylori probably still plays a leading role in the development of gastri aner in young patients. 4 In a ountry suh as India, where.75% of the population are infeted, it has been proved beyond doubt that H pylori infetion is high, espeially in areas of low soioeonomi status and bad hygiene onditions. 5 The vast majority of H pylori-infeted people remain asymptomati throughout their lives with no major linial sequelae, and only a small proportion present with some form of gastri disorder, suh as gastri arinoma and muosa-assoiated lymphoid tissue (MALT) lymphoma. Of those infeted, fewer than 10% present with the disease before 45 years of age, and these younger patients are thought to develop arinomas with a different moleular geneti profile from that of sporadi arinomas ourring at a later age. 6 In support of this notion, a study by Konturek et al 7 showed that the risk of gastri aner in patients with H pylori markedly inreased with infetion with bateria that were ytotoxin-assoiated gene A (aga)- positive in the age group years. Further, a similar study from the Indian sub-ontinent reported a higher risk of developing gastri aner in younger patients (,45 years) infeted with H pylori. 8 Although some studies have rejeted a diret link between H pylori infetion and gastri aner, 8 9 several prospetive studies have supported a positive assoiation between these two entities Perhaps the most onvining evidene for the assoiation between H pylori infetion and gastri arinoma was provided by a Japanese study, 12 whih showed development of gastri aner in 2.9% of H pylori-infeted people, whereas no uninfeted subjets developed it. In addition, a number of environmental fators have been shown to be assoiated with gastri aner, inluding highsalt diets, N-nitrosamines and low intake of dietary antioxidants typially found in fresh fruit and vegetables. Despite the vast array of data on gastri aner, the exat mehanisms leading to this ommon aner are far from understood. Several ofators are believed to ontribute to the overtness of the disease, whih an be broadly ategorised into: (a) Postgrad Med J 2008;84: doi: /pgmj

2 Table 1 Genotype Genotypi distribution of Heliobater pylori in subjets with different linial presentations baterial fators; (b) proinflammatory host fators; () environmental fators. Besides host fators, suh as ylo-oxygenase (COX)-1, COX-2, induible nitri oxide synthase (inos) and p53, and environmental fators, suh as dietary habits, both of whih are impliated in the pathogenesis of gastri aner, reent studies have revealed an enhaned risk of gastri aner in people infeted with more virulent strains of H pylori than those with less pathogeni H pylori. As it is well known that arinogenesis is a multifatorial event and probably takes deades to manifest, it is important to begin to investigate the preise role played by this pathogen in the highly intriate proess. As indiated by some studies, 15 infetion with H pylori is not itself responsible for promoting the proess of aner; instead it ats as a trigger, initiating the arinogeni pathway. This is usually aomplished by the major proteins (eg, CagA and VaA) sereted by H pylori, whih, on injetion into human host ells, undergo a omplex series of modifiations, thereby induing the host ells to serete inflammatory ytokines. These ytokines, in their attempt to eliminate H pylori infetion, ontinuously bombard the host tissue, ausing muosal damage either diretly or indiretly. H pylori strains are diverse, and some strains may have more malignant potential. Patients harbouring aga/vaa-positive strains of H pylori exhibit enhaned expression of various ytokines, thereby ausing major DNA damage, whih over time leads to aumulation of mutations, resulting in aner. 16 These patients have a higher grade of gastri inflammation and aelerated epithelial damage and thus have greater potential to develop gastri metaplasia than patients infeted with aga/ vaa-negative strains. 16 These underlying fats indiate the need to sreen and haraterise H pylori in individual disease onditions. Gastri arinoma (n = 42) Gastri uler (n = 18) NUD (n = 12) Total (n = 72) agt+ve/aga+ve/age+ve/hrga+ve/vaas1+ve 37 (88%)*{ 14 (78%){ 6 (50%) 57 (79%) agt+ve/aga+ve/age+ve/hrga+ve/vaas2+ve 2 (5%) 2 (11%) 4 (33%) 8 (11%) agt ve/aga ve/age ve/hrga+ve/vaas2+ve 0 1 (6%) 1 (8%) 2 (3%) agt+ve/aga ve/age ve/hrga+ve/vaas2+ve (8%) 1 (1%) agt+ve/aga ve/age+ve/hrga+ve/vaas1+ve 3 (7%) 1 (6%) 0 4 (6%) *x 2 = for prevalene ompared with gastri uler (p,0.01). {x 2 = for prevalene ompared with non-uler dyspepsia (p,0.01). {x 2 = for prevalene ompared with non-uler dyspepsia (p,0.01). NUD, non-uler dyspepsia. Figure 1 Invasion of stroma; dysplasia assoiated with invasive intestinal-type gastri adenoarinoma. Several riteria based on the different genes have been used to assess the virulene trait of this pathogen, the most aeptable hypothesis being one based on the presene of aga. 17 A large number of studies have shown inreased risk of gastri aner in people with aga+ve H pylori. However, other data have revealed that the ourrene of gastri malignany is independent of aga status. 