Pathogenic potential of Helicobacter pylori strains can explain differences in H. pylori associated diseases rates from Chile and Cuba

Background: The prevalence of Helicobacter pylori-related diseases varies geographically and it is partially determined by the virulence of the circulating strains. Cuba and Chile exhibit different gastric cancer rates, on despite of very similar H. pylori infection rates. We determined if differences in the pathogenic potential of H. pylori isolates from Chile and Cuba could explain the disease outcome in each population. Methods: H. pylori isolates from 78 Chilean and 71 Cuban patients were analyzed using PCR for the presence of cagA, babA2, vacA alleles and the pattern of EPIYA motifs. Results: cagA was detected in 94.9 % of Chilean and 64.7 % of Cuban isolates (P < 0.001) and was significantly associated with duodenal ulcer (DU) in Cuba (P < 0.01) but not in Chile. The presence of cagA with multiple EPIYA-C motifs was 18.2 % higher in Chile than in Cuba (P < 0.05). Also, an association was observed between GU (P ≤ 0.05) and premalignant lesions (P < 0.001) with the multiple EPIYA-C motif status of the strains in Chile, but not in Cuba. The prevalence of vacA s2m2 genotype was predominant in Chile (66.7 %), while in Cuba was prevalent the s1m1 genotype (56.8 %); and the last one was significantly associated with the presence of DU in Cuban patients. Conclusions: The cagA status and the EPIYA pattern found in Chilean and Cuban H. pylori clinical isolates partially explain the differences in disease prevalence between both countries. The high proportion of vacA s2m2 genotype in Chile was an unexpected result, needing further studies.


Introduction
Helicobacter pylori (H. pylori) is a Gram-negative spiral-shaped bacterium that colonizes the human stomach. In this sense, colonization constitutes an established risk factor in the pathogenesis of functional dyspepsia, peptic ulceration, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma 1,2 . The prevalence of these pathologies varies considerably throughout the world. Among the multiple factors responsible of this dissimilar prevalence, H. pylori strain heterogeneity seems to be a crucial factor 1,3,4 . Although many virulence factors have been reported to H. pylori, only few of them have been linked with disease, and particularly with gastric cancer 4 . CagA protein is the most recognized H. pylori virulence marker 3,5 . The cagA-positive strains are associated with increased risk of peptic ulceration, atrophic gastritis and gastric adenocarcinoma, 6 . The CagA protein is injected into to the eukaryotic cells where is phosphorylated and binds to SHP-2 phosphatase. CagA-SHP-2 complexes disrupt signal transduction pathways developing cell atrophy 5,6 . Phosphorylation of CagA occurs in the C-terminal region within the EPIYA motifs, which are classified as A, B, C and D 7 . The EPIYA-A and B motifs appear in almost all Western and East Asian H. pylori strains while EPIYA-C, in a single or multiple repeat, appears in Western isolates and EPIYA-D is restricted to Eastern strains. The EPIYA-D motif exhibits the greatest levels of CagA phosphorylation and SHP-2 binding follow by strains carrying multiple EPIYA-C motifs 8,9 . Consequently, both genotypes have been associated with higher GC rates in several studies 9, 10,11 . Therefore, detection of the EPIYA patterns has become an important tool to determine the carcinogenic potential of H. pylori isolates. Another major H. pylori virulence marker is the vacuolating cytotoxin (VacA), which causes induction of cytoplasmatic vacuoles, mitochondrial damage and inhibition of T cell activation 3,12 . Polymorphisms among vacA gene regions result in different levels of vacuolation and damage. The major variations are presented within signal (s1 and s2), intermediate (i1 and i2) and middle (m1 and m2) regions. It has been determined that s region is related with vacuolation while the m region determines the cell specificity 13,14 . In general, the vacA s1m1 genotype exhibits the highest vacuolation activity, s1m2 a moderate activity and s2m2 and s2m1 combinations are non-toxic 14 .
