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VacA and CagA Status as Biomarker of Two Opposite End Outcomes of Helicobacter pylori Infection (Gastric Cancer and Duodenal Ulcer) in a Moroccan Population

  • Mounia El Khadir,

    Affiliations Laboratoire de Microbiologie et de Biologie Moléculaire, Equipe Micro-organismes, Génomique et Facteurs Oncogènes, Faculté de Médecine et de Pharmacie de Fès (FMPF), Université Sidi Mohammed Ben Abdellah (USMBA), Fez, Morocco, Laboratoire de Biotechnologie, Faculté des Sciences Dhar El Mehraz, USMBA, Fez, Morocco

  • Samia Alaoui Boukhris,

    Affiliation Laboratoire de Microbiologie et de Biologie Moléculaire, Equipe Micro-organismes, Génomique et Facteurs Oncogènes, Faculté de Médecine et de Pharmacie de Fès (FMPF), Université Sidi Mohammed Ben Abdellah (USMBA), Fez, Morocco

  • Dafr-Allah Benajah,

    Affiliations Service d’Hépato Gastro-entérologie, CHU Hassan II de Fès, Equipe Maladies de l’Appareil Digestif, FMPF, Fez, Morocco, Laboratoire de Pathologie Humaine, Biomédecine et Environnement, FMPF, USMBA, Fez, Morocco

  • Karima El Rhazi,

    Affiliation Laboratoire d’Epidémiologie et de Recherche Clinique, FMPF, USMBA, Fez, Morocco

  • Sidi Adil Ibrahimi,

    Affiliations Service d’Hépato Gastro-entérologie, CHU Hassan II de Fès, Equipe Maladies de l’Appareil Digestif, FMPF, Fez, Morocco, Laboratoire de Pathologie Humaine, Biomédecine et Environnement, FMPF, USMBA, Fez, Morocco

  • Mohamed El Abkari,

    Affiliations Service d’Hépato Gastro-entérologie, CHU Hassan II de Fès, Equipe Maladies de l’Appareil Digestif, FMPF, Fez, Morocco, Laboratoire de Pathologie Humaine, Biomédecine et Environnement, FMPF, USMBA, Fez, Morocco

  • Taoufiq Harmouch,

    Affiliation Service d’Anatomie Pathologique CHU Hassan II, Fez, Morocco

  • Chakib Nejjari,

    Affiliation Laboratoire d’Epidémiologie et de Recherche Clinique, FMPF, USMBA, Fez, Morocco

  • Mustapha Mahmoud,

    Affiliation Service de Bactériologie CHU Hassan II, Fez, Morocco

  • Mohamed Benlemlih,

    Affiliation Laboratoire de Biotechnologie, Faculté des Sciences Dhar El Mehraz, USMBA, Fez, Morocco

  • Bahia Bennani

    bahia.bennani@usmba.ac.ma

    Affiliations Laboratoire de Microbiologie et de Biologie Moléculaire, Equipe Micro-organismes, Génomique et Facteurs Oncogènes, Faculté de Médecine et de Pharmacie de Fès (FMPF), Université Sidi Mohammed Ben Abdellah (USMBA), Fez, Morocco, Laboratoire de Pathologie Humaine, Biomédecine et Environnement, FMPF, USMBA, Fez, Morocco

VacA and CagA Status as Biomarker of Two Opposite End Outcomes of Helicobacter pylori Infection (Gastric Cancer and Duodenal Ulcer) in a Moroccan Population

  • Mounia El Khadir, 
  • Samia Alaoui Boukhris, 
  • Dafr-Allah Benajah, 
  • Karima El Rhazi, 
  • Sidi Adil Ibrahimi, 
  • Mohamed El Abkari, 
  • Taoufiq Harmouch, 
  • Chakib Nejjari, 
  • Mustapha Mahmoud, 
  • Mohamed Benlemlih
PLOS
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Abstract

Helicobacter pylori (H. pylori) infection induces inflammation of the gastric mucosa, which may progress to precancerous lesions leading to gastric cancer. Pathological determinism is associated to some virulence genes of the bacterium, notably the vacA and cagA genes. The present study aimed to determine the H. pylori genotypes distribution and their association with sex, age and gastric diseases in a Moroccan population. Gastric biopsy was taken from 1079 consenting patients. The specimens were processed by PCR to identify H. pylori and to determine the genotypic profile by PCR characterizing vacA s, vacA m and vacA i regions directly from biopsies H. pylori positives. VacA genotyping revealed the predominance of vacA m2 (53.2%), vacA s2 (52.9%) and vacA i2 (52%). The most virulent vacA alleles (s1, i1 and m1) are more predominant in men (47.3%, 41.9% and 46.1% respectively) than in women (38.3%, 33.3% and 37% respectively). However, the association between vacA genotypes and age did not reach a statistical significant value. Logistic regression analysis results show that vacA i1m1 and vacA i1m2 genotypes were strongly associated with the risk of GC, the Odds Ratio (95% confidence interval) was 29.73 [5.08–173.73] and 9.17 [2.06–40.82] respectively, while vacAs1/cagA+ seems to be a risk factor for DU since it is inversely associated with GC (OR was 0.13 [0.02–0.75]. The results of this study suggest that vacA i1 genotype independently to vacAm status may be of a clinical usefulness and will help to identify patients at a high risk of GC development.