18 A few studies have also impliated pivotal roles of other virulene fators (age, agt, vaa, baba and hrga) in the aetiology of gastri aner. 19 Several genotypes of H pylori have been reported to possess higher preditive value for the development of the severe form of the disease. Genotype studies from India have reported on the ommon genotypes of H pylori in patients with gastritis and pepti uler and a few ases of gastri arinoma. 22 Therefore the present study was designed mainly to haraterise the strains of H pylori found in patients with gastri arinoma in and around Hyderabad, India. MATERIALS AND METHODS Patients and lassifiation of endosopi findings Ninety-two patients (70 male, 22 female; age range years, mean age years) referred for routine endosopy at the Department of Gastroenterology, Dean College of Medial Sienes, Hyderabad, India and Department of Gastroenterology, Osmania General Hospital, Hyderabad, India during May Otober 2007 were reruited into the study. Endosopi findings were reorded and used to separate H pyloripositive patients into three groups: group 1 (n = 48), patients with gastri aner; group 2 (n = 20), patients with gastri uler; group 3 (n = 24), patients with non-uler dyspepsia. The institutional ethis ommittee approved the study protool, and all the patients gave informed onsent to partiipate in the Figure 2 Gastri muosal biopsy speimen showing presene of signet ring ell suggestive of diffuse-type arinoma. 194 Postgrad Med J 2008;84: doi: /pgmj

3 study. Patients taking proton pump inhibitors and/or nonsteroidal anti-inflammatory drugs in the preeding month, with a history of gastretomy, and/or had previous H pylori eradiation were exluded from the study. Methods During eah endosopi proedure, two antral and orpus muosal biopsy speimens were obtained using biopsy foreps, whih were leaned with detergent and disinfeted after eah use. One of eah speimen was plaed in Bruella broth supplemented with 2% fetal alf serum and phosphate-buffered saline (ph 7) for ulture and DNA isolation. If there was any growth in the stomah, an additional two speimens from the mass were olleted in 10% buffered formalin for histopathologial analysis of the nature of the growth. H pylori ultivation and DNA isolation H pylori was ultured routinely on Bruella agar (Beton Dikinson, Franklin Lakes, New Jersey, USA) with 7% sheep blood and antibioti supplement for 3 days at 37uC in a miroaerophili atmosphere (5% O 2, 10%CO 2 and 85% N 2 ). In subjets from whom live bateria ould not be ultured, genomi DNA was isolated the modified C TAB method. 20 Harvested ulture and/or biopsy sample was suspended in 0.1 M NaCl/10 mm Tris/HCl/1 mm EDTA (ph 8.0), sodium dodeyl sulphate (1%, w/v) was added, and the mixture was heated at 65uC and then extrated with phenol/hloroform/ isoamyl alohol. Genotyping based on aga, age, agt, vaa and hrga genes of H pylori Isolated genomi DNA (1 ml) was used to obtain the genotypes of H pylori from individual patients using a multiplex PCR assay as previously reported. 20 The list of oligonuleotide primers used in the study is reported. 20 Sequene analysis For sequening, four samples from eah group were randomly seleted, and amplions of eah target gene were sequened diretly, after purifiation with the QIAquik gel extration kit (Qiagen, Hilden, Germany) using the Big Dye Terminator V3.1 yle sequening kit and injeted into an ABI 3730xl Geneti Analyzer (Applied Biosystems, Foster City, California, USA). Sequenes were analysed and ompared with data published in the NCBI database. Figure 3 Infiltrative adenoarinoma. Histopathologial analysis Gastri biopsy speimens olleted in 10% buffered formalin were used to assess the nature (benign or malignant) of the lesions. Only ases with adequately sized speimens were aepted for histologial assessment by an experiened pathologist (ZA), who was blinded to the linial information on eah patient. The following histopathologial variables were sored on an ordinal sale (0 3) using the riteria desribed in the updated Sydney lassifiation system 23 : H pylori density, hroni inflammation, glandular atrophy, intestinal metaplasia and dysplasia. When