Although the iceA (induced by contact with epithelium) allelic types of Helicobacter pylori have been reported to be associated with peptic ulcer, the importance of iceA on clinical outcomes based on subsequent studies is controversial. The aim of this study was to estimate the magnitude of the risk for clinical outcomes associated with iceA.
A literature search was performed using the PubMed and EMBASE databases for articles published through April 2011. Published case-control studies examining the relationship between iceA and clinical outcomes (gastritis, peptic ulcer, including gastric ulcer and duodenal ulcer, and gastric cancer) were included.
Fifty studies with a total of 5,357 patients were identified in the search. Infection with iceA1-positive H. pylori increased the overall risk for peptic ulcer by 1.26-fold (95% confidence interval [CI], 1.09–1.45). However, the test for heterogeneity was significant among these studies. Sensitivity analysis showed that the presence of iceA1 was significantly associated with peptic ulcer (odds ratio [OR] = 1.25, 95% CI = 1.08–1.44). The presence of iceA2 was inversely associated with peptic ulcer (OR = 0.76, 95% CI = 0.65–0.89). The presence of iceA was not associated with gastric cancer. Most studies examined the cagA status; however, only 15 studies examined the correlation and only 2 showed a positive correlation between the presence of cagA and iceA1.
Our meta-analysis confirmed the importance of the presence of iceA for peptic ulcer, although the significance was marginal.
Citation: Shiota S, Watada M, Matsunari O, Iwatani S, Suzuki R, Yamaoka Y (2012) Helicobacter pylori iceA, Clinical Outcomes, and Correlation with cagA: A Meta-Analysis. PLoS ONE 7(1): e30354. doi:10.1371/journal.pone.0030354
Editor: Masaru Katoh, National Cancer Center, Japan
Received: July 19, 2011; Accepted: December 14, 2011; Published: January 18, 2012
Copyright: © 2012 Shiota et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This report is based on work supported in part by grants from the National Institutes of Health (DK62813) (YY), and grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (22390085 and 22659087)(YY). The funders had no role in study design data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Helicobacter pylori infection is now accepted as the major cause of chronic gastritis. Several epidemiological studies have shown that H. pylori infection is also linked to severe gastritis-associated diseases, including peptic ulcer disease (PUD) and gastric cancer (GC) . In 1994, the International Agency for Research on Cancer categorized H. pylori infection as a group I carcinogen (definite carcinogen) . The infection remains latent in the majority of infected patients, with only approximately 20% of infected individuals developing severe diseases. In addition to host, environmental, and dietary factors, another possible reason for the various outcomes of H. pylori infection relates to differences in the virulence of H. pylori strains. Several H. pylori virulence factors have been reported to be associated with peptic ulcer and GC, including cagA, vacA, babA, and oipA , –. We recently reported the importance of the duodenal ulcer-promoting gene (dupA) for developing duodenal ulcer (DU) in a meta-analysis model .
An initial series of studies showed that iceA (induced by contact with epithelium) has 2 main allelic variants, iceA1 and iceA2 , . iceA1 demonstrated sequence homology with a gene from Neisseria lactamica, nlaIIIR, which encodes a CTAG-specific restriction endonuclease . On the other hand, iceA2 has no homology to known genes and the function of the iceA2 product remains unclear. van Doorn et al. reported that the iceA allelic type was independent of the cagA and vacA status, and there was a significant association between the presence of the iceA1 allele and PUD . The expression of iceA1 was upregulated on contact between H. pylori and human epithelial cells, and the iceA1 genotype was linked with enhanced mucosal interleukin (IL)-8 expression and acute antral inflammation , .
However, the role of iceA was controversial subsequently since several studies were not able to reproduce the observation in other populations, including Japanese –. Such discrepant results between the iceA allelic type and clinical outcomes could be explained by the genetic heterogeneity or differences in the geographic location, which were previously reported for other virulence genes . So far, there is no report on the significance of iceA using meta-analysis. In this study, we aimed to perform a meta-analysis to examine the relationship between the iceA allelic type and clinical outcomes.
Materials and Methods
A literature search was performed using the PubMed and EMBASE databases for articles published through April 2011. The following text words were used: 1) iceA, iceA1, or iceA2 and 2) pylori or Helicobacter. We did not include abstracts alone or unpublished articles.