Introduction

Gastric cancer is the third common cause of cancer mortality in the world. Multiple epidemiological studies have documented an increased incidence of gastric cancer with increased prevalence of H. pylori infection. In Morocco, as in other African and South East Asian countries, there is a paradox between the high prevalence of H. pylori infection (59.7%) [1], and low incidence of gastric cancer (5.6%) [2]. The difference of the geographic distribution of H. pylori infection and gastric cancer incidence suggests the presence of the determining factors which could influence the interaction between pathogen and host. Those factors include: human genetic polymorphism, environmental influences and the high genomic diversity of H. pylori. Overall, the genetic diversity of the bacteria occurs mainly in the virulence factors such as the cytotoxin-associated gene (cagA) and the vacuolating cytotoxin (vacA). CagA protein is a 120- to 140-kDa protein that is translocated into host cells by the type IV cag secretion system after bacterial attachment, altering thereafter the cell-signaling mechanisms in gastric cells. VacA protein is a cytotoxin inducing vacuolation of the epithelial cell; the gene is present in all strains but only some of them have a vacuolating activity. This variation is attributed to variations in vacA gene structures within the three regions: the signal sequence region (s-region) (s1 or s2), mid-region (m-region) (m1 or m2) and the intermediate-region (i-region) (i1, i2 or i3) [35]. The polymorphism of these two virulent factors has been the subject of numerous investigations [69].

In Morocco, we previously studied the status of cagA and the genotypes of vacA-s, -m regions. The preliminary results show the predominance of vacA s2m2 which was significantly associated with gastritis while the vacA s1m1 was significantly associated with peptic ulcer diseases[10]. Although, there was no significant difference in the prevalence of these two genotypes in gastric cancer [10]. When the H. pylori genotypes were correlated to histological lesions, significant associations between gastric cancer, cagA+ and vacA m1 genotypes were obtained in older patients [1]. Indeed, these preliminary results need to be confirmed in larger sampling to better characterize isolates that may lead to severe diseases and by exploring another vacA intermediate region (vacA i) described as strong marker of H. pylori associated disease among the vacA alleles [5]. To the best of our knowledge, this is the first study reported in Morocco and in the North Africa that aimed to evaluate the genetic polymorphism among H. pylori strains isolated from Moroccan patients on the basis of their vacA genotypes (s, m, and i) and cagA status. It aimed also to find the association of the vacA genotypes with sex, age and gastric diseases.

Materials and Methods

Patients and sampling

This study was conducted between May 2009 and January 2015. The biopsies of 801 patients, previously characterized on the basis of vacA-s, -m regions and cagA status were used to determine vacA i genotype and 278 patients were prospectively recruited and added to the existing cases to be analyzed.The total of consenting patients aged 15 years or more, who were attending the gastroenterology department of Hospital University (CHU) Hassan II of Fez, Morocco, and who had undergone endoscopy for the diagnosis of abdominal pain or discomfort were included in this study. However, patients aged less than 15 years or who were on medications (antibiotics, proton pump inhibitors) for the last 3 months and also pregnant or nursing women were excluded. The recruited patients have an average age of 49.30 ± 16.29 years, ranging from 15 to 99 years, and had a personal interview, where they were asked about individual characteristics. A total of three biopsies were collected from each patient during the endoscopy: one biopsy from the antrum which was directly used for molecular detection of H. pylori, cagA status and vacA genotyping by polymerase chain reaction (PCR). The other biopsies (one from antrum and the other from corpus) were examined independently by an experimented anatomopathologists.

All participants were informed about the study objectives, methods, confidentiality, and potential outcomes and they provided written informed consent for their participation. Also, parental consent was obtained on the behalf of the participants under the age of 18. In the case of illiterate or semi-literate patients, the written consent was read to them by the interviewer.This study was approved by the Institutional Review Board of the Hassan II University Hospital of Fez, Morocco.

DNA extraction

Using the protocol previously described (10), DNA was extracted from the gastric antrum biopsy specimens and stored at -20°C until molecular analysis.

Polymerase chain reaction (PCR)

H. pylori was detected in biopsies by PCR using glmM primers as described previously [11]. Positive Samples were subjected to multiplex PCR in order to determine the cagA status, vacA s and vacA m subtypes [12,13] and also to simple PCR to determine the vacA i polymorphisms using specific primers as previously described [5].

Samples with non identified genotypes (vacA s and m alleles) in multiplex PCR were subjected to PCR reactions using the same primer sets but in single reactions. Likewise, all non amplified vacA i cases were subjected to another PCR using the sense primer performed by Ferreira [14] and two antisense primers designed by Rhead [5].

The cagA status has been verified using two primer sets as previously described [6] in order to increase the sensitivity of detection. In fact, cagA was considered positive if it shows positive results with one of the three primers pairs.

Negative and positive controls were used for each reaction.

Statistical analysis

The statistical analysis was done using SPSS (Statistical product and services solutions, version 20, SPSS Inc. Chicago, Illinois, USA) software. It consisted primarily to describe the study population, for this, the results were presented as mean ± standard deviation for quantitative variables and number (percentage) for qualitative variables. Then, an univariate analysis was performed to establish all associations between clinical diseases, age, gender, H. pylori infection, and H. pylori genotypes (Only samples with single-strain infection (identified on the basis of vacuolating cytotoxin gene (vacA alleles) and complete vacA alleles were considered), Chi-square or Fisher’s exact tests were applied to establish all statistically significant associations; A p value < 0.05 was considered as significant. The multivariate analysis was performed to highlight the most incriminated factors in the occurrence of GC using simple logistic regression analysis. All variables with p ≤ 0.20 were included in the initial model. The final model was obtained using a stepwise elimination method to identify potential independent factor(s) associated with GC. The results were expressed as odds ratio (OR), 95% confidence intervals (CIs) and p-values.

Results

A total of 1079 patients from urban and rural areas of north center of Morocco and consulting for abdominal pain or discomfort were included in this study. They were 519 women (48.1%) and 560 men (51.9%). The clinical exam shows that 738 patients were with chronic gastritis, 260 with peptic ulcer disease (PUD), 119 with gastric ulcer [GU], 133 duodenal ulcers [DU], 8 with GU and DU and 81 patients were with gastric cancer (GC) [adenocarcinoma or gastric lymphoma]. All GC cases were confirmed by histological exam.