present, intestinal metaplasia was typed as omplete, mixed (omplete and inomplete), or inomplete, and dysplasti hanges were typed as low grade and high grade. Statistial analysis x 2 and Student t tests were used as and where appliable for statistial alulation. p,0.05 was onsidered signifiant. RESULTS H pylori infetion was diagnosed in 72 of the 92 subjets inluded in the study (gastri adenoarinoma, 42; gastri uler, 18; non-uler dyspepsia, 12). H pylori infetion was onfirmed if any of the three tests that is, ulture, 16S ribosomal RNA PCR and histopathology were found to be positive. H pylori genotypes In the 72 subjets, PCR assay revealed genotype agt+ve/ hrga+ve/aga+ve/age+ve/vaas1+ve to be predominant (table 1). Overall, this genotype was found in 79% of all subjets inluded in the study. Genotypes of H pylori infetion in the three linial groups When the genotype data were orrelated with individual disease status, age+ve/vaas1+ve had a higher prevalene in group 1 (gastri adenoarinoma; 37 (88%)) than in group 2 (gastri uleration; 14 (78%)) (p,0.01). In group 3 (non-uler dyspepsia), six (50%) patients harboured H pylori with the above genotype; this prevalene is also signifiantly (p,0.01) different from that in groups 1 and 2. Histopathologial orrelation of genotypes Of the 48 patients with gastri arinoma, 42 (88%) showed H pylori positivity. In the latter, histopathologial examination showed mild intestinal metaplasia in 12 (29%), moderate intestinal metaplasia in seven (17%), low-grade dysplasia in eight (19%), and high-grade dysplasia in 10 (24%). Of the 42 subjets with H pylori positivity, 35 (83%) showed features of intestinal-type arinoma (fig 1), three (7%) showed patterns suggestive of diffuse arinoma (fig 2), two (5%) had infiltrative Table 2 Relationship between Heliobater pylori genotype and type of gastri adenoarinoma Genotype age+ve/vaas1+ve age+ve/vaas2+ve age2ve/vaas1+ve Intestinal (n = 35) Diffuse (n = 3) Infiltrative (n = 2) Undifferentiated (n = 2) Postgrad Med J 2008;84: doi: /pgmj

4 adenoarinoma (fig 3), and in two (5%) the type of arinoma ould not be differentiated. On analysis, a strong orrelation was found between the presene of intestinal-type aner and the genotype agt+ve/ hrga+ve/aga+ve/age+ve/vaas1+ve. Table 2 gives the details of the genotype and histologial orrelations. Sequene analysis Sequenes obtained from PCR produts of four randomly seleted samples from eah patient group were subsequently analysed using BLAST n. The nuleotide sequenes of the amplified produts shared % homology with the aga, agt, age, hrga and vaa genes of H pylori. DISCUSSION This study shows a higher prevalene of aga/age/agt/hrga/ vaas1-positive H pylori in patients with gastri adenoarinoma, espeially the intestinal type, than those with other gastri disorders. These findings are onsistent with previous reports by Koehler et al, 21 Plummer et al 24 and Soltermann et al, 25 suggesting the pivotal role of H pylori genotype in the linial outome of gastroduodenal diseases. This study mainly emphasises the genotypi variants of H pylori in patients with gastri adenoarinoma, as only a few previous studies have been able to haraterise H pylori speifially in patients with gastri arinoma Five virulent markers aga, age and agt of the ytotoxin-assoiated gene pathogeniity island (ag-pai), the signal peptide region of the vauolating-assoiated ytotoxin (vaa), and H pylori restrition endonulease-replaing gene (hrga) of this pathogen were seleted on the basis of previous reports from different ountries and sreened using speifi oligonuleotide primers by a multiplex PCR assay developed by our group. 20 Analysis of the 72 H pylori-positive subjets revealed agt+ve/ hrga+ve/aga+ve/age+ve/vaas1+ve to be the predominant genotype in 79% of them. Correlation of the genotype data with the linial findings indiated that this genotype is highly prevalent in group 1 (88%), followed by group 2 (78%), with subjets with non-uler dyspepsia showing the lowest prevalene (50%). These differenes in prevalene are highly signifiant (p,0.01). We also attempted to assoiate H pylori genotype with the type of gastri arinoma. Of the 42 subjets with endosopially onfirmed gastri aner, histologial analysis showed intestinal type in 35 (83%), diffuse type in three (7%), and infiltrative type in two (5%). Male patients had an inreased risk of developing intestinal-type arinoma ompared with female patients in our study population (data not shown). The reason for this may be the higher number Main findings of the study Genotyping Heliobater pylori in younger patients may be a useful strategy for identifying those with inreased