The following criteria were applied to select fully published case-control studies examining the relationship between the iceA type and clinical outcomes (gastritis, PUD, gastric ulcer [GU], DU, and GC) in adult populations: the presence of iceA (iceA1 or iceA2) was examined by polymerase chain reaction (PCR) and original articles were published in English. Studies were excluded if no raw data were presented. When it appeared that the same subjects were presented in multiple reports, the earliest article was selected. All potentially relevant articles were reviewed independently by 2 investigators (S.S and Y.Y) and disagreement was resolved by discussion.
Studies were excluded if no raw data were presented or if no control groups were included. In addition, in vitro studies and studies conducted in child populations were also excluded.
Data were extracted independently from each study by the investigators and entered into a computerized database. The information retrieved covered countries where the study was performed, characteristics of cases and controls, the method for detection of iceA, the number of subjects, and the iceA status according to clinical outcomes. Two studies examined the prevalence of iceA in several countries , ; thus, the data of each country were entered in separate sheets as an independent study.
Summary odds ratios (ORs) and 95% confidence intervals (CIs) were calculated from raw data. The Mantel-Haenszel method was used to test for statistical heterogeneity. When statistical heterogeneity was noted, the proportion of the total heterogeneity variance was calculated from each study using a fixed-effects model to guide the search for sources of methodologically and clinically important variables. To exclude any possible influence of a single study, we performed a sensitivity analysis to evaluate whether the exclusion of any single study substantially altered the magnitude or statistical result of the summary estimate. Publication bias was assessed by funnel plots and regression test by Egger et al . A P value of <0.05 was considered as statistically significant. All analyses were performed using Comprehensive Meta-Analysis software (version 2, Biostat, Englewood, NJ).
The literature searches generated 140 potentially relevant citations. Of these, 93 articles were excluded. The main reasons for exclusion were that the articles were review articles, not case-control studies, conducted in children, and in vitro studies. Therefore, 46 articles met the inclusion criteria (Figure S1 and Table S1). Two articles included several countries (4 in Yamaoka et al. and 2 in Yamaoka et al. ). For these studies, data from different countries in the same article were considered as separate studies (data); therefore, 50 studies with a total of 5,357 patients met the inclusion criteria. Among these studies, 28 were from Asian –, –, 19 from Western , , , , –, and 3 from African countries –. An age- and sex-matched case-control study was conducted in only 1 report . Although 46 articles showed a difference in the iceA type between gastritis and PUD, 14 articles did not show the distribution of GU and DU , –, , , , , , , , , , . Four studies examined the prevalence of iceA types in gastritis and GC, but not in PUD , , , . Six studies examined only the prevalence of iceA1 but not iceA2 , , , , . The prevalence of mixed infection by iceA1 and iceA2 was also different in each study; it ranged from 1.9%  to 36.7% . Overall, the rate of mixed infection was 13.9%. Although strains from mixed infections were excluded in several studies , , , , , most studies included these mixed genotypes as total denominator. In this study, iceA1-positive means only iceA1-positive cases, but not iceA1- and iceA2-positive. Likewise, iceA2-positive means only iceA2-positive cases, but not iceA1- and iceA2-positive.
Association between the iceA1 status and clinical outcomes
The prevalence of iceA1 in PUD patients was examined in 46 studies from 24 countries. The prevalence of iceA1 ranged from 0.0% to 100.0% in PUD patients and from 4.3% to 100.0% in controls. Among 46 studies, a significantly higher prevalence of iceA1 in PUD compared with controls was found in 4 studies , , , . Only 1 study showed a significantly lower prevalence of iceA1 in PUD patients compared with controls . The overall prevalence of iceA1 was 59.3% (1,264 of 2,131) in PUD patients and 51.0% (1,260 of 2,470) in controls. The summary OR in the fixed-effects model was 1.26 (95% CI, 1.09–1.45). However, the test for heterogeneity was significant among these studies (I squared = 35.5, P = 0.01), suggesting the existence of either methodological or clinical heterogeneity. By exploring the sources of heterogeneity, we found that the studies by Peek et al. and Momenah et al.  showed larger differences in the prevalence of iceA1 compared with other studies. They reported a quite low prevalence of iceA1 in controls (4.3% and 5.4%, respectively) compared with PUD patients (47.6% and 100%, respectively). In addition, the study by Yamaoka et al. in the United States showed a lower prevalence of iceA1, especially in PUD patients (0.0%) . The study by Gatti et al. showed 100% prevalence in controls . Sensitivity analysis excluding these 4 studies showed a similar OR (1.25; 95% CI, 1.08–1.44) and the test for heterogeneity was no longer statistically significant (I squared = 4.5, P = 0.38) (Figure 1). Publication bias did not exist (intercept = 0.17; P = 0.36).