The vacA s1 and m1 alleles have been associated with H. pylori-related diseases, including GC 15,16,17 . Finally, the other well-establish virulence marker is the blood group binding antigen (BabA) 3 . The attachment of H. pylori to gastric epithelial cells facilitates colonization and delivery of virulence factors such as CagA and VacA into eukaryotic cells 12,17 . BabA is encoded by the babA2 gene and is important for H. pylori adherence. There are two distinct babA alleles (babA1 and babA2) and one highly homologous gene, babB, but only babA2 is functionally active. The sequence of babA1 and babA2 only differed in a 10 bp deletion in the signal peptide sequence of babA1 that eliminates its translational initiation codon 18 . Several studies have suggested an association between babA2-positive strains and increased risk of developing severe gastric diseases in Western countries 19,20 ; however this association does not appear in others studies 21 . Nevertheless, the detection of this virulence factor remained important to characterize pathogenicity of H. pylori strains. Gastric cancer is a world health burden, ranging as the second cause of cancer death worldwide 22 . However, the rates of incidence and mortality differ markedly from one region to another all over the world 22,23 , even when H. pylori infection prevalence is similar 24,25 . That is the case for the different mortality rates reported for Chile and Cuba; the former is 14/100 000 26 and the latter 7.5/100 000 27 , exhibiting similar estimated H. pylori infection rates, of 74.5 and 73 %, respectively 28,29 . We hypothesized that this difference may be mainly attributed to differences in the pathogenic potential of circulating H. pylori strains. Thus, this study aimed to compare the prevalence of H. pylori virulence factors in two specific populations, from two Latin American countries, and their association with H. pylori-association diseases finding Materials and Methods: Patients and samples Gastric biopsy samples were obtained from 200 consecutively H. pylori-infected patients of both genders that undergoing routine upper gastrointestinal endoscopy at Medical and Surgical Research Hospitals, Havana city, Cuba and Regional Hospital of Talca, Maule, Chile. The mean age of the one hundred Cuban patients was 49.6 (range, 18 to 79) years and that of the one hundred Chilean patients was 52.0 (range, 18 to 82). All Chilean and Cuban patients were from the Region del Maule and from The Havana province, respectively. We included subjects greater than 18 years old with dyspeptic symptoms. Patients with had received previous treatment for H. pylori infection, antibiotics, acidreducing drugs, such as H2-receptor antagonists, acid pump inhibitors, nonsteroidal anti-inflammatory drugs, or bismuth compounds in the last four weeks were excluded. Endoscopic observation and histological confirmations were used to determine patient pathologies as duodenal ulcer (DU), gastric ulcer (GU), gastritis (G) and premalignant lesion (PL). Peptic ulcer and gastric cancer were identified using endoscopy. Gastritis was diagnosed in the absence of peptic ulcer or gastric malignancy. Experienced endoscopists of each country collected four gastric biopsy specimens during endoscopy session: three two samples from the antrum, approximately 2-3 cm from the pyloric ring, and one sample from the greater curvature of the corpus. The antrum specimens were used for H. pylori culture, rapid urease test and histological examination. The corpus specimen was used for histological examination. The biopsies culture for H. pylori isolation where make in each country, and the strains isolated in Cuba were translated to Catholic University of Maule for DNA isolation and PCR analysis. The biopsy materials were fixed in 10% buffered formalin for 24 h and then embedded in paraffin in each country. The paraffin block from both countries were stained and examined in a blind test by the same experienced pathologist at Regional Hospital of Talca, Maule, Chile in order to minimize potential bias. Histology Gastric biopsy specimens for histopathology were stained using hematoxylin and eosin (stain for the detection of H. pylori), and the histological analysis of the gastric mucosa was performed according to the Updated Sydney System 30 . In addition, on the basis of the topographic locations (antrum and corpus), the gastritis stage (the severity and topography of atrophy) was assessed according to the Operative Link on Gastritis Assessment (OLGA) system 31 .