PCR results show that H. pylori infection rate in the studied population is 59.4% (641/1079). VacA and cagA genes were amplified in 99.53% (638/641) and 61.2% (392/641) cases respectively. The vacA s-, i- and m- regions were amplified in 99.06% (635/641), 90.01% (577/641) and 95% (609/641) of the cases respectively. The determined rate of vacA m2, s2 and i2 alleles was 53.2% (324/609), 52.9% (336/635) and 52% (300/577) respectively “Table 1”.

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Table 1. Correlation between vacA mosaicism and cagA status of H.pylori strains and their distribution according to the gender and to the age.

https://doi.org/10.1371/journal.pone.0170616.t001

The distribution of the intermediate region of vacA among the s and m regions was studied in this series and reveals that 89% and 89.4% of vacA s1m1 and vacA s2m2 strains were vacA i1 and vacA i2 respectively. Also, our results show that 70.4% of the vacA s1m2 strains were vacA i2Table 2”.

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Table 2. Association between H. pylori vacA alleles in studied isolates from Moroccan patients with single-strains infections.

https://doi.org/10.1371/journal.pone.0170616.t002

Multiple infections (presence of the 2 alleles of the same vacA region (s, m or i)) and incomplete vacA (lack of the two alleles of the same vacA region) were detected in 14.2% (91) and 11.1% (71) respectively of the 641 subjects evaluated. Excluding these 2 profiles (incomplete vacA and multiple infection), vacA genotype distribution revealed a large predominance of vacA s2i2m2 with rates of 44.3% (211/476) while vacA s1i1m1, vacA s1i2m2, vacA s2i1m2 and vacA s2i1m1 were detected in 28.8% (137/476), 8% (38/476), 5.3% (25/476) and 4.6% (22/476) respectively. The rate of each genotype is reported in Table 1.

The correlation between cagA and vacA mosaic genotypes in only samples with simple infection and with complete vacA genotypes (1: vacA s1i1m1; 2: vacA s2i2m2; 3: Other less prevalent genotypes of vacA) was studied. The results show a significant association between vacA s1i1m1 and cagA+ and between vacA s2i2m2 and cagA- (p < 0.001) “Table 1”.

Correlation between age and sex with vacA genotypes and cagA status was also tested in two groups: the group 1 includes young patients aged less than 50 years while the group 2 includes those with 50 years and older. The statistical analysis revealed that the most virulent vacA alleles (s1, i1 and m1) are more predominant in men (47.3%, 41.9% and 46.1% respectively) than in women (38.3%, 33.3% and 37% respectively) (p = 0.005, 0.020 and 0.009 respectively), while cagA+ is more frequent in age group 1 (67.3%) than in age group 2 (54.5%) (p = 0.001) “Table 1”. However, the association between vacA genotypes and age did not reach a statistical significant value.

The distribution of the pathologies according to the patients gender, age, H. pylori infection, and H. pylori genotypes was also studied. The results show that GC, GU, and DU were predominant in men (63%, 69.7% and 73.7% cases respectively) while a high prevalence of gastritis was found among women (56.5%). According to the PCR results, H. pylori prevalence is high in all gastric disease with predominance in patients with GC (92.6%). The association of each of the three vacA regions with clinical outcomes shows that vacA s2, vacA i2 and vacA m2 were predominant in gastritis cases (59.5%, 59.9% and 58.7.5% respectively). VacA s1 was present in 57.4% of patients with DU (p <0.001), vacA i1 and vacA m1 were present in GC patients with rates of 73.3% and 65.7% respectively (p <0.001) (Table 3).

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Table 3. Distribution of Demographic and risk factors of the gastro duodenal diseases.

https://doi.org/10.1371/journal.pone.0170616.t003

To find out a possible association between vacA alleles (s, m and i) combination and the studied diseases, only the most predominant genotypes of the simple infections (vacA s2i2m2, vacA s1i1m1 and the other combination allelic of vacA) were considered in the statistical analysis and the results show that the rate of vacA s1i1m1 in GC patients was higher than the other vacA combinations. Also, a high prevalence of vacA s2i2m2 in gastritis patients was noted. While according to cagA gene status, cagA+ was higher in DU cases (78.7%) than the other pathologies (Table 3).

To evaluate the effect of each genotype and demographic characteristics on the occurrence of GC, we have considered the DU cases as reference control. Initially, univariate analysis was done and is shown in Table 4 and Table 5.

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Table 4. Distribution of demographic and vacA alleles (single and associated) of H. pylori in duodenal ulcers and gastric cancer cases.

https://doi.org/10.1371/journal.pone.0170616.t004

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Table 5. Distribution of vacA and cagA combination of H. pylori in duodenal ulcers and gastric cancer cases.

https://doi.org/10.1371/journal.pone.0170616.t005

Thereafter, logistic regression analysis was performed and the results show that vacA i1m1 and vacA i1m2 genotypes were strongly associated with the risk of GC, the Odds Ratio (95% confidence interval) was 29.73 [5.08–173.73]) and 9.17 [2.06–40.82] respectively while vacAs1/cagA+ seem to be more associated to DU since it’s less present in GC cases, (OR: 0.13 [0.02–0.75] when compared with DU group “Table 6”.

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Table 6. Risk for GC in relation to vacAs/cagA combination and i- and m-region of vacA gene in a simple logistic regression analysis.

https://doi.org/10.1371/journal.pone.0170616.t006

Discussion

The H. pylori infection is strongly associated with peptic ulcer disease (PUD), gastric carcinoma, and gastric mucosa-associated lymphoid tissue (MALT) [15]. This bacterium is known for its high genetic diversity that occurs mainly in the bacterium virulence factors as the cytotoxin-associated gene (cagA) and the vacuolating cytotoxin (vacA) gene. The variability that affects these two genes may be useful to better understand the differences in the pathogenesis and the role of each genotype in the occurrence of the pathology. In fact, polymorphisms within vacA have been analyzed in several previous studies, but they have primarily focused on the signal region (s region) and the mid-region (m region), both of which have been used to predict the clinical outcome of infected patients. In Morocco, we have previously studied the polymorphism of vacA in those two regions (signal sequences (s) and mid-region (m)) and established their correlation to clinical outcomes and also to histological lesions[1,10]. Of note, in this study, we confirmed that the studied population was predominantly infected by the less virulent H. pylori strains (vacA s2m2) and we confirmed a significant association between vacA m1 and GC. However, it will be for interest to study vacA i alone and in combination to cagA status and to s and m vacA region in a large sampling to better characterize isolates that may lead to severe diseases, mainly the GC.