Odds ratios (ORs) and their 95% confidence intervals (CI) in summary and for each study are presented using a fixed-effect model. PUD: peptic ulcer disease.
Subgroup analysis by 2 areas (Asian or Western countries) was also performed. The prevalence of iceA1 was 68.9% (840 of 1,218) in PUD patients and 58.1% (676 of 1,163) in controls in Asian countries. It was 43.5% (348 of 799) in PUD patients and 41.8% (447 of 1,068) in controls in Western countries. The summary OR was 1.19 (95% CI, 0.97–1.45) in Asian countries and 1.28 (95% CI, 1.02–1.61) in Western countries. Subgroup analysis was also performed according to the distribution of DU and GU. The prevalence of iceA1 in DU patients was examined in 32 studies. When 4 studies , , ,  of larger effect size were excluded to limit heterogeneity, iceA1 was significantly associated with DU compared with controls (OR = 1.35, 95% CI = 1.11–1.63) (Figure S2). This finding was significant in Asian countries (OR = 1.38, 95% CI = 1.06–1.79). The prevalence of iceA1 was examined in GU patients from 15 studies. One study included only 1 patient with GU ; therefore, this study was excluded from the statistical analysis. There was no association between iceA1 and GU compared with controls (OR = 0.96, 95% CI = 0.69–1.35) (Figure S3). The prevalence of iceA1 was examined in GC patients from 23 studies. One study included only 1 patient with GC ; therefore, this study was excluded from the statistical analysis. There was no association between iceA1 and GC compared with controls (OR = 1.08, 95% CI = 0.86–1.37) (Figure S4).
Association between the iceA2 status and clinical outcomes
The prevalence of iceA2 was examined in PUD patients from 41 studies (24 countries). The prevalence of iceA2 ranged from 0.0% to 91.7% in PUD patients and from 0.0% to 95.7% in controls. Among the 41 studies, a significantly lower prevalence of iceA2 in PUD patients compared with controls was found in 6 studies , , , , , . The overall prevalence of iceA2 was 30.1% (585 of 1,944) in PUD patients and 37.2% (841 of 2,261) in controls. The summary OR in the fixed-effects model was 0.76 (95% CI, 0.65–0.89). However, the test for heterogeneity was significant among these studies (I squared = 40.7, P = 0.004). The studies by Peek et al. and Momenah et al.  showed larger differences in the prevalence of iceA2 compared with other studies. They reported a quiet high prevalence of iceA2 in controls (95.7% and 94.6%, respectively) compared with PUD patients (47.6% and 0.0%, respectively). In addition, the study by Yamaoka et al. in Korea showed a lower prevalence of iceA2, especially in gastritis patients (4.5%) . The study by Gatti et al. showed 0.0% prevalence in controls . Sensitivity analysis excluding these 4 studies showed a similar OR (0.78; 95% CI, 0.66–0.91) and the test for heterogeneity was no longer statistically significant (I squared = 19.9, P = 0.14) (Figure 2). Publication bias did not exist (intercept, 0.35; P = 0.27).
Subgroup analysis by 2 areas (Asian or Western countries) was also performed. The prevalence of iceA2 was 23.4% (269 of 1,148) in PUD patients and 30.2% (318 of 1,051) in controls in Asian countries. It was 41.8% (280 of 669) in PUD patients and 47.7% (456 of 956) in controls in Western countries. The summary OR was 0.91 (95% CI, 0.73–1.13) in Asian countries and 0.64 (95% CI, 0.50–0.82) in Western countries. Subgroup analysis was performed according to the distribution of DU and GU. The prevalence of iceA2 in DU patients was examined in 27 studies. When 4 studies , , ,  of larger effect size were excluded to limit heterogeneity, iceA2 was significantly associated with DU compared with controls (OR = 0.76, 95% CI = 0.60- 0.92) (Figure S5). This finding was significant in Western countries (OR = 0.66, 95% CI = 0.48–0.91). The prevalence of iceA2 was examined in GU patients from 13 studies. There was no association between iceA2 and GU compared with controls (OR = 1.01, 95% CI = 0.69–1.46) (Figure S6). The prevalence of iceA2 was examined in GC patients from 18 studies. One study included only 1 patient with GC ; therefore, this study was excluded from the statistical analysis. There was no association between iceA2 and GC compared with controls (OR = 0.84, 95% CI = 064–1.11) (Figure S7). Subgroup analysis according to the area showed that the OR was 1.10 (95% CI, 0.72–1.67) in Asian countries and 0.70 (95% CI, 0.49–1.00) in Western countries.
