Histo-epidemiological aspect of Helicobacter pylori chronic gastritis in a Moroccan population

. Helicobacter pylori ( H. Pylori ) is the best example of the implication of chronical infection in carcinogenesis. The World Health Organization recognized it as a class I carcinogen since it triggers the progression of premalignant gastric lesions. The aim of this study is to define the prevalence of H. Pylori infection, related risk factors, and explore the histological features of the chronic gastritis. This is a retrospective study of 248 gastric specimens, examinated and evaluated according Sydney to system. The prevalence of H. Pylori is 67 %, chronic gastritis is observed in all the biopsies (100%). Age and gender were not a risk factors for the H. Pylori infection. Prevalence of H. Pylori was 71.93% in low socio-economic class. The severity of the chronic gastritis increases if the colonization of H. Pylori increases. In 74, 58% of cases chronic gastritis was active. Glandular atrophy presented 10.37% and in 80% was related to H. Pylori infection (p=0,004) and 12 % related to autoimmune diseases. The prevalence of Intestinal metaplasia is 10.53%, and H. Pylori was observed in 42.31% of cases (p=0.001). The dysplasia is detected in one case, in a 72 years old patient. We also observed one case of gastric adenocarcinoma, of an 80 years. Follicular gastritis are in 32% of cases, and they were more frequent in H. Pylori infected slides (82% of subjects) (p< 0.005).According to this study, H. Pylori Chronic Gastritis is very common in our population, and a coherent relationship exists between H. Pylori colonization and pre-cancerous lesions. An early eradicating should be considered as a health goal.


Introduction
H. Pylori is the best example of the implication of chronic infection in carcinogenesis, we estimate that approximately 50 % of the word population is affected by this bacteria [1], While the infection is usually occurs in childhood, there is typically a long period of latency with disease manifestations not appearing until adulthood. Gastric cancer does not usually manifest until old age [2]. A significant differences in prevalence exist both within and between countries. Generally, the overall prevalence is higher in countries of underdeveloped regions, such as Africa and Asia, than in the more developed countries like Western Europe and North America [3]. It has now been well established that infection of the human host with H. Pylori is an important factor in the etiology of gastric-related diseases [4] ranging from active chronical gastritis to gastric cancer. The World Health Organization (WHO) recognized H. Pylori as a class I carcinogen, and estimates that gastric cancer will be the 11th leading cause of mortality by 2030 [5].
Correa proposed a human model of gastric cancer based on histological precancerous lesions in the 1975 [6], in fact gastric cancer is the end result of progressive evolution of changes in the gastric mucosa, starting with chronic gastritis, followed by atrophic gastritis then intestinal metaplasia, dysplasia and finally the point of no return, the invasive adenocarcinoma. The present study was undertaken to define the prevalence of H. Pylori infection, on 248 subject suffering from different gastric pathology that required an endoscopy, studying the effect of different epidemiological factors such as, age, gender, socioeconomic status, and explore the histological features of chronic gastritis, with special emphasis on the presence of premalignant changes of the gastric mucosa, such as IM, atrophy, and dysplasia presence or absence of H. Pylori, activity (neutrophilic infiltration) of gastric mucosa and presence or absence of lymphoid aggregates.

Study identification and extraction of data
It's a retrospective descriptive study performed on all gastric biopsies received in the pathological anatomy and cytology laboratory of EL Idrissi Public Hospital, Kenitra, Morocco, between March 2017 and March 2019. 248 specimens were collected, belonging to patients who had Esophagogastroduodenoscopy (EGD) due to different gastric disorders. Data including age, gender, indication for EGD, and pathology reports were acquired from the laboratory medical records.

Histopathology
Biopsy specimens, fixed in 10% buffered formaldehyde solution, embedded in paraffin, and cut in sequential 4μm sections, and stained with hematoxylin-eosin (HE). All the cases were evaluated according updated Sydney scoring system. The following items were evaluated separately: H. Pylori colonization, presence of an inflammatory component (the amount of neutrophilic infiltration), presence of a chronic inflammatory component (the amount of lymphoplasmacytic infiltration), mucosal atrophy, intestinal metaplasia, lymphoid follicles, dysplasia, and cell changes. All these items were scored to mild, moderate, severe.

Statistical Analysis
The data were entered and analyzed using the SPSS software (Version 25, SPSS Inc., Chicago, IL, USA). Chi-square test (Pearson value) was used. A p-value less than 0.05 was considered statistically significant.