H. pylori is known to colonize more than half of the world’s population but the infection rate varies across geographic regions. Its prevalence differs by age, race, and socioeconomic level. In this series, H. pylori infection prevalence was 59.4%, this rate is comparable to our previous study (59.7%) [1] and to other developing countries but it is higher than in developed ones [16].

It is well known that almost all H. pylori strains contain the vacA gene, likewise in the current study we found it in 99.06% of cases. This rate is higher than those reported from Ethiopia (90%) and Netherlands (93%) [9,17]. The failure on vacA gene detection in 0.94% cases can be due to the genomic diversity of the bacteria which can be due to point mutations in the conserved genes. In fact, the multiple infection contribute to the genetic diversity by the extensive inter strain gene transfer and recombination that include large insertions or deletions and chromosomal rearrangements [18]. In this study, the presence of multiple H. pylori strains in a single biopsy specimen was found in 14.2% of cases. This was similar to the results obtained in Senegal (14%), Netherlands (11%), North of America (11%), Brazil (13%), India (15.9%) and Spain (19.2%) [7,1922], and it was lower compared to the reported results in Tunisia (31.4%) Chile (32%) and Portugal (37.3%) [2325]. The presence of multiple infections in developed countries as in the developing countries is totally inconsistent with the idea that mixed infections predominate only in the developing countries by the fact that it can be related to the high prevalence of H. pylori [26].

The polymorphism in the vacA gene sequence has been studied in three variable regions, and the vacA genotyping revealed the predominance of vacA s2, vacA i2 and vacA m2 alleles which were present in 52.9%, 52% and 53.2% of the cases respectively (Table 1). This distribution is almost similar to the follow-up study conducted on Spanish population [7,27] where these alleles (vacA s2, vacA i2 and vacA m2) were present in 53.6%, 55.7% and 52%, and to the epidemiological study conducted on Kenyan population [28], where the vacA s2, vacA i2 and vacA m2 alleles were found at 49%, 52% and 65%, respectively. However, it’s totally different from the profile reported in Bulgaria and Senegal, where vacA s1, vacA i1 and vacA m1 alleles were predominant [8,29]. The results are also different from those obtained in Vietnam and marked by the predominance of vacA s1, vacA i1 and vacA m2 alleles [30].Those results show a great diversity of the bacterium.

All possible combinations of the three vacA regions “Table 1” have been reported, showing a high genetic diversity in our H. pylori clinical isolates. Excluding the multiple infection and the incomplete vacA, vacA genotype distribution reveals a large predominance of vacA s2i2m2 with a rate of 44.3% (n = 211), while vacA s1i1m1, vacA s1i2m2, vacA s2i1m2 and vacA s2i1m1 were detected in 28.8% (n = 137), 8% (n = 38), 5.3% (n = 25) and 4.6% (n = 22) of cases respectively. Unfortunately, there is no data from the North African countries regarding the polymorphism of the three region of vacA. In our study, the determined distribution is almost similar to the one determined among Sicilian and Kenyan patients [28,31] and differs from the others reported among European, Senegalese, Iranian, Malaysian and the South Korean ones, where vacAs1i1m1 was the most predominant genotype [8,3234], while vacA s1i2m2 was the prevalent genotype in Iranian population [35]. This difference may explain the predominance of severe diseases in these regions. In effect, it is well established that the countries with high rates of severe diseases have strains carrying the vacA active forms [5]. This was confirmed by our moderate rate of vacA s1i1m1 strains in the studied population related to the lowest rate of the serious diseases like gastric cancer.

In the present study, the distribution of the intermediate region of vacA among the s and m regions reveals that 89% and 89.4% of vacA s1m1 and vacA s2m2 strains were vacA i1 and vacA i2 respectively (Table 2), which is similar to the results of alleles association in most studies [5,8,36]. Also, our results show that 29.62% of the vacA s1m2 strains were vacA i1, when it was 66.7% in Uruguayan, 92% in Chinese, 71.4% in U.S.African strains and 66.7% in East Asian [36,37]. This might be one hypothesis to explain the African enigma, since as it was approached by Rhead and Coll, strains of vacA s1i1m2 genotype are more virulent, and more associated with GC than the vacA s1i2m2 genotype [5].

The prevalence of H. pylori cagA+ (61.2%) confirm the data obtained in our previous study (59.6%)on smaller sampling [1].

Our study also demonstrates that vacA i1 was strongly associated with the vacA s1, vacA m1 and cagA+ genotypes. As well, vacA i2 was strongly associated with the vacA s2, vacA m2 and cagA- genotypes (p<0.001), suggesting that the polymorphism of vacA gene may affect the functional interaction with cagA [38]. Those results are in agreement with numerous data from Chinese, U.S. African, Uruguayan, Iranian, Iraqi, Jordanian and Turkish studies reporting that H. pylori strains containing the active vacA gene (s1i1m1) carry also cagA gene, and H. pylori strains that carry the inactive vacA gene (s2i2m2) lack the cagA gene [5,37,3941]; this clustering of active virulence factors within H. pylori strains classified H. pylori to be virulent or nonvirulent.