Difference in prevalence in Asian and Western countries
The overall prevalence of iceA1 was 64.6% (1,791 of 2,771) in Asian countries and 42.1% (935 of 2,218) in Western countries. It was significantly higher in Asian countries than in Western countries (P<0.0001). On the other hand, the prevalence of iceA2 was 25.8% (651 of 2,522) in Asian countries and 45.1% (844 of 1,871) in Western countries. It was significantly higher in Western countries than in Asian countries (P<0.0001).
Correlation between cagA and iceA
Fifteen studies examined the correlation between the cagA and iceA status. Only 1 study showed a significant positive association between iceA1 and the presence of cagA . One study showed a positive trend . Another study showed a significant positive association between iceA2 and the presence of cagA . Twelve studies showed no association between iceA1 and cagA status , , –, , , , .
Our present meta-analysis shows that the presence of iceA1 was significantly associated with PUD. Although several studies failed to show a positive association between the iceA status and clinical outcomes, this meta-analysis supported the original report from 1998 . However, this association was not very strong.
The mechanism of the development of PUD induced by iceA remains unclear. iceA1 demonstrated sequence homology with a gene from Neisseria lactamica, nlaIIIR, which encodes a CTAG-specific restriction endonuclease . However, studies on the genetic organization of the iceA locus indicated that a full-length nlaIIIR-like open reading frame has only been observed in 10 (20.4%) of 49 iceA1 H. pylori strains and only full-length iceA1 was a functional endonuclease gene . These data indicate that mutations in iceA1 are common, resulting in protein products with poor or no endonuclease activity. It remains to be determined whether iceA1 plays a role other than encoding an nlaIII-like endonuclease or not.
Most isolates with an iceA2 allele could be divided into 2 types according to the presence of repeated sequences of 105 nucleotides and the size of the PCR products (229 bp for iceA2-1 or 334 bp for iceA2-2) . Ashour et al. reported that no association was observed between the size of the iceA2 amplicon and diseases . On the other hand, Kidd et al. reported that the 334-bp iceA2 amplicon was more prevalent in strains from patients with PUD . These changes may translate into differential binding or function of the protein. The function of iceA2 remains unclear.
The prevalence of mixed infection by iceA1 and iceA2 was also different in each study. The strains of mixed infections were excluded in several studies , , , , . Figueiredo et al. reported that 36.7% of strains were positive for both iceA1 and iceA2, and 53.8% of these strains also contained multiple vacA genotypes . In our previous study, the rate of both iceA1 and iceA2 positivity was significantly lower in the United States than in Korea, Japan, and Columbia (4.3% vs. 20.0, 17.0, and 22.4%, respectively) . Multiple genotypes indicate the presence of multiple strains because there is no full-sequenced strain containing both iceA1 and iceA2 genes in Genbank (data not shown). It may be speculated that more than 1 strain may be acquired in childhood, especially in countries with a high prevalence of H. pylori. Mixed infection by more than 1 strain in the same individual may reflect the capacity of H. pylori to evolve genetic variations during long-term colonization from childhood . The high prevalence of mixed iceA-type strains may obscure any potential relationship between the allele and clinical outcomes.
Fourteen studies combined DU with GU as PUD. However, DU and GU are linked to entirely different patterns of gastric inflammation, such that it would seem they should be examined separately . Tham et al. indicated that in patients with H. pylori infection, those with DU have a higher degree of acute and chronic inflammation in the gastric antrum and higher H. pylori density than those with GU , which illustrates the different pathogenic processes of DU and GU. Therefore, a study to examine the roles of virulence factors needs to be conducted according to DU and GU, respectively. Furthermore, we should pay attention to the fact that patients with only gastritis at the time of endoscopy may develop ulcer diseases later in life and, therefore, may have been misclassified in the present study .
