Helicobacter pylori genotypes, salt intake, and sociodemographic factors associated with premalignant gastric lesions in a Colombian population. A case control study.

An analytical study was conducted including cases (patients with gastric atrophy, intestinal metaplasia, and gastric dysplasia) and controls (patients with nonatrophic gastritis). Sociodemographic information and information about salt intake were obtained using a questionnaire. Histopathological diagnosis was performed according to the Sydney System. The cagA and vacA genotypes were established using polymerase chain reaction in paraffin blocks. ANOVA was used for analyzing quantitative variables. Categorical variables are presented as proportions and absolute frequencies. The effect of each variable on the study outcome (premalignant lesion) is presented as odds ratio (OR) and 95% CI. A p -value of <0.05 was considered as statistically significant. The genotype, and salt intake are indicators of the risk of developing premalignant lesions of the stomach in the study population.

It is challenging to predict the risk of progression, and this risk can be modulated by various genetic and environmental factors including salt intake habits and genetic variability of Helicobacter pylori (9,10). For example, the vacA/s1m1 and cagA+ genotypes have been shown to be associated with an increased risk of presenting precursor lesions of gastric malignancy (11)(12)(13)(14)(15)(16).
Few studies have been conducted in Colombia in which bacterial genotypes (17)(18)(19) or salt intake (20) has been associated with the onset of premalignant lesions of the stomach. In these studies, the approach is based on comparing genotype frequencies, and the results showed contradictory findings. However, to the best of the authors' knowledge, to date, no investigation has evaluated the interactive effect of the genetic variability of H. pylori on salt intake and sociodemographic factors. Therefore, the aim of the present study was to estimate the association among H. pylori genotypes, salt intake, and sociodemographic factors with regard to the precursor lesions of gastric malignancy (atrophy, metaplasia, and dysplasia) in the Cauca population.