risk of gastri aner. The genotype age+ve/ vaas1+ve had a higher preditive value for the overt form of gastri disease in our population. This retrospetive study suggests that the ombined presene of different virulent genes of H pylori is the most effetive for identifying patients at inreased risk of developing gastri adenoarinoma rather than sreening only one or two genes. of male than female partiipants in the study. No orrelation ould be established between genotype and diffuse-type arinoma beause of the small sample size (n = 3) (table 2); however, the existene of suh a relationship annot be ruled out. Sequening analysis of the four samples randomly seleted from eah group did not show any strain-speifi variation in relation to disease status. This signifies that H pylori genotype has a definitive role in early-onset gastri aner; the final outome also depends on the age at aquisition, topographi distribution of H pylori in the stomah, aid seretion, and assoiated pattern of gastritis. This finding agrees with those of Soltermann et al, 25 who reported that virulene genotype of the mirobe and age are important fators in the severity of gastritis and the development of intestinal metaplasia. Although many previous reports have attempted to delineate the preise assoiation between H pylori genotype and gastri arinoma, only a few have been able to provide any information on the usefulness of any of the genes of this pathogen for prediting gastri arinoma. The reason may be the genes seleted. We used a ombination of five different virulent markers for orrelating genotype and linial findings. We were not only able to orrelate the genotype data with the linial data, but we were also able to show the high risk of intestinal-type arinoma in patients infeted with the aga/ age/agt/hrga/vaas1 genotype. Another important aspet of this study is that we inluded subjets in the age range years; this differs from some previous studies 27 in whih the subjets were.50 years of age. There are ertain limitations of this study. The first is the small sample size. Seondly, although this study shows a high preditive value of aga/age/agt/hrga/vaas1-positive H pylori in patients with gastri aner in our study population, it would be best for researhers worldwide to evaluate the prevalene of eah individual gene, as the virulene of H pylori varies from one population to another in different geographi areas. The reason may be the extreme geneti diversity of H pylori at both the transriptional and translational level, whih in turn affets the funtion and antigeniity of the virulene fators assoiated with baterial infetion and ultimately the disease outome. Over-interpretation of the data would therefore be avoided, and the genotypes of H pylori that ause different diseases in different regions of the world would be determined. Thirdly, external fators suh as dietary intake of antioxidants may also alter the linial outome of infetion. In onlusion, gastri aner is a multistep, multifatorial disease, whih involves the umulative interplay of host, environmental and baterial fators and probably takes several deades to manifest. Genotyping H pylori infetion may therefore be an ideal tool for identifying subjets at inreased risk of developing gastri arinoma, espeially in young age groups. Finally, prospetive studies evaluating expression profiles of the seleted genes in different populations are needed to establish a link between H pylori genotype and the entire spetrum of H pylori-related gastroduodenal diseases inluding gastri arinoma. Aknowledgements: We thank T Neeraj for ritial analysis during the ourse of the study, and other staff of the Osmania General Hospital for their help in the olletion of biopsy samples. Funding: This work was not supported by any grant from any soure. Competing interests: None. Ethis approval: Ethis approval was obtained. 196 Postgrad Med J 2008;84: doi: /pgmj

5 REFERENCES 1. Ferlay J, Bray F, Pisani P, et al. GLOBOCAN 2000: aner inidene, mortality and prevalene worldwide, version 1.0. IARC Caner Base No 5. Lyon: IARC Press, Crew KD, Neuget AI. Epidemiology of gastri aner. World J Gastroenterol 2006;12: Ries LAG, Kosary CL, Hankey BF, et al. International Ageny for Researh on Caner Working Group on the Evaluation of Carinogeni Risks to Humans. Shistosomes, liver flukes, and Heliobater pylori. Lyon: International Ageny for Researh on Caner, 1994: Azuma T, Yamazaki S, Yamakawa A, et al. Assoiation between diversity in the Sr homology 2 domain ontaining tyrosine phosphatase binding site of Heliobater pylori CagA protein and gastri atrophy and aner. J Infet Dis 2004;189: Ahmed KS, Khan