The association between the iceA and cagA status remains unclear. Several virulence factors of H. pylori correlated with the presence of cagA , , . In this study, we found that 15 studies examined the association between the iceA and cagA status. As a result, most of them showed no association, indicating that iceA1 might be a risk factor for PUD, independent of cagA. In addition, our recent meta-analysis showed that dupA, which induces DU and has a suppressive action on GC, was significantly associated with DU . The association between dupA and iceA has not been clarified yet; however, 1 study showed that there was no association . To confirm the significance of iceA, it is better to perform a multivariate analysis adjusted for the cagA status and other risk factors for peptic ulcer. However, unfortunately, we could not obtain the raw data from each study. It is difficult to perform a multivariate analysis adjusted for these factors without the raw data. In addition, most studies did not consider other risk factors in their papers. Further study is necessary to examine which factors are true virulence factors and which are just confounding factors. However, it might be better to hypothesize that these factors interact synergistically with each other and induce serious diseases than to discuss which of these factors is the most virulent. A recent study showed that groupings by multi-locus sequence typing (MLST) using 7 housekeeping genes were associated with the prevalence of GC , although we reported a problem of this interpretation . It may be better to classify H. pylori according to the structure of the bacteria instead of each virulence factor.
iceA1 was weakly, but significantly associated with PUD especially DU, whereas iceA2 was inversely associated with PUD. A relationship between iceA and GC and GU was not found in this meta-analysis. It is possible that iceA is a discriminating factor for PUD, independent of cagA.
Flow diagram of study selection.
Results of the meta-analysis for the risk of duodenal ulcer in iceA1-positive H. pylori infections.
Results of the meta-analysis for the risk of gastric ulcer in iceA1-positive H. pylori infections.
Results of the meta-analysis for the risk of gastric cancer in iceA1-positive H. pylori infections.
Results of the meta-analysis for the risk of duodenal ulcer in iceA2-positive H. pylori infections.
Results of the meta-analysis for the risk of gastric ulcer in iceA2-positive H. pylori infections.
Results of the meta-analysis for the risk of gastric cancer in iceA2-positive H. pylori infections.
Characteristics of studies included in the meta-analysis.
Conceived and designed the experiments: SS YY SI RS. Performed the experiments: SS YY MW OM. Analyzed the data: SS YY. Contributed reagents/materials/analysis tools: SS YY MW OM. Wrote the paper: SS YY.
- 1. Suerbaum S, Michetti P (2002) Helicobacter pylori infection. N Engl J Med 347: 1175–1186.
- 2. Schistosomes, liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon (1994) IARC Monogr Eval Carcinog Risks Hum 61: 1–241.
- 3. Atherton J, Cao P, Peek RJ, Tummuru M, Blaser M, et al. (1995) Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 270: 17771–17777.
- 4. Basso D, Zambon C, Letley D, Stranges A, Marchet A, et al. (2008) Clinical relevance of Helicobacter pylori cagA and vacA gene polymorphisms. Gastroenterology 135: 91–99.
- 5. Gerhard M, Lehn N, Neumayer N, Borén T, Rad R, et al. (1999) Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin. Proc Natl Acad Sci U S A 96: 12778–12783.
- 6. Yamaoka Y, Kikuchi S, el-Zimaity H, Gutierrez O, Osato M, et al. (2002) Importance of Helicobacter pylori oipA in clinical presentation, gastric inflammation, and mucosal interleukin 8 production. Gastroenterology 123: 414–424.
- 7. Shiota S, Matsunari O, Watada M, Hanada K, Yamaoka Y (2010) Systematic review and meta-analysis: the relationship between the Helicobacter pylori dupA gene and clinical outcomes. Gut Pathog 2: 13.
- 8. van Doorn L, Figueiredo C, Sanna R, Plaisier A, Schneeberger P, et al. (1998) Clinical relevance of the cagA, vacA, and iceA status of Helicobacter pylori. Gastroenterology 115: 58–66.
- 9. Peek RM, Thompson SA, Donahue JP, Tham KT, Atherton JC, et al. (1998) Adherence to gastric epithelial cells induces expression of a Helicobacter pylori gene, iceA, that is associated with clinical outcome. Proc Assoc Am Physicians 110: 531–544.
- 10. Xu Q, Morgan RD, Roberts RJ, Xu SY, van Doorn LJ, et al. (2002) Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori. Nucleic Acids Res 30: 3839–3847.
- 11. Yamaoka Y, Kodama T, Gutierrez O, Kim JG, Kashima K, et al. (1999) Relationship between Helicobacter pylori iceA, cagA, and vacA status and clinical outcome: studies in four different countries. J Clin Microbiol 37: 2274–2279.