Epidemiological data
248 patient were included in this study. The main age was 49.45 ± 17.41(n = 248, range = 16-80 years), the male to female ratio was 0.7, there was 107 males (43.1%) and 141 Females (56.9%). When we investigated the presence of H. Pylori in our specimens, we found that 171 subjects are affected with a prevalence of 67%.

Gender
The repartition according to gender showed that the prevalence of H. Pylori infection was higher in females comparing to males (39.77% vs. 60.23%) (Figure1). Despite of that finding, there was no statistically significant relationship between gender and intensity of H. Pylori infection. (p=0.109).

Age
We performed an analysis to the prevalence of H. pylori by age groups, For this analysis, the age was divided into five subgroups as follows: ≤19 years, 20 to 39 years, 40  Pylori as well as chronic gastritis seems, to be present in all age groups and there was no statistical difference concerning the prediction to H. Pylori infection (p=0.07). Hence the factor age was not associated to an increased risk of infection. (Figure 2).

Socioeconomic status
In this study we divided patients into two groups according to their national health coverage system, and we investigated the frequency of H. Pylori infection in each group. The first group is patients who paid their tests or benefiting from a mandatory healthcare insurance (AMO). The other group 60.23 %

Frequency of Helicobacter Pylori
Age groups comprises patients holding RAMED cards, which is a medical assistance plan for the neediest, allowing them to benefit from free public healthcare services. National health coverage system has been used as a proxy marker of socioeconomic status and an important determinant of H. Pylori prevalence in resource limited settings, we considered that patients that were benefiting from RAMED belong to low socioeconomic class and those who were benefiting from AMO or cash paying their medical cares are considered as middle to upper social class. The results as in figure 3, revealed a statistically significant difference between the two groups since the first group was highly infected comparing to the second one (71.93% Vs 28.07%) (p=0.04) Fig.3. Distribution of H. Pylori according to national health coverage system

Histological Features
All 248 analyzed samples showed all histological chronic gastritis, otherwise 100 % of our population is suffering from chronic gastritis, the prevalence of H. Pylori is 67 %.
x Gastritis Intensity Among 248 subjects with chronic chronic gastritis, 80 (32.26%) had mild intensity, 134 (54.03%) moderate gastritis, and 34 (13.71%) had severe gastritis. We investigated the relation between density of Helicobacter pylori colonization in gastric mucosa and the gastric mucosal inflammation severity, we found out that, the severity of chronic gastritis was mostly reported as mild in people that are not infected or had mild H. pylori colonization respectively 61.04% and 52.27%, As shown in figure 4, the severity of the chronic gastritis tends to increase as long as the colonization of H. Pylori increases. There was a significant statistical relationship between the incidence of H. pylori infection and the severity of chronic gastritis (p < 0.001).
x Gastritis Activity In term of activity, the active chronic gastritis presented 74.58% of our samples, with different grade, 35.42% mild, 28.33% moderate, and 10.83% severe. The evaluation of the association between the CG activity and the H. Pylori infection revealed a significant difference.  Figure 5, we notice that patients with no Helicobacter Pylori infection have a highest rate of nonactive gastritis (74.29%), in contrast those with severe H. Pylori colonization are mostly moderate to severely active; respectively 42.11% and 31.58%. The level of activity increases by increased intensity of colonization there was a statistically significant association between the severity of H. pylori infection and the activity of chronic gastritis P < 0.005).

Fig.5. Association between the activity of the gastritis and intensity of H. Pylori infection
x Glandular atrophy In our study glandular atrophy presented 10.37% (n = 25), and 80% of those atrophic gastritis were associated with H. Pylori infection (p = 0.004) and 12% related to autoimmune diseases (Pernicious anemia), in our study, there was no statistical correlation between Atrophic Gastritis and autoimmune diseases (p = 0.68). x Intestinal metaplasia 10.53% gastric biopsies have shown Intestinal metaplasia (n = 26), and H. Pylori was observed in 42. 31% of cases (n = 11). Also, there was a statistically significant relationship between H. pylori infection and the intestinal metaplasia (p = 0.001).
x Epithelial dysplasia The dysplasia is detected in one case, in a 72 years old patient, accompanied with a metaplasia and it had very low density of H. Pylori.
x Adenocarcinoma We also observed one case of gastric adenocarcinoma, of an 80 years old woman, the specimen had a low density of H. Pylori.
x Lymphoid follicles Follicular gastritis (lymphoid follicles formations) are in 32% of cases (n = 79), and they were more frequent in H. Pylori infected slides. 82% of subject carrying H. Pylori have shown presence of lymphoid follicles. There was a statistically significant association between the two last variables (p < 0.005).