In this series, vacA genotypes were significantly associated to the patient’s gender. Effectively, the rate of vacA s1i1m1 was higher in men than in women while the rate of vacA s2i2m2 was higher in women than in men (p = 0.009). This finding confirms that men are more susceptible to develop severe disease than women; these results are confirmed in the present study in which GC, GU, and DU were predominantly present in men (63%, 69.7% and 73.7% cases respectively). These results confirmed those reported in Bulgarian patients, who have reported a high rate of strains containing the less virulent genotype (vacA s1i2m2) in women than in men [29].

In our results, cagA is significantly associated with age and is predominant in younger patients (67.3%) vs (54.5%) older ones (p = 0.001). And despite the absence of significant difference in vacA genotypes distribution according to the age, we noted that in contrary to vacA i1 allele, vacA s1 and vacA m1 are increased slightly in age group 1 than in age group 2 which is in disagreement with the work of Alarcón and his colleagues who report that the prevalence of cagA, vacA s1, vacA i1 and vacA m1 were more frequent in older patients than in younger [42]. This raises the hypothesis on the evolution of vacA H. pylori genes in our population towards virulent form, presumably, due to adaptation of the bacteria to the host immune responses and to the environmental change. In fact, Gangwer et al., supposed in 2010 that H. pylori cagA and vacA can evolved separately from the bacteria core genome [43].

The results show also that patients with severe diseases (GU, DU and GC) are commonly infected with vacA s1, vacA m1 and vacA i1 strains (except of patients with gastric cancer who are frequently infected with vacA s2, vacA m1 and vacA i1 alleles) in contrast to patients with gastritis who are commonly infected with vacA s2, vacA m2 and vacA i2 strains (Table 3). Similar results were found in Western populations, while in East Asian population, vacA s1, vacA m1 and vacA i1 alleles were common in both dyspeptic and non-dyspeptic patients. In this regard, the profile of our strains and their association to the pathologies are similar to those isolated in Western populations, since the active form of vacA is not so prevalent and is more frequently found in ulcer or gastric cancer patients from Western populations, conversely, in the Eastern populations, these genotypes has been detected in almost all H. pylori strains. This finding will be confirmed by the studying of cagA EPIYA motifs polymorphism.

Since gastritis can evolved towards GU which predispose to GC disease, and since DU and GC seem to be mutually exclusive outcomes of H. pylori infection [44], only this two pathologies were used in statistical analysis to determine their risk factors. In univariate analysis, vacA s2 allele (the least virulent) is significantly associated with gastric cancer (Table 4). Also high prevalent of this allele was reported in GC cases in our previous study [10]. Likewise, the less virulent genotypes (vacA s2, vacA m2 and vacA s2m2) were reported in patients with MALT lymphoma among Mexican, Germany and French populations respectively [4547]. Those results are in disagreement with several previous studies that report a high rate of the virulent alleles (vacA s1, vacA m1, and vacA i1) in patient with sever diseases [4850]. Indeed, vacA s2 genotype encodes a short extension of the N-terminal peptide on the mature protein, which blocks the vacuolating activity [51]. So, the occurrence of vacA s2 in GC lets suppose that: i) the possible involvement of other factors including human genetic polymorphisms and diet in the gastric cancer genesis [10] ii) a high bacterial load in patients is a factor of gastric cancer genesis independently of the bacterial genotype. In effect, some studies reported an association between an high rate of the bacterium and the development of severe diseases independently to their virulence factors [52,53].

H. pylori infection is an etiological factor common to GC and DU diseases and may be one of the major causative factors of those opposite end outcome of the infection. So it is practical to find the H. pylori genotypes associated to each disease. Despite the common distribution of the active form of vacA and cagA genes among different H. pylori isolates in patients with DU and GC and the difficulty to distinguish H. pylori strains associated to GC from those associated to DU in the previous studies, the regression analysis results of the present study (Table 6) shows that:

  • Patients infected with vacA i1m1 and vacA i1m2 have a significant 29.73 fold (95%, CI, 5.05–173.73) and 9.17 fold (95%, CI, 2.06–40.82) respectively increased risk of GC in comparison with those infected with vacA i2m2 strains. This significant association let’s suppose that vacA i is more important in predicting the risk of GC in our series. This result support those reported in Iran, Spain, United States and Colombia [5,27,32,37,54] and is inconsistent with studies that report the non usefulness of this gene as a risk factor for GC [5557].
  • The infected patients with strains carrying the vacA s1 allele and cagA gene have a greater probability of developing DU than GC since we have compared GC with DU. This finding supports the role of vacA s1 and cagA+ in the development of ulceration, confirming previous studies in western countries [3,19]. However, a study conducted on Portuguese patients reported the associations of this genotypes with both of GC and DU diseases [25].

These results suggest the presence of separate pathways to discriminate the risk of GC development from DU and suggest that H. pylori genotypes may play an important role in the infection outcomes. However, the apparent discrepancy between our results and those reported in other studies could be related to geographic variations between H. pylori strains, or probably by differences in the population origins or inflammatory responses governed by host genetics. Overall, the association between virulence factors and disease outcomes can be hampered by the difference in food habits and living conditions in each region as well as by dissimilarity of the compared gastric diseases. In fact, none of the previous studies has compared the GC with DU but they used either nonulcer disease or chronic gastritis as control groups. Those groups could be evolved towards GC or DU, inversely DU and GG seems to be two “mutually exclusive” end outcomes of H. pylori infection [44]. Indeed, conclusions data derived from a single geographic region will not be considered as representative of what will be found in other geographical regions.

Conclusions

This study indicates that: i) H. pylori strains isolated from Moroccan patients are extremely diverse, ii) the less virulent strain vacA s2i2m2 is the most predominant in Morocco which can partially explain the African enigma, iii) vacAs1/cagA+ seems to be a risk factor for DU, iv) vacA i1 genotype (independently to vacA m status) isstrongly associated to the GC. So, patients infected with this genotype may need more attention. Therefore, vacA i1 may be of a clinical usefulness to identify patients at a high risk of GC development.