- 12. Ito Y, Azuma T, Ito S, Suto H, Miyaji H, et al. (2000) Sequence analysis and clinical significance of the iceA gene from Helicobacter pylori strains in Japan. J Clin Microbiol 38: 483–488.
- 13. Nishiya D, Shimoyama T, Fukuda S, Yoshimura T, Tanaka M, et al. (2000) Evaluation of the clinical relevance of the iceA1 gene in patients with Helicobacter pylori infection in Japan. Scand J Gastroenterol 35: 36–39.
- 14. Maeda S, Amarsanaa J, Mitsuno Y, Hirata Y, Akanuma M, et al. (2002) Relationship between nuclear factor-kappaB activation and virulence factors of Helicobacter pylori in Japanese clinical isolates. J Gastroenterol Hepatol 17: 556–562.
- 15. Ando T, Tsuzuki T, Mizuno T, Minami M, Ina K, et al. (2004) Characteristics of Helicobacter pylori-induced gastritis and the effect of H. pylori eradication in patients with chronic idiopathic thrombocytopenic purpura. Helicobacter 9: 443–452.
- 16. Yamaoka Y, Orito E, Mizokami M, Gutierrez O, Saitou N, et al. (2002) Helicobacter pylori in North and South America before Columbus. FEBS Lett 517: 180–184.
- 17. Egger M, Davey Smith G, Schneider M, Minder C (1997) Bias in meta-analysis detected by a simple, graphical test. BMJ 315: 629–634.
- 18. Mukhopadhyay AK, Kersulyte D, Jeong JY, Datta S, Ito Y, et al. (2000) Distinctiveness of genotypes of Helicobacter pylori in Calcutta, India. J Bacteriol 182: 3219–3227.
- 19. Zheng P, Hua J, Yeoh K, Ho B (2000) Association of peptic ulcer with increased expression of Lewis antigens but not cagA, iceA, and vacA in Helicobacter pylori isolates in an Asian population. Gut 47: 18–22.
- 20. Kim SY, Woo CW, Lee YM, Son BR, Kim JW, et al. (2001) Genotyping CagA, VacA subtype, IceA1, and BabA of Helicobacter pylori isolates from Korean patients, and their association with gastroduodenal diseases. J Korean Med Sci 16: 579–584.
- 21. Wong BC, Yin Y, Berg DE, Xia HH, Zhang JZ, et al. (2001) Distribution of distinct vacA, cagA and iceA alleles in Helicobacter pylori in Hong Kong. Helicobacter 6: 317–324.
- 22. Sheu SM, Sheu BS, Yang HB, Li C, Chu TC, et al. (2002) Presence of iceA1 but not cagA, cagC, cagE, cagF, cagN, cagT, or orf13 genes of Helicobacter pylori is associated with more severe gastric inflammation in Taiwanese. J Formos Med Assoc 101: 18–23.
- 23. Chattopadhyay S, Datta S, Chowdhury A, Chowdhury S, Mukhopadhyay AK, et al. (2002) Virulence genes in Helicobacter pylori strains from West Bengal residents with overt H. pylori-associated disease and healthy volunteers. J Clin Microbiol 40: 2622–2625.
- 24. Rahman M, Mukhopadhyay AK, Nahar S, Datta S, Ahmad MM, et al. (2003) DNA-level characterization of Helicobacter pylori strains from patients with overt disease and with benign infections in Bangladesh. J Clin Microbiol 41: 2008–2014.
- 25. Perng CL, Lin HJ, Sun IC, Tseng GY, Facg (2003) Helicobacter pylori cagA, iceA and vacA status in Taiwanese patients with peptic ulcer and gastritis. J Gastroenterol Hepatol 18: 1244–1249.
- 26. Lin HJ, Perng CL, Lo WC, Wu CW, Tseng GY, et al. (2004) Helicobacter pylori cagA, iceA and vacA genotypes in patients with gastric cancer in Taiwan. World J Gastroenterol 10: 2493–2497.
- 27. Chang YJ, Wu MS, Lin JT, Sheu BS, Muta T, et al. (2004) Induction of cyclooxygenase-2 overexpression in human gastric epithelial cells by Helicobacter pylori involves TLR2/TLR9 and c-Src-dependent nuclear factor-kappaB activation. Mol Pharmacol 66: 1465–1477.