Materials And Methods
An unpaired case-control analytical study was conducted with patients admitted to the Gastroenterology Units of the San José University Hospital and Endovideo in the city of Popayán (located in southwestern Colombia, in the mountainous area of the Andes, and is considered a high-risk area of gastric cancer) from January 2008 to December 2011.
Samples were collected by convenience sampling, and included patients were aged >18 years with a histopathological diagnosis of nonatrophic gastritis (controls) and those with precursor lesions of gastric malignancy (cases). Inclusion criteria included the following: participants should be born in a municipality of Cauca; should be children of parents from Cauca; and should be diagnosed with H. pylori infection determined by histopathological tests and corroborated by molecular diagnostics [polymerase chain reaction (PCR)].
Participants with a history of gastric surgery, who received treatment for H. pylori infection, who had an HIV infection, and who had gastric adenocarcinomas other than of the intestinal histotype were excluded.
Prior to sample collection, participants voluntarily signed an informed consent form and were interviewed via a survey to obtain information on sociodemographic and clinical variables. They were also queried regarding their salt intake habit using the question "Do you add additional salt to the food served on the table?" and three response categories were assigned-always, sometimes, or never.
Gastric samples were collected by experienced gastroenterologists via gastrointestinal endoscopy. Patients underwent gastrointestinal endoscopy following referral for dyspeptic symptoms and after fasting for at least 8 h. Although participants were not sedated, they received topical oropharyngeal anesthesia. Five samples corresponding to two antral biopsies (major and minor curvature), two corpus biopsies (major and minor curvature), and one incisura angularis biopsy were obtained. These samples were fixed in buffered formalin and stained with hematoxylin-eosin and Giemsa stains. To control biases associated with sociodemographic information and salt intake, biologists and doctors who belonged to the GIGHA group were trained for standardizing questions in a closed questionnaire, which was completed before gastrointestinal endoscopy.
To reduce biases associated with histopathological information, diagnoses were validated by a different pathologist who was unaware of the previous diagnosis. In cases wherein diagnosis differed, the case was jointly re-evaluated to reach diagnostic consensus. To limit disagreement in cases of gastric dysplasia, they were grouped into a single category that included low-and high-grade gastric dysplasia. On the other hand, the histopathological diagnosis of H. pylori infection was corroborated using Giemsa staining and PCR.
Molecular diagnostics were conducted according to globally accepted protocols; the equipment was calibrated, and pilot tests were performed to verify the quality of reagents and extraction kits.
To determine the behavior of variables for each histopathological lesion, the frequencies of each precursor lesion of gastric malignancy were compared with the control group.
Thereafter, patients with precursor lesions were regrouped as a single category for comparison with the control group.
Mean differences in age were evaluated using one-way ANOVA, along with post-hoc tests.
Differences in proportions were evaluated using the chi-squared test of independence. The average patient age was 43 years for the nonatrophic gastritis group, 52 years for the atrophic gastritis and metaplasia groups, and 63 years for the dysplasia group. When average age was compared using ANOVA single-factor test, significant differences were observed among all groups (p = 0.000). Post-hoc analysis showed significant differences between each precursor lesion of gastric malignancy and gastric cancer with chronic nonatrophic gastritis (p = 0.000).
Regarding age, the most prevalent age group in the control category was 18-40 years; in the atrophic gastritis and metaplasia group, the prevalent age group was 41-60 years, whereas in the dysplasia group, the prevalent age group was >60 years. Regarding salt intake, 235 participants including 100 (43%) with nonatrophic gastritis denied the use of additional salt in their meals. On the other hand, 63 participants including only 19 (30%) with nonatrophic gastritis stated that they always added additional salt to their meals.
Patient distribution according to age, sex, and salt intake is shown in Table 2. Each precursor lesion of gastric malignancy and cancer was compared with the chronic nonatrophic gastritis group to estimate the measures of association. Female sex, age of 18-40 years, and the "never add salt to meals" groups were selected as control categories. Analyses showed significant associations between the age groups of >40 and >60 years. Salt intake was associated with the development of intestinal metaplasia, whereas the income category lower than one minimum wage was associated with gastric dysplasia (Table 3).  Table 4. To facilitate analyses and considering the absence of the s2m2 genotype in the dysplasia category, patients with gastric atrophy, intestinal metaplasia, and gastric dysplasia were grouped into a single category (precursor lesions of gastric malignancy) and compared with the control group. Chi-squared test showed significant differences among the vacA genotypes (p = 0.025) and showed no differences regarding the cagA genotype (p > 0.05).
To analyze the measures of association, the vacA/s2m2 and cagA-genotypes were selected as control categories (Table 5).  Table 6 illustrates the risk of developing precursor lesions of gastric malignancy according to age-adjusted vacA genotypes and the interactive effect enhancer of salt intake.  (22,23), showing a direct correlation between the severity of precursor lesions of gastric malignancy and age.