Acknowledgments

We would thank the staffs of the gastroenterology unit and anatomo-pathology department for their help in the collection and preparation of specimens.

Author Contributions

  1. Conceptualization: BB DAB SAI CN.
  2. Data curation: BB MEK SAB.
  3. Formal analysis: KER CN MEK BB.
  4. Investigation: MEK SAB DAB SAI MEA.
  5. Methodology: BB MEK MB.
  6. Project administration: BB.
  7. Resources: BB SAI MB TH MM.
  8. Supervision: BB DAB CN SAI TH MM.
  9. Validation: BB MEK SAB.
  10. Visualization: MEK SAB BB.
  11. Writing – original draft: MEK BB MB SAB.
  12. Writing – review & editing: BB CN KER DAB MM.

References

  1. 1. Alaoui Boukhris S, Amarti A, El Rhazi K, El Khadir M, Benajah D-A, Ibrahimi SA, et al. Helicobacter pylori genotypes associated with gastric histo-pathological damages in a Moroccan population. PloS One. 2013;8(12):e82646. pmid:24349327
  2. 2. Registre des Cancers de la Région du Grand Casablanca 2005–2007 [Internet]. [cited 2016 Aug 10]. Available from: http://www.contrelecancer.ma/fr/documents/registre-des-cancers-de-la-region-du-grand-casab-2/
  3. 3. Atherton JC, Cao P, Peek RM, Tummuru MK, Blaser MJ, Cover TL. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem. 1995 Jul 28;270(30):17771–7. pmid:7629077
  4. 4. Falush D, Wirth T, Linz B, Pritchard JK, Stephens M, Kidd M, et al. Traces of Human Migrations in Helicobacter pylori Populations. Science. 2003 Mar 7;299(5612):1582–5. pmid:12624269
  5. 5. Rhead JL, Letley DP, Mohammadi M, Hussein N, Mohagheghi MA, Eshagh Hosseini M, et al. A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer. Gastroenterology. 2007 Sep;133(3):926–36. pmid:17854597
  6. 6. Ortiz-Princz D, Guariglia-Oropeza V, Avila M, Correnti M, Perrone M, Gutierrez B, et al. Helicobacter pylori cagA and vacA genotypes in Cuban and Venezuelan populations. Mem Inst Oswaldo Cruz. 2010 May;105(3):331–5. pmid:20512250
  7. 7. González CA, Figueiredo C, Lic CB, Ferreira RM, Pardo ML, Ruiz Liso JM, et al. Helicobacter pylori cagA and vacA genotypes as predictors of progression of gastric preneoplastic lesions: a long-term follow-up in a high-risk area in Spain. Am J Gastroenterol. 2011 May;106(5):867–74. pmid:21285949
  8. 8. 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 Off Publ Eur Soc Clin Microbiol Infect Dis. 2012 Feb;18(2):153–9.
  9. 9. Asrat D, Nilsson I, Mengistu Y, Kassa E, Ashenafi S, Ayenew K, et al. Prevalence of Helicobacter pylori vacA and cagA Genotypes in Ethiopian Dyspeptic Patients. J Clin Microbiol. 2004 Jun;42(6):2682–4. pmid:15184452
  10. 10. Alaoui Boukhris S, Benajah D -a., Rhazi K, Ibrahimi SA, Nejjari C, Amarti A, et al. Erratum to: Prevalence and distribution of Helicobacter pylori cagA and vacA genotypes in the Moroccan population with gastric disease. Eur J Clin Microbiol Infect Dis. 2013 Jul;32(7):969–969.
  11. 11. Lage AP, Godfroid E, Fauconnier A, Burette A, Butzler JP, Bollen A, et al. Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens. J Clin Microbiol. 1995 Oct;33(10):2752–6. pmid:8567918
  12. 12. Chattopadhyay S, Patra R, Ramamurthy T, Chowdhury A, Santra A, Dhali GK, et al. Multiplex PCR Assay for Rapid Detection and Genotyping of Helicobacter pylori Directly from Biopsy Specimens. J Clin Microbiol. 2004 Jun;42(6):2821–4. pmid:15184482
  13. 13. Bolek BK, Salih BA, Sander E. Genotyping of Helicobacter pylori strains from gastric biopsies by multiplex polymerase chain reaction. How advantageous is it? Diagn Microbiol Infect Dis. 2007 May;58(1):67–70. pmid:17300903
  14. 14. Ferreira RM, Machado JC, Letley D, Atherton JC, Pardo ML, Gonzalez CA, et al. A novel method for genotyping the Helicobacter pylori vacA intermediate region directly in gastric biopsy specimens. J Clin Microbiol. 2012 Dec;50(12):3983–9. pmid:23035185
  15. 15. Atherton JC. The pathogenesis of Helicobacter pylori-induced gastro-duodenal diseases. Annu Rev Pathol. 2006;1:63–96. pmid:18039108
  16. 16. Frenck RW, Clemens J. Helicobacter in the developing world. Microbes Infect Inst Pasteur. 2003 Jul;5(8):705–13.
  17. 17. Scholte GHA, van Doorn L-J, Cats A, Bloemena E, Lindeman J, Quint WGV, et al. Genotyping of Helicobacter pylori in paraffin-embedded gastric biopsy specimens: relation to histological parameters and effects on therapy. Am J Gastroenterol. 2002 Jul;97(7):1687–95. pmid:12135019
  18. 18. Danon SJ, Luria BJ, Mankoski RE, Eaton KA. RFLP and RAPD analysis of in vivo genetic interactions between strains of Helicobacter pylori. Helicobacter. 1998 Dec;3(4):254–9. pmid:9844066
  19. 19. van Doorn LJ, Figueiredo C, Sanna R, Pena S, Midolo P, Ng EK, et al. Expanding allelic diversity of Helicobacter pylori vacA. J Clin Microbiol. 1998 Sep;36(9):2597–603. pmid:9705399