- 28. Ho YW, Ho KY, Ascencio F, Ho B (2004) Neither gastric topological distribution nor principle virulence genes of Helicobacter pylori contributes to clinical outcomes. World J Gastroenterol 10: 3274–3277.
- 29. Han YH, Liu WZ, Zhu HY, Xiao SD (2004) Clinical relevance of iceA and babA2 genotypes of Helicobacter pylori in a Shanghai population. Chin J Dig Dis 5: 181–185.
- 30. Baglan PH, Bozdayi G, Ozkan M, Ahmed K, Bozdayi AM, et al. (2006) Clarithromycin resistance prevalence and Icea gene status in Helicobacter Pylori clinical isolates in Turkish patients with duodenal ulcer and functional dyspepsia. J Microbiol 44: 409–416.
- 31. Erzin Y, Koksal V, Altun S, Dobrucali A, Aslan M, et al. (2006) Prevalence of Helicobacter pylori vacA, cagA, cagE, iceA, babA2 genotypes and correlation with clinical outcome in Turkish patients with dyspepsia. Helicobacter 11: 574–580.
- 32. Momenah AM, Tayeb MT (2007) Helicobacter pylori cagA and iceA genotypes status and risk of peptic ulcer in Saudi patients. Saudi Med J 28: 382–385.
- 33. Linpisarn S, Suwan W, Lertprasertsuk N, Koosirirat C, Steger HF, et al. (2007) Helicobacter pylori cagA, vacA and iceA genotypes in northern Thai patients with gastric disease. Southeast Asian J Trop Med Public Health 38: 356–362.
- 34. Chomvarin C, Namwat W, Chaicumpar K, Mairiang P, Sangchan A, et al. (2008) Prevalence of Helicobacter pylori vacA, cagA, cagE, iceA and babA2 genotypes in Thai dyspeptic patients. Int J Infect Dis 12: 30–36.
- 35. Caner V, Yilmaz M, Yonetci N, Zencir S, Karagenc N, et al. (2007) H pylori iceA alleles are disease-specific virulence factors. World J Gastroenterol 13: 2581–2585.
- 36. Dharne MS, Munot H, Pujari R, Kakrani AL, Patole MS, et al. (2007) Helicobacter pylori cagA, vacA and iceA genotypes in western Indian population of Maharashtra with varied gastroduodenal diseases. Indian J Pathol Microbiol 50: 740–748.
- 37. Zhang Z, Zheng Q, Chen X, Xiao S, Liu W, et al. (2008) The Helicobacter pylori duodenal ulcer promoting gene, dupA in China. BMC Gastroenterol 8: 49.
- 38. Saha DR, Datta S, Chattopadhyay S, Patra R, De R, et al. (2009) Indistinguishable cellular changes in gastric mucosa between Helicobacter pylori infected asymptomatic tribal and duodenal ulcer patients. World J Gastroenterol 15: 1105–1112.
- 39. Amjad N, Osman HA, Razak NA, Kassian J, Din J, et al. (2010) Clinical significance of Helicobacter pylori cagA and iceA genotype status. World J Gastroenterol 16: 4443–4447.
- 40. Figueiredo C, Van Doorn LJ, Nogueira C, Soares JM, Pinho C, et al. (2001) Helicobacter pylori genotypes are associated with clinical outcome in Portuguese patients and show a high prevalence of infections with multiple strains. Scand J Gastroenterol 36: 128–135.
- 41. Miehlke S, Yu J, Schuppler M, Frings C, Kirsch C, et al. (2001) Helicobacter pylori vacA, iceA, and cagA status and pattern of gastritis in patients with malignant and benign gastroduodenal disease. Am J Gastroenterol 96: 1008–1013.
- 42. Ashour AA, Collares GB, Mendes EN, de Gusmão VR, Queiroz DM, et al. (2001) iceA genotypes of Helicobacter pylori strains isolated from Brazilian children and adults. J Clin Microbiol 39: 1746–1750.
- 43. Arents NL, van Zwet AA, Thijs JC, Kooistra-Smid AM, van Slochteren KR, et al. (2001) The importance of vacA, cagA, and iceA genotypes of Helicobacter pylori infection in peptic ulcer disease and gastroesophageal reflux disease. Am J Gastroenterol 96: 2603–2608.
- 44. Koehler CI, Mues MB, Dienes HP, Kriegsmann J, Schirmacher P, et al. (2003) Helicobacter pylori genotyping in gastric adenocarcinoma and MALT lymphoma by multiplex PCR analyses of paraffin wax embedded tissues. Mol Pathol 56: 36–42.