Discussion
H. Pylori is known to colonize more than half of the world's population, but it prevalence varies depending on different contributing factors: socioeconomic status, living standards, ethnicity and geographical location [7]. The present study showed that the prevalence of H. Pylori infection is 67%, similar result was observed in other Moroccan studies [8][9][10], and in other developing countries like Saudi Arabia 65.9, and India 63.5. But it's too quite low compared to neighboring countries like Tunisia (72%) [11], Mauritania (76.4%) [12], Algeria (78%) [13].this number has been declining in developed and industrialized countries of the western world. Switzerland had the lowest reported H. Pylori prevalence (18.9%), 35% in the United Kingdom, and 26% in Sweden. [11] The prevalence of H. Pylori infection according to age is joining several studies that reported no significant difference of H. Pylori infection between different adult age groups [14][15][16][17], Nevertheless some studies reported that this prevalence increases with the age [18,19]. Zamani et al [20] explained this tendency by either birth cohort effect or accumulated risk of infection by age, as the risk of exposure increases with the age, and the infection occurs at a young age. Few studies were found about the H. Pylori infection age decreasing. [21,22] In terms of gender, our findings indicated that, there were no statistically significant differences in the prevalence of H. Pylori between men and women, both are exposed similarly to be infected by this bacteria and several studies sustain this result. [16,20,21]. Although there are controversial reports about this relation, Some authors reported a high prevalence of H. pylori among females [18]. While other authors reported a high rate of H. pylori infection among males [19]. Gender disparity in H. pylori infection is an intriguing topic. Our results revealed that low socio-economic status is a predictive factor to helicobacter pylori infection, 71.93% of low-income patients were infected (p = 0.04). Indeed, a large part of the population that goes to Provincial Kenitra Public Hospital are coming from rural and surrounding communes where the poverty rate (9.7%) is quiet higher than the national average (4.8%) [23]. Lifestyle and basic sanitation conditions favors person-to-person transmission of the microorganism. Hence low socio-economic status families with low income had a higher likelihood of carrying H. Pylori infection. More than few Moroccan and international studies confirmed this fact [10,16,24,25]. CG has been histologically confirmed in all the specimens (100%), which is a very high value. This prevalence can be explained by the fact that all the patient suffered from different clinical gastric disorders before getting gastro-oduodeno-fibroscopy. H. Pylori is found in 67 % of cases , the only similar study, which has been conducted in the same region by Attaf et al. in 2004 reported the same prevalence [26], nevertheless our finding is in the limit of a lot of recent Moroccan studies [8,27,28]. The degree of H. Pylori colonization was significantly associated to the severity of CG. In fact ,The host Immune response to H. pylori invasion make up a complex network of inflammatory mediators (cytokines and chemotactic factors) that will have a direct cytopathic effect and will initiate the gastric inflammation that will result on lymphocyte and plasmocyte cell infiltration [29]. A meta-analysis conducted by Rokkas et al. in 2017, affirms, that H. pylori is a significant risk factor for GC and that its eradication is associated with a reduction in the incidence of GC [30]. Few studies rejected this notion of association [31]. H. Pylori induces epithelial cells to release chemokines that are chemotactic for neutrophils that infiltrates the lamina propria of the gastric mucosa and that defines the activity [32]. Neutrophil infiltration in the lamina propria is generally accepted to be the unanimous indicator of H. Pylori gastritis. A study conducted by Ghasemi et al. in 2017, reported that there was a statistically significant correlation between the intensity of H. Pylori colonization and chronic gastritis [33], and this is in agreement with our findings and those of other researchers on the topic [32,34], that confirm a coherent relationship. Though the literature shows some disagreeing results, both Choudhary et al. and Park et al. are attesting that H. Pylori infection doesn't have any effect on activity of chronic gastritis, which can be related to genetic differences, nutritional habits, and environmental factors between the studied populations [31,35]. The development of CG constitutes a complex progression of events, characterized mainly by interactions between H. Pylori infection, host factors  [32,45]. In fact The significant association of H. Pylori and lymphoid follicles in this study indicates that the bacterium may induce local humoral and T-cell response in the gastric mucosa. H. Pylori infection triggers the development of MALT tissue, which can act as a nest for the potential development of a lymphoma.

Conclusion
In the model of gastric carcinogenesis, H. pylori plays a pivotal role in the initiation and the progression of chronic active gastritis. The infection progress over years through the sequential stages of atrophic gastritis, Intestinal Metaplasia, dysplasia and to gastric adenocarcinoma. Therefore, it seems that an early screening and an early eradicating at an early stages of the infection is the most viable strategy to prevent the long term lesions of the chronic gastritis, it should be considered as a primordial health goal.