  20. 20. Ashour AAR, Magalhães PP, Mendes EN, Collares GB, de Gusmão VR, Queiroz DMM, et al. Distribution of vacA genotypes in Helicobacter pylori strains isolated from Brazilian adult patients with gastritis, duodenal ulcer or gastric carcinoma. FEMS Immunol Med Microbiol. 2002 Jul 12;33(3):173–8. pmid:12110479
  21. 21. Talarico S, Gold BD, Fero J, Thompson DT, Guarner J, Czinn S, et al. Pediatric Helicobacter pylori isolates display distinct gene coding capacities and virulence gene marker profiles. J Clin Microbiol. 2009 Jun;47(6):1680–8. pmid:19386830
  22. 22. Kumar S, Kumar A, Dixit VK. Direct detection and analysis of vacA genotypes and cagA gene of Helicobacter pylori from gastric biopsies by a novel multiplex polymerase chain reaction assay. Diagn Microbiol Infect Dis. 2008 Dec;62(4):366–73. pmid:18842375
  23. 23. Ben Mansour K, Fendri C, Zribi M, Masmoudi A, Labbene M, Fillali A, et al. Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients. Ann Clin Microbiol Antimicrob. 2010;9:10. pmid:20302630
  24. 24. Kim JJ, Kim JG, Kwon DH. Mixed-infection of antibiotic susceptible and resistant Helicobacter pylori isolates in a single patient and underestimation of antimicrobial susceptibility testing. Helicobacter. 2003 Jun;8(3):202–6. pmid:12752732
  25. 25. Figueiredo C, Van Doorn LJ, Nogueira C, Soares JM, Pinho C, Figueira P, et al. Helicobacter pylori genotypes are associated with clinical outcome in Portuguese patients and show a high prevalence of infections with multiple strains. Scand J Gastroenterol. 2001 Feb;36(2):128–35. pmid:11252403
  26. 26. Kibria KMK, Hossain ME, Sultana J, Sarker SA, Bardhan PK, Rahman M, et al. The Prevalence of Mixed Helicobacter pylori Infections in Symptomatic and Asymptomatic Subjects in Dhaka, Bangladesh. Helicobacter. 2015 Oct;20(5):397–404. pmid:25827337
  27. 27. Ferreira RM, Figueiredo C, Bonet C, Pardo ML, Liso JMR, Alonso P, et al. Helicobacter pylori vacA intermediate region genotyping and progression of gastric preneoplastic lesions. Am J Gastroenterol. 2012 Jan;107(1):145–6. pmid:22218041
  28. 28. M’itonga LG, Kimanga AN, Ngugi CW, Mutie TM. Association of Helicobacter Pylori VacA Gene Polymorphisms and CagA Gene with Clinical Outcome in Dyspeptic Patients. Int J Health Sci Res IJHSR. 2015;5(9):436–44.
  29. 29. Yordanov D, Boyanova L, Markovska R, Gergova G, Mitov I. Significance of Helicobacter pylori vacA intermediate region genotyping-a Bulgarian study. Diagn Microbiol Infect Dis. 2012 Nov;74(3):253–7. pmid:22951332
  30. 30. Nguyen TL, Uchida T, Tsukamoto Y, Trinh DT, Ta L, Mai BH, et al. Helicobacter pylori infection and gastroduodenal diseases in Vietnam: a cross-sectional, hospital-based study. BMC Gastroenterol. 2010;10:114. pmid:20920280
  31. 31. Fasciana T, Calà C, Bonura C, Di Carlo E, Matranga D, Scarpulla G, et al. Resistance to clarithromycin and genotypes in Helicobacter pylori strains isolated in Sicily. J Med Microbiol. 2015 Nov;64(11):1408–14. pmid:26338221
  32. 32. Basso D, Zambon C-F, Letley DP, Stranges A, Marchet A, Rhead JL, et al. Clinical relevance of Helicobacter pylori cagA and vacA gene polymorphisms. Gastroenterology. 2008 Jul;135(1):91–9. pmid:18474244
  33. 33. Markovska R, Boyanova L, Yordanov D, Gergova G, Mitov I. Helicobacter pylori oipA genetic diversity and its associations with both disease and cagA, vacA s, m, and i alleles among Bulgarian patients. Diagn Microbiol Infect Dis. 2011 Dec;71(4):335–40. pmid:21937185
  34. 34. Schmidt H-MA, Andres S, Nilsson C, Kovach Z, Kaakoush NO, Engstrand L, et al. The cag PAI is intact and functional but HP0521 varies significantly in Helicobacter pylori isolates from Malaysia and Singapore. Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol. 2010 Apr;29(4):439–51.
  35. 35. Hussein NR, Tunjel I, Majed HS, Yousif ST, Aswad SI, Assafi MS. Duodenal ulcer promoting gene 1 (dupA1) is associated with A2147G clarithromycin-resistance mutation but not interleukin-8 secretion from gastric mucosa in Iraqi patients. New Microbes New Infect. 2015 Jul;6:5–10. pmid:26042186
  36. 36. Ogiwara H, Sugimoto M, Ohno T, Vilaichone R-K, Mahachai V, Graham DY, et al. Role of Deletion Located between the Intermediate and Middle Regions of the Helicobacter pylori vacA Gene in Cases of Gastroduodenal Diseases. J Clin Microbiol. 2009 Nov 1;47(11):3493–500. pmid:19726606
  37. 37. Chung C, Olivares A, Torres E, Yilmaz O, Cohen H, Perez-Perez G. Diversity of VacA Intermediate Region among Helicobacter pylori Strains from Several Regions of the World. J Clin Microbiol. 2010 Mar 1;48(3):690–6. pmid:20053862
  38. 38. Boquet P, Ricci V. Intoxication strategy of Helicobacter pylori VacA toxin. Trends Microbiol. 2012 Apr;20(4):165–74. pmid:22364673