- 45. Russo F, Berloco P, Cuomo R, Caruso M, Di Matteo G, et al. (2003) Helicobacter pylori strains and histologically-related lesions affect the outcome of triple eradication therapy: a study from southern Italy. Aliment Pharmacol Ther 17: 421–428.
- 46. Ribeiro ML, Godoy AP, Benvengo YH, Mendonça S, Pedrazzoli J (2003) Clinical relevance of the cagA, vacA and iceA genotypes of Helicobacter pylori in Brazilian clinical isolates. FEMS Immunol Med Microbiol 36: 181–185.
- 47. Leodolter A, Wolle K, Peitz U, Ebert M, Günther T, et al. (2003) Helicobacter pylori genotypes and expression of gastritis in erosive gastro-oesophageal reflux disease. Scand J Gastroenterol 38: 498–502.
- 48. Leanza AG, Matteo MJ, Crespo O, Antelo P, Olmos J, et al. (2004) Genetic characterisation of Helicobacter pylori isolates from an Argentinean adult population based on cag pathogenicity island right-end motifs, lspA-glmM polymorphism and iceA and vacA genotypes. Clin Microbiol Infect 10: 811–819.
- 49. Gatti LL, Módena JL, Payão SL, Smith MeA, Fukuhara Y, et al. (2006) Prevalence of Helicobacter pylori cagA, iceA and babA2 alleles in Brazilian patients with upper gastrointestinal diseases. Acta Trop 100: 232–240.
- 50. Miciuleviciene J, Calkauskas H, Jonaitis L, Kiudelis G, Tamosiūnas V, et al. (2008) Helicobacter pylori genotypes in Lithuanian patients with chronic gastritis and duodenal ulcer. Medicina (Kaunas) 44: 449–454.
- 51. Bartchewsky W, Martini MR, Masiero M, Squassoni AC, Alvarez MC, et al. (2009) Effect of Helicobacter pylori infection on IL-8, IL-1beta and COX-2 expression in patients with chronic gastritis and gastric cancer. Scand J Gastroenterol 44: 153–161.
- 52. Vega AE, Cortiñas TI, Puig ON, Silva HJ (2010) Molecular characterization and susceptibility testing of Helicobacter pylori strains isolated in western Argentina. Int J Infect Dis 14(Suppl 3): e85–92.
- 53. Kidd M, Peek RM, Lastovica AJ, Israel DA, Kummer AF, et al. (2001) Analysis of iceA genotypes in South African Helicobacter pylori strains and relationship to clinically significant disease. Gut 49: 629–635.
- 54. Smith SI, Kirsch C, Oyedeji KS, Arigbabu AO, Coker AO, et al. (2002) Prevalence of Helicobacter pylori vacA, cagA and iceA genotypes in Nigerian patients with duodenal ulcer disease. J Med Microbiol 51: 851–854.
- 55. Ben Mansour K, Fendri C, Zribi M, Masmoudi A, Labbene M, et al. (2010) Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients. Ann Clin Microbiol Antimicrob 9: 10.
- 56. Kersulyte D, Chalkauskas H, Berg DE (1999) Emergence of recombinant strains of Helicobacter pylori during human infection. Mol Microbiol 31: 31–43.
- 57. Tham KT, Peek RM, Atherton JC, Cover TL, Perez-Perez GI, et al. (2001) Helicobacter pylori genotypes, host factors, and gastric mucosal histopathology in peptic ulcer disease. Hum Pathol 32: 264–273.
- 58. Maeda S, Ogura K, Yoshida H, Kanai F, Ikenoue T, et al. (1998) Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan. Gut 42: 338–343.
- 59. Mizushima T, Sugiyama T, Komatsu Y, Ishizuka J, Kato M, et al. (2001) Clinical relevance of the babA2 genotype of Helicobacter pylori in Japanese clinical isolates. J Clin Microbiol 39: 2463–2465.
- 60. de Sablet T, Piazuelo MB, Shaffer CL, Schneider BG, Asim M, et al. (2011) Phylogeographic origin of Helicobacter pylori is a determinant of gastric cancer risk. Gut 60(9): 1189–95.
- 61. Shiota S, Matsunari O, Watada M, Yamaoka Y (2011) Virulence factors or ancestral origin of Helicobacter pylori: which is a better predictor of gastric cancer risk? Gut. (in press).