H. pylori Culture and Genotyping
Antral biopsy specimens were obtained for the isolation of H. pylori using standard culture methods as previously described 18,29 . H. pylori identified based on their typical morphology, gram staining, and positive reaction for urease, oxidase and catalase test. The reference strains J99 and CCUG17874, were kindly provided by Professor Francis Mégraud from Pellegrin Hospital, Bourdeaux, France and Professor Ann-Mary Svennerholma from Gothenburg University, Sweden. Genomic DNA was extracted and purified from each H. pylori isolate. PCR was used to detect the babA, cagA, vacA genes and the cagA EPIYA motifs. Primer pairs for babA, cagA and vacA are presented in Table I. The number and type of EPIYA motifs were determined as previously reported [32]. PCR were performed in a 25µL reaction containing 1.25 U Taq polymerase (Roche, Germany), 50 ng of genomic DNA and 0.6 mM of each primer. PCR products were visualized by agarose gel electrophoresis. Reference strains with previously characterized genotypes were used as positive controls. Data analysis Variables such as gender, age and the presence of each candidate gene were evaluated. The univariate association between each genotype and the clinical presentations were quantified by the Chi-square (χ2) or Fisher's exact (FE) tests. Independent samples t and one way ANOVA tests were used to compare quantity variables. A P value of less than 0.05 was accepted as statistically significant. The SPSS statistical software package version 16.0 was used for all statistical analyses. Mixed vacA genotypes were excluded from comparisons. Ethical clearance: The study protocol was approved by the ethics and research committees of Cuban a Chilean Hospitals, The National Centre for Scientific Research, Havana, Cuba and Catholic University of Maule, Chile. All patients gave an informed consent for endoscopy and participation in the study.

Results
In the present study, 71 Cuban and 78 Chilean patients were H. pylori positive. From endoscopic observations: 53.5 % of Cuban and 62 % of Chilean patients were diagnosed with gastritis (G), 14.1 % of Cuban and 38 % of Chilean patients had gastric ulcer (GU) and 32.4 % of Cuban patient had duodenal ulcer (DU). Histological analysis revealed intestinal metaplasia and dysplasia in 39.8 % and 4 % of Chilean patients, respectively. Statistical differences between these populations were found for the appearance of GU, DU and premalignant lesions, in all cases with P < 0.001.

Prevalence of cagA-positive strains among Cuban and Chilean populations
The cagA gene was present in 64.7% (46/71) and 94.9% (74/78) of the H. pylori strains from Cuba and Chile (P < 0.001), respectively (Table II). Furthermore, a significant association was found between cagA status and DU (P < 0.01) for Cuban patients. No association was found between the presence of cagA and any pathology in the Chilean group, due to the high cagA positivity in these isolates.

CagA phosphorilation motifs
All the cagA-positive isolates were evaluated to determine their EPIYA motifs pattern. We turned our attention mainly in the number of EPIYA-C motifs. Among Cuban isolates, the number of EPIYA-C motifs ranged from 0 to 3; there were 33 with 1 (71.7 %), 12 with 2 (26.1 %) and 2 with 3 (4.3 %), and thus 30.4 % (14/46) of cagA-positive strains harbored 2 or more EPIYA-C motifs. In the case of Chile, the number of EPIYA-C motifs ranged also from 0 to 3; 34 with 1 (35.4 %), 26 with 2 (35.1 %) and 10 with 3 (13.5 %), and thus 48.6 % (36/74) carried 2 or more EPIYA-C motifs. This was a higher proportion than the founded in Cuban isolates (P < 0.05). No association was found between the presence of more than two EPIYA-C motifs and any pathology present in Cuban patients. On the contrary, it was found a correlation between this status and GU (P ≤ 0.05) and a more strong association with premalignant lesions (P < 0.001), which included dysplasia and intestinal metaplasia, in the Chilean group (Table III).   (Table II). Significant correlation was only found between the presence of vacA s1 and vacA s1m1 genotypes and DU (P < 0 .05) in Cuba (Table II), due to the high prevalence of s2, m2 and s2m2 genotypes found in Chilean isolates. babA2 genotype In both countries, the presence of babA2-positive strains was high (Table II); in Chile 97.4 % (76/78) of the strains carried this allele, while 88.7 % (63/71) of Cuban isolates were babA2 positive (P < 0 .05). No significant association was found for the presence of babA2 gene and any pathology observed in both populations (Table III). ND, not detected. a Nine Chilean isolates were not analyzed due to the presence of mixed genotypes. b These genotypes were not detected in any Cuban isolate. ND, not detected. G, Gastritis; GU, Gastric ulcer; PL, Premalignant lesions; DU, Duodenal ulcer. a. Patients with any stage of gastritis (histopathological analysis) were included in these group. b. Data include patients with endoscopic diagnosis of GU, but in some of them the subsequent histopathological analysis also evidenced some degree of PL.