  39. 39. Nimri LF, Matalka I, Bani Hani K, Ibrahim M. Helicobacter pylori genotypes identified in gastric biopsy specimens from Jordanian patients. BMC Gastroenterol. 2006;6:27. pmid:17018159
  40. 40. Hussein NR, Mohammadi M, Talebkhan Y, Doraghi M, Letley DP, Muhammad MK, et al. Differences in virulence markers between Helicobacter pylori strains from Iraq and those from Iran: potential importance of regional differences in H. pylori-associated disease. J Clin Microbiol. 2008 May;46(5):1774–9. pmid:18353934
  41. 41. Erzin Y, Koksal V, Altun S, Dobrucali A, Aslan M, Erdamar S, et al. Prevalence of Helicobacter pylori vacA, cagA, cagE, iceA, babA2 genotypes and correlation with clinical outcome in Turkish patients with dyspepsia. Helicobacter. 2006 Dec;11(6):574–80. pmid:17083380
  42. 42. Alarcón T, Domingo D, Martinez MJ, López-Brea M. cagA gene and vacA alleles in Spanish Helicobacter pylori clinical isolates from patients of different ages. FEMS Immunol Med Microbiol. 1999 Jun;24(2):215–9. pmid:10378423
  43. 43. Gangwer KA, Shaffer CL, Suerbaum S, Lacy DB, Cover TL, Bordenstein SR. Molecular evolution of the Helicobacter pylori vacuolating toxin gene vacA. J Bacteriol. 2010 Dec;192(23):6126–35. pmid:20870762
  44. 44. Hansson LE, Nyrén O, Hsing AW, Bergström R, Josefsson S, Chow WH, et al. The risk of stomach cancer in patients with gastric or duodenal ulcer disease. N Engl J Med. 1996 Jul 25;335(4):242–9. pmid:8657240
  45. 45. López-Vidal Y, Ponce-de-León S, Castillo-Rojas G, Barreto-Zúñiga R, Torre-Delgadillo A. High diversity of vacA and cagA Helicobacter pylori genotypes in patients with and without gastric cancer. PloS One. 2008;3(12):e3849. pmid:19050763
  46. 46. Koehler CI, Mues MB, Dienes HP, Kriegsmann J, Schirmacher P, Odenthal M. Helicobacter pylori genotyping in gastric adenocarcinoma and MALT lymphoma by multiplex PCR analyses of paraffin wax embedded tissues. Mol Pathol MP. 2003 Feb;56(1):36–42. pmid:12560462
  47. 47. Lehours P, Ménard A, Dupouy S, Bergey B, Richy F, Zerbib F, et al. Evaluation of the Association of Nine Helicobacter pylori Virulence Factors with Strains Involved in Low-Grade Gastric Mucosa-Associated Lymphoid Tissue Lymphoma. Infect Immun. 2004 Feb;72(2):880–8. pmid:14742532
  48. 48. Miftahussurur M, Sharma RP, Shrestha PK, Suzuki R, Uchida T, Yamaoka Y. Molecular Epidemiology of Helicobacter pylori Infection in Nepal: Specific Ancestor Root. PLoS ONE [Internet]. 2015 Jul 30 [cited 2016 Aug 11];10(7). Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4520618/
  49. 49. Sugimoto M, Zali MR, Yamaoka Y. The association of vacA genotypes and Helicobacter pylori-related gastroduodenal diseases in the Middle East. Eur J Clin Microbiol Infect Dis Off Publ Eur Soc Clin Microbiol. 2009 Oct;28(10):1227–36.
  50. 50. Sugimoto M, Yamaoka Y. The association of vacA genotype and Helicobacter pylori-related disease in Latin American and African populations. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis. 2009 Sep;15(9):835–42.
  51. 51. Letley DP, Rhead JL, Twells RJ, Dove B, Atherton JC. Determinants of non-toxicity in the gastric pathogen Helicobacter pylori. J Biol Chem. 2003 Jul 18;278(29):26734–41. pmid:12738773
  52. 52. Lai Y-C, Wang T-H, Huang S-H, Yang S-S, Wu C-H, Chen T-K, et al. Density of Helicobacter pylori may affect the efficacy of eradication therapy and ulcer healing in patients with active duodenal ulcers. World J Gastroenterol. 2003 Jul;9(7):1537–40. pmid:12854158
  53. 53. Belda S, Saez J, Santibáñez M, Rodríguez JC, Sola-Vera J, Ruiz-García M, et al. Relationship between bacterial load, morbidity and cagA gene in patients infected by Helicobacter pylori. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis. 2012 Jul;18(7):E251–253.
  54. 54. Jones KR, Jang S, Chang JY, Kim J, Chung I-S, Olsen CH, et al. Polymorphisms in the Intermediate Region of VacA Impact Helicobacter pylori-Induced Disease Development. J Clin Microbiol. 2011 Jan 1;49(1):101–10. pmid:21084502
  55. 55. Karlsson A, Ryberg A, Dehnoei MN, Borch K, Monstein H-J. Association between cagA and vacA genotypes and pathogenesis in a Helicobacter pylori infected population from South-eastern Sweden. BMC Microbiol. 2012 Jul 2;12:129. pmid:22747681
  56. 56. Sgouras DN, Panayotopoulou EG, Papadakos K, Martinez-Gonzalez B, Roumbani A, Panayiotou J, et al. CagA and VacA polymorphisms do not correlate with severity of histopathological lesions in Helicobacter pylori-infected Greek children. J Clin Microbiol. 2009 Aug;47(8):2426–34. pmid:19535517
  57. 57. Yamaoka Y, El-Zimaity HM, Gutierrez O, Figura N, Kim JG, Kodama T, et al. Relationship between the cagA 3’ repeat region of Helicobacter pylori, gastric histology, and susceptibility to low pH. Gastroenterology. 1999 Aug;117(2):342–9. pmid:10419915