The greatest age-related risk is due to genomic instability acquired over the years owing to chronic inflammation, cumulative damage by free radicals, and inefficiency of DNA repair mechanisms (24)(25)(26)(27). On the other hand, normal gastric mucosa reportedly lacks telomerase activity, and a progressive increase in the activity of this enzyme is directly related to premalignant lesions and cancer (28). Other studies and the present investigation suggest that preneoplastic lesions represent histological changes caused by tissue aging and dysfunctional adaptive responses, thereby increasing the risk of tumors.
Regarding bacterial genotypes, the s1m1 genotype was more prevalent in the case group, whereas the s2m2 genotype was more prevalent in the control group. Similar results have been reported by Colombian and foreign authors (29)(30)(31). The role of the s1m1 genotype can be explained via different mechanisms such as the synthesis of a vacuolizing protein, which induces greater epithelial damage, development of a more persistent inflammation, and blockage in the proliferation of T lymphocytes via its arrest in the G1 or S phase of the cell cycle (32)(33).
In a recent meta-analysis, 33 studies were evaluated, which overall included 2697 controls and 1446 cases with gastric cancer and precursor lesions of gastric malignancy. In that study, the s1 allele showed an increased risk of gastric atrophy Similarly, the analysis of genotype distribution by diagnostic category ( Table 4) helps conclude that the prevalence of the s1m1 subtype increases with the increase in the severity of premalignant lesions, whereas the opposite seems to occur for the s2m2 genotype, suggesting a proportional relationship between the severity of the lesion and bacterial genotype. These findings highlight the conceptual value of the carcinogenesis model proposed by Dr. Correa and provide an important theoretical basis for its predictive capacity for cancer risk.
The carcinogenic effect of the cagA gene product is attributable to diverse mechanisms such as the reorganization of the cytoskeleton of epithelial cells, change in cell phenotype, and activation of signaling pathways that stimulate cell proliferation (35)(36)(37).
These mechanisms would partly explain a higher incidence of gastric cancer in populations wherein approximately 90% isolates are cagA+ and a lower incidence of gastric cancer wherein the prevalence of cagA+ is lower (38,39). In the present study, the prevalence of the cagA+ genotype between case and control groups did not significantly differ, and a relationship between the cagA genotype and development of precursor lesions of gastric malignancy was not documented. These results differ from those reported in the literature (16,40,41). A possible explanation for this finding is related to polymorphisms of the cagA gene and phosphorylated EPIYA motifs. For example, it has been proposed that polymorphisms of the cagA gene and phosphorylated EPIYA motifs modulate the risk of diseases such as duodenal ulcer, degree of inflammation, and risk of gastric cancer (42,43).
The role of salt intake in the genesis of precursor lesions of gastric malignancy has been evaluated in other investigations. Although methods to quantify salt intake differ among studies, a positive relationship with gastric pathology has consistently been demonstrated (44). Further, the present study evaluated bacterial coinfection, whose role in the development of gastric pathology is difficult to determine. For example, it has been suggested that coinfection generates a competitive growth disadvantage for the bacteria or favors growth at certain mucosal sites that would serve as niches (29). The present study shows that coinfection increases the risk of developing premalignant lesions and cancer, albeit at a much lower level than the s1m1 genotype. These results are difficult to compare with those in the literature because coinfection is associated with pathologies such as duodenal ulcer but not with the development of precursor lesions of gastric malignancy (52). This challenge is more evident in the inability to assign a particular pathological effect in cases wherein >1 bacteria are detected. However, the adjusted multivariate model showed that the regular salt intake habit increases the risk of developing preneoplastic lesions by >3 folds, suggesting that bacterial coinfection can have a primary injurious effect on gastric mucosa that could further be enhanced via dietary factors. This association of coinfection was evident in the logistic regression model and not in the bivariate model, which is consistent with the multifactorial nature of gastric cancer.
One of the limitations of the present study could be derived from the participants' perception of the harmful effect of salt intake on human health. This perception could eventually modify their responses, thereby generating the Hawthorne effect. In an attempt to limit this effect, the questionnaire was completed before gastrointestinal endoscopy was performed, without the knowledge of the gastrointestinal endoscopic and histopathological diagnoses. Quantification of 24-h urine sodium excretion may be recommended in future studies for quantitative and precise assessment of salt intake.
The study results suggest that the s1m1 genotype is associated with the precursor lesions of gastric malignancy and that this association is strengthened with an increase in age and salt intake. On the other hand, it can be concluded that the severity of premalignant lesions of gastric malignancy is directly correlated with advanced age and cytotoxic H. pylori genotypes.

Declarations
Ethics approval and consent to participate: Participants provided their consent to participate in the study and signed informed consent. The study was approved by the Scientific Research Ethics Committee of the Cauca University.
Availability of data and materials: Data that support the findings of this study are available from Acosta-Astaiza CP (author) but restrictions apply to the availability of these data, which were used under license for the present study and so are not publicly available. However, data are available from the authors upon reasonable request and with permission of Acosta-Astaiza CP.

Financing
This study was funded by the Colciencias Health Program, project code 1103-519-29123.
drafting and revising the manuscript. All authors approved the final version of the manuscript.