Combinations of virulence factors and associations with pathologies
It was observed a significant association between cagA and babA2 genotypes with the presence of vacA s1 and m1 alleles (P < 0.001) in Cuba. Also in this country, a correlation between the cagA and babA2 status of the strains appeared, but in a lower degree (P < 0.05). No correlations between the combinations of virulence factors were observed in Chile, due to the fact that almost all isolates were cagA-and babA2-positive and a great proportion of them were also vacA s2m2. Strains carrying all three virulence factors only had significant association with DU in Cuba (P < 0.005)

Discussion
The incidence of severe gastroduodenal diseases varies between geographical areas. These variations have been attributed to environmental conditions, host immunological factors, and differences of specific virulence markers in the H. pylori circulating strains 2, 3 . More recently, there is a growing interest to study specific virulence factors in circulating strains of countries and/or populations with different GC rates 6,7,13,29,30 . The gastric cancer rate and the appearance of gastroduodenal pathologies in Chile and Cuba exhibit large differences; the first one possesses one of the highest GC death rate in the Latin American region and also in the world (20 / 100 000) 18 and the second has one of the lowest GC death rate in Latin American (7.5 / 100 000) 19 .
In this study, we found that cagA gene was 30 % more prevalent in Chilean than in Cuban isolates. Furthermore, almost half of Chilean cagA-positive strains had two or more EPIYA-C motif, while in Cuba only 30.4 % of the isolates had these patterns (P < 0.05). Taking these results together and the potential of cagA-associated pathogenicity, they may represent important contributors to the differences in GC rates seen between these populations. Both, cagA status and the number of EPIYA-C motifs have been linked with cancer prevalence in a number of populations 7,30 . Additionally, the percentage of cagA positivity (64.7 %) in Cuban isolates is closer to the values for Western countries; however the Chilean percentage (94.9 %) resembles the values for East Asia 3, 31, 32 . Despite of the differences found, isolates from both countries presented the Western types of cagA and consequently the EPIYA-D motif was not found in any isolate studied. Surprisingly, as was previously reported 39 the group of Chilean strains analyzed in this study showed an inverted situation of the expected vacA genotypes for regions in Latin American of high GC rates 13 ; the less pathogenic combination s2m2 was predominant in the Chilean population studied. Even so, a recent study has shown a high proportion of this genotype in a region with populations having elevated GC rates 34 . However, due to the fact that this phenomenon is new, we are encouraged to perform new studies to repeat and extend our investigation in the near future. In Cuban isolates, the more pathogenic vacA s1m1 was predominant, with a prevalence slightly higher than that found in Western countries 35,36 , lower than the reported for Eastern countries 36 and in the middle range of Latin American countries 13,36 .