AA, Ahmed I, et al. Impat of household hygiene and water soure on the prevalene and transmission of Heliobater pylori: a South Indian perspetive. Singapore Med J 2007;48: Carvalho R, Milne ANA, van Rees BP, et al. Early onset gastri arinomas display distint moleular harateristis within gastri arinogenesis. J Pathol 2004;204: Konturek SJ, Starzynska T, Marliz K. Heliobater pylori (HP), CagA status and plasma gastrin in the development of gastri aner. Gut 2000;47(Suppl 1):A Khanna AK, Seth P, Nath G, et al. Correlation of H. pylori and gastri arinoma. J Postgrad Med 2002;48: Eslik GD, Lim LL, Byles JE, et al. Assoiation of H. pylori infetion with gastri arinoma: a meta-analysis. Am J Gastroenterol 1999;94: Parsonnet J, Friedman GD, Vandersteen DP, et al. Heliobater pylori infetion and the risk of gastri arinoma. N Engl J Med 1991;325: Forman D, Newell DG, Fullerton F, et al. Assoiation between infetion with Heliobater pylori and risk of gastri aner: evidene from a prospetive investigation. BMJ 1991;302: Uemura N, Okamoto S, Yamamoto S, et al. Heliobater pylori infetion and the development of gastri aner. N Engl J Med 2001;345: Crowe SE. Heliobater infetion, hroni inflammation, and the development of malignany. Curr Opin Gastroenterol 2005;21: Hertog MG, Bueno-de-Mesquita HB, Fehily AM, et al. Fruit and vegetable onsumption and aner mortality in the Caerphilly Study. Caner Epidemiol Biomarkers Prev 1996;5: Mueller A, Falkow S, Amieva MR. Heliobater pylori and gastri aner: what an be learned by studying the response of gastri epithelial ells to the infetion? Caner Epidemiol Biomarkers Prev 2005;14: Zarrilli R, Rii V, Roman M. Moleular response of gastri epithelial ells to Heliobater pylori-indued ell damage. Cell Mirobiol 1999;1: Shimoyama T, Fukuda S, Tanaka M, et al. CagA seropositivity assoiated with development of gastri aner in a Japanese population. J Clin Pathol 1998;51: Held M, Engstrand L, Hansson LE, et al. Is the assoiation between H. pylori and gastri aner onfined to CagA-positive strains? Heliobater 2004;9: Fishbah W, On-On Chan A, Chun-Yu Wong B. Heliobater pylori and gastri malignany. Heliobater 2005;10: Tiwari SK, Khan AA, Manoj G, et al. A simple multiplex PCR assay for diagnosing virulent Heliobater pylori infetion in human gastri biopsy speimens from subjets with gastri arinoma and other gastro-duodenal diseases. J Appl Mirobiol 2007;103: Koehler CI, Mues MB, Dienes HP, et al. Heliobater pylori genotyping in gastri adenoarinoma and MALT lymphoma by multiplex PCR analyses of paraffin wax embedded tissues. Mol Pathol 2003;56: Ali M, Khan AA, Tiwari SK, et al. Assoiation between ag-pathogeniity island in Heliobater pylori isolates from pepti uler, gastri arinoma, and non uler dyspepsia subjets with histologial hanges. World J Gastroenterol 2005;11: Dixon MF, Genta RM, Yardley JH, et al. Classifiation and grading of gastritis. The updated Sydney system. Am J Surg Pathol 1996;20: Plummer M, van Doorn L-J, Franeshi S, et al. Heliobater pylori ytotoxinassoiated genotype and gastri preanerous lesions. J Natl Caner Inst 2007;99: Soltermann A, Koetzer S, Eigenmann F, et al. Correlation of Heliobater pylori virulene genotypes vaa and aga with histologial parameters of gastritis and patient s age. Mod Pathol 2007;20: Sinha R, Anderson DE, MDonald SS, et al. Caner risk and diet in India. J Postgrad Med 2003;49: Nogueira C, Figueiredo C, Carneiro F, et al. Heliobater pylori genotypes may determine gastri pathology. Am J Pathol 2001;158: Ando T, Wassenaar M, Peek RM, et al. A Heliobater pylori restrition endonulease-replaing gene, hrga, is assoiated with gastri aner in Asian strains. Caner Res 2002;62: Ladeira MS, Rodrigues MA, Salvadori V, et al. Relationships between aga, vaa, and iea genotypes of Heliobater pylori and DNA damage in the gastri muosa. Environ Mol Mutagen 2004;44: Maeda S, Ogura K, Yoshida H, et al. Major virulene fators, vaa and aga, are ommonly positive in Heliobater pylori isolates in Japan. Gut 1998;42: Ogura K, Maeda S, Nakao M, et al. Virulene fators of Heliobater pylori responsible for gastri diseases in Mongolian gerbil. J Exp Med 2000;192: Yamaoka Y, Kodama T, Gutierrez O, et al. Relationship between Heliobater pylori iea, aga, and vaa status and linial outome: studies in four different ountries. J Clin Mirobiol 1999;37: Censini S, Lange C, Xiang Z, et al. ag, a pathogeniity island of Heliobater pylori, enodes type I-speifi and disease-assoiated virulenefators. Pro Natl Aad Si USA 1996;93: Postgrad Med J: first published as /pgmj on 18 April Downloaded from on 21 November 2018 by guest. Proteted by opyright. Postgrad Med J 2008;84: doi: /pgmj