We looked within the Chilean and Cuban isolates for associations between virulence factors and gastric diseases in the studied groups. In Chile the presence of cagA in the strains did not associate with any pathology, due to almost all strains were cagApositive, a situation also observed in several studies from countries and/or populations with the higher GC incidences 3 . We only found an association between the cagA positivity and DU in Cuba, this result is inside to the tendency of positive associations found in previous studies of Cuban dyspeptic patients 21,27 , and resembles others from Western populations; all, with low rates of premalignant and malignant lesions 16, 38 . In the case of the presence of two or more EPIYA-C motifs and its correlation with pathologies, we found in Chile a significant association with GU and premalignant lesions, with a stronger association in the second case. This last phenomenon has been reported in similar studies of Latin American countries, like Colombia and Brazil 7, 30 ; in which, other premalignant and also malignant lesions were associated with the multiplicity of the EPIYA-C motif. It seems to be, as it has been reported already, that the presence of more pathogenic patterns of EPIYA motifs; either, the Western and the Eastern types, leads to the appearance of premalignant and malignant pathologies rather than DU 29,30,37 . Furthermore, the recent Brazilian study, which included a high number of H. pylori strains from patients with GC and DU, showed a strong association between the presence of two or more EPIYA-C motifs and GC but not with DU 30 . All these evidences together and our results reinforce the theory that there are some bacterial strains that may be more likely to cause DU and others that may promote the development of GC 40 . The results for vacA alleles association with diseases were also assessed; the Chilean group showed a very high number of strains with vacA s2 genotype (75.6 %), the determining allele for the lowest VacA citotoxic activity and therefore virulence 3 . Consequently, the reported association between the more pathogenic vacA s1, m1 and s1m1 genotypes of H. pylori strains in countries of high GC rates in Latin American region did not appear 13 . Recently, a high proportion of vacA s2m2 genotype also appeared in a country with populations of high GC incidence 34 . Our results suggest that the above statement should be questioned. Cuban isolates showed a typical, and even higher, prevalence of s1m1 genotypes than the reported for Western strains 35,36 . This more virulent genotype only correlated with the presence of DU, a finding observed in a meta-analysis study for the Latin American region 13 . For babA2, no significant associations were found between the presence of this genotype and any disease presented in both populations due to the high prevalence of this genotype in the two countries. Previous reports have shown a clustering of active virulence factors within H. pylori strains, for example associations between cagA-positive, vacA s1 and babA2-positive genotypes 16, 29 . In agreement with the findings presented in those reports, we found a significant correlation between all these genotypes in Cuban isolates. Additionally, the strains carrying the different combinations of the more pathogenic alleles appeared associated with DU, a result that support the theory that the clustering of virulence factors promotes the cell damage and thus the pathogenicity of H. pylori 16 . As it was mentioned before, Chilean isolates shown a new clustering of the studied virulence factors, in which the more regular pathogenic association of cagA-and babA2positive genotypes was present, but in combination with the less virulent vacA s2m2 instead of the vacA s1m1 genotype. This finding is new, but recently a high proportion of strains carrying together the cagA gene and vacA s2m2 alleles were reported in an Alaskan study, a region with populations of high GC incidence 41 as Chile. Summarizing, in this study we could evidence a high prevalence of virulent strains in both populations studied in terms of the well-established virulent markers of H. pylori and also that isolates are more related to strains from Western countries than to strains from Asian countries. However, there are significant differences between the appearance and combination of these virulent markers. Cuban strains exhibited a relatively high proportion of cagA-positive strains, although high percentage of them possessed only one EPIYA-C motif. On the contrary, almost all Chilean strains were cagA-positive and almost half of them presented more than two EPIYA-C motifs. The above mentioned results in the cagA status from Chile and Cuba appear to be a good partial explanation to the differences in disease prevalence between both countries. Interesting, in the case of vacA s and m alleles, the group of Chilean strains showed an opposite situation of the expected vacA genotypes for regions of high GC rates, a finding that will be study in deep. Also, we will need to include the dupA gene status and the H. pylori related host immune response in future studies.